Use of an integrated MS – multiplexed MS/MS data acquisition strategy for high‐coverage peptide mapping studies

Chakraborty, Asish; Berger, Scott J.; Gebler, John C.

doi:10.1002/rcm.2888

Cited by 70 publications

(62 citation statements)

References 47 publications

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“…Chakraborty and colleagues had applied LC-MS E for analyzing peptide maps of a protein digest. 36 High sequence coverage and good analytical reproducibility were reached in replicated analyses of protein digestion.…”

Section: Resultsmentioning

confidence: 99%

Versatile characterization of glycosylation modification in CTLA4-Ig fusion proteins by liquid chromatography-mass spectrometry

Zhu

Guo

Guo³

et al. 2014

mAbs

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These authors contributed equally to this work.Keywords: characterization, CTLA4-Ig fusion protein, glycan, glycosylation modification, intact protein, mass spectrometry, peptide mapping, similarity CTLA4-Ig is a highly glycosylated therapeutic fusion protein that contains multiple N-and O-glycosylation sites. Glycosylation plays a vital role in protein solubility, stability, serum half-life, activity, and immunogenicity. For a CTLA4-Ig biosimilar development program, comparative analytical data, especially the glycosylation data, can influence decisions about the type and amount of animal and clinical data needed to establish biosimilarity. Because of the limited clinical experience with biosimilars before approval, a comprehensive level of knowledge about the biosimilar candidates is needed to achieve subsequent development. Liquid chromatography-mass spectrometry (LC-MS) is a versatile technique for characterizing N-and O-glycosylation modification of recombinant therapeutic proteins, including 3 levels: intact protein analysis, peptide mapping analysis, and released glycans analysis. In this report, an in-depth characterization of glycosylation of a candidate biosimilar was carried out using a systematic approach: N-and O-linked glycans were identified and electron-transfer dissociation was then used to pinpoint the 4 occupied O-glycosylation sites for the first time. As the results show, the approach provides a set of routine tools that combine accurate intact mass measurement, peptide mapping, and released glycan profiling. This approach can be used to comprehensively research a candidate biosimilar Fc-fusion protein and provides a basis for future studies addressing the similarity of CTLA4-Ig biosimilars.

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Section: Resultsmentioning

confidence: 99%

Versatile characterization of glycosylation modification in CTLA4-Ig fusion proteins by liquid chromatography-mass spectrometry

Zhu

Guo

Guo³

et al. 2014

mAbs

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show abstract

“…LC-MS E can be used for analyzing peptide maps of the protein digest. 31 More than 95% sequence coverage and good analytical reproducibility were reached in replicated analyses of antibody digestion. Consequently, peptide mapping was used to study protein sequences variant and PTM diffrences between the biosimilar and the reference proteins.…”

Section: Identification Of the Ala To Val Variant By Tryptic Peptide mentioning

confidence: 99%

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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“…Furthermore, newly developed methods for label-free quantification as SWATH [70] and MS E [71,72] could gain in importance for neuroproteomics analysis due to high technical reproducibility and accuracy. Both techniques rely on data-independent acquisition of ion spectra.…”

Section: Label-free Quantificationmentioning

confidence: 99%

Neuroproteomics tools in clinical practice

Shevchenko

Konzer

Musunuri

et al. 2015

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) are characterized by neuronal impairment that leads to disease-specific changes in the neuronal proteins. The early diagnosis of these disorders is difficult, thus, the need for identifying, developing and using valid clinically applicable biomarkers that meet the criteria of precision, specificity and repeatability is very vital. The application of rapidly emerging technology such as mass spectrometry (MS) in proteomics has opened new avenues to accelerate biomarker discovery, both for diagnostic as well as for prognostic purposes. This review summarizes the most recent advances in the mass spectrometry-based neuroproteomics and analyses the current and future directions in the biomarker discovery for the neurodegenerative diseases.

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Use of an integrated MS – multiplexed MS/MS data acquisition strategy for high‐coverage peptide mapping studies

Cited by 70 publications

References 47 publications

Versatile characterization of glycosylation modification in CTLA4-Ig fusion proteins by liquid chromatography-mass spectrometry

Versatile characterization of glycosylation modification in CTLA4-Ig fusion proteins by liquid chromatography-mass spectrometry

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

Neuroproteomics tools in clinical practice

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