Logo

ASTM F2131-02(2007)e1

Historical Revision

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

$83.00

$83.00

$149.40


Sub Total (1 Item(s))

$ 0.00

Estimated Shipping

$ 0.00

Total (Pre-Tax)

$ 0.00


...

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.


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.

SDO ASTM: ASTM International
Document Number F2131
Publication Date Feb. 1, 2007
Language en - English
Page Count 6
Revision Level 02(2007)e1
Supercedes
Committee F04.42
Publish Date Document Id Type View
Oct. 1, 2021 F2131-21 Revision
Feb. 1, 2007 F2131-02R07E01 Revision
Sept. 10, 2002 F2131-02 Revision
Oct. 1, 2012 F2131-02R12 Reaffirmation
Feb. 1, 2007 F2131-02R07 Reaffirmation