Hydrophobic Interaction Resins
Adeno-associated virus (AAV) vector is a preferred delivery system for clinical in vivo gene therapy. The GMP-grade AAV particles require purification strategies that support the generation of vectors with high purity and exhibit a good efficacy and safety profile. As a reliable provider in the viral vector market, Creative Biolabs is committed to designing, cloning, and modifying AAV vectors and offers a variety of advanced vector purification services by affinity chromatographic techniques. Particularly, our hydrophobic interaction chromatography is a versatile method for the separation and purification of AAV vectors through the function of hydrophobicity.
Hydrophobic Interaction Chromatography (HIC)
HIC is a powerful tool for the process of purification of biomolecules, based on the retention of proteins on hydrophobic matrices in the presence of salt. This technique takes advantage of accessible hydrophobic regions located on the protein surfaces and their interactions with a weakly hydrophobic stationary phase. Proteins and other targets with hydrophobic domains are attracted to hydrophobic ligands of HIC resins by employing a high-salt flowing phase. The salt condition contributes to lyotropic effects allowing the proteins to bind to hydrophobic ligands, and proteins are eluted by decreasing the salt concentration.
Due to its high versatility and efficiency, HIC is widely applied in the elusion and isolation of desired molecules in their native state. It has been used in isolating AAV, homologous proteins, recombinant proteins, receptors, antibodies, nucleic acids, and other large molecules. Most therapeutic targets are separated in a low salt or no salt washing buffer. Since HIC isolations are exploited under mild eluting conditions, the biological activity of purified molecules is typically retained.
Characterized Hydrophobic Interaction Resins at Creative Biolabs
Figure 1. A paradigm shift in viral vector purification to increase yields and decrease process development timelines. (Terova, 2018)
Today, recombinant AAV (rAAV) vectors have been implemented in a number of gene delivery platforms. The use of these vehicles in clinical gene transfer has increased the need for their production, purification, and characterization. Chromatographic separation is the most versatile and effective approach for large-scale production of rAAV or viral vectors, compared to density gradient centrifugation for small scale production.
HIC is based on the binding of viral capsid proteins to the column matrix through hydrophobic interaction in an aqueous solution, which has proven to be a viable method for AAV purification. To date, a small portion of, mostly single serotype-targeted affinity resins exist, and thus, using one resin to purify multiple serotypes is valuable. At Creative Biolabs, we focus on different chromatographic modes for AAV purifying process development and the utility of different purification steps for pure, concentrated virus production. Notably, we provide hydrophobic interaction resins that can purify multiple AAV subclasses with high binding efficiency. Our HIC resins are available in three functional forms:
- Methacrylate-based HIC Resin -- weak hydrophobicity, for purifying compounds with strong hydrophobic regions;
- t-Butylacrylate-based HIC Resin -- mild hydrophobicity, for purifying compounds with few or weakly hydrophobic regions;
- Beads SM-2 Resin -- nonpolar polystyrene adsorbents, used in aqueous solvents (e.g., alcohols, hexane, petroleum ether, and diethyl ether) or with solvent mixtures, without expansion or contraction of the beads.
By reducing steps and maximizing productivity, our affinity resins present a cost-effective purification strategy for commercial AAV manufacturing. In addition, there're five HIC ligands of diverse degrees of hydrophobicity and selectivity can be provided for the further development of efficient purification methods, including Hexyl, Butyl, Phenyl, PPG (polypropylene glycol), and Ether.
Advantages
- High selectivity and superior resolution capability
- Purify multiple AAV subclasses (e.g., chimeric and recombinant vectors)
- Robust stability with increased AAV yield and purity
- Flow rate-independent performance
The adaptable solution of HIC can be used at all steps of virus vector production, including capture step, intermediate and final polish purification. At Creative Biolabs, our hydrophobic interaction chromatographic resins are designed specifically for intermediate purification to remove contaminants from eluted AAV particles by strong or weak hydrophobic interactions. For more information, please feel free to contact us or directly send us an online inquiry.
Reference
- Terova, O.; et al. (2018). Overcoming downstream purification challenges for viral vector manufacturing: enabling advancement of gene therapies in the clinic. Cell & Gene Therapy Insights. 101-111.
