TW200521143A - Hydroxyalkyl starch derivatives - Google Patents

Abstract

The present invention relates to a method of producing a hydroxyalkyl starch derivative comprising reacting hydroxyalkyl starch of formula (I) at its reducing end which is not oxidized prior to said reaction, with a compound of formula (II) R'-NH-R" (II) wherein R1, R2 and R3 are independently hydrogen or a linear or branched hydroxyalkyl group, and wherein either R' or R" or R' and R" comprise at least one functional group X capable of being reacted with at least one other compound prior to or after the reaction of (I) and (II), as well as to the hydroxyalkyl starch derivative as such , obtain-able by said method, and to a pharmaceutical composition comprising said hydroxyalkyl starch derivative.

Description

200521143 Α7 Β7 五、發明說明（1) 【發明所屬之技術領域】 本發明係關於經基烧基殿粉衍生物，特別為藉由羥基 烧基澱粉與連接子化合物之一級或二級胺基進行反應之方 法而可得之經基烧基澱粉衍生物。根據特別佳之具體例， 本發明係關於羥基烷基澱粉衍生物，其係可根據羥基烷基 澱粉與連接子化合物之一級或二級胺基進行反應，且產生 的反應產物係與多胜肽，較佳係與醣蛋白且特別佳係與紅 血球生成素，經由連接子化合物之至少一個其他反應基图 進行反應的方法而獲得者。經基烧基澱粉之特別佳者為裡 基乙基殿粉。根據本發明’經基烧基殿粉且宜為觀基乙基 澱粉與連接子化合物係在其還原端（其不會在該反應之前 被氧化)上進行反應。 【先前技術】 經濟部智慧財產局員工消費合作社印製 羥基乙基澱粉(HES)係為天然生成之澱粉果膠的衍生 物且係以身體中之α·澱粉酶降解。HES係為經取代之醣分 子聚合物澱粉果膠衍生物，其係以至多95重量％之濃度 存在於玉米澱粉中。HES具有有利的生物性質且被用作為 血容體替代劑及用於臨床血液稀釋治療中（桑馬梅爾等， 1987，醫藥學，8(8)，271-278 ;及威德勒等，1991，藥物 研究，41，494-498)。 澱粉果膠包括葡萄糖部份，其中，於主鏈中存有α-1,4-配糖鍵且於分支部位上發現α-ΐ，6_配糖鍵。該分孑之物 理-化學性質主要係藉配醣鍵之類型來測定。由於多肽鍵 -3- A7 B7 200521143 五、發明說明（2) 蛋白鏈破損(nicked)的α-1，4-配糠鍵，而產生每轉具有大約 六個葡萄糖單體之螺旋結構。聚合物之物理-化學以及生 物化學性質可經由取代而改質。羥基乙基基困之導入係經 由驗經基乙基化作用而達成Q藉著適應反應條件，可能針 對羥基乙基化作用開拓未經取代之葡萄糖單體中分別的羥 基基團之不同的反應性。由於此種事實，精於此方面技藝 之人士可影響取代模式之程度有限。 某些製造羥基烷基澱粉衍生物的方式係說明於此方面 技藝中。 DE 26 16 086揭示血紅朊對羥基乙基澱粉之共軛，其 中，第一步驟中係將交聯劑，例如，bromocyane，連接到 羥基乙基澱粉且接著將血紅朊連接到中間體產物。 HES使用之一重要領域係多胜肽之穩定化，其係例 如，被應用於循環系統以便獲得特別的生理效應。此等多200521143 Α7 Β7 V. Description of the invention (1) [Technical field to which the invention belongs] The present invention relates to a base-based powder derivative, and is particularly carried out by using a hydroxyl-based starch and a linker compound as a primary or secondary amine group. The method is based on a calcined starch derivative. According to a particularly preferred embodiment, the present invention relates to a hydroxyalkyl starch derivative, which can react according to a hydroxyalkyl starch with a primary or secondary amine group of a linker compound, and the reaction product produced is a polypeptide, Preferred are those obtained by reacting with a glycoprotein and particularly preferably with erythropoietin via at least one other reactive group map of a linker compound. The particularly good base-based starch is rythyl ethyl powder. According to the present invention, the reaction is performed on the reduced end (which will not be oxidized before the reaction) of the base powder and preferably a methyethyl starch and a linker compound. [Previous Technology] Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs, Hydroxyethyl Starch (HES) is a derivative of naturally occurring starch pectin and is degraded by alpha amylase in the body. HES is a substituted sugar polymer polymer starch pectin derivative, which is present in corn starch at a concentration of up to 95% by weight. HES has favorable biological properties and has been used as a blood volume replacement agent and in clinical hemodilution therapy (Sanmamel et al., 1987, Medicine, 8 (8), 271-278; and Weidler et al., 1991, Pharmaceutical Research, 41, 494-498). Starch pectin includes a glucose moiety, in which α-1,4-glycoside bonds are stored in the main chain and α-ΐ, 6_glycoside bonds are found at the branching sites. The physico-chemical properties of this tiller are mainly determined by the type of sugar bond. Due to the peptide bond -3- A7 B7 200521143 V. Description of the invention (2) The α-1,4-coordination bond of the protein chain is broken (nickel), resulting in a helical structure with approximately six glucose monomers per revolution. The physical-chemical and biochemical properties of polymers can be modified by substitution. The introduction of hydroxyethyl group is achieved through the test of ethoxylation. By adapting the reaction conditions, it is possible to open up different reactions of the respective hydroxy groups in the unsubstituted glucose monomer for hydroxyethylation. Sex. Due to this fact, there is a limited degree to which persons skilled in this area can influence the replacement model. Certain ways of making hydroxyalkyl starch derivatives are described in this art. DE 26 16 086 discloses the conjugate of hemoglobin to hydroxyethyl starch. In the first step, a cross-linking agent, such as bromocyane, is attached to the hydroxyethyl starch and then hemoglobin is attached to the intermediate product. One important area of use of HES is the stabilization of dopeptides, which are, for example, applied to the circulatory system in order to obtain special physiological effects. So much

胜肽之一特定實例為紅血球生成素，其係為大約34,000kD 之酸醣蛋白，主要係調節循環中紅血細胞的含量。 熟知的多胜肽與酶之施用問題是此等蛋白質經常具有 令人不滿意的穩定性。尤其是紅血球生成素具有相當短的 鏗濟部智慧財產局員工消費合作社印製 血漿半生期（史畢維克與豪根，1989，血液73，90 ;麥克 馬宏等，1990，血液76，1718)。此係指治療血漿含量會 迅速流失且必須進行重複性靜脈給藥。此外，於某些情況 中，觀察到免疫反應對抗胜肽。 一般相信，當多胜肽偶合至聚合分子時可改良多胜肽 之穩定性且減緩免疫反應對抗此等多胜肽。WO 94/28024 -4- A7 B7 200521143 五、發明說明（3 ) 分. 係揭示經聚乙二醇(PEG)改良之生理活性多胜肽具有減低 的致免疫性及抗原力且於血流中之循環比未經共輛之蛋白 質長很多，亦即，具有較長的清潔率。然而，PEG-藥物輛 合物具有許多缺點，例如，其等不具有可被生體内降解途 徑之元素認定之天然結構。因此，除了 PDG-輛合物之 外，其他輛合物與蛋白質聚合體(polymerates)業已產生。 用於交聯不同蛋白質與大分子，例如，聚合酶之多數方法 業經說明於文獻中。（參閲，例如，王氏，蛋白質共輛與 交聯之化學，1993，CRCS公司）。 揭示於此方面技藝中之HES-藥物輛合物具有HES不 在藥物特定部位上共輛的缺點。所以，由於共輛階段中3-因次結構破壞’共輛會造成具有許多組成份可能為不活性 之極龐雜產物。因此，需要具有改良穩定性及/或生物活 性之經進一步改良的HES-多胜肽轆合物。 經濟部智慧財產局員工消費合作社印製 製造此等輛合物之一方法係用HES經氧化的型式作為 起始物質與交聯化合物進行反應，其中，產生的產物係與 多胜肽進行反應或經進一步改良且接著與多胜肽進行反 應。此方法之主要缺點係在第一步驟中原有的HES必需 選擇地被氧化，通常係在其還原端上，藉著將終端的醛基 及/或半縮酸基團氧化成内酯，如此，使得聱個過程更困 難且昂貴。 WO 02/08079 A2係揭示包含活性劑與羥基烷基澱粉之 軛合物的化合物，其中，活性劑及羥基烷基澱粉係直接或 經由連接子化合物連接。就直接鍵合而言，活性劑與羥基One specific example of a peptide is erythropoietin, which is an acid glycoprotein of about 34,000 kD, which mainly regulates the content of red blood cells in the circulation. A known problem with the administration of dopeptides and enzymes is that these proteins often have unsatisfactory stability. In particular, erythropoietin has a relatively short half-life of plasma printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs (Spike and Hagen, 1989, Blood 73, 90; McMahon et al., 1990, Blood 76, 1718 ). This refers to the rapid loss of therapeutic plasma and the need for repeated intravenous administration. In addition, in some cases, an immune response was observed against the peptide. It is generally believed that when peptides are coupled to polymeric molecules, the stability of the peptides can be improved and the immune response can be slowed against these peptides. WO 94/28024 -4- A7 B7 200521143 V. Description of the invention (3) points. It is revealed that the polyethylene glycol (PEG) modified physiologically active peptide has reduced immunogenicity and antigenicity and is in the bloodstream. The cycle is much longer than that of the uncoated protein, that is, it has a longer cleaning rate. However, PEG-drug compounds have many disadvantages, for example, they do not have a natural structure that can be identified by elements that can be degraded in vivo. Therefore, in addition to PDG compounds, other compounds and polymerates have been produced. Most methods for cross-linking different proteins and macromolecules, such as polymerases, have been described in the literature. (See, for example, Wang's, Protein Co. and Cross-linking Chemistry, 1993, CRCS). The HES-pharmaceutical composition disclosed in this aspect of the art has the disadvantage that HES does not share vehicles at specific sites of the drug. Therefore, due to the 3-dimensional structural damage in the common vehicle stage, the common vehicle will result in extremely complex products with many components that may be inactive. Therefore, there is a need for a further improved HES-polypeptide conjugate having improved stability and / or biological activity. One of the methods of printing and manufacturing these compounds by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs is to use the oxidized form of HES as the starting material to react with the cross-linking compound, where the product produced is reacted with the peptide or It was further improved and then reacted with the peptide. The main disadvantage of this method is that the original HES must be selectively oxidized in the first step, usually on its reducing end, by oxidizing the terminal aldehyde group and / or hemiacetic acid group to lactone, so, This makes the process more difficult and expensive. WO 02/08079 A2 discloses a compound comprising a conjugate of an active agent and a hydroxyalkyl starch, wherein the active agent and the hydroxyalkyl starch are linked directly or via a linker compound. For direct bonding, the active agent is

200521143 A7 B7 五、發明說明（4) 烷基殿粉之反應係在包含至少10重量％水之水性介質中 進行。沒有給予直接連接到藉由羥基烷基澱粉於其還原端 上與包含-NH-結構單位之交聯化合物於水性介質中進行反 應而產生之羥基烷基澱粉衍生物的實例。所有的實例係針 對在進一步反應之前被氧化之羥基烷基澱粉，因此WO 02/08079 A2之特定教導具有前文所提的缺點。 因此，本發明之目的係提供製造容許羥基烷基澱粉於 其還原端上與適當的化合物進行反應，其中殿粉之還原端 於該反應前不會被氧化之羥基烷基澱粉衍生物的方法。 本發明另一個目的係提供製造容許羥基烷基澱粉於其 還原端上與一適當的化合物進行反應，其中殿粉之還原端 於該反應前不會被氧化之羥基烷基澱粉衍生物的方法，該 方法另外的特徵在於羥基烷基澱粉於其還原端與適當的化 合物進行反應之反應產物將進一步與至少另一種化合物進 行反應。 本發明仍有另一個目的係提供如上所述的方法，其 中，至少另一種化合物係為多胜肽，較佳為蛋白質，更佳 為紅血球生成素。 經濟部智慧財產局員工消费合作社印製 本發明還有另一個目的係提供藉由前文所述的方法而 可得之羥基烷基澱粉衍生物，其包括羥基烷基澱粉係於其 還原端與適當的化合物進行反應，其中，澱粉的還原端不 會在反應前被氧化。 【發明内容】 因此，本發明係關於製造羥基烷基澱粉衍生物的方 -6- 200521143 A7 B7 五、發明說明（5) 法，其包括將具式(I)之羥基烷基澱粉(HAS)200521143 A7 B7 V. Description of the invention (4) The reaction of the alkyl powder is carried out in an aqueous medium containing at least 10% by weight of water. No examples are given of direct attachment to hydroxyalkyl starch derivatives produced by reacting hydroxyalkyl starch on its reducing end with a cross-linking compound containing -NH- structural unit in an aqueous medium. All examples are directed to hydroxyalkyl starch which is oxidized before further reaction, so the specific teaching of WO 02/08079 A2 has the disadvantages mentioned above. Therefore, an object of the present invention is to provide a method for producing a hydroxyalkyl starch which allows a hydroxyalkyl starch to react with an appropriate compound on the reducing end thereof, wherein the reduction of the powder is based on a hydroxyalkyl starch derivative which will not be oxidized before the reaction. Another object of the present invention is to provide a method for producing a hydroxyalkyl starch derivative that allows a hydroxyalkyl starch to react with an appropriate compound on its reducing end, wherein the reducing end of the powder is not oxidized before the reaction, This method is further characterized in that the reaction product of the hydroxyalkyl starch which reacts with a suitable compound at its reducing end will further react with at least another compound. Still another object of the present invention is to provide the method as described above, wherein at least another compound is a polypeptide, preferably a protein, more preferably erythropoietin. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs, the present invention has another object to provide hydroxyalkyl starch derivatives obtainable by the method described above, including hydroxyalkyl starch at its reducing end and appropriate The compound undergoes a reaction in which the reducing end of the starch is not oxidized before the reaction. [Summary of the Invention] Therefore, the present invention relates to a method for producing a hydroxyalkyl starch derivative -6- 200521143 A7 B7 V. Description of the invention (5) method, which includes hydroxyalkyl starch (HAS) having formula (I)

經濟部智慧財產局員工消費合作社印製 於其不會在該反應之前被氧化之還原端上，與式(II)之化 合物 R，-NH-R” (Π) 進行反應，其中，Ri，R2及R3係各自獨立為氫或為一線 形或分支的羥基烷基基團，且其中R’或R”或者R，與R”包 括至少一個官能基X ’其係能與至少一種其他化合物於⑴ 及(II)之反應前或後進行反應。 本發明說明書中，’’羥基烷基殿粉’’(HAS)—詞係指已 被至少一個羥基烷基基團所取代之澱粉衍生物。因此，本 發明中所用之羥基烷基澱粉一詞不侷限於其終端醣分子部 分包括如式(I)中為簡便起見所描述之羥基烷基基團Rl，r2 及/或R3之化合物，但亦指化合物中，不論係在終端醣分 子部分及/或在激粉分子之剩餘部份HAS’，其任何地方至 少存有一個羥基基團被羥基烷基基團Ri，R2及r3所取 代。 本說明書中，燒基基團可為線形或分支的烧基基團， 其可適當地被取代。較佳者為，經基燒基基圏含有1至10 個碳原子，較佳為由1至6碳原子，更佳為由1至4個碳 200521143 A7 B7 五、發明說明（6) 原子，且甚至更佳為2-4個碳原子。’’羥基烷基澱粉”因此 宜包括羥基乙基澱粉、羥基丙基澱粉及羥基丁基澱粉，其 中，以羥基乙基澱粉及羥基丙基澱粉為特別佳。 包括二種或多種不同的羥基烷基基團之羥基烷基澱粉 亦為可能。 包含於HAS之至少一個羥基烧基基團可含有二或多個 羥基。根據較佳的具體例中，包含HAS之至少一個羥基 烷基基團含有一個羥基基團。 “羥基烷基澱粉”之表示亦包括衍生物，其中，烷基基 團係經單-或多取代。本說明書中，較佳的是烷基係被鹵 素，尤其是氟，或被芳基所取代，設若HAS仍溶解於水 中。此外，羥基烷基基團之終端羥基可被酯化或醚化。 此外，亦可用線形或分支經取代或未經取代之烯基替 代烷基。 經濟部智慧財產局員工消费合作社印製 經基燒基澱粉係為婦之越衍生物。除了該謎衍生物 之外’其㈣粉衍生物亦可祕本發明之制書中。例 如，包含賴化之縣基圏的衍生物係為有用。此等衍生 物可為’例如’具有如個碳原子未經取代之一 _或二幾 酸之衍生物或餘取代之衍錄。_有时為具有2·6 個破2未經取代之1軸生物，_祕酸之衍生 物。本案說明書中，以乙醜美 基澱粉，丁基澱粉及丙基澱粉 為較佳。 ^外，以具有2·6個碳原子未經取代之二_衍生物 為較佳。 * -8- 200521143 A7 B7 五、發明說明（7) 於二羧酸衍生物之情況中，二羧酸之第二個羧基亦被 酯化係為有用。此外，二羧酸之一燒基酯類衍生物亦適合 於本發明說明書中。 於經取代之一-或二羧酸時，取代基較佳為與如前說明 用於經取代之烷基殘質之相同者。 澱粉之酯化作用技術係已知於此方面技藝中（參閱，例 如，克里曼D·等，綜合纖維素化學，第2卷，1998，惠 里-VCH，韋恩漢，紐约，尤其是第4·4章，纖維素之酯化 作用（ISBN 3·527-29489·9)。 本發明所有具體例中以羥基乙基澱粉(HES)為最佳。 因此，本發明亦關於如前說明的方法，其中，輕基烧 基澱粉係為羥基乙基澱粉。 HES之主要特徵在於分子量分佈及取代度。取代度之 說明有二種可能性： 1 ·對所有的葡萄糖單體(DS)而言，取代度可說明係相關 於經取代之葡萄糖單髏的部份。 2 ·取代度可說明為”莫耳取代”(MS),其中係說明每個 經濟部智慧財產局員工消費合作社印製 葡萄糖部份之羥基乙基基圏。 HES溶液係以多分散組成物存在，其中，各分子之聚 合度，分支部位之數量及類型彼此不同。因此，HES係為 具有不同分子量化合物之混合物。所以，特別的HES溶 液係藉統計工具之助以平均分子量來測定。本案說明書 中，Mn係依分子數而定以算數平均值計算。或者，Mw係 為重量平均，代表依HES質量而定的單位。 •9， 200521143The Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs prints on the reducing end that will not be oxidized before the reaction, and reacts with the compound R, -NH-R "(Π) of formula (II), where Ri, R2 And R3 are each independently hydrogen or a linear or branched hydroxyalkyl group, and wherein R 'or R "or R, and R" include at least one functional group X', which is capable of interacting with at least one other compound. And (II) the reaction is carried out before or after the reaction. In the description of the present invention, `` hydroxyalkyl palace powder '' (HAS)-the word refers to a starch derivative which has been replaced by at least one hydroxyalkyl group. Therefore The term hydroxyalkyl starch used in the present invention is not limited to compounds whose terminal sugar molecules include hydroxyalkyl groups R1, r2 and / or R3 as described in Formula (I) for simplicity, but also Refers to the compound, regardless of whether it is in the terminal sugar molecule part and / or in the remaining part of the powder molecule HAS ', at least one of the hydroxyl groups is replaced by hydroxyalkyl groups Ri, R2 and r3. In the description, the alkyl group may be a linear or branched alkyl group It may be appropriately substituted. Preferably, the alkyl group contains 1 to 10 carbon atoms, preferably from 1 to 6 carbon atoms, and more preferably from 1 to 4 carbons. 200521143 A7 B7 5 Description of the invention (6) Atoms, and even more preferably 2-4 carbon atoms. "Hydroxyalkyl starch" therefore preferably includes hydroxyethyl starch, hydroxypropyl starch and hydroxybutyl starch, of which hydroxyethyl starch Base starch and hydroxypropyl starch are particularly preferred. A hydroxyalkyl starch comprising two or more different hydroxyalkyl groups is also possible. At least one hydroxyalkyl group contained in the HAS may contain two or more hydroxy groups. According to a preferred embodiment, at least one hydroxyalkyl group containing HAS contains one hydroxy group. The expression "hydroxyalkyl starch" also includes derivatives in which the alkyl group is mono- or polysubstituted. In the present specification, it is preferable that the alkyl group is substituted with halogen, especially fluorine, or substituted with aryl, provided that HAS is still dissolved in water. In addition, the terminal hydroxyl group of the hydroxyalkyl group may be esterified or etherified. Alternatively, linear or branched substituted or unsubstituted alkenyl may be used in place of alkyl. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. In addition to the mysterious derivative, its powder derivative can also be used in the production of the present invention. For example, derivatives containing the bases of Laihua County are useful. These derivatives may be 'for example' derivatives having unsubstituted one or dicarboxylic acids such as carbon atoms or derivatives of co-substitutions. _Sometimes there are 2.6 broken 2 unsubstituted 1-axis creatures, _ derivative of mysterious acid. In the description of this case, ethacyl starch, butyl starch and propyl starch are preferred. In addition, unsubstituted bis-derivatives having 2.6 carbon atoms are preferred. * -8- 200521143 A7 B7 V. Description of the invention (7) In the case of a dicarboxylic acid derivative, it is useful that the second carboxyl group of the dicarboxylic acid is also esterified. In addition, an alkyl ester derivative, which is a dicarboxylic acid, is also suitable in the present specification. In the case of a substituted one- or dicarboxylic acid, the substituent is preferably the same as that described above for the substituted alkyl residue. Starch esterification techniques are known in this area (see, for example, Creeman D. et al., Comprehensive Cellulose Chemistry, Volume 2, 1998, Werry-VCH, Wayne Han, New York, and more particularly Chapter 4 · 4, Esterification of Cellulose (ISBN 3.527-29489 · 9). Among all the specific examples of the present invention, hydroxyethyl starch (HES) is the best. Therefore, the present invention also relates to the above The illustrated method, wherein the light-based calcined starch is hydroxyethyl starch. The main characteristics of HES are the molecular weight distribution and the degree of substitution. There are two possibilities for the description of the degree of substitution: 1 · For all glucose monomers (DS) In terms of the degree of substitution, it can be explained that it is related to the substituted glucose monogram. 2 · The degree of substitution can be explained as “Mole Substitution” (MS), which states that each consumer ’s cooperative agency of the Intellectual Property Bureau of the Ministry of Economic Affairs has printed HES solution is a polydisperse composition, in which the degree of polymerization of each molecule, the number and type of branch sites are different from each other. Therefore, HES is a mixture of compounds with different molecular weights. So, special HES The liquid system is measured by the average molecular weight with the help of statistical tools. In the description of this case, Mn is calculated by the arithmetic mean depending on the number of molecules. Alternatively, Mw is the weight average and represents the unit based on the mass of the HES. 200521143

五、發明說明（8) 發月說明書中，經基乙基殿粉可具有自1至獅 之=平均刀子量（重量平均），其中，以自5至100 kDa 粉二子量為較佳。就祕乙基基81而言，祕乙基殿 2 步具有自〜1至G·8之取代莫耳度及C2 : C6間自 20範圍内取代之比率。 與々切)之殘基Rl，R2及R3而論，對化合物(I)仍能 ’(Π)之化合物騎反應沒有給予敎之限制。根據 伽的具體例中，Rl，仏及R3係獨立為氫或具有1至10 围後原子之絲料基團，雜芳基基目，祕芳烧基基 或絲料基基團。錢及具㈣1 JL 6健原子之經 /基基目為㈣。㈣，絲，芳、絲及/或烧芳基基 可為線形或分支且適合被取代者。 因此，本發明亦關於如前說明的方法，其中，R〗，R2 及3獨立為氫或具有由！至6個碳原子線形或分支的觀 基烷基基困。 經濟部智慧財產局員工消费合作社印製 因此，Ri，R2及R3可為羥基己基，羥基戊基，羥基 丁基，羥基丙基，例如，1-羥基丙基，孓羥基丙基，3-羥 基丙基，1-羥基異丙基，2-羥基異丙基，羥基乙基例如， 起基乙基，2-羥基乙基，或經基甲基。以氫及經基乙基 為較隹，氫及2-羥基乙基為特別佳。 因此，本發明亦關於如前說明的方法，其中，;^，r2 及反3獨立為氫或2·羥基乙基基圏。 根據本發明，羥基烷基澱粉係與式(II)之化合物進行反 應’其中，化合物(II)可在與化合物(I)進行反應之前與另 -10- 200521143 A7 B7 五、發明說明（9) 一種化合物反應，而得到經基烧基殿粉衍生物。至於化合 物(II)，若化合物(II)能夠經由ΝΗ基橋R’與R”與化合物(I) 之於其不會在該反應之前被氧化之還原端上進行反應，而 得到羥基烷基澱粉衍生物時則無特定的限制。 較佳的化合物(II)之殘基R，為氩及烷基，環烷基，芳 基，芳烷基，芳基環烷基，烷芳基或環烷基芳基殘基，其 中環烷基，芳基，芳烷基，芳基環烷基，烷芳基或環烷基 芳基殘基可直接連接到化合物(II)之ΝΗ基橋R’與R”或， 根據另一種具體例，可藉氧橋連接到化合物(II)iNH基橋 R’與R”。烷基，芳基，芳烷基或烷芳基殘基可適當地被 取代。較佳的取代基，可提及者為_素例如，F，Ci或 Br。尤其佳之殘基R’為氫，炫基及烧氧基，且甚至更佳者 為氫及未經取代之烷基及烷氧基基團。 因此，本發明亦關於如前說明的方法，其中，R，係為 氫或為一線形或分支的烷基或烷氧基基團。 經濟部智慧財產局員工消费合作社印製 烷基及烷氧基基困之中，以具有1，2，3，4，5，或 6個碳原子之基困為較佳。更佳者為甲基，乙基，丙基， 異丙基，甲氧基，乙氧基，丙氧基，及異丙氧基。尤其佳 者為甲基，乙基，甲氧基，乙氧基，且特別隹者為甲基戋 甲氧基。 一 因此，本發明亦關於如前說明的方法，其中，R，係為 氮或甲基或甲氧基基團。 除了官能基X之外，R”可包含至少再一個官能基 W。該至少再一個官能基w —般可在R”中之任何位 •11- 200521143 A7 B7 五、發明說明（l〇) 敉佳者為，W直接連接到NH基團R’所連接者。 通常，有關化合物(I)能與化合物(Π)進行反應之官能基 W沒有特定限制。於較佳的具體例中，官能基W包括結 構單元_NH-及/或結構單元-(OG)-，而G為Ο或S，及/或 結構單元-S02_。根據更佳之具體例，官能基W係選自於 下列的基團，其包括： Η 丫 Η Η τV. Description of the invention (8) In the manual, the ethyl acetate powder can have from 1 to lion = average knife amount (weight average), of which the powder amount from 5 to 100 kDa is preferred. In the case of mythyl group 81, mythyl group has a substitution molar ratio from ~ 1 to G · 8 and a ratio of C2: C6 substitution from 20. With respect to the residues R1, R2 and R3 of 々), there is no restriction on 之 for the compound riding reaction of compound (I). According to the specific examples of Gallium, R1, Y and R3 are independently hydrogen or a silk group, a heteroaryl group, a arylene group or a silk group based on 1 to 10 atoms. The warp / base of money and ㈣1 JL 6 health atoms is ㈣. Rhenium, silk, aryl, silk and / or aryl may be linear or branched and suitable for substitution. Therefore, the present invention also relates to the method as described above, in which R〗, R 2 and 3 are independently hydrogen or have a source! A linear or branched alkyl radical of 6 carbon atoms is trapped. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. Therefore, Ri, R2 and R3 can be hydroxyhexyl, hydroxypentyl, hydroxybutyl, hydroxypropyl, for example, 1-hydroxypropyl, hydroxypropyl, 3-hydroxy A propyl group, a 1-hydroxyisopropyl group, a 2-hydroxyisopropyl group, a hydroxyethyl group, for example, a methyl group, a 2-hydroxyethyl group, or a methyl group. Hydrogen and triethyl are more preferred, and hydrogen and 2-hydroxyethyl are particularly preferred. Therefore, the present invention also relates to the method as described above, wherein; ^, r2 and trans 3 are independently hydrogen or 2 · hydroxyethyl fluorene. According to the present invention, a hydroxyalkyl starch is reacted with a compound of the formula (II) 'wherein the compound (II) may be reacted with another compound before the reaction with the compound (I) -10- 200521143 A7 B7 V. Description of the invention (9) A compound is reacted to obtain a base powder derivative. As for the compound (II), if the compound (II) can be reacted with the reducing end of the compound (I) via the N′yl bridge R ′ and R ″, which will not be oxidized before the reaction, to obtain a hydroxyalkyl starch There are no particular restrictions on derivatives. The residue R of the preferred compound (II) is argon and alkyl, cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or naphthenic Aryl residues, in which cycloalkyl, aryl, aralkyl, arylcycloalkyl, alkaryl or cycloalkylaryl residues can be directly connected to the NH group bridge R 'of compound (II) and R "Or, according to another specific example, it may be connected to the compound (II) iNH radical bridge R 'and R" by an oxygen bridge. The alkyl, aryl, aralkyl or alkaryl residue may be appropriately substituted. Preferred substituents may be mentioned, for example, F, Ci or Br. Particularly preferred residues R 'are hydrogen, xyl and alkoxy, and even more preferred are hydrogen and unsubstituted alkane. Therefore, the present invention also relates to the method described above, wherein R is hydrogen or a linear or branched alkyl or alkoxy group. Ministry of Economic Affairs Among the printed alkyl and alkoxy groups in the consumer cooperative of the Intellectual Property Bureau, it is better to use a group with 1, 2, 3, 4, 5, or 6 carbon atoms. The more preferred is methyl. Ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, and isopropoxy. Particularly preferred are methyl, ethyl, methoxy, ethoxy, and especially Is methylfluorenylmethoxy. Thus, the present invention also relates to the method described above, wherein R is a nitrogen or methyl or methoxy group. In addition to the functional group X, R "may contain at least Yet another functional group W. The at least one further functional group w may generally be in any position in R ”• 11-200521143 A7 B7 V. Description of the invention (10) The preferred one is that W is directly connected to the NH group R ′. Usually There is no particular limitation on the functional group W capable of reacting the compound (I) with the compound (Π). In a preferred embodiment, the functional group W includes a structural unit _NH- and / or a structural unit-(OG)-, G is 0 or S, and / or structural unit -S02_. According to a more specific example, the functional group W is selected from the following groups, which include: Η Η Η Η τ

G ΟG Ο

II —N—S— Η II Ο 其中，若G出現兩次，則其獨立為Ο或S。 根據本發明較佳之具體例，其中R’係為Η且W直接 連接到ΝΗ基橋R’與R”，R’及ΝΗ基橋R’與R”與W — 起形成下列基團之一： 經濟部智慧財產局員工消費合作社印製II —N—S— Η II 〇 Where, if G appears twice, it is independently 0 or S. According to a preferred embodiment of the present invention, wherein R ′ is Η and W is directly connected to the N ′ radical bridges R ′ and R ″, R ′ and the N ′ radical bridge R ′ and R ″ and W together form one of the following groups: Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs

HNHN

HNHN

ONSMO - NHONSMO-NH

HN TG ¥HN TG ¥

HNHN

HNHN

G TGG TG

^nnG A7 B7 11 200521143 五、發明說明 就至少一個官能基X而論，其係包含於R，及/或R” 中，宜為於R”中，無特定限制存在。通常，所有的官能 基係可能容許與至少另一種化合物進行反應。 就該反應與另外的化合物而言，至少一個官能基與至 少另一種化合物之各種相互作用係可能的。尤其，至少一 個官能基X與另外的化合物進行反應可能導致共價鍵合， 離子鍵合及/或凡德瓦鍵合，以共價鍵合為特別佳。 尤其，下列官能基X為可提及者： -C-C雙鍵或C-Ο三鍵或芳族c_C·鍵； _ 硫基或幾基基圈； - 炫基績酸醢餅，芳基確酸醯胼； • 1，2-二烯類(dioles); -1，2-胺基醇類； -胺基_NH2或包含結構單元-NH-，例如胺基烷基，胺基 芳基，胺基芳烷基，或烷芳基胺基之胺基的衍生物； -羥基胺基-0-ΝΗ2或包含結構單元-ONH-，例如羥基烷 基胺基，羥基芳基胺基，羥基芳烷基胺基，或羥基烷 芳基胺基之羥基胺基的衍生物； 經 濟 部 智 慧 財 產 局 員 X 消 费 合 作 社 -烷氧基胺基，芳氧基胺基，芳烷氧基胺基，或烷芳基 氧基胺基，各個包含結構單元_ΝΗββ〇-; -具有羰基，-Q-C(=G)-M的殘基，其中G為0或S，且 Μ為’例如， —_〇Η 或 _SH ; — 烧氧基，芳基氧基，芳烷基氧基，或烷芳基氧 •13- 200521143 A7 B7 五、發明說明（12) 基； — 烷基硫基，芳基硫基，芳烷基硫基，或烷芳基 硫基； — 烷基羰基氧基，芳基羰基氧基，芳烷基羰基氧 基，烷芳基羰基氧基； — 活性酯類，例如，具有亞胺(imid)結構之羥基胺 的酯類，例如，N-羥基琥珀醯亞胺或具有結構 單元0-N其中N為雜芳基化合物的部份，或具 有G=0且Q不存在，例如，具有經取代之芳基 體殘基之芳基氧基化合物，例如，五氟苯基， 對硝基苯基，三氣苯基； 其中，Q或NH或雜原子，例如S或Ο係不存在； -一ΝΗ^ΝΗ2，或-NH-NH面; --Ν〇2， - 腈基； - 幾基’例如’搭基或闕基； - 叛基； 經濟部智慧財產局員工消费合作社印製 --N=C=0 基或-N=C=S 基； - 乙烯基鹵化物，例如，乙烯基碘或乙烯基溴基或乙烯 基三氟甲烷磺酸鹽； --C=C-H $ --(C=NH2Cl)-0 烷基 -基團-(C=0)-CH：rHa卜其中，Hal 為 F，α，Br 或 I ; --ch=ch-so2-; -14- 200521143 A7 B7 五、發明說明（I3 包括結構_S-S·之二硫化物基團 0.^ nnG A7 B7 11 200521143 V. Description of the Invention As far as at least one functional group X is concerned, it is included in R, and / or R ″, preferably in R ″, and there is no specific limitation. In general, all functional groups may allow reaction with at least one other compound. For this reaction with another compound, various interactions of at least one functional group with at least another compound are possible. In particular, the reaction of at least one functional group X with another compound may lead to covalent bonding, ionic bonding and / or van der Waals bonding, and covalent bonding is particularly preferred. In particular, the following functional groups X may be mentioned:-CC double bond or C-O triple bond or aromatic c_C · bond; _ thio group or several group ring;-xylyl group, aryl acid醯 胼; • 1,2-dienes (dioles); 1,2-amino alcohols; -amino_NH2 or containing structural unit -NH-, such as aminoalkyl, aminoaryl, amine Arylalkyl, or derivatives of amines of alkarylamino; -hydroxyamino-0-0N2 or containing the structural unit -ONH-, such as hydroxyalkylamino, hydroxyarylamino, hydroxyarane Derivatives of hydroxyamino groups, or hydroxyalkanoyl groups of hydroxyalkarylamino groups; Member of the Intellectual Property Bureau of the Ministry of Economy X Consumer Cooperatives-alkoxyamino groups, aryloxyamine groups, aralkoxyamine groups, or alkaryl Oxyoxyamino groups, each containing a structural unit _ΝΗββ〇-;-a residue having a carbonyl group, -QC (= G) -M, where G is 0 or S, and M is' for example, -_〇Η or _ SH; — Carbooxy, aryloxy, aralkyloxy, or alkaryloxy • 13- 200521143 A7 B7 5. Description of the invention (12) Group; — Alkylthio, arylthio, aryl Alkylthio, or alkylarylthio ;-Alkylcarbonyloxy, arylcarbonyloxy, aralkylcarbonyloxy, alkarylcarbonyloxy;-reactive esters, for example, esters of hydroxylamines having an imid structure, such as , N-hydroxysuccinimide or a moiety having structural units 0-N where N is a heteroaryl compound, or G = 0 and Q is absent, for example, an aryloxy group having a substituted aryl residue -Based compounds, for example, pentafluorophenyl, p-nitrophenyl, trisoxyphenyl; wherein Q or NH or heteroatoms such as S or O are absent; -NΗ ^ NΗ2, or -NH-NH face --N〇2,-Nitrile group;-A few bases, such as a base or a base;-Betrayal; Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs-N = C = 0 base or -N = C = S group;-vinyl halide, for example, vinyl iodide or vinyl bromide or vinyl trifluoromethanesulfonate; --C = CH $-(C = NH2Cl) -0 alkyl-group -(C = 0) -CH: rHabu where Hal is F, α, Br or I; --ch = ch-so2-; -14- 200521143 A7 B7 V. Description of the invention (I3 includes the structure _SS · 之Disulfide group 0.

基團 - 〇 基團Group-〇 Group

此等基團之中，以硫基，胺基，羥基胺基，烷氧基胺基及 下列基團為特別佳：Among these groups, a thio group, an amine group, a hydroxyamine group, an alkoxyamine group and the following groups are particularly preferred:

N 2 HN 2 H

HNHN

OMSno - NH \ N 2 H h2nOMSno-NH \ N 2 H h2n

HNHN

HN N 2 ΗHN N 2 Η

HNHN

G rG h/n h2n 經濟部智慧財產局員工消费合作社印製 因此，本發明亦關於如前說明的方法，其中，至少一 個官能基X係選自於下列之基團，其包括：-SH，-NH2，-ΟΝΗ2·，-NH-0-烷基，-(OG)-NH-NH2，-G-(C=G)-NH-NH2，-NH-(C=G)-NH_NH2，及-S02_NH-NH2，其中 G 為 Ο 或S且，若G出現兩次，則其獨立為0或S。 就烷氧基胺基而論，特別佳者為丙氧基胺基，乙氧基 胺基及甲氧基胺基，以甲胺基胺基-NH-0-CH3-為尤其佳 者。 -15- 200521143 A7 B7 五、發明說明（U) 根據本發明還有另一觀點，至少一個官能基X可為一 不能直接與給定之另外的化合物進行反應之基團，但其可 經化學改變以便能夠依想要的方式進行反應。該包含於化 合物(II)之官能基X之改質係可於化合物(II)與化合物⑴進 行反應之前或於化合物（II)與化合物⑴進行反應之後進 行。如果化合物(II)包括至少二個選擇地化學性不同的官 能基X，其可能於化合物(II)與化合物(I)進行反應之前改 變至少一個官能基X及於化合物(II)與化合物(I)進行反應 之後改變至少一個官能基X。 官能基X與另外的化合物進行反應之前被改質之實 例’可提及者為1，2-胺基醇或1，2-二醇，其係經例如，氧 4匕以形成醛或酮基而改質。 官能基X與另外的化合物進行反應之前被改質之另一 f例為-NH2-基團，其係藉著與例如，根據下式之化合物 經濟部智慧財產局員工消費合作社印製G rG h / n h2n Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. Therefore, the present invention also relates to the method described above, wherein at least one functional group X is selected from the group consisting of -SH, -NH2, -ONY2, -NH-0-alkyl,-(OG) -NH-NH2, -G- (C = G) -NH-NH2, -NH- (C = G) -NH_NH2, and- S02_NH-NH2, where G is 0 or S and if G appears twice, it is independently 0 or S. As for the alkoxyamino group, particularly preferred are propoxyamino group, ethoxyamino group and methoxyamino group, and methylaminoamino group -NH-0-CH3- is particularly preferred. -15- 200521143 A7 B7 V. Description of the invention (U) According to another aspect of the present invention, at least one functional group X may be a group that cannot directly react with a given other compound, but it may be chemically changed. So that you can react in the way you want. The modification of the functional group X contained in the compound (II) can be performed before the reaction of the compound (II) with the compound VII or after the reaction of the compound (II) with the compound IX. If the compound (II) includes at least two selectively chemically different functional groups X, it may change at least one functional group X before the compound (II) reacts with the compound (I) and the compound (II) and the compound (I ) Change at least one functional group X after carrying out the reaction. Examples of functional groups X that have been modified before reacting with another compound can be mentioned as 1,2-amino alcohols or 1,2-diols, which are subjected to, for example, oxygen radicals to form aldehyde or ketone groups And modified. Another example of f that was modified before the functional group X reacted with another compound was the -NH2- group, which was printed by, for example, a compound according to the formula:

進行反應而改質以獲得下式之結構 〇The reaction is modified to obtain the structure of the following formula.

