TWI504611B - Hyaluronic acid and / or a salt thereof - Google Patents
Hyaluronic acid and / or a salt thereof Download PDFInfo
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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- C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
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Description
本發明係關於一種玻尿酸及/或其鹽之純化方法。The present invention relates to a method for purifying hyaluronic acid and/or a salt thereof.
玻尿酸除作為化妝品之保濕劑以外，亦於眼科、整形外科、皮膚科等中作為醫藥品而使用。雖然玻尿酸可藉由來自動物組織，例如雞之雞冠、牛眼之玻璃體等之萃取物而製造，但由於混入作為夾雜物之硫酸軟骨素等、或因組織內所含之玻尿酸酶等而易低分子量化，故而亦進行培養具有玻尿酸生產能力之微生物而由培養液中製造玻尿酸之方法(醱酵法)(非專利文獻1及專利文獻1)。Hyaluronic acid is used as a moisturizer for cosmetics, and is also used as a medicine in ophthalmology, orthopedics, dermatology, and the like. Although hyaluronic acid can be produced by an extract derived from an animal tissue such as a chicken cockscomb or a vitreous of a bull's eye, it is easy to be low due to the incorporation of chondroitin sulfate as an inclusion or the hyaluronic acid contained in the tissue. Since the molecular weight is increased, a method of producing hyaluronic acid from a culture solution by culturing a microorganism having hyaluronic acid production ability (Non-Patent Document 1 and Patent Document 1) is also carried out.
於藉由萃取法或醱酵法製造之玻尿酸中，由於蛋白質或發熱性物質等以雜質形式存在，故正研究有將該等分離去除以獲得高純度之製品之方法。尤其是製造之初始階段之雜質的去除能減輕以後之純化步驟之負荷，作為獲得亦可作為醫藥品使用之高純度之製品的方法而期待得到開發。作為高純度地純化玻尿酸之方法，已知：為去除例如蛋白質、核酸等副生物、及來自培養基之無機鹽類而於使包含玻尿酸及其鹽與雜質之玻尿酸溶液通過帶正電之過濾器後添加水溶性有機溶劑，而使玻尿酸鈉析出、沈澱並萃取上清液，藉此進行純化之方法(非專利文獻2)。In the hyaluronic acid produced by the extraction method or the fermentation method, since proteins or exothermic substances exist in the form of impurities, a method of separating and removing the products to obtain a high-purity product is being studied. In particular, the removal of impurities in the initial stage of production can reduce the load of the subsequent purification step, and is expected to be developed as a method for obtaining a high-purity product which can be used as a pharmaceutical product. As a method for purifying hyaluronic acid in high purity, it is known to remove a hyaluronic acid solution containing hyaluronic acid, a salt thereof and an impurity, through a positively charged filter, in order to remove a by-product such as a protein or a nucleic acid, and an inorganic salt derived from a medium. A method of purifying by adding a water-soluble organic solvent, precipitating sodium hyaluronic acid, precipitating and extracting the supernatant, thereby purifying (Non-Patent Document 2).
[非專利文獻1]Journal of General Microbiology，85,372-375,1976[Non-Patent Document 1] Journal of General Microbiology, 85, 372-375, 1976
[專利文獻1]日本專利特公平4-12960號公報[Patent Document 1] Japanese Patent Special Publication No. 4-12960
[專利文獻2]日本專利特開平9-324001公報[Patent Document 2] Japanese Patent Laid-Open Publication No. Hei 9-324001
然而，於以工業規模獲得可作為醫藥品使用之高純度之玻尿酸類時，於上述方法中亦存在玻尿酸之回收率低等問題。However, when a high-purity hyaluronic acid which can be used as a pharmaceutical product is obtained on an industrial scale, there is a problem that the recovery rate of hyaluronic acid is low in the above method.
本發明係有鑒於上述情況而完成者，其目的在於提供一種簡便、高產率地以工業規模純化高純度之玻尿酸類之方法。The present invention has been made in view of the above circumstances, and an object thereof is to provide a method for purifying high-purity hyaluronic acid on an industrial scale in a simple and high-yield manner.
本發明者等人為達成上述目的，對將雜質自包含玻尿酸及其鹽與雜質之玻尿酸溶液中高效地分離去除而簡便且高效地純化高純度之玻尿酸類的方法進行了各種研究，結果發現藉由於將玻尿酸類溶液調整至偏酸性之pH值後、利用超濾膜進行透析處理，可有效去除核酸、發熱性物質、蛋白質、乳酸、無機鹽類等，從而完成了本發明。In order to achieve the above object, the inventors of the present invention conducted various studies on a method for efficiently and efficiently purifying high-purity hyaluronic acid by efficiently separating and removing impurities from hyaluronic acid solution containing hyaluronic acid and its salt and impurities, and found that The hyaluronic acid solution is adjusted to a pH value which is acidic, and then subjected to dialysis treatment using an ultrafiltration membrane to efficiently remove nucleic acids, exothermic substances, proteins, lactic acid, inorganic salts, and the like, thereby completing the present invention.
即，根據本發明，提供一種玻尿酸及/或其鹽之純化方法，其包括於將包含玻尿酸及其鹽與雜質之玻尿酸溶液調整至偏酸性之pH值後、利用超濾膜進行透析處理之步驟。根據該製造方法，可高效地去除雜質。That is, according to the present invention, there is provided a method for purifying hyaluronic acid and/or a salt thereof, which comprises the step of dialysis treatment using an ultrafiltration membrane after adjusting a hyaluronic acid solution containing hyaluronic acid, a salt thereof and an impurity to an acidic pH value. . According to this manufacturing method, impurities can be efficiently removed.
本說明書中之「玻尿酸及/或其鹽」與「玻尿酸類」同義，可交換使用，係指游離之玻尿酸、及於無損本發明之目的之範圍內可使用的任意之玻尿酸鹽(並不限定於此，例如鈉鹽、鉀鹽、鈣鹽、鋰鹽等金屬鹽，或鹽酸鹽、磷酸鹽、檸檬酸鹽等酸加成物等)或水合物、該等之混合物。此處，玻尿酸係指N-乙醯基-D-葡萄糖胺與D-葡糖醛酸鍵結之2糖單元重複連鏈而成之高分子量多糖類，各種鹽主要係指葡糖醛酸部分成為鹽之形態者。玻尿酸因可摺疊之鏈部分與D-葡糖醛酸部分之羧基之負電荷的相互作用而易於空間中展開，藉此可與大量水結合而形成凝膠。另外，即便為低濃度，但分子間力亦較強，因此具有相對較高之黏性。由於此種作用，例如具有濕潤關節之作用、柔軟皮膚之作用等，於生理上亦發揮該等作用。In the present specification, "hyaluronic acid and/or its salt" is synonymous with "hyaluronic acid" and is used interchangeably to mean free hyaluronic acid and any hyaluronic acid which can be used within the scope of the object of the present invention. Here, for example, a metal salt such as a sodium salt, a potassium salt, a calcium salt or a lithium salt, or an acid adduct such as a hydrochloride, a phosphate or a citrate, or a hydrate or a mixture thereof. Here, hyaluronic acid refers to a high molecular weight polysaccharide in which N-acetyl-D-glucosamine is repeatedly linked to a D-glucuronic acid-bonded 2-saccharide unit, and various salts mainly refer to a glucuronic acid moiety. Become the form of salt. Hyaluronic acid is easily spread out in space due to the interaction of the foldable chain portion with the negative charge of the carboxyl group of the D-glucuronic acid moiety, whereby it can combine with a large amount of water to form a gel. In addition, even at a low concentration, the intermolecular force is strong, so that it has a relatively high viscosity. Such an action, for example, has the action of moist joints, the action of soft skin, and the like, and also exerts such effects physiologically.
已知於玻尿酸類中，分子量約200萬Da之玻尿酸鈉與分子量約80萬Da者相比，作為醫藥品，對治療變形性膝關節炎、肩關節周圍炎、慢性類風濕性關節炎等發揮出優異之效果(藥理與治療、Vol. 22、No. 9、289(1994)；藥理與治療、Vol. 22、No. 9、319(1994))。另外，除此以外還已知作為外科手術後之防止黏連用之效果、進而亦於皮膚科領域、眼科領域中作為醫藥品之效果，一部分通常於臨床上使用。於作為醫藥品使用之情形時，較理想為使用平均分子量為100萬以上之玻尿酸類。若考慮獲取或處理之容易性，則作為醫藥品更理想為平均分子量為100萬~500萬Da之玻尿酸類，尤其理想為平均分子量為150萬~400萬Da之玻尿酸類。另外，此種高分子量玻尿酸類於作為化妝品用途使用時，亦因其較高之保濕力而發揮優異之效果。It is known that in hyaluronic acid, sodium hyaluronate having a molecular weight of about 2 million Da is used as a pharmaceutical for treating deformed knee arthritis, periarthritis of the shoulder, chronic rheumatoid arthritis, etc. as compared with a molecular weight of about 800,000 Da. Excellent results (Pharmacology and Therapy, Vol. 22, No. 9, 289 (1994); Pharmacology and Therapy, Vol. 22, No. 9, 319 (1994)). In addition, it is known that it is an effect of preventing adhesion after surgery, and is also effective as a pharmaceutical in the field of dermatology and ophthalmology, and some of them are generally used clinically. When it is used as a pharmaceutical, it is preferred to use a hyaluronic acid having an average molecular weight of 1,000,000 or more. In view of the ease of obtaining or handling, it is more preferable that the pharmaceutical is a hyaluronic acid having an average molecular weight of 1,000,000 to 5,000,000 Da, and particularly preferably a hyaluronic acid having an average molecular weight of 1.5 to 4 million Da. Further, when such a high molecular weight hyaluronic acid is used as a cosmetic, it also exerts an excellent effect due to its high moisturizing power.
