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Purity vs identity

Purity and identity are separate quality attributes: an identity test establishes what a material is, while a purity test estimates how much of the material is the intended substance rather than other components. ICH Q6B lists identity and purity and impurities as separate items of a specification, each with its own tests.[1]

How does ICH Q6B describe each attribute?

ICH Q6B states that identity tests should be highly specific, based on unique aspects of the molecular structure or other specific properties, that more than one physicochemical, biological or immunochemical test may be necessary, and that identity tests can be qualitative in nature.[1]

For purity, the guidance states that absolute purity is difficult to determine and that the results are method dependent, so purity is usually estimated by a combination of methods. It defines an impurity as any component that is not the desired product, a product-related substance or an excipient.[1]

How is an identification procedure shown to be specific?

ICH Q2(R2) states that an identification procedure should be able to identify the analyte based on unique aspects of its molecular structure or other specific properties.[2]

That capability can be confirmed by obtaining positive results comparable to a reference material with samples containing the analyte and negative results from samples that do not contain it. The test should also be applied to materials structurally similar or closely related to the analyte to confirm that a positive result is not obtained.[2]

How is a purity procedure shown to be specific?

For a purity or impurity test, ICH Q2(R2) states that discrimination can be established by stressing or spiking a product to reach appropriate levels of impurities or related substances and showing the absence of interference.[2]

Where impurities cannot be prepared or isolated, specificity can be shown by comparing results for samples containing typical impurities with those of an orthogonal procedure. Representative chromatograms, electropherograms or spectra are used as supporting data.[2]

Why can a material meet one attribute and not the other?

D'Hondt and colleagues list impurities of synthetic peptides that include amino acid deletions and insertions, diastereomers from racemization, oxidation products, dimers and oligomers, and counter ions such as trifluoroacetate.[3]

Some of these impurities are isomers of the intended peptide. Isomers share a chemical formula and therefore a mass-to-charge ratio, so a mass-based identity result cannot by itself exclude an isomeric impurity such as a peptide epimer.[3],[4],[5]

Conversely, ICH Q6B lists identity as a separate item from purity, and describes purity results as method dependent, so a purity result does not by itself show that the main component is the labeled substance.[1]

How do identity and purity results work together?

ICH Q6B 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 separation can contribute to more than one attribute.[1]

ICH Q2(R2) states that where a single procedure is not specific or sufficiently selective, an additional procedure should be used to ensure adequate discrimination; one of its examples combines an assay with a suitable test for impurities.[2]

In its LC/MS example, ICH Q2(R2) combines an accurate m/z value with retention time as the basis for specificity.[2]

Limitations

Neither an identity result nor a purity result measures endotoxin or microbial contamination; ICH Q6B lists sterility, endotoxin and microbial limits as separate pharmacopoeial tests.[1]

Both kinds of result depend on the procedure used and on its validation, as described in ICH Q2(R2) and ICH Q6B.[2],[1]

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. ICH Q2(R2) Validation of Analytical Procedures. U.S. FDA guidance for industry, March 2024. FDA docket FDA-2022-D-1503
  3. D'Hondt M, Bracke N, Taevernier L, et al. Related impurities in peptide medicines. J Pharm Biomed Anal. 2014;101:2-30. PMID 25044089 · DOI 10.1016/j.jpba.2014.06.012
  4. Rathahao-Paris E, Abdoun S, Paris A, et al. Innovative direct introduction-ion mobility-mass spectrometry (DI-IM-MS) approach for fast and robust isomer-specific quantification in a complex matrix. J Mass Spectrom. 2024;59(5):e5026. PMID 38656572 · DOI 10.1002/jms.5026
  5. 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