-16- 200521143 A7 B7 五、發明說明（15) 其係，例如，向硫基反應。 官能基X與另外的化合物進行反應之前被改質之另一 實例為-NH2·基困，其係藉著與例如，根據下式之化合物 〇 〇 進行反應而改質以得到下式之結構-16- 200521143 A7 B7 V. Description of the invention (15) This system, for example, reacts to a sulfur group. Another example of the functional group X being modified before reacting with another compound is -NH2 · group, which is modified by reaction with, for example, a compound according to the following formula to obtain a structure of the following formula

經濟部智慧財產局員Η消費合作社印製 其係，例如，向硫基反應。 該至少一個官能基X可直接連接到NH基橋R，與 R。因此’根據本發明之一具體例，官能基X係等於 R。化合物中X係直接连接到NH基橋R，與R”之特定實 例為，尤其是，Members of the Intellectual Property Bureau of the Ministry of Economic Affairs and Consumer Cooperatives have printed their department, for example, reacting to sulfur bases. The at least one functional group X may be directly connected to the NH group bridge R, and R. Therefore, according to a specific example of the present invention, the functional group X is equal to R. In the compound, X is directly connected to the NH group bridge R, and specific examples of R "are, in particular,

S 此等化合物其係亦包含於本發明中者之另一特定實例 為 NHs 〇 -17· 200521143 A7 B7 五、發明說明（16 ) 經濟部智慧財產局員工消費合作社印製 根據本發明另一個具體例，NH基橋R，與R”可藉由線 形或分支的燒基或環燒基或芳基或芳烷基或芳基環烷基或 烷芳基或環烷芳基，其中，此等基團可包括至少一個雜原 子，例如，N，〇，S，且其中此等基團可適當地被取代， 從至少一個官能基X中分離出來。分離NH，基橋R，與 R及至少一個官能基X之基團的大小可適應特定之需 要。通常，分離的基團具有一般由！至6〇,宜為由！至 4〇，更隹為由1至20，更佳為由1至1〇，更佳為由1至6 且尤其佳者為由1至4個碳原子。如果存有雜原子，則該 分離的基團包含一般由1至2〇，較佳為由j至8且尤其佳 為由1至4個雜原子。根據本發明特別佳之具體例，分離 的基團包括1至4個氧原子。分離的基團可包含具有，例 如由5至7個碳原子任意分支的烷基鏈或芳基基團或環烷 基基團，或為芳烷基，烷芳基，其中烷基部份可為線形及 /或環狀烷基基團。根據甚至更佳的具體例，分離的基圏 係為由1至20，宜由1至8，更佳為由！至6，更佳為由 1至4且尤其佳者為由2至4個碳原子之烷基鏈。於存有 雜原子之情況令，以包括1至4個氧原子之鏈為特別隹。 化合物(II)之X係從NH基橋R，與R，，中分離出來者之 特定實例為，尤其是 -18- 200521143 A7 B7 五、發明說明（Γ7 NH〇 HS^ —NH2S Another specific example of these compounds which are also included in the present invention is NHs 〇-17 · 200521143 A7 B7 V. Description of the invention (16) Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs according to another specific example of the present invention For example, the NH-based bridge R, and R "can be formed by linear or branched alkyl or cycloalkyl or aryl or aralkyl or aryl cycloalkyl or alkaryl or cycloalkaryl groups, among which The group may include at least one heteroatom, for example, N, 0, S, and wherein these groups may be appropriately substituted to separate from at least one functional group X. Isolate NH, the radical bridge R, and R and at least The size of a functional group X can be adapted to specific needs. In general, isolated groups have a general range from! To 60, preferably from! To 40, more preferably from 1 to 20, and more preferably from 1. To 10, more preferably from 1 to 6, and particularly preferably from 1 to 4 carbon atoms. If heteroatoms are present, the isolated group contains generally from 1 to 20, preferably from j to 8 and particularly preferably from 1 to 4 heteroatoms. According to a particularly preferred embodiment of the present invention, the isolated group includes 1 to 4 oxygen atoms The isolated group may comprise an alkyl chain or an aryl group or a cycloalkyl group having, for example, an arbitrary branch from 5 to 7 carbon atoms, or an aralkyl group, an alkaryl group, wherein the alkyl portion May be linear and / or cyclic alkyl groups. According to even better specific examples, the isolated radicals are from 1 to 20, preferably from 1 to 8, more preferably! To 6, and more preferably 1 to 4 and particularly preferred are alkyl chains of 2 to 4 carbon atoms. In the case where heteroatoms are present, a chain including 1 to 4 oxygen atoms is particularly preferred. X of the compound (II) Specific examples of those separated from the NH base bridge R, and R, are, in particular, -18-200521143 A7 B7 V. Description of the invention (Γ7 NH〇HS ^ -NH2

h2n〇〜cooh OHh2n〇 ~ cooh OH

NH, OH h2n〜coohNH, OH h2n ~ cooh

H2N 产丫^OH OHH2N ^ OH OH

H2NH2N

h2n"^Y^ NK OH ?H h2n’ ?H /OH h2no’ ?H /OH h2no’ ,nh2 OH H2NO、h2n " ^ Y ^ NK OH? H h2n ’? H / OH h2no’? H / OH h2no ’, nh2 OH H2NO,

H2NO、入 /OH 經濟部智慧財產局員工消費合作社印製 200521143 A7 B7 五、發明說明（18)H2NO, Printed by / OH Consumer Intellectual Property Cooperative of Intellectual Property Bureau of the Ministry of Economic Affairs 200521143 A7 B7 V. Description of Invention (18)

-20- 200521143 A7 B7 五、發明說明（19) 分離NH，橋R’與R”，及至少一個官能基X之基團 可適當地被取代。較佳的取代基為，例如，齒化物例如， F，C卜 Br 或 I。 分離NH，橋R’與R”，及至少一個官能基X之基團 可包括一個或多個裂解部位，例如， -S—S—-20- 200521143 A7 B7 V. Description of the invention (19) Separation of NH, bridges R ′ and R ″, and at least one functional group X may be appropriately substituted. Preferred substituents are, for example, dentates such as , F, C, Br or I. A group that separates NH, bridges R ′ and R ″, and at least one functional group X may include one or more cleavage sites, for example, -S—S—

HO Υα OH 〇 〇 II S一〇 II ο 其係容許所產生的化合物在預先決定的部位上容易裂解。 根據本發明特別佳的具體例，化合物(II)為〇_[2-(2_胺 基氧基-乙氧基)-乙基]-羥基胺HO Υα OH 〇 〇 II S-II II ο It allows the produced compound to be easily cleaved at a predetermined site. According to a particularly preferred embodiment of the present invention, the compound (II) is 0- [2- (2-aminooxy-ethoxy) -ethyl] -hydroxyamine

XL Η2Ν NH, 經濟部智慧財產局員工消費合作社印製 或碳醯胼。 因此，本發明亦關於如前說明的方法，其中，化合物 (II)為〇[2-(2-胺基氧基-乙氧基)-乙基]-羥基胺或碳醯肼。 於化合物(II)包括一或多個對掌中心之情況中，化合物 (II)可存在於R組態或S組態中或作為就各個對掌中心之 -21« 200521143 B7XL Η2Ν NH, printed or carbon-coated by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. Therefore, the present invention also relates to the method as described above, wherein the compound (II) is O [2- (2-aminooxy-ethoxy) -ethyl] -hydroxyamine or carbohydrazine. In the case where compound (II) includes one or more palm centers, compound (II) may exist in the R configuration or S configuration or as a -21 «200521143 B7

消旋化合物。 如前說明，化合物(I)可與如此之化合物⑼或與業已和 至少另一種化合物於與化合物(I)反應之前進行反應之化合 物(Π)進行反應。 化合物⑴與化合物(II)如此之反應可在至少一適當的溶 劑中進行。各別的溶劑或二種或多種溶劑的混合物可適應 化合物⑴與(II)反應條件及化學性質之特定需要。根據本 發明特別佳之具體例，水係用作為溶劑，單獨或與至少一 種其他溶劑一起使用。可提及作為至少一個其他溶劑者為 DMSO，DMF，甲醇及乙醇。除水之外較佳的溶劑為 DMSO，DMF，甲醇及乙醇。 因此，本發明亦關於如前說明的方法，其中，化合物 (I)與化合物(II)之反應係在水性系統中進行。 本發明說明書中所用之，，水性系統，，一詞係指溶劑或溶 劑混合物，其包括每重量由至少10%，較佳為每重量至少 50%，更佳為每重量至少90%至每重量至多ι〇〇〇/0範圍的 水，以所用溶劑之重量計。較佳的反應介質為水。 經濟部智慧財產局員工消费合作社印製 就反應時所應用的溫度而論，並沒有導致想要的羥基 燒基厥粉衍生物的反應之特定限制存在。 於化合物(I)與化合物<π)進行反應之情況中，化合物(II) 為羥基胺或為醯肼時，溫度較佳係在自5至45°C範圍，更 佳為自10至30°C範園且特別佳為自15至25°C範圍内。 於化合物⑴與化合物(II)進行反應之情況中，該反應為 還原性胺化作用時，溫度較佳係在至多l〇0°c範圍，更佳 -22- 200521143 A7 B7 五、發明說明（η) 為自20至95°C範圍，更佳為自25至90°C範圍，更佳為 自70至90°C範圍且特別佳為自75至85°C範圍内。 因此，本發明亦關於如前說明的方法，其令，化合物 (II)為羥基胺或醯肼時，化合物⑴與化合物(II)之反應係在 自5至45°C之溫度下進行。 因此，本發明亦關於如前說明的方法，其中，該反應 為還原性胺化作用時，化合物(I)與化合物(II)之反應係在 自25至90°C之溫度下進行。 於反應過程中溫度可變化，宜在前文給定的範圍内變 >(匕，或維持實質上恆定。 化合物(I)與(II)反應時之反應時間可適應特定之需要且 通常係在自1小時至7天範圍内。 於化合物(II)為羥基胺或醯肼之情況中，反應時間範圍 較佳係自1小時至3天且更佳為自2小時至48小時。 於化合物(I)與化合物(II)之反應為還原性胺化作用之情 況中，反應時間範圍宜自2小時至7天。 化合物⑴與(II)反應之pH值可適應特定之需要，例 如，反應物之化學性。 經濟部智慧財產局員工消费合作社印製 於化合物(II)為羥基胺或醯胼之情況中，pH值宜自4.5 至6.5範圍。 於化合物⑴與化合物(II)反應為還原性胺化作用之情況 中，pH宜自8至12範圍。 因此’本發明亦關於如前說明的方法，其中，化合物 (II)為羥基胺或醯肼時，化合物(I)與化合物(II)之反應係在 -23· 200521143 A7 B7 五、發明說明（22) pH自4.5至6.5下進行。 因此，本發明亦關於如前說明的方法，其中，該反應 為還原性胺化作用時，化合物⑴與化合物(II)之反應係在 pH自8至12下進行。 前文所提反應條件之特定實例為，例如，化合物為經 基胺之情況中，反應溫度大約25°C且pH大約5.5，且化 合物(I)與化合物(II)之反應為還原性胺化作用之情況中， 反應溫度大約80°C且pH大約11。 反應混合物之適當pH值可藉著添加至少一種適合的 緩衝劑來調節。較隹的緩衝劑中，可提及者為醋酸鈉緩衝 劑，磷酸鈉或硼酸鹽緩衝劑。 根據本發明較佳的具趙例中，化合物(I)與化合物(II)進 行反應所產生的反應產物與至少另一種化合物係經由至少 一個官能基X而反應。 經濟部智慧財產局員工消費合作社印製 如果需要，該至少一個官能基X可於化合物(I)與化合 物(II)反應之前被至少一適當的保護基所保護。於此觀點 中，所有想得到的保護基皆可能避免被保護的化合物(π) 經由至少一個官能基X與化合物⑴反應。因此，保護基可 依被保護官能基X之化學性而定，從例如，進行反應之溶 劑或反應混合物之pH中選擇。較佳的保護基為，尤其 是，苄基氧基羰基，第三-丁氧基羰基，甲氧基苯基，2，‘ 二甲氧基苯基，三芳基甲基，三苯f基，一甲氧基三苯甲 基，二甲氧基三苯f基，一 f基三苯甲基，二甲基三苯甲 基，三氟乙酿基，歒敏(phthalimin)化合物，2-(三燒基甲梦 -24- 經濟部智慧財產局員工消费合作社印製 200521143 A7 B7 五、發明說明（23) 烷基）乙氧基羰基化合物，Fmoc，第三-丁基，或三烷基甲 矽烷基。 若二個或多個不同的官能基X出現於化合物(II)中， 則至少一個基困可被保護，反之至少一個其他基團不被保 護。 化合物⑺與化合物(II)反應後，至少一個保護基會留置 在反應產物中或係藉由適當的方法，例如，熟知於此方面 技藝中習用的方法移除。如果二個不同的官能基X被適當 的保護基所保護，則可能將至少一個保護基移除以便可使 至少一個官能基X於進一步與至少另一種化合物反應中有 效，且留下至少一個被保護的其他官能基直到化合物(I)與 化合物(II)之反應產物與另外的化合物進行反應。之後， 仍被保護之官能基的保護基可被移除，使得剩餘的官能基 X有效地與還有另一種化合物進行反應。 使用至少一個保護基以避免反應產生包含化合物 (11)(其業已與二或多個化合物⑴反應，亦即，經多重has 取代的化合物(II))之羥基烷基澱粉衍生物係重要的。然 而，相同的結果可藉著將化合物⑴與過量化合物(II)進行 反應而達成。如果將過量化合物(II)使用於本發明之過程 中，化合物(Π)對化合物(I)之莫耳比宜在自2至1〇〇範園 内0 一旦化合物(I)與化合物(II)反應形成反應產物，其可藉 由至少一適當的方法從反應混合物中單離出來。如果需 要，反應產物可於單離之前藉由至少一適當的方法沉殺出 -25-Racemic compounds. As explained before, the compound (I) can be reacted with such a compound ⑼ or with a compound (Π) which has been reacted with at least another compound before reacting with the compound (I). The reaction of the compound XI with the compound (II) can be carried out in at least one appropriate solvent. Separate solvents or a mixture of two or more solvents can be adapted to the specific needs of the reaction conditions and chemical properties of compound VII and (II). According to a particularly preferred embodiment of the present invention, an aqueous system is used as a solvent, alone or in combination with at least one other solvent. Mention may be made, as at least one other solvent, of DMSO, DMF, methanol and ethanol. Preferred solvents other than water are DMSO, DMF, methanol and ethanol. Therefore, the present invention also relates to the method described above, in which the reaction of the compound (I) and the compound (II) is performed in an aqueous system. As used in the specification of the present invention, the term "aqueous system" refers to a solvent or a solvent mixture, which includes at least 10% per weight, preferably at least 50% per weight, and more preferably at least 90% to per weight Water in the range of at most 1000/0, based on the weight of the solvent used. The preferred reaction medium is water. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs As far as the temperature applied in the reaction is concerned, there are no specific restrictions that lead to the desired reaction of the hydroxy-based pyrofuran derivative. In the case where the compound (I) is reacted with the compound < π), when the compound (II) is a hydroxylamine or a hydrazine, the temperature is preferably in the range from 5 to 45 ° C, more preferably from 10 to 30 ° C Fan Yuan and particularly preferably in the range from 15 to 25 ° C. In the case of the reaction between compound VII and compound (II), when the reaction is a reductive amination, the temperature is preferably in the range of at most 100 ° C, more preferably -22- 200521143 A7 B7 V. Description of the invention ( η) is in the range from 20 to 95 ° C, more preferably in the range from 25 to 90 ° C, more preferably in the range from 70 to 90 ° C and particularly preferably in the range from 75 to 85 ° C. Therefore, the present invention also relates to a method as described above, in which, when the compound (II) is a hydroxylamine or a hydrazine, the reaction between the compound VII and the compound (II) is performed at a temperature of from 5 to 45 ° C. Therefore, the present invention also relates to the method described above, in which, when the reaction is a reductive amination, the reaction of the compound (I) and the compound (II) is performed at a temperature of from 25 to 90 ° C. The temperature may change during the reaction, and it should be changed within the range given above (>, or maintained substantially constant. The reaction time when the compounds (I) and (II) react can be adapted to specific needs and is usually at In the range from 1 hour to 7 days. In the case where the compound (II) is hydroxylamine or hydrazine, the reaction time range is preferably from 1 hour to 3 days and more preferably from 2 hours to 48 hours. In the case where the reaction between I) and compound (II) is reductive amination, the reaction time should range from 2 hours to 7 days. The pH value of the reaction between compound VII and (II) can be adapted to specific needs, for example, the reactants The chemical property is printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. When the compound (II) is hydroxylamine or osmium, the pH value should be in the range of 4.5 to 6.5. It is reductive when the compound (II) reacts with compound (II). In the case of amination, the pH is preferably in the range of 8 to 12. Therefore, the present invention also relates to the method described above, in which, when the compound (II) is hydroxylamine or hydrazine, the compound (I) and the compound (II) The reaction is at -23 · 200521143 A7 B7 V. Description of the invention (22) The pH is carried out from 4.5 to 6.5. Therefore, the present invention also relates to the method described above, in which, when the reaction is a reductive amination, the reaction of the compound VII and the compound (II) is carried out at a pH of 8 to 12 Specific examples of the reaction conditions mentioned above are, for example, in the case where the compound is trisamine, the reaction temperature is about 25 ° C and the pH is about 5.5, and the reaction of compound (I) and compound (II) is reductive amination. In the case of the effect, the reaction temperature is about 80 ° C and the pH is about 11. The appropriate pH value of the reaction mixture can be adjusted by adding at least one suitable buffer. Among the larger buffers, sodium acetate buffer can be mentioned Agent, sodium phosphate or borate buffer. In a preferred embodiment according to the present invention, the reaction product produced by the reaction between compound (I) and compound (II) and at least one other compound are via at least one functional group X. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. If necessary, the at least one functional group X may be protected by at least one appropriate protecting group before the reaction of the compound (I) with the compound (II). In this view In all, the desired protecting group may prevent the protected compound (π) from reacting with the compound ⑴ via at least one functional group X. Therefore, the protecting group may depend on the chemical nature of the protected functional group X, for example, the reaction proceeds The solvent or the pH of the reaction mixture is selected. Preferred protecting groups are, in particular, benzyloxycarbonyl, tertiary-butoxycarbonyl, methoxyphenyl, 2, 'dimethoxyphenyl, Triarylmethyl, triphenylfyl, monomethoxytrityl, dimethoxytriphenylfyl, monofyltrityl, dimethyltrityl, trifluoroethyl, A phthalimin compound, 2- (Trisynylmethyl dream-24)-Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs, 200521143 A7 B7 V. Description of the invention (23) Alkyl) ethoxycarbonyl compounds, Fmoc, No. Tri-butyl, or trialkylsilyl. If two or more different functional groups X are present in the compound (II), at least one group may be protected, and at least one other group may not be protected. After the compound VII is reacted with the compound (II), at least one protecting group is left in the reaction product or removed by an appropriate method, for example, a method well known in the art. If two different functional groups X are protected by a suitable protecting group, it is possible to remove at least one protecting group in order to make at least one functional group X effective for further reaction with at least another compound, leaving at least one protected by The other functional groups are protected until the reaction product of the compound (I) and the compound (II) reacts with another compound. Thereafter, the protecting group of the functional group that is still protected may be removed, so that the remaining functional group X is effectively reacted with another compound. It is important to use at least one protecting group to avoid the reaction to produce a hydroxyalkyl starch derivative comprising compound (11) which has been reacted with two or more compounds hydrazone, that is, compound (II) substituted with multiple has. However, the same result can be achieved by reacting compound VII with an excess of compound (II). If an excessive amount of compound (II) is used in the process of the present invention, the molar ratio of compound (Π) to compound (I) should be within the range from 2 to 100. Once compound (I) reacts with compound (II) A reaction product is formed which can be isolated from the reaction mixture by at least one suitable method. If desired, the reaction products can be eluted by at least one appropriate method before isolation -25-

200521143 A7 B7 五、發明說明（24) 來0 如果反應產物先沉澱出來，可能將，例如，反應混合 物與，至少一種溶劑或溶劑混合物，除了存在於反應混合 物中之溶劑或溶劑混合物之外，於適當的溫度下接觸。根 據本發明之水用作為溶劑之特別佳的具逋例中，反應混合 物係與乙醇及㈣之齡物’线1 : 1混合物，係指該 化合物之等體積，於較佳自·20 S 5G°C範®且制佳自〇 至25°C範園之溫度下接觸。 產物係藉著加入溶劑或溶劑混合物 而沉澱，其中，產 物係不af±或具低溶解性。使產物沉澱之適當溶劑係依產 物之本質ffijd具體例中，產物係藉著加人醇，宜為 2丙醇或乙醇，且於通常自_6〇至耽之溫度較佳為心 度下培育而峨。於糾的具義中，產物係 藉二點極性有機溶劑之醇的混合物，例如，丙嶋沉 澱。適备的溶劑混合物為乙醇與丙嗣，例如，ι:ι之乙 嗣物’係指該溶劑之等體積，且通常係“0 銀 濟 部 智 慧 財 產 局 員 工 消 费 合 作 社 -,^ ^ ' 至2〇c，宜為o°c之低溫下離 二二圭=或至溶劑_，更佳係與醇， 厂、田唐下π 常自至2〇t ,較佳為-20至2〇t 小時常於相同的溫度培㈣至料時，宜為 反應產物之單離可藉自 進行。根據本發明較僅=:項=項步，之適當方法 於瓶例，首先以適當的方法，例 -26- 200521143 A7 B7 五、發明說明（25) 如離心或過濾法將反應產物或反應混合物與例如乙醇-丙 _混合物之混合物分離出來。於第二步驟中，將分離的反 應產物進行進一步處理，例如後_處理，如透析儀，離心 過濾，或壓力過濾，離子交換色層分離法，HPLC， MPLC，凝膠過濾及/或冷凍乾燥。根據丰發明甚至更佳的 具體例，依照想要的產物規格首先將分離的反應產物透 析’宜對水透析，且然後經冷凍乾燥直到反應產物之溶劑 含量足夠低。冷凍乾燥可在自20至35。(：，宜為自25至 30°C之溫度下進行。 經如此單離之化合物(I)與化合物(II)的反應產物可進一 步與至少一種其他化合物經由包含於該反應產物中之至少 一個官能基X進行反應。 依官能基X之►化學本質而定，可使用能與該基困X形 成化學鍵合之每一種可能的化合物。為了此反應，可使用 一種或多種適當的溶劑，且所有的反應參數例如，反應時 之溫度，反應時間，反應比例或反應混合物之pH值可適 應特定之需要。 經濟部智慧財產局員工消費合作社印製 根據本發明特別佳之具體例，能與至少一個官能基X 形成化學鍵合之至少一種化合物係為多胜肽或為至少二種 不同多胜肽的混合物。 因此，本發明亦關於如前說明的方法，其中化合物⑴ 與化合物(Π)之反應產物係與多胜肽經由包含於化合物(II) 中之官能基X進行反應。 根據本發明另一特別佳之具體例，能與至少一個官能 -27- 200521143 A7 _____ B7 五、發明說明（26) 基X形成化學鍵合之至少另一種化合物係為交聯化合物， 其能夠與化合物⑴與化合物(11)之反應產物之至少一個官 能基X形成第一個化學鍵合，且與又一種化合物形成第二 個化學鍵合。 根據本發明甚至更佳之具艎例，又一種化合物係為多 胜肽或至少二種不同的多胜肽混合物。 本發明之該具體例的說明書中，有可能將化合物⑴與 化合物(II)之反應產物，第一羥基烷基澱粉衍生物，與交 聯化合物進行反應而得到第二羥基烷基澱粉衍生物。該第 二經基燒基澱粉衍生物可隨即與宜為多胜肽之又一種化合 物反應，而得到第三羥基烷基澱粉衍生物。 然而，亦可能將反應化合物⑴與化合物(11)之反應產 物’第一羥基烷基澱粉衍生物，與交聯化合物與又一種化 合物之反庳產物，宜為多胜肽進行反應。 經濟部智慧財產局員工消費合作社印製 因此，本發明亦關於如前說明的方法，其中，化合物 ⑴與(II)之反應產物係與另外的化合物進行反應，該另外 的化合物為交聯化合物，係經由包含於交聯化合物中之官 能基V及包含於化合物⑴與(Π)之反應產物中之官能基X 進行反應。200521143 A7 B7 V. Description of the invention (24) 来 0 If the reaction product precipitates out first, it is possible to, for example, react the reaction mixture with, at least one solvent or solvent mixture, in addition to the solvent or solvent mixture present in the reaction mixture, in Contact at the proper temperature. In a particularly good example of the use of water as a solvent according to the present invention, the reaction mixture is a 1: 1 mixture of ethanol and acetone, which refers to an equal volume of the compound, preferably from 20 S 5G. ° C Fan® and make contact at temperatures ranging from 0 to 25 ° C Fan Yuan. The product is precipitated by adding a solvent or a solvent mixture, wherein the product is not af ± or has low solubility. The appropriate solvent for the precipitation of the product is based on the nature of the product. In specific examples, the product is prepared by adding human alcohol, preferably 2 propanol or ethanol, and is cultivated at a temperature usually from _60 to Yan. And e. In the sense of correction, the product is a mixture of alcohols with two polar organic solvents, such as propane precipitation. A suitable solvent mixture is ethanol and propionate. For example, ι: ι's acetic acid 'means an equal volume of the solvent, and is usually "0 Consumer Consumption Cooperative of Intellectual Property Bureau of the Ministry of Banking--^ ^' to 2 〇c, preferably at a low temperature of o ° c = or to the solvent _, more preferably with alcohol, the plant, Tiantangxia π often from 20t, preferably -20 to 20t hours When cultivating at the same temperature, it is preferred that the reaction product can be borrowed separately. According to the present invention, only the appropriate method is used in the bottle example. First, use the appropriate method, example- 26- 200521143 A7 B7 V. Description of the invention (25) If the reaction product or reaction mixture is separated from a mixture of, for example, an ethanol-propane mixture by centrifugation or filtration, in a second step, the separated reaction product is further processed. Such as post-treatment, such as dialyzer, centrifugal filtration, or pressure filtration, ion exchange chromatography, HPLC, MPLC, gel filtration and / or freeze-drying. According to even better specific examples of the invention, according to the desired Product specifications first dialyze the isolated reaction product ' Dialyze water and then freeze-dry until the solvent content of the reaction product is sufficiently low. Freeze-drying can be carried out at a temperature from 20 to 35. (:, preferably from 25 to 30 ° C. The compound thus isolated ( I) The reaction product of the compound (II) may be further reacted with at least one other compound via at least one functional group X contained in the reaction product. Depending on the ►chemical nature of the functional group X, it is possible to use a compound capable of reacting with the group X X forms every possible compound of chemical bonding. For this reaction, one or more appropriate solvents can be used, and all reaction parameters such as temperature, reaction time, reaction ratio or pH of the reaction mixture can be adapted. Specific needs. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. In accordance with the present invention, at least one compound capable of forming a chemical bond with at least one functional group X is a polypeptide or at least two different polypeptides. Therefore, the present invention also relates to the method as described above, in which the reaction product of compound ⑴ and compound (Π) is The peptide is reacted via the functional group X contained in the compound (II). According to another particularly preferred embodiment of the present invention, it is capable of forming a chemical bond with at least one functional -27- 200521143 A7 _____ B7 V. Description of the invention (26) The group X forms a chemical bond At least one other compound is a cross-linking compound capable of forming a first chemical bond with at least one functional group X of a reaction product of compound VII and compound (11), and forming a second chemical bond with another compound. An even better example of the present invention is that another compound is a polypeptide or a mixture of at least two different polypeptides. In the description of this specific example of the present invention, it is possible to react compound ⑴ with compound (II) The product, the first hydroxyalkyl starch derivative, is reacted with a crosslinking compound to obtain a second hydroxyalkyl starch derivative. This second base-based starch derivative can then be reacted with another compound, preferably a polypeptide, to obtain a third hydroxyalkyl starch derivative. However, it is also possible to react the reaction product of the reaction compound VII with the compound (11), the first hydroxyalkyl starch derivative, and the reaction product of the cross-linking compound with another compound, preferably a polypeptide. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. Therefore, the present invention also relates to the method described above, in which the reaction product of compound VII and (II) is reacted with another compound, which is a cross-linked compound. The reaction is carried out via the functional group V contained in the cross-linked compound and the functional group X contained in the reaction product of the compound VII and (Π).

因此，本發明亦關於如前說明的方法，其中，化合物 (I)與(II)之反應產物係與另外的化合物進行反應，該另外 的化合物係為交聯化合物，經由包含於交聯化合物中之官 能基V及包含於化合物⑴與(II)之反應產物中之官能基X 進行反應，該交聯化合物業已與又一種化合物於反應之前 •28- 200521143 A7 B7 五、發明說明（27) 與化合物⑴與(II)之反應產物進行反應。 因此，本發明亦關於如前說明的方法，其中，又一種 化合物係為多胜肽，宜為紅血球生成素，其係與交聯化合 物經由官能基X進行反應，包含於交聯化合物中。 因此，本發明亦關於如前說明的方法，其中，化合物 (II)係與另一種化合物，宜為與交聯化合物進行反應而得 到第一反應產物，該第一反應產物與又一種化合物進行反 應而得到第二反應產物，且該第二反應產物與化合物(I)進 行反應。 因此，本發明亦關於如前說明的方法，其中，另一種 化合物，宜為交聯化合物，係與又一種化合物，宜為多胜 肽進行反應而得到第一反應產物，該第一反應產物係與化 合物(II)進行反應而得到第二反應產物，且該第二反應產 物係與化合物⑴進行反應而得到羥基烷基澱粉衍生物。 經濟部智慧財產局員工消费合作社印製 根據本發明特別佳之具體例，交聯化合物係用於形成 化合物(II)或化合物⑴與(II)之反應產物間的化學橋，且又 一種化合物，其中與交聯化合物進行反應之又一種化合物 之官能基係為-SH基或搭基或酮基，且與交聯化合物進行 反應之化合物(II)或化合物⑴與(II)之反應產物之官能基係 為包含結構-NH-，特別佳為-NH2之基團。 本發明之說明書中，”交聯化合物”一詞係關於能夠在 化合物(II)或化合物⑴與(II)之反應產物，及至少一給定之 又一種化合物間形成鍵合之化合物。依又一種化合物之化 學本質而定，交聯化合物係包括能與包含於化合物(11)或 -29- A7 B7 200521143 五、發明說明（28 ) 化合物⑴與(II)之反應產物之官能基X進行反應之至少一 個官能基V，及能與又一種化合物形成化學鍵合之至少另 一個官能基。該包含於交聯化合物中之至少另一個官能基 可為前文所述有關官能基X類型之官能基。 交聯化合物可用於擴增化合物(I)與又一種化合物，宜 為多胜肽間整體化學橋之長度，及/或用於影響含或不含 又一種化合物之所產生的反應產物的化學本質，及/或提 供形成許多又一種化合物與化合物⑴、（11)之反應產物與 交聯化合物間之鍵合的可能性，及/或用於將包含於化合 物⑴與(II)之反應產物之官能基X化學改質以便使得該反 應產物能與給定之另外化合物進行反應。 因此，前文說明之本發明的具體例及其關於官能基x 為-NH2基，與另外的化合物，例如Therefore, the present invention also relates to the method described above, in which the reaction product of compounds (I) and (II) is reacted with another compound, which is a cross-linked compound, and is included in the cross-linked compound. The functional group V and the functional group X contained in the reaction product of the compound ⑴ and (II) are reacted, and the cross-linked compound has been reacted with another compound before the reaction. • 28- 200521143 A7 B7 V. Description of the invention (27) and Compound VII reacts with the reaction product of (II). Therefore, the present invention also relates to the method described above, in which another compound is a polypeptide, preferably erythropoietin, which reacts with the cross-linked compound via the functional group X and is contained in the cross-linked compound. Therefore, the present invention also relates to the method as described above, wherein the compound (II) is reacted with another compound, preferably a cross-linked compound to obtain a first reaction product, and the first reaction product is reacted with another compound. A second reaction product is obtained, and the second reaction product is reacted with the compound (I). Therefore, the present invention also relates to the method described above, wherein another compound, preferably a cross-linked compound, is reacted with another compound, preferably a polypeptide, to obtain a first reaction product, the first reaction product is The compound (II) is reacted to obtain a second reaction product, and the second reaction product is reacted with the compound ⑴ to obtain a hydroxyalkyl starch derivative. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs, a particularly good example according to the present invention, the cross-linking compound is used to form a chemical bridge between compound (II) or the reaction product of compound VII and (II), and another compound, wherein The functional group of another compound that reacts with a cross-linked compound is -SH group or an alkoxy group or a keto group, and the functional group of the compound (II) or the reaction product of compound ⑴ and (II) which reacts with the cross-linked compound It is a group containing the structure -NH-, particularly preferably -NH2. In the specification of the present invention, the term "cross-linked compound" refers to a compound capable of forming a bond between the compound (II) or the reaction product of the compound VII and (II), and at least one given another compound. Depending on the chemical nature of another compound, the crosslinked compound includes a functional group X capable of reacting with the compound (11) or -29- A7 B7 200521143 V. Description of the invention (28) Compound ⑴ and (II) At least one functional group V that is reacted, and at least one other functional group capable of forming a chemical bond with another compound. The at least one other functional group contained in the crosslinking compound may be a functional group of the functional group X type described above. Cross-linking compounds can be used to amplify compound (I) and another compound, preferably the length of the overall chemical bridge between the peptides, and / or to affect the chemical nature of the reaction product produced with or without another compound And / or provide the possibility of forming many further bonds between compounds and compounds VII, the reaction products of (11) and cross-linking compounds, and / or for the inclusion of the reaction products of compounds VII and (II) The functional group X is chemically modified so that the reaction product can react with a given additional compound. Therefore, the specific examples of the present invention described above and their functional group x is -NH2 group, and other compounds such as

經濟部智慧財產局貝工消费合作社印製 之化學改質係為了提供與包含於又一種化合物，宜為多胜 肽中之-SH基進行反應之可能性者，係為化合物(I)與(π)之 反應產物與交聯化合物進行反應之特定例。 -30- * *·**♦ ―*^ -*·^ ·脅^•良 ，▲，八 •.二 m A7 B7 200521143 五、發明說明（29 ) 根據本發明較佳的具體例，官能基V可為前文所述如 基團X類型之官能基。The chemical modification printed by the Shellfish Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs is to provide the possibility of reacting with the -SH group contained in another compound, preferably the dopeptide, as compounds (I) and ( A specific example of the reaction product of π) and a crosslinking compound. -30- * * · ** ♦ ― * ^-* · ^ · Threat ^ • Good, ▲, Eight • Two m A7 B7 200521143 V. Description of the invention (29) According to a preferred specific example of the present invention, the functional group V may be a functional group of the type X described above.

根據本發明另一較佳的具體例，官能基父或官能芙V 係為硫基且官能基V或官能基X宜選自於包含下列2美 團： &According to another preferred embodiment of the present invention, the functional group parent or functional group V is a sulfur group and the functional group V or the functional group X is preferably selected from the group consisting of the following two groups: &

其中，Hal為a，Br或I，宜為Br或I。 根據本發明還有另一較佳的具體例，官能基乂哎官能 基V係選自於如前文所述包含活性酯之基團或任意轉化成 活性酯之羧基。於該特別之情況中，官能基ν或官能基X 分別包括化學結構-ΝΗ-。 經 濟 部 智 慧 財 產 局 貝 X 消 费 合 作 社 因此’交聯化合物係為具有至少二種相同或不同官能 基的化合物。於二官能基之情況中，交聯化合物可為均_ 二官能或雜-二官能者。均-二官能交聯化合物，例如，係 提供形成化合物⑴與(II)之反應產物與又一種化合物，反 應產物與具有相同類型官能基之另外化合物間的橋之可能 性。雜-二官能交聯化合物，例如，係提供形成化合物⑴ 與(II)之反應產物與又一種化合物，反應產物與具有彼此 間不能作用的官能基之另外化合物間的橋之可能性。 交聯化合物之至少二個官能基可—起直接連接或可被 線形或分支的烧基或環烧基或芳基或芳燒基或芳基環烧^基 -31- 200521143 B7 五、發明說明（3〇) 或烧芳基或環燒基芳基基團所分離，其中，此等基團可包 括至少一雜原子例如N，0, S，且其中此等基團可適當地 被取代。分離交聯化合物之至少二個官能基的基團長度可 適應特定之需要。通常，分離的基團具有自1至60，較佳 為自1至40，更佳為自1至20，更佳為自1至1〇，更佳 為自5至10個碳原子。如果存有雜原子，分離的基團通 常包括自1至20，較佳為自1至8且特別佳為自1至4個 雜原子。根據甚至更佳的具體例，分離的基團為自1至20 個碳原子之燒基或芳烧基鏈。此外，交聯化合物可進一步 包括如前說明有關化合物(II)之至少一個裂解部位。 經濟部智慧財產局員工消费合作社印製 本發明說明書中所提交聯化合物之其他實例可例如， 根據下列清單分類： 交赛化 合物的 類型 嫵舆又一種化合物， 宜為多胜肽進行反應 之官能基 官能基V A 醯肼(醛-反應性） 馬爾亞胺(Maleimido) (SH-反應性） B 醯肼(醛·反應性） 派里代二硫基 (Pydridydithio)(SH-反應性） C 碘烷基(SH-反應性） N-琥珀醯亞胺酯 (胺-反應性） D 瘓烷基(SH-反應性） N-琥珀醯亞胺酯 (胺_反應性） E 馬爾亞胺 (SH-反應性） N-琥珀醯亞胺酯 (胺_反應性） F 派里代二硫基 (SH-反應性） N-琥珀醯亞胺酯 1反應性） G 己稀基艰 (SH-反應性） N-琥珀醢亞胺酯 (胺-反應性） -32- 200521143 A7Among them, Hal is a, Br or I, preferably Br or I. According to still another preferred embodiment of the present invention, the functional group V is a functional group selected from the group containing an active ester as described above or a carboxyl group optionally converted into an active ester. In this particular case, the functional group ν or the functional group X each includes a chemical structure -NΗ-. The Ministry of Economic Affairs and the Intellectual Property Office of the People's Republic of China, X Consumer Co., Ltd. Therefore, the 'cross-linked compound is a compound having at least two kinds of the same or different functional groups. In the case of a difunctional group, the cross-linking compound may be a homo-difunctional or a hetero-difunctional. Homo-bifunctional cross-linked compounds, for example, provide the possibility of forming a bridge between the reaction product of compounds VII and (II) and another compound, the reaction product, and another compound having the same type of functional group. Hetero-bifunctional cross-linking compounds, for example, provide the possibility of forming a bridge between the reaction product of compounds VII and (II) and another compound, the reaction product, and another compound having functional groups that cannot interact with each other. At least two functional groups of the cross-linking compound may be directly connected or may be linear or branched alkyl or cycloalkyl or aryl or aromatic alkyl or arylcycloalkyl ^ -31- 200521143 B7 V. Description of the invention (30) or an aryl or cycloalkyl aryl group, wherein such groups may include at least one heteroatom such as N, 0, S, and wherein these groups may be appropriately substituted. The length of the group separating at least two functional groups of the cross-linked compound can be adapted to specific needs. Generally, the isolated group has from 1 to 60, preferably from 1 to 40, more preferably from 1 to 20, more preferably from 1 to 10, even more preferably from 5 to 10 carbon atoms. If heteroatoms are present, the isolated groups usually include from 1 to 20, preferably from 1 to 8, and particularly preferably from 1 to 4 heteroatoms. According to even better specific examples, the isolated group is an alkyl or arylalkyl chain of from 1 to 20 carbon atoms. Further, the crosslinking compound may further include at least one cleavage site for the compound (II) as described above. Other examples of compound compounds submitted in the description of the present invention printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economics can be classified, for example, according to the following list: Types of competing compounds: Another compound, preferably a functional group that reacts with a peptide Functional group VA hydrazine (aldehyde-reactive) Maleimido (SH-reactive) B hydrazine (aldehyde-reactive) Pydridydithio (SH-reactive) C iodoane (SH-reactive) N-succinimide (amine-reactive) D Paralyzed alkyl (SH-reactive) N-succinimide (amine-reactive) E Malimine (SH- Reactivity) N-succinimide (amine_reactive) F Peridiodithio (SH-reactive) N-succinimide 1 reactive) G Hexyl dialkyl (SH-reactive ) N-succinimide (amine-reactive) -32- 200521143 A7

本說明書最後部份之表1中列舉了 —些較佳的交聯化 合物實例。 於至少另-種化合物之情況中，就各對掌中心例如， 交聯化合物，其包括一或多個對掌中心，至少另一種化合 物可存在於R組態中或S組態中或作為消旋化合物。 本發明之說明書中所用的，，多胜肽，，一詞係指化合物其 包括至少二個經由胜肽鍵，亦即具結構_(c哪nh_的鍵連 接的胺基酸。多胜肽可為天然生成的化合物或為非天然生 成的多胜肽，後者包括天然生成的胺基酸及/或至少一種 非天然生成的胺基酸。多胜肽之主幹，多胜肽鏈可進一步 被至少一個適當的取代基所取代，如此而具有至少一個側 鏈。至少一個官能基γ可為多胜肽主幹的部份或主幹之至 少一個取代基的部份，其中，具體例可能包括至少一個官 能基’其係為多胜肽主幹之部份且至少一個官能基係為多 胜肽主幹之至少一個取代基的部份。 就多胜肽而言，並沒有限制多胜肽包括至少一個官能 基Y。該官能基Y可直接連接到多胜肽主幹或為主幹側鏈 的部份。側鏈或官能基Y或二者可為天然生成之多胜肽的 部份或可被導入天然生成的多胜肽中或於與官能基x進行 反應之前被導入至少部份非天然生成的多胜肽中。 此外’至少大部伤之多胜肽可為任何人類或動物來源 者。於較佳的具體例中，多胜肽係為人類來源者。 多胜肽可為細胞素’尤其為紅血球生成告，抗凝企酶 (AT)例如，at m，白血球間素，尤其是白血球間素-2， -33-Some examples of preferred crosslinking compounds are listed in Table 1 at the end of this specification. In the case of at least one other compound, for each pair of palm centers, for example, a crosslinked compound that includes one or more palm centers, at least one other compound may exist in the R configuration or the S configuration or as a consumer Rotating compounds. The term "polypeptide" as used in the specification of the present invention refers to a compound that includes at least two amino acids connected via a peptide bond, that is, a bond having the structure _ (cnh_). Polypeptide It can be a naturally occurring compound or a non-naturally occurring polypeptide, the latter including naturally occurring amino acids and / or at least one non-naturally occurring amino acid. The backbone of the polypeptide, the polypeptide chain can be further Substituted by at least one suitable substituent, thus having at least one side chain. At least one functional group γ may be part of the polypeptide backbone or part of at least one substituent of the backbone, of which specific examples may include at least one The functional group is a part of the polypeptide backbone and at least one functional group is a part of the polypeptide backbone. As far as the polypeptide is concerned, the polypeptide is not limited to include at least one functional group. Y. The functional group Y can be directly connected to the backbone of the peptide or part of the backbone side chain. The side chain or the functional group Y or both can be part of the naturally occurring peptide or can be introduced into the naturally occurring In the peptide The functional group x is introduced into at least part of the non-naturally occurring polypeptide before the reaction. In addition, at least most of the polypeptide of the wound can be of any human or animal origin. In a preferred embodiment, the polypeptide It is of human origin. Polypeptides can be cytokines, especially red blood cells, anticoagulant enzymes (AT), such as at m, interleukins, especially interleukin-2, -33-

