Chemistry Reference
In-Depth Information
can be carried out to intentionally modify the target protein, 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 is observed. Peptides modi-
fied by oxidation, deamidation, or other mutations usually have reported LODs in
the range of 2 to 15 mole percent [40]. The particular chemical modifications chosen
to evaluate LOD should take into account both the protein and the expression host.
7.6.2.3 precision
Precision in peptide mapping is measured on two different levels: repeatability, and
reproducibility intratest and intertest reproducibility experiments. Repeatability is
measured by running six replicate injections of a single pooled digest of the ref-
erence 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 intertest measurements are the more important parameters to be evalu-
ated during validation, however. Intratest precision is the reproducibility of the frag-
mentation (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 predetermined specified acceptance criterion.
Intertest precision is what has been traditionally referred to as intermediate preci-
sion 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, and different column lots. For intertest precision, the experi-
mental design should include comparisons using peak retention times and areas rela-
tive 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 intertest compared to the intratest precision, which in turn will be
greater than the repeatability results. Additional general information on determining
precision can be found in Chapter 4, Section 4.3.2.
7.6.2.4 system suitability
System suitability guidance can be found in the USP chapter on chromatography
[(32); and Chapter 5]. Similar to any other method, the acceptance criteria for system
suitability of a peptide map depend on the identification of the critical test param-
eters that affect data interpretation and acceptance. System suitability limits (for
both recovery and chromatography) are determined by running a reference stan-
dard in parallel with the test protein and looking for indicators that monitor, for
example, that the desired endpoint was reached in the digestion, normally selectiv-
ity and precision. However, the consistency of the pattern obtained is best defined
