Processing bovine intestinal mucosa to active heparin removes spiked BSE agent.

Heparin is an anticoagulant sourced from animal tissues. In the 1990s, bovine-sourced heparin was withdrawn from the U.S. market due to a theoretical concern that the bovine spongiform encephalopathy (BSE) agent might contaminate crude heparin and spread to humans as variant Creutzfeldt-Jakob disease. Only porcine intestinal heparin is now marketed in the U.S. FDA has encouraged the reintroduction of bovine heparin. We applied a scaled-down laboratory model process to produce heparin as an active pharmaceutical ingredient (API) starting from bovine intestinal mucosa. The process consisted of two phases. To model the first phase, we applied enzymatic proteolysis, anionic resin separation and methanol precipitation of crude heparin. Bovine intestinal mucosa was spiked with BSE or scrapie agents. We assayed BSE- or scrapie-associated prion protein (PrPTSE) using the Real-Time Quaking-Induced Conversion (RT-QuIC) assay at each step. The process reduced PrPTSE by 4 log10 and 6 log10 from BSE-spiked and scrapie-spiked mucosa, respectively. To model the entire process, we spiked mucosa with scrapie agent and produced heparin API, reducing PrPTSE by 6.7 log10. The purification processes removed large amounts of PrPTSE from the final products. Heparin purification together with careful sourcing of raw materials should allow safely reintroducing bovine heparin in the U.S.

Au nanoparticles as label-free competitive reporters for sensitivity enhanced fiber-optic SPR heparin sensor.

A label-free Au NPs-enhanced surface plasmon resonance (SPR) sensor was developed for the ultrasensitive detection of heparin based on competitive adsorption behavior of heparin and Au NPs on the poly (dimethyl-diallylammonium chloride) (PDDA)-modified optical fiber surface and the corresponding change in the resonance wavelength of SPR. Due to the high affinity between heparin and PDDA, the present senor shows good analytical performance with respect to heparin detection. Two obvious advantages of the proposed heparin sensor over other reported methods are: its much wider linear concentration range (10-6-10-10 g/mL) and lower limit of detection (0.0257 ng/mL). The analysis of heparin in serum demonstrated that the present sensor exhibited high sensitivity and selectivity. It should be noted that the sensing strategy takes advantage of a portable fiber-optic SPR sensing system and avoids the need for complex processes for labeled-Au NPs, and thus the present sensor promises to be a practical tool for the point-of-care monitoring of heparin.

Isolation and Characterization of a Heparin-Like Compound with Potent Anticoagulant and Fibrinolytic Activity from the Clam Coelomactra antiquata.

Heparin from mollusks with unique sulfated glycosaminoglycan exhibits strong anti-thrombotic activities. This study reports on a purified heparinoid from Coelomactra antiquata, which shows potent anticoagulant and fibrinolytic abilities. Its structure was characterized by infrared spectroscopy, high-performance liquid chromatography, and one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy. Its fibrinolytic activity was determined in vitro and in vivo. Its anticoagulant activity was determined by activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT). The results indicated that clam heparinoid was a homogeneous glycosaminoglycan with a molecular weight of 30.99 kDa, mainly composed of →4)-α-IdoA2S-(1→4)-α-GlcNS3S6S (or GlcNS6S)-(1→4)-ß-GlcA-(1→4)-α-GlcNS6S (or GlcNAC)-(1→. Furthermore, this heparinoid showed a highly anticoagulant titer and fibrinolytic value of 149.63 IU/mg and 1.96 IU/mg, respectively. In summary, clam heparinoid shows great potential for application in the clinic and antithrombotic drugs industry.

Salt-free fractionation of complex isomeric mixtures of glycosaminoglycan oligosaccharides compatible with ESI-MS and microarray analysis.

Heparin and heparan sulfate (Hp/HS) are linear complex glycosaminoglycans which are involved in diverse biological processes. The structural complexity brings difficulties in separation, making the study of structure-function relationships challenging. Here we present a separation method for Hp/HS oligosaccharide fractionation with cross-compatible solvent and conditions, combining size exclusion chromatography (SEC), ion-pair reversed phase chromatography (IPRP), and hydrophilic interaction chromatography (HILIC) as three orthogonal separation methods that do not require desalting or extensive sample handling. With this method, the final eluent is suitable for structure-function relationship studies, including tandem mass spectrometry and microarray printing. Our data indicate that high resolution is achieved on both IPRP and HILIC for Hp/HS isomers. In addition, the fractions co-eluted in IPRP could be further separated by HILIC, with both separation dimensions capable of resolving some isomeric oligosaccharides. We demonstrate this method using both unpurified reaction products from isomeric synthetic hexasaccharides and an octasaccharide fraction from enoxaparin, identifying isomers resolved by this multi-dimensional separation method. We demonstrate both structural analysis by MS, as well as functional analysis by microarray printing and screening using a prototypical Hp/HS binding protein: basic-fibroblast growth factor (FGF2). Collectively, this method provides a strategy for efficient Hp/HS structure-function characterization.

