Robert Cartee, PhD- Senior Director, Biopharmaceutical Services, SGS North America, Inc.
The safe and effective introduction of a transgene into a cell to cure or reduce disease severity is one of the key challenges facing the field of gene therapy. Viral vectors have proven to be one of the most efficient means to achieve this goal by taking advantage of a virus’s natural ability to invade and introduce its genetic material into human cells. As a result, viral vectors are being developed to treat a variety of diseases including monogenic syndromes and cancers, and can be delivered both in vivo, in situ, and ex vivo (cell therapy).
Several different viruses have been used as the basis for viral vectors including adenoviruses (AV), adeno-associated viruses (AAV), retroviruses, lentiviruses (LV), and herpes simplex viruses (HSV), with each virus having its advantages and limitations. Similar to the actual virus, viral vectors consist of genetic material encased in a protective protein shell or capsid and may be infectious. However, unlike the virus from which they are derived, viral vectors are engineered to be replication defective through deleting genetic sequences from the viral genome necessary for viral replication and/or production of a capsid. This deletion also serves to make room in the viral vector genome for the transgene.
The development and manufacture of viral vector-based gene therapy products is an intricate process with rigorous quality standards. As shown in Figure 1, the manufacture of a viral vector for gene therapy begins with plasmids that are usually produced and purified from bacteria. One of these plasmids contains the therapeutic transgene with any necessary capsid packaging sequences. The other plasmids, often referred to as helper plasmids, contain the genes that encode the capsid proteins and any replication machinery. These plasmids are transfected into a host cell, where through the encoded sequences, the transgene is packaged into the viral capsid to produce the viral vector. Following purification of the viral vector drug substance particles, they are formulated and filled into containers for either in vivo delivery or for transducing allogenic or autologous cells from subjects or patients for use as cell therapy.
Throughout the development and manufacture of a viral vector, rigorous testing of the raw materials, any ancillary materials, plasmids, cell lines, and viral vector drug substance and drug product is performed to determine identity, content/viability/potency, quality, safety, and stability. These are all critical steps in meeting regulatory requirements and getting viral vector-based drug products to market safely. This intricate testing requires specialized scientific expertise and state-of-the-art instrumentation.
Identity Testing is performed to confirm that the purified viral vector drug substance or drug product contains the correct nucleic acid sequence and viral capsid proteins. The nucleic acid component can be identified through sequencing (Sanger or Next Generation [NGS]) or by a quantitative polymerase chain reaction (qPCR). The identity of the viral vector capsid protein can be determined using either Western blots or liquid chromatography with detection by absorbance or mass spectrometry.
Content/Potency Testing is performed to determine the amount of infectious viral vector particles present in the drug substance or drug product. This can be achieved by quantitating the number of viral genomes contained in capsid particles using qPCR. While this method will indicate the number of genomes it may not accurately reflect the potency of the viral vector which is dependent on both the infectivity of the particle and the expression/ function of the transgene. To determine potency, infectious titer assays and cell-based assays are employed that examine the ability of the viral vector product to infect cultured cells, express their transgene, and show functionality. The ultimate readout from these assays is dependent on the transgene and can be assessed through a variety of techniques including qPCR, ELISAs, or cell reporter systems.
Quality Testing involves evaluating the physicochemical properties of the viral vector and determining the level of process and product-related impurities. The physicochemical properties evaluated usually include appearance, pH, and osmolality. Process-related impurities include residual host cell protein that is usually determined by ELISA and residual host cell DNA and plasmid DNA that are analyzed using qPCR methods. Other process-related impurities are residual compounds that come from the host cell growth media (i.e. antibiotics, raw materials) or the purification procedure (i.e. detergents, column leachable) and are evaluated using a variety of techniques including ELISAs, liquid chromatography, and mass spectrometry. Assessment of key product-related impurities includes determining the ratio of viral vector particles that contain nucleic acid (full capsids) to those that do not contain nucleic acid (empty capsids) or that contain incorrect nucleic acid (illegitimate capsids). For determining the ratio of full to empty viral particles a variety of methods can be utilized including liquid chromatography, analytical ultracentrifugation, electrophoresis, qPCR, and electron microscopy. Illegitimate capsid levels can be determined using qPCR or sequencing methods.
Safety Testing involves determining the presence of any harmful microbial contaminants including bacteria, molds, fungi, mycoplasma, and adventitious viruses. Furthermore, the viral vector product is evaluated for the presence of bacterial endotoxins as well as viral vector particles that are replication-competent. The presence of any bacteria, mold, and fungi is determined by a sterility test while for mycoplasma either a culture method or qPCR method can be utilized. There are multiple methods for the detection of adventitious viruses, including PCR methods, ELISA methods, and electron microscopy that detect specific viruses, however, non-specific methods that look for cytopathic effects in a cell-based assay may also be employed. To detect the presence of replication-competent viral vectors, a permissive cell line is treated with the viral vector, and the presence of the sequences unique to the viral vector is detected using qPCR or Southern blotting.
Containing Closure Integrity Testing (CCIT) and Extractable and Leachables Testing. In addition to the testing described above, viral vector drug products are also evaluated for the integrity of the container closure system and in later stage clinical development any potential compounds that could migrate from the container closure system. There are many methods for evaluating CCIT including dye ingress, high voltage leak detection, oxygen headspace, and helium leak detection. Extractable and leachable components are determined using liquid and/or gas chromatography with mass spectrometry detection.
Stability Testing is a critical component of any drug development program and involves analysis of the potency and quality attributes of the product over time at the storage temperature, accelerated temperatures, and stress temperatures. The goals of a stability study are to determine the shelf life of the product but also refine the attributes of the product that are predictive of stability and potency. For viral vectors, stability studies often examine characteristics such as appearance, pH, content, structural features, degree of aggregation, sterility, and potency.
Overall, viral vectors are complex biologics that require sophisticated testing throughout the manufacturing process to ensure that they are safe and effective for use in patients. With many biopharma companies in the race to release gene therapies to market, it is important to choose a CRO with specialized viral vector and other gene therapy expertise, deep regulatory knowledge, ample capacity and agility, and consistent, transparent communication to support your viral vector product from development to market.
Subscribe to our e-Newsletters
Stay up to date with the latest news, articles, and events. Plus, get special
offers from American Pharmaceutical Review delivered to your inbox!
Sign up now!