(T0930-06-32) Assessing Key Attributes of Adeno-Associated Viral Proteins Using HPLC-FLD-Intact Accurate Mass Analysis

Purpose: Recombinant Adeno-associated viral (rAAV) vectors have emerged as the leading gene delivery vehicles for gene therapy due to their high-efficiency transduction and safety. AAV capsid proteins (VPs) are critical for viral infectivity and vector potency and their key attributes, such as identity and relative ratio of VPs and their PTMs need to be fully characterized and monitored during the viral vector development and manufacturing to ensure the safety, quality, and efficacy of AAV products. To address this analytical need, we developed a HPLC-FLD-HRAM MS method for simultaneous measurement of relative expression ratios of VP1, VP2 and VP3 using FLD and direct accurate intact mass measurement of the VPs and their truncated protein forms using HRAM mass spectrometer.

Methods: Multiple AAV serotype samples expressed via transient transfection in HEK293 cells using the Gibco™ AAV-MAX Helper-Free AAV Production System and a commercially available AAV6 sample were used. The AAV samples were buffer exchanged and concentrated into 80% H₂O/20% acetonitrile containing 5 mM TCEP and 0.1% formic acid using 30K centrifugal filter. The collected sample was incubated at room temperature for 2 hours and used for HPLC-FLD-MS analysis. All data were collected on a Thermo Scientific™ Orbitrap Exploris™ MX mass detector coupled with a Thermo Scientific™ Vanquish™ Horizon UHPLC system and a Thermo Scientific™ Vanquish™ Fluorescence Detector F. The MS data were processed with intact mass analysis workflow in the Thermo Scientific™ BioPharma Finder™ 4.1 software

Results: The goal of this work is to develop a rapid and robust HPLC-FLD-HRAM MS method to monitor several key quality attributes of VPs for helping to optimize the AAV product development and control the quality of the AAV products during the manufacturing process. The Orbitrap Exploris MX mass detector, a simple to operate system which offers high resolution accurate mass (HRAM) and compliance-ready Chromeleon chromatography data system (CDS) software was used in the developed method. As initial work, we first analyzed an AAV9 sample in triplicate for evaluating the intact mass measurement accuracy and reproducibility of the observed relative expression ratios of VP1, VP2 and VP3. The relative expression ratios were observed as 1/0.9/10 (VP1/VP2/VP3) consistently over the three replicate runs. The observed intact average mass accuracies for the VP1, VP2 and VP3 were below 10 ppm. As the potential PTM identification based on the accurate intact mass measurement, deamidation and phosphorylation were observed on the acetylated VP1, phosphorylation on the VP2 and deamidation on the acetylated VP3. Three low abundant truncated VP proteoforms (50470 Da, 47192 Da and 39475 Da) were detected from the AAV9 sample. To mimic AAV products with different production methods, we further analyzed two AAV9 samples (one was commercially available and another one was expressed via transient transfection in HEK293 cells using the Gibco AAV-MAX Helper-Free AAV Production System) with the developed method. The observed intact average masses and the PTMs of VP1, VP2 and VP3 were comparable between the two AAV6 samples. The truncated VP proteoform (43221 Da) was also detected in both AAV6 samples. However, the commercial AAV6 sample showed lower relative expression ratios of VP1/VP2/VP3 (0.7/0.6/10) compared to another AAV6 sample which had relative expression ratios of VP1/VP2/VP3 at 1.3/0.9/10.

Conclusion: An UHPLC-FLD-HRAM Orbitrap MS method was developed for rapid critical quality attributes monitoring. Excellent spectral quality enabled by the Orbitrap Exploris MX mass spectrometer enabled great mass accuracy ( < 10 ppm) for the AAV capsid protein intact mass analysis. The accurate intact mass measurement allowed rapid AAV serotype confirmation. The accurate intact mass measurement allowed rapid AAV viral protein conformation and their associated PTM identification. The sensitive fluorescence detection allowed rapid assessment of the ratio of the AAV structural proteins VP1:VP2:VP3. The UHPLC-FLD-HRAM Orbitrap MS method enabled rapid monitoring of the AAV product quality attributes across different batches, manufacturing processes and over different AAV serotypes.