1 12 October 2023

2 EMA/CHMP/CVMP/QWP/387541/2023
3 Committee for Medicinal Products for Human Use (CHMP)
4 Committee for Veterinary Medicinal Products (CVMP)

5 Guideline on the Development and Manufacture of

6 Synthetic Peptides
7 Draft

Draft agreed by Quality Working Party 6 September 2023

Adopted by CHMP for release for consultation 12 October 2023

Adopted by CVMP for release for consultation 5 October 2023

Start of public consultation 18 October 2023

End of consultation (deadline for comments) 30 April 2024

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Comments should be provided using this EUSurvey form. For any technical issues, please contact
the EUSurvey Support.

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Keywords Guideline, synthetic peptide, solid phase synthesis, liquid phase synthesis,
fragment condensation, comparability, amino acids, solid support resin,
linker, conjugation, deprotection, coupling, capping, cleavage, pooling
strategy, stereoisomers, deletion sequence, truncated sequence, insertion
sequence, immunogenicity, sterilisation, generics

12

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13 Guideline on the Development and Manufacture of
14 Synthetic Peptides
15 Table of contents
16 Executive summary ..................................................................................... 3
17 1. Introduction (background) ...................................................................... 3
18 2. Scope....................................................................................................... 3
19 3. Legal basis and relevant guidelines ......................................................... 4
20 4. Active substance ..................................................................................... 4
21 4.1. General Information 3.2.S.1 ................................................................................ 4
22 4.1.1. Nomenclature 3.2.S.1.1 ................................................................................... 4
23 4.1.2. Structure 3.2.S.1.2 .......................................................................................... 4
24 4.1.3. General Properties 3.2.S.1.3 ............................................................................. 5
25 4.2. Manufacture 3.2.S.2 ........................................................................................... 5
26 4.2.1. Manufacturer(s) 3.2.S.2.1 ................................................................................ 5
27 4.2.2. Description of Manufacturing Process and Process Controls 3.2.S.2.2 ...................... 5
28 4.2.3. Control of Materials 3.2.S.2.3 ............................................................................ 6
29 4.2.4. Control of Critical Steps and Intermediates 3.2.S.2.4 ............................................ 8
30 4.2.5. Process Validation and/or Evaluation 3.2.S.2.5 .................................................... 9
31 4.2.6. Manufacturing Process Development 3.2.S.2.6..................................................... 9
32 4.3. Characterisation 3.2.S.3 ...................................................................................... 9
33 4.3.1. Elucidation of Structure and other Characteristics 3.2.S.3.1 ................................... 9
34 4.3.2. Impurities 3.2.S.3.2....................................................................................... 11
35 4.4. Control of the Active Substance 3.2.S.4............................................................... 13
36 4.4.1. Specification 3.2.S.4.1 ................................................................................... 13
37 4.4.2. Analytical Procedures 3.2.S.4.2 ....................................................................... 14
38 4.4.3. Validation of Analytical Procedures 3.2.S.4.3 ..................................................... 15
39 4.4.4. Batch Analyses 3.2.S.4.4 ................................................................................ 15
40 4.4.5. Justification of Specification 3.2.S.4.5 .............................................................. 15
41 4.5. Reference Standards or Materials 3.2.S.5 ............................................................ 16
42 4.6. Container Closure System 3.2.S.6 ...................................................................... 16
43 4.7. Stability 3.2.S.7 ............................................................................................... 16
44 4.7.1. Stability Summary and Conclusions 3.2.S.7.1 .................................................... 16
45 4.7.2. Post-approval Stability Protocol and Stability Commitment 3.2.S.7.2 .................... 17
46 4.7.3. Stability Data 3.2.S.7.3 .................................................................................. 17
47 4.8. Conjugation..................................................................................................... 17

48 5. Medicinal Product Considerations .......................................................... 18

49 6. Synthetic Peptide Development Programmes Using a Biological Medicinal
50 Product as a European Reference Medicinal Product (human products only)
51 .................................................................................................................. 20
52 7. Requirements for Clinical Trial Applications (human products only) ..... 22
53

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54 Executive summary
55 This guideline addresses specific aspects regarding the manufacturing process, characterisation,
56 specifications and analytical control for synthetic peptides which are not covered in the Guideline on
57 the Chemistry of Active Substances (EMA/454576/2016) or Chemistry of Active Substances for
58 Veterinary Medicinal Products (EMA/CVMP/QWP/707366/2017). It also contains requirements and
59 considerations related to conjugation, to medicinal product development, to synthetic peptide
60 development using biological peptides as European reference medicinal product, and to clinical trial
61 applications (human products only).

62 1. Introduction (background)
63 This guideline has been prepared in accordance with the structure agreed for the quality part of the
64 dossier for human medicinal products (Format ICH-CTD). The subheadings have been included for the
65 sake of clarity.

66 2. Scope
67 The purpose of this guideline is to set out the type of information required for the development,
68 manufacture and control of synthetic peptides (existing or new chemical entities) used in a medicinal
69 product.

70 Synthetic peptides are at the interface of small molecules and proteins and, from a quality point of
71 view, specific considerations apply to this class of therapeutics.

72 Synthetic peptides are fully or partially (i.e. depending on their size 1) excluded from the scope of ICH
73 Q3A/B (VICH GL10/GL11), ICH Q6A/B (VICH GL39/GL40) and ICH M7
74 (EMA/CVMP/SWP/377245/2016). This guideline addresses those specific aspects regarding the
75 manufacturing process (solid phase peptide synthesis, fragment condensation), characterisation,
76 specifications and analytical control for synthetic peptides which are not covered in the Guideline on
77 the Chemistry of Active Substances (EMA/454576/2016) and Chemistry of Active Substances for
78 Veterinary Medicinal Products (EMA/CVMP/QWP/707366/2017), and is to be considered complementary
79 to the latter guidelines.

80 This guideline is not applicable to biological and biotechnological products manufactured by

81 recombinant technologies, radiopharmaceuticals and radiolabelled products containing peptides.

82 For synthetic peptides used in radiopharmaceuticals, the principles of this guideline apply regarding
83 synthesis and starting materials. However, for other aspects reference is made to the guideline on
84 radiopharmaceuticals and to the applicable monographs in Ph. Eur. for radiopharmaceuticals.

85 Whilst veterinary products are outside the scope of ICH guidelines there are no corresponding VICH
86 guidelines on certain topics. Nevertheless, the principles outlined in these ICH guidelines may also be
87 relevant to veterinary products to facilitate flexibility and to allow the applicant the option of using
88 different approaches to product development.

