Sub Topic | Secondary Topic: Biotherapeutics and Biotechnology - Cell-Based Assay | Functional Response-Pharmacodynamic
Authors: Angela Kirik, EAG - Life Sciences (Presenting Author); Charlie Britten, ADC Therapeutics; Karin Havenith, ADC Therapeutics; Mike Whalon, EAG - Life Sciences; Glenn Petrie, EAG - Life Sciences; Esohe Edusogie, ADC Therapeutics; Michael Mulkerrin, ADC Therapeutics; Loretta Sukhu, EAG - Life Sciences (Main Author)
Presenting Author: Angela Kirik
Purpose: Antibody drug conjugates (ADCs) are comprised of a monoclonal antibody coupled to a highly cytotoxic drug. A cell based assay was used to determine the relative binding potency of the monoclonal antibody component of an ADC. The antibody was targeted to an antigen expressed on the surface of B lymphocytes.
Methods: A competitive cell-based binding assay with electrochemiluminescence (ECL) detection was used to determine the binding activity of the ADC relative to a reference standard. The assay utilized CD19 expressing Ramos cells (RA-1, ATCC ® CRL1596™); a SULFO-TAG® anti-CD19 antibody was used as a competitor to unlabeled ADC. Conjugated and unconjugated anti-CD19 antibodies were recognized to a similar extent by the antigen. Luminescence was measured by a Mesoscale Discovery (MSD) Sector Imager plate reader and was proportional to the competition by ADC of the binding of SULFO-TAG® antibody to the CD19 antigen.
Results: A number of critical assay parameters were evaluated during GMP qualification exercises; including Accuracy, Precision (intra assay and intermediate), Specificity, Linearity, Range, and effect of cell passage number on binding activity. As part of the acceptance criteria, a reference standard was compared to an internal assay control in each run of the assay. A 4 parameter logistic (4 PL) non-linear curve was used for data analysis. The coefficient of determination (R2), slope, and upper and lower asymptotic values were monitored as part of the assay acceptance criteria. Demonstration of parallelism between reference standard and internal assay control was also part of the acceptance criteria. Relative potency was determined by the ratio of the C values of reference over test material.
Conclusion: Data obtained during the course of method qualification and analysis of system suitability criteria of reference standard and internal control tracked through the course of a year demonstrated the assay’s suitability and robustness for determining the potency of binding activity.
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