Autophagy and intracellular product degradation genes identified by systems biology analysis reduce aggregation of bispecific antibody in CHO cells

Aggregation of therapeutic bispecific antibodies negatively affects the yield, shelf-life, efficacy and safety of these products. Pairs of stable Chinese hamster ovary (CHO) cell lines produced two difficult-to-express bispecific antibodies with different levels of aggregated product (10-75% aggregate) in a miniaturised bioreactor system. Here, transcriptome analysis was used to interpret the biological causes for the aggregation and to identify strategies to improve product yield and quality. Differential expression- and gene set analysis revealed upregulated proteasomal degradation, unfolded protein response and autophagy processes to be correlated with reduced protein aggregation. Fourteen candidate genes with the potential to reduce aggregation were co-expressed in the stable clones for validation. Of these, HSP90B1, DDIT3, AKT1S1, and ATG16L1, were found to significantly lower aggregation in the stable producers and two (HSP90B1 and DNAJC3) increased titres of the anti-HER2 monoclonal antibody trastuzumab by 50% during transient expression. It is suggested that this approach could be of general use for defining aggregation bottlenecks in CHO cells.