1、Designation: F 2131 02 (Reapproved 2007)e1Standard 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 F 2131; the number immediately following the designation
2、 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 (e) indicates an editorial change since the last revision or reapproval.e1NOTEFormatting and grammar were corrected edit
3、orially throughout in April 2007.1. 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
4、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 i
5、n 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
6、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
7、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 an
8、d 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 for
9、med 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 tha
10、t 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.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 sta
11、ndard 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 mous
12、e 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 en
13、zyme level. The testmethod is performed in a 96-well plate format. A similar test1This 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 edi
14、tion approved Feb. 1, 2007. Published February 2007. Originallyapproved in 2002. Last previous edition approved in 2002 as F 2131 02.2Thies, R. S., Bauduy, M., Ashton, B. A., Kurtzberg, L., Wozney, J.M., andRosen, V., “Recombinant Human Bone Morphogenetic Protein-2 Induces Osteo-blastic Differentiat
15、ion in W-20-17 Stromal Cells,” Endocrinology, 130, 1992, pp.1318-1324.3Guideline 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.1Copyright ASTM International, 100 Barr Ha
16、rbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.method based upon the same cell line has been developed usingchemiluminescent detection of alkaline phosphatase.44. Significance and Use4.1 Although the test method can be used for assessment ofthe bioactivity of crude preparat
17、ions of rhBMP-2, it has onlybeen validated for use with highly pure (98 % by weightprotein purity) preparations of rhBMP-2.5. Interferences5.1 There have been no systematic studies of interferingsubstances for this test method. There is anecdotal evidencethat trypsin and some rhBMP-2 formulation buf
18、fers can inter-fere with the assay. Additionally, the source of fetal bovineserum is an important variable. Each lot should be tested in allparts of the assay where it is required to determine theappropriateness of the lot. This is particularly important if fetalbovine serum vendor is changed.6. App
19、aratus6.1 Polypropylene conical tubes, 15 mL and 50 mL.6.2 Cryovials (Corning or equivalent), sterile 2 mL.6.3 Eppendorf vials, sterilized.6.4 Variable pipets, (range 20 to 1000 L) and Multichannelpipets (range 50 to 300 L).6.5 Biosafety cabinet.6.6 96 Well flat bottom sterile tissue culture microti
20、terplates, (Falcon 3072 or equivalent).6.7 IEC Centra-7R Centrifuge, or equivalent.6.8 CO2humidified tissue culture incubator.6.9 Spectrophotometric microplate reader, (VMAX/Spectramax, Molecular Devices, or equivalent).6.10 Hemacytometer, or automatic cell counter.6.11 Inverted microscope.6.12 Tiss
21、ue culture flasks, Falcon T175 or equivalent.6.13 Sterilized paper towels, or equivalent.6.14 Sterile filter units, (0.2 m).6.15 Sterile pipets, (1 mL, 5 mL, 10 mL, 25 mL, 50 mL).6.16 9 in. Pasteur pipets, sterilized.6.17 Sterilized pipet tips, (1-300 L and 200-1000 L).6.18 Sterile reagent reservoir
22、s.6.19 80C freezer.6.20 96 Well U-Bottom polypropylene sterile tissue culturemicrotiter plates, (Costar 3790 or equivalent).6.21 Water bath.6.22 Orbital shaker.7. Reagents and Materials7.1 W-20-17 Mouse Stromal Cells.57.2 Dulbeccos modified Eagles medium with 4500 mg/Lglucose and 4.0 mM L-glutamine,
23、 without sodium bicarbonate(DME/High, JRH Biosciences, 56439 or equivalent).67.3 Sodium bicarbonate (SigmaAldrich S4019 or equiva-lent).77.4 5 M hydrochloric acid.7.5 Heat inactivated (Hi) fetal bovine serum (FBS).NOTE 1Each new lot of fetal bovine serum must be evaluated in theassay before use.7.6
