201012865 九、發明說明： 【發明所屬之技術領域】 本發明係有關一種製造交聯透明質酸（cross-linked hyaluronic acid ’ cross-linked HA)之技術。詳言之，本發 ' 明係關於一種降低產品中交聯劑含量之交聯透明質酸製造 方法* 【先前技術】 透明質酸為一多醣’其結構由β_1，3·葡萄糖乙醯胺（β_ Φ l，3-N-acetyl glucosamine)及 β-1，4-葡萄糖酸（β-ΐ，4-glucuronic acid)以β-1-4鍵結形成分子量約為400 D之雙醣 重覆單位，此雙醣重覆單位再以β_ U鍵結重覆連結，形成 之直鏈高分子聚合物。其商業來源可由菌種如鏈球菌屬的 撥酵以及動物組織如雞冠中萃取得到。 透明質酸、其鹽類或其衍生物由於不具有種屬特異性， 且具有良好的生物相容性、黏彈性、保濕性及生物可降解 性，而廣泛應用於化妝品、生物醫學、醫療器材及製藥 ❹ 上。 -直鏈的透明質酸在生物體内易因酵素（如透明質酸水解 ，酵素）及自由基的作用而發生降解，因而降低在生物體内 的滯留時間，亦因缺乏機械強度而限制其應用範圍，故於 實際應用時，常將透明質酸進行交聯’製備成可溶於水的 交聯透明質酸溶液或不溶於水的交聯透明質酸凝膠 (hydrogel)，或介於兩者之間的物質，亦可視需要混合兩 者使用。 133837.doc 201012865 然而，藉由交聯劑進行透明質酸的交聯反應以製備交聯 透明質酸時，常有大量交聯劑殘留於產物中，而不利於應 用於生物體内，目前最常用以去除或降低殘留交聯劑的方 法包括以透析法或以水或緩衝溶液清洗。但此類純化方法 效果有限，同時對於一端已是鍵結態而另一端仍為自由態 g能基的交聯劑而言，並無法以透析或清洗的方式去除， 且此種含有自由態官能基之交聯劑具有反應性，如美國發 明專利第5808050號所示，於動物體中使用時具有風險。 另一方面’以透析或清洗交聯透明質酸尚有製程放大不 易之問題，且因於中性或接近中性環境下純化，無菌條件 難以控制，再者，若交聯透明質酸為凝膠狀態時，交聯程 度低的交聯透明質酸因膨潤大，清洗不易，得到的產物之 透明質酸含量低；交聯程度高的交聯透明質酸則因膨潤 低，殘留的交聯劑更加不易去除。再者，於清洗過程中， 交聯程度低的透明質酸易流失，而喪失潤滑性質，對於需 充填於針劑中的應用，需另外添加直鏈或交聯的透明質酸 溶液以增加其潤滑性質。 美國發明專利第4716154號揭露一種無菌、不含熱原的 交聯的透明質酸製法，可作為眼球的玻璃狀液（vhre〇us humor)取代物。此合成方式是以多官能基交聯劑，在驗存 在下’於高溫如50°C進行交聯反應2小時，反應後置於室 溫過夜’由於殘留有未反應之交聯劑’接著將所得膠體切 成小塊後’以蒸餾水清洗24小時，再以沸騰的生理食鹽水 清洗8小時，得到固含量0.23%〜1.2%的膠體。該專利文獻 133S37.doc 201012865 強調因此方法殘留有未反應之交聯劑，徹底清洗所合成的 膠體極端重要且不可缺少。此方法的缺點包括有：（丨）需有 複雜的膠體純化步驟；（2)因以沸騰的生理食鹽水去除交聯 劑’使膠體膨潤而僅得到固含量偏低的膠體，如需提高固 含量’則需額外步驟；（3)需重新於生理緩衝溶液中膨潤， 以調整其滲透壓及酸鹼值，不利於工業上使用。 美國發明公開第2006/0194758A1號將高、低分子量的透 明質酸混合反應，產生一種單相的具良好機械性質的膠 丨體，改良了可注射性，但此方法於5(rc交聯反應結束後， 未經透析純化前殘留大量未反應的交聯劑，其濃度超過 300 ppm以上，之後藉透析法以純化試圖去除交聯劑但 其效果不佳，難以有效去除未反應且自由態的交聯劑，亦 無法去除已為鍵結狀態但交聯劑的另一端尚具自由態官能 基的交聯劑。 美國發明公開第2005/028 1880A1號揭露一種可注射凝膠 丨力製備方法，其步驟包含：在一個密閉容器内，交聯一種 或多種聚合物形成膠體；清洗該膠體；純化該膠體；均質 該膠體形成凝膠。該方法使用了高濃度雙官能基或多官能 主交聯劑，有上述鍵結態交聯劑殘留的問題。再者，膠體 、.純化的時間需2至3天，由於此時溶液的酸驗值已接 近中性’產生微生物汙染的風險。 美國發明公開第200湖26G7GA1號揭示—種㈣交聯凝 翁方法’包括在-驗性溶液巾，使多㈣三或多官能基 衣化物接觸，以提供一種以環氧化物交聯的多酷，其中 133837.doc 201012865 環氧化物基本上是以醚鍵與多醣聯結；在實質上未從驗性 介質中移除環氧化物的情況下’乾燥該環氧化物交聯的多 聽；以水可溶的溶劑合理清洗交聯的多醣；中和交聯的多 糖基質’形成交聯的多醣凝膠。此方法同樣具有已是鍵結 狀態但尚含自由態官能基的交聯劑殘留問題。 如要製備交聯的透明質酸固態物，常用去除交聯劑的方 法是沉澱法，如美國專利第4716224號，但此方法仍有上 述尚含自由態官能基之鍵結態交聯劑殘留的風險存在。 另一方面，於鹼性環境下之交聯反應時，透明質酸同時 也被水解，故鹼性環境下之反應是交聯反應與水解反應相 互競爭，如 Y. Tokita及 A_ Okamoto, “ Hydrolytic Degration of Hyaluronic Acid”，Polymer Degration and Stability，v〇l. 48，pp· 269-273(1995)文獻所述。在鹼性環境反應初期， 交聯劑的濃度最高，故反應以交聯反應為主，但當反應一 段時間交聯劑消耗一定量後，若以同樣溫度繼續進行反 應，則反應以水解反應為主，同時也會破壞膠體的性質， 使膠體性質劣化，造成發生膠體裂解、顏色變成深黃色或 甚至變為褐色而無法使用，故於鹼性環境反應時，進行一 段時間的反應後就必須終止反應，雖然此時還殘留有大量 的交聯劑。以雙官能基或多官能基環氧化物在鹼性條件下 進行交聯反應時，大多在25。〇至6(rc下進行，反應時間則 在10分鐘至24小時之間，但實際的反應時間與所使用的溫 度及鹼濃度有關。而這些反應下所得的膠體，大多有去除 交聯劑的純化步驟或雖無純化的程序，但仍殘留有相當量 133837.doc 201012865 或大量的交聯劑。 目前文獻中已知之去除交聯劑的方法都有其缺點，且都 無法有效去除交聯劑或降低交聯劑含量，尤其無法去除— 端已疋鍵結態另一端仍為自由惡官能基的交聯劑。業界亟 需一種降低產品中交聯劑含量之交聯透明質酸製造方法。 【發明内容】 發明概述 本發明提供一種交聯透明質酸之製造方法，其包含於自 約10 C至約3 0 c之低反應溫度，使包含交聯劑與透明質 酸、其金屬鹽類、其衍生物或其混合物之溶液於鹼性環境 進行交聯反應超過約48小時。 根據本發明之方法不需經由純化步驟，即可製得於產物 中父聯劑含量低、生物相容性高且可供動物體應用之交聯 透明質酸’以克服習知技術中使用純化步驟之缺點。 發明詳細說明 本發明提供一種交聯透明質酸之製造方法’其包含於自 約1 〇 C至約3〇。(：之低反應溫度，使包含交聯劑與透明質 酸、其金屬鹽類、其衍生物、或其混合物之溶液於鹼性環 境進行交聯反應超過約48小時。 本發明所言之「透明質酸」又稱為玻尿酸，係指一多醣 化〇物，其結構由β-1，3-葡萄糖乙醯胺及β-1，4-葡萄糖酸以 β-1-4鍵結形成分子量約為4〇〇 D之雙醣重覆單位此雙醣 重覆單位再以β·^鍵結重覆連結，形成之直鏈高分子聚合 物0 133837.doc 201012865 本發明所言之「透明質酸金屬鹽類」係指由透明質酸與 金屬離子所形成之鹽類，較佳地，其係為鉀鹽、鈉鹽或鋅 鹽0 本發明所言之「透明質酸衍生物」係指含有羥基之多醣 類例如但不限於幾·甲基纖維素（carboxymethylcellulose， CMC)、褐藻酸鹽（alginate)、軟骨素-4-硫酸鹽 (chondroitin_4-Sulfate)、軟骨素-6-硫酸鹽（ch〇ndroitin-6-Sulfate)、咕頓樹膠（xanthane gum)、殼聚糖（chitosan)、果 膠（pectin)、瓊脂（agar)、鹿角菜膠（carrageenan)或瓜耳樹 膠（guar gum)。 本發明所言之「交聯透明質酸」係指在含有交聯劑存在 下’使相同分子或不同分子的透明質酸、其金屬鹽類、其 衍生物或其混合物間，發生部分交聯或完全交聯後，所形 成之分子網狀結構型態的交聯透明質酸。其物理型態可為 固態、液態（指溶於水或緩衝溶液的交聯透明質酸）、凝膠 (指被水或緩衝溶液膨潤的交聯透明質酸膠體）、介於液態 和凝膠間之混合物或是水不溶的固體，此物理型態可由加 入反應的交聯劑含量和合成方法而定。 本發明所言之「交聯反應」係指使兩透明質酸、其金屬 鹽類、其衍生物或其混合物直鏈間產生鍵結之反應。在鹼 性環境下’根據本發明之交聯反應係以醚鍵形成交聯，不 產生副產物，理論上反應可完全進行。 於本發明之一具體實施例中，鹼性環境中鹼濃度為自約 0.05 N至約1.5 N ;較佳為自約0.05 N至約1 N ;更佳為自 133837.doc 201012865 、-、N至約0.5 N ’尤佳係為自約〇 25 N至約〇 5 N 〇另一 方面難環境較佳係由無機驗提供，其巾該無機驗較佳 係選自由氫氧化鈉及氫氧化鉀所組成之群，其中「鹼濃 度」的疋義係指i升的反應物中的液體之體積（包含溶劑、 驗及交聯劑之體積，但不包含透明質酸、其金屬鹽類、其 何生物或其混合物之體積）中所含鹼的當量數。 根據本發明之方法’該交聯反應係使用交聯劑進行。本 發明中所言之「交聯劑」係指可將兩透明質酸、其金屬鹽 類、其衍生物或其混合物直鏈間產生鍵結之物質。通常該 交聯劑具有多個官能基可分別與多個之透明質酸、其金屬 鹽類、其衍生物或其混合物直鏈產生鍵結，並進一步彼此 進行交聯反應。較佳地，該交聯劑係為多官能基環氧化 物，更佳為雙官能基環氧化物。於本發明之一較佳具體實 施例中’該交聯劑係選自由1，4- 丁二醇二縮水甘油鍵（丨,4_ butanediol diglycidyl ether)、乙二醇二縮水甘油醚 (ethylene glycol diglycidyl ether)、1,6-己二醇二縮水甘油 醚（1,6-hexanediol diglycidyl ether)、多丙二醇二縮水甘油 酸（polypropylene glycol diglycidyl ether)、多四甲撐二醇 二縮水甘油謎（polytetramethylene glycol diglycidyl ether)、新戊二醇二縮水甘油鍵（neopentyl glycol diglycidyl ether)、多甘油多縮水甘油醚（polyglycerol polyglycidyl ether)、二甘油多縮水甘油趟（diglycerol polyglycidyl ether)、甘油多縮水甘油醚（glycerol polyglycidyl ether)、三經甲基丙燒多縮水甘油醚（tri- 133837.doc -12- 201012865 methylolpropane polyglycidyl ether)、季戊四醇多縮水甘油 醚（pentaerythritol polyglycidyl ether)、山梨糖醇多縮水甘 油醚（sorbitol polyglycidyl ether)、1，2,7,8-二環氧辛烷 (l，2,7,8-diepoxyoctane)及 1，3-二環氧丁烷（1，3_butadiene diepoxide)及其混合物所組成之群。 本發明所屬技術領域中具通常知識者可視所需之交聯透 明質酸之交聯程度及交聯劑種類選擇合宜之交聯劑濃度， 於本發明之一較佳具體實施例中，交聯劑之濃度為自約 〇.〇5至約2 w/v% ;更佳為自約0」至約丨5 w/v% ;尤佳為自 約0.1至約1.0 w/ν%，最佳為自約〇·6至約i 〇 w/v〇/〇。 本發明之技術特徵在於使用長時間之低溫反應溫度條件 進行交聯反應，根據本發明之低反應溫度係指約1〇。〇至約 30°C ;較佳係自約15°C至約30。(：；最佳為自約2〇。(：至約30 C °於此低反應溫度下進行之交聯反應，所形成之交聯透 明質酸不會因驗的水解作用而快速劣化，於使交聯劑消耗 至合理的含量後即終止反應。本發明中所言之低反應溫度 不限於以單一溫度反應，亦可為多種低反應溫度之組合。 本發明所言之「使交聯劑消耗至合理的含量」是指在最後 所得的交聯產物中交聯劑含量可為後續繼續利用之濃度， 例如1 ppm以下、2 ppm以下、5 ppm以下、10 ppm以下、 15 ppm以下或2〇 ppm以下，但並不意謂限定本發明，根據 本發明交聯劑的消耗並不侷限於因交聯反應而消耗，亦可 能因水解作用而消耗，也可能交聯作用及水解作用一起消 耗。於本發明之具體實施例中，可控制所得的產物的未反 133837.doc -13- 201012865 應完全之交聯劑含量為低於1 ppm、2 ppm、2至5 ppm、5 至10 ppm、10至15 ppm或15至20 ppm。本發明所言「具有 自由態官能基之交聯劑」係包含未與透明質酸、其金屬鹽 類、其衍生物或其混合物反應之交聯劑及部分官能基與透 明質酸、其金屬鹽類、其衍生物或其混合物反應，使交聯 劑官能基與透明質酸、其金屬鹽類、其衍生物或其混合物 間產生鍵結但部分官能基未反應而為自由態時之交聯劑。 較佳地，該交聯反應係於交聯透明質酸劣化前即停止本 發明所言「劣化」係指經交聯反應後的所得之交聯透明質 酸，因為鹼的水解作用變成深黃色或甚至深褐色，使其無 法應用，或是指所合成的產物開始發生明顯降解。通常交 聯反應之時間與交聯反應之溫度相關，溫度越高，所需之 時間越短’溫度越低，所需之時間越長；反應時間亦與交 聯劑之濃度及鹼濃度相關，綜合上述，根據本發明之反應 時間係超過48小時，較佳係約3至約28天，更佳係約3至約 11天，最佳係約3至約7天。 根據本發明，包含透明質酸、其金屬鹽類、其衍生物或 其混合物之反應溶液中透明質酸、其金屬鹽類、其衍生物 或其混合物之濃度較佳為自約2至約4〇 wv%;更佳為自約 10至約30 wv/〇，尤佳為自約15至約2〇 wv%。根據本發明 之透明質酸、其金屬鹽類或其料物分子量理論上並未有 限制’可單獨為低分子量、高分子量，也可為兩種高、低 分子量的透明質酸、其金屬鹽類或其衍生物之任意混合。 其分子量例如為10萬以下、10至50萬之間、50萬至100萬 133837.doc 201012865 之間、1〇0萬至150萬之間、15〇萬至200萬之間、250萬至 300萬之間。較佳為混合高、低分子量之透明質酸、其金 屬鹽類、其衍生物或其混合物進行交聯反應，具有反應溶 液黏度低及適合製程放大的優點。 於本發明之一較佳具體實施例中，當使用M-丁二醇二 縮水甘油醚1v/v%作為交聯劑時，包含透明質酸、其金屬 鹽類、其衍生物或其混合物溶液中透明質酸、其金屬鹽 類、其衍生物或其混合物之濃度為2〇 w/v%、鹼濃度為 〇_25 1^時，所需的反應時間為3至28天。 根據本發明之方法較佳係於無菌條件下進行，但不限於 僅在無菌條件下進行，亦可於一般的環境下進行，只要最 終產物的交聯劑可降低至合理的範圍以下及具有良好的生 物相容性即可，其中生物相容性的性質可藉細胞毒性試驗 進行測試。實際上，根據本發明於低反應溫度下進行交聯 反應雖需較長之反樣時間，但由於是在鹼性的環境中，不 容易有微生物的污染’亦無先前技術中於中性緩衝溶液中 清洗膠體所產生微生物的污染的高風險。 較佳地’根據本發明之方法係於低反應溫度中進行交聯 反應前’另包含一於高反應溫度中進行交聯反應之步驟， 其中該高反應溫度為自約35°c至約6〇〇c ;較佳為自約35C>c 至約50°C ;更佳自約35〇c至約4〇〇c。於此態樣中，其係於 高反應溫度進行交聯反應一段時間，使透明質酸、其金屬 鹽類、其衍生物或其混合物先進行部份的交聯反應，在其 性質尚未劣化，或未開始有明顯降解發生前，再置於低反 133837.doc •15· 201012865 應酿度繼續進行反應，使交聯劑消耗至合理的含量後終止 反應。本發明中所言之高反應溫度不限於以單一溫度反 應，亦可為多種高反應溫度的組合。 於本發明之較佳具體實施例中，當高反應溫度為約35Ό 時，進行交聯反應小於約72小時；較佳為自約4至約48小 時’更佳為自約6至約12小時。