Introduction & Market

Heparin is one of the oldest biological drugs used in treating thrombosis and hemostasis. It is a naturally occurring polysaccharide in the glycosaminoglycan (GAG) family, found primarily in mast cells. McLean discovered heparin in 1916 while attempting to isolate thromboplastic agent. After clinical trials in the 1930s and 1940s, it became the primary treatment for preventing and treating venous thromboembolism (VTE).

Unfractionated heparin (UFH) is the most basic form of natural GAG, purified from animal tissue—most commonly porcine intestine. Examples of UFH salts include Sodium Heparin and Calcium Heparin. While UFH has both polydisperse and heterogeneous sequences, it primarily consists of trisulfated disaccharide repeating units. The development of low-molecular-weight heparin (LMWH) in the 1980s expanded this drug class's utility, with LMWH now replacing UFH for many uses. As of 2013, the annual heparin market (UFH and LMWH combined) reached approximately $7 billion, representing 52% of the anticoagulant market and about 100 tons of active pharmaceutical ingredient.

LMWH: low molecular weight heparin; UFH: unfractionated heparin; VKA: vitamin K antagonists; DTI: direct thrombin inhibitors; DXI: direct factor Xa inhibitors.

src: Onishi, A., St Ange, K., Dordick, J. S. & Linhardt, R. J. Heparin and anticoagulation. Front Biosci (Landmark Ed) 21, 1372–1392 (2016).

All heparin preparations are polydisperse linear polymers, meaning their molecular weights (MWs) cannot be expressed as a single number. This polydisperse nature becomes significant when measuring properties that depend on molar concentration, including binding and kinetic constants. While UFH's physicochemical characteristics remain fairly consistent between products, subtle variations can occur, such as changes in mean MW and specific activity.

LMWHs, including enoxaparin, dalteparin, and nadropain, are produced through controlled chemical or enzymatic cleavage of UFH in a depolymerization reaction. The most common production methods are oxidation, deamination, and β-elimination. Oxidation creates polysaccharide molecules with both even and odd numbers of residues. Deaminative processes form terminal anhydromannitol residues at the reducing end, while elimination methods create an unsaturated uronic residue at the nonreducing end. More than ten LMWHs are in clinical use, all showing similar biological properties.

All LMWH products have mean molecular weights less than half that of UFH, with their key defining feature being that at least 60% of their weight must be below 8,000. Nevertheless, there are significant variations in molecular weight parameters among different LMWH products.

LMWH	Average Mw	Ratio anti-Xa/anti-IIa activity
Enoxaparin	4500	3.9
Dalteparin	6000	2.5
Nadropain	4300	3.3
Tinzaparin	6500	1.6
Parnaparin	5000	2.3
Certoparin	5400	2.4
Bemiparin	3600	9.7
Reviparin	4400	4.2

Mechanism of Action

UFH's anticoagulant activity in vitro primarily depends on a specific sequence that binds strongly to plasma serpin antithrombin. This sequence features a crucial pentasaccharide motif containing an unusual 3,6 di-O-sulfated, 2-N-sulfated glucosamine residue, though not all heparin molecules contain it. 

Generalized structure of a porcine intestinal heparin chain

src: Onishi, A., St Ange, K., Dordick, J. S. & Linhardt, R. J. Heparin and anticoagulation. Front Biosci (Landmark Ed) 21, 1372–1392 (2016).

Heparin works by binding to antithrombin III (AT) and enhancing AT's inhibitory effect on thrombin and activated factor X (factor Xa). While only UFH chains with at least 18 saccharide sequences can affect AT's action on thrombin, UFH fragments of any length containing the specific pentasaccharide sequence can inhibit factor Xa's action.

Unlike thrombin inhibition, FXa inhibition doesn't require a template mechanism, allowing even very low molecular weight fragments containing the pentasaccharide to maintain anti-Xa activity. However, anti-Xa activity measurements typically occur in vitro without calcium ions, using either citrated plasma or purified antithrombin. Ellis et al. demonstrated that calcium ions' presence increases anti-Xa activity consistently across fragments of 5–30 saccharides. Laboratory studies confirmed that calcium ions enhance heparin's anti-Xa activity, with greater potentiation at higher molecular weights. While FXa binding to heparin isn't essential for activity, physiological calcium levels appear to enhance this binding in higher molecular weight chains. Wagenvoord et al.'s recent work found molecular weight dependence in anti-Xa activity even without calcium ions. In vivo, the situation becomes more complex as FXa typically exists within the prothrombinase complex rather than freely. Research shows that while heparin's inhibition of prothrombinase-bound FXa increases with molecular weight, it requires higher concentrations than for free FXa.

