TWI352120B - A method of using gs as a selectable marker in glu - Google Patents

Description

1352120 九、發明說明： 【發明所屬之技術領域】 …本發明係關於新穎的能夠產生蛋白質和能夠生長在無 ，穀乳酿胺培養基之穀氨醒胺營養缺陷的人類細胞。此外， ^其，關於新賴的產生蛋白質的方法和關於在穀氨酿胺營養 、 類』胞中把榖氨醯胺合成酶（GS)當作可選擇標記 的用途。 ° Φ 【先前技術】 由哺乳動物細胞培養所產生的蛋白質係用來提供治療 和。，斷應用的蛋白質。現今，哺乳動物細胞培養是用於人 類和動物醫藥之許多重要蛋白質的較佳來源，尤其那些相 當大的、複雜的和糖基化的（N. B. Finter等人，在大規模 哺乳動物細胞培養技術，1990， ed. A. S. Lubiniecki » Marcel Dekker > Inc.，紐約）。 例如’藉細胞培養產生人類蛋白質紅血球生成素（Ep〇) • 已在WO 93/09222中描述。使用以可編碼人類EP〇外生基 因轉染的人類纖維組織母細胞，人類EP◦已獲得可觀的比 生成速率。在進一步產生人類EPO的方法（WO 94/12650)， 係使用以能夠活化内生編碼的EPO之DNA序列轉染的人 類纖維肉瘤細胞系HT1080。類似的方法已於WO 99/09268 中描述。永生的（Immortalised)人類細胞如Namalwa、Hela S3 及HT 1 080細胞係以能夠活化内生編碼的EPO之DNA序 列轉染。 5 1352120 描述於 WO 93/09222、WO 94/12650 及 WO 99/09268 用 來產生人類EPO的細胞係都在含有榖氨醯胺的培養基中培 養。這是不利的’因為當榖氧酿胺被培養的細胞當作能量 基質時，會產生氨這種代謝產物，其具有細胞毒性及會抑 制細胞生長。再者，因為它在細胞的高基氏體内對pH的 影響，而會抑制蛋白質糖基化。 產生大量組織血漿素原活化劑（一種糖基化蛋白質）已 在WO 87/04462中描述。GS在其中已被用來作為放大系 統，在榖氨醯胺原質營養的（prototrophic)中國倉鼠卵巢 (CHO)細胞用於共放大可編碼組織血漿素原活化劑（tPA)的 基因’其係藉由以可編碼G S的基因轉染細胞。然而，如 同在EP-A 148 605發現的，用CHO細胞來產生糖基化的 人類蛋白質係不利的。由CHO細胞合成的蛋白質其平均 碳水化合物成分會與自然生成的糖基化人類蛋白質有所不 同，這是因為人類細胞含有α2·3唾液酿基轉移酶 (sialyltransferase)及α2.6唾液醯基轉移酶酵素。CHO細胞 只具有α2.3唾液醯基轉移酶，所以不能產生α2.6端唾液醯 基與寡糖部分（moieties)的鍵結，CHO細胞缺乏硫化 (sulphation)碳水化合物結構的酵素，CHO細胞雖然有〇cl-6 巖藻糖轉移酶（fucosyltransferase)(附著中心的巖藻糖殘 基），亦缺乏al-3巖藻糖轉移酶（附著末端的巖藻糖殘基）。 人類細胞則具有兩者嚴蕩糖轉移酶（Cumming D.Α.，1 991， GlycobiologyVo. 1，No. 2，115 - 130，JenkinsN·及 Curling E.M.A.，1 994，enzyme and Micorbial Technology Vol· 1 6， 1352120 354-364 ; Lee 等人，1989 ’ Journal of Biological Chemistry，V〇1 264，13848· 13855)。所以，由CHO細胞合成之糖基化的蛋白質不會 '有希望得到的特徵’例如如於人類細胞產生的活體内生物 活性。 ♦ 在WO 89/10404中，報告了製造骨髓瘤細胞例如鼠 融合瘤、鼠漿細胞瘤細胞及大鼠融合瘤細胞榖氨醯胺獨立 的方法，其藉由以GS轉化他們。其進一步證明GS可以用 •來共放大可編碼免疫球蛋白分子輕及重鏈的基因及用來共 放大在骨髓瘤細胞系中可編碼纖維蛋白分解酵素的基因。 然而，齧齒類細胞系具有缺點，即以N•乙醇醯基神經氨酸 殘基的附著代替N-乙酿神經氨酸、無法進行硫化及以丄3半 乳糖轉移酶酵素的存在。在齧齒類細胞中合成之糖蛋白的 寡糖結構因此可以在人類產生免疫性。 【發明内容】 本發明之目的係提供一種改進的方法，其不具有用於 產生蛋白質時的上述缺點（尤其當產生糖蛋白時），及可獲 得南蛋白質效價。 根據本發明，可得到榖氨醯胺營養缺陷之人類細胞， 其係以（第一）可編碼蛋白質之外生DNA序列或能夠改變可 編碼蛋白質之内生基因表現的外生DNA序列轉染，及進 一步以（第二）可編碼榖氨醯胺合成酶（GS)(較佳的為哺乳動 物GS)之外生DNA序列轉染，其中這些外生的dna序列 7 1352120 係位於-或-個以上DNA構築體，該轉染的 生該蛋白質及能夠生長在無榖氨醯胺的培養基上。 DNA序列 蛋白m白質的外生亀序列"或"能夠改變可編碼 :白質之内生基因表現的外生DNA序列"及” 位於_構築體上， 感染性㈣。質體可以用來作為表現載體。1352120 IX. Description of the Invention: [Technical Field to Which the Invention Is Applicable] The present invention relates to novel human cells capable of producing proteins and auxotrophs which are capable of growing in the glutamine-free amine medium. In addition, ^, it relates to the method of protein production by Xinlai and the use of guanamine-synthesizing enzyme (GS) as a selectable marker in glutamine-rich nutrition. ° Φ [Prior Art] Proteins produced by mammalian cell culture are used to provide treatment and . , break the applied protein. Today, mammalian cell culture is a preferred source of many important proteins for human and animal medicine, especially those that are quite large, complex, and glycosylated (NB Finter et al., in large-scale mammalian cell culture techniques, 1990, ed. AS Lubiniecki » Marcel Dekker > Inc., New York). For example, 'human cell erythropoietin (Ep〇) produced by cell culture has been described in WO 93/09222. Using human fibroblasts that are transfected with human EP 〇 exogenous genes, human EP ◦ has achieved considerable rate of specific production. In a method for further producing human EPO (WO 94/12650), a human fibrosarcoma cell line HT1080 transfected with a DNA sequence capable of activating endogenously encoded EPO is used. A similar method has been described in WO 99/09268. Immortalised human cells such as the Namalwa, Hela S3 and HT 1 080 cell lines are transfected with a DNA sequence capable of activating endogenously encoded EPO. 5 1352120 Cell lines for the production of human EPO described in WO 93/09222, WO 94/12650 and WO 99/09268 are all cultured in a medium containing guanamine. This is unfavorable because the ammonia-derived cell produces a metabolite of ammonia which is cytotoxic and inhibits cell growth when the cultured cells are used as an energy matrix. Furthermore, it inhibits protein glycosylation because of its effect on pH in the high-kilogram of cells. The production of a large amount of tissue plasminogen activator (a glycosylated protein) has been described in WO 87/04462. GS has been used as an amplification system in prototrophic Chinese hamster ovary (CHO) cells for co-amplification of genes encoding tissue plasminogen activator (tPA). The cells are transfected with a gene encoding GS. However, as found in EP-A 148 605, the use of CHO cells to produce glycosylated human proteins is disadvantageous. The average carbohydrate component of proteins synthesized by CHO cells differs from naturally occurring glycosylated human proteins because human cells contain α2·3 salivary sialyltransferase and α2.6 salivary thiol transfer. Enzyme enzyme. CHO cells only have α2.3 salivary thiotransferase, so they cannot produce linkages between α2.6-terminal salivary sulfhydryl groups and oligosaccharide moieties. CHO cells lack enzymes that sulphate carbohydrate structure, although CHO cells There is a cl-6 fucosyltransferase (fucose residue at the center of attachment) and an al-3 fucosyltransferase (fucose residue attached to the end). Human cells have both sugar transferases (Cumming D.Α., 1 991, Glycobiology Vo. 1, No. 2, 115-130, Jenkins N· and Curling EMA, 994, enzyme and Micorbial Technology Vol. , 1352120 354-364; Lee et al., 1989 'Journal of Biological Chemistry, V〇1 264, 13848·13855). Therefore, glycosylated proteins synthesized by CHO cells do not have 'promising characteristics' such as, for example, in vivo biological activity produced by human cells. ♦ In WO 89/10404, methods for making myeloma cells, such as murine fusion tumors, murine plasmacytoma cells, and rat fusion tumor cells, are reported as being independent by GS. It further demonstrates that GS can be used to amplify genes encoding light and heavy chains of immunoglobulin molecules and to amplify genes encoding fibrinolytic enzymes in myeloma cell lines. However, rodent cell lines have the disadvantage that N-ethyl-neuraminic acid is replaced by the attachment of N•ethanol-neuraminic acid residues, vulcanization is not possible, and the presence of 丄3 galactosyltransferase enzyme is present. The oligosaccharide structure of glycoproteins synthesized in rodent cells can thus be immunogenic in humans. SUMMARY OF THE INVENTION An object of the present invention is to provide an improved method which does not have the above disadvantages for producing a protein (especially when a glycoprotein is produced), and which can obtain a south protein titer. According to the present invention, a glutamine-deficient human cell can be obtained which is transfected with a (first) exogenous DNA sequence encoding a protein or an exogenous DNA sequence capable of altering the expression of an endogenous gene encoding a protein, And further transfected with an exogenous DNA sequence encoding a (second) glutamine synthase (GS), preferably a mammalian GS, wherein the exogenous dna sequence 7 1352120 is located at - or - The above DNA construct, which is transfected with the protein and can be grown on a medium without amidoxime. The exogenous sputum sequence of the DNA sequence protein m white matter" or " can change the exogenous DNA sequence that encodes the endogenous gene expression of the white matter" and is located on the _ construct, infectivity (4). The plastid can be used As a performance carrier.

