201139464 六、發明說明： 【發明所屬之技術領域】 本發明係關於玻尿酸類之純化方法、該純化方法所用之 純化劑及活性碳、該等之製造方法。 【先前技術】 玻尿酸除作為化妝品之保濕劑以外’亦作為眼科、整形 外科、皮膚科等之醫藥品而使用。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製造而成， 但因混入作為夾雜物之硫酸軟骨素等，或因組織内所包含 之玻尿酸酶（hyaluronidase)等易造成分子量降低，因此亦進 行培養具有玻尿酸生產能力之微生物而由培養液製造玻尿 酸（醱酵法）（非專利文獻1及專利文獻丨）。 藉由萃取法或醱酵法所製造之玻尿酸中，存在作為雜質 之蛋白質或發熱性物質等，因此研究將該等分離去除而獲 取高純度之製品之方法。尤其是製造之初始階段的雜質之 去除能減輕後序之純化步驟之負荷，而期待開發獲取亦可 作為醫藥品使用之高純度之製品的方法。作為其例，例如 揭示有藉由於醱酵液中添加醇等有機溶劑而析出玻尿酸並 分離雜質之純化法，或使用陰離子交換樹脂自醱酵液中去 除發熱性物質或蛋白質等之玻尿酸之純化法（專利文獻2)。 [非專利文獻 1] J〇urnal 〇f General Micr〇bi〇l〇gy，85, 3乃，Η% [專利文獻1]曰本專利特公平4_1296〇號公報 [專利文獻2]日本專利特開昭63_12293號公報 【發明内容】 154810.doc 201139464 然而，於以工業規模獲取可作為注射液等醫藥品使用之 高純度之玻尿酸類時，存在以下期望進一步改善之問題： (1)因於初始階段中雜質之去除不充分，而導致後序步驟之 負荷較大，（2)操作煩雜而且玻尿酸類之回收率較低等。 本發明係鑒於上述情況而完成者，其目的在於提供一種 簡便且同產率、以工業規模純化高純度之玻尿酸類之方 矢另外目的在於k供§亥純化方法所使用之純化劑、活 性碳等及該等之製造方法。 本發明者等人為達成上述目的，對自玻尿酸類含有液高 效率地分離去除雜質，簡便且高效率地純化高純度之玻尿 酸類之方法進行各種研究，結果發現藉由使用利用特定預 處理方法所製備之活性碳，可高效率地吸附去除雜質，最 終完成本發明。 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：使含有玻尿酸及/或其鹽及雜質之玻尿酸類溶 液與活性碳懸浮液相接觸之步驟。利用該純化方法，藉由 使用活性碳，可高效率地吸附去除雜質。 即，根據本發明，提供一種玻尿酸類之純化方法，其包 括·使活性碳懸浮於水性液中而獲取活性碳懸浮液之步 驟；將該活性碳懸浮液於1〇〇<t以上加熱2〇分鐘以上之步 驟；將該加熱後之活性碳懸浮液攪拌冷卻之步驟；使包含 玻尿酸類及雜質之玻尿酸類溶液與攪拌冷卻後之活性碳懸 浮液相接觸之步驟。利用該純化方法’藉由使用已預處理 成適合於玻尿酸類之純化之狀態的活性碳，可高效率地吸 154810.doc201139464 VI. Description of the Invention: [Technical Field of the Invention] The present invention relates to a method for purifying hyaluronic acid, a purifying agent used for the purification method, activated carbon, and the like. [Prior Art] Hyaluronic acid is used as a moisturizer for cosmetics, and is also used as a medicine for ophthalmology, orthopedics, dermatology, and the like. Hyaluronic acid can be produced by extracting from an animal tissue such as a chicken cockscomb or a vitreous of a bull's eye, but it is easy to incorporate chondroitin sulfate as an inclusion or a hyaluronidase contained in the tissue. Since the molecular weight is lowered, the microorganism having hyaluronic acid production ability is cultured, and hyaluronic acid (fermentation method) is produced from the culture solution (Non-Patent Document 1 and Patent Document). In the hyaluronic acid produced by the extraction method or the fermentation method, there are proteins or exothermic substances as impurities, and therefore, a method of separating and removing the products to obtain a high-purity product has been studied. In particular, the removal of impurities in the initial stage of production can reduce the load of the subsequent purification step, and it is expected to develop a method for obtaining a high-purity product which can also be used as a pharmaceutical product. For example, a purification method in which hyaluronic acid is precipitated by adding an organic solvent such as alcohol to a fermentation broth, and impurities are separated, or a hyaluronic acid which removes exothermic substances or proteins from a mash by an anion exchange resin is disclosed. (Patent Document 2). [Non-Patent Document 1] J〇urnal 〇f General Micr〇bi〇l〇gy, 85, 3, Η% [Patent Document 1] Japanese Patent Laid-Open No. Hei 4_1296 No. [Patent Document 2] Japanese Patent Laid-Open Japanese Patent Publication No. 133810.doc 201139464 However, when obtaining high-purity hyaluronic acid which can be used as a pharmaceutical product such as an injection solution on an industrial scale, there are the following problems which are expected to be further improved: (1) Due to the initial stage The removal of impurities is insufficient, resulting in a large load in the subsequent steps, (2) troublesome operation and low recovery of hyaluronic acid. The present invention has been made in view of the above circumstances, and an object thereof is to provide a simple and same-yield, high-purity hyaluronic acid which is purified on an industrial scale, and another purpose is to use a purifying agent and activated carbon used in the purification method. And the manufacturing methods of these. In order to achieve the above object, the inventors of the present invention conducted various studies on methods for efficiently separating and removing impurities from hyaluronic acid-containing liquids, and purifying high-purity hyaluronic acid easily and efficiently, and found that by using a specific pretreatment method, The prepared activated carbon can efficiently adsorb and remove impurities, and finally completes the present invention. 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 bringing a hyaluronic acid solution containing hyaluronic acid and/or a salt thereof and an impurity into contact with an activated carbon suspension. By using this purification method, impurities can be efficiently adsorbed and removed by using activated carbon. That is, according to the present invention, there is provided a method for purifying hyaluronic acid comprising the steps of: suspending activated carbon in an aqueous liquid to obtain an activated carbon suspension; heating the activated carbon suspension at a temperature above 1 Torr; a step of 〇min or more; a step of stirring and cooling the heated activated carbon suspension; and a step of contacting the hyaluronic acid solution containing hyaluronic acid and impurities with the stirred and cooled activated carbon suspension. By using this purification method, 154810.doc can be efficiently sucked by using activated carbon which has been pretreated to a state suitable for purification of hyaluronic acid.