關於本說明書中之「平均分子量」，只要無特別說明，則於表示玻尿酸類之平均分子量時，係指黏度平均分子量者。黏度平均分子量可藉由業者通常使用之方法而求出。較佳為可藉由各國之藥典等中通常所使用之測定方法而求出，更佳為可藉由日本藥典中所使用之測定方法而求出。作為一例，例如於期望玻尿酸鈉具有與本申請案發明接近之平均分子量(150萬~390萬)之情形時，其平均分子量可使用極限黏度[η]並藉由下式而求出，但並不限定於此。The "average molecular weight" in the present specification means the viscosity average molecular weight when it means the average molecular weight of hyaluronic acid unless otherwise specified. The viscosity average molecular weight can be determined by a method generally used by the manufacturer. It is preferably determined by a measurement method generally used in various Pharmacopoeias and the like, and more preferably, it can be obtained by a measurement method used in the Japanese Pharmacopoeia. As an example, for example, when sodium hyaluronate is desired to have an average molecular weight (1.5 million to 3.9 million) close to the invention of the present application, the average molecular weight can be obtained by using the following formula using the ultimate viscosity [η], but It is not limited to this.
[式1][Formula 1]
溶解玻尿酸類作為醫藥品之注射用溶解液可適當使用通常所使用之注射用溶解液(例如，各國藥典中所確認者)，該注射用溶解液係於注射用水、生理鹽水等中加入含有酸、鹼、磷酸鹽之類之緩衝劑的pH值調整劑等者。Dissolved hyaluronic acid as a solution for injection for pharmaceutical use can be suitably used as a solution for injection (for example, as confirmed in the National Pharmacopoeia), which is added to water for injection, physiological saline, or the like, and contains an acid. , pH adjusters such as alkali and phosphate buffers, and the like.
該等玻尿酸類可為藉由自動物組織中萃取之萃取法而製造者，亦可為藉由使用玻尿酸生產微生物菌株使其醱酵而獲得之醱酵法而製造者。然而，於自動物組織所萃取者中，其他黏多糖等雜質相對較多，且玻尿酸類之分子量亦較小，故而較理想為使用藉由醱酵法而獲得者。於適於本發明之醱酵法之一例中，例如可使用鏈球菌屬之微生物並藉由已知之方法獲得玻尿酸類。These hyaluronic acids may be produced by an extraction method extracted from an animal structure, or may be produced by a fermentation method obtained by producing a microbial strain using hyaluronic acid to ferment. However, among the extracts from the animal tissues, other impurities such as mucopolysaccharides are relatively large, and the molecular weight of the hyaluronic acid is also small, so it is preferable to use those obtained by the fermentation method. In one example of the fermentation method suitable for the present invention, for example, a microorganism of the genus Streptococcus can be used and a hyaluronic acid can be obtained by a known method.
於將藉由醱酵法而獲得之醱酵液用於本發明之方法等中之情形時，較理想為使用藉由已知之方法、例如離心分離或過濾處理等殺菌之溶液者。視情況，亦可使用添加乙醇等水溶性有機溶劑而將玻尿酸析出純化者。另外，亦可使用經氧化鋁等處理者。In the case where the fermentation broth obtained by the fermentation method is used in the method of the present invention or the like, it is preferred to use a solution which is sterilized by a known method such as centrifugation or filtration treatment. The hyaluronic acid may be precipitated and purified by adding a water-soluble organic solvent such as ethanol, as the case may be. Further, a processor such as alumina can also be used.
本說明書中之「鏈球菌」包括：可生產玻尿酸之鏈球菌(Streptococcus)屬之任意細菌‧其變異株。尤其較理想為使用如專利文獻2中所記載之馬鏈球菌(Streptococcus equi)FM-100(微工研菌寄第9027號)、日本專利特開平2-234689號公報中所記載之馬鏈球菌FM-300(微工研菌寄第2319號)之高產率且穩定地生產玻尿酸之變異株。作為適合生產玻尿酸之鏈球菌屬細菌之例，其他例如可列舉：馬鏈球菌(Streptococcus equi)、流行性鏈球菌(Streptococcus zooepidemicus)、類馬鏈球菌(Streptococcus equisimilis)、壞乳鏈球菌(Streptococcus dysgalactiae)、釀膿鏈球菌(Streptococcus pyogenes)及該等之變異株等，但並不限定於此。The "Streptococcus" in the present specification includes any strain which is a bacterium of the genus Streptococcus which can produce hyaluronic acid. In particular, it is preferable to use Streptococcus equium FM-100 (Microtechnology Research No. 9027) as described in Patent Document 2, and Streptococcus equi subsp. The high yield and stable production of hyaluronic acid mutants by FM-300 (Microtech Research No. 2319). As an example of a bacterium belonging to the genus Streptococcus which is suitable for producing hyaluronic acid, for example, Streptococcus equi, Streptococcus zooepidemicus, Streptococcus equisimilis, Streptococcus dysgalactiae ), Streptococcus pyogenes, and the like, but are not limited thereto.
所謂本說明書中之「超濾膜」，係指孔徑為0.001~0.01 μm之過濾膜及/或截留分子量為1000~300000左右之過濾膜。超濾膜之材質大致分為無機膜與有機膜，進而分為疏水性與親水性。作為疏水性有機膜，可列舉聚碸、聚醚碸、聚醚、聚偏二氟乙烯、聚乙烯、聚丙烯等，但並不限定於此。作為親水性有機膜，可列舉聚丙烯腈、聚醯胺、聚醯亞胺、醋酸纖維素等，但並不限定於此。其過濾介質之形狀包括平板膜、管狀膜、捲式膜、中空纖維(Hollow fiber)膜等所有模組形式。The "ultrafiltration membrane" in the present specification refers to a filtration membrane having a pore diameter of 0.001 to 0.01 μm and/or a filtration membrane having a molecular weight cut off of about 1,000 to 300,000. The material of the ultrafiltration membrane is roughly classified into an inorganic membrane and an organic membrane, and is further classified into hydrophobicity and hydrophilicity. Examples of the hydrophobic organic film include polyfluorene, polyether oxime, polyether, polyvinylidene fluoride, polyethylene, and polypropylene, but are not limited thereto. Examples of the hydrophilic organic film include polyacrylonitrile, polyamine, polyimine, cellulose acetate, and the like, but are not limited thereto. The shape of the filter medium includes all the form of a flat film, a tubular film, a roll film, a hollow fiber film, and the like.
過濾方式包括端點過濾方式與掃流方式。端點過濾方式係指將供給於膜之全部水過濾之方式。相對於此，掃流方式係指藉由相對於膜面平行流動而一面抑制供給於膜之水中所含之懸浮物或膠體堆積於膜面之現象一面進行過濾的方式。於掃流方式中，包括單程方式(One-pass method)、反沖方式(Backlash method)及反向方式(Reverse method)，但並不限定於此。所謂單程方式，係指如圖1之A所示不循環利用來自超濾膜之透過液而進行過濾之方式。所謂反沖方式，係指如圖1之B所示包括將來自超濾膜之透過液儲存於透過液槽中、自透過液槽轉移至超濾膜上、沖洗附著於過濾膜表面之玻尿酸類之步驟的過濾方式。所謂反向方式，係指如圖1之C所示包括藉由關閉透過液閥門使來自超濾膜之透過液逆流而沖洗附著於過濾膜表面之玻尿酸類之步驟的過濾方式。Filtering methods include endpoint filtering and sweeping. The endpoint filtration method refers to the manner in which all of the water supplied to the membrane is filtered. On the other hand, the sweeping method refers to a method of filtering while suppressing the phenomenon in which suspended matter or colloid contained in water supplied to the film is deposited on the film surface while flowing in parallel with the film surface. The sweep mode includes a one-pass method, a backlash method, and a reverse method, but is not limited thereto. The single-pass method refers to a method of filtering without using a permeate from an ultrafiltration membrane as shown in FIG. 1A. The so-called recoil method means that the permeate from the ultrafiltration membrane is stored in the permeate tank, transferred from the permeate tank to the ultrafiltration membrane, and the hyaluronic acid attached to the surface of the membrane is washed as shown in FIG. 1B. The filtering method of the steps. The reverse mode refers to a filtration method including a step of rinsing hyaluronic acid adhering to the surface of the filtration membrane by backflowing the permeate from the ultrafiltration membrane by closing the permeate valve as shown in FIG.
