Anita Szajek is principal scientific liaison at the US Pharmacopeia.

Anita Szajek

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?

The US Pharmacopeia (USP) Therapeutic Peptides Expert Panel was formed in 2013 to evaluate quality attributes for synthetic peptide APIs based on currently available regulatory guidance and expectations. This series of three articles by the panel explores the current manufacturing and regulatory landscape and provides a comprehensive overview of quality attributes to be considered for successful synthetic peptide API development from manufacturing to lot release. The first article covered analytical characterization methods, lot release tests and points to consider for synthetic peptide API manufacturers entering the market (1).

This second article discusses raw materials used in the chemical synthesis of peptides and their potential impact on the quality attributes of the final API. A special focus of this article will be on protected amino acid derivatives, setting their specifications and the types of quality control tests necessary to ensure consistent quality of the final API. The need for establishing close ties to manufacturers will also be discussed. Finally, the requirement for source verification of raw materials, in the context of transmissible spongiform encephalopathies (TSEs), will be presented.


  The term “raw materials” encompasses the starting materials, reagents, and solvents used in the manufacture of an API.  Of these, starting materials-defined as “a raw material, an intermediate or an API that is used in the production of an API and that is incorporated as a significant structural fragment into the structure of the API” (2)—are generally considered to be the most crucial, because they are actually incorporated into the final product during the manufacturing process. For the manufacture of peptide APIs, these “critical raw materials” are usually protected amino acid derivatives, although other substances may be proposed and justified.

The implementation of International Conference on Harmonization (ICH) Q11, which has been in effect since 2012 (3), has placed an increased emphasis on the control of raw materials, particularly the justification of starting material selection, with focus on the ability to detect and control impurities in starting materials, understand their fate and purge during the manufacturing process, and their relationship to the impurities in the final API.

Based on their justification, API manufacturers may define starting materials for synthetic peptides as amino acids, protected amino acid derivatives, or peptide fragments. The latter are typically assembled using the former; therefore, protected amino acid derivatives will be referred to as “critical raw materials” in this article, because they are the most commonly designated starting materials and their quality may have a significant impact on the quality of the API.

Impurities in peptide APIs are generally classified as follows (4):

Depending on their levels in the final substance at the time of registration and the requirement to impose limits, impurities from the first two classes may be included in the final release specification as “specified impurities,” together with limits for individual and total impurities from the third class.

The final impurity specifications for a peptide API are established on the basis of knowledge of the manufacturing process and also through “qualification” of individual impurities by virtue of their presence in the API during toxicological studies and clinical trials. In general, regulatory agencies are more willing to accept higher levels of qualified impurities (i.e., those in the first category), because their safety profile is known. To a lesser extent, the same is true of the second category of impurities, whose safety may be assessed through knowledge of their structure, even in the absence of toxicological data. In the case of the third category, however, where the structure of the impurity is unknown, allowed limits in the final substance must, necessarily, be lower. While ICH Q3A (5) limits such impurities to 0.1%, peptides are excluded from the guidance and, depending on the regulatory agency and the therapeutic dose of the API, higher limits of unidentified impurities may be permitted.


  The current expectation of regulatory agencies is that levels of peptide-related impurities, which may be derived directly from the starting materials through transformation of impurities in those materials, or from side reactions during the process used to manufacture the API, are strictly controlled. This discussion will focus on the impurities derived from critical raw materials, while those derived from the manufacturing process will be discussed in a subsequent article.  


  Virtually all of the likely impurities in critical raw materials (with the exception of non-amino acid residues from their manufacturing, such as most reagents and solvents) have the potential to react and, therefore, be incorporated as impurities during the manufacture of a peptide API. Such impurities can include:

 Free amino acids or amino acid derivatives (i.e., without the Nα-protecting group and/or the side-chain protecting group, if applicable, or containing an incorrect protecting group)

Amino acid contaminants (i.e., amino acids other than the desired amino acid)

Isomeric contaminants, such as isoleucine in place of leucine; or isomers of aspartic and glutamic acid derivatives with the protecting group on the α-carboxyl group instead of the side chain

Dipeptides or oligopeptides in 9-fluorenylmethyloxycarbonyl- (Fmoc-) amino acid derivatives (6)

 β-alanine containing contaminants in Fmoc-amino acid derivatives (7).
  

