Bivalent bispecific antibodies (BsAb) are a novel class of therapeutics that allow for the binding of two distinct epitopes due to the presence of an additional single chain fragment (scFv) that is expressed with the intact IgG molecule. High aggregate content (10-30%) at the end of cell culture is often observed and requires yield compromises in order to meet purity targets. Furthermore, the high aggregate levels pose robustness risks to a conventional mAb three column platform purification process which uses only the polishing steps (e.g. cation exchange chromatography (CEX) for aggregate removal. As the mechanism of aggregation in BsAbs is not well-understood, developing solutions to address aggregation becomes challenging. Root cause investigations were performed for different BsAb constructs which demonstrated that BsAb aggregates are primarily covalent species arising from cross-linkage between the engineered cysteines on the heavy and light chains of the scFv. The aggregates are also formed within the cell before being secreted into the culture media. Alternative means of introducing orthogonal aggregate removal capabilities into other aspects of the purification process such as Protein A chromatography with pH gradient elution, high performance tangential flow filtration (HP-TFF) and calcium phosphate precipitation were evaluated. Protein A pH gradient elution followed by calcium phosphate precipitation was also evaluated at pilot scale, demonstrating comparable performance. With the implementation of Protein A chromatography with gradient elution and/ or calcium phosphate precipitation, a sufficient portion of the aggregate burden was removed prior to the CEX final polishing step, enabling robust operation under conditions favoring higher monomer yield. From starting aggregate levels ranging from 15-23% in the condition media, levels were reduced to between 2-3% at the end of the CEX step, while achieving an overall yield of 71%. The findings from this study suggest an improved three-column mAb platform-like purification process for high aggregate scFv-IgG bispecific antibodies is feasible.

Integrated approach to understanding and controlling for scFv bispecific antibody aggregation during process development

2019 Spring National Meeting

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.