1 Tetrapeptides and below will be considered as small molecules to which the quoted (V)ICH guidelines apply

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 89 3. Legal basis and relevant guidelines
90 This guideline should be read in conjunction with the introduction and general principles of Annex I to
91 Directive 2001/83/EC as amended for human medicinal products and Annex II of Regulation (EU)
92 2019/6 as amended for veterinary medicinal products, and all other relevant EU and (V)ICH guidelines.
93 These include, but are not limited to:

94 Guideline on the Chemistry of Active Substances (EMA/454576/2016) and Chemistry of Active

95 Substances for Veterinary Medicinal Products (EMA/CVMP/QWP/707366/2017)

96 EU GMP Part II: Basic Requirements for Active Substances used as Starting Materials

97 ICH Q2 Validation of analytical procedures (veterinary VICH GL1 and GL2)

98 ICH Q3A Impurities in new drug substances (veterinary VICH GL10)

99 ICH Q3B Impurities in new drug products (veterinary VICH GL11)

100 ICH Q3C Residual solvents (veterinary VICH GL18)

101 ICH Q3D Elemental impurities CHMP/ICH/353369/2013 (veterinary Reflection paper

102 EMA/CVMP/QWP/153641/2018)

103 ICH Q6A Specifications – Test Procedure and Acceptance Criteria for New Drug Substances and New
104 Drug Products – Chemical Substances (veterinary VICH GL39)

105 ICH Q9 Quality risk management

106 ICH Q11 guideline on development and manufacture of drug substances (chemical entities and
107 biotechnological/ biological entities)

108 ICH Q13 Continuous manufacturing of drug substances and drug products

109 ICH M7 Assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit
110 potential carcinogenic risk (veterinary EMA/CVMP/SWP/377245/2016)

111 Investigation of Chiral Active Substances 3CC29a, EMEA/CVMP/128/95

112 Note for Guidance on Minimizing the Risk of Transmitting Animal Spongiform Encephalopathy Agents
113 via Human and Veterinary Medicinal Products - EMA/410/01

114 Ph. Eur. Monograph 2902 ‘Precursors for radiopharmaceutical preparations’

115 Reflection paper on statistical methodology for the comparative assessment of quality attributes in
116 drug development - EMA/CHMP/138502/2017

117 4. Active substance

118 4.1. General Information 3.2.S.1

119 4.1.1. Nomenclature 3.2.S.1.1

120 No additional requirements.

121 4.1.2. Structure 3.2.S.1.2

122 Letter codes may be used for the primary structure of the active substance, i.e. 3-letter amino acid
123 codes for the natural amino acids. If also unnatural amino acids or substituents are part of the

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124 structure and shown with letter codes, the used codes have to be accompanied with a legend. In case
125 of peptide chains, the N-terminus and the C-terminus of the chain have to be clearly indicated.

126 In case the molecule only contains amino acids with the natural L-configuration (eventually in
127 conjunction with unnatural amino acids without chiral center), a structural formula with indication of
128 the chiral centers is not needed (i.e. the 3-letter codes are sufficient). If the molecule contains amino
129 acids with both D- and L-configuration (eventually in conjunction with unnatural amino acids without
130 chiral center), a chemical formula with clear indication of the chiral centers and their configuration is
131 necessary. In the latter case, a structure consisting of 3-letter codes only preceded by the appropriate
132 D- or L- letters for unnatural amino acid may be helpful (in addition to the structural formula) for
133 further use throughout the dossier.

134 Full chemical structure of non-peptidic side chains and linkers is expected.

135 4.1.3. General Properties 3.2.S.1.3

136 Relevant general properties of the peptide in question should be listed. In most cases, water content,
137 isoelectric point, pH of a solution of the peptide, optical rotation, biological activity and solubility in
138 different media would be expected. Hygroscopicity needs to be indicated, e.g. with moisture sorption
139 isotherms, or reference could be made to 3.2.S.3.1 where such information could be provided more in
140 detail.

141 Most peptides are amorphic powders, therefore melting point and polymorphic form are generally not
142 applicable.

143 The counter ion needs to be indicated, if relevant, and whether it is present in a stoichiometric or non-
144 stoichiometric ratio.

145 4.2. Manufacture 3.2.S.2

146 4.2.1. Manufacturer(s) 3.2.S.2.1

147 No additional requirements.

148 4.2.2. Description of Manufacturing Process and Process Controls 3.2.S.2.2

149 Schematic representation of the manufacturing process

150 For the graphical presentations of the synthetic process(es) for peptides, it is considered acceptable to
151 replace chemical structures by (three) letter codes in the reaction schemes to improve legibility,
152 especially in case of longer peptide sequences. Letter codes for unnatural amino acids and substituents
153 should be accompanied with a legend.

154 Sequential procedural narrative

155 The sequential procedural narrative should describe each step in the manufacturing process. During
156 peptide synthesis the same standardized steps may be used several times, e.g. in Solid Phase Peptide
157 Synthesis (SPPS), the peptide sequence is built up on a solid support by repeated cycles of
158 deprotection, washing and coupling steps. These standardized steps with their associated Proven
159 Acceptable Ranges (PARs) need not be described in detail each time they are used, provided clear
160 descriptions of the used conditions (e.g. reagents, solvents, reaction times, …) are given, and provided
161 it is clearly indicated where these steps are used in the overall manufacturing process. The final
162 deprotection step should be described in detail, including any use of scavengers and other reagents, in

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163 case of which a discussion of their genotoxic potential should be included in 3.2.S.3.2. Amounts can be
164 described as weights/volumes or equivalents.

165 Splitting or combining of sub-batches/multiple cycles may be performed at different stages during
166 manufacturing, e.g. based on equipment capacity or operational efficiency in SPPS. The criteria applied
167 in the decision on splitting or pooling of sub-batches and/or multiple cycles should be provided, along
168 with an adequate justification for the selected approach. Moreover, material traceability from the
169 synthesis steps through the final drug substance is expected and S.2.2 should contain an unambiguous
170 definition of the commercial batch size (range).

171 Hybrid manufacturing approaches i.e. manufacturing of peptide fragments by solid phase peptide
172 synthesis followed by fragment condensation (liquid phase synthesis) have been used in the past and
173 may become more popular e.g. for large scale production. For the manufacturing of the peptide
174 fragments it is referred to the section above. The fragment condensation steps should be described in
175 adequate detail including the purification steps.

176 When continuous manufacturing approaches are intended, the requirements of ICH Q13 on the
177 description of the manufacturing process should be considered.

178 In case two drug substance manufacturing processes will be used in parallel (e.g. solid phase synthesis
179 and a hybrid process), results from comparability studies on drug substance and drug product level
180 should be provided.

181 Reprocessing, recovery and rework

182 The terms should be used and understood as defined in ICH Q7.

183 Synthetic peptides are generally purified using chromatographic techniques, often starting from a
184 relatively complex crude intermediate. It is recognized that the crude peptide typically contains pre-
185 and post-eluting structurally related impurities. It is acceptable to perform repeated purification steps
186 of these side-fractions resulting in eluate meeting the purity requirements of the main fractions. When
187 routine repurification is carried out this is not considered reprocessing but part of the regular
188 manufacturing process and should be justified accordingly. Clear description of the repurification
189 procedure and the criteria for deciding when it can be performed should be provided (see also ‘4.2.3.
190 Control of Critical Steps and Intermediates 3.2.S.2.4’ below). The routine repurification process of the
191 side fractions (if used) should be part of the manufacturing process qualification/validation. Likewise, if
192 repetition of the coupling reaction is part of the routine manufacturing process, it is not considered
193 reprocessing.

194 Appropriate measures to prevent cross-contamination due to the successive purification of different
195 peptides using the same column should be in place, as required by GMP.

196 Lyophilisation

197 Lyophilisation of synthetic peptides is considered common practice. Lyophilisation process parameters
198 should be described.

199 4.2.3. Control of Materials 3.2.S.2.3

200 Active Substance (AS) Starting Material(s)

201 The requirements of ICH Q11 and its associated Q&A in relation to the selection of starting materials
202 are relevant to synthetic peptides. The name and address of all starting material manufacturers should
203 be provided. The addition of manufacturers for the starting materials needs to be approved by a
204 variation according to European legislation. Information, in the form of flowcharts, indicating the

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205 synthetic process(es) of all starting materials including details of reagents, solvents and catalysts used,
206 should be provided, followed by a criticality assessment of which starting material impurities may have
207 an impact on the impurity profile of the peptide.

