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Beyond PTMs: Novel Charge Variants Discovered in icIEF Profiling of PEGylated Proteins

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Abstract

icIEF (imaged capillary isoelectric focusing) charge variants characterization of biologics is important for understanding product charge isoforms and how they affect product quality. Protein post translational modifications (PTMs), such as deamidation, sialylation, glycation, c-terminal lysine cleavage and c-terminal glycine amidation, are common sources of acidic and basic charge variants of proteins and antibodies. Our PEGylated protein was expressed in E. coli which significantly reduced the PTMs, only deamidation and carbamylation were observed as charge variants related PTMs by LC–MS peptide mapping analysis. Deamidation introduces negative charge and was experimentally confirmed to be the major contributor of the acidic peaks in icIEF charge profiling of our PEGylated protein. The possible PTMs for basic variants were screened, but they were not observed by LC–MS peptide mapping. Beyond PTM induced charge heterogeneity, we found that N terminal methionine excision (NME) and residual un-PEGylated protein contributed to the basic peaks. NME is common in E. coli expression which is often used for PEGylated proteins; thus, it could be leveraged to charge variants characterization of other PEGylated proteins. Our finding of residual un-PEGylated protein contributing to basic peaks is being reported for the first time and could broaden the knowledge of possible charge variants in PEGylated proteins.

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Data Availability

All data and materials are available in a data repository.

Abbreviations

AEX:

Anion exchange chromatography

FC:

Fluorocarbon

HMW:

High molecular weight

IEF:

Isoelectric focusing

icIEF:

Imaged capillary isoelectric focusing

IEX:

Ion exchange chromatography

mAb:

Monoclonal antibody

MAP:

Methionine aminopeptidase

Met:

Methionine

NME:

N terminal methionine excision

PEG:

Polyethylene glycol

pI:

Isoelectric point

QC:

Quality control

v/v:

Volume to volume

w/v:

Weight to volume

w/w:

Weight to weight

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Acknowledgements

We thank Dr. Jeff Meyer for scientific discussions.

Funding

The authors declare that no funds and grants were received during the preparation of this manuscript.

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Authors and Affiliations

  1. Analytical Development and Analytical Attribute Science, Bristol-Myers Squibb, Devens, USA

    Xin Zhang & Richard Ludwig

  2. Global Process Analytical Science, Bristol-Myers Squibb, Devens, USA

    Letha Chemmalil, Chun Shao & Julia Ding

Authors
  1. Xin Zhang
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  2. Richard Ludwig
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  3. Letha Chemmalil
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  4. Chun Shao
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  5. Julia Ding
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Contributions

XZ conceived and carried out the experiments. XZ took the lead in writing the manuscript. RL, JD helped shape the research, analysis, and manuscript. All authors (RL, LC, CS, JD) provided critical review, comments, and scientific discussion.

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Correspondence to Xin Zhang.

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Zhang, X., Ludwig, R., Chemmalil, L. et al. Beyond PTMs: Novel Charge Variants Discovered in icIEF Profiling of PEGylated Proteins. Chromatographia 85, 1065–1073 (2022). https://doi.org/10.1007/s10337-022-04215-2

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  • DOI: https://doi.org/10.1007/s10337-022-04215-2

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