S 201139464 附去除雜質β 方φ，甘根據本發明’提供一種玻尿酸類之純化劑之製造 ^ 、包括·使活性碳懸浮於水性液中而獲取活性碳賤 洋液之步驟^㈣性钱浮液㈣代以上加㈣分鐘以 上之㈣；將該加熱後之活性碳懸浮液授拌冷卻之步驟。 利用β製造方法，藉由將活性碳預處理成適合於玻尿酸類 屯化之狀態，而可製造能高效率地吸附去除雜質之玻尿 酸類之純化劑。 另外，根據本發明，提供一種活性碳之預處理方法，其 包括.使活性碳懸浮於水性液中而獲取活性碳懸浮液之步 驟；將該活性碳懸浮液於10(rc以上加熱2〇分鐘以上之步 驟，將該加熱後之活性碳懸浮液攪拌冷卻之步驟。利用該 預處理方法，可將活性碳預處理成適合於玻尿酸類之純化 之狀態。 另外’根據本發明，提供一種玻尿酸類之純化劑，其包 含藉由包括以下步驟之方法而製造之活性碳：使活性碳懸 浮於水性液中而獲取活性碳懸浮液之步驟；將該活性碳懸 浮液於100°c以上加熱20分鐘以上之步驟；將該加熱後之活 性碳懸浮液攪拌冷卻之步驟^該純化劑藉由包含已預處理 成適合於玻尿酸類之純化之狀態的活性碳，而可高效率地 吸附去除雜質》 【實施方式】 [術語之說明] 本說明書中之「玻尿酸類」包括：游離之玻尿酸及不損 154810.doc -5- 201139464 害本發明之目的之範圍内的可使用之任意玻尿酸鹽(例如 納鹽、if鹽、鈣鹽、锂鹽等金屬鹽，或鹽酸鹽、磷蛟鹽、 咎松馱鹽等酸加成物等，但並不限定於此）或水合物、其等 之混合物。此處，所謂玻尿酸，係指N_乙醯_D_葡糖胺與〇一 葡糖齡酸鍵έ士夕-Α也gg 壬、—Α “ 更、之一糖皁兀重禝連鏈而成之高分子量多糖 類’各種鹽主要指葡糖醛酸部分形成鹽之形態者。玻尿酸 藉由可摺疊之鏈部分與D_葡糖醛酸部分之羧基的負電荷之 相互作用’而容易於空間内展開’藉此可與大量之水結合 而形成凝膠。另外’即便為低濃度，但分子間力亦較強， 故仍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，於生理性上亦發揮該等 作用。 已知於玻尿酸類之中，分子量約200萬Da之玻尿酸鈉與分 子里約8G萬Da者相&，作為醫藥品，對治療變形性膝關節 病、肩周炎、慢性類風濕性關節炎等發揮出優異之效果（藥 理與’。療 ’ Vol.22、No.9、289(1994);藥理與治療，Vol.22、 N〇.9、319(1994))。另外’除此之外，已知有：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，-部分於臨床上f遍使用。於作 為语藥品使用之情形時，較理想為使用平均分子量為1〇〇 萬以上之玻尿酸類。進而，考慮到獲取或操作之容易性， 作為醫藥品較理想為平均分子量為1〇〇萬〜5〇〇萬Da之玻尿 酉文類，尤其理想為平均分子量為15〇萬〜4〇〇萬之玻尿酸 類。另外，此種高分子量之玻尿酸類作為化妝品之用途使 1548l0.docS 201139464 Attaching impurities β square φ, according to the invention 'providing a production of a hyaluronic acid purifying agent ^, including · suspending activated carbon in an aqueous liquid to obtain an activated carbon hydrazine liquid step ^ (4) sex money floating liquid (4) Adding (four) minutes or more to the above (4); and heating and cooling the activated carbon suspension to the step of cooling. By the β production method, by purifying the activated carbon into a state suitable for hyaluronic acid deuteration, a purifying agent of hyaluronic acid capable of efficiently adsorbing and removing impurities can be produced. Further, according to the present invention, there is provided a method for pretreating activated carbon comprising the steps of: suspending activated carbon in an aqueous liquid to obtain an activated carbon suspension; heating the activated carbon suspension at 10 (rc or more for 2 minutes) In the above step, the heated activated carbon suspension is stirred and cooled. The pretreatment method can pretreat the activated carbon into a state suitable for purification of hyaluronic acid. Further, according to the present invention, a hyaluronic acid is provided. a purifying agent comprising activated carbon produced by a method comprising the steps of: suspending activated carbon in an aqueous liquid to obtain an activated carbon suspension; heating the activated carbon suspension at 100 ° C or more for 20 minutes The above step; the step of stirring and cooling the heated activated carbon suspension; the purifying agent can efficiently adsorb and remove impurities by including activated carbon which has been pretreated to a state suitable for purification of hyaluronic acid. Embodiments] [Description of terms] The "hyaluronic acid" in this specification includes: free hyaluronic acid and no damage 154810.doc -5- 201139464 Any hyaluronic acid that can be used within the scope of the purpose of the invention (for example, a metal salt such as a sodium salt, an if salt, a calcium salt or a lithium salt, or an acid adduct such as a hydrochloride, a phosphonium salt or a cesium salt), but It is not limited to this) or a mixture of hydrates, etc. Here, hyaluronic acid means N_acetamidine_D_glucosamine and glucosinolate-acid bond έ Α Α Α gg gg 壬, —Α “More, one of the high-molecular-weight polysaccharides made from the saponins of the saponins. The various salts mainly refer to the form of the salt formed by the glucuronic acid. The hyaluronic acid is separated from the D-glucose by the chain part. The negative charge interaction of the carboxyl group of the aldehyde acid moiety is easy to expand in space', thereby forming a gel in combination with a large amount of water. In addition, even at low concentrations, the intermolecular force is strong, so Relatively high viscosity. Because of this action, for example, the action of moist joints, the action of soft skin, etc., it also plays a physiological role. It is known that hyaluronic acid with a molecular weight of about 2 million Da is among hyaluronic acids. Sodium and the molecule in the molecule of about 8G million Da, as a pharmaceutical, treatment Deformable knee disease, frozen shoulder, chronic rheumatoid arthritis, etc. (Pharmacology and 'Therapy' Vol.22, No.9, 289 (1994); Pharmacology and Therapy, Vol.22, N〇.9, 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. In some cases, it is preferable to use hyaluronic acid having an average molecular weight of 10,000 or more. In view of the ease of acquisition or handling, it is preferable to use it as a pharmaceutical product. The hyaluronic acid having an average molecular weight of 1 million to 50,000 Da is particularly desirable as a hyaluronic acid having an average molecular weight of 15 million to 4 million. In addition, the use of such high molecular weight hyaluronic acid as a cosmetic makes 1548l0.doc
S * 6 · 201139464 用時，亦因其較高之保濕力而發揮優異之效果。 溶解玻尿酸類作為醫藥品之注射用溶解液可適當使用： 於注射用水、生理鹽水等中添加含有如酸、鹼、磷酸鹽之 緩衝劑之pH值調整劑的普遍使用之注射用溶解液（例如各 國藥典中所確認者）。 。亥等玻尿酸類可為藉由自動物組織萃取之萃取法所製造 者，亦可為藉由使用玻尿酸生產微生物菌株醱酵而得之醱 酵法所製造者。然而，於自動物組織萃取者中，其他黏多 糖等雜質相對較多、分子量亦較小，故較理想為使用藉由 醱酵法所獲取者。作為適合於本發明之醱酵法之一例’例 如可使用鏈球g屬之微生物以已知之方法獲取玻尿酸類。 本發明之方法等所使用之溶液較理想為原樣地使用培養 液田J亦可使用藉由已知之方法例如離心分離或過濾 處理等除菌之溶液。根據情況，亦可進行藉由透析處理去 除低刀子化合物、藉由微濾處理去除水不溶微粒子等操 作，亦可使用添加料水溶性有機溶劑而析出純化玻尿酸 者。另外，亦可使用藉由氧化鋁等所處理者。 本說明書中之「鏈球菌」包括：可生產玻尿酸之鍵球菌 (Streptococcus)屬之任意的細菌·其變異株。尤其較理想為 使用如專利文獻2所揭示之馬鏈球菌FM_1〇〇(微工研菌寄第 9027號）、日本專利特開平2_234689號公報所揭示之馬鍵球 菌FM-300(微I研菌寄第2319號）之類高產率且穩定地生產 玻尿酉夂之1異株。作為適合於生產玻尿酸之鏈球菌屬之細 菌之例子’其他例如可列舉：馬鏈球菌（Strept〇e。咖s q叫、 154810.doc 201139464 動物流行鏈球g (Strept()coecus z〇〇epidemicus)、類馬鍵球菌 (Streptococcus equisimilis)、壞乳鏈球菌 咖㈣Ctiae) 1膿鍵球菌（Streptoc〇ccus刚㈣⑷及該等 之變異株等，但並不僅限定於此。 關於本說明書中之「平均分子量」，只要無特別敍述，於 表示玻尿酸類之平均分子量時，係指黏度平均分子量。可 藉由業者通常進行之方法求得。較佳為可藉由各國之藥典 等所普遍使用之敎方法求得，更佳為可藉由日本藥典所 使用之敎方法求得。作為—例，例如其平均分子量可利 用極限黏度[由下式求得，但並稀定於此。 [式1] 平均分子量 I 22.8 ) 本說明書中之「活性碳」係、指市f、f遍所使用之分離 去除·純化用之多孔狀碳，不限定於煤或木材等原料之來源 或化學活化或水蒸氣活化等製法。進而，亦不限定於粉末 活性碳、粒狀活性碳等形狀’或乾式、濕式等性狀。於本 發明之方法等中，較理想為使用來源於木材之活性碳，但 並不限定於此。另夕卜’同樣較理想為使用藉由水蒸氣活化 所製造者。另外，同樣較理想為使用粉末活性碳。 