title: Development and validation of novel enzyme activity methods to assess inhibition of matrix metalloproteinases (MMPs) in human serum by antibodies against enzyme therapeutics.
abstract: This paper summarizes the development and validation of five enzyme activity methods to assess the specific inhibition of human endogenous matrix metalloproteinases MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin 1), MMP-8 (collagenase 2) and MMP-13 (collagenase 3) by anti-Collagenase Clostridium histolyticum (CCH) antibodies in human serum. These MMPs are of interest since antibodies against a therapeutic enzyme may cross-react with, and inactivate, the MMPs. The validated methods utilize spiked exogenous individual MMPs added to serum to determine if the serum inhibits MMP enzyme activity. Factors evaluated and optimized during development include pH, reaction time and temperature, inhibitor concentration for the positive control, and substrate and serum concentration. Characteristics established during validation for each MMP activity inhibition method included intra- and inter-assay precision and recovery, recovery in the pooled normal human serum samples, bench-top stability at room temperature and on wet ice, and assay cut-point determination. Precision results ranged from ~1 to 12% CV, recoveries of the activities of the exogenous MMPs ranged from ~84 to 90% and cut-point values ranged from 67 to 91%.
authors.affiliation: Consultant to Auxilium, Edkins Pharma Services, LLC, Wayne, PA 19087-3229, USA.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Edkins
authors.forename: Jack A
authors.initials: JA
authors.lastname: Alhadeff
authors.forename: Vincent
authors.initials: V
authors.lastname: Kwok
authors.forename: Charles
authors.initials: C
authors.lastname: Kalensky
authors.forename: Marie T
authors.initials: MT
authors.lastname: Rock
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Vidmar
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Del Tito
mesh_terms: D000907:Antibodies, Bacterial / Q000097:blood*; D000918:Antibody Specificity; D001681:Biological Assay / Q000379:methods / Q000592:standards; D002138:Calibration; D047009:Hathewaya histolytica / Q000201:enzymology*; D003429:Cross Reactions; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020781:Matrix Metalloproteinase 1 / Q000032:analysis / Q000276:immunology; D053509:Matrix Metalloproteinase 13 / Q000032:analysis / Q000276:immunology; D020778:Matrix Metalloproteinase 2 / Q000032:analysis / Q000276:immunology; D019278:Matrix Metalloproteinase 3 / Q000032:analysis / Q000276:immunology; D020784:Matrix Metalloproteinase 8 / Q000032:analysis / Q000276:immunology; D061965:Matrix Metalloproteinase Inhibitors / Q000494:pharmacology*; D020782:Matrix Metalloproteinases / Q000032:analysis* / Q000276:immunology; D003012:Microbial Collagenase / Q000276:immunology* / Q000627:therapeutic use; D011994:Recombinant Proteins / Q000032:analysis / Q000037:antagonists & inhibitors / Q000276:immunology; D012015:Reference Standards; D015203:Reproducibility of Results; D013696:Temperature; D013997:Time Factors
chemical_list: D000907:Antibodies, Bacterial; D061965:Matrix Metalloproteinase Inhibitors; D011994:Recombinant Proteins; C507079:MMP13 protein, human; D053509:Matrix Metalloproteinase 13; D020782:Matrix Metalloproteinases; C511768:MMP3 protein, human; D019278:Matrix Metalloproteinase 3; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2; D003012:Microbial Collagenase; C488762:MMP8 protein, human; D020784:Matrix Metalloproteinase 8; C522360:MMP1 protein, human; D020781:Matrix Metalloproteinase 1