Tina S. Morris, PhD, is vice president of Biologics & Biotechnology at United States Pharmacopeia (USP) 

Tina S. Morris, PhD

Heparin regulates hemostasis at various points of the coagulation cascade mainly through its interaction with antithrombin and heparin cofactor II. Because of these properties, heparin is a life-saving anticoagulant drug used in renal dialysis, cardiac surgery, and treatment for deep vein thrombosis. The drug also binds to platelets, inhibiting platelet function and contributing to the hemorrhagic effects of heparin. Bovine heparin, first approved in 1939, was widely used in the United States for more than 50 years (see 

). Like all drugs, heparin can cause adverse effects, but overall, bovine heparin products were found to be safe and effective during that period.


	
	In the late 1980s, bovine spongiform encephalopathy (BSE, or “mad cow disease”) was reported first in the United Kingdom and later in several other countries, raising concerns about the use of bovine-sourced heparin products in humans. Because of these concerns, manufacturers of bovine heparin products voluntarily withdrew them from the US market in the 1990s. Since then, heparin products approved for use in the US and Europe have been sourced solely from pigs, with approximately 60% of the supply of the drug coming from China. 

 illustrates steps involved in manufacturing heparin from porcine intestinal mucosa and potential impurities that are inactivated and/or removed from each manufacturing step. 

	Heparin is a lifesaving drug that was safely used since the 1940s. However, in 2007, contaminated heparin caused a number of deaths in the US and hundreds of adverse reactions worldwide (1). The contaminated heparin was found to contain over-sulfated chondroitin sulfate (OSCS). OSCS was an inexpensive synthetic adulterant that had some anticoagulant activity and was presumably added to heparin to increase profit when the drug was in short supply due to a pig disease outbreak. This “heparin crisis” demonstrated the vulnerability of drug supplies produced from increasingly global manufacturing chains and highlighted the risks inherent in reliance on one country and one animal species as the primary source for a crucial drug.

	To mitigate these concerns by diversifying the sources of heparin drugs, FDA is considering reintroduction of bovine heparin drug product to the US market. In August 2015, the US Pharmacopeial Convention (USP) hosted the 6th Workshop on the Characterization of Heparin Products in São Paulo, Brazil, with co-sponsors, the National Institute for Biological Standards and Control (NIBSC, UK), National Health Surveillance Agency (ANVISA, Brazil), and Sao Paulo State Pharmaceutical Manufacturers Association (SINDUSFARMA, Brazil).  The focus of the workshop was an examination of the global heparin supply chain, specifically the risks of heparin shortages, adulteration, and contamination.

	The following is an overview of scientific research and clinical experience presented at the workshop to generate improved understanding of the differences between porcine and bovine heparins, the clinical implications of reintroducing bovine heparin in the US, and the broader ramifications of bovine heparin in the US market and worldwide.


	Heparin is a natural product, extracted from animals. Just as pork and beef are different from each other, heparin products made from pigs and cattle are similar but not identical. Two sessions of the USP workshop focused on laboratory tests used to understand the differences in structure and biological activity between bovine and porcine heparin. 

	The structure of heparin is that of a linear polysaccharide consisting of repeating disaccharide motifs in which uronic acids alternate with glucosamine. The polysaccharide chains can vary in length and in substitution with sulfates and N-acetyl groups. Structural analysis techniques range from relatively simple, straightforward spectroscopic and chromatographic analyses to sophisticated applications of techniques in nuclear magnetic resonance and mass spectrometry.

	The biological activity of heparin, including its abilities to inhibit the enzymes of blood clot formation 

, can be quantified by several methods. Research suggests that molecular weight and disaccharide composition both play an important role in biological activity. High molecular weight fractions of heparin, for example, have a greater effect than do lower molecular weight fractions on anticoagulation potency.

	Overall, the combination of structural and functional information available provides a clear picture of heparin products from different species. Numerous samples have been tested by heparin manufacturers and academic and regulatory labs revealing clear and consistent differences in the structures (see 

) and biological activity profiles of porcine mucosal and bovine mucosal heparin. In addition, the few samples of bovine lung heparin tested were quite distinct from either mucosal sample type. 

	Importantly, the data presented show that bovine heparin was significantly less potent, weight for weight, than porcine heparin. The relationship between laboratory testing and clinical experience is not straightforward for such complex products, yet a difference in potency could have important clinical relevance (2). Therefore, further investigation including clinical research may be warranted. 

 
	There are two main safety concerns associated with bovine heparin. The first is heparin-induced thrombocytopenia (HIT), an infrequent but potentially devastating adverse event (3, 4). HIT is an immune response in which the body makes antibodies to large complexes formed between the highly sulfated heparin chains and platelet factor 4 (5). HIT occurs in 0.2-5% of patients regardless of the type of heparin administered; porcine and bovine heparin appear similar in terms of HIT risk (6, 7).  Another common adverse event associated with heparin is bleeding, which can be controlled through the neutralization of heparin by protamine sulfate (8). 

	The second safety concern, specific for bovine heparin, is the possible presence of BSE infectious agents (9).  During the BSE epidemic in the UK, some people consumed BSE-infected beef. From 1999-2000, after a long incubation period, some of these individuals developed variant Creutzfeldt-Jakob disease (vCJD); 229 cases have occurred worldwide as of May 28, 2015 (10).  Since its peak in 2000, vCJD has declined significantly but has not been eradicated, as a few cases are still detected every year. No known cases of vCJD, however, have been linked to use of bovine heparin. In addition, in India, Brazil, and Argentina--where bovine heparin products have been in continuous use--no cows have tested positive for BSE and no cases of vCJD have been observed. In the US, only three atypical BSE cases (i.e., different from the distinct BSE strain from the UK that causes vCJD) in cattle have been identified (11).  Since the 1990s, much has been learned about how BSE leads to vCJD in humans. In addition, methods for minimizing BSE risk in bovine materials have advanced (12, 13). Generally, risks from tissue spongiform encephalopathy (TSE) agents are controlled in three steps: animal origin of species and supply chain control, tissue harvest controls, and chemical treatments that remove infectious agents (11).  If bovine heparin is reintroduced, these steps will be instrumental for ensuring patient safety. Because of the safety concerns noted previously, bovine heparin is likely to have its own USP monograph and a separate label (Physician Labelling Rule) that differentiates bovine heparin from porcine heparin.

