Continuous Manufacture of Lentiviral Vectors for Therapeutic Vaccine Applications
Lead Research Organisation:
University College London
Department Name: Biochemical Engineering
Abstract
Gene-modified cell therapies, such as chimeric antigen receptor T-cell therapies, have emerged as a promising therapeutic modality for the treatment of a variety of indications. The manufacture of these therapies often employs a lentiviral vector (LV) to mediate gene transfer. LVs provide a higher safety profile than live viral vectored therapies, as they are non-replicative due to modifications to the virus.
The production of LVs for research applications and pre-clinical studies is achieved through the co-transfection of mammalian cells with plasmid DNA. The high costs of the plasmid DNA and the transfection regent coupled with batch-to-batch variability and limited scalability make transient transfection unsuitable for large-scale LV production. To address this, the creation of a stable packaging cell line to produce LVs has been reported. However, this cell line uses VSV-G envelope protein that is cytotoxic to the cell and has historically led to poor reproducibility and difficulties in scale-up. The WinPac-RDpro stable cell line developed at UCL has replaced VSV-G with RDpro, which is less toxic and has enabled reproducible expression of LVs (1). During LV production, adherent producer cells are usually grown in static culture vessels, with the virus being harvested through the surface irrigation of confluent cells. It is necessary to concentrate the harvested viral-containing material prior to further purification steps. This project will address the requirements for scalable production of lentiviral vectors by investigating a scalable bioreactor system, Pall's iCELLis bioreactor, for adherent producer cell lines like the WinPac-RDpro and its integration with a novel scale-down in-line concentrator based on Pall's Cadence in-line concentration system and UCL's ultra scale-down design methodology.
The iCELLis bioreactor is a fixed-bed bioreactor capable of growing adherent cell lines to high densities has previously been applied for clinical-grade LV production using transient transfection (2). The Cadence in-line concentrator offers a potential option for single pass tangential flow filtration, which may be capable of concentrating the viral vectors as media is continuously drawn out of the reactor.
This project will create a scale-down system for the perfusion production and concentration of lentiviral vectors from a fix bed reactor. The project specifically aims to:
- Investigate and characterise the bioreactor performance of the iCellis nano during batch operation and benchmark against T-flask production using the WinPac producer cell line.
- Establish a perfusion process using the WinPac producer cell line and determine the impact of key parameters on LV production.
- Establish a scale-down TFF for the continuous concentration of lentiviral vectors
- Demonstrate the continuous production of lentiviral vectors.
References
1. Sanber, K., Knight, S., Stephen, S., Bailey, R., Escors, D., Minshull, J., Santilli, G., Thrasher, A., Collins, M. and Takeuchi, Y., 2015. Construction of stable packaging cell lines for clinical lentiviral vector production. Scientific Reports, 5(1).
2. Valkama, A., Leinonen, H., Lipponen, E., Turkki, V., Malinen, J., Heikura, T., Ylä-Herttuala, S. and Lesch, H., 2017. Optimization of lentiviral vector production for scale-up in fixed-bed bioreactor. Gene Therapy, 25(1), pp.39-46.
3. Casey, C., Gallos, T., Alekseev, Y., Ayturk, E. and Pearl, S., 2011. Protein concentration with single-pass tangential flow filtration (SPTFF). Journal of Membrane Science, 384(1-2), pp.82-88.
The production of LVs for research applications and pre-clinical studies is achieved through the co-transfection of mammalian cells with plasmid DNA. The high costs of the plasmid DNA and the transfection regent coupled with batch-to-batch variability and limited scalability make transient transfection unsuitable for large-scale LV production. To address this, the creation of a stable packaging cell line to produce LVs has been reported. However, this cell line uses VSV-G envelope protein that is cytotoxic to the cell and has historically led to poor reproducibility and difficulties in scale-up. The WinPac-RDpro stable cell line developed at UCL has replaced VSV-G with RDpro, which is less toxic and has enabled reproducible expression of LVs (1). During LV production, adherent producer cells are usually grown in static culture vessels, with the virus being harvested through the surface irrigation of confluent cells. It is necessary to concentrate the harvested viral-containing material prior to further purification steps. This project will address the requirements for scalable production of lentiviral vectors by investigating a scalable bioreactor system, Pall's iCELLis bioreactor, for adherent producer cell lines like the WinPac-RDpro and its integration with a novel scale-down in-line concentrator based on Pall's Cadence in-line concentration system and UCL's ultra scale-down design methodology.
