title: Complement dependent cytotoxicity (CDC) activity of a humanized anti Lewis-Y antibody: FACS-based assay versus the 'classical' radioactive method -- qualification, comparison and application of the FACS-based approach.
abstract: The fully humanized Lewis-Y carbohydrate specific monoclonal antibody (mAb) IGN311 is currently tested in a passive immunotherapy approach in a clinical phase I trail and therefore regulatory requirements demand qualified assays for product analysis. To demonstrate the functionality of its Fc-region, the capacity of IGN311 to mediate complement dependent cytotoxicity (CDC) against human breast cancer cells was evaluated. The "classical" radioactive method using chromium-51 and a FACS-based assay were established and qualified according to ICH guidelines. Parameters evaluated were specificity, response function, bias, repeatability (intra-day precision), intermediate precision (operator-time different), and linearity (assay range). In the course of a fully nested design, a four-parameter logistic equation was identified as appropriate calibration model for both methods. For the radioactive assay, the bias ranged from -6.1% to -3.6%. The intermediate precision for future means of duplicate measurements revealed values from 12.5% to 15.9% and the total error (beta-expectation tolerance interval) of the method was found to be <40%. For the FACS-based assay, the bias ranged from -8.3% to 0.6% and the intermediate precision for future means of duplicate measurements revealed values from 4.2% to 8.0%. The total error of the method was found to be <25%. The presented data demonstrate that the FACS-based CDC is more accurate than the radioactive assay. Also, the elimination of radioactivity and the 'real-time' counting of apoptotic cells further justifies the implementation of this method which was subsequently applied for testing the influence of storage at 4 degrees C and 25 degrees C ('stability testing') on the potency of IGN311 drug product. The obtained results demonstrate that the qualified functional assay represents a stability indicating test method.
authors.affiliation: Vela Pharmazeutische Entwicklung und Laboranalytik GmbH, Vienna, Austria. a.nechansky@vela-labs.at
authors.forename: A
authors.initials: A
authors.lastname: Nechansky
authors.forename: O H J
authors.initials: OH
authors.lastname: Szolar
authors.forename: P
authors.initials: P
authors.lastname: Siegl
authors.forename: I
authors.initials: I
authors.lastname: Zinoecker
authors.forename: N
authors.initials: N
authors.lastname: Halanek
authors.forename: S
authors.initials: S
authors.lastname: Wiederkum
authors.forename: R
authors.initials: R
authors.lastname: Kircheis
mesh_terms: D000465:Algorithms; D000906:Antibodies / Q000633:toxicity*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001681:Biological Assay; D045744:Cell Line, Tumor; D002470:Cell Survival; D004396:Coloring Agents; D003165:Complement System Proteins / Q000502:physiology*; D003627:Data Interpretation, Statistical; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007983:Lewis Blood Group Antigens / Q000276:immunology*; D016014:Linear Models; D015203:Reproducibility of Results; D018570:Risk Assessment
chemical_list: D000906:Antibodies; D000911:Antibodies, Monoclonal; D004396:Coloring Agents; C510956:IGN 311; D007983:Lewis Blood Group Antigens; C094162:Lewis Y antigen; D003165:Complement System Proteins