本說明書中之「雜質」係指玻尿酸類、水以外之溶劑成分、無機鹽以外之物質，尤其係指於使用作為最終產品之玻尿酸類時可帶來不利之物質(發熱性物質等)。作為主要雜質源，可列舉：於玻尿酸類之生產階段中源自組織、微生物或培養液(培養基)者、或者於其後之純化階段等中混入者。作為本說明書中之雜質之例，可列舉組織或菌體、蛋白質、核酸、多糖類、低分子化合物、或內毒素等，但並不限定於此。作為雜質之組織或菌體分別包括：源自於萃取法中所使用之作為萃取原料之組織的組織片等，或於醱酵法中所使用之微生物之菌體或菌體片等，但並不限定於此。作為雜質之蛋白質包括：源自上述組織、菌之蛋白質及於生產後之步驟中混入之蛋白質等，但並不限定於此。作為雜質之內毒素包括源自上述菌之脂多糖類等，但並不限定於此。In the present specification, the term "impurity" means a substance other than a hyaluronic acid, a solvent component other than water, or an inorganic salt, and particularly refers to a substance (heat generating substance or the like) which may be disadvantageous when using hyaluronic acid as a final product. Examples of the main impurity source include those derived from tissues, microorganisms, or culture liquids (medium) in the production stage of hyaluronic acid, or in a subsequent purification stage or the like. Examples of the impurities in the present specification include a tissue, a bacterial cell, a protein, a nucleic acid, a polysaccharide, a low molecular compound, or an endotoxin, but are not limited thereto. The tissue or the bacterial body as the impurity includes a tissue piece derived from a tissue used as an extraction raw material used in the extraction method, or a microbial cell or a bacterial cell sheet used in the fermentation method, but It is not limited to this. The protein as an impurity includes, but is not limited to, a protein derived from the above-mentioned tissues, bacteria, and a protein mixed in the step after production. The endotoxin as an impurity includes lipopolysaccharides derived from the above-mentioned bacteria, but is not limited thereto.
所謂本說明書中之「低分子化合物」，係指與玻尿酸類相比分子量相對較小之化合物，例如分子量為2000 Da以下、或分子量為1000 Da或分子量為500 Da以下之化合物，但並不限定於此。此種低分子化合物包括各種胺基酸、有機酸(例如乳酸)、糖(例如葡萄糖)等。The "low molecular compound" in the present specification means a compound having a relatively small molecular weight compared with hyaluronic acid, for example, a compound having a molecular weight of 2000 Da or less, or a molecular weight of 1000 Da or a molecular weight of 500 Da or less, but is not limited herein. Such low molecular compounds include various amino acids, organic acids such as lactic acid, sugars such as glucose, and the like.
本說明書中之「去除」除包括完全去除對象物質以外，亦包括部分去除(減少該物質之量)。本說明書中之「純化」包括去除任意或特定之雜質。In the present specification, "removal" includes partial removal (reduction of the amount of the substance) in addition to complete removal of the target substance. "Purification" in this specification includes the removal of any or specific impurities.
關於本說明書中之各個數值範圍，分別包含由「~」表示之上限值及下限值。Each numerical range in the present specification includes the upper limit and the lower limit by "~".
本發明例如係關於以下之實施態樣，但並不限定於此。The present invention is, for example, the following embodiments, but is not limited thereto.
實施態樣1.Implementation aspect
一種玻尿酸及/或其鹽之純化方法，其包括藉由於將包含玻尿酸及/或其鹽與雜質之玻尿酸溶液調整至偏酸性之pH值後、利用超濾膜進行透析處理而去除雜質之步驟。A method for purifying hyaluronic acid and/or a salt thereof, which comprises the step of removing impurities by dialysis treatment using an ultrafiltration membrane after adjusting a hyaluronic acid solution containing hyaluronic acid and/or a salt thereof and an impurity to an acidic pH value.
實施態樣2.Implementation aspect 2.
如實施態樣1之方法，其中於超濾膜之截留分子量與利用超濾膜進行透析處理時之pH值滿足下式之條件下，利用超濾膜進行透析處理：pH值≦-5×10-5 ×(截留分子量)+4.4978。The method according to the first aspect, wherein the dialysis treatment is performed by using an ultrafiltration membrane under the condition that the molecular weight of the ultrafiltration membrane and the pH value of the dialysis treatment using the ultrafiltration membrane satisfy the following formula: pH ≦-5×10 -5 x (molecular weight cut off) + 4.4978.
實施態樣3.Implementation aspect 3.
如實施態樣1或2之方法，其中超濾膜之截留分子量為25000~35000，透析處理時之pH值為3.3以下。According to the method of the first aspect or the second aspect, the molecular weight cut off of the ultrafiltration membrane is 25,000 to 35,000, and the pH value during the dialysis treatment is 3.3 or less.
實施態樣4.Implementation aspect 4.
如實施態樣1或2之方法，其中上述超濾膜之截留分子量為12000~14000，透析處理時之pH值為3.9以下。The method according to Embodiment 1 or 2, wherein the ultrafiltration membrane has a molecular weight cut off of 12,000 to 14,000, and the pH value during dialysis treatment is 3.9 or less.
實施態樣5.Implementation aspect 5.
如實施態樣1或2之方法，其中上述超濾膜之截留分子量為9000~11000，透析處理時之pH值為4.1以下。The method of the aspect 1 or 2, wherein the ultrafiltration membrane has a molecular weight cut off of 9000 to 11,000, and the pH of the dialysis treatment is 4.1 or less.
實施態樣6.Implementation aspect 6.
如實施態樣1或2之方法，其中上述超濾膜之截留分子量為6000~8000，透析處理時之pH值為4.2以下。The method according to Embodiment 1 or 2, wherein the ultrafiltration membrane has a molecular weight cut off of 6000 to 8000, and the pH of the dialysis treatment is 4.2 or less.
實施態樣7.Implementation aspect 7.
如實施態樣1或2之方法，其中上述超濾膜之截留分子量為4000~5000，透析處理時之pH值為4.3以下。The method according to Embodiment 1 or 2, wherein the ultrafiltration membrane has a molecular weight cut off of 4,000 to 5,000, and the pH of the dialysis treatment is 4.3 or less.
實施態樣8.Implementation aspect 8.
如實施態樣1至7中任一項之方法，其中上述超濾膜為疏水性有機膜。The method of any one of embodiments 1 to 7, wherein the ultrafiltration membrane is a hydrophobic organic membrane.
實施態樣9.Implementation aspect 9.
如實施態樣1至8中任一項之方法，其中上述處理之過濾方式為反向方式。The method of any of embodiments 1 to 8, wherein the filtering of the above treatment is in a reverse manner.
實施態樣10.Implementation aspect
如實施態樣1至9中任一項之方法，其中上述處理時之透過流速為20~50 L/m2 ‧hr。The method of any one of the embodiments 1 to 9, wherein the permeation flow rate in the above treatment is 20 to 50 L/m 2 ‧ hr.
實施態樣11.Implementation aspect 11.
如實施態樣1至10中任一項之方法，其中上述雜質包括菌體、蛋白質、核酸、低分子化合物或內毒素。The method of any one of embodiments 1 to 10, wherein the impurities include bacteria, proteins, nucleic acids, low molecular compounds or endotoxins.
實施態樣12.Implementation aspect 12.
如實施態樣1至11中任一項之方法，其中純化後之上述玻尿酸及/或其鹽之平均分子量為350萬~700萬Da。The method of any one of the aspects 1 to 11, wherein the hyaluronic acid and/or its salt after purification has an average molecular weight of 3.5 to 7 million Da.
實施態樣13.Implementation aspect 13.
如實施態樣1至12中任一項之方法，其中上述玻尿酸類溶液中之玻尿酸及/或其鹽之濃度為1~5 g/L。The method according to any one of the aspects 1 to 12, wherein the concentration of hyaluronic acid and/or a salt thereof in the hyaluronic acid solution is 1 to 5 g/L.
以下對本發明之態樣進行說明。The aspects of the invention are described below.
本發明之態樣(例如實施態樣1)係一種玻尿酸及/或其鹽之純化方法，其包括藉由於將包含玻尿酸及/或其鹽與雜質之玻尿酸類溶液調整至偏酸性之pH值後、利用超濾膜進行透析處理而去除雜質之步驟。根據該純化方法，可藉由調整至偏酸性之pH值而減少利用超濾膜進行過濾時之玻尿酸類之損失，而高效地去除雜質。The aspect of the present invention (for example, the embodiment 1) is a method for purifying hyaluronic acid and/or a salt thereof, which comprises adjusting a hyaluronic acid solution containing hyaluronic acid and/or a salt thereof and an impurity to an acidic pH value. The step of removing the impurities by performing dialysis treatment using an ultrafiltration membrane. According to this purification method, the loss of hyaluronic acid when filtering by the ultrafiltration membrane can be reduced by adjusting to a pH value which is acidic, and impurities can be efficiently removed.
