Bispecific antibody process development: Assembly and purification of knob and hole bispecific antibodies.

Production of knob and hole dual light chain bispecific antibodies poses several unique challenges for development of a feasible industrial scale manufacturing process. We developed an efficient process for the assembly and purification of knob and hole dual light chain bispecific antibodies. Two distinct half-antibodies targeting two different antigens were expressed separately in Escherichia coli cells and captured independently using Protein A chromatography.

Improved assembly of bispecific antibodies from knob and hole half-antibodies.

A process was developed for large-scale assembly of IgG1 and IgG4 bispecific antibodies from knob and hole half-antibodies. We optimized assembly conditions such as pH, temperature, stabilizers, and reducing agent. We also identified and exploited structural changes unique to knob and hole half-antibodies with the result of improving assembly outcome, specifically storing half-antibodies at higher pH will condition them to assemble more rapidly and produce fewer high molecular-weight species (HMWS).

Purification of antibodies by precipitating impurities using Polyethylene Glycol to enable a two chromatography step process.

The purification of antibodies by precipitating impurities using Polyethylene Glycol (PEG) was assessed with the objective of developing a two chromatography column purification process. A PEG precipitation method was evaluated for use in the industrial purification of recombinant monoclonal antibodies (MAbs). Effective and robust precipitation conditions including PEG concentration, pH, temperature, time, and protein concentration were identified for several different MAbs.