作為活性碳之具體例子，例如可列舉：Μ職i㈤ GL-30S、Tsurumi c〇al H(%3〇s[以上為粒狀，鹤見煤炭 (Tsurumi Coal)(股）製造]; LH2C20/48、白驚WH2C8/32SS[以 白鷺WH5C8/32 、白驚 上為粒狀’武田藥品工業 154810.doc 201139464 (股）製造];白鷺A、白鷺p、純化白鷺、特性白鷺、 Carbonraffin[以上為粉狀，武田藥品工業（股）製造];北越 SD、北越GSA[以上為粉狀，北越碳素工業（股）製造 SLIVER A花F、雪A[以上為粉狀，大三工業（股）製造]等， 但並不限定於此。 本說明書巾之「雜質」係指除玻尿_、水以外之其他 溶劑成分、無機鹽以外之物#，尤其係指於使用作為最终 製品之玻尿酸類時可帶來不利之„(發熱性物質朴作為 主要之雜質源’可列舉：於玻尿酸類之生產階段中來源於 組織、微生物或培養液（培養基）者，或其後之純化階段中所 混入者。作為本說明書中之雜質之例子，可列舉：组織或 菌體、蛋白質、核酸、多糖類、低分子化合物、或内毒素 等’但並不限定於此。作為雜質之組織或g體分別包括： 來源於萃取法中所使用之作為萃取原料之組織的組織片 等’及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：來源於上述組織、 菌之蛋白f ’或生產後之步驟中所混入之蛋白質等，但並 不限定於此。作為雜質之内毒素包括來源於上述菌之脂多 糖類等’但並不限定於此。 本。兒明書中之「低分子化合物」係指較之玻尿酸類分 子量相對車乂小之化合物，例如指分子量為⑽&以下，或 ^子量l_Da’或分子量5GGDa以下之化合物，但並不限 疋；此ilt種低刀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。 154810.doc •9- 201139464 本說明書中之「水性液」係、指將水作為主體（溶劑）之液 體者，亦包括含有各種鹽、緩衝劑、少量有機溶劑等者。 作為水性液，只要不損害本發明之目的，則可使用任意者， 但就於使用作為最終製品之玻尿酸類時防止混入不必要之 物質之觀點而言，較理想為水。 本說明書中之「攪拌冷卻 攪拌而進行冷卻。此處，將 卻結束溫度。 」係指藉由將對象之液體連續 完成攪拌冷卻後之溫度設為冷 本說明書中之「無塵環境」係指藉由進行空氣調節管理， 使來源於環境之菌或内毒素等雜f對製品進行污染之可能 性為極低之環境’尤其係指相當於醫藥： 製品之製造環境。 "、、門毋素） 作為無塵環境之-例，例如可列 物質之環境。 卜万式規疋對象 [表1] 微粒子數[個/m3] 生物數[cfii/90mmf/4hrl 本說明書中之「去除」 包括部分去除（使該物質之量減^物/兀全去除以外，亦 包括去除任意或特定之雜質。 砘化」 本說明書中之「純化劑你 」係各吸附劑等用於 所謂「物質A之純化劑 屯化之樂劑’ 劑。純化劑亦可為純化用έ 質Α之樂 用組合物之形態。另外H“ 除劑、分離劑等亦分別係指同樣地去 去除之樂劑、用於分離之 I54810.doc 201139464 藥劑，例如，可為包括吸附劑或載體等之術語，亦可為去 除用組合物、分離用組合物等形態。 關於本說明書中之各個數值範圍，分別包含「〜」所表示 之上限值及下限值。例如，「A〜B」之表述係指A以上且B以 下。另外’「含有…」包括「實質上由·_.形成」及「包含」。 [實施之形態] 本發明係關於例如以下之實施形態，但並不限定於此。 實施形態1 -種玻尿酸及/或其鹽之純化方法’其包括：使活性碳懸 浮於水性液中而獲取活性碳懸浮液之步驟； 將該活性碳懸浮液於10(rc以上加熱20分鐘以上之步驟； 將該加熱後之活性碳懸浮液攪拌冷卻之步驟； 使包含玻尿酸及/或其鹽及雜質之玻尿酸類溶液與攪拌 冷卻後之活性碳懸浮液相接觸之步驟。 實施形態2 一種玻尿酸及/或其鹽之純化劑之製造方法，其包括：使 活性碳懸浮於水性液中而獲取活性碳懸浮液之步驟； 將該活性碳懸浮液於loot以上加熱20分鐘以上之步驟； 將該加熱後之活性碳懸浮液攪拌冷卻之步驟。 實施形態3 -種活性碳之預處理方法，其包括：使活性碳_於水 性液中而獲取活性碳懸浮液之步驟； 將該活性碳懸浮液於100。(：以上加熱2〇分鐘以上之步驟； 將該加熱後之活性碳懸滓液攪拌冷卻之步驟。 ’ 154810.doc -11 · 201139464 實施形態4 其中於上述攪拌冷卻 如實施形態1至3中任一項之方法， 之步驟中’冷卻結束溫度為60它以下 實施形態5 如實施形態1至4中任-項之方法，其中於上述搜掉冷卻 之步驟中，攪拌冷卻係於溫度1〇〜6(rc下進行丨小時以上。 實施形態6 如實施形態1至5中任一項之方法 一步驟以上係於無塵環境下進行。 其中上述步驟之至少 實施形態7 如實施形態1至6中任-項之方法，其中玻尿酸及/或其鹽 係藉由醱酵法而製造者。 實施形態8 其中上述玻尿酸及/或 如實施形態1至7中任一項之方法 其鹽之分子量為350萬〜7〇〇萬Da。 實施形態9 -種玻尿酸及/或其鹽之純化劑，其包含藉由包括以下步 驟而製造之活性碳：使活性碳懸浮於水性液中而獲取活性 碳懸浮液之步驟； 將該活性碳懸浮液於1 〇(rc u L以上加熱2〇分鐘以上之步驟 將該加熱後之活性碳懸浮液攪拌冷卻之步驟。 以下就本發明之形態進行說明。 本發明之第一形態（例如實施形態1)係-種玻尿酸類之 純化方法’其包括：使活性碳料於水㈣中而獲取活性 1548I0.docS * 6 · 201139464 It also exerts excellent results due to its high moisturizing power. The dissolved hyaluronic acid can be suitably used as a solution for injection for injection of a pharmaceutical product: a commonly used injection solution containing a pH adjuster such as an acid, a base or a phosphate buffer is added to water for injection, physiological saline, or the like (for example, Confirmed in the Pharmacopoeia of each country). . The hyaluronic acid such as Hai may be produced by an extraction method of automatic tissue extraction or by a fermentation method obtained by fermenting a microbial strain using hyaluronic acid. However, in the automatic tissue extractor, other impurities such as mucopolysaccharides are relatively large and the molecular weight is small, so it is preferable to use those obtained by the fermentation method. As an example of the fermentation method suitable for the present invention, for example, a hyaluronic acid can be obtained by a known method using a microorganism of the genus Streptococcus. The solution used in the method of the present invention or the like is preferably used as it is, and a solution which is sterilized by a known method such as centrifugation or filtration treatment can also be used. Depending on the situation, it is also possible to remove the low-knife compound by dialysis treatment, remove the water-insoluble fine particles by microfiltration, or use a water-soluble organic solvent to precipitate and purify the hyaluronic acid. Further, those processed by alumina or the like can also be used. The "streptococcus" in the present specification includes any strain of bacteria belonging to the genus Streptococcus of hyaluronic acid. In particular, it is preferable to use the Streptococcus equi subsp. FM_1 〇〇 (Microtechnology Research No. 9027) disclosed in Japanese Patent Laid-Open No. Hei No. No. 9027, and the Japanese Patent Publication No. Hei No. 2-234689. High yield and stable production of a different strain of hyaluronic acid, such as No. 2319. Examples of the bacteria which are suitable for the production of hyaluronic acid of the genus Streptococcus include, for example, Streptococcus elegans (Strept〇e, café sq, 154810.doc 201139464 animal epictag g (Strept () coecus z〇〇epidemicus), Streptococcus equisimilis, C. serrata (C), C. elegans (Streptoc〇ccus Gang (4) (4) and the variants thereof, etc., but are not limited thereto. About the "average molecular weight" in this specification Unless otherwise specified, when referring to the average molecular weight of hyaluronic acid, it means the average molecular weight of viscosity. It can be obtained by a method generally carried out by the manufacturer. It is preferably obtained by a method commonly used by Pharmacopoeias of various countries and the like. More preferably, it can be obtained by the hydrazine method used in the Japanese Pharmacopoeia. For example, the average molecular weight can be obtained by using the ultimate viscosity [determined by the following formula, but is also diluted thereto. [Equation 1] Average molecular weight I 22.8) The "activated carbon" in the present specification refers to the porous carbon used for separation, removal and purification used in the city f and f, and is not limited to the source or the source of raw materials such as coal or wood. Activation or like system steam activation method. Further, it is not limited to a shape such as powder activated carbon or granular activated carbon, or a dry type or a wet