藉由於超濾膜之截留分子量與利用超濾膜進行透析處理時之pH值滿足下式之條件下利用超濾膜進行透析處理，可於實質上不損失玻尿酸類之情況下進行純化：pH值≦-5×10-5 ×(截留分子量)+4.4978。作為滿足上述之式的超濾膜之截留分子量與利用超濾膜進行透析處理時之pH值，例如可列舉：超濾膜之截留分子量為35000，pH值為2.7；超濾膜之截留分子量為30000，pH值為3.0；超濾膜之截留分子量為25000，pH值為3.3；超濾膜之截留分子量為20000，pH值為3.5；超濾膜之截留分子量為14000，pH值為3.8；超濾膜之截留分子量為13000，pH值為3.9；超濾膜之截留分子量為12000，pH值為3.9；超濾膜之截留分子量為11000，pH值為4.0；超濾膜之截留分子量為10000，pH值為4.0；超濾膜之截留分子量為9000，pH值為4.1；超濾膜之截留分子量為8000，pH值為4.1；超濾膜之截留分子量為7000，pH值為4.2；超濾膜之截留分子量為6000，pH值為4.2；超濾膜之截留分子量為5000，pH值為4.3；超濾膜之截留分子量為4000，pH值為4.3。By using the ultrafiltration membrane for dialysis treatment because the molecular weight of the ultrafiltration membrane and the pH value of the ultrafiltration membrane are dialysis treatment, the purification can be carried out without substantially losing hyaluronic acid: pH value ≦-5×10 -5 × (molecular weight cut off) +4.4978. The pH value of the ultrafiltration membrane satisfying the above formula and the pH value of the dialysis treatment using the ultrafiltration membrane include, for example, an ultrafiltration membrane having a molecular weight cutoff of 35,000 and a pH of 2.7; and the molecular weight cutoff of the ultrafiltration membrane is 30000, pH value is 3.0; the ultrafiltration membrane has a molecular weight cutoff of 25000 and a pH value of 3.3; the ultrafiltration membrane has a molecular weight cutoff of 20,000 and a pH of 3.5; the ultrafiltration membrane has a molecular weight cutoff of 14,000 and a pH of 3.8; The molecular weight cut-off of the filter membrane is 13000, the pH value is 3.9; the molecular weight cut-off of the ultrafiltration membrane is 12000, the pH value is 3.9; the molecular weight cut-off of the ultrafiltration membrane is 11000, the pH value is 4.0; and the molecular weight cutoff of the ultrafiltration membrane is 10,000. The pH value is 4.0; the molecular weight cut-off of the ultrafiltration membrane is 9000, the pH value is 4.1; the molecular weight cutoff of the ultrafiltration membrane is 8000, the pH value is 4.1; the molecular weight cutoff of the ultrafiltration membrane is 7000, the pH value is 4.2; ultrafiltration membrane The molecular weight cut-off is 6000, the pH value is 4.2; the molecular weight cut-off of the ultrafiltration membrane is 5000, the pH value is 4.3; the molecular weight cutoff of the ultrafiltration membrane is 4000, and the pH value is 4.3.
此處，所謂「實質上不損失玻尿酸類」，係指玻尿酸類之損失率成為7%以下(回收率成為93%以上)、6%以下(回收率成為94%以上)、5%以下(回收率成為95%以上)、4%以下(回收率成為96%以上)或3%以下(回收率成為97%以上)。Here, "there is no loss of hyaluronic acid" means that the loss rate of hyaluronic acid is 7% or less (recovery rate is 93% or more), 6% or less (recovery rate is 94% or more), and 5% or less (recycling) The rate is 95% or more, 4% or less (recovery rate is 96% or more) or 3% or less (recovery rate is 97% or more).
若使用截留分子量較高之膜，則玻尿酸類透過超濾膜而產生損失之風險增高，另一方面若使用截留分子量較低之膜，則蛋白質等相對高分子之雜質之去除效率下降。根據上述實施態樣之方法，使用截留分子量較高之膜、與使用截留分子量較低之膜均可藉由調整至與膜之截留分子量對應之pH值而於不損失玻尿酸類之情況下去除雜質，因此於上述態樣之方法中使用之超濾膜的截留分子量並無特別限定。例如，於使用具有25000~35000之截留分子量之超濾膜之情形時，可藉由將pH值調整至3.3以下而於不損失玻尿酸類之情況下進行純化。於使用具有12000~14000之截留分子量之超濾膜之情形時，可藉由將pH值調整至3.9以下而於不損失玻尿酸類之情況下進行純化。於使用具有9000~11000之截留分子量之超濾膜之情形時，可藉由將pH值調整至4.1以下而於不損失玻尿酸類之情況下進行純化。於使用具有6000~8000之截留分子量之超濾膜之情形時，可藉由將pH值調整至4.2以下而於不損失玻尿酸類之情況下進行純化。於使用具有4000~5000之截留分子量之超濾膜之情形時，可藉由將pH值調整至4.3以下而於不損失玻尿酸類之情況下進行純化。When a film having a high molecular weight cut off is used, the risk of loss of hyaluronic acid through the ultrafiltration membrane is increased. On the other hand, when a membrane having a low molecular weight cut off is used, the removal efficiency of impurities such as proteins with respect to a polymer is lowered. According to the method of the above embodiment, the use of a membrane having a higher molecular weight cut-off and the use of a membrane having a lower molecular weight cut-off can be carried out by adjusting the pH corresponding to the molecular weight cut off from the membrane without loss of hyaluronic acid. Therefore, the molecular weight cut off of the ultrafiltration membrane used in the method of the above aspect is not particularly limited. For example, in the case of using an ultrafiltration membrane having a molecular weight cut off of 25,000 to 35,000, purification can be carried out without loss of hyaluronic acid by adjusting the pH to 3.3 or less. In the case of using an ultrafiltration membrane having a molecular weight cut off of 12,000 to 14,000, purification can be carried out without loss of hyaluronic acid by adjusting the pH to 3.9 or less. In the case of using an ultrafiltration membrane having a molecular weight cut off of 9000 to 11,000, purification can be carried out without loss of hyaluronic acid by adjusting the pH to 4.1 or less. In the case of using an ultrafiltration membrane having a molecular weight cut off of 6000 to 8000, purification can be carried out without loss of hyaluronic acid by adjusting the pH to 4.2 or less. In the case of using an ultrafiltration membrane having a molecular weight cut off of 4000 to 5,000, purification can be carried out without loss of hyaluronic acid by adjusting the pH to 4.3 or less.
此處，超濾膜之截留分子量可藉由使用如表1所示之目標物質進行過濾並調查各自之阻塞率相當於90%之分子量而確定。Here, the molecular weight cut-off of the ultrafiltration membrane can be determined by filtering using the target substance shown in Table 1 and investigating the respective blocking ratios corresponding to a molecular weight of 90%.
於上述態樣之方法中使用之超濾膜之材質並無特別限定，就雜質之去除之觀點而言較理想為疏水性有機膜，更理想為聚碸、聚醚碸、聚醚、聚偏二氟乙烯、聚乙烯、聚丙烯。The material of the ultrafiltration membrane used in the method of the above aspect is not particularly limited, and is preferably a hydrophobic organic membrane from the viewpoint of removal of impurities, and more preferably polyfluorene, polyether oxime, polyether, and polyposition. Difluoroethylene, polyethylene, polypropylene.
作為於上述態樣之方法中使用之超濾膜，例如可使用：PM-10、PM-50、PM-100(Koch公司製造)，NTU-3050(日東電工公司製造)，IRIS3065(羅納‧普朗克公司製造)，FS-10(旭化成公司製造)，MU-6303(可樂麗公司製造)、DUSO400(泰金化學工業公司製造)，SLP-3053(旭化成化學公司製造)等，但並不限定於該等。As the ultrafiltration membrane used in the method of the above aspect, for example, PM-10, PM-50, PM-100 (manufactured by Koch Co., Ltd.), NTU-3050 (manufactured by Nitto Denko Corporation), IRIS 3065 (Rona ‧ can be used) Made by Planck, FS-10 (made by Asahi Kasei Corporation), MU-6303 (made by Kuraray Co., Ltd.), DUSO400 (made by Taijin Chemical Industry Co., Ltd.), SLP-3053 (made by Asahi Kasei Chemicals Co., Ltd.), etc. Limited to these.
上述態樣之方法中之過濾方式並無特別限定，但由於水透過膜之流速易穩定化、過濾膜之壽命延長等，故而較理想為掃流方式，其中尤其理想為反向方式。The filtration method in the method of the above aspect is not particularly limited. However, since the flow rate of the water permeable membrane is easily stabilized, the life of the filtration membrane is prolonged, and the like, it is preferable to use a sweeping method, and particularly preferably a reverse mode.
於上述態樣之方法中，玻尿酸類溶液於超濾膜處理時之透過流速根據玻尿酸類溶液之性狀或超濾膜之種類而不同，不能一概而論。例如，於以工業規模純化玻尿酸類時，較理想為20 L/m2 ‧hr以上，更理想為25 L/m2 ‧hr以上，尤其理想為30 L/m2 ‧hr以上。因存在剪切玻尿酸類而引起分子量降低之可能性，故玻尿酸類溶液於過濾時之透過流速較佳為100 L/m2 ‧hr以下，進而較佳為50 L/m2 ‧hr以下。In the above method, the flow rate of the hyaluronic acid solution in the ultrafiltration membrane treatment varies depending on the properties of the hyaluronic acid solution or the type of the ultrafiltration membrane, and cannot be generalized. For example, when purifying hyaluronic acid on an industrial scale, it is preferably 20 L/m 2 ‧ hr or more, more preferably 25 L/m 2 ‧ hr or more, and particularly preferably 30 L/m 2 ‧ hr or more The flow rate of the hyaluronic acid solution during filtration is preferably 100 L/m 2 ‧ hr or less, and more preferably 50 L/m 2 ‧ hr or less due to the possibility of a decrease in molecular weight due to shearing hyaluronic acid.