208 Amino acids

209 Protected amino acid derivatives (with terminal and side-chain protection as relevant) are generally
210 acceptable as starting materials in the manufacturing process of synthetic peptides. Nevertheless, a
211 justification on the designation of starting materials needs to be provided. For D-amino acid and
212 unnatural amino acid derivatives, more detailed information regarding their manufacture (e.g.,
213 precursors and used reagents) and impurity profile is required than for standard L-amino acid
214 derivatives.

215 In justified cases, short peptide segments such as protected dipeptide building blocks, may be
216 acceptable as starting materials. Examples are dipeptides containing glycine or other dipeptides whose
217 use will reduce the formation of diketopiperazine by-products compared to consecutive couplings of the
218 individual amino acid derivatives. Other examples include Dmb-Glycine and pseudoproline-dipeptides
219 which can be used to minimize aggregation during peptide assembly. However, polypeptide segments
220 that undergo further modification (e.g. cyclisation or conjugation) are generally not acceptable as
221 starting materials but are considered intermediates.

222 More complex peptides could be acceptable as starting materials in duly justified cases (e.g. in
223 fragment condensation manufacturing processes). Companies are recommended to go for scientific
224 advice to discuss their proposal well in advance.

225 Quality attributes for amino acid derivatives used as starting materials for synthetic peptides generally
226 include: appearance, identification, related impurities, other impurities and assay. For the protected
227 amino acid derivatives used as starting materials during peptide synthesis several typical related
228 impurities may be present, these include: enantiomeric and diastereomeric impurities, (partially)
229 unprotected amino acids, dipeptides and β-alanyl impurities. Other impurities may include e.g. residual
230 solvents, water content and elemental impurities. Since the impurities of the amino acid derivatives,
231 which can react like the parent compound during coupling, can accumulate in the final drug substance,
232 the relevant impurities should be adequately controlled and limited in the starting material
233 specifications. The impurity profiles of the starting materials and their potential impact on the quality
234 of the final drug substance should be investigated during manufacturing process development. This
235 should include a fate and purge assessment of the impurities that may be formed downstream in the
236 manufacturing process.

237 Amino acids from human or animal origin should be avoided where possible. If used, Ph. Eur. chapter
238 5.2.8 on ‘Minimizing the Risk of Transmitting Animal Spongiform Encephalopathy Agents via Medicinal
239 Products,’ and the ‘Note for Guidance on Minimizing the Risk of Transmitting Animal Spongiform
240 Encephalopathy Agents via Human and Veterinary Medicinal Products’ (EMA/410/01) should be
241 considered and TSE safety should be addressed.

242 Peptide synthesis resins preloaded with the first amino acid of the peptide sequence (through a linker)
243 are also considered starting materials. However, the unloaded solid support resin itself is not
244 considered a starting material as it is not incorporated as a significant structural fragment into the
245 structure of the drug substance.

246 For solid support resins preloaded with amino acids, quality attributes related to the (chiral) purity and
247 potential impurities of the loaded amino acid derivative are recommended. For the resin itself,
248 reference is made to section ‘Other materials used in the manufacturing process’.

249 Non-peptidic structural moieties

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250 Conjugation and other derivatisations of peptides are commonly used. In these cases, sometimes
251 complex structures are added to the peptide sequence. The classification of these materials will be
252 handled on a case-by-case basis and early interaction (scientific advice) with the regulatory agencies is
253 recommended. However, also for starting materials of non-peptide structural moieties (e.g. PEG-
254 chains), compliance with the requirements as laid down in ICHQ11 and its associated Q&A is expected
255 and its selection as starting material should be justified. For instance, sufficient subsequent chemical
256 transformation steps after the starting material should be performed under GMP.

257 Other materials used in the manufacturing process

258 A list of all other reagents, such as resins, solvents and chromatographic materials used in the
259 manufacturing process of a synthetic peptide should be provided. Adequate specifications for all
260 materials should be laid down considering their role in the process but covering as a minimum identity
261 as well as purity and/or assay where applicable.

262 The solid support resin is a key component of the SPPS process, typical quality attributes of the resin
263 include: appearance, identification, cross-linking, swelling volume, mesh size and loading.

264 4.2.4. Control of Critical Steps and Intermediates 3.2.S.2.4

265 The criticality of the manufacturing steps for peptides made by solid phase synthesis should be
266 evaluated during development according to the principles described in ICH Q9–Q11. In-process
267 controls should be defined. The control of critical steps can be achieved by a combination of analytical
268 tests and process control. During SPPS critical steps could include, e.g., 9-fluorenylmethoxycarbonyl
269 (Fmoc) deprotection, control of washing steps, coupling or capping reaction monitoring, control of
270 cleavage steps and drying steps. The most common test for the monitoring of coupling, capping and
271 deprotection reactions is the Kaiser test, which is a colorimetric test based on the reaction of ninhydrin
272 with primary amines.

273 During peptide purification by preparative chromatography, individually collected fractions are usually
274 combined into a pool of fractions. The pooling strategy should be defined and acceptance criteria for
275 the purity of individual fractions and the main pool should be stated. These criteria for purity usually
276 include overall purity and criteria for individual impurities. In case secondary purification is proposed
277 in the manufacturing process, adequate requirements for side-fractions that are allowed to undergo
278 such purification, and the conditions thereof, should be defined. It should be stated which fractions are
279 discarded. Filtration and lyophilisation steps should also be adequately controlled.

280 Intermediates as defined in the manufacturing process are tested before use in the next stage of the
281 manufacturing process. The methods used for in-process control and/or intermediate testing should be
282 described and confirmation of validation provided where applicable.

283 The ICH Q7 definition of intermediate should be considered, i.e., “A material produced during steps of
284 the processing of an API that undergoes further molecular change or purification before it becomes an
285 API. Intermediates may or may not be isolated.” For all intermediates, justified specifications should be
286 presented. Only intermediates complying with specifications may be pooled.

287 For the crude peptide after cleavage and deprotection adequate and justified specifications should be
288 presented.

289 For solution-phase synthesis or fragment condensation processes, other requirements may apply for
290 control of intermediates compared to SPPS.

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291 4.2.5. Process Validation and/or Evaluation 3.2.S.2.5

292 No additional requirements (i.e. process validation data would normally not be expected in the dossier).

293 4.2.6. Manufacturing Process Development 3.2.S.2.6

294 It is acknowledged that there are general aspects of peptide synthesis where established knowledge
295 and manufacturing experience may be extrapolated between different processes. Also, the starting
296 materials and their properties are mostly well-known. If justified, manufacturers may make reference
297 to prior knowledge for general aspects of the manufacturing process (e.g. choice of resin or coupling
298 reagents). However, it is expected that substance specific aspects such as e.g. reaction times,
299 temperatures and molar equivalents are addressed in the development section. If in-house knowledge
300 from related products is referred to, the data and source should be identified as appropriate and
301 differentiated from product-specific data. A discussion of how the prior knowledge data is to be used
302 should be integrated with the relevant product-specific data to provide an overall understanding of
303 product development and control. If prior knowledge from scientific papers is quoted, copies of the
304 paper should be provided as appropriate.

305 4.3. Characterisation 3.2.S.3

306 4.3.1. Elucidation of Structure and other Characteristics 3.2.S.3.1

307 The structure of the peptide should be confirmed by analytical data, this includes the primary,
308 secondary, tertiary and quaternary structure where relevant. Mass spectrometry (MS) is a powerful
309 analytical tool for the structure elucidation of peptides. Variants of the MS technique can be used to
310 determine the molecular mass of a peptide and to confirm its amino acid sequence. Typical
311 representative spectra and interpretation of the fragmentation data, including assignments and tables
312 with theoretical and observed mass values, should be provided.

