ASTM F2131-21
(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="ital"> 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, health, and environmental practices and determine the applicability of regulatory limitations prior to use.
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.
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Standards Content (Sample)
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.
Designation: F2131 − 21
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 (´) 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
2
bone marrow stromal cells of the W++ mouse strain.
UT assays. These data suggest that a single receptor binding
1.2 This test method (assay) has been qualified and vali-
domainonrhBMP-2isresponsibleforboth in-vitroand in-vivo
dated based upon the International Committee on Harmoniza-
activity and that the W-20 bioassay is a relevant predictor of
3
tion assay validation guidelines (with the exception of inter-
the bone-forming activity of rhBMP-2.
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,
has been correlated with the ectopic bone-forming capacity of
1.5 This standard does not purport to address all of the
rhBMP-2 in the in-vivo Use Test (UT). rhBMP-2 that was
safety concerns, if any, associated with its use. It is the
partially or fully inactivated by targeted peracetic acid oxida-
responsibility of the user of this standard to establish appro-
tion of the two methionines was used as a tool to compare the
priate safety, health, and environmental practices and deter-
activities. Oxidation of rhBMP-2 with peracetic acid was
mine the applicability of regulatory limitations prior to use.
showntobespecificallytargetedtothemethioninesbypeptide
1.6 This international standard was developed in accor-
mappingandmassspectrometry.Thesemethioninesresideina
dance with internationally recognized principles on standard-
hydrophobic receptor binding pocket on rhBMP-2. Oxidized
ization established in the Decision on Principles for the
samples were compared alongside an incubation control and a
Development of International Standards, Guides and Recom-
nativecontrol.The62,87,98,and100%oxidizedsampleshad
mendations issued by the World Trade Organization Technical
W-20 activity levels of 62, 20, 7, and 5%, respectively. The
Barriers to Trade (TBT) Committee.
incubation and native control samples maintained 100% ac-
tivity. Samples were evaluated in the UTand showed a similar
effect of inactivation on bone-forming activity. The samples 2. Terminology
with 62% and 20% activity in the W-20 assay demonstrated
2.1 rhBMP—recombinant human bone morphogenetic pro-
reduced levels of bone formation, similar in level with the
tein.
2.2 GDF—growth and differentiation factor.
1
ThistestmethodisunderthejurisdictionofASTMCommitteeF04onMedical
andSurgicalMaterialsandDevicesandisthedirectresponsibilityofSubcommittee
F04.42 on Biomaterials and Biomolecules for TEMPs.
3. Summary of Test Method
Current edition approved Oct. 1, 2021. Published November 2021. Originally
approved in 2002. Last previous edition approved in 2012 as F2131–02 (2012).
3.1 Inthistestmethod,themousestromalcelllineW-20-17
DOI: 10.1520/F2131-21.
is used as a target cell line for rhBMP-2. The W-20-17 cells
2
Thies, R. S., Bauduy, M., Ashton, B. A., Kurtzberg, L., Wozney, J. M., and
exhibit increased alkaline phosphatase activity in response to
Rosen, V., “Recombinant Human Bone Morphogenetic Protein-2 Induces Osteo-
blastic Differentiation in W-20-17 Stromal Cells,” Endocrinology, Vol 130, 1992,
rhBMP-2. Optical density at 405 nm of the p-nitrophenol
pp. 1318–1324.
generated from the alkaline phosphatase substrate is used as a
3
Guideline for Industry, ICH-Q2AText on Validation ofAnalytical Procedures,
measure of alkaline phosphatase enzyme level. The test
November 1996, International Committee on Harmonization, March 1995, htt
...
This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
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
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. 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
2
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
3
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.
1
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.
2
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.
3
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
1
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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 Org
...
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