Figure 5 . Regression plot depicting the tradeoff between GOI genome recovery and full-rAAV enrichment. Circles correspond to step elution, whereas the squares correspond to gradient elution. Open markers correspond to AEX chromatography runs using NaCl in process buffers. Closed markers correspond to AEX chromatography runs that employ an isocratic TEA-Ac wash, followed by either a step or gradient elution using NaCl and MgCl2. The genome recovery is calculated using the genome copy determined by qPCR assay, while the full-rAAV percentage is determined by AUC assay.
4. DISCUSSION
Chromatographic separation of empty- and full-AAV particles is a resolution challenge. This combination of TEA and Ac displays the behavior of a weak eluent and is observed to be more effective to discriminate empty- and full-rAAV species in AEX chromatography. In a qualitative sense, if the eluting power of a modifier is too high, the eluent may fail to distinguish the species retained on the stationary phase. In addition, a strong eluent may produce retention times that are too short with the result of poorly resolved elution peaks. Conversely, a weaker eluent is more likely to discriminate between the species retained on the stationary phase and increase the retention time, and in return increase resolution. Similarly, the weak AEX stationary phase, whose ligand is derived from a weak acid and partially ionized in a narrow pH range, may also be able to provide enhanced selectivity between empty- and full-rAAV particles. In line with this notion, Zhou et al.reported that a DEAE-based weak AEX stationary phase, POROS 50D, provided markedly improved resolution between empty and full-rAAV particles of AAV-6, when compared with other strong AEX stationary phases.[27] It is foreseeable that a more systematic study around AEX stationary phase may provide further insight on the delicate separation of empty and full-rAAV in AEX chromatography.
Most recently, it was reported that the empty-rAAV species may have preferential binding to a process-related impurity, chromatin, in Sf9 cell line generated rAAV viral vector product.[28]As chromatin consists of histone octamers whose protruded tails carry positive charged lysine residues, it is possible that these positively charged histones could preferentially bind empty-rAAV. In line with this thought, it is also possible that TEA-Ac preferentially interacts with empty-rAAV, either through electrostatic interaction using TEA positive amine group or through enforced charge pairing [24] using both TEA positive amine group and TEA hydrophobic alkyl-chain. Both of these interactions may lead to the decrease of net charge of the empty-rAAV species and thus benefiting empty- and full-rAAV separation in AEX chromatography.
An important consideration of using QA salt in purification process is that its sufficient clearance needs to be demonstrated to assure product safety. As aforementioned, since sodium ion has stronger elution power than that of TEA ion, the second wash with NaCl could replace and flush out residual TEA-Ac salt from AEX column prior to elution phase. In addition, the volume of the second wash buffer could be further optimized to achieve extra clearance of TEA ions. Furthermore, the final concentration and buffer exchange step in downstream process usually could achieve >99.9% buffer exchange efficiency,[29] which offers at least 3 more log reduction value (LRV) for TEA-Ac, providing feasibility for TEA-Ac to be used as a potent process buffer salt to enhance full-rAAV percentage.
Regarding the implementation of AEX step elution method, the lot-to-lot variability (in terms of peak elution retention volume) of monolithic column may add one more layer of complexity when converting LGE to isocratic elution, as the isocratic elution buffer may not consistently elute full-rAAV species when AEX column changed, resulting in either low full-rAAV percentage or low genome recovery. As a mitigation, packed column with AEX resin may provide more consistent peak elution behavior, although the resolution of empty- and full-rAAV separation using AEX resin may suffer certain level of decrease when compared to AEX monolithic column. In addition, it has been communicated that a new line of AEX monolithic column product will be lunched by CIMultus-QA vendor whose manufacturing procedure was specifically improved to reduce lot-to-lot variability (personal communication), which may offer process developer an extra option to achieve enhanced process robustness while still maintaining optimal empty full separation in AEX monolithic column operation.
Furthermore, AEX chromatography is usually employed to clear process related impurities, such as host cell protein (HCP).[30] In AEX LGE run, the HCP species are eluted off column by higher ionic strength post full peak, thus delicate peak cutting criteria need to be implemented to exclude HCP from entering product stream. Similarly, in AEX step elution run, elution salt concentration needs to be carefully optimized to ensure equivalent HCP clearance as achieved by LGE. When implementing TEA-Ac in an AEX step, due to the unique property of TEA ion comparing to other cations in process salts, TEA ion may be able to interact with HCP through its hydrophobic alkyl-chain. Therefore, the HCP reduction capability of AEX step needs to be re-evaluated to assure both product-related impurity and process-related impurity are in control.
