Continuous Affinity Purification of Adeno-Associated Virus Using Periodic Counter-Current Chromatography

Abstract

Replacing batch unit operations of biopharmaceuticals by continuous manufacturing is a maturing concept, with periodic counter-current chromatography (PCC) favoured to replace batch chromatography. Continuous affinity capture of adeno-associated virus (AAV) using PCC has the potential to cope with the high doses required for AAV therapies thanks to its inherent high throughput. The implementation of continuous AAV affinity capture using a four-column PCC process is described herein. First, elution buffer screening was used to optimize virus recovery. Second, breakthrough curves were generated and described using a mechanistic model, which was later used to characterize the loading zone of the PCC. The experimental runs achieved a stable cyclic steady state yielding virus recoveries in line with the optimized batch process (>82%), with almost a three-fold improvement in productivity. The PCC affinity capture process developed here can bolster further improvements to process economics and manufacturing footprint, thereby contributing to the integrated continuous manufacturing concept.

Implementation of Novel Affinity Ligand for Lentiviral Vector Purification

“…The presence of several charged groups in its structure (positively charged amines and negatively charged C-terminal carboxylic acid) could be the explanation for the good performance of arginine across the different affinity ligands [ 46 ]. This strategy has also been applied to other viral vectors; for instance, in the purification of adeno-associated viruses [ 47 ]. The effect of the pH on viral release was also investigated for the selected buffer.…”

Efficient adeno-associated virus serotype 5 capture with affinity functionalized nanofiber adsorbents

“…The nanofiber adsorbents reveal a good dynamic binding capacity and recovery yields even at low residence times in comparison to packed beds. Mendes et al, using periodic counter-current chromatography, reported an AAVs recovery of 82.8% using Capto AVB™ of 0.2 mL, with 10-fold higher residence times in comparison to the used nanofibers ( Mendes et al, 2022 ). With respect to the experimental binding capacity, the highest value (6.08 × 10 14 TP mL −1 ) was obtained for a residence time of 4.8 s. Although capacity is in the same order of magnitude as what is reported by some manufacturers there is a substantial improvement in diminished residence times Cytiva, 2018 ; Cytiva, 2020a ; Fisher Scientific, 2017 ; Repligen Corporation, 2022 ).…”

Reviewing the process intensification landscape through the introduction of a novel, multitiered classification for downstream processing

“…Lastly, dedicated systems for Level 1 process intensification are beginning to gain traction, both due to the diversity of systems offered and user familiarity to concepts such as continuous chromatography, simulated moving bed, and so forth (Baur et al, 2016; Girard et al, 2015). While these sorts of systems have been typically limited to protein‐based therapies, recent work has been done using intensified systems for advanced therapies (Mendes, Bergman, et al, 2022). However, most of the work published in the literature today has been done by academic groups, and the translation into GMP processes is still to be seen.…”