  While they are not related to the critical raw materials themselves, it is important to note that residues of carboxylic acids, such as acetic acid, in the starting material can also lead to impurities in the final substance, because they may react during the coupling of the critical raw material to the growing peptide chain. Because of the potential for impurities such as those described above to be the origin of impurities in the final API, it is important that the specifications for these critical raw materials, including their impurity profiles, be tightly controlled.  Methods used for their analysis should be validated.

Based on the considerations outlined above, the recommended minimum quality attributes for critical raw materials (protected amino acid derivatives) are summarized in 

. Similar quality attributes are recommended for other types of starting materials.

Table I: Recommended minimum quality attributes for critical raw materials. HPLC is high-performance liquid chromatography. TLC is thin layer chromatography. GC-MS is gas chromatography-mass spectrometry.   

Refers   to amino acids other than the one contained in the amino acid derivative   which is the subject of the specification 

Identity (if applicable) and limits   set on the basis of batch history and the outcome of process characterization   and a risk assessment 

Includes potential reactive   impurities, such as carboxylic acids determined during risk assessment

Limits for specified impurities, content of the incorrect enantiomer, free amino acid, or other amino acids can be justified through prior batch history, supported by process characterization and a process risk assessment. This assumes that analytical methods exist that can establish a correlation between the content of an impurity in the critical raw material and the content of a related impurity in the API; and that the process is well understood in terms of increasing or reducing or eliminating this impurity. Furthermore, the limit may be adjusted based on the process risk assessment.

For unidentified impurities, setting limits for acceptance criteria in the absence of data is based mainly on the process risk assessment. The API specification for unspecified impurities may be used as a starting point with an appropriate safety factor, which would include the following considerations:

 The peak area % by high-performance liquid chromatography (HPLC) may underestimate the true content of an impurity due to its potentially lower extinction coefficient

The coupling rate of an impurity during synthesis may be higher than that of the targeted critical raw material, leading to its enrichment in the crude API

 The resulting peptide-related impurity may co-elute with the API during purification, thus making it difficult to reduce or eliminate

The likelihood that a new, peptide-related impurity may not be detectable by routine API release methods.
  

Consequently, to minimize the potential impact on the API, the limits for this type of impurity should be very stringent.  

It is important to note that an assay method based on titration is preferred, because the amino acid derivative may contain varying levels of carboxylate salt forms of the material (which may not be reactive in coupling steps), and other assay methods, such as those based on HPLC or elemental analysis, may not be sufficiently sensitive or accurate.  


  Because all other raw materials, including the reagents and solvents used in the manufacturing process for peptides, are unlikely to be incorporated into the final product, there is generally less emphasis on their quality attributes. Thus, while the process risk assessment discussed previously should consider these raw materials, in general, most are considered to be non-critical, with only a very low risk of impacting the quality of the final product.  Therefore, specifications are usually set based on those for the available materials used in the process.  As a general rule, materials of the highest available grade, such as 

 grade, should be used, unless materials of a lower grade are qualified through use in the validated manufacturing process.

An exception to this is the materials used in the final purification and isolation steps of the manufacturing process. In contrast to earlier steps in the process, where the raw materials are not considered to be final product contacting because downstream purification is performed, the materials used in the final steps of the process can be considered critical, because no further purification is performed.  Furthermore, if the final manufacturing steps include concentration processes, such as evaporation or lyophilization, any impurities, especially those which are less volatile, may actually be concentrated in the final substance.  Therefore, the solvents and reagents (e.g., acetonitrile, methanol, isopropanol, acetic acid, trifluoroacetic acid), which may typically be used during final, reverse-phase HPLC purification or precipitation steps of peptide APIs, should be identified as having a potentially high risk of impacting the quality of the final API.  Quality attributes for these raw materials should take into account not only the identity and purity of the material but also, for example, tight limits for residue on evaporation.

 <232> and <233>) (8) and ICH Q3D, all APIs, including peptides, must be evaluated for their potential contamination by elemental impurities. While these may be introduced during the manufacturing process through use of metal catalysts or by leaching of metals from equipment, the possibility also exists for their introduction through the raw materials used in the process.  As part of risk assessment, the manufacturing process must be evaluated for its ability to remove potential elemental impurities in raw materials through appropriate validation. If this cannot be demonstrated, appropriate limits for elemental impurities in raw materials must be established.


