ASTM F2131-02
(Test Method)Standard Test Method forIn Vitro Biological Activity of Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2) Using the W-20 Mouse Stromal Cell Line
Standard Test Method for<bdit>In Vitro</bdit> Biological Activity of Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2) Using the W-20 Mouse Stromal Cell Line
SCOPE
1.1 This test method describes the method used and the calculation of results for the determination of the in vitro biological activity of rhBMP-2 using the mouse stromal cell line W-20 clone 17 (W-20-17). This clone was derived from bone marrow stromal cells of the W++ mouse strain.
1.2 This test method (assay) has been qualified and validated based upon the International Committee on Harmonization assay validation guidelines (with the exception of interlaboratory precision) for the assessment of the biological activity of rhBMP-2. The relevance of this in vitro test method to in vivo bone formation has also been studied. The measured response in the W-20 bioassay, alkaline phosphatase induction, has been correlated with the ectopic bone-forming capacity of rhBMP-2 in the in vivo Use Test (UT). rhBMP-2 that was partially or fully inactivated by targeted peracetic acid oxidation of the two methionines was used as a tool to compare the activities. Oxidation of rhBMP-2 with peracetic acid was shown to be specifically targeted to the methionines by peptide mapping and mass spectrometry. These methionines reside in a hydrophobic receptor binding pocket on rhBMP-2. Oxidized samples were compared alongside an incubation control and a native control. The 62, 87, 98, and 100 % oxidized samples had W-20 activity levels of 62, 20, 7, and 5 %, respectively. The incubation and native control samples maintained 100 % activity. Samples were evaluated in the UT and showed a similar effect of inactivation on bone-forming activity. The samples with 62 % and 20 % activity in the W-20 assay demonstrated reduced levels of bone formation, similar in level with the reduction in W-20 specific activity, relative to the incubation control. 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 the receptor binding site decrease the activity in both the W-20 and UT assays. These data suggest that a single receptor binding domain on rhBMP-2 is responsible for both in vitro and in vivo activity and that the W-20 bioassay is a relevant predictor of the bone-forming activity of rhBMP-2.
1.4 The values stated in SI units are to be regarded as standard.
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
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Designation:F2131–02
Standard Test Method for
In Vitro Biological Activity of Recombinant Human Bone
Morphogenetic Protein-2 (rhBMP-2) Using the W-20 Mouse
Stromal Cell Line
This standard is issued under the fixed designation F2131; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
1. Scope control. Little or no ectopic bone was formed in the 7 and 5%
active rhBMP-2 implants.
1.1 This test method describes the method used and the
1.3 Thus, modifications to the rhBMP-2 molecule in the
calculation of results for the determination of the in vitro
receptorbindingsitedecreasetheactivityinboththeW-20and
biological activity of rhBMP-2 using the mouse stromal cell
UT assays. These data suggest that a single receptor binding
line W-20 clone 17 (W-20-17). This clone was derived from
2 domainonrhBMP-2isresponsibleforbothinvitroandinvivo
bone marrow stromal cells of the W++ mouse strain.
activity and that the W-20 bioassay is a relevant predictor of
1.2 This test method (assay) has been qualified and vali-
the bone-forming activity of rhBMP-2.
dated based upon the International Committee on Harmoniza-
1.4 The values stated in SI units are to be regarded as
tion assay validation guidelines (with the exception of inter-
standard.
laboratory precision) for the assessment of the biological
1.5 This standard does not purport to address all of the
activity of rhBMP-2.The relevance of this in vitro test method
safety concerns, if any, associated with its use. It is the
to in vivo bone formation has also been studied. The measured
responsibility of the user of this standard to establish appro-
responseintheW-20bioassay,alkalinephosphataseinduction,
priate safety and health practices and determine the applica-
has been correlated with the ectopic bone-forming capacity of
bility of regulatory limitations prior to use.
rhBMP-2 in the in vivo Use Test (UT). rhBMP-2 that was
partially or fully inactivated by targeted peracetic acid oxida-
2. Terminology
tion of the two methionines was used as a tool to compare the
2.1 rhBMP—recombinant human bone morphogenetic pro-
activities. Oxidation of rhBMP-2 with peracetic acid was
tein.
showntobespecificallytargetedtothemethioninesbypeptide
2.2 GDF—growth and differentiation factor.
mappingandmassspectrometry.Thesemethioninesresideina
hydrophobic receptor binding pocket on rhBMP-2. Oxidized
3. Summary of Test Method
samples were compared alongside an incubation control and a
3.1 Inthistestmethod,themousestromalcelllineW-20-17
nativecontrol.The62,87,98,and100%oxidizedsampleshad
is used as a target cell line for rhBMP-2. The W-20-17 cells
W-20 activity levels of 62, 20, 7, and 5%, respectively. The
exhibit increased alkaline phosphatase activity in response to
incubation and native control samples maintained 100% ac-
rhBMP-2. Optical density at 405 nm of the p-nitrophenol
tivity. Samples were evaluated in the UTand showed a similar
generated from the alkaline phosphatase substrate is used as a
effect of inactivation on bone-forming activity. The samples
measure of alkaline phosphatase enzyme level. The test
with 62% and 20% activity in the W-20 assay demonstrated
method is performed in a 96-well plate format. A similar test
reduced levels of bone formation, similar in level with the
methodbaseduponthesamecelllinehasbeendevelopedusing
reduction in W-20 specific activity, relative to the incubation
chemiluminescent detection of alkaline phosphatase.
