Global Protein Science & Supply, DECS, AstraZeneca R&D Södertälje, Building 841, 151 85, Sodertalje, Sweden.
Cytotechnology. 2008 Feb;56(2):123-36. doi: 10.1007/s10616-008-9135-2. Epub 2008 Feb 22.
We have developed a generic transient transfection process at 100 L scale, using HEK293-EBNA cells and PEI as the transfection reagent for the production of recombinant IgG. The process, including large-scale plasmid preparation, expression at bioreactor scale, capture, purification and, if necessary, endotoxin removal allows reproducible production of more than 0.5 g IgG for in vitro and in vivo studies. We compared the performance of two HEK cell lines, investigated the effect of conditioned medium, optimized the DNA:PEI ratio and implemented a feed strategy to prolong the culture time to increase product yield. The transient transfection protocol developed enables a closed process from seeding culture to protein capture. The challenge of performing a medium exchange before transfection at large scale is solved by applying a continuous centrifugation step between the seeding bioreactor and the production bioreactor. After 7-8 days the harvest and capture is performed in a one-step operation using a Streamline expanded bed chromatography system. Following a polishing step the purified antibody is transferred to the final formulation buffer. The method has shown to be reproducible at 10, 50, and 100 L scale expressing between 5 and 8 mg L(-1) IgG.
我们开发了一种通用的瞬时转染工艺，规模为 100L，使用 HEK293-EBNA 细胞和 PEI 作为转染试剂生产重组 IgG。该工艺包括大规模质粒制备、生物反应器规模表达、捕获、纯化，如果需要，还可以去除内毒素，可重复性地生产超过 0.5g 的 IgG，用于体外和体内研究。我们比较了两种 HEK 细胞系的性能，研究了条件培养基的影响，优化了 DNA:PEI 比例，并实施了补料策略，以延长培养时间来提高产物产量。所开发的瞬时转染方案可实现从接种培养到蛋白捕获的封闭工艺。通过在接种生物反应器和生产生物反应器之间应用连续离心步骤，解决了在大规模转染前进行培养基交换的挑战。7-8 天后，采用 Streamline 扩展床层析系统一步法进行收获和捕获。经过抛光步骤后，将纯化的抗体转移到最终的制剂缓冲液中。该方法在 10、50 和 100L 规模上均表现出可重复性，表达量在 5 至 8mg/L 之间。