載體則可以使用如反轉錄病毒、泡療病毒、腺病毒：腺病 毒伴隨病毒、流行性聪腺炎病毒及脊髓灰質炎病毒載體。 較佳的為表現載體，尤其使用質體。 "可編碼蛋白質的外生DNA序列”可以包括額外序列， 例如調控序列如啟動子及/或增強子、多聚腺苷酸區域 (P〇 丨 yadenylati〇n site)及剪接接頭（splice juncti叫通常 用於表現外生基因或可以包括額外_或多個分開的乾向性 (targeting)序列及視需要的可編碼可選擇的標記之dna(如 WO 93/09222中所描述）。 "能夠改變可編碼蛋白質之内生基因表現的外生dna 序列”可以包括外生的DNA序列，其不編碼蛋白質的基因 產物，但可編碼部分該基因產物（如外顯子），及可以包括 額外序列，例如調控序列及剪接接頭，通常用於表現外生 DNA序列。他們可以進一步包括乾向性序列及視需要的可 編碼可選擇的標記之DNA(如w〇93/〇9222中所描述 通吊’旎夠改變可編碼蛋白質之内生基因表現的外生 DNA序列"係在轉染進細胞後***細胞的染色體dna。同 8 1352120 源重組或乾向性係在此用來取代或使與含有調控序列之内 生基因相關的調控區域失去作用。作為調控序列可以使用 如啟動子及/或增強子，其造成基因表現量高於相對岸之盈 轉染的細胞（如W0 93/09222所描述），合適的啟動子可以 是可調控的或持續表現的啟動子。合適的啟動子可以是強 啟動子，其係取決於所使用的細胞系，如人類巨細胞病毒 主要立即早期啟動子（maj〇r immediate丨 iaic early promoter » hCMV-MIE)、SV 4〇早期及晚期啟叙$ ^ 丁4汉光期啟動子、其他腺病毒啟動 子、任何多瘤病毒或乳多泡病毒早期及晚期啟動子、干擾 素cxi啟動子、鼠金屬硫因（metall()thi()nein)啟動子、r_ 氏肉瘤病毒長端重複啟動子、β_珠蛋白啟動子、伴白蛋白 $動子4白蛋白啟動子、鼠β•珠蛋白啟動子及人類卜珠 蛋白啟動子。 根據本發明之，，可編碼GS之外生_α序列"可以受強 啟動子及弱啟動子的控制，如果外生的職序列係單單 需要表現可編㉙GS的基因，則使用強啟動子，當外生的 魏序列係作為可選擇的標記，以及若gs係用於放大作 用時’則使用弱啟動子。合適的啟動子可以是可調控的或 持續表現的啟動子。啟動子可以選擇，例如，仍係在足夠 使轉染的細胞生長的潰庚φ主 王长的/晨度中表現，但細胞培養時並不產生 高量穀氨酿胺代謝產物氨，通常不大於4 mM，較佳的不 大於2 mM ’更佳的少於2 mM氨。 "可選擇的標記”提供了可選擇的表現型，使鑑別及分 離接受㈣―叫細胞成為可能。GS可以在本發明用來當 1352120 作可選擇的標記，以筛選出成功轉染的穀氨酿胺營養缺陷 人類細胞，其併入和表現可編碼Gs的外生dna序列。 根據所用的細胞系，強啟動子可以是如hcMv侧、 SV 40早期及晚期啟動子、其他腺病毒啟動子、任何多瘤 =毒或乳多泡病毒早期及晚期啟動子、干擾素α1啟動子、 鼠金屬硫因（metaIl〇thionein)啟動子、R〇us肉瘤病毒長端 重複啟動子、β-珠蛋白啟動子、伴白蛋白啟動子、卵白蛋 白啟動子、鼠β-珠蛋白啟動子及人類卜珠蛋白啟動子。 根據所用的細胞系，弱啟動子可以是如鼠白血病病毒 長端重複、單純疱疹病毒胸苷激酶及鼠***腫瘤病毒長端 重複，較佳的，可編碼GS的基因係在強啟動子控制下，更 佳的係在hCMV-MIE啟動子控制下。一項可能的具體實 例，種來自倉鼠之可放大的（amplifiable)哺乳動物GS序 列及其在哺乳動物細胞中作為可選擇的標記的用途已為該The vector may be used, for example, a retrovirus, a bubble virus, an adenovirus: an adeno-associated virus, an epidemic adeno-inflammatory virus, and a poliovirus vector. Preferred are expression carriers, especially plastids. "exclusive DNA sequences encoding proteins" may include additional sequences, such as regulatory sequences such as promoters and/or enhancers, polyadenylation regions (P〇丨yadenylati〇n site), and splice junctions (splice juncti Typically used to represent exogenous genes or may include additional _ or multiple separate targeting sequences and optionally DNA-encoded selectable markers (as described in WO 93/09222). An exogenous DNA sequence that alters the expression of an endogenous gene encoding a protein may comprise an exogenous DNA sequence that does not encode a gene product of the protein, but may encode a portion of the gene product (eg, an exon), and may include additional sequences , for example, regulatory sequences and splice junctions, typically used to express exogenous DNA sequences. They may further comprise a tropic sequence and optionally a DNA encoding a selectable marker (eg, as described in w〇93/〇9222) 'Exogenous DNA sequences that change the expression of endogenous genes that encode proteins" are chromosomal DNAs that are inserted into cells after transfection into cells. Same as 8 1352120 Source recombination or isotropic lines This serves to replace or disable the regulatory regions associated with the endogenous gene containing the regulatory sequences. As a regulatory sequence, promoters and/or enhancers can be used, which result in a higher gene expression than the oppositely transfected cells. (As described in WO 93/09222), a suitable promoter may be a promoter that is regulated or sustained. Suitable promoters may be strong promoters depending on the cell line used, such as human giant cells. The virus is mainly immediate early promoter (maj〇r immediate丨iaic early promoter » hCMV-MIE), SV 4〇 early and late initiation of the ^ ^ 4 Hanguang promoter, other adenovirus promoters, any polyomavirus or Early and late promoters of papovavirus, interferon cxi promoter, mouse metallothionein (metall() thi() nein) promoter, r_sarcoma virus long-end repeat promoter, β-globin promoter, Albumin $ mover 4 albumin promoter, murine β• globin promoter and human globin promoter. According to the present invention, GS can be encoded as an exogenous _α sequence " can be strongly promoter and weak Promoter Control, if the exogenous sequence requires a gene that can be programmed to encode 29GS, use a strong promoter, when the exogenous Wei sequence is used as a selectable marker, and if the gs is used for amplification, then use weak Promoter. A suitable promoter may be a promoter that is regulated or sustained. The promoter may be selected, for example, to be expressed in a morning/morning degree sufficient to allow the transfected cells to grow, However, cell culture does not produce high amounts of ammonia in the glutamine amine metabolite, typically no greater than 4 mM, preferably no greater than 2 mM 'better than less than 2 mM ammonia. The "optional marker" provides an alternative phenotype that allows for identification and separation to accept (4) - called cells. GS can be used in the present invention as a selectable marker for 1352120 to screen for successfully transfected valleys. Amine-lactam auxotrophic human cells that incorporate and express exogenous DNA sequences encoding Gs. Depending on the cell line used, strong promoters may be, for example, the hcMv side, the SV 40 early and late promoters, and other adenoviral promoters. , any multi-tumor = virulence or papovavirus early and late promoter, interferon alpha 1 promoter, murine metallothionein (metaIl〇thionein) promoter, R〇us sarcoma virus long-end repeat promoter, β-globin Promoter, accompanied by albumin promoter, ovalbumin promoter, murine β-globin promoter and human globin promoter. Depending on the cell line used, the weak promoter may be a long-end repeat such as murine leukemia virus, herpes simplex The viral thymidine kinase and the murine mammary tumor virus are long-end repeats. Preferably, the gene encoding the GS is under the control of a strong promoter, and more preferably under the control of the hCMV-MIE promoter. Specific examples of use, species (amplifiable) sequence derived from a mammal can be amplified GS hamster and in mammalian cells has been used as a selectable marker for

項技術所熟知，且如WO 87/04462、WO 91/06657及WO 89/0 1 036所描述；本發明實施例使用此倉鼠gs表現單元 及個別的篩選方法如參考文獻所提及。 ”可編碼蛋白質之外生DNA序列"或"能夠改變可編碼 蛋白質之内生基因表現的外生DNA序列"及"可編碼GS之 外生DNA序列”係位於一或多於一個DNA構築體上，較佳 的’這些外生的DNA序列係位於多於一個，更佳的位於 兩個DNA構築體上。若這些外生的DNA序列位於一個 DNA構築體上，他們可能功能上相組合’例如他們的表現 會受同一個調控序列，如啟動子及/或增強子（如w〇 1352120 89/10404所描述）驅策。 人類細胞 榖氨醯胺營養缺陷人類細胞係指所有不表現GS或表 . 現GS很差的人類細胞，所以能夠生長在含有榖氨醯胺的 v 培養基，但在無榖氨醯胺培養基不能生長或生長很差。用 於本發明之榖氨醯胺營養缺陷人類細胞係必死的榖氨醯胺 營養缺陷人類細胞或永生的榖氨醯胺營養缺陷人類細胞。 必死的榖氨醯胺營養缺陷人類細胞係在培養基中具有有限 I 哥命的榖氣酿胺營養缺陷人類細胞’永生的（又稱永久的或 建立的）榖氨醯胺營養缺陷人類細胞係在培養基中如習於該 項技術者所熟知以適當地繼代培養和次培養，具有明顯無 限壽命之榖氨醯胺營養缺陷細胞。 必死的榖氨醯胺營養缺陷人類細胞的例子可以是人類 纖維組織母細胞及人類胎肺組織細胞，永生的榖氣醯胺營 養缺陷人類細胞的例子可以是人類纖維肉瘤細胞，如 HT1080 細胞系（如 DSMZNo. ACC-315 或 ATCCNo. CCL 121)及B-淋巴母細胞人類細胞如HL60 (DSMZ No. Acc-3) 或Namalwa (DSMZ Acc-24)細胞系。用於本發明較佳的係 永生的榖氨醯胺營養缺陷人類細胞，更佳的，此永生的榖 氨醯胺營養缺陷人類細胞係用B-淋巴母細胞或纖維肉瘤細 胞’更佳的係用人類纖維肉瘤細胞’最佳的係用ΗΤ1080 細胞系（如 ATCC No. CCL 121)。 轉染 榖氨醯胺營養缺陷人類細胞可藉由已知的基因工程技 1352120 術以外生的DNA序列轉染。 以外生的DNA序列轉染取決於序列是否位於一或多 於一個DNA構築體上’若序列位於多於一個DNA構築體 上’轉染可隨各序列分別發生或共轉染。當轉染隨各序列 分別發生時，序列轉染順序通常是隨意的，隨各序列分別 發生的轉染較佳先以"可編碼該蛋白質的外生DNA序列" 或"能夠改變可編碼該蛋白質之内生基因表現的外生DNA 序列”轉染，然後再以”可編碼GS的外生DNA序列"轉染， 轉染的穀氨醯胺營養缺陷細胞可以在各別轉染之後培養並 评估蛋白質生成。 為了成功篩選轉染的細胞’這些係生長在無榖氨醯胺 的培養基。細胞可以直接生長在無榖氨醯胺培養基或先 在含有榖氨醯胺的培養基，然後逐步稀釋成無榖氨醯胺的The art is well known and described in WO 87/04462, WO 91/06657 and WO 89/0 1 036; the present invention uses this hamster gs expression unit and individual screening methods as mentioned in the references. An exogenous DNA sequence that encodes a protein-exclusive DNA sequence"or" an exogenous DNA sequence that can alter the expression of an endogenous gene encoding a protein" and a codeable GS-existing DNA sequence is located in one or more than one Preferably, these exogenous DNA sequences are located on more than one DNA construct, and more preferably on two DNA constructs. If these exogenous DNA sequences are located on a DNA construct, they may be functionally combined 'eg their expression will be subject to the same regulatory sequence, such as a promoter and/or enhancer (as described by w〇1352120 89/10404) ) Drive. Human cell glutamine auxotrophic human cell line refers to all human cells that do not express GS or GS. Therefore, they can grow in v medium containing guanamine, but can not grow in glutamine-free medium. Or growing very poorly. The indoleamine auxotrophic human cell line of the present invention is a mortal guanamine derivative auxotrophic human cell or immortal guanamine-deficient human cell. The mortal guanamine-deficient human cell line has a limited I sputum in the culture medium. The eutrophication of human cells is 'immortal (also known as permanent or established) glutamine auxotrophic human cell line The media is well known to those skilled in the art to suitably subculture and subculture, with a significantly infinite life span of guanamine-deficient cells. Examples of mortal guanamine-deficient human cells may be human fibroblasts and human fetal lung tissue cells, and examples of immortal sputum guanamine-deficient human cells may be human fibrosarcoma cells, such as the HT1080 cell line ( For example, DSMZ No. ACC-315 or ATCC No. CCL 121) and B-lymphocyte human cells such as HL60 (DSMZ No. Acc-3) or Namalwa (DSMZ Acc-24) cell lines. Preferred for use in the present invention is an immortalized guanamine-deficient human cell, and more preferably, the immortal guanamine-deficient human cell line uses a better system of B-lymphocyte or fibrosarcoma cells. The best line for human fibrosarcoma cells is the ΗΤ1080 cell line (eg ATCC No. CCL 121). Transfection of indoleamine auxotrophic human cells can be transfected by DNA sequences that are known to be genetically engineered to use the genetic engineering technique 1352120. Transfection of an exogenous DNA sequence depends on whether the sequence is located on one or more DNA constructs. If the sequence is located on more than one DNA construct, transfection can occur separately or co-transfect with each sequence. When transfection occurs with each sequence, the sequence of transfection is usually random, and the transfection with each sequence is preferably preceded by "external DNA sequences encoding the protein" or " The exogenous DNA sequence encoding the endogenous gene expression of the protein is transfected and then transfected with the GS-expressing exogenous DNA sequence & transfected, transfected glutamine-deficient cells can be transfected separately Protein production is then cultured and evaluated. In order to successfully screen for transfected cells, these lines were grown in a medium without amidoxime. The cells can be grown directly in the medium without guanamine or in a medium containing guanamine, and then gradually diluted into no guanamine.

