Generating Multispecific Antibodies Through Column-Based Redox Reactions: Part I

Multispecific antibodies are increasingly being explored in the pharmaceutical industry for unmet patient needs. This study focuses on generating these molecules through an electrostatic-steering strategy, where two separate parent homodimer antibodies are expressed and purified, then combined into the heterodimer multispecific through reduction and oxidation chemistry. Traditional manufacturing operations for electrostatic steering multispecifics can include complex processing steps. Therefore, a novel redox process to generate the multispecific has been explored. This process involves a column-based reduction reaction and a spike of oxidant in the elution pool to form the heterodimer. This new strategy can simplify the downstream purification process for electrostatic-steering based molecules. The method consists of simultaneously binding two separate parental homodimers to the protein A chromatography resin and applying a reductant wash to reduce the interchain disulfide bonds. The molecules are then eluted, neutralized, and oxidized to form the intact heterodimer. The mechanism and rates of reduction, heterodimerization, and oxidation have been characterized to maximize conversion and product quality. This strategy has been demonstrated successfully for five multispecifics with diverse specificity and IgG subclasses. Implementing this method for pharmaceutical bioprocesses in the production of multispecific molecules offers the potential for the reduction in manufacturing complexity while maintaining acceptable product quality and yield.