313 Amino acid analysis as described in the Ph. Eur. general chapter 2.2.56 usually complements the
314 characterisation of synthetic peptides.

315 Elemental analysis may be used in view of structure confirmation.

316 NMR spectrometry is described in Ph. Eur. general chapter 2.2.64, ‘Peptide Identification by Nuclear
317 Magnetic Resonance Spectrometry.’ However, the scope described in that general chapter is qualitative
318 and consists of comparing the NMR spectrum of a test sample with that of a reference sample acquired
319 under identical conditions. Furthermore, the scope is restricted to one-dimensional NMR spectrometry.
320 NMR experiments are recommended to be part of the characterisation studies. One- and two-
321 dimensional techniques should be used to assign the structure by means of 1H, 13C and 15N NMR data
322 where relevant.

323 NMR can be used for

324 • determination of the number and types of proton nuclei;

325 • determination of the peptide sequence;

326 • identification of amino acids;

327 • assignment of carbon atoms;

328 • assignment of nitrogen atoms;

329 • secondary and tertiary structure elucidation.

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330 Chiral gas chromatography (GC) is often used to identify and quantify the enantiomers of the different
331 amino acids after acid hydrolysis. As the hydrolysis is known to induce some level of racemisation, it is
332 carried out in deuterated hydrochloric acid, yielding deuterated amino acids if the racemisation takes
333 place at this stage; deuterated and non-deuterated amino acid residues are detected separately by a
334 mass spectrometric detector placed in tandem with the chiral GC system. Enantiomeric purity can be
335 controlled by several means during the manufacture of a synthetic peptide. However, it should be
336 justified that it is sufficient to perform the test on enantiomeric purity as a characterisation test and
337 that no routine release control is required.

338 Ultraviolet (UV) and infrared (IR) spectroscopy are part of the standard characterisation programme.
339 Additional information on the secondary structure can be gathered from these techniques.

340 Circular dichroism (CD) spectroscopy can be used to determine the absorption, e.g. in the presence of
341 chromophores such as tryptophan, tyrosine, phenylalanine, disulfide bonds and peptide bonds. It
342 measures differences in absorbance between left and right circularly polarized light and hence
343 asymmetric properties of the chromophores. Changes in the structure and hence aromatic
344 environments result in different CD spectra. Near-UV CD spectroscopy determines the tertiary
345 structure due to asymmetric environments of tryptophan, tyrosine, phenylalanine and disulfide.

346 Far-UV CD spectroscopy determines the secondary structure due to asymmetric environments of the
347 peptide.

348 Usually, no biological assay is required for the routine release of synthetic peptides. Nevertheless,
349 biological assays can serve as additional tools for the characterisation of synthetic peptides.

350 Peptide mapping may be applicable for longer peptides. Ph. Eur. General chapter 2.2.55, ‘Peptide
351 Mapping’, should be considered.

352 Peptides can also be characterized by Thioflavin T (ThT) dye assays and intrinsic tryptophan
353 fluorescence to investigate whether a peptide can form fibrillary aggregates.

354 Where relevant disulfide bridge confirmation should be part of the characterisation studies.

355 Evidence of chemical structure

356 The information will normally include such evidence as:

357 List of characterisation techniques used for synthetic peptides (example table):

Test Analytical technique

Molecular mass MS, LC-MS

Amino acid sequence confirmation LC-MS/MS of intact molecule

LC-MS of enzymatically treated material (peptide

mapping for long peptides)

Enantiomeric purity Chiral GC-MS

Identity of potential counter ions RP-HPLC and Ion Chromatography

Extinction coefficient UV spectroscopy

Secondary structure Far-UV circular dichroism (CD) spectroscopy

FT-IR spectroscopy

Tertiary structure* Near UV CD spectroscopy

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 Test Analytical technique

Quaternary structure / association state* Composition gradient multiangle light scattering

(CG-MALS)

Biological characterisation Cell-based and other biological assays

358 * In rare cases, tertiary structures or the association state (e.g. in the form of oligomers) may be relevant and
359 should be addressed on a case by case basis

360 The relevance of the eventual or possible isomers regarding biological/pharmacological activity should
361 be discussed (for veterinary products see Investigation of Chiral Active Substances 3CC29a,
362 EMEA/CVMP/128/95).

363 Physico-chemical Characteristics

364 Physicochemical characterisation of the drug substance could include solubility and hygroscopicity
365 studies, determination of the isoelectric point (pI) and thermogravimetric studies. The morphology
366 may be examined by powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC) and light
367 microscopy.

368 4.3.2. Impurities 3.2.S.3.2

369 Purity is one of the most important critical quality attributes (CQAs) for synthetic peptides. Impurities
370 are often categorized as either peptide-related impurities or non-peptide impurities. Peptide-related
371 impurities contain structural elements of the synthetic peptide sequence. Non-peptide impurities
372 include process reagents and their potential by-products, residual solvents, elemental impurities and
373 potential genotoxic impurities.

374 Peptide-related impurities may originate from different sources:

375 • starting materials;

376 • formation during the manufacturing process;

377 • resulting from degradation during the manufacturing process or during storage.

378 The levels of certain related substances may be the result of combined contributions from these
379 sources.

380 Related substances resulting from starting materials

381 Impurities present in the starting materials may cause the formation of peptide-related impurities.
382 Examples of such impurities include incorrect enantiomers, β-Ala residues, dipeptides, single amino
383 acid derivatives in dipeptides and amino acids with incorrect or without protecting groups. These
384 impurities may be incorporated in the sequence during the assembly of the peptide by SPPS. In
385 addition, with the exception of glycine, all natural amino acids constituting synthetic peptides are
386 optically active with at least one stereogenic centre each. The presence of isomeric impurities in these
387 starting materials can lead to the formation of stereoisomers of the final peptide.

388 Narrow acceptance criteria for those impurities should be set for each starting material used in the
389 manufacture of the peptide.

390 Related substances formed during the manufacturing process

391 Related substances designated as process impurities may be a result of undesired or incomplete
392 reactions during SPPS or cleavage.

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393 Stereoisomers

394 In addition to starting materials, the manufacturing process can be a source of stereoisomers
395 formation, for example by epimerisation. Racemisation of amino acids during the manufacturing
396 process is possible. Hence, in theory, a multitude of different diastereoisomers of the target peptide
397 can be formed. However, SPPS conditions should be optimised to ensure minimized racemisation.
398 Amino acid derivatives with urethane-type protecting groups (e.g. Fmoc) are rather resistant to
399 oxazolone formation which leads to racemisation at the stereocentre. Additionally, additives and bases
400 used in the manufacturing process also suppress the potential racemisation of utilized amino acids.

401 Deletion sequences and truncated sequences

402 Deletion sequences are peptides with one or several amino acids missing. Their formation is often
403 prevented by repetition of the coupling procedure to complete the coupling reaction and systematic
404 acetylation to cap unreacted coupling sites. The acetylation procedure results in acetylated peptide
405 fragments (truncated sequences).

406 Insertion sequences

407 If a particular amino acid is coupled more than once during one coupling step, an insertion sequence is
408 generated. Insertion sequences may occur as the result of premature deprotection during prolonged
409 coupling reactions or incomplete removal of excess amino acid derivative prior to the next deprotection
410 step.