Lastly, certain challenges still remain in purification of certain AAV serotype. For instance, Joshi et al . reported that AAV9 empty and full peaks could be separated using POROS HQ column with 0.93 peak resolution, compared to 0.91 for rAAV8 serotype, indicating that the rAAV8 and rAAV9 viruses behave similarly in their study in terms of empty full separation.[10] However, Lock and Alvira found that their rAAV9 product possess two empty species with distinct chromatography behavior on CIMmultus QA column using salt LGE, with the major empty peak (early elution species) almost co-eluted with full peak.[31] The discrepancy observed in rAAV9 Empty and Full separation may be contributed by the variation of multiple factors between research labs, including DNA genome sequence, DNA genome size, viral protein amino acid sequence, viral protein post-translation modification,[13] and other factors, which may warrant researchers to consider a case-by-case implementation and optimization of this TEA-Ac and step elution AEX technology.
AUTHOR CONTRIBUTIONS
D.P.C. : Conceptualization, Methodology, Investigation, Writing – Original Draft, Writing – Review Editing. C.H. : Supervision, Resources, Writing – Original Draft, Writing – Review Editing.J.C.W. : Resources, Writing – Review Editing.
CONFLICTS OF INTEREST
D.P.C. , C.H., and J.C.W. are inventors on a patent that includes the work in this manuscript
Data Availability Statement
The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding author.
ACKNOWLEDGEMENTS
The authors thank the following members of Ultragenyx’s Pharmaceutical Development, Analytical Development team for assay support: Will Beyer and Sara Forman for supporting vector genome titer quantification through qPCR assays; Brittany Brancato and Adriana Kita for supporting capsid content quantification through SV-AUC; and Matt Lotti for supporting AAV-particle concentration quantification through Gyrolab xPand assays. The authors are grateful to Ultragenyx’s Pharmaceutical Development, Pilot Plant team for their support in generating the AAV8 samples used in this study; in particular, the authors thank Amy Medeiros for coordinating the in-process intermediate materials generation and sample handoff.
5. REFERENCES
1. Daya, S., & Berns, K. I. (2008). Gene therapy using adeno-associated virus vectors. Clinical Microbiology Reviews , 21 (4), 583–593. https://doi.org/10.1128/CMR.00008-08
2. Asokan, A., Schaffer, D. V, & Jude Samulski, R. (2012). The AAV Vector Toolkit: Poised at the Clinical Crossroads. Molecular Therapy , 20 (4), 699–708. https://doi.org/https://doi.org/10.1038/mt.2011.287
3. Samulski, R. J., & Muzyczka, N. (2014). AAV-Mediated Gene Therapy for Research and Therapeutic Purposes. Annual Review of Virology ,1 (1), 427–451. https://doi.org/10.1146/annurev-virology-031413-085355
4. Naso, M. F., Tomkowicz, B., Perry, W. L., & Strohl, W. R. (2017). Adeno-Associated Virus (AAV) as a Vector for Gene Therapy.BioDrugs , 31 (4), 317–334. https://doi.org/10.1007/s40259-017-0234-5
5. Gao, K., Li, M., Zhong, L., Su, Q., Li, J., Li, S., He, R., Zhang, Y., Hendricks, G., Wang, J., & Gao, G. (2014). Empty Virions In AAV8 Vector Preparations Reduce Transduction Efficiency And May Cause Total Viral Particle Dose-Limiting Side-Effects. Molecular Therapy. Methods & Clinical Development , 1 (9), 20139. https://doi.org/10.1038/mtm.2013.9
6. Kishimoto, T. K., Samulski, R. J., & Samulski, R. J. (2022). Expert Opinion on Biological Therapy Addressing high dose AAV toxicity – ‘ one and done ’ or ‘ slower and lower ’? Expert Opinion on Biological Therapy , 00 (00), 1–5. https://doi.org/10.1080/14712598.2022.2060737
7. Hermens, W. T. J. M. C., Brake, O. Ter, Dijkhuizen, P. A., Sonnemans, M. A. F., Grimm, D., Kleinschmidt, J. A., & Verhaagen, J. (1999). Purification of recombinant adeno-associated virus by iodixanol gradient ultracentrifugation allows rapid and reproducible preparation of vector stocks for gene transfer in the nervous system. Human Gene Therapy , 10 (11), 1885–1891. https://doi.org/10.1089/10430349950017563
