ASTM F2131-02(2012)
(Test Method)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
Standard Test Method for <emph type="bdit">In Vitro</emph> Biological Activity of Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2) Using the W-20 Mouse Stromal Cell Line
SIGNIFICANCE AND USE
4.1 Although the test method can be used for assessment of the bioactivity of crude preparations of rhBMP-2, it has only been validated for use with highly pure (>98 % by weight protein purity) preparations of rhBMP-2.
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.2
1.2 This test method (assay) has been qualified and validated based upon the International Committee on Harmonization assay validation guidelines3 (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. No other units of measurement are included in this 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.
General Information
Standards Content (Sample)
NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: F2131 − 02 (Reapproved 2012)
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 (´) indicates an editorial change since the last revision or reapproval.
1. Scope reduction in W-20 specific activity, relative to the incubation
control. Little or no ectopic bone was formed in the 7 and 5%
1.1 This test method describes the method used and the
active rhBMP-2 implants.
calculation of results for the determination of the in-vitro
biological activity of rhBMP-2 using the mouse stromal cell
1.3 Thus, modifications to the rhBMP-2 molecule in the
line W-20 clone 17 (W-20-17). This clone was derived from
receptorbindingsitedecreasetheactivityinboththeW-20and
bone marrow stromal cells of the W++ mouse strain.
UT assays. These data suggest that a single receptor binding
domainonrhBMP-2isresponsibleforboth in-vitroand in-vivo
1.2 This test method (assay) has been qualified and vali-
activity and that the W-20 bioassay is a relevant predictor of
dated based upon the International Committee on Harmoniza-
the bone-forming activity of rhBMP-2.
tion assay validation guidelines (with the exception of inter-
laboratory precision) for the assessment of the biological
1.4 The values stated in SI units are to be regarded as
activity of rhBMP-2.The relevance of this in vitro test method
standard. No other units of measurement are included in this
to in vivo bone formation has also been studied.The measured
standard.
responseintheW-20bioassay,alkalinephosphataseinduction,
1.5 This standard does not purport to address all of the
has been correlated with the ectopic bone-forming capacity of
safety concerns, if any, associated with its use. It is the
rhBMP-2 in the in vivo Use Test (UT). rhBMP-2 that was
responsibility of the user of this standard to establish appro-
partially or fully inactivated by targeted peracetic acid oxida-
priate safety and health practices and determine the applica-
tion of the two methionines was used as a tool to compare the
bility of regulatory limitations prior to use.
activities. Oxidation of rhBMP-2 with peracetic acid was
showntobespecificallytargetedtothemethioninesbypeptide
2. Terminology
mappingandmassspectrometry.Thesemethioninesresideina
hydrophobic receptor binding pocket on rhBMP-2. Oxidized
2.1 rhBMP—recombinant human bone morphogenetic pro-
samples were compared alongside an incubation control and a
tein.
nativecontrol.The62,87,98,and100%oxidizedsampleshad
2.2 GDF—growth and differentiation factor.
W-20 activity levels of 62, 20, 7, and 5%, respectively. The
incubation and native control samples maintained 100% ac-
3. Summary of Test Method
tivity. Samples were evaluated in the UTand showed a similar
effect of inactivation on bone-forming activity. The samples
3.1 Inthistestmethod,themousestromalcelllineW-20-17
with 62% and 20% activity in the W-20 assay demonstrated
is used as a target cell line for rhBMP-2. The W-20-17 cells
reduced levels of bone formation, similar in level with the
exhibit increased alkaline phosphatase activity in response to
rhBMP-2. Optical density at 405 nm of the p-nitrophenol
generated from the alkaline phosphatase substrate is used as a
ThistestmethodisunderthejurisdictionofASTMCommitteeF04onMedical
measure of alkaline phosphatase enzyme level. The test
andSurgicalMaterialsandDevicesandisthedirectresponsibilityofSubcommittee
F04.42 on Biomaterials and Biomolecules for TEMPs.
method is performed in a 96-well plate format. A similar test
Current edition approved Oct. 1, 2012. Published October 2012. Originally
methodbaseduponthesamecelllinehasbeendevelopedusing
ε1
approved in 2002. Last previous edition approved in 2007 as F2131–02 (2007) .
chemiluminescent detection of alkaline phosphatase.
