Rapid and refined separation of human IgG2 disulfide isomers using superficially porous particles.

A rapid reversed-phase HPLC separation of recombinant human immunoglobulin gamma 2 (IgG2) disulfide isomers using columns packed with superficially porous particles is reported. Under optimal conditions, a separation of monoclonal IgG2 disulfide isomers was achieved in 10 min using a Poroshell™ 300SB-C8 column via a combination of high column temperature (85°C), mobile phases with high eluotropic strength (e.g. isopropanol) and high flow rate (1.5 mL/min). Thermodynamic stability analyses of chromatographically enriched IgG2 disulfide isomers revealed differences in their individual denaturation temperatures, which correlate with the observed temperature-dependent refinement of peak profiles by reversed-phase HPLC. This reversed-phase HPLC method in conjunction with other orthogonal analytical techniques (e.g. capillary gel electrophoresis, peptide mapping, ion exchange chromatography, etc.) is being used to characterize disulfide isomers in the development of therapeutic IgG2 antibodies.

Development of a capillary gel electrophoresis method for monitoring disulfide isomer heterogeneity in IgG2 antibodies.

A CGE method for monitoring the disulfide isomer distribution characteristic of IgG2 MAbs is presented. Disulfide heterogeneity of MAbs has been studied using various chromatographic and electrophoretic methods. Although CGE operates using a different selectivity mechanism from that of sorption chromatographic techniques, similar trends are present in the data, which allow the CGE method to be used as a complementary method for studying disulfide isomer distribution. This article focuses on the optimization of a capillary-based gel electrophoresis method that can be used to support antibody development including bioprocess optimization, antibody characterization, release, and formulation stability assessment.

Mutations within a human IgG2 antibody form distinct and homogeneous disulfide isomers but do not affect Fc gamma receptor or C1q binding.

Human IgG2 antibodies may exist in at least three distinct structural isomers due to disulfide shuffling within the upper hinge region. Antibody interactions with Fc gamma receptors and the complement component C1q contribute to immune effector functions. These interactions could be impacted by the accessibility and structure of the hinge region. To examine the role structural isomers may have on effector functions, a series of cysteine to serine mutations were made on a human IgG2 backbone. We observed structural homogeneity with these mutants and mapped the locations of their disulfide bonds. Importantly, there was no observed difference in binding to any of the Fc gamma receptors or C1q between the mutants and the wild-type IgG2. However, differences were seen in the apparent binding affinity of these antibodies that were dependent on the selection of the secondary detection antibody used.