Potency.
The pharmacopeial heparin potency assay traditionally was performed using sheep plasma. The rationale for this method is
that it resembles as closely as possible the actual situation in which heparin typically is used (i.e., in human blood). Unfortunately,
the results of the assay in plasma are affected by other proteins that are present in sheep plasma. Heparin can bind to a
large variety of plasma proteins such as histidine-rich glycoprotein, platelet factor 4 (PF-4), and heparin cofactor II (HCII).
The large enhancement of antifactor IIa activity for OSCS is an HCII-mediated event (4). This phenomenon explains how contaminated
heparin batches successfully passed the USP sheep plasma clotting activity assay. Based on the known modes of action of heparin,
USPC proposes to replace the current potency assay based on sheep plasma with a chromogenic antifactor IIa assay that uses
purified reagents.
Organic impurities.
An Impurity test has been added to quantify the percent galactosamine of total hexosamine in heparin. Heparins are salts
of sulfated glucosamino-glycans composed of polymers of alternating D-glucosamine (N-sulfated or N-acetylated) and uronic acid (L-iduronic or D-glucuronic acid) joined by glycosidic linkages. This procedure involves the hydrolysis of heparin into the monomers (galactosamine
and glucosamine) by hydrochloric acid and analysis by high-performance ion chromatography. The percent (w/w) of galactosamine
with respect to the total amount of both galactosamine and glucosamine (hexosamine) is quantified using the relative response
of galactosamine to glucosamine from the chromatograph's peak areas. This test provides a measure of dermatan sulfate and
other galactosamine-containing impurities in heparin.
A quantitative Lowry method has replaced the old turbidity test for protein impurities. Nucleotidic impurities and residual
solvents have been added to control residual DNA and residual-solvent levels in heparin.
Conclusion
The proposed heparin sodium and heparin sodium injection monographs were posted on the USPC website on Feb. 4, 2009. These
Stage 2 monographs will be presented in the March–April Pharmacopeial Forum as proposed Interim Revision Announcements. After review of comments and approval by the BBP EC, the new heparin monographs
are expected to become official Aug. 1, 2009.
USPC plans to ensure that any revisions to the monographs are suitable and will not disrupt the supply of safe and effective
medicines for patients. Comments on the standards should be sent to heparin@usp.org
by May 15, 2009.
Anita Y. Szajek, PhD,* is senior scientist, Tina S. Morris, PhD, is director of biologics & biotechnology, William F. Koch, PhD, is chief metrology officer, Darrell R. Abernethy, MD, PhD, is chief science officer, and Roger L. Williams, MD, is chief executive officer, all at the US Pharmacopeia, 12601 Twinbrook Parkway, Rockville, MD 20852-1790, tel. 301.816.8325;
aey@usp.org
.
*To whom all correspondence should be addressed.
References
1. FDA, "Information on Heparin (2009),"
http://www.fda.gov/cder/drug/infopage/heparin/default.htm, accessed Jan. 29, 2009.
2. M. Guerrini et al., "Oversulfated Chondroitin Sulfate Is a Contaminant in Heparin Associated with Adverse Clinical Events,"
Nature Biotechnol.
26 (6), 669–675 (2008).
3. USP 31–NF 26 <1225>, "Validation of Compendial Procedures," 683–687 (2008).
4. T. Maruyama et al., "Conformational Changes and Anticoagulation Activity of Chondroitin Sulfate Following its O-Sulfonation,"
Carbohydr. Res.,
306, 35–43 (1998).
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