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Virus production

For vaccine production, gene therapy & oncolytic cancer treatment

Producing viruses or virus-like particles (VLPs) is necessary on a large-scale for vaccine production, gene therapy and oncolytic cancer treatments. Virus manufacturing is a complex multistep process, involving: (1) replication of the viral genome, (2) production of the viral proteins (the capsid) and sometimes enveloping lipids, and (3) the assembly of the units into the functional viral particle.

In virus production, manipulations of the genetic code will allow for the virus to produce modified viral particles, which will behave as per design, for example to immunize people given the modified virus or VLP (vaccinations), or deliver the modified viral vector to target cells, which will then alter their behavior (gene therapy).

In cancer treatment, oncolytic (cancer-killing) agents are delivered by nonreplicating, modified viruses, including Adenoviruses1, Herpes simplexvirus2, morbillivirus (measles)3 and Poxviruses4. Learn more about methods for studying cancers.

Doctor injecting a vaccine into a patient intramuscularly.

Vaccine development: viruses for COVID-19 treatment

After many years of receiving little interest, spotlight or research funding, vaccine development has been at almost a stand-still for the past several decades. With the emergence of SARS-CoV-2, the virus responsible for the COVID-19 pandemic that started in 2019, we have seen technology advancement and collaboration surge to an unparalleled level.

Vaccines and treatments against the coronavirus SARS-CoV-2 are mainly based on three technologies:

  1. Viral vector technology (mRNA) delivering genetic material into host cells for the production of antigens which then elicit immune response5
  2. Modified/inefficient virus or virus-like particles (VLPs) used to elicit an immune response by presenting antigens directly, without having to rely on infection of host cells and production of viral proteins6
  3. Using CRISPR-Cas technology both for diagnosis and as a possible cure7

Learn more about the development of specific vaccines and treatments in the fight against COVID-19:

Culture systems for virus manufacturing & scaling up

When used in vivo for immune response activation and patient treatments, viruses often show a limited affinity for their target cells. This means that to achieve a therapeutic effect, large titer counts of up to 1012 active virus particles per dose are needed8. Therefore, the upstream production steps and downstream clarification, purification and formulation steps must be carefully optimized and made efficient. Since the production is based on virus/host interactions that differ with each formulation/treatment type, optimization of culture conditions and vessels, and the timing of each step in the process mode are key to success.

The manufacturing systems for virus or VLP generation are cell lines from host species like insect (S2 and Sf9 cells), mammals (Vero and CHO cell lines) and human (HEK293, HER96 and MRC-5 cell lines9). The viral systems used in commercial manufacturing vary based on the purpose thereof, but mainly include retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), herpes simplex virus, and Sendai virus.

Another means of virus production is through using animals as live hosts. The virus is harvested from the animal eggs or blood. The virus antigen is then purified and is ready for the next stage of the manufacturing process.

Illustration of static, semi-dynamic, and dynamic cell culture systems.

There are many types of culture vessels and each has its pros and cons. Small-scale cultures are easier to control but produce virus particles at a scale which is not feasible for profitable manufacturing. Large-scale bioreactors typically include static systems, single-use systems, continuous stirred-tank bioreactors or fixed-bed bioreactors10,11.

Other factors that aid our understanding and application of virus production include cell concentration at infection, process mode, multiplicity of infection, process control and safety.

Learn how AdaptVac and ExpreS2ion Technologies fast-tracked a vaccine for SARS-CoV-19 based on their VLP-technology and producing in S2 insect cells.

Solutions for virus production

The NucleoCounter® automated cell counters are widely used in the cell therapy industry. Our automated cell counters are now making a name in the virus production industry, helping even more customers achieve optimal results. Recently, our instruments have helped the development of vaccines and treatments against COVID-19: ChAdOx1 nCoV-19 (renamed AZD1222) from the Jenner Institute; work by the PREVENT-nCoV Consortium; and a treatment for Severe Acute Respiratory Syndrome (caused by SARS-CoV-2) from the Méary Center APHP.

NucleoCounter® NC-202™ ensures robust cell counting and viability data. Our instruments do not require calibration or manual adjustments while in use. The Via2-Cassette™ technology eliminates human error that can occur when cell counting.

Since the NC-202™ has a 21 CFR Part 11/GMP-compliant script, it is transferable throughout your production pipeline from research, through development, process scaling, manufacturing and quality control (QC). With the instrument’s cell counting parameter, the DebrisIndex™, together with its accuracy and precision, the NucleoCounter® demonstrates unparalleled reliability and robustness in protocol transfer without any downtime.

The NucleoCounter® has a two-minute cell counting and analysis protocol for primary cells including chicken embryo fibroblasts (CEFs) and cells grown on microcarriers or as aggregates. It is so easy to use, that operators at any laboratory experience level could be responsible for process monitoring. Furthermore, with a low counting variation, the NucleoCounter® mitigates the risk of production deviation.

References

  1. EE Cohen and CM Rudin: ONYX-015. Onyx Pharmaceuticals. Curr Opin Investig Drugs. 2001; 2(12):1770-5.
  2. H Kasuya, Y Kodera, A Nakao et al.: Phase I Dose-escalation Clinical Trial of HF10 Oncolytic Herpes Virus in 17 Japanese Patients with Advanced Cancer. Hepatogastroenterology. 2014; 61(131):599-605.
  3. MF Leber, S Neault, E Jirovec et al.: Engineering and combining oncolytic measles virus for cancer therapy. Cytokine & Growth Factor Reviews. 2020; Volume 56, 39-48
  4. KA Parato, CJ Breitbach, F Le Boeuf et al.: The oncolytic poxvirus JX-594 selectively replicates in and destroys cancer cells driven by genetic pathways commonly activated in cancers. Mol Ther. 2012; 20(4):749-58.
  5. JS Putter: Immunotherapy for COVID-19: Evolving treatment of viral infection and associated adverse immunological reactions. Published online. 2021.
  6. C Fougeroux, L Goksøyr, M Idorn et al.: Capsid-like particles decorated with the SARS-CoV-2 receptor-binding domain elicit strong virus neutralization activity. Nat Commun. 2021; 12(1):324.
  7. L Fernandez-Garcia, O Pacios, M González-Bardanca et al.: Viral Related Tools against SARS-CoV-2. Viruses. 2020; 12(10):1172.
  8. TA Grein, T Weidner and P Czermak: Concepts for the Production of Viruses and Viral Vectors in Cell Cultures. Open access peer-reviewed chapter. 2017.
  9. AF Rodrigues, HR Soares, MR Guerreiro et al.: Viral vaccines and their manufacturing cell substrates: New trends and designs in modern vaccinology. Biotechnol J. 2015; 10(9): 1329–1344
  10. DM Berrie, RC Waters, C Montoya et al.: Development of a high-yield live-virus vaccine production platform using a novel fixed-bed bioreactor. Vaccine. 2020; 38(20), 3639-3645.
  11. LE Gallo-Ramírez, A Nikolay, Y Genzel et al.: Bioreactor concepts for cell culture-based viral vaccine production. Expert Rev Vaccines, 2015; 14(9):1181-95.