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

Abstract：

: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.

journal_name

Adv Exp Med Biol

journal_title

Advances in experimental medicine and biology

authors

Xu CF,Wang Y,Bryngelson P,Sosic Z,Zang L

doi

10.1007/978-3-030-15950-4_12

subject

Has Abstract

pub_date

2019-01-01 00:00:00

pages

225-236

eissn

0065-2598

issn

2214-8019

journal_volume

1140

pub_type

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