Serene W Chen, Darryl Tan, Yuan Sheng Yang and Wei Zhang
Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore
Published in mAbs 2020 12(1): 1718440 (Online Version)
Bispecific antibodies, as a new drug modality, have yielded remarkable promise in improving drug therapeutic efficacy through the recognition and targeting of two different antigens. Compared to their parental monoclonal antibodies that bind to a single target, bispecific antibodies possess great potential in addressing the multifactorial nature of complex diseases. The importance and promise of bispecific antibodies is further exemplified by the increase in recent reports proposing combinations of antibodies as a vaccine for COVID-19. The enormous therapeutic potential of bispecific antibodies has led to its massive development of different bispecific antibody formats with 2 approved drugs available in the market thus far. The tandem single-chain variable fragment (scFv) format stands out as one of the most promising reported.
Little is known about the scalable purification development of tandem scFv bispecific antibodies. Due to its unique structure, a tandem scFv antibody cannot be easily purified using protocols commonly used for monoclonal antibodies. Furthermore, bispecific antibodies are much more prone to aggregation compared to the parental monoclonal antibodies. The overall small sizes and high impurity content due to low expression level of this particular type of bispecific antibodies also pose additional challenges in the downstream purification process to achieve products of high purity within a limited number of purification steps.
Research Fellow Dr Serene Chen and Staff Scientist, Head of Downstream Processing Group Dr Zhang Wei from Bioprocessing Technology Institute (BTI) discovered a novel mechanism of salt additive effect which regulates the separation of tandem scFv bispecific antibody monomers and aggregates during affinity chromatography. Through systematic investigation using Protein L affinity chromatography, they significantly improved product purity from 18.8% to 98.8% in a scalable and economical two-step process.
This novel mechanism was revealed to be governed by an interplay between electrostatic interactions as well as other interactions such as hydrophobicity, cation-π interaction and hydrogen bonding. It is completely different from the widely reported chaotropic effect exerted by salt additives observed in Protein A affinity chromatography, which is extensively used for monoclonal antibody purification. The use of Protein L affinity chromatography opened new opportunities and uncovered a novel mechanism that can guide purification of future bispecific antibodies.