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Characterization of Difficult-to-Remove Host Cell Proteins in Adeno-Associated Virus Downstream Processing

ABSTRACT: A scalable, serotype-agnostic, and fully chromatographic downstream platform was developed for purification of adeno-associated virus (AAV) based on affinity (AC), ion-exchange (AEX), and multi-modal (MMC) chromatography. Quantitative proteomic profiling across each purification stage was performed using sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS) to characterize residual host cell protein (HCP) retention for four AAV serotypes (AAV2, -5, -8, and -9) produced with suspension HEK293 cells. The downstream process showed cumulative vector genome (VG) yields ranging from 44.6 – 69.2% with full capsid enrichments ranging from 2.62 – 5.93-fold across all AAV samples. 880, 99, and 21 HCPs were identified in all AAV samples after AC, AEX, and MMC, respectively. The characterization of HCP retention described here advances the understanding of process-related impurity clearance in scalable AAV downstream systems, with implications for both purification process design and AAV interactions with host cell factors.

ORGANISM(S): Cellular Organisms

SUBMITTER: Kelvin H. Lee

PROVIDER: PXD067503 | JPOST Repository | Sat Nov 15 00:00:00 GMT 2025

REPOSITORIES: jPOST

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Project description:Host cell proteins (HCPs) are process-related impurities that are generated by the host organism and are typically present at low levels in recombinant biopharmaceutical products, such as therapeutic antibodies. While overall HCP levels are usually monitored by enzyme-linked immunosorbent assay (ELISA), liquid chromatography coupled to mass spectrometry (LC-MS) is emerging as a powerful tool that can provide both qualitative and quantitative information about HCP levels during purification process development. However, a major challenge for LC-MS-based methods is that there can be a more than 5 orders of magnitude difference in the concentration between HCPs and therapeutic antibody in solution, which precludes the effective identification of low abundance HCPs in antibody product. This work reports a simple and powerful strategy to identify HCPs in antibody drug substance by applying molecular weight cut-off (MWCO) filtration step followed by shotgun proteomic analysis. After dissociating the interaction between HCPs and antibody with an anionic detergent, the depletion of antibody from HCPs can be easily achieved with the MWCO filtration step. Using this method, we observed that the dynamic range across proteins in the HCP samples was significantly decreased by 1,000-fold. In addition, by spiking in known amounts of HCPs to purified antibody drug substance with low levels of HCPs, we demonstrated that our method could detect HCP at a concentration as low as 1 ppm. When applying this methodology to the study of HCPs in NIST monoclonal antibody (NISTmAb), more than 150 HCPs were confidently identified, which doubles the number of identified HCPs that have been previously reported. Parallel reaction monitoring (PRM) results confirmed that the novel HCPs found using this method were present in very low abundance (0.01-8 ppm), highlighting that our method reduces the dynamic range by removing antibody interference and improving the sensitivity of HCP identification and quantification.

			Action	DRS
	01_AX_AAV2R1.zip	Other
	02_AX_AAV2R2.zip	Other
	03_AX_AAV5R1.zip	Other
	04_AX_AAV5R2.zip	Other
	05_AX_AAV8R1.zip	Other

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Characterization of Difficult-to-Remove Host Cell Proteins in Adeno-Associated Virus Downstream Processing

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