Guide To Viral Vector Production: From Bench to Bedside. Faster.
Viral vector technologies are taking center stage in the development of novel vaccines and therapies to combat a wide spectrum of human diseases. Efficient development of safe, cost-effective, and robust methods for viral vector manufacture is therefore of vital importance for patients to benefit from these potentially life changing new medicines.
Can I use my cell line for clinical production? What is the quickest route to clinical trials? How can we effectively de-risk and accelerate viral vector development? These are just a few of the many questions we address in this go-to resource for anyone navigating the complexities of viral vector process development, scale-up, and cGMP manufacturing.
Viral vector technologies
The use of viral vectors in vaccines and targeted therapies is revolutionizing the way we tackle some of nature’s most intractable human diseases and infectious pathogens. From rare diseases to cancer to COVID-19: viral vectors are becoming indispensible weapons in the armory of drug modalities?
What are viral vectors
Viral vectors are viruses that have been engineered in the lab to efficiently infect target cells and deliver a genetic payload. These properties make them highly attractive for a wide variety of clinical applications.
When genetically engineered with the code for an immunogenic antigen, viral vectors essentially hijack the cell’s own machinery to produce large amounts of the antigen, which is then effectively presented to the host immune system. This makes them an attractive platform for development of novel prophylactic and therapeutic viral vector vaccines.
Viral vectors also offer tremendous promise for the development of novel cell and gene therapies. An estimated 70% of gene therapy trials worldwide involve the use of viral vectors.
The scope of viral vector technology further extends to the development of oncolytic viruses, a rapidly evolving class of anticancer agents that are tailored to specifically target cancer cells for destruction, working in combination with the patient’s immune system.
Viral vector vaccines
The rising global threat of infectious diseases and cancers has been a major driver in the evolution of next-generation vaccines.
Milestones in preventative and therapeutic viral vector vaccines
Viral vector technologies in particular have emerged as an enabling platform for vaccine development. Notably, the technology gave rise to the first FDA-approved vaccine for Ebola virus in late 2019, and in December 2020 the first viral vector vaccines for SARS-CoV-2 / COVID-19 were granted emergency use authorization (EUA) less than a year after the genetic sequence for SARS-CoV-2 was shared.
Milestones like these, underpinned by significant progress in addressing safety and scale-up challenges over the past few years, are paving the way to more widespread adoption of viral vectors in vaccine development.
Table 1. Human clinical trials in progress with viral vectored vaccines1,2
| Virus Vector | Phase I Clinical Trial |
| Adenoviruses (Ad) | |
| ChAd3 (Chimpanzee adenovirus) | Ebola Zaire, Hepatitis C, Ebola Sudan, Ebola Marburg |
| ChAdOx (Chimpanzee adenovirus) | Tuberculosis, Chikungunya, MERS-CoV |
| Ad5 (Adenovirus type 5) | Cystic fibrosis, HIV, Ebola Zaire |
| VXA (Replication-deficient Ad5) | Respiratory syncytial virus, Norovirus, Influenza |
| rAd26 (Recombinant Ad 26) | HIV, Ebola Zaire |
| Ad35 | Tuberculosis, HIV |
| Ad4 | HIV, Anthrax |
| Alphaviruses | |
| VEE Replicon (Venezuelan equine encephalitis) | CMV |
| Measles Virus (MeV) | |
| Measles virus | COVID-19 |
| Poxviruses | |
| MVA (Modified vaccinia virus Ankara) | Ebola, HIV, Hepatitis C, MERS-CoV |
| FPV (Fowlpox vector) | HIV |
| ALVAC (canarypox vector) | HIV |
| Vesicular Stomatitis Virus (VSV) | |
| Replication-competent VSV rVSV | HIV Lassa virus |
| Virus Vector | Phase II Clinical Trial |
| Adenoviruses | |
| ChAdOx1 | Malaria, SARS-CoV-2 |
| Poxviruses | |
| MVA | CMV, Tuberculosis |
Gene and cell therapies
According to the Alliance for Regenerative Medicine, at the end of 2020 there were 1,220 ongoing regenerative medicine and advanced gene and cell therapy trials worldwide.3 Of these, 152 were in phase 3 clinical trials, in line with EMA and FDA predictions that by 2025 the annual rate of cell and gene therapy approvals will reach 10-20 per year.
