October 1, 2017 (Vol. 37, No. 17)

Next-Generation Sequencing (NGS) and Quality by Design (QbD)

      Lab scientist testing raw materials.

Viral safety is a key component to assuring the safety of biopharmaceuticals for human use. Manufacturers need to verify that their products are free of harmful viruses, while regulatory agencies are tasked with upholding standards for viral safety for all types of biopharmaceutical products. As live cells are used to express the biopharmaceutical product, and many of the components used in cell growth are of animal origin, the risks of virus contamination are high. New technologies and methodologies are emerging for both producing and testing biopharmaceuticals, so the industry is entering a new era of viral safety assurance.

The High Cost and Likely Suspects of Viral Contamination

 “If virus is detected anywhere in a biomanufacturing process, the entire process must be shut down and an intensive investigation embarked upon,” says Bala Raghunath, Ph.D., director of global manufacturing sciences and technology at MilliporeSigma. A contamination incident in 2009 triggered a facility decontamination, extensive investigations of root cause, and penalties from concerned health authorities, not to mention the significant loss in revenues as a result of the plant shutdown. “This impacted supply of critical drugs which, in turn, affected patient access,” says Dr. Raghunath. Several reports detailed the incidence of this contamination.

Raw materials have often been implicated in viral contamination incidents and there is a general acceptance of their vulnerability to virus contamination. Testing cell culture media for the presence of virus is inherently constrained by assay sensitivity and an inability to detect low levels of virus contaminants. As a consequence, manufacturers are considering adding steps to inactivate or remove potential virus contaminants from cell culture medium and other raw materials used in upstream manufacturing processes.

“Some companies have evaluated high temperature short time (HTST) pasteurization methods to kill
pathogens in cell culture media,” says Dr. Raghunath. “However, not all media components are stable at high temperatures. Further, HTST requires some significant initial investment and equipment, so only a few companies have taken that approach. More recently, companies have developed novel filters designed for cell culture-media treatment. Cell culture media is at risk for introducing viral contamination into a bioreactor, so having that viral filtration step is considered a good way to enhance viral safety assurance in the manufacturing process.”

In addition to cell culture media, cell banks used for manufacturing biotherapeutics are also considered high risk for introducing contamination and need to be well characterized before starting manufacturing.

“There is extensive characterization of the master, working, and end of production cell banks before bioprocessing,” said Kathryn Remington, Ph.D., principal scientist focusing on the BioReliance® portfolio of MilliporeSigma. “Companies need to verify that the cells are the type that they think they are and also look for purity of the cells. They’ll then need to be screened for bacteria, fungi, mycoplasma, and viruses.”

Martin Wisher Ph.D., global head of regulatory affairs focusing on the BioReliance® portfolio of MilliporeSigma added that “in vivo studies need to be completed in the master cell bank and end of production cell line. You want to be sure that your cell banks are free of viral contamination. Testing the bulk harvest with in vitro assays gives you further evidence that the cell banks were clear and gives you assurance that there is no other contamination coming in through the process.”

Regulatory testing standards for cell banks and bulk harvest are outlined in the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH Q5A) guidelines. The ICH brings together the regulatory authorities from Europe, Japan, and the U.S. as well as industry experts with a purpose of providing harmonized recommendations and technical guidelines for product registration. Current guidelines recommend extensive in vivo testing for viral safety purposes in bioprocessing. But, with the development of new technologies, the industry is questioning the utility and relevance of these more traditional tests.

Scientists involved in performing cell line characterization testing.

In with NGS, out with In Vivo Analyses

“One technique that the industry is hoping to get rid of is the in vivo assay,” says Dr. Wisher. “In the last few months, there has been a move to rewrite specific ICH guidelines to remove the requirement for in vivo testing. The industry wants to reduce the number of animals used in pharmaceutical testing in general. The old, classical in vivo techniques made sense when there wasn’t an option to use tissue culture, but they’re not as sensitive as people thought they were.”

An emerging technique that could help to replace at least some in vivo testing is next-generation sequencing (NGS), otherwise known as massively parallel sequencing or “deep” sequencing. NGS is a technique that allows unbiased sequencing of all DNA or RNA in a given sample, and has been commonly used in a research setting for over a decade, but is only more recently being implemented for viral safety testing.

