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Early detection and more sensitive methods of detecting adventitious agents are becoming increasingly critical in bioprocessing.
The importance of adventitious agent testing, a routine aspect of biopharmaceutical production, cannot be overlooked as it is a critical component in ensuring the purity and quality of biological product from the start. The growing complexity of newer biologic modalities, however, requires more highly sensitive testing methods and poses challenges to current analytical methods.
Generally speaking, adventitious agents are the microorganisms (bacteria, fungi, protozoa, mycoplasma, viruses, etc.) that may be introduced into the product during the normal course of the biomanufacturing process. They can be introduced through product processing or through process material used for production.
“These contaminants are living organisms that can adversely affect manufacturing efficiency, product quality but more importantly pose a threat to patient safety,” notes Marian L. McKee, PhD, vice-president, Biosafety, Eurofins BioPharma Product Testing.
Typically, endogenous agents are differentiated from adventitious agents as they are integrated pathogens of the production system, says Horst Ruppach, scientific and portfolio director, Global Biologics, Charles River. The Chinese hamster ovary (CHO) production cell line, for example, contains retrovirus-like particles that are typically regarded as endogenously.
“As biologics are produced to treat patients, it is highly important to ensure pathogens are not co-purified with the product,” Ruppach states, adding that “even though a pathogen might be harmless for healthy people, it can seriously affect patients. It is, therefore, essential to detect and eliminate pathogens, ideally at an early stage of the product development.”
Contamination of the manufacturing process can also have significant business and medicine supply consequences. “Facilities must be closed and fully sanitized and a deep route cause investigation is required. The supply of urgent medicine is probably impacted. It takes a minimum of six months and can go much beyond to get back to production,” Ruppach adds, emphasizing some of the consequences that can result from adventitious agents.
To date, many guidelines and compendial methods recommend different approaches to screen for and identify pathogens in biopharmaceuticals, Ruppach states. “General screening methods for viruses include inoculation of manufacturing test articles into susceptible animal systems and/or indicator cell lines to amplify outgrowth of adventitious contaminants. The infection is detected via symptoms, including death or the production of virus-specific antibodies in animals, changes of cell-line phenotypes, including cell disruption, or detection of virus-specific components in the supernatant of the inoculated cell lines,” Ruppach adds.
Single polymerase chain reaction (PCR) or multiplex PCR are frequently used to screen for suspected specific contaminants. While in-vivo and in-vitro methods detect infectious viruses, molecular biology-based methods detect viral genomes that include both infectious and non-infectious viruses, which cannot be differentiated, Ruppach points out. “The combination of a cell-based assay with PCR readout combines the benefit of both methods and is, for instance, applied when screening adeno-associated virus (AAV) preparations for replication-competent viruses. In any case, the choice of methods must be adapted to the test item and the characteristics of the test item can affect the assay sensitivity significantly,” he says. “For instance, test items can be toxic for animals or cell lines and must be pre-diluted to bypass the toxicity, and there is a risk that low contaminant concentrations are diluted out before analysis. That said, low contamination levels can be difficult to detect, which can be a specific risk for recombinant products on cell lines. Minimal viral contaminants can be introduced via media into the fermentation process, and minimal amounts of viruses can amplify to high levels during fermentation with all the aforementioned consequences.”
Traditionally, and in accordance with regulatory guidance, an orthogonal approach to detecting the presence of adventitious agents has been taken, McKee states. The analytical methods used include in-vitro and/or in-vivo culture as well as molecular methods for detection. “In-vitro culture under permissive conditions for the suspected adventitious agents, taking into consideration the environmental conditions of the manufacturing space, is the long standing or ‘old school’ method used for detection of many adventitious contaminants. The benefit of in-vitro culture-based methods is that they will detect a broad range of agents. In the case of the IVAA [in-vitro adventitious agent] method for virus detection, various endpoints (for example, hemagglutination and hemadsorption of various species of red blood cells) are used to widen the range of detected viruses even further,” says McKee.
McKee notes that in the last decade, PCR-based methods have taken hold in the industry as a more specific and sensitive means for detecting adventitious agents. “As new viruses are found to contaminate raw materials, specific PCR-based test methods can easily and rapidly be deployed for use. A good example is the advent of PCR methods against the minute virus of mice (MVM), which came on the scene after a contamination event in the mid-90s. Next generation sequencing (NGS) is also taking off as a method for detection of contaminants in bioprocessing,” she states.
NGS technologies or other disruptive multiplex technologies are increasinglyconsidered for testing or replacing the standard methods and the in-vivo assays specifically, says Ruppach, who sees that these new technologies offer many benefits. “Different from PCR or multiplex PCR, NGS can also detect unknown viruses. In fact, NGS can detect any pathogen in a sample—bacteria, mycoplasma, and viruses—and makes it a multi-attribute method. Instead of applying many different methods, one method can be used. Like all nucleic acid-based detection methods, NGS can typically not differentiate infectious from non-infectious pathogens, but approaches such as whole transcriptomic analysis have been developed to circumnavigate this downside. Detection of pathogen-specific mRNA in cell substrates is a clear indicator of active replicating virus,” Ruppach explains.
Ruppach goes on to say that, while NGS is being progressively considered for pathogen testing in the biopharmaceutical industry, there is an emerging need for methods that attempt to offer the benefits of shorter turnaround times, small sample volume, and low equipment requirements to address specific challenges inherent to next generation biologics.