type. In the method and the like of the present invention, it is preferable to use activated carbon derived from wood, but it is not limited thereto. It is also desirable to use a person activated by steam activation. In addition, it is also preferred to use powdered activated carbon. Specific examples of the activated carbon include, for example, Μ i i (5) GL-30S, Tsurumi c〇al H (%3 〇s [above granulated, manufactured by Tsurumi Coal); LH2C20/48 , 白惊 WH2C8/32SS [Egret WH5C8/32, white shocked as granular] Takeda Pharmaceutical Industry 154810.doc 201139464 (share)]; Egret A, Egret p, purified egret, characteristic egret, Carbonraffin [above for powder Manufacture, Takeda Pharmaceutical Co., Ltd.]; North Vietnam SD, North Vietnam GSA [above is powdery, North Vietnam Carbon Industry Co., Ltd. manufactures SLIVER A flower F, snow A [above powdery, junior industrial (stock) manufacturing The term "impurity" as used in the specification refers to a substance other than the glass uranium, water, and other inorganic salts, especially when using the hyaluronic acid as the final product. It can be unfavorable (the febrile substance is the main source of impurities), which can be exemplified by those derived from tissues, microorganisms or culture solutions (medium) in the production stage of hyaluronic acid, or in the subsequent purification stage. As an example of the impurities in this specification, Illustrative: a tissue or a bacterial cell, a protein, a nucleic acid, a polysaccharide, a low molecular compound, or an endotoxin, etc. 'but is not limited thereto. The tissue or the g body as an impurity respectively includes: The tissue sheet of the structure of the raw material is extracted, and the microbial cell or the bacterial cell sheet used in the fermentation method is not limited thereto. The protein as an impurity includes: a protein derived from the above-mentioned tissue and bacteria The protein or the like mixed in the step after the production is not limited thereto. The endotoxin as an impurity includes a lipopolysaccharide derived from the above-mentioned bacteria, but is not limited thereto. "Low-molecular compound" means a compound which is smaller than the molecular weight of hyaluronic acid relative to ruthenium, for example, a compound having a molecular weight of (10) & or less, or a molecular weight of 1_Da' or a molecular weight of 5 GGDa or less, but is not limited to 疋; The compounds include various amino acids, organic acids (such as lactic acid), sugars (such as glucose), etc. 154810.doc •9-201139464 The term "aqueous liquid" in this specification refers to the use of water as The liquid of the body (solvent) also includes various salts, a buffering agent, a small amount of an organic solvent, etc. As the aqueous liquid, any one may be used as long as the object of the present invention is not impaired, but the hyaluronic acid used as the final product is used. In the case of preventing the mixing of unnecessary substances, it is preferable to use water. In the present specification, "cooling is carried out by stirring and cooling, and here, the temperature is terminated." means that the liquid of the object is continuously stirred. The temperature after cooling is set to be cold. The “dust-free environment” in this manual refers to the environment where the environmentally-friendly bacteria or endotoxin can contaminate the product with extremely low energy by air conditioning management. In particular, it refers to the manufacturing environment equivalent to pharmaceuticals: products. ",, thresholds.) As an example of a dust-free environment, for example, an environment in which substances can be listed. Objects of the Buwan type [Table 1] Number of microparticles [/m3] Bionumber [cfii/90mmf/4hrl "Removal" in this specification includes partial removal (to reduce the amount of the substance/all removed) It also includes the removal of any or specific impurities. "Chemicals" in this manual are used as so-called "purifiers for purifying agents of substance A." Purifiers may also be used for purification.形态 形态 Α Α 。 。 。 。 。 。 。 。 。 。 “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ I I I I I I I I I I I I I I The terms of the carrier and the like may be in the form of a composition for removal, a composition for separation, etc. Each numerical range in the present specification includes the upper limit and the lower limit indicated by "~". For example, "A~ The expression "B" means A or more and B or less. In addition, "including" includes "substantially formed by _." and "includes". [Embodiment] The present invention relates to, for example, the following embodiments, but It is not limited to this. Embodiment 1 - Glass a method for purifying an acid and/or a salt thereof, comprising the steps of: suspending an activated carbon in an aqueous liquid to obtain an activated carbon suspension; and heating the activated carbon suspension at 10 (rc or more for 20 minutes or more; a step of stirring and cooling the heated activated carbon suspension; and contacting the hyaluronic acid solution containing hyaluronic acid and/or its salt and impurities with the stirred and cooled activated carbon suspension. Embodiment 2 A hyaluronic acid and/or a salt thereof a method for producing a purifying agent, comprising: a step of suspending activated carbon in an aqueous liquid to obtain an activated carbon suspension; and heating the activated carbon suspension above a loot for 20 minutes or more; and heating the activated carbon The step of stirring and cooling the suspension. Embodiment 3 - A method for pretreating activated carbon, comprising: a step of obtaining activated carbon in an aqueous solution to obtain an activated carbon suspension; and the activated carbon suspension is at 100. (: The above step of heating for more than 2 minutes; the