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Utility of High Resolution NMR Methods to Probe the Impact of Chemical Modifications on Higher Order Structure of Monoclonal Antibodies in Relation to Antigen Binding

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Abstract

Purpose

An understanding of higher order structure (HOS) of monoclonal antibodies (mAbs) could be critical to predicting its function. Amongst the various factors that can potentially affect HOS of mAbs, chemical modifications that are routinely encountered during production and long-term storage are of significant interest.

Methods

To this end, two Pfizer mAbs were subjected to forced deamidation stress for a period of eight weeks. Samples were aliquoted at various time points and high resolution accurate mass liquid chromatography-mass spectrometry (LC-MS/MS) was performed using low-artifact trypsin digestion (LATD) peptide mapping to identify and quantify chemical modifications. 2D backbone amide and sidechain methyl NMR spectra were acquired to gauge the effect of HOS changes upon chemical modification. Differential scanning calorimetry was also performed to assess the effect of thermal stability of mAbs upon modification. Finally, functional studies via target-binding based ELISA were performed to connect HOS changes to any loss of potency.

Results

The extent of deamidation in the mAb domains were quantified by LC-MS/MS. The HOS changes as obtained from 2D NMR were mostly localized around the affected sites leaving the overall structure relatively unchanged. The antigen-antibody binding of the mAbs, in spite of deamidation in the Fab region, remains unchanged.

Conclusion

This case study provides an integrated approach of relating chemical modifications in mAb domains with possible changes in HOS. This can be potentially used to assess a possible loss of potency within the structure-function paradigm of proteins in an orthogonal manner.

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Acknowledgments and Disclosures

SM and AAI acknowledge the support from the Pfizer Worldwide R&D postdoctoral program and the leadership team in Pfizer Biotherapeutics Pharmaceutical Sciences and Pharmaceutical R&D. The authors gratefully acknowledge Dr. Markus Voehler in scheduling and assisting with NMR experiments at the high resolution Biomolecular NMR facility at Vanderbilt University. Sincere thanks to Hai-Young Kim and David Cirelli for their critical review of the manuscript. The authors thank Jamie Lee and Jason Kennedy for generating ELISA data.

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Authors and Affiliations

  1. Pharmaceutical Research and Development, BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., Chesterfield, MO, 63017, USA

    Subhabrata Majumder & Arun Alphonse Ignatius

  2. Analytical Research and Development, BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., Andover, MA, 01810, USA

    Andrew Saati, Shibu Philip, Lucy L. Liu, Elaine Stephens & Jason C. Rouse

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  1. Subhabrata Majumder
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  2. Andrew Saati
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  3. Shibu Philip
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Majumder, S., Saati, A., Philip, S. et al. Utility of High Resolution NMR Methods to Probe the Impact of Chemical Modifications on Higher Order Structure of Monoclonal Antibodies in Relation to Antigen Binding. Pharm Res 36, 130 (2019). https://doi.org/10.1007/s11095-019-2652-1

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