當高反應溫度為約机 時’進行交聯反應小於約48小時；較佳為自約2至24小 ❹201012865 IX. Description of the Invention: [Technical Field of the Invention] The present invention relates to a technique for producing cross-linked hyaluronic acid (cross-linked HA). In particular, the present invention relates to a method for producing crosslinked hyaluronic acid which reduces the content of a crosslinking agent in a product. * [Prior Art] Hyaluronic acid is a polysaccharide whose structure is composed of β_1,3·glucosamine (β_ Φ l,3-N-acetyl glucosamine) and β-1,4-gluconic acid (β-ΐ, 4-glucuronic acid) are β-1-4 bonded to form a double sugar repeat with a molecular weight of about 400 D. In this unit, the double sugar repeating unit is repeatedly connected by a β_U bond to form a linear polymer. Commercial sources thereof can be extracted from strains such as streptococci and animal tissues such as cockscombs. Hyaluronic acid, its salts or its derivatives are widely used in cosmetics, biomedicine, medical equipment because they are not species-specific, have good biocompatibility, viscoelasticity, moisture retention and biodegradability. And pharmaceuticals. - Straight-chain hyaluronic acid is easily degraded in the living body by enzymes (such as hyaluronic acid hydrolysis, enzymes) and free radicals, thereby reducing the residence time in the living body and limiting its lack of mechanical strength. Scope of application, so in practical applications, hyaluronic acid is often cross-linked to prepare a water-soluble cross-linked hyaluronic acid solution or a water-insoluble cross-linked hyaluronic acid gel (hydrogel), or The substance between the two can also be used in combination. 133837.doc 201012865 However, when cross-linking reaction of hyaluronic acid is carried out by a crosslinking agent to prepare cross-linked hyaluronic acid, a large amount of cross-linking agent is often left in the product, which is unfavorable for application in living organisms. Methods commonly used to remove or reduce residual crosslinkers include dialysis or washing with water or a buffer solution. However, such purification methods have limited effectiveness, and at the same time, for a cross-linking agent having a bonded state at one end and a free-standing g-energy group at the other end, it cannot be removed by dialysis or washing, and the free-state functional group is contained. The cross-linking agent is reactive, as shown in U.S. Patent No. 5,808,050, and is of risk when used in animals. On the other hand, the use of dialysis or cleaning cross-linking hyaluronic acid is not easy to amplify the process, and because of neutral or near-neutral environment purification, aseptic conditions are difficult to control, and further, if cross-linked hyaluronic acid is condensed In the gel state, the crosslinked hyaluronic acid having a low degree of crosslinking is large in swelling, difficult to clean, and the hyaluronic acid content of the obtained product is low; the crosslinked hyaluronic acid having a high degree of crosslinking is low in swelling and residual cross-linking. The agent is more difficult to remove. Furthermore, during the cleaning process, the hyaluronic acid with low cross-linking degree is easily lost, and the lubricating property is lost. For the application to be filled in the injection, a linear or cross-linked hyaluronic acid solution is additionally added to increase the lubrication. nature. U.S. Patent No. 4,716,154 discloses a sterile, pyrogen-free, crosslinked hyaluronic acid process which can be used as a replacement for the eyeball's vhre〇us humor. This synthesis is carried out by using a polyfunctional crosslinking agent in the presence of a 'crosslinking reaction at a high temperature such as 50 ° C for 2 hours, and then leaving it at room temperature overnight after the reaction. 'Because the residual unreacted crosslinking agent remains' After the obtained colloid was cut into small pieces, it was washed with distilled water for 24 hours, and then washed with boiling physiological saline for 8 hours to obtain a colloid having a solid content of 0.23% to 1.2%. This patent document 133S37.doc 201012865 emphasizes that the method remains unreacted cross-linking agent, and thorough cleaning of the synthesized colloid is extremely important and indispensable. Disadvantages of this method include: (丨) need to have a complicated colloid purification step; (2) removal of the cross-linking agent by boiling physiological saline to make the colloid swell and only obtain a colloid having a low solid content, if it is necessary to improve the solid The content 'is an extra step; (3) it needs to be swelled in the physiological buffer solution to adjust its osmotic pressure and pH value, which is not suitable for industrial use. U.S. Patent Publication No. 2006/0194758A1 combines high- and low-molecular-weight hyaluronic acid to produce a single-phase colloidal body with good mechanical properties, which improves injectability, but this method is 5 (rc cross-linking reaction). After the end, a large amount of unreacted cross-linking agent remains before the dialysis purification, and its concentration exceeds 300 ppm. Then, by dialysis, purification is attempted to remove the cross-linking agent, but the effect is not good, and it is difficult to effectively remove the unreacted and free state. The cross-linking agent is also incapable of removing a cross-linking agent which is in a bonded state but has a free-standing functional group at the other end of the cross-linking agent. U.S. Patent Publication No. 2005/028 1880A1 discloses a method for preparing an injectable gel. The method comprises: crosslinking a polymer or a plurality of polymers into a colloid in a closed container; washing the colloid; purifying the colloid; homogenizing the colloid to form a gel. The method uses a high concentration of difunctional or polyfunctional primary crosslinks. The agent has the problem of residual bonding of the above-mentioned bonded state. Further, the colloid and purification time takes 2 to 3 days, because the acidity of the solution is close to the neutral 'production microorganism. The risk of dyeing. The US invention discloses that the 200th Lake 26G7GA1 discloses that the species (four) cross-linking method includes 'in-testing solution towel, contacting the poly(tetra)tri- or polyfunctional coating to provide an epoxide How cool is the joint, wherein 133837.doc 201012865 epoxide is basically linked with polysaccharides by ether bonds; in the case where epoxide is not substantially removed from the test medium, 'drying the epoxide crosslinks more Listening; reasonably washing the cross-linked polysaccharide with a water-soluble solvent; neutralizing the cross-linked polysaccharide matrix to form a crosslinked polysaccharide gel. This method also has cross-linking which is already bonded but still contains free-state functional groups. Residue problem of the agent. If a crosslinked hyaluronic acid solid is to be prepared, the method for removing the crosslinking agent is usually a precipitation method, such as U.S. Patent No. 4,716,224, but the method still has the above-mentioned bonding state containing a free functional group. The risk of residual cross-linking is present. On the other hand, hyaluronic acid is also hydrolyzed in the cross-linking reaction in an alkaline environment, so the reaction in an alkaline environment is a competition between the cross-linking reaction and the hydrolysis reaction, such as Y. Tokita A_ Okamoto, "Hydrolytic Degration of Hyaluronic Acid", Polymer Degration and Stability, v.l. 48, pp. 269-273 (1995). In the initial stage of alkaline reaction, the concentration of the crosslinking agent is the highest, so the reaction The cross-linking reaction is mainly used, but when the cross-linking agent consumes a certain amount for a certain period of time, if the reaction is continued at the same temperature, the reaction is mainly hydrolyzed, and the properties of the colloid are also destroyed, thereby deteriorating the colloidal properties, resulting in deterioration. Colloidal cleavage occurs, the color becomes dark yellow or even brown, and it cannot be used. Therefore, when reacting in an alkaline environment, the reaction must be terminated after a period of reaction, although a large amount of crosslinking agent remains at this time. When the cross-linking reaction is carried out under a basic condition with a difunctional or polyfunctional epoxide, it is mostly at 25. 