step of stirring and cooling the heated activated carbon suspension is carried out. '154810.doc -11 · 201139464 Embodiment 4 The method of any one of Embodiments 1 to 3, wherein the step of cooling the temperature is 60, and the method of any of Embodiments 1 to 4, wherein the step of searching for cooling is performed. The stirring cooling is carried out at a temperature of 1 〇 to 6 (r 下 丨 以上 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 The method of any one of Embodiments 1 to 6, wherein the hyaluronic acid and/or a salt thereof is produced by a fermentation method. Embodiment 8 wherein the hyaluronic acid and/or any of Embodiments 1 to 7 The method has a molecular weight of 3.5 million to 7 million Da. Embodiment 9 A purifying agent for hyaluronic acid and/or a salt thereof, comprising the step of obtaining activated carbon by suspending activated carbon in an aqueous liquid to obtain an activated carbon suspension by suspending the activated carbon; The step of heating and heating the heated activated carbon suspension in a step of heating at 1 Torr (rc u L or more for 2 〇 minutes or more) will be described below. The first aspect of the present invention (for example, Embodiment 1) a method for purifying a hyaluronic acid, which comprises: obtaining an active carbonaceous material in water (IV) to obtain an activity 1548I0.doc
S *12- 201139464 碳懸洋液之步驟；將該活性碳懸浮液於1〇〇(>c以上加熱2〇 分鐘以上之步驟·’將該加熱後之活性碳懸浮液攪拌冷卻之 步驟；使包含玻尿酸及/或其鹽及雜質之玻尿酸類溶液與攪 拌冷部後之活性碳懸浮液相接觸之步驟。根據該純化方 法，藉由使用已預處理成適合於玻尿酸類之純化之狀態的 活性碳，而可高效率地吸附去除雜質。 本發明之第二形態（例如實施形態2)係一種玻尿酸類之 純化劑之製造方法，其包括：使活性碳懸浮於水性液中而 獲取活性碳懸#液之步冑；m性碳懸浮液於丨⑼t以上 加熱20分鐘以上之步驟；將該加熱後之活性碳懸浮液攪拌 冷卻之步驟。根據該製造方法，藉由將活性碳預處理成適 口於玻尿酸類之純化之狀態，而可製造能高效率地吸附去 除雜質之玻尿酸類之純化劑。 本發明之第三形態（例如實施形態3)係一種活性碳之預 處理方法’其包括：使活性碳懸浮於水性液中而獲取活性 碳懸浮液之步驟·，將該活性碳懸浮液於1〇〇它以上加熱2〇 分鐘以上之步驟；將該加熱後之活性碳懸浮液攪拌冷卻之 步驟。根據該預處理方法，可將活性碳預處理成適合於玻 尿酸類之純化之狀態。 本發明之第四形態（例如實施形態9)係—種玻尿㈣之 純化劑，#包含藉由包括以下步驟而製造之活性碳：使活 性碳懸浮於水性液中而獲取活性碳料液之步驟；將該活 性碳懸浮液於HKTC以上加熱2G分鐘以上之㈣；將該加熱 後之活性碳懸浮液授拌冷卻之步驟。該純化劑藉由包含已 1548l0.doc -13· 201139464 預處理成適合於玻尿酸類之純化之狀態的活性碳，而可高 效率地吸附去除雜質。 藉由上述第一形態之純化方法、第二形態之製造方法而 製造之純化劑，藉由第三形態之預處理方法而預處理之活 性碳或使用第四形態之純化劑之純化方法（以下稱為「上述 形態之純化方法」），較之使用未經處理之活性碳之純化方 法，具有更高之雜質吸附力，故可進行更為有效的雜質去除。 即，若使用±述形態之純化方法，則可將破尿酸類之消 失抑制在較低水平，並且不僅可將作為雜質之通常藉由活 性碳而可去除之高分子化合物（例如菌體、蛋白質、核酸、 内毒素）’甚至連低分子化合物（例如胺基酸、糖、有機酸） 亦可高效地分離·去除。另外’上述形態之純化方法較之使 用未經處理之活性碳之純化方法，尤其對於活菌、蛋白質、 内毒素有顯著之去除效果。 另外’上述形態之純化方法與使用未經處理之活性碳之 情形相比’可減少玻尿酸類之分子量之降低。可減少純化 時之玻尿酸類之分子量之降低，尤其於高分子量(例如350 萬〜700萬Da)之玻展酸類之純化時，發揮優異之效果。另 外，對於先前難以處理之濃度相對較高⑽Μ 之玻尿酸類溶液，亦可高效地處理。 進而’若使用上述形態之 理、透析處理、純化處理等 進行純化（去除雜質.菌體等） 尿酸類時亦發揮優異之效果 純化方法，則可不進行除菌處 ，而將培養液本身作為對象而 ’於自培養液本身分離.純化玻 。藉由不進行離心分離或過濾 154810.doc 201139464 處理等煩雜之操作，而直接使用培養液，從而可獲取更加 效率化及削減成本等效果。 進行上述形態之純化方法時之玻尿酸類溶液之玻尿酸類 浪度，就因溶液黏度高引起之操作之困難度及破尿酸類之 溶解度的觀點而言，較理想為〇.1〜2〇§/[，更理想為〇5〜15 g/L ’最理想為1〜1 〇 g/L，但並不限定於此。 成為上述形態之純化方法之對象的玻尿酸類溶液 值，就防止玻尿酸分解、防止分子量降低之觀點而言，較 理想為3〜12，最理想為3〜9，但並不限定於此。進而，就進 一步提高純化（吸附）效果之觀點而言，pH值較理想為3.5以 上及/或7以下’但並不限定於此。 使用上述形態之純化方法時之玻尿酸類溶液之溫度，較 理想為0〜80Ϊ，但並不限定於此。若溫度為8〇〇c以下，則 可強力抑制處理中之玻尿酸類之分解及分子量之降低。 上述形態之純化方法中，將活性碳懸浮液與純化對象之 玻尿酸溶液相接觸時，雖可一次性懸浮並保持原樣，但較 理想為持續懸浮.攪拌。進行攪拌之情形時，較理想為攪拌 10分鐘〜1小時，更理想為攪拌20〜40分鐘，但並不限定於此。 於上述形態之各方法中之活性碳懸浮液之加熱處理中， 藉由將活性碳懸浮液於100艽以上加熱20分鐘以上而提高 去除雜質之能力。然而，為了提高加熱處理之效果，溫度 更理想為120°c以上，最理想為121力或其以上，但並不限 定於此。另外，為提高加熱處理之效果，較.理想為加熱時 之氣壓較同。作為此種較高之氣壓，例如較理想為〇 i 154810.doc 15 201139464 以亡丄更理想為0.2奶以上，但並不限定於此。另外，為 了提南加熱處理之效果’加熱時間更理想為30分鐘以上， 最理想以小時以上，但並不限定於此。另外，加熱時較理 想為持續攪拌並且進行加熱，此時，較理想為以H咖 rpm進行攪拌、更理想為以1〇〇〜6〇〇 rpm進行攪拌但並不 限定於此。 ^ 於上述形態之各方法中’將加熱後之活性碳懸浮液授掉 冷卻時’藉由將經加熱之活性碳懸浮液連續㈣而進行冷 卻。於預處理中進行攪拌並且冷卻之情形，與不進行攪： 而冷卻之情形相比’可更大地減少玻尿酸類於純化時之分 子量之降低。攪拌冷卻時之周圍之溫度較理想為 〇°C〜80°c，更理想為nrc〜6(rc，最理想為15〜4〇艽，但並 不限定於此。另外，尤其亦可於不進行操作之溫度即室溫 下操作ϋ半時間較理想為H、時以上，更佳理想為2小時 以上，尤其理想為3小時以上，最理想為4小時以上，但並 不限定於此。即便冷卻結束溫度為同一溫度，以適當之攪 拌時間進行攪拌，則用於純化時之玻尿酸類的分子量之降 低效果較大。另外，就冷卻結束溫度對玻尿酸之分子量降 低等所造成之影響而言，較理想為較低。較理想之冷卻結 束溫度為80°C以下，更理想之冷卻結束溫度為6〇π以下 (0°C以上），但並不限定於此。 上述形態之純化方法所使用之活性碳之量，相對於玻尿 酸類溶液100重量％，較理想為〇·5〜2〇 〇重量％，更理想為 5.0〜20.0重量°/〇，但並不限定於此。於該等範圍内，可期望 • 16 - 1S4810.docS*12-201139464 carbon suspension step; the step of heating the activated carbon suspension by stirring the activated carbon suspension at a temperature of 1 Torr (>c or more for 2 〇 minutes or more); a step of contacting a hyaluronic acid solution containing hyaluronic acid and/or its salt and impurities with an activated carbon suspension liquid after stirring the cold portion. According to the purification method, by using a state which has been pretreated to be suitable for purification of hyaluronic acid The second aspect of the present invention (for example, the second embodiment) is a method for producing a hyaluronic acid purifying agent, which comprises: suspending activated carbon in an aqueous liquid to obtain activated carbon. a step of suspending the liquid solution; heating the m-type carbon suspension to a temperature of 丨(9)t or more for 20 minutes or more; and cooling the heated activated carbon suspension by stirring. According to the manufacturing method, the activated carbon is pretreated by The purified form of hyaluronic acid capable of efficiently adsorbing and removing impurities can be produced in a state of purification of hyaluronic acid. The third aspect of the present invention (for example, embodiment 3) is an activity. The pretreatment method includes the steps of: suspending activated carbon in an aqueous liquid to obtain an activated carbon suspension, and heating the activated carbon suspension over 1 Torr for more than 2 minutes; The activated carbon suspension is stirred and cooled. According to the pretreatment method, the activated carbon can be pretreated to a state suitable for purification of hyaluronic acid. The fourth aspect of the present invention (for example, embodiment 9) is a type of hyaluronic (4) a purifying agent, comprising: an activated carbon produced by the following steps: a step of obtaining an activated carbon liquid by suspending activated carbon in an