1、Designation: F2131 02 (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 ind
2、icates 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 use
3、d 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 ba
4、sed 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 measuredrespon
5、se 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 theacti
6、vities. 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 con
7、trol.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 %
8、 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 mole
9、cule 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
10、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-pr
11、iate 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-20-17
12、is 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
13、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 respo
14、nsibility 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., Kurtzber
15、g, 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 Comm
16、ittee 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 Bioch
17、em,Vol 80, 2001, pp. 532537.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States14. 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 f
18、or 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 buffers can inter-fere with the assay. Addition
19、ally, 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. Apparatus6.1 Polypropylene conical tubes, 15 mL
20、 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 microtiterplates, (Falcon 3072 or equivalent).6.7 I
21、EC 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 Tissue culture flasks, Falcon T175 or equivalent
22、.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 reservoirs.6.19 80C freezer.6.20 96 Well U-Bottom pol
23、ypropylene 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, without sodium bicarbonate(DME/High, JRH Bi
24、osciences, 56439 or equivalent).7.3 Sodium bicarbonate (SigmaAldrich S4019 or equiva-lent).7.4 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.7.6 200 mM L-Glutamine (Invitrogen Life Technologi
25、es,25030081 or equivalent).7.7 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 streptomycin sulfate in0.85 % saline (Invitrogen Li
26、fe Technologies, #15140122 orequivalent).7.9 Phosphate Buffered Saline, Calcium and MagnesiumFree, 1x (PBS-CMF), (Invitrogen Life Technologies (cat.#20012050 or equivalent).7.10 Dimethyl sulfoxide (DMSO), cell culture grade(Sigma-Aldrich or equivalent).7.11 Trypsin-EDTA(0.05 % trypsin, 0.53 mM EDTA
27、4Na)(1X), liquid (Invitrogen Life Technologies 25300054 orequivalent).7.12 Glycine (Sigma Aldrich or equivalent).7.13 Sodium Hydroxide (NaOH) 0.2 N and 10 N.7.14 Triton X-100 (J.T. Baker Cat. No. X198-05 or equiva-lent).7.15 Magnesium Chloride, Crystalline (MgCl26H2O).7.16 p-Nitrophenol phosphate (P
28、NPP, SigmaAldrich104(R) phosphatase substrate, product # 1040 or equivalent).7.17 NaCl.7.18 Purified water.5This cell line has been deposited in mid-2001. The sole source of supply of theapparatus known to the committee at this time isAmerican Type Culture Collection,10801 University Blvd., Manassas
29、, VA 20110-2209, U.S., http:/www.atcc.org Ifyou are aware of alternative suppliers, please provide this information to ASTMInternational Headquarters. Your comments will receive careful consideration at ameeting of the responsible technical committee,1which you may attend.F2131 02 (2012)27.19 rhBMP-
30、2, 1st WHO Reference Reagent 1997 (5000Units per ampoule, cat. # 93/574, National Institute forBiological Standards and Control).67.20 rhBMP-2 internal control, 1 mg/mL (stored at80C).8. Procedure8.1 Solution Preparation:8.1.1 DME Low Bicarb:8.1.1.1 Dissolve 66.87 g DME/High and 11.13 g sodiumbicarb
31、onate in 4.5 L 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 a
32、mbienttemperature, 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 wate
33、r bath and allow 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 co
34、rre-sponding 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 co
35、mponents in the 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 %
36、 w/v:8.1.5.1 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
37、.8.1.6.2 Filter 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 Mediu
38、m 80 80 mLDMSO 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 mLGly
39、cine 7.5 gMgCl26H2O 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 i
40、s, 5 mL glycine 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
41、Store the cells in 1 mL aliquots in 2 mL cryovials at 5105cells/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 Ameri-can Type Culture Collection (ATCC) or other source followingthe procedure described i
42、n 8.3.8.2.4 In order to obtain the expected cell 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 proce
43、dure 8.2.2. Label theappropriate number of cryovials 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 wit
44、hout DMSO.The volume should be one half of the appropriate volume for6The sole source of supply of the material known to the committee at this timeis National Institute for Biological Standards and Control (NIBSC), Blanche Ln.,South Mimms, Potters Bar, Herts, EN6 3QG, U.K., http:/www.nibsc.ac.uk. .
45、If youare aware of alternative suppliers, please provide this information to ASTMInternational Headquarters. Your comments will receive careful consideration at ameeting of the responsible technical committee,1which you may attend.F2131 02 (2012)3the desired cell suspension for freezing. The second
46、half of thecold freezing medium should be made with culture medium(see 8.1.7, 20 % DMSO). The final DMSO concentration shallbe 10 %.8.2.8 Slowly add the half-volume of culture medium with20 % DMSO to the other half of the volume of the cellsuspension.8.2.9 Using a sterile pipet, transfer 1 mL of cel
47、l 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.NOTE 4The period of time from the addition of the DMSO-containingmedium to the start of the freezing process should not exc
48、eed 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 Perform test thaws to check the viability and assayperformance of cells.NOTE 5It is recommende
49、d 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 to makea1in2dilution in trypan blue (that is, 50 Lcells + 50 L trypan blue). Add 10 mL growth medium to theremaining cells. Live cells (that is, cells no
