Biosynthetic production of anticoagulant heparin polysaccharides through metabolic and sulfotransferases engineering strategies.

Heparin is an important anticoagulant drug, and microbial heparin biosynthesis is a potential alternative to animal-derived heparin production. However, effectively using heparin synthesis enzymes faces challenges, especially with microbial recombinant expression of active heparan sulfate N-deacetylase/N-sulfotransferase. Here, we introduce the monosaccharide N-trifluoroacetylglucosamine into Escherichia coli K5 to facilitate sulfation modification. The Protein Repair One-Stop Service-Focused Rational Iterative Site-specific Mutagenesis (PROSS-FRISM) platform is used to enhance sulfotransferase efficiency, resulting in the engineered NST-M8 enzyme with significantly improved stability (11.32-fold) and activity (2.53-fold) compared to the wild-type N-sulfotransferase. This approach can be applied to engineering various sulfotransferases. The multienzyme cascade reaction enables the production of active heparin from bioengineered heparosan, demonstrating anti-FXa (246.09 IU/mg) and anti-FIIa (48.62 IU/mg) activities. This study offers insights into overcoming challenges in heparin synthesis and modification, paving the way for the future development of animal-free heparins using a cellular system-based semisynthetic strategy.

Saturated tetrasaccharide profile of enoxaparin. An additional piece to the heparin biosynthesis puzzle.

Enoxaparin, widely used antithrombotic drug, is a polydisperse glycosaminoglycan with highly microheterogeneous structure dictated by both parent heparin heterogeneity and depolymerization conditions. While the process-related modifications of internal and terminal sequences of enoxaparin have been extensively studied, very little is known about the authentic non-reducing ends (NRE). In the present study a multi-step isolation and thorough structural elucidation by NMR and LC/MS allowed to identify 16 saturated tetramers along with 23 unsaturated ones in the complex enoxaparin tetrasaccharide fraction. Altogether the elucidated structures represent a unique enoxaparin signature, whereas the composition of saturated tetramers provides a structural readout strictly related to the biosynthesis of parent heparin NRE. In particular, both glucuronic and iduronic acids were detected at the NRE of macromolecular heparin. The tetrasaccharides bearing glucosamine at the NRE are most likely associated with the heparanase hydrolytic action. High sulfation degree and 3-O-sulfation are characteristic for both types of NRE.

ZNF263 is a transcriptional regulator of heparin and heparan sulfate biosynthesis.

Heparin is the most widely prescribed biopharmaceutical in production globally. Its potent anticoagulant activity and safety makes it the drug of choice for preventing deep vein thrombosis and pulmonary embolism. In 2008, adulterated material was introduced into the heparin supply chain, resulting in several hundred deaths and demonstrating the need for alternate sources of heparin. Heparin is a fractionated form of heparan sulfate derived from animal sources, predominantly from connective tissue mast cells in pig mucosa. While the enzymes involved in heparin biosynthesis are identical to those for heparan sulfate, the factors regulating these enzymes are not understood. Examination of the promoter regions of all genes involved in heparin/heparan sulfate assembly uncovered a transcription factor-binding motif for ZNF263, a C2H2 zinc finger protein. CRISPR-mediated targeting and siRNA knockdown of ZNF263 in mammalian cell lines and human primary cells led to dramatically increased expression levels of HS3ST1, a key enzyme involved in imparting anticoagulant activity to heparin, and HS3ST3A1, another glucosaminyl 3-O-sulfotransferase expressed in cells. Enhanced 3-O-sulfation increased binding to antithrombin, which enhanced Factor Xa inhibition, and binding of neuropilin-1. Analysis of transcriptomics data showed distinctively low expression of ZNF263 in mast cells compared with other (non-heparin-producing) immune cells. These findings demonstrate a novel regulatory factor in heparan sulfate modification that could further advance the possibility of bioengineering anticoagulant heparin in cultured cells.

High-throughput method for in process monitoring of 3-O-sulfotransferase catalyzed sulfonation in bioengineered heparin synthesis.

Bioengineered heparin (BEH) offers a potential alternative for the preparation of a safer pharmacological heparin. Construction of in-process control assays for tracking each enzymatic step during bioengineered heparin synthesis remains a challenge. Here, we report a high-throughput sensing platform based on enzyme-linked immunosorbent assay (ELISA) and enzymatic signal amplification that allows the rapid and accurate monitoring of the 3-OST sulfonation in BEH synthesis process. The anticoagulant activity of target BEH was measured to reflect the degree of sulfonation by testing its competitive antithrombin (AT) binding ability. BEH samples with different sulfonation degrees show different AT protein binding capacity and thus changes the UV response to a different extent. This BEH-induced signal can be conveniently and sensitively monitored by the plate sensing system, which benefits from its high sensitivity brought in by the enzymatic signal amplification. Furthermore, modification convenience and mechanical robustness also ensure the stability of the test platform. This proposed strategy exhibits excellent analytical performance in both BEH activity analysis and 3-OST sulfonation evaluation. The simple and sensitive plate system shows great potential in developing on-chip, high-throughput methods for fundamental biochemical process research, drug discovery, and clinic diagnostics.

