Implementing Chromatographic Methods For Evaluating Large-Scale Monolithic Columns For AAV Capsid Separation

By S. Rotar, T. Simčič, R. Miklavčič, I. Bergoč, D. Kukanja, M. Makarovič Hrvatin, D. Pavlovič, J. Vidič, U. Černigoj, and A. Štrancar

Quaternary amine (QA)-modified monolithic columns are frequently used for the purification of therapeutically-relevant adeno-associated virus (AAV) vectors, used in gene therapy. Besides process- and sample-derived variability, the variability of chromatographic material can influence the efficiency and scalability of downstream AAV processing. This study presents the evaluation of highly reproducible (HR) QA-modified CIM® monolithic columns. The aim was to elute AAV capsids in a very narrow conductivity range, regardless of the batch and size of the column used.

We developed a chromatographic test method for demonstrating the intra- and inter-batch homogeneity of the material through different column sizes. The method is based on the separation of AAV2 | 8 capsids in an ascending KCl gradient on CIMmultus® QA 1 mL columns and specimen 0.2 mL units, taken from large-scale CIMmultus® QA monoliths up to 8,000 mL in size.