Valued at $4.4 billion in 2020, the global gene and cell therapy market is projected to reach $15.48 billion by 2025 at a growth rate of 28.7%.4 Viral vectors are playing a central role in this rapidly expanding market.
Although in vivo gene therapies have had their ups and downs recently, there are over 400 viral vector gene therapy assets in preclinical and clinical phases of development.5 The vast majority of these utilize adenovirus, adeno-associated virus (AAV), and lentivirus vectors.
While cancer is by far the predominant disease area, viral vector gene and cell therapies are under investigation for a wide variety of other indications including cardiovascular and monogenic disorders, as well as metabolic, inflammatory, neurological and ocular diseases.
In late 2020, two new viral vector-based therapies gained regulatory approval: Libmeldy (Orchard Therapeutics), approved by the European Medical Agency for treatment of metachromatic leukodystrophy (MLD), and Tecartus (Kite), the first CAR-T treatment approved by the FDA for relapsed or refractory mantle cell lymphoma. This was followed in February 2021 by FDA approval of Breyanzi, a lentiviral vector-based CAR-T therapy for relapsed or refractory Large B-cell Lymphoma.
Viral vectors in gene therapy trials globally
Data source: Gene Therapy Clinical Trials Worldwide database, The Journal of Gene Medicine, John Wileys and Sons LTD.
Oncolytic viruses
Targeted eradication of cancer cells using oncolytic viruses is another exciting and constantly evolving application area for viral vector technologies.
Oncolytic viruses are able to selectively infect, replicate in and lyse cancer cells and/or deliver a genetic payload, leaving healthy cells untouched. In addition to directly mediating cell death, virus-induced lysis of the target cell can activate cytotoxic T cells. This wakes up the immune system to actively seek out and destroy tumor cells.
Many different virus families and subfamilies have been investigated for use in oncolytic therapies, including: adenovirus, coxsackievirus, herpes simplex, vaccinia, measles, reovirus and parvovirus. This diversity presents a potential manufacturing challenge in terms of the breadth of manufacturing strategies and capabilities required.
Roadmap for viral vector process development
While global markets for viral vector vaccines and therapies continue to expand, the journey from bench to clinic is rarely straightforward. Strategic end-to-end planning of the entire process development journey is an important first step to avoid common development and scale-up pitfalls, mitigate risks across the development program, and identify the most cost-effective strategies for each stage of development.
Viral vector process development road map
Common roadblocks in viral vector production
While the promise of viral vector-based vaccines and therapies is great, so too are the development risks. This is largely because of the complexities of working with viral particles and living cells, including the challenges of scaling up production for clinical manufacture. At the same time, rigorous safety standards must be met to ensure products are safe for patients and the environment.
The bottom line is that many potentially life-changing viral vector products never make it to market. It is not uncommon to invest considerable resources in viral vector development, only to discover at a late stage that the production process is not sufficiently scalable, robust or cost-effective to be commercially viable. As these statistics from the vaccine industry illustrate, the price of failure is high:
The high cost of failure in vaccine development
5 frequently reported challenges in viral vector manufacturing
Recent business surveys10 have identified some of the main problems respondents mention when asked about the difficulties they face in viral vector manufacturing. These include:
- Complexity of working with viral particles and living cells
- Scaling up manufacturing processes
- Gaps in process engineering and regulatory expertise
- Temperature sensitivity and related storage and logistics challenges
- High capital investments required to establish and maintain production facilities
To address issues like these and de-risk development, many viral vector product developers are turning to contract manufacturing and development organizations (CDMOs) that have the track record, specialized expertise, and facilities needed to bypass these issues.
