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Complicated analysis, especially for sequences which are N-terminally blocked; largely superseded by LC–MS–MS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

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

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

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