A Polishing Strategy For Removing Impurities In Bispecific Antibody Purification

Bispecific antibodies (bsAbs) are therapeutically promising due to their ability to bind to two different antigens. Although bsAb molecules provide huge therapeutic advantages over traditional monoclonal antibodies (MAbs), their complex product-and-process-related impurities pose unique challenges to their downstream processing.

Here, using two knob-into-hole (KiH) based asymmetric IgG-like bsAb and one symmetric IgG-like bsAb constructs as model molecules, we demonstrated an effective bsAb polishing strategy leveraging on multimodal CHT™ Ceramic Hydroxyapatite chromatography media. Under optimized conditions, excellent removal of bsAb byproducts and impurities was achieved in a single chromatographic step, with eluate purity in all products over 97% and HMW (high molecular weight) species less than 0.6%. In addition, the chromatography step yielded purified products with low HCP contents (below 100 ppm), and HCDNA levels for all products post-CHT resins were lower than 10 ppb. The study illustrates that CHT chromatography can be utilized as an effective polishing step for not only MAbs, but also bsAbs, covering both asymmetric and symmetric formats.

The results presented here would be insightful guidance to all researchers working on the purification process development to produce bsAbs, especially for asymmetric and symmetric IgG-like bsAb therapeutics.