Chromatography
Optimising Viral Vector Purifi cation Strategies with Multimodal Chromatography
Mark A. Snyder and Laura Kronbetter, Bio-Rad Laboratories
The global vaccine market is expected to surpass $85 billion USD by 2027, with an annual growth rate of c. 9% [1]. At the same time, the global gene therapy market is expected to surpass $5 billion USD, with an even faster annual growth rate of approximately 34% [2]. The successful use of virus preparations as nucleic acid delivery vectors or vaccines has been one of the most signifi cant contributors to these rapidly expanding biomedical sectors. As the diversity and clinical application of viral-based products continues to grow, so do challenges during downstream processing. Taking clinical application, manufacturing scalability and inherent viral properties into consideration, chromatography can be utilised to streamline downstream processes, thus helping meet current needs.
Challenges in Downstream Virus Purifi cation
Viruses are employed in numerous clinical applications ranging from gene therapy to vaccine development and cancer treatment. Contemporary downstream processes for viral particle purifi cation have mostly been adapted from protocols originally developed for the purifi cation of recombinant proteins and biologics, such as monoclonal antibodies (mAbs). Similar to protein isolation, downstream processes for virus purifi cation must also ensure that essential virus aspects such as activity and stability remain intact throughout the entire production process [3]. However, the complex biophysical properties of viral particles pose some serious challenges that conventional protein purifi cation protocols cannot effectively address. Unlike individual proteins, viral particles are much larger and are composed of a complex matrix of multiple proteins arranged in unique three- dimensional structures, known as capsids. Additionally, they are susceptible to damage by fl uid shear stress conditions, non-physiological pH, surfactants and interfacial stress (Figure 1). Enveloped viral particles such as viral-like particles, retro- and lentiviruses are particularly susceptible to damage and possess additional surface characteristics (e.g., a lipid bilayer) and/or have undergone post-translational surface modifi cations (e.g., glycosylation) which can further complicate downstream processes [4].
This inevitably introduces cell debris and other impurities into the virus preparation that need to be removed prior to product isolation, such as host cell DNA and proteins, endotoxins, foreign viral contaminants, and virus-DNA aggregates [5], which may compromise safety and effi cacy. Upstream processes can also result in the production of damaged or empty viral particles that lack therapeutic payload or activity and viruses that encapsulate the wrong genetic material. Downstream purifi cation processes have to be tailored to the needs of a particular product, taking into consideration the properties of specifi c viruses and impurities resulting from upstream virus production and harvesting approaches.
Traditionally, downstream viral purifi cation protocols often involved laborious steps such as density gradient ultracentrifugation - while suffi cient for proof-of-principle experiments, such techniques can be diffi cult to scale and may not meet the stringent purity standards of therapeutic products [6]. Consequently, the biopharmaceutical industry is leveraging the capabilities of chromatography in downstream processes to help meet large-scale virus purifi cation requirements and achieve high viral recovery yields [5, 6].
Scaling-Up Downstream Processes Using Chromatography
Often, downstream processes for protein purifi cation are comprised of an initial protein capture step, which for mAbs includes affi nity chromatography (AC) using Protein A resin, followed by two polishing steps that take into consideration the physicochemical properties of the protein of interest, and help remove any remaining impurities prior to product isolation. Mirroring protein purifi cation approaches, optimal virus purifi cation could be obtained by combining steps based on distinct interaction modes between the stationary (resin) and mobile (solute) phase - for example, one step based on viral particle charge and another step based on its hydrophobicity.
AC could be applied in downstream virus purifi cation processes by leveraging the ability of viral surface protein motifs to bind to specifi c cellular ligands, thus helping to isolate the viral particle of interest. A signifi cant limitation of this approach is that a ligand used for initial AC capture is directed toward existing serotypes, resulting in time-consuming ligand design every time a new viral serotype or platform emerges - making such an approach unsuitable for large-scale viral vector and vaccine manufacturing [3].
Ion Exchange Chromatography Figure 1: Average size comparison between biomolecules and cells
Typical cell culture impurities resulting from upstream processes should also be taken into consideration when determining which purifi cation approach to follow. For example, adenoviruses and adeno-associated viruses (AAV), some of the most extensively researched vectors, are harvested from lysed cell cultures.
Ion exchange chromatography (IEX) is an effective alternative to AC and is commonly used for initial mass virus capture across the biopharmaceutical industry [4,5,6]. An ion-exchanger exploits the differences in net surface charge between the viral particle of interest and other biomolecules present in the solution. Nucleic acids for example, will always be negatively charged due to the presence of ionised phosphate groups in the backbone, while virus net charge depends on the proportion of surface charged amino acids at a particular pH [5].
INTERNATIONAL LABMATE - NOVEMBER 2022
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