February 21, 2020

In the cell and gene therapy industry people often say that manufacturing is 80% of the business. Naturally, I continue to look for nuggets of wisdom from manufacturing experts. Recently, I had an opportunity to observe release testing for a fresh CAR T product at an academic institution with a state-of-the art cGMP cell therapy unit. In the process, technologists shared with me their guidelines, learnings, and rules of thumb. Clearly, it is impossible to ignore commercial and business development implications of the manufacturing process and quality control in this space.

Each product has pre-determined tests and release specifications. However, not all testing is mandatory for providing a product to patients. Mandatory tests include

• Cell viability
• % cells expressing CAR
• Endotoxin
• Microorganisms
• Mycoplasma
• Replication Competent Retrovirus (RCR)

The PDF table below describes key Tests, rough time needed for each test completion (under an hour, under a day, etc.), purpose of each test, and Release Specifications. Of these parameters, Tests and Release Specifications, along with results, are recorded in a Certificate of Analysis (CoA) for each product lot.

Table links: 7-AAD dye, LAL.

Cell viability precedes CAR+% analysis to include only viable cells into further phenotyping.

To calculate the volume of CAR T cells needed for injections, medical staff uses total % of CAR-positive cells and the dose per label or for a specific Dose Cohort per the IND Clinical Protocol.

Quantification of CAR-positive T cells in the product by flow cytometry
Additionally, phenotyping of CAR-T cells using other surface markers is performed, but is not required for product release to patients. Notably, such surface markers may include CD14, CD20, CD16/56, and so on.

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