DOI: 10.1520/F2131-02R12.
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, Vol 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. Vol 80, 2001, pp. 532–537.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F2131 − 02 (2012)
4. Significance and Use 7. Reagents and Materials
4.1 Although the test method can be used for assessment of
7.1 W-20-17 Mouse Stromal Cells.
the bioactivity of crude preparations of rhBMP-2, it has only
7.2 Dulbecco’s modified Eagle’s medium with 4500 mg/L
been validated for use with highly pure (>98% by weight
glucose and 4.0 mM L-glutamine, without sodium bicarbonate
protein purity) preparations of rhBMP-2.
(DME/High, JRH Biosciences, 56439 or equivalent).
5. Interferences
7.3 Sodium bicarbonate (Sigma—Aldrich S4019 or equiva-
lent).
5.1 There have been no systematic studies of interfering
substances for this test method. There is anecdotal evidence
7.4 5 M hydrochloric acid.
that trypsin and some rhBMP-2 formulation buffers can inter-
7.5 Heat inactivated (Hi) fetal bovine serum (FBS).
fere with the assay. Additionally, the source of fetal bovine
NOTE 1—Each new lot of fetal bovine serum must be evaluated in the
serum is an important variable. Each lot should be tested in all
assay before use.
parts of the assay where it is required to determine the
appropriatenessofthelot.Thisisparticularlyimportantiffetal
7.6 200 mM L-Glutamine (Invitrogen Life Technologies,
bovine serum vendor is changed.
25030081 or equivalent).
7.7 Gentamicin Gibco sterile filtered: 10 mg/mL or equiva-
6. Apparatus
lent.
6.1 Polypropylene conical tubes, 15 mL and 50 mL.
7.8 Penicillin Streptomycin (PS), contains 10 000 units of
6.2 Cryovials (Corning or equivalent), sterile 2 mL.
penicillin(base)/mLand10000µgofstreptomycin(base)/mL,
6.3 Eppendorf vials, sterilized. utilizing penicillin G (sodium salt) and streptomycin sulfate in
0.85% saline (Invitrogen Life Technologies, #15140122 or
6.4 Variable pipets,(range20to1000µL)and Multichannel
equivalent).
pipets (range 50 to 300 µL).
7.9 Phosphate Buffered Saline, Calcium and Magnesium
6.5 Biosafety cabinet.
Free, 1x (PBS-CMF), (Invitrogen Life Technologies (cat.
6.6 96 Well flat bottom sterile tissue culture microtiter
#20012050 or equivalent).
plates, (Falcon 3072 or equivalent).
7.10 Dimethyl sulfoxide (DMSO), cell culture grade
6.7 IEC Centra-7R Centrifuge, or equivalent.
(Sigma-Aldrich or equivalent).
6.8 CO humidified tissue culture incubator.
7.11 Trypsin-EDTA(0.05%trypsin,0.53mMEDTA·4Na)
(1X), liquid (Invitrogen Life Technologies 25300054 or
6.9 Spectrophotometric microplate reader, (VMAX/
equivalent).
Spectramax, Molecular Devices, or equivalent).
6.10 Hemacytometer, or automatic cell counter. 7.12 Glycine (Sigma —Aldrich or equivalent).
6.11 Inverted microscope.
7.13 Sodium Hydroxide (NaOH) 0.2 N and 10 N.
6.12 Tissue culture flasks, Falcon T175 or equivalent.
7.14 Triton X-100 (J.T. Baker Cat. No. X198-05 or equiva-
lent).
6.13 Sterilized paper towels, or equivalent.
7.15 Magnesium Chloride, Crystalline (MgCl ·6H O).
2 2
6.14 Sterile filter units, (0.2 µm).
7.16 p-Nitrophenol phosphate (PNPP, Sigma—Aldrich
6.15 Sterile pipets, (1 mL, 5 mL, 10 mL, 25 mL, 50 mL).
104(R) phosphatase substrate, product # 1040 or equivalent).
