Controlling the Quality of Peptides

Peptides based custom compounds production on large scale engage high level of expertise as well as instrumentation to achieve controlled level of peptide mixture in industrial processing systems with having the potential quality parameters to determine the purity and qualitative status of customized peptide end-product.

The peptides are the highly unstable chemical substances and are more prone to structure breakdown due to addition of minute impurities during the manufacturing as well storage processes. The leading peptide research and manufacturing companies and lab institutes implant strict high-level quality control processes, they ensure that each customized peptide is double and tripled checked for quality via both mass spectrometry (MS) and high performance liquid chromatography (HPLC) analyses after each step during peptide purification and quality control (QC) procedures. As the final step, they also perform additional quality assurance (QA) procedures for every custom peptide to further guarantee the delivery of high-quality peptides.

Even the first specification item “identity” presents a challenge. There is no simple, single test available (with the exception of NMR (Nuclear magnetic resonance spectroscopy), which is expensive, time consuming, and requires complex data interpretation) that would unequivocally establish the identity of a peptide with respect to amino acid composition, sequence and chirality. For this reason a combination of techniques is typically used, usually encompassing mass spectroscopy, amino acid analysis and HPLC. Let us brief you how the quality is controlled through popular methods and strategies mentioned above.


The most appropriate characteristic of a peptide that can be established using mass spectroscopy is its mono-isotopic mass (the mass of the isotopic peak whose elemental composition is composed of the most abundant isotopes of those elements). Mass spectroscopy can be utilized further, in order to confirm the sequence of amino acids in the peptide. Mass fragmentrometry has in many instances replaced the traditional method of Edman degradation, especially for peptides that are longer than 20 residues.

Sequencing is particularly useful for authenticating the identity of reference standards, although it is not necessary as a release criterion for each lot of peptide produced. Very long sequences may require the use of partial proteolytic digestion of the peptide using an enzyme selected to generate limited fragmentation, prior to separation of the fragments on HPLC and mass spectral analysis.


The purity of the peptide is typically determined using HPLC, which is the most universally used method in quality control of peptides. Typically, for a first GMP lot, an HPLC purity specification greater than 97% with no single impurity greater than 1% will be set. Correctly developed chromatographic method must enable the separation and subsequent determination of the most common impurities in peptide products, such as enantiomers, deletion sequences, and products of deamidation or acetylation.

Method development, however, is not a trivial task because it is difficult to determine which potential impurities should be included as separation targets. Usually, the different impurities are not readily available as separate compounds and may need to be synthesized separately at considerable cost. It is often necessary to perform the initial method development without access to these compounds. Thus, method development becomes an iterative process, in which the peptide product is screened using a variety of buffer systems based on different salts, pH values and columns.

Typically, reverse-phase systems are attempted first followed by ion exchange, ion-pairing or size exclusion methods. Under special circumstances HILIC (hydrophilic interaction chromatography), which is a variation of normal-phase chromatography using water as a mobile phase modifier, may be used. As impurities are revealed, isolated and identified, further optimization of the HPLC method can be undertaken. The best way forward is to synthesize the identified impurities. Perform final method development using a well-defined mixture of peptides.


Amino acid analysis, first introduced by Stein and Moore [1], involves hydrolysis of the peptide (typically using acid conditions) to its individual amino acid residues, followed by chromatographic separation on a column and detection/quantitation. The classical method employing ion exchange chromatography and ninhydrin post-column derivatization is still the technique of choice despite the emergence of numerous pre-column derivatization methods, using reagents such as AQC4 [2], OPA5 [3], PITC6 [4], etc., which typically offer increased sensitivity and accessibility to commonly available HPLC equipment. These more recently introduced methods are not as accurate and the derivatives are not always stable.

1. S. Moore and W.H. Stein, Methods in Enzymology, 6, 819 (1958)
2. S.A. Cohen and D.P. Michaud, Anal. Biochem., 211, 279 (1993)
3. D.W. Hill, F.H. Walters, T.D. Wilson and J.D. Stuart, Anal. Chem., 51 1338 (1979)
4. S. A. Cohen and D. J. Strydom, Anal. Biochem., 174, 1 (1988)