24、200 mM L-Glutamine (Invitrogen Life Technologies,25030081 or equivalent).87.7 Gentamicin Gibco sterile filtered: 10 mg/mL or equiva-lent.7.8 Penicillin Streptomycin (PS), contains 10 000 units ofpenicillin (base)/mL and 10 000 g of streptomycin (base)/mL,utilizing penicillin G (sodium salt) and stre
25、ptomycin sulfate in0.85 % saline (Invitrogen Life Technologies, #15140122 orequivalent).87.9 Phosphate Buffered Saline, Calcium and MagnesiumFree, 1x (PBS-CMF), (Invitrogen Life Technologies (cat.#20012050 or equivalent).87.10 Dimethyl sulfoxide (DMSO), cell culture grade(Sigma-Aldrich or equivalent
26、).77.11 Trypsin-EDTA(0.05 % trypsin, 0.53 mM EDTA 4Na)(1X), liquid (Invitrogen Life Technologies 25300054 orequivalent).87.12 Glycine (Sigma Aldrich or equivalent).77.13 Sodium Hydroxide (NaOH) 0.2 N and 10 N.7.14 Triton X-100 (J.T. Baker Cat. No. X198-05 or equiva-lent).97.15 Magnesium Chloride, Cr
27、ystalline (MgCl26H2O).7.16 p-Nitrophenol phosphate (PNPP, SigmaAldrich104(R) phosphatase substrate, product # 1040 or equivalent).77.17 NaCl.7.18 Purified water.7.19 rhBMP-2, 1st WHO Reference Reagent 1997 (5000Units per ampoule, cat. # 93/574, National Institute forBiological Standards and Control)
28、.107.20 rhBMP-2 internal control, 1 mg/mL (stored at80C).8. Procedure8.1 Solution Preparation:8.1.1 DME Low Bicarb: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 Morphogen
29、etic Protein 2,” Jour. Cellular Biochem.,80, 2001, pp. 532-537.5This cell line has been deposited in mid-2001 at the American Type CultureCollection, 10801 University Blvd., Manassas, VA 20110-2209, U.S., http:/www.atcc.org.6This medium has been found satisfactory for this purpose and is available f
30、romJRH Biosciences, P.O. Box 14848, Lenexa, KS 66215, U.S., http:/.7This material has been found satisfactory for this purpose and is available fromSigma-Aldrich Corp., 3050 Spruce St., St. Louis, MO 63103, U.S., http:/.8This material has been found satisfactory for this purpose and is available fro
31、mInvitrogen Life Technologies, 1600 Faraday Ave., P.O. Box 6482, Carlsbad, CA92008, U.S., http:/.9This material has been found satisfactory for this purpose and is available fromJ. T. Baker, (Mallinckrodt Baker, Inc.), 222 Red School Ln., Phillipsburg, NJ 08865,U.S., http:/.10This material has been
32、found satisfactory for this purpose and is available fromthe National Institute for Biological Standards and Control (NIBSC), Blanche Ln.,South Mimms, Potters Bar, Herts, EN6 3QG, U.K., http:/www.nibsc.ac.uk.F 2131 02 (2007)e128.1.1.1 Dissolve 66.87 g DME/High and 11.13 g sodiumbicarbonate in 4.5 L
33、of purified water.8.1.1.2 Adjust the pH to 7.3 6 0.10 with 5 M HCl and bringsolution to 5 L with purified water.8.1.1.3 Filter through a 0.2 m filter into sterile bottles.8.1.1.4 Store at 2 to 8C. The solution expires in 8 weeks.8.1.2 Hi FBS:8.1.2.1 Thaw the desired amount of FBS at ambient tem-pera
34、ture, or 2 to 8C.8.1.2.2 Adjust the water bath to a temperature of 56 6 2C.8.1.2.3 Place the bottle of FBS into the water bath so thatthe entire contents of the bottle are immersed in water.8.1.2.4 Heat the bottle for 45 min, swirling periodically.8.1.2.5 Remove the bottle from the water bath and al
35、low tocool to room temperature. Aliquot 50 mL of the FBS in sterile50-mL conical tubes.8.1.2.6 Label each container with name, lot number, expi-ration date, and the heat inactivation date. Store at 20 6 10Cor 2 to 8C.8.1.3 Growth Medium:8.1.3.1 Combine the following components in the corre-sponding
36、proportions (v/v):Component Proportion (% v/v) Example: 500 mL (mL)DME Low Bicarb 85.5 427.5Hi FBS 10.0 50.0L-Glutamine (200 mM) 4.0 20.0Gentamicin 0.5 2.58.1.3.2 Filter through a 0.2 m filter and store at 2 to 8C ina sterile container.8.1.4 Assay Medium:8.1.4.1 Combine the following components in t
