Development and Validation of AAV Capsids Separation on Specimen Columns for Reproducibility Evaluation of Large-Scale Chromatographic Monoliths.

One of the key challenges in adeno-associated virus (AAV) viral vector manufacturing is the effective and consistent separation of full (F) AAV capsids from undesired non-functional (empty = E, partially filled, etc.) capsids. Typically, at least one chromatography step is used for this purpose in AAV manufacturing. Due to the complexity of viral capsids separation, even a small change in the chromatographic process is reflected in unreproducible results. One solution for robust polishing of full AAV capsids is the highly reproducible (HR) design of chromatographic columns used in this step. Implementation of such columns requires the development of control tests, which efficiently predict column performance for AAV separation. In this paper, the methodology for reproducible separation of empty and full recombinant AAV2/8 (E/F rAAV2/8) capsids was defined using quaternary amine (QA) chromatographic monoliths in a linear potassium chloride (KCl) gradient. The scalability of the procedure was experimentally confirmed on 1, 80, and 800 mL CIMmultus QA columns, where empty capsids eluted at a KCl concentration range of 89.4-91.4 mM. A sampling of the monolith material from the 800 mL CIMmultus QA column and testing it for E/F rAAV2/8 capsid separation in the form of a 200 µL column resulted in a highly comparable elution pattern as obtained with the parent 800 mL column. The principle of sampling material by cutting the parent monolith, packing it in 200 µL columns (specimens) and testing them for E/F rAAV2/8 capsid separation was further developed to demonstrate intra-column homogeneity; batch-to-batch homogeneity; and scalability of CIM QA monoliths. Finally, specimens testing using a validated E/F rAAV2/8 separation method was used to monitor 28 CIMmultus QA production batches (bed volumes between 1 and 8000 mL). E rAAV2/8 capsids eluted at KCl concentration between 89.3 and 95.3 mM for 28 batches, paving the way for commercialization of highly reproducible preparative QA chromatographic monoliths (CIMmultus QA HR).