Crude Heparin Preparations Unveil the Presence of Structurally Diverse Oversulfated Contaminants.

Nowadays, pharmaceutical heparin is purified from porcine and bovine intestinal mucosa. In the past decade there has been an ongoing concern about the safety of heparin, since in 2008, adverse effects associated with the presence of an oversulfated chondroitin sulfate (OSCS) were observed in preparations of pharmaceutical porcine heparin, which led to the death of patients, causing a global public health crisis. However, it has not been clarified whether OSCS has been added to the purified heparin preparation, or whether it has already been introduced during the production of the raw heparin. Using a combination of different analytical methods, we investigate both crude and final heparin products and we are able to demonstrate that the sulfated contaminants are intentionally introduced in the initial steps of heparin preparation. Furthermore, the results show that the oversulfated compounds are not structurally homogeneous. In addition, we show that these contaminants are able to bind to cells in using well known heparin binding sites. Together, the data highlights the importance of heparin quality control even at the initial stages of its production.

Detection of coralyne and heparin by polymerase extension reaction using SYBR Green I.

Polydeoxyadenosine (poly (dA)) has been extensively applied for detecting many drug molecules. Herein, we developed a sensitive method for detecting coralyne and heparin using a modified DNA probe with poly (dA) at one end. In the absence of coralyne, the DNA probe was digested by the Exonuclease I (Exo I), and therefore the SYBR Green I (SG I) emitted an extremely low fluorescent signal. While coralyne specifically binding to poly (dA) with strong propensity could remarkably restrain the disintegration of the DNA probe, through which as a template the second strand of DNA sequence was formed with the introduction of DNA polymerase. Therefore, the fluorescent signal of SG I was intensified to quantify coralyne. Based on this method, heparin can be determined due to its strong affinity towards coralyne. This method showed a linear range from 2 to 500 nM for coralyne with a low detection limit of 0.98 nM, and the linear range of heparin was from 1 to 100 nM when 1.25 nm was the detection limit. The proposed method was also implemented successfully in biological samples and showed a potential application for screening potential therapeutic molecules.

Imminent risk of a global shortage of heparin caused by the African Swine Fever afflicting the Chinese pig herd.

Most of the unfractionated and low-molecular-weight heparins available worldwide are produced by Chinese companies from porcine mucosa. China is the world's largest producer of pork and thus has plenty of raw material to produce heparins. However, the deadly African Swine Fever (ASF) outbreaks afflicting China since August 2018 may cause extensive losses to the pig herd, with serious consequences for the global supply of heparins. In 2008, a sudden shortage of heparin's raw material resulting from a viral disease in Chinese pigs prompted adulterations responsible for 80 deaths and hundreds of adverse events. This incident revealed the fragility of such a supply chain, which is mostly based on raw material from a single animal from a single country. A worldwide introduction of bovine mucosa heparins manufactured in different countries certainly is a feasible way to mitigate eventual shortages of these life-saving anticoagulants caused by local veterinary problems such as the ASF threatening China now.

Adenosine-Related Compounds as an Enhancer for Peroxidase-Mimicking Activity of Nanomaterials: Application to Sensing of Heparin Level in Human Plasma and Total Sulfate Glycosaminoglycan Content in Synthetic Cerebrospinal Fluid.