〇 to 6 (r, the reaction time is between 10 minutes and 24 hours, but the actual reaction time is related to the temperature and alkali concentration used. The colloids obtained under these reactions mostly have the cross-linking agent removed. The purification step or although there is no purification procedure, but there is still a considerable amount of 133837.doc 201012865 or a large amount of cross-linking agent. The methods for removing cross-linking agents known in the literature have their disadvantages, and they are unable to effectively remove the cross-linking agent. Or reduce the content of cross-linking agent, especially the non-removable cross-linking agent. The other end of the bonded state is still free-radical-functional cross-linking agent. There is a need in the industry for a cross-linked hyaluronic acid manufacturing method for reducing the content of cross-linking agent in the product. SUMMARY OF THE INVENTION The present invention provides a method for producing crosslinked hyaluronic acid comprising a crosslinking reaction with hyaluronic acid, a metal salt thereof, at a low reaction temperature of from about 10 C to about 30 c. The solution of the derivative thereof or a mixture thereof is subjected to a crosslinking reaction in an alkaline environment for more than about 48 hours. According to the method of the present invention, the content of the parent-linked agent in the product can be obtained without a purification step. Cross-linking hyaluronic acid which is highly compatible and suitable for animal use to overcome the disadvantages of the purification step used in the prior art. DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for producing cross-linked hyaluronic acid, which is included in From about 1 〇C to about 3 〇. (: a low reaction temperature, the crosslinking reaction comprising a crosslinking agent with hyaluronic acid, a metal salt thereof, a derivative thereof, or a mixture thereof is carried out in an alkaline environment for more than about 48 hours. The term "hyaluronic acid" as used in the present invention is also called hyaluronic acid, and refers to a polysaccharideized sputum whose structure is composed of β-1,3-glucopyridylamine and β-1,4-gluconic acid. The β-1-4 bond forms a disaccharide repeating unit having a molecular weight of about 4〇〇D. The double sugar repeating unit is repeatedly connected by a β·^ bond to form a linear polymer. 0 133837.doc 201012865 The term "hyaluronic acid metal salt" as used in the present invention means a salt formed of hyaluronic acid and a metal ion, preferably a potassium salt, a sodium salt or a zinc salt. "Hyaluronic acid derivative" means a polysaccharide containing a hydroxyl group such as, but not limited to, a few methyl fibers Carboxymethylcellulose (CMC), alginate, chondroitin-44-ulfate, ch〇ndroitin-6-Sulfate, xanthane gum ), chitosan, pectin, agar, carrageenan or guar gum. The term "crosslinked hyaluronic acid" as used in the present invention means Molecular network structure formed after partial crosslinking or complete crosslinking between hyaluronic acid, its metal salt, its derivative or a mixture thereof of the same molecule or different molecules in the presence of a crosslinking agent Cross-linked hyaluronic acid. The physical form can be solid, liquid (referred to as cross-linked hyaluronic acid dissolved in water or buffer solution), gel (refers to cross-linked hyaluronic acid colloid swollen by water or buffer solution), between liquid and gel The mixture is either a water-insoluble solid, and the physical form can be determined by the amount of the crosslinking agent added to the reaction and the synthesis method. The "crosslinking reaction" as used in the present invention means a reaction in which a bond between two hyaluronic acids, a metal salt thereof, a derivative thereof or a mixture thereof is linearly bonded. In the alkaline environment, the crosslinking reaction according to the present invention forms crosslinks by ether bonds without generating by-products, and the theoretical reaction can be completely carried out. In one embodiment of the invention, the alkali concentration in the alkaline environment is from about 0.05 N to about 1.5 N; preferably from about 0.05 N to about 1 N; more preferably from 133837.doc 201012865, -, N To about 0.5 N ', especially from about 25 N to about 5 N 〇 on the other hand, it is better to provide an environmental test, and the inorganic test is preferably selected from sodium hydroxide and potassium hydroxide. The group consisting of "base concentration" refers to the volume of the liquid in the i liter of the reaction (including the solvent, the volume of the cross-linking agent, but does not contain hyaluronic acid, its metal salt, The number of equivalents of the base contained in the organism or the mixture thereof. According to the method of the present invention, the crosslinking reaction is carried out using a crosslinking agent. The term "crosslinking agent" as used in the present invention means a substance which can bond between two hyaluronic acids, metal salts thereof, derivatives thereof or a mixture thereof. Usually, the crosslinking agent has a plurality of functional groups which are respectively linearly bonded to a plurality of hyaluronic acid, a metal salt thereof, a derivative thereof or a mixture thereof, and further cross-linked with each other. Preferably, the crosslinking agent is a polyfunctional epoxide, more preferably a difunctional epoxide. In a preferred embodiment of the invention, the crosslinking agent is selected from the group consisting of 1,4-butanediol diglycidyl ether, ethylene glycol diglycidyl. Ether), 1,6-hexanediol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol Diglycidyl ether), neopentyl glycol diglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, glycerol polyglycidyl ether Polyglycidyl ether), trimethyl methacrylate polyglycidyl ether (tri- 133837.doc -12- 201012865 methylolpropane polyglycidyl ether), pentaerythritol polyglycidyl ether, sorbitol polyglycidyl Ether), 1,2,7,8-diepoxyoctane (1,2,7,8-diepoxyoctane) and 1 3-butylene oxide (1,3_butadiene diepoxide), and the group consisting of a mixture. In the technical field of the present invention, the degree of crosslinking of the cross-linked hyaluronic acid and the cross-linking agent concentration of the cross-linking hyaluronic acid required by a person skilled in the art can be selected. In a preferred embodiment of the present invention, cross-linking is preferred. The concentration of the agent is from about 〇.〇5 to about 2 w/v%; more preferably from about 0" to about w5 w/v%; more preferably from about 0.1 to about 1.0 w/v%, most preferably It is from about 〇·6 to about i 〇w/v〇/〇. The technical feature of the present invention resides in that a crosslinking reaction is carried out using a long-term low-temperature reaction temperature condition, and the low reaction temperature according to the present invention means about 1 Torr. 