December 1, 2014 (Vol. 34, No. 21)

Don’t be satisfied with existing techniques for detecting and removing viral contaminants from recombinant bioproducts. Regulatory bodies aren’t.

Viral clearance and quality control remain key challenges for biomanufacturers involved in both single-use and/or steel applications.

New approaches and the latest thinking on ways to deal with viral clearance were topics of discussion at the recent Sartorius Stedim Biotech “European Upstream and Downstream Technology Forum,” which was held in Germany.

Thomas R. Kreil, Ph.D., associate professor of virology and senior director for global pathogen safety at Baxter, discussed the ubiquitous presence of viruses versus the limitations of current virus detection technologies.

“It is an inherent property of any test for an infectious agent that their presence cannot be ruled out by a negative result,” he stated. “All one can conclude is that their occurrence is excluded down to the respective limit of detection. This aspect is often missed in discussions of the topic with respect to bioprocessing and in the application of testing to maintain the safety margins of plasma-derived and other blood products.”

According to Dr. Kreil, examples have been observed in which all the assay systems utilized couldn’t identify contaminants that were later found to be present. A few cell lines that had gone through all the required testing and scored negative were later found to harbor viral contaminants to the point where contaminated vaccines manufactured from those cell lines were released to the market, although initially unknowingly.

Dr. Kreil argues that while new technologies such as deep sequencing may not eliminate the problem, they do provide a better probability of detecting, in particular, less well-known microorganisms. Nonetheless, he remains confident that safety margins of biologicals produced by the pharma industry are appropriate.

“There has never been, to our knowledge, a case in which an infectious disease was transmitted by a viral contaminant in a product based on recombinant biotechnology,” he said. “However, we can’t reign in our efforts to develop new and more sensitive detection technologies. As soon as new platforms for quality control are available, we need to put them into use. If you stop your effort, you are falling behind.”

Dr. Kreil proposed the application of the “safety tripod” concept to bioprocessing operations. This protocol was developed by regulators and the representatives from the plasma derivatives industry to deal with the historic contaminations of blood products with HIV, hepatitis C, and other viruses. “For biotechnology products, the safety tripod should consist first of the selection of the appropriate cell lines and related culture materials; second, the testing of appropriate production intermediates; and third, the removal or inactivation of potential harmful agents,” he explained.

According to Dr. Kreil, next-generation sequencing and other advanced technologies need to be seriously considered as possible approaches in viral inactivation activities.

“Industry has a choice of operating at today’s state-of-the-art, i.e., meaning under current-GMP regulations, or act in a proactive fashion rather than be confronted by regulators making decisions. This will mean that the stakeholders for industry must constantly move ahead of the game to generate data supporting the development of a rational regulatory context.”

Sartorius Stedim Biotech has assembled an orthogonal platform for quantitative virus and contaminant clearance The platform encompasses UVivatec® virus inactivation, Sartobind® membrane chromatography, and Virosart® filtration.

Best Practices

The regulatory requirements for viral clearance studies from early- to late-stage product development were reviewed by Horst Ruppach, Ph.D., manager of viral clearance and global coordinator of virology at Charles River Laboratories. He emphasized the point that study design needs to demonstrate the most economical route for carrying out efficient and robust viral removal and inactivation. Effectiveness and robustness are not only defined by the level of the reduction factor, but also by the method of process-step analysis, e.g., sampling mode and application of high-sensitivity assays.

Since companies are responsible for the safety of their products, “the approach that you apply is critical,” advised Dr. Ruppach. “I would not recommend staying with the minimal approach according to the [GMP] guidelines. Keep in mind that regulatory bodies interpret guidelines differently and demand [the best of the current] approaches.”

In accord with the other presentations, Dr. Ruppach stressed the complexity of living systems and the need to constantly evaluate what you are working with. “In employing isolates of the same cell line, you must keep in mind that they evolve in different directions,” he stated. “After a year, a cell line grown in two different labs will have changed, and the same assays used in the two facilities can give different results. This makes standardization of viral clearance studies challenging.”

