Modernization of Enoxaparin Molecular Weight Determination Using Homogeneous Standards

Katelyn Arnold,Stephen Capuzzi,Anita Szajek,Jian Liu,Yongmei Xu,Alexander Tropsha,Kevin Carrick,Eugene Muratov

doi:10.3390/ph10030066

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.

Highlights

Heparin is a mixture of glycosaminoglycan (GAG) chains originating from porcine intestinal mucosa
A total of 27 oligosaccharides were synthesized by a chemoenzymatic approach, and the molecular weight (MW) of each compound was verified by mass spectrometry [10,11]
The determination of the MW distribution and the Mw of enoxaparin is an essential yet challenging quality control step that relies on calibrants to ensure the quality and consistency of the product

Summary

Introduction

Heparin is a mixture of glycosaminoglycan (GAG) chains originating from porcine intestinal mucosa. It is used therapeutically as an anticoagulant for the treatment and prevention of thrombosis [1]. The most common form of LMWH in the U.S is enoxaparin, which is produced by β-eliminative cleavage of the benzyl esters of porcine mucosal heparin under alkaline conditions [2]. This cleavage process leads to the generation of unnatural structures in enoxaparin (Figure 1A). The majority of the resulting chains have an unsaturated uronate residue at the non-reducing end, which can be utilized for UV detection at 232 nm, and up to 25% of chains have a 1,6-anhydro structure at the reducing end [3]

Methods

Results

Discussion

Conclusion