Development of purification processes for fully human bispecific antibodies based upon modification of protein A binding avidity

Abstract

There is strong interest in the design of bispecific monoclonal antibodies (bsAbs) that can simultaneously bind 2 distinct targets or epitopes to achieve novel mechanisms of action and efficacy. Multiple bispecific formats have been proposed and are currently under development. Regeneron's bispecific technology is based upon a standard fully human IgG antibody in order to minimize immunogenicity and improve the pharmacokinetic profile. A single common light chain and 2 distinct heavy chains combine to form the bispecific molecule. One of the heavy chains contains a chimeric Fc sequence form (called Fc*) that ablates binding to Protein A via the constant region. As a result of co-expression of the 2 heavy chains and the common light chain, 3 products are created, 2 of which are homodimeric for the heavy chains and one that is the desired heterodimeric bispecific product. The Fc* sequence allows selective purification of the FcFc* bispecific product on commercially available affinity columns, due to intermediate binding affinity for Protein A compared to the high avidity FcFc heavy chain homodimer, or the weakly binding Fc*Fc* homodimer. This platform requires the use of Protein A chromatography in both a capture and polishing modality. Several challenges, including variable region Protein A binding, resin selection, selective elution optimization, and impacts upon subsequent non-affinity downstream unit operations, were addressed to create a robust and selective manufacturing process.

Keywords: Affinity chromatography; bispecific antibody; downstream process; elution pH; heterodimeric antibody; protein A chromatography; protein purification.

Figure 1.

Diagram illustrating bispecific (FcFc*) and related contaminating antibodies (FcFc, Fc*Fc*) synthesized in the production bioreactor. The theoretical expression ratio assumes equal production of the HC and HC* chain and no thermodynamic preference for the formation of either quaternary structure. The star substitution present on the HC* chain (blue) is indicated via the circle. The Protein A binding HC chain lacking the star substitution is shown as red, and the common light chain is green.

Figure 2.

Representation of the structure of IgG1 subtype bispecific antibody bsAb C, with HC* (blue), HC (red) and light chains (green). Heavy chain dimerization is achieved through 2 heavy chain intermolecular disulfide bonds located within the hinge region. The star substitution on the HC* is denoted by the black circle, with the amino acid residues substituted indicated. Antibody domains are labeled on each chain.

Figure 3.

Expression ratios of bispecific (□), FcFc (○), and Fc*Fc* homodimer (Δ) for bsAb C (A) and bsAb D (B) obtained for 4 (bsAb C) or 3 (bsAb D) replicate bioreactors.

Figure 4.

Chromatograms illustrating pH (dotted) and absorbance at 280 nm (solid) during elution step for purification of bsAb E with a recombinant Protein A resin (MabSelect Xtra, A), and an engineering Protein A-based resin which lacks V_H binding (MabSelect SuRe, B).

Figure 5.

Binding sites of Staphylococcal Protein A (SpA) and the MabSelect SuRe ligand to IgG1, 2 and 4 antibodies. While both ligands bind in the CH2-CH3 interfacial region, only SpA is capable of binding some antibodies of the V_H3 gene family in the V_H region.

Figure 7.

Resolutions obtained between bsAb A and FcFc peaks as a function of residence time during a 30CV gradient elution in (40mM Acetate, 500mM calcium chloride) with either MabSelect SuRe (○), or POROS MabCapture A (□) as the stationary phase.

Figure 6.

Chromatograms illustrating pH (dotted) and absorbance at 280 nm (solid) during elution step for purification of bsAb A with either sodium citrate (A), sodium chloride (B), magnesium chloride (C), or calcium chloride (D) added as modifiers to the elution mobile phase. Bispecific peak fractionation is marked by vertical dotted lines.

Figure 8.

Sweet spot analysis utilizing model obtained from CCD evaluation of the design space for isocratic affinity chromatography for resolution of the bsAb C from the parental homodimers using POROS MabCapture A (A) and MabSelect SuRe (B). The sweet spot in the white region where bispecific yield is > 80% and pool bispecific purity is > 95%.

Figure 9.

Flow scheme of suggested purification processes for star substitution containing bispecific antibodies exhibiting (A) and not exhibiting (B) V_H domain SpA binding.