Based on the risk assessment, a control strategy must be established that identifies the process steps that can impact the critical quality attributes of the final API. The ability of the manufacturing process to control impurities originating from the starting materials determines their purity requirements. Therefore, strict control of the quality of raw materials, especially critical raw materials, together with appropriate in-process controls, is necessary, to ensure consistency in the quality of the final API.


In view of the increasing emphasis by regulatory agencies on raw material controls, it is essential to re-evaluate the approach to the control of the quality of critical raw materials.  While ICH Q7 does not require manufacture of critical raw materials under GMP conditions, it is recommended that suppliers manufacture them under, at a minimum, “GMP-like” conditions with appropriate systems and controls to assure quality, including implementation of stringent specifications for critical raw materials in the process (i.e., the starting amino acids, in particular), together with a system of batch-specific documentation and strict control of process changes, to ensure that manufacturing processes are both reliable and robust, resulting in critical raw materials of consistent quality.  

It is also incumbent on the manufacturers of APIs to implement appropriate procedures to qualify suppliers of starting materials through auditing of their facilities and systems on an ongoing basis at regular intervals of time. During such audits, emphasis should be placed on systems that have been implemented to control product quality, including change control systems, as well as the manufacturer’s knowledge and depth of experience with the manufacture of the products. Such an audit can be a key factor in initiating the process of establishing a level of trust between the parties and building the relationship, with the goal of developing a true partnership.  

It is also essential that the expectations and responsibilities between the two parties should be formalized in a “Quality Agreement” (often referred to as a “Technical Agreement”), to avoid misunderstandings.  In particular, such an agreement should include an obligation for the supplier of the starting materials to inform the peptide manufacturer prior to the introduction of any significant changes in their manufacturing processes, such as a change of a recrystallization solvent, so that the impact of such changes on the manufacturing process for the final API may be assessed. Implementation of an appropriate confidentiality agreement is recommended to facilitate the exchange of confidential information.  If multiple suppliers of starting materials are qualified, the same type of system and agreements should be implemented for each supplier.

While the requirements outlined previously for the tight control of quality may be difficult for some starting material manufacturers to comply with, the authors believe that they are an essential element of the control of the quality of the final peptide API.  An assessment of whether a supplier is able to meet (or potentially is able to meet) these requirements should be an important element of the initial screening and eventual approval of potential suppliers.  


Current regulations in most countries require that all licensed pharmaceutical products must be evaluated for their potential risk of containing Transmissible Spongiform Encephalopathies (TSEs) (9). A risk assessment must be performed for all raw materials used in the manufacture of a peptide API to confirm the absence of TSEs. Raw materials, which are either directly derived from “TSE relevant animal species” or are manufactured using materials derived from such species should be identified and a certificate of suitability demonstrating TSE compliance obtained.   


Based on the guidance provided by ICH Q11, it can be concluded that the starting materials used during the manufacture of peptide APIs play a crucial role in determining the quality of the final substance. This requires the establishment of detailed specifications for these materials, including limits for identified and unidentified related impurities, and the implementation of agreements with starting material suppliers, to minimize the impact of manufacturing process changes on the introduction of new impurities. 

USP evaluates raw materials used in the chemical synthesis of peptides.

Control Strategies for Synthetic Therapeutic Peptide APIs Part II: Raw Material Considerations

The United States Pharmacopeial Convention (USP) Therapeutic Peptides Expert Panel was formed in 2013 to evaluate quality attributes for synthetic peptides based on currently available regulatory guidance and expectations. Public quality standards for drug products and drug substances are developed by USP and enforceable by FDA. This series of three articles by the Panel explores the current manufacturing and regulatory landscape and provides a comprehensive overview of quality attributes to be considered for successful synthetic peptide active pharmaceutical ingredient (API) development from manufacturing to lot release. Specifically, the first article covers analytical characterization methods, lot release tests, and points to consider for synthetic peptide API manufacturers entering the market. The second article will focus on quality control of raw materials and impurities resulting from the starting materials used for peptide synthesis. The last article will be devoted to manufacturing processes and impurity control of synthetic peptide APIs. 