培養基，如先以榖氨醯胺濃度1 〇 mM，然後以2 mM至0 mM 逐步稀釋。合適的篩選方法可以根據所用的細胞系來選 擇。如前述顯知’本發明能夠產生蛋白質及能夠生長在無 榖氨酸胺培養基的榖氨醯胺營養缺陷人類細胞係得自以可 編碼該蛋白質的外生DNA序列或能夠改變可編碼蛋白質 之内生基因表現的外生DNA序列及可編碼榖氨醯胺合成 酶之外生DNA序列轉染該細胞，其中這些外生的DNA序 列係位於一或多於一個DNA構築體上。 放大 可編碼蛋白質的外生DNA序列或能夠改變可編碼蛋 白質之内生基因表現的外生DNA序列可以在轉染後根據 12 1352120 生長因子（EGF)及纖維組織母細胞生長因子（Fgf)家於、 糖基化生長因子及無糖基化荷爾蒙及生長因子受體。…、 糖基化蛋白質的例子係荷爾蒙及荷爾蒙釋放因子， 生長荷爾蒙，包括人類生長荷爾蒙、牛生長荷爾蒙生長 荷爾蒙釋放因子、副甲狀腺素、甲狀腺刺激素、 匕 u、月曰 蛋白質、α -1-抗胰蛋白酶、***、降鈣素、黃體生 成素、騰增血糖素、凝血因子如因子vine、田工 口于IX、組 織因子及von Willebrand氏因子、抗凝血因子例如蛋白質 C、心房利鈉因子、肺表面活性物質、血漿素原活化劑例 如尿激酶或人類尿或組織型血漿素原活化劑（t_pA)、凝血 酶、造血生長因子 '腦啡肽、RANTES(調控活化自然的丁_ 細胞表現及分泌）、人類巨噬細胞發炎蛋白質（Μα」· α )、 血清蛋白素例如人類血清蛋白素、muUerian氏抑制物質、 鬆弛素A鏈、鬆弛素b鏈、前鬆弛素、鼠***相關 肽、微生物蛋白質例如石-lactanase、DNase、抑制素、激 活素、腎素、血管内皮生長因子（VEGF)、荷爾蒙或生長因 子文體、整合素、蛋白質A或D、類風濕性因子、神經營 養因子例如源自骨神經營養因子（BDNF)、神經營養素 (neUr〇tr〇Phin).3、_4、-5 或-6 (NT-3、NT-4、NT-5 或 NT- 6)、或神經生長因子例如NGF_P、源自血小板生長因子 (PDGF)、纖維組織母細胞生長因子例如fgf及bFGF、表 皮生長因子（EGF)、轉化生長因子（TGF)例如TGF- α及TGF- 冷，包括 TGF-βΙ、TGF-p2、TGF-p3、TGF-P4 或 TGF-P5、 類胰島素生長因子d及_„ (WFd及IGF II)、des(1_3)_IGF_ 14 1352120 I (腦IGF-I)、類胰島素生長因子結合蛋白、cd蛋白（一群 分化蛋白質）例如CD-3、CD-4、CD-8及CD-19、骨誘導因 子、免疫毒素、骨形態發生蛋白質（BMP)、細胞活素及其 受體，還有嵌合（chimeric)蛋白質，包含其受體的細胞活素， 包括例如腫瘤壞死因子α及冷、他們的受體（TNFR_丄，Ep 41 7 563及TNFR-2， £P 41 7 014)及其衍生物，一種干擾 素例如干擾素-α、-万、及_ 7，細胞族群刺激因子（CSFs)， 如 M CSF、GM-CSF 及 G-CSF ’ 白介素（iLs)如 IL-1 到 IL· 10 ’超氧化物歧化酶，T·細胞受體、纟面膜蛋白、降解加 速因子、病毒抗原例如aids夾膜的一部份、運輸蛋白、 導引（homing)受體' addressins、調控蛋白質、抗體、嵌合 蛋白如免疫黏連素，及任何上述所列片段之糖基化蛋白 質。較佳的’本發明產生糖基化蛋白質，更佳的本發明產 生N糖基化蛋白質，最佳的糖基化荷爾蒙像π。為N_糖 基化及其生物活性係依賴其上，或尤其產生Ep〇。 細胞培養 任何於該項技術已知之合適的培養程序及培養設備均 :用來生長本發明轉染的人類細胞。添加約〇1至判較 佳〇·5 i 15 %)血清之常見無榖氨酸胺基礎培養基以及益 血清無榖氨醯胺常見基礎培養基都可作為培養基用，此 ^，也可用不含動物來源蛋白質的常見無榖氨醯胺基礎培 、基’較佳的使用無血清無榖氨醯胺常見基礎培養基。 可以用的血清如胎牛Α清或成牛血清，較佳係使用胎 血清。常見之可用的無榖氨醯胺基礎培養基為，例如無 15 1352120 榖氨酿胺Eagle氏最低基本培養基（MEM)培養基、無榖氨 酿胺Dulbecco氏修飾的Eagle氏培養基（DMEM)、無榖氨 酿胺 Iscove 氏 DMEM 培養基（N. Iscove 及 F. Melchers， Journal of Experimental Methods，1978，147，923)、無榖 氨醯胺 Ham 氏 F12 培養基（R.G. Ham，Proceedings of National Academy of Science，1965，53 5 288)、無穀氨 酿胺 L-15 培養基（A. Leibovitz，American Journal of Hygiene，1963，78， 173)、無榖氨醯胺 RPMI 1640 培養The medium, for example, is first diluted with guanamine as 1 〇 mM and then gradually diluted at 2 mM to 0 mM. Suitable screening methods can be selected depending on the cell line used. As described above, it is apparent that the present invention is capable of producing a protein and a guanamine-deficient human cell line capable of growing in a proline-free amine medium derived from an exogenous DNA sequence encoding the protein or capable of altering the encodeable protein. The exogenous DNA sequence of the gene expression and the exogenous DNA sequence encoding the indoleamine synthase are transfected into the cell, wherein the exogenous DNA sequences are located on one or more than one DNA construct. Amplification of an exogenous DNA sequence encoding a protein or an exogenous DNA sequence capable of altering the expression of an endogenous gene encoding a protein can be transfected according to 12 1352120 Growth Factor (EGF) and Fibroblast Growth Factor (Fgf) , glycosylation growth factors and aglycosylated hormones and growth factor receptors. ..., examples of glycosylated proteins are hormones and hormone releasing factors, growth hormones, including human growth hormone, bovine growth hormone, hormone release hormone, parathyroid hormone, thyroid stimulating hormone, 匕u, sputum protein, α -1- Antitrypsin, follicle stimulating hormone, calcitonin, luteinizing hormone, granulocyte, clotting factors such as factor vine, tiangongkou in IX, tissue factor and von Willebrand's factor, anticoagulant factors such as protein C, atrium Natriuretic factors, pulmonary surfactants, plasminogen activators such as urokinase or human urine or tissue plasminogen activator (t_pA), thrombin, hematopoietic growth factor 'enkephalin, RANTES (regulatory activation of natural Ding _ cell expression and secretion), human macrophage inflammatory protein (Μα"· α ), serum albumin such as human serum albumin, muUerian inhibitor, relaxin A chain, relaxin b chain, pre-relaxanthin, rat Gonadal hormone-related peptides, microbial proteins such as stone-lactanase, DNase, inhibin, activin, renin, vascular endothelial growth Sub- (VEGF), hormone or growth factor stroma, integrin, protein A or D, rheumatoid factor, neurotrophic factor, for example, derived from bone neurotrophic factor (BDNF), neurotrophin (neUr〇tr〇Phin). _4, -5 or -6 (NT-3, NT-4, NT-5 or NT-6), or nerve growth factors such as NGF_P, derived from platelet growth factor (PDGF), fibroblast growth factor such as fgf and bFGF, epidermal growth factor (EGF), transforming growth factor (TGF) such as TGF-α and TGF-cold, including TGF-βΙ, TGF-p2, TGF-p3, TGF-P4 or TGF-P5, insulin-like growth factor d And _„ (WFd and IGF II), des(1_3)_IGF_ 14 1352120 I (brain IGF-I), insulin-like growth factor binding protein, cd protein (a group of differentiated proteins) such as CD-3, CD-4, CD- 8 and CD-19, osteoinductive factors, immunotoxins, bone morphogenetic proteins (BMPs), cytokines and their receptors, as well as chimeric proteins, including cytokines of their receptors, including, for example, tumors Necrosis factor alpha and cold, their receptors (TNFR_丄, Ep 41 7 563 and TNFR-2, £P 41 7 014) and their derivatives An interferon such as interferon-α, -10,000, and _ 7, cell population stimulating factors (CSFs), such as M CSF, GM-CSF, and G-CSF 'interleukins (iLs) such as IL-1 to IL·10 'super Oxide dismutase, T·cell receptor, 纟 mask protein, degradation accelerating factor, viral antigen such as a part of aids sandwich, transport protein, homing receptor 'addressins, regulatory proteins, antibodies, chimerism Proteins such as immunoadhesin, and glycosylated proteins of any of the above listed fragments. Preferably, the invention produces a glycosylated protein, and more preferably the invention produces an N-glycosylated protein with an optimal glycosylated hormone like π. It is dependent on, or in particular produces, Ep glycosylation for N-glycosylation and its biological activity. Cell Culture Any suitable culture procedure and culture equipment known in the art is used to grow the transfected human cells of the present invention. Common non-proline amine basal medium supplemented with serum from about 〇1 to 〇5 5 15%) and common basal medium with no serum sulphate can be used as a medium. A common sputum-free sulphate-based basal medium of the source protein is preferably a serum-free sputum-free guanamine. Serum which can be used, such as fetal calf serum or bovine serum, is preferably a fetal serum. Commonly available guanamine-free basal mediums are, for example, no 15 1352120 榖 酿 E E Eagle's Minimum Essential Medium (MEM) medium, no hydrazine-enriched Dulbecco's modified Eagle's medium (DMEM), no hydrazine Iscove's DMEM medium (N. Iscove and F. Melchers, Journal of Experimental Methods, 1978, 147, 923), glutamine-free Ham's F12 medium (RG Ham, Proceedings of National Academy of Science, 1965, 53) 5 288), glutamine-free amine L-15 medium (A. Leibovitz, American Journal of Hygiene, 1963, 78, 173), glutamine-free RPMI 1640 culture

基（G.E. Morre 專人，The Journal of the American MedicalG.E. Morre, The Journal of the American Medical

