Manufacture of viral vectors

Viral vector manufacture for therapeutic purposes involves initial viral propagation in appropriate animal cell lines, viral recovery, concentration, purification and formulation. A generalized manufacturing scenario for adenoviral-based vectors is outlined in Figure 14.7. The manufacture of alternative viral vectors likely follows a substantially similar approach.

Master and working banks of both the viral vector and the animal cell line will have been constructed during the drug development process (see Chapter 4). Manufacture of a batch of vector, therefore, will be initiated by the culture of packing cells in suitable animal cell bioreactors. The

Seed vith vector

Seed vith vector

Figure 14.7 Large-scale manufacture of adenoviral vectors for use for gene-therapy-based clinical protocols. Refer to text for details

principles and practice of animal cell culture have been overviewed in Chapter 5. To date, bioreactor size of 100 l or less have been used, which are sufficient to satisfy clinical trial demand. The packing cells are then seeded with the replication-deficient virus, allowing vector propagation (see also Figure 14.5). After a fixed time the viral-infected cells are collected (harvested) by microfiltration or centrifugation and the cells are then homogenized in order to release the viral vector. Traditionally, adenoviral vectors (and indeed many other animal cell viruses) were purified from such a crude mixture by caesium chloride density-gradient centrifugation. While appropriate to laboratory-scale operations, this method is unsuitable for large-scale viral recovery due to scale-up issues and cost.

Alternative purification methods based upon column chromatography are thus employed on an industrial scale. Major 'contaminants' present in this crude viral vector preparation include some intact animal cells, cellular debris, and intracellular molecules, most notably animal cell protein and nucleic acid. Intact cells/cellular debris is removed by filtration. The release of large amounts of cellular DNA increases the solution viscosity and complicates downstream processing. The purification protocol, therefore, usually entails the physical degradation of DNA by addition of a nuclease enzyme. A solvent/detergent treatment step is then undertaken as a safety step in order to inactivate any enveloped contaminant viruses that might also be present. High-resolution purification is usually achieved by a combination of ion-exchange and gel-filtration chromatography, with product concentrations steps being undertaken by ultrafiltration if necessary. The final product is then filter sterilized and filled into glass vials. The purified vectors generally may be stored either refrigerated or frozen, and they display useful shelf lives of 2 years or more.

Supplements For Diabetics

Supplements For Diabetics

All you need is a proper diet of fresh fruits and vegetables and get plenty of exercise and you'll be fine. Ever heard those words from your doctor? If that's all heshe recommends then you're missing out an important ingredient for health that he's not telling you. Fact is that you can adhere to the strictest diet, watch everything you eat and get the exercise of amarathon runner and still come down with diabetic complications. Diet, exercise and standard drug treatments simply aren't enough to help keep your diabetes under control.

Get My Free Ebook


Post a comment