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Mitigation of pre-existing immunogenicity in ADA method development.

Jihua Chen1, Henry Zhao1, Chris Gardner1

  • 1Bioanalytical Development, Alexion Pharmaceuticals, AstraZeneca Rare Disease, New Haven, CT, USA.

Bioanalysis
|February 3, 2026
PubMed
Summary

Preexisting immune reactivity in patients can challenge the development of anti-drug antibody (ADA) assays for biological therapeutics. A modified ADA assay effectively mitigates this interference, ensuring reliable immunogenicity assessments.

Keywords:
ADA assay cut pointPreexisting reactivityanti-drug antibody (ADA)immunogenicitytreatment-boosted ADA responsetreatment-emergent ADA response

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Area of Science:

  • Biologics
  • Immunology
  • Assay Development

Background:

  • Immunogenicity assessments are crucial for biological therapeutics.
  • Preexisting immune reactivity complicates anti-drug antibody (ADA) assay development and cut point determination.
  • In the ALXN-X program, ~50% of samples showed significant preexisting reactivity, challenging assay methods.

Purpose of the Study:

  • To characterize the nature of preexisting reactivity against ALXN-X.
  • To develop a modified ADA assay to mitigate interference from preexisting reactivity.
  • To ensure reliable immunogenicity assessment of ALXN-X therapeutics.

Main Methods:

  • Competitive inhibition experiments were used to map binding sites.
  • Depletion with protein A/G agarose characterized reactivity.
  • A modified ADA assay utilized an engineered drug molecule.

Main Results:

  • Preexisting reactivity targets the C-terminal conserved framework sequence of ALXN-X.
  • This reactivity is likely immunoglobulin-mediated.
  • The modified ADA assay effectively mitigated interference while maintaining sensitivity for ALXN-X-specific ADA.

Conclusions:

  • Preexisting immunoglobulin-mediated reactivity against ALXN-X's C-terminal sequence complicates ADA assays.
  • A modified ADA assay successfully mitigates this interference.
  • The modified assay supports reliable immunogenicity assessment by detecting ALXN-X-specific ADA.