Possible reintroduction of bovine heparin into the US market


	The only approved source of heparin in most of the world is pig intestine, but the global pig supply is limited geographically. In addition, there is little growth potential for porcine heparin products to be manufactured in other parts of the world. Thus, FDA is concerned about potential shortages due to pig disease or possible geo-political instability. 

	After considering the available options, FDA hosted a meeting with its Science Board in June 2014 to discuss the possible reintroduction of bovine heparin in the US. Reintroduction of bovine heparin would no longer limit the source to one animal species and would extend the geographic distribution of source animals. If disease occurs in one animal source and/or there is geo-political instability in a major source country, the risk of supply shortages could be more readily mitigated.

	The original US-approved heparin drugs from the 1930s were from a bovine source (cow lung) and upon approval of porcine heparin products, both were used interchangeably until the 1990s without major safety risks or concerns. Notably, bovine mucosa heparin drug product is currently available and manufactured in South America (particularly Brazil and Argentina) and India. In some countries, bovine heparin is preferred for religious reasons. Thus, there have been more than 50 years of safe and effective use of bovine lung heparin in patients in the US, and bovine mucosal heparin has been used safely in South America and India. However, because the heparin characterization technology has advanced in the interim, the specifications for the proposed bovine heparin should be modernized in a manner similar to the recent update of the USP porcine heparin monograph.


	Heparin derived from bovine lungs was in general use in Europe and the US until it gradually fell out of use for two reasons: the commercial advantages of the more potent porcine product, and concerns in the 1990s about transmission of BSE to humans through contaminated beef products.

	Workshop speakers from Argentina, Brazil, and India emphasized that bovine heparin has been approved and used for decades in their countries. The Argentinian Pharmacopeia is discussing how to manage bovine and porcine heparins, and the Brazilian Pharmacopeia is currently devising specific monographs for bovine and porcine mucosal heparin.

	The availability of bovine and porcine heparins in Brazil has varied considerably in recent years. In 2008, 42% of heparin was of bovine origin, but this was followed by a decrease and then total removal from the market in 2013. In Argentina, the bovine source accounts for 70% of the total heparin; this has remained unchanged in recent years.

	No serious adverse effects have been associated with the use of bovine heparin in these two countries or in India. Some excess adverse events associated with heparin in cardiovascular surgery, however, were reported to ANVISA in 2008 when, on short notice, bovine heparin replaced porcine heparin for cardiovascular surgery in Brazil. The Brazilian Society of Cardiovascular Surgery (14) and ANVISA (15) published warning notes suggesting careful monitoring of anticoagulant levels when using bovine heparin.

	There are no reports of clinical trials comparing heparins from bovine versus porcine intestine. Therefore, although bovine heparin has been used successfully in several countries for many years, caution may be required when porcine heparin is replaced with bovine heparin without warning.

Bovine heparin use in the manufacture of LMWHs


	Low molecular weight heparins (LMWHs) are manufactured from heparin sodium (also known as unfractionated heparin sodium, or UFH) using chemical or enzymatic depolymerization methods (16). Currently, in the US, all forms of LMWH are made from porcine UFH. Therefore, their composition and properties are known based on their biological starting material. 

	The structure and composition of bovine UFHs differ from that of porcine; therefore, a LMWH product made from bovine UFH could have different properties from the same product made from porcine UFH. For example, if enoxaparin (a LMWH) is produced from bovine heparin in the future, this product could not be called enoxaparin because the structure and properties would be different and the activity would probably be different as well. 


	The current USP Heparin Sodium monograph describes system suitability and acceptance criteria for identity, purity, and strength that are designed strictly for porcine heparin (17, see 

	The workshop presentations showed clear differences in the structures and biological activity profiles of porcine mucosal, bovine lung, and bovine mucosal heparin samples, thereby suggesting the need to have a separate monograph for bovine heparin sodium. The structural differences would necessitate separate identification reference standards (RS) for 1H NMR, chromatographic identity, and molecular weight determination tests for bovine heparin (see Table I). Depending on the tissue source(s) of bovine heparin, there may be a need to establish separate identification RS for bovine lung and bovine mucosal heparins.  

	Based on the testing of numerous samples, the anticoagulant activity of bovine heparin is significantly lower than that of porcine in the laboratory. Most of the current bovine products gave potencies of about 100 IU/mg and some batches were estimated to be as low as 70 IU/mg.  Considering the monograph specification for porcine heparin is 180 IU/mg, it is likely that the clinicians will need to give more bovine heparin than porcine heparin by weight. Many years of safe bovine heparin use suggest that the higher amounts needed, as compared with porcine heparin, do not impact clinical efficacy. Clinical issues with bovine heparin, however, will need to be monitored carefully because wider clinical use could identify unforeseen differences between porcine and bovine heparin. Furthermore, bovine heparin requires higher doses of protamine for neutralization.

	Future bovine heparin production sites (both drug substance and drug product) should be under cGMP compliance. Supply chains including farms, slaughterhouses, and facilities that isolate, treat, store, and ship the bovine tissue need to follow the same steps and tests described for porcine heparin (18). These steps include, but are not limited to:

		Determine the species origin to verify that the ingredient comes only from the correct species.

		Confirm the absence of OSCS and ruminant material contaminants from another species. 

		Ensure that all heparin suppliers are audited and inspected regularly regarding their documentation practices and compliance with cGMP. 

		Reject any lots containing mucosa from another species.


	Heparin is an essential, life-saving drug that is needed worldwide. To avoid drug shortages, FDA is considering reintroduction of bovine heparin, which would diversify the supply chain by adding a bovine source to the currently used porcine heparin. Sourcing heparin from two species could greatly reduce vulnerability to shortages when disease strikes one species, and could also reduce reliance on one country as the primary source. The risks involved in reliance on one species from one country were clearly illustrated by the heparin crisis of 2007-2008, when adulterated porcine heparin from China caused numerous deaths and hundreds more adverse effects (1). Currently, China is the source for roughly 60% of crude porcine heparin used in the US and Europe.

