A quantitative mass spectrometry method to differentiate bovine and ovine heparins from pharmaceutical porcine heparin

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Abstract

Heparin is a polysaccharide extracted from animal tissues and is used widely as an anticoagulant. In most countries, porcine intestine mucosa is the only legal source for producing heparin. It is challenging to differentiate heparins derived from porcine, ovine and bovine, especially when low amounts of ruminant heparin are adulterated into porcine heparin. Herein, we find that two marker saccharides, ΔUA2S-GlcNS6S-HexA2S (ΔISH) and ΔUA2S-GlcNAc6S (ΔIA), show significant differences in the basic building blocks of porcine heparin obtained from ruminant heparin. A quantitative mass spectrometry (MS) method was then established to selectively monitor these two marker saccharides. By using the ΔISH to ΔIA ratio, porcine heparin adulterated with a low amount of ruminant heparin (10 % ovine heparin or 5 % bovine heparin) can be differentiated. This represents a robust and sensitive method for ensuring the authenticity and safety of heparin drugs.

Introduction

Heparin is one of the most important polysaccharides and has been used widely in the clinic for over 80 years (Alaez-Verson et al., 2017; Hao et al., 2019). As an anticoagulant, heparin drugs extracted from natural animal sources are irreplaceable, although many different classes of synthetic small molecule anticoagulant drugs have been developed. Initially, pharmaceutical heparin was extracted from the tissues of food animals, such as porcine intestines, ovine intestines and bovine lungs (Jasper et al., 2015). Since the 1990s, with the outbreak of bovine spongiform encephalopathy (mad cow disease) in Europe, which was caused by prions, the production and use of bovine heparin has decreased. A similar situation occurs with ovine heparin, which presents a potential risk of causing Creutzfeldt-Jakob disease, a consequence of residual prions in heparin products from scrapie-infected sheep (Fu et al., 2013). Currently, although the use of bovine-derived heparin is still allowed in Brazil, Argentina, India and a few Islamic countries (van der Meer et al., 2017), porcine intestine is designated as the only legal source for extracting pharmaceutical heparin in a majority of countries and regions, including the United States of America, the European Union and China, to avoid the risk of prion diseases and ensure the drug safety of heparin.

However, serious problems have emerged due to the use of a single animal source for producing pharmaceutical heparin. For example, the population size of pigs is often affected by disease outbreaks and the demands from the meat market, which results in an unstable supply of porcine intestines and therefore crude heparin. The shortage of crude heparin makes it susceptible to adulteration with contaminant polysaccharides, such as oversulfated chondroitin sulfate (OSCS) (H. Liu et al., 2009), or nonporcine heparin, such as bovine or ovine heparins (Ouyang et al., 2019). The adulteration ingredients can cause severe consequences. For example, OSCS-contaminated heparin led to the death of nearly 100 patients (H. Liu et al., 2009). In Brazil, the mixed use of bovine heparin and porcine heparin has led to serious bleeding events (Vilanova et al., 2019). Since heparin obtained from different animals or tissues shows certain differences in physicochemical properties (molecular weight distribution, disaccharide composition) (Fu et al., 2013), anticoagulant activity (Gotti et al., 2013) and pharmacology (in vivo efficacy, pharmacodynamics and side effects) (Jasper et al., 2015), it is important to ensure the origin and purity of pharmaceutical heparin. However, while the detection of OSCS in heparin using strong ion exchange liquid chromatography (LC) has become a routine test, there is a lack of an efficient method to differentiate bovine and ovine heparins from pharmaceutical porcine heparin, especially when part of the ruminant heparin is adulterated with porcine heparin.

Some indirect methods, such as real-time quantitative polymerase chain reaction (qPCR) analysis to detect residual DNA (Concannon et al., 2011) and immunochemical analysis to detect residual proteins (Levieux et al., 2002), have been developed. However, since heparin manufacturing involves several steps under harsh conditions, the undamaged DNA and proteins remaining in heparin usually exist in trace amounts. Furthermore, heparin itself is a DNA amplification enzyme inhibitor, which easily leads to false negative results (Schrader et al., 2012). Therefore, there is still a need for an efficient and sensitive method capable of determining the animal origins of heparin for industry as well as regulatory agencies.

