Ivo Eggen is section lead global technical operations API chemistry at Aspen Pharma.

Ivo Eggen

The United States Pharmacopeia (USP) Therapeutic Peptides Expert Panel was formed in 2013 at the direction of the Monographs-Biologics & Biotechnology Expert Committee to evaluate quality attributes for synthetic peptides 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 from development through manufacturing to lot release. The first article (1) covered analytical characterization methods, lot-release tests and points to consider for synthetic peptide API manufacturers entering the market. The second article (2) focused on raw materials used in the chemical synthesis of peptides. This last article in the series is devoted to manufacturing processes and impurity control for synthetic peptide APIs.  

In the 1950s, pioneers in the field, such as Bodanszky and Du Vigneaud, produced the first bioactive peptides by purely synthetic methods in solution (3). Synthetic peptide chemistry received a big boost in 1963, when Bruce Merrifield developed the method for synthesis on a solid support (solid-phase peptide synthesis [SPPS]) (4). Nowadays, solid-phase techniques and materials have evolved to the point that sequences exceeding 100 amino acids (AAs) in length have become feasible. Besides various hybrid techniques based on extractive methods and solution- and solid-phase synthesis methods, the scope of synthetic peptide chemistry has been further expanded in the past decade by the development of native chemical ligation techniques, which allow the coupling of unprotected peptide fragments to even larger assemblies (5). 


  The chemical manufacturing of peptides generally involves the following sequence of operations:

Assembly of the protected peptide sequence

Removal of the semi-permanent protecting groups

Modifications such as disulfide bond formation and fragment couplings

Purification of the crude peptide by preparative chromatography followed by salt exchange

Isolation of the final, purified peptide.
  

Assembly of the protected peptide sequence.

 The assembly of the peptide backbone involves a series of cycles involving a coupling and a deprotection step. During the coupling step, an activated AA is coupled, usually in molar excess to ensure complete conversion, to the N-terminal amino acid of the growing peptide chain. This AA is protected by a temporary protecting group on its Nα-amino function and, if the side chain of the AA is reactive under the coupling conditions, it may also be protected by a semi-permanent protecting group. Following coupling, the Nα-amino function of the AA is selectively deprotected, leaving side chain protecting groups on the growing peptide intact and liberating the N-terminus of the growing peptide for further elongation. Removal of excess AA derivative is essential to prevent impurity formation. The same principles apply to both classical solution-phase synthesis and solid-phase synthesis. However, in the latter approach, the growing peptide chain is anchored at the C-terminus to a solid support, which allows removal of excess amino acid derivatives and coupling reagents by washing and filtration. 

Removal of the semi-permanent protecting groups.

 Following assembly of the protected peptide sequence, the semi-permanent protecting groups are removed by acidolysis and the peptide is simultaneously cleaved from the resin support in the case of SPPS. Carbocations originating from the cleaved protecting groups are generated under the harsh conditions of acidolysis, requiring the use of scavengers to minimize impurities resulting from the modification of the sensitive peptide chain. In orthogonal protecting schemes involving only one acidolytic deprotection step at the end of the backbone assembly (Z and Fmoc chemistry), the integrity of the peptide sequence is better preserved than in a protecting scheme, which involves acidolysis in every cycle of the peptide assembly, followed by a harsh acidolysis after the backbone assembly (Boc chemistry).

Modifications such as disulfide bond formation and fragment couplings.

 A peptide may contain one or more generally intramolecular and well-defined disulfide bonds between Cysteine residues. Formation of disulfide bonds may be achieved during assembly of the peptide backbone, or after the removal of the semi-permanent protecting groups, with some approaches employing orthogonal thiol protecting groups to selectively affect the desired connectivity, particularly when multiple disulfide bonds are required. 

Fragment couplings, as well as other modifications of specific functions on the peptide sequence, usually involve the selective deprotection of the functional groups to be modified. Several types of protecting groups for the various AA functional groups have been developed, which are either orthogonal to the normal protecting schemes or display a higher sensitivity towards acidolysis.
  
  

Purification of the crude peptide by preparative chromatography and salt exchange.

 Protected peptide fragments have a low propensity to crystallize and, contrary to other classes of compounds, impurities formed in the course of peptide synthesis usually accumulate up to the stage of the crude peptide. This is particularly the case for peptides assembled on a solid support. The crude peptide is, therefore, unlikely to meet the purity specifications set for the API and must be purified. Purification typically involves preparative chromatography, which may comprise sequential purification steps that are based on different retention principles, such as ion exchange or reversed phase. Purification processes developed for peptide APIs typically result from an extensive initial screening of various purification methods on the laboratory scale and rely on a meticulously developed and validated in-process control analytical high-performance liquid chromatography (HPLC) method, which is able to discern actual impurities generated by the synthesis process. This requires an in-depth knowledge of the impurity profile and an understanding of the ability of the selected purification process to purge the impurities.

