Chemistry Reference
In-Depth Information
chromatographic separation and is capable of identifying single amino acid differ-
ences. This “fingerprint,” sensitive to even the smallest change in a protein's struc-
ture, makes it an extremely valuable tool for identity testing and process monitoring.
Because of this sensitivity, a peptide map can be used not just for the identification
of proteins based on the elution pattern of the peptide fragments in the separation,
but also for the determination of posttranslational modifications, the confirmation
of genetic stability, and the analysis of protein sequence when interfaced to a mass
spectrometer.
While it is necessary to resolve each peptide fragment into a single peak, peptide
mapping also represents a significant chromatographic challenge due to the large
number of peptides that are generated from the enzymatic digest of a protein, and the
significant number of alternative peptide structures (posttranslational modifications
from proteolysis, phosphorylation,
N
-terminal acetylation and glycosylation, oxida-
tions, etc.) that can also be obtained.
Similar to any other analytical procedure, when used in a biopharmaceuti-
cal laboratory, the method used to generate or evaluate the peptide map must be
validated.
Applicable guidance is available from both the United States Pharmacopeia
(USP) and the International Conference on Harmonization (ICH) on method vali-
dation in general [1,2]. However, more specific USP guidance for peptide mapping
validation is also available, and should certainly be examined for more detailed
information [38]. Section 7.6 highlights the USP guidance; however, for more infor-
mation and background, the reader is encouraged to review the appropriate USP
chapters.
7.6.1 b
IochArActerIzAtIon
of
P
ePtIdeS
Peptide mapping involves comparative testing of specific maps for each unique pro-
tein (the test sample) against a reference standard or reference material treated in an
identical fashion. It is the end product of one of several potential chemical processes
that ultimately provide information about the protein under study. The process of
generating a peptide map consists of four steps: isolation and purification of the pro-
tein, selective cleavage into the resulting peptides, the chromatographic separation,
and the final analysis and identification of the peptides.
Isolation and purification are necessary for dosage forms or bulk drugs that may
have excipients or additional active ingredients that may interfere with the protein
of interest. When an isolation or purification step is employed, quantitative recovery
should be validated against a reference standard.
7.6.1.1 selective cleavage of the protein peptide bonds
The cleavage approach used is very dependent on the protein test sample. Cleavage
can be either enzymatic or chemical, and each type has multiple cleavage agents, as
summarized in Table 7.7. Complete cleavage is more likely to occur with enzymes
compared to chemical agents. However, the overall goal is simply to have enough
peptide fragments to be meaningful. If there are too many fragments, the map will