4. Significance and Use
ThistestmethodisunderthejurisdictionofASTMCommitteeF04onMedical
andSurgicalMaterialsandDevicesandisthedirectresponsibilityofSubcommittee
4.1 Although the test method can be used for assessment of
F04.42 on Biomaterials and Biomolecules for TEMPs.
the bioactivity of crude preparations of rhBMP-2, it has only
Current edition approved Sept. 10, 2002. Published December 2002.
Thies, R. S., Bauduy, M., Ashton, B. A., Kurtzberg, L., Wozney, J.M., and
Rosen, V., “Recombinant Human Bone Morphogenetic Protein-2 Induces Osteo-
blastic Differentiation in W-20-17 Stromal Cells,” Endocrinology, 130, 1992, pp.
1318-1324. Blum, R. S., Li, R. H., Mikos,A.G., and Barry, M.A., “An Optimized Method
Guideline for Industry, ICH-Q2AText on Validation ofAnalytical Procedures, for the Chemiluminescent Detection of Alkaline Phosphatase Levels During
November 1996, International Committee on Harmonization, March 1995, http:// Osteodifferentiation by Bone Morphogenetic Protein 2,” Jour. Cellular Biochem.,
www.fda.gov/cder/guidance/index/htm. 80, 2001, pp. 532-537.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
F2131–02
NOTE 1—Each new lot of fetal bovine serum must be evaluated in the
been validated for use with highly pure (>98% by weight
assay before use.
protein purity) preparations of rhBMP-2.
7.6 200 mM L-Glutamine (Invitrogen Life Technologies,
5. Interferences
25030081 or equivalent).
5.1 There have been no systematic studies of interfering 7.7 Gentamicin Gibco sterile filtered: 10 mg/mL or equiva-
substances for this test method. There is anecdotal evidence lent.
that trypsin and some rhBMP-2 formulation buffers can inter- 7.8 Penicillin Streptomycin (PS), contains 10 000 units of
fere with the assay. Additionally, the source of fetal bovine penicillin(base)/mLand10000µgofstreptomycin(base)/mL,
serum is an important variable. Each lot should be tested in all utilizing penicillin G (sodium salt) and streptomycin sulfate in
parts of the assay where it is required to determine the 0.85 % saline (Invitrogen Life Technologies, #15140122 or
appropriateness of the lot. This is particularly needed if fetal equivalent).
bovine serum vendor is changed. 7.9 Phosphate Buffered Saline, Calcium and Magnesium
Free, 1x (PBS-CMF), (Invitrogen Life Technologies (cat.
6. Apparatus
#20012050 or equivalent).
7.10 Dimethyl sulfoxide (DMSO), cell culture grade
6.1 Polypropylene conical tubes, 15 mL and 50 mL.
(Sigma-Aldrich or equivalent).
6.2 Cryovials (Corning or equivalent), sterile 2 mL.
7.11 Trypsin-EDTA(0.05%trypsin,0.53mMEDTA·4Na)
6.3 Eppendorf vials, sterilized.
(1X), liquid (Invitrogen Life Technologies 25300054 or
6.4 Variablepipets,(range20to1000µL)andMultichannel
equivalent).
pipets (range 50 to 300 µL).
7.12 Glycine (Sigma —Aldrich or equivalent).
6.5 Biosafety cabinet.
7.13 Sodium Hydroxide (NaOH) 0.2 N and 10 N.
6.6 96 Well flat bottom sterile tissue culture microtiter
7.14 Triton X-100 (J.T. Baker Cat. No. X198-05 or equiva-
plates, (Falcon 3072 or equivalent).
lent).
6.7 IEC Centra-7R Centrifuge, or equivalent.
7.15 Magnesium Chloride, Crystalline (MgCl ·6H O).
6.8 CO humidified tissue culture incubator.
2 2
7.16 p-Nitrophenol phosphate (PNPP, Sigma—Aldrich
6.9 Spectrophotometric microplate reader, (VMAX/
104(R) phosphatase substrate, product # 1040 or equivalent).
Spectramax, Molecular Devices, or equivalent).