411 Related substances formed during cleavage

412 Certain side-chain protecting groups are released as reactive cationic species during cleavage. These
413 can react with the nucleophilic side chains of sensitive amino acids (e.g. Trp, Tyr, Met). To prevent the
414 formation of related substances through such side reactions, suitable scavengers are added to the
415 cleavage mixture. Potential impurities could also result from incomplete cleavage of the amino acid
416 side-chain protecting groups.

417 Related substances resulting from degradation during the manufacturing process or during storage

418 Degradation products of synthetic peptides may also occur as process impurities. Their content may
419 increase during storage. Generally, the following pathways can contribute to the formation of
420 degradation products of synthetic peptides:

421 • oxidation;

422 • hydrolysis;

423 • deamidation;

424 • diketopiperazine and pyroglutamic acid formation;

425 • β-elimination;

426 • condensation and formation of dehydropeptides;

427 • disulfide cleavage/exchange.

428 Aggregation may occur for synthetic peptides and could potentially be related to safety issues,
429 including immunogenicity and should therefore be investigated.

430 Possible routes of degradation should be discussed - see section 3.2.S.7.1.

431 Analytical methods

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432 Highly specific analytical methods (with appropriate limits of detection (LOD) and limits of quantitation
433 (LOQ) used to detect each of the likely impurities considered above, or other related impurities, the
434 exact identities of which may be unknown, should be described. Copies of relevant chromatograms
435 should be provided. A summary should be given on the nature and levels of the actual impurities
436 detected in the batch samples of the material.

437 Process-related impurities

438 Non-peptide impurities include process reagents, by-products, residual solvents and elemental
439 impurities. The solid phase synthesis process requires extensive washing of the resin with solvents.
440 Reagents and solvents used for the coupling steps are washed with incremental quantities of a suitable
441 solvent. Nevertheless, for all reagents and solvents used in the manufacturing process, the depletion
442 should be addressed in the dossier by either data and/or risk analysis. Any residuals of reagents
443 and/or solvents should either comply with ICH M7 / EMA/CVMP/SWP/377245/2016 (if genotoxic), or –
444 if not genotoxic - ICH Q3C/VICH GL18 thresholds, or (in absence of ICHQ3C/VICH GL18 thresholds),
445 be toxicologically qualified.

446 Synthetic peptides themselves and peptide-related impurities are not within the scope of ICH M7 /
447 EMA/CVMP/SWP/377245/2016).

448 4.4. Control of the Active Substance 3.2.S.4

449 4.4.1. Specification 3.2.S.4.1

450 The active substance specification should be provided.

451 Typical specification tests included as an attribute in the specification are as follows (non-exhaustive
452 list):

453 • appearance (+ appearance of solution if relevant);

454 • identification;

455 • amino acid analysis;

456 • purity (total impurities; individual impurities (unspecified/specified);

457 • aggregates / oligomers by SEC-HPLC (if relevant);

458 • assay/content; e.g. by HPLC or elemental analysis;

459 • acetic acid content/counter-ion content*;

460 • (residual) TFA content*;

461 • pH of solution;

462 • water content;

463 • mass balance;

464 • residual solvents;

465 • elemental impurities (e.g. in case of use of metal catalyst);

466 • bacterial endotoxins;

467 • microbiological purity.

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468 The acceptance criteria laid down in the drug substance specification are identical with the limits that
469 apply for stability studies.

470 Synthetic peptides are excluded from the scope of ICH Guideline Q3A/VICH GL10, ‘Impurities in New
471 Drug Substances’, and consequently the limits laid down in this guideline are not applicable. Specific
472 thresholds for peptide-related impurities are defined in the Ph. Eur. According to the Ph. Eur. general
473 monograph ‘Substances for Pharmaceutical Use’, peptide-related impurities should be reported above
474 0.1%, identified above 0.5% and qualified above 1.0%.

475 * The type of counter ion can affect the biological and physicochemical properties of the peptide and
476 related final formulations. For synthetic peptides, usually acetate is used as counter ion, however,
477 other counter-ions are also possible (e.g. trifluoroacetic acid or TFA). The type of counter ion should be
478 justified, and the amount of counter ions should be controlled in the drug substance specification with
479 an upper and lower limit.

480 4.4.2. Analytical Procedures 3.2.S.4.2

481 Analytical Development

482 Ph. Eur. general chapters applicable to peptides (e.g. 2.2.55 Peptide mapping, 2.2.56. Amino acid
483 analysis, 2.5.34 Acetic acid in synthetic peptides, 2.2.64 Peptide identification by NMR) and the “EDQM
484 Technical guide for the elaboration of monographs on synthetic peptides and recombinant DNA
485 proteins” may be also helpful for the development of the analytical methods.

486 The development of analytical procedures to control the quality of peptides, specifically the identity
487 and the peptide-related impurities, could be a challenge due to the complexity of the structure of these
488 molecules and the risk of co-eluting impurities.

489 Identification

490 The evidence of chemical structure should be discussed under Section 3.2.S.3.1. For the identification
491 of the peptide as part of the specification and release, use of at least two orthogonal methods is
492 recommended. Identification by mass, relative retention time (RRT), LC-MS, LC-MS/MS, peptide
493 mapping, bioactivity, amino acid analysis or NMR are considered appropriate.

494 The applicant has to ensure that the proposed identification test or combination of tests is suitable to
495 unambiguously confirm the sequence of the peptide.

496 Purity

497 The suitability of analytical procedures to detect and quantify impurities discussed under Section
498 3.2.S.3.2 should be demonstrated. At least, the analytical methods used for the control of purity
499 should be suitable to fulfil the requirement for the Ph. Eur. reporting threshold of 0.1% for synthetic
500 peptides.

501 If one analytical method for detection and quantification of all the peptide-related impurities is not
502 appropriate to separate all peaks, additional independent method(s) may be needed. The combination
503 of methods may enable measurement of structurally related impurities measured in drug substance.
504 Stability indicating properties and mass balance of the method(s) need to be studied.

505 Control of diastereomers may require the development of specific chiral methods.

506 Changes of the analytical methods during development

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507 The level of detail of the commercial analytical procedures used for testing peptides should be
508 described in the dossier in such a way that they can be repeated by an Official Medicines Control
509 Laboratory.

510 If different methods have been used to generate data during development, a brief description and
511 comparison of data should be included in appropriate place in the dossier.

512 4.4.3. Validation of Analytical Procedures 3.2.S.4.3

513 The analytical procedures used for the control of the drug substance, including the analytical
514 procedures used during the development of the peptide, should be fully validated. In general, the
515 validation of analytical tests concerning the active substance should be performed according to the
516 requirements of the current Guidelines (ICH Q2, VICH GL1 and GL2).

517 In case the quality of the peptide is covered by a monograph in the Ph. Eur. and the methods of the
518 monograph are used, it is not necessary to present the validation of these analytical procedures in the
519 dossier. In case the peptide is described in the Ph. Eur. monograph but the company applies its own
520 methods, then (cross)validation data is needed in the dossier.

521 4.4.4. Batch Analyses 3.2.S.4.4

522 This section should summarize the batch analysis data for all the peptide batches described in the
523 dossier.

524 Apart from the analytical determination, for each batch the following information should be provided:
525 date of manufacture, batch size and number, scale (laboratory/pilot/commercial), route of synthesis
526 (commercial or not), place of manufacture, use of batches.

527 As recommended in other guidelines, presentation of this information in tabular form is recommended
528 for improved clarity. Usually, early development batches are tested using a slightly different
529 specification. The differences in the results obtained in the batches used in earlier development and
530 pilot/commercial batches should be explained and justified.

