IgG Aggregation Mechanism for CHO Cell Lines Expressing Excess Heavy Chains

Aggregates in protein therapeutics like IgG monoclonal antibodies (mAb) are detrimental to product safety and efficacy. It has been reported that aggregates form in Chinese hamster ovary (CHO) cell lines expressing greater amount of heavy chain (HC) than light chain (LC). In this study, we observed that aggregates could form within the cells with excess HC and were partially secreted into the supernatant. The aggregates in the supernatant consisted of mainly HC and were partially dissociated under either reducing or denaturing conditions. Mutation of a predicted free cysteine on HC to prevent disulfide bonding did not reduce aggregation. Re-transfecting CHO cells with excess HC with more BiP, an important IgG molecular chaperone, partially reduced unwanted aggregates and fragments possibly by helping retain more incomplete products within the cell for either proper assembly or degradation. A second transfection of LC into CHO cells with excess HC to increase the LC expression to a level greater than the HC expression successfully removed all aggregates and fragments. mAb product aggregation in CHO cells with excess HC occur due to a combination of limited chaperones and LC:HC ratio. These results provide added insights to aggregate formation and would be useful for development of mAb cell lines with reduced aggregates.