7.17 NaCl.
6.10 Hemacytometer, or automatic cell counter.
7.18 Purified water.
6.11 Inverted microscope.
7.19 rhBMP-2, 1st WHO Reference Reagent 1997 (5000
6.12 Tissue culture flasks, Falcon T175 or equivalent.
Units per ampoule, cat. # 93/574, National Institute for
6.13 Sterilized paper towels, or equivalent.
Biological Standards and Control).
6.14 Sterile filter units, (0.2 µm).
7.20 rhBMP-2 internal control, >1 mg/mL (stored at
6.15 Sterile pipets, (1 mL, 5 mL, 10 mL, 25 mL, 50 mL).
−80°C).
6.16 9 in. Pasteur pipets, sterilized.
6.17 Sterilized pipet tips, (1-300 µL and 200-1000 µL).
8. Procedure
6.18 Sterile reagent reservoirs.
6.19 −80°C freezer.
8.1 Solution Preparation:
6.20 96 Well U-Bottom polypropylene sterile tissue culture
8.1.1 DME Low Bicarb:
microtiter plates, (Costar 3790 or equivalent).
8.1.1.1 Dissolve 66.87 g DME/High and 11.13 g sodium
6.21 Water bath.
bicarbonate in 4.5 L of purified water.
6.22 Orbital shaker.
8.1.1.2 Adjust pH to 7.3 6 0.10 with 5 M HCl and bring
solution to 5 L with purified water.
7. Reagents and Materials
8.1.1.3 Filter through a 0.2 µm filter into sterile bottles.
7.1 W-20-17 Mouse Stromal Cells. 8.1.1.4 Store at 2 to 8°C, expires in 8 weeks.
7.2 Dulbecco’s modified Eagle’s medium with 4500 mg/L 8.1.2 Hi FBS:
glucose and 4.0 mM L-glutamine, without sodium bicarbonate 8.1.2.1 Thaw the desired amount of FBS at ambient tem-
(DME/High, JRH Biosciences, 56439 or equivalent). perature, or 2 to 8°C.
7.3 Sodium bicarbonate (Sigma—Aldrich S4019 or equiva- 8.1.2.2 Adjust the waterbath to a temperature of 56 6 2°C.
lent). 8.1.2.3 PlacethebottleofFBSintothewaterbathsothatthe
7.4 5 M hydrochloric acid. entire contents of the bottle are immersed in water.
7.5 Heat inactivated (Hi) fetal bovine serum (FBS).
Thismaterialhasbeenfoundsatisfactoryforthispurposeandisavailablefrom
This cell line has been deposited in mid-2001 at the American Type Culture Invitrogen Life Technologies, 1600 Faraday Ave., P.O. Box 6482, Carlsbad, CA
Collection, 10801 University Blvd., Manassas, VA 20110-2209, U.S., http:// 92008, U.S., http://www.invitrogen.com.
www.atcc.org. Thismaterialhasbeenfoundsatisfactoryforthispurposeandisavailablefrom
Thismediumhasbeenfoundsatisfactoryforthispurposeandisavailablefrom J.T.Baker,(MallinckrodtBaker,Inc.),222RedSchoolLn.,Phillipsburg,NJ08865,
JRHBiosciences,P.O.Box14848,Lenexa,KS66215,U.S.,http://www.jrhbio.com. U.S., http://www.jtbaker.com.
7 10
Thismaterialhasbeenfoundsatisfactoryforthispurposeandisavailablefrom Thismaterialhasbeenfoundsatisfactoryforthispurposeandisavailablefrom
Sigma-Aldrich Corp., 3050 Spruce St., St. Louis, MO 63103, U.S., http:// the National Institute for Biological Standards and Control (NIBSC), Blanche Ln.,
www.sigmaaldrich.com. South Mimms, Potters Bar, Herts, EN6 3QG, U.K., http://www.nibsc.ac.uk.
F2131–02
8.1.2.4 Heat the bottle for 45 min, swirling periodically. 8.1.9.1 Take a sufficient volume of the glycine buffer to
coverdevelopingneeds(thatis,5mLglycinebufferperplate).
8.1.2.5 Remove the bottle from the waterbath, allow to cool
8.1.9.2 Add 0.34% (w/v) p-nitrophenol phosphate within
to room temperature. Aliquot 50 mL of the FBS in sterile 50
one (1) h of use and mix well.
mL conical tubes.
8.1.2.6 Label each container with name, lot number, expi-
NOTE 2—Assay mix must be made on day of use.
rationdate,andtheheatinactivationdate.Storeat−20 610°C
Component Example: 50 mL for 10 plates
or 2 to 8°C.
Glycine buffer 50 mL
PNPP substrate 170 mg
8.1.3 Growth Medium:
8.2 Cell Line Storage and Cell Banking Procedure:
8.1.3.1 Combine the following components in the corre-
8.2.1 Store the cells in 1 mLaliquots in 2 mLcryovials at 5
sponding proportions (v/v):
3 10 cells/mL in freezing medium (see 8.1.7).