〇 to about 30 ° C; preferably from about 15 ° C to about 30. (:; optimally from about 2 〇. (: to about 30 C ° cross-linking reaction at this low reaction temperature, the formed cross-linked hyaluronic acid does not rapidly deteriorate due to the hydrolysis of the test, The reaction is terminated after the crosslinking agent is consumed to a reasonable content. The low reaction temperature mentioned in the present invention is not limited to a single temperature reaction, and may be a combination of a plurality of low reaction temperatures. "Consumable to a reasonable content" means that the cross-linking product obtained in the final cross-linked product may be a concentration that is subsequently used, for example, 1 ppm or less, 2 ppm or less, 5 ppm or less, 10 ppm or less, 15 ppm or less or 2 The present invention is not limited to 〇ppm, and the consumption of the crosslinking agent according to the present invention is not limited to being consumed by the crosslinking reaction, may be consumed by hydrolysis, or may be consumed by crosslinking and hydrolysis. In a specific embodiment of the invention, the resulting product may be controlled to be unreacted 133837.doc -13 - 201012865 should have a complete crosslinker content of less than 1 ppm, 2 ppm, 2 to 5 ppm, 5 to 10 ppm, 10 to 15 ppm or 15 to 20 ppm. The "crosslinking agent having a free functional group" includes a crosslinking agent and a partial functional group which are not reacted with hyaluronic acid, a metal salt thereof, a derivative thereof or a mixture thereof, and hyaluronic acid, a metal salt thereof , a derivative thereof or a mixture thereof, which reacts with a crosslinker functional group to form a bond between hyaluronic acid, a metal salt thereof, a derivative thereof or a mixture thereof, but a part of the functional group is unreacted and is in a free state. Preferably, the crosslinking reaction is stopped before the degradation of the cross-linked hyaluronic acid, and the term "deterioration" as used in the present invention means the cross-linked hyaluronic acid obtained after the crosslinking reaction because the hydrolysis of the alkali becomes deep. Yellow or even dark brown, making it unusable, or indicating that the synthesized product begins to undergo significant degradation. Usually the time of the crosslinking reaction is related to the temperature of the crosslinking reaction. The higher the temperature, the shorter the time required. Low, the longer the time required; the reaction time is also related to the concentration of the cross-linking agent and the alkali concentration. In summary, the reaction time according to the present invention is more than 48 hours, preferably about 3 to about 28 days, more preferably About 3 to about 1 1 day, preferably from about 3 to about 7 days. According to the present invention, hyaluronic acid, a metal salt thereof, a derivative thereof or a reaction solution containing hyaluronic acid, a metal salt thereof, a derivative thereof or a mixture thereof The concentration of the mixture is preferably from about 2 to about 4 〇 wv%; more preferably from about 10 to about 30 wv/〇, more preferably from about 15 to about 2 〇 wv%. Hyaluronic acid according to the present invention. The molecular weight of the metal salt or its material is theoretically not limited to 'low molecular weight, high molecular weight, or two high and low molecular weight hyaluronic acid, metal salt or derivative thereof. The molecular weight is, for example, 100,000 or less, between 100,000 and 500,000, between 500,000 and 1 million, 133,837, doc, 2010, 12,865, between 10,000 and 1.5 million, between 150,000 and 2 million, and between 2.5 million. Between 3 million. It is preferred to mix a high- and low-molecular-weight hyaluronic acid, a metal salt thereof, a derivative thereof or a mixture thereof for crosslinking reaction, which has the advantages of low reaction solution viscosity and suitable for process amplification. In a preferred embodiment of the present invention, when M-butylene glycol diglycidyl ether 1v/v% is used as a crosslinking agent, a solution containing hyaluronic acid, a metal salt thereof, a derivative thereof or a mixture thereof is contained. When the concentration of the hyaluronic acid, the metal salt thereof, the derivative thereof or a mixture thereof is 2 〇w/v%, and the alkali concentration is 〇_25 1 , the reaction time required is 3 to 28 days. The method according to the present invention is preferably carried out under aseptic conditions, but is not limited to being carried out only under aseptic conditions, or in a general environment, as long as the crosslinking agent of the final product can be lowered to a reasonable range and has good The biocompatibility is sufficient, and the nature of biocompatibility can be tested by cytotoxicity test. In fact, according to the present invention, the cross-linking reaction at a low reaction temperature requires a long anti-sampling time, but since it is in an alkaline environment, it is not easy to have microbial contamination. There is no neutral buffer in the prior art. There is a high risk of contamination of the microorganisms produced by the cleaning of the colloid in the solution. Preferably, the method according to the present invention is preceded by a crosslinking reaction at a low reaction temperature, and further comprises a step of crosslinking reaction at a high reaction temperature, wherein the high reaction temperature is from about 35 ° C to about 6 〇〇c; preferably from about 35C > c to about 50 ° C; more preferably from about 35 ° C to about 4 ° c. In this aspect, the crosslinking reaction is carried out at a high reaction temperature for a period of time, and the hyaluronic acid, the metal salt thereof, the derivative thereof or a mixture thereof is partially subjected to a crosslinking reaction, and the properties thereof have not deteriorated. Or before the obvious degradation occurs, put it on the low side 133837.doc •15· 201012865 The reaction should be continued, and the reaction should be stopped after the cross-linking agent is consumed to a reasonable level. The high reaction temperature as referred to in the present invention is not limited to being reacted at a single temperature, but may be a combination of various high reaction temperatures. In a preferred embodiment of the invention, the crosslinking reaction is carried out for less than about 72 hours when the high reaction temperature is about 35 Torr; preferably from about 4 to about 48 hours, more preferably from about 6 to about 12 hours. . When the high reaction temperature is about 约, the crosslinking reaction is carried out for less than about 48 hours; preferably from about 2 to 24 hours.
