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HPLC purity

HPLC purity is a purity result produced by high-performance liquid chromatography (HPLC), a separation technique that a methods review describes as extremely versatile for the isolation and purification of peptides. ICH Q6B lists reverse-phase, size exclusion, ion-exchange and affinity liquid chromatography among the procedures from which chromatographic data on identity, homogeneity and purity can be obtained.[1],[2]

Which HPLC modes are used for peptides?

A methods review by Mant and colleagues covers the major HPLC modes used for peptides: size-exclusion, ion-exchange and reversed-phase chromatography. The same review also describes capillary electrophoresis for peptide separations.[1]

The same review also introduces preparative reversed-phase purification of peptides and recombinant proteins on analytical columns and instrumentation.[1]

What is a purity analysis trying to separate?

ICH Q6B states that the choice and optimization of analytical procedures should be directed at the separation of the desired product from product-related substances and from impurities. It defines an impurity as any component that is not the desired product, a product-related substance or an excipient.[2]

For synthetic peptides, a review by D'Hondt and colleagues groups impurities arising from solid-phase synthesis: amino acid deletions and insertions, diastereomers from racemization, protection adducts, oxidation of side chains, dimers and oligomers, and counter ions such as trifluoroacetate. A second group arises from degradation, including beta-elimination, diketopiperazine, pyroglutamate and succinimide formation.[3]

Why can two laboratories report different purity figures for the same material?

ICH Q6B states that absolute purity is difficult to determine and that results are method dependent; purity is therefore usually estimated by a combination of methods.[2]

ICH Q2(R2) describes specificity or selectivity as the ability to show that identification or quantitation of an analyte is not affected by other substances present, and recommends combining two or more procedures where one does not discriminate enough.[4]

ICH Q2(R2) defines accuracy as the closeness of agreement between an accepted reference value and the value measured, and precision as the closeness of agreement between a series of measurements obtained from multiple samplings of the same homogeneous sample. Both are demonstrated during validation across the reportable range of the procedure.[4]

How are peptide impurities identified?

A review by Lian and colleagues describes synthetic peptides as subject to many complex structural modifications from starting materials, the manufacturing process and storage conditions, and discusses liquid chromatography-mass spectrometry (LC-MS) for identifying impurities, with emphasis on structural isomers and stereoisomers such as peptide epimers.[5]

What does the specification on a certificate mean?

An acceptance criterion is a numerical limit, range or other measure for accepting the results of an analytical procedure, as defined in ICH Q6B. A reported purity is compared against the stated criterion for that lot.[2]

Limitations

An HPLC purity figure describes separation by one procedure. It does not by itself confirm the identity of the main peak, and impurities that are not resolved from it are not counted separately.[2],[4]

D'Hondt and colleagues note that peptide-related impurities can greatly influence initial functionality studies during early drug discovery, possibly resulting in erroneous conclusions, which is one reason a purity figure is read alongside the method and specification.[3]

Purity by HPLC is not a measure of endotoxin or of microbial contamination, which are measured by different tests.[2],[6]

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

Research peptide profiles in this library. What this page describes applies to peptide lots in general; none of these profiles reports a result of it for any lot.

References

  1. Mant CT, Chen Y, Yan Z, et al. HPLC analysis and purification of peptides. Methods Mol Biol. 2007;386:3-55. PMID 18604941 · DOI 10.1007/978-1-59745-430-8_1 · PMC7119934
  2. 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
  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. ICH Q2(R2) Validation of Analytical Procedures. U.S. FDA guidance for industry, March 2024. FDA docket FDA-2022-D-1503
  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
  6. U.S. FDA. Pyrogen and Endotoxins Testing: Questions and Answers. Guidance for industry. FDA docket FDA-2013-S-0610