ASTM F2131-02(2007)
(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.
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 (Reapproved 2007)
Standard Test Method for
In Vitro Biological Activity of Recombinant Human Bone
Morphogenetic Protein-2 (rhBMP-2) Using the W-20 Mouse
1
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-
3
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
4
chemiluminescent detection of alkaline phosphatase.
4. Significance and Use
1
ThistestmethodisunderthejurisdictionofASTMCommitteeF04onMedical
4.1 Although the test method can be used for assessment of
andSurgicalMaterialsandDevicesandisthedirectresponsibilityofSubcommittee
the bioactivity of crude preparations of rhBMP-2, it has only
F04.42 on Biomaterials and Biomolecules for TEMPs.
been validated for use with highly pure (>98% by weight
Current edition approved Feb. 1, 2007. Published February 2007. Originally
approved in 2002. Last previous edition approved in 2002 as F2131–02.
protein purity) preparations of rhBMP-2.
2
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.
4
1318-1324. Blum, R. S., Li, R. H., Mikos,A.G., and Barry, M.A., “An Optimized Method
3
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
...
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