Iodixanol Gradient Centrifugation

Adeno-associated virus (AAV) is a small, non-pathogenic parvovirus that can stably infect quiescent and replicating host cells while eliciting a minimal immune response, further enlarging its potential as a gene therapy vector. The application of AAV vector in clinical gene transfer has increased the request for viral characterization, production, and purification. As a famous expert in gene therapy, Creative Biolabs has developed a method of iodixanol gradient centrifugation for a rapid AAV preparation and purification, followed by column chromatography. It is a simple protocol that can be used to purify the AAV particle of any serotype and reliably serves to boost virus production efficiency.

Introduction of Iodixanol Gradient Centrifugation

Iodixanol (C₃₅H₄₄I₆N₆O₁₅) is an iodine-containing non-ionic dimeric medium. In medicine, it's used as an intravenous isomolar radiocontrast agent. In research, it's used as an isomolar density gradient medium appropriate for virus isolation and purification of cells, organelles, and other macromolecules (e.g., lipoproteins). More importantly, iodixanol is an inert substance having non-toxic to mammalian cells. As such, it's reported not necessary to remove iodixanol before the use of purified viruses, unlike other density gradient matrixes. In 1999, iodixanol gradient purification of AAV was first proposed by Dr.Zolotukhin's team, and today it's still one of the most common purification methods.

Iodixanol Gradient Centrifugation Services

AAV is a promising gene therapy system that has been widely used as a gene delivery vector for basic research. Recombinant AAV (rAAV) has been arranged to treat a variety of diseases in animal models, such as Hemophilia B, Leber congenital amaurosis, Duchenne muscular dystrophy, and Parkinson's disease. As AAV becomes the vector of choice, it is increasingly critical to have access to a simplified production and purification protocol for GMP-grade rAAV.

There're two main AAV purification techniques, namely density gradient centrifugation and chromatography purification. In most cases, one method, iodixanol density gradient centrifugation, is sufficient to purify and harvest laboratory-grade rAAV. This technique is serotype independent, minimizing variance between varying AAV preps and is more affordable than affinity and ion-exchange chromatography. Also, the range of iodixanol gradient is wide so that it can be used for virus concentration.

Figure 1. Iodixanol step gradient for the purification of rAAV. (Zolotukhin, 1999)

At Creative Biolabs, we have established a rapid, reproducible approach to purify and concentrate rAAV under physiological conditions. This method is based on the use of iodixanol as a density gradient medium. Due to its molecular mass and high solubility (1.32 g/ml), iodixanol is readily able to form self-generating isosmotic gradients whose density profiles can be regulated by alterations to run parameters (e.g., sedimentation path length of the rotor, relative centrifugal force, centrifugation time, and temperature) and starting concentration. As long as keeping the gradient to sample ratio constant, our current strategy results in reproducible rAAV purification and concentration, which would be performed in less than one day.

Advantages

• Highly reproducible, easy to execute, and serotype independent
• A simplified, step by step protocol for rAAV production
• Purification and buffer exchange, concentration could be completed in 1 day
• High quantities of laboratory-grade AAV particles (>5x10¹²) could be completed in 1 week

AAV vectors are an attractive tool for direct in vivo gene therapy and numerous methods have been developed to generate and characterize them over the past few years. As a reliable partner in the vector purification area, Creative Biolabs provides an up-to-date, detailed iodixanol gradient centrifugation services to isolate and purify hundreds of AAV serotypes and variants as requests. With the help of us, clients will pave the way to a better understanding of the viral vector and, finally, its use in clinical applications. For more information, please feel free to contact us or directly send us an online inquiry.

Reference

1. Zolotukhin, S.; et al. (1999). Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther. 6(6): 973-985.