Association ’ 1967，199，519)、無榖氨醯胺專屬培養基和 其合適比例之混合物。用於高密度細胞培養之強化 (Fortification)常見細胞培養生長培養基已在該項技術所熟 知，且已描述於如GB 2251249，其也非常適合作為本發明 之無榖氨醯胺培養基。 基。常 對細胞 中的蛋 可以對 大豆脂 以依所 酸、白 榖胺酸 加至1j常 見的添加物可以加到常見的無榖氨醯胺基礎培養 加入的添加物包括血清中的蛋白質以及視需要可以 生長及/或細胞生存有正面影響之進一步成分。血清 白質如牛血清白蛋白（BSA)、運鐵蛋白及/或胰島素； 細胞生長及/或細胞生存有正面影響之進一步成分如 西及乙醇胺。取代榖氨醯胺及/或核苦的胺基酸可 用的細胞系加到培養基中’胺基酸的例子如異白胺 胺酸、纈胺酸、離胺酸、天冬醯胺、天門冬胺酸、 、絲胺酸、丙胺酸。視需要’榖氨醯胺可以低濃度 見的無榖氨醯胺基礎培養基中’通常少於丄mg/1， 16 1352120 干又压的，於υ·5 mg/】，以支持其生物合成作用（如轉胺反 應）。 若可編碼蛋白質的外生DNA序列或能夠改變可編碼 蛋白貝之内生基因表現的外生DNA序列在轉染後以可放 大的（amphfiable)基因放大，相關的選擇劑可以加到常見 的無榖氨醯胺基礎培養基中，選擇劑施用的濃度範圍依所 用的細胞系而定，通常用1〇 μΜ及更高的濃度。 懸浮生長於無血清培養基之適應作用 榖氨醯胺營養缺陷人類細胞可以是固著依賴或固著獨 立，其可以作為起始材料來獲得根據本發明之轉染的榖氨 醯胺營養缺陷人類細胞，根據本發明，該轉染的細胞能夠 產生蛋白質及能夠生長在無榖氨醯胺培養基。若使用固著 依賴人類細胞，如ΗΤ1080細胞系（ATCC No. CCL· 121)， 其可以適應成為固著獨立HT1〇8〇細胞系，能夠懸浮生長 於無血清培養基，尚未於文獻中描述。 適應作用可以發生在以可編碼蛋白質的外生DNA序 列或旎夠改變可編碼蛋白質之内生基因表現的外生dna 序列及可編碼GS的外生DNA序列轉染之前或之後。較佳 的，細胞先以可編碼蛋白質的外生DNA序列或能夠改變 可編碼蛋白質之内生基因表現的外生DNA序列轉染，再 使其適應懸洋生長於無血清培養基，然後進一步以可編碼 GS之外生DNA序列轉染。若需要，轉染的細胞可以再使 其適應懸浮生長於無血清無榖氨醯胺的培養基。 本發明之轉染的榖氨醯胺營養缺陷人類細胞可以是固 17 著依賴或固著獨立， 的培養義^ 並月b夠懸子生長於無血清無榖氨醯胺 著獨Γ :的轉染的穀氨醒胺營養缺陷人類細胞係固 ⑽夠懸子生長在無血清無㉟氨酿胺的培養基。 著猶^應作用使其成為能夠懸浮生長於無血清培養基之固 細胞而：以藉由在第一步用含有▲清的培養基適應 、。14可以經由如姨蛋白酶處理細胞，然後接著 動或猎搖動釋放細胞來達成。細胞然後在第二步藉接著 減少血清含量來適應無血清培養基，在適應時，若^用選 擇劑，可以減少其用量’以避免抑制細胞生長、然而，細 胞也可以在第-步藉由接著減少血清含量適應生長於無血 清培養基，第二步適應成為能夠懸浮生長的固著獨立細 胞，藉由如胰蛋白酶處理細胞然後接著搖動或藉搖動釋放 細胞來達成，兩個步驟也可以同時施行。 較佳的，細胞係在第一步含血清培養基適應成為固著 獨立細胞用，藉由搖動釋放細胞然後第二步經由接著減少 血清含量來適應於無血清培養基。 如上所描述的培養基可以做為含血清培養基的基本。 在此所定義之選擇劑可以加到培養基中，選擇劑施用的濃 度範圍取決於所用的細胞系’通常使用1 〇 μΜ及更高濃度。 含有血清的培養基通常添加約〇. 1到20 %，較佳的〇 2到 10 %，最佳的〇.5到5 %血清’可以使用的血清如上所提 及。接著減少血清含量可以藉由逐步減少血清含量，例如 從10 %到1 %到0 %來達成。 1352120 本發明轉染的榖氨酿胺營養缺陷人類細胞係用在產生 蛋白貝的方法令，藉由在適合用來表現該蛋白質及取得 0-C〇vering)該蛋白質的條件下之培養基培養該細胞，產生 的蛋白質係描述如上。如上所描述之常見的無榖氨醯胺基 礎培養基及常見的添加物可以作為培養基，合適的培養條 件係如WO 96/39488所描述之習知的用於哺乳動物細胞體 外培養者。 蛋白質取得 蛋白質可以從細胞培養物中以習知的分離技術分離， 例如免疫親和或離子交換管柱分劃、沉澱、逆相Hplc、 色層分析、色層焦集法、SDS-PAGE、膠體過濾。習於該 項技術者應了解，適合用於有興趣的多肽之純化方法，可 能需要根據多肽在重組細胞培養中表現的特性來做修飾。 【實施方式】 實施例1 準備人類鐵維肉瘤細胞系HT1080-R223 含有多人類EPO基因複製（copies)的固著依賴人類 HT1080-R223細胞系是產業上用來生產EPO的細胞，一開 始係由 Transkaryotic Therapies 公司所創造（劍橋，MA 02139 (US))，其係源自固著依賴人類纖維肉瘤HT 1080細胞系。 親本HT1080細胞系（ATCC No. CCL 121)具有產生EP0的 能力，其係藉由以類似DNA構築體pREP018(描述於WO 95/3 1 560)的 DNA 構築體 pREP022 轉染，除了 DHFR 基 1352120 因係在相反方向以及pREP022含有比pREP018少約6〇〇 鹼基對同源序列。此細胞系接下來將以R223細胞系提及。 實施例2 R223細胞系的適應作用以飽夠懸浮生長於無血清培養基中 藉由騰蛋白酶處理釋放在靜置瓶中以附著培養生長的 細胞，懸浮於專屬（proprietory)含有榖氨醯胺之無血清培 養基（進一步以“HM9”提及），添加1〇 %透析的胎牛血清 (dFBS)及500 nM MTX，然後以和震盪瓶培養。生長在六 天後開始，細胞次培養到同樣的培養基中（圖1}。一旦可 仏的生長模式建立了’就減少培養基的血清含量至1 %， 再一次，可信的生長可在添加的血清完全用盡之前被再建 立。 使添加血清和無血清的培養物過度生長以評估產量， 結果顯示於表丨，並附有附著培養的數據，足夠的適應作 用之後，懸浮培養的比生長速率（即使沒有血清）與添加血 清之附著培養一樣。 在添加血清的培養基中從附著培養到懸浮培養時的適 應作用，EP◦合成比速率下降5Q%，從24爿12Ευ/ι〇6細 胞/小時，然而，此速率在適應無血清生長之後，大體上會 恢復至18 EU/106細胞/小時。 20 1352120 表1 在適應無血清培養基之前或之後，R223鉍胞系在附著培養 及懸浮培養的生長及產董。 最大細胞 數/mL X 10·6 比生長速 率h·1 累積細胞 h/ml X 10·6 EPO EU/ml q epo EU/106 細 胞/h 附著培養 (DMEM + 10 % dFBS 500 nM MTX) 不適用 0.0157 不適用 不適用 24 懸浮培養（HM9 + 10 % dFBS 500 nM MTX) 1.4 0.0185 334 4199 12 懸浮培養（HM9 (無血清）500 nM MTX) 1.1 0.0166 210 3600 18 實施例3 HT1080 (ATCC CCL121)的適應作用以能夠懸浮生長在無 血清培養基中 HT1080 細胞系 ATCC CCL121 是從 American Type culture Collection (Rockville， Mary land，US A)取得，一 開始細胞係以附著培養生長於含有10 %胎牛血清（FBS)的 DMEM 中。 懸浮和無血清的適應作用可以根據圖2的程序進行。 為了開始懸浮培養，細胞係藉由胰蛋白酶處理從附著培養 中釋放，而釋放的細胞再懸浮於加了 2 % FBS的HM9， 21 1352120 绝些接著以震盪培養，一旦細胞在加了 2%fbs的hm9 的懸浮生長建立了，以相同的培養基稀釋培養物作子代培 養，可信的ΗΤ1080細胞系懸浮生長在3()天後建立（圖 3)。在那之後培養基血清含量減少，最後完全不見❶細胞 持續在無血清中繼續次培養生長。 資施例4 φ 着•對R223細胞系生長及產量的影響 當以榖氨醯胺作為能量基質時，氨是培養的細胞產生 的代謝產物，具有細胞毒性並會抑制細胞生長，除此之外， 它會藉由對細胞高基氏體pH的影響’抑制蛋白質糖基化。 在瓶中無供應之培養的R223細胞通常產生5 氨，在有 養分供應之發酵槽培養則產生1〇 mM。 f 一開始評估氨的影響時，R223細胞係生長在震盡瓶 中，沒有加氨或加氨（2、5或1〇 mM)，重複培養之各氨濃 _ 度係在不同的PH值下得到，藉改變施加氣體之c〇2含量。 此處之主要目標係、決定氨對生長抑制的程冑，及測試減低 的PH能不能克服此生長抑制。 氨被發現會抑制細胞生長（表2)，而減低的pH並不 能緩和氨所造成的抑制作用，雖然用來減少？1^的升高之 pC〇2本身可能造成一些生長抑制。 培養在僅3天後結束，加長培養是無效的，因為不揮 發性的酸代謝產物累積會造成所有培養物pHT降數個 單位。 < S ) 22 1352120 表2 在不同的培養pH值中，氨對R223細胞生長及產量的影 響· pH係以施加氣髏之c〇2含量調整〇 pC〇2 % 加入的氨 mM 起始pH 最大細胞 數/ml X W6 比生長速 率 h'1 EPO EU/ml q.EP〇 EU/106 細胞/h 5 0 7.26 0.9 0.020 1719 35 10 0 6.93 0.7 0.017 1580 40 15 0 ^6J5 0.4 0.012 ill 86 56 5 2 7.15 0.8 0.018 1796 38 10 2 6.88 0.7 0.017 1928 42 15 2 6.80 0.3 0.009 1404 78 5 5 7.18 0.6 0.018 2639 40 10 5 6.91 0.6 0.014 1663 51 15 5 6.75 0.3 0.016 1626 45 5 10 7.17 0.5 0.016 2222 43 10 10 6.90 0.6 0.014 1966 50 15 10 6.75 0.2 0.008 1891 在接下來的實驗（表3) pH係藉由調整培養基NaHC03 的3里來調整，並維持施加氣體的c〇2含量在無抑制作Association ' 1967, 199, 519), a mixture of the indefinite aminoguanamine-free medium and its suitable ratio. Fortification common cell culture growth media for high density cell culture are well known in the art and have been described, for example, in GB 2251249, which is also well suited as the amoxicillin-free medium of the present invention. base. Often the eggs in the cells can be added to the soy fat with the acid, and the common addition of megluminic acid to 1j can be added to the common non-amidoxime base culture added additives including serum proteins and as needed Further ingredients that can grow and/or have a positive effect on cell survival. Serum White matter such as bovine serum albumin (BSA), transferrin and/or insulin; further components that have a positive effect on cell growth and/or cell survival, such as West and ethanolamine. A cell line which can be used in place of the amino acid of glutamine and/or nucleoside is added to the medium. Examples of amino acids such as isoleucine, valine, lysine, aspartame, asparagus Aminic acid, serine, alanine.榖 榖 榖 视 可以 可以 可以 可以 可以 可以 可以 可以 可以 可以 可以 可以 可以 可以 可以 可以 可以 可以 可以 ' ' ' ' ' ' ' 通常 通常 通常 通常 通常 通常 通常 通常 通常 通常 通常 通常 通常 通常 通常 通常 通常 通常 通常 通常 通常 通常(such as transamination reaction). If an exogenous DNA sequence encoding a protein or an exogenous DNA sequence capable of altering the expression of an endogenous gene encoding a protein can be amplified by an amplify gene after transfection, the relevant selection agent can be added to the common In the guanamine buffer basal medium, the concentration range of the selective agent administration depends on the cell line used, and is usually 1 〇μΜ and higher. Adaptation of Suspension Growth in Serum-Free Medium The glutamine-deficient human cells can be anchor-dependent or fixation-independent, which can be used as a starting material to obtain transfected indoleamine-deficient human cells according to the present invention. According to the present invention, the transfected cells are capable of producing protein and are capable of growing in a non-ammoniumamine-free medium. If fixation-dependent human cells, such as the ΗΤ1080 cell line (ATCC No. CCL. 121), can be adapted to become a fixed independent HT1〇8〇 cell line, it can be suspended in serum-free medium and has not been described in the literature. Adaptation can occur either before or after transfection with an exogenous DNA sequence encoding the protein or an exogenous DNA sequence that alters the expression of the endogenous gene encoding the protein and an exogenous DNA sequence encoding the GS. Preferably, the cells are first transfected with an exogenous DNA sequence encoding a protein or an exogenous DNA sequence capable of altering the expression of an endogenous gene encoding the protein, and then adapted to the growth of the suspension in a serum-free medium, and then further Transfection of a DNA sequence encoding GS. If desired, the transfected cells can be adapted to suspension growth in serum-free, sulphate-free medium. The transfected indoleamine auxotrophic human cells of the present invention may be solid-dependent or sessile independent, and the growth of the sputum is sufficient for the growth of the serum-free serotonin-free transfection The glutamine amine auxotrophic human cell line is solid (10) enough to grow in a serum-free medium without 35 ammonia-nitramine. It acts as a solid cell capable of being suspended in a serum-free medium: by adapting it to a medium containing ▲ clear in the first step. 14 can be achieved by treating the cells with, for example, chymotrypsin followed by shaking or releasing the cells. The cells then adapt to the serum-free medium in the second step by reducing the serum content. If the selection agent is used, the amount can be reduced to avoid inhibiting cell growth. However, the cells can also be followed in the first step. The serum content is adapted to grow in serum-free medium, and the second step is adapted to become a sessile independent cell capable of suspension growth. The two steps can also be performed simultaneously by treating the cells with trypsin and then shaking or shaking the cells. Preferably, the cell line is adapted to become a fixation-independent cell in the first step of the serum-containing medium, adapted to the serum-free medium by shaking to release the cells and then in the second step by subsequently reducing the serum content. The medium as described above can be used as a basic medium for serum-containing medium. The selection agent defined herein can be added to the medium, and the concentration range of the selection agent administration depends on the cell line used, which is usually used at a concentration of 1 〇 μΜ and higher. The serum-containing medium is usually added in an amount of about 1 to 20%, preferably 2 to 10%, and optimal 〇5 to 5% of serum can be used as mentioned above. Subsequent reduction in serum levels can be achieved by gradually reducing serum levels, for example from 10% to 1% to 0%. 1352120 The transfected lysine-deficient human cell line of the present invention is used in a method for producing a protein shell, which is cultured in a medium suitable for expressing the protein and obtaining 0-C〇vering the protein. The cells produced are described above. The common amoxicillin-based medium and common additives as described above can be used as a medium, and suitable culture conditions are as described in WO 96/39488 for use in mammalian cell body culture. Protein-derived proteins can be isolated from cell cultures using conventional separation techniques such as immunoaffinity or ion exchange column partitioning, precipitation, reverse phase Hplc, chromatography, chromatographic methods, SDS-PAGE, colloidal filtration . Those skilled in the art will appreciate that purification methods suitable for use in polypeptides of interest may require modification based on the properties of the polypeptide in recombinant cell culture. [Embodiment] Example 1 Preparation of human iron sarcoma cell line HT1080-R223 Fixation-dependent human HT1080-R223 cell line containing multi-human EPO gene copies is an industrial cell for producing EPO, initially Created by Transkaryotic Therapies (Cambridge, MA 02139 (US)), which is derived from a fixation-dependent human fibrosarcoma HT 1080 cell line. The parental HT1080 cell line (ATCC No. CCL 121) has the ability to produce EP0 by transfection with a DNA construct pREP022 similar to the DNA construct pREP018 (described in WO 95/3 1 560), except for DHFR-based 1352120 Because the line is in the opposite direction and pREP022 contains about 6 〇〇 base pair homologous sequences less than pREP018. This cell line will next be referred to in the R223 cell line. Example 2 Adaptation of the R223 cell line was carried out in a serum-free medium in a serum-supplemented medium to release cells grown in a stationary vial in an adherent culture, suspended in a proprietary (proprietory) containing nothracene. Serum medium (further referred to as "HM9"), 1% dialysis fetal bovine serum (dFBS) and 500 nM MTX were added and then cultured in a shake flask. Growth begins six days later, and the cells are cultured in the same medium (Fig. 1}. Once the growth pattern of the cockroach is established, the serum content of the medium is reduced to 1%. Once again, the reliable growth can be added. The serum was completely re-established before it was completely used. The serum- and serum-free cultures were overgrowth to assess the yield, and the results were shown in the table, with attached culture data, sufficient growth rate, and specific growth rate of suspension culture. (even if there is no serum) as in the serum-attached adherent culture. In the serum-added medium from the attachment culture to the suspension culture, the EP◦ synthesis rate decreased by 5Q% from 24爿12Ευ/ι〇6 cells/hour. However, this rate will generally return to 18 EU/106 cells/hour after adaptation to serum-free growth. 20 1352120 Table 1 Growth of R223 cell line in adherent and suspension cultures before or after adaptation to serum-free medium And production of Dong. Maximum number of cells / mL X 10·6 Specific growth rate h·1 Cumulative cells h/ml X 10·6 EPO EU/ml q epo EU/106 cells/h Attachment culture (DM EM + 10 % dFBS 500 nM MTX) Not applicable 0.0157 Not applicable Not applicable 24 Suspension culture (HM9 + 10 % dFBS 500 nM MTX) 1.4 0.0185 334 4199 12 Suspension culture (HM9 (serum free) 500 nM MTX) 1.1 0.0166 210 3600 18 Example 3 Adaptation of HT1080 (ATCC CCL121) to enable suspension growth in serum-free medium HT1080 cell line ATCC CCL121 was obtained from American Type Culture Collection (Rockville, Mary land, US A), initially cell line adherent culture Growth in DMEM containing 10% fetal bovine serum (FBS). Suspension and serum-free adaptation can be performed according to the procedure of Figure 2. To initiate suspension culture, the cell line is released from the adherent culture by trypsin treatment and released. The cells were resuspended in HM9 supplemented with 2% FBS, 21 1352120, and then cultured in a shock. Once the cells were established in suspension growth with 2% fbs of hm9, the cultures were diluted in the same medium for subculture. Susceptible growth of the ΗΤ1080 cell line was established after 3 () days (Fig. 3). After that, the serum content of the medium was reduced, and finally the cells were completely absent. Continued to continue the growth in serum-free medium. Example 4 Effect of φ on the growth and yield of R223 cell line When guanamine is used as the energy matrix, ammonia is a metabolite produced by cultured cells and has cytotoxicity. It also inhibits cell growth, and in addition, it inhibits protein glycosylation by affecting the pH of the cell's high base. R223 cells cultured in unsupplied bottles typically produce 5 ammonia, which is 1 mM in a fermenter with nutrient supply. f When initially assessing the effects of ammonia, the R223 cell line was grown in a shock bottle without ammonia or ammonia (2, 5 or 1 mM), and the concentration of each ammonia in repeated cultures was at different pH values. Obtained by changing the c〇2 content of the applied gas. The main target here is to determine the extent to which ammonia inhibits growth, and to test whether the reduced pH can overcome this growth inhibition. Ammonia was found to inhibit cell growth (Table 2), and the reduced pH did not alleviate the inhibition caused by ammonia, although it was used to reduce it? The elevated pC〇2 of 1^ itself may cause some growth inhibition. The culture is terminated after only 3 days, and lengthening the culture is ineffective because the accumulation of non-volatile acid metabolites causes all cultures to drop several units of pHT. <S) 22 1352120 Table 2 Effect of ammonia on growth and yield of R223 cells in different culture pH values. pH system is adjusted by c〇2 content of gas 〇pC〇2 % added ammonia mM initial pH Maximum number of cells/ml X W6 Specific growth rate h'1 EPO EU/ml q.EP〇EU/106 Cells/h 5 0 7.26 0.9 0.020 1719 35 10 0 6.93 0.7 0.017 1580 40 15 0 ^6J5 0.4 0.012 ill 86 56 5 2 7.15 0.8 0.018 1796 38 10 2 6.88 0.7 0.017 1928 42 15 2 6.80 0.3 0.009 1404 78 5 5 7.18 0.6 0.018 2639 40 10 5 6.91 0.6 0.014 1663 51 15 5 6.75 0.3 0.016 1626 45 5 10 7.17 0.5 0.016 2222 43 10 10 6.90 0.6 0.014 1966 50 15 10 6.75 0.2 0.008 1891 In the next experiment (Table 3), the pH system was adjusted by adjusting the medium NaHC03 for 3 liters, and the c〇2 content of the applied gas was maintained without inhibition.

