Genentech, Inc., South San Francisco, California 94080, USA.
Biotechnol Bioeng. 2010 Jun 15;106(3):452-61. doi: 10.1002/bit.22699.
In the biopharmaceutical industry, therapeutic monoclonal antibodies are primarily produced in mammalian cell culture systems. During the scale-up of a monoclonal antibody production process, we observed excessive mechanical cell shear as well as significant reduction of the antibody's interchain disulfide bonds during harvest operations. This antibody reduction event was catastrophic as the product failed to meet the drug substance specifications and the bulk product was lost. Subsequent laboratory studies have demonstrated that cells subjected to mechanical shear release cellular enzymes that contribute to this antibody reduction phenomenon (manuscript submitted; Kao et al., 2009). Several methods to prevent this antibody reduction event were developed using a lab-scale model to reproduce the lysis and reduction events. These methods included modifications to the cell culture media with chemicals (e.g., cupric sulfate (CuSO(4))), pre- and post-harvest chemical additions to the cell culture fluid (CCF) (e.g., CuSO(4), EDTA, L-cystine), as well as lowering the pH and air sparging of the harvested CCF (HCCF). These methods were evaluated for their effectiveness in preventing disulfide bond reduction and their impact to product quality. Effective prevention methods, which yielded acceptable product quality were evaluated for their potential to be implemented at manufacturing-scale. The work described here identifies numerous effective reduction prevention measures from lab-scale studies; several of these methods were then successfully translated into manufacturing processes.
在生物制药行业，治疗性单克隆抗体主要在哺乳动物细胞培养系统中生产。在单克隆抗体生产工艺放大过程中，我们在收获操作过程中观察到过度的机械细胞剪切以及抗体间二硫键的显著减少。这种抗体减少事件是灾难性的，因为产品未能达到药物物质规格，大量产品损失。随后的实验室研究表明，受到机械剪切的细胞释放出细胞酶，这些酶导致了这种抗体减少现象（已提交手稿；Kao 等人，2009 年）。使用实验室规模模型重现裂解和减少事件，开发了几种防止这种抗体减少事件的方法。这些方法包括用化学物质（例如硫酸铜 (CuSO(4)))修改细胞培养基、在细胞培养液 (CCF) 中进行收获前和收获后的化学添加（例如 CuSO(4)、EDTA、L-半胱氨酸），以及降低 pH 值和收获的 CCF（HCCF）中的空气吹气。评估了这些方法在防止二硫键减少方面的有效性及其对产品质量的影响。评估了有效的预防方法，以确定其在制造规模上实施的潜力，这些方法产生了可接受的产品质量。这里描述的工作从实验室研究中确定了许多有效的减少预防措施；然后成功地将其中几种方法转化为制造工艺。
