Peptide Knowledge Center

Peptide Characterisation methods (2)

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. Lyophilised peptides are often hygroscopic, therefore samples for assay should be handled in a controlled humidity environment.


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.

>> Peptide impurity synthesis link

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 characterisation 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 bondslend 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 derivatisation 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 derivatisation 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 racemisation during hydrolysis or subsequent derivatisation. An approach that addresses this issue involves the use of deuterated reagents (e.g., deuterium chloride [DCl]/deuterium oxide [D 2 O fmoc-osu]). Substitution of the α-carbon hydrogen with deuterium allows for correction for racemisation when using MS-based detection methods.

Infrared (IR) spectrophotometry is not a useful characterisation 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.

Quantitative NMR is potentially useful for assay determination

The characterisation 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 emphasise 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 lyophilised 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.

>> salmon calcitonin impurities link

Assay of peptides has traditionally been determined by quantitative AAA, which compares the peak areas of AAs in a hydrolysed 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.