時，更佳為自約3至約6小時。當高反應溫度為約机時， 進打交聯反應小於約8小時；較佳為自約Q1至約2小時； 更佳為自約〇.2至約1小時。當高反應溫度為約6〇t時，進 行交聯反應小於約2小時；較佳為自約0.1至約0.5小時；更 佳為自約0.2至約〇.3小時。 較佳地，根據本發明之方法另包含—稀釋步驟。其係將 交聯反應中所得之交聯透明f酸含量，經稀釋為所欲之濃 度及生理上可接受之滲透壓或酸驗值。稀釋之方法例如使 用水、中性溶液、緩衝溶液、鹽類溶液或其混合物加入反 應液中而得。例如調整交聯透明f酸之含量於在5叫化 至60 mg/mL之間，較佳在1〇 mg/mLjL4() 之間，_ 佳在20 mg/mL至30 mg/mL之間，滲透壓為28〇至34 mOsm/kg之間。 較佳地’根據本發明之方法另包含一中和步驟其係將 交聯反應中所需之非中性酸鹼環境，中和為中性環境，較 佳為pH值在6.5至7.5之間，俾刺於私从地 评引於動物體内應用。於本發 明之一具體實施例中，稀釋步驟 π鄉興中和步驟可同時進行。 133837.doc 201012865 較佳地，根據本發明之方法另包含一均質步驟其係將 最後產物碾碎、壓碎而均勻分散成顆粒或溶液者，例如可 使用均質機或其它碾碎或壓碎之機器進行均質。 根據本發明之方法所製得之交聯透明質酸，依其型態可 為溶液、凝膠、溶液與凝膠之混合物或進一步乾燥或冷凍 乾燥為顆粒或固體，固體的形狀可為但不限於海綿狀、片 狀、條狀、球狀或橢圓狀。根據本發明之方法不需經由純 化步驟，即可製得於產物中交聯劑含量低、生物相容性高 且可供動物體應用之交聯透明質酸，以克服習知技術中使 用純化步驟之缺點。 兹以下列實例予以詳細說明本發明’唯其並不意謂本發 明僅侷限於此等實例所揭示之内容。 【實施方式】 實例一：透明質酸鈉濃度=20 w/v% HHA、驗濃度=〇 5 N、 反應溫度=30°C、交聯劑漢度=1 v/v%之l，2,7,8-二環氧 辛烷 取8.9 mL的去離子水加入1 mL之5 N氫氧化鈉水溶液及 O. 1 mL交聯劑1,2,7,8-二環氧辛烷，在磁石攪拌下加入2克 (乾重）平均分子量為135萬之透明質酸鈉（high mQleeuiaf weight hyaluronic acid ’ HHA)，室溫下攪拌 5 分鐘後，置 於30°C恆溫箱中反應如表1所示之反應時間。反應後，所 得反應物中加入79.2 mL之pH為7.0±0.2之0.073 Μ鱗酸鹽緩 衝溶液及0.8 mL之6 Ν的氣化氫水溶液，以使得均質後至 生理上可接受的pH值及滲透壓，再將膠體溶液均質後，即 133837.doc • 17· 201012865 可得交聯透明質酸產物。 均質後之交聯透明質酸產物之具有自由態官能基之交聯 劑含量測定是將產物經透明質酸水解酵素水解，再依據 Nelis和 Sinsheimer所使用的方法（A Sensitive Fluorimetric for the Determination of Aliphatic Epoxides under Physiological Conditions, Anal. Biochem., Vol. 115, pp. 151-157’ 1981)’於吸收波長為3 7〇nrn下進行測定，此測 定方法在具有自由態官能基之交聯劑含量為1至3〇〇 ppm時 檢量線具良好之線性關係；產物的交聯透明質酸含量測定 則先取精秤後的產物約0.1克置於5〇 mL的定量瓶中，加入 3 mL的6 N的硫酸水溶液先行溶解產物後，以去離子水稀 釋溶液至定量瓶刻度’再依據歐洲藥典第四版（2〇〇2版）所 使用的方法進行測定醛醣酸（glucuronic acid)含量後，再換 算得交聯透明質酸含量。不同反應時間下所得的產物其交 聯透明質酸含量和具有自由態官能基之交聯劑含量如表i 所示。 實施例二：透明質酸鈉濃度=20 w/v% HHA、鹼濃度=05 N、反應溫度=3〇。(3、交聯刺濃度=1 v/v%之ι，3_二環氧丁燒 除交聯劑改為1，3-二環氧丁烷外，其餘反應條件及含量 的測定方法同實施例一。不同反應時間下所得的產物其交 聯透明質酸含量和具有自由態官能基之交聯劑含量如表1 所示。 實施例三：透明質酸鈉濃度=2〇 W/V% HHA、鹼濃度=〇25 N、反應溫度=1〇。(：、交聯劑濃度=1 丁二趙一 133837.doc -18 - 201012865 縮水甘油醚 取9.4 mL的去離子水加入0.5 mL之5 N氫氧化鈉水溶液 及0· 1 mL交聯劑1，4-丁二醇二縮水甘油韆，在磁石授拌下 加入2克（乾重）平均分子量為135萬之透明質酸納（hhA)， 室溫下攪拌5分鐘後’置於1 〇°C恆溫箱中反應。反應後， 所得反應物中加入79.6 mL之pH為7.0土0.2之0.1 Μ填酸鹽緩 溶液及0.4 mL之6 Ν的氣化氫水溶液，以使得均質後至生 理上可接受的pH值及滲透壓，再將膠體溶液均質後，即可 ® 得到交聯透明質酸產物。產物之交聯透明質酸含量和具有 自由態官能基之交聯劑含量的測定方法同實施例一。不同 反應時間下所得的產物其交聯透明質酸含量和具有自由態 官能基之交聯劑含量如表1所示。 實施例四：透明質酸鈉濃度=2〇 w/v% HHA、鹼濃度=0.25 N、反應溫度=20°C、交聯刺濃度=1 v/v%之1，4 丁二醇二縮 水甘油醚 除反應溫度改為20°C外’其餘反應條件與測定方法同實 Ο 施例三。不同反應時間下所得的產物其交聯透明質酸含量 * 和具有自由態官能基之交聯劑含量如表1所示。 實施例五：透明質酸鈉濃度=20 w/v% HHA、鹼濃度=0.25 N、反應溫度=30°C、交聯劑激度=1 v/v%之ι,4丁二酵二縮 水甘油鍵 除反應溫度改為30°C外’其餘反應條件與測定方法同實 施例三。不同反應時間下所得的產物其交聯透明質酸含量 和具有自由態官能基之交聯劑含量如表1所示。 133837.doc • 19· 201012865 實施例六：透明質酸鈉濃度=20 w/v〇/〇 HHA、驗滚度=〇.2 N、 反應溫度=30°C、交聯刺滚度=1 v/v%之1，4- 丁二酵二 縮水甘油醚 取9.5 mL的去離子水加入0.4 mL之5 N氫氧化鈉水溶液 及0.1 mL交聯劑1,4-丁二醇二縮水甘油謎，在磁石攪拌下 加入平均分子量為135萬之透明質酸鈉（Ηηα)2·0克（乾 重），攪拌5分鐘後置於可控制溫度的恆溫箱中以3〇。〇反應 一定時間後（反應時間如表1所示）’所得反應物中加入 O. 32 mL·之ό Ν氣化氫水溶液及79.68 mL pH為7.0 士0.2之0.10 Μ填酸鹽緩溶液，以使得均質後至生理上可接受的pH值及 滲透壓’再將膠體溶液均質後，即可得到交聯透明質酸產 物。產物的交聯透明質酸含量和具有自由態官能基之交聯 劑含量的測定方法同實施例一。不同反應時間下所得的產 物其交聯透明質酸含量和具有自由態官能基之交聯劑含量 如表1所示。 由表1之結果可得知當於低反應溫度進行交聯反應超過2 天時’其產品交聯劑之含量低，可應用於生物體内。 133837.doc 201012865More preferably, it is from about 3 to about 6 hours. When the high reaction temperature is about 约约, the crosslinking reaction is less than about 8 hours; preferably from about Q1 to about 2 hours; more preferably from about 〇2 to about 1 hour. When the high reaction temperature is about 6 Torr, the crosslinking reaction is carried out for less than about 2 hours; preferably from about 0.1 to about 0.5 hours; more preferably from about 0.2 to about 0.3 hours. Preferably, the method according to the invention further comprises a dilution step. It is a cross-linked transparent f-acid content obtained in a cross-linking reaction which is diluted to a desired concentration and a physiologically acceptable osmotic pressure or acid value. The method of dilution is, for example, obtained by adding water, a neutral solution, a buffer solution, a salt solution or a mixture thereof to the reaction solution. For example, adjusting the content of the cross-linked transparent f acid is between 5 and 60 mg/mL, preferably between 1 〇 mg/mLjL4(), and preferably between 20 mg/mL and 30 mg/mL. The osmotic pressure is between 28 〇 and 34 mOsm/kg. Preferably, the method according to the present invention further comprises a neutralization step which neutralizes the non-neutral acid-base environment required in the crosslinking reaction, neutralizing to a neutral environment, preferably having a pH between 6.5 and 7.5. The spurs are privately evaluated in animal applications. In one embodiment of the invention, the dilution step π xiangxing neutralization step can be carried out simultaneously. 133837.doc 201012865 Preferably, the method according to the invention further comprises a homogenization step of crushing, crushing and uniformly dispersing the final product into granules or solutions, for example using a homogenizer or other crushing or crushing. The machine is homogenized. The crosslinked hyaluronic acid prepared according to the method of the present invention may be a solution, a gel, a mixture of a solution and a gel or further dried or freeze-dried into particles or solids according to the form thereof, and the shape of the solid may be but not Limited to sponge, sheet, strip, sphere or ellipse. According to the method of the present invention, cross-linking hyaluronic acid having low cross-linking agent content, high biocompatibility and suitable for animal application can be obtained without using a purification step, thereby overcoming the purification in the prior art. The disadvantages of the steps. The present invention is described in detail by the following examples, which are not intended to be construed as limiting. [Examples] Example 1: Sodium hyaluronate concentration = 20 w / v% HHA, test concentration = 〇 5 N, reaction temperature = 30 ° C, cross-linker Han = 1 v / v% l, 2, 7,8-diepoxyoctane 8.9 mL of deionized water was added to 1 mL of 5 N aqueous sodium hydroxide solution and 0.1 mL of crosslinker 1,2,7,8-diepoxyoctane in magnet 2 g (dry weight) of high mQleeuiaf weight hyaluronic acid 'HHA' was added under stirring, stirred at room temperature for 5 minutes, and placed in a 30 ° C incubator as shown in Table 1. Show the reaction time. After the reaction, 79.2 mL of a 0.073 cerium sulphate buffer solution having a pH of 7.0 ± 0.2 and 0.8 mL of a 6 Ν aqueous solution of hydrogen hydride were added to the resulting reaction to achieve a physiologically acceptable pH and penetration after homogenization. After pressing and then homogenizing the colloidal solution, 133837.doc • 17· 201012865 can obtain cross-linked hyaluronic acid product. The content of the crosslinker having a free functional group of the crosslinked hyaluronic acid product after homogenization is determined by hydrolyzing the product via hyaluronic acid hydrolyzing enzyme, and then according to the method used by Nelis and Sinsheimer (A Sensitive Fluorimetric for the Determination of Aliphatic Epoxides under Physiological Conditions, Anal. Biochem., Vol. 115, pp. 151-157' 1981) is measured at an absorption wavelength of 3 7 〇 nrn. The determination method has a crosslinker content of a free functional group. The calibration line has a good linear relationship at 1 to 3 〇〇ppm; the cross-linked hyaluronic acid content of the product is determined by taking about 0.1 gram of the product after the precision scale, placing it in a 5 〇mL quantitative bottle, and adding 3 mL of 6 After the N-sulfuric acid aqueous solution is dissolved first, the solution is diluted with deionized water to the quantitative bottle scale, and then the content of glucuronic acid is determined according to the method used in the fourth edition of the European Pharmacopoeia (2〇〇2 version). The cross-linked hyaluronic acid content is further converted. The crosslinked hyaluronic acid content of the product obtained at