	Bovine heparin is currently being used in some countries and was used safely for more than 50 years in the US before manufacturers voluntarily withdrew it from the market during the BSE crisis in the UK. Despite concerns about bovine products, there are no known cases of BSE contamination of bovine heparin. If bovine heparin is reintroduced in the US, methods for inactivating BSE could be applied to further reduce any risk. The other main safety issue with heparin is a severe adverse effect called HIT, but bovine heparin does not appear to have higher rates of HIT than does porcine heparin.

	Porcine and bovine heparins are distinctly different in terms of their structures and biological activities. These complex products may behave differently in clinical use than in laboratory testing, but if potency does in fact differ significantly in clinical use, this will need careful evaluation and perhaps dosage adjustment to avoid giving patients too much or too little heparin. If bovine-sourced heparin is reintroduced, supply-chain control will be critical, with frequent inspections of slaughterhouses and processing facilities for cGMP compliance. From the data presented at the meeting, bovine heparin and porcine heparin are not equivalent drugs, and therefore, they will require two different compendial monographs. The next steps are for manufacturers to bring bovine products to the regulatory agencies for evaluation and possible reintroduction to the market after a 15-year absence. 


	The participants in the USP 6th Workshop on the Characterization of Heparin Products are acknowledged with gratitude. 

, 19, 921-933 (2010).
	2. A. Tovar et al., 

 6, 230 (2013)
	3. T.E. Warkentin et al., 

106, 3791-3796 (2005).
	4. T.E. Warkentin and A. Greinacher, 

76, 2121-2131 (2003).
	5. A. Greinacher et al., Arterioscler Thromb Vasc Biol 26, 2386-2393 (2006).
	6. J. E. Ansell et al., 

 88, 878-882 (1985).
	7. J.L. Francis et al., 

 75, 17-22 (2003).
	8. G. Costantino et al., 

 7, e44553 (2012).
	9. J.L. Harman and C.J. Silva, 

Journal of the American Veterinary Medical Association

 234, 59-72 (2009).
	10.   EuroCJD Network, 

Creutzfeldt-Jakob Disease International Surveillance Network

 (2015).
	12. I. DeVeau et al., “Analytical Microbiology Expert Committee. The USP Perspective to Minimize the Potential Risk of TSE Infectivity in Bovine-Derived Articles Used in the Manufacture of Medical Products.,” 

 30, 1911-1921 (2004).
	13. D. Taylor,  

 325, 75-76 (2002).
	14. W. J. Gomes and D.M. Braile. 

 (2008), , accessed Aug. 27, 2015.
	16. H. Liu et al., 

, First Supplement, Heparin Sodium monograph, p.3748 (USP, Rockville, MD).
	18. FDA, 

Heparin for Drug and Medical Device Use:  Monitoring Crude Heparin for Quality


	This article reflects the views of the authors and should not be construed to represent US FDA’s views or policies.


	David Keire is acting laboratory chief, Branch I, Division of Pharmaceutical Analysis, FDA.
	Barbara Mulloy is visiting professor at Institute of Pharmaceutical Sciences, King’s College London.
	Christina Chase is senior scientific writer at US Pharmacopeial Convention (USP).
	Ali Al-Hakim is acting director of Division of New Drug API at the FDA.
	Damian Cairatti is head of Latin American Regulatory Affairs at USP.
	Elaine Gray is principal scientist at National Institute of Biological Standards and Control (NIBSC) in the UK.
	John Hogwood is research scientist at NIBSC.
	Tina Morris is senior VP of Science Global Biologics at USP.
	Paulo Mourão is professor at Federal University of Rio de Janeiro.
	Monica Da Luz Carvalho Soares is a member of the Deliberative Council of the Brazilian Pharmacopeia at ANVISA and a visiting fellow at the University of Maryland Baltimore County (UMBC).
	Anita Szajek is principal scientific liaison at USP.

 When referring to this article, please cite it as D. Keire et al., “Diversifying the Global Heparin Supply Chain: Reintroduction of Bovine Heparin in the United States?,” 

Articles

The global supply chain for bovine and porcine heparin and regulatory considerations are examined.

Diversifying the Global Heparin Supply Chain: Reintroduction of  Bovine Heparin in the United States?

	As FDA gears up towards approving biosimilar drugs in the United States, it is unquestionable that the role of biologics has rapidly expanded in healthcare, raising questions and presenting crucial issues associated with their quality and efficacy. Unlike small-molecule medicines, which have more easily defined quality attributes and are relatively easy to characterize, biologics are large-molecule drugs, typically manufactured using living cells. Because biologic medicines range from simple structures like certain peptides to complex mixtures, such as vaccines, establishing meaningful quality attributes for each specific product can present challenges.

	Besides serving a much reduced patient population because of their specific applications, biologics manufacturing requires living material and a much more complicated process than small-molecule synthesis, making a biologic drug naturally more costly. 


	Currently, licensed monoclonal antibody (mAb) therapeutics include those involved in the activation of effector cells, cell killing, cross-linked induced apoptosis, antagonism against several targets, and agonist antibodies. Because of structural similarities between antibodies of the same class, the United States Pharmacopeial Convention’s (USP) expert panel decided to focus its efforts in developing standards for mAbs on a class of molecules that would be more amenable to a “platform approach” both in terms of manufacturing and analytical development. 

	USP has been involved in developing quality standards for biologics as part of its expanding portfolio of monographs and general chapters for a long time. In 2012, USP started working on a clearly defined set of quality expectations for recombinant therapeutic mAbs, 

) General Chapter <129> “Analytical Procedures for Recombinant Therapeutic Monoclonal Antibodies,” which is set to become official in USP’s compendia of standards, 

	As individual product monographs will possibly describe specific quality attributes of individual drugs, USP deemed it necessary to have a broad foundation of general standards to set minimum quality requirements for molecule classes, and that is what it hopes to achieve with the publication of the new general chapter. 

