Quantitative LC–MS/MS Method Refines rAAV9 Capsid Protein Stoichiometry

Researchers from the United Kingdom’s National Measurement Laboratory (NML) developed and evaluated a tailored mass spectrometry (MS)-based workflow for the detailed characterization and quantification of rAAV serotype 9 (rAAV9) capsid proteins. The approach integrates liquid chromatography (LC)-MS and LC-tandem mass spectrometry (MS/MS) analyses to achieve multiple objectives: confirm the amino acid sequence of the rAAV9 capsid proteins, identify and map potential PTMs, accurately quantify the VP1, VP2, and VP3 proteins to determine overall capsid stoichiometry, and identify host cell proteins in rAAV samples. To ensure high measurement confidence, data were made traceable to the International System of Units (SI) through amino acid analysis (AAA) of peptides used as quantification standards.

Their results demonstrated that the measured stoichiometry of the rAAV9 capsid deviates from the widely assumed 1:1:10 ratio, underscoring the importance of robust analytical methods for precise vector characterization. The MS-based strategy described here offers a comprehensive framework for standardizing rAAV capsid quantification and could serve as the foundation for developing reference methods and materials for gene therapy manufacturing and quality control. This work represents one of the first International System of Units (SI)-traceable, MS-based quantification strategies for rAAV capsid proteins, providing an essential step toward harmonized analytical standards in gene therapy development. By establishing a metrologically sound approach to rAAV characterization, the study contributes to improving measurement reproducibility across laboratories and supports the reliable production of safe and effective rAAV-based therapeutics.

LCGC International spoke to Theodoros Kontogiannis from the NML and the lead author of the paper (1) that resulted from this work, about the group’s findings.

How does liquid chromatography (LC) contribute to the separation and quantification of viral capsid proteins (VP1, VP2, VP3) in recombinant AAV analysis compared to electrophoretic methods such as sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)?

SDS-PAGE is a semi-quantitative method at best and has a limited linear dynamic range, which restricts its accuracy in determining capsid stoichiometry. As a result, staining intensity, especially when bands of very different intensities are observed such as VP3 compared to VP1 and VP2, does not reliably correlate with protein quantity across a wide range. Furthermore, staining intensity does not necessarily reflect true protein abundance due to differences in dye binding efficiencies and variability in protein stain interactions. In contrast, LC coupled to a triple quadrupole mass spectrometer enables precise quantification of signature peptides belonging to viral proteins, offering higher sensitivity, specificity, and reproducibility than SDS-PAGE for accurate capsid protein quantification, particularly when used with isotopically labeled peptides.

What are the key considerations when developing an LC–tandem mass spectrometry (MS/MS) method for quantifying rAAV capsid proteins, and how do these differ from traditional ultraviolet (UV)- or fluorescence-based LC methods?

When separating AAV capsid proteins using an LC method coupled to a detector, the analysis can be affected by issues such as peak tailing, overlapping peaks, and differential analyte recovery, all of which can compromise quantitative accuracy. Due to the very similar hydrophobicity of VP1, VP2, and VP3, achieving complete chromatographic separation of the three capsid proteins is particularly challenging, often resulting in partial peak overlap. In contrast, LC–MS/MS methods overcome this limitation by quantifying capsid proteins through specific peptides associated with each VP. These peptides have unique mass-to-charge ratios which, upon fragmentation, produce highly predictable and specific product ions known as “transitions.” By monitoring multiple transitions per target peptide, the method achieves far greater specificity and selectivity than approaches that rely on UV or fluorescence detection, which is less selective and more susceptible to interference from non-target analytes. Furthermore, the use of isotopically labeled internal standard peptides improves accuracy and reproducibility by accounting for variability in recovery and ionization efficiency.

In your study (1), isotopically labeled peptides were used for quantification. How does LC–MS/MS exploit these labeled standards to achieve accurate and reproducible measurements?

By employing isotopically labeled peptides, we account for biases in LC recovery, ionization efficiency, and sample preparation.Quantification is based on calculating the peak area ratio between each endogenous peptide and its isotopically labeled counterpart. Because both peptides share identical physicochemical properties and co-elute under the same chromatographic conditions, any bias in LC recovery, ionization efficiency, or sample handling affects them equally. For example, if one peptide shows low LC recovery, then the isotopically labeled counterpart will also show low LC recovery, and this will affect the peak intensities of both. Therefore, regardless of whether the LC recovery of a peptide is low or high, this variation will equally affect the corresponding labeled peptide. As a result, the peak area ratio between the endogenous and labeled peptide, which is used for quantitation by interpolating the value in the relevant standard curve, remains consistent and reliable.

