Fast and high-throughput LC-MS characterization, and peptide mapping of engineered AAV capsids using LC-MS/MS

Adeno-associated virus (AAV) has emerged as a leading platform for gene therapy. With the skyrocketing rate of AAV research and the prevalence of many new engineered capsids being investigated in preclinical and clinical trials, capsid characterization plays a vital role in serotype confirmation and quality control. Further, peptide mapping the capsid proteins might inevitably be a future requirement by regulatory agencies since it is a critical step in good manufacturing practice (GMP) for biotherapeutic characterization. To overcome many challenges that traditional methods like SDS-PAGE and western blots carry, liquid chromatography and mass spectrometry (LC-MS) allows high resolution and sensitivity with great accuracy in characterizing the AAV capsid proteins. Our optimized LC-MS method provides quick sample preparation, a fast and high-throughput 4-min run, and high sensitivity, which allows for very efficient characterization of wild-type and engineered capsids. This study also reports the usage of LC-MS/MS peptide mapping of AAV capsid proteins to determine the most accessible lysine residues targeted by chemical modifications. Our detailed protocols are anticipated to promote the development and discovery of AAV variants with high accuracy and efficiency.

• Publication year

  2022

• Venue

  Molecular Therapy: Methods & Clinical Development

• Publication date

  2022-09-01

• Fields of study

  Medicine, Engineering

• Identifiers
  DOI 10.1016/j.omtm.2022.09.008PMID 36284765PMCID 9563341
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• Genomic Designs of rAAVs Contribute to Pathological Changes in the Livers and Spleens of Mice

  2022cited by this paper
• Gene Therapy Advances: A Meta-Analysis of AAV Usage in Clinical Settings

  2022cited by this paper
• Optimized Reversed-Phase Liquid Chromatography/Mass Spectrometry Methods for Intact Protein Analysis and Peptide Mapping of Adeno-Associated Virus Proteins

  2021cited by this paper
• Chemical Modifications of the Capsid for Redirecting and Improving the Efficacy of Adeno-Associated Virus Vectors

  2021cited by this paper
• The clinical landscape for AAV gene therapies

  2021cited by this paper
• Optimizing Adeno-Associated Virus (AAV) Capsid Protein Analysis Using UPLC and UPLC-MS : Waters

  2020cited by this paper
• Capsid Modifications for Targeting and Improving the Efficacy of AAV Vectors

  2019cited by this paper
• Adeno-associated virus vector as a platform for gene therapy delivery

  2019cited by this paper
• Ligand Coupling to the AAV Capsid for Cell-Specific Gene Transfer.

  2019cited by this paper
• Prevalence of Anti-Adeno-Associated Virus Immune Responses in International Cohorts of Healthy Donors

  2019cited by this paper
• Site-Specific Glycation and Chemo-enzymatic Antibody Sortagging for the Retargeting of rAAV6 to Inflamed Endothelium

  2019cited by this paper
• A systematic capsid evolution approach performed in vivo for the design of AAV vectors with tailored properties and tropism

  2019cited by this paper
• Oligonucleotide conjugated multi-functional adeno-associated viruses

  2018cited by this paper
• Structure-guided evolution of antigenically distinct adeno-associated virus variants for immune evasion

  2017cited by this paper
• Direct Liquid Chromatography/Mass Spectrometry Analysis for Complete Characterization of Recombinant Adeno-Associated Virus Capsid Proteins

  2017cited by this paper
• Site-Specific PEGylated Adeno-Associated Viruses with Increased Serum Stability and Reduced Immunogenicity

  2017cited by this paper
• Syngeneic AAV Pseudo-particles Potentiate Gene Transduction of AAV Vectors

  2016cited by this paper
• A Precise Chemical Strategy To Alter the Receptor Specificity of the Adeno-Associated Virus.

  2016cited by this paper
• Vector design Tour de Force: integrating combinatorial and rational approaches to derive novel adeno-associated virus variants.

  2014cited by this paper
• Structural Insight into the Unique Properties of Adeno-Associated Virus Serotype 9

  2012cited by this paper
• PEG conjugation moderately protects adeno-associated viral vectors against antibody neutralization.

  2005cited by this paper

• Altering VP1 and VP2 expression in trans affects the transduction efficiency of AAV9

  2026cites this paper
• Identification of Recombinant Adeno-Associated Virus Serotypes by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.

  2026cites this paper
• Enabling icIEF Peak Identification of AAV Capsid Proteins by Fractionation on MauriceFlex and Subsequent Analysis by LC–MS

  2025cites this paper
• Development of an HEK293 Suspension Cell Culture Medium, Transient Transfection Optimization Workflow, and Analytics for Batch rAAV Manufacturing

  2025cites this paper
• Combination of hydrophilic interaction liquid chromatography and top-down mass spectrometry for characterisation of adeno-associated virus capsid proteins

  2025cites this paper
• Mass photometry as a robust method for characterizing adeno-associated virus critical quality attributes in gene therapy vector

  2025cites this paper
• Insights into Adeno-Associated Virus Capsid Charge Heterogeneity

  2025cites this paper
• Development and implementation of an LC-MS-based multi-attribute method for adeno-associated virus

  2025cites this paper
• Development of LC-MS methods for AAV capsid protein quantification and host cell protein profiling

  2025cites this paper
• Microflow LC-MS/MS reveals platform-specific post-translational modification signatures in recombinant adeno-associated virus capsids linked to enhanced potency and stability: A quality control framework for gene therapy.

  2025cites this paper
• Peptide Mapping for Sequence Confirmation of Therapeutic Proteins and Recombinant Vaccine Antigens by High-Resolution Mass Spectrometry: Software Limitations, Pitfalls, and Lessons Learned

  2025cites this paper
• Systematic formulation optimisation to enhance buffer exchange recovery and storage stability for adeno-associated virus (AAV2) vectors.

  2025cites this paper
• A recombinant lectin-like protein (MgLec-1) from Magallana gigas: Antimicrobial, antioxidant, and wound-healing promotion in infected diabetic wounds.

  2025cites this paper
• The state of technological advancement to address challenges in the manufacture of rAAV gene therapies.

  2024cites this paper
• Assessment of adeno-associated virus purity by capillary electrophoresis-based western

  2024cites this paper
• Quantitative proteomic analysis of residual host cell protein retention across adeno-associated virus affinity chromatography

  2024cites this paper
• Reviewing the process intensification landscape through the introduction of a novel, multitiered classification for downstream processing

  2024cites this paper
• B cell focused transient immune suppression protocol for efficient AAV readministration to the liver

  2024cites this paper
• Gene Doping Control Analysis of Human Erythropoietin Transgene in Equine Plasma by PCR-Liquid Chromatography High-Resolution Tandem Mass Spectrometry.

  2024cites this paper
• Drug Product Formulation and Fill/Finish Manufacturing Process Considerations for AAV-based Genomic Medicines.

  2024cites this paper
• Characterization of AAV vectors: A review of analytical techniques and critical quality attributes

  2024cites this paper
• Development of an icIEF assay for monitoring AAV capsid proteins and application to gene therapy products.

  2023cites this paper
• Chemical modification of AAV9 capsid with N-ethyl maleimide alters vector tissue tropism

  2023cites this paper
• Comprehensive Comparison of AAV Purification Methods: Iodixanol Gradient Centrifugation vs. Immuno-Affinity Chromatography

  2023cites this paper
• Characterization of Adeno-Associated Virus (AAV) Gene Therapy Products

  2023cites this paper