531 The improvement in the analytical methods during development of the peptide could lead to the
532 observation of new impurities in pilot/commercial batches. In those cases, comparison of the batch
533 analysis data should be performed, and the impact on the quality of the drug substance and or
534 clinical/preclinical data should be discussed.

535 4.4.5. Justification of Specification 3.2.S.4.5

536 The proposed specification should be supported by batch data from preclinical, clinical and production
537 scale batches combined with an adequate understanding of the manufacturing process of the peptide.

538 The limits applied for peptide-related impurities should be based on the general monograph of the
539 European Pharmacopoeia ‘Substances for Pharmaceutical Use (2034)’. In case that the limit for
540 identified or unidentified impurities is above the prescribed Ph. Eur. qualification threshold,
541 qualification of these impurities is expected. Nevertheless, the acceptance criteria for the peptide-
542 related impurities and total impurities should not only be based on the qualified limit but on data
543 obtained from the batches used to support the quality of the drug substance.

544 Grouping of impurities which can be analytically separated (e.g. pre- and post-eluting groups) is not
545 recommended, and can only be accepted in duly justified cases, based on demonstrated analytic
546 efforts.

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547 In case of a very complex impurity profile or where two or more impurities are very similar, it may not
548 be technically feasible to obtain peak separation. In such cases it may be necessary to set a limit for a
549 combination of unresolved peaks. In this case, thresholds should be applied for the combination of
550 peaks. For qualification, the impurity profile of the batches used in the toxicological studies should be
551 taken into account.

552 The assay and how it is calculated should be clearly defined. The assay limits are typically expressed in
553 terms of the counter-ion free, anhydrous substance, unless otherwise justified. The limits for assay are
554 typically asymmetric, the upper limit being 100 per cent + the permitted assay reproducibility, and the
555 lower limit being 100 per cent – (the permitted assay reproducibility + the maximum permitted level of
556 impurity).

557 The absence of a biological assay should be justified.

558 4.5. Reference Standards or Materials 3.2.S.5

559 Peptides are often very hygroscopic powders, therefore appropriate precautions against moisture
560 uptake by the reference standard during storage and during analysis should be taken when relevant.

561 The origin of the reference standards should be briefly indicated (e.g. batch synthesised according to
562 the commercial process). If a 2-tiered system is used (primary reference standard and working
563 reference standard) the preparation and qualification strategy should be briefly explained, and the
564 characterisation results obtained for the reference standard batches, the approach to periodically
565 requalify the reference standards, as well as the approach that will be followed to qualify future
566 batches of reference standards, including the measures that will be taken to prevent drift in peptide
567 content, should be presented.

568 If reference standards are used for certain impurities, a short description on how these were prepared
569 and characterised/qualified for use should be provided.

570 4.6. Container Closure System 3.2.S.6

571 The container closure system should be suitable, considering the substance properties, storage
572 conditions and use: e.g. for hygroscopic powders, appropriate desiccant should be included.
573 Alternatively, storage under inert atmosphere could be considered.

574 4.7. Stability 3.2.S.7

575 4.7.1. Stability Summary and Conclusions 3.2.S.7.1

576 The principles outlined in EMA's and (V)ICH scientific guidelines on the stability of drug substances
577 should be followed with regards to aspects such as the types of studies conducted, protocols used,
578 selection of batches, container closure system and storage conditions.

579 The choice of test conditions applied during stability storage (temperature and humidity) should be
580 justified. Generally, to prevent or minimize degradation, peptides are stored under refrigerated (5°C ±
581 3°C) or freeze conditions (-20°C ± 5°C), but the use of higher temperatures/humidities are also
582 expected not only to address short term excursions, but to obtain a comprehensive overview of the
583 degradation pathways of the drug substance; these data might be especially important for the
584 development of the drug product.

585 The potential degradation pathways of the peptide should be discussed taking into account the amino
586 acids composition and sequence: e.g. oxidation of Cys and Met residues, deamidation, hydrolysis, β-

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587 Asp-containing sequences. Forced degradation studies are foreseen to evaluate both, the degradation
588 of the peptide and the ability of the analytical procedures to detect the degradation.

589 For hygroscopic powders, it is expected that water content should be part of the stability protocols.

590 Aggregation may also occur for synthetic peptides and could potentially be related to safety issues,
591 including immunogenicity and should therefore be investigated during stability.

592 The retest period and storage conditions should be justified following EMA's and (V)ICH scientific
593 guidelines on the stability of drug substances.

594 Variability in stability testing results should be avoided by establishing appropriate handling procedures
595 during analytical testing.

596 4.7.2. Post-approval Stability Protocol and Stability Commitment 3.2.S.7.2

597 General principles outlined in EMA's and (V)ICH scientific guidelines should be followed.

598 4.7.3. Stability Data 3.2.S.7.3

599 In some cases, the analytical procedures used during stability, especially during earlier development
600 stages, are not identical to the analytical procedures which are part of the drug substance
601 specification. The changes between these methods and the commercial analytical methods should be
602 justified, and the impact on generated stability results to be discussed.

603 4.8. Conjugation

604 Conjugation has emerged as a popular mechanism to alter or enhance the properties of peptide drug
605 candidates. Conjugation to poly(ethylene glycol) (PEG), lipids and proteins has been used as a half-life
606 extension strategy. Conjugation can also be used to deliver a cytotoxic payload or imaging agent to
607 specific cell types targeted by the peptide.

608 However, there is added complexity with respect to the characterisation and control of these
609 conjugates. The control of the unconjugated peptide which is usually classified as an intermediate is
610 essential. Adequate specifications and control methods should be established for these intermediates.
611 In cases where no intermediate is isolated these approaches should be justified and an adequate
612 control strategy should be developed (see also 4.2.3).

613 The underlying conjugation chemistry should be described in the manufacturing process development
614 section. Conjugatable versus non-conjugatable impurities should be identified by means of a risk
615 analysis and the incorporation into the target molecule should be investigated. Purging of process-
616 related impurities from the conjugation process should be investigated.

617 An additional quality attribute for conjugated peptides is the amount of the free unconjugated peptide
618 and the free form of the conjugate moiety (e.g. free PEG/linker). Di-PEGylation or multi-PEGylation (or
619 other conjugation moieties) may also occur and should be adequately controlled.

620 The choice of the starting material of the conjugation component needs to be justified according to ICH
621 Q11, ‘Questions & Answers: Selection and Justification of Starting Materials for the Manufacture of
622 Drug Substances’. It has to be assured that all steps of the intermediate synthesis starting from the
623 defined starting material are performed under good manufacturing practice (GMP). Consequently, e.g.
624 the activation of the suitable PEG starting material is considered a part of the manufacturing process
625 and an activated PEG derivative (e.g. in the form of an N-hydroxysuccinimide (NHS) ester) may not be
626 suitable as starting material and is considered to be an intermediate itself.

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627 Full information should be provided in Section 3.2.S.2.2 of Module 3, including flowchart, process
628 description with all process steps, raw materials and manufacturing process controls.

629 In numerous development programmes, polymers or other conjugation moieties are coupled to the
630 peptide via a chemical linker. The points mentioned above are also applicable for such chemical linkers,
631 especially for the selection of suitable starting materials and the control of the impurity profile. The
632 critical attributes should be evaluated and a justification for the specification attributes should be
633 provided. The basic principles of ICH M7/EMA/CVMP/SWP/377245/2016 regarding a mutagenic
634 impurities assessment should be considered for chemical linkers and conjugation moieties.