Chemoenzymatic quantification for monitoring unpurified polysaccharide in rich medium.

The heparosan polysaccharide serves as the starting carbon backbone for the chemoenzymatic synthesis of heparin, a widely used clinical anticoagulant drug. The previous quantification methods for heparosan rely on time-consuming purification or expensive instruments not readily available for many labs. Here, a chemoenzymatic approach is developed to monitor the production of heparosan in rich medium without purification. After removing the interfering small molecules by ultrafiltration, heparosan was decomposed into oligosaccharides using heparin lyase III. The oligosaccharides were separated from large molecules by ultrafiltration and quantitatively determined by the anthrone-sulfuric acid assay using a spectrophotometer. Based on the different substrate specificity of heparin lyases, the study showed that the concentration of heparosan and heparin in a mixture was discriminatively determined by the two-step chemoenzymatic assay. Furthermore, the anthrone-sulfuric acid assay was observed to be more reliable than the phenol-sulfuric acid assay under these conditions. Besides heparosan and heparin, the chemoenzymatic assay may be adapted to quantify other types of polysaccharides if the specific lyases were available.

By-Products of Heparin Production Provide a Diverse Source of Heparin-like and Heparan Sulfate Glycosaminoglycans.

Global production of pharmaceutical heparin (Hp) is increasing, and the production process from raw mucosal material results in large amounts of waste by-products. These contain lower sulfated Hp-like and heparan sulfate (HS), as well as other glycosaminoglycans, which are bioactive entities with pharmaceutical potential. Here we describe the first purification, structural and functional characterisation of Hp-like and HS polysaccharides from the four major by-product fractions of standard heparin production. Analysis of the by-products by disaccharide composition analysis and NMR demonstrated a range of structural characteristics which differentiate them from Hp (particularly reduced sulfation and sulfated disaccharide content), and that they are each distinct. Functional properties of the purified by-products varied, each displaying distinct anticoagulant profiles in different assays, and all exhibiting significantly lower global and specific inhibition of the coagulation pathway than Hp. The by-products retained the ability to promote cell proliferation via fibroblast growth factor receptor signalling, with only minor differences between them. These collective analyses indicate that they represent an untapped and economical source of structurally-diverse Hp-like and HS polysaccharides with the potential for enhancing future structure-activity studies and uncovering new biomedical applications of these important natural products.

Advances in Heparins and Related Research. An Epilogue.

The discovery of heparin in 1916 by Jay McLean, a medical student at Johns Hopkins University, not only provided a universal anticoagulant, but also laid the foundation for the discipline of hemostasis and thrombosis[...].

Enzymatic Generation of Highly Anticoagulant Bovine Intestinal Heparin.

Unlike USP porcine heparin, bovine intestinal heparin (BIH) has a low anticoagulant activity. Treatment with 6-OST-1, -3, and/or 3-OST-1 afforded two remodeled heparins that met USP heparin activity and Mw specifications. We explored the pharmacodynamics and pharmacokinetics in a rabbit model. We conclude that a modest increase in the content of 3-O-sulfo groups in BIH increases the number of antithrombin III binding sites, making remodeled BIH behave similarly to pharmaceutical heparin.

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.

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.

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.

Expression and secretion of glycosylated heparin biosynthetic enzymes using Komagataella pastoris.

Heparin, an anticoagulant drug, is biosynthesized in selected animal cells. The heparin biosynthetic enzymes mainly consist of sulfotransferases and all are integral transmembrane glycoproteins. These enzymes are generally produced in engineered Escherichia coli as without their transmembrane domains as non-glycosylated fusion proteins. In this study, we used the yeast, Komagataella pastoris, to prepare four sulfotransferases involved in heparin biosynthesis as glycoproteins. While the yields of these yeast-expressed enzymes were considerably lower than E. coli-expressed enzymes, these enzymes were secreted into the fermentation media simplifying their purification and were endotoxin free. The activities of these sulfotransferases, expressed as glycoproteins in yeast, were compared to the bacterially expressed proteins. The yeast-expressed sulfotransferase glycoproteins showed improved kinetic properties than the bacterially expressed proteins.

