This workflow is to produce the recombinant virus vector, carrying the gene of interest (GOI), which will be used to infect the patient's cells e.g. a target tissue, such as the eye, or the patient's T cells in CAR-T.
Currently, for companies in the early stages of gene therapy, most vector production is still carried out via transient transfection. However, for clinical trials, much larger amounts of material are required. Not only is this costly using current approaches, there are also more requirements around batch-to-batch consistency, higher titres needed, and improved vector quality and safety for GMP.
Gene therapy companies increasingly need “stable producer” cell lines.
As these cell lines will also be producing a therapeutic/biologic, there is an implied requirement that they must be shown to be clonal for regulatory approval by the FDA or other regulator.
Using the tools developed by Solentim for stable cell line development, some of our first gene therapy customers have already developed and banked stable high producer clones, with demonstrated monoclonality, in less than 3 months.
Our existing gene therapy customers predominantly work with two primary methods of viral gene delivery: Lentivirus (LV) or Adeno-Associated Virus (AAV) as follows:
The packaging cells line will produce the capsid or empty virus particle. This is effectively the “mother” cell line for future producer cell lines. They are generally used for making smaller amounts of material by transient transfection.
The producer cell line is a stable cell line for the GOI and is used to manufacture the final clinical materials.
The producer cells will be able to infect isolated patient cells (ex-vivo) e.g. T cells or have the possibility to be directly injected into patients allowing for long term in-vivo vector production.
Single cell seeding
The requirements are to dispense single cells from a population into wells of 96 well microtiter plates.
This should be done at high efficiency so that most wells contain a single cell and they need to be viable, so they grow into a colony over the next few days.
Depending on virus type, this seeding process will be required for the packaging cells line and the producer cell lines.
Cell types used by customers can be either adherent or suspension-adapted e.g. HEK293, 293T, HeLa.
Based on the cell types used, FACS and Cell Printers are not very good for adherent cells, and hence many customers still use a limiting dilutions approach.
Solentim has developed the VIPS as the preferred approach for single cell seeding of these adherent cells. The VIPS will generate a resultant plate with high seeding efficiencies in around 10 minutes and due to the gentle nature of the dispensing method, most of these wells survive and grow up successfully into colonies.
Solentim have now developed a system called the VIPS as the potentially preferred approach for cell line development groups.
The VIPS will generate a resultant plate with high cloning efficiency in around 10 minutes and due to the gentle nature of the dispensing method, most of these wells will go on and grow up successfully.
Assurance of Clonality and Documentation for the Regulator
After seeding single cells into 96 well plates, cells can be allowed to passively settle to the bottom of the well or can be centrifuged.
Imaging is continued for subsequent days of growth and ultimately colony formation. This can be 10-21 days depending on cell types.
A library of images is created for each well and it is possible to track back in time from the colony all the way back to Day 0 and confirm whether it started from a single cell or not.
For the clonal wells, images can rapidly be assembled into a Clonality Report for the Regulator.
Screening to select for Manufacturing
Clones which have been shown to be monoclonal, are adapted to serum-free media and grown in 24 DWP suspension culture. Clones are screened and ranked for virus yield.
Finally, clones are subject to scale down models (shake flasks or ambr) at higher cell densities and screened for best media and culture conditions as a predictor for manufacturing conditions.
Top 3-4 clones will typically be selected for genetic stability studies, bioreactor yield and vector quality assessments.