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Important 10 questions about Large-scale production of viral vectors
Important 10 questions about Large-scale production of viral vectors. Viral vector production is a key part of most gene therapy and genetically modified cell therapy production processes. Lentiviral vectors are commonly used vectors for CAR-T gene-modified cell therapy and are the key to the progress of these treatments.
In addition, lentiviral vectors are also used in gene therapy to treat some genetic diseases. Due to the importance of these carriers to production, it is necessary to improve the production process. Areas for improvement include stable cell line development, scalability, upstream and downstream process optimization, and ensuring that the production process is ready for commercialization of gene therapy. Below experts to help us seize these process improvement opportunities. At this expert meeting, we convened a group of experts to answer questions about viral vector production and process improvement.
Areas to be explored further include:
- Cell line development for lentiviral vector production
- Upstream process development of lentiviral vector production
- Scale up the upstream process into a disposable bioreactor
- Lentiviral vector downstream process optimization
- Prepare for the commercialization of the vector production process
Dr. Lisa Freeman Cook, Head of Technology Operations, MilliporeSigma Virus and Gene Therapy Production
Dr. Lisa Freeman Cook is the head of technical operations for Millipore Sigma’s virus and gene therapy manufacturing facility in Carlsbad, California. She received a bachelor’s degree in biology from Carleton College in Northfield, Minnesota, and a PhD from the University of Colorado in the Lorraine Pillus laboratory, where she studied chromatin structure and epigenetic gene regulation. She received postdoctoral training in the laboratory of Daniel Di Maio at Yale University, and her research interests focused on papillomavirus oncogenes.
After that, she worked at Invitrogen and Life Technologies (now Thermo Fisher Scientific), managing contract services, production, and R&D for the human protein microarray business. Lisa then transferred to GenMark Diagnostics, where she managed analytical development and technical operations, developed a sample response platform and four FDA-approved molecular diagnostic assays, and served as the vice president of analytical development. Currently, Dr. Cook is leading the process development, technology transfer, technical failure investigation and verification engineering of MilliporeSigma viral vector and gene therapy contract production business.
MilliporeSigma viral vector production cell line, gene editing and new model development, Henry J. George, director of process solution research and development
Henry J. George brings expertise in research and product development through his professional experience in small-scale biotechnology and large-scale pharmaceutical industries. Henry received a bachelor’s degree in biology from Southern Illinois University in Edwardsville in 1976, and conducted genetics research at Florida State University and biochemical research at St. Louis University. Since 1981, Henry has been committed to the fields of biotechnology and pharmaceuticals, developing recombinant protein expression systems for bacteria, insects and mammalian hosts.
In 1981, he joined Minnetonka (MN), an early biotechnology leader, to develop bacterial expression systems and produce recombinant proteins for agricultural purposes (such as animal vaccines and animal growth hormones). In 1988, he joined R&D Systems, Inc (Mpls, MN), a leader in the development and production of research reagents today, and established a gene expression team responsible for the production of various mammalian cytokines in bacteria and insect systems .
His team has produced many initial products currently sold by the R&D department. From 1990 to the beginning of 2001, he was the principal investigator at DuPont Pharmaceuticals (now part of Bristol-Myers Squibb, Wilmington). His laboratory work at DuPont continues to focus on the development and use of high-level expression systems to produce (analytical and large-scale) essential proteins for drugs. In early 2001, he joined Sigma-Aldrich as the R&D manager, developing FLAG® protein expression system and Mission™ lentiviral shRNA library for gene silencing of human and mouse genes.
In 2010, he joined Sigma’s SAFC department as Group Manager of Cell Science and Development (CSD). He led a team to develop an improved new CHO cell line (CHOZN)™ ZFN technology for the monoclonal antibody production platform through Sigma’s CompoZr. In 2016, he joined MilliporeSigma’s gene editing and new model R&D team, leading a new team (viral vector production cell line), focusing on improving viral vector production systems and bioprocesses for gene therapy applications.
The team developed a new suspension cell-based production platform (Virus Express™) for large-scale production-lentiviral vectors. Henry’s research experience covers multiple scientific fields, including molecular biology, gene expression, cell biology, virology and protein chemistry. The results of his work have been published in journals such as “Science”, “Proceedings of the National Academy of Sciences”, “Journal of Biological Chemistry”, “Biochemistry” and “DNA”.
Question 1 What is your opinion on adhesion and suspension in lentivirus production?
Of course, there are many limitations to growing cells in adherent cultures, mainly around the scalability of these systems, because all of them depend on the surface area of the culture substrate. Therefore, only an expansion process can achieve larger and larger scales, requiring larger production space, equipment, and so on. Another responsibility of the adhesion system is the use of undefined media and the use of animal serum/components to grow cells. These reagents increase the chances of introducing foreign agents into products and workflows. The suspension growth process is a true “scale-up” process, which can utilize the growth of cells in bioreactor systems of various scales, so that the production process can reflect the production process established in the monoclonal antibody industry. These cells can also be grown in a medium without animal components and chemical definitions, which will reduce the chance of introducing contaminants.
Question 2 Do you see that the suspension quickly reaches a certain point where the productivity is better than the adhesive system?