於上述態樣之方法中，用以輸送液體之壓力並無限定，通常較佳為於加壓下通過過濾膜。尤佳為利用泵等加壓而輸送之方法。當於輸送液體時利用泵施加壓力時，只要不損壞或堵塞過濾膜而使性能劣化，則無特別限定。施加於過濾膜上之壓力較佳為0.01 MPa以上0.30 MPa以下、更佳為0.03 MPa以上0.20 MPa以下、尤佳為0.05 MPa以上0.10 MPa以下。In the above method, the pressure for transporting the liquid is not limited, and it is usually preferred to pass through the filtration membrane under pressure. It is a method of transporting by means of a pump or the like. When the pressure is applied by the pump when the liquid is transported, there is no particular limitation as long as the performance is deteriorated without damaging or clogging the filter membrane. The pressure applied to the filtration membrane is preferably 0.01 MPa or more and 0.30 MPa or less, more preferably 0.03 MPa or more and 0.20 MPa or less, and particularly preferably 0.05 MPa or more and 0.10 MPa or less.
進而，根據上述態樣之方法，可減少純化時玻尿酸類之分子量之下降，尤其是於高分子量(例如，純化後之平均分子量為350萬~700萬Da)玻尿酸類之純化中，發揮優異之效果。另外，即便對先前難以處理之相對高濃度(例如0.1~20 g/L、0.5~15 g/L、1~10 g/L)之玻尿酸類溶液，亦可高效地處理。Further, according to the method of the above aspect, the decrease in the molecular weight of hyaluronic acid at the time of purification can be reduced, and in particular, in the purification of hyaluronic acid having a high molecular weight (for example, an average molecular weight after purification of 3.5 to 7 million Da), it is excellent. effect. In addition, even a relatively high concentration (for example, 0.1 to 20 g/L, 0.5 to 15 g/L, and 1 to 10 g/L) of hyaluronic acid solution which was previously difficult to handle can be efficiently treated.
另外，根據上述態樣之方法，雖然玻尿酸類之消失受到較低的抑制，但作為雜質，不僅通常可利用超濾膜去除之低分子化合物(例如胺基酸、糖、有機酸)而且高分子化合物(例如核酸、內毒素、蛋白質)亦可高效地分離‧去除。另外，上述態樣之方法於核酸、內毒素及或蛋白質之去除中亦發揮優異之效果。Further, according to the method of the above aspect, although the disappearance of hyaluronic acid is suppressed to a low level, as an impurity, not only a low molecular compound (for example, an amino acid, a sugar, an organic acid) but also a polymer which can be removed by an ultrafiltration membrane can be used. Compounds (eg, nucleic acids, endotoxins, proteins) can also be efficiently separated and removed. Further, the above method also exerts an excellent effect in the removal of nucleic acids, endotoxins, and proteins.
使用上述態樣之方法時之玻尿酸類溶液之玻尿酸類濃度就由溶液黏度之高低引起之處理之難易度及玻尿酸類之溶解度之觀點而言，較理想為0.1~20 g/L，最理想為1~10 g/L，但並不限定於此。The hyaluronic acid concentration of the hyaluronic acid solution when using the above method is preferably 0.1 to 20 g/L from the viewpoint of the ease of treatment caused by the viscosity of the solution and the solubility of hyaluronic acid. 1~10 g/L, but it is not limited to this.
另外，於上述態樣之方法中，為降低玻尿酸類溶液之黏度，亦可於玻尿酸類溶液中共存氯化鈉等鹽類。於此情形時，較理想為避免高濃度鹽之共存使得純化效果不受損壞。作為此種鹽之共存之具體例，可列舉於玻尿酸類溶液中添加0.1~5重量%之氯化鈉。Further, in the method of the above aspect, in order to reduce the viscosity of the hyaluronic acid solution, a salt such as sodium chloride may be coexisted in the hyaluronic acid solution. In this case, it is desirable to avoid the coexistence of high concentration salts so that the purification effect is not damaged. Specific examples of the coexistence of such a salt include the addition of 0.1 to 5% by weight of sodium chloride to the hyaluronic acid solution.
使用上述態樣之方法時之玻尿酸類溶液之溫度較理想為0~80℃，但並不限定於此。若溫度為80℃以下，則可較強地抑制處理時之玻尿酸類之分解及分子量之降低。The temperature of the hyaluronic acid solution when the method of the above aspect is used is preferably 0 to 80 ° C, but is not limited thereto. When the temperature is 80 ° C or lower, the decomposition of hyaluronic acid and the decrease in molecular weight during the treatment can be strongly suppressed.
另外，於進行超濾透析處理時，作為膜前處理，較理想為藉由利用2%以下之鹼(例如氫氧化鈉水溶液)、過氧化物(例如次氯酸鈉水溶液)、界面活性劑、檸檬酸、檸檬酸銨‧酵素清潔劑等藥劑對膜進行清洗處理之化學方法，或藉由沖洗、海綿球、空氣注入法等物理方法進行。Further, in the ultrafiltration dialysis treatment, it is preferred to use 2% or less of a base (for example, an aqueous sodium hydroxide solution), a peroxide (for example, an aqueous sodium hypochlorite solution), a surfactant, citric acid, or the like as a pretreatment for the membrane. A chemical method in which a film such as ammonium citrate or an enzyme cleaner is cleaned or treated by a physical method such as rinsing, sponge ball or air injection.
本發明之進一步之態樣，於上述第一態樣中，除上述步驟以外，亦可進而包含使玻尿酸類溶液與無機吸附劑、有機吸附劑及/或活性碳接觸之步驟。In a further aspect of the present invention, in the first aspect, in addition to the above steps, the step of contacting the hyaluronic acid solution with the inorganic adsorbent, the organic adsorbent and/or the activated carbon may be further included.
另外，若考慮其後所必須之分離‧純化步驟等，則較理想為避免需要追加之純化步驟之成分的混入。即，為避免混入新雜質之本發明之其他態樣，係於上述第一態樣中不包括於玻尿酸類溶液之超濾後，使其與無機吸附劑或有機吸附劑接觸之步驟的純化方法。關於本發明之進一步之態樣，於上述第一態樣中不包括使玻尿酸類溶液與無機吸附劑或有機吸附劑接觸之步驟。於本發明中，即便為超濾亦可發揮充分之純化效果，故而於重視避免新雜質之混入之情形時，較理想為單獨利用超濾進行處理。當然，於此情形時，除超濾以外亦可進行其他純化處理等步驟。Further, in consideration of the separation, purification step, and the like which are necessary thereafter, it is preferable to avoid the incorporation of components requiring an additional purification step. That is, in order to avoid the other aspect of the present invention in which new impurities are mixed, the purification method of the step of contacting the inorganic adsorbent or the organic adsorbent after the ultrafiltration of the hyaluronic acid solution in the first aspect is not included. . In a further aspect of the invention, the step of contacting the hyaluronic acid solution with the inorganic adsorbent or organic adsorbent is not included in the first aspect described above. In the present invention, even if it is ultrafiltration, a sufficient purification effect can be exhibited. Therefore, when it is important to avoid the incorporation of new impurities, it is preferred to carry out the treatment by ultrafiltration alone. Of course, in this case, other purification treatments and the like may be performed in addition to ultrafiltration.
另外，關於本發明之進一步之態樣，係於上述態樣中藉由馬鏈球菌FM-100(微工研條寄第9027號)或馬鏈球菌FM-300(微工研條寄第2319號)生產玻尿酸類。藉由使用由該等微生物生產之玻尿酸類作為純化對象，可獲得雜質更少且高分子量之玻尿酸類純化物，尤其是於作為醫藥使用時發揮優異之效果。In addition, in a further aspect of the present invention, in the above aspect, the Streptococcus equi subsp. FM-100 (Microtech Research No. 9027) or Streptococcus equi subsp. FM-300 (Microtech Research No. 2319) No.) Production of hyaluronic acid. By using hyaluronic acid produced by such microorganisms as a purification target, a purified hyaluronic acid having a small amount of impurities and a high molecular weight can be obtained, and in particular, it is excellent in use as a medicine.
藉由使用上述態樣之純化方法可減輕玻尿酸類之分離‧純化步驟之負荷，因此有關上述態樣之純化方法於製造之工業製程的較初始階段中使用尤為有效。By using the above-described purification method, the separation of the hyaluronic acid and the loading step can be alleviated, so that the purification method of the above aspect is particularly effective in the initial stage of the industrial process of manufacturing.