aqueous liquid; heating the activated carbon suspension above HKTC for 2 G minutes or more (4); The heated activated carbon suspension is subjected to a step of cooling. The purified agent can be efficiently adsorbed by using activated carbon which has been pretreated to a state suitable for purification of hyaluronic acid by 1548 l0.doc -13 · 201139464. Removal of impurities. Purification agent prepared by the purification method of the first aspect and the production method of the second aspect, and the activated carbon pretreated by the pretreatment method of the third aspect The purification method using the purification agent of the fourth aspect (hereinafter referred to as "the purification method of the above form") has a higher impurity adsorption force than the purification method using the untreated activated carbon, so that it can be more effective The impurity removal is performed. That is, if the purification method of the above-described form is used, the disappearance of the uric acid can be suppressed to a low level, and not only a polymer compound which is usually removed by activated carbon as an impurity (for example, Bacteria, proteins, nucleic acids, endotoxins) even low-molecular compounds (such as amino acids, sugars, organic acids) can be efficiently separated and removed. In addition, the above-mentioned purification method uses untreated activity. The purification method of carbon has a remarkable removal effect especially for living bacteria, protein and endotoxin. In addition, the purification method of the above form can reduce the decrease of the molecular weight of hyaluronic acid compared with the case of using untreated activated carbon. It can reduce the decrease in the molecular weight of hyaluronic acid during purification, and is particularly effective in purifying a high molecular weight (e.g., 3.5 million to 7 million Da) of aluminexic acid. In addition, the hyaluronic acid solution having a relatively high concentration (10) 先前 which was previously difficult to handle can be efficiently treated. Furthermore, when the uric acid is used to purify the uric acid by using the above-described method, the dialysis treatment, the purification treatment, and the like, the purification method is excellent, and the culture solution itself can be used as the target. And 'separate from the culture solution itself. Purify the glass. By using the broth without using centrifugation or filtration, the cultivating solution can be used more efficiently, and the cost can be further improved and the cost can be reduced. The hyaluronic acid wave degree of the hyaluronic acid solution in the above-described purification method is preferably 1/2〇§/ from the viewpoint of difficulty in handling due to high viscosity of the solution and solubility of uric acid. [More preferably, 〇5 to 15 g/L ' is most preferably 1 to 1 〇g/L, but is not limited thereto. The hyaluronic acid solution value which is a target of the purification method of the above-described form is preferably from 3 to 12, and most preferably from 3 to 9, from the viewpoint of preventing decomposition of hyaluronic acid and preventing a decrease in molecular weight, but is not limited thereto. Further, from the viewpoint of further improving the purification (adsorption) effect, the pH is preferably 3.5 or more and/or 7 or less, but is not limited thereto. The temperature of the hyaluronic acid solution in the case of using the above-described purification method is preferably 0 to 80 Å, but is not limited thereto. When the temperature is 8 〇〇c or less, the decomposition of hyaluronic acid and the decrease in molecular weight during the treatment can be strongly suppressed. In the purification method of the above aspect, when the activated carbon suspension is brought into contact with the hyaluronic acid solution to be purified, it may be suspended and kept as it is, but it is preferably continuously suspended and stirred. In the case of stirring, it is preferably stirred for 10 minutes to 1 hour, more preferably 20 to 40 minutes, but is not limited thereto. In the heat treatment of the activated carbon suspension in each of the above methods, the ability to remove impurities is improved by heating the activated carbon suspension at 100 Torr or more for 20 minutes or more. However, in order to increase the effect of the heat treatment, the temperature is more preferably 120 ° C or more, and most preferably 121 force or more, but is not limited thereto. Further, in order to improve the effect of the heat treatment, it is preferable to use the same pressure at the time of heating. As such a higher gas pressure, for example, it is preferable that 〇 i 154810.doc 15 201139464 is more preferably 0.2 milk or more, but is not limited thereto. Further, in order to enhance the effect of the heat treatment in the south, the heating time is more preferably 30 minutes or longer, and most preferably hours or more, but is not limited thereto. Further, in the case of heating, it is preferable to continuously stir and heat. In this case, it is preferred to carry out the stirring at H rpm, and it is more preferable to carry out the stirring at 1 Torr to 6 rpm, but is not limited thereto. ^ In each of the above-described methods, 'heating of the activated carbon suspension after cooling off' is carried out by continuously heating the activated activated carbon suspension (four). In the case of stirring and cooling in the pretreatment, the decrease in the molecular weight of the hyaluronic acid at the time of purification can be more greatly reduced than in the case of no stirring: in the case of cooling. The temperature around the stirring and cooling is preferably 〇 ° C to 80 ° C, more preferably nrc 〜 6 (rc, most preferably 15 〜 4 〇艽, but is not limited thereto. In addition, especially The