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Cynthia A. Challener is a contributing editor to Pharmaceutical Technology.
There is intense focus on the prevention and control of viral contaminants during viral vector manufacturing, which is driving the need for more accurate and powerful analytical methods for detection.
Viral vectors must be free of viral contaminants, both adventitious agents introduced via raw materials and those generated during processing. Because conventional viral clearance treatments would degrade or destroy many viral vectors, assessment of the potential risk for contamination and the implementation of prevention and control strategies are essential. Detection can be an issue, too. Multiple analytical methods must be used, many of which have performance limitations, adding to the challenge.
Regulatory authorities require testing to be performed at every stage of the manufacturing process. Cell substrate banks, viral seed banks, raw materials of animal origin, bulk harvests, and the batches of the final manufactured clinical product all have to be tested, according to Francesca Vitelli, head of process development, for viral vectors at Lonza. Both adventitious viral agents and replication-competent viruses are a concern, adds Tony Hitchcock, technical director with Cobra Biologics.
While there are no regulations solely dedicated to the viral safety of viral vector products, several guidance documents from various agencies provide relevant information on ensuring the viral safety of biological products. There are also guidance documents specifically for cell and gene therapies, according to Karen Tiano, spokesperson for MilliporeSigma, and some do mention that testing for adventitious viruses is expected. Select guidelines for viral contamination testing are listed in Table I.
Q5A (R1) Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines
of Human or Animal Origin (September 1999)
Content and Review of Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs) (April 2008)
Characterization and Qualification of Cell Substrates and Other Biological Materials Used
in the Production of Viral Vaccines for Infectious Disease Indications, Guidance for Industry (February 2010)
EP 5.2.3 Cell Substrates for the Production of Vaccines for Human Use
EP 2.6.16 Test for Extraneous Agents in Viral Vaccines for Human Use
EudraLex, The Rules Governing Medicinal Products in the European Union, Volume 4,
Good Manufacturing Practice, Guidelines on Good Manufacturing Practice Specific
to Advanced Therapy Medicinal Products (Effective May 2018)
FDA Draft Guidance
Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy
Investigational New Drug Applications (INDs) (July 2018)
FDA Draft Guidance
Testing of Retroviral Vector-Based Human Gene Therapy Products for Replication Competent Retrovirus During Product Manufacture and Patient Follow-up (July 2018)
Many viral contaminants may be present in viral vector products. The risk for specific contaminants is dependent on the raw materials and the process, according to Lorraine Borland, product manager for regenerative medicine–cell and gene therapy in the cell line, media, and testing solutions business of Sartorius Stedim Biotech.
“The highest risk pathways for introduction of adventitious viruses are typically raw materials used in the upstream portion of the manufacturing process,” asserts Morven McAlister, senior director of regulatory and validation consultancy at Pall Biotech. “Raw materials such as cell-culture media and trypsin typically cannot be autoclaved and so represent the highest risk of entry of these viruses. This risk is heightened with the use of serum. The cell line is also a potential source of adventitious viruses. To reduce the risk associated with the cell line, it must be very well characterized and both the master and working cell banks thoroughly tested,” she adds.
“The manufacturing process, if not carried out in a closed system, may also have multiple aseptic process steps where breaks in the process or transfer of the intermediate material may increase contamination risk,” Borland notes. For instance, Hitchcock notes that producer cells and viral seed stocks can also become contaminated when used. The generation of replication-competent viral vectors during the manufacturing process is also a key concern.
For raw materials, McAlister adds that contaminants can be placed into two categories: adventitious viruses that enter the viral preparation inadvertently through raw materials or poorly characterized cell lines and helper viruses such as baculovirus, adenovirus, or herpes simplex virus, which are deliberately used to stimulate expression of the virus of interest (generally adeno-associated virus [AAV] produced via transient routes).
Efforts to control for the presence of viral contaminants have focused on testing for retroviral contamination and species-specific viruses when animal-derived raw materials are used, according to Vitelli. Examples include bovine or porcine adventitious agents and species-specific viruses such as rhabdovirus, polyomavirus SV40, and arboviruses for other animal and insect cells.
“Where bovine serum or porcine trypsin has been used in the process, there is a risk of introducing bovine and porcine viruses such as bovine polyomavirus and bovine and porcine circovirus,” notes Borland. “Minute virus of mice and vesivirus are viruses that have emerged in recent years as contaminants of bioreactors, and screening for both is now included as standard for rodent cell lines.”
Detection is more complicated with viral vectors compared to antibodies, according to McAlister, because human cell lines are generally used for their manufacture. A typical panel for a human cell line could include herpes simplex virus, HIV-1, hepatitis A virus, porcine parvovirus, and possibly encephalomyocarditis virus. “When working with human cell lines-for example HEK293 cells-besides standard viral safety issues, special precautions should be taken because this cell line is susceptible to infection by human viruses,” she observes. Other human viruses that may need to be tested for include cytomegalovirus, human T-lymphotropic virus, Epstein-Barr virus, hepatitis B virus, human papillomavirus, and hepatitis C virus.
