Sequence Variant and Posttranslational Modification Analysis During Cell Line Selection via High-Throughput Peptide Mapping

  • Chong-Feng XuEmail author
  • Yan Wang
  • Pete Bryngelson
  • Zoran Sosic
  • Li Zang
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 1140)


Selection of high-producing lead and backup cell lines with high-fidelity primary structure is a major goal of cell line development of protein therapeutics. Conventional techniques for sequence variant analysis, such as mass spectrometry (MS) and next-generation sequencing (NGS) have limitations on the sample number and turnaround time, thus often are only applied at the final stages of development, where an undesired lead or backup clone could cause a significant delay in project timeline. Here we presented a high-throughput (HT) peptide mapping workflow which can be applied at early stages of cell line selection for testing of a batch of 30–40 clones within 2-week turnaround while reporting valuable information on sequence variants and posttranslational modifications (PTMs). The successful application of this workflow was demonstrated for two mAb programs. Multiple clones were removed from a total of 33 mAb-1 clones using various criteria: nine clones contained at least one >1% upregulated unknown peptide ions, 11 clones contained at least eight >0.1% upregulated unknowns, and six clones contained upregulated critical PTMs. For mAb-2, light chain (LC) sequence extension of approximately 30 amino acids were detected in 6 out of 36 clones at levels up to 11%. Besides, a Q to H mutation at ~30% was detected in the heavy chain (HC) of a single clone. Q to H mutation has mass change of 9.00 Da and failed to be detected by intact mass analysis. Rapid PTM quantitation also facilitated the selection of clones with desirable quality attributes, such as N-glycan profile. Hence, we demonstrated a risk-reducing strategy where abnormal clones could be detected at earlier stages of cell line selection, which should result in reduced and more predictable timeline of cell line development.


Sequence variant Posttranslational modification High throughput peptide mapping Clone selection Mutation Amino acid substitution Amino acid misincorporation Mass spectrometry Antibody 





Complementarity Determining Region


Cation exchange


Chinese hamster ovary






Heavy chain


Light chain


Mass spectrometer


Tandem mass spectrometry


Molecular weight


Parts per million


Reduced capillary electrophoresis-sodium dodecyl sulfate


Reversed phase ultra-high pressure liquid chromatography


Size exclusion chromatography


Trifluoracetic acid





We thank Dingyi Wen, Monika Vecchi, Susan Foley and Yaping Sun for detailed mass spectrometric characterization of mAb reference standards, Dan Cage for manuscript editing, Christina Alves and Shelly Martin for providing cell material and Kanvasri Jonnalagadda for protein purification. The authors declare no competing financial interest.


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Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Chong-Feng Xu
    • 1
    Email author
  • Yan Wang
    • 1
  • Pete Bryngelson
    • 1
  • Zoran Sosic
    • 1
  • Li Zang
    • 1
  1. 1.Analytical DevelopmentBiogenCambridgeUSA

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