	General Chapter <129> provides analytical procedures for murine, chimeric, and humanized IgG isotype mAbs and subtypes (e.g., IgG1 and IgG2). Because subclasses show differences in amino acid sequence and in the number of disulfide bonds, in some cases they require subclass-specific analysis. IgG-type monoclonal antibodies have a molecular weight of approximately 150 kDa. Each molecule consists of two heavy and two light polypeptide chains that have a molecular weight of approximately 50 and 25 kDa, respectively. 

	This chapter includes purity assessments by chromatographic separation of size variants and capillary sodium dodecyl sulfate (CE

SDS) electrophoresis. In addition, it includes procedures for analysis of oligosaccharides in mAbs. The chapter also provides validated procedures and associated performance criteria for each of these.

	Although the general chapter is focused on IgG-type mAbs, the principles of the tests included can apply to other related molecules, such as IgM or other isotype antibodies, antibody fragments, and Fc-fusion proteins. When the active substance is a conjugated antibody, such tests can be performed on the purified intact antibody before modification or conjugation.
	
	The proposed new general chapter does not apply to mAbs used as reagents in the manufacture of medicinal products. Applicable requirements for those are determined by the appropriate regulatory agency. 
	
	


	Accompanying the new general chapter is a recently-developed reference standard (RS). While this standard is not to be used for identification or to assign content, it has been developed to establish IgG system suitability. The material is an IgG1, 2mg/vial lyophilized presentation, which is used in the size-exclusion chromatography assay (SEC) and the capillary sodium dodecyl sulfate (SDS) electrophoresis (reduced and non-reduced) assays (CE-SDS), described in General Chapter <129>. 
	
	For SEC, the monoclonal IgG system suitability RS solution must meet the following system suitability requirements: the RS injections must bracket the injections of sample solutions and the RS chromatograms must be consistent with the typical chromatogram provided in the USP certificate; the area percent for the high molecular weight species (HMWS), the main peak, and the low molecular weight species (LMWS) must meet the criteria.  
	
	For CE-SDS (reduced and non-reduced), the Monoclonal IgG System Suitability RS solution must meet the following system suitability requirements: 1) the RS electropherograms must be consistent with the typical electropherograms provided in the USP certificate; 2) for reducing conditions, the main peak of the heavy chain (HC) must be clearly identified, the resolution criteria between nonglycoslylated HC and intact HC must be met, and the ratio of nonglycosylated HC to intact HC must be met; and 3) for nonreducing conditions, the IgG main peak must be clearly identified, the resolution criteria between IgG main peak and Fragment 1 must be met, and the area percent of main IgG peak must be met.

 When referring to this article, please cite it as D. Schmidt, et al., “USP Publishes Monoclonal Antibody Guidelines,” 

A General Chapter on mAbs will be published in USP-NF as biologics increase their role in healthcare.

USP Publishes Monoclonal Antibody Guidelines

Adverse health events associated with the use of adulterated or counterfeit heparin prompted the Centers for Disease Control and Prevention (CDC) and FDA to begin a nationwide investigation in January 2008 of the commonly used blood-thinning drug. FDA confirmed that serious injuries and deaths had been associated with the use of heparin manufactured with an API sourced from China (1). During this investigation, FDA scientists also identified a previously unknown contaminant, oversulfated chondroitin sulfate (OSCS), in the heparin (2). 

Working with FDA, manufacturers, and other stakeholders, the US Pharmacopeial Convention (USP) responded to this public health crisis by embarking on a revision of its public quality standards for heparin to enable the identification of OSCS and related contaminants. USP, a scientific nonprofit organization, develops standards that test for the identity, strength, quality, and purity of medicines, including biologic drugs like heparin. Published in its compendia, 

), USP's documentary or written standards (i.e., monographs) are developed for individual drugs and their ingredients. 

Since 2008, revisions of the heparin standards have taken place in three stages in order to better protect the US supply in a timely manner and help prevent future adulteration (see 

). To address the immediate public health risk posed in 2008, Stage 1 monograph revisions consisted of validating and implementing proton nuclear magnetic resonance spectroscopy (

H NMR) and capillary electrophoresis (CE) procedures initially developed at FDA to detect OSCS. Stage 2 monograph revisions included new identification, potency, and impurity tests to better control the quality of heparin API (3). The Stage 2 revised heparin monographs became official on October 1, 2009 (4).  

Table I: Changes to heparin sodium monograph during Stage 1 through Stage 3 revisions. 

Stage 3 revisions focus on further optimization of identification tests and a protein impurities procedure as well as the introduction of brand new molecular weight determination and nucleotidic impurities procedures. To decide on specifications for the new molecular weight determination and impurities tests, USP engaged stakeholders in two round-robin studies. The studies were designed to gather feedback on the proposed procedures prior to their publication in 

—USP's free-access, online publication for posting and receiving public comments on standards in development. Round-robin participants were requested to test their in-house heparin API batches using the proposed procedures. Final acceptance criteria for the tests were based on the batch data received during the studies.  

Heparin Sodium monograph Stage 3—specifics

Immediately after the implementation of the Stage 2-revised Heparin Sodium monograph, USP collaborated with stakeholders and FDA to further strengthen the standard and help ensure continued access to heparin of good and acceptable quality. USP's third stage of revisions aimed to optimize and reinforce the Heparin Sodium monograph against OSCS and process impurities.  

The USP Unfractionated Heparin Expert Panel played a crucial role in modernizing the Heparin Sodium monograph and updating the associated specifications. The panel consists of industry, academic, and regulatory experts from around the world and is advisory to USP's Monographs—Biologics & Biotechnology 1 Expert Committee (Monographs-BB1 EC), which is responsible for establishing and updating heparin standards and is part of USP's Council of Experts. With FDA input, panel members reviewed submissions regarding new and improved analytical procedures for the characterization of heparin. These procedures first were evaluated for suitability as compendial methods; when needed, further validation work was carried out.  