It is easier to identify residual helper viruses such as baculovirus, adenovirus, and herpes simplex virus because they have been deliberately introduced and are also usually present at much higher concentration, according to McAlister. Due to that fact, however, the challenge becomes removal rather than detection, she comments.
In addition to raw material and process risks, the facility, equipment, utilities, and people involved in the manufacturing the process are all potential sources of viral contamination, according to Borland.
“The facility itself may be a point of entry for rodents, providing a pathway for viral and bacterial contamination to transfer to the process stream. If using stainless-steel bioreactors, the cleaning and sterilization regimes must be validated to ensure no cross-contamination of product. Such a risk is driving the movement to sterile, single-use systems. Water systems and non-sterile gas systems must also be taken into consideration,” Borland says. In multi-product facilities, there can also be opportunities for cross contamination by adventitious agents, according to Hitchcock.
Operators, Borland asserts, may present the biggest risk to the process. “Viral manufacturing processes require aseptic manipulation within cleanrooms to controlled standards. People provide perfect reservoirs of bacteria, mycoplasma, and viruses, and therefore, the controls around people working on the processes must be to the highest standards,” she explains.
One of the biggest challenges to assuring that viral vectors contain no viral contaminants is the fact that traditional viral clearance steps such as filtration and treatment with heat or strong acids cannot be used with many viral vectors such as enveloped viruses (e.g., lentiviruses), because they are too fragile and cannot withstand these processes. AAV is one exception; it is a relatively robust virus and so typically tolerates standard virus hold strategies to eliminate unknown adventitious agents in the preparation, according to McAlister.
Neutralization of viral vector products without impacting the viral contaminants is also difficult, Hitchcock says. As a result, detection of unknown viral contaminants present at low levels is highly challenging.
The range of potential viral contaminants and numerous sources, meanwhile, creates the need for a holistic approach to viral contaminant detection using a combination of methodologies including both general and specific assays, observes Vitelli. Examples are adventitious agent tests, usually non-specific tests capable of detecting a broad range of viruses; species-specific assays designed to detect the presence of identified potential contaminants (e.g., specific bovine or porcine viruses in serum or trypsin, respectively, or murine viruses in mouse cells); and tests for retroviruses.
The limitations of the testing methods provide a huge challenge to the industry, Borland agrees. “Traditional in-vitro and in-vivo methods are broadly specific and will detect viruses that infect a range of cell types but may not detect viruses that do not cause cytopathic effects (CPE). Molecular methods such as real-time polymerase chain reaction (PCR) are more specific and have the ability to target those viruses that do not cause CPE in cell culture; however, they are limited by the fact they are specific. Methods such as [transmission electron microscopy] TEM, which is used to detect retroviruses, are limited by their sensitivity,” she explains.
There are few tools available for accurate, efficient, and reliably precise assays to determine the activity of residual viruses, agrees Vitelli. “Generally, the limit of detection for these assays tends to be low, and more sensitive methods such as PCR-based assays can sometime result in false positives. In addition, risks are posed by potentially unknown viral contaminants that may be undetectable using current analytical tools,” she states.
Borland notes that next-generation sequencing has been applied for general viral detection in biologics; technology advances and validation of this technique may bring it into routine testing use.
Another concern for McAlister is the time required to test for the absence of viruses. “Current methods are laborious and may require more time to perform than the actual product is stable for. This issue is one of the biggest drivers for the development and acceptance of rapid alternative microbiology methods to allow testing within the shortest timeframe possible,” she comments.
Once adequate testing is established, Vitelli notes that another substantial challenge is obtaining enough purified viral material for use in formal viral clearance studies, which is costly and time-consuming to generate. “These tests also typically require purified material that must be redirected from other potential uses that are critical to the product development cycle and timeline,” she observes.
Tiano adds that there is only a finite amount of sample available for testing, which creates challenges, particularly when material is required for cytotoxicity pre-studies and viral interference testing in addition to virus spiking studies. “Reduction of the sample size for pre-studies is being made possible, however, with the development of sensitive molecular assays useful for testing cells, virus stocks, and raw materials used in the production of viral vectors,” she notes.
The traditional approach to assuring that no viral contaminants are present in biologic products involves prevention, detection, and removal. Prevention involves ensuring that only high-quality, pure, contaminant-free raw materials are purchased from reliable suppliers, detection involves verification of the absence of contaminants in raw materials and process intermediates, and removal involves steps to remove or inactivate any potential viral contaminants that may be present.
