Combining NMR Spectroscopy and Chemometrics to Monitor Structural Features of Crude Hep-arin

Abstract

Because of the complexity and global nature of the heparin supply chain, the control of heparin quality during manufacturing steps is essential to ensure the safety of the final active pharmaceutical ingredient (API). For this reason, there is a need to develop consistent analytical methods able to assess the quality of heparin early in production (i.e., as the crude heparin before it is purified to API under cGMP conditions). Although a number of analytical techniques have been applied to characterize heparin APIs, few of them have been applied for crude heparin structure and composition analyses. Here, to address this issue, NMR spectroscopy and chemometrics were applied to characterize 88 crude heparin samples. The samples were also analyzed by strong anion exchange HPLC (SAX-HPLC) as an orthogonal check of the purity levels of the crudes analyzed by NMR. The HPLC data showed that the chemometric analysis of the NMR data differentiated the samples based on their purity. These orthogonal approaches differentiated samples according their glycosaminoglycan (GAG) composition and their mono and disaccharide composition and structure for each GAG family (e.g., heparin/heparan, dermatan sulfate, and chondroitin sulfate A). Moreover, quantitative HSQC and multivariate analysis (PCA) were used to distinguish between crude heparin of different animal and tissue sources.

The use of ATR-FTIR spectroscopy to characterise crude heparin samples by composition and structural features

“…This means that analysts can use loading plots to identify regions associated with specific functional groups or interactions. Furthermore, the band assignments made in Table 1 agree with the ATR-FTIR loading plots, when combined with information from NMR/SAX-HPLC analysis 7 , allowing greater confidence in the band assignments and the results shown here.…”

“…The sulphation of disaccharide units may vary throughout the polysaccharide and a measure of the average sulphation of each disaccharide unit,dubbed degree of sulfation (DoS) is often used to compare samples, however, heparin disaccharides typically consist of 70-80 % I2S residues and GNS and 6S residues, mixed with lower levels of other constituents including D-glucuronate, unsulphated I (IOH), GNAc and some G3S, to generate considerable sequence diversity 3,4 , 5 . The structural diversity of pharmaceutical heparin is compounded further by variations in molecular weight, compositional differences between heparin from different organs, individual animals and different species 6 , and is amplified by the practice of amalgamating the material obtained from many individual animals before processing 7 .…”

“…While the steps that create pharmaceutical quality heparin from crude heparin are performed under cGMP regulations, the traceability of the porcine intestine suppliers that create the crude heparin is poor and, since many sites are involved, the risk of contamination from an unknown source is greatly increased 7 . This is particularly pertinent in modern heparin manufacture, following the contamination of pharmaceutical heparin in 2007-8, dubbed the "heparin crisis", which was caused by the introduction of material, much of which was an unnatural, chemically modified chondroitin sulphate (over-sulfated chondroitin sulfate; OSCS) into the supply chain, and resulting in approximately 150 deaths and 350 other adverse events in the USA alone 8,9 .…”

“…Many studies have been undertaken with the aim of detecting contamination (almost always with OSCS [12][13][14][15][16] , even though a very wide range of other potential future contaminants, whether accidentally or deliberately introduced, can be envisaged 17 ) in pharmaceutical heparin samples, without first defining what level of structural variation is observed between bona fide heparin samples. In the case of pharmaceutical heparin, this issue was first identified and addressed in 2011 using proton NMR 18,19 and, in 2017 the first steps in this direction were taken for crude heparin using NMR spectroscopy and strong anion exchange HPLC (SAX-HPLC), coupled with principal component analysis (PCA) 7 . Using this approach, it was possible to characterise crude samples by their composition and structural features.…”

“…Here, 73 of the original 75 crude heparin samples (taken from those used in the 2017 study 7 ) were analysed with ATR-FTIR, and the viability of this method for use in the characterisation of crude heparin was examined. This approach is particularly pertinent because, when the dispersed nature of the heparin supply chain is considered, the simple machine set-up, straightforward sample preparation, short acquisition time and relatively undemanding level of training or expertise required (compared with NMR and SAX-HPLC) makes ATR-FTIR a strong candidate for practical use by manufacturer and regulator alike.…”