Quantification of Human Papillomavirus (HPV) Antigens in the Nonavalent Vaccine GARDASIL 9

Importance of the Topic

Accurate quantification and identity testing of vaccine antigens are critical for quality control and batch release in vaccine manufacturing. Traditional assays like total protein tests and ELISA often suffer from limited specificity, slow development times, and poor multiplexing, especially for complex multivalent vaccines such as GARDASIL 9. Implementing a targeted LC-MS/MS workflow addresses these challenges by providing selective, simultaneous measurement of multiple protein antigens.

Objectives and Study Overview

This study aimed to develop and validate a robust, reproducible LC-MS/MS method for the simultaneous quantification of nine HPV L1 protein antigens in the nonavalent GARDASIL 9 vaccine. Key goals included optimizing sample preparation (adjuvant desorption and tryptic digestion), selecting surrogate peptides for each HPV strain, and establishing MRM transitions for quantitative monitoring over the expected concentration range.

Methodology and Instrumentation

A complete sample preparation workflow was implemented:

• Adjuvant desorption: MS-compatible surfactant (RapiGest SF) at pH 10.0 to disrupt aluminum hydroxyphosphate sulfate binding and VLP structure.
• Tryptic digestion: Overnight digestion at a 1:10 trypsin:protein ratio to ensure complete peptide generation and minimize missed cleavages.
• Peptide mapping: LC-MSE analysis on a UPLC-QTof system to identify high-intensity, modification-free surrogate peptides for each HPV L1 protein.
• MRM assay development: Optimization of precursor/product ion transitions and collision energies via a MassLynx–Skyline interface.

Used Instrumentation

• ACQUITY UPLC I-Class PLUS with PREMIER Peptide BEH C18 column (2.1 × 100 mm, 1.7 µm, 300 Å)
• Xevo TQ-S micro Triple Quadrupole MS (ESI+ mode)
• Xevo G2-XS QTof Mass Spectrometer
• QuanRecovery 96-well plates with MaxPeak HPS surfaces
• MassLynx v4.2, TargetLynx XS v4.2 and UNIFI v1.9.4 software

Main Results and Discussion

The finalized LC-MS/MS method achieved simultaneous quantification of all nine HPV L1 antigens in a single 7-minute run time. Calibration curves were linear from 10–200 µg/mL (16-strain up to 20–200 µg/mL), with QC precision and accuracy CVs <8%. Inter- and intra-day reproducibility across three days of validation also yielded CVs <8%. The PREMIER column with MaxPeak HPS technology doubled peptide peak areas and accelerated column passivation. High-pH desorption and optimized digestion parameters were essential to recover antigens from the aluminum adjuvant and generate reproducible surrogate peptides.

Benefits and Practical Applications

• High selectivity and multiplexing: Quantify nine HPV antigens simultaneously versus single-analyte ELISAs.
• Rapid analysis: Seven-minute LC-MS/MS run enabling high throughput.
• Robust performance: CVs <8% ensure reliable QC data for batch release.
• Adaptable workflow: The approach can be tailored to other adjuvanted vaccines and large-molecule assays.

Future Trends and Potential Applications

The described LC-MS/MS strategy is poised for further automation and integration into GMP environments. Future developments may include expanding the panel to additional antigen targets, shorter chromatographic gradients, and coupling with high-resolution MS for simultaneous identity confirmation. The surrogate peptide approach can be extended to multi-component biologics beyond HPV vaccines, enabling streamlined vaccine characterization and regulatory compliance.

Conclusion

This study demonstrates a validated LC-MS/MS method for the selective, reproducible quantification of nine HPV L1 protein antigens in GARDASIL 9. Optimized desorption, digestion, chromatographic separation, and MRM detection deliver rapid, multiplexed measurements with high precision and accuracy, supporting efficient vaccine quality control and batch release workflows.

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