Glen Giese

Development of a human IgG4 bispecific antibody for dual targeting of interleukin-4 (IL-4) and interleukin-13 (IL-13) cytokines

Background: Dual neutralization of IL-4 and IL-13 is a promising therapeutic approach for asthma and allergy. Results: Knobs-into-holes IgG1 and IgG4 bispecific antibodies targeting both cytokines were developed. Conclusion: Bispecific antibodies of both isotypes have comparable in vitro potencies, in vivo pharmacokinetics, and lung partitioning. Significance: Further extension of knobs-into-holes technology to human IgG4 isotype as reported here provides greater options for therapeutics. Human bispecific antibodies have great potential for the treatment of human diseases. Although human IgG1 bispecific antibodies have been generated, few attempts have been reported in the scientific literature that extend bispecific antibodies to other human antibody isotypes. In this paper, we report our work expanding the knobs-into-holes bispecific antibody technology to the human IgG4 isotype. We apply this approach to generate a bispecific antibody that targets IL-4 and IL-13, two cytokines that play roles in type 2 inflammation. We show that IgG4 bispecific antibodies can be generated in large quantities with equivalent efficiency and quality and have comparable pharmacokinetic properties and lung partitioning, compared with the IgG1 isotype. This work broadens the range of published therapeutic bispecific antibodies with natural surface architecture and provides additional options for the generation of bispecific antibodies with differing effector functions through the use of different antibody isotypes.

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. A recovery process using two chromatography steps in combination with PEG precipitation gave acceptable yield and purity levels for IgG1 and IgG4 antibodies with a broad range of isoelectric points (pI). PEG precipitation removed host cell proteins (HCPs), high molecular weight species (HMWS), leached Protein A ligand, and host cell DNA to acceptable levels when run under appropriate conditions, and some endogenous virus removal was achieved.

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). Application of heat to the assemblies resulted in an acceleration of assembly rate, with optimal formation of bispecific achieved at 37°C. IgG4 half‐antibodies were unusually sensitive to temperature‐dependent formation of HMWS in pre‐assembly conditioning as well as during assembly. We selected l‐histidine and Polyvinylpyrrolidone (PVP) as stabilizers to prevent HMWS formation in IgG4, and achieved rapid and high‐efficiency assemblies. Using optimized assembly conditions, we developed and scaled up a method for assembling bispecific antibody with 90% assembly efficiency over 6 h with minimal impact to product quality, generating a pool with bispecific antibody for downstream processing.

Process Design for Bispecific Antibodies

Abstract Bispecific antibodies provide an opportunity to target two different antigens with a single therapeutic. Applications range from combining two drug activities into one molecule to unique modes of action like the recruiting of immune cells to tumors. However, this advancement in antibody engineering brings new process challenges for cell line development, assembly, purification, and analytics. Creation of bispecific antibody from four individual amino acid sequences (different light and heavy chains) is a process which must be optimized to minimize burden on downstream processing. Knob-and-hole mutations in the Fc region ensure correct hetero-dimerization of heavy chains, however to create bispecific with correct pairing of light chains with heavy chains, two upstream strategies exist. We discuss assembly of bispecific antibody from separately-expressed half-antibodies, as well as an alternative domain-crossover approach called CrossMab, which is produced in a single production fermentation. Bispecific antibodies from both approaches may present product-related variants which must be eliminated in the purification process. Removal of product-related variants requires new purification development approaches, because their structures can resemble that of the bispecific antibody. Case studies from two different bispecific antibody formats will be discussed.