Why is peptide mapping using LC–MS/MS an essential step in confirming the amino acid sequence and identifying post-translational modifications (PTMs) in rAAV capsid proteins?

Peptide mapping using LC-MS/MS is used to verify the protein sequence and detect any deviations or modifications that could impact the capsid’s properties and the quantification strategy. Specifically, PTMs or sequence deviations can alter capsid interactions with host-cell components, affecting infectivity, stability, and tropism, and ultimately impacting the safety and efficacy of viral vectors.

What role does chromatographic peak area integration play in determining the VP1:VP2:VP3 stoichiometry and how can differences in chromatographic behavior between peptides affect quantitative accuracy?

Peak area integration is essential to determining capsid stoichiometry because the peak areas directly reflect the abundance of each peptide. Accurate integration is critical, as issues such as peak tailing or overlapping peaks can distort the measured areas and compromise measurement accuracy. Selecting transitions with low background noise and minimal tailing is therefore essential for reliable quantification. Additionally, peptides can exhibit different chromatographic behaviors due to their different physicochemical properties, leading to differential LC recovery. For that reason, using isotopically labeled peptides as internal standards accounts for these differences, improving the accuracy and reproducibility of the quantification.

How does LC–MS/MS improve accuracy and traceability in capsid protein quantification when compared to enzyme-linked immunosorbent assay (ELISA) and SDS-PAGE?

SDS-PAGE is only semi-quantitative and ELISA, depending upon the antibody, and kit available, normally measures only total capsid content without distinguishing individual capsid proteins. Therefore, ELISA cannot be used to determine capsid protein stoichiometry.In contrast, the developed LC–MS/MS method quantifies each capsid protein by measuring the abundance of signature unique peptides in SI units. The method’s traceability is established by measuring the concentration of synthesized natural peptides that were used to generate the standard curves through amino acid analysis, linking the quantification to known standards. Therefore, the method provides traceability to SI and enables data comparability across laboratories. However, a peptide-based quantification method will also quantify free protein not associated with an intact capsid.

What chromatographic parameters would you monitor or optimize to ensure the reliable detection and separation of closely related viral proteins or peptide variants in rAAV samples?

For the LC–MS/MS method, it is important to monitor multiple transitions and ideally select three with high intensity to improve the sensitivity of the method. One transition is the quantifier, which is used for the quantification of the target peptide, and two of them should ideally be used as qualifier transitions to ensure that the correct target is being quantified. Low background noise and peak tailing are also important to enhance the specificity and reliability of the method. For the separation of intact capsid proteins, the selection of the appropriate column, mobile phase composition, and optimization of the gradient profile are very important. It is important to note that with the use of a multiple reaction monitoring (MRM) method, peaks may co-elute without contributing to accurate quantification of separate VPs, unlike in UV-based methods.

How can LC–MS–based characterization of host cell proteins (HCPs) and deamidation sites inform the purification strategy and quality control of rAAV production?

Identification and quantification of HCPs allow us to see which HCPs are commonly present in AAV batches, and if they have a high risk of immunogenicity. This information is important for the development of tailored purification strategies to remove these HCPs and ensure the safety of the product. Similarly, monitoring deamidation is important because it can affect the stability of the viral capsid and the infectivity of the vector. Identifying deamidation sites and tracking their occurrence under different storage conditions, such as variations in temperature, light, and pH, enables optimization of storage and formulation to minimize deamidation and thus maintain infectivity.

Given the observed deviation from the expected 1:1:10 capsid stoichiometry, what chromatographic or analytical factors might contribute to measurement discrepancies between different analytical methods?

The capsid stoichiometry is not fixed but instead depends on the expression levels of each capsid protein. Therefore, variations in the production methods used can result in altered expression levels of the VPs between studies, leading to changes in stoichiometry. Additionally, the measured stoichiometry can be affected by the analytical method used. As mentioned previously, the semi-quantitative nature and inherent biases of SDS-PAGE, as well as recovery biases and peak tailing in LC methods, can skew the results. Effective separation of the three capsid proteins is therefore essential for obtaining more reliable measurements.

References

1. Kontogiannis, T.; McElroy, C.; Quaglia, M. et al. Development of LC-MS Methods for AAV Capsid Protein Quantification and Host Cell Protein Profiling. Mol. Ther. Methods Clin. Dev. 2025, 33 (3), 101562. DOI: 10.1016/j.omtm.2025.101562