How to test master cell banks as starting material for gene therapies

By BioPhorum

The gene therapy (GT) modality offers an exciting new way to impact patients and GT manufacturers are looking to leverage understanding of previous modalities to streamline the ramp-up of Good Manufacturing Practices (GMP) for the GT.

BioPhorum’s Cell and Gene Therapy Raw Materials Team offers a platform approach to testing an essential starting material for GTs – master cell banks (MCBs) – and wishes to identify, share and improve a framework for MCB testing and release. This will increase the speed of learning and ultimately influence and/or assist regulators when developing guidelines or policies regarding the development and manufacture of GTs.

Ideas here are limited to release assays and specifications of human-derived (HeLa and HEK293/293T) and insect-derived (Sf9) MCBs used for generation of adeno-associated virus (AAV) or lentivirus (LV) for use in GTs. Cells used in cell therapy applications, for example, autologous (patient) and allogeneic (donor) cells, are out of reach, as are assays used solely for characterization of cell lines.

We propose the following platform framework for testing HeLa, HEK293/293T and Sf9 MCBs containing parental host cells. This platform is not intended to define the methods or specifications that to have to be used, but those who are generally used. A new test can be used if validated and agreed with the appropriate regulatory authorities.

The platform’s testing methodology mimics the approach outlined in the recent BioPhorum paper Critical starting material for cell and gene therapy: a discussion to help establish the release specifications of plasmids and the bacterial stem cell banks used to produce them.

Table 1 of the document (an excerpt is shown below) shows the Critical Quality Attributes (CQAs) for BCMs, which have been grouped into the following sections:

Division 1 – Sterility and mycoplasma

Division 2 – Growth and viability, and performance, identity and appearance

Division 3 – Adventitious agents (not specific to the host cell; classified by cell type)

Section 3.1 – Tests related to cattle and pigs (or process-related tests; classified by cell type)

Section 3.2 – Host cell-specific tests (categorized by cell type)

Division 4 – Retroviral contaminants (classified by cell type)

Additional proprietary testing is likely required for each treatment.

Table 1: Proposed MCB Plasmid and Plasmid DNA Testing and Release Platform (excerpt only)

Phase-appropriate approach

The approaches in Table 1 should be compendial or validated for non-compendial methods and the need depends on the process or phase (preclinical, early/late clinical). This is because the MCB is established at the start of project development work and is used to generate hardware or a work cell bank (WCB) that is used throughout the multiple phases of a project.

Testing of a WCB may be a subset of the tests performed on the MCB if the MCB is well characterized by the methods in Table 1. At a minimum, the WCB should be confirmed as safe to use by demonstrating that it is free from mycoplasmas, bacteria, fungi, and adventitious viruses (the latter usually only requires in vitro test). The tests carried out must be compendial or validated if they are not compendial.

Phenotypic Stability Considerations

Generally, cell banks do not need to undergo a formal stability study. Instead, the stability of MCB or WCB during long-term storage can be assessed by monitoring the viability of one or more cell bank vials when thawed for use in routine production. and by the constant production of an active substance of the intended quality.

A protocol should be established to confirm the phenotypic stability of cell banks when not used in routine production for long periods of time. This testing interval should depend on the stability of cell banks established during product development and the expected life of the cell bank. Examples of parameters that can be taken into account include viability after thawing and doubling time. Release specifications for post-thaw doubling time and viability established for CAT should be met at all future time points.

This article is a summary of a recent BioPhorum publication on the subject. To learn more, read the full report, A discussion to help establish release specifications for master cell banks of parental host cells.

What is your opinion?

BioPhorum welcomes feedback on key questions included in this article via a confidential survey. Before responding, readers are encouraged to review the 2010 FDA Cell Substrate Guidance Document Characterization and Qualification of Cellular Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications: Guidelines for Industry.

The high level questions are:

  1. Do you agree with the ambition of a platform approach for MCB testing of parental cell lines?
  2. Are there any attributes missing?
  3. Testing requirements may change due to different phases of product development. By process step, when do you need GMPs to be applied?
  4. What checks or documentation are important at each stage?
  5. Over time, monitoring of phenotypic stability may be necessary to reduce commercial risk. Which is better practice: a formal stability program or tracking a trend over time as part of normal project work?
  6. What attributes should be tested when monitoring the phenotypic stability of GMP cell banks? When monitoring the phenotypic stability of GMP cell banks, what is your sample size?
  7. When monitoring the phenotypic stability of GMP cell banks, what is the best practice for the number of time points?
  8. Do you see this platform approach being applicable to allogeneic cell therapy? What modifications may be necessary?

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