title: A sensitive enzyme immunoassay for the quantitation of human CTLA4Ig fusion protein in mouse serum: pharmacokinetic application to optimizing cell line selection.
abstract: A sensitive, accurate, and precise enzyme immunoassay (EIA) for the quantitation of human CTLA4Ig in mouse serum was validated. The EIA method employed a technique in which a monoclonal anti-CTLA4 antibody was adsorbed onto 96-well polystyrene microtiter plates and used to capture the CTLA4Ig in mouse serum samples. The captured CTLA4Ig was then detected using a goat anti-human IgGFc antiserum conjugated to the enzyme horseradish peroxidase. The validation included assessments of method accuracy and precision, range of reliable response, lower limit of quantitation (LLQ), inter-analyst robustness, storage stability in mouse serum and assay specificity. The results indicate that this validated assay is precise, accurate, and reproducible. This EIA has a range of reliable response in 10% mouse serum of 0.14-4.58 ng ml-1 resulting in a 100% serum equivalent curve of 1.4-45.8 ng ml-1. Assessment of individual standard curve variations indicated a reproducible response with R2 values of > or = 0.995. The LLQ was established at 1.4 ng ml-1. The accuracy and precision estimates, based on the quality control values, were within 3.8% and 5.2% respectively, for CTLA4Ig. Stability of CTLA4Ig was established in mouse serum for 5 days at both 4 degrees C and room temperature, for 2 months at -70 degrees C and through five freeze-thaw cycles. This validated assay was successfully employed in the assessment of pharmacokinetic characteristics of CTLA4Ig in mice and to aid in the selection of an optimal CTLA4Ig-producing cell line.
authors.affiliation: Department of Metabolism and Pharmacokinetics, Bristol-Myers Squibb Pharmaceutical Research Institute, Syracuse, NY 13221, USA.
authors.forename: R S
authors.initials: RS
authors.lastname: Weiner
authors.forename: N R
authors.initials: NR
authors.lastname: Srinivas
authors.forename: J D
authors.initials: JD
authors.lastname: Calore
authors.forename: C G
authors.initials: CG
authors.lastname: Fadrowski
authors.forename: W C
authors.initials: WC
authors.lastname: Shyu
authors.forename: L K
authors.initials: LK
authors.lastname: Tay
mesh_terms: D000069594:Abatacept; D000818:Animals; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000378:metabolism*; D016466:CHO Cells / Q000378:metabolism; D060908:CTLA-4 Antigen; D002460:Cell Line; D006224:Cricetinae; D005260:Female; D006801:Humans; D018796:Immunoconjugates; D007124:Immunoenzyme Techniques; D007166:Immunosuppressive Agents / Q000097:blood* / Q000493:pharmacokinetics*; D007275:Injections, Intravenous; D051379:Mice; D008815:Mice, Inbred Strains; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D015703:Antigens, CD; D000943:Antigens, Differentiation; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; C556707:Ctla4 protein, mouse; D018796:Immunoconjugates; D007166:Immunosuppressive Agents; D000069594:Abatacept