Cell Line Considerations for Manufacturing…
With growing interest to develop treatments that circumvent the drawbacks of traditional first-line tr…
Oncolytic viruses (OVs) are a versatile class of therapeutic agents offering the ability to selectively target cancerous cells and mediate anti-tumor activity via direct immunogenic cell death and induction of anti-tumor immune responses. However, development and manufacturing of OVs are not simple. Careful consideration is needed at every step to overcome the many challenges that can arise throughout while ensuring regulatory compliance.
In the second of our blog series “Oncolytic virus manufacturing: challenges, considerations, and opportunities” Kai Lipinski, Chief Scientific Officer of Vibalogics follows on from the first installment which focused on cell line selection. Here, he explores the critical considerations for de novo generation and recovery of infectious viruses (virus rescue) through a reverse genetics and cloning system.
Establishing a Virus Clone
Virus rescue is the starting point in the production of recombinant viral clones which are used in multiple applications in discovery for early candidate screening, and then later in early process development. In most cases, it will involve the design of a set of bacterial plasmids, and transfection of a producer cell substrate with one or more plasmids encoding the viral genome. The genes for trans elements, such as core structural proteins, can be encoded on separate plasmids to improve safety.
Following transfection, replication of the viral genome and expression of viral proteins will enable initial recombinant virus recovery. By using this kind of reverse-genetics approach, it is now possible to recover RNA viruses with negative-strand RNA genomes, such as influenza viruses and the rabies virus.
Ensuring monoclonality
Following generation of the first virus passage, virus cloning is required to produce the clonal pre-master virus seed stock (pre-MVSS). Plaque isolation and suitable plaques will be “picked” for virus amplification. If plaque cloning cannot be carried out on an adherent producer cell line, limited dilution cloning will be performed in combination with an adherent virus-indicator cell line. Three rounds of cloning will be needed to statistically ensure monoclonality.
Pre-Characterization
Depending on how extensive virus characterization and evaluation is at each round of cloning, the process of evaluation and identification of the final pre-MVSS can take 4–7 months. During this stage initial clones are assessed for ideal characteristics such as infectivity and titer, transgene expression (as appropriate), and sequenced for genetic integrity. Once the pre-MVSS is selected it will be used to later establish the cGMP Master Virus Seed Stock or Virus Bank for production.
Critical Considerations for Rescue and Cloning
Manufacturing of replication-competent virus products like OVs necessitates the minimization of potential harm to the patients. These can arise from natural genetic mutations of the virus throughout passage or contamination of the raw materials used. Developers must show compliance with regulations that have been introduced to minimize potential risks to patients.
Genetic stability
It is essential that the passage level (the number of times a culture has been split or re-seeded since initial establishment) is determined and the genetic stability of the virus product is investigated during development and prior to BLA (biologics license application) filing.
Genetic stability studies, including genome sequencing and restriction digests with gel electrophoresis, should be carried out using the pre-MVSS. These can also be used to demonstrate the absence of unwanted viral constructs.
Proving genetic stability is particularly important for RNA viruses, as the error rate of RNA polymerase is higher as compared with DNA polymerase, resulting in increased potential for genetic instability.
Use of animal-derived raw materials
The use of animal-derived components in virus rescue and cloning should be kept to a minimum to reduce risk of potential adventitious agent contamination. However, growth of many producer cell lines currently relies on the use of fetal bovine serum (FBS) and serum-free options are not always available.
If animal-derived components must be used, transmissible/bovine spongiform encephalopathy (TSE/BSE) vendor compliance certificates from the European Directorate for the Quality of Medicines & Healthcare are required. It is also recommended that FBS is sourced from countries with negligible risk and established control over BSE such as New Zealand. The serum should be triple-filtered and irradiated with certificates available that demonstrate compliance with E.P 2262 monograph and importation regulations of the producing country.
Contamination risk
Care must be taken to follow practices and procedures to manage and minimize contamination. Although virus rescue and cloning are generally not performed under GMP conditions, these activities should be performed in a fully-segregated laboratory with enhanced hygiene measures. This provides both security for the work being conducted in the room itself, as well as other studies ongoing within multi-product facilities or CDMOs. Where possible, it is recommended to perform activities in dedicated areas on a campaign basis to reduce cross-contamination.
Procedural control
Developers must ensure that optimal documentation and data integrity is upheld throughout development of virus products, including fully-traceable raw materials and their specific lot numbers, to ensure regulatory compliance and application approval. Regulatory bodies can require an extensive recorded history of the cell substrate including the conditions under which it was passaged, and details of other agents handled within the same biosafety cabinets or incubators.
As a critical step toward the production of OVs, virus rescue and cloning has many important considerations that drug developers and manufacturers must keep in mind to ensure successful production of the product and delivery to patients. Partnering with a contract service partner with expertise and experience in OV development and manufacturing, can help support a successful outcome and the timeliness to clinic and marketization.
Explore our Virotherapy Insider: Deep Dive into GMP Manufacturing of Oncolytic Viruses, to review the critical steps involved in OV development, manufacturing, testing, and the important considerations that must be made throughout.
About Vibalogics
Since 2003, Vibalogics has operated as a specialized global Contract Development and Manufacturing Organization (CDMO) offering process and analytical development, manufacturing, testing, and fill-finish services to innovators developing transformational virotherapy products. From its headquarters and commercial facility in Boxborough, MA, USA and operations in Cuxhaven, Germany, Vibalogics supports its customers in the development and commercialization of oncolytic viruses, gene therapies and viral vaccines..
