15
In the case of negatively charged retroviruses and adenoviruses, nuclease treatment of the feedstock followed by single-step chromatography utilising anion exchange chromatography (AEX) resins (e.g., Nuvia HP-Q) has been shown to be an effective mass capture step well-suited for the production of clinical-grade viral vectors [4, 9]. Unlike cation-exchangers, anion-exchangers typically bind viruses under pH and ionic strength (salt concentration) conditions consistent with those in upstream bioreactor feed- streams improving both process economics and effi ciency. AEX can also be employed as a polishing step, to help separate empty and full viral capsids based on subtle charge differences and remove negatively charged impurities from the feedstock [8, 9]. Cation exchange chromatography (CEX) resins (e.g., Nuvia S) can be used to capture viruses, and are also commonly used in polishing steps, to successfully remove the majority of process- and product-related impurities [5, 10].
Considering that viral particle integrity is susceptible to environmental fl uctuations which includes pH changes, different buffer composition, temperature alterations and shear stress, it is crucial to design a downstream processing platform capable of achieving the highest levels of purity with the minimal number of steps, to maximise quality, effi ciency and safety.
Improving Downstream Process Effi ciency with Multimodal Chromatography
The emergence of multimodal (or mixed-mode) chromatography has given rise to highly selective media designed to simplify the purifi cation process, reduce downstream processing time and improve overall process economics. Mixed-mode resins combine ligands capable of at least two modes of interaction, such as affi nity, size exclusion, ion exchange, and hydrophobic interactions in a single purifi cation step. Multimodal chromatography allows for the optimal purifi cation of viral particles that would be ineffectively purifi ed by sequential single-mode approaches.
For example, combining the properties of hydrophobic interaction and cation exchange in a single hydrophobic cation exchange resin allowed for optimised capture of recombinant adenovirus [7]. Specifi cally, a hydrophobic cation exchange resin (e.g., Nuvia cPrime) was used in a capture step as it offered the best clearance from negatively charged feed-stream contaminants (e.g., animal serum) and resulted in a tenfold reduction in processing volume. Eluate from this resin could then be loaded into the second and fi nal anion exchange column (e.g., Nuvia Q) which resulted in an additional two-fold reduction of product volume and optimised product purity comparable to clinical grade products [7]. Leveraging multiple modes of interaction, such two-step approaches enhance separation of viral particles from impurities in a more cost- and time-effective manner.
Similarly, hydroxyapatite chromatography (HAC) mixed-mode media, with the ability to bind a wide range of viral particles, can be used effectively for intermediate or fi nal polishing. The mixed-mode capabilities of HAC can separate molecules based on calcium affi nity and cation exchange interactions. Single-step HAC has successfully been used for the separation of both enveloped (e.g., dengue virus, Japanese encephalitis virus, infl uenza, and mouse hepatitis virus) and non-enveloped (e.g., adenovirus, feline calicivirus and poliovirus) mammalian viruses from impurities, resulting in concentrated preparation of highly active virus [11, 12].
Conclusions
As the application of viral vectors in gene therapy and vaccine development continues to grow, so does the demand for faster product development and manufacturing. Complexities associated with the physicochemical properties of viral particles present unique purifi cation challenges, especially in a large-scale manufacturing setting. Chromatography has emerged as a highly selective and yet robust scalable method for virus purifi cation - specifi cally, mixed-mode media innovations can be the driving force behind faster and more cost-effective product development and manufacturing. Unlike single-mode chromatography resins, mixed-mode resins allow for (i) effective separation of molecules that appear homogeneous using other chromatographic methods, (ii) selective single-step removal of impurities, (iii) establishment of optimal binding and elution conditions due to large design space, (iv) minimal feedstock processing due to robust salt tolerance, and (v) large capacity high-titre feed-streams [13].
Application of multimodal chromatography in downstream purifi cation processes can signifi cantly improve the manufacturing process of such therapeutic modalities to help meet the ever-increasing demands for safe and effective gene therapies and viral vector vaccines.
References
1. Global Vaccine Market,
https://www.researchandmarkets.com/reports/5576812/global-vaccine-market- size-global-forecast-2022 2. Gene Therapy Market,
https://brandessenceresearch.com/healthcare/gene-therapy-market-global
3. Nestola P et al (2015) Improved virus purifi cation processes for vaccines and gene therapy. Biotechnology and Bioengineering.
4. Fitchmun M, Low-Cost, Fast and Scalable Downstream Purifi cation Process Development for a Clinical- Stage Retrovirus-Like Particle,
https://www.bio-rad.com/webroot/web/pdf/psd/literature/Bulletin_7200.pdf
5. Singh et al (2022) Challenges in downstream purifi cation of gene therapy viral vectors. Current Opinion in Chemical Engineering. 35.
6. Merten et al (2014) Manufacturing of viral vectors: part II. Downstream processing and safety aspects. Pharmaceutical Bioprocessing.
7. Fitchmun M et al, Development of an Effi cient Manufacturing Process for Adenovirus,
https://www.bio-rad. com/webroot/web/pdf/psd/literature/Bulletin_6719.pdf
8. Joshi PRH et al (2021) Development of a scalable and robust AEX method for enriched rAAV preparations in genome-containing VCs of serotypes 5, 6, 8, and 9. Molecular therapy. Methods & clinical development. 21. 341–356.
9. Quick purifi cation Strategy – H1N1 Purifi cation,
https://www.bio-rad.com/webroot/web/pdf/lsr/literature/ Bulletin_7375.pdf
10. Nuvia™ S Media — A High-Capacity Cation Exchanger for Biomolecule Purifi cation and Downstream Processing,
https://www.bioradiations.com/nuvia-s-media-a-high-capacity-cation-exchanger-for-biomolecule- purifi cation-and-downstream-processing/
11. Kurosawa et al, Mammalian Virus Purifi cation Using Ceramic Hydroxyapatite,
https://www.bio-rad.com/ webroot/web/pdf/lsr/literature/Bulletin_6549.pdf
12. Kurosawa et al, Single-Step Infl uenza and Dengue Virus Purifi cation with Mixed-Mode CHT Ceramic Hydroxyapatite XT Media,
https://www.bio-rad.com/webroot/web/pdf/psd/literature/Bulletin_7115.pdf
13. Scott C (2020) Mixed-mode chromatography for purifi cation of biopharmaceuticals, BioProcess International eBooks,
https://bioprocessintl.com/sponsored-content/mixed-mode-chromatography-resins-for- purifi cation-of-biopharmaceuticals/
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