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. 2017 Jul 22;10(3):66.
doi: 10.3390/ph10030066.

Modernization of Enoxaparin Molecular Weight Determination Using Homogeneous Standards

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Free PMC article

Modernization of Enoxaparin Molecular Weight Determination Using Homogeneous Standards

Katelyn M Arnold et al. Pharmaceuticals (Basel). .
Free PMC article

Abstract

Enoxaparin is a low-molecular weight heparin used to treat thrombotic disorders. Following the fatal contamination of the heparin supply chain in 2007-2008, the U.S. Pharmacopeia (USP) and U.S. Food and Drug Administration (FDA) have worked extensively to modernize the unfractionated heparin and enoxaparin monographs. As a result, the determination of molecular weight (MW) has been added to the monograph as a measure to strengthen the quality testing and to increase the protection of the global supply of this life-saving drug. The current USP calibrant materials used for enoxaparin MW determination are composed of a mixture of oligosaccharides; however, they are difficult to reproduce as the calibrants have ill-defined structures due to the heterogeneity of the heparin parent material. To address this issue, we describe a promising approach consisting of a predictive computational model built from a library of chemoenzymatically synthesized heparin oligosaccharides for enoxaparin MW determination. Here, we demonstrate that this test can be performed with greater efficiency by coupling synthetic oligosaccharides with the power of computational modeling. Our approach is expected to improve the MW measurement for enoxaparin.

Keywords: HPLC; MW determination; USP; compendial test; computational modeling; enoxaparin; oligosaccharide calibrants.

Conflict of interest statement

The authors declare no conflict of interest. This work was prepared while Anita Szajek was employed at United States Pharmacopeia. The opinions expressed in this article are the author’s own and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government.

Figures

Figure 1
Figure 1
(A) Enoxaparin structure. The 1,6-anhydro reducing end is depicted in the box on the right; (B) the structure of 12-mer NS6S (19); (C) representative size exclusion chromatography (SEC) chromatogram of Lovenox and 12-mer NS6S (19). Enoxaparin’s heterogeneity and complexity is evident from multiple peaks with poor base-line separation.
Figure 2
Figure 2
Calibration curves for determining Mw generated from different structural groups: (A) NAc series (group A); (B) NS series (group B); (C) NS2S series (group C); (D) NS6S series (group D); (E) NS6S2S series (group E); (F) USP Calibrant A and B (group G). The nonlinear regression R2 value is listed in upper right corner of each panel.
Figure 3
Figure 3
Comparison of two structural series and SEC behavior. Blue diamonds are group E (NS6S2S series). Black circles are group B (NS series).
Figure 4
Figure 4
(A) USP calibrants (red squares) and all synthetic oligosaccharides (black open circles); (B) Correlation of the molecular weight (MW) and retention time (RT) of compounds below 3000 Da annotated. Compounds enclosed in the circle, 8-mer NAc (1), 10-mer NAc (2), and 8-mer NS (7), were the outliers and were excluded from further analysis.
Figure 5
Figure 5
Predicted log(MW) versus observed log(MW) to demonstrate the strength of the model’s prediction. For predicted log(MW), only external set MW predictions are used. Observed MW is determined by MS for the pure oligosaccharides. For dp4, dp6, and USP calibrants, the provided MW values were used as the observed MW. The model’s predictions highly correlate to the expected results (R2 = 0.96).
Figure 6
Figure 6
(A) 232 nm trace of enoxaparin mixed with three oligosaccharides. Although primary detection of the synthetic oligosaccharides is at 310 nm, there is some residual absorbance also at 232 nm, as evidenced by the abnormal profile compared to Figure 1C; (B) Overlay of 310 nm and 232 nm trace from a single run of the enoxaparin/oligosaccharide mixture. 310 nm peak RTs (red trace) were used to determine the suitability of the HPLC system by comparing them with the predetermined 310 nm chromatogram from oligosaccharides alone (Figure S4); (C) Overlay of predetermined 232 nm trace (blue trace); (D) The resulting 232 nm trace when the oligosaccharide contribution is removed. The peak RTs and area values are input into the model for analysis.

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