表达全人源抗人IgE单抗的候选细胞株鉴定及筛选

Characterization and selection of recombinant cell line candidates expressing fully-human m Abs against human IgE

目的:鉴定及筛选表达原创性全人源抗人Ig E单克隆抗体的候选工程细胞株。方法:对实验室前期筛选到的Mab1#及Mab2#2个候选细胞株的3 L摇瓶流加培养12 d并经Protein A一步柱层析纯化的抗体样品,进行紫外光谱全波长扫描,采用LC-MS测定精确相对分子质量进行鉴别,采用SDS-PAGE(还原型及非还原型)进行分子完整性分析,采用SEC-HPLC进行集聚倾向性分析,采用IEF进行等电点(p I)测定及对N-糖苷酶(PNGase F)酶切前后样品的电荷进行比较分析,采用CEX-HPLC进行电荷异质性分析。同时,对Mab1#及Mab2#2个候选细胞株的3个月传代稳定性进行比较研究。结果:Mab1#及Mab2#2个候选细胞株表达抗体的紫外最大吸收波长均为280 nm;LC-MS轻链相对分子质量分别为23 464.82和23 465.06,重链(G0F糖型)相对分子质量分别为50 807.50 Da和50 807.48 Da,均与理论预期相符;SDS-PAGE(还原型及非还原型)电泳结果显示表达的抗体分子完整性好;SEC-HPLC聚集倾向分析显示2株单抗经一步Protein A亲和柱层析后的可溶性聚合体的含量均小于2%;IEF结果显示Mab1#和Mab2#p I分别为8.38和8.44,PNGase F酶切前后未见明显的由于糖基化修饰引起的电荷变异体。CEX-HPLC结果显示2株单抗的电荷均一性好,酸性变体及碱性变体含量之和均小于4%。完成的3个月细胞株稳定性研究结果显示Mab1#及Mab2#2株克隆均稳定。结论:Mab1#及Mab2#2个候选细胞株具有相似的目标抗体表达量、表达抗体的质量(分子完整性、聚集倾向、电荷异质性、亲和力)、以及细胞稳定性特征和细胞生长代谢特征等质量属性,均符合制定的阶段筛选目标。

Objective： To characterize and screen candidate cell lines originally expressing fully-human anti-human Ig E monoclonal antibody. Methods： The protein A column chromatography purified antibody samples of our previously screened two candidate cell lines,Mab1#and Mab2#,which were taken from 12 days fed-batch culture in 3 L flask. A full wavelength UV spectroscopy scan were performed,and accurate molecular weight was identified by LC-MS. Then,the molecular integrity was analyzed by SES-PAGE,the aggregation by SEC-HPLC,and the isoelectric point（ p I） by IEF. The sample charges before and after the N-glycosidase digestion were compared,and the charge heterogeneity was analyzed by CEX-HPLC. Meanwhile,passage stability of the two candidate cell lines was compared for a period of 3 months. Results： The maximal UV absorption wavelengths of the expression antibodies of the two candidate cell lines（ Mab1#and Mab2#） were 280 nm. The LC-MS light chain molecularweights were 23 464. 82 Da and 23 465. 06 Da,respectively,and molecular weights of the heavy chain（ G0 F glycoforms） were 50 807. 50 Da and 50 807. 48 Da,respectively,which are consistent with the theoretical predictions.The expressed antibodies were integral molecules from the result of SDS-PAGE（ reduced and non-reduced） electrophoresis. SEC-HPLC analysis showed that the content of the soluble polymers of the two monoclonal antibody candidate cell lines after the protein A affinity chromatography was less than 2%. IEF showed that p I of Mab1#and Mab2#was 8. 38 and 8. 44,respectively. There was no significant charge variant caused by the glycosylation before and after the PNGase F digestion. CEX-HPLC showed that the two monoclonal charges were homogeneous,and the sum contents of the acid and basic variants were less than 4%. The 3-month stability study showed that the cell lines Mab1#and Mab2#were stable clones. Conclusion： The two candidate cell lines Mab1#and Mab2#have similar expression levels of target antibodies,antibody quality（ molecular integrity,aggregation tendency,the charge heterogeneity,and affinity） as well as the stability of the cell metabolism and cell growth characteristic features,which are in line with the stage screening goals.