Peptide characterisation (APIs) Test Methods
Comprehensive characterisation of a peptide API is required to obtain regulatory approval for marketing; the reference standard is fully characterised 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.
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.
HPLC co-elution with reference standard
Method same as for related substances
Monoisotopic mass ± 1.0 mass units
Amino acid analysis
Hydrolysis protocol should be specified
May be complicated for longer sequences
Complex interpretation for longer sequences
For longer sequences (>20 AAs); complementary to MS–MS
Chiral amino acid analysis
N-terminal sequence analysis
by Edman Degradation
Complicated analysis, especially for sequences which are N-terminally blocked; largely superseded by LC–MS–MS
Limited use for peptides
Higher order structure
For investigation of secondary or tertiary structure in aqueous solution
Not a routine test for most quality control laboratories; may be required for longer, complex sequences
When considering assay, it is important to emphasise that many peptides are quite hygroscopic and should be handled in a controlled- humidity environment
Method same as for related substances; based on comparison with a reference standard
Peptide content by amino acid analysis
Hydrolysis protocol must be validated, only stable amino acids should be included in calculation
Peptide content by nitrogen analysis
Direct analysis by elemental analysis or Kjeldahl or using HPLC with a chemiluminescence nitrogen detector (CLND)
Only useful for products containing Trp, Tyr, or Phe
A stable internal standard is required
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.
Specific method for substance; must be validated for both process-related impurities and degradation products; limits for total and individual impurities should be specified
May be limited to those used in the final step of the manufacturing process
<(231), 232, 233>
Requirement if metal catalysts used in the process; may be required because of contact with metals in processing equipment
Residual trifluoroacetic acid (TFA)
Only required if TFA used during the manufacturing process; <1065> used for peptides which are not soluble in acid.
<1065> or ion-selective electrode
Only required if hydrofluoric acid (HF) used during the manufacturingprocess (Boc-chemistry)
Other small molecule impurities
Non-peptide impurity limits are required to follow the ICH Q3A guideline; potential genotoxic impurities require additional evaluation
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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.
<1065> may be required for acetate if peptide not soluble in acid; titration with AgNO 3 may be used to determine chloride
Coulometric titration (method Ic) most commonly used
Information only of limited utility
Only required if the reduced form(s) of a peptide containing disulfide bonds cannot be determined using the method for related substances
Often required for APIs used in the manufacture of parenteral drug products
Requirement for APIs used in the manufacture of parenteral drug products
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*Recommended test to be performed as part of lot release. HPLC is high-performance liquid chromatography. MS is mass spectroscopy. NMR is nuclear magnetic resonance. FTIR is fourier transform infrared.
1. FDA, FDA FY2012 Innovative Drug Approvals (December 2012), www.fda.gov/downloads/AboutFDA/ReportsManualsForms/Reports/UCM330859.pdf, accessed 7 Feb. 2014.
2. P. Vlieghe et al., Drug Discovery Today15(1/2) 40-56 (2010).
3. P. Van Arnum, Pharm. Technol. 37(8)48-50 (2013).
4. Ph. Eur., General Guidance 2034,Substances for Pharmaceutical Use
5. March 2014,Pharmaceutical Technology Europe
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