CAJ | 학술논문

目的：：以抗HER2抗体为模型，研究抗体在糖基工程酵母菌中的表达及工程菌发酵技术。方法：首先通过摇瓶试验分析诱导用甲醇浓度对抗体表达的影响，并用高表达HER2的SK-BR-3细胞分析抗HER2抗体的抗原结合活性。以此为基础，在5 L发酵罐中研究甲醇-山梨醇混合碳源流加诱导对抗HER2抗体表达水平的影响；收集发酵培养液，采用阳离子交换层析对目标产物进行纯化；利用SDS-PAGE、Western印迹、Lowry法对抗体的相对分子质量、浓度等进行分析。结果：摇瓶试验结果表明，甲醇浓度为0.5%时抗体表达量最高，且糖基工程毕赤酵母菌表达的抗HER2抗体具有与SK-BR-3细胞抗原结合的活性；在5 L发酵罐中，利用甲醇和山梨醇混合诱导方式发酵表达抗体，其表达量可提高至0.6 g/L，比摇瓶诱导表达的抗体产量提高了近10倍；非还原SDS-PAGE及Western印迹表明抗体相对分子质量为1.5×105，与商业化抗体Herceptin的大小一致；经过一步阳离子交换层析纯化，纯化后抗体浓度为0.365 g/L。结论：采用甲醇-山梨醇混合碳源诱导方式在5 L发酵罐中进行发酵表达，能够提高抗HER2抗体在糖基工程酵母菌中的表达量，本研究可为抗体在酵母中的规模发酵技术提供重要参考。
목적：：이항HER2항체위모형，연구항체재당기공정효모균중적표체급공정균발효기술。방법：수선통과요병시험분석유도용갑순농도대항체표체적영향，병용고표체HER2적SK-BR-3세포분석항HER2항체적항원결합활성。이차위기출，재5 L발효관중연구갑순-산리순혼합탄원류가유도대항HER2항체표체수평적영향；수집발효배양액，채용양리자교환층석대목표산물진행순화；이용SDS-PAGE、Western인적、Lowry법대항체적상대분자질량、농도등진행분석。결과：요병시험결과표명，갑순농도위0.5%시항체표체량최고，차당기공정필적효모균표체적항HER2항체구유여SK-BR-3세포항원결합적활성；재5 L발효관중，이용갑순화산리순혼합유도방식발효표체항체，기표체량가제고지0.6 g/L，비요병유도표체적항체산량제고료근10배；비환원SDS-PAGE급Western인적표명항체상대분자질량위1.5×105，여상업화항체Herceptin적대소일치；경과일보양리자교환층석순화，순화후항체농도위0.365 g/L。결론：채용갑순-산리순혼합탄원유도방식재5 L발효관중진행발효표체，능구제고항HER2항체재당기공정효모균중적표체량，본연구가위항체재효모중적규모발효기술제공중요삼고。
Objective: In this work, a study of the fermentation technique of engineered antibodies in glycosyl-ation engineered yeast using anti-HER2 monoclonal antibody(mAb) as model was presented. Methods: The opti-mal methanol induction concentration was confirmed by flask trial. The antigen binding affinity of anti-HER2 mAb was tested with the high HER2 expression breast cancer cell line SK-BR-3. The methanol/sorbitol co-feeding in-duction strategy for antibody production was carried out in a 5 L bioreactor on the basis of flask experiment. The medium was collected and subjected to purification with cation exchange chromatography. The molecular weight was analyzed by reducing and non-reducing SDS-PAGE. The antibody was identified by Western blotting and the purity was determined by Lowry method. Results: The highest expression level of anti-HER2 antibody was in-duced by 0.5% methanol in flask culture. Expressed antibody can bind to antigen on the cell surface of the SK-BR-3. The production of antibody in methanol/sorbitol co-feeding fermentation reached about 0.6 g/L, which was about ten times than in flask culture. The molecular weight of antibody was 1.5×105 in non-reducing SDS- PAGE which demonstrates that light chain and heavy chain could be assembled the right antibody structure. The final concentration of the antibody was 0.365 g/L after one step purification by cation exchange chromatography. Conclu-sion: Using the co-feeding strategy in 5 L bioreactor, the production of antibody expressed in glycoengineering Pi-chia pastoris was improved and this will be reference for a platform of large-scale antibody fermentation.