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Cynthia A. Challener, PhD, is a contributing editor to BioPharm International.
Advances in adventitious agent detection methodology are bringing benefits, but more work needs to be done.
The raw materials used in the manufacture of biologic drugs come from different sources: animals (although use of such materials is decreasing), plants, and-more frequently-chemically derived ingredients. As a result, it is not possible to know all of the possible viral contaminants that may be present. The biopharmaceutical industry has consequently taken extensive measures to prevent contamination and continues to develop advanced analytical methods to detect both known and unknown viruses. Traditional cell-based assays are broad-based and generally effective, but do suffer limitations, such as lengthy test times. Rapid nucleic acid-based techniques have been developed as alternatives, but they often only target specific viral agents. The methods under investigation today enable the identification of multiple viruses, but issues must be addressed before they can be fully adopted by industry.
Technology advances drive interest in new methods
Adventitious agents are microbial contaminants that are introduced inadvertently into a biopharmaceutical manufacturing process. Even if they are not harmful to patients, they are impurities and are undesired. Although current industry processes and analytical techniques have been effective in keeping biologic drugs safe from viral contamination, there has been significant scientific progress with respect to viral detection, and these advances are attracting the interest of the biopharmaceutical industry.
Several important steps have been taken by the industry to prevent contamination, including the movement away from animal-based to chemically derived materials, the use of well-characterized cell lines, and the implementation of risk-mitigation and control strategies for sourcing activities. The implementation of process steps that minimize the risk of viral infection, such as heat treatment and viral filtration, has also been highly effective.
In fact, it is important that these approaches be combined with extensive analytical testing, according to Ivar Kljavin, director of adventitious agent management with Genentech. “It is not possible to prove a negative result with analytical testing, and therefore, testing, while a critical part of an effective solution for viral contamination prevention, is not sufficient alone,” he asserts.
The conventional in vitro adventitious virus assay, which uses various indicator cell lines for detection of viruses, is sensitive and can potentially detect one infectious virus particle, but the virus must replicate in at least one of the indicator cell types and produce some type of detectable effect (visual for cytopathic effects and/or hemadsorption or hemagglutination for specific red blood cells). False negatives are possible if a virus replicates but does not cause a detectable effect or is present but does not replicate in the specific indicator cells. There is also some variability with cell-based assays, and these tests typically take 14–28 days, during which time contaminated materials may be sent downstream for further processing. The need to decontaminate a production facility can lead to significant disruption of the drug supply, a situation that is unacceptable, according to Kljavin.
Despite these limitations, when used in conjunction with other risk-mitigating steps like viral clearance validation, cell-based assays are effective. New, broader, more sensitive, and more rapid assays have been developed, however, that are attracting the interest of the biopharmaceutical industry.
State of the art
The Parenteral Drug Association (PDA) has been working on a technical report representing some of the best thinking of technology developers, users, and regulators on viral detection methods. According to Paul Duncan, senior principal scientist for vaccine analytical development in vaccine bioprocess R&D with Merck Research Laboratories, based on this work, it appears that the profile for the polymerase chain reaction (PCR) mass spectrometry approach has decreased somewhat in recent years, perhaps due to a shift in focus to clinical diagnostic applications. He also notes that various microarray options exist and have interesting advantages, including rapid turnaround, but their accessibility is limited. A compelling ‘product presentation’ that would fit into factories as in-process rapid decision-making tools (not necessarily for product release, though) is still lacking. “On the other hand,” he says, “massively parallel sequencing is now readily available, and some commercial testing labs are quite good at processing the datasets and reaching reasonable conclusions. Of course, several large pharma companies are also developing their own expertise and capabilities.” He does note, though, that the turnaround time for the sequencing approach is longer, and bioinformatic analysis is still an area of active development.
GMP validation a key issue
Use of new viral detection methods in commercial biopharmaceutical manufacturing in a GMP testing environment may require system validation, lockdown, and archival, business and regulatory acceptance, which are challenging issues to address at this time. “There is a need to continue evolving these systems to keep up with expanding databases, improvements in computing infrastructure, analysis tools, and algorithms, yet at the same time maintain GMP change control,” Duncan explains. In addition, he points out that replacing existing tests with any of these newer techniques involves the added complexity of defending the suitability (breadth and sensitivity of detection) of the new methods to regulators in not just one, but every intended market. Manufacturers would also have to come to terms with new types of specifications and manage the risk of false positives.
