While incidents of biotherapeutic viral contamination have occurred, due to well-established knowledge of the potential sources of contamination and procedures to prevent it, the industry has an impressive track record.
- Donors and their tissues
- Raw materials of human or animal origin
- Cell culture media and reagents
- Starting materials like cells, cell banks, plasmids, and viral banks
- Manufacturing operations, equipment, or facilities
For the more established classes of biologics, viral safety procedures are well understood. Broadly, the following three areas work together to assure therapeutics are safe from contamination:
1) Careful consideration of source materials
2) Testing of processes and products for viral contaminants at appropriate manufacturing steps
3) Downstream viral clearance protocols to remove or inactivate viral contaminants
This three-prong approach works quite well especially for the production of recombinant protein and mAb therapies. However, viral safety for gene therapies is trickier given that viral vectors are the most common therapeutic delivery vehicle. Therefore, the issue becomes, how can adventitious (contaminating) virus be effectively differentiated from the viral vectors inherent to the proper functioning of the therapeutic?
Potential adventitious viruses are often not distinctly different in size from therapeutic viral vectors, often prohibiting effective particle size-based filtration strategies. Additionally, downstream methods commonly used to inactivate viruses, such as low-pH incubation and gamma- or UV-irradiation treatment, are typically too harsh for a gene therapeutic— processes that would inactivate a contaminating virus would also likely inactivate a therapeutic virus.
Viral Clearance Studies Are Required for Gene Therapies
“There can be a misconception that you do not need to perform viral clearance studies for gene therapy products, but in fact, regulators expect to see this study performed on those virus types that can be purified, for example, enveloped viruses,” commented Andrew Bulpin, PhD, head of process solutions at MilliporeSigma, in a December 2020 GEN article.
Virus clearance studies are required for gene therapies, but there is no one size fits all solution. Classic culture-based testing methods can work, but to work within a gene therapy context, neutralizing antiserum is required to neutralize the viral vector to effectively detect active adventitious virus. Unfortunately, developers cannot always access neutralizing antiserum, and it is in particularly short supply amid aggressive SARS-CoV-2 vaccine development efforts.
Additionally, developers can apply common viral clearance methods to adeno-associated virus (AAV) and lentiviral vector applications. The chart below shares general applications of approaches.
Common viral clearance methods, their suitability for use with adeno-associated virus (AAV) and lentiviral vectors, and potential approaches to viral clearance
|Clearance Method||AAV (Size 20 nm; non-enveloped)||Lentivirus (Size 80–100 nm; enveloped)|
|Heat||Heat stable; application of heat will inactivate heat-sensitive viruses with minimal impact on AAV||Heat sensitive; may not be a suitable clearance method|
|Low pH||Low-pH stable; a hold at low pH will inactivate pH-sensitive viruses with minimal impact on AAV||pH sensitive; may not be a suitable clearance method|
|Solvent/detergent||Non-enveloped virus; detergent can be used to inactivate enveloped virus with minimal impact on AAV||Enveloped virus sensitive to detergent; not a suitable clearance method|
|Chromatography||Differences in surface charge can allow AAV to be separated from other viruses||Differences in surface charge can allow lentivirus to be separated from other viruses|
|Nanofiltration||AAV will pass through, for example, 35-nm filters, allowing separation of AAV from larger viral contaminants||Lentivirus will be retained by nanofilters (pore size, for example, 35 or 50 nm), potentially allowing separation from smaller viral contaminants|
Emerging Adventitious Virus Detection Approach—High-throughput Sequencing
Although there are well-established viral clearance methods that can be used on appropriate viral vectors, new virus detection approaches are needed as the gene therapy sector continues to grow and mature. High-throughput sequencing (HTS) is one such solution. It is a non-specific technique with the potential to detect both known and unknown adventitious viruses. In fact, high-throughput molecular biology methods (HTS combined with a pan-viral microarray) succeeded in detecting the contamination of Rotarix vaccine by a porcine circovirus.
Although HTS results for adventitious virus testing may differ between laboratories, the development of well-characterized model virus stocks would support standardization and validation of the different HTS platforms. Today, there are no established viral reference standards with corresponding in vivo data for assessing new techniques for the detection of adventitious viruses. For this technique to be adopted, viral reference standards would need to be established.
To learn more about applying HTS for adventitious virus detection, read Nature’s article published in July 2020—”Sensitivity and breadth of detection of high-throughput sequencing for adventitious virus detection.”
Addressing Gene Therapy Safety Challenges Ahead
For the gene therapy sector to continue to mature, viral clearance is one, among many safety concerns, that must advance. However, a distinct challenge is that each gene therapeutic has specific quality attributes that ultimately must be comparable for each lot of material released.
Quality attributes will differ based on the targeted disease state and the modality of the treatment. However, the industry must continue to advance testing approaches to extend the available portfolio of therapeutic modalities while doing so safely.