In 2012, the number of peptide drugs approved by FDA surpassed the number of approved monoclonal antibodies and enzymes (1). These approvals serve to highlight the recent revival of interest in peptides, which have generally been considered to be poor drug candidates due to their low oral bioavailability and propensity to be rapidly metabolized. However, new formulation and conjugation strategies for alternative routes of administration and overcoming short half-lives have emerged, resulting in a larger number of marketed peptide-based drugs, some of which have reached blockbuster status (2, 3).  Despite these successes, some challenges still remain. Due to varying sizes and amino acid sequences, synthetic peptides are not easily classified into either small molecule or biologic categories. Therein lie the regulatory challenges with peptides, especially with respect to impurities and bioassay requirements. Understanding these challenges can help shape and create consistency among USP’s quality standards for this growing class of drugs.


Comprehensive characterization of a peptide API is required to obtain regulatory approval for marketing; the reference standard is fully characterized to ensure unequivocal identity of the material, its purity and API content.  Besides evaluation of appearance, a selection of tests that fall into four categories is performed: identification, assay, purity, and specific tests.  The evidence obtained from these studies also may be used in regulatory submissions, for example in Module 3 of the Common Technical Document. Available methods and USP chapters for characterization of peptide APIs are summarized in 

 For routine identification of peptides, a high-performance liquid chromatography (HPLC) method is recommended. This method should be capable of distinguishing between the peptide and its closely related impurities and, therefore, usually is identical to the method used to determine impurities. The preferred identification procedure involves a comparison of retention times between the main peak in the standard and samples, as well as a coinjection of an equal mixture of both, with a requirement to obtain a single peak.  

Identification by mass spectrometry (MS) involves determination of the monoisotopic mass, which should be within ±1.0 mass units of theoretical.  For peptides larger than 2 kDa, the use of high resolution instruments may be necessary and determination of the average mass may be appropriate for larger molecules.  The sample concentration and solvent should be specified if sample introduction to the instrument via direct infusion is used.  MS cannot distinguish between isomers (e.g., isoleucine and leucine) or D- and L-amino acid substitutions.

Information on amino acid (AA) composition can be obtained from amino acid analysis (AAA). The hydrolysis protocol should be established, because those factors may significantly impact the recovery of certain amino acids.

Further characterization should involve determination of the peptide sequence using MS-MS. At a minimum, fragmentation by collision-induced dissociation (CID) should be used, but if sequence coverage is poor, alternative fragmentation techniques such as electron-transfer dissociation (ETD) may provide better quality data. The latter technique is particularly useful for peptides that contain a significant number of basic residues and are highly positively charged. Furthermore, peptides containing multiple disulfide bonds lend themselves well to ETD, which can selectively cleave S-S linkages, something CID is not capable of.  Prior to MS analysis, the disulfide bonds may be reduced using suitable reducing agents, such as dithioerythritol (DTE), dithiothreitol (DTT), or tris(2-carboxyethyl)phosphine (TCEP).  The linear peptide can then be subjected to MS/MS, which will allow confirmation of sequence but not disulfide bond assignment.

For most peptides, nuclear magnetic resonance (NMR) spectroscopy should be attempted, although interpretation of spectra may be challenging for peptides containing more than 10 AAs.  If comprehensive structure elucidation by NMR is not possible, then peptide mapping may be used as an alternative.  The proteases Trypsin or Lys-C are recommended for peptides containing a sufficient number of Arg and Lys residues, whereas for peptides containing Glu residues, Glu-C is useful. The resulting peptide fragments are separated by HPLC and identified by MS. Peptide mapping, without prior peptide reduction, is a potential approach for disulfide bond assignment. For routine use, peptide mapping can involve comparison with a standard.