200521143 A7 B7 五、發明說明（32) IFN-β，IFN-α，G-CSF，CSF，白金球間素-6及治療性抗 體。 根據較佳的具體例，多胜肽係為抗凝血酶(AT)，宜為 AT III(里維JH，威辛袼A，錫梅克CA，艾奇拉γ，重組 體抗凝血酶：心血管疾病中之製造與任務，栓塞與止血法 研討會27，4(2001) 405-416 ;艾德木T，泛巴登SM，波 洛克J，韓森E，柏納可尼R，錫金E，馬納瓦蘭p，錫梅 克C’美第Η ’麥費森J’柯爾ES，基因轉殖製造的人類 抗凝jk酶：對人類ok漿所衍生之抗凝赢酶之結構與功能的 比較，血液91，12(1998) 4661-4671 ;明尼馬MC，張氏 ACK，杰森PM，魯柏YTP，普拉特BM，惠塔克BG，泰 勒FB，哈克CE，佛迪曼B，重組體人類抗凝金酶iij改 進大腸桿菌致命激發發炎反應中之狒狒的生存與減弱，血 液 95 ’ 4 (2000) 1117-1123 ;泛巴登 SM，韓森 EH，柏納 可尼R’江氏K，馬納瓦蘭P，柯爾ES，麥費森JM，艾 德木T，甲琉胺酸殘質於抗凝血酶中之氧化作用，生物化 學期刊 274，15 (1999) 10268-10276)。 經濟部智慧財產局員工消费合作社印製 根據另一較佳的具體例，多胜肽係為人類IFN-β，特 別為 IFN-β la (參閱 Avonex®，REBIFd))及 IFN-β lb (參閱 BETASERON®) 〇 另外較佳的多胜肽為人類G-CSF(粒細胞菌落刺激因 子）。參閱，例如，長田等，人類粒細胞菌落刺激因子之 染色體基因結構與二種mRNAs，EMBO J. 5 : 575-581， -34- 200521143 A7 B7 五、發明說明（33) 1986 ;索薩等；重組體人類粒細胞菌落刺激因子··於正常 及白血病骨髓細胞上的影響，科學232 (1986) 61-65，•及 賀曼等，Neupogen®(費爾佳斯定）之特徵、調配、及穩定 性，重組體人類粒細胞菌落刺激因子，於：蛋白質藥物中 之調配、特徵、及穩定性，羅德尼貝爾曼與孓王約翰編 輯，普利南出版社，紐約，1996，303-328。 若係用至少二種不同多胜肽的混合物，則至少二種多 胜肽可在，例如，莫耳質量，胺基酸數量及/或次序上不 同’不同的醣化度，取代基數量及/或化學本質或被適當 化學鍵，例如，二硫化物橋連接之多胜肽鏈數量。 根據本發明較佳的具體例，選擇地與交聯化合物進一 步反應之化合物(I)與化合物(II)之反應產物係經單離，宜 根據至少一種前文說明的方法，且然後與具有至少一個官 能基Y能與化合物(I)與化合物(II)之反應產物之至少一個 官能基X進行反應之多胜肽進行反應，選擇地進一步與交 聯化合物進行反應而形成至少一個化學鍵合。多胜肽例如 蛋白質之官能基Y為，例如：200521143 A7 B7 V. Description of the invention (32) IFN-β, IFN-α, G-CSF, CSF, platinum-interleukin-6 and therapeutic antibodies. According to a preferred specific example, the polypeptide system is antithrombin (AT), preferably AT III (Rivier JH, Wessinger A, Simec CA, Aigira γ, recombinant antithrombin : Manufacturing and Tasks in Cardiovascular Diseases, Symposium on Embolism and Hemostasis 27, 4 (2001) 405-416; Edmund T, Panbaden SM, Pollock J, Hansen E, Bernaconi R, Sikkim E, Manawalan p, Simek C 'Medellin' Mefferson J 'Kohl ES, human anticoagulant jk enzymes produced by genetic transformation: anti-coagulant enzymes derived from human ok plasma Comparison of Structure and Function, Blood 91, 12 (1998) 4661-4671; Minnema MC, Zhang ACK, Jason PM, Ruby YTP, Pratt BM, Whitaker BG, Taylor FB, Hack CE, Friedman B, Recombinant human anticoagulant enzyme iij improves survival and attenuation of baboons in lethal inflammatory response to E. coli, blood 95 '4 (2000) 1117-1123; Pan-Baden SM, Hansen EH, Bernard Koni R 'Jiang's K, Manawalan P, Cole ES, Macpherson JM, Edmund T, Oxalinic Acid Residues in Antithrombin, Journal of Biochemistry 274, 15 (1999) 10268-10276). Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. According to another preferred embodiment, the peptides are human IFN-β, especially IFN-βla (see Avonex®, REBIFd) and IFN-β lb (see BETASERON®) 〇 Another preferred peptide is human G-CSF (granulocyte colony-stimulating factor). See, for example, Nagata et al., Chromosomal gene structure of human granulocyte colony-stimulating factor and two types of mRNAs, EMBO J. 5: 575-581, -34- 200521143 A7 B7 V. Description of the invention (33) 1986; Sosa et al .; Effect of recombinant human granulocyte colony-stimulating factor on normal and leukemia bone marrow cells, Science 232 (1986) 61-65, and Herman et al. Characteristics, formulation, and distribution of Neupogen® Stability, Recombinant Human Granulocyte Colony Stimulating Factor, In: Formulation, Characteristics, and Stability in Protein Drugs, Edited by Rodney Bellman and King John, Purinan Press, New York, 1996, 303-328 . If a mixture of at least two different polypeptides is used, the at least two polypeptides may differ in, for example, molar mass, amino acid quantity and / or order. 'Different degree of saccharification, number of substituents and / Either the chemical nature or the number of peptide chains linked by an appropriate chemical bond, for example, a disulfide bridge. According to a preferred embodiment of the present invention, the reaction product of the compound (I) and the compound (II) which is further reacted with the cross-linking compound is selectively isolated, preferably according to at least one of the methods described above, and then with at least one The functional group Y can react with a polypeptide that reacts with at least one functional group X of the reaction product of the compound (I) and the compound (II), and optionally further reacts with a cross-linking compound to form at least one chemical bond. The functional group Y of a peptide such as a protein is, for example:

——SH —〇H——SH —〇H

-35- 200521143 A7 B7 五、發明說明（34) 或為可藉N-醣化作甩或〇醣化作用連接到多胜肽之醣分 子部分。 本發明之說明書中，，，醣分子部分，，一詞係指羥基醛類 或羥基酮類以及其化學改變（參閱，羅普化學詞典，-35- 200521143 A7 B7 V. Description of the invention (34) Or it can be linked to the polysaccharide moiety of the polypeptide by N-glycosylation or glycosylation. In the description of the present invention, the term, a sugar molecule portion, refers to a hydroxy aldehyde or a hydroxy ketone and a chemical change thereof (see, Rope Chemical Dictionary,

Thieme Verlag Stuttgart，德國，1990 第九版，第 9 卷， 2281_2285頁及其中所引述的文獻）。此外，亦指天然生成 之醣分子部分的衍生物，例如，葡萄糖，半乳糖，甘露 糖’唾液酸等。該詞亦包括經化學氧化天然生成的酷分子 部分。經氧化的醣分子部分之結構可為環狀或線形。 醣分子部分可直接連接到多胜肽主幹。較佳為，醣分 子部分係為醣分子側鏈的一部份。更佳為，醣分子部分係 為酷分子侧鏈之終端部分。 於甚至更佳的具體例中，醣分子部分係為醣分子侧鏈 之半乳糖殘基，宜為酷分子側鏈之終端半乳糖殘基。該半 乳糖殘基可藉著將終端延酸移除，接著藉氧化作用，如下 文所說明者，而有效與化合物⑴與化合物之反應產物 進行反應。 經濟部智慧財產局員工消费合作社印製 於還有另外較佳的具體例中，化合物⑴與(π)之反應產 物係連接到醣分子側鏈之唾液酸殘基，宜為醣分子側鏈之 終端唾液酸殘基。 終端醣分子基團之氧化可以化學或酵素地進行。 多胜狀_分子部分之化學氧化的方法係已知於此方面 技藝中且包括用過破酸鹽處理（查謀等，1992，生物化學 期刊，267，15916-15922)。 -36- 200521143 A7 B7 五、發明說明（35) ^ 以化學氧化，原則上可能氧化任何醣分子部分，在終 知位或否。然而’藉著選擇的溫和條件（lmM過破酸鹽，〇 C相對於惡劣的條件：1〇mM過碘酸鹽於室溫下i小時）， 可能宜將醣分子侧鏈之終端延酸氧化。 或者’酷分子部分可被酵素氧化。用於氧化個別的醣 分子部分之酵素係已知於此方面之技藝中，例如，於*** 糖之清況中’酵素係為半乳糖氧化酶。如果想要氧化終端 之半乳糖部分，則實質上需要將終端唾液酸移除(部份的 或完全的）’若細胞中已產生能夠使唾液酸附著於醣分子 鏈之多胜肤’例如，於哺乳動物細胞中或於通常業經改變 使延酸能夠附著於糖分子鏈之細胞中的話。用於移除唾液 酸之化學或酵素的方法係已知於此方面之技藝中（查普林 與甘乃迪(編輯），1996，醣分子分析：實用探討，尤其是 第5章說明，醣蛋白，175_177頁；IRL出版實用探討系 列(ISBN 0-947946-44-3))。 經濟部智慧財產局員工消费合作社印製 因此，本發明亦關於如前說明的方法，其中，化合物 ⑴與化合物(II)之反應產物與多胜肽係經由包含於多胜肽 中之氧化的醣分子部分進行反應。 根據本發明另一個較佳的具體例，多胜肽之官能基係 為硫基基團。因此，化合物⑴與化合物(II)之反應產物可 經由硫基醚基團連接到多胜肽，其中，s原子可從任何包 含於多胜肽中之硫基衍生出來。 硫基基團可像這樣存在於多胜狀中。此外，可能依照 適當的方法將硫基導入多胜肽中。尤其，以化學法為可被 -37- 200521143 A7 B7 五、發明說明（36) 提及者。若二硫化物存在於多胜肽中，可能將-S-S結構減 少以得到硫基基團。亦可能藉著將多胜肽經由胺基酸基團 與具有至少二個不同官能基的化合物，，其中之一能夠與 胺基酸進行反應及另一個為SH基或為SH基團之前質進 行反應而將存在於多胜肽中之胺基酸轉換成SH基。 該胺基酸之改質可視為一實例，其係首先將蛋白質與 具有至少二個不同官能基之化合物(L)，其中之一能夠與胺 基酸進行反應且另一係為SH基進行反應，且產生的反應 產物然後與例如，包含HAS與化合物(D)之HAS衍生物進 行反應，該衍生物包含一能與SH基團起作用之官能基。 亦可能將SH基困藉由多胜肽之突變，例如，藉著將半胱 胺酸或適當的SH官能性胺基酸導入多胜肽中，或例如將 半胱胺酸從多胜肽中移除使得多胜肽中之另一個半胱胺酸 不能形成二硫化物橋而導入。 該具體例之說明書中，特別佳者係將多胜肽與從化合 物⑴與(II)之反應產物與交聯化合物之反應中所產生之反 應產物進行反應。 經濟部智慧財產局員工消费合作社印製 因此，本發明亦關於如前說明的方法，其中，化合物 ⑴與化合物(II)之反應產物係與交聯化合物進行反應，且 所產生的反應產物係經由包含於多胜肽中之氧化的醣分子 部分及/或硫基基團進一步與多胜肽進行反應。 尤其佳之多胜肽係用紅血球生成素(EPO)。 因此，本發明亦關於如前說明的方法，其中多胜肽係 為紅血球生成素。 -38- A7 B7 200521143 五、發明說明（37) 經濟部智慧財產局員工消费合作社印叛 EPO可為任何人類（參閲’例如，井上，和田，竹内， 1994，以貧血病患尿液中之高生體内活性純化人類紅血球 生成素之改良方法，生物醫藥報導17(2)，180-4 ;三宅， 康氏’金瓦瑟，1977，人類紅血球生成素之純化，生物化 學期刊，252(15)，5558_64)或另一種哺乳動物來源及可從 天然生成的來源如人類腎臟，胚胎人類肝臟或動物，宜為 猴子腎臟藉純化而獲得者。此外，”紅企球生成素，， 或’ΈΡΟ”之表示亦涵蓋EPO變異體，其中一或多個胺基酸 (例如1至25，較佳為1至1〇，更佳為1至5，最佳為i 或2個）業已被另一個胺基酸所改變且其具有紅血球生成 素的活性(參閱’例如EP 640 619 B1)。紅血球生成素之測 定係說明於此方面技藝中（試管内活性之測定，參閱，例 如，費畢等1991，血液，77，1203ff;北村等，1989 ,細 胞生理期刊；140，323-334 ;生體内EPO活性之測定，參 閱，歐洲藥典2001，911-917 ;歐洲藥典2000，1316紅血 球生成素濃縮液，78〇_785 ;歐洲藥典（1996/2000);歐洲 藥典，1996，紅血球生成素濃縮液，Pharmaeur〇pa , 8， 371-377 ;費畢，贺門仃，保利，勞夫，札特美索； 1995，選自於BHK-21細胞之重組體人類紅血球生成素之 N-與〇-醣化作用突變蛋白紅血球生成素，血液，85(5)， 1229-36 ;(將EPO與改質的EPO型式注入雌NMRI老鼠 中（等量蛋白質50毫克/老鼠），於第1，2及3天採血樣， 於第4天並測定網狀血球)）。另外的公開案中，Ep〇活性 測定之試驗係說明於巴邦，阿帕里西，安德森，納塔拉 -39- 200521143 A7 B7 五、發明說明（3〇 加，里奇，1994，網狀血球測定作為紅血球生成素之生物 分析，醫藥生物醫學年鑑期刊，12(4)，515-22 ;波維恩， 古利根，貝金，肯達爾，威利斯，1994，以自動裝置之網 狀血球參數，用血清運鐵蛋白受體及紅血球生成素濃縮物 估計脊髓發育不良中之有效與總的紅產球生成素，白血 病，8(1)，151-5 ;戴洛米，羅倫西尼，吉芬，馬丁，賈柯 柏森，布尼，艾略特，1992，醣化作用在紅血球生成素之 分泌與生物活性上的任務，生物化學，31(41)，9871-6 ; 樋口，戸田，久保庭，友野，下仲，越智，1992 ;糖鏈於 人類紅血球生成素生物活性表現中的任務，生物化學期 刊，267(11)，7703-9 ;山口，赤井，川西，上田，增田， 佐佐木，1991，N_醣化作用部位之定位移除在人類紅血 球生成素中於其製造與生物性質上的影響，生物化學期 刊，266(30) ’ 20434_9，竹内，井上，史崔克蘭，久保 田，和田，清水，保志，庫津，高崎，古細，1989，糖鏈 結構與中國倉鼠卵巢細胞中所製造之重組體人類紅血球生 成素生物活性間的關係’美國國家科學院研討會， 經濟部智慧財產局員工消费合作社印製 85(20)，7819-22 ;克滋，艾卡達，1989，紅灰球生成素的 分析法，尼弗隆，51(1)，11-4(德國）；袓卡利，史科斯基 1985 ,人類尿紅血球生成素於控制孔玻璃及唾液酸上之純 化，血液實驗，13(3)，833_7 ;克里斯朵，1983，母紅血 球增強因子(EEF)之物理與生物特性，血清中不同於紅血 球生成素之遲生效製造紅血球的刺激質，血液實驗， 11(1) > 18-31 〇 -40- 200521143 A7 B7 五、發明說明（39 ) 較佳者為，EPO係重組體地製成。此包括於真核狀態 或原核性細胞，宜為哺乳動物，昆蟲，酵母，細菌細胞中 或於任何其他便利EPO重組體製造之細胞類型中製造。 此外，EPO可在基因轉殖的動物中（例如，於體液中，像 牛奶，血液等），在基因轉殖之烏蛋中，尤其是家禽，較 佳為雞，或在基因轉殖的植物中表現。 多胜肽之重組體製造係已知於此方面之技藝中。通 常，此包括用適當的表現載體轉染寄主細胞，寄主細胞在 能從寄主細胞製造多胜肽及純化多胜肽之條件下培養。詳 細資料請參閱例如，克里斯朵、潘卡拉茲、法柏、史馬 特，1986 ,以快速五-階段過程純化人類紅血球生成素成 同質性，血液，67(1)，71·9 ;奎爾，卡斯拉克，柏克特， 沃朝斯基，1989，使用桿狀病毒媒介製造重組體人類紅血 球生成素之高度表現與純化，血液，74(2)，652-7 ; ΕΡ 640 619 Β1 與 ΕΡ 668 351 Β1。 於較佳的具體例中，ΕΡΟ具有人類ΕΡΟ之胺基酸次序 (參閱 ΕΡ 148 605 Β2)。 經濟部智慧財產局員工消费合作社印製 ΕΡΟ可包含一個或多個醣分子側鏈，較佳為1至12 個，更佳為1至9個，甚至更佳為丨至6個且特別佳為j 至4個’尤其佳者為4個聽分子侧鏈，經由及/或〇_連 接的醣化作用附著於ΕΡΟ，亦即ΕΡΟ係經醣化。一般， 當ΕΡΟ係在真核狀態的細胞中產生時，多胜肽係經後轉 移醣化。所以，於哺乳動物，尤其是人類，昆蟲或酵母細 胞中生物合成時醣分子側鏈已連接到Ερ〇。經醣化之Ερ〇 -41- 200521143 A7 B7 五、發明說明（4〇) 的結構或性質業已廣泛地在此方面技藝中研究（參閱，EP 428 267 Bl ; EP 640 619 B1 ;盧斯，德比，史密斯，瑪 琍，羅捷，羅德，卡達，1995，紅血球醣分子結構之微異 :質性，化學年鑑，67(8)，1442-52 ;竹内，古佃，1991， > · 人類紅血球生成素之糖鏈的結構與功能性任務，酷生物 學，1(4)，337-46(回顧）。Thieme Verlag Stuttgart, Germany, 1990, 9th edition, volume 9, p. 2281_2285 and references cited therein). In addition, it also refers to derivatives of naturally occurring sugar molecules, such as glucose, galactose, mannose 'sialic acid, and the like. The term also includes cool molecular parts that are naturally produced by chemical oxidation. The structure of the oxidized sugar molecule portion may be circular or linear. The sugar molecule portion can be directly connected to the polypeptide backbone. Preferably, the sugar moiety is part of a side chain of a sugar molecule. More preferably, the sugar molecule portion is a terminal portion of a cool molecular side chain. In an even better specific example, the sugar molecule part is a galactose residue of a sugar molecule side chain, and preferably a terminal galactose residue of a cool molecule side chain. The galactose residue can be effectively reacted with the reaction product of the compound VII and the compound by removing the terminal extended acid and then by oxidation, as described below. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. In another preferred embodiment, the reaction product of compound ⑴ and (π) is a sialic acid residue connected to the side chain of the sugar molecule. Terminal sialic acid residue. The oxidation of the terminal sugar molecule group can be carried out chemically or enzymatically. The method of chemical oxidation of the polysaccharide-like molecular part is known in the art and includes the treatment with salt-breaking salts (Chamou et al., 1992, Journal of Biochemistry, 267, 15916-15922). -36- 200521143 A7 B7 V. Description of the invention (35) ^ With chemical oxidation, in principle, it is possible to oxidize any sugar molecule part, in the final position or not. However, 'with the choice of mild conditions (lmM peroxoate, 0C vs. harsh conditions: 10mM periodate for 1 hour at room temperature), it may be appropriate to oxidize the terminal end acid of the sugar molecule side chain . Alternatively, the 'cool molecule part can be oxidized by an enzyme. Enzymes for oxidizing individual sugar molecules are known in the art, for example, in the case of breast milk, the enzyme is galactose oxidase. If it is desired to oxidize the terminal galactose moiety, the terminal sialic acid essentially needs to be removed (partially or completely) 'if there are already many cells in the cell capable of attaching sialic acid to the sugar molecule chain', for example, In mammalian cells, or in cells that are usually altered to enable the attachment of dextrin to sugar molecule chains. Chemical or enzyme methods for removing sialic acid are known in the art (Chapulin and Kennedy (eds.), 1996, Analysis of Glycomolecules: A Practical Discussion, especially Chapter 5, glycoproteins, 175_177 pages; IRL Publishes Practical Discussion Series (ISBN 0-947946-44-3)). Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. Therefore, the present invention also relates to the method described above, in which the reaction product of compound VII and compound (II) and the polypeptide are via the oxidized sugar contained in the polypeptide. The molecular part reacts. According to another preferred embodiment of the present invention, the functional group of the polypeptide is a thio group. Therefore, the reaction product of compound VII and compound (II) can be connected to the polypeptide via a thioether group, in which the s atom can be derived from any sulfide group contained in the polypeptide. The thio group can exist in multiple forms like this. In addition, it is possible to introduce a thio group into a polypeptide according to an appropriate method. In particular, the chemical method can be mentioned by -37- 200521143 A7 B7 V. Description of Invention (36). If a disulfide is present in the polypeptide, the -S-S structure may be reduced to obtain a thio group. It is also possible to carry out the polypeptide via an amino acid group with a compound having at least two different functional groups, one of which can react with the amino acid and the other is an SH group or a precursor of the SH group The reaction converts the amino acid present in the peptide into the SH group. The modification of the amino acid can be regarded as an example. The protein is first reacted with a compound (L) having at least two different functional groups, one of which can react with the amino acid and the other with the SH group. The resulting reaction product is then reacted with, for example, a HAS derivative comprising HAS and compound (D), the derivative comprising a functional group capable of interacting with the SH group. It is also possible to trap SH groups by mutations in the peptide, for example, by introducing cysteine or an appropriate SH-functional amino acid into the peptide, or for example, cysteine from the peptide Removal prevents the introduction of another cysteine in the polypeptide to form a disulfide bridge. In the description of this specific example, a particularly preferred one is to react a polypeptide with a reaction product generated from a reaction product of the compound VII and (II) with a cross-linked compound. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. Therefore, the present invention also relates to the method described above, in which the reaction product of compound VII and compound (II) is reacted with a crosslinking compound, and the reaction product produced is The oxidized sugar molecule portion and / or thio group contained in the polypeptide further reacts with the polypeptide. Particularly preferred peptides are erythropoietin (EPO). Therefore, the present invention also relates to the method described above, wherein the polypeptide is erythropoietin. -38- A7 B7 200521143 V. Description of the Invention (37) The Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs, the Consumer Cooperative EPO can be any human being (see 'For example, Inoue, Hotan, Takeuchi, 1994, the Improved method for in vivo purification of human erythropoietin, Biomedical Reports 17 (2), 180-4; Miyake, Kang''Kimwasser, 1977, Purification of human erythropoietin, Journal of Biochemistry, 252 (15 ), 5558_64) or another mammalian source and can be obtained from monkey kidney by purification from naturally occurring sources such as human kidney, embryonic human liver or animal. In addition, the expression of "erythropoietin, or" HPPO "also covers EPO variants in which one or more amino acids (for example, 1 to 25, preferably 1 to 10, and more preferably 1 to 5) , Preferably i or 2) have been altered by another amino acid and have erythropoietin activity (see 'e.g. EP 640 619 B1). The determination of erythropoietin is described in the art of this aspect (for the measurement of the activity in a test tube, see, for example, Fei et al. 1991, Blood, 77, 1203ff; Kitamura et al., 1989, Journal of Cell Physiology; 140, 323-334; Health For the determination of in vivo EPO activity, see, European Pharmacopoeia 2001, 911-917; European Pharmacopoeia 2000, 1316 Erythropoietin Concentrate, 78-785; European Pharmacopoeia (1996/2000); European Pharmacopoeia, 1996, Erythropoietin Concentration Solution, Pharmaeuropa, 8, 371-377; Fei Bi, He Menji, Poly, Lauf, Zartemexol; 1995, selected from recombinant human erythropoietin's N- and BHK-21 cells. -Glycosylation mutant protein erythropoietin, blood, 85 (5), 1229-36; (inject EPO and modified EPO type into female NMRI mice (equivalent protein 50 mg / mouse), in sections 1, 2 and Blood samples were taken on 3 days, and reticulocytes were measured on day 4)). In other publications, the test of EpO activity was described in Babang, Aparisi, Anderson, Natala-39-200521143 A7 B7 5. Description of the invention (30 plus, Ridge, 1994, Reticulate Bioanalysis of Hematocrit as Red Blood Cells, Journal of Medical Biomedical Yearbook, 12 (4), 515-22; Bowen, Gulligen, Beckin, Kendall, Willis, 1994, with a mesh of automatic devices Hematocrit parameters, using serum transferrin receptor and erythropoietin concentrates to estimate the effectiveness and total erythropoietin in spinal dysplasia, Leukemia, 8 (1), 151-5; Delomi, Lorenzie Benedict, Giffin, Martin, Jacobson, Bunney, Eliot, 1992, The role of saccharification in the secretion and biological activity of erythropoietin, Biochemistry, 31 (41), 9871-6; Horiguchi, Putian , Kuboting, Tono, Shimonaka, Ochi, 1992; Tasks of sugar chains in the expression of human erythropoietin bioactivity, Journal of Biochemistry, 267 (11), 7703-9; Yamaguchi, Akai, West Sichuan, Ueda, Masuda, Sasaki, 1991, N_Saccharification site determination Impact of Removal in Human Erythropoietin on Its Manufacturing and Biological Properties, Journal of Biochemistry, 266 (30) '20434_9, Takeuchi, Inoue, Strikeline, Kubota, Wada, Shimizu, Baozhi, Kujin, Takasaki, Gu Xi, 1989, The relationship between sugar chain structure and the biological activity of recombinant human erythropoietin produced in Chinese hamster ovary cells. Proceedings of the National Academy of Sciences, Ministry of Economic Affairs, Intellectual Property Bureau, Employee Consumption Cooperative, 85 (20), 7819-22; Kezi, Ikada, 1989, Analytical method of red-grey globulin, Nefron, 51 (1), 11-4 (Germany); Scully, Skoksky 1985, Human urinary red blood cells Purification of auxin in control well glass and sialic acid, blood experiment, 13 (3), 833_7; Kristol, 1983, physical and biological characteristics of mother red blood cell enhancement factor (EEF), serum is different from erythropoietin Late-acting erythrocyte stimulator, blood test, 11 (1) > 18-31 〇-40- 200521143 A7 B7 V. Description of the invention (39) Preferably, EPO is made by recombinant. This includes in Eukaryotic state or Nuclear cells, preferably mammalian, insect, yeast, bacterial cells, or any other cell type that facilitates the manufacture of EPO recombinants. In addition, EPO can be produced in genetically transgenic animals (for example, in body fluids, like Milk, blood, etc.), expressed in transgenic black eggs, especially poultry, preferably chicken, or in transgenic plants. Recombinant manufacturing of peptides is known in the art in this regard In general, this includes transfection of the host cells with an appropriate expression vector, and the host cells are cultured under conditions capable of producing the polypeptide from the host cell and purifying the polypeptide. For details, see, for example, Cristo, Pankaraz, Faber, and Smart, 1986, Purifying human erythropoietin into homogeneity by a fast five-stage process, Blood, 67 (1), 71.9; Kui , Caslak, Beckett, Wochaoski, 1989, High expression and purification of recombinant human erythropoietin using baculovirus vector, Blood, 74 (2), 652-7; EP 640 619 Β1 With EP 668 351 B1. In a preferred embodiment, EPO has the amino acid sequence of human EPO (see EP 148 605 B2). EPO printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs may contain one or more sugar molecule side chains, preferably from 1 to 12, more preferably from 1 to 9, even more preferably from 丨 to 6 and particularly preferably Particularly preferred are j to 4 ′, which are 4 hearing molecule side chains, which are attached to EPO through saccharification and / or 0-linked, that is, EPO is saccharified. Generally, when the EPO line is produced in cells in a eukaryotic state, the polypeptide line is post-transglycated. Therefore, in mammals, especially humans, insects or yeast cells, the side chains of sugar molecules have been linked to Epo during biosynthesis. Saccharified Ερ〇-41- 200521143 A7 B7 V. The structure or nature of the invention description (40) has been extensively studied in this area (see EP 428 267 Bl; EP 640 619 B1; Ruth, Derby, Smith, Ma Er, Luo Jie, Rhodes, Qatar, 1995, Slight differences in the molecular structure of erythrocytes: qualitative nature, Chronicle of Chemistry, 67 (8), 1442-52; Takeuchi, Guru, 1991, > · Human Structural and Functional Tasks of Erythropoietin Sugar Chains, Cool Biology, 1 (4), 337-46 (Review).