8. Ayuso, E., Mingozzi, F., Montane, J., Leon, X., Anguela, X. M., Haurigot, V., Edmonson, S. A., Africa, L., Zhou, S., High, K. A., Bosch, F., & Wright, J. F. (2010). High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency.Gene Therapy , 17 (4), 503–510. https://doi.org/10.1038/gt.2009.157
9. Crosson, S. M., Dib, P., Smith, J. K., & Zolotukhin, S. (2018). Helper-free Production of Laboratory Grade AAV and Purification by Iodixanol Density Gradient Centrifugation. Molecular Therapy - Methods and Clinical Development , 10 (September), 1–7. https://doi.org/10.1016/j.omtm.2018.05.001
10. Joshi, P. R. H., Bernier, A., Moço, P. D., Schrag, J., Chahal, P. S., & Kamen, A. (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 (June), 341–356. https://doi.org/10.1016/j.omtm.2021.03.016
11. Wang, C., Mulagapati, S. H. R., Chen, Z., Du, J., Zhao, X., Xi, G., Chen, L., Linke, T., Gao, C., Schmelzer, A. E., & Liu, D. (2019). Developing an Anion Exchange Chromatography Assay for Determining Empty and Full Capsid Contents in AAV6.2. Molecular Therapy - Methods and Clinical Development , 15 (December), 257–263. https://doi.org/10.1016/j.omtm.2019.09.006
12. Wright, J. F., Le, T., Prado, J., Bahr-Davidson, J., Smith, P. H., Zhen, Z., Sommer, J. M., Pierce, G. F., & Qu, G. (2005). Identification of factors that contribute to recombinant AAV2 particle aggregation and methods to prevent its occurrence during vector purification and formulation. Molecular Therapy , 12 (1), 171–178. https://doi.org/10.1016/j.ymthe.2005.02.021
13. Mary, B., Maurya, S., Arumugam, S., Kumar, V., & Jayandharan, G. R. (2019). Post-translational modifications in capsid proteins of recombinant adeno-associated virus (AAV) 1-rh10 serotypes. FEBS Journal , 286 (24), 4964–4981. https://doi.org/10.1111/febs.15013
14. Grieger, J. C., & Samulski, R. J. (2005). Packaging Capacity of Adeno-Associated Virus Serotypes: Impact of Larger Genomes on Infectivity and Postentry Steps. Journal of Virology ,79 (15), 9933–9944. https://doi.org/10.1128/jvi.79.15.9933-9944.2005
15. Giles, A. R., Sims, J. J., Turner, K. B., Govindasamy, L., Alvira, M. R., Lock, M., & Wilson, J. M. (2018). Deamidation of Amino Acids on the Surface of Adeno-Associated Virus Capsids Leads to Charge Heterogeneity and Altered Vector Function. Molecular Therapy ,26 (12), 2848–2862. https://doi.org/10.1016/j.ymthe.2018.09.013
16. Dickerson, R., Argento, C., Pieracci, J., & Bakhshayeshi, M. (2021). Separating Empty and Full Recombinant Adeno-Associated Virus Particles Using Isocratic Anion Exchange Chromatography.Biotechnology Journal , 16 (1). https://doi.org/https://doi.org/10.1002/biot.202000015
17. Johnson, A. R., & Vitha, M. F. (2011). Chromatographic selectivity triangles. Journal of Chromatography. A , 1218 (4), 556–586. https://doi.org/10.1016/j.chroma.2010.09.046
18. Qu, G., Bahr-Davidson, J., Prado, J., Tai, A., Cataniag, F., McDonnell, J., Zhou, J., Hauck, B., Luna, J., Sommer, J. M., Smith, P., Zhou, S., Colosi, P., High, K. A., Pierce, G. F., & Wright, J. F. (2007). Separation of adeno-associated virus type 2 empty particles from genome containing vectors by anion-exchange column chromatography.Journal of Virological Methods , 140 (1–2), 183–192. https://doi.org/10.1016/j.jviromet.2006.11.019
19. Khatwani, S. L., Pavlova, A., & Pirot, Z. (2021). Anion-exchange HPLC assay for separation and quantification of empty and full capsids in multiple adeno-associated virus serotypes. Molecular Therapy: Methods & Clinical Development , 21 (June), 548–558. https://doi.org/10.1016/j.omtm.2021.04.003
20. Urabe, M., Xin, K. Q., Obara, Y., Nakakura, T., Mizukami, H., Kume, A., Okuda, K., & Ozawa, K. (2006). Removal of empty capsids from type 1 adeno-associated virus vector stocks by anion-exchange chromatography potentiates transgene expression. Molecular Therapy ,13 (4), 823–828. https://doi.org/10.1016/j.ymthe.2005.11.024
21. Yang, H., Koza, S., & Chen, W. (2020). Anion-Exchange Chromatography for Determining Empty and Full Capsid Contents in Adeno-Associated Virus. Waters Application Note , 1–7.