Enantiomeric purity is determined using chiral AAA. The method is based on acid hydrolysis of the peptide, suitable derivatization of the resulting AAs and determination of the optical isomers of the constituent AAs by chiral gas chromatography with mass spectrometric detection (GC-MS).  HPLC-based methods of chiral AAA involve pre- or post-column derivatization of AA hydrolysates, for example using Marfey’s reagent or o-phthalaldehyde, respectively.  Regardless of the hydrolysis procedure employed, the method should be validated to account for potential racemization during hydrolysis or subsequent derivatization.  An approach that addresses this issue involves the use of deuterated reagents (e.g., deuterium chloride [DCl])/deuterium oxide [D

O]). Substitution of the α-carbon hydrogen with deuterium allows for correction for racemization when using MS-based detection methods. 

Infrared (IR) spectrophotometry is not a useful characterization or identification tool, because spectra tend to be dominated by water vibrations from residual water, and carbonyl bands, arising from the peptide backbone and acetate, if present as a counterion.  

The characterization of secondary or tertiary structure in aqueous solution may be relevant for peptides, particularly in the context of determining whether a bioassay is required.  The most commonly used methods for this purpose are circular dichroism (CD), fourier transform intrared  (FTIR) analysis of amide bond vibration regions, and NMR. For the latter technique, additional information can be obtained using deuterium exchange. 
  
  

 When considering assay, it is important to emphasize that many peptides are quite hygroscopic and should be handled in a controlled-humidity environment (e.g., a glove box). Failure to do so can lead to erroneous assay results because moisture exchange of the peptide material with the environment can be very rapid. This process is especially fast if high surface area lyophilized material is being weighed. Establishing a sorption isotherm under ambient conditions is useful to determine the optimum relative humidity conditions for handling the API.   

Assay by HPLC is the preferred approach, assuming that a quantitative standard has been established. In the absence of a quantitative standard, a “gravimetric” method, in which both the standard and the sample are corrected for counterion content and water content (determined as part of the assay method), may be used.

Assay of peptides has traditionally been determined by quantitative AAA, which compares the peak areas of AAs in a hydrolyzed sample with the areas of the peaks for the same AAs in an external standard. The peptide hydrolysis procedure should be validated to ensure good recovery, and only AAs stable to the hydrolysis conditions should be used in the calculation. Some AA sequences containing multiple, adjacent hydrophobic residues, such as Ile, Phe, Leu and Val, may give lower recovery than expected.  Because the method involves a relatively complex sequence of operations, the results may be variable.

Assay by nitrogen content is also used routinely, most frequently determined from elemental analysis.  Kjeldahl analysis or chromatographic methods with chemiluminescence nitrogen detection may also be used.  

Only peptides containing Trp, Tyr, or Phe residues may lend themselves to assay determination using UV absorbance measurement at 280 nm. The molar absorption coefficient can be calculated theoretically based on knowledge of the sequence; however, the method should be independently validated using one of the techniques described previously.  At lower wavelengths, common counterions such as acetate may interfere with this assay.

Quantitative NMR is potentially useful for assay determination, although the method requires a stable internal standard with signals that fully resolve from bands associated with the peptide.  
  
  

Purity/related impurities determination by HPLC and control strategy

. The purity of a peptide API is usually determined using HPLC. The method should be capable of separating impurities introduced through starting materials, process-related impurities, and impurities resulting from degradation of the peptide. One or more methods may be required to accomplish this objective. When using multiple methods, it is recommended that orthogonal separation mechanisms be used. At least one of the methods should be stability-indicating and methods should be validated per 

 <1225> Validation of Compendial Procedures. In addition, use of LC-MS compatible methods may be advantageous for the identification of impurities.

For peptides containing hydrophobic residues two reversed-phase (RP) methods may be appropriate, although alternatives, such as mixed mode RP/ion-exchange (IEX) stationary phases, should be considered. For hydrophilic peptides, a RP method or mixed-mode RP/IEX stationary phase may be complemented by an IEX or hydrophilic interaction chromatography (HILIC) type method.

A single HPLC method may be appropriate based on the sequence of the peptide and knowledge of its potential impurities. A control strategy, built on process characterization, should be implemented that accounts for the fate of impurities introduced with starting materials and generated during the synthesis process and purification of the peptide.  Additional, appropriately validated in-process control methods may be used to ensure quality of the final product.

Non-peptide, process-related impurities, such as residual solvents and heavy metals (especially if metal catalysts are used in the process), should also be monitored. To support the overall control strategy, additional methods should be used, such as ion chromatography, inductively coupled plasma (ICP)-MS and NMR. The latter is particularly useful during process characterization, because diffusion experiments allow for rapid differentiation between small-molecule organic impurities and peptide-bound modifications.  

