Oligosaccharide Chromatographic Techniques for Quantitation of Structural Process-Related Impurities in Heparin Resulting From 2-O Desulfation.

Heparin is a widely-used intravenous anticoagulant comprising a complex mixture of highly-sulfated linear polysaccharides of repeating sequences of uronic acids (either iduronic or glucuronic) 1->4 linked to D-glucosamine with specific sulfation patterns. Preparation of crude heparin from mammalian mucosa involves protease digestion with alcalase under basic conditions (pH ≥ 9) and high temperature (>50°C) and also oxidation. Under such conditions, side reactions including the ubiquitous 2-O desulfation occur on the heparin backbone yielding non-endogenous disaccharides within polysaccharide chains. Whatever the process used for its manufacture, some level of corresponding degradation impurities is therefore expected to be found in heparin and the derived Low Molecular Weight Heparins. These impurities should be monitored to control the quality of the final therapeutic product. Two anion exchange chromatography techniques were used to analyze heparin samples exhaustively or partially depolymerized with heparinases and determine the proportions of non-endogenous disaccharides generated by side reactions during the manufacturing process (epoxides and galacturonic moieties). We also present data from a case study of marketed heparin. Current heparin sodium monographs do not directly address process impurities related to modification of the structure of heparin. Although desulfation reduces the overall biological potency, we found that heparin with an average of one modified disaccharide per chain can still comply with the USP or Ph. Eur. heparin sodium monographs requirements. We have implemented disaccharide analysis to monitor the quality of this product on a risk basis.

Reproducibility of the anti-Factor Xa and anti-Factor IIa assays applied to enoxaparin solution.

Enoxaparin is a widely used subcutaneously administered antithrombotic agent comprising a complex mixture of glycosaminoglycan chains. Owing to this complexity, its antithrombotic potency cannot be defined by physicochemical methods and is therefore evaluated using an enzymatic assay of anti-Xa and anti-IIa activity. Maintaining consistent anti-Xa activity in the final medicinal product allows physicians to ensure administration of the appropriate dosage to their patients. Bioassays are usually complex and display poorer reproducibility than physicochemical tests such as HPLC assays. Here, we describe the implementation of a common robotic platform and standard release potency testing procedures for enoxaparin sodium injection (Lovenox, Sanofi, Paris, France) products at seven quality control sites within Sanofi. Qualification and analytical procedures, as well as data handling, were optimized and harmonized to improve assay reproducibility. An inter-laboratory study was performed in routine-release conditions. The coefficients of variation for repeatability and reproducibility in assessments of anti-Xa activity were 1.0% and 1.2%, respectively. The tolerance interval in reproducibility precision conditions, expressed as percentage potency, was 96.8-103.2% of the drug product target of 10,000 IU/ml, comparing favorably with the United States of America Pharmacopeia specification (90-110%). The maximum difference between assays in two different laboratories is expected to be 4.1%. The reproducibility characteristics of anti-IIa activity assessments were found to be similar. These results demonstrate the effectiveness of the standardization process established and allow for further improvements to quality control in Lovenox manufacture. This process guarantees closeness between actual and target potencies, as exemplified by the results of release assays obtained during a three-year period.

New developments in quantitative polymerase chain reaction applied to control the quality of heparins.

Heparin is a widely used intravenous anticoagulant comprised of a very complex mixture of glucosaminoglycan chains, mainly derived from porcine intestinal mucosa. Recent contamination of heparin with oversulfated (OS) chondroitin sulfate resulted in a significant number of deaths, triggering a rapid revision of product monographs and the introduction of new analytical methods to limit as far as possible the chances of another occurrence of such a phenomenon. The distribution of heparin-processing units across the globe prevents their complete fool-proof auditing. Therefore, the implementation of additional orthogonal analytical techniques for quality control (QC) of heparin batches is highly important. We perform routine quantitative polymerase chain reaction (Q-PCR) release tests to confirm the quality of all crude heparin batches received by sanofi-aventis. The routine test used provides information on the animal species of origin as requested by the US Pharmacopoeia (USP) and European Pharmacopoiea monographs. Here, we demonstrate that the Q-PCR test is inhibited by OS glycosaminoglycans at concentrations as low as 0.5% (w/w versus heparin) and can be used as an additional safeguard to monitor levels of potentially harmful contaminants without any increased workload. In response to a request from the USP, we also describe the development of a Q-PCR method for monitoring nucleotidic impurities in pure heparin, which is able to detect amplifiable DNA at concentrations lower than 0.1 ng DNA per milligram of heparin. This increased sensitivity makes this modified Q-PCR method a potential candidate for inclusion as a QC requirement in future monographs.

Isolation and characterization of contaminants in recalled unfractionated heparin and low-molecular-weight heparin.

Recently, a contaminant was found in some clinically used unfractionated heparin (UFH) preparations. Administration of this UFH was associated with an increased risk of developing a wide range of adverse effects including death. To further investigate the chemical profile of the contaminant, contaminated batches of UFH were treated by exhaustive nitrous acid depolymerization followed by methanol precipitation to remove heparin oligosaccharides. Because contaminated heparins may have been used as starting material in the production of low-molecular-weight heparins (LMWHs), a similar procedure was carried out using an experimental batch of enoxaparin prepared from contaminated heparin. While high-pressure liquid chromatography (HPLC) analysis of contaminated heparin did not distinguish the presence of the contaminant, it could readily be observed as a high-molecular weight shoulder in the elution profile of contaminated enoxaparin. Digesting contaminated heparin with heparinase-I prior to HPLC analysis showed the presence of a nondigestible component (15%-30% of the mixture). This contaminant was also resistant to degradation by chondroitinases A, B, and C. Proton nuclear magnetic resonance (NMR) indicated that the contaminant was oversulfated chondroitin sulfate (OSCS). Size-exclusion chromatography indicated that the mean molecular weight of the OSCS was 16.8 kD, comparable to that of a synthetic porcine cartilage OSCS preparation that was used as a reference material (17.2 kD). While varying degrees of high-molecular weight dermatan sulfate and other minor impurities were detected, OSCS appeared to be the major contaminant in these preparations. The process involved in the production of enoxaparin does not significantly degrade OSCS.