A variety of compounds, such as DNA and protein, have been demonstrated to be effective in suppressing the catalytic activity of peroxidase-like nanomaterials. However, little investigations have been conducted to discover new chemical compounds for amplifying the catalytic activity of peroxidase-mimicking nanomaterials. This study discloses that adenosine analogues were useful as a universal enhancer for peroxidase-mimicking nanomaterials in the hydrogen peroxide-mediated oxidation of amplex ultrared at neutral pH. The optimal adenosine analogues for improving the peroxidase-like performance of citrate-stabilized gold nanoparticles (Au NPs), citrate-capped platinum NPs, bovine serum albumin-encapsulated gold nanoclusters, and unmodified magnetite NPs were found to be adenosine diphosphate (ADP), ADP, ADP, and adenosine monophosphate, respectively. The results show that adenosine analogue-induced enhancement in the peroxidase-like activity of nanomaterials was heavily associated with the number of adsorbed adenosine analogues onto the nanomaterial surface. The analysis of ADP-modified Au NPs by electron paramagnetic resonance spectroscopy indicates that the adsorbed ADP molecules on the Au NP surface not only activated H2O2 but also strengthened the interaction between hydroxyl radicals and nanomaterials. By integrating the ADP-boosted catalytic activity of peroxidase-like Au NPs, surfen-triggered NP aggregation, and specific surfen-sulfated glycosaminoglycan (GAG) interaction, a turn-on fluorescent probe was constructed to quantify the heparin level in human plasma and total sulfate GAG content in synthetic cerebrospinal fluid.

Heparin: role in protein purification and substitution with animal-component free material.

Heparin is a highly sulfated polysaccharide which belongs to the family of glycosaminoglycans. It is involved in various important biological activities. The major biological purpose is the inhibition of the coagulation cascade to maintain the blood flow in the vasculature. These properties are employed in several therapeutic drugs. Heparin's activities are associated with its interaction to various proteins. To date, the structural heparin-protein interactions are not completely understood. This review gives a general overview of specific patterns and functional groups which are involved in the heparin-protein binding. An understanding of the heparin-protein interactions at the molecular level is not only advantageous in the therapeutic application but also in biotechnological application of heparin for downstreaming. This review focuses on the heparin affinity chromatography. Diverse recombinant proteins can be successfully purified by this method. While effective, it is disadvantageous that heparin is an animal-derived material. Animal-based components carry the risk of contamination. Therefore, they are liable to strict quality controls and the validation of effective good manufacturing practice (GMP) implementation. Hence, adequate alternatives to animal-derived components are needed. This review examines strategies to avoid these disadvantages. Thereby, alternatives for the provision of heparin such as chemical synthesized heparin, chemoenzymatic heparin, and bioengineered heparin are discussed. Moreover, the usage of other chromatographic systems mimetic the heparin effect is reviewed.

B'reshith.

The Hebrew word "b'reshith" (בְּרֵאשִׁית) means "in the beginning". It is the first word and title of the Book of Genesis, and it describes a process of creation. The four authors were present at the beginning of Langer labs, and the purpose of this essay is to convey the scientific and technological zeitgeist that existed in the late 1970s and early 1980s, when Bob Langer began his exceptionally creative work. While Langer labs has branched into many other areas, Bob's unique ability to recognize important problems and entice people to look beyond their own disciplines to solve them was evident from the start. We focus on the two areas of most interest to Bob at the time, namely controlled release of macromolecules from polymers, and removal of heparin in order to prevent uncontrolled bleeding during surgery.

Chemical and pharmacological aspects of neutralization of heparins from different animal sources by protamine.

Essentials Bovine (HBI) and porcine (HPI) heparins differ in structure and anticoagulant activity. Protamine-neutralization was evaluated on a variety of physical-chemical methods. HBI requires more protamine than HPI to fully neutralize its anticoagulant activity. Protamine preferentially removes higher-sulfated chains of HBI while HPI is evenly precipitated. SUMMARY: Background Protamine neutralization is an essential step for the safe use and inactivation of the unfractionated heparin (UFH) that is widely employed in surgical and non-surgical procedures involving extracorporeal circulation. Objective To compare protamine neutralization of different pharmaceutical-grade UFHs prepared from porcine or bovine intestine (HPI and HBI, respectively). HBI has approximately half the anticoagulant potency of HPI, mostly as consequence of its fraction enriched with N-sulfated α-glucosamine disaccharides. Methods Protamine neutralization of HPI and HBI was evaluated with in vitro, ex vivo and in vivo assays. We also performed in-depth assessments of the complexation of protamine with these distinct UFHs by using nuclear magnetic resonance and mass spectroscopy. Results HPI and HBI interact similarly with protamine on a mass/mass basis; however, HBI requires more protamine than HPI to have its anticoagulant activity fully neutralized, because of its lower potency, which entails the use of higher doses. Nuclear magnetic resonance spectra revealed that HPI precipitates homogeneously with protamine. On the other hand, the low-sulfated fraction of HBI, enriched with N-sulfated α-glucosamine, precipitates at higher concentrations of protamine than the fraction more like HPI, with a preponderance of N,6-disulfated α-glucosamine disaccharides. Finally, mass spectroscopy spectra showed that some of the different peptide components of protamine interact preferentially with the heparins, irrespective of their animal origin. Conclusion Our results have important medical implications, indicating that protamine neutralization of HBI, determined exclusively by point-of-care coagulation assessments, must fail because of its lower-sulfated fraction with reduced anticoagulant activity that could remain in the circulation after the neutralization procedure.