635 In many cases, the conjugation moiety and the linkers are manufactured by a different manufacturer
636 than the synthetic peptide. In the case of multiple suppliers of the conjugation moiety and/or linker,
637 for each supplier separate documentation is expected, and a compiled specification for the conjugation
638 moiety should be elaborated by the manufacturer of the peptide-conjugate.

639 Peptide-conjugated material from all suppliers of the conjugation moiety and/or linker should be
640 manufactured and batch analysis and stability data should be generated.

641 It is recommended to consider the legal framework for cases where a New Active Substance status is
642 claimed and an unconjugated or differently conjugated product is already approved.

643 Conjugation-specific aspects regarding the SPC and labelling may be discussed with the Competent
644 Authorities prior to submission.

645 5. Medicinal Product Considerations

646 The quality target product profile (QTPP) relates to quality, safety and efficacy, considering e.g. the
647 route of administration, dosage form, bioavailability, strength and stability of a medicinal product
648 containing a synthetic peptide as active substance.

649 (V)ICH Guidelines ICH Q3B (VICH GL11) and ICH Q6A (VICH GL39) are not or only partly applicable to
650 synthetic peptides. The thresholds for impurities as defined in the general monograph of the Ph. Eur.
651 ‘Substances for Pharmaceutical Use’ may also be applicable to the resulting medicinal products. Limits
652 should be justified on a case-by-case basis considering the batch analysis history and qualification
653 data.

654 Synthetic peptides are included in the scope of ICH Q3D ‘Guideline for Elemental Impurities’ (Reflection
655 paper EMA/CVMP/QWP/153641/2018 for veterinary products), thus the requirements laid down in this
656 guideline are applicable for medicinal products containing synthetic peptides as active substances. Also
657 the risk considerations and requirements for nitrosamine impurities are applicable to synthetic peptide
658 active substances that are used in finished products for human use.

659 Potential interactions of the peptide with the excipients present in the formulation and leachables that
660 could result from manufacturing materials and packaging materials such as stoppers should be
661 evaluated during pharmaceutical development.

662 If the mode of action is based on the primary structure and the content (quantity) of the peptide only,
663 no potency assay is needed for release and stability testing of the finished product. Applicants are
664 encouraged to give more details on the possible (absence of) 3-D (secondary) structure, e.g. based on
665 NMR and FTIR, as well as computation investigations. Additionally, experiments on the 3-D structure
666 stability characteristics in the formulation, with techniques such as CD or others are recommended as
667 characterisation data, to justify the omission of such analysis in the routine control strategy.

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668 Furthermore, where relevant, formulation development should address the aggregation propensity and
669 the nature of the aggregates formed, especially under stress conditions including terminal sterilisation
670 (see below), in which case the immunogenicity risk should be discussed.

671 Most of the medicinal product formulations containing synthetic peptides as active substance are for
672 parenteral use. The principles for the choice of sterilisation process for finished products and containers
673 are presented in the form of decision trees in the ‘Guideline on the Sterilisation of the Medicinal
674 Product, Active Substance, Excipient and Primary Container’ are also relevant for synthetic peptides.
675 Terminal sterilisation provides the highest sterility assurance level, thus this should be the method of
676 choice unless demonstrated unsuitable.

677 A combination of sterile filtration, pre-sterilised container closure system and aseptic processing is only
678 acceptable if the applicant demonstrates by data that the use of a terminal steam sterilisation process
679 under the least stressful conditions (F0 ≥ 8 minutes) causes significant degradation. In case of
680 moderate degradation, exceeding the qualification threshold is not a valid argument in itself to reject
681 terminal sterilisation. Formulation optimisation efforts (e.g. pH, buffer system, osmolality), and choice
682 of container closure system should be made during pharmaceutical development in view of enabling
683 terminal sterilisation.

684 If synthetic peptide drug products in development show moderate degradation towards heat stress,
685 feasibility of terminal sterilisation should be addressed from early-development onwards. At that point,
686 assay loss and increase in impurities/degradations products at levels that would not be observed with
687 aseptic processing, may still be qualified in toxicological and pivotal clinical studies, including those
688 impurities that exceed the qualification threshold. Such studies should address the physicochemical
689 properties, biological activity, and if relevant the immunogenicity risk of the product after terminal
690 sterilisation. All of this with due consideration of the potential issues that may occur during formulation
691 development (e.g. pH and buffering range) and further upscaling towards the commercial-scale
692 terminal sterilisation process. To this extent, timely availability of stability indicating analytical
693 methods with good resolution of peaks and good mass balance, are a pre-requisite. If needed
694 complementary/orthogonal methods should be established to detect and quantify difficult to detect
695 impurities.

696 Thresholds for peptide-related impurities as defined in the Ph. Eur. general monograph ‘Substances for
697 Pharmaceutical Use’, also apply to finished products: peptide-related impurities should be reported
698 above 0.1%, identified above 0.5% and qualified above 1.0%. If aggregation/oligomerisation occurs
699 during finished product manufacture and/or storage, aggregates/oligomers should be included in the
700 finished product release and stability specification, unless otherwise justified.

701 Manufacturing processes should take into account any special characteristics such as hygroscopicity of
702 (lyophilised) active substance, as well as any temperature and/or light sensitivity of the active
703 substance, as relevant.

704 If correction factors are applied during dispensing (e.g. based on assay, purity, moisture content,
705 residual solvent content, and/or salt content of active substance) to achieve a specific declared
706 (labelled) amount of active in the formulation, these have to be described in the dossier.

707 The label claim strategy should be conclusively described and justified, including (where relevant)
708 calculation of active substance assay, any correction factors applied during dispensing, any in-process
709 controls for assay adjustment during drug product manufacturing, and assay calculation for release-
710 and stability testing. Any changes in label claim strategy during development have to be described in
711 detail and justified carefully, to ensure that the dose definition used in clinical trial(s) can be bridged
712 unequivocally to the proposed commercial product with label claim as per the SmPC/labelling.

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713 For medicinal products where European product-specific guidance on the demonstration of the
714 bioequivalence has been published the generic product should comply with the quality requirements
715 described therein e.g. for comparability studies.

716 Additional characteristics for complex finished product dosage forms should be considered on a case-
717 by-case basis.

718 6. Synthetic Peptide Development Programmes Using a

719 Biological Medicinal Product as a European Reference
720 Medicinal Product (human products only)
721 The European legislation for medicinal products clearly differentiates between biological medicinal
722 products (‘biologicals’) and chemically derived molecules.

723 The quality of biological medicines is tightly controlled and specific guidelines and regulations with
724 additional requirements apply. The level of information that needs to be submitted in the dossier is
725 more extensive than for chemically derived molecules.

726 A biosimilar is a biological medicine highly similar to another biological medicine already approved in
727 the EU (called 'reference medicine') in terms of structure, biological activity and efficacy, safety and
728 immunogenicity profile.

729 A biosimilar is not regarded as a generic of a biological medicine. This is mostly because the natural
730 variability and more complex manufacturing of biological medicines do not allow an exact replication of
731 the molecular micro-heterogeneity.

732 Most biological medicines in current clinical use contain active substances made of proteins. Some
733 smaller proteins can be produced either by using a biological manufacturing process or through
734 chemical synthesis (e.g. solid phase synthesis). Typically, structural heterogeneity and post-
735 translational modifications are not relevant for these molecules.

736 The biosimilar regulatory pathway is not possible for chemically synthesised peptides since these fall
737 outside the definition of a biological substance.