Traces of reagents used in the process, such as trifluoroacetic acid (TFA), which is commonly used in chromatographic purification, and inorganic fluoride [from the use of liquid hydrogen fluoride in products manufactured using t-butyloxycarbonyl- (Boc-) based chemistry], should also be monitored.

 A number of tests are universally applicable to quality control testing of peptide APIs, of which the most important are counterion content and residual water content.  As discussed in the assay section, the hygroscopicity of the peptide may affect the results.

APIs intended for use in injectables require bioburden and endotoxin determination. Finally, a mass balance calculation is recommended which, at a minimum, requires a summation of the percentages of major sample components (i.e., assay [peptide content], counterion content, and water content).  The result should approach 100%.

Lot release methods
  While it is, obviously, not necessary to perform all of the tests described as part of routine lot release, the testing performed must comply with regulatory requirements for, at a minimum, establishment of identity, purity, and assay for the peptide API.  The Panel’s recommendations for the test methods to be performed as part of lot release, as well as the quality attributes necessary for inclusion in monographs for this class of APIs, are highlighted with an asterisk in 

The use of two separate identification methods is required per the International Conference on Harmonization (ICH), because a single method is rarely unequivocal. HPLC, for example, may be used together with MS; or HPLC together with AAA, based on method complementarity considerations.  Other methods may also be necessary. An assay method based on HPLC is preferred, but other methods may also be used if appropriately validated.

Guidance for methods and levels of impurities exist (see references provided in 

) except for peptide-related impurities and TFA, where no generally accepted guidance exists. Levels for these are, therefore, set during the regulatory approval process, based on safety data for the product. Specifications for counterion content, water content, and other specific tests are product-related and are set based on process capability and batch history.

Points to consider for API manufacturers entering the market


  It should be recognized that USP monograph requirements list the minimum quality attributes that APIs must satisfy.  For synthetic peptides, the suitability of a monograph to adequately control the quality of the API must be closely evaluated based on the manufacturing process used. While the HPLC impurities methods described in monographs are demonstrated to be stability-indicating for the subject API and, therefore, should be expected to universally control potential degradation products, the same is not automatically true for process-related impurities. It is necessary that manufacturers demonstrate the suitability of the HPLC impurities methods to control actual and potential synthetic impurities specific to their process.  

Other process related residuals may include starting materials, intermediates, solvents, reagents, catalysts, and metals. They may be adequately controlled by the manufacturing process through in-process controls and the design of the process. Synthetic peptide API manufacturers should, therefore, assess the ability of their manufacturing process to control potential process residuals and identify which residuals may be present at levels that require monitoring and control at the final API stage.  If the monograph methods are inadequate to control such identified process residuals, then the manufacturer must use additional validated methods, as appropriate.  The manufacturer must also establish appropriate limits for process residuals not controlled by the monograph that are justified based on the capabilities of the manufacturing process and a toxicological safety assessment of the process residuals.  


  As a class of APIs, peptides are extremely diverse, not only in terms of their pharmacological properties, but also in terms of their size, AA sequence, and potential for secondary and tertiary structures. Possibly because of this, peptides are specifically excluded from virtually all international guidance documents. Furthermore, while peptides are generally considered to be non-toxic, there is no generally accepted guidance for the requirements for characterization and toxicological characterization of product-related impurities. Limits proposed by the European Medicines Agency (EMA) (4) are wider than those required by the guidance in ICH Q3A, but the former are not recognized by FDA, which therefore, considers the specifications for new peptide APIs on a case-by-case basis.

The USP hopes that, through the work of the Therapeutic Peptides Expert Panel and this series of articles, more consistent guidance may be provided, both for the minimum, acceptable quality attributes for peptide API monographs, and also to industry for the characterization and quality control testing of peptide APIs.

 (December 2012), accessed Feb. 7, 2014.
  2. 	P. Vlieghe et al., 

 15(1/2) 40-56 (2010).
  3. 	P. Van Arnum, 

General Guidance 2034, Substances for Pharmaceutical Use

USP evaluates quality attributes for synthetic peptides.