temperature at which the operation is performed, that is, the half-time operation at room temperature is preferably H or more, more preferably 2 hours or longer, more preferably 3 hours or longer, and most preferably 4 hours or longer, but is not limited thereto. When the cooling completion temperature is the same temperature and the stirring is carried out under an appropriate stirring time, the molecular weight of the hyaluronic acid used for the purification is greatly reduced, and the effect of the cooling end temperature on the molecular weight of the hyaluronic acid is lowered. Preferably, the cooling end temperature is 80° C. or less, and more preferably the cooling end temperature is 6 〇 π or less (0° C. or more), but is not limited thereto. The amount of the activated carbon is preferably from 5% to 2% by weight, more preferably from 5.0 to 20.0% by weight, based on 100% by weight of the hyaluronic acid solution, but is not limited thereto. Inside, can be expected 16 - 1S4810.doc
S 201139464 更高之純化（吸附）作用。 進而’作為活性碳之原料’尤其就用於生物體時能引發 問題之雜質等之觀點而言，較理想為來源於木材，其形狀 就为散性或吸附效率之觀點而言，較理想為粉末狀。 另外，本發明之進一步之形態係於上述形態中玻尿酸類 係藉由馬鏈球菌FM-10〇(微工研條寄第9〇27號）或馬鏈球菌 FM-300(微工研條寄第2319號）而生產。藉由使用由該等微 生物所生產之玻尿酸類作為純化對象，可獲取雜質更少、 高分子量之玻尿酸類純化物，尤其於作為醫藥而使用時會 發揮優異之效果。 藉由使用上述形態之純化方法，可減輕玻尿酸類之分離· 純化步驟之負#，因此，上述形態之相關純化方法尤其有 效的是於製造之工業製程之相對較為初始階段中使用。 進而’藉由於無塵環境下進行上述形態之純化方法，而 可實現先前難以實現之醫藥品所要求的高品質之玻尿酸類 以工業規模之高效純化。此種純化方法於#藥品用之玻尿 酸類之生產中，具有極高之產業有用性。 再者，上述實施形態、藉由形態所說明之純化方法等並 不限定本發明’而係旨在例示而進行揭示者。本發明之技 術範圍係由申請專利範圍之揭示所規定，業者可於申請專 利範圍所揭*之發明之技術範圍内’進行各種^計變更。 例如，上述純化方法等亦可為進-步包含其他步驟、或 繼上述純化方法而進_步實施其他步驟·方法而製造玻尿 酸财之方法。作為.此種步驟·方法，例如可列舉同時地或 154810.doc -17-S 201139464 Higher purification (adsorption) effect. Further, from the viewpoint of being used as a raw material of activated carbon, particularly in the case of causing a problem in the case of using a living body, it is preferable that it is derived from wood, and its shape is a dispersive property or an adsorption efficiency. Powdery. Further, in a further aspect of the present invention, the hyaluronic acid is administered by Streptococcus equi subsp. FM-10 〇 (Microtech Research No. 9〇27) or Streptococcus equi subsp. FM-300 (Microtech Produced by No. 2319). By using hyaluronic acid produced by these microorganisms as a purification target, a purified hyaluronic acid having a low impurity and a high molecular weight can be obtained, and it is excellent in the use as a medicine. By using the purification method of the above form, the negative # of the separation and purification steps of the hyaluronic acid can be alleviated. Therefore, the purification method of the above aspect is particularly effective in the relatively initial stage of the industrial process of manufacturing. Further, by performing the above-described purification method in a dust-free environment, high-quality hyaluronic acid required for pharmaceuticals which have been previously difficult to realize can be efficiently purified on an industrial scale. This purification method has extremely high industrial usefulness in the production of hyaluronic acid for pharmaceutical products. Further, the above-described embodiments, the purification methods described by the aspects, and the like are not intended to limit the invention, and are disclosed as examples. The technical scope of the present invention is defined by the disclosure of the scope of the patent application, and the various modifications can be made within the technical scope of the invention as disclosed in the patent application. For example, the above purification method or the like may be a method of producing a hyaluronic acid by further including other steps, or performing the other steps and methods in accordance with the above purification method. As such a step and method, for example, simultaneous or 154810.doc -17-
S 201139464 分開進行··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滤液之步驟、將純化對象 液離心分離之步驟、將對象液中和之步驟、將鹽添加於對 象液之步驟、將純化對象液進行微遽之步驟、將對象液進 行透析處理之步驟、將芳香族系吸附樹脂添加於純化對象 液之步驟、將對象液進行超渡之步驟、利用層析法將對象 液進行純化之步驟、將活性碳之吸附劑添力口於對象液之步 驟、將活性碳自對象液中分離之步驟、將活性碳自對象液 中去除之步冑、添加有冑溶劑而使玻尿酸類沈殿之步驟、 使玻尿酸類結晶化之步驟、使玻尿酸類乾燥之步驟等。 [實施例] 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。 將使用馬鍵球菌FM.100(微工研菌寄第9〇27號）所培養之 醱酵液150 ml(玻尿酸鈉浪度35 g/L、pH值4〇)採集至则 mL之玻璃燒杯中’藉由以表_所示之條件所製備之吸附 劑（活性碳等）進行處理，使用磁力授拌器以400 rpm進行30 分鐘懸浮授拌處理。將該處理液使用滤紙過據，將氯化納 4.5 g(3.0重量％)添加於15〇机滤液中之後利用乙醇_ μ 使玻尿&鈉析出。將該析出之玻尿酸鈉利用乙醇则 洗後於40 C下進行真空乾燥而獲取玻尿酸納〇5g。 使用表4所示之評價項目、評價基準，對以此方式所獲取 之玻尿酸納進行6平價。將純化條件及評價結果示於表2及 3(實驗例1〜17)。 154810.doc 201139464S 201139464 The steps of cultivating a hyaluronic acid producing microbial strain, the step of producing a culture filtrate from a hyaluronic acid-producing microbial strain culture solution, the step of centrifuging the purification target liquid, the step of neutralizing the target liquid, and adding the salt to the target liquid a step of performing a micro-purification of the purification target liquid, a step of subjecting the target liquid to dialysis treatment, a step of adding an aromatic-based adsorption resin to the purification target liquid, a step of superposing the target liquid, and using a chromatography method a step of purifying the target liquid, a step of adding the activated carbon adsorbent to the target liquid, a step of separating the activated carbon from the target liquid, a step of removing the activated carbon from the target liquid, and adding a hydrazine solvent. a step of causing hyaluronic acid to smear, a step of crystallizing hyaluronic acid, a step of drying hyaluronic acid, and the like. [Examples] The present invention will be more specifically illustrated by the examples, but the invention is not limited thereto. The glass beaker of the mL is collected using 150 ml of the broth (sodium hyaluronate wave 35 g/L, pH 4 培养) cultured in the bacterium of the bacterium of the bacterium of the genus The medium was treated by an adsorbent (activated carbon or the like) prepared under the conditions shown in Table _, and subjected to a suspension stirring treatment at 400 rpm for 30 minutes using a magnetic stirrer. The treatment liquid was passed through a filter paper, and 4.5 g (3.0% by weight) of sodium chloride was added to the filtrate of 15 Torr, and then hyaluronic acid & sodium was precipitated by using ethanol_μ. The precipitated sodium hyaluronate was washed with ethanol and vacuum-dried at 40 C to obtain 5 g of sodium hyaluronate. Using the evaluation items and evaluation criteria shown in Table 4, the hyaluronic acid obtained in this manner was subjected to 6 parity. The purification conditions and evaluation results are shown in Tables 2 and 3 (Experimental Examples 1 to 17). 154810.doc 201139464