用的濃度。測試的PH範圍⑨PH 7.0至，j 7.5(要強調的是， 在瓶中培養的培養基pH不能被控制，而且會大大的下降， P使最先兩天的培養，所指的值係各培養之起始pH)。 比生長速率在pH 7.5 (NaHc〇3於3 g/i)時最大，但在這 p下，因為10 mM氨的存在使生長速率 度減半EP〇合成比速率減少四倍（表3)。 當PH7.25(NaHC〇3於…⑴時，相較於pH” 培養’比生長速率減少，Ep〇合成比速率增加然而因 有1〇ΙΏΜ氨存在，生長速率較不受影響，EP〇合成比速 23 1352120 不受影響。 在最低的研究pH，pH 7.0 (NaHC03於0·75 g/l)，比 生長速率進一步減少但仍比pH 7.5時較不受氨影響。EPO 合成比速率因為加入的氨之存在而增加（這可能是因為生 長速率降低）。 表3 在不同的培養pH值下，氨對R223細胞生長及產量的影 響。pH係藉由改變培養基中NaHC03含量來調整。 最大細胞 比生長速 EPO q Ερο 起始 數/ml 率 pH X ΙΟ'6 h'1 EU/ml EU/106個細 胞/hThe concentration used. The pH range of the test was 9PH 7.0 to j 7.5 (it is important to emphasize that the pH of the medium cultured in the bottle cannot be controlled and will be greatly reduced. P makes the first two days of cultivation, and the values indicated are for each culture. Starting pH). The specific growth rate was greatest at pH 7.5 (NaHc〇3 at 3 g/i), but at this p, the growth rate was reduced by half the EP〇 synthesis ratio by a factor of four mM ammonia (Table 3). When PH7.25 (NaHC〇3 is at (1), the specific growth rate is decreased compared with the pH “culture”, and the Ep〇 synthesis ratio is increased. However, the growth rate is unaffected due to the presence of 1 〇ΙΏΜ ammonia. Specific speed 23 1352120 is not affected. At the lowest research pH, pH 7.0 (NaHC03 at 0.75 g/l), the specific growth rate is further reduced but still less affected by ammonia than pH 7.5. EPO synthesis ratio rate is added The presence of ammonia increased (this may be due to a decrease in growth rate). Table 3 Effect of ammonia on the growth and yield of R223 cells at different culture pH values. The pH system was adjusted by changing the NaHC03 content in the medium. Specific growth rate EPO q Ερο starting number /ml rate pH X ΙΟ'6 h'1 EU/ml EU/106 cells/h

NaHC03 3 g/l 7.49 1.3 0.030 951 16 NaHC03 3 g/l 10 mM 氨 7.51 0.5 0.016 256 4NaHC03 3 g/l 7.49 1.3 0.030 951 16 NaHC03 3 g/l 10 mM ammonia 7.51 0.5 0.016 256 4

NaHC03 1.5 g/l 7.26 1.1 0.027 1151 24 NaHC03 1.5 g/l 10 mM 氨 7.27 0.7 0.021 1191 26NaHC03 1.5 g/l 7.26 1.1 0.027 1151 24 NaHC03 1.5 g/l 10 mM ammonia 7.27 0.7 0.021 1191 26

NaHC03 0.75 g/l 7.02 0.9 0.020 995 26 NaHC03 0.75 g/l lOmM 氨 7.01 0.4 0.016 1009 42 24 1352120 實施例5 R223細胞系在5公升規模發酵槽培養的產量 對於R223細胞系，實施了三個5公升的發酵，控制 在介於6.95和7.15的不同pH值（參見表4)，每一個培養 得到濃縮的營養供應，其含有胺基酸和葡萄糖，設計來維 « 持主要的消耗養分在足夠濃度。培養物在pH 7.15及pH 7.05 生長的很好，但在pH 6.95則無法生長，可能是因為用來 控制該pH之高濃度C02。 鲁表4 發酵槽培養之R223細胞系的產量及代謝. 發酵條件 最大細 胞數/ml x ΙΟ'6 倍殖時 間 小時 累積的細 胞 小時/ml X 10-6 EPO EU/ml q Ερο EU/106 細胞數 /h 最終氨 mg/1 pH 7.15 NaHC03 於 2 g/1 1.0 46 197 10795 58 163 pH 7.05 NaHC03 於 2 g/1 1.0 51 195 11414 64 157 pH 6.95 NaHC03 於 2 g/1 0.2 105 62 2995 70 未做 實施例6.以GS轉染的R223及GS轉染子在附著培養 的產量。 用來轉染的細胞係來自實施例 2之R223細胞系懸浮 25 1352120 適應的無血清儲備溶液，當這些細胞長到像大的多細胞聚 集時，再將他們以胰蛋白酶處理使其成為實質上的單細胞 懸浮。 轉染從實施例2獲得的約！〇7單懸浮適應R223細胞 系分液（在不含弼或鎂的磷酸緩衝鹽）係混合以2〇 含有 GS基因之線狀DNA(DNA序列pCMGS Bam Η1描述於 Bebbington 等人 ’ 1992’ Biotechnology 10，169-175)及使 用 Biorad Gene Pulser (450 伏特，250 pF)施以電穿孔。 作為控制組者，等量分液之細胞在不加DNA下作電穿孔。NaHC03 0.75 g/l 7.02 0.9 0.020 995 26 NaHC03 0.75 g/l lOmM ammonia 7.01 0.4 0.016 1009 42 24 1352120 Example 5 Yield of R223 cell line cultured in a 5 liter scale fermentation tank Three 5 liters were applied to the R223 cell line. The fermentation, controlled at different pH values between 6.95 and 7.15 (see Table 4), each cultured to obtain a concentrated nutrient supply containing amino acids and glucose, designed to maintain a major concentration of nutrients at a sufficient concentration. The culture grew well at pH 7.15 and pH 7.05, but did not grow at pH 6.95, probably because of the high concentration of CO 2 used to control the pH. Yield and metabolism of R223 cell line cultured in Luwan 4 fermentation tank. Maximum number of cells in fermentation conditions/ml x ΙΟ'6 Cell accumulation hours/ml accumulated at the time of colonization X 10-6 EPO EU/ml q Ερο EU/106 cells Number / h Final ammonia mg / 1 pH 7.15 NaHC03 at 2 g / 1 1.0 46 197 10795 58 163 pH 7.05 NaHC03 at 2 g / 1 1.0 51 195 11414 64 157 pH 6.95 NaHC03 at 2 g / 1 0.2 105 62 2995 70 Example 6. Yield of adherent culture of R223 and GS transfectants transfected with GS. The cell line used for transfection was obtained from the R223 cell line of Example 2, suspended in a serum-free stock solution of 25 1352120. When these cells grew to a large multicellular aggregate, they were trypsinized to make them essentially Single cell suspension. Transfection was obtained from Example 2! 〇7 single suspension-adapted R223 cell line (in phosphate buffered saline containing no strontium or magnesium) mixed with 2 线 DNA containing GS gene (DNA sequence pCMGS Bam Η1 described in Bebbington et al '1992' Biotechnology 10 , 169-175) and electroporation using a Biorad Gene Pulser (450 volts, 250 pF). As a control group, cells that were equally divided were electroporated without DNA.