Foundations and guiding principles for success
Designing for quality, manufacturability, compliance and cost-efficiency are key foundations for success when developing any commercial product. This becomes all the more important when manufacturing viral vectors, which are especially complex, difficult to handle, and subject to stringent regulatory oversight.
Batavia’s guiding principles in viral vector process development
At Batavia Biosciences, we follow three guiding principles in viral vector process development:
- Right first time. Getting process development right the first time around starts with a thorough understanding of the expected commercial manufacturing process. This means ensuring that all raw materials, equipment and process architecture will allow seamless extrapolation to commercial scales. This is supported by Design of Experiment (DoE) approaches, scale-down models, assay platforms, and cost modeling tools that enable timely data-driven decision-making at every stage of the process.
- Cost-efficiency. A cost-efficient manufacturing process is vital to ensuring you meet the Cost of Goods (CoG) requirements for your product. This includes fine-tuning upstream and downstream processes to maximize viral yield in bioreactors and obtain high recovery from purification steps.
- Bullet-proof documentation. Robust documentation is essential at every stage of development: from establishing a sound development roadmap, to submitting an IND or IMPD dossier that will hold up to regulatory scrutiny, to ensuring that QA release and technology transfers happen without a hitch. Batavia’s expert QA team provides vital support throughout the entire development cycle.
Step by step: troubleshooting process development
Process evaluation and design
The starting point for clinical process development is usually a thorough evaluation of the current process for vector production. Raw materials, including plasmids, virus seeds, cell lines, media and supplements, are assessed for cGMP compliance and suitability for clinical manufacture, as well as ability to achieve the target product profile (TPP).
Both the upstream and downstream processes are assessed for overall design, scalability and anticipated CoG. Time constraints and investment milestones are also factored into the mix to map out the most phase-appropriate and cost-efficient strategies. In addition, critical supply chains and any necessary licensing for raw materials or technology IP needs to be secured.
Batavia Biosciences has a strong track record in cGMP process development for clinical manufacturing of viral vectors. Our experts can evaluate your project from every angle – scientific, technical, operational, financial and regulatory. An early assessment will maximize the number of design options available and ensure that strategic gaps, issues, and potential stumbling blocks are spotted early, before they become problematic.
Cell line selection: Can I use my cell line for clinical production?
One of the most crucial steps in the evaluation phase is ensuring that a suitable cell line has been selected.
Some of the questions asked at this stage include:
- Does the cell line comply with cGMP requirements? For example, is the history of the cell line known? Has it been properly tested for purity and viral safety?
- Is a cGMP-produced master cell bank (MCB) available?
- How robust does the cell line need to be to tolerate upstream processing
- Does the cell line support viral stability and yields?
- Does it make sense to switch from an adherent to a suspension format?
HEK293 cells and their derivatives are a common choice for clinical manufacture of AAV, adenovirus and lentivirus vectors. Vero cell lines are also widely used, particular for production of measles and VSV vectors.
Cell and virus banking
Once an appropriate cell line has been chosen, the MCB can be established. To do this, the cells are expanded under cGMP conditions and quality tested to demonstrate identity, purity and viral safety. From the MCB, working cell banks (WCB) can be generated. These are the banks that will be used for expansion to support production of the final product, without the chance of depleting the MCB.
Similarly, a robust, traceable and regulatory-compliant master virus seed (MVS) must be established using the MCB or WCB. Once tested to confirm viral identity, purity and stability, the MVS gives rise to the working virus seed (WVS) used in the production process.
Upstream process (USP)
Cell cultures are inherently heterogeneous, sensitive to mechanical and environmental disturbances, and susceptible to contamination. Factors such as these mean that the USP portion of viral vector manufacturing is particularly challenging to develop, optimize and control.
During USP development, many parameters need to be investigated to identify the critical process parameters. These must then be optimized to find the “sweet spot” for a robust, reproducible and cost-efficient design.
