Chromatographic Methods
Virus-mediated gene transfer has great potential as a strategy for treating various inherited and acquired human diseases. Among the current viral vectors, adeno-associated virus (AAV) has become the preferred vector for many gene therapy applications. With the clinical application of AAV-based vectors, the demand for scalable production and purification is increasing. Creative Biolabs has accumulated enough experience in purifying AAV by chromatography and is confident in providing customers with the best service.
Chromatographic Purification for AAV Vector
AAV is the smallest known particulate gene delivery vector. AAV virions are relatively stable to heat, non-ionic detergents and proteolytic enzymes, so they can be well purified by chromatography. At present, the use of chromatography to purify viral vectors has made progress, and is becoming a mature method. The vector can be purified by its net charge, hydrophobicity, affinity to ligands, size or other physical properties. Chromatographic columns can be reused many times and are considered to be more scalable and cost-effective than centrifugation. Meanwhile, based on the different serotypes of AAVs, chromatographic columns can run in parallel or in series with different strategies. In addition, chromatography is an effective means to remove potential uncertainties and a key aspect of late-stage development.
Chromatography for AAV Vector Purification Service at Creative Biolabs
Viral vectors differ greatly in physical properties, depending on the type of virus, the serotype of the same virus, and can sometimes vary with different transgene inserts within the same vector (AAV). Therefore, the method of vector purification should make full use of the physical characteristics of virus particles and determine the purification scheme according to local conditions.
Figure 1. Schematic of various approaches to the purification of AAV particles from HEK293 cells. (Sandoval, 2019)
Creative Biolabs has established several chromatographic methods for the purification of AAV vectors, including but not limited to:
Ion Exchange Chromatography
Ion exchange chromatography (IEC) is a simple and economical technique based on surface charge, which can be applied to many types of viruses. Using AAV vectors, IEC can work across multiple serotypes, and can also be used to isolate auxiliary viruses (such as adenovirus and baculovirus) from AAV preparations. Compared with the most polluted impurities, recombinant AAVs particles contain multiple sites binding to anion exchange, so anion exchange chromatography (AEC) is a common chromatography for AAV purification. In addition, IEC is one of the few methods that can separate the whole particle from the empty AAV particles due to the different charges of the whole particle. In the presence of full-length DNA, IEC can also be effective in this respect, because studies have shown that the elimination rate of empty particles is more than 90%.
Figure 2. The rAAV separation by IEC. (Qu, 2015)
Hydrophobic Interaction Chromatography
Hydrophobic amino acids of proteins are usually far from the surface of molecules. Full exposure of hydrophobic groups allows the protein to interact with hydrophobic ligands in the medium. Hydrophobic interaction chromatography (HIC) has been used for virus capture/removal in the vector and recombinant protein industries for many years. HIC is a good purification step after ammonium sulfate precipitation or high salt elution. In the purification scheme, it has been proven that the AAV solution precipitated by ammonium sulfate can be loaded on HIC to achieve a good purification effect, particularly suitable for the purification of rAAV1, rAAV2 serotypes.
Heparin Affinity Chromatography
Affinity chromatography (AC) depends on the interaction between viral particles and ligands. Heparin AC (HAC) has been used to purify the left ventricle and other viruses and has been reported to be able to handle a large number of them. In AAV, the surface structure of capsid protein is an important part binding to target cells. Researchers have found that heparin sulfate proteoglycan (HSPG) is a receptor of AAV2. AAV2 heparin binding sites and adjacent alkaline residues are located on the trimer of VP3 subunit on the surface of capsid. At present, heparin-based AC has been successfully used to purify rAAV2 vectors.
Sialic Acid Affinity Chromatography
The development of recombinant gene therapy vectors based on other AAV serotypes has attracted much attention to the preparation of AAV. Many AAV serotypes, such as AAV 1, 4 and 5, fail to bind the heparin column effectively, thus preventing the application of the same production process to all serotypes. Therefore, new types of AAV vectors need new purification methods. It has been observed that the entrance of AAV4 and AAV5 requires 2,3-linked sialic acid, which leads to the development of AC using sialic acid. Sialic acid-based AC has been proved to be very effective in the purification of AAV5.
Features of Our Services
- High selectivity and ability of viral vector purification;
- According to different AAV serotypes, the customized chromatographic purification method can be developed;
- Fast, strong, high yield and purity, from raw materials in one step, purification process, provide scalability and processing consistency;
- Improving downstream processing significantly by reducing purification steps and maximizing productivity.
If you are interested in our services, please feel free to contact us or send us an inquiry.
References
- Sandoval, I.M.; et al. (2019). Multimodal Production of Adeno-Associated Virus. Viral Vectors for Gene Therapy. Humana Press, New York, NY. 101-124.
- Qu, W.; et al. (2015). Scalable downstream strategies for purification of recombinant adeno-associated virus vectors in light of the properties. Current pharmaceutical biotechnology. 16(8): 684-695.