【<Ν<Ι_—I 1實施例9 1 S 〇 寸 爸30 m 濕潤 νή 寸 Ο 寸 一般 -B- 4、 -δ- 〇 實施例8 1 1 1 1 I 1 濕潤 ΓΠ 寸 1 400 一般 < 乞 4< 實施例7 § 400 ^30 yr) ο 濕潤 CO 寸 400 一般 夺 -δ- 4、 -9- m 實施例6 〇 寸 客80 in ο 濕潤 CO 寸 t—Η ν*) ι> 400 一般 备 •δ- +、 -ft- 沄 實施例5 120 S 400 S30 v〇 濕潤 in rn 寸 »η 400 一般 4、 备 m 實施例4 r·· § 400 I ^30 cn 乾燥 rn 寸 400 一般 -δ- 4、 B- m 實施例3 〇 寸 130 乾燥 in co νη ι> ! 400 一般 •δ- 4、 -θ- 沄 實施例2 1 1 1 1 1 t |乾燥| rn 寸 400 一般 < 4、 -κ (N 實施例1 1 1 1 1 1 1 1 1 1 1 I 1 1 無塵 原料位準 原料位準 原料位準 原料位準 實施例 活性碳之預處理(熱殺菌） 加熱溫度 [°C] 加熱時間 [min] 攪拌轉速 [rpm] 冷卻結束溫度[°c] 冷卻時間 [hr] 有無實施攪拌 活性碳之狀態 HA 濃度 [g/L] pH值 培養液量 [mL] AC(白鷺 Ρ) [%] 攪拌轉速 [rpm] 攪拌時間 [min] 環境 活菌數 蛋白質 胺基酸 内毒素 極限黏度 [dL/g] 加熱條件 冷卻條件 預處理 吸附條件 154810.doc -19- 201139464 Γ—Ιεΐ I54810.doc 幸邀铀螭®够tiHS 實施例17 V? 400 含30 濕潤 rn 寸 <n 400 無塵 七、 七、 士、 〇 實施例16 1 1 1 1 1 1 I濕潤| cn 寸 400 無塵 夺 -a- 4、 實施例15 120 g 400 S30 l〇 Ο 濕潤 yr) rn 寸 S 400 無塵 4、 4、 4、 4、 實施例14 400 含80 ο 濕潤 的 ΓΛ 寸 400 無塵 +、 4、 4、 七、 沄 實施例13 § 400 含30 ν〇 濕潤 rn in 400 無塵 4、 七、 士、 七、 cn 實施例12 ； 400 各30 m 乾燥 in rn yr) 400 無塵 4、 4、 士、 實施例11 S ； 400 S30 乾燥 CO 寸 § l> 400 無塵 士、 4、 4、 4、 沄 實施例10 1 t 1 1 1 1 乾燥 寸 400 無塵 -6- 七、 4- <N 實施例 活性碳之預處理(熱殺菌） 加熱溫度 [°C] 加熱時間 [min] 授拌轉速 [rprn] 冷卻結束溫度[°C] 冷卻時間 [hr] 有無實施攪拌 活性碳之狀態 HA 濃度 [g/L] pH值 培養液量 [mL] AC(白鷺 P)[%] 授拌轉速 [rpm] 攪拌時間 [min] 環境 活菌數 蛋白質 胺基酸 内毒素 極限黏度 [dL/g] 加熱條件 1 冷卻條件 i 預處理 吸附條件 -20- s 201139464 [表4] 評價項目 價 少 中 多 原料位準 活菌數 [cfb/mL] 含100 100〈菌 <1000 1000$ 菌 <5000 姿 5000 漏菌 [cfu/mL] iioo 100〈菌 <1000 1000爸菌 <5000 15000 蛋白質 〇g/mL] 筌 0.04 0.04<蛋白質 <0.20 0.20笤蛋白質<0.5 S0.5 胺基酸 [pg/mL] 爸5 5<胺基酸<1000 1000S 胺基酸 <150000 § 150000 内毒素 [EU/mL] 笤 0.001 0.001<ET*<0.01 0.01ίΕΤ*<50000 爸 50000 *ET :内毒素 根據以上實驗之結果可確定：藉由使用以一定之預處理 方法所預處理之活性碳進行純化，可自玻尿酸類溶液將雜 質有效地吸附去除而進行純化。 以上，基於實施例對本發明進行說明。業者應理解：該 實施例僅僅為例示，可實施各種變形例，另外該等之變形 例亦屬於本發明範圍。 -21 - 154810.doc[<Ν<Ι_—I 1 Example 9 1 S 〇 inch dad 30 m wet νή inch Ο inch general-B-4, -δ- 〇 Example 8 1 1 1 1 I 1 Wet ΓΠ inch 1 400 lm ; 乞 4 < Example 7 § 400 ^ 30 yr) ο Wet CO inch 400 General - δ - 4, -9- m Example 6 〇 inch guest 80 in ο Wet CO inch t-Η ν*) ι> 400 General preparation δ-+, -ft- 沄Example 5 120 S 400 S30 v〇wetting in rn inch»η 400 General 4, preparation m Example 4 r·· § 400 I ^30 cn Dry rn inch 400 General - Δ-4, B-m Example 3 130130 drying in co νη ι> ! 400 General • δ- 4, -θ- 沄 Example 2 1 1 1 1 1 t | Drying | rn Inch 400 General < 4 - κ (N Example 1 1 1 1 1 1 1 1 1 1 1 I I 1 1 dust-free raw material level raw material level raw material level raw material level example pretreatment of activated carbon (heat sterilization) heating temperature [° C] Heating time [min] Stirring speed [rpm] Cooling end temperature [°c] Cooling time [hr] Whether or not to stir the activated carbon state HA concentration [g/L] pH value Nutrient volume [mL] AC (Egret Ρ) [%] Stirring speed [rpm] Stirring time [min] Ambient viable count protein amino acid endotoxin ultimate viscosity [dL/g] Heating conditions Cooling conditions Pretreatment adsorption conditions 154810 .doc -19- 201139464 Γ—Ιεΐ I54810.doc Fortunately, uranium strontium® is sufficient for tiHS Example 17 V? 400 with 30 wet rn inches <n 400 dust-free VII, VII, 士, 〇 Example 16 1 1 1 1 1 1 I humid | cn inch 400 dust-free - a- 4, example 15 120 g 400 S30 l〇Ο wet yr) rn inch S 400 dust-free 4, 4, 4, 4, embodiment 14 400 contains 80 ο Wet ΓΛ inch 400 dust-free +, 4, 4, VII, 沄 Example 13 § 400 with 30 ν 〇 wet rn in 400 dust-free 4, seven, s, seven, cn Example 12; 400 each 30 m dry in Rn yr) 400 Dust free 4, 4, Shi, Example 11 S; 400 S30 Dry CO inch § l> 400 No dust, 4, 4, 4, 沄 Example 10 1 t 1 1 1 1 Dry inch 400 Dust-6- VII, 4- <N Example Pretreatment of Activated Carbon (Hot Sterilization) Heating Temperature [°C] Heating time [min] Mixing speed [rprn] Cooling end temperature [°C] Cooling time [hr] Whether or not to stir the activated carbon state HA concentration [g/L] pH culture fluid volume [mL] AC (Egret P) [%] Mixing speed [rpm] Stirring time [min] Environmental viable count protein amino acid endotoxin ultimate viscosity [dL/g] Heating condition 1 Cooling condition i Pretreatment adsorption condition -20- s 201139464 [Table 4] The number of viable bacteria in the multi-feedstocks in the evaluation item price is small [cfb/mL] 100 100 <bacteria<1000 1000$ bacteria<5000 posture 5000 leaking bacteria [cfu/mL] iioo 100 <bacteria< 1000 1000 Daddy <5000 15000 Protein 〇g/mL] 筌0.04 0.04<protein<0.20 0.20 笤 protein <0.5 S0.5 Amino acid [pg/mL] Daddy 5 5 <Amino acid <1000 1000S Amino Acid <150000 § 150000 Endotoxin [EU/mL] 笤0.001 0.001<ET*<0.01 0.01ίΕΤ*<50000 Dad 50000 *ET: Endotoxin can be determined based on the results of the above experiment: by using Purification by activated carbon pretreated by a certain pretreatment method, the impurities can be effectively adsorbed and removed from the hyaluronic acid solution to be pure Chemical. Hereinabove, the present invention has been described based on the embodiments. It is to be understood that the embodiments are merely illustrative and that various modifications can be made and that such modifications are also within the scope of the invention. -21 - 154810.doc