Upstream processing steps
Given the large number of USP variables, the numbers of experimental conditions and replicates that need to be tested can quickly become unmanageable or require too much time and materials. Design of Experiments (DoE) provides a systematic way to maximize the information gained about the test system, while limiting the number of experiments. It can also uncover hidden relationships between variables that can have profound effects on process outcomes. In a nutshell, DoE approaches can enable you to more quickly hone in on the most optimal system and process settings. This significantly shortens the development cycle and reduces experimental costs.
Batavia Biosciences combines DoE with its proprietary SCOUT® platform to facilitate high-throughput cell culture in mini bioreactors that closely mimic what happens at clinical production scales. Not only does this reduce the amount of time and materials required for testing, it also allows parallel development of both upstream and downstream processes.
Downstream process (DSP)
One of the many challenges of DSP development is that processing needs are highly product-specific and dependent on the type of vector produced and USP choices. This means that the nature and order of processing steps can vary widely, there are no standardized solutions for vectors, and it can be difficult to develop both USP and DSP in parallel.
Despite product-specific variations, there are certain commonalities in downstream workflows, usually starting with clarification of the harvest to remove large impurities like cell debris. This is typically followed by tangential flow filtration (TFF) to concentrate the virus and transfer it to the appropriate buffer.
If depth filtration is used, it is sometimes possible to streamline DSP by combining particle purification with removal of impurities such as host-cell or plasmid-derived nucleic acids. A number of chromatography steps may be needed to remove any remaining impurities. If the viral vector particle size is not prohibitive, a sterile filtration step is performed after formulating in the final buffer. Otherwise, aseptic process validation may be a necessity.
Downstream processing steps
Analytical methods and assay development
Success of viral vector process development and manufacturing strongly depends on having suitable analytical assays for a variety of different purposes:
- Qualification of MCB and MVS
- Materials testing and control
- Process development
- Monitoring and QC during manufacturing
- Quality assurance and release testing
To develop the appropriate assays product release, the process must first be well characterized. This means being able to generate representative material. Assays intended for release testing must then be qualified for phase 1/2 clinical trials. Regulatory guidelines such as those issued by the ICH and FDA for chemistry, manufacturing and control (CMC) require demonstration of suitable testing for key critical quality attributes relating to: identity, potency, purity, safety and stability.
Common abbreviations and terms
CDMO
(Contract Development and Manufacturing Organization) – A company that provides development and manufacturing services on a contract basis.
cGMP
(Current Good Manufacturing Practice) – Refers to the Current Good Manufacturing Practice regulations issued by the United States Food and Drug Administration. cGMP regulations aim to assure the identity, strength, quality and purity of drug products by requiring that manufacturers to adequately control manufacturing operations. The “c” of cGMP stands for “current”, signifying the requirement for companies to use up-to-date technologies and systems that are fully compliant with the latest regulations.
CMC
(Chemistry, Manufacturing and Controls) – The body of information that defines product characteristics, manufacturing processes and product testing to ensure the safety, efficacy and batch consistency of pharmaceutical products.
CoG
(Cost of Goods) – In manufacturing, the abbreviation CoG is often used to refer to the cost of goods manufactured, comprising the total direct manufacturing cost of a product, including materials, labor and factory overhead.
CPP
(Critical Process Parameter) – A key variable in pharmaceutical manufacturing affecting the production process. CPPs impact critical quality attributes (CQA) and should therefore be controlled within a proven acceptable range to ensure the drug product meets its quality specifications.
CQA
(Critical Quality Attribute) – Any physical, chemical, biological or microbiological property or characteristic that must be maintained within a defined range, limit or distribution in order to ensure a product’s quality.
DoE
(Design of Experiments) – A systematic method of determining cause-and-effect relationships between factors affecting a process and the output of that process.
DP
(Drug Product) – A specific drug in dosage form.
DS
(Drug Substance) – An active ingredient that is intended to furnish pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease or to affect the structure or any function of the human body, but does not include intermediates used in the synthesis of such ingredient.
DSP
(Downstream Processing) – Describes the steps in biopharmaceutical manufacturing required to purify the desired ingredient, from the production harvest up to purified Drug Substance Bulk.