Heparin mechanisms within the coagulation cascade

src: Oduah, E., Linhardt, R. & Sharfstein, S. Heparin: Past, Present, and Future. Pharmaceuticals 9, 38 (2016).

Clinical Effects of UFH vs LMWH

UFH

UFH is one of the oldest established antithrombotic drugs. The first clinical studies, conducted in Sweden during the 1930s and 1940s, demonstrated that heparin effectively prevented postoperative thrombosis. Its use later expanded to treat established venous thrombosis. In a landmark 1960 clinical trial, Barritt and Jordan proved heparin's high efficacy in treating pulmonary embolism. The concept of prophylaxis advanced in the 1970s when Kakkar introduced low-dose heparin, administered subcutaneously three times daily before and after surgery. A comprehensive 1988 review by Collins et al., analyzing over 70 randomized trials with more than 16,000 patients in general, orthopedic, and urological surgery, found that perioperative subcutaneous heparin prevented approximately half of all pulmonary emboli and two-thirds of deep vein thromboses (DVTs). They also discovered that twice-daily injections matched the effectiveness of thrice-daily doses, though both carried a slightly higher bleeding risk compared to no heparin use.

The primary clinical indications for UFH include preventing and treating VTE, managing certain types of coronary artery syndrome (particularly unstable angina), and treating thrombotic stroke. It serves several other minor purposes, including use in hemodialysis. LMWHs have been studied for all these indications. Despite UFH's proven efficacy, LMWHs might offer improvements in three areas: greater efficacy (lower thrombotic event rates), improved safety (reduced bleeding or other side effects), and enhanced convenience (fewer injections).

LMWH

For general surgery, early studies in the 1980s showed that LMWH was as effective as UFH in preventing DVT. However, the bleeding rate was not reduced and could be higher with excessive dosing. The main advantage of LMWH was its convenience—requiring only one injection daily instead of two or three. For example, Bergqvist et al. conducted two double-blind studies of surgical prophylaxis. In the first study, 5,000 anti-Xa units of LMWH once daily showed equivalent efficacy to twice-daily UFH, but with a significantly higher bleeding rate (11.6% vs. 4.6%). In the second study using the same doses but with a longer interval between the first dose and surgery, LMWH's bleeding rates were lower (5.9%) but still exceeded UFH's (3%).

Is LMWH better than UFH for prevention and treatment of DVT?

Current evidence shows that LMWH is at least as effective as UFH in preventing DVT in both general and orthopedic surgery. While some trials found greater effectiveness in orthopedic surgery, particularly in preventing proximal thrombosis, meta-analyses have not shown this trend to be significant. Though individual studies have reported reduced bleeding with LMWH, overall data show no safety improvements regarding hemorrhage. The clear advantage of LMWH lies in its convenience, requiring just one daily injection instead of two or three with UFH.

Perhaps LMWH's greatest advantage over UFH is its simplified dosing regimen—fixed doses based on body weight, administered subcutaneously without monitoring. This contrasts with UFH's requirement for continuous infusion, frequent monitoring, and dose adjustments. This advancement has enabled home treatment for many patients, benefiting both patients and healthcare economics.

Are all LMWH products the same in clinical terms?

The debate over similarities and differences among LMWH products has persisted since early clinical studies, periodically resurfacing as new clinical evidence emerges. The two critical properties of any LMWH are its molecular weight distribution and anticoagulant activities. Significant differences exist among products in these aspects. Each manufacturer uses its own production process, and regulatory authorities treat each product as a distinct drug requiring separate toxicology, pharmacology, and clinical studies.

LMWHs, as a group, share the same essential mechanism of action (binding to antithrombin) and show the following differences from UFHs:

• higher anti-Xa than anti-IIa activity;

• bioavailability approaching 100%, leading to administration once or twice daily;

• lesser interaction with heparin-binding proteins (PF4, protamine, lipase, histidine-rich glycoprotein etc).

Product differences are less pronounced than their collective differences from UFH. After subcutaneous injection, the in vitro differences in molecular weight range diminish, as higher molecular weight fractions are less readily absorbed. This results in more similar circulating components than their in vitro profiles suggest. While manufacturing methods create some chemical variations, no evidence indicates these affect the products' overall biological activities.

Side Effects

Over many years of clinical use, heparin has proven to be remarkably safe, especially considering its biological origin and heterogeneity. The main concern, as with all anticoagulants, is excessive bleeding. The manufacturing methods of LMWHs do not introduce additional safety concerns. Any differences in side effects between LMWH and UFH likely stem from molecular weight differences, with LMWH showing less interaction with heparin-binding proteins and cells.

