长效生长激素生产用工程细胞株稳定性分析  

Stability Analysis of Engineering Cell Line for Production of Long-acting Growth Hormone

作　　者：魏利[1] 刘道琴[1] 徐婷 黄伟 唐瑜 俞安安 于雪莲 杨文静 叶洪涛 许文娟 倪旭婷 范清林[1] WEI Li;LIU Daoqin;XU Ting;HUANG Wei;TANG Yu;YU Anan;YU Xuelian;YANG Wenjing;YE Hongtao;XU Wenjuan;NI Xuting;FAN Qinglin(Anhui Anke Biotechnology(Group)Co.,Ltd.,Hefei 23000,China)

机构地区：[1]安徽安科生物工程(集团)股份有限公司,安徽合肥230000

出　　处：《药物生物技术》2024年第5期464-470,共7页Pharmaceutical Biotechnology

基　　金：安徽省重点研究与开发计划(No.2023s07020005)。

摘　　要：rhGH-Fc融合蛋白(AK2017)是治疗生长激素缺乏症的长效化生长激素药物,通过其C端融合Fc蛋白来延长药物的半衰期,从而达到长效化的目的。建立适合生产的稳定的CHO工程细胞株是rhGH-Fc融合蛋白商业化生产的前提,也是所有重组蛋白类药物在开发的过程中所面临的重要问题。为了分析rhGH-Fc融合蛋白生产用工程细胞株CHO-rhGH-Fc的稳定性,通过细胞株筛选的单克隆源性、传代稳定性和GMP条件下三批次样品生产质量一致性的三个维度来对生产用工程细胞株CHO-rhGH-Fc的稳定性进行评估。结果表明CHO-rhGH-Fc细胞株的单克隆源性约96.7%,传代过程中细胞形态和倍增速率与传代前工作细胞库细胞一致;传代至G54代目的蛋白表达量和目的基因拷贝数分别为G8代的78%和87.8%,培养过程中不同代次细胞生长趋势稳定,目的基因核苷酸序列与原始理论序列保持一致;GMP条件下连续三批次200 L生物反应器规模培养的细胞生长和代谢趋势均相似,纯化后的三批rhGH-Fc融合蛋白样品的纯度、翻译后修饰和相对生物学活性相似。数据显示长效生长激素生产用工程细胞株CHO-rhGH-Fc的传代稳定性和批间稳定性良好,细胞在商业化生产规模的培养周期内保持稳定,可满足商业化生产需要。The rhGH-Fcfusion protein(AK2017)is a long-acting growth hormone drug for the treatment of growth hormone deficiency.The half-life of the drug is prolonged by fusion of Fc protein at the C-terminus of rhGH,so as to achieve the purpose of long-acting efficacy.The establishment of a stable CHO cell line suitable for production is the prerequisite for the commercial production of rhGH-Fc fusion protein,and it is also an important problem in the development faced by all recombinant protein drugs.To analyze the stability of engineering cell line CHO-rhGH-Fc for production of rhGH-Fc fusion protein,the stability of the engineered cell line CHO-rhGH-Fc for production was evaluated from three aspects of monoclonal origin of cell line screening,passage stability and quality consistency of three batches samples under GMP conditions.The results showed that the monoclonality of CHO-rhGH-Fc cell line for production was about 96.7%,and cell morphology and multiplication rate during passage were consistent with those of the working cell bank before passage.The rhGH-Fc fusion protein titer in supernatant and copy number of target gene in G54 generation were 78%and 87.8%of those in G8 generation,respectively.The growth trend of different generation cells was stable during the whole culture process,and the nucleotide sequence of the target gene was consistent with the original theoretical sequence.Furthermore,during three consecutive batches of 200 L bioreactor scale cultures under GMP conditions,cells growth and metabolic parameters were similar.The purity,post-translational modifications and relative biological activity of three consecutive batches of purified rhGH-Fc fusion protein samples were essentially identical.The results shows that CHO-rhGH-Fc cell line for the production of long-acting growth hormone have good passaging stability and inter-batch stability,and the cells remained identical during the culture cycle of commercial production scale,which could meet the needs of commercial production.

关 键 词：长效生长激素 CHO-rhGH-Fc 稳定性分析 拷贝数 翻译后修饰 生物学活性 

分 类 号：R977.1[医药卫生—药品]