因此，根據本發明之羥·基燒基殿粉句"生物，每個EPO 分子可包含至少一個，較佳為1至12個，更佳為1至9 個，甚至更佳為1至6個且特別隹為1至4個HAS分 子。每個EPO分子HAS_分子之數量可於產物水解且產生 的單醣衍生後用GC_MS藉由定量醣分子組成分析來測定 tl (參閱查普林與甘乃迪(編輯），1986，醣分子分析：實用探 討，IRL出版實用探討系列(ISBN 0-947946_44-3)，尤其是 第1章，單醣類，1-36頁；第2章，寡醣類，37-53頁， 第3章，中性多酷類’ 55_96頁）。 根據本發明尤其佳之具體例，連接到EPO之醣分子部 分係為醣分子側鏈的一部份。更佳為，醣分子部分係為醣 經濟部智慧財產局員工消费合作社印製 分子側鏈之終端基團。於甚至更佳的具體例中，醣分子部 分係為雜分子側鏈之半乳糖殘基’較佳為酶分子側鏈之終 端半乳糖殘基。該半乳糖殘基可藉著將終端唾液酸移除， 接著藉氧化作用，如下文所說明者而可得，用於與化合物 (I)與化合物(II)之反應產物進行反應。於另外較佳的具體 例中，化合物(I)與(Π)之反應產物係連接到醣分子侧鏈之 唾液酸殘基，宜為醣分子側鏈之終端唾液酸殘基。唾液酸 -42- A7 B7 200521143 五、發明說明（41) 係被氧化，如本文中所說明者。 特別佳考為，該半乳糖殘基係經由官能基χ藉著將終 端诞酸移除，接著藉氧化作用而可得，用於與化合物⑴與 化合物(II)之反應產物或與化合物(I)與化合物(Η)之反應產 物與交聯化合物之反應產物進行反應。 如前文所提，選擇地與交聯化合物進行反應之化合物 (I)與化合物(II)之反應產物可與包含於ΕΡ0中之硫基進行 反應。 亦可能將選擇地與交聯化合物之化合物⑴與化合物(Η) 之反應產物，與硫基以及與酷分子部分進行反應，其各個 係包含於至少另一種化合物，宜為多胜肽，更佳為紅血球 生成素中。 根據較佳的具體例，該SH基可，例如，藉由經基衍 生物，例如，2*(胺基氧基）乙基硫醇氫氣化物（包爾L 等，1965，有機化學期刊，30，949)或藉由醯肼衍生物， 例如，硫代甘醇酸醯肼（懷塞德等，1977，有機化學期 刊，42，332)連接到較佳係經氧化的醣分子部分。 經濟部智慧財產局員工消费合作社印製 根據另外較佳的具體例，較佳係將硫基導入ΕΡ0之經 氧化的醣分子部分，更佳為ΕΡ0醣側鏈一部份之經氧化 的酷分子部分。 較佳者為，硫基係從天然生成的半胱胺酸或從附加的 半胱胺酸所衍生。更隹者為，ΕΡ0具有人類ΕΡΟ之胺基 酸次序且天然生成的半胱胺酸係為半胱胺酸29及/或33。 於更佳的具體例中，選擇地與交聯化合物作用之化合物⑴ -43- 200521143 Α7 Β7 五、發明說明（42) 與化合物(II)之反應產物係與半胱胺酸29進行反應，然 而，半胱胺酸33係被另一個胺基酸所替代。或者，選擇 地與交聯化合物作用之化合物⑴與化合物(ΪΙ)之反應產物 係與半胱胺酸33進行反應，然而，半胱胺酸29係被另一 個胺基酸所替代。 本發明之說明書中，’’附加的半耽胺酸”一詞係指多胜 肽，宜為EPO，其包括不出現於野生型多胜肽中之半胱胺 酸殘基。 本發明該觀點之說明書中，半胱胺綾可為一加在EPO 之N-或C-端上之另外的胺基酸。 此外，附加的半胱胺酸可藉著將半胱胺酸或經適當取 代之半胱胺酸替代天然生成的胺基睃而加入。較佳者為， 本發明該觀點之說明書中，ΕΡΟ係為人類ΕΡΟ且經替代 的胺基酸殘基係為絲胺酸126。 經濟部智慧財產局員工消费合作社印製 選擇地與交聯化合物作用之化合物⑴與化合物(II)之反 應產物與至少另一種化合物進行反應之反應條件可適應個 別反應之特定需要，例如，於至少另一種化合物係為多胜 肽之情況中或於至少另一種化合物係為交聯化合物之情況 中’或於至少另一種化合物係為交聯化合物與多胜肽之反 應產物之情況中。為緩衝化合物時，較好使用上述所提化 合物之至少一種。為溶劑或溶劑混合物時，較好使用上述 所提溶劑之至少一種。可進行單離及/或後_處理，其中， 較佳的方法係選自於前文所述的方法。 若化合物(I)與化合物(II)之反應產物係，例如，進一步 -44- 200521143 A7 B7 五、發明說明（43) 與多胜肽作為另外的化合物，宜為EPO，進行反應，於該 反應較好係使用水作為溶劑。除水之外，可存有至少一稚 另外的溶劑。可提及之較佳可能的另外溶劑為DMSO， DMF，甲醇或乙醇。 因此，本發明亦關於如前說明的方法，其中，化合物 ⑴與化合物(II)之反應產物與多胜肽，宜為EPO，之反應 係在水性介質中進行。 就該反應時所用的溫度而言，對生成想要的羥基烷基 澱粉衍生物之反應沒有給予特定的限制，其包括化合物⑴ 與(II)之反應產物與多胜肽經由至少一個官能基X之反 應。反應的溫度較佳係自4至37°C範圍，更佳係自1〇至 30°C範圍且特別佳係自15至25°C範圍。 因此，本發明亦關於如前說明的方法，其中，化合物 (I)與化合物(II)之反應產物與多胜肽之反應係在自4至37 它之溫度下進行。 於反應過程中溫度可變化，宜在前文給定的範圍内變 化，或維持實質上恆定。 經濟部智慧財產局員工消费合作社印製 化合物(I)與化合物(II)之反應產物與多胜肽作用之反應 時間可適應特定之需要且通常係在自0.5至48小時之範圍 内，較佳係自2至24小時範園且特別佳係自1〇至20小 時範圍。 化合物(I)與化合物(II)之反應產物與多胜肽進行反應之 pH值可適應特定之需要，例如，反應物之化學本質。 如果，例如，化合物⑴與(II)之反應產物與另外的化合 -45- 200521143 A7 B7 五、發明說明（44) 物係經由為羥基胺基基團-0-NH2之官能基X與包含於多 胜肽中之至少一個醛基之反應進行反應，則pH值宜在自 4 5至6之範園内，更佳係在大約5.5。 右化合物(I)與化合物(II)之反應產物係，例如，進一步 與交聯化合物作為另外的化合物，宜為EP0，進行反應， 則該反應宜用水作為溶劑。除水之外，可存有至少另一種 溶劑。可提及之較佳可能的另外溶劑為DMSO，DMF，甲 醇或乙醇。 因此’本發明亦關於如前說明的方法，其中，化合物 (1)與化合物(U)之反應產物與交聯化合物之反應係在水性 系統中進行。 就該反應時所用的溫度而言，對生成想要的羥基烷基 澱粉衍生物之反應沒有給予特定的限制，其包括化合物⑴ 與(II)之反應產物與交聯化合物經由至少一個官能基X之 反應。反應的溫度較佳係在自4至37°c之範圍内，更佳係 自10至30°C範圍且特別佳係自15至25°C範圍。 經濟部智慧財產局員工消費合作社印製 因此，本發明亦關於如前說明的方法，其中，化合物 (I)與化合物(II)之反應產物與交聯化合物之反應係在自4 至37 C之溫度下進行。 於反應過程中溫度可變化，宜在前文給定的範圍内變 化，或維持實質上恆定。 化合物⑴與化合物(II)之反應產物與交聯化合物作用之 反應時間可適應特定之需要且通常係自10分鐘至10小 時，較佳係自20分鐘至5小時且特別佳係自30分鐘至2 -46- 200521143 A7 ______ B7 五、發明說明（45) 小時範圍。 化合物(I)與化合物(Π)之反應產物與交聯化合物進行反 應之pH值可適應特定之需要，例如，反應物之化學本 質。 如果例如，化合物⑴與(Π)之反應產物與交聯化合物， 其係交聯化合物經由包含於化合物⑴與(11)之反應產物中 且係為胺基基團_NH2之官能基X進行反應，則pH值宜在 自7至8·5之範圍内，更佳係在大約72。 如果化合物(I)與(II)之反應產物與交聯化合物作用之反 應產物*例如，進一步與多胜肽，宜為ΕΡΟ進行反應， 則該反應宜用水作為溶劑。除水之外，可存有至少另一種 溶劑。可提及之較佳可能的另外溶劑為DMSO，DMF，甲 醇或乙醇。 因此，本發明亦關於如前說明的方法，其中，化合物 (I)與化合物(II)之反應產物進一步與交聯北合物，與多胜 肽進行反應之反應係在水性系統中進行。 經濟部智慧財產局員工消费合作社印製 就該反應時所用的溫度而言，對生成想要的羥基烷基 澱粉衍生物之反應沒有給予特定的限制，其包括化合物(工) 與(II)之反應產物與交聯化合物進行反應且進一步經由包 含於交聯化合物中之至少一個官能基X與多胜肽進行反 應。反應的溫度較佳係在自4至37°c之範圍内，更佳係自 10至30°C範園且特別佳係自15至25°C範圍。 因此，本發明亦關於如前說明的方法，其中，化合物 ⑴與化合物(Π)之反應產物進一步與交聯化合物，與多胜 -47- 200521143 A7 ______ B7_ _. 五、發明說明（46) 肽進行反應之反應係在自4至37°C之溫度下進行。 於反應過程中溫度可變化，宜在前文給定的範圍内變 化，或維持實質上恆定。 化合物(I)與化合物(Π)之反應產物進一步與交聯化合 物，與多胜肽進行反應之反應時間可適應特定之需要且通 常係在自0·5至48小時，較佳係自2至24小時且特別佳 係自10至20小時範圍。 化合物⑴與化合物(II)之反應產物進一步與交聯化合 物，與多胜肽進行反應之pH值可適應特定之需要，例 如，反應物之化學本質。 如果，例如，化合物⑴與(II)之反應產物係與交聯化合 物進一步進行反應，係經由包含於交聯化合物中且係為胺 基基團-NH2之官能基X與多胜狀進行反應，則pH值宜 在自7至8.5之範圍内，更佳係在大約7.2。 反應混合物之適當pH值於各情況中可藉著添加至少 一適當的緩衝劑而調節。較佳的緩衝劑中，可提及者為酷 酸鈉緩衝劑，磷酸鈉緩衝劑，或硼酸鈉緩衝劑。 經 濟 部 智 慧 財 產 局 員 消 费 合 作 社 從化合物(I)與化合物(Π)之反應產物與至少另一種化合 物之反應中所產生的反應產物，該至少另一種化合物係為 多胜肽或為交聯化合物，且反應產物進一步包括從化合物 ⑴、化合物(II)、交聯化合物與多胜肽之反應中所產生的 化合物，可藉由至少一適當的方法從反應混合物中單離出 來且進行至少另一項處理，例如，後-處理，例如透析及/ 或冷;東乾燥。 -48- 200521143 A7 A7 B7 五、發明說明（47) 一旦前文所提之反應產物形成了，其可藉由至少一適 當的方法從反應混合物中單離出來。 反應產物之單離可藉包含一或多項步驟之適當過程來 進行。 根據本發明較佳的具體例，其中反庳產物不包括多胜 肽者，首先將反應產物從反應混合物或反應混合物之混合 物中分離出來，較佳係藉離心過濾。第二步驟中，將分離 的反應產物進行進一步處理例如，後-處理，像透析及/或 冷凍乾燥。根據甚至更佳的具體例，依照想要的產物規格 首先將分離的反應產物透析，宜對水透析，且然後經冷凍 乾燥直到反應產物之溶劑含量足夠低。 根據本發明另一個具體例，其中反應產物包括多胜 肽，反應產物較隹係經實例7.8中所說明者單離出來。 經濟部智慧財產局員工消费合作社印製 根據本發明另外的具體例，化合物(II)與另外的化合物 係在與化合物(I)反應之前進行反應，亦即，化合物(II)之 衍生物係藉化合物(II)經由至少一個官能基X與包含至少 一個官能基Y之至少另一種化合物，如前說明者，於與化 合物⑴作用之前進行反應所產生。 如果，化合物(II)首先係與另外的化合物，宜為多胜 肽’更隹為EPO，進行反應，則該反應宜用水作為溶劑。 除水之外，可存有至少另一種溶劑。可提及之較佳可能的 另外溶劑為DMSO，DMF，甲醇或乙醇。 因此，本發明亦關於如前說明的方法，其中，化合物 (II)，於與化合物⑴反應之前，與另外的化合物，宜為多 -49- 200521143 A7 B7 五、發明說明（48) 胜肽，甚至更佳為EPO，之反應係在水性系統中進行。 就該反應時所用的溫度而言，對生成想要的化合物(Π) 之衍生物之反應沒有給予特定的限制，其包括化合物(II) 之反應產物與至少另一種化合物經由至少一個官能基X， 宜為多胜肽，更佳為EPO進行反應。反應的溫度較佳係 在自4至37°C之範圍内，更佳係自10至30°C範圍且特別 佳係自15至25°C範圍。 因此’本發明亦關於如前說明的方法，其中，化合物 (II)與至少另一種化合物之反應係在自4至37°C之溫度下 進行。 於反應過程中溫度可變化，宜在前文給定的範圍内變 化，或維持實質上恆定。 化合物(II)與至少另一種化合物進行反應之反應時間， pH值可適應特定之需要，例如，反應物之化學本質。反 應混合物之適當pH值可藉著加入至少一適當的緩衝劑來 調節。較佳的緩衝劑中，可提及者為醋酸鹽，磷酸鹽，或 领酸鹽緩衝劑，例如，醋酸鈉，磷酸鈉，或硼酸鈉緩衝 劑。 經濟部智慧財產局員工消费合作社印製 化合物(II)與至少另一種化合物之反應中所產生的反應 產物可藉由至少一適當的方法從反應混合物中單離出來且 進行至少另一項處理，例如，後·處理，例如透析及/或冷 柬乾燥。 一旦化合物(π)與至少另一種化合物進行反應之反應產 物形成了 ’其可藉由至少一適當的方法從反應混合物中單 -50- 200521143 A7 B7 五、發明說明（49) 離出來。 反應產物之單離可藉如前文中已說明之包含一或多項 步驟之適當過程來進行。 如果想要及/或需要，化合物(II)之NH基橋R’與R”可 於化合物(II)與至少另一種化合物進行反應之前被一適當 的保護基所保護。可用前文提及保護基之一種作為保護 基。化合物(II)與至少另一種化合物，例如，多胜肽，宜 為EPO，之反應產物與化合物⑴進行反應之前，保護基可 被至少一適當的方法移除。 如果，首先將化合物(II)與交聯化合物或交聯化合物與 多胜肽之反應產物進行反應，則所有的反應條件可調節至 此等反應之特定需要。尤其，可使用前文提及之緩衝系統 及/或溶劑。 於第二步驟中，化合物(II)與至少另一種化合物之反應 的反應產物係與化合物⑴進行反應α 於該反應時，所有的反應條件可調節到此等反應之特 定需要。尤其，可使用前文提及之緩衝系統及/或溶劑。 經濟部智慧財產局員工消费合作社印製 化合物(II)與至少另一種化合物炙反應產物與化合物⑴ 之反應中所產生的反應產物，可藉由至少一適當的方法從 個別的反應混合物甲單離出來且進行至少另一種處理，例 如’後-處理，例如透析及/或冷凍乾燥。本說明書中，前 文所述之每一種適當的方法皆可使用。 通常，HAS-多胜肽軛合物之單離，含或不含交聯化合 物’係可使用已知於純化天然及重組體多胜肽的方法進 -51- 200521143 A7 B7 五、發明說明（5〇) 行，例如，斥濾層析法，離子交換層析法，RP-HPLC，羥 基磷灰石層析法，疏水交互層析法或其至少二種方法之組 合。 HAS附著於多胜肽之共輟可於經改質的蛋白質水解後 藉醣分子組成分析而確認。 HAS在多胜肽之N-連接的寡醣分子上改質之證明可 藉著將經HAS改質的N-多醣移除及觀察於SDS-PAGE+/-西方墨點法中預期位移至較高的移動而完成。 多胜肽於半胱胺酸殘基之HAS改質可藉著未能於RP-HPLC中測定到相關解朊的半胱胺酸-胜肽及未能於經 HAS-改質的產物之解朊斷片中測定到MAld/YOF-MS而 證實（周氏等，1988，毛細管電泳法，液相層析法，電喷 灑質譜法及介質輔助雷射脫附離子化時間飛行式質譜儀_ 對重組體人類紅血球生成素特性之質譜儀，電泳法之應 用，19(13)，2348_55)。經半胱胺酸改質的多胜肽解朊消 化後’將包含HAS-餾份單離出來能使相關胜肽中之該餾 份藉由習用胺基酸組成分析而確認。 經濟部智慧財產局員工消费合作社印製 前文所揭示之所有具體例係針對本發明之HAS-多胜 肽關於多胜肽之性質或HAS亦應用於本發明的方法製造 HAS_多胜肽轆合物。此外，前文揭示之所有具體例係針對 HAS-EPO或其製法，其係關於多胜肽或關於HAS亦應用 本發明的方法製造HAS-多胜肽軛合物。 根據特別佳之具體例，羥基烷基澱粉衍生物係與化合 物(Π)進行反應，較佳係選自於前文說明之均-及雜二官能 -52- 200521143 Α7 Β7 五、發明說明（Μ ) 化合物，且產生的反應產物係與醣蛋白，宜為紅血球生成 素，較佳係與EPO醣分子側鏈之經氧化的終端醣分子部 分進行反應。 根據本發明另一個尤其佳之具體例，羥基燒基殿粉衍 生物係與化合物(II)，較佳係選自於前文說明之均-及雜二 官能化合物進行反應而得到第一羥基乙基澱粉衍生物。該 第一羥基乙基澱粉衍生物隨即與交聯化合物進行反應而得 到第二羥基乙基殿粉衍生物。該第二羥基乙基澱粉衍生物 隨即與醣蛋白，宜為紅血球生成素，宜與包含於醣蛋白中 之-SH基進行反應而得到第三羥基乙基澱粉衍生物。較佳 者為，交聯化合物係為雜二官能化合物。更佳者為，交聯 化合物係與包含結構-NH-之官能基進行反應，該_ΝΗ·結構 係包含於第一幾基乙基澱粉衍生物中。更佳者為，該官能 基係為-ΝΗ2-。 經濟部智慧財產局員工消费合作社印製 本發明之一優點係在至少一個反應階段中，較佳係在 所有的反應階段，所涉及之反應階段中不需要使用毒物學 方面重要的溶劑，且因此，於製造過程中不需要將此等溶 劑移除以避免產物被溶劑污染。此外，不需要進行另外有 關殘留毒物學方面重要溶劑之品質控制。如果使用有機溶 劑，宜除水之外，宜使用毒物學方面重要的溶劑，例如， 乙醇及/或丙二醇。 本發明另一個優點係於步驟中以水性系統用作為溶 劑’而避免可能由有機溶劑引起之不可逆或可逆的結構性 改變。所以，根據本發明的方法所獲得的多胜肽衍生物係 -53- 200521143 Α7 Β7 五、發明說明（52) 不同於彼等於有機滚剩’例如DMSO中所製得者。 此外，令人驚奇的觀察到HAS對多胜肽，例如EPO 共軛於水性溶液中會減少或避免副作用。所以，本發明的 方法之該具體例會造成具高純度經改良之羥基烷基澱粉衍 生物。 根據另一觀點，本發明亦關於藉由方法而可得之羥基 烷基殿粉衍生物’該方法包括將式（I)之羥基烷基澱粉 (HAS)Therefore, according to the hydroxy-based burning base powder sentence of the present invention, each EPO molecule may contain at least one, preferably 1 to 12, more preferably 1 to 9, and even more preferably 1 to 6. And especially 隹 are 1 to 4 HAS molecules. The number of HAS_ molecules per EPO molecule can be determined by GC_MS by quantitative analysis of sugar molecular composition after the product is hydrolyzed and the resulting monosaccharide is derivatized (see Chaplin and Kennedy (eds.), 1986, Sugar Molecule Analysis: Practical Discussion, IRL publishes Practical Discussion Series (ISBN 0-947946_44-3), especially Chapter 1, Monosaccharides, pages 1-36; Chapter 2, Oligosaccharides, pages 37-53, Chapter 3, Neutral How cool class' 55_96). According to a particularly preferred embodiment of the present invention, the sugar molecule portion linked to the EPO is a part of the side chain of the sugar molecule. More preferably, the sugar molecule is a terminal group of the molecular side chain printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. In an even more preferred embodiment, the sugar molecule portion is a galactose residue ' of the side chain of the heteromolecule, and preferably the terminal galactose residue of the side chain of the enzyme molecule. The galactose residue can be obtained by removing terminal sialic acid and then by oxidation, as described below, and is used to react with the reaction product of compound (I) and compound (II). In another preferred embodiment, the reaction product of compounds (I) and (Π) is a sialic acid residue attached to the side chain of the sugar molecule, and is preferably a terminal sialic acid residue of the side chain of the sugar molecule. Sialic acid -42- A7 B7 200521143 V. Description of the invention (41) is oxidized, as described in this article. A particularly good test is that the galactose residue is obtained by removing the terminal acid through the functional group χ and then by oxidation, and is used for the reaction product with compound ⑴ and compound (II) or with compound (I ) The reaction product with the compound (VII) is reacted with the reaction product of the crosslinking compound. As mentioned above, the reaction product of the compound (I) and the compound (II) selectively reacted with the cross-linking compound can be reacted with a sulfur group contained in EP0. It is also possible to react the reaction product of the compound ⑴ with the compound (Η) selectively with the cross-linking compound, with the thio group and with the cool molecular moiety, each of which is contained in at least one other compound, preferably a polypeptide, more preferably For erythropoietin. According to a preferred embodiment, the SH group can be, for example, via a derivative such as 2 * (aminooxy) ethyl mercaptan hydrogenide (Bauer et al., 1965, Journal of Organic Chemistry, 30 949) or via a hydrazine derivative, for example, hydrazide thioglycolate (Wyside et al., 1977, Journal of Organic Chemistry, 42, 332) is attached to the preferably oxidized sugar molecule moiety. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. According to another preferred specific example, it is preferred that the sulfur group is introduced into the oxidized sugar molecule part of EPO, and more preferably an oxidized cool molecule part of the EPO sugar side chain. section. Preferably, the thio group is derived from naturally occurring cysteine or from an additional cysteine. What is more, EPO has the amino acid sequence of human EPO and the naturally occurring cysteine system is cysteine 29 and / or 33. In a more specific example, a compound which selectively interacts with a cross-linking compound 7 -43- 200521143 A7 B7 V. Description of the invention (42) The reaction product of compound (II) reacts with cysteine 29, however Cysteine 33 was replaced by another amino acid. Alternatively, the reaction product of compound VII and compound (XI) which selectively interacts with a cross-linking compound is reacted with cysteine 33, however, cysteine 29 is replaced with another amino acid. In the description of the present invention, the term "additional hemamic acid" refers to a polypeptide, preferably EPO, which includes cysteine residues that do not occur in wild-type polypeptides. This aspect of the present invention In the description, cysteamine may be an additional amino acid added to the N- or C-terminus of EPO. In addition, additional cysteine may be obtained by replacing cysteine or by appropriately replacing it. Cysteine is added in place of the naturally occurring aminoamidine. Preferably, in the description of this aspect of the invention, EPO is human EPO and the substituted amino acid residue is serine 126. Ministry of Economic Affairs The Intellectual Property Bureau employee consumer cooperative prints compounds that selectively interact with cross-linking compounds. The reaction product of the reaction product of compound (II) with at least one other compound can be adapted to the specific needs of individual reactions, for example, in at least one other compound. In the case where the compound is a polypeptide or in the case where at least another compound is a cross-linked compound 'or in the case where at least another compound is a reaction product of a cross-linked compound and a polypeptide. It is a buffer compound Preferably, at least one of the above-mentioned compounds is preferably used. When it is a solvent or a solvent mixture, at least one of the above-mentioned solvents is preferably used. Single isolation and / or post-treatment may be performed, and the preferred method is selected from The method described in the foregoing. If the reaction product of compound (I) and compound (II) is, for example, further -44- 200521143 A7 B7 V. Description of the invention (43) and polypeptide as another compound, it is preferably EPO The reaction is carried out. In this reaction, water is preferably used as a solvent. In addition to water, there may be at least one additional solvent. The other possible solvents that may be mentioned are DMSO, DMF, methanol, or ethanol. Therefore The present invention also relates to the method described above, in which the reaction product of compound VII and compound (II) and a polypeptide, preferably EPO, is performed in an aqueous medium. As far as the temperature used in this reaction is concerned No particular limitation is imposed on the reaction to produce the desired hydroxyalkyl starch derivative, and it includes the reaction of the reaction product of compound VII and (II) with a polypeptide via at least one functional group X. The temperature of the reaction It is preferably in the range from 4 to 37 ° C, more preferably in the range from 10 to 30 ° C and particularly preferably in the range from 15 to 25 ° C. Therefore, the present invention also relates to the method described above, wherein the compound ( I) The reaction product of the reaction product with the compound (II) and the polypeptide are carried out at a temperature from 4 to 37. The temperature can be changed during the reaction, and it should be changed within the range given above, or maintained substantially Constant. The reaction time of the reaction product of compound (I) and compound (II) printed by the consumer cooperative of employees of the Intellectual Property Bureau of the Ministry of Economic Affairs and the reaction of polypeptide can be adapted to specific needs and is usually within the range of 0.5 to 48 hours. It is preferably from 2 to 24 hours, and particularly preferably from 10 to 20 hours. The reaction product of the reaction product of compound (I) and compound (II) with polypeptide can be adapted to specific needs, such as , The chemical nature of the reactants. If, for example, the reaction product of compound VII and (II) is combined with another compound -45- 200521143 A7 B7 V. Description of the invention (44) The system is via a functional group X which is a hydroxylamine group-0-NH2 and is contained in The reaction of at least one aldehyde group in the polypeptide is carried out, and the pH value is preferably in the range from 45 to 6, and more preferably about 5.5. The reaction product of the right compound (I) and the compound (II) is, for example, further reacted with a cross-linked compound as another compound, preferably EP0, and the reaction is preferably water as a solvent. In addition to water, at least one other solvent may be present. Other solvents which may be mentioned as being preferred are DMSO, DMF, methanol or ethanol. Therefore, the present invention also relates to the method described above, in which the reaction product of the reaction product of the compound (1) with the compound (U) and the cross-linked compound are carried out in an aqueous system. As far as the temperature used in this reaction is concerned, no particular limitation is placed on the reaction to produce the desired hydroxyalkyl starch derivative, which includes the reaction product of compounds ⑴ and (II) and a cross-linked compound via at least one functional group X Response. The reaction temperature is preferably in the range from 4 to 37 ° C, more preferably in the range from 10 to 30 ° C and particularly preferably in the range from 15 to 25 ° C. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. Therefore, the present invention also relates to the method described above, in which the reaction product of the reaction product of the compound (I) and the compound (II) and the cross-linked compound are between 4 and 37 C. Performed at temperature. The temperature may change during the reaction, and it should be changed within the range given above, or maintained substantially constant. The reaction time of the reaction product of the compound ⑴ with the compound (II) and the crosslinking compound can be adapted to the specific needs and is usually from 10 minutes to 10 hours, preferably from 20 minutes to 5 hours and particularly preferably from 30 minutes to 2 -46- 200521143 A7 ______ B7 V. Description of invention (45) hours. The pH of the reaction product of the compound (I) with the compound (Π) and the cross-linked compound can be adapted to specific needs, for example, the chemical nature of the reactants. If, for example, the reaction product of compound VII and (Π) and a crosslinked compound, it is a crosslinked compound that reacts via functional group X that is included in the reaction product of compound IX and (11) and is an amine group _NH2 , Then the pH value should be in the range from 7 to 8.5, more preferably about 72. If the reaction product of compounds (I) and (II) reacts with a cross-linked compound, for example, a reaction product *, for example, further reacts with a polypeptide, preferably EPO, the reaction is preferably water. In addition to water, at least one other solvent may be present. Other solvents which may be mentioned as being preferred are DMSO, DMF, methanol or ethanol. Therefore, the present invention also relates to the method described above, in which the reaction product of the compound (I) and the compound (II) further reacts with a cross-linked compound and reacts with a dopeptide in an aqueous system. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs As far as the temperature used in this reaction is concerned, there is no specific restriction on the reaction to produce the desired hydroxyalkyl starch derivative, which includes compounds The reaction product reacts with the crosslinking compound and further reacts with the polypeptide via at least one functional group X contained in the crosslinking compound. The reaction temperature is preferably in the range from 4 to 37 ° C, more preferably in the range from 10 to 30 ° C and particularly preferably in the range from 15 to 25 ° C. Therefore, the present invention also relates to the method as described above, in which the reaction product of compound VII and compound (Π) further interacts with a cross-linked compound, and Ducson-47- 200521143 A7 ______ B7_ _. V. Description of the invention (46) Peptide The reaction is carried out at a temperature of from 4 to 37 ° C. The temperature may change during the reaction, and it should be changed within the range given above, or maintained substantially constant. The reaction product of the compound (I) and the compound (Π) further reacts with the cross-linked compound, and the reaction time for the reaction with the polypeptide can be adapted to the specific needs and is usually from 0.5 to 48 hours, preferably from 2 to 24 hours and particularly preferred ranges from 10 to 20 hours. The reaction product of compound VII and compound (II) further reacts with the cross-linked compound, and the pH value of the reaction with the polypeptide can be adapted to specific needs, for example, the chemical nature of the reactant. If, for example, the reaction product of the compound VII and (II) is further reacted with a cross-linked compound, is reacted with a polysaccharide via a functional group X contained in the cross-linked compound and is an amine group -NH 2, The pH should then be in the range from 7 to 8.5, more preferably about 7.2. The appropriate pH of the reaction mixture can be adjusted in each case by adding at least one suitable buffer. Among the preferred buffers, mention may be made of sodium buffer, sodium phosphate buffer, or sodium borate buffer. The reaction product produced by the consumer cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs from the reaction product of compound (I) and compound (Π) with at least one other compound that is a polypeptide or a cross-linked compound, The reaction product further includes a compound produced from the reaction of compound IX, compound (II), a cross-linked compound, and a polypeptide, which can be isolated from the reaction mixture by at least one appropriate method and carried out at least one other item. Treatment, for example, post-treatment, such as dialysis and / or cold; drying. -48- 200521143 A7 A7 B7 V. Description of the invention (47) Once the reaction product mentioned above is formed, it can be isolated from the reaction mixture by at least one suitable method. Isolation of the reaction product can be performed by a suitable process comprising one or more steps. According to a preferred embodiment of the present invention, where the reaction product does not include a polypeptide, the reaction product is first separated from the reaction mixture or a mixture of the reaction mixture, preferably by centrifugation. In the second step, the separated reaction product is subjected to further processing such as post-processing, such as dialysis and / or freeze-drying. According to even better specific examples, the isolated reaction product is first dialyzed according to the desired product specifications, preferably with water, and then freeze-dried until the solvent content of the reaction product is sufficiently low. According to another embodiment of the present invention, wherein the reaction product includes a polypeptide, the reaction product is more isolated than that described in Example 7.8. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs According to another specific example of the present invention, compound (II) and another compound are reacted before reacting with compound (I), that is, derivatives of compound (II) are borrowed Compound (II) is produced by reacting at least one functional group X with at least one other compound containing at least one functional group Y, as described above, before reacting with compound VII. If the compound (II) is first reacted with another compound, preferably a polypeptide ', more preferably EPO, the reaction is preferably performed with water as a solvent. In addition to water, at least one other solvent may be present. Other solvents which may be mentioned as being preferred are DMSO, DMF, methanol or ethanol. Therefore, the present invention also relates to the method described above, in which the compound (II) is reacted with another compound, preferably poly-49-200521143 A7 B7, before the reaction with compound VII. 5. Description of the invention (48) peptide, Even more preferably EPO, the reaction is carried out in an aqueous system. As far as the temperature used in this reaction is concerned, no particular restriction is given to the reaction to form a derivative of the desired compound (Π), which includes the reaction product of compound (II) and at least one other compound via at least one functional group X Polypeptide is preferred, and EPO is more preferred. The reaction temperature is preferably in the range from 4 to 37 ° C, more preferably in the range from 10 to 30 ° C and particularly preferably in the range from 15 to 25 ° C. Therefore, the present invention also relates to the method as described above, wherein the reaction of the compound (II) with at least another compound is carried out at a temperature from 4 to 37 ° C. The temperature may change during the reaction, and it should be changed within the range given above, or maintained substantially constant. The reaction time of the reaction of the compound (II) with at least one other compound, the pH value can be adapted to specific needs, for example, the chemical nature of the reactants. The appropriate pH of the reaction mixture can be adjusted by adding at least one suitable buffer. Among the preferred buffers, mention may be made of acetate, phosphate, or collarate buffers, such as sodium acetate, sodium phosphate, or sodium borate buffers. The reaction product produced in the reaction of the printed compound (II) and the at least one other compound by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs may be separated from the reaction mixture by at least one appropriate method and subjected to at least one other treatment, For example, post-treatments, such as dialysis and / or cold card drying. Once the reaction product of compound (π) and at least one other compound is formed, it can be separated from the reaction mixture by at least one appropriate method. -50- 200521143 A7 B7 V. Description of the invention (49). Isolation of the reaction product can be carried out by a suitable process comprising one or more steps as described above. If desired and / or required, the NH group bridges R 'and R "of compound (II) may be protected by a suitable protecting group before the reaction of compound (II) with at least one other compound. The protecting group may be mentioned above One serves as a protecting group. Compound (II) can be removed by at least one suitable method before reacting the reaction product of compound (II) with at least one other compound, for example, polypeptide, preferably EPO, with compound VII. If, The compound (II) is first reacted with the cross-linked compound or the reaction product of the cross-linked compound and the polypeptide, then all the reaction conditions can be adjusted to the specific needs of these reactions. In particular, the buffer system mentioned above and / Or solvent. In the second step, the reaction product of the reaction of compound (II) with at least one other compound is reacted with compound ⑴. In this reaction, all reaction conditions can be adjusted to the specific needs of these reactions. Especially The buffer system and / or solvent mentioned above can be used. Compound (II) printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs and at least one other compound The reaction product produced in the reaction between the product and compound VII can be isolated from the individual reaction mixture A by at least one suitable method and subjected to at least another treatment, such as' post-treatment, such as dialysis and / or lyophilization In this specification, each of the appropriate methods described above can be used. In general, the HAS-polypeptide conjugate is isolated, with or without cross-linking compounds, and can be used for purification of natural and recombinant Method for peptides in vivo-51-200521143 A7 B7 V. Description of the invention (50) Line, for example, repulsion chromatography, ion exchange chromatography, RP-HPLC, hydroxyapatite chromatography, hydrophobic Cross chromatography or a combination of at least two methods. The co-drop of HAS attached to the peptide can be confirmed by analysis of the sugar molecule composition after the modified protein is hydrolyzed. HAS is an N-linked oligosaccharide of the peptide The proof of the modification of the sugar molecule can be completed by removing the HA-modified N-polysaccharide and observing the expected shift to a higher shift in the SDS-PAGE +/- Western blot method. Polypeptide in Cysteine HAS modification of amino acid residues can be achieved by Correlation-determined cysteine-peptide and the failure to detect MAld / YOF-MS in HAS-modified product fragments (Zhou et al., 1988, capillary electrophoresis) , Liquid chromatography, electrospray mass spectrometry, and media-assisted laser desorption ionization time of flight mass spectrometer _ mass spectrometer for the characteristics of recombinant human erythropoietin, application of electrophoresis, 19 (13), 2348_55 ). After digestion with cysteine-modified polypeptide, digestion and separation of the HAS-containing fraction can separate the fraction in the relevant peptide through conventional amino acid composition analysis. Ministry of Economic Affairs All the specific examples disclosed in the foregoing printed by the Intellectual Property Bureau employee consumer cooperatives are directed to the properties of the HAS-polypeptide with respect to the polypeptide of the present invention or HAS is also applied to the method of the present invention to produce the HAS-polypeptide conjugate. In addition, all the specific examples disclosed above are directed to HAS-EPO or a method for preparing the same, which are related to polypeptides or to HAS. The method of the present invention is also used to produce HAS-polypeptide conjugates. According to a particularly preferred specific example, the hydroxyalkyl starch derivative is reacted with the compound (Π), preferably selected from the homo- and heterodifunctional-52- 200521143 Α7 B7 described above. V. Description of the compound (M) The reaction product is glycoprotein, preferably erythropoietin, and preferably reacts with the oxidized terminal sugar molecule portion of the side chain of the sugar molecule of EPO. According to another particularly preferred embodiment of the present invention, the hydroxy-caustic powder derivative is reacted with compound (II), preferably selected from the homo- and heterodifunctional compounds described above to obtain the first hydroxyethyl starch. derivative. The first hydroxyethyl starch derivative is then reacted with a crosslinking compound to obtain a second hydroxyethyl starch powder derivative. The second hydroxyethyl starch derivative is then reacted with a glycoprotein, preferably erythropoietin, and preferably with a -SH group contained in the glycoprotein to obtain a third hydroxyethyl starch derivative. Preferably, the cross-linking compound is a heterobifunctional compound. More preferably, the cross-linking compound is reacted with a functional group containing a structure -NH-, and the ΝΝΗ · structure is contained in the first few ethyl starch derivatives. More preferably, the functional group is -NQ2-. An advantage of the invention printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economics is that it is in at least one reaction stage, preferably in all reaction stages. The involved reaction stages do not require the use of toxicologically important solvents, and therefore It is not necessary to remove these solvents during the manufacturing process to avoid contamination of the product with the solvent. In addition, no additional quality control of important solvents in terms of residual toxicology is required. If organic solvents are used, in addition to water, solvents that are important in terms of toxicology, such as ethanol and / or propylene glycol, should be used. Another advantage of the present invention resides in the use of an aqueous system as a solvent 'in the step to avoid irreversible or reversible structural changes that may be caused by organic solvents. Therefore, the polypeptide derivative obtained according to the method of the present invention is -53- 200521143 A7 B7 5. The description of the invention (52) is different from those obtained by organic rollover, such as those obtained in DMSO. In addition, it has been surprisingly observed that HAS can reduce or avoid side effects on polypeptides such as EPO conjugated in aqueous solutions. Therefore, this specific example of the method of the present invention results in an improved hydroxyalkyl starch derivative with high purity. According to another aspect, the present invention also relates to a hydroxy alkyl powder derived by a method. The method includes converting a hydroxyalkyl starch (HAS) of formula (I)

Η 經濟部智慧財產局員工消费合作社印製 於其不會在該反應之前被氧化之還原端上，與式(π)之化 合物 R’-NH‘R” (Π) 進行反應，其中Ri，&及R3係各自獨立為氫或為一線形 或分支的羥基烷基基圈，且其中R，或R”或者R，與R”包括 至少一個官能基X，其係能與至少一其他化合物於(j)與(H) 之反應前或後進行反應。 〇[2-(2-胺基氧基-乙氧基)_乙基]_經基胺係用作為較佳 的化合物(II)，且羥基乙基澱粉係用作為較佳的羥基烷基 澱粉業已說明於前文本發明的方法之說明書中。 因此’本發明亦關於藉由方法而可得之羥基烷基澱粉 -54- 200521143员工 The consumer cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs printed on the reducing end which will not be oxidized before the reaction, and reacts with the compound of formula (π) R'-NH'R "(Π), where Ri, & amp And R3 are each independently hydrogen or a linear or branched hydroxyalkyl ring, and wherein R, or R "or R, and R" include at least one functional group X, which can interact with at least one other compound in (j) The reaction is carried out before or after the reaction with (H). [2- (2-Aminooxy-ethoxy) _ethyl] -Amine is used as the preferred compound (II), And the use of hydroxyethyl starch as the preferred hydroxyalkyl starch has been described in the description of the method invented in the previous text. Therefore, the present invention also relates to hydroxyalkyl starch obtainable by the method-54- 200521143

五、發明說明（53) 衍生物，其中，羥基烷基濺粉係經由其還原端與 胺基氧基-乙氧基)-乙基l·羥基胺進行反應。 依個別的反應條件，所用之溶劑或溶劑混合物及/威殘 基R，及/或R”而定，藉由方法或前文所述的方法而讦得之 羥基烷基澱粉衍生物可能具有下列構造(HIa):5. Description of the invention (53) A derivative in which a hydroxyalkyl splash powder is reacted with an aminooxy-ethoxy) -ethyl l · hydroxyamine via its reducing end. Depending on the individual reaction conditions, the solvent or solvent mixture used and / or R residues, and / or R ", the hydroxyalkyl starch derivative obtained by the method or the method described above may have the following structure (HIa):

(IHa) 因此，本發明亦關於如前說明具有如式(Ilia)構造之經 基烧基殿粉衍生物。 亦可能，例如，於R’為氳之情況中，藉由方法或前文 說明的方法而可得之經基燒基殿粉衍生物可具有下列構造 (Ilia)或(Illb)，其中(Ilia)及(Illb)二者可於具有某種平衡分 佈之反應混合物中存在：(IHa) Therefore, the present invention also relates to a base-based powder derivative having a structure such as the formula (Ilia) as described above. It is also possible that, for example, in the case where R ′ is 氲, the base-based powder obtained by the method or the method described above may have the following structure (Ilia) or (Illb), where (Ilia) And (Illb) can exist in a reaction mixture with some equilibrium distribution:

經 濟 部 智 慧 財 產 局 貝 合 作 社 -55- 200521143 A7. B7 五、發明說明（54) 經 濟 部 智 慧 財 產 局 員 合 作 社Shell Cooperative of Intellectual Property Bureau of Ministry of Economic Affairs -55- 200521143 A7. B7 V. Invention Description (54) Member Cooperative of Intellectual Property Bureau of Ministry of Economic Affairs

(nib) 因此，本發明亦關於如前說明具有如式(nib)構造之羥 基烧基殿粉衍生物。 此外，本發明亦關於如前說明存在於如式(IIIa)及(IIIb) 構造混合物中之羥基烷基澱粉衍生物。 依反應之反應條件及/或用於反應之化合物(II)的化學 本質而定，如式(Ilia)之化合物可與N原子在橫轴或縱軸 位置上存在，其中兩種型式之混合物亦可以具有特定平衡 分佈存在。 依反應之反應條件及/或用於反應之化合物(II)的化學 本質而定，如式(IIIb)之化合物可與C-N雙鍵存在於E或 2結構中，其中兩種型式之混合物亦可以具有特定平衡分 佈存在。 於某些情況中，想要的是穩定如式(Ilia)之化合物。尤 其是在如式(Ilia)之化合物係在水性溶液中製造及/或使用 之情況中。如式(Ilia)化合物之醯化作用作為穩定的方法係 特別佳，尤其是在R’為氫之情況中。所有適當的試劑可用 作為醯化劑，其會導致想要的如式(IVa)之羥基烷基殿粉衍 生物。 -56- 200521143 A7 B7 五、發明說明（55) HAS，(nib) Therefore, the present invention also relates to a hydroxy-based pentamidine derivative having a structure of the formula (nib) as described above. In addition, the present invention also relates to a hydroxyalkyl starch derivative that exists in a structural mixture of formulae (IIIa) and (IIIb) as described above. Depending on the reaction conditions of the reaction and / or the chemical nature of the compound (II) used for the reaction, for example, a compound of formula (Ilia) may exist on the horizontal or vertical axis with the N atom, and a mixture of the two types is also There may be a specific equilibrium distribution. Depending on the reaction conditions of the reaction and / or the chemical nature of the compound (II) used for the reaction, for example, the compound of formula (IIIb) can exist in the E or 2 structure with the CN double bond, and a mixture of the two types can also A specific equilibrium distribution exists. In some cases, it is desirable to stabilize a compound of formula (Ilia). This is particularly the case when the compound of formula (Ilia) is manufactured and / or used in an aqueous solution. For example, the halogenation of a compound of formula (Ilia) is particularly preferred as a stable method, especially when R 'is hydrogen. All suitable reagents can be used as hydrating agents, which will lead to the desired hydroxyalkyl powder derivatives such as formula (IVa). -56- 200521143 A7 B7 V. Description of the invention (55) HAS,

根據本發明尤其佳的具體例，為醯化劑之一部份之殘 基Ra係為甲基。較好使用羧酸酐，羧酸南化物，及羧酸 活化酯作為醯化劑。 因此，本發明亦關於藉如前說明的方法而可得之羥基 烷基澱粉衍生物，其中，該衍生物具有如式(IVa)之構造。 醯化作用係在自0至30°C範圍内，較佳係自2至20 C範園且尤其佳係自4至l〇°c範圍之溫度下進行。 於其他情況中，想要的是穩定如式(mb)之化合物。尤 其是在如式(Illb)之化合物係在水性溶液中製造及/或使用 之情況中。如式(Ilia)化合物之還原作用作為穩定的方法者 係特別佳，尤其是在R，為氫之情況中。所有適當的試劑可 經濟部智慧財產局員工消费合作社印製 用作為還原劑，其會導致想要的如式(IVb)之羥基烷基澱粉 衍生物αAccording to a particularly preferred embodiment of the present invention, the residue Ra which is a part of the amidine is a methyl group. Carboxylic anhydrides, carboxylates, and carboxylic acid activated esters are preferably used as the halogenating agent. Therefore, the present invention also relates to a hydroxyalkyl starch derivative obtainable by the method described above, wherein the derivative has a structure as shown in formula (IVa). The tritiation is carried out at a temperature ranging from 0 to 30 ° C, preferably from 2 to 20 ° C and particularly preferably from 4 to 10 ° C. In other cases, it is desirable to stabilize compounds of formula (mb). This is particularly the case when the compound of formula (Illb) is manufactured and / or used in an aqueous solution. Reduction of a compound of formula (Ilia) is particularly preferred as a stable method, especially in the case where R is hydrogen. All suitable reagents can be printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs as a reducing agent, which will lead to the desired hydroxyalkyl starch derivative of formula (IVb) α

