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Forced Degradation Testing as Complementary Tool for Biosimilarity Assessment.

Yan Felix Karl Dyck1,2, Daniel Rehm1,3, Jan Felix Joseph1,4

  • 1Department of Pharmaceutical & Medicinal Chemistry, Institute of Pharmacy, Freie Universität Berlin, Königin-Luise-Straße 2+4, 14195 Berlin, Germany.

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Summary
This summary is machine-generated.

Oxidation of monoclonal antibodies (mAbs) impacts efficacy. A middle-up LC-QTOF-MS approach revealed oxidation sites in bevacizumab and infliximab, aiding biosimilarity assessment and quality control.

Keywords:
QTOF-MSbevacizumabbiopharmaceuticalsbiosimilarforced stability testinginfliximabliquid chromatography-mass spectrometry (LC-MS)middle-up approachstructure reactivity relationship

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Area of Science:

  • Biopharmaceutical analysis
  • Protein chemistry
  • Analytical chemistry

Background:

  • Oxidation of monoclonal antibodies (mAbs) can affect their efficacy and function.
  • Oxidation is a critical quality attribute (CQA) requiring careful evaluation in protein drug development.
  • Understanding oxidation susceptibility is crucial for ensuring drug quality and consistency.

Purpose of the Study:

  • To investigate the oxidation susceptibility of bevacizumab and infliximab under forced oxidative stress.
  • To identify specific oxidation sites within these monoclonal antibodies using a middle-up approach.
  • To compare the oxidation profiles of reference products (RP) and biosimilar (BS) versions of bevacizumab and infliximab.

Main Methods:

  • Subjecting bevacizumab and infliximab to oxidative stress using hydrogen peroxide (H2O2) for varying durations (24, 48, 72 h).
  • Employing a middle-up approach with liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) for detailed analysis.
  • Comparing post-translational modification profiles of unstressed and stressed reference products (RP) and biosimilars (BS).

Main Results:

  • The Fc/2 subunit of both mAbs was completely oxidized under stress conditions.
  • Specific oxidation sites were identified in the light chain (LC) and Fd' subunit of infliximab (M55 and M18, respectively), but not in bevacizumab.
  • While unstressed products showed high similarity, bevacizumab biosimilar (BS) exhibited higher oxidation susceptibility than its reference product (RP) after forced oxidation.

Conclusions:

  • The middle-up LC-QTOF-MS approach effectively probes mAb oxidation susceptibility.
  • Forced oxidation can reveal subtle differences in oxidation profiles between reference and biosimilar products.
  • This methodology is valuable for biopharmaceutical engineering, biosimilarity assessment, and quality control of protein therapeutics.