H NMR procedure generally was positive, but commenters did recommend minor modifications to the experimental procedure. These include the option of adding ethylenediaminetetraacetic acid (EDTA), reduced trimethylsilylpropionic acid sodium salt (TSP) concentration in a sample solution, as well as adjustments to the temperature range, acquisition time, and longer delay time. The major change was the reduced percentage—0.3% (w/w) instead of 1% (w/w)—of OSCS in system suitability solution to ensure a higher sensitivity of the method for OSCS. 

Also based on public comments, the expert panel optimized and validated an improved anion-exchange high-performance liquid chromatography (HPLC) procedure that provides a shorter run time, higher resolution between heparin and dermatan sulfate, and improved sensitivity for impurities.  

The third identification test that has been introduced is molecular weight determinations. The molecular weight profile of heparin is an intrinsic property that depends on the starting material and the manufacturing process. The resulting molecular weight distribution of finished heparin API in turn influences its potency, its ease of neutralization by protamine sulfate, and its propensity to interact with plasma proteins (e.g., platelet factor 4, leading to heparin-induced thrombocytopenia [HIT]). Therefore, the molecular weight distribution of heparin API could be used to ensure manufacturing process consistency as well as the safety of heparin API.   

Nucleotidic impurities and protein impurities. 

A new quantitative HPLC-based method has been added to control residual DNA in heparin. A quantitative Lowry method replaced the old turbidity test for protein impurities during Stage 2 revision. The improved procedure proposed in Stage 3 incorporates an additional step for removing interfering substances from heparin sample solution. A lower acceptance limit is proposed for both impurities procedures based on the batch data received during the round-robin studies. 

The proposed revisions to the Heparin Sodium monograph—which include the two optimized identification procedures, the new molecular weight determinations procedure, the optimized method for protein impurities, and the new method for nucleotidic impurities—will be published in 

38(6) (November–December 2012). Comments will be accepted until Jan. 31, 2013. After review of the comments and further consideration by the monographs-BB1 EC, the Heparin Sodium Monograph is expected to be approved and become official in 

. Comments on the standards should be sent to 

In light of the significant changes introduced in the Stage 3 revisions, USP will continue to update stakeholders in a timely manner about the current status of the monograph revisions and ensure that sufficient time is provided for a public evaluation of the procedures, specifications, and implementation date. Working in close partnership with FDA, industry, and other stakeholders, USP will work to ensure that revisions to the monographs are suitable and will support the overall framework for delivering a safe and effective supply of medicines for patients.  

 is vice-president of biologics & biotechnology, both at the US Pharmacopeial Convention (USP), 

www.fda.gov/cder/drug/infopage/heparin/default.htm

, Heparin Sodium monograph, pp. 3403–3406 (USP, Rockville, MD). 

USP optimizes identification tests and impurities procedures.

USP Further Strengthens Quality Standards for Heparin

In a study released in May 2011, US regulatory approvals for biologic drugs and biotechnology products (collectively referred to as "biologics") were reported to have nearly doubled in the past decade, compared to the 1990s. This growing market emphasizes the need for consistent approaches to product quality and public standards. In its upcoming Science & Standards Symposium on Biologics & Biotechnology, "Advancing Quality Standards through Analytics and Assays," taking place October 3-6 in Seattle, the US Pharmacopeial Convention (USP) will lead discussions on standards-setting initiatives related to biologics and the materials critical for their manufacture as a growing number of these types of drugs enter the therapeutic space.  

Setting the stage for the symposium are keynote speakers Steve Kozlowski, director of the FDA Office of Biotechnology Products, and Gillian Woollett, chief scientist at the Washington, DC, law firm Engel & Novitt. Each will discuss the regulation of biologics. The following two-and-a-half days of technical sessions will include presentations from an international panel of industrial, academic, and regulatory stakeholders engaged in the development of quality standards for biologics. USP topic experts will discuss general analytical approaches and what they can do for biologics; quality attributes of biologic drugs by product class; and the US regulatory landscape for these products. An entire track of sessions will focus on bioassays and the assessment of biological potency, critical to all biological medicines. 

Quality standards set by the USP—and enforceable by FDA—have traditionally been developed using what's called a "vertical approach," which is particularly suited for the characterization of small-molecule drugs. Under this approach, documentary standards (or product monographs) have been created for one drug product at a time, with associated reference materials (i.e., pure physical samples used as reference chemicals for testing) developed in tandem with monographs. General chapters in USP's official compendium, 

US Pharmacopeia-National Formulary (USP-NF),

 represent overarching information and testing procedures that apply across all or groups of monographs. These chapters broadly support product-specific (vertical) standards in the determination of identity, strength, and purity.  

Compared with their small-molecule counterparts, biologics are heterogeneous and more complex in their makeup. Thus, analysis and characterization are more challenging and require an even greater portfolio of good analytical procedures that can underpin product characterization.  

Another important issue for biologics quality is the use of ancillary and process materials that are also complex biologics. Common examples are fetal bovine serum (FBS) widely used in vaccine manufacturing; cytokines like interleukin-4 (IL-4) used in the cell-therapy arena; and materials like protein A, used in monoclonal-antibody manufacturing. The quality of these materials going into the manufacturing process can have a direct impact on the quality of the medicinal product itself, especially when the end product is not highly purified and residual amounts of the ancillary material have to remain. 

Working through expert committees and advisory expert panels, the USP biologics and biotechnology department has been actively revising and developing new chapters in USP-NF. Work has focused on general guidance in the form of information chapters, and also on specific test and product-class chapters that speak to the analysis of quality attributes for certain biologics product classes and the analytical procedures necessary to measure them. The goal with these "horizontal" standards is to provide more broadly applicable quality expectations in areas where analytical platform approaches already exist.   

In deploying a horizontal approach to develop standards for biologics, USP's activities in the following areas are reflected in the themes of session tracks for the Science & Standards Symposium. 