Following purification, a salt exchange step is typically implemented to remove salts originating from the buffers used during purification to convert the peptide to the desired counterion and set pH. This salt exchange step may be achieved by an additional preparative chromatography step or by use of ion exchange resins. 

The isolation of the peptide API usually occurs through lyophilization of the concentrated aqueous peptide solution following purification and salt exchange. Alternatively, precipitation may be used as a more economical process, although this may not be feasible for all sequences and is likely to involve significant development and engineering to ensure that the API meets the desired solid-state properties and is fit for use in the drug product. Although not yet widely applied in peptide manufacturing, other techniques, such as spray drying, may be used.

Types of impurities in synthetic peptides


  Several types of impurities may be encountered in synthetic peptides, which either originate from the raw materials, from the manufacturing process, or are formed by degradation during the manufacturing process or during storage (

). The various types of manufacturing process impurities, together with their origins, are presented in 

, while degradation impurities are presented in 

. Identification methods and detectability are indicated in both tables.

 Deletion sequences lack one or more amino acid residues. These sequences originate either from incomplete coupling or from incomplete Nα-deprotection steps, especially around so-called “difficult sequences” in SPPS, and hence require careful in-process controls during backbone assembly. The Kaiser colorimetric test (6), typically applied in SPPS to monitor completion of deprotection and coupling reactions, is rapid and straightforward but not always sensitive enough to determine quantitative completion of coupling and especially deprotection reactions. Hence, use of the Kaiser test may not completely prevent the formation of deletion sequences. In such instances, chromatographic analysis can provide quantitative results, but requires a soluble intermediate or, in the case of SPPS, requires cleavage of a resin sample. Sequences lacking the C-terminal residue may arise from incomplete coupling of the first AA residue to a solid-phase resin, when this step is not followed by an efficient capping protocol for residual, active anchoring sites. Removal of a specific deletion sequence impurity during purification is typically more difficult when the missing amino acid is relatively simple (e.g., Glycine or Alanine).

Table I: Potential synthetic peptide process-related impurities. 

HPLC spiking with synthesized diastereomeric   analogs

HPLC spiking with synthesized analogs or isolation/AAA

LC-MS is liquid chromatography-mass spectrometry. LC-MS/MS is liquid chromatography-tandem mass spectrometry. HPLC is high performance liquid chromatography. AAA is amino acid analysis. 
Leu is Leucine. Ile is Isoleucine.

Insertion sequences contain one or more “double” AA residues. The presence of either Nα-unprotected AA derivatives or dipeptides in the starting AA derivatives leads to the formation of insertion sequences and can be controlled by setting appropriate specifications for these materials. Alternatively, during peptide backbone assembly, incomplete removal of excess AA derivative prior to the next deprotection and coupling cycle will lead to the formation of insertion sequences. To prevent process-related insertion sequences, following the coupling step, excess activated AA derivative must be inactivated by quenching or extraction prior to the subsequent deprotection step, and the excess of un-activated AA derivative must be removed prior to the next coupling step. Removal of a specific insertion sequence impurity during purification is typically more difficult when the inserted amino acid is relatively simple. 

Table II: Potential peptide degradation impurities.

 N-terminally truncated sequences may be generated when capping is used as part of the synthesis protocol to prevent deletion sequences that result from incomplete couplings, which is often the case in SPPS. C-terminally truncated sequences, on the other hand, may be generated when quenching is part of the synthesis protocol to prevent insertion sequences. The structure of truncated sequences is usually sufficiently different from the target sequence to allow for efficient removal during purification.

Diastereomeric sequences contain one or more AA residues in the undesired chiral form. Diastereomers are usually more difficult to remove during purification and typically present a greater separation challenge by analytical HPLC than other types of impurities. Their identification usually depends on HPLC spiking experiments of the peptide product with the synthesized diastereomeric analogs. Such spiking experiments may also corroborate the suitability of the analytical release method(s).

Diastereomers may originate from the presence of the optical antipode in starting AA derivatives, requiring the establishment of appropriate specification limits for these raw materials. Alternatively, diastereomers may be formed during peptide backbone assembly through epimerization. Several epimerization mechanisms are known that either involve 5(4H)-oxazolone formation during activation of carboxylic functions (during coupling) or direct Cα-proton abstraction under basic conditions. AA residues with a relatively acidic Cα-proton such as Cysteine and Histidine are especially sensitive to epimerization.