Having said that, at the present time newer viral detection analyses do have a use in biopharmaceutical development and perhaps even manufacturing as characterization and investigation tools, according to Duncan. “These new analytical tools certainly have a place where scientific validity through suitable controls is the main concern,” he says.
Preparation is of primary importance
Sample selection and processing determine what is possible to detect and how results must be interpreted, according to Duncan. In particular, sample selection requires understanding the different ‘compartments’ and the types and packaging of nucleic acids to be expected in each. Cell pellets and whole culture lysates present different challenges and opportunities than cell-free supernatants or raw material solutions, for instance. “There are tradeoffs in the selection of any given compartment, and also tradeoffs in sampling all compartments at once,” he observes.
In addition, the selection of just one sample type without adequate controls can make interpretation of results unnecessarily complex. On the other hand, the preparation of separate RNA and DNA extractions instead of combined total nucleic acid extractions may help differentiate the relevance of some signals. Concentrating samples from some matrices to achieve higher sensitivity can also be surprisingly challenging; there may be, for example, differential effects on recovery of different viruses, according to Duncan.
For each case, there are likely many viable alternative approaches, but defending any selected approach requires a significant effort, because the goal is to detect all possible viruses-or adding even more complexity-the detection of all possible bacteria and fungi as well. Sample processing also creates an opportunity for cross-contamination, and therefore, Duncan notes that significant care is needed to prevent false positives and unnecessary investigation.
Huge role for bioinformatics
Bioinformatic analysis must parse meaningful information from a noisy background. The challenge is detection of not only glaringly obvious viral signatures (high levels of complete coverage of known viral genomes), but also hints of signatures of agents that are unknown and/or unexpected, that may be present at low levels in the midst of tens to hundreds of millions of other sequence reads. The computational infrastructure must be sufficiently scaled, or else even terrific software will have limited success, given the analysis challenges. That isn’t the only issue. “Not all software can be readily translated across different computational platforms, so perhaps bioinformatics analysis solutions may need to be developed to some extent in the context of the specific high-performance computational infrastructure,” Duncan adds. He also notes that once detected, some interpretation of these signatures is then required to determine if they suggest actual infectious contamination or just normal inactivated process residual nucleic acid from input materials.
Beyond further improvement in the newer, advanced viral detection technologies themselves, Duncan believes that there are additional steps the biopharmaceutical manufacturing industry can take to improve their acceptance. Better control of upstream bioprocessing could make it less necessary to rely solely on conventional non-real-time testing for assurance of culture integrity, and perhaps allow for more in-line or near-real-time process monitoring approaches. “Such an approach may be a tougher sell in the viral vaccines space, but could be a realistic opportunity for some biologics where there are extremely robust downstream viral inactivation/clearance systems in place,” he says. “Examples of such advances could arguably include, for instance, single-use processing and otherwise better use of closed systems, chemically-defined media, and a better understanding of other inputs including cell substrates,” Duncan continues. He adds that longer-term perfusion cultures and continuous downstream processing will also require better in-line and near-real-time means for defending culture integrity.
For practical use in the future, Duncan would also like to see factory-friendly presentations of systems that do not rely on de novo sequencing and would like to have the opportunity to evaluate in some detail how well they work at detecting real contaminations, because they might be the most rapid options in the near term. “It is possible that such systems already approach the sensitivity needed to be meaningful and practical,” he notes.
In the longer term, Duncan believes that shorter turnaround times with sequencing might be achievable, but at the cost of generating an astronomical amount of data for which archiving could become a real issue. “For applications of sequencing that may not require such rapid turnaround, such as cell-substrate characterization in early development, there is also still a need to raise the understanding of many technical issues among technology developers, service laboratories, biopharmaceutical companies, and regulators in order to assure that potential adventitious or endogenous agent-related issues in biological production systems can be detected and addressed,” he asserts. An interest group sponsored by PDA is actively pursuing this goal and welcomes active contributors.
Article DetailsBioPharm International
Vol. 28, No. 7
When referring to this article, please cite it as C. Challener, “Viral Detection Technologies Must Continue to Evolve,” BioPharm International 28 (7) 2015.