 USP has developed a new suite of bioassay chapters that includes guidance and information focused on development, analysis and validation of biological assays.  As a companion to these guidance chapters, USP has also developed example bioassay data sets and will make software analyses of the data available to meeting attendees. Case studies on analytical and compliance challenges related to bioassays as well as the assignment to biologics of quantities and values will be discussed. 

Critical quality attributes for biologics. 

In the development of product-class standards, identification of critical quality attributes (CQAs) that apply across products within a designated class is key. 

  <129> Critical Quality Attributes of Recombinant Therapeutic Monoclonal Antibodies is one example of a USP chapter in development which focuses on CQAs. In addition, 

 <90> Fetal Bovine Serum—Quality Attributes and Functionality Tests  became official in 

Ancillary materials and process enzymes. 

Ancillary materials, including growth factors and process enzymes, are necessary for the manufacture of certain biologics such as vaccines and cell-based or tissue-based therapies, but generally must be removed from the final product once the manufacturing process is complete. 

  <1024> Bovine Serum addresses quality issues related to the production, sourcing, and characterization of this class of ancillary materials, with the intent of supporting its users in conducting risk assessment and implementing risk-mitigation measures.  

The functionality of a protein-based therapy is primarily based on its proper structure as well as correct post-translational modifications. 

 General Chapter <1084> Glycoprotein and Glycan Analysis—General Considerations  has been developed to address modifications that result from the process of glycosylation. In addition, USP currently is working on two chapters addressing compendial procedures for oligosaccharide and monosaccharide analysis, important for the manufacture of glycosylated proteins.  

Analysis and regulation of bioassays. As technological improvements emerge, capabilities regarding bioassays must be updated to parallel those changes. Transitioning legacy products away from animal-based potency assays is an important issue for manufacturers. USP guidance chapters include <111> Design and Analysis of Biological Assays, which provides direction to users on developing appropriate strategies regarding biologic potency. 

The topics of the conference speak broadly to industry professionals engaged in the development, analysis and quality control of biological medicines, as well as to professionals interested in the regulatory and compendial issues related to the latest trends in biotechnology.  In support of its nearly 200-year-old mission to advance public health, USP is committed to continuing this global dialog among manufacturers, regulators, and practitioners to address the need for public quality standards for biologic medicines. 

 is vice-president of biologics and biotechnology for the US Pharmacopeial Convention. 

USP promotes horizontal standards for biologics' quality attributes.

Inside USP: International Dialogue on Biologics Standards

Continuing to help ensure the identity, purity, and quality of heparin, the US Pharmacopeial Convention (USP) has revised written and physical standards for the widely used blood thinner. In February 2009, USP released updated heparin standards at the request of the US Food and Drug Administration in response to the 2008 public health crisis in which many patients died as a result of adulterated heparin. A second phase of heparin monograph revisions is reflected in the newly posted standards (1). These developments and new analytical characterization tools for heparin were discussed by scientists and regulators at the Third International Heparin Workshop held at USP headquarters in Rockville, Maryland, July 27–28, 2009 (2). 

The workshop, cosponsored by USP, the British National Institute of Biological Standards and Control (NIBSC), and the European Directorate for the Quality of Medicines (EDQM), reflected the global nature of both the problem of adulteration and the resources available to combat it. Attendees came from more than 17 countries. Workshop deliberations focused on safeguarding heparin supply, pharmacopeial updates, and progress on biosimilar low-molecular-weight heparins (LMWHs). 

In early 2008, FDA confirmed reports of adverse health events associated with the use of heparin that had been contaminated with oversulfated chondroitin sulfate (OSCS) originating in China (3, 4). In June 2008, USP responded to the heparin crisis by revising its heparin sodium and heparin calcium monographs. Stage-1 monograph revision incorporated FDA's analytical methods for the identification of OSCS in heparin: proton nuclear magnetic resonance spectroscopy (

H NMR) and capillary electrophoresis (CE). Other pharmacopeias followed suit. 

Although this short-term measure prevented contaminated heparin from entering the supply chain, USP and stakeholders realized that a thorough modernization of the existing monographs would be needed to ensure the continuing quality of heparin. USP has since undertaken a second stage of revisions to the heparin sodium monograph, adding new identification, potency, and impurity tests that better control the quality of heparin active pharmaceutical ingredient (API). The European Pharmacopoeia (EP) and Japanese Pharmacopoeia (JP) also are taking a stepwise approach to revising their heparin monographs. 

LMWHs are defined as heparin salts that have an average molecular weight of < 8000 Da and for which at least 60% of all chains have a molecular weight of < 8000 Da. LMWHs are prepared from unfractionated heparin (UFH) by various chemical or enzymatic depolymerization processes. Thus, the starting material of LMWHs is of biological origin, and the manufacturing process defines the characteristics of the drug substance. Because the manufacturing process does not necessarily remove all impurities present in UFH, manufacturers should ensure that the starting material complies with the latest regulatory requirements.  

Table I: Stage-2 revisions of heparin monographs from world pharmacopeias. 

Several licensed LMWHs differ in their source material, manufacturing process, pharmacokinetic/pharmacodynamic properties, and therapeutic indications. The development of biosimilar LMWH products has increased globally during the past decade. In anticipation of the arrival of more biosimilar LMWHs in the near future, workshop participants discussed the regulatory framework for follow-on biologic products. Additional topics included regulatory issues (e.g., immunogenicity) and analytical tools aimed at demonstrating comparability to licensed products. 

Heparin is structurally the most complex member of the glycosaminoglycan (GAG) family of polysaccharides, which also includes chondroitin sulfate, dermatan sulfate, and keratin sulfate. Commercially available heparin in the US is derived mostly from porcine intestinal mucosa. Selectivity and yield emphasis in the purification steps affect the impurity profile of the resulting API with regard to common impurities such as dermatan sulfate and residual solvents. Because the different GAGs coexist in their natural sources, it is not uncommon to obtain GAG preparations contaminated with a structurally similar polysaccharide. 

