Client Challenge
A cell therapy developer needed a validated method to confirm the identity of its drug substance under cGMP regulation. The release specification rested on a panel of cell surface markers, with some markers expected to be strongly positive and others weakly positive or negative. The company needed a flow cytometry method that could be shown to detect that pattern specifically and reproducibly across analysts and days.
Avance Biosciences’ Solution
Avance Biosciences validated a multi-color flow cytometry method for surface marker analysis on a CytoFLEX platform. The method quantifies the percentage of positive cells for five surface markers, using isotype controls to set positive gates, a viability dye to exclude non-viable cells, and a stepwise gating strategy that isolates viable, intact, single cells before marker analysis.
Project Overview
The study was run under a pre-approved validation protocol with predefined acceptance criteria. The drug substance was tested alongside a positive control and several negative control cell types, with each stain run in replicate and every sample tracked by a unique ID to maintain traceability.
Assay Validation
Each parameter was selected to answer a question a quality unit will ask of an identity test:
- Specificity: Does the method detect only the intended markers? Antibodies were tested on cells known not to express each target. All negative controls measured below 2% positive against a criterion of <10%, and the positive control met every predefined specification.
- Precision: Will the same sample give the same answer regardless of replicate, run, analyst, or day? Repeatability, intermediate precision, inter-analyst precision, and inter-day precision were all evaluated. Variability stayed below 10% CV in every case, and below 1% CV for the test sample, against acceptance criteria of 20 to 30%.
- System suitability: Is each run valid on its own? Every run met predefined criteria for post-thaw cell viability, daily instrument QC, and background staining.
Customer Outcome
- A validated, cGMP-compliant identity method with documented specificity and precision across all five markers
- A defined identity profile for the drug substance: strong expression of three markers, weak or low expression of one, and negative expression of one
- Reproducible results across three analysts and multiple days
- A signed final validation report supporting use of the method for drug substance identity testing
Client Impact
With the assay validated, the client has a defensible, data-supported identity test for its cell therapy drug substance, ready to support quality control testing under cGMP.
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