Autophagy and intracellular product degradation genes reduce aggregation of bispecific antibody in CHO cells with a high translational burden

Aggregation of therapeutic bispecific antibodies negatively affects the yield, shelf-life, efficacy and safety of the product. Pairs of stable Chinese hamster ovary cell lines produced two difficult-to-express bispecific antibodies with different levels of aggregated product (10-75% aggregate) in a miniaturized bioreactor system. Here, we analyse the cellular response and link to product aggregation by comparative transcriptome analysis of these CHO cells, to define biological causes and infer strategies to improve yield and quality. Differential expression- and gene set analysis revealed upregulated proteosomal degradation, unfolded protein response and autophagy processes to be correlated with reduction of protein aggregation. Fourteen candidate genes with potential to reduce aggregation were co-expressed in the stable clones for validation. Of these, HSP90B1, DDIT3, AK1S1, and ATG16L1, were found to significantly lower aggregation in the stable producers and two (HSP90B1 and DNAJC3) increased trastuzumab titres by 50% each during transient expression. We suggest our approach to be of general use for defining aggregation bottlenecks in CHO.