Early detection of adventitious agents is also important and can benefit the bioprocessing cycle. Early detection of an adventitious agent contamination helps contain the event, for one, McKee says. If the working cell bank or a raw material is found to be contaminated during release testing, for example, it is much less costly to deal with the contamination at that early point, rather than missing detection of that same contaminant and not finding it until after a full bioreactor run has been initiated. “Raw materials, cell, virus or plasmid stocks, and other reagents used in biomanufacturing should all be tested and confirmed free of adventitious agents prior to use in a bioprocessing cycle,” McKee recommends.
As there are multiple points throughout the bioprocessing cycle where sampling for adventitious agent testing occurs, early detection can save bigger headaches further down the line. “Ultimately, it comes down to mitigating the risk to a very costly and time intensive manufacturing process,” McKee adds.
Ideally, Ruppach says, the pathogen risk should be addressed in the product development phase and before stepping into the clinic. “Even though many regulations require pathogen safety testing for clinical material, it is reasonable to address the pathogen risk much earlier. For instance, extended pathogen testing is required for master cell banks (MCB—starting material for cell line-based production systems). However, it would be reasonable to screen any pre-bank or the parental cell bank before MCB preparation by using the most sensitive methods with the widest breadth of detection,” he states.
NGS approaches have these attributes, Ruppach continues, and when applied at early stages they perfectly mitigate the risk for all subsequent steps. NGS significantly reduces the risk that any contaminant is found later in the production cycle, which Ruppach says can save many resources and time. “The same is true for animal- or human-derived raw materials, such as extracts of human platelets in cell therapy products used in the production process,” he adds.
Furthermore, detecting any contaminant in the raw material before it comes into contact with the product is mandatory. Early detection minimizes the risk for the product and avoids subsequent measures to clear the virus from the product, Ruppach says.
With complex biologic modalities, it is necessary to have analytical methods with higher sensitivity for detecting adventitious agents. As McKee points out, sensitivity is important in adventitious agent testing for several reasons. “The burden of the contamination may be quite low early in infection or very high later in the event. As discussed previously, the earlier an adventitious agent contamination is detected, the better. The ability to detect <10 microorganism is important in mitigating the risk by early detection of low-level contaminants,” she says.
Ruppach adds that sensitivity is highly important; however, he points out, one method is not equally sensitive to all potential pathogens. NGS comes closest, he says. Some animal-based assays, however, can be very sensitive for specific contaminants, such as embryonated chicken eggs for Influenza viruses. “These aspects must be considered when selecting testing methods which should be based on a risk assessment,” Ruppach states.
“For some biotherapeutics like viral gene vectors or CAR-T cells [chimeric antigen receptor T cells], the available sample volumes for testing can be limited. The smaller the test sample volume, the more important it is to have a highly sensitive method,” he further explains.
In addition, autologous cell therapy product cannot wait weeks for testing results. “Rapid testing methods must replace established long-lasting assays such as in-vivo and in-vitro test methods, and industry has developed rapid sterility and mycoplasma assays reducing turnaround times from weeks to a few days without impacting the sensitivity,” Ruppach says.
As the modality of therapeutic biological molecules grows increasingly complex, the manufacturing processes for these therapies also evolve. The needs for adventitious agent testing must also respond and change, says McKee.
“The complexity of therapeutics is also driving technology. Viral vectors for gene therapy are not going through the same downstream purification processes that traditional biologics such as monoclonal antibodies do. The viral clearance gained by downstream processing of protein biologics is not an option for cell and gene therapies. Both the need for increased sensitivity and a compressed time to result are becoming more important, especially in the field of autologous cell therapies and regenerative medicine,” McKee explains.
“With personalized medicine and small batch manufacturing, as we see for some rare disease gene therapy clinical trials, the sample volumes available for testing is also becoming smaller. A technology solution that can leverage a small sample to gain high volume information on contamination in a short period of time will be a game-changer in the field,” she adds.
The need for rapid testing assays is also driven by the limited stability of some products or the need to release final products quickly, Ruppach says. In some cases, as in the autologous cell therapy field, the patient cannot wait weeks for testing results before receiving treatment, he points out. “While rapid sterility and mycoplasma methods are available, there is still a need for rapid virus screening methods. PCR based testing is rapid but typically target known viruses or known virus groups. NGS approaches, however, have the potential to detect any contaminant and unknown variants but cannot yet be regarded as rapid, except where one is willing to compromise with respect to the sensitivity,” he further explains.
Ruppach notes that rapid testing requirements can also be met by application of onsite, easy-to-use, and robust assays/kits, rather than shipping material to contract research organizations for testing. “Here is still space for development, even though some of the rapid assays are set up for onsite applications,” he says.
Moreover, there is a trend away from “off-the-shelf” assays to customized assays, Ruppach observes. “Biopharmaceutical products are more and more optimized for specific applications (cell therapy, AAV serotypes, etc.) that require individual adaption of adventitious virus testing methods. For instance, the assay to screen for replication competent AAVs in AAV vector products requires a sensitive cell line capable of supporting amplification of a potential replication competent virus. Not all wild type serotypes grow up on the same cell line and it requires implementation of serotype-specific suitable cell lines in the assay. Ideally, this is addressed by building platform assays which can be adapted to the specific needs of a specific product type,” he explains.
New production methods, such as continuous manufacturing processes where material from the fermenter moves through to fill/finish continuously, require continuous online testing, Ruppach also notes. Continuous online testing is best achieved by automated sampling and testing using devices installed at critical positions of the process flow and that provide results within hours, maximally.
Feliza Mirasol is the science editor for BioPharm International.
Vol. 33, No. 11
When referring to this article, please cite it as F. Mirasol, “Complex Modalities Require More Sensitive Adventitious Agent Testing,” BioPharm International 33 (11) 2020.