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Qualitative and Quantitative Aspects of Peptide Mapping Using UPLC-UV

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As outlined in my previous post, 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).  Because there is a wealth of general validation guidance available, discussion will be restricted here to areas where peptide mapping validation might differ from other types of methods (for example, methods for synthetic drugs).

Robustness

To evaluate a peptide map against a standard, the chromatographic separation must be robust.  Although general chromatographic robustness has to be applied however, there are additional issues to consider in a peptide map method, and these include (enzyme) reagent quality or purity and digest stability.

Column considerations also must be made during a proper robustness study, since it is a well known fact that no two chromatographic columns are created equal.  When determining the robustness of the reagents used for digestion, it is common to evaluate a protein reference standard of known composition with cleavage agents from different lots.  The number of peaks obtained, their shape, and the peak areas all are compared in the resulting chromatograms.  Because in some cases chromatographic run times can by quite long, the length of time and the conditions under which a digest can be stored before being analyzed also must be evaluated as part of a robustness study.  Digest stability usually is evaluated by looking for significant differences in the map resulting from the analysis of several aliquots of a single digest stored at different conditions.  It also can be desirable to investigate stability through to several freeze-thaw cycles.

Lastly, it is a well known fact that no two chromatographic UPLC columns are created equal.  Although the manufacturer Waters today provides much better control of their processes than in the past, minor column differences can have a significant effect on the separation of these complex samples.  It is a good idea to evaluate the reference standard on several different UPLC systems, column lots, and evaluate column life, because as a column ages, the separation can be affected.

Linearity and Limit of Detection

The serial dilution of a peptide mixture should be made in order to demonstrate that there is no significant shift in retention or deterioration in peak shape from low to high levels.  It has to be confirmed that the dynamic range of the chromatographic material and gradient method is sufficient for the analysis of a small amount of one peptide in the presence of much larger amount of another.  The detector response should be linear with the sample amount.

Six replicate injections should be overlaid to demonstrate the reproducibility of UPLC-UV peptide mapping method.  The same sample might subsequently be injected at different levels to test linearity and sensitivity.

The limit of detection (LOD) in a peptide map is determined by the ability of the method to distinguish changes in the map, for example, the presence or absence of a peak.  Experiments can be carried out to modify the target protein intentionally and then a digest of the modified protein is mixed with a control digest or standard reference material in varying proportions.  Ideally, a decrease in peak response for the unmodified peptide and a corresponding increase for the modified peptide should be observed.  Peptides modified by oxidation, deamidation, glycosylation or other mutations usually have reported LOD's in the range of 2-15 mol% .  The sensitivity in combination with robust chromatographic behavior enables detection of low level peptides in a complex digest.

Precision

Precision in peptide mapping is measured on two levels; repeatability and reproducibility from both intra and inter-testing runs. Repeatability is measured by running six replicate injections of a single pooled digest of the reference standard.  When repeatability is performed in this manner, all variability from the sample and reagents are eliminated, and the true instrument or system component of precision can be measured and used to help set system suitability criteria.

Intra and inter measurements are the more important parameters to be evaluated during validation, however. Intra-test precision is the reproducibility of the fragmentation (digestion) and the chromatographic separation.  Acceptable precision is obtained when the peak retention times and areas are constant from chromatograms obtained from consecutive tests of a series of separately prepared digests of the test protein.  The average standard deviation of the retention times and areas should not exceed a pre-determined specified acceptance criterion.

Inter-test precision is what traditionally has been referred to as intermediate precision or true reproducibility.  It is a measure of the reproducibility of the peptide map when the analysis is run according to an experimental design made to measure the effects of the test run on different days, by different analysts, in different laboratories on different systems, different column lots.  For inter-test precision, the experimental design should include comparisons using peak retention times and areas relative to an internal standard peak within the same chromatogram.  By using relative values, the need to make adjustments for things like injection volume differences, column volumes and instrument gradient delay volumes is eliminated.

In general, it can be expected that %RSD for peak retention times and areas will be greater for the inter-test compared with the intra-test precision, which in turn will be greater than the repeatability results.

System Suitability

The guidance on system suitability can be found in the USP chapter on chromatography.  Like any other method, the acceptance criteria for system suitability of a peptide map depends upon the identification of the critical test parameters that affect data interpretation and acceptance.  System suitability limits, for both recovery and chromatography, are determined by running a reference standard in parallel with the test protein and looking for indicators that monitor, for example, that the desired endpoint was reached in the digestion; normally, selectivity and precision.  However, the consistency of the pattern obtained is best defined by peak-to-peak resolution.  Additional chromatographic parameters such as peak width, tailing factors and column efficiency also can be used.

The parallel study (reference standard and test protein) also is used to visually compare each peaks relative retention time, responses (peak retention time and area), the number of peaks and the overall elution pattern.  This comparison often is complemented by mixing the two samples (1:1, v/v) and evaluating the peak response ratios and elution pattern.  If all peaks in this mixed sample have the same relative retention times and peak response ratios, then the identity of the protein test sample can be confirmed.  Significantly different retention times are also an indication of system variability, while the appearance of new or broader peaks indicates non-equivalence.  Computer-aided pattern recognition software and other automated approaches have been used on occasion to examine the degree of difference or similarity when comparing two different peptide maps, but these have not gained routine acceptance.

Conclusion

Although most of the underlying principles still apply, the validation of a peptide map includes some additional considerations when it comes to LOD, robustness, and precision, and depends upon the stage of the regulatory process.  While mostly involving comparative testing, when properly validated, a peptide map can be used to accomplish its intended purposes: to confirm the primary structure of a protein; to detect whether or not alterations have occurred; and to demonstrate process consistency.