(IVb) -57- 200521143 Α7 Α7 Β7 五、發明說明（56) 根據本發明尤其佳的具體例，氮翊化物，例如， NaCNBH3或NaBH4係用作為還原劑。 因此，本發明亦關於藉如前說明的方法而可得之羥基 烷基澱粉衍生物，其中，該衍生物具有如式(lvb)之構造。 還原作用係在自4至lOOt：範圍内，較佳係自1〇至 90°C範圍且尤其佳係自25至80°C範圍之溫度下進行。 本發明又關於化合物（Ilia)與(Illb)，（IVa)與(IVb)， (Ilia)與(IVa)，（Ilia)與(ivb)，（Illb)與(IVa)，（Illb)與 (IVb)，（Ilia)與(Illb)與(IVa)，（Ilia)與(Illb)與(IVb)，（IVa) 與(IVb)與(Ilia)，及(IVa)與(IVb)與(Illb)之混合物，其中， (Ilia)及/或(IVa)可竭立存在於結構中，其中n原子係在橫 轴或縱轴位置中及/或其中(Illb)可與C-N雙鍵存在於E或 Z結構中。 經濟部智慧財產局員工消费合作社印製 根據本發明之一觀點，化合物(I)係與化合物(Π)進行反 應而得到第一反應產物。然後將該第一反應產物根據前文 所述之至少一種方法任意地穩定。然後該第一個經任意穩 定之反應產物然後係與至少另一種化合物經由包含於第一 反應產物之R”中之至少一個官能基X與包含於至少另一 種化合物中之至少一個官能基γ進行反應而得到第二反應 產物。然後將該第二反應產物根據前文所述之至少一種方 法任意ά穩定。 根據本發明還有另一個觀點，至少另一種化合物係為 多胜肽或交聯化合物或交聯化合物與多胜肽之反應產物。 當至少另一種化合物為多胜肽時，官能基Υ係包含於多胜 -58- A7 B7 200521143 五、發明說明（57 肽中且亦選擇地於多胜肽中。當至少另一種化合物為交聯 化合物時，官能基Y係包含於交聯化合物中且任意的亦包 含於多胜肽中。當至少另一種化合物為交聯化合物與多胜 肽之反應產物時，官能基γ係包含於交聯化合物中。 根據本發明之另一觀點，化合物(H)係與至少另一種化 合物經由包含於化合物(II)之R”中之至少一個官能基X與 包含於至少另一種化合物中之至少一個官能基Y進行反應 而得到第一反應產物°該至少另一種化合物宜為多胜肽或 為交聯化合物或為交聯化合物與多胜狀之反應產物，如前 文所述者。 該第一反應產物然後與化合物⑴經由化合物⑴之還原 端與化合物(II)之第一反應產物之橋連原有的殘基R，與R，， 之NH基進行反應而得到第二反應產物。然後將該第二反 應產物根據前文所述之至少一種方法任意地穩定。 經濟部智慧財產局員工消費合作社印製 根據本發明尤其佳之具體例，羥基乙基澱粉係用作為 化0物⑴〇_[2·(2_胺基氧基_乙氧基)·乙基】經基胺係用作 為化合物(II)，且具有醣分子側鏈經氧化之終端醣分子部 分之ΕΡΟ係用作為另一個化合物。更佳者為經基乙基 殿粉係與0-[2·(2-胺基氧基·乙氧基)_乙基]録胺進行反 應而得到第-㈣乙基婦触物，且將該第—衍生物進 -步與具有醣分子側鏈經氧化之終端_分子部分之脚 進行反應叫到第二«乙級贿錄。㈣特定讀 況中，並不需要進行任何穩定反應。 因此’本發明亦關於藉由方法而可得之經基燒基殿粉 -59- 200521143 A7 B7 五、發明說明（58) 衍生物，其中，羥基烷基殿粉係經由其還原端與0-[2-(2_ 胺基氧基-乙氧基)-乙基]-輕基胺進行反應，且反應產物係 經由紅血球生成素之醣分子侧鏈經氧化之終端醣分子部分 與紅血球生成素進行反應。 根據本發明還有另一個尤其佳之具體例，羥基乙基澱 粉係用作為化合物⑴，〇-[2-(2·胺基氧基-乙氧基)_乙基]-羥 基胺係用作為化合物(II)，使用具有順丁烯二醯亞胺基團 及N-幾基破酿亞胺活性醋基之雜二官能交聯化合物， 及具有至少一個-SH基(被認為是硫代EPO)之EPO用作為 多胜肽。更佳者為，羥基乙基殿粉係與〇-[2-(2-胺基氧基_ 乙氧基)-乙基]-羥基胺進行反應而得到第一羥基乙基澱粉 衍生物，該第一羥基乙基澱粉衍生物進一步與交聯化合物 之N-經基破拍醜亞胺活性酿基進行反應而得到第二衍生 物，且該第二衍生物經由順丁烯二醯亞胺與硫代Ep〇進 行反應而得到第三羥基乙基澱粉衍生物。 經濟部智慧財產局員工消费合作社印製 羥基烷基澱粉衍生物於下文中被稱為HAS-EPO概合 物且其係藉由化合物⑴與化合物(II)及可能交聯化合物及 紅血球生成素進行反應而形成，當與共輛前之紅血球生成 素比較時，其具有改良的生物穩定性的優點。這是主要由 於羥基烷基澱粉衍生物較少或甚至不被肝臟及腎臟之移除 系統所認定之事實因此而存留在循環系統中一段較長的時 間。此外，因為HAS係位置-特定地附著，破壞生體内 EPO生物活性之風險藉著HAS共軛至EPO而減低。 本發明之HAS-EPO概合物可實質上具有與試管内相 -60- 200521143 A7 B7 五、發明說明（59) 同之生物活性如重組體天然的EPO,因為試管内生物活性 僅測定EPO受體之束縛親合力。測定試管内生物活性的 方法係已知於此方面技藝中。 此外，HAS-EPO比EPO用作為共輛(未共輛之EPO) 之起動物質時具有較大的生體内活性。測定試管内生物活 性的方法係巳知於此方面技藝中。 若將未共輛之EPO之生體内活性設定為1〇〇%，HAS-EPO軛合物可具有自110%至300%，較佳係自110%至 200%，更佳係自110%至180%或自110至150%，最佳係 自110%至140%之生體内活性。 若將高度延酸化(sialylated)EPO之生體内活性設定為 100%，相較於安根公司（Amgen)之高度涎酸化EPO(參閱 EP 428 267 Bl)，HAS_EP0具有較佳為至少50%，更佳為 至少70%，甚至更佳為至少85%或至少95%，至少 150%，至少200%或至少300%之高度涎酸化EPO的生體 内活性。最佳者為，具有至少95%高度涎酸化EP0的生 體内活性。 經濟部智慧財產局員Η消费合作社印製 本發明之HAS-EPO輛合物之高生體内生物活性主要 係起因於由於較高的分子量HAS_EP0概合物於循環中比 未共輛之EP0保持較長久的事實，因為其比較不被肝臟 之移除系統所認定且因為腎臟清除減低。測定循環中EPO 之生體内半生期的方法係已知於此方面技藝中（塞可斯 基，盧恩，達維斯，費德曼，席克曼，1998，具顯著增強 生體内活性之人類紅血球生成素二元體，美國國家科學院 -61- 200521143 A7 B7 五、發明說明（60) 研討會，95(3)，1184-8)。 所以，本發明之一大優點係提供了 HAS_EPO輛合 物，其在給藥頻率上比目前市售可得之EPO製劑者少。 雖然EPO製劑必須至少每3天給藥，本發明之HAS-EPO 輛合物宜每週給藥二次，更佳係每週給藥一次。 此外，本發明的方法具有可在降低成本下製造有效 EPO衍生物的優點’因為該方法不包括導致低最終產物昂 貴且耗時的純化步驟，例如，其不需要將已知具有低或無 生體内生物活性之在涎酸化下之EPO型式純化除去。 此外，本發明亦關於醫藥組成物，其包括治療有效量 之HAS-多胜肽輛合物，較佳為HAS-EPO軛合物，更佳為 本發明的HES-EPO輛合物。於較佳的具體例中，醫藥組 成物進一步包括有用於紅血球生成素治療中之至少一製藥 上可接受的稀釋劑，輔助劑及/或載劑。 經濟部智慧財產局員工消費合作社印製 因此，本發明亦關於醫藥組成物，其包括治療有效量 如前說明之羥基烷基澱粉衍生物，其中化合物⑴與化合物 (II)之反應產物係經由包含於化合物(H)中之至少一個官能 基X與至少另一種化合物進行反應，或其中化合物(11)係 經由至少一個官能基X與至少另一種化合物於與化合物(1) 之反應前進行反應且其中至少另一種化合物係為多胜肽。 根據本發明較佳的具體例，多胜肽宜為紅血球生成素 與化合物(II)或與化合物⑴與化合物(11)之反應產物經由包 含於多胜肽中之硫基或經氧化的醣分子部分進行反應。 根據本發明甚至更佳的具體例，多胜肽宜為紅血球生 -62- 200521143 A7 B7 五、發明說明（61) 成素與化合物(II)或與化合物⑴與化合物(H)之反應產物經 由經包含於多胜肽中之氧化的醣分子部分進行反應。 因此，本發明係關於如前說明之醫藥組成物，其中， 多胜肽係與化合物(II)或與化合物(I)與化合物(π)之反應產 物經由包含於多胜肽中之經氧化的醣分子部分進行反應。 根據較佳的具體例，多胜肽係為GCS-F，AT III， IFN-β或紅血球生成素，更佳者為紅血球生成素。 因此，本發明亦關於如前說明之醫藥組成物，其中， 多胜肽係為紅血球生成素。 根據本發明尤其佳之具體例，如前說明之醫藥組成物 係藉著將羥基乙基澱粉於水性介質中與如下式之化合物 進行反應且藉著將反應產物與紅血球生成素進行反應而製 經濟部智慧財產局員工消费合作社印製 根據特別隹之具體例，紅血球生成素係於上述所提之 反應前被過峨酸納氧化。 根據另一個特別佳之具體例，紅金球生成素係被部份 二延酸化（desialylated)且隨即於反應之前被過蛾酸納氧 化。 根據本發明又較佳的具體例中，醫藥組成物係包含以 經完全還原之硫代-EPO為基礎，不包含根據實例5而製 -63- 200521143 A7 B7 五、發明說明（62) 得之羥基烷基澱粉衍生物。 根據另一個較佳的具體例，本發明亦關於醫藥組成 物，其包括治療有效量如前說明之羥基烷基澱粉衍生物， 其中，化合物(I)與化合物(II)之反應產物係經由包含於化 合物(II)中之至少一個官能基X與至少另一種化合物進行 反應，或其中化合物(II)係經由至少一個官能基X與至少 另一種化合物於與化合物⑴之反應前進行反應，且其中至 少另一種化合物係為交聯化合物且化合物⑴與(II)之反應 產物與交聯化合物之反應產物係與多胜肽進行反應。 根據另一個較佳的具體例，本發明係關於前文提及之 醫藥組成物，其中，多胜肽係為紅血球生成素。 前文所提之醫藥組成物尤其適用來治療貧血障礙或造 血機能障礙或其相關的疾病。 經濟部智慧財產局員工消费合作社印製 本文中所用之，，治療有效量，，一詞係指提供治療有效於 給定條件及給藥攝取的數量。紅血球生成素異構重整體 (isoform)之給藥宜藉由非經腸胃途徑投服。特定的途徑係 依所治療之病症而定。紅血球生成素異構重整體宜作成包 含適當的載劑，例如，人類血清蛋白素，適當的稀釋劑， 例如，經緩衝的食鹽溶液，及/或適當的辅助劑之調配物 之-部份給藥。所需的劑量係以足夠提昇病患血細胞比容 之量且係依所治療之病症的嚴重性，給藥方法等而定。 以本發明醫藥組成物治療之對象宜為血液中增加的本 紅朊值超過6.8毫莫耳/升。為此，錢組成物可以每週增 加自〇·6毫莫耳/升與I·6毫莫耳/升間之血紅朊值的方式給 -64- 200521143 A7 B7 五、發明說明（63 藥。如果血紅朊值值超過8.7毫莫耳/升，較佳應中斷治療 直到血紅朊值低於8.1毫莫耳/升。 本發明之組成物宜用於適合皮下給藥或靜脈給藥或非 經腸胃給藥注射之調配物中。為此，適當的賦形劑及載劑 為例如，磷酸二氫鈉，磷酸氮二鈉，氯酸鈉，聚山梨糖醇 酯80，HSA及水用於注射。組成物可以每週三次’宜為 每週二次，更佳為每週一次，且最隹為每兩週一次給藥。 較佳者為，醫藥組成物係以001_1()微克/公斤病患體 重之量給藥，更佳為0.1至5微克/公斤，01至1微克公 斤，或0.2-0.9微克/公斤，最隹為0·3_0·7微克/公斤’且 最佳為0.4-0.6微克/公斤體重。 通常，每劑量宜在10微克及200微克間，更佳係在 15微克及1〇〇微克間給藥 本發明又關於根據本發明之HAS_多胜肽於治療人類 或動物體之方法中的用途。 本發明又關於本發明之HAS-EPO輛合物於製備治療 造血機能障礙或其相關疾病之醫藥品的用途。 經濟部智慧財產局員工消费合作社印製 本發明進一步藉下列實例，表列，及圖形來闡明，其 並非用來限制本發明之範疇。 -65- 200521143 B7 五、發明說明（64) 圈形之簡要說明 圖1 圖1顯示根據實例4.1所製得之HES-EPO軛合物的 SDS PAGE 分析。 A行：經預染色之蛋白質標諸Roti®-Mark(卡爾羅斯 GmbH+Co公司，卡爾欺魯厄市，D);蛋白質標誌 從頂到底之分子量（kD)為：245，123，77，42， 30，25.4 及 17 〇 B行：根據實例4.1共軛後之粗產物。 C行：EPO起動物質。 圖2 圖2顯示根據實例4.3所製得之HES_EPO輛合物的 SDS PAGE 分析。 A行：根據實例4.3共軛後之粗產物。 B行：EPO起動物質。 經濟部智慧財產局員工消费合作社印製 C行：經預染色之蛋白質標誌Roti®-Mark(卡爾羅斯 GmbH+Co公司，卡爾欺魯厄市，D);蛋白質標諸 從頂到底之分子量（kt>)為：245，123，77，42， 30，25·4 及 17 〇 圈3 圖3顯示根據實例6.1及6.4所製得之HES-EPO軛合 物的SDS PAGE分析。 A行：經預染色之蛋白質標誌Roti®-Mark(卡爾羅斯 -66- 200521143 A7 B7 五、發明說明（65)(IVb) -57- 200521143 A7 A7 B7 V. Description of the invention (56) According to a particularly preferred embodiment of the present invention, a nitrogen compound such as NaCNBH3 or NaBH4 is used as a reducing agent. Therefore, the present invention also relates to a hydroxyalkyl starch derivative obtainable by the method described above, wherein the derivative has a structure such as the formula (lvb). The reduction is carried out at a temperature ranging from 4 to 100 t :, preferably from 10 to 90 ° C, and particularly preferably from 25 to 80 ° C. The present invention also relates to compounds (Ilia) and (Illb), (IVa) and (IVb), (Ilia) and (IVa), (Ilia) and (ivb), (Illb) and (IVa), (Illb) and ( (IVb), (Ilia) and (Illb) and (IVa), (Ilia) and (Illb) and (IVb), (IVa) and (IVb) and (Ilia), and (IVa) and (IVb) and (Illb ), Where (Ilia) and / or (IVa) can exist in the structure, where the n atom is in the horizontal or vertical axis position and / or (Illb) can exist in the double bond with CN in E Or Z structure. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs According to one aspect of the present invention, the compound (I) reacts with the compound (Π) to obtain a first reaction product. This first reaction product is then optionally stabilized according to at least one of the methods described above. The first arbitrarily stabilized reaction product is then performed with at least another compound via at least one functional group X included in R "of the first reaction product and at least one functional group γ included in at least another compound. The reaction results in a second reaction product. The second reaction product is then arbitrarily stabilized according to at least one of the methods described above. According to another aspect of the present invention, at least another compound is a polypeptide or a cross-linked compound or The reaction product of a cross-linking compound and a polypeptide. When at least one other compound is a polypeptide, the functional group is contained in the polypeptide-58- A7 B7 200521143 V. Description of the invention (57 in the peptide and optionally in polypeptide In peptides, when at least one other compound is a cross-linked compound, the functional group Y is included in the cross-linked compound and any one is also included in the poly-peptide. When at least another compound is a cross-linked compound and the poly-peptide In the reaction product, the functional group γ is included in the crosslinking compound. According to another aspect of the present invention, the compound (H) and at least one other compound At least one functional group X in R "of compound (II) is reacted with at least one functional group Y contained in at least another compound to obtain a first reaction product. The at least another compound is preferably a polypeptide or is The cross-linked compound may be a reaction product of a cross-linked compound and a polysaccharide, as described above. The first reaction product is then bridged with the compound ⑴ via the reducing end of the compound 与 and the first reaction product of the compound (II). The original residue R, is reacted with the NH group of R, to obtain a second reaction product. Then the second reaction product is arbitrarily stabilized according to at least one of the methods described above. Employees' Cooperatives, Intellectual Property Bureau, Ministry of Economic Affairs According to a particularly preferred embodiment of the present invention, hydroxyethyl starch is used as a chemical compound ⑴〇_ [2 · (2-aminooxy_ethoxy) · ethyl] is used as a compound through a amine system ( II), and EPO with a terminal sugar molecule moiety oxidized on the side chain of the sugar molecule is used as another compound. The more preferred is via ethyl ethyl powder and 0- [2 · (2-aminooxy ·· Ethoxy) _ethyl] amine Go to the second ㈣ ethyl feminine object, and further react the first derivative with the foot of the molecular molecule with an oxidized terminal _ molecular part of the side chain of the sugar molecule called the second «class B bribery. ㈣ specific reading In this case, there is no need to perform any stable reaction. Therefore, the present invention also relates to the basic fired powder-59- 200521143 A7 B7 obtained by the method. 5. Description of the invention (58) Derivatives, in which hydroxyalkanes Kedian powder reacts with 0- [2- (2-aminooxy-ethoxy) -ethyl] -light amine via its reducing end, and the reaction product is passed through the sugar molecule side chain of erythropoietin The oxidized terminal sugar molecule part reacts with erythropoietin. According to another particularly preferred embodiment of the present invention, hydroxyethyl starch is used as the compound ⑴, 〇- [2- (2 · aminooxy-ethoxy Group) _ethyl] -hydroxyamine system is used as the compound (II), using a heterobifunctional cross-linking compound having a cis-butenyldiimine group and an N-kispermine imine-active acetic acid group, and having EPO having at least one -SH group (considered as a thio-EPO) is used as a peptide. More preferably, the hydroxyethyl starch powder is reacted with 0- [2- (2-aminooxy_ethoxy) -ethyl] -hydroxyamine to obtain a first hydroxyethyl starch derivative. The first hydroxyethyl starch derivative is further reacted with the N-Cyclo-imine active imine group of the cross-linked compound to obtain a second derivative, and the second derivative is reacted with maleimide The thioep0 was reacted to obtain a third hydroxyethyl starch derivative. The hydroxyalkyl starch derivative printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs is hereinafter referred to as a HAS-EPO complex and it is carried out by compound ⑴ and compound (II), and possibly cross-linked compounds and erythropoietin It is formed by the reaction, and has the advantage of improved biological stability when compared with erythropoietin in front of a common vehicle. This is mainly due to the fact that hydroxyalkyl starch derivatives are few or even not recognized by the liver and kidney removal systems and therefore remain in the circulatory system for a longer period of time. In addition, because the HAS line is site-specifically attached, the risk of disrupting the biological activity of EPO in vivo is reduced by the conjugation of HAS to EPO. The HAS-EPO complex of the present invention may substantially have the same internal phase with the test tube-60-200521143 A7 B7 V. Description of the invention (59) Same biological activity as the recombinant natural EPO, because the biological activity in the test tube only determines the EPO receptor Bondage of the body. Methods for measuring biological activity in a test tube are known in the art. In addition, HAS-EPO has greater in vivo activity than EPO when it is used as a starting material for common vehicles (unshared EPO). Methods for measuring biological activity in a test tube are known in the art. If the in vivo activity of unshared EPO is set to 100%, the HAS-EPO conjugate can have from 110% to 300%, preferably from 110% to 200%, and more preferably from 110% To 180% or from 110 to 150%, the best is from 110% to 140% in vivo activity. If the in vivo activity of highly sialylated EPO is set to 100%, compared to Amgen's highly sialylated EPO (see EP 428 267 Bl), HAS_EP0 has preferably at least 50%, More preferably it is at least 70%, even more preferably at least 85% or at least 95%, at least 150%, at least 200% or at least 300% of the in vivo activity of highly sialylated EPO. The most preferred is in vivo activity with at least 95% highly sialylated EPO. The bioactivity of the HAS-EPO vehicle compound printed by the present invention printed by a member of the Intellectual Property Bureau of the Ministry of Economic Affairs is mainly due to the higher molecular weight of the HAS_EP0 complex in the circulation than the EP0 that has not been shared for a long time. The fact that it is less recognized by the liver removal system and because kidney clearance is reduced. The method of measuring the half-life in vivo of EPO in circulation is known in the art (Sekowski, Luen, Davis, Federman, Hickman, 1998, and has significantly enhanced in vivo activity Human Erythropoietin Binary, National Academy of Sciences-61-200521143 A7 B7 V. Invention Description (60) Symposium, 95 (3), 1184-8). Therefore, one of the great advantages of the present invention is that it provides a HAS_EPO vehicle compound, which is less frequent in administration than those currently available commercially available EPO preparations. Although the EPO preparation must be administered at least every 3 days, the HAS-EPO vehicle composition of the present invention should preferably be administered twice a week, and more preferably once a week. In addition, the method of the present invention has the advantage that effective EPO derivatives can be produced at reduced cost, 'because the method does not include expensive and time-consuming purification steps that lead to low end products, for example, it does not require In vivo biological activity was purified and removed by EPO type under sialic acid. In addition, the present invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of a HAS-polypeptide conjugate, preferably a HAS-EPO conjugate, and more preferably a HES-EPO conjugate of the present invention. In a preferred embodiment, the pharmaceutical composition further includes at least one pharmaceutically acceptable diluent, adjuvant and / or carrier used in the treatment of erythropoietin. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. Therefore, the present invention also relates to a pharmaceutical composition, which includes a therapeutically effective amount of a hydroxyalkyl starch derivative as described above, in which the reaction product of compound VII and compound (II) is At least one functional group X in compound (H) is reacted with at least another compound, or wherein compound (11) is reacted with at least one other compound via at least one functional group X before reaction with compound (1), and At least one other compound is a polypeptide. According to a preferred embodiment of the present invention, the polypeptide is preferably a reaction product of erythropoietin and compound (II) or a compound ⑴ and compound (11) via a sulfur group or an oxidized sugar molecule contained in the polypeptide. Partial reaction. According to an even better specific example of the present invention, the polypeptide is preferably erythrocyte-62-200521143 A7 B7 V. Description of the invention (61) The reaction product of the element with the compound (II) or with the compound ⑴ and the compound (H) passes through The reaction proceeds via the oxidized sugar molecules contained in the polypeptide. Therefore, the present invention relates to the pharmaceutical composition as described above, in which the polypeptide is a reaction product with compound (II) or with compound (I) and compound (π) via an oxidized compound contained in polypeptide. The sugar molecule part reacts. According to a preferred specific example, the polypeptide is GCS-F, AT III, IFN-β or erythropoietin, and more preferably erythropoietin. Therefore, the present invention also relates to the pharmaceutical composition as described above, wherein the polypeptide is erythropoietin. According to a particularly preferred embodiment of the present invention, the pharmaceutical composition as described above is prepared by reacting hydroxyethyl starch in an aqueous medium with a compound of the following formula and by reacting the reaction product with erythropoietin. Printed by the Intellectual Property Bureau's Consumer Cooperatives According to a specific example, red blood cells are oxidized by sodium perenate before the reactions mentioned above. According to another particularly good example, erythropoietin is partially diallylated and immediately oxidized with sodium permoate before the reaction. In a further preferred embodiment according to the present invention, the pharmaceutical composition system is based on the fully reduced thio-EPO, and does not include the preparation according to Example 5.-63- 200521143 A7 B7 V. Description of the Invention (62) Hydroxyalkyl starch derivatives. According to another preferred embodiment, the present invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of a hydroxyalkyl starch derivative as described above, wherein the reaction product of compound (I) and compound (II) is At least one functional group X in compound (II) is reacted with at least another compound, or wherein compound (II) is reacted via at least one functional group X with at least another compound before reaction with compound IX, and wherein At least one other compound is a cross-linked compound and the reaction product of compound VII with (II) and the cross-linked compound are reacted with a polypeptide. According to another preferred embodiment, the present invention relates to the aforementioned medicinal composition, wherein the polypeptide is erythropoietin. The aforementioned pharmaceutical composition is particularly suitable for treating anemia disorder or hematopoietic dysfunction or related diseases. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. As used in this article, the term therapeutically effective amount refers to the amount that provides a treatment effective for a given condition and ingestion. Administration of isoforms of erythropoietin should be administered by parenteral route. The specific route depends on the condition being treated. Erythropoietin isomers should be prepared as part of a formulation containing a suitable carrier, such as human serum protein, a suitable diluent, such as a buffered common salt solution, and / or a suitable adjuvant. medicine. The required dose is an amount sufficient to increase the hematocrit of the patient and depends on the severity of the condition to be treated, the method of administration, and the like. The subject to be treated with the pharmaceutical composition of the present invention preferably has an increased erythrocyte value in the blood of more than 6.8 millimolars / liter. For this reason, the composition of money can increase the blood redness value between 0.6 millimoles / liter and 1.6 millimoles / liter every week by way of -64- 200521143 A7 B7 V. Description of the invention (63 drugs). If the hemoglobin value exceeds 8.7 millimoles / liter, the treatment should preferably be discontinued until the hemoglobin value is less than 8.1 millimoles / liter. The composition of the present invention is suitable for subcutaneous or intravenous or non-menstrual administration. In the formulation for parenteral injection. For this purpose, suitable excipients and carriers are, for example, sodium dihydrogen phosphate, disodium phosphate, sodium chlorate, polysorbate 80, HSA and water for injection. The composition can be administered three times a week, preferably twice a week, more preferably once a week, and most preferably once every two weeks. Preferably, the pharmaceutical composition is 001_1 () micrograms per kilogram of disease The amount of the diseased body is administered, more preferably 0.1 to 5 μg / kg, 01 to 1 μg / kg, or 0.2-0.9 μg / kg, and the maximum is 0 · 3_0 · 7 μg / kg 'and the best is 0.4-0.6 Micrograms per kilogram of body weight. Generally, each dose is preferably between 10 micrograms and 200 micrograms, and more preferably between 15 micrograms and 100 micrograms. According to the use of the HAS-polypeptide of the present invention in a method for treating a human or animal body, the present invention also relates to the use of the HAS-EPO compound of the present invention in the preparation of a medicinal product for treating a hematopoietic dysfunction or a related disease. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs, the present invention is further illustrated by the following examples, tables, and figures, which are not intended to limit the scope of the present invention. -65- 200521143 B7 V. Description of the invention (64) Brief Description Figure 1 Figure 1 shows the SDS PAGE analysis of the HES-EPO conjugate prepared according to Example 4.1. Line A: Pre-stained protein labeled Roti®-Mark (Carlos GmbH + Co, Carl Bull) Ecuador, D); The molecular weight (kD) of the protein marker from top to bottom is: 245, 123, 77, 42, 30, 25.4 and 17 〇 B: The crude product after conjugation according to Example 4.1. Line C: EPO start Substance. Figure 2 Figure 2 shows the SDS PAGE analysis of HES_EPO vehicle compound prepared according to Example 4.3. Line A: The crude product after conjugation according to Example 4.3. Line B: EPO starting substance. Consumption by employees of Intellectual Property Bureau of the Ministry of Economic Affairs Cooperative printed line C: The pre-stained protein marker Roti®-Mark (Carlos GmbH + Co, Karl Bulle, D); the molecular weight of the protein marker from top to bottom (kt >) is: 245, 123, 77, 42, 30, 25.4 and 17.0 Circle 3 Figure 3 shows the SDS PAGE analysis of HES-EPO conjugates prepared according to Examples 6.1 and 6.4. Line A: Pre-stained protein marker Roti®-Mark (Carlos-66- 200521143 A7 B7 V. Description of Invention (65)

GmbH+Co公司，卡爾欺魯厄市，D);蛋白質標誌 從頂到底之分子量（kD)為：245，123，77，42， 30，25.4 及 17。 B行：根據實例6.4共輛後之粗產物。 C行：根據實例6.1共軛後之粗產物。 〇行：EPO起動物質。 圈4 圖4顧示根據實例6.2，6.3，6.5及6.6所製得之 HES-EPO軛合物的SDS PAGE分析。 A行：經預染色之蛋白質標誌Roti®-Mark(卡爾羅斯 GmbH+Co公司，卡爾欺魯厄市，D);蛋白質標誌 從頂到底之分子量（kD)為：245，123，77，42， 30, 25.4 及 17。 B行：以實例1.1b)為基礎，根據實例6.6共軛後之粗產 物。 C行：以實例1.3b)為基礎，根據實例6.5共輊後之粗產 物0 D行：以實例l_3a)為基礎，根據實例6·6共輛後之粗產 經 濟 部 智 慧 財 產 局 員 物0 Ε行··以實例1.1a)為基礎，根據實例6.5共輛後之粗產 物0 消 费 合 作 社 F行··根據實例6.2共軛後之粗產物。 G行：根據實例6.3共軛後之粗產物。 K行：EPO起動物質。 -67- 200521143 A7 五、發明說明（66GmbH + Co, Karl-Rue, D); The molecular weight (kD) of protein markers from top to bottom: 245, 123, 77, 42, 30, 25.4 and 17. Line B: The crude product after a total of vehicles according to Example 6.4. Line C: The crude product after conjugation according to Example 6.1. Line 0: EPO starting material. Circle 4 Figure 4 shows the SDS PAGE analysis of HES-EPO conjugates prepared according to Examples 6.2, 6.3, 6.5 and 6.6. Line A: Pre-stained protein marker Roti®-Mark (Carlos GmbH + Co, Karl de Roue, D); molecular weight (kD) of the protein marker from top to bottom: 245, 123, 77, 42, 30, 25.4 and 17. Line B: Based on Example 1.1b), the crude product after conjugation according to Example 6.6. Line C: Based on Example 1.3b), based on Example 6.5, the crude product is 0. Line D: Based on Example 1-3a), based on Example 6.6, the crude product is from the Intellectual Property Bureau of the Ministry of Economic Affairs. 0 Ε OK ······················································································································ Line G: The crude product after conjugation according to Example 6.3. Line K: EPO starting substance. -67- 200521143 A7 V. Description of the invention (66

圈S EP0-GT-1進行弱酸處理5分鐘：=第2行；10分鐘=第 3行；60分鐘=第4行及未經處理EPO=第1行；顯示移除 N-多醣後EPO移動位移(+PMGASE)i SDS pAGE分析。 圖6 將寡醣之HPAEC-PAD類型從未處理EP0及從epo 在弱酸水解之條件下培育5分鐘，1〇分鐘及60分鐘中單 離出來。羅馬數字Ι-V表示1=二涎酸化(desialylated)二突 觸（diantennary)結構，11=三涎酸化的三突觸結構(二個異構 物），111=四涎酸化的四突觸結構+ 2N-乙醯基乳糖胺重 覆’ IV=四涎酸化的四突觸結構+ ιΝ_乙醯基乳糖胺重覆； 四涎酸化的四突觸結構+不含N_乙醯基乳糖胺重覆之洗 提位置。寡醣類結構不含，含1-4唾液酸之洗提區係以架 橋待號表示。 國7 經濟部智慧財產局員工消費合作社印製 二涎酸化後N-連結的寡醣之HPAEC-PAD; N-乙釀 基神經胺酸之洗提位置顯示出來；數字1-9表示標準寡聽 之洗提位置：1=二突觸；2=三突觸(2-4異構物），3=三突 觸（2,6異構物）；4=四突觸；5=三突觸加上一重覆；“四 突觸加上1次重覆；7=四突觸加上二重覆；8=四突觸加上 二重覆及9=四突觸加上三重覆。 固8 將經EPO溫和處理及未處理之SDS-PAGE分析進行 唾液酸殘基之過峨酸鹽氧化作用。1=經過蛾酸鹽氧化不 _68- 200521143 A? B7 五、發明說明（67) 酸處理，2=經過碘酸鹽氧化酸處理5分鐘；3=經過碘酸鹽 氧化及酸處理10分鐘；4=經過碘酸鹽氧化不含酸處理； 5=BRPEPO標準不含過碘酸鹽氧化且不含酸處理。 圈9 將寡聽類HPAEC-PAD類型從未經處理EPO及從 EPO在弱酸水解之條件下培育5分鐘及1〇分鐘中單離出 來且隨即過碘酸鹽處理。寡醣結構不含及含1-4涎酸之洗 提區以架橋符號1-5表示。 圈10 經濟部智慧財產局員工消费合作社印製 EPO_GT-l-A之HES改變時間過程之SDS-PAGE分 析：將各份20微克EPO_GT-l-A用經輕基胺改質的HES 衍生物X處理30分鐘，2 ’ 4及17小時。第1行=反應時 間30分鐘；第2行=反應時間2小時；第3行=反應時間 4小時；第4行=反應時間17小時；第5行HEPO-GT-1-A 不含HES-改質。左圖顯示EPO-GT-1-A在含經羥基胺改 質之HES衍生物(流速：1毫升。分鐘存在之下於增 加的培育時間中之移動位移：第1行==反應時間30分鐘； 第2行==反應時間2小時；第3行=反應時間4小時；第4 行=反應時間17小時；第5行=EPO-GT_l-A含HES-改 質。右圖顯示相同樣品用N-配醣酶處理其等後之分析。 圈11 HES-EPO軛合物之Q-瓊脂糖凝膠餾份之SDS-PAGE 分析。將高鹽濃度下洗提之每一 1〇/❶浸流(flow_through)及 -69- 200521143 A7 B7 五、發明說明（6〇 1 %餾份於高速真空濃縮器下濃縮且裝载於樣品緩衝液中 之凝膠上。將EPO蛋白質用考瑪斯亮藍(Coomassie mue) 染色。樣品I ; B=樣品II ; Ο樣品III ; K=控制組EPO-GT-1 ; A1，B1，Cl 及 K1 表示浸流的餘份,· A2，B2，C2 及K2表示經高鹽濃縮洗提之餾份。 圈12a 經HES改質的EPO樣品A2(參閱圖7)，控制組EPO 樣品K2及EPO-GT-l_A蛋白質製劑在N-配醣酶存在之下 消化以便移除N-連接的寡醣之SDS-PAGE分析。所有的 EPO樣品顯示出向缺乏或含有0-多醣之低分子量型式移 動位移。觀察到經HES改質的EP0樣品A2於去醣化 作用後經〇醣化的與非經醣化的蛋白質帶之較低比例， 且測定到浸潤蛋白質帶為30Kda左右，大概代表在〇-多 醣殘基之涎酸下之HES-改質（參閱箭頭註上星號標示者）。 圖12b 經濟部智慧財產局員工消費合作社印製 經HES改質的EPO樣品A2(參閱圖11)，控制組EPO 樣品K2及EPO-GT-1A，其係未經處理或在N-配酷酶存 在之下消化以便除N-連接的募醣(參閱圖12a)之弱水解作 用後之SDS-PAGE分析。N_配醣酶處理（參閱含與不含箭 \ 頭之架橋符號)前的A2及處理後的A之高分子量型式二者 於酸處理樣品後消失。比較用之BRP EP0標準表里鼓弱 酸處理。 圈13 從經HES-改質之樣品A，從EP0-GT_1-A及從未經改 -70- 200521143 A7 B7 五、發明說明（69) 質之HES培育控制組epo樣品(κ)中釋放N-鍵結的寡醣 物質之HPAECXPAD分析。羅馬數字ΐ-ν表示1=二涎酸化 (desialyMed)的二突觸結構，11=三涎酸化的三突觸結構(二 個異構物），111=四涎酸化的四突觸結構+ 2N-乙醯棊乳糖 胺重覆，IV=四涎酸化的四突觸結構+ 醯基乳糖胺重 覆；四延酸化的四突觸結構+不含N-乙醯基乳糖胺重覆 之洗提位置；支架符號表示二_，三_及四-涎酸化N-多醣 的洗提區’如圖6及9圖片中所記錄者。 圈14 從經HES-改質之樣品A，從EPOGT-1-A及從控制組 EPO樣品(K)未經改質之HES培育中釋放N-鍵結的寨醣物 質之HPAEC-PAD分析。寡醣類混合物之滯留時間顯示 為：數字1-9表示標準寡醣之洗提位置：1=二突觸；2=三 突觸(2-4異構物），3=三突觸(2-6異構物）；4=四突觸；5= 三突觸加上一重覆；6=四突觸加上一重覆；7=四突觸加上 二重覆；8=四突觸加上二重覆及9=四突觸加上三重覆0 國15至21 經濟部智慧財產局員工消费合作社印製 圖15至21代表經酶催釋放及化學性二涎酸化的多 醣從經HES-改質之EPO與控制組EPO製劑中單離出來之 MALDI/TOF 質譜。主要信號係在 m/z 18〇9 7，2539.9， 2905.0及3270.1 ([m+Na]+)，相對於二-至四突觸絡合物型 式N-多醣結構含無，一或二次]sf_乙醯基乳糖胺重覆伴隨 著弱信號，係由於岩藻糖或半乳糖因用於MS分析之樣品 二涎酸化作用之酸水解作用條件而流失。 -71- 200521143 A7 B7 五 經濟部智慧財產局員工消费合作社印製 發明說明（7〇) 圈is MALDI/TOF質譜：經HES-改質的Ep〇八2之二涎酸 化寡醣類。 國16 MALDI/TOF質譜：EPOGT-l_A之二涎酸化寡醣類。 圖17 MALDI/TOF質譜：EPO K2之二涎酸化寡酶類。 圈18 MALDI/TOF質譜：EPO-GT-1之二涎酸化寡酷類。 圈19 MALDI/TOF質譜：EPO-GT-1進行酸水解$分鐘之二 涎酸化募醣類。 圈20 MALDI/TOF質譜：EPO-GT-1進行酸水解1〇分鐘之 二涎酸化寡醣類。 圖21 MALDI/TOF質譜：EPO-GT-1進行酸水解6〇分鐘之 二涎酸化募醣類。 【實施方式】 實例 實例1 :藉還原性胺化作用形成羥基乙基澱粉衍生物 實例1·1 羥基乙基澱粉舆1，3-二胺基羥基丙烷之反應 -72- A7 B7 200521143 五、發明說明（71) H2N 八γ^ΝΗ2Circle S EP0-GT-1 for weak acid treatment for 5 minutes: = line 2; 10 minutes = line 3; 60 minutes = line 4 and untreated EPO = line 1; showing EPO movement after removal of N-polysaccharide Displacement (+ PMGASE) i SDS pAGE analysis. Figure 6 HPAEC-PAD type of oligosaccharide was isolated from untreated EP0 and incubated from epo under weak acid hydrolysis for 5 minutes, 10 minutes and 60 minutes. The Roman numerals I-V indicate 1 = desialylated didiannary structure, 11 = trisialylated trisynaptic structure (two isomers), 111 = tetrasialylated tetrasynaptic structure + 2N-Ethyl lactosamine repeat 'IV = Tetrasialified tetrasynaptic structure + ιΝ_ethylated lactosamine repeat; Tetrasialylated tetrasynaptic structure + N_Ethyl lactosamine free Repeated elution position. The oligosaccharide structure does not contain, and the elution zone containing 1-4 sialic acid is indicated by the bridge number. HPAEC-PAD of N-linked oligosaccharides printed by disialylated after consumption of disialylated by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs of the State of China. Elution position: 1 = second synapse; 2 = trisynapse (2-4 isomers), 3 = trisynapse (2,6 isomers); 4 = tetrasynapse; 5 = trisynapse Plus one repeat; "four synapses plus one repeat; 7 = four synapses plus two repeats; 8 = four synapses plus two repeats and 9 = four synapses plus three repeats. Solid 8 Peroxidate oxidation of sialic acid residues by mildly treated and untreated SDS-PAGE analysis of EPO. 1 = Not mothate oxidation_68- 200521143 A? B7 V. Description of the invention (67) Acid treatment , 2 = Iodate oxidation acid treatment for 5 minutes; 3 = Iodate oxidation and acid treatment for 10 minutes; 4 = Iodate oxidation without acid treatment; 5 = BRPEPO standard does not contain periodate oxidation and Does not include acid treatment. Circle 9 The oligoacoustic HPAEC-PAD type was isolated from untreated EPO and incubated with EPO under weak acid hydrolysis for 5 minutes and 10 minutes and then treated with periodate. Oligosaccharide structure The elution zone containing 1-4 sialic acids is indicated by the bridging symbol 1-5. Circle 10 SDS-PAGE analysis of the HES change time course of EPO_GT-lA printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs: 20 copies of each Micrograms of EPO_GT-lA were treated with HES derivative X modified with light amine for 30 minutes, 2 '4 and 17 hours. Line 1 = reaction time 30 minutes; Line 2 = reaction time 2 hours; Line 3 = reaction Time 4 hours; line 4 = reaction time 17 hours; line 5 HEPO-GT-1-A does not contain HES-modification. The figure on the left shows that EPO-GT-1-A is derived from HES modified with hydroxylamine (Flow rate: 1 ml. Movement displacement in increasing incubation time in the presence of minutes: line 1 == reaction time 30 minutes; line 2 == reaction time 2 hours; line 3 = reaction time 4 hours; Line 4 = Reaction time 17 hours; Line 5 = EPO-GT_l-A with HES-modified. The figure on the right shows the analysis of the same sample treated with N-glycosidase and the subsequent analysis. Circle 11 HES-EPO conjugate SDS-PAGE analysis of Q-Agarose gel fractions. Each 10 / ❶ effluent (flow_through) and -69- 200521143 A7 B7 eluted at a high salt concentration V. Description of the invention (60% Distill Concentrated under high-speed vacuum concentrator and loaded on gel in sample buffer. EPO protein was stained with Coomassie mue. Sample I; B = Sample II; 〇Sample III; K = Control Group EPO-GT-1; A1, B1, Cl, and K1 represent the remainder of the leaching stream, and · A2, B2, C2, and K2 represent the fractions eluted by high salt concentration. Circle 12a HES modified EPO sample A2 (see Figure 7), control group EPO sample K2 and EPO-GT-1_A protein preparation were digested in the presence of N-glycosidase to remove N-linked oligosaccharide SDS -PAGE analysis. All EPO samples showed shifts to low molecular weight patterns lacking or containing 0-polysaccharides. It was observed that HES-modified EP0 sample A2 had a lower ratio of saccharified and non-saccharified protein bands after deglycosylation, and the infiltrated protein band was determined to be about 30 Kda, which roughly represents the 0-polysaccharide residue. HES-modification under sialic acid (see those marked with an asterisk in the arrow). Figure 12b HEP-modified EPO sample A2 (see Figure 11) printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs, EPO samples K2 and EPO-GT-1A in the control group, which are either untreated or in N-paraben SDS-PAGE analysis after digestion in the presence of weak hydrolysis to remove N-linked sugar-removal (see Figure 12a). Both the high molecular weight form of A2 before N_glycosidase treatment (see the bridge symbol with and without the arrow \ head) and the high molecular weight form of A after treatment disappeared after the acid-treated sample. Compare the BRP EP0 standard surface with weak acid treatment. Circle 13 Release N from HES-modified sample A, EP0-GT_1-A and from unmodified-70- 200521143 A7 B7 V. Description of the invention (69) Quality HES incubation control group epo sample (κ) released N -HPAECXPAD analysis of bonded oligosaccharides. The Roman numeral ΐ-ν indicates 1 = disialylated bisynaptic structure, 11 = trisialylated trisynaptic structure (two isomers), 111 = tetrasialylated tetrasynaptic structure + 2N -Ethyl lactosamine repeat, IV = tetrasialized tetrasynaptic structure + fluorenyl lactosamine repeat; Tetra-acidified tetrasynaptic structure + N-acetylated lactosamine repeat elution Location; the scaffold symbol indicates the elution zone of di-, tri-, and tetra-sialylated N-polysaccharides as shown in the pictures of Figs. 6 and 9. Circle 14 HPAEC-PAD analysis of the release of N-bonded cottage sugar substances from HES-modified sample A, EPOGT-1-A and control group EPO sample (K) unmodified HES incubation. The residence time of the oligosaccharide mixture is shown as: Numbers 1-9 indicate the elution position of standard oligosaccharides: 1 = second synapse; 2 = trisynapse (2-4 isomers), 3 = trisynapse (2 -6 isomers); 4 = four synapses plus one repeat; 6 = four synapses plus one repeat; 7 = four synapses plus two repeats; 8 = four synapses plus The last two repeats and 9 = four synapses plus three repeats. Country 15 to 21 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. Figures 15 to 21 represent enzymatically released and chemically sialylated polysaccharides from HES- MALDI / TOF mass spectra isolated from modified EPO and control group EPO preparations. The main signals are at m / z 18〇9 7, 2539.9, 2905.0 and 3270.1 ([m + Na] +), relative to the di- to tetra-synaptic complex type N-polysaccharide structure containing none, one or two times] The sf_ethosyllactosamine repeat was accompanied by a weak signal due to the fucose or galactose loss due to acid hydrolysis conditions of the sialylation of the samples used for MS analysis. -71- 200521143 A7 B7 Five Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs (70) Circle is MALDI / TOF Mass Spectroscopy: HES-modified Ep0082 sialylated oligosaccharides. National 16 MALDI / TOF mass spectrometry: EPOGT-l_A bissialylated oligosaccharides. Figure 17 MALDI / TOF mass spectrum: diasialyl oligoenzymes of EPO K2. Circle 18 MALDI / TOF mass spectrometry: EPO-GT-1 bis-sialylated oligomeric class. Circle 19 MALDI / TOF mass spectrometry: EPO-GT-1 undergoes acid hydrolysis for two minutes. Salivation of sugars. Circle 20 MALDI / TOF mass spectrometry: EPO-GT-1 was bissialylated oligosaccharides subjected to acid hydrolysis for 10 minutes. Figure 21 MALDI / TOF mass spectrometry: EPO-GT-1 was acid-hydrolyzed for 60 minutes to sialylate to raise sugars. [Embodiment] Example Example 1: Formation of hydroxyethyl starch derivatives by reductive amination Example 1.1 Reaction of hydroxyethyl starch with 1,3-diaminohydroxypropane-72- A7 B7 200521143 V. Invention Explanation (71) H2N Eightγ ^ ΝΗ2

OH a) 將0.83亳莫耳l，t二胺基-2-羥基丙烷及50毫克氰基 硼氫酸鈉NaCNBH3加至含200毫克羥基乙基澱粉 (HES18/0.4 (MW = 18,000 D，DS=0.4))於 5 毫升水中之 溶液。將產生的混合物於80°C培育17小時。將反應 混合物加至160毫升丙酮與乙醇1 : 1之冷混合物(v/v) 中。將沉澱以離心法收集起來且對水透析4天(蛇皮透 析管，3.5KD切斷，貝爾生物科學都史蘭GmbH公 司，柏恩，D)，且冷凍乾燥。 b) 亦可能將0.83毫莫耳1，3·二胺基-2-羥基丙烷及50毫 克氰基硼氫酸鈉NaCNBH3加至含200毫克羥基乙基澱 粉所產生的混合物培育且在25°C下進行3天。 實例1·2 羥基乙基激粉舆1，2-二羥基-3-胺基丙烷之反應 經濟部智慧財產局員工消费合作社印製 ㈣八丫^〇Η ΟΗ a)將0.83毫莫耳1，2-二羥基_3_胺基丙烷及50毫克氰基硼 氳酸鈉NaCNBH3加至含200毫克羥基乙基澱粉 (HES18/0.4 (MW = 18,000 D，DS=0.4))於 5 毫升水中之 溶液。將產生的混合物於80°C培育17小時。將反應混 -73- 200521143 B7 五 '發明說明（72 合物加至160毫升丙_與乙醇1: 1之冷混合物(ν/ν) 中。將沉澱以離心法收集起來且對水透析4天(蛇皮透 析管，3.5KD切斷，貝爾生物科學都史蘭GmbH公 司，柏恩，D)，且冷凍乾燥。 b)亦可能將0·83毫莫耳1，3-二胺基-2-羥基丙烷及50毫克 氰基硼氫酸鈉NaCNBH3加至含200毫克羥基乙基澱粉 所產生的混合物培育且在25°C下進行3天。 U-二羥基_3_胺基丙烷與HES <反應係間接地藉甲醛 之定量來確認，其係由過碘酸鹽引起反應產物中1，2-二烯 類(dioles)之氧化性裂解’如G·阿維加德，於生物化學年 鑑，134(1983) 449-504中所說明者。 實例1.3羥基已基激粉舆1，4_二胺基丁烷之反應OH a) Add 0.83 mol l, t diamino-2-hydroxypropane and 50 mg sodium cyanoborohydride NaCNBH3 to 200 mg hydroxyethyl starch (HES18 / 0.4 (MW = 18,000 D, DS = 0.4)) in 5 ml of water. The resulting mixture was incubated at 80 ° C for 17 hours. The reaction mixture was added to 160 ml of a cold 1: 1 mixture of acetone and ethanol (v / v). The pellet was collected by centrifugation and dialyzed against water for 4 days (snake skin dialysis tube, 3.5 KD cut-off, Bell Biosciences Shihland GmbH, Byrne, D), and freeze-dried. b) It is also possible to add 0.83 millimoles of 1,3-diamino-2-hydroxypropane and 50 mg of sodium cyanoborohydride NaCNBH3 to a mixture containing 200 mg of hydroxyethyl starch and incubate at 25 ° C The next three days. Example 1.2 Reaction of hydroxyethyl phosphonate to 1,2-dihydroxy-3-aminopropane Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs ㈣ 八 丫 ^ 〇Η 〇Η a) 0.83 millimoles1, 2-Dihydroxy_3_aminopropane and 50 mg of sodium cyanoborate NaCNBH3 was added to a solution containing 200 mg of hydroxyethyl starch (HES18 / 0.4 (MW = 18,000 D, DS = 0.4)) in 5 ml of water . The resulting mixture was incubated at 80 ° C for 17 hours. The reaction mixture-73-200521143 B7 five 'invention description (72 compound was added to 160 ml of a cold mixture of propylene and ethanol 1: 1 (ν / ν). The precipitate was collected by centrifugation and dialyzed against water for 4 days (Snake skin dialysis tube, 3.5KD cut-off, Bell Biosciences Shihland GmbH, Byrne, D), and freeze-dried. B) It is also possible to 0.83 millimolar 1,3-diamino-2 -A mixture produced by adding hydroxypropane and 50 mg of sodium cyanoborohydride NaCNBH3 to 200 mg of hydroxyethyl starch was incubated and performed at 25 ° C for 3 days. The reaction between U-dihydroxy_3-aminopropane and HES < was indirectly confirmed by the quantitative determination of formaldehyde, which is caused by the oxidative cleavage of 1,2-dienes (dioles) in the reaction product caused by periodate. 'As described in G. Avigard, Yearbook of Biochemistry, 134 (1983) 449-504. Example 1.3 Reaction of hydroxyhexyl powder with 1,4-diaminobutane

Hjsr 經濟部智慧財產局員工消费合作社印製 a)將0.83毫莫耳1，4_二胺基丁烷及50毫克氰基硼氫酸鈉 NaCNBH3加至含200毫克羥基乙基澱粉(HES18/〇4 (MW = 18,000 D，DS=0.4))於5毫升水中之溶液。將產 生的洗合物於80°C培育17小時。將反應混合物加至 160毫升丙酮與乙醇1 · 1之冷混合物(v/v)中。將沉殿 以離心法收集起來且對水透析4天(蛇皮透析管，$ ^kd 切斷’貝爾生物科學都史蘭GinbH公司，柏恩，d), 且冷凍乾燥。 -74- 200521143 A7 B7 五、發明說明（73) b)亦可能將0.83毫莫耳ι，4-二胺丁烷及50毫克氰基硼氫 酸鈉NaCNBH3加至含200毫克羥基乙基澱粉所產生的 混合物培育且在25°C下進行3天。 實例1·4 羥基乙基激粉舆1_鲅基-2_胺基乙烷之反應 a) 將0.83毫莫耳1-巯基-2-胺基乙烷及50毫克氰基硼氫酸 鈉NaCNBHb加至含200毫克羥基乙基澱粉(HES18/0.4 (MW = 18,000 D，DS=0.4))於5毫升水中之溶液。將產 生的混合物於80°C培育17小時。將反應混合物加至 160毫升丙酮與乙醇ι:ι之冷混合物(v/v)中。將沉殿 以離心法收集起來且對水透析4天(蛇皮透析管，3.5KD 切斷，貝爾生物科學都史蘭GmbH公司，柏恩，D)， 且冷;東乾燥。 經濟部智慧財產局員工消费合作社印製 b) 亦可能將0.83毫莫耳1-巯基-2-胺基乙烷及50毫克氰基 硼氳酸鈉NaCNBH3加至含200毫克羥基乙基澱粉所產 生的混合物培育且在25。〇下進行3天。 1，2-二羥基-3-胺基丙烷與HES之反應係間接地藉甲醛 之定量來確認，其係由過峨酸鹽引起反應產物中1，2_二烯 類(dioles)之氧化性裂解’如g·阿維加德，於生物化學年 鑑，134(1983)449-504中所說明者。 實例2 :藉共軛作用形成羥基乙基激粉衍生物 -75- 200521143 A7 B7 五、發明說明（74 實例2·1 羥基乙基澉粉舆碳醯麟之反應 Η2Ν、人 /ΝΗ2 2 Ν Ν 2 Η Η 將 〇·96 克 HES18/0.4 (MW = 18,000 D，DS=0.4)溶解於 8毫升水性0.1M醋酸鈉緩衝液中，ρΗ5·2，且加入碳醯胼 (席格馬歐德里奇公司，桃夫克成市，D)。於25°C下攪拌 18小時後，將反應混合物加至160毫升丙酮與乙醇1 : 1 之冷混合物(v/v)中。將沉澱產物藉離心法收集起來，再溶 解於40毫升水中，且對水透析4天(蛇皮透析管，3.5KD 切斷，貝爾生物科學都史蘭GmbH公司，柏恩，D)，且冷 象乾燥。 實例2·2羥基乙基澱粉舆阿比酸二醮肼之反應Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs a) Add 0.83 millimolar 1,4-diaminobutane and 50 mg sodium cyanoborohydride NaCNBH3 to 200 mg hydroxyethyl starch (HES18 / 〇 4 (MW = 18,000 D, DS = 0.4)) in 5 ml of water. The resulting wash was incubated at 80 ° C for 17 hours. The reaction mixture was added to 160 ml of a cold mixture (v / v) of acetone and ethanol 1.1. The Shen Dian was collected by centrifugation and dialyzed against water for 4 days (snake skin dialysis tube, $ ^ kd cut off 'Bell Biosciences Shilan GimbH, Byrne, d), and freeze-dried. -74- 200521143 A7 B7 V. Description of the invention (73) b) It is also possible to add 0.83 millimolar, 4-diamine butane and 50 mg of sodium cyanoborohydride NaCNBH3 to 200 mg of hydroxyethyl starch The resulting mixture was incubated and performed at 25 ° C for 3 days. Example 1.4 Reaction of hydroxyethyl phosphate with 1-fluorenyl-2_aminoethane a) 0.83 millimoles of 1-mercapto-2-aminoethane and 50 mg of sodium cyanoborohydride NaCNBHb Add to a solution containing 200 mg of hydroxyethyl starch (HES18 / 0.4 (MW = 18,000 D, DS = 0.4)) in 5 ml of water. The resulting mixture was incubated at 80 ° C for 17 hours. The reaction mixture was added to 160 ml of a cold mixture (v / v) of acetone and ethanol. The Shen Dian was collected by centrifugation and dialyzed against water for 4 days (snake skin dialysis tube, 3.5 KD cut off, Bell Biosciences Shihland GmbH, Byrne, D), and cold; east dry. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs b) It is also possible to add 0.83 millimolar 1-mercapto-2-aminoethane and 50 mg sodium cyanoborate NaCNBH3 to 200 mg The mixture was incubated at 25. 0 ° C for 3 days. The reaction of 1,2-dihydroxy-3-aminopropane with HES was confirmed indirectly by the quantitative determination of formaldehyde, which is caused by the peroxidic acid salt of 1,2-dienes (dioles) in the reaction product. Cleavage 'as described in g. Avigard, Yearbook of Biochemistry, 134 (1983) 449-504. Example 2: Formation of hydroxyethyl radical derivative-75- 200521143 A7 B7 by conjugation V. Description of the invention (74 Example 2.1 Reaction of hydroxyethyl powder with carboline 2N, human / N 2 2 Ν Ν 2 Η Η Dissolve 0.96 g of HES18 / 0.4 (MW = 18,000 D, DS = 0.4) in 8 ml of aqueous 0.1 M sodium acetate buffer, ρ Η 5.2, and add carbon 醯 胼 (Sigma Oedrich Company, Taofucheng, D). After stirring at 25 ° C for 18 hours, the reaction mixture was added to 160 ml of a cold 1: 1 mixture of acetone and ethanol (v / v). The precipitated product was collected by centrifugation Get up, re-dissolve in 40 ml of water, and dialyze against water for 4 days (snake skin dialysis tube, 3.5KD cut off, Bell Biosciences Shihland GmbH, Byrne, D), and freeze dry. Example 2.2 Hydroxyethyl starch

