October 1, 2018 (Vol. 38, No. 17)

Viral clearance is not a new challenge for the biopharmaceutical industry. However, regulatory demand for better data combined with the growing recognition of the shortcomings of current virus removal and inactivation methods are fostering innovation, according to industry experts.

Ensuring biopharmaceutical products are free of viruses is vital. The risk that a patient treated with a contaminated product will become infected by the virus is significant, particularly if they are already immunosuppressed.

Viruses can be inadvertently introduced into biopharmaceutical production lines in a variety of ways. For example, they can be present in raw materials or found on improperly cleaned bioreactors and other processing equipment. As a result, manufacturers follow procedures designed to minimize the risk of viral contamination.

However, viruses are resilient and can be difficult to remove particularly non-enveloped strains.1 As a consequence, biopharmaceutical producers also implement viral inactivation and or removal/clearance steps to ensure the safety of their products.

GEN interviewed several viral clearance experts who recently spoke at the Cambridge Healthtech Bioprocessing Summit in Boston.

Facility for Viral Clearance

Facility and production-line design can play a vital part in viral clearance strategies, according to Paul W. Barone, Ph.D., associate director of the biomanufacturing research program at the MIT Center for Biomedical Innovation.

He tells GEN, “Traditional biotech products, like proteins and vaccines, have well-established processes and approaches to ensure viral safety that has been proven to be effective.”

Dr. Barone cites approaches such as solvent-detergent treatment, low pH activation, and nanofiltration as proven, effective ways of removing viruses.  “Many traditional manufacturing processes have been validated, in total, to reduce potential viruses by 18 logs or more,” he adds.

A theme common to most viral clearance methodologies is to view the process as a continuum, with actions implemented during each unit operation being used to reduce viral load. Such a strategy requires that the manufacturer establishes pre- and post-clearance segregation between each process, which is a technical challenge, according to Dr. Barone.

“Facility segregation is really product and facility dependent and is one part of an overall safety strategy. The most conservative approach is to have the process segregated by walls with separate HVAC systems. This has often been the ‘easiest’ way to get approval from regulators.

“There are a variety of manufacturing facilities which may have constraints on how segregation can be applied, and certain products will have specific processing requirements as well,” he says.

To help address these challenges, Dr. Barone and his colleagues at MIT’s Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) have developed a definition of pre- and post-viral clearance segregation. The definition “would be valuable for the industry and would help companies as they consider their own approach to facility segregation both now and in the future,” says Dr. Barone.

The work has been accepted by the PDA Journal of Pharmaceutical Science and Technology, but it has yet to be published.

Rational Testing

A common approach to viral clearance is to undertake extremely detailed cell-line and reagent characterization. Such a strategy was core to the development and production of the influenza vaccine, Flublok Quadrivalent, according to Penny Post, Ph.D., vice president at Sanofi Protein Sciences.

“The FDA requires extensive safety testing and characterization of cell substrates for the production of vaccines,” she tells GEN. “We followed the FDA’s guidance document, ‘Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications’ to qualify our cell line,” she adds.

To achieve this, Dr. Post and her team performed general adventitious agent testing using traditional in vitro and in vivo methodology set out by the FDA as well as PCR-, biochemical-, and infectivity-based assays for any viruses of concern identified from literature searches.

The group also conducted viral clearance studies to determine how effective purification processes used in the production of Flublok were at removing a selection of model viruses.

“Model viruses were selected to represent a wide range of potential contaminants, including DNA-based, RNA-based, enveloped, non-enveloped, small, and spherical, icosahedral, and rod-shaped viruses,” Dr. Post says.

Expression System Selection

The cell line used can also be used to minimize the viral clearance work required. For example, the protein components from which Flublok is made are produced using a baculovirus-based expression system which, according to Dr. Post, was selected in part to make viral clearance more straightforward.

The process involves culturing the production cell line in a bioreactor, infecting the cells with a baculovirus engineered to contain the genes of interest to be expressed, incubating the infection for the appropriate amount of time to obtain high product yields with minimal host cell proteins, and purifying and formulating the expressed product.

“The baculovirus infection is a lytic process and is highly species-specific; the virus cannot replicate in mammals and therefore has even been used as a biopesticide,” says Dr. Post.

Model Viruses

Mock viruses—such as those used by Dr. Post and her team—are a recent tool to emerge to help pharmaceutical companies fine-tune viral clearance processes. The basic idea is to deliberately introduce virus-like particles (VLPs) into a manufacturing set-up to determine the efficacy of established removal procedures to allow whatever fine-tuning is necessary.

MockV Solutions is seeking to tap into this growing area of demand. CEO David Cetlin tells GEN, the idea to produce VLPs for clearance testing was prompted by the experience of the shortcomings of current testing methods.

“While working as a downstream process development scientist, I was frustrated that often we’d spend years developing and optimizing a scale-down manufacturing step only to see it underperform during viral clearance validation.

“I thought there must be something co

mmercially available that could serve as a predictor for live MVM and other model viruses. At some point it hit me that VLPs could be used for this purpose and I started MockV Solutions,” Cetlin says.

He claims that in contrast to costly live virus spiking studies that require expertise and take months to design and analyze, MockV’s VLPs could allow for the same data to be generated in a fraction of the time and at a lower cost.

David A. Cetlin at work in MockV Solutions’ lab in Rockville, Maryland.

Development work is ongoing, according to Cetlin, who says, “Our current prototype kit, which predicts MVM clearance, is limited by the sensitivity of the MVP quantification assay. As a result, an end user can expect to achieve less than or equal to 3.0 logs of reduction, based on their experimental parameters.”

He adds, “We are currently working to improve the assay, increasing its dynamic range to 4–5 log10. This work is being funded through an SBIR grant with the National Center for Advancing Translational Sciences (NCATS) of the NIH.”

Cetlin adds that the aim is to make the test kits available to the pharmaceutical industry through a range of channels, including deals with companies in the contract research sector.

“Offering a product or service such as the MVM-MVP Kit would allow a CRO to capitalize and engage customers during these phases leading up to validation studies,” he says. “We actually have a service structure worked out with Texcell to do just that.”

Cetlin explains that customers purchase spiking MVP and ship their samples to Texcell for analysis, which he says allows the CRO to build relationships with these customers who may use them for viral clearance validation. MockV will also seek agreements with technology suppliers, according to Cetlin.

“For industry vendors, such as those selling chromatography resins, virus filters, or analytical kits, a product line of predictive viral clearance kits would be of great interest to their existing customers.”



1. World Health Organization WHO Technical Report, Series No. 924, 2004, Guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products

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