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 bsAbs and one symmetric IgG-like bsAb constructs as model molecules, we demonstrated an effective bsAb polishing strategy leveraging multimodal CHT Ceramic Hydroxyapatite chromatography media. Under optimized conditions, excellent removal of bsAb byproducts and impurities were achieved in a single chromatographic step, with eluate purity in all products over 97% and high molecular weight (HMW) species less than 0.6%. In addition, the chromatography step yielded purified products with low host cell protein (HCP) contents (below 100 ppm) and host cell DNA (hcDNA) levels for all products post-CHT media 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.

Key takeaways:

• Three post–Protein A bsAbs were polished by one-step CHT to over 97% purity
• Product-and-process-related impurities were efficiently removed
• Chromatography-induced aggregation was minimized for aggregation-prone bsAbs

About the presenter: Zhang Wei, PhD is a principal scientist heading the Downstream Processing Group (DSP) at the Bioprocessing Technology Institute (BTI), Agency for Science, Technology and Research (A*STAR) in Singapore. Her team focuses on purification process development and product characterization for the production of a variety of biotherapeutics, including monoclonal antibodies, bsAbs, vaccines, viral vectors, mRNA, and more.