I think we have arrived. Our VirusExpress lentivirus production platform has been proven to outperform the adhesion system (up to 5 times) in terms of overall potency and specific productivity. Future improvements, such as optimizing the transfection process, introducing better transfection reagents, and improving cell growth parameters, will continue to increase overall productivity.
Question 3 We are considering transferring to a stable cell line to produce lentivirus. Do you have any suggestions for transfection and production cell lines?
Although we see that the future of LV production is moving towards the use of stable cell lines, mainly due to the reduction in the cost of the reagents required for the plasmid/transfection method currently in use, the length of time to develop such cell lines (>1 Years) and the lower productivity of such systems are currently the main obstacles. The transfection-based production process allows one person to develop and produce within a few weeks, allowing one person to test and get their therapeutic products into preclinical and clinical environments faster.
Question 4 What is the most effective way to establish a stable lentiviral cell line?
Introducing viral gene expression vectors into cells, their respective integration and high-yield selections are relatively difficult and time-consuming (also >1 year). At this time, the team must screen hundreds of clones. Production and commercially useful titers produce LV particles. Unlike the use of monoclonal antibody clones driven by constitutive promoters to drive the transcription of IgG genes, some viral genes are toxic to producing cells, so an inducible promoter system is required to control the production of these toxic proteins in cells.
Question 5 When considering changing the lens from adhesion to suspension, how do you deal with the shear problem?
It boils down to choosing a cell line and medium combination that has been adapted to grow under suspension conditions. This may require cloning and screening hundreds of clones, and then screening these clones for their growth and productivity attributes. Normally, reagents such as Pluronic F-68 are added to the medium to minimize shearing, but the effect of PF68 must be balanced because PF68 may affect the transfection ability when using the whole plasmid production method. Our VirusExpress cell lines and media grow well under suspension conditions, from small shake flasks to large bioreactors.
Question 6 Several articles have studied the application of baculovirus vectors in suspension production. What do you think of this method?
Although we have heard of the use of such cells to produce AAV, no one has yet to use insect cells to produce LV. Since both LV and baculovirus produce budding viruses, I think using this virus to produce LV will result in low titer (if any) because of the competition between cell secretion and assembly factors. For the production of AAV, the use of insect cells is well documented. Several treatments for AAV are now in the clinic. The advantage of the insect cell system is that cells can be grown in suspension culture without the need to produce expensive plasmids or use transient transfection reagents. However, it is now necessary to demonstrate the removal of the large baculovirus used to infect cultures (although size exclusion is relatively simple), and there have been some reports of post-translational modifications and capsid differences that, when applied to target cells/organs, can cause Different transduction efficiency.
Question 7 Our process has problems with stability and yield loss during the production process. We are considering how to reduce the waiting time and/or transfer to perfusion. I would like to hear your thoughts.
LV particles are budding retroviruses, which are known to be relatively “fragile” and have an unstable environment (short half-life at physiological temperature and pH values other than neutral). We have seen some teams cancel the maintenance step and develop a continuous purification process without stopping to minimize losses during the purification and treatment process. A perfusion system may be helpful, although only when using a stable production system, because high levels of VSV-g pseudotyped env-LV particles produced can cause cytotoxicity and reduce productivity 48 hours after transfection.
Question 8 I used anion exchange method to purify LV from the crude extract, but the yield will be lost during the elution step. Are there any good suggestions?
Several factors can be considered here. First of all, you should make sure that you are using the appropriate size column/capsule; if the capacity is too small, you may lose the product during circulation. Second, ionic strength is obviously an important factor to be considered for ion exchange columns. The loading, washing, and elution ionic strength should be optimized to ensure maximum binding and elution. Third, Lenti is not as stable as AAV or adenovirus, so you should be careful to keep the eluate. For example, diluting to restore the eluate to a more neutral pH and/or lower salt concentration is important to maintain the infection titer. .
Question 9 We have been using Hyperstack optimized for gas exchange for production, but we need to expand the scale. We want to maintain the same gas exchange capacity. Any suggestions?
For adhesion processes that need to be expanded beyond the HYPERStack/CellSTACK scale, we used iCELLis® bioreactors. The system uses a disposable fixed-bed bioreactor with a growth area of up to 500 square meters, which is equivalent to nearly 800 cell piles. The system is designed to achieve optimal gas exchange, but some work needs to be done to ensure you have a process that can match or exceed your current productivity/volume. They did provide iCELLis nanosystems (up to 4 square meters of growth area) for small-scale feasibility studies. There are also other suppliers that have or are developing fixed-bed bioreactors. When the adhesion process needs to be expanded beyond the HYPERStack/CellSTACK scale, moving to suspension is not feasible.
Question 10 Do you have any suggestions for the virus removal/sterile process of lentiviral vectors?
Control the contamination of foreign viruses by using well-characterized cell lines and qualified raw materials and consumables that are certified to be free of contaminants. The final product is also tested for the presence of uncertain viruses. Bacterial/microbial contamination of lentivirus products should be controlled as far as possible by using closed systems and sterile disposable consumables. All operations should use aseptic technique as much as possible. Finally, the operation of the downstream unit includes the first clarification through a 0.5 µm filter, and finally a 0.2 µm filter to ensure the sterility of the final product. In addition, during batch harvesting and other downstream device operations, in-process sampling of bioburden and endotoxins is performed, and the final product is tested for sterility and endotoxins.
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