The growing significance of biotherapeutics, particularly monoclonal antibodies (mAbs), assures that increased demand for virus-safety products and services will continue, despite the trend toward a more risk-based approach to virus removal and inactivation.
Virus safety is one component of world-wide regulatory guidelines requiring biomanufacturers to demonstrate that their manufacturing processes remove or inactivate known contaminants, among them viruses, TSEs, DNA, mycoplasma, endotoxins, and bacteria.
Virus safety encompasses two loosely related contamination issues. The better-known involves viruses that could potentially infect human patients after a product hits the market. The other focuses on viruses that are noninfective to humans, but that adversely affect the production cells or organisms.
In June 2009, Genzyme closed down production of its Fabrazyme and Cerezyme enzyme products at a Massachusetts production facility due to infection of CHO production cells with Vesivirus 2117. This was the second time this virus struck Genzyme production facilities, the first being in 2008 at its Geel, Belgium, manufacturing facility.
Vesivirus does not cause human infection but rather interferes with the growth of CHO cells. The company traced the virus to a nutrient additive, and the problem was subsequently resolved.
Genzyme’s contamination problems have led to renewed interest in contaminating viruses, particularly Vesivirus. In February, Life Technologies introduced a rapid (five-hour) molecular test for detecting Vesivirus 2117. The ViralSEQ™ detection kit uses bead-based PCR to isolate viral RNA from process samples and includes a positive control to reduce the possibility of a false positive.
Compared with older infectivity assays, PCR and its variants enable much more rapid, accurate evaluation of downstream steps for their ability to remove viruses. Bioreliance, a biosafety services company, has validated qPCR for the most common viruses affecting bioprocesses and routinely uses the technique in viral clearance studies it runs for clients. The technique is particularly effective for calculating the partitioning of virus and product after chromatography steps.
Bacteriophages, or phages, represent another significant viral contaminant that can wreck a fermentation. Unlike Vesivirus, bacteriophages infect bacteria (for example E. coli, Bacillus, or Pseudomona fermentations) and not mammalian cells, but similarly phages do not cause human disease.
“Bacteriophages are the most numerous organisms on earth,” notes Marcin Los, Ph.D., CEO of Phage Consultants, which consults and performs studies on both common and rare viral contaminants. “All production bacteria are vulnerable because phages are so prevalent in the environment. A single virus particle can destroy a fermentation.”
Perhaps the most vexing attribute of phages is their persistence, which is in good part due to their huge numbers. It is not uncommon for several phages to colonize a manufacturing facility, but one strain usually predominates. Once a bacteriophage infection is detected, preventing subsequent contaminations is difficult. Dr. Los says, “The goal after confirming infection is to prevent phages from becoming a persistent problem.”
Positively identifying bacteriophages and excluding other potential pathogenic contaminants is critical. Phage Consultants employ qPCR-based tests to detect bacteriophages and other viral pathogens. Rapid, reliable methods are essential with bacterial fermentations that tend to be shorter than mammalian cell cultures.
It is sometimes possible to recover product from a phage-infected fermentation, but this should never be done if it increases the likelihood that viruses will survive and continue to colonize the plant. Dr. Los adds, “Each liter of an infected fermentation may hold up to 1013 phage particles.” Phages spread quite easily, so in most cases it is more prudent to shut down the fermentation, kill the viruses and infected cells, take appropriate containment and decontamination measures, and start again.