The iCELLis bioreactor is a fixed-bed bioreactor capable of growing adherent cell lines to high densities has previously been applied for clinical-grade LV production using transient transfection (2). The Cadence in-line concentrator offers a potential option for single pass tangential flow filtration, which may be capable of concentrating the viral vectors as media is continuously drawn out of the reactor.
This project will create a scale-down system for the perfusion production and concentration of lentiviral vectors from a fix bed reactor. The project specifically aims to:
- Investigate and characterise the bioreactor performance of the iCellis nano during batch operation and benchmark against T-flask production using the WinPac producer cell line.
- Establish a perfusion process using the WinPac producer cell line and determine the impact of key parameters on LV production.
- Establish a scale-down TFF for the continuous concentration of lentiviral vectors
- Demonstrate the continuous production of lentiviral vectors.
References
1. Sanber, K., Knight, S., Stephen, S., Bailey, R., Escors, D., Minshull, J., Santilli, G., Thrasher, A., Collins, M. and Takeuchi, Y., 2015. Construction of stable packaging cell lines for clinical lentiviral vector production. Scientific Reports, 5(1).
2. Valkama, A., Leinonen, H., Lipponen, E., Turkki, V., Malinen, J., Heikura, T., Ylä-Herttuala, S. and Lesch, H., 2017. Optimization of lentiviral vector production for scale-up in fixed-bed bioreactor. Gene Therapy, 25(1), pp.39-46.
3. Casey, C., Gallos, T., Alekseev, Y., Ayturk, E. and Pearl, S., 2011. Protein concentration with single-pass tangential flow filtration (SPTFF). Journal of Membrane Science, 384(1-2), pp.82-88.
Planned Impact
The IDC has a proven track record of delivering impact from its research and training activities and this will continue in the new Centre. The main types of impact relate to: (i) provision of highly skilled EngD graduates; (ii) generation of intellectual property (IP) in support of collaborating companies or for new venture creation; (iii) knowledge exchange to the wider bioprocess-using industries; (iv) benefits to patients in terms of new and more cost effective medicines, and (v) benefits to wider society via involvement in public engagement activities and encouraging future generations of researchers.
With regard to training, the provision of future bioindustry leaders is the primary mission of the IDC and some 97% of previous EngD graduates have progressed to relevant bioindustry careers. These highly skilled individuals help catalyse the development and expansion of private sector innovation and biomanufacturing activity. This is of enormous importance to capitalise on emerging markets and to create new jobs and a skilled labour force to underpin the UK economy.
In terms of IP generation each industry-collaborative EngD project will have direct impact on the industry sponsor in terms of new technology generation and improvements to existing processes or procedures. Where substantial IP is generated this has the potential to lead to spin-out company creation and job creation with wider UK economic benefit. IDC research has already led to creation of two UCL spin-out companies focussed on the emerging field of Synthetic Biology (Synthace) and novel nanofibre adsorbents for improved bioseparations (Puridify). Once arising IP is protected the IDC also provides a route for wider dissemination of project outputs and knowledge exchange available to all UK bioprocess-using companies. This occurs via UCL MBI Training Programme modules which have been attended by more than 1000 individuals from over 250 companies to date.