different reaction times and the content of the crosslinker having a free functional group are shown in Table i. Example 2: Sodium hyaluronate concentration = 20 w/v% HHA, alkali concentration = 0 5 N, reaction temperature = 3 Torr. (3, cross-linking concentration = 1 v / v% of ι, 3_ diepoxy butyl burning cross-linking agent changed to 1,3-dibutylene butane, the rest of the reaction conditions and content determination method is the same as the implementation Example 1. The crosslinked hyaluronic acid content of the product obtained at different reaction times and the content of the crosslinker having free functional groups are shown in Table 1. Example 3: Sodium hyaluronate concentration = 2 〇 W / V% HHA, alkali concentration = 〇25 N, reaction temperature = 1 〇. (:, crosslinker concentration = 1 Ding Er Zhao 133837.doc -18 - 201012865 Glycidyl ether Take 9.4 mL of deionized water and add 0.5 mL of 5 N sodium hydroxide aqueous solution and 0.1 mL of cross-linking agent 1,4-butanediol diglycidyl thousand, 2 g (dry weight) of hyaluronic acid sodium (hhA) with an average molecular weight of 1.35 million was added under magnetization. After stirring at room temperature for 5 minutes, the reaction was carried out in a 1 ° C incubator. After the reaction, 79.6 mL of a pH 7.0 soil 0.2 0.1 Μ sulphonate solution and 0.4 mL of 6 Ν were added to the obtained reaction. Aqueous hydrogenated aqueous solution to homogenize to a physiologically acceptable pH and osmotic pressure, and then homogenize the colloidal solution to obtain a crosslinked hyaluronic acid product The method for determining the cross-linked hyaluronic acid content of the product and the content of the cross-linking agent having a free-state functional group is the same as in the first embodiment. The cross-linked hyaluronic acid content of the product obtained at different reaction times and cross-linking with free-state functional groups The content of the agent is shown in Table 1. Example 4: Sodium hyaluronate concentration = 2 〇 w / v% HHA, alkali concentration = 0.25 N, reaction temperature = 20 ° C, cross-linking concentration = 1 v / v% 1,4 butanediol diglycidyl ether except that the reaction temperature was changed to 20 ° C. The rest of the reaction conditions and the determination method are the same as in the third embodiment. The product obtained at different reaction times has a crosslinked hyaluronic acid content* and has The content of the crosslinker of the free functional group is shown in Table 1. Example 5: Sodium hyaluronate concentration = 20 w/v% HHA, alkali concentration = 0.25 N, reaction temperature = 30 ° C, crosslinking agent excitation =1 v / v% of ι, 4 butyl diacetate diglycidyl bond except that the reaction temperature was changed to 30 ° C 'the rest of the reaction conditions and determination methods are the same as in the third embodiment. The product obtained under different reaction times is cross-linked transparent The acid content and the crosslinker content with free functional groups are shown in Table 1. 133837.doc • 19· 201012865 Example 6: Sodium hyaluronate concentration = 20 w / v 〇 / 〇 HHA, rolling degree = 〇. 2 N, reaction temperature = 30 ° C, cross-linking licking degree = 1 v / v% of 1, 4 - Dinger Yeast Diglycidyl Ether 9.5 mL of deionized water is added to 0.4 mL of 5 N aqueous sodium hydroxide solution and 0.1 mL of crosslinker 1,4-butanediol diglycidrene. The average molecular weight is added under magnet stirring. It was 1.35 million sodium hyaluronate (Ηηα) 2·0 g (dry weight), stirred for 5 minutes and placed in a temperature-controlled incubator at 3 Torr. After the reaction for a certain period of time (reaction time is shown in Table 1), the obtained reaction solution was added with O. 32 mL·ό Ν Ν hydrogenated aqueous hydrogen solution and 79.68 mL of a 0.10 Μ sulphate solution with a pH of 7.0 ± 0.2. After homogenization to a physiologically acceptable pH and osmotic pressure, the colloidal solution is homogenized to obtain a crosslinked hyaluronic acid product. The method for determining the crosslinked hyaluronic acid content of the product and the crosslinker content having a free functional group is the same as in the first embodiment. The crosslinked hyaluronic acid content and the crosslinker content having free functional groups of the obtained product at different reaction times are shown in Table 1. From the results of Table 1, it can be seen that when the crosslinking reaction is carried out at a low reaction temperature for more than 2 days, the content of the cross-linking agent of the product is low, and it can be applied to a living body. 133837.doc 201012865
表1 : 實施例 反應溫度 ΓΟ 反應時間 產物之交聯透明 質.含量(w/v%) 具有自由態官能基 之交聯劑含量(ppm) 一 30 1天 1.85 64.64 2天 1.91 5.68 3天 1.89 3.15 4天 1.86 <Γ 二 30 1天 1.94 15.95 2天 1.89 4.25 3天 1.96 <Γ 三 10 14天 2.09 42.67 21天 2.06 12.16 28天 1.95 <Γ 四 20 1天 2.02 441.08* 3天 1.95 157.04 5天 2.05 47.32 7天 1.98 9.26 9天 1.98 1.71 11天 1.98 <Γ 五 30 1天 1.91 167.91 2天 1.97 30.25 3天 1.94 9.87 4天 1.83 1.89 5天 1.95 <Γ 六 30 1天 1.95 247.69 2天 2.00 79.92 3天 2.03 24.38 4天 1.82 9.05 5天 1.95 4.57 6天 1.90 2.51 7天 1.98 <1* 不在檢量線範圍（1〜300 ppm) 133837.doc -21 - 201012865 實施例七：透明質酸鈉濃度=10 w/v% HHA、鹼濃度=〇.os N、高反應溫度=50°C、低反應溫度=3〇°C、交聯劑濃度 v/v%之1,4-丁二酵二縮水甘油醚 取9.8 mL的去離子水加入0.1 mL之5 N氫氧化納水溶液 及0.1 mL交聯劑1，4- 丁二醇二縮水甘油醚，在磁石攪拌下 加入平均分子量為135萬之透明質酸鈉（HHA)，攪拌5分鐘 後置於可控制溫度的恆溫箱中以50°C反應7小時後，再以 30°C下反應一定時間後（反應時間如表2所示），所得反應 ❹ 物中加入去離子水9.87 mL、0.13 mL之6 N氣化氫水溶液 及20 mL pH為7.0±0.2之0_15 Μ磷酸鹽缓溶液，以使得均質 後至生理上可接受的pH值及滲透壓，再將膠體溶液均質 後’即可得到交聯透明質酸產物。產物的交聯透明質酸含 量和具有自由態官能基之交聯劑含量的測定方法同實施例 一。不同反應時間下所得的產物其交聯透明質酸含量和具 有自由態官能基之交聯劑含量如表2所示。 Ο 實施例八：透明質酸鈉濃度=20 w/v% HHA、鹼濃度=〇.5 N、高反應溫度=40°C、低反應溫度=25°C、交聯劑濃度=1 v/v%之1,4-丁二酵二縮水甘油醚 - 取8.9 mL的去離子水加入1 mL之5 N氫氧化納水溶液及 0.1 mL交聯劑ι，4-丁二醇二縮水甘油醚，在磁石攪拌下加 入平均分子量為135萬之透明質酸鈉（HHA)，攪拌5分鐘後 置於可控制溫度的恆溫箱中以4(TC反應3小時後，再以 25°C下反應一定時間後（反應時間如表2所示），所得反應 物中加入0.8 mL之6 N氣化氫水溶液及79.2 mL pH為 133837.doc •22- 201012865 7.0±0.2之0.073 Μ填酸鹽緩溶液，以使得均質後至生理上 可接受的pH值及滲透壓，再將膠體溶液均質後即可得到 • 交聯透明質酸產物。產物的交聯透明質酸含量和具有自由 態官能基之交聯劑殘留量的測定方法同實施例一。不同反 ' 應時間下所得的產物其交聯透明質酸含量和具有自由態官 能基之交聯劑含量如表2所示。 實施例九：透明質酸納激度=20 w/v% HHA、餘漢度=〇 5 N、高反應溫度=40°C、低反應溫度=25°C、交聯劑渡度=1 ® v/v%之1,2,7,8-二環氧辛烷 除交聯劑改為1，2,7,8-二環氧辛烷，其餘反應條件及含 量的測定方法同實施例八。不同反應時間下所得的產物其 交聯透明質酸含量和具有自由態官能基之交聯劑殘留量如 表2所示。 實施例十：透明質睃納瀵度=20 w/v% HA (10 w/v% HHA + 10 w/v%LHA)、驗泼度=0.25 N、高反應溫度=40°C、低 反應溫度=l〇°C、交聯劑濃度=〇·6 v/v%之1，4-丁二酵二縮 水甘油鍵 • 取9.44 mL的去離子水加入0.5 mL之5 N氫氧化鈉水溶液 . 