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Antibody Structure and Classes01:25

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Antibodies, also known as immunoglobulins, are produced by B cells in response to foreign substances, such as bacteria and viruses. These proteins are critical for recognizing and neutralizing these substances, protecting the body from potential harm.
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2D-CEX-FcRn-MS to Study Structure/Function Relation of mAb Charge Variants.

Liesa Verscheure1,2, Isabel Vandenheede1, Eline De Rore1

  • 1RIC group, President Kennedypark 26, Kortrijk B-8500, Belgium.

Analytical Chemistry
|October 29, 2024
PubMed
Summary
This summary is machine-generated.

Automated 2D-LC-MS rapidly analyzes monoclonal antibody (mAb) charge variants and their FcRn binding affinity. Oxidation of heavy chains significantly reduces binding, enabling faster development of antibody therapeutics.

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Area of Science:

  • Biochemistry and Biophysics
  • Analytical Chemistry
  • Protein Therapeutics

Background:

  • Understanding the relationship between monoclonal antibody (mAb) structure and function is crucial for therapeutic development.
  • Assessing charge variants and their impact on neonatal crystallizable fragment receptor (FcRn) binding is essential for mAb efficacy.
  • Existing workflows for mAb characterization are often time-consuming and resource-intensive.

Purpose of the Study:

  • To develop and validate an automated two-dimensional liquid chromatography-mass spectrometry (2D-LC-MS) platform for simultaneous structural and functional analysis of mAb charge variants.
  • To elucidate the impact of specific modifications, particularly oxidation, on mAb binding affinity to FcRn.
  • To improve the efficiency and reduce the resource requirements of mAb characterization compared to established methods.

Main Methods:

  • Utilized a 2D-LC-MS system employing cation-exchange chromatography (CEX) in the first dimension and FcRn affinity chromatography in the second dimension.
  • Employed a multiple heart-cutting valve for automated collection and transfer of charge variants between dimensions.
  • Incorporated post-column denaturing solution addition to enhance MS sensitivity and utilized multidimensional LC-MS (mD-LC-MS) for detailed structural analysis.

Main Results:

  • Successfully resolved and characterized mAb charge variants induced by forced degradation.
  • Identified heavy chain (HC) M253 and M429 oxidation as primary drivers of reduced FcRn binding affinity, with increased oxidation leading to decreased binding.
  • Demonstrated a 7-fold improvement in throughput and a significant reduction in material requirements compared to traditional offline methods, with good correlation to ELISA binding data.

Conclusions:

  • The automated 2D-LC-MS platform provides a streamlined and efficient approach for multiattribute analysis of mAbs, integrating structural and functional assessments.
  • This method enables precise ranking of charge variants based on FcRn binding affinity, highlighting the critical role of oxidation.
  • The developed platform accelerates the development of safer and more effective antibody therapeutics by providing rapid, comprehensive mAb characterization.