Clinical-Grade Oncolytic Adenovirus Purification Using Polysorbate 20 as an Alternative for Cell Lysis

Abstract

Introduction: Oncolytic virus therapy is currently considered as a promising therapeutic ap-proach for cancer treatment. Adenovirus is well-known and extensively characterized as an oncolytic agent. The increasing number of clinical trials using this virus generates the demand for the development of a well-established purification approach. Triton X-100 is commonly used in cell lysis buffer prepara-tions. The addition of this surfactant in the list of substances with the very high concern of the Registra-tion, Evaluation, Authorization and Restriction of Chemicals (REACH) regulation promoted the research for effective alternatives.Methods: In this work, a purification strategy for oncolytic adenovirus compatible with phase I clinical trials, using an approved surfactant – Polysorbate 20 was developed. The proposed downstream train, composed by clarification, concentration using tangential flow filtration, intermediate purification with anion exchange chromatography, followed by a second concentration and a final polishing step was evaluated for both Triton X-100 and Polysorbate 20 processes. The impact of cell lysis with Polysorb-ate20 and Triton X-100 for each downstream step was evaluated in terms of product recovery and impu-rities removal. Overall, 61 ± 4% of infectious viral particles were recovered. Depletion of host cell pro-teins and ds-DNA was 99.9% and 97.1%, respectively.Results & Conclusion: The results indicated that Polysorbate 20 can be used as a replacement for Triton X-100 during cell lysis with no impact on product recovery, potency, and purity. Moreover, the devel-oped process is scalable and able to provide a highly purified product to be used in phase I and II clinical trials.

Current challenges in biotherapeutic particles manufacturing

“…At large scale production, chemical lysis is the strategy of choice, as opposed to homogenization, and is usually done by the addition of low concentrations of surfactants (0.1% Triton X-100 or NP-40) [52][53][54]. More recently, Triton X-100 was included on the list (Annex XIV) of Registration, Evaluation, Authorization, and Restriction of Chemicals (REACH), prohibiting its use from 4 January 2021, leading to the evaluation of different cell lysis detergents [55]. However, when adding surfactants, or even other reagents to the culture, they should be removed or reduced later to decrease the impact on the performance of the next unit operations.…”

“…PES membrane cassettes of 100 or 300 kDa have also been used for lentiviral vectors concentration allowing 66-fold concentration with complete vector recovery [83]. The use of Hollow-fibres devices can result in lower shear rates when compared with membrane cassette devices and have been used in the manufacturing of several viral particles -Adenovirus, Influenza virus, rotavirus like particles and HIV VLPs [53,55,60,63]. Regarding extracellular vesicles, PES or RC membrane devices with 100 kDa have been used, but their behavior in the purification of these particle has not been fully explored yet [76,84,86].…”

Manufacturing Challenges and Rational Formulation Development for AAV Viral Vectors

“…73 It is easy to scaleup; however, recent research has shown that Triton X-100 has created some acute oral toxicity, eye damage, skin irritation, and chronic aquatic toxicity. 74 As a result, the detergent was listed as the substance of very high concern (SVHC) in December 2016 by the European Chemicals Agency under REACH regulations after several years of debate regarding its safety. 74 Therefore, an alternative cell lysis method is needed that is not only able to lyse cells, but can also protect AAV vectors from various stresses during the purification process.…”

Oncolytic virus purification with periodic counter‐current chromatography

“…This selection had in consideration the fact that although working with a chromatographic media with a lower DBC the impact in current work of using a convective media would be mainly in terms of volumetric productivity. Besides, keeping the current chromatography media (Capto Q ImpRes) enabled us to benchmark the current strategy against our previous study (Moleirinho et al, 2018).…”