Clarity in cell line development

Currently, for companies in the early stages (up to Phase 2 clinical trials) of gene therapy, most vector production is still carried out via transient transfection with multiple plasmids. However, for Phase 3 and beyond, 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 as available commercial manufacturing capacity is at a premium, and improved vector quality and safety for GMP.

Gene therapy companies increasingly need “stable producer” cell lines where all vectors are stably integrated into the host cell. This has the following advantages:

  1. Fewer starting materials and operations during manufacture
  2. Simplified manufacturing process
  3. Potential for more reproducible vector production
    • Defined monoclonal cell lines
    • Each batch starts with the same cell bank
  4. More scalable, providing enough material for patient access

 

FDA Guidelines and Genetic Analysis for Gene Therapy Vectors

FDA draft guidelines 2018 state the following:

  • “We recommend that you fully sequence all vectors that are 40kb or smaller”
  • “For integrating viral vectors, we recommend that you perform DNA sequencing on the integrated vector. The material for sequencing can be collected from the producer cell line.”
  • “For stable vector producer cell lines, we recommend that you test the genetic stability of the gene insert in the end of production (EOP) cells.”

Hence to draw parallels with the more mature guidance for cell lines producing mAbs, the stability testing and genetic analysis should be carried out at the clonal cell line level.

Workflow for stable cells lines for vector production

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’s Cell Metric and VIPS products can be used to produce stable cell lines for gene therapy vector production with assurance of clonality

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.

Successful Single Cell Recovery and Outgrowth for Stable HEK 293 Cells

Following single cell cloning, it is recommended to add the animal component-free growth supplement InstiGRO HEK for improved single cell recovery, highest outgrowth and cloning efficiency.

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.

Whole well imaging is then carried out on Day 0 by either the VIPS or the Cell Metric for assurance of clonality.

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.

Top clones selected for the MCB and back-up should also undergo stability testing and full sequencing of the integrated vector

CASE STUDY

Learn how Ultragenyx used CellMetric to Accelerate Scalable rAAV Production