6.16 9 in. Pasteur pipets, sterilized.
7.17 NaCl.
6.17 Sterilized pipet tips, (1-300 µL and 200-1000 µL).
7.18 Purified water.
6.18 Sterile reagent reservoirs.
6.19 −80°C freezer.
6.20 96 Well U-Bottom polypropylene sterile tissue culture This cell line has been deposited in mid-2001.The sole source of supply of the
apparatusknowntothecommitteeatthistimeisAmericanTypeCultureCollection,
microtiter plates, (Costar 3790 or equivalent).
10801 University Blvd., Manassas, VA 20110-2209, U.S., http://www.atcc.org. If
you are aware of alternative suppliers, please provide this information to ASTM
6.21 Water bath.
International Headquarters. Your comments will receive careful consideration at a
6.22 Orbital shaker. meeting of the responsible technical committee, which you may attend.
F2131 − 02 (2012)
7.19 rhBMP-2, 1st WHO Reference Reagent 1997 (5000 8.1.6 12.5 % Triton X-100:
Units per ampoule, cat. # 93/574, National Institute for 8.1.6.1 Mix 12.5 mL Triton X-100 with 87.5 mL of 0.9%
Biological Standards and Control). NaCl.
8.1.6.2 Filter through a 0.2 µm filter and store in a sterilized
7.20 rhBMP-2 internal control, >1 mg/mL (stored at
container at room temperature.
−80°C).
8.1.7 Freezing Medium:
8. Procedure
8.1.7.1 Prepare freezing medium immediately before the
freezing procedure by adding DMSO to growth medium (see
8.1 Solution Preparation:
9.1.3) to 20% v/v.
8.1.1 DME Low Bicarb:
8.1.1.1 Dissolve 66.87 g DME/High and 11.13 g sodium Component Proportion (% v/v) Example: 100 mL
Growth Medium 80 80 mL
bicarbonate in 4.5 L of purified water.
DMSO 20 20 mL
8.1.1.2 AdjustthepHto7.3 60.10with5MHClandbring
8.1.8 Glycine Buffer:
solution to 5 L with purified water.
8.1.8.1 Dissolve0.75%(w/v)glycineinrequiredvolumeof
8.1.1.3 Filter through a 0.2 µm filter into sterile bottles.
purifiedwater.AdjustthepHofthesolutionto10.3 60.1with
8.1.1.4 Store at 2 to 8°C. The solution expires in 8 weeks.
10 N NaOH.
8.1.2 Hi FBS:
8.1.8.2 Add 0.8 %(v⁄v) of 12.5% Triton X-100.
8.1.2.1 Thaw the desired amount of FBS at ambient
8.1.8.3 Add 0.13% (w/v) MgCl ·6H O and mix well.
temperature, or 2 to 8°C. 2 2
8.1.2.2 Adjust the water bath to a temperature of 56 6 2°C. Component Example: 1000 mL
Glycine 7.5 g
8.1.2.3 Place the bottle of FBS into the water bath so that
MgCl ·6H O 1.3 g
2 2
the entire contents of the bottle are immersed in water.
12.5 % Triton X-100 8.0 mL
Water To 1000 mL
8.1.2.4 Heat the bottle for 45 min, swirling periodically.
8.1.2.5 Remove the bottle from the water bath and allow to
8.1.8.4 Filter through a 0.2 µm filter and store in a sterile
cool to room temperature.Aliquot 50 mLof the FBS in sterile
container at room temperature. The solution has a one-month
50-mL conical tubes.
expiration.
8.1.2.6 Label each container with name, lot number, expi-
8.1.9 Assay Mix:
rationdate,andtheheatinactivationdate.Storeat−20 610°C
8.1.9.1 Take a sufficient volume of the glycine buffer to
or 2 to 8°C.
coverdevelopingneeds(thatis,5mLglycinebufferperplate).
8.1.3 Growth Medium:
8.1.9.2 Add 0.34% (w/v) p-nitrophenol phosphate within
8.1.3.1 Combine the following components in the corre-
one (1) h of use and mix well.
sponding proportions (v/v):
NOTE 2—The assay mix must be made on day of use.