“Traditional testing will only test for a panel of viruses that we know can impact human health,” explained Colette Coté, Ph.D., principal scientist focusing on the BioReliance® portfolio of MilliporeSigma. “But the reality is that there may be emerging viruses that we haven’t identified that can also affect human health, Zika being a perfect example. NGS enables a deeper search to look for viruses that we may not have known about.”

As promising as the technique is for giving more insight into potential viral contaminants, regulatory guidelines are slow to adopt.

“The industry is trying to get their hands and minds around the technology,” continued Dr. Coté. “Consistency, robustness, reproducibility and sensitivity need to be shown. They need answers to questions about what the technology can do, what the limitations and advantages are, and how it compares against current technologies.” When these questions are answered, regulatory expectations and guidance will likely change.

The use of NGS for testing biologics poses greater challenges as compared to standard PCR or other molecular tests due to its technical complexities and requirement for Big Data bioinformatics. To address these concerns, the Advanced Virus Detection Technologies Interest Group (AVDTIG) was formed as a joint effort by regulatory and industry scientists to share data and experiences using advanced virus-detection technologies such as NGS. “There are about 30 companies working in the AVDTIG right now,” said Dr. Wisher. “This year, the group is publishing papers that will discuss best practices for NGS methods and data analysis. They’re also developing a curated database of useful sequences for viral safety studies as well as making a number of standard purified virus preparations so that spiking studies can be done to get a better idea of the sensitivity of the technique.”

Although regulatory agencies will not yet accept NGS as a replacement for standard virus detection tests, the technology is already impacting industry expectations and standards. “Last month at a viral safety meeting, Sanofi announced during a presentation that they will be using NGS for screening cell lines and vaccines and submitting that data along with all of the conventional testing that was done for all new products,” said Dr. Wisher.  

Viral Safety Testing in Quality by Design

Another trend in the industry, which has been introduced and consistently encouraged by regulatory authorities is the implementation of Quality by Design (QbD) approach in process operations. The QbD approach encourages an understanding of how raw materials and manufacturing processes impact the critical quality attributes of the product, i.e., attributes that impact the safety, efficacy, and quality of a drug product. Ultimately, a design space is defined, which represents the operating range, within which the process meets the critical quality attributes of the product. The “absence of virus” can be considered a critical quality attribute that impacts the safety of the drug product.

“The QbD approach enhances a manufacturer’s understanding of their process and unit operations as well as the impact of operating parameters on quality attributes,” explained Dr. Raghunath. “Following QbD methodology, we have put together a knowledge base that details the impact of various process conditions and operating parameters on the viral clearance performance of a downstream virus filter. The impact of parameters that can typically change during the process, such as pressure, conductivity, pH, protein concentration, and membrane lots, on virus retention is reported. Understanding the effects of these parameters on quality attributes like virus retention allows those parameters to be controlled within a given range.”

Using the principles of QbD for viral safety testing results in a more thorough understanding of the design space for viral clearance unit operations and the appropriate controls that are needed to maintain the operating parameters. Key to the methodology is upfront identification of risk factors and incorporation of testing protocols to manage those risks, and NGS helps to identify viral contamination risk. “When using novel substrates in bioprocessing, extensive NGS characterization can be performed on several lots of the raw materials to identify potential contaminants that may be present,” said Dr. Remington. “This can help focus testing so that manufacturers have a rational testing strategy for specific contaminants, and appropriate assays can be designed.”

Integrating sensitive new technologies for viral detection into a QbD approach to biomanufacturing provides confidence for both the manufacturer and regulatory agency that the risks of virus entry to the process are minimized, low levels of virus can be detected, and the process parameters are controlled to assure the expected levels of virus removal in the downstream process, thereby assuring the integrity and safety of the drug product.

Read the rest of the Viral Safety supplement.

Advances in Upstream Technologies Reduce Viral-Contamination Risks
Viral Safety in Monoclonal Antibody Manufacturing
Laying the Foundation for Viral Safety
Upstream Virus Safety: Protect Your Bioreactor by Media Filtration
Utility of GMP Next-Generation Sequencing (NGS) for Biosafety Assessment of Biological Products

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