及0.06 mL交聯劑1,4-丁二醇二縮水甘油醚，在磁石攪拌下Table 1: Example reaction temperature ΓΟ Reaction time product crosslinked hyaluronic acid. Content (w/v%) Crosslinker content with free functional groups (ppm) One 30 1 day 1.85 64.64 2 days 1.91 5.68 3 days 1.89 3.15 4 days 1.86 < Γ 2 30 1 day 1.94 15.95 2 days 1.89 4.25 3 days 1.96 < Γ 3 10 14 days 2.09 42.67 21 days 2.06 12.16 28 days 1.95 < Γ 4 20 1 day 2.02 441.08* 3 days 1.95 157.04 5 days 2.05 47.32 7 days 1.98 9.26 9 days 1.98 1.71 11 days 1.98 <Γ 5 30 1 day 1.91 167.91 2 days 1.97 30.25 3 days 1.94 9.87 4 days 1.83 1.89 5 days 1.95 <Γ 6 30 1 day 1.95 247.69 2 days 2.00 79.92 3 days 2.03 24.38 4 days 1.82 9.05 5 days 1.95 4.57 6 days 1.90 2.51 7 days 1.98 <1* Not in the calibration line range (1~300 ppm) 133837.doc -21 - 201012865 Example 7: Hyaluronic acid Sodium concentration=10 w/v% HHA, alkali concentration=〇.os N, high reaction temperature=50°C, low reaction temperature=3〇°C, crosslinker concentration v/v% of 1,4-butane Fermented diglycidyl ether, 9.8 mL of deionized water, 0.1 mL of 5 N aqueous sodium hydroxide solution and 0.1 mL of cross-linking agent 1,4-butane Diglycidyl ether, sodium hyaluronate (HHA) with an average molecular weight of 1.35 million was added under stirring with a magnet. After stirring for 5 minutes, it was placed in a temperature-controlled incubator at 50 ° C for 7 hours and then at 30 °. After a certain reaction time in C (reaction time is shown in Table 2), 9.87 mL of deionized water, 0.13 mL of 6 N aqueous hydrogenation solution and 20 mL of 0_15 yttrium phosphate having a pH of 7.0 ± 0.2 were added to the obtained reaction product. The solution is soaked so that after homogenization to a physiologically acceptable pH and osmotic pressure, and then the colloidal solution is homogenized, a crosslinked hyaluronic acid product can be obtained. The method for determining the crosslinked hyaluronic acid content of the product and the content of the crosslinking agent having a free functional group is the same as in the first embodiment. The crosslinked hyaluronic acid content of the product obtained at different reaction times and the content of the crosslinking agent having a free functional group are shown in Table 2.实施 Example 8: Sodium hyaluronate concentration = 20 w/v% HHA, alkali concentration = 〇.5 N, high reaction temperature = 40 ° C, low reaction temperature = 25 ° C, crosslinker concentration = 1 v / V% of 1,4-butanedihydrate diglycidyl ether - 8.9 mL of deionized water is added to 1 mL of 5 N aqueous sodium hydroxide solution and 0.1 mL of crosslinker ι,4-butanediol diglycidyl ether. Sodium hyaluronate (HHA) with an average molecular weight of 1.35 million was added under stirring with a magnet. After stirring for 5 minutes, it was placed in a temperature-controlled incubator at 4 (TC reaction for 3 hours, and then reacted at 25 ° C for a certain period of time). After the reaction time is as shown in Table 2, 0.8 mL of a 6 N aqueous solution of hydrogen peroxide and 79.2 mL of a 0.073 Μ sulphate solution having a pH of 133837.doc •22-201012865 7.0±0.2 were added to the obtained reactant. After homogenization to a physiologically acceptable pH and osmotic pressure, and then homogenizing the colloidal solution, a cross-linked hyaluronic acid product can be obtained. The cross-linked hyaluronic acid content of the product and the cross-linking agent having a free-state functional group are obtained. The residual amount is determined in the same manner as in the first embodiment. The product obtained under different time is cross-linked hyaluronic acid content and has a free state. The content of the crosslinker of the energy base is shown in Table 2. Example 9: sodium hyaluronic acid = 20 w/v% HHA, Yuhan = 〇5 N, high reaction temperature = 40 ° C, low reaction temperature =25 ° C, cross-linking agent = 1 ® v / v% of 1,2,7,8-diepoxyoctane, except cross-linking agent changed to 1,2,7,8-diepoxyoctane The remaining reaction conditions and content were determined in the same manner as in Example 8. The crosslinked hyaluronic acid content of the product obtained at different reaction times and the residual amount of the crosslinking agent having a free functional group are shown in Table 2. Example 10: Hyalin cancerness = 20 w/v% HA (10 w/v% HHA + 10 w/v% LHA), test flu = 0.25 N, high reaction temperature = 40 ° C, low reaction temperature = l 〇 °C, crosslinker concentration = 〇·6 v/v% of 1,4-butanedihydrate diglycidyl bond • Take 9.44 mL of deionized water and add 0.5 mL of 5 N aqueous sodium hydroxide solution and 0.06 mL of Co-agent 1,4-butanediol diglycidyl ether under magnet stirring
加入平均分子量為44萬之透明質酸納（low molecular weight hyaluronic acid，LHA)及平均分子量為135萬之透 明質酸鈉（HHA)各1克（乾重），攪拌5分鐘後置於可控制溫 度的恆溫箱中以40°C反應4小時後，再以1 〇°C下反應一定 時間後（反應時間如表2所示）’所得反應物中加入〇.4 mL 133837.doc -23- 201012865 之6 N氣化氫水溶液及79 6 mL PH為7·0 士 0·2之0.10 Μ磷酸 鹽緩溶液’以使得均質後至生理上可接受的pH值及滲透 壓’再將膠體溶液均質後，即可得到交聯透明質酸產物。 產物的父聯透明質酸含量和具有自由態官能基之交聯劑含 量的測定方法同實例一。不同反應時間下所得的產物其交 聯透明質酸含量和具有自由態官能基之交聯劑含量如表2 所示。 實施例十一：透明質酸鈉濃度=3〇 w/v% HA (15 w/v% HHA+15 w/v%LHA)、鹼濃度=0.25 N、高反應溫度 =40°C、低反應溫度=25°C、交聯劑濃度=〇·6ν/ν%之M-丁 二酵二縮水甘油鰱 取9.44 mL的去離子水加入〇.5 mL之5 Ν氫氧化納水溶液 及0.06 mL交聯劑1，4·丁二醇二縮水甘油醚，在磁石攪拌下 加入平均分子量為44萬之透明質酸鈉（LHA)及平均分子量 為135萬之透明質酸鈉（HHA)各1.5克（乾重），攪拌5分鐘後 置於可控制溫度的恆溫箱中以40°C反應4小時後，再以 25C下反應一疋時間後（反應時間如表2所示），所得反應 物中加入0.4 mL之6 N氣化氫水溶液及79.6 mL pH為 7_0±0.2之0·10 Μ磷酸鹽緩溶液，以使得均質後至生理上可 接受的pH值及滲透壓，再將膝體溶液均質後，即可得到交 聯透明質酸產物。產物的交聯透明質酸含量和具有自由態 官能基之交聯劑含量的測定方法同實施例一。不同反應時 間下所得的產物其交聯透明質酸含量和具有自由態官能基 之交聯劑殘留量如表2所示。 133837.doc -24· 201012865 實施例十二：透明質酸鈉濃度=20 w/v%LHA、鹼濃度 =0.25 N、高反應溫度=40°C、低反應溫度=25°C、交聯劑 濃度=0.6 v/v%之1，4-丁二酵二縮水甘油醚 除透明質酸改為低分子量透明質酸鈉（LHA)2.0克（乾重） 外’其餘反應條件及含量的測定方法同實施例十一。不同 反應時間下所得的產物其交聯透明質酸含量和具有自由態 官能基之交聯劑殘留量如表2所示。 實施例十三：透明質酸納波度=20 w/v% HA(10 w/v% HHA+10 w/v%LHA)、驗濃度=0.25 N、高反應溫度 =40°C、低反應溫度=25°C、交聯瘌濃度=0.1 Wv%之1，4-丁 二酵二縮水甘油醚 取9.49 mL的去離子水加入0.5 mL之5 N氫氧化鈉水溶液 及0.01 mL交聯劑1,4-丁二醇二縮水甘油醚，在磁石授拌下 加入平均分子量為44萬之透明質酸鈉（LHA)及平均分子量 為135萬之透明質酸鈉（HHA)各1.0克（乾重），攪拌5分鐘後 置於可控制溫度的恆溫箱中以40。(：反應4小時後，再以 2 5 C下反應一疋時間後（反應時間如表2所示），所得反應 物中加入0.4 mL之6 N氣化氫水溶液及79 6 mL pH為 7.0±0.2之0.10 Μ磷酸鹽緩溶液，以使得均質後至生理上可 接受的pH值及滲透壓，再將溶液均質後，即可得到交聯透 明質酸產物。產物的交聯透明質酸含量和具有自由態官能 基之交聯劑含量的測定方法同實例一。不同反應時間下所 得的產物其交聯透明質酸含量和具有自由態官能基之交聯 劑含量如表2所示。 133*37.doc -25- 201012865 實施例十四：透明質酸鈉濃度=20 w/v%LHA、鹼濃度=0.2 N、高反應溫度=40°C、低反應溫度=30°C、交聯劑濃度=1 v/v%之1,4-丁二酵二縮水甘油醚Add 1 gram (dry weight) of dry molecular weight hyaluronic acid (LHA) with an average molecular weight of 440,000 and sodium hyaluronate (HHA) with an average molecular weight of 1.35 million, and place it for 5 minutes after stirring. After reacting at 40 ° C for 4 hours in a temperature incubator, and then reacting at 1 ° C for a certain period of time (reaction time is shown in Table 2), the obtained reaction was added with 〇.4 mL 133837.doc -23- 201012865 6 N hydrogenated aqueous hydrogen solution and 79 6 mL PH is 7. 0 0 0·2 0.10 Μ phosphate slow solution 'to make homogenized to physiologically acceptable pH and osmotic pressure' and then homogenize the colloidal solution After that, a crosslinked hyaluronic acid product can be obtained. The method for determining the content of the parent hyaluronic acid of the product and the content of the crosslinking agent having a free functional group is the same as in the first example. The crosslinked hyaluronic acid content of the product obtained at different reaction times and the crosslinker content having a free functional group are shown in Table 2. Example 11: Sodium hyaluronate concentration = 3 〇 w / v% HA (15 w / v% HHA + 15 w / v% LHA), alkali concentration = 0.25 N, high reaction temperature = 40 ° C, low reaction Temperature = 25 ° C, crosslinker concentration = 〇 · 6 ν / ν% of M - butyl diglycol diglycolate 9.44 mL of deionized water was added to 〇 5 mL of 5 Ν aqueous sodium hydroxide solution and 0.06 mL The 1,4·butanediol diglycidyl ether was added, and 1.5 kg of hyaluronic acid (LHA) having an average molecular weight of 440,000 and sodium hyaluronate (HHA) having an average molecular weight of 1.35 million were added under stirring with a magnet ( Dry weight), stirred for 5 minutes, placed in a temperature-controlled incubator at 40 ° C for 4 hours, and then reacted at 25 ° C for a while (reaction time as shown in Table 2), the reaction was added 0.4 6 6 N hydrogenated aqueous solution of hydrochloric acid and 79.6 mL of 0. 