Hanson Jeffrey, Groff Dan, Carlos Abi, Usman Hans, Fong Kevin, Yu Abigail, Armstrong Stephanie, Dwyer Allison, Masikat Mary Rose, Yuan Dawei, Tran Cuong, Heibeck Tyler, Zawada James, Chen Rishard, Hallam Trevor, Yin Gang
Sutro Biopharma Inc., 111 Oyster Point, South San Francisco, CA 94080, USA.
Bioengineering (Basel). 2023 Feb 28;10(3):304. doi: 10.3390/bioengineering10030304.
The XpressCF+ cell-free protein synthesis system is a robust platform for the production of non-natural amino acids containing antibodies, which enable the site-specific conjugation of homogeneous antibody drug conjugates (ADCs) via click chemistry. Here, we present a robust and scalable means of achieving a 50-100% increase in IgG titers by combining the high productivity of cell-based protein synthesis with the unique ability of XpressCF+ reactions to produce correctly folded and assembled IgGs containing multiple non-natural amino acids at defined positions. This hybrid technology involves the pre-expression of an IgG light-chain (LC) protein in a conventional recombinant expression system, engineered to have an oxidizing cytoplasm. The prefabricated LC subunit is then added as a reagent to the cell-free protein synthesis reaction. Prefabricated LC increases IgG titers primarily by reducing the protein synthesis burden per IgG since the cell free translation machinery is only responsible for synthesizing the HC protein. Titer increases were demonstrated in four IgG products in scales ranging from 100-µL microplate reactions to 0.25-L stirred tank bioreactors. Similar titer increases with prefabricated LC were also demonstrated for a bispecific antibody in the scFvFc-FabFc format, demonstrating the generality of this approach. Prefabricated LC also increases robustness in cell-free reactions since it eliminates the need to fine-tune the HC-to-LC plasmid ratio, a critical parameter influencing IgG assembly and quality when the two IgG subunits are co-expressed in a single reaction. ADCs produced using prefabricated LC were shown to be identical to IgGs produced in cell-free alone by comparing product quality, in vitro cell killing, and FcRn receptor binding assays. This approach represents a significant step towards improving IgG titers and the robustness of cell-free protein synthesis reactions by integrating in vivo and in vitro protein production platforms.
XpressCF+无细胞蛋白质合成系统是用于生产含非天然氨基酸抗体的强大平台，这些抗体能够通过点击化学实现均一抗体药物偶联物（ADC）的位点特异性偶联。在此，我们展示了一种强大且可扩展的方法，通过将基于细胞的蛋白质合成的高生产力与XpressCF+反应在特定位置产生正确折叠和组装的含多个非天然氨基酸的IgG的独特能力相结合，使IgG滴度提高50 - 100%。这种混合技术涉及在传统重组表达系统中预表达IgG轻链（LC）蛋白，该系统经工程改造具有氧化性细胞质。然后将预制的LC亚基作为试剂添加到无细胞蛋白质合成反应中。预制的LC主要通过减轻每个IgG的蛋白质合成负担来提高IgG滴度，因为无细胞翻译机制仅负责合成重链（HC）蛋白。在从100微升微孔板反应到0.25升搅拌罐生物反应器等不同规模的四种IgG产品中都证明了滴度的提高。对于scFvFc - FabFc格式的双特异性抗体，使用预制LC也显示出类似的滴度提高，证明了这种方法的通用性。预制的LC还提高了无细胞反应的稳健性，因为它消除了微调HC与LC质粒比例的需要，当两个IgG亚基在单一反应中共表达时，该比例是影响IgG组装和质量的关键参数。通过比较产品质量、体外细胞杀伤和FcRn受体结合试验，使用预制LC生产的ADC显示与仅在无细胞条件下产生的IgG相同。这种方法通过整合体内和体外蛋白质生产平台，朝着提高IgG滴度和无细胞蛋白质合成反应的稳健性迈出了重要一步。
