ATR-FTIR spectroscopy of heparin: using multivariate analyses for next-generation quality control

Devlin, Anthony James

Abstract

Heparin is a member of the glycosaminoglycan (GAG) family of naturally occurring, polydisperse, linear polysaccharides. Heparin is a ubiquitous anticoagulant drug, a member of the WHO’s list of vital medicines and the second used drug by weight after insulin. The structure of heparin is complex, extremely challenging to sequence - requiring large amounts of pure starting material, and there is currently no way of synthesising viable full-length heparin. Pharmaceutical heparin is therefore a natural product, extracted from the mucosa of pigs and cows. In 2007/2008, batches of heparin were found to have been adulterated with a semi-synthetic analogue (OSCS), which resulted in at least 100 deaths in the US alone. In 2011, Rudd et al. introduced the idea of multivariate analysis for heparin quality control and demonstrated that, through use of NMR and principal components analysis (PCA), pharmaceutical heparins could be defined in N-dimensions and that samples contaminated with OSCS could be discriminated to the level of 1% (w/w).

Here, infrared (IR) spectroscopy, coupled with multivariate analyses, is explored as a potential tool for the quality control of pharmaceutical heparin. Levels of OSCS as low as 0.25% (w/w) were detected and through expansion of the model, levels of 1 - 5% (w/w) of the other GAGs chondroitin sulphate (CS), dermatan sulphate (DS), hyaluronic acid (HA) and the sulphated semi-synthetic analogues thereof were detected. PC-regression (PCR) was applied to IR spectra of crude heparins – 2:1 (w/w) mixes of heparin:CS/DS which are believed to be where OSCS contamination first occurred – and was able to accurately detect and quantify heparin, CS and DS in the sample (RMSE = 2.2, 1.7 and 0.8 respectively).

PCR and partial least squares-regression were applied to the IR spectra of crude heparins, specifically aimed at the quantification of sulphate moieties on the carbon ring and was able to predict the levels of 6-O-, 2-O- and N- sulphate accurately (RMSEaverage = 0.91 and 0.76 respectively). The techniques were applied to heparins extracted from different animal sources and through cluster analysis, discrimination of heparins from pig, cow and sheep was achieved with 99% accuracy. Blends of porcine heparin with bovine and ovine heparin were also examined and detection was facilitated at the level of 10%.

Contained within this thesis is an exploration of the use of IR spectroscopy as both a tool for QC and a tool for GAG characterisation. IR, coupled with multivariate analyses is shown to detect low levels (sub 1%) of contaminants, accurately quantify and discriminate between different GAGs in a mixture and accurately quantify and discriminate between different sulphate moieties, all with accuracies that parallel those of NMR.

Citation

Devlin, A. J. (2023). ATR-FTIR spectroscopy of heparin: using multivariate analyses for next-generation quality control. (Thesis). Keele University

• Thesis Type: Thesis
• Deposit Date: Jul 19, 2023
• Publicly Available Date: Jul 19, 2023
• Award Date: 2023-06