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Manufacturing and Analytical Characterization - Biomolecular

(T1530-02-09) Validating the Measurement of Loss of Cell Surface CD6 on CD4 T-Cell Based Potency Assay Using Flow Cytometry

Tuesday, November 11, 2025
3:30 PM - 4:30 PM CT
Purpose: The development and validation of a potency assay is a critical component in the development control strategy of biopharmaceutical products. It helps in ensuring that the assay meets defined performance standards which support the drug’s safety and efficacy. In this case, a flow-based assay was validated to assess the relative potency of an anti-CD6 monoclonal antibody. CD6, which is a stimulatory receptor on T cells, combines with activated leukocyte cell adhesion molecule (ALCAM) leading to the proliferation of T-cells. Activated pathogenic T effector cells having very high CD6 expression induces excessive secretion of chemokines causing a series of autoimmune disorders. The antibody promotes cleavage of the CD6 receptor from the surface of T cells in the presence of monocytes, resulting in decreased surface expression of CD6. By blocking CD6, the drug reduces T effector cell proliferation and downregulates signaling pathways. 

Methods: The experimental setup involved utilization of commercially available total PBMCs that were incubated with the drug for approximately 24hrs at 37C. To determine a dose dependent response, cells were treated with 12 dose concentrations, then cells were collected and stained with a non-competing CD6 detection antibody. This cocktail was then detected by capturing CD6 expression gating CD4+ cells using CytoFlex. The resultant cells were then analyzed for % CD6 cells that emit the fluorescent signal associated with the target STCs as shown in Figure 1. A series of experiments were conducted to establish assay reliability and define acceptance criteria, including linearity, accuracy, assay range, precision, specificity, robustness, stability, and lot-to-lot consistency. Assay linearity, accuracy and precision were established by testing samples at 3 different NDCs of 50%, 100% and 150% with 3 determinations across 2 analysts. Repeatability was determined by running test samples and assay control at 100% NDC with a minimum of 3 determinations.  A base media was tested twice across duplicate dilution curves by substituting the sample to determine the specificity of the assay. Robustness was measured by monitoring critical incubation times across a wide range of time. Different lots of Reference Standard were also tested for performance of parallelism of curves and comparability with the current lot. 

Results: All data were collected using Beckman Coulter CytExpert software. The Reference Standard, system suitability control, and sample data points were analyzed using both restricted and unrestricted 4-Parameter Logistic (4PL) models in PLA software.  Representative potency assay curves for test samples at 50%, 100%, and 150% of the nominal drug concentration (NDC) are shown in Figure 2. Linearity was achieved at 50%, 100% and 150% NDC with R2 of 0.99. Assay runs showed inter and intra assay precision with less than 10% CV and the sample was stable after 3 freeze thaw and showed substantial relative potency at 37C for 24 hours.  Figure 3 demonstrates lot to lot testing during assay development. This validated method consistently showed a decrease in CD6 expression with increasing drug concentration and maintained stability across various temperatures and amongst different lots. 

Conclusion: In conclusion, a flow-based expression method was successfully validated to confirm the drug's mechanism of action. This functional assay has played a key role in advancing the product to subsequent stages of drug development. The study highlights both a reliable assay and a promising therapeutic candidate with the potential to yield positive outcomes for autoimmune and inflammatory diseases.

References: 21 CFR Part 820 and ICH Q2 (R1) Guidelines

Acknowledgements: Employee of BioAgilytix Labs, LLC. There is no conflict of interest.

Figure 1: Gating strategy on CD4+ cells to determine CD6 expression

Figure 2: Dose Response Curves at 50%, 100% and 150% NDCs

Figure 3: Relative potency of stability test sample in comparison to reference material