22. Nam, H.-J., Lane, M. D., Padron, E., Gurda, B., McKenna, R., Kohlbrenner, E., Aslanidi, G., Byrne, B., Muzyczka, N., Zolotukhin, S., & Agbandje-McKenna, M. (2007). Structure of Adeno-Associated Virus Serotype 8, a Gene Therapy Vector. Journal of Virology ,81 (22), 12260–12271. https://doi.org/10.1128/jvi.01304-07
23. Gagnon, P., Leskovec, M., Prebil, S. D., Žigon, R., Štokelj, M., Raspor, A., Peljhan, S., & Štrancar, A. (2021). Removal of empty capsids from adeno-associated virus preparations by multimodal metal affinity chromatography. Journal of Chromatography A ,1649 , 462210. https://doi.org/10.1016/j.chroma.2021.462210
24. Fritz, J. S. (2005). Factors affecting selectivity in ion chromatography. Journal of Chromatography A , 1085 , 8–17. https://doi.org/10.1016/j.chroma.2004.12.087
25. GRUHZIT, O. M., FISKEN, R. A., & COOPER, B. J. (1948). TETRAETHYLAMMONIUM CHLORIDE. ACUTE AND CHRONIC TOXICITY IN EXPERIMENTAL ANIMALS. Journal of Pharmacology and Experimental Therapeutics ,92 (2), 103 LP – 107. http://jpet.aspetjournals.org/content/92/2/103.abstract
26. Singh, N., & Heldt, C. L. (2022). Challenges in downstream purification of gene therapy viral vectors. Current Opinion in Chemical Engineering , 35 , 100780. https://doi.org/10.1016/j.coche.2021.100780
27. Zhou, J., Hauck, B., Wright, J. F., & High, K. A. (2007). Weak Anion Exchange Column Chromatography Enhances the Resolution of Separation of AAV Empty Capsid and Full Vectors. Molecular Therapy , 15 , S36. https://doi.org/10.1016/s1525-0016(16)44297-8
28. Gagnon, P., Goricar, B., Mencin, N., Zvanut, T., Peljhan, S., Leskovec, M., & Strancar, A. (2021). Multiple-monitor HPLC assays for rapid process development, in-process monitoring, and validation of AAV production and purification. Pharmaceutics , 13 (1), 1–14. https://doi.org/10.3390/pharmaceutics13010113
29. Schwartz, L. (2003). Diafiltration: A Fast, Efficient Method for Desalting or Buffer Exchange of Biological Samples. Pall Scientific & Technical Report , 6. http://www4.pall.com/pdf/02.0629_Buffer_Exchange_STR.pdf
30. Levy, N. E., Valente, K. N., Lee, K. H., & Lenhoff, A. M. (2016). Host cell protein impurities in chromatographic polishing steps for monoclonal antibody purification. Biotechnology and Bioengineering , 113 (6), 1260–1272. https://doi.org/10.1002/bit.25882
31. Lock, M., & Alvira, M. R. (2019). SCALABLE PURIFICATION METHOD FOR AAV9 (Patent No. US 2019 / 0002842 A1). In United States Patent Application Publication (US 2019 / 0002842 A1).
32. Ayuso, E., Mingozzi, F., & Bosch, F. (2010). Production, purification and characterization of adeno-associated vectors.Current Gene Therapy , 10 (6), 423–436. https://doi.org/10.2174/156652310793797685
33. Grieger, J. C., Soltys, S. M., & Samulski, R. J. (2016). Production of recombinant adeno-associated virus vectors using suspension HEK293 cells and continuous harvest of vector from the culture media for GMP FIX and FLT1 clinical vector. Molecular Therapy , 24 (2), 287–297. https://doi.org/10.1038/mt.2015.187
34. Fu, X., Chen, W. C., Argento, C., Clarner, P., Bhatt, V., Dickerson, R., Bou-Assaf, G., Bakhshayeshi, M., Lu, X., Bergelson, S., & Pieracci, J. (2019). Analytical Strategies for Quantification of Adeno-Associated Virus Empty Capsids to Support Process Development. Human Gene Therapy Methods , 30 (4), 144–152. https://doi.org/10.1089/hgtb.2019.088