Apart from bleeding, the primary side effect is heparin-induced thrombocytopenia (HIT). While its incidence varies by clinical indication and treatment duration, it generally affects 1–3% of patients. HIT occurs when antibodies bind to a complex of PF4 and heparin, causing a sharp drop in platelet count.

Heparin-induced skin necrosis is another side effect that may be related to HIT. This condition manifests away from the injection site, and while heparin-induced antibodies appear in most patients, severe thrombocytopenia occurs rarely. Beyond HIT-related mechanisms, other pathogenic processes may be involved. Milder skin reactions, classified as delayed hypersensitivity, commonly occur with subcutaneous UFH administration but are rare with intravenous administration.

Osteoporosis represents another significant side effect, though it rarely occurs and typically only during long-term therapy.

Manufacturing

Approximately 1.2 billion hogs are slaughtered for food annually worldwide. The intestinal mucosa of these hogs is recovered and processed into 100 tons of heparin used each year. Typically, one hog provides three doses of heparin or a single dose of LMWH. China accounts for nearly 60% of annual hog slaughter, followed by the European Union at 20%, the USA at 14%, and the rest of the world at 6%.

Process flow diagram for production of heparin

src: Zhu, Y., Zhang, F. & Linhardt, R. J. Heparin Contamination and Issues Related to Raw Materials and Controls. in The Science and Regulations of Naturally Derived Complex Drugs (eds. Sasisekharan, R., Lee, S. L., Rosenberg, A. & Walker, L. A.) 191–206 (Springer International Publishing, Cham, 2019). doi:10.1007/978-3-030-11751-1_11.

Heparin extraction begins with isolating crude or raw heparin from mast-cell-rich tissues such as liver, lung, or intestine. The extraction process involves grinding tissue, salting out, proteolysis, and anion-exchange resin capture. The resulting raw heparin has molecular weight properties similar to pharmaceutical heparins. Raw heparin from intestinal or lung tissue of food animals—including cows, sheep, and pigs—has been used to manufacture pharmaceutical heparin.

Differences between Bovine Source and Porcine Source

Research has shown that bovine intestinal and lung heparins differ structurally and functionally from porcine intestinal heparin. 

Structurally speaking for both intestine derived heparins:

- Bovine intestinal heparin is less sulfated, more heterogeneous, has a lower molecular weight, and is more polydisperse than porcine heparin, indicating greater physical and chemical variability.

- Porcine intestinal heparin contains less glucuronic acid and more GlcNS3S6S than bovine intestinal heparin, suggesting different biosynthetic modification levels. It also demonstrates significantly higher activity.

To achieve the same antithrombotic effect, bovine intestinal heparin requires double the dose of porcine intestinal heparin, though bleeding risks remain comparable at similar doses. It also needs higher doses of protamine for neutralization.

Other Organ Sources

Bovine lung heparin differs from porcine intestinal heparin in its higher N-sulfo and O-sulfo levels, lower average molecular weight, and reduced anticoagulant activity. Ongoing comparison studies of heparins from different animal species and tissues indicate these are distinct drugs requiring separate monographs and cannot be easily interchanged in clinical practice.

The production of heparin API from different species and tissues presents unique challenges. Different isolation and purification processes can introduce varying impurities. Additionally, cows' susceptibility to bovine spongiform encephalopathy (BSE), or "mad cow disease"—which can cause Creutzfeldt-Jakob disease (CJD) in humans—poses a significant concern. After a BSE and CJD outbreak in Europe in the 1990s, bovine lung heparin was voluntarily withdrawn from the U.S. market. Reintroducing bovine-sourced heparin API to the U.S. market would require new cattle slaughtering requirements. While other food animal sources might be possible, each would likely face similar challenges.

Our Offer

Unibest is partnering with one of the most renowned heparin suppliers to provide global pharmaceutical companies interested in development and/or sales of heparin products:

UFH APIs (both Bovine and Porcine sources are available)

- Sodium Heparin (EP/USP/BP/CP/IP)

- Calcium Heparin (EP/USP/BP/CP)

LMWH APIs

- Enoxaparin Sodium (EP/USP/BP/IP)

- Nadroparin Calcium (EP/BP)

- Dalteparin Sodium (EP/USP/BP)

Heparin FDFs

- Heparin Sodium Injection (2.0ml/12500IU; 5.0ml/5000IU; 5.0ml/25000IU)

- Enoxaparin Sodium Injection (0.2ml/2000IU; 0.4ml/4000IU; 0.6ml/6000IU; 0.8ml/8000IU; 1.0ml/10000IU)

- Nadroparin Calcium Injection (0.4ml/4100IU; 0.6ml/6150 IU)

- Dalteparin Sodium Injection (0.2ml/2500IU; 0.2ml/5000IU; 0.3ml/7500IU)

References