Assessing Genetic Heterogeneity in Production Cell Lines: Detection by Peptide Mapping of a Low Level Tyr to Gln Sequence Variant in a Recombinant Antibody

Harris, Reed J.; Murnane, Amy A.; Utter, S L; Wagner, Karen; Cox, Edward T.; Polastri, Gian; Helder, Judith C.; Sliwkowski, Mary B.

doi:10.1038/nbt1193-1293

Cited by 56 publications

(70 citation statements)

References 26 publications

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“…34,35 For example, Harris et al found a Y376Q variant in the heavy chain of an anti-human epidermal growth factor receptor-2 antibody expressed in CHO cells. 3 Zhang et al reported the mutation of TAA (stop codon) to GAA, which caused light chain extension. 36 Dorai et al reported that a Phe to Leu sequence variant was observed in the recombinant peptide-antibody fusion protein, expressed in CHO cells.…”

Section: Discussionmentioning

confidence: 99%

“…2 However, in addition to expressing the product of interest, cells may also generate an array of sequence variants, which regarded as "product-related impurities." 3 Sequence variants, which result from unintended amino acid substitutions, are protein isoforms containing undesired amino acid sequences that may cause concern during the production of mAbs and other therapeutic proteins. [2][3][4][5][6][7] Therefore, it is necessary to detect potential sequence variants to confirm the purity and safety of the product during clone selection and bioprocess development.…”

Section: Introductionmentioning

confidence: 99%

“…3 Sequence variants, which result from unintended amino acid substitutions, are protein isoforms containing undesired amino acid sequences that may cause concern during the production of mAbs and other therapeutic proteins. [2][3][4][5][6][7] Therefore, it is necessary to detect potential sequence variants to confirm the purity and safety of the product during clone selection and bioprocess development. To ensure product safety, efficacy and consistency, manufacturers who produce the therapeutic mAbs are increasingly using advanced analytical methods and technologies during product characterization and development.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of alanine to valine sequence variants in the Fc region of nivolumab biosimilar produced in Chinese hamster ovary cells

Liu

et al. 2016

mAbs

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(2016) Characterization of alanine to valine sequence variants in the Fc region of nivolumab biosimilar produced in Chinese hamster ovary cells, mAbs, 8:5, 951-960, DOI: 10.1080/19420862.2016 To link to this article: https://doi.org/10. 1080/19420862.2016 ABSTRACTNivolumab is a therapeutic fully human IgG4 antibody to programmed death 1 (PD-1). In this study, a nivolumab biosimilar, which was produced in our laboratory, was analyzed and characterized. Sequence variants that contain undesired amino acid sequences may cause concern during biosimilar bioprocess development. We found that low levels of sequence variants were detected in the heavy chain of the nivolumab biosimilar by ultra performance liquid chromatography (UPLC) and tandem mass spectrometry. It was further identified with UPLC-MS/MS by IdeS or trypsin digestion. The sequence variant was confirmed through addition of synthetic mutant peptide. Subsequently, the mixing base signal of normal and mutant sequence was detected through DNA sequencing. The relative levels of mutant A424V in the Fc region of the heavy chain have been detected and demonstrated to be 12.25% and 13.54%, via base peak intensity (BPI) and UV chromatography of the tryptic peptide mapping, respectively. A424V variant was also quantified by real-time PCR (RT-PCR) at the DNA and RNA level, which was 19.2% and 16.8%, respectively. The relative content of the mutant was consistent at the DNA, RNA and protein level, indicating that the A424V mutation may have little influence at transcriptional or translational levels. These results demonstrate that orthogonal state-of-the-art techniques such as LC-UV-MS and RT-PCR should be implemented to characterize recombinant proteins and cell lines for development of biosimilars. Our study suggests that it is important to establish an integrated and effective analytical method to monitor and characterize sequence variants during antibody drug development, especially for antibody biosimilar products.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Characterization of alanine to valine sequence variants in the Fc region of nivolumab biosimilar produced in Chinese hamster ovary cells

Liu

et al. 2016

mAbs

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“…In recent years, numerous examples of such expression errors in production of recombinant proteins at various stages of the manufacturing process have been reported. [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] These reported examples are predominately single amino acid substitution caused by either DNA mutations [3][4][5][6][7][8][9][10][11] or misincorporation during translation, [12][13][14][15][16] which are commonly referred as sequence variants. Expression errors leading to alteration of longer segments of amino acid sequence have also been reported.…”