A Turn-on Fluorescence Sensor for Heparin Detection Based on a Release of Taiwan Cobra Cardiotoxin from a DNA Aptamer or Adenosine-Based Molecular Beacon.

This study presents two sensitive fluorescent assays for sensing heparin on the basis of the electrostatic interaction between heparin and Naja naja atra cardiotoxin 3 (CTX3). Owing to CTX3-induced folded structure of an adenosine-based molecular beacon (MB) or a DNA aptamer against CTX3, a reduction in the fluorescent signal of the aptamer or MB 5'-end labeled with carboxyfluorescein (FAM) and 3'-end labeled with 4-([4-(dimethylamino)phenyl]azo)-benzoic acid (DABCYL) was observed upon the addition of CTX3. The presence of heparin and formation of the CTX3-heparin complex caused CTX3 detachment from the MB or aptamer, and restoration of FAM fluorescence of the 5'-FAM-and-3'-DABCYL-labeled MB and aptamer was subsequently noted. Moreover, the detection of heparin with these CTX3-aptamer and CTX3-MB sensors showed high sensitivity and selectivity toward heparin over chondroitin sulfate and hyaluronic acid regardless of the presence of plasma. The limit of detection for heparin in plasma was determined to be 16 ng/mL and 15 ng/mL, respectively, at a signal-to-noise ratio of 3. This study validates the practical utility of the CTX3-aptamer and CTX3-MB systems for determining the concentration of heparin in a biological matrix.

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

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.

Quantification and comparison of acidic polysaccharides in edible fish intestines and livers using HPLC-MS/MS.

Fish intestines and livers are usually considered as delicious and nutritious food in China. Acidic polysaccharides are important nutrients in these food of animal origin, but there is currently little information regarding their quantitative distributions. The present study demonstrated a method to quantify acidic polysaccharides simultaneously by analyzing their disaccharides produced from the acid hydrolysis using high-performance liquid chromatography (HPLC) coupled with triple quadrupole mass spectrometry. The recoveries for these acidic polysaccharides were all 97%-115% with relative standard deviation of 3.0%-9.0%. All of the acidic polysaccharides had good linearities. Then this method was applied to determine the composition of acidic polysaccharides in 5 edible fish livers and intestines. Besides well-known glycosaminoglycans (GAGs) including hyaluronic acid (HA), Chondroitin sulfate (CS), dermatan sulfate (DS) and heparin (HP), 4 novel acidic polysaccharides including 2 GAGs and 2 non-GAGs comprised of hexose-hexuronic acid repeating units were also found. CS and HP were the major acidic polysaccharides components in fish intestines and livers, respectively. The absolute amounts of acidic polysaccharides differed greatly in these fish tissues, but their proportions showed similarity in the same type of tissues. The present study demonstrated an effective method for acidic polysaccharides quantification, and revealed acidic polysaccharides compositions of edible fish livers and intestines.

From Farm to Pharma: An Overview of Industrial Heparin Manufacturing Methods.

The purification of heparin from offal is an old industrial process for which commercial recipes date back to 1922. Although chemical, chemoenzymatic, and biotechnological alternatives for this production method have been published in the academic literature, animal-tissue is still the sole source for commercial heparin production in industry. Heparin purification methods are closely guarded industrial secrets which are not available to the general (scientific) public. However by reviewing the academic and patent literature, we aim to provide a comprehensive overview of the general methods used in industry for the extraction of heparin from animal tissue.

Heparan sulfate disaccharide measurement from biological samples using pre-column derivatization, UPLC-MS and single ion monitoring.

