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LC–MS Approach to Decipher a Light Chain Chromatographic Peak Splitting of a Monoclonal Antibody

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Abstract

Purpose

Monoclonal antibodies (mAbs), like other protein therapeutics, are prone to various forms of degradation, some of which are difficult to distinguish from the native form yet may alter potency. A generalizable LC–MS approach was developed to enable quantitative analysis of isoAsp. In-depth understanding of product quality attributes (PQAs) enables optimization of the manufacturing process, better formulation selection, and decreases risk associated with product handling in the clinic or during shipment.

Methods

Reversed-phase chromatographic peak splitting was observed when a mAb was exposed to elevated temperatures. Multiple LC–MS based methods were applied to identify the reason for peak splitting. The approach involved the use of complementary HPLC columns, multiple enzymatic digestions and different MS/MS ion dissociation methods. In addition, mAb potency was measured by enzyme-linked immunosorbent assay (ELISA).

Results

The split peaks had identical masses, and the root cause of the peak splitting was identified as isomerization of an aspartic acid located in the complementarity-determining region (CDR) of the light chain. And the early eluting and late eluting peaks were collected and performed enzymatic digestion to confirm the isoAsp enrichment in the early eluting peak. In addition, decreased potency was observed in the same heat-stressed sample, and the increased isoAsp levels in the CDR correlate well with a decrease of potency.

Conclusion

Liquid chromatography-mass spectrometry (LC–MS) has been utilized extensively to assess PQAs of biological therapeutics. In this study, a generalizable LC–MS-based approach was developed to enable identification and quantitation of the isoAsp-containing peptides.

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Data Availability

The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. LC-MS raw data were generated at ProtaGene US, Inc.and ELISA raw data were generated at Analytical Research & Development, Merck & Co., Inc.. Due to the research privacy and legal/commercial restrictions, raw data are not available. Please contact the corresponding authors for questions and concerns.

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Acknowledgements

This work was funded and supported by Analytical Research & Development, Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA. The authors would like to thank Dr. Christopher Barton, Hongxia Wang and Rahul Upadhya for their helpful discussions and/or suggestions about the manuscript. We also thank Dr. Wanlu Qu for Intact LC-MS data collection of the stability study. The authors thank Ping Zhou for assistance with data collection of stability samples.

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Author notes

  1. Yue Zhang

    Present address: Biogen, 225 Binney Street, Cambridge, MA, 02142, USA

  2. Dongdong Wang

    Present address: Takeda Pharmaceutical Company, 35 Landsdowne St, Cambridge, MA, 02139, USA

Authors and Affiliations

  1. ProtaGene US, Inc. was Formerly BioAnalytix Inc., 4 Burlington Woods Dr., Burlington, MA, 01803, USA

    Yanjun Liu, Yue Zhang, Dongdong Wang & Jennifer S. Chadwick

  2. Analytical Research & Development, Merck & Co., Inc., 126 E. Lincoln Ave, Rahway, NJ, 07065, USA

    Zac VanAernum, Xinliu Gao, Mariana Vlad, Bo Feng, Robert Cross, Bruce Kilgore, Alice Newman, Hillary A. Schuessler & Douglas D. Richardson

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  1. Yanjun Liu
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Correspondence to Yanjun Liu or Zac VanAernum.

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Liu, Y., VanAernum, Z., Zhang, Y. et al. LC–MS Approach to Decipher a Light Chain Chromatographic Peak Splitting of a Monoclonal Antibody. Pharm Res 40, 3087–3098 (2023). https://doi.org/10.1007/s11095-023-03631-9

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