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Antibodies, also known as immunoglobulins (Ig), are essential players of the adaptive immune system. These antigen-binding proteins are produced by B cells and make up 20 percent of the total blood plasma by weight. In mammals, antibodies fall into five different classes, which each elicits a different biological response upon antigen binding.
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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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Cooperative allosteric transitions can occur in multimeric proteins, where each subunit of the protein has its own ligand-binding site. When a ligand binds to any of these subunits, it triggers a conformational change that affects the binding sites in the other subunits; this can change the affinity of the other sites for their respective ligands. The ability of the protein to change the shape of its binding site is attributed to the presence of a mix of flexible and stable segments in the...
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Antibodies, or immunoglobulins, are critical players in the immune system's arsenal against invading pathogens. Produced by B cells and plasma cells, their primary role is to detect and bind to specific antigens, molecules found on the surface of pathogens like bacteria or viruses. Beyond antigen recognition, antibodies perform several vital functions that contribute to immune defense.
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Heterotrimeric G proteins are guanine nucleotide-binding proteins. As the name suggests, heterotrimeric G proteins are composed of three subunits: alpha, beta, and gamma. They remain GDP-bound or GTP-bound inside the cells and switch between inactive/active states. The Gα subunit possesses the nucleotide-binding pocket that binds guanine nucleotides and switches between GDP or GTP-bound states. In contrast, the Gꞵ and Gγ subunits are always bound together with high...
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Immunoprecipitation, or IP, is a widely used technique that employs protein-antibody interactions to isolate proteins or protein complexes in their native state for studying protein-protein interactions, quaternary structures, or supramolecular complexes. Various modifications of the technique, including chromatin IP, cross-linking IP, and fluorescence IP, are commonly used.
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Protein A does not induce allosteric structural changes in an IgG1 antibody during binding.

Juan P Rincon Pabon1, Brent A Kochert2, Yan-Hui Liu2

  • 1Department of Chemistry and the Ralph N. Adams Institute for Bioanalytical Chemistry, University of Kansas, Lawrence, KS, United States.

Journal of Pharmaceutical Sciences
|February 28, 2021
PubMed
Summary

Protein A affinity chromatography can alter antibody structure. Researchers used HX-MS to show that while Protein A resin does not cause reversible allosteric changes in NIST mAb, irreversible effects are still possible.

Keywords:
Deuterium exchangeMass spectrometry (MS)Monoclonal antibody(s)Protein binding

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Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions
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Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions

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Area of Science:

  • Biochemistry
  • Protein Chemistry
  • Analytical Chemistry

Background:

  • Affinity chromatography, particularly with Protein A (ProA), is crucial for antibody purification in biopharmaceutical manufacturing.
  • Potential structural alterations in antibodies during affinity chromatography, specifically allosteric changes, remain underexplored.

Purpose of the Study:

  • To investigate conformational changes in NIST mAb induced by Protein A (ProA) binding using hydrogen exchange-mass spectrometry (HX-MS).
  • To differentiate between allosteric effects in solution versus on-resin binding conditions.

Main Methods:

  • Hydrogen exchange-mass spectrometry (HX-MS) was employed to monitor antibody backbone dynamics.
  • NIST mAb was studied in complex with both in-solution and resin-bound forms of Protein A (ProA) under various molar ratios.

Main Results:

  • ProA binding to NIST mAb in solution and on resin protected the CH2 and CH3 domains from hydrogen exchange (HX), confirming the known binding site.
  • Increased ProA concentration in solution (2:1 ProA:mAb ratio) induced higher HX uptake in the Fab regions, suggesting allosteric effects and increased flexibility.
  • These allosteric effects were not observed at lower ProA concentrations or with ProA resin, indicating a potential change in binding mode.

Conclusions:

  • Reversible allosteric structural changes in NIST mAb are unlikely during interaction with ProA resin under typical pharmaceutical purification conditions.
  • The possibility of irreversible structural modifications to NIST mAb from prior ProA exposure during initial purification cannot be excluded.