Component Proportion (% v/v) Example: 500 mL (mL)
Component Example: 50 mL for 10 plates
DME Low Bicarb 85.5 427.5
Glycine buffer 50 mL
Hi FBS 10.0 50.0
PNPP substrate 170 mg
L-Glutamine (200 mM) 4.0 20.0
Gentamicin 0.5 2.5
8.2 Cell Line Storage and Cell Banking Procedure:
8.1.3.2 Filter through a 0.2 µm filter and store at 2 to 8°C in
8.2.1 Store the cells in 1 mLaliquots in 2 mLcryovials at 5
a sterile container.
×10 cells/mL in freezing medium (see 8.1.7).
8.1.4 Assay Medium:
8.2.2 Prepare cells to make a working cell bank (100+
8.1.4.1 Combine the following components in the corre-
vials).
sponding proportions (v/v):
8.2.3 Thaw the vial of W-20-17 cells obtained fromAmeri-
canType Culture Collection (ATCC) or other source following
Component Proportion (% v/v) Example: 1000 mL (mL)
DME Low Bicarb 87.0 870.0
the procedure described in 8.3.
Hi FBS 10.0 100.0
8.2.4 Inordertoobtaintheexpectedcellnumber,subculture
L-Glutamine (200 mM) 2.0 20.0
Penicillin/streptomycin 1.0 10.0 the cells by expanding them through one or two additional
passages (repeat steps in 8.3).
8.1.4.2 Filter through a 0.2 µm filter and store at 2 to 8°C in
a sterile container.
NOTE 3—The viability should be in the range ≥80%.
8.1.5 NaCl, 0.9 % w/v:
8.2.5 Determine the number of vials to be made based on
8.1.5.1 Dissolve 9 g NaCl in approximately 800 mL of
totalcellnumberobtainedfollowingprocedure8.2.2.Labelthe
purified water and bring to a final volume of 1 Lwith purified
appropriate number of cryovials as follows:
water.
Cell Line Name WCB
8.1.5.2 Filter through a 0.2 µm filter and store in a sterile
Passage Number
container at room temperature.
Freezing Date
Preparation Reference Number
6 Initials
The sole source of supply of the material known to the committee at this time
is National Institute for Biological Standards and Control (NIBSC), Blanche Ln.,
8.2.6 Decap the cryovials in the biosafety cabinet.
South Mimms, Potters Bar, Herts, EN6 3QG, U.K., http://www.nibsc.ac.uk. . If you
8.2.7 Dilute the cell suspension to one half the appropriate
are aware of alternative suppliers, please provide this information to ASTM
volume with 2 to 8°C cold freezing medium without DMSO.
International Headquarters. Your comments will receive careful consideration at a
meeting of the responsible technical committee, which you may attend. The volume should be one half of the appropriate volume for
F2131 − 02 (2012)
the desired cell suspension for freezing.The second half of the 8.3.6 Place the flasks in a 37 6 2°C, 5 6 0.5% CO
cold freezing medium should be made with culture medium humidified incubator for 4 days.
(see 8.1.7, 20% DMSO). The final DMSO concentration shall
NOTE 6—A final volume of 30 mL per T175 flask is required.
be 10%.
NOTE 7—Do not prepare partial flasks, that is, less than2×10
8.2.8 Slowly add the half-volume of culture medium with cells/flask.
20% DMSO to the other half of the volume of the cell
8.4 Plating of Cells:
suspension.
8.4.1 After 4 days, aspirate the medium from the flasks.
8.2.9 Using a sterile pipet, transfer 1 mLof cell suspension
8.4.2 Add 10 mLPBS-CMF to each flask; swirl and lay the
toeachofthelabeledcryovialsonice.Repeatuntilallvialsare
flasks flat to cover the monolayer. Remove the PBS-CMF.
filled. Gently mix the cell suspension during the filling process
8.4.3 Add 10 mL trypsin to each flask; swirl and lay the
to prevent settling of the cells.
flasks flat, making sure the monolayer is coated. Allow the
flasks to sit for approxima
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