At Charles River Laboratories, it is recognized that study design needs to demonstrate not only economy in carrying out viral removal and deactivation, but also effectiveness and robustness. These last two qualities are defined by both the level of the reduction factor as well as the method of process-step analysis.

Increasing QA Complexity

Conference participants acknowledged the complexity of monitoring bioproducts for viral contaminants and of designing technologies adequate to eliminate them. Anika Manzke, product manager for viral clearance at Sartorius Stedim Biotech, affirmed that revised regulatory guidance standards are now much more highly detailed, as put forward by the European Medicines Agency. As the technology for the detection of viral contaminants has become more sophisticated and sensitive, many incidents of contamination of plasma and bioproducts have surfaced in the United States and the European Union.

If these threats are to be managed, the overall focus on monitoring must be implemented earlier in the process train, according to Manzke. Selection of efficient technologies with the ability to clear both small and large viruses will be paramount, and a single clearance step will no longer be accepted by authorities. In accord with these guidelines, the 20 nm filter is now considered state-of-the-art technology.

UV Inactivation

The use of nanofiltration to remove potential viral contaminants from large protein molecules has its limitations, according to Tobias O’Neill, senior scientist at Gallus Biopharmaceuticals. Although viral filters with small (20 nm) pore size are effective in separating typical protein molecules such as IgG antibodies from viral contaminants, they can trap and retain larger molecules, including IgM antibodies. These proteins are damaged by the standard viral-inactivation strategies of chemical and heat treatments.

O’Neill stated that it is necessary to closely monitor the UV process, during which protein aggregates may be formed. If the energy application is too high, aggregation or possible degradation may inactivate the protein. As the energy input is adjusted, the starting material absorption at 254 nm (UV-C radiation) needs to be subtracted out in order to obtain accurate dosage quantities. IgMs can be quite finicky proteins and tend to denature in low conductivity or low pH solutions, causing them to precipitate out of solution.

Parvoviruses are particularly problematic in viral-clearance strategies, as they are often resistant to other forms of inactivation, and their small size makes them challenging to remove via filtration. The UV-C technology not only worked in initial small-scale parvovirus experiments, but O’Neill found that the method also performed well at higher scales.

“We’re seeing pretty much the same purities on scaleup of production,” he said.

O’Neill indicated that he has used Sartorius Stedim Biotech’s UVivatec® technology, which employs UV-C irradiation to bring about viral inactivation. According to O’Neill, the product technique can deliver >4 logs of inactivation of small nonenveloped viruses (20 nm) such as the porcine parvovirus and minute virus of mice. He added that the UVivatec technology can also be effective with larger enveloped viruses of >50 nm dimensions. 

Maximizing Viral Clearance for High-Titer Validations

Virus filtration is one of the most robust viral-clearance processes, and contemporary small virus filters reliably achieve greater than 4 logs of reduction of small nonenveloped viruses such as parvoviruses. Recent efforts to demonstrate high levels of viral clearance have led to the development of high-titer spiking stocks, but exceptional viral clearance is not guaranteed.

To provide a solution for consistently achieving very high logarithmic reduction value (LRV) on Planova™ 20N filters, Daniel Strauss, et al. from Asahi Kasei Bioprocess performed clearance studies using PP7, a small nonenveloped bacteriophage, under various filtration conditions.

In their recent poster entitled, “Maximizing Viral Clearance for High Titer Validations”, they demonstrated the dependence of PP7 LRV on overall challenge titer. Standard levels of PP7 LRV could be achieved with high-titer spiking stock, but for filtrations exceeding the recommended total virus challenge of 11 log PFU/m2, residual virus increased in the filtrate.

For those processes for which higher LRV is critical, they offer a filters-in-series process instead of using a single Planova filter.

“Using this filters-in-series strategy, we were able to achieve over fivefold increase of product throughput with complete PP7  clearance (LRV ≥ 7),” wrote the scientists. “This alternate process, with an optimized control strategy, takes full advantage of the reliability and performance of Planova filters while achieving high viral clearance that is only possible with high-titer virus spiking.”

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