ASTM F2131-21

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Designation: F2131 − 02 (Reapproved 2012) F2131 − 21
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. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. 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 vitroin-vitro test method to in vivoin-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 vivoin-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 safety, health, and healthenvironmental practices and determine the
applicability of regulatory limitations prior to use.
This test method is under the jurisdiction of ASTM Committee F04 on Medical and Surgical Materials and Devices and is the direct responsibility of Subcommittee
F04.42 on Biomaterials and Biomolecules for TEMPs.
Current edition approved Oct. 1, 2012Oct. 1, 2021. Published October 2012November 2021. Originally approved in 2002. Last previous edition approved in 20072012
ε1
as F2131 – 02 (2007)(2012). . DOI: 10.1520/F2131-02R12.10.1520/F2131-21.
Thies, R. S., Bauduy, M., Ashton, B. A., Kurtzberg, L., Wozney, J.M., J. M., and Rosen, V., “Recombinant Human Bone Morphogenetic Protein-2 Induces Osteoblastic
Differentiation in W-20-17 Stromal Cells,” Endocrinology, Vol 130, 1992, pp. 1318–1324.
Guideline 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.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F2131 − 21
1.6 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Terminology
2.1 rhBMP—recombinant human bone morphogenetic protein.
2.2 GDF—growth and differentiation factor.
3. Summary of Test Method
3.1 In this test method, the mouse stromal cell line W-20-17 is used as a target cell line for rhBMP-2. The W-20-17 cells 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 measure of alkaline phosphatase enzyme level. The test method is performed in a
96-well plate format. A similar test method based upon the same cell line has been developed using chemiluminescent detection
of alkaline phosphatase.
4. 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.
5. Interferences
5.1 There have been no systematic studies of interfering substances for this test method. There is anecdotal evidence that trypsin
and some rhBMP-2 formulation buffers can interfere with the assay. Additionally, the source of fetal bovine serum is an important
variable. Each lot should be tested in all parts of the assay where it is required to determine the appropriateness of the lot. This
is particularly important if the fetal bovine serum vendor is changed.
6. Apparatus
6.1 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 μL) and Multichannel pipets (range 50 to 300 μL).
6.5 Biosafety cabinet.
6.6 96 Well 96-Well flat bottom sterile tissue culture microtiter plates, (FalconFalcon 3072 or equivalent).equivalent.
6.7 IEC Centra-7R Centrifuge, or equivalent.
6.8 CO humidified tissue culture incubator.
6.9 Spectrophotometric microplate reader, (VMAX/Spectramax,VMAX/Spectramax, Molecular Devices, or equivalent).equiva-
lent.
6.10 Hemacytometer, or automatic cell counter.
Blum, R. S., Li, R. H., Mikos, A.G., A. G., and Barry, M.A., M. A., “An Optimized Method for the Chemiluminescent Detection of Alkaline Phosphatase Levels During
Osteodifferentiation by Bone Morphogenetic Protein 2,” Jour. Cellular Biochem, Vol 80, 2001, pp. 532–537.
F2131 − 21
6.11 Inverted microscope.
6.12 Tissue culture flasks, Falcon T175 or equivalent.
6.13 Sterilized paper towels, or equivalent.
6.14 Sterile filter units, (0.2 μm).0.2 μm.
6.15 Sterile pipets, (11 mL, 5 mL, 10 mL, 25 mL, 50 mL).mL.
6.16 9 in. Pasteur pipets, sterilized.
6.17 Sterilized pipet tips, (1-300 (1 to 300 μL and 200-1000 200 to 1000 μL).
6.18 Sterile reagent reservoirs.
6.19 −80°C−80 °C freezer.
6.20 96 Well U-Bottom 96-Well U-bottom polypropylene sterile tissue culture microtiter plates, (CostarCostar 3790 or
equivalent).equivalent.
6.21 Water bath.
6.22 Orbital shaker.
7. Reagents and Materials
7.1 W-20-17 Mouse Stromal Cells.mouse stromal cells.
7.2 Dulbecco’s modified Eagle’s medium with 4500 mg/L glucose and 4.0 mM L-glutamine, without sodium bicarbonate
(DME/High, JRH Biosciences, 56439 or equivalent).
7.3 Sodium bicarbonate (Sigma—Aldrich(Sigma-Aldrich S4019 or equivalent).
7.4 5 M hydrochloric acid.
7.5 Heat inactivated (Hi) fetal bovine serum (FBS).
NOTE 1—Each new lot of fetal bovine serum must be evaluated in the assay before use.
7.6 200 mM L-Glutamine (Invitrogen Life Technologies, 25030081 or equivalent).
7.7 Gentamicin Gibco sterile filtered: 10 mg/mL or equivalent.
7.8 Penicillin Streptomycin (PS), contains 10 000 units of penicillin (base)/mL and 10 000 μg of streptomycin (base)/mL, utilizing
penicillin G (sodium salt) and streptomycin sulfate in 0.85 % saline (Invitrogen Life Technologies, #15140122 or equivalent).
This cell line has been deposited in mid-2001. The sole source of supply of the apparatus known to the committee at this time is American Type Culture Collection, 10801
University Blvd., Manassas, VA 20110-2209, U.S., http://www.atcc.org.http://www.atcc.org. If you are aware of alternative suppliers, please provide this information to
ASTM International Headquarters. Your comments will receive careful consideration at a meeting of the responsible technical committee, which you may attend.
F2131 − 21
7.9 Phosphate Buffered Saline, Calcium and Magnesium Free,buffered saline, calcium and magnesium free, 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 · 4Na) (1X), liquid (Invitrogen Life Technologies 25300054 or equivalent).
