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.
Identification
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 test | Method | Comments |
HPLC co-elution with reference standard | <621> | Method same as for related substances |
Mass spectrometry | <736> | Monoisotopic mass ± 1.0 mass units |
Amino acid analysis | <1052> | Hydrolysis protocol should be specified |
MS–MS sequencing | <736> | May be complicated for longer sequences |
NMR spectroscopy | <761> | Complex interpretation for longer sequences |
Peptide mapping | <1055> | For longer sequences (>20 AAs); complementary to MS–MS |
Enantiomeric purity | Chiral amino acid analysis | |
N-terminal sequence analysis by Edman Degradation | <1045> | Complicated analysis, especially for sequences which are N-terminally blocked; largely superseded by LC–MS–MS |
Infrared spectroscopy | <197, 851> | Limited use for peptides |
Higher order structure | Circular dichroism, NMR, FTIR | For investigation of secondary or tertiary structure in aqueous solution |
Bio-identity | ELISA | Not a routine test for most quality control laboratories; may be required for longer, complex sequences |
Peptide Assay
When considering assay, it is important to emphasise that many peptides are quite hygroscopic and should be handled in a controlled- humidity environment
Assay test | Method | Comments |
HPLC assay | <621> | Method same as for related substances; based on comparison with a reference standard |
Peptide content by amino acid analysis | <1052> | Hydrolysis protocol must be validated, only stable amino acids should be included in calculation |
Peptide content by nitrogen analysis | CHN analysis | Direct analysis by elemental analysis or Kjeldahl or using HPLC with a chemiluminescence nitrogen detector (CLND) |
UV spectroscopy | <197, 851> | Only useful for products containing Trp, Tyr, or Phe |
Quantitative NMR | <761>, <1761> | 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.
Peptide Impurities | Method | Comments |
Peptide-related substances | <621> | Specific method for substance; must be validated for both process-related impurities and degradation products; limits for total and individual impurities should be specified |
Residual solvents | <467> | May be limited to those used in the final step of the manufacturing process |
Heavy metals | <(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) | <503.1>/ <1065> | Only required if TFA used during the manufacturing process; <1065> used for peptides which are not soluble in acid. |
Residual fluoride | <1065> or ion-selective electrode | Only required if hydrofluoric acid (HF) used during the manufacturingprocess (Boc-chemistry) |
Other small molecule impurities | Impurity dependent | Non-peptide impurity limits are required to follow the ICH Q3A guideline; potential genotoxic impurities require additional evaluation |
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Specific tests
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.
Specific tests | Method | Comments |
Counterion content | <503> (Acetate) <1065> (Others) | <1065> may be required for acetate if peptide not soluble in acid; titration with AgNO 3 may be used to determine chloride |
Water content (Karl Fischer) | <921> | Coulometric titration (method Ic) most commonly used |
Specific rotation | <781> | Information only of limited utility |
Ellman test | N/A | Only required if the reduced form(s) of a peptide containing disulfide bonds cannot be determined using the method for related substances |
Bioburden | <61, 62> | Often required for APIs used in the manufacture of parenteral drug products |
Bacterial endotoxins | <85> | Requirement for APIs used in the manufacture of parenteral drug products |
Mass balance | N/A | Calculation |
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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.
References
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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