To better purify immunoglobulin 2 disulfide isoforms, Pfizer researchers developed their own method based around using a pH-gradient cation exchange separation. Their research and manufacturing processes were published in the Journal of Chromatography A (1).
Monoclonal antibodies (mAbs) are laboratory-made proteins made to stimulate peoples’ immune systems (2). Since their first approval in 1986, various immunoglobulin (IgG)-based therapies have been approved for treating various conditions, including cardiovascular disease, respiratory illnesses, and cancer. As of December 31st, 2023, 96IgG-based antibodies have been approved by the United States Food and Drug Administration (U.S. FDA) (3). IgG2 subclass mAbs can neutralize antigens, such as soluble cytokines, while inducing limited Fc-mediated cytotoxicity. IgG2 antibodies are unique compared to IgG1 antibodies, in that they can exhibit disulfide bond heterogeneity in the hinge, which leads to different isoforms. These isoforms have product-specific impacts on molecular functions.
For IgG2-based therapies under development, isoform distribution is determined by what manufacturing processes are employed. As these therapies advance through clinical trials, this process is typically modified or scaled up to meet increasing supply demands. Isoform content can vary between different manufacturing processes, and because of this, researchers must understand their impact on potency.
The researchers created a pH-gradient cation exchange separation to purify IgG2 disulfide isoforms in their native state. The IgG2 mAb used in this experiment was not compatible with previously reported purification methods, which used salt-gradient cation exchange. As such, collected fractions were analyzed using high-resolution denaturing reversed phase chromatography, while isoform content was determined with fluorescence detection. Fractions were combined to generate solutions with varying amounts of IgG2-B isoforms, ranging from 20.3% to 80.8% IgG2-B isoform. Across the different options for IgG2-B isoform content abundances, every sample retained similar levels of product-related impurities while being amenable to potency testing.
The impact of IgG2-A and IgG2-B isoforms on potency is product-dependent. IgG2-A has shown to increase potency due to increased flexibility in the Fab region, while IgG2-B was associated with increased potency through FcyR-independent agonist activity as reported in previous studies. Characterizing IgG2 disulfide isoforms requires sufficient approaches to both isolate and enrich species while retaining their native structures. The novel pH-gradient cation exchange purification process was created and applied to an IgG2 antibody. Ultimately, the purified IgG2-B isoform pools were determined to be of sufficient quality, having consistent levels of acidic/basic species, aggregates, and fragments. The only difference between IgG2-B isoform pools was IgG2-B content, as intended.
While the work described above was used for a specific biotherapeutic, the pH-gradient cation exchange method can be used as a potential alternative for IgG2 antibodies that are resistant to purification by cation exchange with salt elution. Future efforts should be used to understand the applicability of other antibodies and the impact of charged separation variants. Additionally, implementing high-throughput mass spectrometry-based multi-attribute methods could help to understand later eluting IgG2 isoform subpopulations. Continued development of approaches to purify and study IgG2 isoforms will continue to be an important characterization tool to employ during product development.
(1) Chipley, M.; Wells, K.; DuMontelle, J. L.; Smith, J.; Powers, T. W.; Lerch, T. F. pH-Gradient Cation Exchange Purification of IgG2 Disulfide Isoforms. J. Chromatogr. B 2025, 1264, 124716. DOI:
(2) Acevedo, A. Tracking Antibody Changes with Circular Dichroism and Fluorescence. LCGC International 2025.
(3) Strohl, W. R. Structure and Function of Therapeutic Antibodies Approved by the US FDA in 2023. Antib. Ther. 2024, 7 (2), 132–156. DOI:
