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ABSTRACT
Charge variants are important attributes of monoclonal antibodies, including antibody-drug conjugates (ADCs), because charge variants can potentially influence the stability and biological activity of these molecules. Ion exchange chromatography (IEX) is widely used for charge variants analysis of mAbs and offers the feasibility of fractionation for in-depth characterization. However, the conjugated linker-drug on ADCs could potentially affect the separation performance of IEX, considering IEX separation relies on surface charge distribution of analyte and involves the interaction between analyte surface and IEX stationary phase. Here, we investigated weak cation exchange chromatography (WCX) for its application in analyzing three ADCs (two broad distribution ADCs and an ADC with controlled conjugation sites) and the 2-drug/4-drug loaded species isolated from the two broad distribution ADCs using hydrophobic interaction chromatography. The major peaks in WCX profile were characterized via fraction collection followed by capillary electrophoresis-sodium dodecyl sulfate or peptide mapping. Results suggested that both the number of drug loads and conjugation sites could impact WCX separation of an ADC. The hypothesis was that the linker drugs could interfere with the ionic interaction between its surrounding amino acids on the mAb surface and column resin, which reduced the retention of ADCs on WCX column in this study. Our results further revealed that WCX brings good selectivity towards positional isomers, but limited resolution for different drug load, which causes the peak compositions of the two broad-distribution ADCs to be highly complex. We also compared results from WCX and imaged capillary isoelectric focusing (icIEF). Results showed that separation in icIEF was less influenced by conjugated linker drugs for the ADCs studied in this work, and better alignment was found between the two techniques for the ADC with controlled conjugate sites. Overall, this work provides insights into the complexity of WCX analysis of ADCs, which should be considered during method development and sample characterization.
Abbreviations.
| ADC | = | Antibody-drug conjugate |
| AEX | = | Anion exchange chromatography |
| CE-SDS | = | Capillary electrophoresis sodium dodecyl sulfate |
| CEX | = | Cation exchange chromatography |
| cIEF | = | Capillary isoelectric focusing |
| CZE | = | Capillary zone electrophoresis |
| H | = | Heavy chain |
| HIC | = | Hydrophobic interaction chromatography |
| icIEF | = | Imaged capillary isoelectric focusing |
| IEX | = | Ion exchange chromatography |
| IgG | = | Immunoglobin gamma |
| L | = | Light chain |
| LC | = | Liquid chromatography |
| mAb | = | Monoclonal antibody |
| mc-vc-MMAE | = | Maleimidocaproyl-valine-citrulline-monomethyl auristatin E |
| MS | = | Mass spectrometry |
| WCX | = | Weak cation exchange chromatography |
Acknowledgments
We thank Jorge Gandarilla, Xiaoqiang Shen, Brad Gates and Xiaoli Liao from AbbVie Process Chemistry for providing us with the mAb and ADC samples used in this study and helpful discussions.
Disclosure
This study was sponsored by AbbVie Inc. AbbVie contributed to the design, study conduct, financial support and interpretation of data, writing, reviewing, and approving the publication. Zhaorui Zhang, Shiyue Zhou, Linjie Han, Qunying Zhang, and Wayne Pritts are employees of AbbVie Inc.
Supplementary Materials
Supplemental materials data for this article can be accessed here.
Correction Statement
This article has been republished with minor changes. These changes do not impact the academic content of the article.