經濟部智慧財產局員工消费合作社印製 將 0·96 克 HES18/0.4 (MW = 18,000 D，DS=0.4)溶解於 8毫升水性0.1M醋酸鈉緩衝液中，pH5.2，且加入8毫莫 耳阿比酸二醯肼(adepic dihydrazide)(蘭卡斯德合成公司， 法蘭克福/緬因，D)。於25°C下攪拌18小時後，將反應 混合物加至160毫升丙酮與乙醇1 : 1之冷混合物(v/v) -76- A7 B7 200521143 五、發明說明（75 ) 中。將沉澱產物藉離心法收集起來，再溶解於4〇毫升水 中，且對水透析3天(蛇皮透析管’3 5KD切斷’貝爾生 物科學都史蘭GmbH公司，柏恩，D)，且冷涞乾燥。 實例2.3羥基乙基澱粉舆h4·伸苯基··雙-3-硫代半辛酹之 反應Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. 0.96 grams of HES18 / 0.4 (MW = 18,000 D, DS = 0.4) was dissolved in 8 ml of aqueous 0.1M sodium acetate buffer, pH 5.2, and 8 mmol was added. Adepic dihydrazide (Lancaster Synthetic, Frankfurt / Maine, D). After stirring at 25 ° C for 18 hours, the reaction mixture was added to 160 ml of a cold 1: 1 mixture of acetone and ethanol (v / v) -76- A7 B7 200521143 V. Description of the invention (75). The precipitated product was collected by centrifugation, redissolved in 40 ml of water, and dialyzed against water for 3 days (snake skin dialysis tube '3 5KD cut-off' Bell Biosciences Shihland GmbH, Byrne, D), Chill and dry. Example 2.3 Reaction of hydroxyethyl starch with h4 · phenylene ·· bis-3-thiosemi-octylamine

經濟部智慧財產局員工消费合作社印製 將 0.96 克 HES18/0.4 (MW = 18,0〇〇 d，DS=0.4)溶解於 8毫升水性0.1M醋酸鈉緩衝液中，ρΗ5·2，且加入8亳莫 耳1，4-伸苯基_雙-3_硫代半苄肼（蘭卡斯德合成公司，法蘭 克福/緬因，D)。於25°C下攪拌18小時後，將8毫升水 加至反應混合物中，且將懸浮液以4,5〇〇轉數/秒離心15 分鐘。將透明的上澄液傾析出來且隨即加至160毫升丙陶 與乙醇1 : 1之冷混合物(v/v)中。將沉澱的產物藉離心法 收集起來，再溶解於40毫升水中，且以4,500轉數/秒離 心15分鐘。將透明的上澄液對水透析3天(蛇皮透析管， 3.5KD切斷，貝爾生物科學都史蘭GmbH公司，柏恩， D) ’且冷;東乾燥。 實例2.4羥基乙基激粉舆〇•丨2-(2_胺基氧基_乙氧基>_乙 基1-幾基联之反應 -77- 200521143 A7 B7 五、發明說明（76) H2N/〇V^O 八ν〇、ΝΗ2 〇_[2-(2-胺基氧基-乙氧基)-乙基]-羥基胺係如波吐來等 於四面體53 (1997) 5485-5492頁中所說明者以2階段從市 售可得之物質合成。 將 0·96 克 HES18/0_4 (MW = 18,000 D，DS=0.4)溶解於 8毫升水性0.1M醋酸鈉緩衝液中，pH5.2，且加入8毫莫 耳〇-[2-(2-胺基氧基-乙氧基)-乙基]-羥基胺。於25°C下攪 拌18小時後，將反應混合物加至160毫升丙酮與乙醇1 : 1之冷混合物(v/v)中。將沉澱的產物藉離心法收集起來， 再溶解於40毫升水中，且對水透析3天(蛇皮透析管， 3.5KD切斷，貝爾生物科學都史蘭GmbH公司，柏恩， D)，且冷凍乾燥。 資例2.4(a)羥基乙基激粉與〇-[2-(2-胺基氧基·已氡基)-已 基】-轰基胺之反應 經濟部智慧財產局員工消费合作社印製 經氧化之HES係如DE 196 28 705 A1中之說明製 備。將 200 亳克經氧化的 HEsi8/0.4 (MW = 18,000 D， DS=0.4)於80°C真空中加熱17小時且溶解於2毫升無水 DMSO中（弗祿卡，席格馬歐德里奇公司’桃夫克成市， D)。將2毫莫耳〇-[2-(2-胺基氧基-乙氧基)_乙基Hi基胺 加至溶液。於65°C培育5天後，將反應混合物加至20毫 升冷丙醇中且於-20°C培育1小時。於4°c時將沉澱的產 -78- 200521143 A7 B7 五、發明說明（Τ7) 物藉離心法收集起來，用42毫升冰冷的2-丙醇清洗，再 溶解於10毫升水中，對水透析27小時（蛇皮透析管， 3.5KD切斷，貝爾生物科學都史蘭GmbH公司，柏恩， D)，且冷柬乾燥。 實例3紅血球生成素之氧化作用 經氧化的紅血球生成素係如實例7中之說明製得。使 用如實例7.11(c )中說明之EPO-GT-1-A作為經氧化的紅 血球生成素(EPO_GT-l未經酸水解，用弱過碘酸鹽氧化作 用處理）。 實例4羥基乙基澱粉舆實例3經氧化的紅轰球生成素之 共輕 實例4·1經氡化的紅也球生成素舆實例2.1反應產物之共 輛 經濟部智慧財產局員工消費合作社印製 將含氧化的ΕΡΟ(1.〇55微克/微升)於20 mM PBS緩衝 液用5M醋酸鈉緩衝液pH 5.2調節到pH 5·3。將18微升 如實例2.1(MW 18 Kd ; 18.7微克/微升於0.1M醋酸鈉緩 衝液中，pH 5.2)所製造之HES衍生物溶液加入，且將混 合物於25°C培育16小時。冷束乾燥後，將粗產物用安維 特吉公司給予之通如中所說明之NuPAGE 10%雙-三凝膠 /MOPS緩衝液（安維特吉公司，卡斯巴，加州，美國）藉 SDS_Page分析。凝勝係用洛提-藍考瑪斯染劑（羅斯，卡斯 盧，D)染色過夜。 實驗結果顯示於圖1中。成功的共輛作用係以蛋白質 帶移向較高的分子量來表示。帶-寬係由於所用之HES衍 -79- 200521143 A7 B7 五、發明說明（78) 生物分子量分佈及HES衍生物連接到蛋白質之數量而增 加0 實例4·2經氧化的紅血球生成素與實例2·3反應產物之反 應 將含氧化的ΕΡΟ(1.055微克/微升)於20 mM PBS之緩 衝液用5M醋酸鈉緩衝液pH 5.2調節到pH 5.3。將18微 升如實例2.3(MW 18 kD ; 18,7微克/微升於〇·1Μ醋酸鈉 緩衝液中，pH 5.2)所製造之HES衍生物溶液加至19微升 EPO溶液中，且將混合物於25°C培育16小時。冷凍乾燥 後，將粗產物用安維特吉公司給予之通知中所說明的 NuPAGE 10%雙-三凝膠/MOPS緩衝液(安維特吉公司，卡 斯巴，加州，美國）藉SDS-Page分析。 實例4·3經氧化的紅血球生成素舆實例2.4反應產物之反 應 經濟部智慧財產局員工消費合作社印製 將含經氧化的ΕΡΟ(1_055微克/微升)於20 mM PBS之 緩衝液用5M醋酸鈉缓衝液PH 5.2調節到pH 5.3。將18 微升如實例2J(MW 18 kD ; 18.7微克/微升於0.1M醋酸 鈉緩衝液中，pH 5.2)所製造之HES衍生物溶液加至19微 升EPO溶液中，且將混合物於25°C培育16小時。冷凍乾 燥後’將粗產物用安維特吉公司給予之通知中所說明的 NuPAGE 10%雙-三凝膠/MOPS緩衝液(安維特吉公司，卡 斯巴，加州，美國）藉SDS-Page分析。凝膠係用洛提-藍考 瑪斯染劑(羅斯，卡斯盧，D)染色過夜。 實驗結果顯示於圖2中。成功的共軛作用係以蛋白質 -80- 200521143 Α7 Β7 五、發明說明（79) 帶移向較高的分子量來表示。帶_寬係由於所用之HES衍 生物分子量分佈及HES衍生物連接到蛋白質之數量而增 加0Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. 0.96 grams of HES18 / 0.4 (MW = 18,000 d, DS = 0.4) was dissolved in 8 ml of aqueous 0.1M sodium acetate buffer, ρΗ5.2, and 8 was added. Anamol 1,4-phenylene_bis-3_thiosemi-benzylhydrazine (Lancaster Synthetic, Frankfurt / Maine, D). After stirring at 25 ° C for 18 hours, 8 ml of water was added to the reaction mixture, and the suspension was centrifuged at 4,500 rpm for 15 minutes. The clear supernatant was decanted and added to 160 ml of a cold 1: 1 mixture of propionol and ethanol (v / v). The precipitated product was collected by centrifugation, redissolved in 40 ml of water, and centrifuged at 4,500 rpm for 15 minutes. The clear supernatant was dialyzed against water for 3 days (snake skin dialysis tube, 3.5 KD cut off, Bell Biosciences Shihland GmbH, Byrne, D) 'and cold; dry. Example 2.4 Hydroxyethyl radical powder 〇 2- 丨 2-aminooxy_ethoxy > _ethyl 1-quinyl reaction-77- 200521143 A7 B7 V. Description of the invention (76) H2N / 〇V ^ O νν〇, ΝΗ2 〇_ [2- (2-aminooxy-ethoxy) -ethyl] -hydroxylamine system such as Botol is equal to tetrahedron 53 (1997) 5485-5492 The ones described in the above were synthesized in two stages from commercially available substances. 0.96 g of HES18 / 0_4 (MW = 18,000 D, DS = 0.4) was dissolved in 8 ml of aqueous 0.1 M sodium acetate buffer, pH 5.2 And 8 millimoles of 0- [2- (2-aminooxy-ethoxy) -ethyl] -hydroxyamine was added. After stirring at 25 ° C for 18 hours, the reaction mixture was added to 160 ml of acetone 1: 1 cold mixture (v / v) with ethanol. The precipitated product was collected by centrifugation, redissolved in 40 ml of water, and dialyzed against water for 3 days (snake skin dialysis tube, 3.5KD cut, Bell The biological sciences are Shilan GmbH, Byrne, D), and freeze-dried. Example 2.4 (a) Hydroxyethyl powder and 0- [2- (2-aminooxy · hexyl) -hexyl 】 -Response to the oxidation of HES by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs Prepared according to the instructions in DE 196 28 705 A1. 200 氧化 g of oxidized HEsi8 / 0.4 (MW = 18,000 D, DS = 0.4) was heated in a vacuum at 80 ° C for 17 hours and dissolved in 2 ml of anhydrous DMSO (Flu) Card, Sigma-Aldrich, Inc., "Taufke City, D). 2 millimoles of 0- [2- (2-aminooxy-ethoxy) -ethyl Hiylamine was added to the solution. After 5 days of incubation at 65 ° C, the reaction mixture was added to 20 ml of cold propanol and incubated for 1 hour at -20 ° C. The precipitated product was produced at 4 ° c -78- 200521143 A7 B7 V. Description of the invention ( Τ7) The material was collected by centrifugation, washed with 42 ml of ice-cold 2-propanol, dissolved in 10 ml of water, and dialyzed against water for 27 hours (snake skin dialysis tube, 3.5KD cut-off, Bell Bioscience Shilan GmbH Company, Byrne, D), and cold-dried. Example 3 Oxidation of erythropoietin Oxidized erythropoietin was prepared as described in Example 7. Use EPO-GT as described in Example 7.11 (c) -1-A is used as oxidized erythropoietin (EPO_GT-1 is not treated with acid and treated with weak periodate oxidation). Example 4 Hydroxyethyl starch 3 An example of the oxidized red blast pheromone. 4.1 An example of the oxidized red sphere pheromone. 2.1 The reaction product is printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. .05 μg / μl) in 20 mM PBS buffer was adjusted to pH 5.3 with 5M sodium acetate buffer pH 5.2. 18 microliters of the HES derivative solution prepared in Example 2.1 (MW 18 Kd; 18.7 micrograms / microliter in 0.1M sodium acetate buffer, pH 5.2) was added, and the mixture was incubated at 25 ° C for 16 hours. After cold beam drying, the crude product was analyzed by NuPage 10% Bi-Trigel / MOPS Buffer given by Avitej Corporation (Aventuji, Caspar, California, USA) and analyzed by SDS_Page . Ningsheng was stained with Loti-Lancoomas (Ross, Kaslow, D) overnight. The experimental results are shown in Figure 1. Successful co-transportation is represented by the shift of protein bands to higher molecular weights. The band-width is increased due to the use of HES derivatives-79- 200521143 A7 B7 V. Description of the invention (78) Biological molecular weight distribution and the number of HES derivatives linked to proteins 0 Example 4.2 Oxidized erythropoietin and Example 2 • Reaction of 3 reaction products The buffer containing oxidized EPO (1.055 μg / μl) in 20 mM PBS was adjusted to pH 5.3 with 5M sodium acetate buffer pH 5.2. 18 microliters of the HES derivative solution prepared in Example 2.3 (MW 18 kD; 18.7 micrograms / microliter in 0.1M sodium acetate buffer, pH 5.2) was added to 19 microliters of EPO solution, and The mixture was incubated at 25 ° C for 16 hours. After freeze-drying, the crude product was analyzed by NuPAGE 10% double-triple gel / MOPS buffer (Avjet, Kasba, California, USA) as described in the notice given by Avitjet. . Example 4.3 Oxidized erythropoietin Example 2.4 Reaction of reaction products Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs The oxidized EPO (1_055 μg / μl) in 20 mM PBS buffer with 5M acetic acid Sodium buffer pH 5.2 was adjusted to pH 5.3. 18 microliters of the HES derivative solution prepared in Example 2J (MW 18 kD; 18.7 micrograms / microliter in 0.1M sodium acetate buffer, pH 5.2) was added to 19 microliters of EPO solution, and the mixture was mixed at 25 ° C for 16 hours. After freeze-drying 'the crude product was analyzed by NuPAGE 10% Bi-Trigel / MOPS Buffer (Avitec, Caspar, California, USA) as described in the notice given by Avitec. . The gel system was stained overnight with Loti-Lancomas stain (Ross, Kaslu, D). The experimental results are shown in FIG. 2. The successful conjugation is represented by protein -80- 200521143 Α7 Β7 V. Description of the invention (79) The band moves to a higher molecular weight. Band_width is increased by 0 due to the molecular weight distribution of the HES derivative used and the number of HES derivatives attached to the protein

實例5以紅丘球生成素之還原作用形成硫代-EPO 將含241·5微克紅血球生成素(EP0_GT_i，參閱實例7) 於500微升o iM硼酸鈉緩衝液，5mM EDTA，10mM DTT(蘭卡斯特，莫卡貝，英國），pH 8.3於37°C培育1小 時。用VIVASPIN 0·5毫升濃縮器，10KD MWCO (萬歲科 學公司，漢諾威，D)藉離心過濾法以13,000轉數/秒將 DTT移除，隨即用硼酸鹽緩衝液清洗三次且用磷酸鹽緩衝 液(0.1Μ，9.15MNaC卜 50 mMEDTA，ρΗ7·2)清洗二次。 實例6:羥基乙基激粉衍生物舆硫代-紅Α球生成素使用 交麟化合物之共軛作用 於各個下列實例中，Ν-(α·馬爾亞胺(Maleimido)乙醯 氧基)琥珀醯亞胺酯(AMAS) ΟExample 5 Formation of thio-EPO by reduction of red hill globulin, 241.5 micrograms of erythropoietin (EP0_GT_i, see Example 7) in 500 microliters of iM sodium borate buffer, 5mM EDTA, 10mM DTT (blue Custer, Mocabe, UK), pH 8.3, incubated at 37 ° C for 1 hour. DTT was removed using a VIVASPIN 0.5 ml concentrator, 10KD MWCO (Viva Scientific, Hanover, D) at 13,000 rpm by centrifugal filtration, then washed three times with borate buffer and phosphate buffer ( 0.1M, 9.15M NaC, 50 mMEDTA, ρΗ7.2 ·) were washed twice. Example 6: A hydroxyethyl excimer derivative and a thio-red A globulin are conjugated with a compound of cyanine to each of the following examples, N- (α · Maleimido ethoxy) amber Amidoimide (AMAS) 〇

經濟部智慧財產局員工讀费合作社印製 係用作為交聯化合物。 實例6·1 ··經氧化的紅血球生成素舆實例2·1及交麟化合 物之反應產物的反應 -81- A7 B7Printed by the Intellectual Property Office of the Ministry of Economic Affairs for the fee-paying cooperatives. Used as a cross-linking compound. Example 6.1 · Reaction of oxidized erythropoietin Example 2-1 and reaction products of cross-linked compounds -81- A7 B7

200521143 將10微升2·5毫微莫耳AMAS(席格馬歐德里奇公 司’桃夫克成市，D)於DMSO中之溶液加至50毫微莫耳 根據實例2.1所製得的HES衍生物且溶解於200微升 0·1Μ 磷酸鈉緩衝液(〇·1Μ，9.15M NaC卜 50mM EDTA， pH 7·2)中。將透明的溶液於25°C培育80分鐘及於40°C培 育20分鐘。剩餘的AMAS用VIVASPIN 0·5毫升漢縮 器’ 5KD MWCO (萬歲科學公司，漢諾威，d)藉離心過濾 法以13,000轉數/秒移除，各個用磷酸鹽緩衝液清洗四次 及30分鐘。 於殘留的溶液中加入15微克根據實例5(1微克/微升 於磷酸鹽緩衝液中）所製得之硫代EPO，且將混合物於25 °C培育16小時。冷凍乾燥後，將粗產物用安維特吉公司 給予之通知中所說明的NuPAGE 10%雙-三凝膠/mops緩 衝液(安維特吉公司，卡斯巴，加州，美國）藉SDS-Page分 析。凝膠係用洛提-藍考瑪斯染劑（羅斯，卡斯盧，D)染色 過夜。 經濟部智慧財產局員工消费合作社印製 實驗結果顯示於圖3中。成功的共輛作用係以蛋白質 帶移向較高的分子量來表示。帶-寬係由於所用之HES衍 生物分子量分佈及HES衍生物連接到蛋白質之數量而增 加0 實例6.2:經氧化的紅血球生成素舆實例2.2及交赛化合 物之反應產物的反應 將10微升2·5毫微莫耳AMAS(席袼馬歐德里奇公 司，桃夫克成市，D)於DMSO中之溶液加至50毫微莫耳 -82- 200521143 A7 A7 B7 五、發明說明（81) 根據實例2·2所製得的HES衍生物且溶解於200微升， 0.1Μ 磷酸鈉緩衝液(〇 1Μ，9.15Μ NaCl，50mM EDTA， pH 7·2)中。將透明的溶液於25°C培育80分鐘及於40°C培 育20分鐘。剩餘的AMAS用VIVASPIN 0.5毫升濃縮 器’ 5KD MWCO (萬歲科學公司，漢諾威，D)藉離心過濾 法以13,000轉數/秒移除，各個用磷酸鹽緩衝液清洗四次 及30分鐘。 於殘留的溶液中加入15微克根據實例5(1微克/微升 於磷酸鹽緩衝液中）所製得之硫代EPO,且將混合物於25 °C培育16小時。冷凍乾燥後，將粗產物用安維特吉公司 給予之通知中所說明的NuPAGE 10%雙·三凝膠/MOPS緩 衝液(安維特吉公司，卡斯巴，加州，美國）藉SDS-Page分 析。凝膠係用洛提-藍考瑪斯染劑（羅斯，卡斯盧，D)染色 過夜。 實驗結果顯示於圖4中。成功的共軛作用係以蛋白質 帶移向較高的分子量來表示。帶-寬係由於所用之HES衍 生物分子量分佈及HES衍生物連接到蛋白質之數量而增 加0 經濟部智慧財產局員工消费合作社印製 實例6·3:經氧化的紅血球生成素舆實例2.3及交耱化合 物之反應產物的反應 將10微升2.5毫微莫耳AMAS(席格馬歐德里奇公 司，桃夫克成市，D)於DMSO中之溶液加至50毫微莫耳 根據實例2.3所製得的HES衍生物且溶解於200微升 0·1Μ 磷酸鈉緩衝液(〇·ΐΜ，9.15Μ NaC卜 50mM EDTA， -83- 200521143 A7 A7 B7 五、發明說明（82) pH 7.2)中。將透明的溶液於25°C培育80分鐘及於40°C培 育20分鐘。剩餘的AMAS用VIVASPIN 0.5毫升濃縮 器，5KD MWCO (萬歲科學公司，漢諾威，D)藉離心過濾 法以13,000轉數/秒移除，各個用磷酸鹽緩衝液清洗四次 及30分鐘。 於殘留的溶液中加入15微克根據實例5(1微克/微升 於磷酸鹽緩衝液中）所製得之硫代EPO，且將混合物於25 °C培育16小時。冷凍乾燥後，將粗產物用安維特吉公司 給予之通知中所說明的NuPAGE 10%雙-三凝膠/MOPS緩 衝液(安維特吉公司，卡斯巴，加州，美國）藉SDS-Page分 析。凝膠係用洛提_藍考瑪斯染劑（羅斯，卡斯盧，D)染色 過夜。 實驗結果顯示於圖4中。成功的共輛作用係以蛋白質 帶移向較高的分子量來表示。帶-寬係由於所用之HES衍 生物分子量分佈及HES衍生物連接到蛋白質之數量而增 加0 經濟部智慧財產局員工消費合作社印製 實例6.4:實例2·4及交礴化合物之反應產物舆經氡化的 紅血球生成素之反應 將含10微升2.5毫微莫耳AMAS(席格馬歐德里奇公 司’桃夫克成市，D)於DMSO中之溶液加至50毫微莫耳 根據實例2·4所製得的HES衍生物且溶解於200微升， 01M 碟酸鈉緩衝液(0.1Μ，9.15Μ NaCl，50mM EDTA， pH 7·2)中。將透明的溶液於25幻培育8〇分鐘及於仞它培 育20分鐘。剩餘的AjyjAs用VIVASPIN 〇 5毫升濃縮 -84- A7 B7 200521143 五、發明說明（83 ) 器，5KD MWCO (萬歲科學公司，漢諾威，D)藉離心過濾 法以13,000轉數/秒移除，各個用磷酸鹽緩衝液清洗四次 及30分鐘。 於殘留的溶液中加入15微克根據實例5(1微克/微升 於磷酸鹽緩衝液中）所製得之硫代EPO,且將混合物於25 °C培育16小時。冷凍乾燥後，將粗產物用安維特吉公司 給予之通知中所說明的NuPAGE 10%雙-三凝膠/MOPS緩 衝液(安維特吉公司，卡斯巴，加州，美國）藉SDS-Page分 析◎凝膠係用洛提-藍考瑪斯染劑（羅斯，卡斯盧，D)染色 過夜。 實驗結果顧示於圖3中。成功的共軛作用係以蛋白質 帶移向較高的分子量來表示。帶-寬係由於所用之HES衍 生物分子量分佈及HES衍生物連接到蛋白質之數量而增 加0 實例6.5:綏氧化的紅jk球生成素舆實例1#1及交礴化合 物之反應產物的反應 經濟部智慧財產局員工消費合作社印製 將10微升2·5毫微莫耳AMAS(席格馬歐德里奇公 司’桃夫克成市，D)於DMSO中之溶液加至50毫微莫耳 根據實例1·1所製得的HES衍生物，於8〇t及17小時(實 例U.a)以及25°C及三天(實例1.1b)之培育條件且溶解於 2〇〇微升01M磷酸鈉緩衝液(〇 1M，9 15M Naa，5〇mM EDTA，pH 7.2)中。將透明的溶液於2Γ(：培育80分鐘及 於4〇C培育2〇分鐘。剩备的AMAS用VIVASPIN 0.5毫 升濃縮器，5KD MWCO (萬歲科學公司，漢諾威，D)藉離 -85- 200521143200521143 Add a solution of 10 μl 2.5 nanomolar AMAS (Sigma O'Dridge Co., Inc., “Taofucheng, D) to 50 nanomolar of the HES derivative prepared according to Example 2.1 And dissolved in 200 microliters of 0.1M sodium phosphate buffer (0.1M, 9.15M NaC, 50mM EDTA, pH 7.2). The clear solution was incubated at 25 ° C for 80 minutes and at 40 ° C for 20 minutes. The remaining AMAS was removed with a VIVASPIN 0.5 ml Han shrink condenser ' 5KD MWCO (Viva Scientific, Hanover, d) by centrifugal filtration at 13,000 rpm, each washed four times with phosphate buffer and 30 minutes. To the residual solution was added 15 µg of the thio-EPO prepared according to Example 5 (1 µg / µl in phosphate buffer), and the mixture was incubated at 25 ° C for 16 hours. After freeze-drying, the crude product was analyzed by SDS-Page using NuPAGE 10% Bi-Trigel / Mops Buffer (Aventji, Caspar, California, USA) as described in the notice given by Avitjet. . The gel system was stained with Loti-Laccoma Stain (Rose, Kaslou, D) overnight. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. The experimental results are shown in Figure 3. Successful co-transportation is represented by the shift of protein bands to higher molecular weights. The band-width increases due to the molecular weight distribution of the HES derivative used and the number of HES derivatives attached to the protein. Example 6.2: Oxidized erythropoietin Example 2.2 and the reaction product of the reaction product will be 10 μl 2 Add 5 nanomolar AMAS (Ximao Aldridge, Taofucheng, D) in DMSO to 50 nanomolar -82- 200521143 A7 A7 B7 V. Description of the invention (81) According to The HES derivative prepared in Example 2.2 was dissolved in 200 microliters of 0.1 M sodium phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2). The clear solution was incubated at 25 ° C for 80 minutes and at 40 ° C for 20 minutes. The remaining AMAS was removed with a VIVASPIN 0.5 ml concentrator ′ 5KD MWCO (Viva Scientific, Hanover, D) by centrifugal filtration at 13,000 rpm, and each was washed four times with phosphate buffer and 30 minutes. To the residual solution was added 15 μg of the thio-EPO prepared according to Example 5 (1 μg / μl in phosphate buffer), and the mixture was incubated at 25 ° C. for 16 hours. After freeze-drying, the crude product was analyzed by NuPAGE 10% double · triple gel / MOPS buffer (Avitegi, Caspar, California, USA) as described in the notice given by Avitjet. . The gel system was stained with Loti-Laccoma Stain (Rose, Kaslou, D) overnight. The experimental results are shown in FIG. 4. Successful conjugation is represented by the shift of protein bands to higher molecular weights. The band-width is increased due to the molecular weight distribution of the HES derivative used and the number of HES derivatives attached to the protein. Reaction of the reaction product of the hydrazone compound 10 microliters of 2.5 nanomolar AMAS (Sigma Oedrich, Taofuke, D) in DMSO was added to 50 nanomolar according to Example 2.3 The obtained HES derivative was dissolved in 200 μl of a 0.1 M sodium phosphate buffer solution (0 μM, 9.15 M NaC, 50 mM EDTA, -83- 200521143 A7 A7 B7 V. Description of the invention (82) pH 7.2). The clear solution was incubated at 25 ° C for 80 minutes and at 40 ° C for 20 minutes. The remaining AMAS was removed with a VIVASPIN 0.5 ml concentrator, 5KD MWCO (Viva Scientific, Hanover, D) by centrifugal filtration at 13,000 rpm, and each was washed four times with phosphate buffer and 30 minutes. To the residual solution was added 15 µg of the thio-EPO prepared according to Example 5 (1 µg / µl in phosphate buffer), and the mixture was incubated at 25 ° C for 16 hours. After freeze-drying, the crude product was analyzed by NuPAGE 10% double-triple gel / MOPS buffer (Avjet, Kasba, California, USA) as described in the notice given by Avitjet. . The gel system was stained with Lotte-Laccomas stain (Ross, Kaslu, D) overnight. The experimental results are shown in FIG. 4. Successful co-transportation is represented by the shift of protein bands to higher molecular weights. The band-width is increased due to the molecular weight distribution of the HES derivative used and the number of HES derivatives attached to the protein. Example 6.4: Example 2. 4 and the reaction products of cross-linked compounds Reaction of tritiated erythropoietin. A solution containing 10 microliters of 2.5 nanomolar AMAS (Sigma O'Dridge Co., Inc., "Tofukcheng, D) in DMSO was added to 50 nanomolar according to Example 2. • The HES derivative prepared in 4 and dissolved in 200 μl of a 01 M sodium dish buffer (0.1M, 9.15M NaCl, 50 mM EDTA, pH 7.2). The clear solution was incubated at 80 ° C for 80 minutes and incubated at 20 minutes. The remaining AjyjAs were concentrated with VIVASPIN 〇5ml-84- A7 B7 200521143 V. Description of the invention (83), 5KD MWCO (Long live science company, Hanover, D) was removed by centrifugal filtration at 13,000 rpm, each with Wash with phosphate buffer four times and 30 minutes. To the residual solution was added 15 μg of the thio-EPO prepared according to Example 5 (1 μg / μl in phosphate buffer), and the mixture was incubated at 25 ° C. for 16 hours. After freeze-drying, the crude product was analyzed by NuPAGE 10% double-triple gel / MOPS buffer (Avjet, Kasba, California, USA) as described in the notice given by Avitjet. ◎ The gel system was stained with Loti-Larkomas stain (Ross, Kaslu, D) overnight. The experimental results are shown in FIG. 3. Successful conjugation is represented by the shift of protein bands to higher molecular weights. The band-width increases due to the molecular weight distribution of the HES derivative used and the number of HES derivatives attached to the protein. Example 6.5: Sulfoxidized red jk globulin, Example 1 # 1 and the reaction economy of the reaction product of the compound Printed by the Ministry of Intellectual Property Bureau's Consumer Cooperative, adding 10 microliters of 2.5 nanomolar AMAS (Sigma O'Dridge Co., Inc.'s "Taofu Kecheng, D) in DMSO to 50 nanomolar according to The HES derivative prepared in Example 1.1 was incubated at 80 t and 17 hours (Example Ua) and incubation conditions of 25 ° C and three days (Example 1.1b) and dissolved in 200 μl of 01M sodium phosphate buffer. (0 1M, 9 15M Naa, 50 mM EDTA, pH 7.2). The transparent solution was incubated at 2Γ (: incubation for 80 minutes and incubation at 40 ° C for 20 minutes. The remaining AMAS was loaned with VIVASPIN 0.5 ml concentrator, 5KD MWCO (Viva Scientific Corporation, Hanover, D) -85- 200521143