再者，藉由上述實施態樣、態樣而說明之純化方法等並非限定本發明者，而旨在舉例說明而揭示者。本發明之技術範圍係由申請專利範圍所規定，業者可於申請專利範圍所揭示之發明之技術範圍內進行各種設計變更。In addition, the purification method and the like described by the above-described embodiment and aspects are not intended to limit the inventors, and are intended to be illustrative. The technical scope of the present invention is defined by the scope of the patent application, and various design changes can be made within the technical scope of the invention disclosed in the scope of the patent application.
例如，上述純化方法亦可為進一步包括其他步驟、或者繼上述純化方法之後進一步實施其他之步驟‧方法，而製造玻尿酸類等之方法。作為此種步驟‧方法，例如可列舉：培養玻尿酸生產微生物菌株之步驟；由玻尿酸產生微生物菌株培養液製造培養濾液之步驟；離心分離純化對象液之步驟；中和對象液之步驟；微濾純化對象液之步驟；透析處理對象液之步驟；於純化對象液中添加芳香族系吸附樹脂並攪拌及超濾之步驟；藉由層析法純化對象液之步驟；將活性碳自對象液中分離之步驟；將活性碳自對象液中去除之步驟；添加有機溶劑使玻尿酸類沈澱之步驟；將玻尿酸類結晶化之步驟；使玻尿酸類乾燥之步驟等。For example, the above purification method may be a method of further preparing a hyaluronic acid or the like by further including other steps or further performing the other steps ‧ methods after the above purification method. As such a step ‧ method, for example, a step of culturing a hyaluronic acid producing microorganism strain; a step of producing a culture filtrate from a hyaluronic acid-producing microbial strain culture solution; a step of centrifuging the purification target liquid; a step of neutralizing the target liquid; and a microfiltration purification a step of the target liquid; a step of dialysis treatment of the target liquid; a step of adding an aromatic adsorption resin to the purification target liquid, stirring and ultrafiltration; a step of purifying the target liquid by chromatography; and separating the activated carbon from the target liquid a step of removing activated carbon from the target liquid; a step of adding an organic solvent to precipitate hyaluronic acid; a step of crystallizing hyaluronic acid; a step of drying hyaluronic acid; and the like.
以下，藉由實施例更具體地說明本發明，但本發明並不限定於該等。Hereinafter, the present invention will be specifically described by way of Examples, but the present invention is not limited thereto.
實施例1Example 1
利用純水將使用馬鏈菌球FM-100(微工研寄第9027號)培養之培養液45 L稀釋為80 L(玻尿酸鈉濃度2.0 g/L)並藉由離心分離去除菌體。將所得之粗製玻尿酸調整至pH 2.9後，利用截留分子量為30000且材質為聚碸之超濾膜(Koch公司製造‧PM-100)2 m2 ，以30 L/m2 ‧hr之透過流速反覆進行容積比為2倍之濃縮、等倍之稀釋操作，並以透析次數10次、反向方式進行處理。將食鹽2.4 kg溶解於80 L所得之溶液中，調整至pH 7後以乙醇240 L使其析出並以乙醇8 L進行清洗，於40℃下進行真空乾燥，而獲得玻尿酸鈉。將分析結果及玻尿酸回收率示於表2。The culture medium (45 mL of sodium hyaluronic acid concentration: 2.0 g/L) cultured in a spheromycin ball FM-100 (Microtechnical Research No. 9027) was diluted with pure water and the cells were removed by centrifugation. After the obtained crude hyaluronic acid was adjusted to pH 2.9, it was repeated at a flow rate of 30 L/m 2 ‧ hr using an ultrafiltration membrane (manufactured by Koch Co., Ltd., ‧PM-100) 2 m 2 having a molecular weight cutoff of 30,000 and a polypyrene A concentration-equal dilution operation of 2 times the volume ratio was performed, and the treatment was performed in the reverse manner with 10 times of dialysis. 2.4 kg of salt was dissolved in 80 L of the solution, adjusted to pH 7, precipitated with 240 L of ethanol, washed with 8 L of ethanol, and vacuum dried at 40 ° C to obtain sodium hyaluronate. The analysis results and the hyaluronic acid recovery rate are shown in Table 2.
實施例2Example 2
將實施例1中所使用之粗製玻尿酸調整至pH 2.9後，利用截留分子量為30000且材質為聚醚碸之超濾膜(旭化成公司製造‧FS-10)5 m2 ，以30 L/m2 ‧hr之透過流速反覆進行容積比為2倍之濃縮、等倍之純水稀釋，並以透析次數11次、反向方式進行處理。所得之溶液係以與實施例1相同之方式進行處理而獲得玻尿酸鈉。將分析結果及玻尿酸回收率示於表2。After the crude hyaluronic acid used in Example 1 was adjusted to pH 2.9, an ultrafiltration membrane (manufactured by Asahi Kasei Co., Ltd. ‧ FS-10) 5 m 2 having a molecular weight cut off of 30,000 and a polyether oxime was used, and 30 L/m 2 was used. The flow rate of ‧ hr was repeatedly diluted with a volume ratio of 2 times, diluted with pure water, and treated in the reverse direction with 11 times of dialysis. The resulting solution was treated in the same manner as in Example 1 to obtain sodium hyaluronate. The analysis results and the hyaluronic acid recovery rate are shown in Table 2.
實施例3Example 3
將實施例1中所使用之粗製玻尿酸調整至pH 3.3後，以利用截留分子量20000材質聚碸之超濾膜(日東電工公司製造‧NTU-3050)3 m2 ，以30 L/m2 ‧hr之透過流速反覆進行容積比為2倍之濃縮、等倍之純水稀釋，並以透析次數8次、反向方式進行處理。所得之溶液係以與實施例1相同之方式進行處理而獲得玻尿酸鈉。將分析結果及玻尿酸鈉之回收率示於表2。After the crude hyaluronic acid used in Example 1 was adjusted to pH 3.3, it was made up of an ultrafiltration membrane (manufactured by Nitto Denko Corporation ‧ NTU-3050) 3 m 2 using a molecular weight cut off of 20,000, to 30 L/m 2 ‧ hr The flow rate was repeatedly diluted by a volume ratio of 2 times, diluted with pure water of equal magnification, and treated in the reverse manner with 8 times of dialysis. The resulting solution was treated in the same manner as in Example 1 to obtain sodium hyaluronate. The analysis results and the recovery rate of sodium hyaluronate are shown in Table 2.
實施例4Example 4
將實施例1中所使用之粗製玻尿酸調整至pH 2.9後，利用截留分子量為30000且材質為聚偏二氟乙烯之超濾膜(羅納‧普朗克公司製造‧IRIS3065)5 m2 ，以30 L/m2 ‧hr之透過流速反覆進行容積比為2倍之濃縮、等倍之純水稀釋，並以透析次數9次、反向方式進行處理。所得之溶液係以與實施例1相同之方式進行處理而獲得玻尿酸鈉。將分析結果及玻尿酸鈉之回收率示於表2。After the crude hyaluronic acid used in Example 1 was adjusted to pH 2.9, an ultrafiltration membrane (manufactured by Rhone-Planck Co., Ltd. ‧IRIS3065) 5 m 2 having a molecular weight cutoff of 30,000 and a material of polyvinylidene fluoride was used. The flow rate of 30 L/m 2 ‧ hr was repeatedly diluted with a volume ratio of 2 times, diluted with pure water, and treated in the reverse direction with 9 times of dialysis. The resulting solution was treated in the same manner as in Example 1 to obtain sodium hyaluronate. The analysis results and the recovery rate of sodium hyaluronate are shown in Table 2.
實施例5Example 5
將實施例1中所使用之粗製玻尿酸調整至pH 2.7後，利用截留分子量為40000且材質為聚醚碸之超濾膜(泰金化學工業公司製造‧DUSO400)5 m2 ，以30 L/m2 ‧hr之透過流速反覆進行容積比為2倍之濃縮、等倍之純水稀釋，並以透析次數11次、反向方式進行處理。所得之液係以與實施例1相同之方式進行處理而獲得150 g之玻尿酸鈉。將分析結果及玻尿酸鈉之回收率示於表2。After the crude hyaluronic acid used in Example 1 was adjusted to pH 2.7, an ultrafiltration membrane (manufactured by Taijin Chemical Industry Co., Ltd. ‧DUSO400) 5 m 2 with a molecular weight cut off of 40,000 and a polyether oxime was used, at 30 L/m. 2 ‧ hr of the permeation flow rate was repeated to dilute the volume ratio of 2 times, diluted with pure water, and treated in the reverse direction with 11 times of dialysis. The resulting liquid was treated in the same manner as in Example 1 to obtain 150 g of sodium hyaluronate. The analysis results and the recovery rate of sodium hyaluronate are shown in Table 2.
