ABSTRACT
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. When combined, the knob and hole mutations in the CH3 domains promoted heterodimer formation. The hinge region disulfides were reduced and reoxidized to form the disulfide bridge between the two complementary half antibodies. Unreacted half antibodies, noncovalently linked homodimers, covalently linked homodimers, and noncovalently linked heterodimers are impurities closely related to the product of interest and are challenging to remove by standard processes. Characterization of the molecular properties of the half antibodies and high-throughput screening predicted column chromatography performance and allowed for rapid development of downstream purification steps for removal of unique product-related and process-related impurities. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:397-404, 2018.
Subject(s)
Antibodies, Bispecific/isolation & purification , High-Throughput Screening Assays/methods , Protein Engineering/methods , Antibodies, Bispecific/genetics , Chromatography, Gel , Chromatography, Reverse-Phase , Escherichia coli/genetics , Immunoglobulin G/genetics , Mutation , Protein Domains , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Staphylococcal Protein A/chemistryABSTRACT
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.
Subject(s)
Antibodies, Bispecific/chemistry , Protein Engineering/methods , Glutathione , Histidine/chemistry , Hot Temperature , Hydrogen-Ion Concentration , Immunoglobulin G/chemistry , Molecular Weight , Povidone/chemistryABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/isolation & purification , Chromatography, Affinity/methods , Polyethylene Glycols/chemistry , Recombinant Proteins/isolation & purification , Animals , Antibodies, Monoclonal/analysis , Antibodies, Monoclonal/chemistry , CHO Cells , Chemical Precipitation , Chromatography, Ion Exchange/methods , Cricetinae , Cricetulus , Hydrogen-Ion Concentration , Isoelectric Point , Recombinant Proteins/analysis , Recombinant Proteins/chemistry , Temperature , Viruses/isolation & purificationABSTRACT
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.
