ASTM_F_2131_-_02_2012.pdf

Designation:F213102(Reapproved 2012)Standard Test Method forIn Vitro Biological Activity of Recombinant Human BoneMorphogenetic Protein-2(rhBMP-2)Using the W-20 MouseStromal Cell Line1This standard is issued under the fixed designation F2131;the number immediately following the designation indicates the year oforiginal adoption or,in the case of revision,the year of last revision.A number in parentheses indicates the year of last reapproval.Asuperscript epsilon()indicates an editorial change since the last revision or reapproval.1.Scope1.1 This test method describes the method used and thecalculation of results for the determination of the in-vitrobiological activity of rhBMP-2 using the mouse stromal cellline W-20 clone 17(W-20-17).This clone was derived frombone marrow stromal cells of the W+mouse strain.21.2 This test method(assay)has been qualified and vali-dated based upon the International Committee on Harmoniza-tion assay validation guidelines3(with the exception of inter-laboratory precision)for the assessment of the biologicalactivity of rhBMP-2.The relevance of this in vitro test methodto in vivo bone formation has also been studied.The measuredresponse in the W-20 bioassay,alkaline phosphatase induction,has been correlated with the ectopic bone-forming capacity ofrhBMP-2 in the in vivo Use Test(UT).rhBMP-2 that waspartially or fully inactivated by targeted peracetic acid oxida-tion of the two methionines was used as a tool to compare theactivities.Oxidation of rhBMP-2 with peracetic acid wasshown to be specifically targeted to the methionines by peptidemapping and mass spectrometry.These methionines reside in ahydrophobic receptor binding pocket on rhBMP-2.Oxidizedsamples were compared alongside an incubation control and anative control.The 62,87,98,and 100%oxidized samples hadW-20 activity levels of 62,20,7,and 5%,respectively.Theincubation and native control samples maintained 100%ac-tivity.Samples were evaluated in the UT and showed a similareffect of inactivation on bone-forming activity.The sampleswith 62%and 20%activity in the W-20 assay demonstratedreduced levels of bone formation,similar in level with thereduction in W-20 specific activity,relative to the incubationcontrol.Little or no ectopic bone was formed in the 7 and 5%active rhBMP-2 implants.1.3 Thus,modifications to the rhBMP-2 molecule in thereceptor binding site decrease the activity in both the W-20 andUT assays.These data suggest that a single receptor bindingdomain on rhBMP-2 is responsible for both in-vitro and in-vivoactivity and that the W-20 bioassay is a relevant predictor ofthe bone-forming activity of rhBMP-2.1.4 The values stated in SI units are to be regarded asstandard.No other units of measurement are included in thisstandard.1.5 This standard does not purport to address all of thesafety concerns,if any,associated with its use.It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2.Terminology2.1 rhBMPrecombinant human bone morphogenetic pro-tein.2.2 GDFgrowth and differentiation factor.3.Summary of Test Method3.1 In this test method,the mouse stromal cell line W-20-17is used as a target cell line for rhBMP-2.The W-20-17 cellsexhibit increased alkaline phosphatase activity in response torhBMP-2.Optical density at 405 nm of the p-nitrophenolgenerated from the alkaline phosphatase substrate is used as ameasure of alkaline phosphatase enzyme level.The testmethod is performed in a 96-well plate format.A similar testmethod based upon the same cell line has been developed usingchemiluminescent detection of alkaline phosphatase.41This test method is under the jurisdiction ofASTM Committee F04 on Medicaland Surgical Materials and Devices and is the direct responsibility of SubcommitteeF04.42 on Biomaterials and Biomolecules for TEMPs.Current edition approved Oct.1,2012.Published October 2012.Originallyapproved in 2002.Last previous edition approved in 2007 as F2131 02(2007)1.DOI:10.1520/F2131-02R12.2Thies,R.S.,Bauduy,M.,Ashton,B.A.,Kurtzberg,L.,Wozney,J.M.,andRosen,V.,“Recombinant Human Bone Morphogenetic Protein-2 Induces Osteo-blastic Differentiation in W-20-17 Stromal Cells,”Endocrinology,Vol 130,1992,pp.13181324.3Guideline for Industry,ICH-Q2A Text on Validation of Analytical Procedures,November 1996,International Committee on Harmonization,March 1995,http:/www.fda.gov/cder/guidance/index/htm.4Blum,R.S.,Li,R.H.,Mikos,A.G.,and Barry,M.A.,“An Optimized Methodfor the Chemiluminescent Detection of Alkaline Phosphatase Levels DuringOsteodifferentiation by Bone Morphogenetic Protein 2,”Jour.Cellular Biochem,Vol 80,2001,pp.532537.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 4.Significance and Use4.1 Although the test method can be used for assessment ofthe bioactivity of crude preparations of rhBMP-2,it has onlybeen validated for use with h