實施例6Example 6
將實施例1中所使用之粗製玻尿酸調整至pH 3.7後，利用截留分子量為13000且材質為聚碸之超濾膜(可樂麗公司製造‧MU-6303)5 m2 ，以30 L/m2 ‧hr之透過流速反覆進行容積比為2倍之濃縮、等倍之純水稀釋，並以透析次數11次、反向方式進行處理。所得之溶液係以與實施例1相同之方式進行處理而獲得玻尿酸鈉。將分析結果及玻尿酸鈉之回收率示於表2。After the crude hyaluronic acid used in Example 1 was adjusted to pH 3.7, an ultrafiltration membrane (manufactured by Kuraray ‧ MU-6303) 5 m 2 with a molecular weight cut off of 13,000 and a molecular weight of 13,000 was used, and 30 L/m 2 was used. The flow rate of ‧ hr was repeatedly diluted with a volume ratio of 2 times, diluted with pure water, and treated in the reverse direction with 11 times of dialysis. The resulting solution was treated in the same manner as in Example 1 to obtain sodium hyaluronate. The analysis results and the recovery rate of sodium hyaluronate are shown in Table 2.
實施例7Example 7
將實施例1中所使用之粗製玻尿酸調整至pH 3.7後，利用截留分子量為10000且材質為聚碸之超濾膜(旭化成化學公司製造‧SLP-3053)4.5 m2 ，以30 L/m2 ‧hr之透過流速反覆進行容積比為2倍之濃縮、等倍之純水稀釋，並以透析次數11次、反向方式進行處理。所得之溶液係以與實施例1相同之方式進行處理而獲得玻尿酸鈉。將分析結果及玻尿酸鈉之回收率示於表2。After the crude hyaluronic acid used in Example 1 was adjusted to pH 3.7, an ultrafiltration membrane (manufactured by Asahi Kasei Chemicals Co., Ltd. ‧SLP-3053) 4.5 m 2 , having a molecular weight cut off of 10,000 and having a molecular weight of 10,000, was used at 30 L/m 2 . The flow rate of ‧ hr was repeatedly diluted with a volume ratio of 2 times, diluted with pure water, and treated in the reverse direction with 11 times of dialysis. The resulting solution was treated in the same manner as in Example 1 to obtain sodium hyaluronate. The analysis results and the recovery rate of sodium hyaluronate are shown in Table 2.
比較例1Comparative example 1
將實施例1中所使用之粗製玻尿酸調整至pH 5.5後，利用截留分子量為10000且材質為聚碸之疏水性超濾膜(Koch公司製造‧PM-10)2 m2 ，以30 L/m2 ‧hr之透過流速反覆進行容積比為2倍之濃縮、等倍之稀釋操作，並以透析次數15次、反向方式進行處理。所得之溶液係以與實施例1相同之方式進行處理而獲得玻尿酸鈉。將分析結果及玻尿酸回收率示於表2。After adjusting the crude hyaluronic acid used in Example 1 to pH 5.5, a hydrophobic ultrafiltration membrane (manufactured by Koch Co., Ltd. ‧PM-10) 2 m 2 having a molecular weight cut off of 10,000 and having a molecular weight of 10,000 was used, and 30 L/m was used. 2 ‧ hr of the permeation flow rate was repeated twice the volume ratio of the concentration, equal dilution operation, and the number of dialysis times 15 times, reverse processing. The resulting solution was treated in the same manner as in Example 1 to obtain sodium hyaluronate. The analysis results and the hyaluronic acid recovery rate are shown in Table 2.
比較例2Comparative example 2
將實施例1中所使用之粗製玻尿酸調整至pH 3.6後，利用截留分子量為20000且材質為乙酸纖維素之親水性超濾膜(DDS公司製造‧CA600PP)4.5 m2 ，以30 L/m2 ‧hr之透過流速反覆進行容積比為2倍之濃縮、等倍之純水稀釋，並以透析次數10次、反向方式進行處理。所得之溶液係以與實施例1相同之方式進行處理而獲得玻尿酸鈉。將分析結果及玻尿酸回收率示於表2。After the crude hyaluronic acid used in Example 1 was adjusted to pH 3.6, a hydrophilic ultrafiltration membrane (manufactured by DDS Co., Ltd. ‧ CA600PP) 4.5 m 2 having a molecular weight cut off of 20,000 and a cellulose acetate content of 30 m/m 2 was used. The flow rate of ‧ hr was repeatedly diluted with a volume ratio of 2 times, diluted with pure water, and treated in the reverse direction with 10 times of dialysis. The resulting solution was treated in the same manner as in Example 1 to obtain sodium hyaluronate. The analysis results and the hyaluronic acid recovery rate are shown in Table 2.
比較例3Comparative example 3
將實施例1中所得之粗製玻尿酸調整至pH 3.3後，利用截留分子量為20000且材質為乙酸聚醯亞胺之親水性超濾膜(日東電工公司製造‧NTU-4220)5 m2 ，以30 L/m2 ‧hr之透過流速反覆進行容積比為2倍之濃縮、等倍之純水稀釋，並以透析次數9次、反向方式進行處理。所得之溶液係以與實施例1相同之方式進行處理而獲得玻尿酸鈉。將分析結果及玻尿酸回收率示於表2。After the crude hyaluronic acid obtained in Example 1 was adjusted to pH 3.3, a hydrophilic ultrafiltration membrane (manufactured by Nitto Denko Corporation ‧ NTU-4220) 5 m 2 with a molecular weight cut off of 20,000 and a polyacetimimine acetate was used, and 30 was used. The permeation flow rate of L/m 2 ‧ hr was repeated to dilute the volume ratio by 2 times, diluted with pure water, and treated in the reverse manner with 9 times of dialysis. The resulting solution was treated in the same manner as in Example 1 to obtain sodium hyaluronate. The analysis results and the hyaluronic acid recovery rate are shown in Table 2.
比較例4Comparative example 4
將實施例1中所得之粗製玻尿酸調整至pH 3.7後，利用截留分子量為13000且材質為乙酸聚碸之疏水性超濾膜(可樂麗公司製造‧MU-6303)5 m2 ，以5 L/m2 ‧hr之透過流速反覆進行容積比為2倍之濃縮、等倍之純水稀釋，並以透析次數11次、單程方式進行處理。將所得之溶液以與實施例1相同之方式進行處理而獲得玻尿酸鈉。將分析結果及玻尿酸回收率示於表2。After the crude hyaluronic acid obtained in Example 1 was adjusted to pH 3.7, a hydrophobic ultrafiltration membrane (manufactured by Kuraray Co., Ltd. ‧ MU-6303) 5 m 2 with a molecular weight cut off of 13,000 and a polyacetate of acetic acid was used, 5 L / The flow rate of m 2 ‧ hr was repeatedly diluted with a volume ratio of 2 times, diluted with pure water, and treated in a single pass with 11 times of dialysis. The resulting solution was treated in the same manner as in Example 1 to obtain sodium hyaluronate. The analysis results and the hyaluronic acid recovery rate are shown in Table 2.
比較例5Comparative Example 5
將實施例1中所使用之粗製玻尿酸調整至pH 3.7後，利用截留分子量為13000且材質為乙酸聚碸之疏水性超濾膜(可樂麗公司製造‧MU-6303)5 m2 ，以15 L/m2 ‧hr之透過流速反覆進行容積比為2倍之濃縮、等倍之純水稀釋，並以透析次數11次、反沖方式進行處理。所得之溶液係以與實施例1相同之方式進行處理而獲得玻尿酸鈉。將分析結果及玻尿酸回收率示於表2。After adjusting the crude hyaluronic acid used in Example 1 to pH 3.7, a hydrophobic ultrafiltration membrane (manufactured by Kuraray Co., Ltd. ‧ MU-6303) 5 m 2 with a molecular weight cutoff of 13,000 and a molecular weight of 13,000 was used, and 15 L was used. The flow rate of /m 2 ‧ hr was reversed and the volume ratio was doubled, and the diluted water was diluted twice, and the number of dialysis was 11 times and the backflushing method was used. The resulting solution was treated in the same manner as in Example 1 to obtain sodium hyaluronate. The analysis results and the hyaluronic acid recovery rate are shown in Table 2.
測定方法test methods
(1)核酸含量：對0.1%之玻尿酸鈉於260 nm下之吸光度進行測定。(1) Nucleic acid content: The absorbance of 0.1% sodium hyaluronate at 260 nm was measured.
(2)蛋白質含量：將玻尿酸鈉溶解於0.1 N之氫氧化鈉中，以洛利法進行測定。(2) Protein content: The sodium hyaluronate was dissolved in 0.1 N sodium hydroxide and measured by the Loley method.
(3)乳酸：以0.1%之濃度溶解玻尿酸鈉，以L-LDH(L-Lactate Dehydrogenase，L-乳酸脫氫酶)法進行測定。(3) Lactic acid: Sodium hyaluronate was dissolved at a concentration of 0.1%, and it was measured by L-LDH (L-Lactate Dehydrogenase, L-lactate dehydrogenase) method.
(4)金屬：將玻尿酸鈉以0.05之濃度溶解於8 N之硝酸中，進行ICP(Inductively Coupled Plasma，電感耦合等離子體)發射光譜分析。(4) Metal: Sodium hyaluronate was dissolved in 8 N nitric acid at a concentration of 0.05, and subjected to ICP (Inductively Coupled Plasma) emission spectrum analysis.
(5)極限黏度：將玻尿酸鈉以0.02%之濃度溶解於0.2 M之氯化鈉中，對30℃下之極限黏度進行測定。(5) Ultimate viscosity: Sodium hyaluronate was dissolved in 0.2 M sodium chloride at a concentration of 0.02%, and the ultimate viscosity at 30 ° C was measured.