10 Μ phosphate slow solution with a pH of 7_0±0.2, so that after homogenization to physiologically acceptable pH and osmotic pressure, the knee solution is homogenized. A crosslinked hyaluronic acid product is obtained. The method for determining the crosslinked hyaluronic acid content of the product and the content of the crosslinking agent having a free functional group is the same as in the first embodiment. The crosslinked hyaluronic acid content of the product obtained at different reaction times and the residual amount of the crosslinking agent having a free functional group are shown in Table 2. 133837.doc -24· 201012865 Example 12: Sodium hyaluronate concentration = 20 w/v% LHA, alkali concentration = 0.25 N, high reaction temperature = 40 ° C, low reaction temperature = 25 ° C, crosslinking agent Concentration = 0.6 v / v% of 1,4-butanedihydrate diglycidyl ether in addition to hyaluronic acid changed to low molecular weight sodium hyaluronate (LHA) 2.0 g (dry weight) outside 'other reaction conditions and content determination method Same as Embodiment 11. The crosslinked hyaluronic acid content of the obtained product at different reaction times and the residual amount of the crosslinking agent having a free functional group are shown in Table 2. Example 13: Hyaluronic acid nanopod = 20 w / v% HA (10 w / v% HHA + 10 w / v% LHA), test concentration = 0.25 N, high reaction temperature = 40 ° C, low reaction Temperature = 25 ° C, cross-linking enthalpy concentration = 0.1 Wv% of 1,4-butanedihydrate diglycidyl ether Take 9.49 mL of deionized water, add 0.5 mL of 5 N aqueous sodium hydroxide solution and 0.01 mL of crosslinker 1 , 4-butanediol diglycidyl ether, and added 1.0 gram of sodium hyaluronate (LHA) having an average molecular weight of 440,000 and sodium hyaluronate (HHA) having an average molecular weight of 1.35 million (magnetic weight) ), after stirring for 5 minutes, placed in an oven capable of controlling temperature to 40. (: After reacting for 4 hours, and then reacting at 25 C for a while (reaction time is shown in Table 2), 0.4 mL of 6 N aqueous hydrogenation solution and 79 6 mL of pH 7.0±0.2 were added to the obtained reactant. 0.10 Μ phosphate slow solution, so that after homogenization to physiologically acceptable pH and osmotic pressure, and then homogenizing the solution, the cross-linked hyaluronic acid product can be obtained. The cross-linked hyaluronic acid content of the product has The method for determining the content of the cross-linking agent of the free-state functional group is the same as in the first example. The cross-linked hyaluronic acid content of the product obtained at different reaction times and the cross-linking agent content having a free-state functional group are shown in Table 2. 133*37 .doc -25- 201012865 Example 14: Sodium hyaluronate concentration = 20 w/v% LHA, alkali concentration = 0.2 N, high reaction temperature = 40 ° C, low reaction temperature = 30 ° C, crosslinker concentration =1 v/v% of 1,4-butanedihydrate diglycidyl ether
取9.5 mL的去離子水加入0.4 mL之5 N氫氧化鈉水溶液 及0.1 mL交聯劑1,4-丁二醇二縮水甘油醚，在磁石攪拌下 加入平均分子量為44萬之透明質酸鈉（LHA)2_0克（乾重）， 攪拌5分鐘後置於可控制溫度的恆溫箱中以40°C反應4小時 後，再以30°C下反應一定時間後（反應時間如表2所示）， © 所得反應物中加入0.32 mL之6 N氣化氫水溶液及79.68 mL pH為7·0±0.2之0.10 Μ磷酸鹽緩溶液，以使得均質後至生理 上可接受的pH值及渗透壓，再將膠體溶液均質後，即可得 到交聯透明質酸產物。產物的交聯透明質酸含量和具有自 由態官能基之交聯劑含量的測定方法同實施例一。不同反 應時間下所得的產物其交聯透明質酸含量和具有自由態官 能基之交聯劑含量如表2所示。 ⑩ 由表2之結果可得知當於高反應溫度反應一段時間後繼 續進行低反應溫度交聯反應超過2天時，其產品具有自由 • 態官能基之交聯劑之含量低，可應用於生物體内。 133837.doc • 26- 201012865 表2 實施例 南反應溫度 低反應溫度 產物之交聯透明 具有自由態官能基 °c 時間 °C 時間 質政含量(w/v%) 之交聯劑含量(ppm) 1天 2.11 425.01* 3天 2.23 346.10* 5天 2.25 189.35 9天 1.82 145.49 七 50 7小時 30 11天 2.15 64.34 13天 2.10 54.45 15天 2.07 27.14 17天 2.09 16.41 24天 2.04 15.50 28天 2.14 11.81 0天 1.82 313.53* 八 40 3小時 25 1天 1.92 24.54 2天 1.83 4.38 3天 1.96 <1* 0天 1.86 365.13* 1天 1.81 111.77 九 40 3小時 25 2天 1.88 23.37 3天 2.04 5.90 4天 1.91 <1* 0天 1.82 297.81 4- 40 4小時 10 14天 1.87 7.67 丁 18天 2.01 2.97 23天 1.89 <1 0天 2.04 218.05 1天 2.05 95.08 十一 40 4小時 25 2天 1.79 37.50 3天 2.07 15.96 4天 1.93 8.66 5天 1.90 <1 0天 1.98 331.94* 1天 1.86 123.51 -1-- 40 4小時 25 2天 1.99 54.89 丁'— 3天 1.81 21.44 4天 1.77 10.58 5天 1.87 <1* 0天 1.91 46.27 十三 40 4小時 25 1天 1.91 19.62 2天 1.94 5.37 3天 1.79 <1 1天 1.86 162.98 2天 1.87 50.66 十四 40 4小時 30 3天 1.97 16.09 4天 1.81 5.42 5天 1.80 1.32 6天 1.78 <1* * :不在檢量線範圍（1〜300 ppm) 133837.doc •27· 201012865 上述實施例僅為說明本發明之原理及其功效，而非限制 本發明。因此，習於此技術之人士對上述實施例所做之修 改及變化仍不違背本發明之精神。本發明之權利範圍應如 後述之申請專利範圍所列。Add 9.5 mL of deionized water to 0.4 mL of 5 N aqueous sodium hydroxide solution and 0.1 mL of crosslinker 1,4-butanediol diglycidyl ether, and add sodium hyaluronate with an average molecular weight of 440,000 under stirring with a magnet. (LHA) 2_0g (dry weight), stirred for 5 minutes, placed in a temperature-controlled incubator at 40 ° C for 4 hours, and then reacted at 30 ° C for a certain period of time (reaction time as shown in Table 2 ), © 0.32 mL of 6 N aqueous hydrogenation solution and 79.68 mL of 0.10 Μ phosphate slow solution at pH 7.00 ± 0.2 to achieve a physiologically acceptable pH and osmotic pressure. After homogenizing the colloidal solution, a crosslinked hyaluronic acid product can be obtained. The method for determining the crosslinked hyaluronic acid content of the product and the content of the crosslinking agent having a free functional group is the same as in the first embodiment. The crosslinked hyaluronic acid content of the product obtained at different reaction times and the crosslinker content having a free state functional group are shown in Table 2. 10 From the results of Table 2, it can be seen that when the reaction temperature is continued for a period of time after the reaction at a high reaction temperature for a period of more than 2 days, the product has a low content of a free functional group crosslinking agent and can be applied. In vivo. 133837.doc • 26- 201012865 Table 2 Example South Reaction Temperature Low Reaction Temperature Product Crosslinking Transparent with Free State Functional Group °c Time °C Time Quality Content (w/v%) Crosslinker Content (ppm) 1 day 2.11 425.01* 3 days 2.23 346.10* 5 days 2.25 189.35 9 days 1.82 145.49 7 50 7 hours 30 11 days 2.15 64.34 13 days 2.10 54.45 15 days 2.07 27.14 17 days 2.09 16.41 24 days 2.04 15.50 28 days 2.14 11.81 0 days 1.82 313.53* 八40 3 hours 25 1 day 1.92 24.54 2 days 1.83 4.38 3 days 1.96 <1* 0 days 1.86 365.13* 1 day 1.81 111.77 9 40 3 hours 25 2 days 1.88 23.37 3 days 2.04 5.90 4 days 1.91 <1 * 0 days 1.82 297.81 4- 40 4 hours 10 14 days 1.87 7.67 Ding 18 days 2.01 2.97 23 days 1.89 <1 0 days 2.04 218.05 1 day 2.05 95.08 11 40 4 hours 25 2 days 1.79 37.50 3 days 2.07 15.96 4 days 1.93 8.66 5 days 1.90 <1 0 days 1.98 331.94* 1 day 1.86 123.51 -1-- 40 4 hours 25 2 days 1.99 54.89 Ding '— 3 days 1.81 21.44 4 days 1.77 10 .58 5 days 1.87 <1* 0 days 1.91 46.27 13 40 4 hours 25 1 day 1.91 19.62 2 days 1.94 5.37 3 days 1.79 <1 1 day 1.86 162.98 2 days 1.87 50.66 1440 4 hours 30 3 days 1.97 16.09 4 days 1.81 5.42 5 days 1.80 1.32 6 days 1.78 <1* * : not in the calibration line range (1 to 300 ppm) 133837.doc • 27· 201012865 The above embodiments are merely illustrative of the principles and effects of the present invention. It is not intended to limit the invention. Therefore, modifications and variations of the embodiments described above will be made without departing from the spirit of the invention. The scope of the invention should be as set forth in the appended claims.
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