37、he corre-sponding proportions (v/v):Component Proportion (% v/v) Example: 1000 mL (mL)DME Low Bicarb 87.0 870.0Hi FBS 10.0 100.0L-Glutamine (200 mM) 2.0 20.0Penicillin/streptomycin 1.0 10.08.1.4.2 Filter through a 0.2 m filter and store at 2 to 8C ina sterile container.8.1.5 NaCl, 0.9 % w/v:8.1.5.1
38、Dissolve 9 g NaCl in approximately 800 mL ofpurified water and bring to a final volume of 1 L with purifiedwater.8.1.5.2 Filter through a 0.2 m filter and store in a sterilecontainer at room temperature.8.1.6 12.5 % Triton X-100:8.1.6.1 Mix 12.5 mL Triton X-100 with 87.5 mL of 0.9 %NaCl.8.1.6.2 Filt
39、er through a 0.2 m filter and store in a sterilizedcontainer at room temperature.8.1.7 Freezing Medium:8.1.7.1 Prepare freezing medium immediately before thefreezing procedure by adding DMSO to growth medium (see9.1.3) to 20 % v/v.Component Proportion (% v/v) Example: 100 mLGrowth Medium 80 80 mLDMS
40、O 20 20 mL8.1.8 Glycine Buffer:8.1.8.1 Dissolve 0.75 % (w/v) glycine in required volume ofpurified water.Adjust the pH of the solution to 10.3 6 0.1 with10 N NaOH.8.1.8.2 Add 0.8 % (v/v) of 12.5 % Triton X-100.8.1.8.3 Add 0.13 % (w/v) MgCl26H2O and mix well.Component Example: 1000 mLGlycine 7.5 gMgC
41、l26H2O 1.3 g12.5 % Triton X-100 8.0 mLWater To 1000 mL8.1.8.4 Filter through a 0.2 m filter and store in a sterilecontainer at room temperature. The solution has a one monthexpiration.8.1.9 Assay Mix:8.1.9.1 Take a sufficient volume of the glycine buffer tocover developing needs (that is, 5 mL glyci
42、ne buffer per plate).8.1.9.2 Add 0.34 % (w/v) p-nitrophenol phosphate withinone (1) h of use and mix well.NOTE 2The assay mix must be made on day of use.Component Example: 50 mL for 10 platesGlycine buffer 50 mLPNPP substrate 170 mg8.2 Cell Line Storage and Cell Banking Procedure:8.2.1 Store the cel
43、ls in 1 mL aliquots in 2 mL cryovials at 53 105cells/mL in freezing medium (see 8.1.7).8.2.2 Prepare cells to make a working cell bank (100+vials).8.2.3 Thaw the vial of W-20-17 cells obtained from ATCCor other source following the procedure described in 8.3.8.2.4 In order to obtain the expected cel
44、l number, subculturethe cells by expanding them through one or two additionalpassages (repeat steps in 8.3).NOTE 3The viability should be in the range $80 %.8.2.5 Determine the number of vials to be made based ontotal cell number obtained following procedure 8.2.2. Label theappropriate number of cry
45、ovials as follows:Cell Line Name WCBPassage NumberFreezing DatePreparation Reference NumberInitials8.2.6 Decap the cryovials in the biosafety cabinet.8.2.7 Dilute the cell suspension to one half the appropriatevolume with 2 to 8C cold freezing medium without DMSO.Volume should be one half of the app
46、ropriate volume for thedesired cell suspension for freezing. The second half of thecold freezing medium should be made with culture medium(see 8.1.7, 20 % DMSO). The final DMSO concentration mustbe 10 %.8.2.8 Slowly add the half-volume of culture medium with20 % DMSO to the other half of the volume
47、of the cellsuspension.8.2.9 Using a sterile pipet, transfer 1 mL of cell suspensionto each of the labeled cryovials on ice. Repeat until all vials arefilled. Gently mix the cell suspension during the filling processto prevent settling of the cells.F 2131 02 (2007)e13NOTE 4The period of time from the
48、 addition of the DMSO containingmedium to the start of the freezing process should not exceed 45 to 60min.8.2.10 Transfer the cryovials to an insulated box or rack.Store the box or rack at 80C for 20 to 24 h.8.2.11 After 20 to 24 h, transfer the vials to a liquid nitrogendewar or freezer.8.2.12 Perf
49、orm test thaws to check the viability and assayperformance of cells.NOTE 5It is recommended to perform mycoplasma and sterilitytesting on the new bank.8.3 Preparation of Cells for the Assay:8.3.1 Two vials of cells (W-20-17, stored in liquid nitrogen)are quick thawed in a 37 6 2C waterbath. The contents of thevials are combined in a 15-mL conical tube and mixedthoroughly. The total volume is recorded.8.3.2 An aliquot of the undiluted cell suspension is takenand used