Because of this complex milieu, the challenges are to sufficiently quantify process impurities and naturally occurring impurities that are not cleared by the purification steps and to concurrently detect any potential contaminants intentionally added to heparin. Workshop partcipants agreed that the quality of crude heparin can only be achieved by complete traceability and good manufacturing practices (GMPs) throughout the manufacturing life cycle and by supplier qualification. 

Workshop participants strongly agreed that modernization of heparin monographs was necessary but grappled with the questions of methods, specifications, and speed of implementation. USP announced that its Stage-2 revision of heparin monographs will become official on Oct. 1, 2009. To support these extensive revisions, USP released five new reference standards (RS): USP galatosamine hydrochloride RS, USP glucosamine hydrochloride RS, USP oversulfated chondroitin sulfate RS, USP dermatan sulfate RS, and USP heparin sodium for assays RS. 

As part of the secondary revisions to the heparin sodium monograph, USP has adopted a new potency assay for heparin, the chromogenic antifactor IIa test. The high specificity of this assay provides an additional safeguard against potential adulterants that may display heparin-like activity in the current USP plasma-based assay. 

H NMR test to detect potential contaminants, including OSCS. One possibility under consideration is a 2-D NMR procedure. Like USP, EP recommends adopting the antifactor IIa assay and increasing the potency specification to not less than (NLT) 180 IU/mg. For related substances, EP is evaluating eight procedures submitted by industry and academic laboratories. A crucial criterion for the selection of a related-substance procedure is the ability to differentiate between natural impurities and chemically modified contaminants, but it is not deemed necessary to differentiate naturally occurring impurities. EP plans to set the limit for dermatan sulfate, a heparin-related substance, at not more than (NMT) 2.0%. 

For JP's heparin sodium monograph, planned revisions include two new identification tests, weak anion–exchange highperformance liquid chromatography (HPLC) and 

H NMR, and two new impurity tests, a test for galactosamine and an updated 

H NMR test for the absence of OSCS. Additional revisions for protein impurities and nucleotidic impurities are planned as well. For the heparin calcium monograph, JP plans to incorporate the 

H NMR identification test and the galactosamine impurity at a later stage. Note that the potency assay in JP heparin monographs is an antifactor Xa-based assay. 

At the workshop, industry stakeholders were invited to present their experiences with the proposed USP Stage-2 revised heparin monographs. In general, industry concurred with and supported the modernization efforts, but manufacturers are concerned about the lack of harmonization between pharmacopeias. Several recommendations were raised, including further optimization of protein impurities and chromatographic identity tests to improve protocols and resolution requirements. An improved CE procedure was presented as an alternative to the proposed anion-exchange HPLC procedure. Several manufacturers expressed concerns regarding the implementation timeline for these revisions. Industry noted that the lack of a new potency RS, USP heparin sodium for assays RS, during the public comment period contributed to its inability to evaluate USP's new potency requirement (NLT 180 USP heparin units/mg). (USP released the new potency RS in late July.) In addition, many comments made at the workshop and during the public comment period were incorporated in the final USP Stage-2 revised monographs. USP Stage-3 revisions of heparin monographs will include further optimization of procedures as suggested by the workshop participants. 

The workshop reviewed the European Medicines Agency (EMEA) guidance document for biosimilar LMWHs, which establishes the nonclinical and clinical requirements for LMWH-containing medicinal products that claim to be similar to another one already marketed (5). EMEA's current recommendation is that the major burden of demonstrating that two LMWHs have similar biological medicinal properties is by means of a clinical trial. Clinical trials are preferred because the product's mode of action is not completely understood, and it is uncertain whether the pharmacodynamic markers are representative for the clinical outcome. 

In contrast, the US has not yet finalized a regulatory pathway for approval of follow-on biologics. Workshop participants advocated that FDA be granted the authority to approve biologics that reference a previously approved Public Health Service Act biologic and that the burden remain on sponsors to demonstrate safety, purity, and potency. This pathway should be immediately available, apply consistent regulatory standards, allow an interchangeability designation, and be flexible enough to incorporate scientific developments. 

Regulatory expectations regarding the quality of UFH have increased since the global heparin crisis of 2008. Industry has witnessed modernization of pharmacopeial methods that demand higher criteria for all quality attributes. Orthogonal tests have been implemented to identify heparin and to demonstrate lack of contaminants. At the workshop, USP and FDA jointly announced their intent to further revise heparin monographs to ensure the continued supply of safe and effectve heparin for patients. USP will update heparin stakeholders about the status of the monograph revisions and will ensure sufficient time for a public evaluation of the procedures, specifications, and implementation date. 

is vice-president of biologics & biotechnology, both at the US Pharmacopeia, 12601 Twinbrook Parkway, Rockville, MD 20852-1790, tel. 301.816.8325, 

www.usp.org/meetings/workshops/heparinWorkshop2009.html

www.fda.gov/Drugs/DrugSafety/PostmarketDrugSafetyInformationforPatientsandProviders/UCM112597

4. M. Guerrini et al., "Oversulfated Chondroitin Sulfate Is a Contaminant in Heparin Associated with Adverse Clinical Events," 

Guideline on Similar Biological Medicinal Products Containing Low-Molecular-Weight-Heparins 

USP workshop participants support new methods to safeguard heparin products but desire international harmonization. This article contains bonus online-exclusive material.

Inside USP: Characterization of Heparin Products

In January 2008, the US Centers for Disease Control and Prevention (CDC), along with the Food and Drug Administration, began a nationwide investigation of adverse health events associated with the use of heparin. FDA confirmed that serious injuries and hundreds of deaths have been associated with heparin, a blood-thinning drug that had been manufactured with an active pharmaceutical ingredient (API) sourced from China (1). In February 2008, Baxter Healthcare recalled multidose and single-dose vials of heparin sodium for injection, as well as "HEP-Lock" heparin flush products. During this investigation, FDA scientists identified a previously unknown contaminant, oversulfated chondroitin sulfate (OSCS), in the heparin (2).