Bioengineered human heparin with anticoagulant activity.

Heparin is a carbohydrate anticoagulant used clinically to prevent thrombosis, however impurities can limit its efficacy. Here we report the biosynthesis of heparin-like heparan sulfate via the recombinant expression of human serglycin in human cells. The expressed serglycin was also decorated with chondroitin/dermatan sulfate chains and the relative abundance of these glycosaminoglycan chains changed under different concentrations of glucose in the culture medium. The recombinantly expressed serglycin produced with 25mM glucose present in the culture medium was found to possess anticoagulant activity one-seventh of that of porcine unfractionated heparin, demonstrating that bioengineered human heparin-like heparan sulfate may be a safe next-generation pharmaceutical heparin.

Targeting of Proteoglycan Synthesis Pathway: A New Strategy to Counteract Excessive Matrix Proteoglycan Deposition and Transforming Growth Factor-ß1-Induced Fibrotic Phenotype in Lung Fibroblasts.

Stimulation of proteoglycan (PG) synthesis and deposition plays an important role in the pathophysiology of fibrosis and is an early and dominant feature of pulmonary fibrosis. Transforming growth factor-ß1 (TGF-ß1) is a major cytokine associated with fibrosis that induces excessive synthesis of matrix proteins, particularly PGs. Owing to the importance of PGs in matrix assembly and in mediating cytokine and growth factor signaling, a strategy based on the inhibition of PG synthesis may prevent excessive matrix PG deposition and attenuates profibrotic effects of TGF-ß1 in lung fibroblasts. Here, we showed that 4-MU4-deoxy-ß-D-xylopyranoside, a competitive inhibitor of ß4-galactosyltransferase7, inhibited PG synthesis and secretion in a dose-dependent manner by decreasing the level of both chondroitin/dermatan- and heparin-sulfate PG in primary lung fibroblasts. Importantly, 4-MU4-deoxy-xyloside was able to counteract TGF-ß1-induced synthesis of PGs, activation of fibroblast proliferation and fibroblast-myofibroblast differentiation. Mechanistically, 4-MU4-deoxy-xyloside treatment inhibited TGF-ß1-induced activation of canonical Smads2/3 signaling pathway in lung primary fibroblasts. The knockdown of ß4-galactosyltransferase7 mimicked 4-MU4-deoxy-xyloside effects, indicating selective inhibition of ß4-galactosyltransferase7 by this compound. Collectively, this study reveals the anti-fibrotic activity of 4-MU4-deoxy-xyloside and indicates that inhibition of PG synthesis represents a novel strategy for the treatment of lung fibrosis.

Diverse Roles of Heparan Sulfate and Heparin in Wound Repair.

Heparan sulfate (HS) and heparin (Hp) are linear polysaccharide chains composed of repeating (1→4) linked pyrosulfuric acid and 2-amino-2-deoxy glucopyranose (glucosamine) residue. Mentioned glycosaminoglycans chains are covalently O-linked to serine residues within the core proteins creating heparan sulfate/heparin proteoglycans (HSPG). The latter ones participate in many physiological and pathological phenomena impacting both the plethora of ligands such as cytokines, growth factors, and adhesion molecules and the variety of the ECM constituents. Moreover, HS/Hp determine the effective wound healing process. Initial growth of HS and Hp amount is pivotal during the early phase of tissue repair; however heparan sulfate and heparin also participate in further stages of tissue regeneration.

A purification process for heparin and precursor polysaccharides using the pH responsive behavior of chitosan.

The contamination crisis of 2008 has brought to light several risks associated with use of animal tissue derived heparin. Because the total chemical synthesis of heparin is not feasible, a bioengineered approach has been proposed, relying on recombinant enzymes derived from the heparin/HS biosynthetic pathway and Escherichia coli K5 capsular polysaccharide. Intensive process engineering efforts are required to achieve a cost-competitive process for bioengineered heparin compared to commercially available porcine heparins. Towards this goal, we have used 96-well plate based screening for development of a chitosan-based purification process for heparin and precursor polysaccharides. The unique pH responsive behavior of chitosan enables simplified capture of target heparin or related polysaccharides, under low pH and complex solution conditions, followed by elution under mildly basic conditions. The use of mild, basic recovery conditions are compatible with the chemical N-deacetylation/N-sulfonation step used in the bioengineered heparin process. Selective precipitation of glycosaminoglycans (GAGs) leads to significant removal of process related impurities such as proteins, DNA and endotoxins. Use of highly sensitive liquid chromatography-mass spectrometry and nuclear magnetic resonance analytical techniques reveal a minimum impact of chitosan-based purification on heparin product composition.

