A comprehensive guide on FDA requirements, validation standards, and best practices for using flow cytometry in regulatory submissions.
Some of the most common drug products use flow cytometry as an identity assay to determine what the drug product looks like. To the best of my knowledge, here are the some tips to get your flow cytometry assay up to speed, and in good shape.
- Choose the right antibodies for you. Not all antibodies or manufacturers are created equal.
- Determine if you need to compensate your fluorochromes. This should always be step two after you decide what you want to use.
- Always include a live dead stain even if you think it’s unnecessary.
- TITRATE YOUR ANTIBODIES.
- Determine a gating strategy. This should be based off evidence, not a feeling.
- Decide between FMOs or isotypes. I find FMOs to suffice EXCEPT for intracellular staining in most situations.
- Once steps 1-6 are completed, a written procedure should be established to ensure repeatability. In addition to a procedure, every replicate adds to data in determining gating strategy and acceptance criteria for your assay.
In addition to the list, if working on CAR-T products, here is an excerpt from the FDA guidance, “Considerations for the development of CAR-T cell products”:
“We recommend direct detection of the CAR to determine the
percentage of CAR-positive cells. If the CAR is detected by
surrogate protein expression (e.g., detection of a co-expressed
gene) or other broad-specificity reagents (e.g., protein L), you
should evaluate the correlation with CAR expression.
Assessment of the sensitivity and specificity of the surrogate
marker should be included as part of the justification for use.
v. A comprehensive validation study for lot release flow
cytometry assay(s) must be conducted to support licensure.8
(21 CFR 211.165(e)). This validation study should be
conducted per International Conference on Harmonisation
(ICH) Q2 (Ref. 30) and include validation of each fluorescently
labeled marker in the flow cytometry panel on the flow
cytometer(s) used for CAR T cell release. Robustness studies,
including defining the maximum holding time for samples
before staining and between staining and acquisition, should be
included.”
If you’re unfamiliar, validation is not synonymous with qualification, but this excerpt provides feedback on what should be expected of a drug product in the future.
-Kyler Aqueche. Flow Cytometry SME.