IMP
(Investigational Medicinal Product) – A pharmaceutical drug or active substance or placebo being tested or used as a reference in a clinical trial.
IMPD
(Investigational Medicinal Product Dossier) – A document that must be submitted to regulatory authorities in the European Union in order to gain approval for use of an IMP in clinical trials.
IND
(Investigational New Drug) – A drug that has not been approved by the US FDA, but is under investigation for use in human clinical trials. An IND application must be submitted to the FDA to obtain authorization to administer an IND or biological product to humans in a clinical trial.
MCB
(Master Cell Bank) – A bank of a cell substrate from which all subsequent cell banks will be derived. The MCB represents a collection of cells of uniform composition derived from a single source prepared under defined culture conditions.
MVS
(Master Virus Seed) – A viral seed of a selected vaccine virus, from which all future vaccine production will be derived—either directly or via Working Virus Seeds.
Scale-down model
– A scaled down or miniaturized model that mimics a larger scale unit operation in the manufacturing process. Scale-down models are developed and used to support process development studies. Models should account for scale effects and be representative of the proposed commercial process. A scientifically justified model can enable a prediction of quality, and can be used to support the extrapolation of operating conditions across multiple scales and equipment.
SCOUT®
– A scale-down model using a miniaturized production and purification platform to rapidly develop multivariate processes. The SCOUT® technology provides for an effective tool for “Design-of-Experiments” (DoE) approaches.
SIDUS®
– The SIDUS® platform combines biological materials (cell lines and vector systems) with in-depth experience and complete protocol systems for manufacturing viral vector-based vaccine, oncolytic and gene therapy products.
USP
(Upstream Processing) – The entire process from early cell isolation and cultivation, to cell banking and culture expansion of the cells, and production of the desired biological substance until final harvest.
WCB
(Working Cell Bank) – A cell bank derived by propagation of cells from MCB under defined conditions and used to initiate host cell cultures required for virus production on a lot-by-lot basis.
WVS
(Working Virus Seed) – A viral seed derived by propagation of virus from the MVS under defined conditions and used to initiate production virus production lot-by-lot.
1 Vrba SM et al. Development and applications of viral vectored vaccines to combat zoonotic and emerging public health threats. Vaccines (Basel) (2020) 8(4):680.
2 ClinicalTrials.gov database. US National Library of Medicine, NIH, https://clinicaltrials.gov/ct2/home.
3 Alliance for Regenerative Medicine. (2021). 2020: Growth & Resilience in Regenerative Medicine. Annual Report. https://alliancerm.org/sector-report/2020-annual-report
4 Gene Therapy accounts for a major portion of the cell and gene therapy market and it is expected to have the most growth. The Business Research Company, Intrado GlobeNewswire, 23 February 2021, https://www.globenewswire.com/news-release/2021/02/23/2180767/0/en/Gene-Therapy-Accounts-For-A-Major-Portion-Of-The-Cell-And-Gene-Therapy-Market-And-It-Is-Expected-To-Have-The-Most-Growth.html.
5 Capra E et al. (17 May 2021). Gene-therapy innovation: Unlocking the promise of viral vectors. McKinsey & Company web post. Retrieved from https://www.mckinsey.com/industries/pharmaceuticals-and-medical-products/our-insights/gene-therapy-innovation-unlocking-the-promise-of-viral-
6 D’amore T and Yang Y-P. Advances and Challenges in Vaccine Development and Manufacture. BioProcess International (2019) Volume 17, September issue.
7 This is how much it costs to develop a vaccine. The Cost of Things. MarketWatch, 1 October
8 Pronker ES et al. Risk in vaccine research and development quantified. PLOS One (2013) 8(3): e57755.
9 Gouglas D, et al. Estimating the cost of vaccine development against epidemic infectious diseases: a cost minimisation study. The Lancet (2018) 6(12): E1386-1396.
10 Viral Vectors, Non-Viral Vectors and Gene Therapy Manufacturing Market (3rd and 4th Editions)Roots Analysis.
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