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Peptide Mapping Using UPLC-UV Method Development Techniques

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Peptide mapping or peptide finger printing produced is characteristic for a particular protein and the UPLC technique can be used to separate a mixture of peptides.

Normally, recombinant proteins are developed for therapeutic purposes.  Peptide mapping is used to confirm the primary structure of a protein, identify post-translational modification, to demonstrate generic stability and analyze potential impurities.  Any difference in the structure of a protein should be reflected in a change in retention time for the peptide containing the modification.  The relative amounts of the peptide with and without a particular modification are used to measure the fraction of the protein in the particular sample that carries that modification.  Changes in area proportions correspond to the fraction of the protein molecules in the sample having a particular modification.  Using a UPLC technique, peptide analysis has been shown to give consistent chromatographic separations and reproducible quantitation for peptide mapping in combination with UV, MS or MS/MS detection.

In the initial characterization of a protein, it's important to develop a peptide mapping method that resolves modified peptides from native peptides so that all possible modifications may be detected. As development of the biopharmaceutical advances, these peptides must be quantitated.  Quantitation is generally expressed as an area or height percent of the native peptides.  In this way, the peptide map can provide information on the mixture of protein forms in each sample so that safety and efficacy of the preparation may be assured.  The method must, therefore, exhibit excellent sensitivity and linearity for quantitative work.

The strategy of peptide quantification includes adding a known amount of a specific peptide (peptide standards) to an actual protein digest to test estimates of quantification.  This peptide serves as a surrogate, illustrating the behavior of modified peptides in the digest.

Method validation Viewpoint

At the Investigational New Drug (IND) phase, limited validation is necessary; typically only an approved test procedure that includes system suitability as a test control.  Sometimes termed qualification, complete characterization of the individual peaks is not needed.  As the regulatory process proceeds, a partial validation might be needed to give assurance that the method performs as intended in the development of a map for the test protein.  However, validation of peptide mapping in support of further regulatory submissions requires a rigorous characterization of each of the individual peaks in the map.  Methods that are used to characterize the peaks in a map commonly use mass spectrometry (MS).

There are several critical factors that must be considered to validate a method used for peptide mapping, and each of the factors, along with the acceptance criteria, should be designed into a protocol or Standard Operating Procedure (SOP).

The critical factors include robustness, the limit of detection, specificity, linearity, range, accuracy, and precision. Recovery and reagent stability are also important to consider during method validation.  Recovery can be addressed by performing either quantitative amino acid analysis, spiking studies, or radiolabeling.  Many of the validation parameters must not only address the separation, but also the fragmentation or digestion, particularly when considering robustness studies.  The protocol also should include written test procedures that give a detailed description of the analytical method.