Synthetic approaches have been developed to minimize epimerization, the most basic being that the peptide backbone is assembled from the C-terminus to the N-terminus by stepwise coupling of Nα-urethane protected AAs. Some AA derivatives, such as Cysteine and Histidine derivatives, may require special coupling protocols. Concerning fragment couplings, the only safe options to prevent epimerization are the use of a Glycine or Proline residue in the C-terminal position of the fragment being activated in the fragment condensation. All other residues in this position will lead to some degree of epimerization, which can only be minimized by applying special coupling protocols. Furthermore, the esterification of AA derivatives under basic conditions, with the exception of Glycine, will result in some degree of epimerization, the most frequent examples arising from esterification of the first AA to a solid-phase resin. Finally, peptides with a C-terminal Cysteine ester are prone to epimerization during base treatment, and therefore, direct esterification to a solid-phase resin for Fmoc synthesis should be avoided. Numerous coupling reagents have been developed during the years to promote fast and epimerization-minimizing coupling conditions.

 Substitution sequences occur when one or more AA residues have been substituted by another AA residue, the most common being an Isoleucine ↔ Leucine substitution. Substitution sequences originate from the presence of contaminants in the starting AA derivatives and can consequently be controlled by setting appropriate specifications for these raw materials. The same purification and analytical HPLC challenges described for diastereomer sequences may apply to substitution sequences, especially for the Isoleucine ↔ Leucine substitution.

Modifications of functional groups and disulfide bonds. 

Several AA side chains are susceptible to modification, either during synthesis or during storage. AAs may undergo rearrangements during coupling (e.g., Asparagine, Aspartic acid, and Glutamine) or may be prone to degradation or electrophilic substitution during acidolysis (e.g., primary amides, Tryptophan, Tyrosine, and Methionine). Alternatively, modifications of functional groups may arise from incomplete removal of protecting groups.

Impurities related to disulfide bond modification include reduced (linear) monomers, oxidized (parallel and anti-parallel) dimers and higher polymers, isomers arising from scrambling of disulfide bonds, and oxidized disulfides (thiosulfinates). The desired isomer is usually obtained under strictly controlled and optimized process conditions. The handling of concentrated solution of the peptide product following the purification process should be carefully controlled to avoid polymerization. 

The most common degradation mechanisms include deamidation of Asparagine, Glutamine and the C-terminal amide function, acetylation of amino functions by residual acetate, and disulfide modification (i.e., polymerization). 
  The identification of critical process parameters, as well as extensive stability studies, provide process and product understanding, allowing for optimization of the manufacturing process and definition of hold-times and storage conditions, thereby contributing to the preservation of the integrity of the target peptide.


  Synthetic peptide-related impurities may result from impurities in AA derivatives used as raw materials, from the manufacturing process itself, and from degradation of the peptide during manufacturing or upon storage. Based on a thorough understanding of the peptide, its stability characteristics and its manufacturing process, including identification of raw material attributes and process parameters that affect API quality, an appropriate control strategy for peptide-related impurities can be developed to achieve a manufacturing process that is both economically feasible and also able to yield a peptide API meeting predetermined quality attributes. An understanding of the ability of the purification process to remove peptide impurities can be used to define appropriate impurity specifications for AA derivatives used as raw materials, as well as to define and control critical process parameters during manufacturing. Increased process knowledge may provide an understanding of the ability of the purification process to tolerate variability in the quality of raw materials as well as variability in the crude peptide resulting from peptide backbone assembly, removal of protecting groups, and modification of peptide functional groups. 

USP hopes that the work of the Therapeutic Peptides Expert Panel and this series of articles will provide more consistent guidance to help support efforts to create a sustainable platform for the future of peptide-based drugs.

 38(4) (2014).
  3.	Bodanszky, M. and du Vigneaud, V. J. 

. 81 5688-5691 (1959).
  4.	Merrifield, R.B. J. 

 85 2149-2154 (1963).
  5.	P.E. Dawson et al., 

 266 776-778 (1994).
  6.	E. Kaiser et al., 

 is section lead global technical operations API chemistry at Aspen Pharma, 

 is chief operating officer at Bachem Americas,

 is a consultant at Rode Biologics Consulting,

 is technical advisor at PolyPeptide Group, and 

 is principal scientific liaison at the US Pharmacopeia.

Articles

USP's Therapeutic Peptides Expert Panel discusses manufacturing processes and impurity control for synthetic peptide APIs.

Control Strategies for Synthetic Therapeutic Peptide APIs Part III: Manufacturing Process Considerations

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.