Development and application of a mechanistic model for protein A resolution of a bispecific antibody for process characterization and optimization

Abstract Regeneron's bispecific technology, based on a fully human IgG antibody construct, consists of two distinct heavy chains and a common light chain co‐expressed in a single cell. The co‐expression of the two distinct heavy chains, one of which contains a dipeptide substitution that ablates Protein A binding, leads to the formation of three species with varying degrees of avidity to Protein A: the heterodimer bispecific and two parental homodimer impurities. The avidity difference between the three species can be leveraged for both bulk capture of the bispecific and resolution of the bispecific from the homodimer impurities via a ternary resolving Protein A chromatographic separation on commercially available resins amenable to large scale Good Manufacturing Practice (GMP) production. This case study highlights the efficiency of a mechanistic modeling approach for the optimization and characterization of ternary resolving Protein A unit operations. A mechanistic model, comprised of a general rate transport model and colloidal particle adsorption (CPA) affinity isotherm, was developed to describe this ternary separation for a model bispecific. The model was calibrated and validated with 10 laboratory experiments and demonstrated an ability to predict bispecific yield within 3% and bispecific purity within 1% on average. The model was then used to optimize the process and characterize the design space, while providing mechanistic insight into the process.