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Development and implementation of an LC-MS-based multi-attribute method for adeno-associated virus.

Thomas W Powers1, Shawn Mariani1, Halyna Narepekha1

  • 1Pfizer Inc., Analytical Research and Development, 875 Chesterfield Pkwy. West, Chesterfield, MO 63017, USA.

Molecular Therapy. Methods & Clinical Development
|September 8, 2025
PubMed
Summary
This summary is machine-generated.

A multi-attribute method (MAM) quantifies peptide modifications in adeno-associated virus (AAV) therapeutics. This validated method accurately measures deamidation and other modifications, crucial for biotherapeutic development and quality control.

Keywords:
adeno-associated virus, AAVcapsid deamidationcapsid modificationsgene therapymulti-attribute method

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Area of Science:

  • Biopharmaceutical analysis
  • Mass spectrometry
  • Protein characterization

Background:

  • Multi-attribute methods (MAM) are increasingly vital for biotherapeutic characterization.
  • Adeno-associated virus (AAV) therapeutics require precise quantification of capsid modifications impacting transduction efficiency.

Purpose of the Study:

  • To develop and validate a robust peptide map MAM for quantifying modifications in AAV capsid proteins.
  • To assess the suitability of the MAM for monitoring deamidation and other modifications throughout biotherapeutic development.

Main Methods:

  • Characterization of an AAV product to identify capsid sequence liabilities.
  • Development and validation of a peptide map MAM according to ICH Q2(R2) guidelines.
  • Application of the validated MAM in stability, process development, and comparability studies.

Main Results:

  • A peptide map MAM was successfully developed and validated for quantifying specific sites of deamidation on AAV capsid proteins.
  • The method demonstrated suitability for monitoring deamidation during various stages of biotherapeutic development.
  • Preliminary findings indicated the MAM's applicability to a broader range of post-translational and chemical modifications.

Conclusions:

  • The validated MAM is a suitable and precise tool for quantifying AAV capsid modifications, particularly deamidation.
  • This analytical approach supports quality control and development of AAV-based biotherapeutics.
  • The MAM shows potential for monitoring diverse modifications beyond deamidation in biopharmaceutical applications.