AT B7 五、發明說明（84) ---- 過慮法以13,〇〇〇轉數/秒移除，各個用磷酸鹽缓衝液清 洗四次及3〇分鐘。 於殘留的溶液中加入15微克根據實例5(1微克/微升 於磷酸鹽緩衝液巾）所製得之硫代EpQ，且將混合物於^ C培育16小時。冷凍乾燥後，將粗產物用安維特吉公司 給予之通知中所說明的NuPAGE 1〇%雙_三凝膠/1^〇1&gt;§緩 衝液(安維特吉公司，卡斯巴，美國）藉SDS«page分析。凝 膠係用洛提-藍考瑪斯染劑（羅斯，卡斯盧，D)染色過夜。 實驗結果顯示於圖4中。成功的共輛作用係以蛋白質 帶移向較高的分子量來表示。帶-寬係由於所用之HES衍 生物分子量分佈及HES衍生物連接到蛋白質之數量而增 加0 實例6·6 :經氧化的紅血球生成素舆實例ι·3及交聯化合 物之反應產物的反應 經濟部智慧財產局員工消費合作社印製 將10微升2·5毫微莫耳AMAS(席格馬歐德里奇公 司，桃夫克成市，D)於DMSO中之溶液加至50毫微莫耳 根據實例1.3所製得的HES衍生物，於80°C及17小時(實 例1.3.a)以及25°C及三天(實例1.3b)之培育條件且溶解於 200微升，0.1M磷酸鈉緩衝液(OJM，9.15MNaC卜50mM EDTA，pH 7·2)中。將透明的溶液於25°C埼育80分鐘及 於40°C培育20分鐘。剩餘的AMAS用VIVASPIN 0·5毫 升濃縮器，5KD MWCO (萬歲科學公司，漢諾威，D)藉離 心過濾法以13,000轉數/秒移除，各個用磷酸鹽緩衝液清 洗四次及30分鐘。 -86- A7 B7 200521143 五、發明說明（85) 於殘留的溶液中加入15微克根據實例5(1微克/微升 於磷酸鹽緩衝液中）所製得之硫代EPO，且將混合物於25 °C培育16小時。冷凍乾燥後，將粗產物用安維特吉公司 給予之通知中所說明的NuPAGE 10%雙-三凝膠/MOPS緩 衝液(安維特吉公司，卡斯巴，美國）藉SDS-Page分析。凝 膠係用洛提-藍考瑪斯染劑(羅斯，卡斯盧，D)染色過夜。 實驗結果顯示於圖4中。成功的共輛作用係以蛋白質 帶移向較高的分子量來表示。帶-寬係由於所用之HES衍 生物分子量分佈及HES衍生物連接到蛋白質之數量而增 加。 實例7 HES-EPO軛合物之製備性生成 摘要 經濟部智慧財產局員工消费合作社印製 HES_EPO輛合物係藉HES衍生物（平均分子量為 18,000道爾頓；羥基乙基取代度為〇.4)偶合至重組體人類 EPO之寡醣鏈上經部份(弱過碘酸鹽）氧化的唾液酸殘基而 合成。以醣分子結構分析為基礎引入的改質不會影響核心 寡醣鏈之結構完整，因為經弱酸處理經HES-改質的多醣 之MALDI/TOF-MS顯露出完整的中性N-乙醯基乳糖胺-類 型鏈，其係不能從彼等未經改質之EPO產物所觀察者中 辨別出來。所獲得的結果指出於EPO製劑係在無需移除 部份涎酸之前進行改質之情況中，每EPO分子附著至少3 個經改質的HES-殘基。於SDS-PAGE(60-110Kda對 40Kda用於BRP EPO標準）中缺乏大約50%前者蛋白質之 唾液酸殘基的EPO變異體顯示出類似的表觀高分子量移 -87- 200521143 A7 B7 五、發明說明（86) 動性。經HES改質的EPO於室溫，pH 3_1〇時於標準離子 交換色層分離條件下係穩定的。 於血球正常狀態(normocythaemic)老鼠系統中之EPO-生物鑑定中指出於談分析中，當經HES-改質的EPO與以 蛋白質測定為基礎，用來自歐洲藥典之吸收值及Rp-HpLC EPO測定法經Brp EPO標準製備比對校正之國際BRP EPO參考標準相比較時，其特定活性高出2.5-3.0倍。 實例7·1原料舆方法 (a) Ν-鍵結的寡醣類藉Ν_配醣酶消化之游離 經濟部智慧財產局員工消費合作社印製 將樣品與25單位(根據德國羅氏診斷法之製造商說明 書）重組體PNG酶F於37°C培育過夜。完全消化係藉蛋白 質於SDS-PAGE中特定的移動位移來監測。釋放的％多 醣係從多胜肽中藉著添加3倍體積冷100〇/〇乙醇而分離出 來且培育於-20°C至少2小時（史洛特s等，1999)。沉澱的 蛋白質係於4°C以13000轉數/秒離心分鐘而移除。然 後將小丸用500微升冰冷的75%乙醇再清洗二次。將含寡 醣類之集中上澄液於真空分離中乾燥（快速真空濃縮器， 薩凡儀器公司，美國）。多醣樣品係用海霸卡(Hypercarb)匣 (25毫克或100毫克海霸卡)於使用前依照下列除去鹽分： 管柱係用3x5〇0毫微升8〇〇/0乙腈(v/v)於含〇 之水 清洗，接著用3x500毫微升水清洗。將樣品裝載於經水劇 烈清洗之匣上之刖，用水稀釋至最終體積為3〇〇微升·6〇〇 微升。寡醣類係用1·2毫升(25毫克匣；於1〇〇毫克匣之 情況中用1·8毫升）25%6腈於含〇.1〇/0三氟醋_/ν)之水 -88- 200521143 A7 B7 五、發明說明（87) 稀釋。將稀釋的寡醣用2MNH4OH中和且於快速真空濃縮 器中乾燥。於某些情況中，N-配醣酶釋放的寡醣係藉著將 消化混合物從總(醣)蛋白質&lt;100微克之樣品吸附到1〇〇毫 克海霸卡匣上而去鹽。 (b)藉由介質辅助雷射脫附/離子化時間飛行式質譜儀 (MALDI/TOF/IOF-MS)之寡醣分析 使用布魯克ULTRAFLEX時間飛行式(TOF/TOF)儀 器：天然的二涎酸化寡醣類係用2,5_二羥基苯甲酸作為正 性以及負性離子模式中之UV_吸收物質，於兩者情況中使 用反射而分析。於MS-MS時，將選擇的母離子進行由雷 射所誘導的解離(LID)且將產生的斷片離子藉第二TOF階 (LIFT)儀器分離出來。將1微升及大約1-1〇 pmol.er1濃 度之樣品溶液與等量分別的基質混合。將該混合物點在不 銹鋼目標上且分析前於室溫下乾燥。 經濟部智慧財產局員工消費合作社印製 實例7·2 重组艟人類EPO(EPO-GT-l)之製備舆特徵 EPO係從重組體CHO細胞表現如（莫勒PP等， 1999，多納AJ等，1984)所說明者且製備具有如歐洲藥典 (PH· Eur. 4，專題論文01/2002 : 1316 :紅血球生成素濃縮 的溶液）中所述方法之特性。最終產物具有每毫莫耳蛋白 質12毫莫耳(±1.5毫莫耳)之唾液酸含量。N-鍵結的寡醣 結構係藉由HPAEC-PAD及藉由MALDI/TOF-MS如(尼姆 茲等，1999，葛拉本賀斯特，1999)所說明者來測定。所 獲得的EPO製劑含有二-，三-及四涎酸化的寡醣類（分別 為2-12%，15-28%及60-80%，亦存有少量硫酸鹽化與五 •89- A7 B7 200521143 五、發明說明（88 ) 延酸化的鏈）。EPO製劑之整體醣化作用特性類似於國際 BRPEPO標準製備者。 重組體EPO之等電聚焦類型係相當於國際BRP參考 EPO標準製備者，其顯示出相關的異構重整體(isoform)。 25%EPO蛋白質於多胜肽鏈之Ser〗26上缺乏〇-醣化作用。 實例7.3 部份二涎酸化EPQ型式之製備 將EPO GT_1蛋白質(2.84毫克/毫升)於20mM磷酸鈉 緩衝液ρΗ7·0中之加熱至80°C且然後每1毫升EPO溶液 加入100微升IN H2S04 ;分別培育持績5分鐘，10分鐘 及60分鐘，產生不同涎酸化作用程度之EPO製劑。寡醣 以0-4唾液酸之定量係在以多胜肽N-配醣酶游離寡餹後進 行且N-鍵結鏈之單離係使用海霸卡匣(25毫克Heper Sep Hypercarb ; ThermoHypersil-Keystone，英國）藉去鹽而進 行。將EPO製劑加入IN NaOH中和且冷;束於液態N2中 且貯存於-20°C直到進一步使用。 實例7·4 涎酸化EPO型式之過碘酸鹽氧化作用 經濟部智慧財產局員工消費合作社印製 將1.5毫升0.1M醋酸鈉緩衝液pH 5.5加至10毫克未 處理或經弱酸處理之EPO溶解於3·5毫升20mM雄酸納 pH 7.0緩衝液中且將混合物於冰浴中冷卻至；加入 500微升lOmM過碘酸鈉且將反應混合物保持在0°c黑暗 中60分鐘。然後將1〇微升甘油加入且繼續於黑暗中再培 育10分鐘。部份氧化之EPO型式係從試劑中藉著使用 VWASPIN 濃縮器（1〇,〇〇〇MWCO，PES Vivascience AG， 漢諾威，德國），根據製造商建議以3〇〇〇轉數/秒於配備固 •90- 200521143 A7 ____________ B7 五、發明說明（89) 定角轉片之實驗室離心器中去鹽而分離出來。於液態氮中 冷凍後，將EPO製劑貯存於_2〇°C4毫升之最終體積中ό 將等份100微克經部份氧化之ΕΡΟ製劑進行N-配醣 酶處理且用如前所述之海霸卡匣將募醣單離出來。寡醣係 藉弱酸處理而二诞酸化且用HPAEC-PAD分析且將其等之 滯留時間與如所述(尼姆茲等，Β90及1993)可靠的標準寡 醣者相比較。 實例7·5 ΕΡΟ二碴化物以二硫代艾里瑞妥還原 將5毫克EPO-GT-1培育於5亳升0.1Μ Tris/HCl緩衝 液 ρΗ8·1 中，於 30mlV[二硫代艾里瑞妥(dithioerythreitol) (DTT)存在之下於37°C時培育60分鐘；DTT係藉著使用 Vivaspm濃縮器於4°C時4次緩衝液交換循環而完成移 除。最終還原的ΕΡΘ製劑係於液態氮中冷凍且貯存於_2〇 °0 5〇1111\/1醋酸鈉緩衝液？115.5中。 實例7·6 EPO蛋白質測定 經濟部智慧財產局員工消費合作社印製 ΕΡ0蛋白質之定量測定係根據歐洲藥典(歐洲藥典4， 專論01/2002 : 1316 :紅血球生成素濃縮的溶液)於具有j 公分徑長之比色器中藉著測量280毫微米時之UV吸收而 進行。此外，EP0係藉著應用RP-HPLC法使用RP-C4管 柱（維達克蛋白質C4，貨號·# 214TP5410，葛蕾絲維達克 公司，加州，美國）而定量；HPLC法係用紅血球生成素 BRP1參考標準(歐洲藥典，歐洲會議B p 9〇7-F67〇29，史 達司堡郵遞區號1)校正。 實例7.7二涎酸化的EPO以半乳糖氡化酶氧化 -91- 200521143 Α7 Β7 五、發明說明（90) 將4·485毫克完全二延酸化的EPO於20mM填酸納缓 衝液中pH 6.8，於16微升催化酶(6214單位/200毫升)及 80微升半乳糖氧化酶(2250單位/毫升，來自達提里丹多 (席格馬歐德里奇公司’祧夫克成市，D)存在之下培育； 於37°C培育期過夜；培育起始4小時及8小時後加入2次 20微升半乳糖氧化酶。 實例7.8製儀生物鑣定之EPO樣品 EPO從碘酸鹽或經半乳糖_氧化酶-氧化之EPO蛋白質 製劑與活性HE S培育中之純化 訂 經濟部智慧財產局員工消費合作社印製 EPO樣品之純化(移除未反應之HES衍生物)係在室溫 下進行。將EPO培育混合物(大約5毫克EPO蛋白質）以 1 : 1〇緩衝液A(20mM N-嗎福啉丙烷磺酸[MOPS/NaOH]於 HbO水浴中，pH8.0)稀釋且施用於含3毫升Q-瓊脂糖凝膠 HP(製藥編號Π-1014-03，批號220211)以10管柱體積 (CV)緩衝液A平衡，流速為0·5毫升/分鐘之管柱。將管 柱用6_8 CV緩衝液Α (流速=0·8毫升/分鐘)清洗且用緩衝 液Β (20毫莫耳嗎福啉乙烷磺酸[MES/NaOH]，0.5Μ NaCl 於H20水浴中，Ph6.5)以0.5毫升/分鐘流速進行洗提。 EPO係藉280毫微米時之UV吸收來檢測且於大約6毫 升中洗提。藉著使用3CV緩衝液C (流速=0.8毫升/分鐘） 清洗且用緩衝液B (20毫莫耳MES，1.5M NaCl於H20水 浴中調節到Ph6.5)將管柱再處理且再用10CV緩衝液A (流速=0·7毫升/分鐘)再-平衡。 從Q-瓊脂糖凝膠步驟獲得之ΕΡΟ洗出液之緩衝交換 -92- A7 B7 200521143 五、發明說明（91) 係用Vivaspin濃縮器進行且每個樣品以鱗酸鹽緩衝食鹽水 (PBS)離心循環3次；以PBS將樣品調節到2毫升且於-20 °(：下貯存。 將得自於Q-瓊脂糖凝膠洗提經部份二涎酸化且隨即經 弱過碘酸鹽氧化之EPO型式進行HES-改變者僅有25%， 因為在使用鹼性EPO型式之條件下不會結合Q-瓊脂糖凝 膠且與未反應之HES衍生物在浸流中一起被發現。 實例7·9具脉衝性電流檢測之高-pH陰難子·交換色層分 離法(HPAEC-PAD&gt; 經濟部智慧財產局員工消費合作社印製 鲽純化之天然與二涎酸化的寡醣係藉由高-pH陰離子-交換（HPAE)色層分離法使用配備CarboPac PA1管柱(0.4 X 25公分)之Dionex BioLC系統(迪歐尼公司，美國）與脉 衝性電流檢測器(PAD)(舒洛特等，1999 ;尼姆茲等，1999) 合併而分析。檢測器電位(E)及脉衝期間（T)為：El : +50 mV，T1 : 480 ms ; E2 : +500 mV，T2 : 120 ms ; E3 : -500 mV，T3 : 60 ms，且輸出範圍為500-1500 nA。然後將寡 醣注射在以100%溶劑平衡之CarboPac PA1管柱上面。二 誕酸化寡酷洗提(流速：1毫升。分鐘-1)係應用溶劑B之線 性梯度(0-20%)於40分鐘期間接著以自20-100%溶劑B於 5分鐘之線性增加而進行。溶劑A係為0.2M NaOH於二 蒸餾的H20中，溶劑B包括0.6M NaOAc於溶劑A中。 於天然的寡醣類時，管柱係以100%溶劑C(0.1M NaOH於 二蒸餾的H20中）平衡且洗提(流速：1毫升。分鐘-1)係應 用溶劑D之線性梯度(0-35%)於48分鐘期間接著以自35- -93- 200521143 A7 B7 五、發明說明（92) 100%溶劑D於10分鐘之線性增加而進行。溶鋼c包括 0.6 M NaAc於溶劑C中。 實例7·10 N-多醣，經HES-改質的N·多醣舆Ep〇蛋白 質藉由GC-MS之單醣组成物分析 單醣係於甲醇醇解，N-再乙醯化作用及三甲基甲梦燒 化作用後藉GC/MS分析為相關的甲基配醣體[查普林， MR(1982)醣分子分析之快速且敏銳的方法。生物化學年 鑑123，336-341]。該分析係在芬尼根GCQ離子陷位質譜 儀（ion trap mass spectrometer)(芬尼根 MAT 公司，聖荷 西，加州）上於配備30米DB5毛細管柱之正性離子m模 式中運轉而進行。溫度程式：於80°C等溫2分鐘，然後每 分鐘10度至300°C。 單醣係藉其等之滞留時間及斷片圖樣特徵而被確認。 電子尖峰累計之未校正結果係用於定量。由於殊碳糖及/ 或存有呋喃糖素(furanoid)及吡喃糖素(pyran〇id)型式，產 量生超過一個尖峰之單醣係將所有主要的尖峰加起來而定 量。〇·5微克之肌-肌醇係用作為内標準化合物。 經濟部智慧財產局員工消費合作社印製 實例7.11結果 實例7.11(a)經弱酸處理之（部份二涎酸化)Ep〇_GT_i之 N-多酶的特徵 經5，10或60分鐘弱酸處理之EP0-GT-1製劑係藉 SDS-PAGE於游離沁鍵結之寡醣前或後藉著與N_配醣酶 培育而進行分析，如圖5中所示者。將鍵結之寡醣進 行HPAEC_PAD寡醣繪圖（圖6)。未處理之EP0-GT-1包含 -94- 200521143 A7 B7 五、發明說明（93) 90%含有3或4個唾液酸殘基之N-鍵結的寡醣，然而於 弱鰊存在之下培育5分鐘後，&lt;40%之醣分子鏈具有3或4 個唾液酸殘基。經二涎酸化N-多醣之HPAEC-PAD顯露出 用於檢測未處理EPO-GT-1之中性寡醣的比例且於進行 5，10或60分鐘弱酸處理之製劑中保持穩定。二涎酸化多 醣之MALDI/TOF-MS顯露出於蛋白質弱酸處理後存有 &lt;90%近侧的岩藻糖。 實例7.11(b)經過碘酸鹽處理之EPO-GT-1的特徵 前項進行5及10分鐘酸處理或未經處理之經弱過蛾 酸鹽處理EPO型式之SDS-PAGE移動性係於圖8中比 較。用於涎酸之過破酸鹽氧化條件並未改變EPO製劑之 SDS-PAGE圖樣（比較圖5)。唾液酸之氧化作用導致寡酶 於HPAEC-PAD分析中對較早的洗提時間之位移（比較圖6 及9) 〇 實例7，ll(c)經HES改質的EPO衍生物的特徵 (aa) EPO-GT-1-A舆根據實例2.4所產生的經羥基胺改質 的HES衍生物X之HES改質之時間行裎 經濟部智慧財產局員工消費合作社印數 將400微克經羥基胺改質的HES衍生物χ加至2〇微 克EPO-GT-l_A(溫和過碘酸鹽氧化的Ep〇，於溫和過碘酸 鹽氧化之前非酸水解)於20微升，〇·5Μ NaAc ΡΗ5·5緩衝 液中，反應分別於30分鐘，2 , 4及17小時後藉著將樣品 冷凍於液態氮中而停止。隨即將樣品於_2〇t下貯存直仃 進一步分析。 將SDS-PAGE樣品緩衝液加入且將樣品加熱至9〇艽 -95- 200521143 A? _—__________ B7 五、發明說明（94) 且施用於SDS-凝膠上。如圖1〇中所示，增加的培育時間 導致向較高分子量蛋白質之位移增加。於經羥基胺-改質 的HES衍生物X存在之下培育17小時後，檢測出浸潤性 經考瑪斯染色的蛋白質帶於6〇與u KDa間之區域中移 行，以分子量標準之位置為基礎(參閱圖1〇之左邊）。當 以N-配醣體處理時多數蛋白質係向經去醣化作用的 EPO位置位移(參閱圖1〇 ,右膠；箭頭a係指n-配醣酶之 移行位堇，箭頭B係指N-醣化EPO之移行位置；於假設 代表經HES及分子之〇-醣化作用部位改質之Ερ〇-型式 28 KDa與36 KDa莫耳重舉標準間之區域中可見浸潤蛋 白質帶。鑒於N-配醣酶之特性，吾人從該結果得到結 論：事實上改質係在EPO蛋白質之多醣之經過碘酸鹽氧 化的唾液酸殘基上發生。 (bb) HES-EPO耗合物之特徵 經濟部智慧財產局員工消費合作杜印製 將HES-EPO輛合物I (源自於弱過碘酸鹽氧化後之 EPO-GT]，亦即，來自於EPO-GT-1-A)，11(源自於進行5 分鐘酸水解及弱過碘酸鹽氧化作用之EPO-GT-1)，111(源 自於進行10分鐘酸水解及弱過碘酸鹽氧化作用之EPO_ GT-1)如前說明合成◊控制培育(K)包括在相同緩衝條件 下，相等量未改質的HES係加至含未改質的EPO-GT-1 中。將培育混合物進行進一步純化用於後績之HES-EPO 衍生物之生物化學分析。 為了移除在離子交換管枉浸流中所預期之過量未反應 的HES-試劑，將培育HES-EPO軛合物I，Π及III以及控 -96y 200521143 A7 B7 五、發明說明 制培月K進行如”原料與方法，，(實例7 8)下所說明之Q-瓊 脂糖凝膠、iMb㈣。由於高㈣性Ep〇型从含於前項 經酸處理之樣品η及m中，吾人期望於浸流中有相當量 來自此等培育之經改質的EPO產物。如圖u中所示，幾 乎所有來自樣品I之EP〇物質被Q_瓊脂糖凝膠管柱留 住，然而，僅約20-30%之樣品m及π於高鹽濃度洗提之 餾份中被重新找到。來自培育之所有的蛋白質物質與HES 衍生物X二者於浸流及高鹽洗提之镏份中，當與控制組 EPO比較時具有明顯高於於SDS-PAGE中之分子量。 經濟部智慧財產局員工消費合作社印製 為了記述更詳細的特徵，經HES-改質的EPO樣品A 及K(參閱圖η)係與經過蛾酸鹽氧化的型式EPO-GT-1-A 比較。將樣品進行Ν-配醣酶處理且表述於圖i2a及12b 中，N-多醣之釋放導致在標準Epo製劑之〇_醣化及之非 醣化的EPO型式的位置上具二低分子量帶。於樣品a之 情況中，另外的帶移行在28KI&gt;a mw標準位置上被檢測 出來而主張在該EPO變異體（參閱實例7.11(c )(aa))之 HES-改質。該帶（以及Ν·醣化的EPO之大量HES-改質的 高分子量型式，參閱圖12a及12b)於樣品進行弱水解後消 失，其係與HES改質係在紅血球生成素之經過碘酸鹽氧 化的唾液酸殘基上達成之觀點一致。 各份N-配醣酶培育混合物係用能從募醣類中完全移除 唾液酸殘基(以及涎酸鍵結的HES衍生物）之條件水解；中 和後，然後將混合物吸附在小海霸卡管上以利其等去鹽。 將管柱用水劇烈清洗接著用4〇%乙腈於含01°/〇三氟醋酸 -97- A7 B7 200521143 五、發明說明（96) 之H2〇結合中性寡醣洗提。將產生的寡_進行 MALDI/TOF-MS。得自於樣品A之二延酸化募傭斷片、 EPO-GT-1-A與樣品K之光譜顯示絡合物型募醣於m/z= 1810 Da (二突觸），2175=三突觸，2540=四突觸，2906=四 突觸加上1 N-乙醯基乳糖胺重複及3271=四突觸加上2N-乙醯基乳糖胺重複具相同的質譜；相關於缺少岩藻糖(-146) 與半乳糖(-162)之小信號被檢測出來，其可歸因於施用於 涎酸移除之酸水解條件(參閱MALDI-圖15，16及Π)。 經濟部智慧財產局員工消費合作社印製 於平行實驗中，將N-配醣酶消化混合物吸附至1毫升 RP-C18匣上（不超先前之寡醣的酸水解)且用5%乙腈於含 0.1%TFA之H20洗提於此等條件下，EPO蛋白質完全留在 RP-物質上且用5%乙腈於含0.1%TFA之H20將寡醣從管 柱中被清洗出來。經去醣化的EPO蛋白質係用70%乙 腈於含0.1%TFA之H20洗提。來自於經N-配醣酶處理之 樣品A，EPO GT-1-A及樣品K之RP-C18步驟之寡醣斷 片中和且用如前說明之海霸卡匣去鹽。將單離的寡醣於弱 酸處理前（參閱圖13)及後，於能使涎酸從多醣中定量移除 之條件下(參閱圖14)進行HPAEC-PAD繪圖。 從HES-改質的樣品A中所獲得的天然物質之 HPAEC-PAD態樣顧示出募醣僅有可忽略量之信號，而 EPO GT-1-A所衍生的寡醣具有相同的多醣態樣，如圖9 中所示者（樣品於弱酸處理後稱為EPO-GT·1)。從控制組 EPO樣品(K)所獲得之寡醣的洗提態樣產生預期的圖樣（比 較圖6中之態樣）。為比較時，將國際BRP-EPO標準之天 -98- 200521143 A7 B7 五、發明說明（97) 然寡醣態樣用於比較且作為參考標準。 弱酸水解後’所有的寡聽製劑顯示出具有如用本研究 中作為起動物質之EPO製劑的方法章節中所說明之二·， 三-及四突觸絡合物-型醣分子鏈之預期品質與定量組成物 之相同中性募醣結構之洗提態樣(參閱圖14)。該結果證實 EPO樣品之HES-改質會導致HES衍生物之共價鍵合，其 係藉N-配醣酶從EPO蛋白質中分離且係為酸-不穩定的， 因為其係用已知的弱酸處理條件從N-多醣移除以二涎酸 化醣分子(參閲圖12a+b)。 (cc) HES-EPO及HES-EPO N-多醣藉GC-MS之單醣組成 物分析 為了進一步確認EPO於莫耳之N-多醣之HES_改質， 將EPO樣品用N-配醣酶消化且將EPO蛋白質吸附到RP-c18匣上，而將寡醣物質如前說明者清洗出來。如表2中 所示者，葡萄糖及羥基乙基化的葡萄糖衍生物僅於EPO 蛋白質，其係在半胱胺酸進行HES-改質且於EPO樣品A2 之募醣斷片中檢測出來。 經濟部智慧財產局員工消費合作社印製 實例7.11(d)經HES改質之EPO的生物活性之生髏内分 析 於血球正常狀態（normocythaemic)老鼠系統指示中之 EP〇_生物鑑定係根據歐洲藥典中所說明之步驟進行；EPO 分析之實驗係用國際BRP EPO參考標準製備進行。於製 備經HES-改質的EPO A2時，所測定蛋白質之特定活性之 平均值為每毫克EPO 294,600單位，當與供活性分析用之 -99- A7 B7 200521143 五、發明說明（98 ) 包含於樣品中之國際BRP EPO參考標準製備之特定活性 相比較時，高出大約為3-倍。 研究結果概述於表3中。 經濟部智慧財產局員工消費合作社印製 -100 200521143 A7 B7 五、發明說明（99) 參考文獻： 尼姆茲，諾爾巴克斯EP，康拉德HS。 人類組織纖維蛋白溶酶原活化劑表現於重組體中國倉鼠卵 巢細胞中之醣分子結構 FEBS 手札，1990，10 月 1 日；271(1-2) ·· 14-8 多納AJ，華斯里LC，考夫曼RJ. 經丁酸酯處理之中國倉鼠卵巢細胞中經調節葡萄糖蛋白質 之隱密性蛋白質誘發表現增加的合成法 生物化學期刊1989年12月5日；264(34) : 20602-7 莫勒PP，史蘭克P，尼姆茲M，康拉德HS，豪瑟Η 增生-控制之ΒΗΚ-21細胞中之重組體醣蛋白質品質 生物技術生物工程1999年12月5日；65(5) : 529-36AT B7 V. Description of the invention (84) ---- The cathodic method was removed at 13,000 rpm, and each was washed four times with phosphate buffer and 30 minutes. To the residual solution was added 15 μg of the thio-EpQ prepared according to Example 5 (1 μg / μl in a phosphate buffered towel), and the mixture was incubated at ^ C for 16 hours. After freeze-drying, the crude product was borrowed with NuPAGE 10% Bi-Trigel / 1 ^ 〇1 &gt; § buffer solution (Annavite, Kasba, USA) as described in the notice given by Avitej. SDS «page analysis. The gel system was stained with Loti-Larkomas stain (Ross, Kaslow, D) overnight. The experimental results are shown in FIG. 4. Successful co-transportation is represented by the shift of protein bands to higher molecular weights. The band-width is increased due to the molecular weight distribution of the HES derivative used and the number of HES derivatives attached to the protein. Example 6.6: Oxidized erythropoietin Example ι · 3 and the reaction product of the reaction product of a cross-linked compound Printed by the Ministry of Intellectual Property Bureau's Consumer Cooperative, adding 10 microliters of 2.5 nanomolar AMAS (Sigma Oedrich, Taofucheng, D) to 50 nanomolar based on DMSO The HES derivative obtained in Example 1.3 was incubated at 80 ° C and 17 hours (Example 1.3.a) and at 25 ° C and three days (Example 1.3b). It was dissolved in 200 μl of 0.1M sodium phosphate buffer. (OJM, 9.15 M NaC, 50 mM EDTA, pH 7.2). The clear solution was incubated for 80 minutes at 25 ° C and 20 minutes at 40 ° C. The remaining AMAS was removed with a VIVASPIN 0.5 ml concentrator, 5KD MWCO (Viva Scientific, Hanover, D) by centrifugal filtration at 13,000 rpm, and each was washed four times with phosphate buffer and 30 minutes. -86- A7 B7 200521143 V. Description of the invention (85) 15 micrograms of thio-EPO prepared according to Example 5 (1 microgram / microliter in phosphate buffer solution) was added to the residual solution, and the mixture was added at 25 ° C for 16 hours. After lyophilization, the crude product was analyzed by SDS-Page using NuPAGE 10% Bi-Trigel / MOPS buffer (Aventji, Caspar, USA) as described in the notice given by Aventji. Gel systems were stained overnight with Loti-Lancoomas stain (Ross, Kaslow, D). The experimental results are shown in FIG. 4. Successful co-transportation is represented by the shift of protein bands to higher molecular weights. Band-width increases due to the molecular weight distribution of the HES derivative used and the number of HES derivatives attached to the protein. Example 7 Summary of the Preparative Production of HES-EPO Conjugate The HES_EPO printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs is a HES derivative (average molecular weight is 18,000 Daltons; hydroxyethyl substitution degree is 0.4 ) Synthesized by partial (weak periodate) oxidation of sialic acid residues coupled to the oligosaccharide chain of recombinant human EPO. Modifications introduced on the basis of sugar molecular structure analysis will not affect the structural integrity of the core oligosaccharide chain, as MALDI / TOF-MS of HES-modified polysaccharides treated with weak acid reveals intact neutral N-acetamyl groups Lactosamine-type chain, which is indistinguishable from observers of their unmodified EPO products. The results obtained indicate that in the case where the EPO preparation was modified without removing a part of the sialic acid, at least 3 modified HES-residues were attached per EPO molecule. EPO variants lacking sialic acid residues of approximately 50% of the former protein in SDS-PAGE (60-110Kda vs. 40Kda for BRP EPO standards) show similar apparent high molecular weight shifts -87- 200521143 A7 B7 V. Invention Note (86) Mobility. EPO modified by HES is stable at room temperature and pH 3-10 under standard ion exchange chromatography separation conditions. It was pointed out in the EPO-biological identification in normocythaemic mouse system that in the talk analysis, when HES-modified EPO and protein-based measurement were used, the absorption value from the European Pharmacopoeia and Rp-HpLC EPO measurement were used. When compared with the BRP EPO standard prepared by the Brp EPO standard comparison method, its specific activity is 2.5-3.0 times higher. Example 7.1 Method of raw materials (a) N-linked oligosaccharides were digested by N-glycosidase and released by the Consumers ’Cooperative of the Intellectual Property Bureau of the Ministry of Economics. Samples and 25 units (manufactured according to the German Roche diagnostic method) Commercial instructions) Recombinant PNGase F was incubated at 37 ° C overnight. Complete digestion is monitored by specific movement of proteins on SDS-PAGE. The released% polysaccharides were isolated from the polypeptides by adding 3 times the volume of cold 100/0 ethanol and incubated at -20 ° C for at least 2 hours (Schlott et al., 1999). The precipitated protein was removed by centrifugation at 13,000 rpm for 1 minute at 4 ° C. The pellets were then washed twice with 500 µl of ice-cold 75% ethanol. The concentrated supernatant containing oligosaccharides was dried in a vacuum separation (rapid vacuum concentrator, Savant Instruments, USA). Polysaccharide samples were prepared using a Hypercarb cartridge (25 mg or 100 mg), prior to use, to remove salts as follows: 3 x 5000 nanoliters of 800/0 acetonitrile (v / v) for the column system Rinse in water containing 0, followed by 3x500 nanoliters of water. The sample was loaded on a water-washed box and diluted with water to a final volume of 300 µl · 600 µl. For oligosaccharides, use 1.2 ml (25 mg box; 1.8 ml in the case of 100 mg box) 25% 6 nitrile in water containing 0.10 / 0 trifluoroacetic acid. -88- 200521143 A7 B7 V. Description of the invention (87) Dilution. The diluted oligosaccharide was neutralized with 2MNH4OH and dried in a flash vacuum concentrator. In some cases, oligosaccharides released by N-glycosidase were desalted by adsorbing the digestion mixture from a total (glyco) protein &lt; 100 microgram sample onto a 100 milligram Seamaster cartridge. (b) Oligosaccharide analysis by medium-assisted laser desorption / ionization time-of-flight mass spectrometer (MALDI / TOF / IOF-MS) using Bruker's ULTRAFLEX time-of-flight (TOF / TOF) instrument: natural sialylation Oligosaccharides use 2,5-dihydroxybenzoic acid as a UV-absorbing substance in positive and negative ion modes, and in both cases are analyzed using reflection. In MS-MS, the selected parent ion is subjected to laser-induced dissociation (LID) and the fragment fragments generated are separated by a second TOF stage (LIFT) instrument. Mix 1 microliter and a sample solution of about 1-10 pmol.er1 with equal amounts of each matrix. The mixture was spotted on a stainless steel target and dried at room temperature before analysis. Printed by the Consumer Property Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 7.2 Recombination 制备 Preparation of human EPO (EPO-GT-1) Characteristics EPO is expressed from recombinant CHO cells such as (Mole PP et al. 1999, Dona AJ et al. , 1984) and the preparation has the characteristics as described in the European Pharmacopoeia (PH · Eur. 4, monograph 01/2002: 1316: concentrated solution of erythropoietin). The final product had a sialic acid content of 12 millimoles (± 1.5 millimoles) per millimolar protein. The N-bonded oligosaccharide structure was determined by HPAEC-PAD and by MALDI / TOF-MS as described (Nimz et al., 1999, Grabenhurst, 1999). The obtained EPO preparation contains di-, tri-, and tetrasialylated oligosaccharides (2-12%, 15-28%, and 60-80%, respectively), and there is also a small amount of sulfated and 5 • 89- A7 B7 200521143 V. Description of the invention (88) Ducrated chain). The overall saccharification characteristics of the EPO preparation are similar to those of the international BRPEPO standard maker. The isoelectric focusing type of the recombinant EPO is equivalent to the preparation of the international BRP reference EPO standard, which shows the related isoform. 25% EPO protein lacks O-glycation on Ser 26 of the polypeptide chain. Example 7.3 Preparation of Partially Salicylated EPQ Forms Heat EPO GT_1 protein (2.84 mg / ml) in 20 mM sodium phosphate buffer pH 7 · 0 to 80 ° C and then add 100 μl IN H2S04 per 1 ml of EPO solution ; Cultivate EPO preparations with different salivation levels for 5 minutes, 10 minutes and 60 minutes, respectively. The oligosaccharides were quantified with 0-4 sialic acid after free oligosaccharides with polypeptide N-glycosidase and the N-linked chains were isolated using a Seamaster cassette (25 mg Heper Sep Hypercarb; ThermoHypersil- (Keystone, UK) by borrowing salt. The EPO formulation was neutralized and cold with IN NaOH; bundled in liquid N2 and stored at -20 ° C until further use. Example 7.4 Periodic acid oxidation of sialic acid EPO type Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 1.5 ml of 0.1M sodium acetate buffer pH 5.5 was added to 10 mg of untreated or weakly acid-treated EPO dissolved in 3.5 ml of 20 mM sodium androsate buffer pH 7.0 and the mixture was cooled to in an ice bath; 500 μl of 10 mM sodium periodate was added and the reaction mixture was kept at 0 ° C. in the dark for 60 minutes. 10 microliters of glycerol was then added and the incubation was continued for another 10 minutes in the dark. Partially oxidized EPO types were obtained from the reagents by using a VWASPIN concentrator (10,000 MWCO, PES Vivascience AG, Hanover, Germany) according to the manufacturer's recommendations at a speed of 3,000 rpm in the equipment. • 90- 200521143 A7 ____________ B7 V. Description of the invention (89) Desalted and separated in laboratory centrifuge with fixed angle rotor. After freezing in liquid nitrogen, the EPO preparation was stored in a final volume of 4 ml at -20 ° C. An aliquot of 100 micrograms of the partially oxidized EPO preparation was N-glycosylated and treated with the sea as previously described. The Pa card box separates the sugar collection. Oligosaccharides were acidified by weak acid treatment and analyzed by HPAEC-PAD and their retention times were compared with standard oligosaccharides that were reliable as described (Nimz et al., B90 and 1993). Example 7.5 Reduction of EpPO dihalide with dithioiririto 5 mg of EPO-GT-1 was incubated in 5 μl of 0.1M Tris / HCl buffer ρΗ8.1, in 30 ml of V [dithioairyl Incubate for 60 minutes at 37 ° C in the presence of dithioerythreitol (DTT); DTT was removed by 4 buffer exchange cycles at 4 ° C using a Vivaspm concentrator. The final reduced EpPΘ formulation is frozen in liquid nitrogen and stored in _2 ° 0 501111 \ / 1 sodium acetate buffer? 115.5. Example 7.6 EPO protein determination The quantitative determination of EPO protein printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs was based on the European Pharmacopoeia (European Pharmacopoeia 4, Monograph 01/2002: 1316: Erythropoietin-concentrated solution) in j cm. The diameter-length colorimeter was performed by measuring UV absorption at 280 nm. In addition, EP0 was quantified by RP-HPLC using an RP-C4 column (Vidak Protein C4, Cat ## 214TP5410, Grace Vidak, California, USA); HPLC was used for erythropoietin The BRP1 reference standard (European Pharmacopoeia, European Conference B p 107-F67〇29, Staples postal code 1) was corrected. Example 7.7 Disialylated EPO is oxidized with galactosylase-91- 200521143 A7 B7 V. Description of the invention (90) 4.485 mg of fully di-extended acidified EPO was dissolved in 20 mM sodium acid buffer pH 6.8. 16 microliters of catalytic enzymes (6214 units / 200 ml) and 80 microliters of galactose oxidase (2250 units / ml) from Datiri Dando (Sigma Oedrich Corporation's Fifth City, D) Incubate at 37 ° C overnight; add 20 microliters of galactose oxidase 2 times after the initial 4 hours and 8 hours of incubation. Example 7.8 EPO sample of biodegraded EPO from iodate or galactose_ Oxidase-oxidized EPO protein preparation and purification during the cultivation of active HE S. Purification (removal of unreacted HES derivatives) of EPO samples printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs was performed at room temperature. EPO The incubation mixture (approximately 5 mg of EPO protein) was diluted with 1: 1 buffer A (20 mM N-morpholine propanesulfonic acid [MOPS / NaOH] in a HbO water bath, pH 8.0) and applied to 3 ml of Q- Agarose gel HP (pharmaceutical number Π-1014-03, lot number 220211) was used at 10 column volumes (CV) in buffer A The column with a flow rate of 0.5 ml / min. The column was washed with 6_8 CV buffer A (flow rate = 0.8 ml / min) and buffer B (20 millimolar morpholine ethanesulfonic acid) [MES / NaOH], 0.5M NaCl in H20 water bath, Ph6.5) was eluted at a flow rate of 0.5 ml / min. EPO was detected by UV absorption at 280 nm and eluted in approximately 6 ml. Borrow Wash the column with 3CV Buffer C (flow rate = 0.8 ml / min) and use Buffer B (20 millimoles MES, 1.5M NaCl in H20 water bath to adjust to Ph6.5) and reprocess the column with 10CV buffer Solution A (flow rate = 0.7 ml / min) was re-equilibrated. Buffer exchange of the EPO eluate obtained from the Q-sepharose step-92- A7 B7 200521143 V. Description of the invention (91) Concentrated with Vivaspin The centrifuge was performed and each sample was centrifuged 3 times with phosphonate-buffered saline (PBS); the sample was adjusted to 2 ml with PBS and stored at -20 ° (:). Washed from Q-Agarose gel washes Only 25% of the di-sialic acid extracts and HES-changes of the EPO type that were subsequently oxidized by weak periodate were used, because Q-Jon would not be combined under the condition of using the basic EPO type. The liposugar gel was found in the immersion stream together with the unreacted HES derivative. Example 7.9 High-pH cationic with pulsed current detection · Exchange chromatography separation method (HPAEC-PAD &gt; Ministry of Economic Affairs Printed by the Intellectual Property Bureau's Consumer Cooperative, purified natural and disialylated oligosaccharides were purified by high-pH anion-exchange (HPAE) chromatography using Dionex BioLC equipped with CarboPac PA1 column (0.4 X 25 cm). The system (Dionys Corporation, USA) was analyzed by merging with a pulsed current detector (PAD) (Schulot et al., 1999; Nimitz et al., 1999). Detector potential (E) and pulse period (T): El: +50 mV, T1: 480 ms; E2: +500 mV, T2: 120 ms; E3: -500 mV, T3: 60 ms, and output The range is 500-1500 nA. Oligosaccharides were then injected onto a CarboPac PA1 column equilibrated with 100% solvent. Dioxin acidified oligo extract (flow rate: 1 ml. Min-1) was applied using a linear gradient (0-20%) of solvent B over a period of 40 minutes followed by a linear increase from 20-100% of solvent B over 5 minutes. get on. Solvent A is 0.2M NaOH in distillate H20, and solvent B includes 0.6M NaOAc in solvent A. For natural oligosaccharides, the column system is equilibrated with 100% solvent C (0.1M NaOH in distillate H20) and eluted (flow rate: 1 ml. Min-1) using a linear gradient of solvent D (0 -35%) followed by a linear increase from 35-93-2005-200021143 A7 B7 within 48 minutes. (92) 100% solvent D increased linearly at 10 minutes. Solvent steel c includes 0.6 M NaAc in solvent C. Example 7.10 N-polysaccharide, HES-modified N · polysaccharide and Ep 0 protein. Monosaccharide composition was analyzed by methanol-glycolysis, GC-MS monosaccharide system in methanol alcoholysis, N-reacetylation and trimethylamine. GC / MS analysis was used to correlate methyl glycosides after the calcination of methacrylam [Chapulin, MR (1982). A fast and sensitive method for sugar molecule analysis. Biochemical Yearbook 123, 336-341]. The analysis was performed on a Finnig GCQ ion trap mass spectrometer (Finigan MAT, San Jose, CA) in positive ion m mode equipped with a 30 m DB5 capillary column. . Temperature program: Isothermal at 80 ° C for 2 minutes, then 10 degrees to 300 ° C per minute. Monosaccharides are identified by their residence time and fragment pattern characteristics. The uncorrected results of electronic spike accumulation are used for quantification. Due to the presence of carbohydrates and / or furanoid and pyranoid patterns, monosaccharides with a yield of more than one spike are determined by adding all the major spikes together. 0.5 microgram of myo-inositol was used as the internal standard compound. Printed by the Consumers' Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs Example 7.11 Results Example 7.11 (a) Characteristics of N-polyenzyme of Ep〇_GT_i treated with weak acid (partial sialylation) Ep0_GT_i The EP0-GT-1 preparation was analyzed by SDS-PAGE before or after free Qin-linked oligosaccharides by incubation with N-glycosidase, as shown in FIG. 5. HPAEC_PAD oligosaccharide mapping was performed on the bound oligosaccharides (Figure 6). Untreated EP0-GT-1 contains -94- 200521143 A7 B7 V. Description of the invention (93) 90% of N-bonded oligosaccharides containing 3 or 4 sialic acid residues, but cultivated in the presence of weak pupae After 5 minutes, &lt; 40% of the sugar molecular chains had 3 or 4 sialic acid residues. The HPAEC-PAD of the sialylated N-polysaccharide was revealed. It was used to measure the proportion of untreated EPO-GT-1 neutral oligosaccharides and remained stable in formulations that were treated with weak acid for 5, 10 or 60 minutes. The MALDI / TOF-MS of the sialylated polysaccharide revealed that <90% of the fucose on the near side was stored after the protein was treated with weak acid. Example 7.11 (b) Characteristics of iodate-treated EPO-GT-1 The SDS-PAGE mobility of EPO type with 5 or 10 minutes of acid treatment or untreated weakly mothate-treated EPO in the preceding paragraph is shown in Figure 8 Medium comparison. The oxidation conditions of peroxyacid for sialic acid did not change the SDS-PAGE pattern of the EPO preparation (compare Fig. 5). Oxidation of sialic acid leads to shift of the oligozyme to the earlier elution time in HPAEC-PAD analysis (compare Figs. 6 and 9). Example 7, 11 (c) Characteristics of HES modified EPO derivatives (aa ) EPO-GT-1-A According to Example 2.4, the time of HES modification of the HES derivative X modified with hydroxylamine produced in Example 2.4. The consumer cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs will print 400 micrograms of modified hydroxylamine. The quality HES derivative χ was added to 20 μg of EPO-GT-l_A (Ep 0 mildly periodate oxidized, non-acid hydrolyzed before mild periodate oxidation) in 20 μl, 0.5 M NaAc PF 5. In 5 buffer solution, the reaction was stopped by freezing the sample in liquid nitrogen at 30 minutes, 2, 4, and 17 hours later. Immediately after the samples were stored at -20 t for further analysis. SDS-PAGE sample buffer was added and the sample was heated to 90-2005-200521143 A? ___________ B7 V. Description of the invention (94) and applied to the SDS-gel. As shown in Figure 10, the increased incubation time resulted in increased displacement to higher molecular weight proteins. After incubation for 17 hours in the presence of hydroxylamine-modified HES derivative X, the infiltrating coomassie-stained protein band was detected to migrate in the region between 60 and u KDa. Basic (see left of Figure 10). When treated with N-glycosides, most proteins are shifted to the deglycosylated EPO position (see Figure 10, right glue; arrow a refers to the translocation of n-glycosidase, and arrow B refers to N- The migration position of glycosylated EPO; infiltrating protein bands can be seen in the region between the hypothesized Eρ〇-type 28 KDa and 36 KDa Moire weighting standards modified by HES and the 0-saccharification site of the molecule. Given the N-glycoside The characteristics of the enzyme, we can draw conclusions from this result: In fact, the modification occurred on the sialic acid residues of the polysaccharide of EPO protein after iodate oxidation. (Bb) Characteristics of HES-EPO consumables Intellectual property of the Ministry of Economic Affairs Bureau ’s consumer cooperation, Du printed, HES-EPO Vehicle Compound I (derived from EPO-GT after weak periodate oxidation), that is, from EPO-GT-1-A), 11 (derived from EPO-GT-1 after 5 minutes acid hydrolysis and weak periodate oxidation), 111 (derived from EPO_GT-1 after 10 minute acid hydrolysis and weak periodate oxidation) were synthesized as described above ◊ Controlled breeding (K) involves adding equal amounts of unmodified HES lines to the unmodified EPO-GT-1 under the same buffer conditions. The compounds were further purified for biochemical analysis of HES-EPO derivatives for later performance. In order to remove excess unreacted HES-reagents expected in the ion exchange tube immersion stream, HES-EPO conjugates will be cultivated I, Π and III, and control-96y 200521143 A7 B7 V. Description of the invention The preparation of culturing month K is performed as "raw materials and methods," Q-Agarose gel, iMb㈣ described in (Example 7 8). Ep0 type From the acid-treated samples η and m contained in the previous item, we expect a considerable amount of the modified EPO products from these cultivations in the immersion stream. As shown in Figure u, almost all from Sample I The EP0 material was retained by the Q_sepharose column, however, only about 20-30% of the sample m and π were rediscovered in the fractions eluted at high salt concentration. All the protein material from the incubation Both in the leaching and high-salt extraction fractions with HES derivative X, when compared with the control group EPO, the molecular weight is significantly higher than that in the SDS-PAGE. Describe more detailed characteristics, HES-modified EPO samples A and K (see Figure η) It is compared with the mothate-oxidized version EPO-GT-1-A. Samples were treated with N-glycosidase and expressed in Figures i2a and 12b. The release of N-polysaccharide resulted in _saccharification in standard Epo preparations. The non-glycosylated EPO pattern has two low molecular weight bands in the position. In the case of sample a, another band migration was detected at the 28KI &gt; a mw standard position and it is claimed that the EPO variant (see Example 7.11 ( c) (aa)) HES-modified. This band (and a large number of HES-modified high molecular weight versions of glycosylated EPO, see Figures 12a and 12b) disappeared after the sample was subjected to weak hydrolysis. It is the same as HES modified system in erythropoietin through iodate The consensus was reached on oxidized sialic acid residues. Each portion of the N-glycosidase incubation mixture is hydrolyzed with conditions that completely remove sialic acid residues (and sialic acid-bonded HES derivatives) from the sugars; after neutralization, the mixture is then adsorbed to Xiaohaiba Stick on the tube to allow it to desalinate. The column was vigorously washed with water and then eluted with 40% acetonitrile in 01 ° / 〇 trifluoroacetic acid -97- A7 B7 200521143 5. H20-bound neutral oligosaccharides in the description of the invention (96). The resulting oligos were subjected to MALDI / TOF-MS. Spectra obtained from the di-acidated dimerized fragment of sample A, EPO-GT-1-A, and sample K show that complex-type sugar is raised at m / z = 1810 Da (two synapses), 2175 = three synapses , 2540 = tetrasynaptic, 2906 = tetrasynaptic plus 1 N-acetylsyllactosamine repeat and 3271 = tetrasynaptic plus 2N-acetylsyllactosamine repeat have the same mass spectrum; related to lack of fucose Small signals of (-146) and galactose (-162) were detected, which can be attributed to acid hydrolysis conditions applied to sialic acid removal (see MALDI-Figures 15, 16 and Π). The Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs printed a parallel experiment in which the N-glycosidase digestion mixture was adsorbed onto a 1 ml RP-C18 box (not more than the acid hydrolysis of the previous oligosaccharide) and 5% acetonitrile was added to Elution of H20 with 0.1% TFA Under these conditions, the EPO protein remained completely on the RP-substance and the oligosaccharide was washed out of the column with 5% acetonitrile in H20 containing 0.1% TFA. The deglycosylated EPO protein was eluted with 70% acetonitrile in H20 containing 0.1% TFA. Oligosaccharide fragments from the RP-C18 step of sample A, EPO GT-1-A and sample K treated with N-glycosidase were neutralized and desalted with a Seamaster cartridge as described previously. The isolated oligosaccharides were subjected to HPAEC-PAD mapping before and after weak acid treatment (see Figure 13) and under conditions that allowed quantitative removal of sialic acid from the polysaccharide (see Figure 14). The HPAEC-PAD state of natural substances obtained from HES-modified sample A shows that there is only a negligible signal for sugar recruitment, and the oligosaccharides derived from EPO GT-1-A have the same polysaccharide state. As shown in Figure 9 (sample is called EPO-GT · 1 after weak acid treatment). The elution pattern of the oligosaccharide obtained from the control group EPO sample (K) produced the expected pattern (compared to the pattern in Figure 6). For comparison, the days of the international BRP-EPO standard -98- 200521143 A7 B7 V. Description of the invention (97) Of course, the oligosaccharide form is used for comparison and as a reference standard. After weak-acid hydrolysis, 'all oligo-acoustic preparations exhibited the expected qualities of tri- and tetra-synaptic complex-type sugar molecular chains as explained in the method section of the EPO preparations used as starting materials in this study. An elution pattern of the same neutral sugar-producing structure as the quantitative composition (see FIG. 14). This result confirms that the HES-modification of EPO samples will lead to covalent bonding of HES derivatives, which are isolated from the EPO protein by N-glycosidase and are acid-labile because they use known Weak acid treatment conditions are removed from the N-polysaccharide to disialosaccharide molecules (see Figure 12a + b). (cc) Analysis of HES-EPO and HES-EPO N-polysaccharides by GC-MS monosaccharide composition To further confirm the HES modification of EPO in Moore's N-polysaccharides, EPO samples were digested with N-glycosidase And the EPO protein was adsorbed on the RP-c18 cassette, and the oligosaccharide material was washed out as described above. As shown in Table 2, glucose and hydroxyethylated glucose derivatives are only found in EPO proteins, which were detected by HES-modification of cysteine and detected in sugar-removing sections of EPO sample A2. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. Example 7.11 (d) The biological activity of the EPO modified by HES was analyzed in the internal skull in the normal blood cell (normocythaemic) mouse system instruction EP0. The steps described in the description were performed; the EPO analysis experiments were performed using international BRP EPO reference standards. In the preparation of HES-modified EPO A2, the average value of the specific activity of the measured protein was 294,600 units per milligram of EPO. When compared with -99- A7 B7 200521143 for activity analysis, V. Description of the invention (98) is included in The specific activity prepared by the international BRP EPO reference standard in the sample is approximately 3-fold higher when compared. The results of the study are summarized in Table 3. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs -100 200521143 A7 B7 V. Description of Invention (99) References: Nimz, Norrbach EP, Conrad HS. Human tissue plasminogen activator is shown in the sugar molecular structure of recombinant Chinese hamster ovary cells. FEBS Manual, 1990, October 1; 271 (1-2) · 14-8 Dona AJ, Vasri LC, Kauffman RJ. Synthetic Biochemical Journal of Butyrate-treated Chinese Hamster Ovary Cells with Increased Secretory Protein-induced Expression of Regulated Glucose Proteins December 5, 1989; 264 (34): 20602- 7 Moeller PP, Shrank P, Nimz M, Conrad HS, Hauser Η Recombinant Glycoprotein Quality in Proliferative-Controlled Beta-K-21 Cells Biotechnology Biotechnology December 5, 1999; 65 (5): 529-36