Because the latter step cannot be performed for many viral vector products, a risk-based approach must be taken, according to Borland. “Performing a detailed and relevant risk assessment is key to ensuring that an appropriate and comprehensive virus control strategy is employed,” adds McAlister. She also notes that because few viral vector processes have been performed at large scale, knowing how to do this risk assessment can be a challenge. “The good news is,” she observes, “that many years of experience have been developed in closely related processes such as monoclonal antibodies and vaccines, and this experience can be leveraged for these processes.”
In addition, Hitchcock believes the best strategies clearly rely on understanding the vector design; selection of the appropriate producer cell line and manufacturing process, including the use of closed manufacturing systems, especially with regards to the production of replication-competent vectors; implementation of a stringent quality system and management of suppliers to ensure that the required control measures are in place to minimize the risks of adventitious viral contamination and for multi-product areas/facilities, adoption of procedures for prevention of cross-contamination and that demonstration of the removal of previously manufactured product from the cleanroom facility and shared equipment.
“The over-arching requirement,” states Vitelli, “centers on good facility design, process design, material control, and strict compliance with good manufacturing practices to ensure safety and success. Fundamental facility controls are typically established during the facility design phase, such as the segregation of personnel, material and waste flows, and selection of cleanable materials of construction, and are propagated by validated facility management practices to include robust environmental monitoring, product changeover, and visual inspection.”
Following the general viral safety strategy that incorporates prevention through safe sourcing, detection, and removal has resulted in safe biological products for many years, says Tiano. “Cell and gene therapies is an exciting and rapidly developing area. Although the therapies may be new, using this strategy for guaranteeing viral safety will help ensure that these new therapies are as safe as more traditional biological therapies.”
There are limited options for viral clearance other than extensive testing of materials going into the process. Many of the same strategies used to inactivate/remove contaminating viruses can impact the quality/potency of the final viral gene therapy product. Manufacturers can look to adopting viral removal steps into their raw material entry points, Borland suggests. She points to next-generation virus filters that have been developed to offer a cost-efficient strategy for media filtration but notes that this technology is not yet adopted throughout the entire industry.
Techniques such as nanofiltration, ultraviolet-treatment, gamma irradiation, and high temperature, short-time treatment can also be used to ensure that no viruses are present in starting materials, according to McAlister. She stresses, though, that it is important to ensure during process development that subjecting raw materials to these techniques does not affect their performance.
Viral clearance technologies that are used in the manufacturing process for viral vectors that can withstand them are engineered into the manufacturing process and are most commonly heat inactivation and the use of viral retentive filtration, according to Vitelli. In some cases, such as for AAV products, affinity chromatography and exposure to acidic pH conditions (< pH3) may be employed.
For removal of helper virus, McAlister notes that nanofiltration works well because there is a large difference in diameter between the virus of interest and the contaminating virus. She notes, however, that direct-flow filtration is not well adapted for the removal of viruses at high concentration. “It is generally recommended, therefore, that an initial virus removal step that can remove significant amounts of the helper virus, such as membrane chromatography, be performed first.”
Some chromatography steps that are used in the purification of non-enveloped viral vectors may contribute to viral clearance for the vector as well, according to Tiano. “These steps may include an affinity resin or an anion exchange resin, which are optimized to purify the viral vector product and coincidentally may remove potential viral contaminants. Since the chromatography steps purify the vector product, viral clearance is only achieved when there is a difference in partitioning between the vector and potential viral contaminants,” she explains.
Lonza’s R&D group is evaluating chemical additives and exploring different lysis reagents that have viral inactivation capabilities without risking impact on the quality of the viral vector product.
As next-generation processes move toward commercialization, there has been a general shift away from adherent cell-culture processes in open, manual-intensive, flatware systems that are difficult to scale to automated, suspension cell-culture processes in closed bioreactors that are more efficient and readily scalable. This shift is also enabling the use of sterile, disposable systems, which is in turn enabling improved risk-mitigation strategies, according to Borland.
Hitchcock notes that as viral vector manufacturing processes are scaled, the likelihood of the generation of replication competent viruses increases, which poses challenges for purification.
Improving overall process yield will be an issue, in fact. McAlister points to downstream processing yields, typically around 30%, for AAV as an example. “Nanofiltration, if not done properly, can further reduce this yield. Appropriate filterability trials must be performed to ensure that a scalable scheme for virus control is designed,” she observes. Vitelli adds that as the per-dose demand for viruses increases, notably AAV, vendors of viral-retentive filters should continue to work closely with product developers and contract development and manufacturing organizations to provide scaling recommendations and new product sizing.
For viral vectors that can withstand heat inactivation, these steps may also pose unique challenges with respect to scale up due to the need to adequately control both the temperature and duration of exposure in the liquid state, according Vitelli.
Vol. 32, No. 12
When referring to this article, please cite it as C. Challener, “Prevent, Detect, and Remove: Viral Control for Viral Vectors,"BioPharm International 32 (12) 2019.