7.12 Glycine (Sigma —Aldrich (Sigma-Aldrich or equivalent).
7.13 Sodium Hydroxidehydroxide (NaOH) 0.2 N and 10 N.
7.14 Triton X-100 (J.T. Baker Cat. No. X198-05 or equivalent).
7.15 Magnesium Chloride, Crystallinechloride, crystalline (MgCl · 6 H O).
2 2
7.16 p-Nitrophenol phosphate (PNPP, Sigma—AldrichSigma-Aldrich 104(R) phosphatase substrate, product # 1040 or equiva-
lent).
7.17 NaCl.
7.18 Purified water.
7.19 rhBMP-2, 1st WHO Reference Reagent 1997 (5000 Unitsunits per ampoule, cat. # 93/574, National Institute for Biological
Standards and Control).
7.20 rhBMP-2 internal control, >1 mg/mL (stored at −80°C).−80 °C).
8. Procedure
8.1 Solution Preparation:
8.1.1 DME Low Bicarb:
8.1.1.1 Dissolve 66.87 g DME/High and 11.13 g sodium bicarbonate 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 bring solution to 5 L with purified water.
8.1.1.3 Filter through a 0.2 μm filter into sterile bottles.
8.1.1.4 Store at 2 to 8°C.8 °C. The solution expires in 8eight weeks.
8.1.2 Hi FBS:
8.1.2.1 Thaw the desired amount of FBS at ambient temperature, or 2 to 8°C.8 °C.
8.1.2.2 Adjust the water bath to a temperature of 56 6 2°C.2 °C.
8.1.2.3 Place the bottle of FBS into the water bath so that the entire contents of the bottle are immersed in water.
8.1.2.4 Heat the bottle for 45 min, swirling periodically.
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., South
Mimms, Potters Bar, Herts, EN6 3QG, U.K., http://www.nibsc.ac.uk. . If you are aware of alternative suppliers, please provide this information to ASTM International
Headquarters. Your comments will receive careful consideration at a meeting of the responsible technical committee, which you may attend.
F2131 − 21
8.1.2.5 Remove the bottle from the water bath and allow to cool to room temperature. Aliquot 50 mL of the FBS in sterile 50-mL
conical tubes.
8.1.2.6 Label each container with name, lot number, expiration date, and the heat inactivation date. Store at −20 6 10°C5 °C or
2 to 8°C.8 °C.
8.1.3 Growth Medium:
8.1.3.1 Combine the following components in the corresponding proportions (v/v):
Component Proportion (% v/v) Example: 500 mL (mL)
DME Low Bicarb 85.5 427.5
Hi FBS 10.0 50.0
L-Glutamine (200 mM) 4.0 20.0
Gentamicin 0.5 2.5
8.1.3.2 Filter through a 0.2 μm filter and store at 2 to 8°C8 °C in a sterile container.
8.1.4 Assay Medium:
8.1.4.1 Combine the following components in the corresponding proportions (v/v):
Component Proportion (% v/v) Example: 1000 mL (mL)
DME Low Bicarb 87.0 870.0
Hi FBS 10.0 100.0
L-Glutamine (200 mM) 2.0 20.0
Penicillin/streptomycin 1.0 10.0
8.1.4.2 Filter through a 0.2 μm filter and store at 2 to 8°C8 °C in a sterile container.
8.1.5 NaCl, 0.9 % w/v:w ⁄v:
8.1.5.1 Dissolve 9 g NaCl in approximately 800 mL of purified water and bring to a final volume of 1 L with purified water.
8.1.5.2 Filter through a 0.2 μm filter and store in a sterile container 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 Filter through a 0.2 μm filter and store in a sterilized container at room temperature.
8.1.7 Freezing Medium:
8.1.7.1 Prepare freezing medium immediately before the freezing procedure by adding DMSO to growth medium (see 8.1.39.1.3))
to 20 % v/v.
Component Proportion (% v/v) Example: 100 mL
Growth Medium 80 80 mL
DMSO 20 20 mL
8.1.8 Glycine Buffer:
8.1.8.1 Dissolve 0.75 % (w/v) glycine in required volume of purified water. Adjust the pH of the solution to 10.3 6 0.1 with 10
N NaOH.
8.1.8.2 Add 0.8 %(v0.8 %⁄v) (v/v) of 12.5 % Triton X-100.
F2131 − 21
8.1.8.3 Add 0.13 % (w/v) MgCl · 6H O and mix well.
2 2
Component Example: 1000 mL
Glycine 7.5 g
MgCl · 6H O 1.3 g
2 2
12.5 % Triton X-100 8.0 mL
Water To 1000 mL
8.1.8.4 Filter through a 0.2 μm filter and store in a sterile container at room temperature. The solution has a one-month expiration.
8.1.9 Assay Mix:
8.1.9.1 Take a sufficient volume of the glycine buffer to cover developing needs (that is, 5 mL glycine buffer per plate).
8.1.9.2 Add 0.34 % (w/v) p-nitrophenol phosphate within one (1) h of use and mix well.
NOTE 2—The assay mix must be made on the day of use.
Component Example: 50 mL for 10 plates
Glycine buffer 50 mL
PNPP substrate 170 mg
8.2 Cell Line Storage and Cell Banking Procedure:
8.2.1 Store the cells in 1 mL aliquots in 2 mL cryovials at 51.0 × 10 cells/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 American Type Culture Collection (ATCC) or other source following the
procedure described in 8.3.
8.2.4 In order to obtain the expected cell number, subculture the cells by expanding them through one or two additional passages
(repeat steps in 8.3).
NOTE 3—The viability should be in the range ≥80 %.≥85 %.
8.2.5 Determine the number of vials to be made based on total cell number obtained following proce
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