Oncolytic virus purification with periodic counter‐current chromatography

Abstract

Virus‐based biologicals are one of the most promising biopharmaceuticals of the 21st century medicine and play a significant role in the development of innovative therapeutic, prophylactic, and clinical applications. Oncolytic virus manufacturing scale can range from 5 L in research and development up to 50 L for clinical studies and reach hundreds of liters for commercial scale. The inherent productivity and high integration potential of periodic counter‐current chromatography (PCC) offer a transversal solution to decrease equipment footprint and the reduction of several non‐value‐added unit operations. We report on the design of an efficient PCC process applied to the intermediate purification of oncolytic adenovirus. The developed ion‐exchange chromatographic purification method was carried out using a four‐column setup for three different scenarios: (i) variation in the feedstock, (ii) potential use of a post‐load washing step to improve virus recovery, and (iii) stability during extended operation. Obtained virus recoveries (57%–86%) and impurity reductions (>80% DNA, and >70% total protein) match or overcome batch purification. Regarding process stability and automation, our results show that not only the dynamic control strategy used is able to suppress perturbations in the sample inlet but also allows for unattended operation in the case of ion exchange capture.

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

“…As expected, results show that overloading the nanofibers or decreasing residence time is reflected in flowthrough losses as adsorptive capacity is exhausted. This can be minimized with applications such as continuous chromatography where media saturation is desired and loading zones composed of several interconnected adsorptive beds are used ( Araújo et al, 2007 ; Silva et al, 2015 ; Mendes et al, 2021 ).…”

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

“…This resulted in a PCC process using four columns during six cycles. Additionally, due to the high UV signal found in the sample detector and no visible breakthrough of AAV found in the frontal experiments, the dynamic control capabilities of the ÄKTA pcc (as reported elsewhere [ 19 , 27 , 28 ]) were not used. In this sense, a time-based loading was performed.…”

“…Over the last two decades, several research groups have reported on improvements to the chromatographic process realized by replacing batch operation with continuous processing alternatives. This has been demonstrated for the purification of recombinant proteins [ 10 , 11 ], mAbs [ 12 , 13 ], mAb fragments [ 14 ], viruses [ 15 , 16 , 17 , 18 , 19 ], and exosomes [ 20 ], for which different multi-column chromatography processes have been utilized. These processes operate in continuous or semi-continuous mode with distinct column configurations, enabling significant improvements regarding productivity, resin utilization, and buffer consumption while keeping product quality and yield at least similar to that obtained in batch processes.…”

Improved adeno‐associated virus empty and full capsid separation using weak partitioning multi‐column AEX chromatography

“…Mendes et al reported a case of four-column MCC-based AEX chromatography (Capto Q) on capturing Adenovirus 5 (Ad5) and claimed a 24% increase in purification efficiency and a 78% decrease in column usage. [18] Additionally, Nestola et al reported a two-column SEC method to efficiently purify Ad5, which resulted in a six-fold productivity increase and > 10% overall yield increase. [19] In addition to Ad5, most recently, Mendes et al demonstrated that rAAV also could be purified continuously through a four-column MCC affinity chromatography and the authors claimed that the MCC method achieved a three-fold improvement in affinity productivity with similar virus recoveries.…”

“…reported a case of four‐column MCC‐based AEX chromatography (Capto Q) on capturing Adenovirus 5 (Ad5) and claimed a 24% increase in purification efficiency and a 78% decrease in column usage. [ 18 ] Additionally, Nestola et al. reported a two‐column SEC method to efficiently purify Ad5, which resulted in a six‐fold productivity increase and > 10% overall yield increase.…”