尼姆茲Μ，馬丁 W，雷萊V，克洛培KD，歐斯丁 J，康 拉德HS 經濟部智慧財產局員工消費合作社印製 人重組體表現於重組體ΒΗΚ-21細胞中之經涎液化寡醣的 結構 歐洲生物化學期刊1993年4月1日；213(1) : 39-56Nimz M, Martin W, Leila V, Clope KD, Austin J, Conrad HS, Intellectual Property Bureau, Ministry of Economic Affairs, Consumer Consumption Cooperative, printed human recombinants expressed in recombinant β-K-21 cells Structure of salivated oligosaccharides. European Journal of Biochemistry, 1993.1; 213 (1): 39-56

贺曼丁 Ρ，維茲R，維利根哈JF，卡默林JP，尼姆茲Μ， 康拉德HS 以脉衝性電流檢測法藉高-ph陰離子交換色層分離用於圖 -101- 200521143 A7 B7 五、發明說明（100 ) 繪N-多醣之策略 生物化學年鑑1992年6月；203(2) : 281-9Hermantin P, Wiz R, Wieligenha JF, Carmelin JP, Nimz M, Conrad HS. High-ph anion exchange color separation was used for pulse-current detection for Figure-101- 200521143 A7 B7 V. Description of the Invention (100) Strategy for Mapping N-Polysaccharide Biochemical Yearbook June 1992; 203 (2): 281-9

舒洛特S，戴爾P，康拉德HS，尼姆茲Μ，海爾G，克次 霍夫C 人類CD52之雄性特定改變 生物化學期刊1999年10月15日；274(42) ·· 29862-73 經濟部智慧財產局員工消費合作社印製 表1 縮寫 化學名稱 種類 AMAS Ν_(α -馬爾亞胺基(Maleimido)破珀 醯亞胺酯 Ε ΒΜΡΗ 馬爾亞胺基丙酸)醯肼.TFA A BMPS Ν·(点-馬爾亞胺基丙氧基)琥珀醯亞 胺酯 E EMCH Ν-(ε -馬爾亞胺基己酸)醯肼 A EMCS Ν-(ε -馬爾亞胺基己醯基氧基)琥珀 醯亞胺酯 Ε GMBS Ν- 7 -馬爾亞胺基丁酿基氧基-破ίό 醯亞胺酯 Ε -102- 200521143 at B7 經濟部智慧財產局員工消費合作社印製 五、 發明說明 (101) 縮寫 化學名稱 種類 KMUH N-（/c-馬爾亞胺基十一烷酸)醯肼 A LC-SMCC 4-(N-馬爾亞胺基甲基)環己烷-1-羧 基-(6-醯胺基-己酸琥珀醯亞胺酯） E LC-SPDP 6-(3’-[2-哎咬基-二硫基]-丙釀胺基) 己醇酸琥珀醯亞胺酯 F MBS 間-馬爾亞胺基苄醯基-N，羥基琥珀 醯亞胺酯 E m2c2h ‘(N-馬爾亞胺基甲基)-環己烷-1-羧 基-酶肼HC1.1/2二哼烷 A ΜΡΒΗ 4-(4项_馬爾亞胺基苯基）-丁酸醯 肼.HC1 A 縮寫 化學名稱 種類 SATA S-乙醯基硫基·醋酸N-琥珀醯亞胺 酯 Η SATP S-乙醯基硫基·丙酸N_琥珀醯亞胺 酯 Η SBAP 3-(溴乙醯胺基）丙酸琥珀醯亞胺酯 D SIA N-琥珀醯亞胺基碘基醋酸酯 C -103- 200521143 A7 B7 經濟部智慧財產局員工消費合作社印製 五、 發明說明（102) SIAB (4-碘基乙醯基)胺基苯甲酸N-琥珀 醯亞胺酯 C SMCC 4-(N-馬爾亞胺基甲基)環己烷-1-羧 酸琥珀醯亞胺酯 E 縮寫 化學名稱 種類 SMPB 4-(對-馬爾亞胺基苯基)丁酸琥珀醯 亞胺酯 E SMPH 號ίό釀亞胺基-6-(沒-馬爾亞胺基丙 醯胺基）己醇酸酯 E SMPT 4-破ίό驢亞胺基氧基-幾基-甲基-CK (2-吡啶基二硫基）甲苯 F SPDP 3·(2-吡啶基二硫基）丙酸Ν-琥珀醯 亞胺酯 F 績基- EMCS Ν-( ε -馬爾亞胺基己醯基氧基)磺基 破ίό酿亞胺醋 E 確基_ GMBS Ν- r -馬爾亞胺基丁醯基氧基-磺基 琥珀醯亞胺酯 E 縮寫 化學名稱 種類 績基- KMUS N-(/c -馬爾亞胺基十一烷醯基氧基)-磺基琥珀醯亞胺酯 E -104- 200521143 A7 B7 五、發明說明（l〇3 )Shulot S, Dale P, Conrad HS, Nimz M, Haier G, Ketthofer C, Male Specific Changes in Human CD52, Biochemical Journal, October 15, 1999; 274 (42) · 29862- 73 Printed by the Employees' Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. Table 1 Abbreviated chemical name type AMAS Ν_ (α-Maleimido) Polpoimide E ΒΜΡΗ Malimidopropionic acid) hydrazine. TFA A BMPS Ν · (dot-malimidopropoxy) succinic imide E EMCH Ν- (ε-malimidohexanoic acid) hydrazine A EMCS Ν- (ε-malimidohexanoyloxy ) Succinimide E GMBS Ν-7 -Malimidobutyryloxy-degraded Eimide E -102- 200521143 at B7 Printed by the Consumers' Cooperative of Intellectual Property Bureau of the Ministry of Economic Affairs 101) Abbreviation Chemical Name Kind KMUH N-(/ c-Malliminododecanoic acid) hydrazine A LC-SMCC 4- (N-Malliminomethyl) cyclohexane-1-carboxyl- (6 -Amido-hexanoic acid succinimide ester) E LC-SPDP 6- (3 '-[2-Acetyl-dithio] -propanamine) Hexanoate succinimide F MBS Room-horse Iminobenzylidene-N, hydroxysuccinimide E m2c2h '(N-malimidomethyl) -cyclohexane-1-carboxy-enzyme HC1.1 / 2 dihumane A MPBΗ 4- (4 items_Malimidophenyl) -Hydrazine butyrate. HC1 A Abbreviated Chemical Name Type SATA S-Ethylthiothio · N-Succinimidyl Acetate Η SATP S-Ethylthio N-succinimide propionate Η SBAP 3- (Bromoacetamido) succinimide propionate D SIA N-succinimide iodoacetate C -103- 200521143 A7 B7 Economy Printed by the Intellectual Property Cooperative of the Ministry of Intellectual Property, V. Description of the invention (102) SIAB (4-iodoethylamido) aminobenzoic acid N-succinimide C SMCC 4- (N-malimidomethyl ) Cyclohexane-1-carboxylic acid succinimide E Abbreviated chemical name type SMPB 4- (p-Malimidophenyl) butyric acid succinimide E SMPH No. (N-Malimidopropylamido) hexanolate E SMPT 4-Hydroxyimidooxy-kisyl-methyl-CK (2-pyridyldithio) toluene F SPDP 3. · (2-pyridyl disulfide ) N-succinimide propionate F-Methyl group-EMCS Ν- (ε-Malimiminylhexyloxy) sulfolysis Ethyl imine vinegar _ GMBS NR- r-Malimine Butylsulfenyloxy-sulfosuccinimide E Abbreviation Chemical name Kind-KMUS N-(/ c -Malliminundecyloxy) -sulfosuccinimide E-104 -200521143 A7 B7 V. Description of the invention (l03)

磺基-LC-SPDP 6-(3’-[2-吡啶基-二硫基]丙醯胺基）己 醇酸磺基琥珀醯亞胺酯 F 磺基-MBS 間·馬爾亞胺基琥苄醯基-N-羥基磺基 琥珀醯亞胺酯 E 磺基-SIAB (4-硪基乙醢基)胺基苯甲酸磺基破拍 醯亞胺酯 C 磺基- SMCC 4-(N-馬爾亞胺基甲基)環己烷-1-羧酸 磺基琥珀醯亞胺酯 E 績基 SMPB 4-(對-馬爾亞胺基苯基)丁酸磺基琥珀 醯亞胺酯 ESulfo-LC-SPDP 6- (3 '-[2-pyridyl-dithio] propanamido) hexanol sulfosuccinimide F sulfo-MBS m-malimidosuccinyl Fluorenyl-N-hydroxysulfosuccinimide imide E sulfo-SIAB (4-fluorenylacetamido) aminobenzoic acid sulfo burst imidate C sulfo-SMCC 4- (N-Mal Iminomethyl) cyclohexane-1-carboxylic acid sulfosuccinimide E EMP SMPB 4- (p-mariminophenyl) butyrate sulfosuccinimide E

縮寫 化學名稱 種類 績基-LC_ SMPT 6-(α -甲基-α -[2-吼咬基二硫 基]-甲苯醯胺基）己醇酸酯 F SVSB Ν-破ίό釀亞胺基-(4-乙婦基績釀 基)苯甲酸酯 G 經濟部智慧財產局員工消費合作社印製 -105- A7 B7 200521143 五、發明說明（l〇4) 表2 來自經HES-改質的EPO與控制樣品之多醣 的單醣组成物分析 經濟部智慧財產局員工消費合作社印製 **單醣 I· 來自A2 之多醣 II· 來自 EPO· GT-1A 之多醣 III. 來自K2 之多醣 III· 來自A2 之多醣 IV. 來自 EPO- GT-1A 之多醣 V· 來自K2 之多醣 VI· 經半胱 胺酸改 質的 EPO蛋 白質* 果糖 1,935 3,924 2,602 2,246 4,461 2,601 2,181 甘露糖 6,028 11,020 9,198 6,379 11,668 6,117 6,260 半乳糖 8,886 19,935 14,427 10,570 16,911 11,555 10,386 葡萄糖 17,968 — 21,193 微量 微量 33,021 GlcNAc 7,839 21，310 14,440 11,360 15,953 10,503 10,498 GlcHel 5,583 •— 5,926 … 14,857 GlcHe2 1,380 —- 1,552 — 一 _ 3,775 NeuNAc 5,461 822 4,504 3,895 4,871 13,562 13,003 肌醇 1,230 2,310 1,620 2,050 1，320 1,134 1,087 *將等量經Cys_HES-改質的EPO 蛋白質進行組成物分 析；EPO蛋白質係從HES-培育混合物中藉如前說明之 色層分離法在Q-瓊脂糖凝膠管柱上單離出來且使用 Vivaspin5分離裝置藉離心法去鹽。 **單醣測定係從過三甲基涎酸化的甲基配醣體之單一 GC 運轉中進行；尖峰之電子積分值係於各化合物衍生過程 及回收時未經流失校正而得到。 -106- 200521143 五、發明說明（l〇5 ) A7 B7 表3 樣品號碼 樣品說明 EPO樣品之經計算的特 定活性（以A280亳微米 及RP-HPLC測定計） 850247 1.經HES-改質的EPO A2 344,000U/毫克 850248 2 · EPO-GT-1-A 82,268 U/毫克 850249 3.控制組EPOK2 121，410 U/毫克 850250 4 · BR1&gt; EOD 標準 86,702 U/亳克 850251 1·以4倍體積PBS稀釋 309,129 U/亳克 850252 2.以4倍體積PBS稀釋 94,500 U/亳克 850253 3·以4倍體積PBS稀釋 114，100U/亳克 850254 4.以4倍體積PBS #釋 81，200 U/毫克 850255 1.以4倍體積PBS稀釋 230,720 U/毫克 經濟部智慧財產局員工消費合作社印製 -107-Abbreviation Chemical Name Kind-LC_ SMPT 6- (α -Methyl-α-[2-Syringyldithio] -toluenylamino) hexanolate F SVSB Ν- breakdown imimine- (4-Ethylbenzyl) Base Benzoate G Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs-105- A7 B7 200521143 V. Description of Invention (104) Table 2 From HES-modified EPO Analysis of monosaccharide composition of polysaccharides from control samples Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs ** Monosaccharide I · Polysaccharide from A2 II · Polysaccharide from EPO · GT-1A III. Polysaccharide III from K2 · From Polysaccharides from A2 IV. Polysaccharides from EPO- GT-1A V Polysaccharides from K2 VI Ecysteine-modified EPO protein * Fructose 1,935 3,924 2,602 2,246 4,461 2,601 2,181 Mannose 6,028 11,020 9,198 6,379 11,668 6,117 6,260 half Lactose 8,886 19,935 14,427 10,570 16,911 11,555 10,386 Glucose 17,968 — 21,193 Traces 33,021 GlcNAc 7,839 21,310 14,440 11,360 15,953 10,503 10,498 GlcHel 5,583 • — 5,926… 14,857 GlcHe2 1,380 —- 1,552 — one_ 3,775 NeuNA c 5,461 822 4,504 3,895 4,871 13,562 13,003 Inositol 1,230 2,310 1,620 2,050 1,320 1,134 1,087 * Composition analysis of the same amount of Cys_HES-modified EPO protein; EPO protein was extracted from the HES-incubation mixture as previously explained The chromatographic separation method was isolated on a Q-sepharose column and desalted by centrifugation using a Vivaspin 5 separation device. ** Monosaccharide determination was performed from a single GC run of trimethylsialylated methylglycosides; the peak electronic integration value was obtained without bleed correction during the derivatization of each compound and recovery. -106- 200521143 V. Description of the invention (105) A7 B7 Table 3 Sample No. Sample Description The calculated specific activity of the EPO sample (measured by A280 μm and RP-HPLC) 850247 1. Hes-modified EPO A2 344,000U / mg 850248 2EPO-GT-1-A 82,268 U / mg 850249 3.Control group EPOK2 121,410 U / mg 850250 4BR1> EOD standard 86,702 U / gram 850251 1 4 times 309,129 U / Ugram 850252 diluted in volume PBS 2.94,500 U / 亳 gram 850253 diluted in 4 times volume PBS 3.114,100 U / 亳 gram 850254 diluted in 4 times volume PBS 4.81,200 in 4 times volume PBS U / mg 850255 1.Diluted 230,720 with 4 times the volume of PBS. U / mg printed by the Consumer Cooperative of Intellectual Property Bureau of the Ministry of Economic Affairs -107-

Claims (1)

8 8^8 A B CD 200521143 六、申請專利範圍 1 · 一種製造羥基烷基澱粉衍生物的方法，其係包括具 式(I)之經基烧基殿粉8 8 ^ 8 A B CD 200521143 VI. Scope of patent application 1 · A method for producing a hydroxyalkyl starch derivative, which comprises a basal base powder of formula (I) 經濟部智慧財產局員工消費合作社印製 於其不會在該反應之前被氧化之還原端上，與式(II) 之化合物 R，-NH_R” (II) 進行反應，其中，Ri，R2及R3係各自獨立為氳或為 一線形或分支的羥基烷基基團，且其中R’或R”或者 R’與R”包括至少一個官能基X，其係能與至少一種其 他化合物於(I)與(II)之反應前或後進行反應。 2 · 如申請專利範圍第1項中的方法，其中，羥基烷基 澱粉係為羥基乙基澱粉。 3· 如申請專利範圍第1或2項中的方法，其中，R!， R2及R3係各自獨立為氫或2-羥基乙基基團。 4 · 如申請專利範圍第1至3項中任一項中的方法，其 中，R’係為氫或為一線形或分支的烷基或烷氧基基 團。 5· 如申請專利範圍第4項中的方法，其中，R’係為氫 或甲基或甲氧基基團。 6· 如申請專利範圍第1至5項中任一項中的方法，其 -108 200521143 A8 B8 C8The Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs printed on the reducing end which will not be oxidized before the reaction, and reacts with the compound of formula (II) R, -NH_R "(II), among which Ri, R2 and R3 Are each independently fluorene or a linear or branched hydroxyalkyl group, and wherein R 'or R "or R' and R" includes at least one functional group X, which is capable of interacting with at least one other compound in (I) The reaction is performed before or after the reaction with (II). 2 · As in the method in the scope of patent application, item 1, wherein the hydroxyalkyl starch is hydroxyethyl starch. 3. · As in the scope of patent application, item 1 or 2 , Wherein R !, R2 and R3 are each independently hydrogen or 2-hydroxyethyl group. 4 · The method according to any one of claims 1 to 3, wherein R 'is Hydrogen may be a linear or branched alkyl or alkoxy group. 5. The method as in item 4 of the scope of the patent application, wherein R 'is hydrogen or a methyl or methoxy group. 6 · Such Apply for the method in any one of items 1 to 5, which is -108 200521143 A8 B8 C8 訂Order A8B8C8D8 200521143 六、申請專利範圍 括：-SH，-NH2，-0-NH2-，-ΝΗ-Ο-烷基，-(〇=〇)-ΝΗ·ΝΗ2，-G-(〇G)-NH_NH2，_NH-(C=G)-NH-NH2， 及-S02-NH-NH2，其中G為O或S且，若G出現兩 次，則其獨立為〇或s。 9 · 如申請專利範圍第1至6或8項中任一項中的方 法，其中，如式(II)之化合物係為〇-[2-(2-胺基氧基_ 乙氧基)-乙基]-經基胺或碳醯肼。 10·如申請專利範圍第1至9項中任一項中的方法，其 中，化合物(I)係與化合物(II)進行反應，該化合物(II) 與化合物（I)進行反應之前不會與另外的化合物作 用。 11 ·如申請專利範圍第10項中的方法，其中，化合物(11) 係為經基胺或醯肼時，化合物(I)與化合物(π)之反應 係在溫度自5至45°C及pH自4.5至6.5範圍於水性 介質中進行。 12 ·如申請專利範圍第1〇項中的方法，其中，該反應為 還原性胺化作用時，化合物⑴與化合物(π)之反應係 經濟部智慧財產局員工消費合作社印製 在溫度自25至90°C及ΡΗ自8至12範圍於水性介質 中進行。 13如申請專利範圍第1〇至12項中任一項中的方法， 其中，化合物(I)與化合物(H)之反應產物係經由至少 一個S能基X與另外的化合物進行反應。 14 ·如中請專利範圍第13項中的方法，其中，至少另一 種化合物係經由包含於至少另一種化合物中之硫基 -110 200521143 A8 B8 C8 D8 六、申請專利範圍 或經氧化的醣分子部分進行反應。 15 ·如申請專利範圍第14項中的方法，其中，至少另一 種化合物係為多胜肽。 16 ·如申請專利範圍第15項中的方法，其中，多胜肽係 為紅血球生成素。 17 ·如申請專利範圍第13至16項中任一項中的方法， 其中，反應係在自4至37°C範圍之溫度下進行。 18 ·如申請專利範圍第13至17項中任一項中的方法， 其中，反應係在水性介質中進行。 19 ·如申請專利範圍第13項中的方法，其中，另外的化 合物係為交聯化合物。 20 ·如申請專利範圍第19項中的方法，其中，化合物(I) 與(Π)之反應產物與交聯化合物之反應產物係與又一 種化合物進行反應。 21 ·如申請專利範圍第20項中的方法，其中，又一種化 合物係為多胜狀。 22 ·如申請專利範圍第20或21項中的方法，其中，又 經濟部智慧財產局員工消費合作社印製 一種化合物係經由包含於至少另一種化合物中之硫 基或經氧化的醣分子部分進行反應。 23 ·如申請專利範圍第13項中的方法，其中，另外的化 合物係為交聯化合物與又一種化合物之反應產物p 24 ·如申請專利範圍第23項中的方法，其中，又一種化 合物係為多胜肽。 25 ·如申請專利範圍第1至3項中任一項中的方法，其 -111 200521143 A8 B8 C8 D8 六、申請專利範圍 中，化合物(II)係在與化合物(I)進行反應之前與另外 的化合物進行反應。 26 ·如申請專利範圍第25項中的方法，其中，至少另一 種化合物係經由包含於至少另一種化合物中之硫基 或經氧化的醣分子部分進行反應。 27 ·如申請專利範圍第25或26項中的方法，其中，至 少另一種化合物係為多胜肽。 28 ·如申請專利範圍第27項中的方法，其中，多胜肽係 為紅血球生成素。 29 ·如申請專利範圍第25至28項中任一項中的方法， 其中，反應係在自4至37°C範圍之溫度下進行。 30 ·如申請專利範圍第25至29項中任一項中的方法， 其中，反應係在水性介質中進行。 31 ·如申請專利範圍第25項中的方法，其中，另外的 化合物係為交聯化合物。 經濟部智慧財產局員工消費合作社印製 32·如申請專利範圍第31項中的方法，其中，交聯化 合物係在與化合物(II)進行反應之前與又一種化合物 進行反應。 33 ·如申請專利範圍第25項中的方法，其中，另外的 化合物係為交聯化合物與又一種化合物之反應產 物。 34·如申請專利範圍第33項中的方法，其中，又一種 化合物係為多胜肽。 35· —種製造羥基烷基澱粉衍生物的方法，其係包括具 -112 A8B8C8D8 200521143 六、申請專利範圍 式(I)之羥基烷基澱粉 OR, Η / 1 HES，A8B8C8D8 200521143 6. The scope of patent application includes: -SH, -NH2, -0-NH2-, -NΗ-O-alkyl,-(〇 = 〇) -ΝΗ · ΝΗ2, -G- (〇G) -NH_NH2, _NH- (C = G) -NH-NH2, and -S02-NH-NH2, where G is O or S and if G appears twice, it is independently 0 or s. 9 · The method according to any one of claims 1 to 6 or 8, wherein the compound of formula (II) is 0- [2- (2-aminooxy_ethoxy)- Ethyl] -Amine or Carbazine. 10. The method according to any one of claims 1 to 9, wherein the compound (I) is reacted with the compound (II), and the compound (II) does not react with the compound (I) before the reaction. Additional compounds act. 11 · The method according to item 10 of the scope of patent application, wherein when the compound (11) is based on amine or hydrazine, the reaction of the compound (I) and the compound (π) is at a temperature of 5 to 45 ° C and The pH ranges from 4.5 to 6.5 in aqueous media. 12 · The method in item 10 of the scope of patent application, wherein, when the reaction is a reductive amination, the reaction between compound ⑴ and compound (π) is printed by a consumer cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs at a temperature from 25 To 90 ° C and pH from 8 to 12 in aqueous media. 13. The method according to any one of claims 10 to 12, in which the reaction product of the compound (I) and the compound (H) is reacted with another compound via at least one S energy group X. 14 · The method in item 13 of the patent scope, wherein at least one other compound is via a thio group contained in at least one other compound-110 200521143 A8 B8 C8 D8 Six. Patent application scope or oxidized sugar Partial reaction. 15-The method according to item 14 of the scope of patent application, wherein at least one other compound is a polypeptide. 16. The method according to item 15 of the application, wherein the polypeptide is erythropoietin. 17. The method according to any one of claims 13 to 16, wherein the reaction is performed at a temperature ranging from 4 to 37 ° C. 18. The method according to any one of claims 13 to 17, wherein the reaction is performed in an aqueous medium. 19-The method according to item 13 of the scope of patent application, wherein the additional compound is a crosslinked compound. 20. The method according to item 19 of the scope of the patent application, wherein the reaction product of the compounds (I) and (Π) and the cross-linked compound are reacted with another compound. 21-The method as described in claim 20 of the scope of patent application, wherein another compound is multi-victory. 22 · The method in the scope of application for patents No. 20 or 21, wherein the printing of a compound by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs is performed through a sulfur group or an oxidized sugar molecule portion contained in at least another compound reaction. 23 · The method according to item 13 of the patent application, wherein the other compound is a reaction product p 24 of a crosslinked compound and another compound. · The method according to item 23 of the patent application, wherein another compound is Is a polypeptide. 25 · As in the method of any one of claims 1 to 3, which is -111 200521143 A8 B8 C8 D8 6. In the scope of patent application, compound (II) is reacted with compound (I) before The compounds are reacted. 26. The method of claim 25, wherein at least one other compound is reacted via a sulfur group or an oxidized sugar molecule portion contained in the at least one other compound. 27. The method as claimed in claim 25 or 26, wherein at least one other compound is a polypeptide. 28. The method as described in claim 27, wherein the polypeptide is erythropoietin. 29. The method according to any one of claims 25 to 28, wherein the reaction is performed at a temperature ranging from 4 to 37 ° C. 30. The method according to any one of claims 25 to 29, wherein the reaction is performed in an aqueous medium. 31. The method as described in claim 25, wherein the other compound is a crosslinked compound. Printed by the Employees' Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 32. The method in item 31 of the scope of patent application, wherein the crosslinked compound is reacted with another compound before reacting with compound (II). 33. The method of claim 25, wherein the other compound is a reaction product of a crosslinked compound and another compound. 34. The method according to item 33 of the patent application scope, wherein the further compound is a polypeptide. 35 · —A method for producing a hydroxyalkyl starch derivative, which comprises -112 A8B8C8D8 200521143 6. Application scope of the patent A hydroxyalkyl starch of formula (I) OR, Η / 1 HES, (I) OH ·: 於其不會在該反應之前被氧化之還原端上’於水性介 質中與式(II)之化合物 R’-NH_R” （II) 經濟部智慧財產局員工消費合作社印製 進行反應，其中，Ri，R2及R3係各自獨立為氫或2-羥基乙基基團，其中，R’係為氳或甲基或甲氧基基 團，且其申R”包含至少一個官能基X，其係能與至 少一種其他化合物於式⑴及(II)之反應前或後進行反 應，該官能基X係選自於下列的基團，其包括：_ SH，-ΝΗ2，-〇-ΝΗ2-，-ΝΗ-0 烷基，-(0=0)-1«1-NH2，_G-(C=G)-NH-NH2，_NH-(OG)-NH-NH2，及_ S〇2-NH_NH2，其中G為0或S且，若G出現兩次， 則其獨立為G或S。 36 ·如申請專利範圍第35項中的方法，其中，如式(I) 之化合物係為〇-[2-(2-胺基氧基-乙氧基)-乙基]-羥 基胺或碳醯肼。 37 ·如申請專利範圍第35或36項中的方法，其中，化 合物(Ϊ)與化合物(Π)之反應產物係在水性介質中經由 -113 A8 B8 C8 D8 200521143 六、申請專利範圍 包含於化合物(II)中之至少一個官能基X及包含於多 胜肽中之硫基或經氧化的醣分子部分與多胜肽進行 反應。 38 ·如申請專利範圍第37項中的方法，其中，多胜肽 係為紅血球生成素。 39 ·如申請專利範園第35項中的方法，其中，化合物 (Π)係於水性介質中經由包含於化合物(π)中之至少一 個官能基X及包含於多胜肽中之硫基或經氧化的醣 分子部分與多胜肽進行反應，且產生的反應產物係 與化合物(I)進行反應。 40 ·如申請專利範圍第39項中的方法，其中，多胜肽 係為紅血球生成素。 41 · 一種製造羥基烷基澱粉衍生物的方法，其係包括具 式⑴之羥基烷基澱粉(I) OH ·: On the reducing end which will not be oxidized before the reaction, 'in aqueous solution with compound R'-NH_R of formula (II)' (II) Printed by the Consumer Cooperative of Intellectual Property Bureau, Ministry of Economic Affairs The reaction is performed, wherein Ri, R2 and R3 are each independently hydrogen or 2-hydroxyethyl group, wherein R ′ is fluorene or methyl or methoxy group, and its application R ”includes at least one function The functional group X is capable of reacting with at least one other compound before or after the reaction of formulas XI and (II). The functional group X is selected from the group consisting of:-SH, -N2,-. -ΝΗ2-, -ΝΗ-0 alkyl,-(0 = 0) -1 «1-NH2, _G- (C = G) -NH-NH2, _NH- (OG) -NH-NH2, and _S. 2-NH_NH2, where G is 0 or S and if G occurs twice, it is independently G or S. 36. The method as described in claim 35, wherein the compound of formula (I) is 0- [2- (2-aminooxy-ethoxy) -ethyl] -hydroxyamine or carbon Hydrazine. 37. The method according to item 35 or 36 of the scope of patent application, wherein the reaction product of compound (ii) and compound (Π) is in an aqueous medium via -113 A8 B8 C8 D8 200521143 6. The scope of patent application is included in the compound At least one functional group X in (II) and a sulfur group or an oxidized sugar molecule portion contained in the polypeptide react with the polypeptide. 38. The method according to item 37 of the scope of patent application, wherein the polypeptide is erythropoietin. 39. The method according to item 35 of the patent application park, wherein the compound (Π) is in an aqueous medium via at least one functional group X contained in the compound (π) and a sulfur group contained in the polypeptide or The oxidized sugar molecule part reacts with the polypeptide, and the resulting reaction product reacts with the compound (I). 40. The method of claim 39, wherein the polypeptide is erythropoietin. 41. A method for producing a hydroxyalkyl starch derivative, comprising a hydroxyalkyl starch of formula ⑴ Η 經濟部智慧財產局員工消費合作社印製 於其不會在該反應之前被氧化之還原端上，於水性介 質中與0-[2〇胺基氧基_乙氧基分羥基胺進行反應， 其中，R!，R2及R3係各自獨立為氩或孓羥基乙基基 團，且反應產物係於水性介質中經由包含於該紅血球 -114 200521143 A8 B8 C8 D8 六、申請專利範圍 生成素中之經氧化的醣分子部分與紅血球生成素進行 反應。 42· —種可藉由方法而得之羥基烷基澱粉衍生物，該方 法係包括具式(I)之羥基烷基澱粉(HAS)员工 Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs on the reducing end which will not be oxidized before the reaction, and reacted with 0- [2〇 aminooxy_ethoxy hydroxylamine in aqueous medium Among them, R !, R2 and R3 are each independently argon or fluorene hydroxyethyl group, and the reaction product is contained in the red blood cell via the red blood cell-114 200521143 A8 B8 C8 D8 VI. The oxidized sugar molecule part reacts with erythropoietin. 42 · —A hydroxyalkyl starch derivative obtainable by a method comprising a hydroxyalkyl starch (HAS) of formula (I) 經 濟 部 智 慧 財 產 局 員 工 消 費 合 作 社 印 於其不會在該反應之前被氧化之還原端上，與式(II) 之化合物 R，_NH-R” (II) 進行反應，其中Ri，R2及R3係各自獨立為氫或為一 線形或分支的羥基烷基基團，且其中R’或R”或者R’ 與R”包括至少一個官能基X，其係能與至少一種其 他化合物於(I)及(II)之反應前或後進行反應。 43·如申請專利範圍第42項中之羥基烷基澱粉衍生 物，其中，羥基烷基澱粉係為羥基乙基澱粉。 44·如申請專利範圍第42或43項中之羥基烷基澱粉衍 生物，其中，心，112及R3係各自獨立為氫或2-羥基 乙基基團。 45·如申請專利範圍第42至44項中任一項中之羥基烷 基澱粉衍生物，其中，R’係為氳或為一線形或分支 -115 200521143 A8 B8 C8 D8 六、申請專利範圍 ~ 的烷基或為烷氧基基團。 46·如申請專利範圍第45項中之羥基烷基澱粉衍生 物，其中，R’為氫或甲基或甲氧基基團。 47·如申請專利範圍第42至46項中任一項中之羥基烧 基澱粉衍生物，其中，除官能基X之外，R”包含 至少再一個直接連接到NH基橋R’與R”之官能基 W，該官能基W係選自於下列的基圓，其包括：The Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs is printed on the reducing end which will not be oxidized before the reaction, and reacts with the compound of formula (II) R, _NH-R "(II), where Ri, R2 and R3 are Each is independently hydrogen or a linear or branched hydroxyalkyl group, and wherein R 'or R "or R' and R" includes at least one functional group X, which is capable of interacting with at least one other compound in (I) and (II) The reaction is performed before or after the reaction. 43. As the hydroxyalkyl starch derivative in item 42 of the scope of patent application, wherein the hydroxyalkyl starch is hydroxyethyl starch. 44. As the scope of patent application, 42 Or the hydroxyalkyl starch derivative according to item 43, wherein, Xin, 112 and R3 are each independently hydrogen or 2-hydroxyethyl group. 45. As described in any one of claims 42 to 44 A hydroxyalkyl starch derivative, in which R ′ is fluorene or is linear or branched-115 200521143 A8 B8 C8 D8 6. Alkyl or alkoxy group in the scope of patent application 46. If the scope of patent application The hydroxyalkyl starch derivative in item 45, Wherein R ′ is hydrogen or a methyl or methoxy group. 47. The hydroxyalkyl starch derivative according to any one of claims 42 to 46 of the scope of application for a patent, wherein, in addition to the functional group X, R "Comprising at least one further functional group W directly connected to the NH radical bridges R 'and R", the functional group W is selected from the group consisting of: 其中G為Ο或S且，若出現兩次，則G獨立為Ο或 S 〇 48 ·如申請專利範圍第47項中的方法，其中，若R，為 Η，則R’及NH基橋R’及R”與W —起形成下列基 經濟部智慧財產局員工消費合作社印製 之 團 ΗΝ OHSNO I ΝΗ ΗΝ YMG ΗΝ ΗΝ G YG -116 200521143 B8 C8 D8 Η2Ν-Νγ- G 49 50 51 如申請專利範圍第42至48項中任一項中之羥基烷 基凝粉衍生物，其中，至少一個官能基X係選自於 下列之基團，其包括：-SH，-NH2，-0-NH2-，-NH-烷基，-(〇G)-NH-NH2，-G-(OG)-NH-NH2，-NH-&lt;e&gt;G)-NH_NH2，及-S02_NH-NH2，其中 G 為 O 或 S 且’若G出現兩次，則其獨立為cj或S。 &amp;申請專利範圍第42項中之羥基烷基澱粉衍生 物’其中，如式(II)之化合物係為0-[2-(2-胺基氧基-乙氣基基】-羥基胺或碳醯肼。 如申請專利範圍第42項中之羥基烷基澱粉衍生物， 其中，該衍生物具有如式(Ilia)之結構。Where G is 0 or S and if it occurs twice, G is independently 0 or S 〇 48. As in the method in the 47th scope of the patent application, where R is Η, then R ′ and NH radical bridge R 'And R' and W — form the following group printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs: OΝ OHSNO I ΝΗ ΗΝ YMG ΗΝ ΗΝ G YG -116 200521143 B8 C8 D8 Η2Ν-Νγ- G 49 50 51 The hydroxyalkyl gel powder derivative according to any one of the items 42 to 48, wherein at least one functional group X is a group selected from the group consisting of -SH, -NH2, -0-NH2- , -NH-alkyl,-(〇G) -NH-NH2, -G- (OG) -NH-NH2, -NH- &lt; e &gt; G) -NH_NH2, and -S02_NH-NH2, where G is O Or S and 'if G occurs twice, it is independently cj or S. &amp; hydroxyalkyl starch derivative in item 42 of the patent application' wherein the compound of formula (II) is 0- [2 -(2-aminooxy-ethoxy) -hydroxyamine or carbohydrazine. For example, the hydroxyalkyl starch derivative in item 42 of the scope of patent application, wherein the derivative has the formula (Ilia) structure. -117 200521143 六、申請專利範園 52·如申請專利範圍第42項中之羥基烷基殿粉衍生 物，其中，R’係為氫且其中衍生物係為式(Ilia)之化 合物或式(Illb)之化合物或式(nia)及(Illb)化合物之 混合物。 HAS-117 200521143 VI. Patent Application Fanyuan 52. For example, the hydroxyalkyl palace powder derivative in item 42 of the patent application scope, wherein R 'is hydrogen and wherein the derivative is a compound of formula (Ilia) or formula ( Illb) or a mixture of compounds of formulae (nia) and (Illb). HAS N ,H (Ilia) R·， HAS，N, H (Ilia) R ·, HAS, R，， (mb) 53 經濟部智慧財產局員工消費合作社印製 54 如申請專利範圍第42項中之羥基烷基澱粉衍生 物，其中，化合物(I)與化合物(II)之反應產物係經由 包含於化合物(II)中之至少一個官能基X與至少另一 種化合物進行反應，或其中，化合物(II)係經由至少 一個官能基X與至少另一種化合物於與化合物(I)反 應之前進行反應。 如申請專利範圍第53項中之羥基烷基澱粉衍生 物，其中，至少另一種化合物係與化合物(II)或與 化合物⑴及化合物(II)之反應產物經由包含於至少另 -118 2 L88 8» A B c D 200521143 六、申請專利範圍 一種化合物中之硫基或經氧化的醣分子部分進行反 應。 55·如申請專利範圍第54項中之羥基烷基澱粉衍生 物，其t，至少另一種化合物係為多胜肽。 56 ·如申請專利範圍第55項中之羥基烷基澱粉衍生 物，其中，多胜肽係為紅血球生成素。 57·如申請專利範圍第53項中之羥基烷基澱粉衍生 物，其中，另外的化合物係為交聯化合物。 58·如申請專利範圍第57項中之羥基烷基澱粉衍生 物，其中，化合物(I)與(II)之反應產物與交聯化合物 之反應產物係與又一種化合物進行反應。 59·如申請專利範圍第57項中之羥基烷基澱粉衍生 物’其中’又一種化合物係為多胜狀。 經濟部智慧財產局員工消费合作社印製 60·如申請專利範圍第58項中之羥基烷基澱粉衍生 物，其中，又一種化合物係經由包含於至少另一種 化合物中之硫基或經氧化的醣分子部分進行反應。 61·如申請專利範圍第53項中之羥基烷基澱粉衍生 物，其中，另外的化合物係為交聯化合物與又一種 化合物之反應產物。 62·如申請專利範圍第61項中之羥基烷基澱粉衍生 物，其中，又一種化合物係為多胜肽。 63· —種醫藥組成物，其係包括治療有效量如申請專利 範圍第42項中之羥基烷基澱粉衍生物，其中，化 合物⑴與化合物(II)之反應產物係經由包含於化合物 -119 200521143 A8B8C8D8 、申請專利範圍 (π)中之至少一個官能基X與至少另一種化合物進行 反應，或其中化合物(π)係經由至少一個官能基χ與 至少另一種化合物於與化合物(I)反應之前進行反應 且其中至少另一種化合物係為多胜肽。 64 ·如申請專利範圍第63項中之醫藥組成物，其中， 多胜肽係與化合物(II)或與化合物（I)及化合物(II)之 反應產物經由包含於多胜肽中之經氧化的醣分子部 分進行反應。 65 ·如申請專利範圍第64項中之醫藥組成物，其中， 多胜狀係為紅金球生成素。 66 ·如申請專利範圍第65項中之醫藥組成物，其中， 羥基乙基澱粉係於水性介質中與如下式之化合物 Η2Ν/〇^^〇^^〇、ΝΗ2 進行反應’且反應產物與紅jk球生成素進行反應。 67·如申請專利範圍第66項中之羥基烷基澱粉衍生 物，其中，紅血球生成素係於反應之前被過碘酸納 氧化。 68 ·如申請專利範圍第66項中之醫藥組成物，其中， 紅血球生成素係於反應之前被部份二延酸化 (desialylated)且接著被過蛾酸納氧化。 69 · —種醫藥組成物，其係包括治療有效量如申請專利R ,, (mb) 53 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 54 As the hydroxyalkyl starch derivative in item 42 of the scope of patent application, wherein the reaction product of compound (I) and compound (II) is via At least one functional group X contained in compound (II) is reacted with at least another compound, or wherein compound (II) is reacted with at least one other compound via at least one functional group X before reacting with compound (I) . For example, the hydroxyalkyl starch derivative in item 53 of the scope of patent application, wherein the reaction product of at least one other compound with compound (II) or with compound IX and compound (II) is contained in at least another -118 2 L88 8 »AB c D 200521143 VI. Application for Patent Range A sulfur or oxidized sugar molecule in a compound reacts. 55. The hydroxyalkyl starch derivative according to item 54 of the application, wherein t, at least one other compound is a polypeptide. 56. The hydroxyalkyl starch derivative according to item 55 of the application, wherein the polypeptide is erythropoietin. 57. The hydroxyalkyl starch derivative according to item 53 of the application, wherein the other compound is a crosslinked compound. 58. The hydroxyalkyl starch derivative according to item 57 of the application, wherein the reaction product of the reaction product of the compounds (I) and (II) and the cross-linked compound is reacted with another compound. 59. The hydroxyalkyl starch derivative 'wherein' as described in item 57 of the scope of application for a patent is further multifunctional. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 60. For example, the hydroxyalkyl starch derivative in Item 58 of the scope of patent application, wherein another compound is via a sulfur group or an oxidized sugar contained in at least one other compound The molecular part reacts. 61. The hydroxyalkyl starch derivative according to item 53 of the application, wherein the other compound is a reaction product of a crosslinked compound and another compound. 62. The hydroxyalkyl starch derivative according to item 61 of the application, wherein another compound is a polypeptide. 63. A pharmaceutical composition comprising a therapeutically effective amount of a hydroxyalkyl starch derivative as described in item 42 of the patent application, wherein the reaction product of compound VII and compound (II) is contained in compound-119 200521143 A8B8C8D8, at least one functional group X in the patent application range (π) is reacted with at least another compound, or compound (π) is reacted with at least one other compound through at least one functional group χ before reacting with compound (I) And where at least one other compound is a polypeptide. 64. The pharmaceutical composition according to item 63 in the scope of the patent application, wherein the reaction product of dopeptide with compound (II) or with compound (I) and compound (II) is oxidized by inclusion in dopeptide The sugar molecule is partially reacted. 65. The pharmaceutical composition according to item 64 of the patent application scope, wherein the victorious system is erythropoietin. 66. The pharmaceutical composition according to item 65 in the scope of the patent application, wherein the hydroxyethyl starch is reacted in an aqueous medium with a compound of the formula: Η2Ν / 〇 ^^ 〇 ^^ 〇, ΝΗ2, and the reaction product reacts with red jk spheroidin reacts. 67. The hydroxyalkyl starch derivative according to item 66 of the application, wherein the erythropoietin is oxidized by sodium periodate before the reaction. 68. The pharmaceutical composition according to item 66 of the scope of application, wherein the erythropoietin is partially diallylated before the reaction and then is oxidized by sodium permothate. 69.-a pharmaceutical composition, including a therapeutically effective amount such as a patent application 經濟部智慧財產局員工消費合作社印製 •120 200521143 A8 B8 C8 D8 申請專利範圍 70 經濟部智慧財產局員工消費合作社印製 範圍第42項中之羥基烷基澱粉衍生物，其中，化 合物(I)與化合物(II)之反應產物係經由包含於化合物 (II)中之至少一個官能基X與至少另一種化合物埃 行反應，或其中化合物（II)係經由至少一個官能基 X與至少另一種化合物於與化合物⑴反應之前進行 反應，且其中至少另一種化合物係為交聯化合物且 化合物(I)與(Π)之反應產物與交聯化合物之反應產物 係與多胜肽進行反應。 如申請專利範園第69項中之醫藥組成物，其中，多 胜狀係為紅血球生成素。 -121 、 ▲分sPrinted by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs • 120 200521143 A8 B8 C8 D8 Application for a patent scope 70 The hydroxyalkyl starch derivative in Item 42 printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs, where compound (I) The reaction product with the compound (II) is reacted with at least one other compound via at least one functional group X contained in the compound (II), or wherein the compound (II) is reacted with at least one other compound via at least one functional group X. The reaction is performed before the reaction with the compound VII, and at least one other compound is a crosslinked compound and the reaction product of the compounds (I) and (Π) and the reaction product of the crosslinked compound are reacted with a polypeptide. For example, the pharmaceutical composition in item 69 of the patent application park, wherein the winnowing system is erythropoietin. -121, ▲ minutes