細胞以HM9培養基稀釋，不加MTX，含有0或10% dFBS ’及分布在96孔培養盤或25 cm2瓶，35.5至37 0C 培養。 HM9培養基一開始含有2mM榖氨醯胺，但1天後稀 釋成0.5 mM ’然後十天後換成無榖氨醯胺HM9培養基， 並且在第1天或第1〇天要再加入mtx(5〇〇 nM)於培養物 中 。 至於在不加DNA下作電穿孔的控制組細胞培養，沒 有生長。 以DNA電穿孔的細胞’有μ個GS轉染子可以在96The cells were diluted in HM9 medium, without MTX, containing 0 or 10% dFBS ' and distributed in 96-well plates or 25 cm 2 bottles, cultured at 35.5 to 37 0C. The HM9 medium initially contained 2 mM guanamine, but was diluted to 0.5 mM after 1 day. Then, after 10 days, it was replaced with guanamine-free HM9 medium, and mtx (5 was added on the first day or the first day). 〇〇nM) in culture. As for the control group cell culture for electroporation without DNA, there was no growth. Cells electroporated with DNA 'with μ GS transfectants can be found at 96

孔盤鑑別出來，不管起始培養基有沒有MTX，都可獲得GS 轉染子。1 5個GS轉染子中，有7個成功的進展到瓶培養（表 5) ’剩下的8個GS轉染子呈現異常的細胞型態，或生長 的不好，遺棄之。 分離出的每一個起始的七個GS轉染子，設置一組複 26 1352120 製靜置瓶培養，使用1 〇 %添加血清之無榖氨醯胺HM9培 養基，每隔1到4天，犧牲培養物以計數細胞數，並以ΕΡΟ ELISA測量ΕΡΟ濃度。從這些數據集成可以估計每一個GS 轉染子的EPO合成比速率，數據整理於表 5及（作為比較） 數據也包括無轉染的R223細胞系，所有GS轉染子相較於 無轉染的R223細胞系呈現提高的EPO合成比速率，最佳 的GS轉染子，3E10，具有高於無轉染R223細胞系五到 六倍之合成速率。 籲表5 R223細胞系GS轉染子及無轉染的R223細胞系之EPO 合成比速率》 數據來自生長在含有10% dFBS之附著培養，在製造 轉染子的培養基中不加甲氨。票呤（Methotrexate)，但在轉染 後第一天或第十天再加到培養物中。 GS轉染子 添加 MTX (500 nM)到 轉染盤的曰子 q epo (附者） EU/106細胞數/h 3B3 10 59 3E10 10 139 3E11 10 45 4D9 10 35 8F11 1 47 8F12 1 79 8G3 1 52 評估附著培養中無轉染的 R223細胞系 不適用 24 實施例Ί 適應GS轉染子於懸浮培養及無血清培養基。 由實施例6所得到的七個GS轉染子，GS轉染子#3E10 27 1352120 及GS轉染子#8G3進行懸浮培養。 細胞係以附著培養生長於靜置瓶，内含無榖氨醯胺 HM9培養基，添加10 % dFBS及500nM MTX，於35.5至 3 7 °C。細胞在五或六天後藉搖動釋放，再懸浮於同樣的培 養基中，於相同溫度以震盪瓶培養，生長在二或三天後開 始，細胞於相同培養基次培養一次，然後過度生長以評估 產量。效價及產物合成速率至少高於生長在1 〇 % dFBS及 5 00 nM MTX的無轉染R223細胞系兩倍（表 6)。 籲表6 GS轉染子3E10及8G3在震盪瓶懸浮培養的生長及產量。 無榖氨醯胺HM9培養基含有10%dFBS及 500 nMMTX。 GS轉染子 最大細胞數/ ml X 10-6 最大EPO效 價 EU/ml q.EP〇 (懸浮） EU/ 106個細 胞/h 3E10 0.7 11113 57 8G3 1.2 10171 33 評估在懸浮培養生長之無 轉染的R223細胞系，於 10% dFBS 及 500 nM MTX 中 1.4 4199 12 GS轉染子3E10及8G3的懸浮培養，無榖氨醯胺HM9 培養基中dFBS含量係逐步減少，3E10從10 °/〇至2 %至1 % 至0.2 %至0.1 °/。至0 %，8G3從10 %至2 %至1 %，並在 28 1352120 各dFBS濃度進一步減少前，建立可信的細胞生長，8G3 沒有進一步適應於1 % dFBS。 實施例8 GS轉染子3E10在無血清懸浮培養的產董及代謝。 實施例 7中適應於無血清生長的GS轉染子3E10係 以懸浮培養培養於無企清無榖氨醯胺之HM9培養基中， 35.5到37。(：。對於GS轉染子 3E10細胞系，培養在相同 • 條件於含有榖氨醯胺的HM9培養基，其具有比無轉染的 R223細胞系快2到3倍的EPO合成比速率（表7)。 表7 GS轉染子3E10細胞在無血清懸浮培養的生長及產量。 最大細胞數/ml X 1〇-6 EPO EU/ml q.EP〇 EU/ 106/h 3E10 (無榖氨醯胺 HM9培養基） 1.1-1.2 9731 - 14234 40-67 R223 (HM9培養基加6 mM榖氨醯胺） 1.0-1.6 3075-5818 13-24 GS轉染的細胞的高EPO產量伴隨代謝氨釋放的減少， 不同於在含有6 mM榖氨醯胺HM9培養基中瓶培養典型會 產生5 mM氨的無轉染之R223細胞系，GS轉染子3E10 於無榖氨醯胺HM9培養基只產生1.8 mM氨（表8)。 29 1352120 電荷“Z”，表10沒有決定無轉染的R223細胞系之“Z”， 然而’ GS轉染子3E10 (273於最大細胞濃度及265於收集 時）之“Z”值超過瓶培養（183-228)無轉染R223細胞系的值。 “Z”係根據 Hermentin 等人於 Glycobiology，1996, 6,217-230 決定；將個別％-唾液酿化的（sialyated)異構物之部分乘以 對應的該異構物負電荷，可得Z，視其是否缺乏唾液醯基/ 中性、單涎、三·、四或五涎而定。該乘積之數學總合術語 係Z。Z數字係關於活體内給予的治療上糖蛋白質 (Hermentin，ibd_)之清除速率。 表10 來自無轉染R223細胞系及GS轉染子3E10之EPO產物 品質分析· 效價 EU / mL 異構物相對 活性 ，Ζ· R223親本(加6mM榖氨醯胺之HM9 培養基） 高峰 收集 3518 25.2 not done 6150 202~~ not done 3E10 (無榖氨醯胺之HM9培養基)高峰 收集 3921 416 273 6862 3ό!〇~~~ 265 實施例10 在6 a升發酵槽無血清懸浮培養之Gs轉染子的產 量、代謝及產物品質· GS轉染子3E10係生長在分批培養於氣舉式㈨川⑴發 酵槽，培養基係無穀氨醯胺HM9，不含血清，每一個培養 有濃縮的養分供應，其含有胺基酸及葡萄糖，設計使主要 32 1352120 消耗之養分維持在足夠濃度。結果顯示在表11，也包括 無轉染的親本R223發酵數據，用於比較。 GS轉染子呈現延長的生存能力，所以培養期間增加， 產物合成的比速率相同於無轉染的親本細胞系，但較長培 養壽命造成最大產物濃度增加，氨累積至少低於GS轉染 子四倍，GS轉染子產物品質（以IRA測量）改善。 表11 GS轉染子3E10細胞在無血清》浮培養的生長及產量，產 鲁 生的EPO產物品質係以IRA定量。 發酵條件 最大 最大 收集日 氨 收集 整體 收集時 細胞數 CCH*/ mg/L 效價 q epo 的IRA /ml ml EU/106 X 10'6 X ΙΟ'6 EU/ml 細胞數/h 無榖氨醯胺 1.03 306 21 41 20936 69 43.6 無榖氨醯胺 1.13 360 24 20 20150 52 42.6 無榖氨醯胺 0.94 319 24 15 18680 49 48.5 以標準方法 用榖氨醯胺 之親本 R223 1.11 216 13 165 12225 58 28 *累積細胞時數，細胞濃度的時間積分[Renard 等人， 1988，Biotechnology Letters 10 (9) : 1-96]。 實施例11 在無血清懸浮培養 Iscove 氏為基的培養基之GS轉染子的 產量及代謝 實施例 7中適應於無血清生長的GS轉染3E10係培 33 1352120 養在震盈瓶，在無血清無榖氨醯胺的I sco ve氏培養基，親 本細胞系R223係對應生長在相等的添加榖氨醯胺之培養 基。 所用的培養基係經Iscove氏修飾之Dulbecco氏培養 基，含有Iscove氏的添加物（牛血清白蛋白0.4 g/L、人類 全（holo)運鐵蛋白30 mg/L)、重組人類胰島素10 mg/L、 Lutrol F68 1 g/L及乙醇胺60 pL/L。用於培養R223細胞系 的培養基含有4mM榖氨醯胺，但用於GS轉染的細胞系則 ® 不加榖氨醯胺’改以4 mM榖氨酸鈉加4 mM天冬醯胺。 GS轉染的細胞系3E10之產物合成比速率大約50%高 於無轉染的親本系R223(表12)，轉染的細胞系3E10之氨 生成比速率低七倍（表 13)。 產物係從這些個別培養純化而來，並在等電點聚焦法 (IEF)膠體上分析，此分析結果顯示於圖5，其顯示染色後 的IEF膠。IEF膠在pH 2.5到6.5變性條件下跑，並以 Coomassie Blue 染色。 圈5解說 第卜3、6及7行 空白 第2及7行 pi標記 第4行 從在含有榖氨醢胺的Iscove氏培養 基中生長的R223免疫親和純化的產 物 第5行 免疫親和純化的產物，其係從從在無 榖氨醯胺Iscoves培養基中生長的GS 轉染的R223細胞系之轉殖3E10 34 1352120 來自R223的產物至少有13條可見的亮帶，分布在整 條膠體上；來自GS轉染的細胞系3E10生成的產物，較鹼 的亮帶（膠體上方）強度比從細胞系R223產物亮帶弱，但細 胞系3E 1 0較酸的亮帶（膠體下方）則強度增加。有至少一條 額外的酸性亮帶可在GS轉染子3E10產物中可偵測到，但 親本系R223產物偵測不到，這顯示來自GS轉染的3E10 系之產物唾液醯化（sialylation)增加。 表12 ® 在無血清》浮培養於 Iscove 氏培養基中的GS轉染子3E10 細跑之生長及產量 最大存活知胞 it/mL EPO EU/mL Qepo EU/106細胞數 /h 生長在無榖氨醯胺 Iscove氏培養基之 3E10 0.55 1340 11.5 生長在含有榖氨醯胺 Iscove氏培養基之 R223 0.72 993 7.3 • 表 13 在無血清懸浮培養於 Iscove 氏培養基中GS轉染子3E10 細胞之氨生成減少 最大存活麵跑 氣mM <1氨 it/mL η莫耳/106細胞 難 生長在無榖氨醯胺 Iscove氏培養基之 3E10 0.55 0.61 2.6 生長在含有榖氨醯胺 Iscove氏培養基之 R223 0.72 2.39 17.4 35 1352120 【圖式簡單說明】 >月培養基之適 圖1顯示細胞系R223懸浮培養於 • J3ZZ. 滑培養基之適 應作用的細胞濃度圖’經由重複連續的次培養 圖2顯示HT1080細胞懸浮培養於無血、 應作用概要。 圖3顯示細胞系HT1080懸浮培養於無血清培養基之 適應作用的細胞濃度圖’經由重複連續的次培養。The wells were identified and GS transfectants were obtained regardless of the presence or absence of MTX in the starting medium. Of the 15 GS transfectants, 7 successfully progressed to bottle culture (Table 5). The remaining 8 GS transfectants showed abnormal cell types, or poor growth, and abandoned. Each of the first seven GS transfectants isolated was set up, and a set of complex 26 1352120 static flask cultures was set up, using 1% 添加% serum-free guanamine-free HM9 medium, every 1 to 4 days, sacrificed. Cultures were counted for cell count and sputum concentration was measured by ΕΡΟ ELISA. From these data integrations, the EPO synthesis ratio of each GS transfectant can be estimated. The data is compiled in Table 5 and (as a comparison). The data also includes the untransfected R223 cell line. All GS transfectants are compared to no transfection. The R223 cell line exhibited an increased rate of EPO synthesis, and the best GS transfectant, 3E10, had a synthesis rate five to six times higher than that of the untransfected R223 cell line. The EPO synthesis ratio of the R223 cell line GS transfectants and the untransfected R223 cell line was derived from the growth culture in 10% dFBS, and no methylamine was added to the medium in which the transfectants were produced. Methotrexate, but added to the culture on the first or tenth day after transfection. GS transfectants add MTX (500 nM) to the transfected disk of the scorpion q epo (attached) EU/106 cell number / h 3B3 10 59 3E10 10 139 3E11 10 45 4D9 10 35 8F11 1 47 8F12 1 79 8G3 1 52 Evaluation of non-transfected R223 cell line in adherent culture Not applicable 24 Example 适应 Adapted to GS transfectants in suspension culture and serum-free medium. The seven GS transfectants obtained in Example 6, GS transfectants #3E10 27 1352120 and GS transfectants #8G3 were subjected to suspension culture. The cell line was grown in an adherent culture in a static vial containing hydrazine-free HM9 medium supplemented with 10% dFBS and 500 nM MTX at 35.5 to 37 °C. The cells were released by shaking after five or six days, resuspended in the same medium, cultured in a vibrating flask at the same temperature, grown in two or three days, cells were cultured once in the same medium, and then overgrowth to assess yield. . The titer and product synthesis rate were at least twice higher than the untransfected R223 cell line grown at 1 〇 % dFBS and 500 mM MTX (Table 6). Call for the growth and yield of GS transfectants 3E10 and 8G3 in suspension culture in suspension flasks. The guanamine-free HM9 medium contains 10% dFBS and 500 nMMTX. Maximum number of cells in GS transfectants / ml X 10-6 Maximum EPO titer EU/ml q.EP〇 (suspended) EU/106 cells/h 3E10 0.7 11113 57 8G3 1.2 10171 33 Evaluation of growth in suspension culture The stained R223 cell line was suspended in 10% dFBS and 500 nM MTX in 1.4 4199 12 GS transfectants 3E10 and 8G3. The dFBS content in the HM9-free medium was gradually reduced, and 3E10 was from 10 °/〇 to 2% to 1% to 0.2% to 0.1 °/. To 0%, 8G3 was from 10% to 2% to 1%, and before the concentration of 28 1352120 dFBS was further reduced, a reliable cell growth was established, and 8G3 was not further adapted to 1% dFBS. Example 8 GS Transfectant 3E10 was produced in serum-free suspension culture and metabolized. The GS transfectant 3E10 strain adapted to serum-free growth in Example 7 was cultured in suspension culture in HM9 medium without chloramphenicol, 35.5 to 37. (: For the GS transfectant 3E10 cell line, cultured in the same • conditional HM9 medium containing guanamine, which has a 2 to 3 times faster EPO synthesis rate than the untransfected R223 cell line (Table 7). Table 7 Growth and yield of GS transfectant 3E10 cells in serum-free suspension culture. Maximum number of cells/ml X 1〇-6 EPO EU/ml q.EP〇EU/ 106/h 3E10 (no guanamine) HM9 medium) 1.1-1.2 9731 - 14234 40-67 R223 (HM9 medium plus 6 mM guanamine) 1.0-1.6 3075-5818 13-24 GS transfected cells with high EPO production accompanied by a decrease in metabolic ammonia release, The untransfected R223 cell line, which typically produced 5 mM ammonia, was cultured in a bottle containing 6 mM guanamine amine HM9 medium. GS transfectant 3E10 produced only 1.8 mM ammonia in the guanamine-free HM9 medium (Table 8). 