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Identity testing

Identity testing is analysis that confirms a material is the substance it is labeled as. ICH Q6B states that identity tests should be highly specific, based on unique aspects of the molecular structure or other specific properties, and that more than one physicochemical, biological or immunochemical test may be necessary to establish identity.[1]

Which structural methods does ICH Q6B describe?

ICH Q6B describes amino acid sequence determination, amino acid composition, terminal amino acid sequence, peptide mapping and molecular size determination by methods including size exclusion chromatography, electrophoresis and mass spectrometry.[1]

In a peptide map, the product is fragmented into discrete peptides using enzymes or chemicals and the fragments are analyzed by HPLC or other procedures. ICH Q6B notes that peptide mapping with a validated procedure is frequently used to confirm product structure for lot release.[1]

How are amino acid composition and terminal sequence used?

ICH Q6B describes terminal amino acid analysis as performed to identify the nature and homogeneity of the amino- and carboxy-terminal amino acids, with the result compared against the expected terminal sequence.[1]

ICH Q6B states that amino acid composition analysis provides useful structural information for peptides and small proteins, but that such data are generally less definitive for large proteins.[1]

Where does mass spectrometry fit?

A review by Lian and colleagues describes liquid chromatography-mass spectrometry (LC-MS) as increasingly used to characterize synthetic peptides and their impurities, including structural isomers and stereoisomers, and proposes an LC-MS workflow for that characterization.[2]

What makes an identity method specific?

ICH Q2(R2) lists specificity as the characteristic to be demonstrated for an identity procedure. It states that specificity can be shown through absence of interference or by comparison with an orthogonal procedure, and that a combination of two or more procedures is recommended where one does not discriminate enough.[3]

ICH Q2(R2) adds that specificity can be verified by showing that the measured result is comparable to that of a second, well-characterized procedure that ideally applies a different measurement principle.[3]

Identity results are usually obtained by comparison with a reference material. ICH Q6B describes an appropriately characterized in-house primary reference material, against which the in-house working reference materials used to test production lots are calibrated, and calibration against an international or national standard where one is available.[1]

Why is identity tested separately from purity?

ICH Q6B lists identity and purity and impurities as separate specification items, each with its own tests. A purity result does not establish identity, and an identity result does not establish purity.[1]

ICH Q6B also notes that chromatographic patterns and data on identity, homogeneity and purity can be obtained by size exclusion, reverse-phase, ion-exchange or affinity chromatography, so one chromatographic run can contribute to both items when the method is suited to each.[1]

Limitations

An identity result is qualitative. ICH Q6B states that identity tests can be qualitative in nature; a positive identity result does not state how much of the substance is present or how pure it is.[1]

Stereoisomers such as peptide epimers share a mass, so a mass measurement alone may not distinguish them.[2]

This page describes what the cited documents say. It is not a statement that any PepGenex material has been manufactured, tested or released under any of them.

Compound profiles

Compound profiles whose identity section records the sequence and molecular formula that an identity result is compared against.

References

  1. ICH Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products. U.S. FDA guidance for industry, August 1999. FDA docket FDA-1998-D-0003
  2. Lian Z, Wang N, Tian Y, Huang L. Characterization of Synthetic Peptide Therapeutics Using Liquid Chromatography-Mass Spectrometry: Challenges, Solutions, Pitfalls, and Future Perspectives. J Am Soc Mass Spectrom. 2021;32(8):1852-1860. PMID 34110145 · DOI 10.1021/jasms.0c00479
  3. ICH Q2(R2) Validation of Analytical Procedures. U.S. FDA guidance for industry, March 2024. FDA docket FDA-2022-D-1503