One of the key challenges in adeno-associated virus (AAV) viral vector manufacturing is effective and consistent separation of full AAV particles (containing the full recombinant DNA of the gene of interest) from non-functional (empty, partially filled etc.) capsids. Small deviations in chromatographic process performance arise from variability in upstream sample, variability in chromatographic conditions and variability in the chromatography media and can change the AAV capsid separation into a nightmare. One part of the solution is providing highly reproducible (HR) chromatographic columns intended for empty and full AAV capsid separation.
For this purpose we have first set up reproducible and highly controlled chromatographic conditions for separation of AAV8 capsids in an ascending KCl gradient using quaternary amine (QA) modified monolithic columns. Separation of empty and full AAV8 capsids on 1, 8, 80 and 800 mL CIMmultus QA columns demonstrated reproducible elution conditions as empty capsids eluted at KCl concentration in the range of 89.4–91.4 mM. Extracting 200 μL units from the 800 mL monolith and testing them for AAV8 separation with the same method confirmed the same elution pattern as obtained with the parental column. The principle of extracting small units (specimen) and testing them for AAV separation was further developed to prove excellent scalability and homogeneity of CIM QA chromatographic monoliths. Specimen (200 μL or 1 mL bed volume) enabled implementation of AAV8 separation as one of column release test methods in the production of the commercial CIMmultus QA HR monolithic line (1–8000 mL). All columns in this new column line fit a strict release criteria of empty AAV8 capsid elution at KCl concentration between 89.5 and 95.1 mM and demonstrate good batch-to-batch and scale-to-scale material consistency.
