Evaluation of restricted access media for the purification of cell culture‐derived Orf viruses

Abstract

Recently, multimodal chromatography using restricted access media (RAM) for the purification of nanoparticles, such as viruses has regained increasing attention. These chromatography resins combine size exclusion on the particle shell and adsorptive interaction within the core. Accordingly, smaller process‐related impurities, for example, DNA and proteins, can be retained, while larger product viruses can pass unhindered. We evaluated a range of currently available RAM, differing in the shells’ pore cut‐off and the core chemistry, for the purification of a cell culture‐derived clarified model virus, namely the Orf virus (ORFV). We examined impurity depletion and product recovery as relevant criteria for the evaluation of column performance, as well as scale‐up robustness and regeneration potential for evaluating a multiple use application. The results indicate that some columns, for example, the Capto Core, enable both a high DNA and protein removal, while others, for example, the Monomix Core 60 (MC60), are more suitable for DNA depletion. Furthermore, column regeneration is facilitated by using columns with larger shell pores (5000 vs. 700 kDa) and weaker binding interactions (anion exchange vs. multimodal). According to these findings, the choice of RAM resins should be selected according to the respective feed sample composition and the planned number of application cycles.

Affinity and Pseudo-Affinity Membrane Chromatography for Viral Vector and Vaccine Purifications: A Review

“…Possible approaches include charge- and pH-driven principles using ion exchange chromatography [ 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 ], hydrophobic interaction chromatography [ 12 , 13 , 14 , 15 ], size-dependent separation principles such as size exclusion [ 16 , 17 , 18 , 19 ] or steric exclusion chromatography [ 20 , 21 , 22 , 23 ], and affinity applications [ 24 , 25 ]. In addition, combined approaches using mixed-mode resins have recently been described, with restricted access media (mixed-mode size exclusion chromatography) resins as the most promising option for separating large virus particles from smaller DNA and protein contaminants [ 6 , 26 , 27 , 28 ]. These methods differ in their specificity and selectivity for separating product viruses or VLPs from process-related contaminants, namely host cell-derived proteins and DNA or from other particles produced alongside with the products, e.g., extracellular vesicles [ 29 , 30 ].…”