Giese Glen, Williams Ambrose, Rodriguez Maricel, Persson Josefine
Department of Purification Development, Genentech, Inc., 1 DNA Way, South San Francisco, CA, 94080.
Biotechnol Prog. 2018 Mar;34(2):397-404. doi: 10.1002/btpr.2590. Epub 2018 Jan 17.
Production of knob and hole dual light chain bispecific antibodies poses several unique challenges for development of a feasible industrial scale manufacturing process. We developed an efficient process for the assembly and purification of knob and hole dual light chain bispecific antibodies. Two distinct half-antibodies targeting two different antigens were expressed separately in Escherichia coli cells and captured independently using Protein A chromatography. When combined, the knob and hole mutations in the CH3 domains promoted heterodimer formation. The hinge region disulfides were reduced and reoxidized to form the disulfide bridge between the two complementary half antibodies. Unreacted half antibodies, noncovalently linked homodimers, covalently linked homodimers, and noncovalently linked heterodimers are impurities closely related to the product of interest and are challenging to remove by standard processes. Characterization of the molecular properties of the half antibodies and high-throughput screening predicted column chromatography performance and allowed for rapid development of downstream purification steps for removal of unique product-related and process-related impurities. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:397-404, 2018.
旋钮和孔双轻链双特异性抗体的生产在开发可行的工业规模制造工艺方面带来了几个独特的挑战。我们开发了一种用于组装和纯化旋钮和孔双轻链双特异性抗体的高效工艺。两种靶向不同抗原的不同半抗体在大肠杆菌细胞中分别表达，并使用蛋白A色谱法独立捕获。当结合时，CH3结构域中的旋钮和孔突变促进异二聚体形成。铰链区二硫键被还原并重新氧化，以在两个互补半抗体之间形成二硫桥。未反应的半抗体、非共价连接的同二聚体、共价连接的同二聚体和非共价连接的异二聚体是与目标产物密切相关的杂质，通过标准工艺去除具有挑战性。对半抗体分子特性的表征和高通量筛选预测了柱色谱性能，并允许快速开发下游纯化步骤以去除独特的与产物相关和与工艺相关的杂质。©2018美国化学工程师学会生物技术进展，34:397 - 404，2018。