The majority of IDC projects address production of new medicines or process improvements for pharmaceutical or biopharmaceutical manufacture which directly benefit healthcare providers and patients. Examples arising from previous EngD projects have included: engineered enzymes used in the synthesis of a novel pharmaceutical; early stage bioprocess development for a new meningitis vaccine; redevelopment of the bioprocess for manufacture of the UK anthrax vaccine; and establishment of a cGMP process for manufacture of a tissue-engineered trachea (this was subsequently transplanted into a child with airway disease and the EngD researcher was featured preparing the trachea in the BBC's Great Ormond Street series). Each of these examples demonstrates IDC impact on the development of cost-effective new medicines and therapies. These will benefit society and provide new tools for the NHS to meet the changing requirements for 21st Century healthcare provision.
Finally, in terms of wider public engagement and society, the IDC has achieved substantial impact via involvement of staff and researchers in activities with schools (STEMnet, HeadStart courses), presentations at science fairs (Big Bang, Cheltenham), delivery of high profile public lectures (Wellcome Trust, Royal Institution) as well as TV and radio presentations. The next generation of IDC researchers will be increasingly involved in such outreach activities to explain how the potential economic and environmental benefits of Synthetic Biology can be delivered safely and responsibly.
With regard to training, the provision of future bioindustry leaders is the primary mission of the IDC and some 97% of previous EngD graduates have progressed to relevant bioindustry careers. These highly skilled individuals help catalyse the development and expansion of private sector innovation and biomanufacturing activity. This is of enormous importance to capitalise on emerging markets and to create new jobs and a skilled labour force to underpin the UK economy.
In terms of IP generation each industry-collaborative EngD project will have direct impact on the industry sponsor in terms of new technology generation and improvements to existing processes or procedures. Where substantial IP is generated this has the potential to lead to spin-out company creation and job creation with wider UK economic benefit. IDC research has already led to creation of two UCL spin-out companies focussed on the emerging field of Synthetic Biology (Synthace) and novel nanofibre adsorbents for improved bioseparations (Puridify). Once arising IP is protected the IDC also provides a route for wider dissemination of project outputs and knowledge exchange available to all UK bioprocess-using companies. This occurs via UCL MBI Training Programme modules which have been attended by more than 1000 individuals from over 250 companies to date.
The majority of IDC projects address production of new medicines or process improvements for pharmaceutical or biopharmaceutical manufacture which directly benefit healthcare providers and patients. Examples arising from previous EngD projects have included: engineered enzymes used in the synthesis of a novel pharmaceutical; early stage bioprocess development for a new meningitis vaccine; redevelopment of the bioprocess for manufacture of the UK anthrax vaccine; and establishment of a cGMP process for manufacture of a tissue-engineered trachea (this was subsequently transplanted into a child with airway disease and the EngD researcher was featured preparing the trachea in the BBC's Great Ormond Street series). Each of these examples demonstrates IDC impact on the development of cost-effective new medicines and therapies. These will benefit society and provide new tools for the NHS to meet the changing requirements for 21st Century healthcare provision.
Finally, in terms of wider public engagement and society, the IDC has achieved substantial impact via involvement of staff and researchers in activities with schools (STEMnet, HeadStart courses), presentations at science fairs (Big Bang, Cheltenham), delivery of high profile public lectures (Wellcome Trust, Royal Institution) as well as TV and radio presentations. The next generation of IDC researchers will be increasingly involved in such outreach activities to explain how the potential economic and environmental benefits of Synthetic Biology can be delivered safely and responsibly.
| Description | - Design a modular, microscale single-pass tangential flow filtration system that will facilitate the continuous concentration of biological materials. Initial results indicate that the system that has been designed enables the continuous concentration of proteins. - Additionally, a continuous process for the production of lentiviral (LV) vectors, which are indispensable tools for cell therapy applications, has been established in a fixed-bed bioreactor. This process will facilitate the economical production of high titre LVs. |
| Exploitation Route | - The filtration system is intended to be a mimic of an existing technology developed by the product sponsor company, Pall Biotech. If it can be successfully demonstrated that the system provides representative insight into the performance of the large-scale system, this will facilitate early stage process development. - Similarly, the continuous LV production process employs a fixed-bed bioreactor marketed by the sponsor company. The established process would be of interest to organisations interested in the production of viral vectors. |
| Sectors | Pharmaceuticals and Medical Biotechnology |