Abstract:
Monoclonal antibodies (mAbs) are often engineered at the sequence level for improved clinical performance yet are rarely evaluated prior to candidate selection for their \"developability\" characteristics, namely expression, which can necessitate additional resource investments to improve the manufacturing processes for problematic mAbs. A strong relationship between primary sequence and expression has emerged, with slight differences in amino acid sequence resulting in titers differing by up to an order of magnitude. Previous work on these \"difficult-to-express\" (DTE) mAbs has shown that these phenotypes are driven by post-translational bottlenecks in antibody folding, assembly, and secretion processes. However, it has been difficult to translate these findings across cell lines and products. This work presents a systematic approach to study the impact of sequence variation on mAb expression at a larger scale and under more industrially relevant conditions. The analysis found 91 mutations that decreased transient expression of an IgG1κ in Chinese hamster ovary (CHO) cells and revealed that mutations at inaccessible residues, especially those leading to decreases in residue hydrophobicity, are not favorable for high expression. This workflow can be used to better understand sequence determinants of mAb expression to improve candidate selection procedures and reduce process development timelines.
摘要:
单克隆抗体(mAb)通常在序列水平上进行工程改造,以改善临床性能,但在候选选择之前很少对其“发育性”特征进行评估。即表达式,这可能需要额外的资源投资来改善有问题的单克隆抗体的制造工艺。一级序列和表达之间的强烈关系已经出现,氨基酸序列略有差异,导致滴度相差高达一个数量级。先前对这些“难以表达”(DTE)mAb的研究表明,这些表型是由抗体折叠的翻译后瓶颈驱动的,装配,和分泌过程。然而,很难在细胞系和产品中翻译这些发现。这项工作提出了一种系统的方法,可以在更大规模和更工业相关的条件下研究序列变异对mAb表达的影响。分析发现91个突变降低了中国仓鼠卵巢(CHO)细胞中IgG1κ的瞬时表达,并揭示了无法进入的残基的突变,尤其是那些导致残留物疏水性降低的物质,不利于高表达。该工作流程可用于更好地理解mAb表达的序列决定因素,以改善候选选择程序并减少过程开发时间。