Heparin is a linear polysaccharide but has complex and heterogeneous sequences. Unlike nucleic acids and proteins, which are template-directed synthesized molecules with defined and unique sequences distinguishable by use of qPCR method or mass spectrometry (MS)-based proteomic method, a signature sequence indicating the tissue or animal origin of heparin has not yet been reported. For example, collagens from pigs, cows, horses and donkeys can be unambiguously determined by monitoring their corresponding marker peptides using a LC-MS multiple reaction monitoring (MRM) approach (Li et al., 2017). However, the same strategy cannot be applied directly to differentiate the origin of heparin due to the lack of a marker oligosaccharide. Heparins derived from porcine, bovine and ovine are all comprised of repeating disaccharide units of hexuronic acid residue (HexA), either iduronic acid residue (IdoA) or glucuronic acid residue (GlcA), 1 → 4 linked to glucosamine residue (GlcN). Substitution groups, including the 2-O-sulfo group at HexA and the 3-O-sulfo group, 6-O-sulfo group, N-sulfo group, and/or N-acetylation group at GlcN, are distributed along the heparin backbone. A pentasaccharide sequence, GlcNAc6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S, occurs at a low frequency but is responsible for the anticoagulant activity of heparin due to specific binding to and activation of the serine protease inhibitor antithrombin III (Lindahl et al., 1980). The composition of repeating disaccharides and substitution patterns are different within porcine, bovine and ovine heparin, which can be observed by nuclear magnetic resonance (NMR) spectroscopy, showing that the N-acetylation level of porcine heparin is significantly higher than that in bovine and ovine heparin (Fu et al., 2013). Furthermore, a higher resolution quantitative 2D-NMR method was established to distinguish the origin of heparin and evaluate the quality of industrial production process based on the percentage of mono- and disaccharides (Mauri et al., 2019). Ouyang et al. detected approximately 25 % bovine or ovine heparin blended in porcine heparin by NMR method combined with principal component analysis (PCA) analysis, and when correlating the data of HPLC-MS disaccharides and tetrasaccharides to clustering analysis, this method can detect heparin contamination by other sources down to 10 % contamination levels (Ouyang et al., 2019). Colombo et al. also reported an NMR method with stoichiometric analysis, which further pushed the limit of discriminating porcine heparin with 8 % of ovine or bovine heparin (Colombo et al., 2022). In addition, the multiplex analytical method (including HPLC, SEC, NMR and statistical analysis) established by Sargison et al. to distinguish the very closely related heparan sulfates is also well worth trying to find markers that distinguish heparins from different animals (Sargison et al., 2022). The NMR technique is promising to solve the identification issue of heparin origin. Meanwhile, it is interesting to explore the use of MS technique to see if an optional or better method could be established to help to understand the essential structural difference of heparin from different sources.

In this study, we examined multiple lots of heparins derived from bovine, ovine and porcine animals. Since heparin is constituted by repeating disaccharides, we use exhaustive enzyme digestion to break the polysaccharide chains into their basic building blocks. Most of them are unsaturated disaccharides with different substitution patterns, reflecting the backbone structure of heparin. Some of them are tetrasaccharides, which contain the 3-O-sulfated GlcN related to the heparin anticoagulant activity and are enzyme-resistant. Odd numbered oligosaccharides, including monosaccharides and trisaccharides, are also present and derived from the terminus of the original heparin chain. By analyzing the composition of these basic building blocks using LC-MS, differences were found in the structure of natural polysaccharides as well as in modified structures during the manufacturing process within heparins obtained from different animal species. Based on the difference of building blocks, A LC-MS/MS MRM method was then established to quantify the heparin basic blocks that reflect the structural differences, serving as markers to differentiate bovine and ovine heparins from porcine heparin. The enzyme mediated depolymerization and quantification of the basic building blocks permits the quantification of the composition and the detection of adulteration in heparins due to the different composition of differently sourced heparin materials.

Section snippets

Materials and chemicals

Porcine intestines, ovine intestines, and bovine intestines were purchased from farmers in Inner Mongolia, Henan, Shandong, Hubei and Zhejiang Province, China. Strong anion exchange resin S5428 was purchased from Lanxess (Cologne, Germany). Alcalase was purchased from Novozymes (Bagsværd, Denmark). Standard heparin disaccharides, including eight nature heparin disaccharides (ΔIA to ΔIVA and ΔIS to ΔIVS), were purchased from Iduron (Manchester, UK). Heparinase I, II and III were purchased from

Preparation of heparins from porcine, ovine and bovine intestine mucosa

Six parallel lots of porcine intestine mucosa heparin (PMH), ovine intestine mucosa heparin (OMH) and bovine intestine mucosa heparin (BMH) were prepared from corresponding animal tissues of location diversity. The animal tissues were collected by farmers from different provinces in China to reflect the possible structural diversity of heparins from different regions. Each batch of animal tissues were from multiple animals with the same species and same location. Other information, such as the

Conclusion

Since heparin is the most important anticoagulant in the pharmaceutical industry, drug safety and a stable supply of heparin are priority concerns. A single animal source, pig intestinal mucosa, makes the supply chain for heparin very fragile. On the one hand, possible adulteration of ruminant heparins, including OMH and BMH, leads to more difficulties for the surveillance of heparin for both drug regulatory agencies and heparin manufacturers. On the other hand, the Food and Drug Administration

CRediT authorship contribution statement

Conceptualization: Lianli Chi, Feng Shi

Methodology: Bin Zhang, Deling Shi, Mengmeng Li

Validation: Bin Zhang, Deling Shi

Formal analysis: Bin Zhang, Deling Shi

Resources: Lianli Chi, Feng Shi

Writing - Original Draft: Bin Zhang

Writing - Review & Editing: Lianli Chi, Feng Shi

Funding acquisition: Lianli Chi

All authors have read and agreed to the published version of the manuscript.

Declaration of competing interest

All authors declare that there are no conflicts of interest in this work.

Acknowledgements

This work was supported by the grant from the National Key Research and Development Program of China (2021YFC2103104) and the National Natural Science Foundation of China (21877072).

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These authors have contributed equally to this work as co-first authors.

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