29 1352120 Charge "Z", Table 10 did not determine the "Z" of the untransfected R223 cell line, whereas the "Z" value of 'GS transfectant 3E10 (273 at maximum cell concentration and 265 at collection) exceeded Bottle culture (183-228) was not transfected with the value of the R223 cell line. The "Z" line was determined according to Hermentin et al. in Glycobiology, 1996, 6, 217-230. By multiplying the fraction of the individual %-saliva-stained isomer by the corresponding negative charge of the isomer, Z can be obtained depending on whether it lacks salivary sulfhydryl / neutral, monoterpene, tri-, The mathematical total term for this product is Z. The Z number is the rate of removal of the therapeutic glycoprotein (Hermentin, ibd_) administered in vivo. Table 10 From the untransfected R223 cell line and GS transgene EPO product quality analysis of dye 3E10 · titer EU / mL isomer relative activity, Ζ · R223 parent (addition of 6mM guanamine HM9 medium) peak collection 3518 25.2 not done 6150 202~~ not done 3E10 ( HM9 medium without indoleamine) Peak collection 3921 416 273 6862 3ό!〇~~~ 265 Example 10 Yield, metabolism and product quality of Gs transfectants in serum suspension culture in 6 aliter fermentation tank · GS conversion Stain 3E10 is grown in batch culture in a gas lift (Jiu) Chuan (1) fermentation tank. The medium is glutamine-free HM9, free of serum. Each culture has a concentrated nutrient supply containing amino acids and glucose. Keep the main 32 1352120 consumed nutrients strong enough The results are shown in Table 11, which also includes non-transfected parental R223 fermentation data for comparison. GS transfectants exhibit prolonged viability, so the growth rate during the culture period is the same as that of the non-transfected parental cell line, but the longer the culture life leads to the increase of the maximum product concentration, and the ammonia accumulation is at least lower than the GS transfection. Four times the GS transfection product quality (measured by IRA) improved. Table 11 Growth and yield of GS transfectant 3E10 cells in serum-free suspension culture. The quality of EPO products produced by R&D was quantified by IRA. Fermentation conditions Maximum maximum collection Daily ammonia collection Overall collection number of cells CCH*/mg/L Potency q epo IRA /ml ml EU/106 X 10'6 X ΙΟ'6 EU/ml Cell number/h No ammonia Amine 1.03 306 21 41 20936 69 43.6 Anthraquinone-free amine 1.13 360 24 20 20150 52 42.6 Anthraquinone-free amine 0.94 319 24 15 18680 49 48.5 Parental R223 using a guanamine in a standard method 1.11 216 13 165 12225 58 28 * Cumulative cell hours, time integral of cell concentration [Renard et al., 1988, Biotechnology Letters 10 (9): 1-96]. Example 11 Yield and metabolism of GS transfectants in serum-free suspension culture of Iscove's medium. Example 7 Adapted to serum-free growth of GS transfected with 3E10 lineage 33 1352120 Raised in a shake flask, in serum-free I sco ve's medium without samariumamine, and the parental cell line R223 was grown in an equal medium containing guanamine. The medium used was Iscove's modified Dulbecco's medium containing Iscove's supplement (bovine serum albumin 0.4 g/L, human whole (holo) transferrin 30 mg/L), recombinant human insulin 10 mg/L Lutrol F68 1 g/L and ethanolamine 60 pL/L. The medium used to culture the R223 cell line contained 4 mM guanamine, but the cell line used for GS transfection was replaced with 4 mM sodium citrate plus 4 mM aspartate. The rate of synthesis of the product of the GS-transfected cell line 3E10 was approximately 50% higher than that of the untransfected parental line R223 (Table 12), and the rate of ammonia formation by the transfected cell line 3E10 was seven times lower (Table 13). The product was purified from these individual cultures and analyzed on an isoelectric focusing (IEF) gel. The results of this analysis are shown in Figure 5, which shows the dyed IEF gel. The IEF gel was run under denaturing conditions of pH 2.5 to 6.5 and stained with Coomassie Blue. Circle 5 illustrates blanks 3, 6 and 7 blanks 2 and 7 lines pi labeling 4th line of immunoaffinity purified product from the R223 immunoaffinity purified product grown in Iscove's medium containing guanamine. , which is derived from the RG transfected R223 cell line grown in serotonin-free Iscoves medium. 3E10 34 1352120 The product from R223 has at least 13 visible bright bands distributed over the entire colloid; The product produced by the GS-transfected cell line 3E10 was weaker than the bright band of the base (above the colloid) than the bright band of the cell line R223, but the cell line 3E 10 was stronger than the bright band of the acid (below the colloid). At least one additional acidic bright band was detectable in the GS transfectant 3E10 product, but the parental line R223 product was undetectable, indicating saliva sialylation from the GS transfected 3E10 line. increase. Table 12 ® Growth and Yield of GS Transfectant 3E10 in No-Serum Float Culture in Iscove's Medium Maximum Survival Cell I/mL EPO EU/mL Qepo EU/106 Cell Count/h Growth in Ammonia-Free Ammonia 3E10 0.55 1340 11.5 of indole Iscove's medium was grown on R223 0.72 993 7.3 containing indoleamine Iscove's medium. Table 13 Ammonia production in GS transfectants 3E10 cells in serum-free suspension culture in Iscove's medium reduced maximal survival Noodles running mM <1 ammoniait/mL η mol/106 cells difficult to grow in 3E10 without sulphonamide Iscove's medium 0.55 0.61 2.6 Growing in R223 0.72 2.39 17.4 35 1352120 containing Isoprofen Iscove's medium [Simplified description of the schema] > Appropriate Figure 1 shows the cell line R223 suspension culture in • J3ZZ. The cell concentration map of the adaptation of the slide medium 'via repeated repeated subcultures Figure 2 shows that HT1080 cells are cultured in suspension without blood, A summary of the role. Figure 3 shows a cell concentration map of cell line HT1080 suspension cultured in serum-free medium' via repeated sequential subcultures.

圖4顯示從GS轉染子3E10及從無轉染的細胞 系中免疫純化的EPO之IEF分析’其係生長在產業高密度 生長培養基。行2:收集的（收集）3Ε1〇;行3:3E10峰； 行4 :無轉染的R223細胞系峰；行5 :收集的無轉染的R223 細胞系。 圖5顯示R223細胞系之GS-R223轉染子3E10及從無 轉染的R223細胞系免疫純化的EPO之IEF分析，生長在 傳統的Iscove氏培養基。行4:自添加榖氨醯胺的Iscove 氏收集的無轉染的R223細胞系；行5 :自無榖氨醯胺Iscove 氏收集的GS-223細胞系3E10。 【主要元件符號說明】 »»»> 36Figure 4 shows an IEF analysis of EPO immunostained from GS transfectants 3E10 and from untransfected cell lines. The lines were grown in an industrial high density growth medium. Row 2: collected (collected) 3Ε1〇; row 3: 3E10 peak; row 4: untransfected R223 cell line peak; row 5: collected untransfected R223 cell line. Figure 5 shows the IEF analysis of RG-R223 transfectant 3E10 of the R223 cell line and EPO immunopurified from the untransfected R223 cell line, grown in a traditional Iscove's medium. Lane 4: Non-transfected R223 cell line collected from Iscove's addition of guanamine; row 5: GS-223 cell line 3E10 collected from no squalamine Iscove. [Main component symbol description] »»»> 36

