Viral Bioprocessing

Gene and cell therapy are currently two of the most promising therapeutic areas, potentially allowing novel treatment of a variety of inherited and acquired diseases. Both therapies employ various types of engineered viral vectors to selectively deliver genetic material to certain tissues, and subsequently replace the erroneous genetic expression causing the disease. Among the most promising tools in human gene therapy are viral vectors based on adeno viruses, adeno-associated virus (AAV) and Lentiviruses. The increase in effective treatments using viral vectors have stressed the importance of scalable, high-yield manufacturing methods.

Image

SAN HQ and M-SAN HQ

are especially developed to suit the high quality and regulatory needs for production of therapeutics, including viral vectors

Image

“Given its optimal activity in high salt conditions, SAN High Quality improves downstream processes and reduces purification time without loss of vector yield and activity. SAN High Quality’s improved efficiency is serotype independent”.

Lab Director of viral vector core at a major academic centre in the mid-Atlantic region

Regulators have imposed limits on the amount and length of DNA residues in final dosages for human administration, and viral vector manufacturers therefore seek to minimize DNA impurities (typically host-cell DNA and plasmids from transfection) in the final product. The industry standard for removing residual DNA is to use a bioprocessing-grade nuclease in the downstream process.

The conditions for the nuclease treatment vary across vectors, serotypes and media, and also due to downstream considerations. In some cases, typically with fragile enveloped vectors such as Lentivirus, digestion of DNA under close to physiological conditions may be preferred. When manufacturing more robust vectors such as AAV, the downstream process can benefit from a higher salt concentration. One particular benefit is due to the high surface charge of some types of viral capsids, which increases their tendency to associate with residual DNA. This results in aggregation, which ultimately reduces the viral titer.[1,2] Using a high salt buffer can reduce the affinity of capsids to DNA and thus also the viral aggregation. An additional benefit of increasing the salt concentration is that it makes the DNA more accessible for digestion by similar mechanisms.

Several commercially available endonucleases used for DNA removal in bioprocessing originate from Serratia marcescens. These nucleases do not perform optimally at the physiological conditions found in cell media, nor do they tolerate high salt concentrations. The conditions in many conventional viral vector manufacturing processes, inhibits the activity of the nucleases. To compensate for the loss of activity, a possible solution is to adapt the workflow to the nuclease (e.g by introducing a desalting step), or to increase the amount of nuclease used. By instead using nucleases that are tailor made for viral bioprocessing, this can result in a less expensive and more efficient process.

SAN High Quality® is the ultimate solution for efficient removal of nucleic acids in high-salt manufacturing and bioprocessing workflows. This non-specific endonuclease has optimum activity at salt concentrations between 400 – 600 mM, allowing significant improvements in efficiency and yield. A small-scale protocol using this approach in AAV production has been published.[3] SAN High Quality is also used to eliminate exogenous DNA contamination for in-process qPCR titer measurements. Following the efficient contamination clearance, the nuclease can easily be inactivated at PCR-friendly conditions. Viral DNA is protected by capsids, and remains intact also after the viral lysis.

M-SAN High Quality® is a novel, nonspecific endonuclease with excellent performance at physiological conditions found in typical cell media. M-SAN HQ outperforms other commercially available nucleases in removal of DNA impurities at conditions often used for manufacture of fragile viral vectors such as Lentivirus.

Image

Detection of SAN High Quality with SAN HQ ELISA Kit. SAN HQ ELISA is used for the detection and quantification of SAN High Quality. The kit consists of monoclonal antibodies, specifically capturing trace amounts of SAN High Quality present in the sample.