The production of bispecific molecules through controlled Fab-Arm-Exchange (cFAE) is a multistep process. First, two parental monoclonal antibodies (mAbs) are expressed by independent Chinese hamster ovary (CHO) cell lines and purified by Protein A chromatography. Second, parental mAbs are subjected to a mild reducing agent, resulting in reduction of inter-heavy chain disulfide bonds at the hinge region and separation of parental mAb Fab-arms (heavy chain-light chain pair). Next, each Fab-arm combines with the complementary Fab-arm forming a heterodimer bispecific antibody. Last, the reformation of disulfide bonds between Fab-arms is achieved upon removal of the reducing agent. Multiple process parameters during the cFAE (e.g. temperature, pH, reducing agent concentration, antibody concentration, and dissolved oxygen level) and UF/DF (e.g. cross flow rate, transmembrane pressure, diafiltration concentration, membrane load ratio) may impact product quality and process performance attributes. Although the individual effects of each parameter cannot be known, because of the complex nature of the reaction (reduction and dissociation of Fab homodimers, association of Fab heterodimers, and oxidation of disulfide bonds), it can be difficult to optimize and predict the outcome of the reaction under a set of specific conditions. Two eight-factor, three-level definitive screening designs (DSD) and one four-factor, three-level design of experiments (DOE) were executed using two different IgG4 antibodies to establish a robust design space for bispecific manufacturing. Results demonstrated that under the parametric space studied, greater than 90% overall process yield and bispecific formation were achieved with no significant impact on critical quality attributes. The DSD and DOE responses included process performance such as yield and processing time and quality attributes such as charge heterogeneity, purity, residual reducing agent, and post-translational modifications.

Robust design space for bispecific manufacturing using controlled fab-arm-exchange (cFAE): Implementing DSD and DOE designs

2019 Spring National Meeting

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