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Detailed mass analysis of structural heterogeneity in monoclonal antibodies using native mass spectrometry

Nature Protocols volume 9, pages 967–976 (2014)Cite this article

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Abstract

The molecular complexity of biopharmaceuticals puts severe demands on the bioanalytical techniques required for their comprehensive structural characterization. Mass spectrometry (MS) has gained importance in the analysis of biopharmaceuticals, taking different complementary approaches ranging from peptide-based sequencing to direct analysis of intact proteins and protein assemblies. In this protocol, we describe procedures optimized to perform the analysis of monoclonal antibodies (mAbs) at the intact protein level under pseudo-native conditions, using native MS. Some of the strengths of native MS in the analysis of biopharmaceuticals are its analysis speed, sensitivity and specificity: for most experiments, the whole protocol requires one working day, whereby tens of samples can be analyzed in a multiplexed manner, making it suitable for high-throughput analysis. This method can be used for different applications such as the analysis of mixtures of mAbs, drug-antibody conjugates and the analysis of mAb post-translational modifications, including the qualitative and quantitative analysis of mAb glycosylation.

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Figure 1: Schematic structure of an IgG1 antibody.
Figure 2: Schematic of two mass spectrometers applied in the native analysis of mAbs.
Figure 3: Typical workflow for native MS analysis of mAbs.
Figure 4: Specific glycan digestion under nondenaturing conditions on intact mAbs.
Figure 5: Detailed images of sample loading before injection.
Figure 6: Qualitative and quantitative analysis of a mixture of four different mAbs.
Figure 7: Detailed mass analysis of heterogeneous glycosylation occurring on an IgG1 Y407E mutant antibody.

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Acknowledgements

This work, and in particular that of S.R., A.B. and A.J.R.H., was supported in part by Stichting voor de Technische Wetenschappen (STW) (project 10805) and Y.Y. and A.J.R.H. are supported by the ManiFold project, grant agreement number 317371. We further acknowledge support of the PRIME-XS project, grant agreement number 262067, funded by the European Union Seventh Framework Program. The Netherlands Proteomics Centre, embedded in The Netherlands Genomics Initiative, is acknowledged for funding.

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Author notes

  1. Sara Rosati and Yang Yang: These authors contributed equally to this work.

Authors and Affiliations

  1. Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands

    Sara Rosati, Yang Yang, Arjan Barendregt & Albert J R Heck

  2. Netherlands Proteomics Centre, Utrecht, The Netherlands

    Sara Rosati, Yang Yang, Arjan Barendregt & Albert J R Heck

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Contributions

S.R., Y.Y., A.B. and A.J.R.H. developed the protocols described here, obtained the presented data and wrote the manuscript.

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Correspondence to Albert J R Heck.

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Rosati, S., Yang, Y., Barendregt, A. et al. Detailed mass analysis of structural heterogeneity in monoclonal antibodies using native mass spectrometry. Nat Protoc 9, 967–976 (2014). https://doi.org/10.1038/nprot.2014.057

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