Glycosaminoglycans are a heterogeneous family of linear polysaccharides comprised of repeating disaccharide subunits that mediate many effects at the cellular level. There is increasing evidence that the nature of these effects is determined by differences in disaccharide composition. However, the determination of GAG disaccharide composition in biological samples remains challenging and time-consuming. We have developed a method that uses derivatization and selected ion recording and RP-UPLCMS resulting in rapid separation and quantification of twelve heparin/heparin sulfate disaccharides from 5 µg GAG. Limits of detection and quantitation were 0.02-0.15 and 0.07-0.31 µg/ml respectively. We have applied this method to the novel analysis of disaccharide levels extracted from heparan sulfate and human cancer cell lines. Heparan sulfate disaccharides extracted from biological samples following actinase and heparinase incubation and derivatized using reductive amination with 2-aminoacridone. Derivatized disaccharides were analyzed used UPLC-MS with single ion monitoring. Eight HS disaccharide subunits were separated and quantified from HS and cell lines in eleven minutes per sample. In all samples the most abundant subunits present were the unsulfated ΔUA-GlcNAc, ΔUA-GlcNAc,6S and ΔUA,2S-GlcNS,6S. There was considerable variation in the proportions and concentrations of disaccharides between different cell lines. Further studies are needed to examine the significance of these differences.

Optimization of bioengineered heparin/heparan sulfate production for therapeutic applications.

Heparin has been used clinically as an anti-coagulant for more than 100 y and the major source of this therapeutic is still animal tissues. Contamination issues in some batches of heparin over 10 y ago have highlighted the need to develop alternative methods of production of this essential drug. 1 Bioengineering heparin by expressing serglycin in mammalian cells is a promising approach that was recently reported by the authors. 2 This addendum explores the approaches that the authors are taking to increase the yield of recombinantly expressed serglycin decorated with heparin/heparan sulfate focusing on cell culture and bioreactor conditions and proposes that the cell microenvironment is a key modulator of heparin biosynthesis.

A Heparin Purification Process Removes Spiked Transmissible Spongiform Encephalopathy Agent.

In 2000, bovine heparin was withdrawn from the US market for fear of contamination with bovine spongiform encephalopathy (BSE) agent, the cause of variant Creutzfeldt-Jakob disease in humans. Thus, US heparin is currently sourced only from pig intestines. Availability of alternative sources of crude heparin, a life-saving drug, would benefit public health. Bovine heparin is an obvious option, but BSE clearance by the bovine heparin manufacturing process should be evaluated. To this end, using hamster 263K scrapie as a surrogate for BSE agent, we applied a four-step bench-scale heparin purification protocol resembling a typical heparin manufacturing process to investigate removal of the spiked scrapie agent. We removed aliquots from each step and analyzed them for residual abnormal prion protein (PrPTSE) using a sensitive in vitro method, real-time quaking-induced conversion (RT-QuIC) assay, and for infectivity using animal bioassays. The purification process reduced infectivity by 3.6 log10 and removed PrPTSE, measured as seeding activity, by 3.4 log10. NaOH treatment was the most effective removal step tested. We also investigated NaOH at different concentrations and pH: the results showed that as much as 5.2 log10 of PrPTSE seeding activity was removed at pH 12.5. Thus, changes to the concentration, treatment time, and temperature of alkaline extraction might further improve removal. Our results, using a basic heparin manufacturing process, inform efforts to reintroduce safe bovine heparin in the USA.

Surprising absence of heparin in the intestinal mucosa of baby pigs.

Heparin, a member of a family of molecules called glycosaminoglycans, is biosynthesized in mucosal mast cells. This important anticoagulant polysaccharide is primarily produced by extraction of the mast cell-rich intestinal mucosa of hogs. There is concern about our continued ability to supply sufficient heparin to support the worldwide growth of advanced medical procedures from the static population of adult hogs used as food animals. While the intestinal mucosa of adult pigs is rich in anticoagulant heparin (containing a few hundred milligrams per animal), little is known about how the content of heparin changes with animal age. Using sophisticated mass spectral analysis we discovered that heparin was largely absent from the intestinal mucosa of piglets. Moreover, while the related, nonanticoagulant heparan sulfate glycosaminoglycan was present in significant amounts we found little chondroitin sulfate E also associated with mast cells. Histological evaluation of piglet intestinal mucosa showed a very low mast cell content. Respiratory mast cells have been reported in baby pigs suggesting that there was something unique about the piglets used in the current study. These piglets were raised in the relatively clean environment of a university animal facility and treated with antibiotics over their lifetime resulting in a depleted microbiome that greatly reduced the number of mast cells and heparin content of the intestinal mucosal in these animals. Thus, from the current study it remains unclear whether the lack of intestinal mast cell-derived heparin results from the young age of these animals or their exposure to their depleted microbiome.