Claims (1)

i.—種増加由縠氨醯胺營養缺陷 Λ ^ ^ w s脣职咍人類細胞所表現的糖 土 Ί匕蛋白質之唾液醯化及 於#叙& 夕電何之方法，其特徵在 、。Λ权氣醯胺營養缺陷人類胞俜 貝、腮係Λ、扁碼榖氨醯胺合成酶 的外生DNA轉染。 ^根據中請專利範μ w的方法，其中該糖基化蛋白 二外生DNA編碼且係自該穀氨醯胺營養缺陷人類 之培養回收。i.—Growth and auxotrophy by glutamine Λ ^ ^ w s lip 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍 咍Exogenous DNA transfection of Λ 醯 醯 营养 营养 营养 人类 人类 人类 人类 人类 人类 人类 人类 人类 人类 。 。 。 。 。 。 。 。 。 。 。 ^ According to the method of the patent application, wherein the glycosylated protein is exogenous DNA-encoded and recovered from the culture of the glutamine-deficient human. 胺技羞 專利範圍第1或2項的方法，其中該穀氨醯 :養缺陷人類細胞係進—步以編碼—酵素的可放大的基 ，轉染’其中該酵素係二氫葉酸還原酶（MFR)、腺苦脫 :酶、天冬醯胺合成酶、天門冬胺酸氨曱醯基轉移酶、金 硫因-1 (metallothionein])、鳥氨酸脫_、p-糖蛋白 質、核糖核找還原酶、胸錢酶或黃以令票呤填酸核 糖基轉移酶。The method of claim 1, wherein the glutamine:following human cell line is further transfected with a amplifiable group encoding an enzyme, wherein the enzyme is dihydrofolate reductase ( MFR), adenine bitterness: enzyme, aspartate synthase, aspartate aminopurine transferase, metallothionein, ornithine de-, p-glycoprotein, ribonucleoside Look for reductase, chestnut enzyme or yellow to make the acid ribosyltransferase. 4.根據申請專利範圍第 胺營養缺陷人類細胞係永 胞。 1或2項的方法，其中該穀氨醢 生的榖氨醯胺營養缺陷人類細 5. 根據中請專利範圍第3項的方法，其中該縠氨酿胺與 養缺陷人類細胞係永生的榖氨醯胺營養缺陷人類細胞。呂 6. 根據中請專利範圍第4項的方法，其中該永生的穀氨 醯胺營養缺陷人類細胞係人類纖維肉瘤細胞。 ★ 7. 根射請專利範圍第5項的方法，其中該永生的穀氨 醯胺營養缺陷人類細胞係人類纖維肉瘤細胞。 37 1352120 '0么八 瘤細胞係Η T1 0 8 0細胞系。 9.根據申請專利範圍第7項的方法，其 瘤細胞係HT1 080細胞系。 人類纖維肉 1〇.根據申請專利範圍第…項的方法，其 仇 的細胞係固著獨立及能夠料生長在無血 Λ木 養汽中。 月…、祓虱醯胺培 方法’其中該轉染的細 血清無榖氨醯胺培養基4. According to the scope of the patent application, the auxotrophic human cell line ternary cells. The method of 1 or 2, wherein the glutamine-derived glutamine-deficient auxotrophic human is 5. According to the method of claim 3, wherein the hydrazine-derived amine and the deficiencies of the human cell line are immortalized. Aminoguanamine auxotrophic human cells. Lu 6. According to the method of claim 4, wherein the immortalized glutamine auxotrophic human cell line is a human fibrosarcoma cell. ★ 7. Root shots of the patent scope of the fifth method, wherein the immortalized glutamine auxotrophic human cell line human fibrosarcoma cells. 37 1352120 '0 What is the tumor cell line Η T1 0 8 0 cell line. 9. The tumor cell line HT1 080 cell line according to the method of claim 7 of the patent application. Human fiber meat 1〇. According to the method of the patent application scope item, the cell line of the hate is fixed independently and can be grown in the bloodless eucalyptus. Month..., amidoxime method, wherein the transfected fine serum is free of amidine 11·根據申請專利範圍第3項的 胞係固著獨立及能夠懸浮生長在無 1^7 〇 5項的方法， 長在無血清I ’ , *\\\ 1 2.根據申請專利範圍第4 胞係固著獨立及能夠懸浮生長 〇 1 3 ·根據申請專利範圍第 胞係固著獨立及能夠懸浮生 中 〇 項的方法’其中該轉染的細 在無也清無榖氨醯胺培養基 其中該轉染的細 榖氨醯胺培養基11. According to the third paragraph of the patent application scope, the cell line fixation is independent and can be suspended and grown in the absence of 1^7 〇5 items, and is long in serum-free I ', *\\\ 1 2. According to the patent application scope 4 Cell line fixation independent and capable of suspension growth 〇1 3 · According to the patent application range, the cell line fixation is independent and capable of suspending the sputum in the raw sputum', wherein the transfected fine is in the sputum-free amoxicillin medium. Transfected fine guanamine buffer medium •根據申請專利範圍第6項的$ 胞係固著獨立及能夠懸浮生長在無血 十 〇 ，其中該轉染的細 無榖氨醯胺培養基 I5.根據申請專利範圍第7項的方 胞係固著獨立及能夠懸浮生長在無血 中 〇 法，其中該轉染的細 清無榖氨醯胺培養基 •低踝申請專利範圍第8 J 胞係固著猶古芬一 词立及此夠懸洋生長名 法，其中該轉染的細 清無榖氨醯胺培養基 38 1352120 中 。 i / _很據申請專利範圍 , --- 六· T皤锝栄的細 胞係固著獨立及能夠懸浮生長在無血清無榖氨酿胺培養基 中 〇• According to the scope of the patent application, the cell line is fixed independently and can be suspended and grown in the bloodless scorpion, wherein the transfected fine sputum-free amino amide medium I5. According to the scope of claim 7 Independent and capable of suspending growth in a bloodless sputum method, wherein the transfected fine clear sputum-free amino guanamine medium • low 踝 patent application range 8 J cell fixation yugufen word and this enough for the growth of the ocean The name method, wherein the transfected fine clear sputum-free amino amide medium 38 1352120. i / _ very according to the scope of the patent application, --- hexa-T皤锝栄 cell line fixation independent and capable of suspension growth in serum-free and sulphur-free ammonia-branched amine medium 〇 18. 根據申請專利範圍第 化蛋白貝係紅血球生成素。 19. 根據申請專利範圍第 白質係紅血球生成素。 2 〇.根據申請專利範圍第 白質係紅血球生成素。 2 1_根據申請專利範圍第 白質係紅血球生成素。 22. 根據申請專利範圍第 白質係紅血球生成素。 23. 根據申請專利範圍第 白質係紅血球生成素。 24. 根據申請專利範圍第 白質係紅血球生成素。 2 5 ·根據申請專利範圍第 白質係紅血球生成素。 26.根據申請專利範圍第 白質係紅血球生成素。 1或2項的方法，其中該糖基 3項的方法1中該糖基化蛋 4項的方法’纟中該糖基化蛋 5項的方法，其中該糖基化蛋 6項的方法，其中該糖基化蛋 7項的方法，其中該糖基化蛋 8項的方法，其中該糖基化蛋 9項的方法，其中該糖基化蛋 10項的方法’其中該糖基化蛋 其中該糖基化蛋 2 7.根據申請專利範圍第1 1項的方法 白質係紅血球生成素。 39 丄Ju 28·根據申請專利範圍第12項的 白質係紅jk球生成素。 29. 根據申請專利範圍第13項的 白質係紅血球生成素。 30. 根據申請專利範圍第14項的方 白質係紅血球生成素。 3 1.根據申請專利範圍第丨5項的方 白質係紅企球生成素。 32. 根據申請專利範圍第16項的方 白質係紅血球生成素。 33. 根據申請專利範圍第1 7項的方 白質係紅企球生成素。 34. 根據申請專利範圍第18項的方 成素係人類紅血球生成素。 3 5 .根據申請專利範圍第1 9項的方 成素係人類紅血球生成素。 36·根據申請專利範圍第20項的方 成素係人類紅血球生成素。 37·根據申請專利範圍第21項的方 成素係人類紅血球生成素。 38.根據申請專利範圍第22項的方 成素係人類紅血球生成素。 39_根據申請專利範圍第23項的方 成素係人類紅血球生成素。 法，其中該糖基化蛋 法，其中該糖基化蛋 法，其中該糖基化蛋 法，其中該糖基化蛋 法’其中該糖基化蛋 法’其中該糖基化蛋 法，其中該紅血球生 法，其中該紅血球生 法’其中該紅血球生 法’其中該紅血球生 法’其中該紅血球生 法，其中該紅血球生 40 S 40. 根據申請專利範圍第 成素係人類紅血球生成素。 41. 根據申請專利範圍第 成素係人類紅血球生成素。 42. 根據申請專利範圍第 成素係人類紅血球生成素。 43 ·根據申請專利範圍第 • 成素係人類紅血球生成素。 44.根據申請專利範圍第 成素係人類紅血球生成素。 45_根據申請專利範圍第 成素係人類紅血球生成素。 46·根據申請專利範圍第 成素係人類紅血球生成素。 47. 根據申請專利範圍第 _ 成素係人類紅血球生成素。 48. 根據申請專利範圍第 成素係人類紅血球生成素。 49_根據申請專利範圍第 成素係人類紅血球生成素。 十一、圖式： 如次頁 項的方法，其中該紅血球生 項的方法，其中該紅血球生 項的方法，其中該紅血球生 項的方法，其中該紅血球生 項的方法，其中該紅血球生 項的方法，其中該紅血球生 項的方法，其中該紅血球生 項的方法，其中該紅血球生 項的方法，其中該紅血球生 項的方法，其中該紅血球生 4118. According to the scope of the patent application, the protein is erythropoietin. 19. According to the scope of the patent application, the white matter erythropoietin. 2 〇. According to the scope of the patent application, the white matter erythropoietin. 2 1_ According to the scope of the patent application, the white matter erythropoietin. 22. According to the scope of the patent application, the white matter erythropoietin. 23. According to the scope of the patent application, the white matter erythropoietin. 24. According to the scope of the patent application, the white matter erythropoietin. 2 5 · White matter erythropoietin according to the scope of patent application. 26. According to the scope of the patent application, the white matter erythropoietin. The method of Item 1 or 2, wherein the method of the glycosylated egg 4 in the method 1 of the glycosyl group 3, the method of the glycosylated egg 5, wherein the method of glycosylated egg 6 The method of the glycosylated egg 7 item, wherein the glycosylated egg 8 item method, wherein the glycosylated egg item 9 item, wherein the glycosylated egg item 10 item, wherein the glycosylated egg Wherein the glycated egg 2 is a white matter erythropoietin according to the method of claim 11 of the patent application. 39 丄Ju 28· White matter red jk globogenin according to item 12 of the patent application. 29. White matter erythropoietin according to item 13 of the patent application. 30. White matter erythropoietin according to item 14 of the patent application. 3 1. According to the fifth paragraph of the patent application scope, the white matter is a red globulin. 32. The white matter erythropoietin according to item 16 of the patent application. 33. According to Article 17 of the scope of patent application, the white matter is a red globulin. 34. According to the 18th article of the patent application, the formula is human erythropoietin. 3 5. According to the scope of patent application No. 19, the formula is human erythropoietin. 36. According to the 20th article of the patent application, the formula is human erythropoietin. 37. According to the 21st patent application, the formula is human erythropoietin. 38. According to the 22nd aspect of the patent application, the formula is human erythropoietin. 39_ According to the 23rd item of the patent application, the formula is human erythropoietin. a method of the glycosylated egg method, wherein the glycosylated egg method, wherein the glycosylated egg method, wherein the glycosylated egg method, wherein the glycosylated egg method, wherein the glycosylated egg method, Wherein the red blood cell production method, wherein the red blood cell production method, wherein the red blood cell production method, wherein the red blood cell production method, wherein the red blood cell production method, wherein the red blood cell is 40 S 40. According to the patent application range, the adult system is human erythropoietin . 41. According to the scope of the patent application, the adult is erythropoietin. 42. According to the scope of the patent application, the adult is erythropoietin. 43 · According to the scope of the patent application, the adult is erythropoietin. 44. According to the scope of the patent application, the adult system is human erythropoietin. 45_ According to the scope of the patent application, the adult system is human erythropoietin. 46. According to the scope of the patent application, the adult system is human erythropoietin. 47. According to the scope of the patent application, the _ adult system is human erythropoietin. 48. According to the scope of the patent application, the adult is erythropoietin. 49_ According to the scope of the patent application, the adult system is human erythropoietin. Eleven: a method of a secondary page item, wherein the method for producing a red blood cell, wherein the method for producing a red blood cell, wherein the method for producing a red blood cell, wherein the red blood cell is a method, wherein the red blood cell is The method of the red blood cell birth method, wherein the red blood cell birth method, wherein the red blood cell birth method, wherein the red blood cell birth method, wherein the red blood cell birth 41