Effective impurities removal to simplify and accelerate bispecific antibody (bsAb) production

2022_08 Wei Grp Photo
Dr Zhang Wei (left) conceived and designed project, Mr Hoi Kong Meng (right) conducted experiments.



The bsAb are a fast-growing group of therapeutic antibodies that is therapeutically promising due to their ability to bind to two different epitopes/antigens simultaneously. However, the various by-products and impurities generated during bsAb cell culture process are very difficult to remove, posing unique and significant challenges to bsAb production.


Societal Impact

Due to their ability to bind more than one target, bsAbs have demonstrated improved therapeutic efficacy and hold great promise in therapeutic applications that are difficult to achieve by single-targeting monospecific antibodies. However, the unique challenges in bsAb purification have posed a big hurdle in the development of bsAb as a therapeutic. Our study serves as an insightful guide for researchers working on the bsAb purification process development, enabling effective impurities removal in bsAb production, thereby simplifying and accelerating bsAb development as therapeutics.


Technical Summary

The bsAb by-products and impurities, especially mispaired homodimers, half antibodies and high levels of high molecular weight species, pose unique challenges to the downstream processing of bsAbs. One of the most employed strategies to reduce mis-paired products is the generation of bsAbs via the knob-into-hole approach. This approach favoured the formation of the desired heterodimer bsAb over the mispaired products. However, hole-hole homodimerization can still occur at low levels. These mispaired hole-hole dimers are physiochemically similar to the target bsAbs, thus making it challenging to remove.

Using two knob-into-hole constructs of bsAbs as model molecules, we demonstrated the excellent removal of bsAb by-products and impurities, including the removal of mispaired hole-hole homodimer products, using a single optimised Protein A chromatography. An overall high monomer purity of 92.1 – 93.2 % can be achieved with good recovery of 78.4 – 90.6 % within one capture step, which is a significant improvement from a monomer purity of ~ 30 % in the bsAb cell culture supernatant.

2022_08 Wei Fig 1
Figure 1. Excellent removal of bispecific antibody by-products and impurities in a single step



Chen, S.W., Hoi, K.M., Mahfut, F.B. et al. Excellent removal of knob-into-hole bispecific antibody byproducts and impurities in a single-capture chromatography. Bioresour. Bioprocess. 9, 72 (2022).