Analytical Tools for Improving Up- and Downstream AAV Process Development

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During a May 2022 Ask the Expert presentation, Ivana Petrović Koshmak (head of upstream process development at BIA Separations, part of Sartorius) highlighted the gene- therapy industry’s need for fast, reliable analytics that work with both up- and downstream samples. High-performance liquid chromatography (HPLC) methods can fulfill that need, but process development (PD) scientists would need to account for the complexity of bioreactor samples to make such methods feasible for end-to-end analytics. Koshmak described a novel instrument that is designed to characterize adenoassociated virus (AAV)-vector quality attributes from transfection through fill–finish.

Koshmak’s Presentation
Koshmak emphasized the advantages of monolith chromatography columns for AAV process analytics. In bead-based media, diffusion limits mass transfer. Thus, samples take a long time to process. Beads also provide little capacity for large biomolecules and generate countercurrent flow, causing shear stress that diminishes product recovery. CIMac monoliths rely on convective mass transport with laminar flow that minimizes shear stress. Compared with porous particles, monolith microchannels provide much more surface area for AAV binding. Unlike dead-end pores, open-ended channels keep binding capacity and resolution high at fast flow rates.

PATfix HPLC valve-switching instruments leverage the rapidity of monolith-based processing to analyze upstream and early downstream AAV samples. Each instrument comprises a cation-exchange (CEX) and an anion-exchange (AEX) monolith joined by a valve. A sample is loaded onto the CEX column for AAV binding. Elution by a pH gradient produces material that then passes through the valve to the AEX column, in which a salt gradient separates full and empty capsids. Using multiangle light scattering (MALS), ultraviolet spectroscopy (UV) at 260 nm and 280 nm, and peak-integration algorithms, the system software determines relative AAV concentration, absolute titer (when a calibration curve is available), and full:empty capsid ratios. Additional detectors are available — e.g., for identification of pDNA using PicoGreen staining. Samples can come from bioreactor cultures (adherent or suspension systems), spent media, cell pellets, whole harvests, clarified and nonclarified lysates, and even fractions from tangential-flow filtration (TFF) steps. The system requires 10 mL of sample per run and generates results within an hour.

Koshmak demonstrated several applications for AAV PD. Her first example showed how the PATfix valve-switching system could be used to compare AAV8 titers and full:empty capsid ratios across samples from different processes. In this case, materials represented BIA’s AAV8 reference standard, harvests from an established process based on insect cells, and harvests from a nonoptimized process using human embryonic kidney (HEK) cells. Results revealed that the nonoptimized process generated high percentages of full capsids but at low bioreactor titers. Further PD could increase those values.

BIA has used the PATfix valve-switching instrument to study plasmid DNA–transfection reagent (pDNA-TR) complex formation, including the influences of incubation time and temperature, pDNA concentrations, and TR:pDNA ratios on production of full and empty capsids. The system also has been used to measure capsid secretion kinetics throughout an upstream process.

BIA is developing a distinct method for measuring AAV subpopulations. The current workflow involves switch-valve HPLC analysis of fractions from an ultracentrifugation process. That method holds promise for distinguishing rapidly between full and partially filled capsids.

Koshmak emphasized the importance of applying orthogonal methods to AAV analysis. The PATfix valve-switching instrument is designed to provide a holistic solution for PD that addresses up- and downstream needs. It also can be used to complement results from ddPCR methods and enzyme-linked immunosorbent assays (ELISAs) for measurement of AAV titers and full:empty capsid ratios.

Questions and Answers
What other analytics are needed for upstream AAV PD? To assess upstream full:empty capsid ratios, BIA uses not only the valve-switching method, but also ddPCR assays and size-exclusion analytics. CIMac SO3 CEX columns are ideal for AAV quantitation, and pDNA content can be measured by chromatographically separating samples stained with PicoGreen dye. Many plate assays are available for total DNA and protein analysis.

Can AAV be quantified without using MALS? The method provides good sensitivity, which is helpful when samples have low AAV concentrations. UV readings suffice when samples have high titers, although analysts must use correction factors to account for differences in extinction coefficients between full and empty capsids.

Find the full webinar online at