738 Nevertheless, the basic principles to demonstrate biosimilarity – high similarity in terms of structure,
739 biological activity and efficacy, safety and immunogenicity profile – should be considered for synthetic
740 peptide development programmes using a biological medicinal product as a European Reference
741 Medicinal Product. (Reference to: ‘Guideline on similar biological medicinal products containing
742 biotechnology-derived proteins as active substance: quality issues (revision 1)).

743 Analytical comparability testing, comprising physicochemical (structural) and biological (functional)
744 assays and conventional analytical testing, forms the basis of the demonstration of comparability.

745 A broad panel of analytical methods (see section 4.1.3 Characterisation 3.2.S.3) to demonstrate the
746 comparability between the recombinant reference product and the synthetic version is required for the
747 side-by-side comparability studies. It is important to note that the reference product used in the
748 comparability studies should be sourced from the European market.

749 The applicants will need to fully quantify all differences in peptides produced by chemical synthesis and
750 peptides produced by recombinant technology and demonstrate that both products are comparable.
751 The applicants should consider what analytical tests might be used to confirm comparability and to
752 define and justify, prior to conducting these studies, the acceptance range to conclude comparability.
753 Any observed differences from the reference medicinal product should be evaluated and justified.

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754 The primary structure should be confirmed by, e.g., MS, peptide mapping with MS/MS, capillary
755 isoelectric focusing (IEF), western blotting, etc. Higher-order structures should be addressed by CD
756 analysis, Fourier transform infrared (FTIR) spectroscopy, 2D NMR spectrometry, fluorescence
757 spectroscopy and DSC as appropriate. For certain peptides the oligomeric state should be investigated.

758 Characterisation of purity should be addressed using an orthogonal approach, i.e. size-based, charge-
759 based and hydrophobicity-based separation techniques. It is the responsibility of the applicant to
760 demonstrate that the purity methods are suitable to cover the complete impurity profile of the peptide
761 or whether additional purity testing with additional supplementary methods is necessary. Aggregation
762 propensity should also be investigated by suitable techniques detecting fibrillary aggregates such as
763 Thioflavin T (ThT) assay. When differences in the impurity profiles are observed it should be
764 demonstrated that the impurities in the synthetic peptide not present in the biological reference
765 product are qualified and do not raise concerns regarding immunogenicity.

766 Regarding the assessment of impurity related immunogenicity, experience has shown that
767 immunogenicity of peptides is of lesser concern than that of proteins due to their size. Furthermore,
768 changes or modifications (e.g. deamidations) of a small number of amino acids are not noticeably
769 immunogenic. If the total amount of peptide-related impurities does not exceed the respective amount
770 of peptide-related impurities of the originator product, this is not considered as a concern even if a
771 given peptide-related impurity is not present in the originator preparation. In case a novel type of
772 impurity occurs, i.e. differing from the drug substance in more than a few amino acid modifications,
773 this novel impurity should be reduced as far as possible since reliable prediction of immunogenicity is
774 not feasible.

775 In-silico prediction of immunogenicity, e.g. based on predicted binding to T-cell receptors (TCR), or in-
776 vitro tests of T-cell activation are not considered useful since also T-cell independent immune
777 responses are described (e.g. heparin-induced thrombocytopenia (HIT)). Mainly intended for vaccine
778 development, their predictive value for impurities appears to be low.

779 In general, these synthetic peptides have to comply with the requirements of the Ph. Eur. General
780 monograph ‘Substances for Pharmaceutical Use’. This monograph allows an identification threshold of
781 0.5%. However, for comparability purposes a full evaluation of the impurity profile also covering
782 impurities lower than 0.5% is expected. A limit of quantification (LOQ) of 0.1% for HPLC purity testing
783 is required.

784 Comparative forced degradation studies are also recommended and the suitability of the analytical
785 purity methods to fully characterize the impurity profiles of both products should be demonstrated.

786 Process-related impurities from the cell construct (e.g. host cell protein (HCP), DNA) or resulting from
787 the manufacturing process (e.g. antibiotics and other media components) do not need to be part of the
788 comparability studies. Clearance of reagents, residual solvents, elemental impurities and potential
789 genotoxic impurities for the synthetic peptides should be addressed as described above under 4.1.3.

790 Functional assays (e.g. cell based assays using appropriate cell lines) should be developed and used in
791 the comparability studies. It depends on the mechanism of action which additional functional assays
792 may be needed to demonstrate similarity (e.g. binding kinetics).

793 The absence of a biological assay in the release specifications for drug substance and drug product
794 should be appropriately justified, e.g. by commercial-scale batch biological assay data and, in addition,
795 by appropriate characterisation of higher-order structure by physicochemical testing.

796 The analytical methods used in the comparability exercise should be suitable, sufficiently qualified
797 and/or validated and sensitive to detect potential differences between both products. In the case that

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798 statistical models are used to demonstrate comparability they should be adequately described and
799 justified.

800 Batches preferably from the commercial process should be used for the side-by-side analyses. The
801 number of batches used in the comparability studies should be adequately justified. (Reference to:
802 ‘Reflection paper on statistical methodology for the comparative assessment of quality attributes in
803 drug development’ - EMA/CHMP/138502/2017)

804 Comparative stability studies can be useful for detecting potential differences in the stability profile of
805 the peptides manufactured either by chemical synthesis or by recombinant techniques. Stability and
806 shelf-life claims cannot be derived from the reference product without their own data.

807 7. Requirements for Clinical Trial Applications (human

808 products only)
809 The requirements for peptides intended to be used in the course of clinical studies are evolving
810 depending on the stage of development, with increasing expectations going towards Phase 3 and in
811 preparation of MAA. The main focus should be on the safety of the synthetic peptide, especially in the
812 early stages of development.

813 It is acknowledged that most peptides will be manufactured by solid phase supported synthesis hence
814 a platform similar to other peptides will be used; nevertheless, details regarding the type of resin used,
815 as well as coupling agents and the use of capping will be expected.

816 With regard to starting materials of the active substance, it is expected that from an early-stage,
817 individual impurities will be monitored in the amino acid building blocks in order to allow understanding
818 and control of the impurity profile of the final peptide. Setting of limits for certain impurities may be
819 expected for later development.

820 Similar expectations also apply to isolated intermediates (e.g. crude peptide after cleavage from the
821 resin) and critical purification steps (e.g. preparative chromatography on the crude peptide).

822 The changes introduced during manufacturing process development should be described in terms of
823 potential impact on the quality of the active substance; particular attention should be paid to
824 differences in impurity profile compared to preclinical batches used for qualification of impurities.

825 The novel peptide should be fully characterised in terms of primary structure; particular attention
826 should be paid to its potential for aggregation from as early as possible in the development in order to
827 avoid problems during formulation of the drug product. The propensity toward racemisation should also
828 be investigated.

829 Impurities in the peptide active substances should be identified in the course of development. Peptide-
830 related impurities above the threshold of 1.0%, should be identified and qualified in preclinical studies.
831 Orthogonal/complementary analytical procedures should be employed also in the early development
832 stages in order to minimise the risk of co-elution of impurities and to adequately characterise the
833 impurity profile of the synthetic peptide; if it can be shown in the course of development that one
834 analytical procedure is sufficient to control all impurities, the other(s) could be omitted.

835 With regard to the stability studies to be conducted on the synthetic peptide, it is essential that
836 stability-indicating analytical procedures are employed given that peptides tend to be thermally labile
837 hence degradation should be detected at inappropriate storage conditions.

Guideline on the Development and Manufacture of Synthetic Peptides

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