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Poster Presentations
Day 1, June 22（Sun.）
Room P (Maesato East, Foyer, Ocean Wing)
- 1P-PM-23
Identification and quantification of host cell proteins in recombinant adeno-associated virus by data-independent acquisition mass spectrometry
(Osaka Univ.)
oYuma Furuta, Yuki Yamaguchi, Yasuo Tsunaka, Mitsuko Fukuhara, Tetsuo Torisu, Susumu Uchiyama
Host cell proteins (HCPs) are protein impurities originating from the host cells during biopharmaceutical manufacturing. Residual HCPs affect the efficiency and safety of biopharmaceuticals and must be effectively removed. In this study, identification and relative quantification of HCPs in recombinant adeno-associated virus (rAAV) were carried out using data-independent acquisition mass spectrometry (DIA-MS). DIA-MS, which performs comprehensive fragmentation for all precursor ions within predefined isolation windows, allows for high sensitivity and reproducibility in identifying and quantifying low-abundance HCPs that might be overlooked by conventional methods data-dependent acquisition mass spectrometry (DDA-MS). After the optimization and analytical validation of spectral library-based DIA-MS, rAAV purified by affinity chromatography and multiple cesium chloride equilibrium density gradient ultracentrifugation (DG-UC) were analyzed. A spectral library containing more than 8000 proteins was created by analyzing rAAV samples collected before purification using DDA-MS. More than 500 kinds of HCPs were identified even after twice DG-UC steps. In addition, quantitative HCP analysis suggested that some HCPs remain in the rAAV formulation due to interactions with rAAV particles. This study demonstrates that DIA-MS is effective for monitoring HCPs in rAAV, providing both high sensitivity and comprehensiveness of low-abundance HCPs even after rigorous purification steps.