Optimization of bioprocess conditions improves production of a CHO cell-derived, bioengineered heparin.

Heparin is the most widely used anticoagulant drug in the world today. Heparin is currently produced from animal tissues, primarily porcine intestines. A recent contamination crisis motivated development of a non-animal-derived source of this critical drug. We hypothesized that Chinese hamster ovary (CHO) cells could be metabolically engineered to produce a bioengineered heparin, equivalent to current pharmaceutical heparin. We previously engineered CHO-S cells to overexpress two exogenous enzymes from the heparin/heparan sulfate biosynthetic pathway, increasing the anticoagulant activity ∼100-fold and the heparin/heparan sulfate yield ∼10-fold. Here, we explored the effects of bioprocess parameters on the yield and anticoagulant activity of the bioengineered GAGs. Fed-batch shaker-flask studies using a proprietary, chemically-defined feed, resulted in ∼two-fold increase in integrated viable cell density and a 70% increase in specific productivity, resulting in nearly three-fold increase in product titer. Transferring the process to a stirred-tank bioreactor increased the productivity further, yielding a final product concentration of ∼90 µg/mL. Unfortunately, the product composition still differs from pharmaceutical heparin, suggesting that additional metabolic engineering will be required. However, these studies clearly demonstrate bioprocess optimization, in parallel with metabolic engineering refinements, will play a substantial role in developing a bioengineered heparin to replace the current animal-derived drug.

Combinatorial one-pot chemoenzymatic synthesis of heparin.

Contamination in heparin batches during early 2008 has resulted in a significant effort to develop a safer bioengineered heparin using bacterial capsular polysaccharide heparosan and recombinant enzymes derived from the heparin/heparan sulfate biosynthetic pathway. This requires controlled chemical N-deacetylation/N-sulfonation of heparosan followed by epimerization of most of its glucuronic acid residues to iduronic acid and O-sulfation of the C2 position of iduronic acid and the C3 and C6 positions of the glucosamine residues. A combinatorial study of multi-enzyme, one-pot, in vitro biocatalytic synthesis, carried out in tandem with sensitive analytical techniques, reveals controlled structural changes leading to heparin products similar to animal-derived heparin active pharmaceutical ingredients. Liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy analysis confirms an abundance of heparin's characteristic trisulfated disaccharide, as well as 3-O-sulfo containing residues critical for heparin binding to antithrombin III and its anticoagulant activity. The bioengineered heparins prepared using this simplified one-pot chemoenzymatic synthesis also show in vitro anticoagulant activity.

High cell density cultivation of recombinant Escherichia coli strains expressing 2-O-sulfotransferase and C5-epimerase for the production of bioengineered heparin.

Bioengineered heparin is being investigated as a potential substitute for the animal-sourced anticoagulant drug. One step in the current process to prepare bioengineered heparin involves the conversion of N-sulfo heparosan, rich in → 4)GlcNS(1 → 4) GlcA(1 → sequences (where S is sulfo, GlcN is α-D-glucosamine, and GlcA is ß-D-glucuronic acid), to a critical intermediate, rich in → 4)GlcNS(1 → 4) IdoA2S(1 → sequences (where S is sulfo and IdoA is α-L-iduronic acid), using 2-O-sulfotransferase (2-OST) and C5 epimerase (C5-epi). Until now, these heparan sulfate biosynthetic enzymes have been expressed in Escherichia coli grown in shake flask culture as fusion proteins. The current study is focused on the high cell density fed-batch cultivation of recombinant E. coli strains expressing both enzymes. We report the high productivity expression of active 2-OST and C5-epi enzymes of 6.0 and 2.2 mg/g dry cell weight, respectively.

Viral diversity in swine intestinal mucus used for the manufacture of heparin as analyzed by high-throughput sequencing.

Heparin is one of the main pharmaceutical products manufactured from raw animal material. In order to describe the viral burden associated with this raw material, we performed high-throughput sequencing (HTS) on mucus samples destined for heparin manufacturing, which were collected from European pigs. We identified Circoviridae and Parvoviridae members as the most prevalent contaminating viruses, together with viruses from the Picornaviridae, Astroviridae, Reoviridae, Caliciviridae, Adenoviridae, Birnaviridae, and Anelloviridae families. Putative new viral species were also identified. The load of several known or novel small non-enveloped viruses, which are particularly difficult to inactivate or eliminate during heparin processing, was quantified by qPCR. Analysis of the combined HTS and specific qPCR results will influence the refining and validation of inactivation procedures, as well as aiding in risk analysis of viral heparin contamination.