In response to this public health crisis, the US Pharmacopeial Convention (USPC), in collaboration with FDA, manufacturers, and other stakeholders, revised the USP heparin monographs. The revision took place in two stages in order to better protect the US heparin supply and to help prevent future adulteration. To address the immediate public health risk, Stage 1 monograph revisions, completed in June 2008, consisted of validating and implementing proton nuclear magnetic resonance spectroscopy (

H NMR) and capillary electrophoresis (CE) procedures initially developed at FDA to detect OSCS. Stage 2 proposed monograph revisions include new Identification, Potency, and Impurity tests to better control the quality of heparin API.

The Stage 1 monograph revisions of heparin sodium and heparin calcium incorporated FDA's analytical methods for identifying OSCS in heparin: 

 H NMR and CE. USPC laboratories validated these methods according to procedures outlined in General Information Chapter <1225> 

(3). In addition, USPC proposed appropriate system suitability criteria and specifications and developed two new reference standards (RS) to support the revised monographs: 

 heparin sodium identification reference standard (RS) and 

The revised monographs were posted June 18, 2008, on the USPC website (

) as Revision Bulletins and became official immediately. These monographs are referenced on the FDA website as well and have played an important role in controlling the quality of heparin API in the US (1).

Before posting the heparin Revision Bulletins, USPC solicited feedback from stakeholders by hosting two online meetings in spring 2008. Industry comments as well as submissions of new and improved analytical methods for characterization of heparin were critical in shaping the next stage of the monograph revision.

USPC and involved stakeholders realized that a thorough modernization of the existing monographs would be needed to effectively ensure the continuing quality of heparin. With this objective, USPC has undertaken a second stage of revisions that will completely revise the heparin sodium monograph (see Table I).

Table I: Changes to heparin sodium monograph.			

The USPC Heparin Ad Hoc Advisory Panel played a critical role in revising the heparin sodium monograph and updating the associated specifications. The panel consists of industry, academia, and regulatory experts from around the world and is advisory to USP's Biologics & Biotechnology Blood and Blood Products Expert Committee (BBP EC), which is part of USPC's Council of Experts. Working closely with FDA, panel members reviewed numerous submissions regarding new and improved analytical procedures for the characterization of heparin. These procedures first were evaluated for suitability as compendial methods, and when needed, further validation work was carried out.

H NMR method generally was positive, but commenters also recommended that USPC state a clear pass/fail criterion while expanding the procedure to cover the entire spectral range. The revised procedure covers a spectral window of at least 10 to–2 ppm and requires identification of four main heparin signals: H1 of 

-sulfated glucosamine, 6S (signal 1), H1 of iduronic acid 2S (signal 2), H2 of 

-sulfated glucosamine S (signal 3), and methyl H of GlcNAc (signal 4). In addition, clear acceptance criteria are spelled out to control both known and potential contaminants in heparin.

Based on public comments, the advisory panel recommended replacing the CE procedure with an anion-exchange high-performance liquid chromatography (HPLC) procedure that provides higher resolution between heparin and other impurities. Another consideration favoring the change is the wide availability of HPLC instruments.

The third Identification test that has been introduced is 

. This ratio of activities is routinely used to characterize unfractionated heparin as well as to distinguish different forms of low-molecular weight heparins. For high-quality heparin, the ratio of anti-factor Xa to antifactor IIa should be not less than 0.9 and not more than 1.1. Manufacturers must perform all Identification tests because they serve orthogonal purposes.

 The pharmacopeial heparin potency assay traditionally was performed using sheep plasma. The rationale for this method is that it resembles as closely as possible the actual situation in which heparin typically is used (i.e., in human blood). Unfortunately, the results of the assay in plasma are affected by other proteins that are present in sheep plasma. Heparin can bind to a large variety of plasma proteins such as histidine-rich glycoprotein, platelet factor 4 (PF-4), and heparin cofactor II (HCII). The large enhancement of antifactor IIa activity for OSCS is an HCII-mediated event (4). This phenomenon explains how contaminated heparin batches successfully passed the USP sheep plasma clotting activity assay. Based on the known modes of action of heparin, USPC proposes to replace the current potency assay based on sheep plasma with a chromogenic antifactor IIa assay that uses purified reagents.

 An Impurity test has been added to quantify the percent galactosamine of total hexosamine in heparin. Heparins are salts of sulfated glucosamino-glycans composed of polymers of alternating 

-glucuronic acid) joined by glycosidic linkages. This procedure involves the hydrolysis of heparin into the monomers (galactosamine and glucosamine) by hydrochloric acid and analysis by high-performance ion chromatography. The percent (w/w) of galactosamine with respect to the total amount of both galactosamine and glucosamine (hexosamine) is quantified using the relative response of galactosamine to glucosamine from the chromatograph's peak areas. This test provides a measure of dermatan sulfate and other galactosamine-containing impurities in heparin.

A quantitative Lowry method has replaced the old turbidity test for protein impurities. Nucleotidic impurities and residual solvents have been added to control residual DNA and residual-solvent levels in heparin.

The proposed heparin sodium and heparin sodium injection monographs were posted on the USPC website on Feb. 4, 2009. These Stage 2 monographs will be presented in the March–April 

as proposed Interim Revision Announcements. After review of comments and approval by the BBP EC, the new heparin monographs are expected to become official Aug. 1, 2009.

USPC plans to ensure that any revisions to the monographs are suitable and will not disrupt the supply of safe and effective medicines for patients. Comments on the standards should be sent to 

is director of biologics & biotechnology, 

 is chief executive officer, all at the US Pharmacopeia, 12601 Twinbrook Parkway, Rockville, MD 20852-1790, tel. 301.816.8325; 

*To whom all correspondence should be addressed.

1.  FDA, "Information on Heparin (2009)," 

2.  M. Guerrini et al., "Oversulfated Chondroitin Sulfate Is a Contaminant in Heparin Associated with Adverse Clinical Events," 

26 <1225>, "Validation of Compendial Procedures," 683–687 (2008).

4. T. Maruyama et al., "Conformational Changes and Anticoagulation Activity of Chondroitin Sulfate Following its O-Sulfonation," 

USP's Stage 2 heparin monograph revisions address identification, potency, and impurities.