BMC Biotechnol. 2013 Jun 26;13:52. doi: 10.1186/1472-6750-13-52.
The demand of monospecific high affinity binding reagents, particularly monoclonal antibodies, has been steadily increasing over the last years. Enhanced throughput of antibody generation has been addressed by optimizing in vitro selection using phage display which moved the major bottleneck to the production and purification of recombinant antibodies in an end-user friendly format. Single chain (sc)Fv antibody fragments require additional tags for detection and are not as suitable as immunoglobulins (Ig)G in many immunoassays. In contrast, the bivalent scFv-Fc antibody format shares many properties with IgG and has a very high application compatibility.
In this study transient expression of scFv-Fc antibodies in human embryonic kidney (HEK) 293 cells was optimized. Production levels of 10-20 mg/L scFv-Fc antibody were achieved in adherent HEK293T cells. Employment of HEK293-6E suspension cells expressing a truncated variant of the Epstein Barr virus (EBV) nuclear antigen (EBNA) 1 in combination with production under serum free conditions increased the volumetric yield up to 10-fold to more than 140 mg/L scFv-Fc antibody. After vector optimization and process optimization the yield of an scFv-Fc antibody and a cytotoxic antibody-RNase fusion protein further increased 3-4-fold to more than 450 mg/L. Finally, an entirely new mammalian expression vector was constructed for single step in frame cloning of scFv genes from antibody phage display libraries. Transient expression of more than 20 different scFv-Fc antibodies resulted in volumetric yields of up to 600 mg/L and 400 mg/L in average.
Transient production of recombinant scFv-Fc antibodies in HEK293-6E in combination with optimized vectors and fed batch shake flasks cultivation is efficient and robust, and integrates well into a high-throughput recombinant antibody generation pipeline.
近年来，对单特异性高亲和力结合试剂（特别是单克隆抗体）的需求稳步增长。通过使用噬菌体展示技术优化体外选择，提高了抗体生成的通量，这将主要瓶颈转移到了以用户友好的格式生产和纯化重组抗体上。单链（sc）Fv 抗体片段需要额外的标签进行检测，并且在许多免疫测定中不如 IgG 适合。相比之下，二价 scFv-Fc 抗体形式与 IgG 具有许多共同特性，并且具有非常高的应用兼容性。
本研究优化了人胚肾（HEK）293 细胞中 scFv-Fc 抗体的瞬时表达。在贴壁 HEK293T 细胞中实现了 10-20mg/L scFv-Fc 抗体的生产水平。与无血清条件下表达 EBV 核抗原（EBNA）1 截断变体的 HEK293-6E 悬浮细胞一起使用，可将体积产率提高 10 倍以上，达到超过 140mg/L scFv-Fc 抗体。经过载体优化和工艺优化，scFv-Fc 抗体和细胞毒性抗体-RNase 融合蛋白的产量进一步提高了 3-4 倍，达到了 450mg/L 以上。最后，构建了一种全新的哺乳动物表达载体，用于从抗体噬菌体展示文库中一步法框内克隆 scFv 基因。瞬时表达超过 20 种不同的 scFv-Fc 抗体，体积产率高达 600mg/L，平均产率为 400mg/L。
HEK293-6E 中瞬时生产重组 scFv-Fc 抗体与优化的载体和补料分批摇瓶培养相结合，效率高且稳健，并且很好地集成到高通量重组抗体生成管道中。