實施例8Example 8
使用具有各種截留分子量之聚碸之疏水性超濾膜、於以下條件下將實施例1所使用之粗製玻尿酸純化，調查超濾時之pH值與玻尿酸之回收率的關係。The crude hyaluronic acid used in Example 1 was purified using a hydrophobic ultrafiltration membrane having various molecular weight cut-off molecular weights under the following conditions, and the relationship between the pH value at the time of ultrafiltration and the recovery rate of hyaluronic acid was examined.
HA溶液條件HA solution conditions
HA濃度：2 g/LHA concentration: 2 g/L
分子量：440萬(極限黏度：55 dL/g)Molecular weight: 4.4 million (limit viscosity: 55 dL/g)
過濾條件Filter condition
線速：1 m/sLine speed: 1 m/s
透過流速：30 L/(m2 ‧hr)Transmission flow rate: 30 L/(m 2 ‧hr)
濃縮：2倍濃縮Concentrate: 2 times concentrated
溫度：25℃Temperature: 25 ° C
於圖2中表示超濾時之pH值與玻尿酸回收率之關係之圖表。另外，關於各截留分子量之膜，獲得表示超濾時之pH值與玻尿酸損失率之關係的式1~5。使用下述式1~5算出實質上不引起各截留分子量之超濾膜中之玻尿酸類之損失的pH值，於該pH值之範圍內(最適pH值)利用超濾膜進行純化，藉此可於不損失玻尿酸類之情況下進行純化。A graph showing the relationship between the pH value at the time of ultrafiltration and the hyaluronic acid recovery rate is shown in Fig. 2 . Further, regarding the membranes each having a molecular weight cut off, Formulas 1 to 5 indicating the relationship between the pH value at the time of ultrafiltration and the hyaluronic acid loss rate were obtained. The pH values of the hyaluronic acid in the ultrafiltration membranes which do not cause the respective molecular weight cutoffs are calculated by the following formulas 1 to 5, and are purified by using an ultrafiltration membrane within the pH range (optimum pH value). Purification can be carried out without loss of hyaluronic acid.
於使用截留分子量為30,000之超濾膜之情形時When using an ultrafiltration membrane with a molecular weight cutoff of 30,000
(式1)玻尿酸類之損失率(%)=44.86×(超濾時之pH值)-131.79(Formula 1) Loss rate of hyaluronic acid (%) = 44.86 × (pH value during ultrafiltration) - 131.79
(最適pH值≦2.9)(Optimum pH ≦ 2.9)
於使用截留分子量為13,000之超濾膜之情形時When using an ultrafiltration membrane with a molecular weight cutoff of 13,000
(式2)玻尿酸類之損失率(%)=40.84×(超濾時之pH值)-86.92(Formula 2) Loss rate of hyaluronic acid (%) = 40.84 × (pH value during ultrafiltration) - 86.92
(最適pH值≦3.7)(Optimum pH ≦3.7)
於使用截留分子量為10,000之超濾膜之情形時When using an ultrafiltration membrane with a molecular weight cutoff of 10,000
(式3)玻尿酸類之損失率(%)=24.36×(超濾時之pH值)-97.19(Formula 3) Loss rate of hyaluronic acid (%) = 24.36 × (pH value during ultrafiltration) - 97.19
(最適pH值≦4.0)(Optimum pH ≦4.0)
於使用截留分子量為7,000之超濾膜之情形時When using an ultrafiltration membrane with a molecular weight cutoff of 7,000
(式4)玻尿酸類之損失率(%)=7.09×(超濾時之pH值)-29.02(Formula 4) Loss rate of hyaluronic acid (%) = 7.09 × (pH value during ultrafiltration) - 29.02
(最適pH值≦4.1)(Optimum pH ≦4.1)
於使用截留分子量為5,000之超濾膜之情形時When using an ultrafiltration membrane with a molecular weight cut off of 5,000
(式5)玻尿酸類之損失率(%)=0.79×(超濾時之pH值)-2.01(Formula 5) Loss rate of hyaluronic acid (%) = 0.79 × (pH at ultrafiltration) - 2.01
(最適pH值≦4.2)(Optimum pH value ≦ 4.2)
將玻尿酸類之損失率成為3%以下之pH值與超濾膜之截留分子量的關係示於圖3。另外，可明確玻尿酸類之損失率成為3%以下之pH值與超濾膜之截留分子量之關係滿足以下之式6。The relationship between the pH value at which the loss rate of hyaluronic acid is 3% or less and the molecular weight cut off of the ultrafiltration membrane is shown in Fig. 3 . In addition, it is understood that the relationship between the pH value at which the loss rate of hyaluronic acid is 3% or less and the molecular weight cut off of the ultrafiltration membrane satisfies the following formula 6.
(式6)玻尿酸類之損失率成為3%以下之pH值=-5×10-5 ×(截留分子量)+4.4978(Formula 6) The loss rate of hyaluronic acid is 3% or less pH = -5 × 10 -5 × (molecular weight cut off) + 4.4978
藉由使用式6，可求出最適pH值相對於超濾膜之截留分子量之上限，藉由以最適pH值利用超濾膜進行透析，可以3%以下之損失率純化玻尿酸類。By using Formula 6, the upper limit of the optimum pH value relative to the molecular weight cut-off of the ultrafiltration membrane can be determined, and by dialysis using an ultrafiltration membrane at an optimum pH value, hyaluronic acid can be purified at a loss rate of 3% or less.
根據以上之實驗可確認，若使用本發明之純化方法，則可自玻尿酸類溶液中高效地去除雜質而純化高分子量之玻尿酸類。According to the above experiment, it was confirmed that the high molecular weight hyaluronic acid can be purified by efficiently removing impurities from the hyaluronic acid solution by using the purification method of the present invention.
以上，基於實施例進行了本發明。該實施例始終為示例，業者明白可實施各種變形例，且此種變形例亦包含於本發明之範圍內。Hereinabove, the present invention has been carried out based on the examples. This embodiment is always an example, and it is understood that various modifications can be made and such modifications are also included in the scope of the invention.
圖1係表示掃流方式所含之單程方式(A)、反沖方式(B)及反向方式(C)之過濾方式的圖；1 is a view showing a filtering method of a one-way mode (A), a backflushing mode (B), and a reverse mode (C) included in a sweeping mode;
圖2係表示超濾時之pH值與玻尿酸損失率之關係的圖表；及Figure 2 is a graph showing the relationship between the pH value at the time of ultrafiltration and the hyaluronic acid loss rate;
圖3係表示玻尿酸類之損失率成為3%以下之pH值與超濾膜之截留分子量之關係的圖表。Fig. 3 is a graph showing the relationship between the pH value of hyaluronic acid loss rate of 3% or less and the molecular weight cut off of the ultrafiltration membrane.
(無元件符號說明)(no component symbol description)
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| CN105837705A (en) * | 2016-03-04 | 2016-08-10 | 冯翠军 | Method for removing heat source in hyaluronic acid |
| CN105949478B (en) * | 2016-05-05 | 2021-10-01 | 宜昌东阳光生化制药有限公司 | Method for removing cross-linking agent in cross-linked hyaluronic acid |
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| JPH04158796A (en) * | 1990-10-23 | 1992-06-01 | Chisso Corp | Production of aqueous solution of sodium hyaluronate |
| US6489467B1 (en) * | 1999-01-28 | 2002-12-03 | Chemedica S.A. | Process for purifying high molecular weight hyaluronic acid |
| US20100137579A1 (en) * | 2008-12-01 | 2010-06-03 | Sustineo Biotechnology Co., Ltd. | Process for purifying medical grade hyaluronic acid |
| CN103319627A (en) * | 2013-07-10 | 2013-09-25 | 南宁中诺生物工程有限责任公司 | Method for grading molecule weight of sodium hyaluronate |
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| JP2728542B2 (en) | 1990-05-07 | 1998-03-18 | 株式会社日立製作所 | Link overload detection device for simultaneous biaxial stretching device |
| JPH09324001A (en) | 1996-04-02 | 1997-12-16 | Kyowa Hakko Kogyo Co Ltd | Purification method for sodium hyaluronate |
| JP4151092B2 (en) * | 1997-10-22 | 2008-09-17 | チッソ株式会社 | Method for producing oligohyaluronic acid or a salt thereof |
| KR101509139B1 (en) * | 2006-11-23 | 2015-04-08 | 주식회사 엘지생명과학 | Method for purifying hyaluronic acid |
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| JPH04158796A (en) * | 1990-10-23 | 1992-06-01 | Chisso Corp | Production of aqueous solution of sodium hyaluronate |
| US6489467B1 (en) * | 1999-01-28 | 2002-12-03 | Chemedica S.A. | Process for purifying high molecular weight hyaluronic acid |
| US20100137579A1 (en) * | 2008-12-01 | 2010-06-03 | Sustineo Biotechnology Co., Ltd. | Process for purifying medical grade hyaluronic acid |
| CN103319627A (en) * | 2013-07-10 | 2013-09-25 | 南宁中诺生物工程有限责任公司 | Method for grading molecule weight of sodium hyaluronate |
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