Component Proportion (% v/v) Example: 500 mL (mL)
DME Low Bicarb 85.5 427.5 8.2.2 Prepare cells to make a working cell bank (100+
Hi FBS 10.0 50.0
vials).
L-Glutamine (200 mM) 4.0 20.0
8.2.3 Thaw the vial of W-20-17 cells obtained from ATCC
Gentamicin 0.5 2.5
or other source following the procedure described in 8.3.
8.1.3.2 Filter through a 0.2 µm filter and store at 2 to 8°C in
8.2.4 Inordertoobtaintheexpectedcellnumber,subculture
a sterile container.
the cells by expanding them through one or two additional
8.1.4 Assay Medium:
passages (repeat steps in 8.3).
8.1.4.1 Combine the following components in the corre-
NOTE 3—The viability should be in the range$80%.
sponding proportions (v/v):
Component Proportion (% v/v) Example: 1000 mL (mL) 8.2.5 Determine the number of vials to be made based on
DME Low Bicarb 87.0 870.0
totalcellnumberobtainedfollowingprocedure8.2.2.Labelthe
Hi FBS 10.0 100.0
appropriate number of cryovials as follows:
L-Glutamine (200 mM) 2.0 20.0
Penicillin/streptomycin 1.0 10.0
Cell Line Name WCB
Passage Number
8.1.4.2 Filter through a 0.2 µm filter and store at 2 to 8°C in
Freezing Date
Preparation Reference Number
a sterile container.
Initials
8.1.5 NaCl, 0.9% w/v:
8.2.6 Decap the cryovials in the biosafety cabinet.
8.1.5.1 Dissolve 9 g NaCl in approximately 800 mL of
8.2.7 Dilute the cell suspension to one half the appropriate
purified water and bring to a final volume of 1 Lwith purified
volume with 2 to 8°C cold freezing medium without DMSO.
water.
Volume should be one half of the appropriate volume for the
8.1.5.2 Filter through a 0.2 µm filter and store in a sterile
desired cell suspension for freezing. The second half of the
container at room temperature.
cold freezing medium should be made with culture medium
8.1.6 12.5% Triton X-100:
(see 8.1.7, 20% DMSO, the final DMSO concentration must
8.1.6.1 Mix 12.5 mL Triton X-100 with 87.5 mL of 0.9%
be 10%).
NaCl.
8.2.8 Slowly add the half-volume of culture medium with
8.1.6.2 Filter through a 0.2 µm filter and store in a sterilized
20% DMSO to the other half of the volume of the cell
container at room temperature.
suspension.
8.1.7 Freezing Medium:
8.2.9 Using a sterile pipet, transfer 1 mL of cell suspension
8.1.7.1 Prepare freezing medium immediately before freez-
toeachofthelabeledcryovialsonice.Repeatuntilallvialsare
ing procedure by adding DMSO to growth medium (see 9.1.3)
filled. Gently mix the cell suspension during the filling process
to 20% v/v.
to prevent settling of the cells.
Component Proportion (% v/v) Example: 100 mL
Growth Medium 80 80 mL NOTE 4—TheperiodoftimefromtheadditionoftheDMSOcontaining
DMSO 20 20 mL
medium to the start of freezing process should not exceed 45 to 60 min.
8.1.8 Glycine Buffer:
8.2.10 Transfer the cryovials to an insulated box or rack.
8.1.8.1 Dissolve0.75%(w/v)glycineinrequiredvolumeof
Store the box or rack at −80°C for 20 to 24 h.
purifiedwater.AdjustthepHofthesolutionto10.3 60.1with 8.2.11 After20to24h,transferthevialstoaliquidnitrogen
10 N NaOH.
dewar or freezer.
8.2.12 Perform test thaws to check the viability and assay
8.1.8.2 Add 0.8% (v/v) of 12.5% Triton X-100.
performance of cells.
8.1.8.3 Add 0.13% (w/v) MgCl·6H O and mix well.
2 2
Component Example: 1000 mL
NOTE 5—It is recommended to perform mycoplasma and sterility
Glycine 7.5 g
testing on the new bank.
MgCl·6H O 1.3 g
2 2
12.5 % Triton X-100 8.0 mL
8.3 Preparation of Cells for the Assay:
Water To 1000 mL
8.3.1 Two vials of cells (W-20-17, stored in liquid nitrogen)
8.1.8.4 Filter through a 0.2 µm filter and store in a sterile
are quick thawed in a 37 6 2°C waterbath.The contents of the
container at room temperature. One month expiration.
vials are combined in a 15 mL conical tube and mixed
8.1.9 Assay Mix: thoroughly. The total volume is recorded.
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