Section: Introductionmentioning

confidence: 99%

Identification and characterization of a -1 reading frameshift in the heavy chain constant region of an IgG1 recombinant monoclonal antibody produced in CHO cells

Lian

Wang

2015

mAbs

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Frameshifts lead to complete alteration of the intended amino acid sequences, and therefore may affect the biological activities of protein therapeutics and pose potential immunogenicity risks. We report here the identification and characterization of a novel -1 frameshift variant in a recombinant IgG1 therapeutic monoclonal antibody (mAb) produced in Chinese hamster ovary cells during the cell line selection studies. The variant was initially observed as an atypical post-monomer fragment peak in size exclusion chromatography. Characterization of the fragment peak using intact and reduced liquid chromatographymass spectrometry (LC-MS) analyses determined that the fragment consisted of a normal light chain disulfide-linked to an aberrant 26 kDa fragment that could not be assigned to any HC fragment even after considering common modifications. Further analysis using LC-MS/MS peptide mapping revealed that the aberrant fragment contained the expected HC amino acid sequence (1-232) followed by a 20-mer novel sequence corresponding to expression of heavy chain DNA sequence in the -1 reading frame. Examination of the DNA sequence around the frameshift initiation site revealed that a mononucleotide repeat GGGGGG located in the IgG1 HC constant region was most likely the structural root cause of the frameshift. Rapid identification of the frameshift allowed us to avoid use of a problematic cell line containing the frameshift as the production cell line. The frameshift reported here may be observed in other mAb products and the hypothesis-driven analytical approaches employed here may be valuable for rapid identification and characterization of frameshift variants in other recombinant proteins.

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“…In recent years, improvements in mass spectrometry (MS)-based characterization assays and associated analysis software has enabled detection of sequence variants, even when present at very low levels. Sequence variants can be classified into 3 general groups: N-terminal variants due to incomplete cleavage of the signal sequence, 1 single amino acid changes due to DNA mutations 2,3 or protein translation errors, [4][5][6][7] and sequence variants due to DNA re-arrangement and mis-splicing. 8 Detection of sequence variants typically employs the use of highresolution mass spectrometry assays, with peptide map analysis followed by computer algorithm searching of the data (such as programs like Mascot 9 or Mass Analyzer 10 ).…”

Section: Introductionmentioning

confidence: 99%

Rapid identification of an antibody DNA construct rearrangement sequence variant by mass spectrometry

Scott

Rogers

Balland

et al. 2014

mAbs

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During cell line development for an IgG1 antibody candidate (mAb1), a C-terminal extension was identified in 2 product candidate clones expressed in CHO-K1 cell line. The extension was initially observed as the presence of anomalous new peaks in these clones after analysis by cation exchange chromatography (CEX-HPLC) and reduced capillary electrophoresis (rCE-SDS). Reduced mass analysis of these CHO-K1 clones revealed that a larger than expected mass was present on a sub-population of the heavy chain species, which could not be explained by any known chemical or post-translational modifications. It was suspected that this additional mass on the heavy chain was due to the presence of an additional amino acid sequence. To identify the suspected additional sequence, de novo sequencing in combination with proteomic searching was performed against translated DNA vectors for the heavy chain and light chain. Peptides unique to the clones containing the extension were identified matching short sequences (corresponding to 9 and 35 amino acids, respectively) from 2 non-coding sections of the light chain vector construct. After investigation, this extension was observed to be due to the re-arrangement of the DNA construct, with the addition of amino acids derived from the light chain vector non-translated sequence to the C-terminus of the heavy chain. This observation showed the power of proteomic mass spectrometric techniques to identify an unexpected antibody sequence variant using de novo sequencing combined with database searching, and allowed for rapid identification of the root cause for new peaks in the cation exchange and rCE-SDS assays.

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Assessing Genetic Heterogeneity in Production Cell Lines: Detection by Peptide Mapping of a Low Level Tyr to Gln Sequence Variant in a Recombinant Antibody

Cited by 56 publications

References 26 publications

Characterization of alanine to valine sequence variants in the Fc region of nivolumab biosimilar produced in Chinese hamster ovary cells

Characterization of alanine to valine sequence variants in the Fc region of nivolumab biosimilar produced in Chinese hamster ovary cells

Identification and characterization of a -1 reading frameshift in the heavy chain constant region of an IgG1 recombinant monoclonal antibody produced in CHO cells

Rapid identification of an antibody DNA construct rearrangement sequence variant by mass spectrometry

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