Multispecific antibodies are increasingly being explored in the pharmaceutical industry for unmet patient needs. This work 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 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.

Multispecifics are increasingly being evaluated in the pharmaceutical industry due to the unique mechanisms of action of the molecules, which is enabled by the multiple antigen binding capability. The complexity of these molecules can make production difficult, therefore different approaches for the generation of these molecules have been developed. The approach employed in this study utilizes electrostatic-steering, wherein charge-based differences between two parental homodimer antibodies are used to drive correct heterodimerization during a redox reaction of the partially purified parental homodimers. This strategy results in high conversion to the heterodimer with minimal product-related impurities; however, this method also requires separate bioreactors for each parental homodimer, resulting in complex manufacturing campaigns. This work describes a new bioprocess for electrostatic steering-based multispecifics. This strategy couples two unique components. First, the two separate cell lines are co-cultured, resulting in simultaneous production of both parental homodimers in a single bioreactor. The second component utilizes a column-based redox reaction, wherein the homodimers are captured and the disulfide bonds reduced while bound to the protein A resin using a reductant wash. The column is then eluted and neutralized to allow the reduced parental homodimers to heterodimerize and finally, the addition of an oxidant enables the disulfide bond reformation to complete the formation of the multispecific. This new process is robust and efficient across bench and manufacturing scales, with well-controlled impurity profiles. Through this strategy, the new multispecific bioprocess is more similar to a typical antibody-like bioprocess, enabling more efficient use of clinical and commercial manufacturing resources, while resulting in the production of complex multispecific molecules with minimal product-related impurities.