Validation by Type of Method - Analytical Validation - page 152

tAble 7.7
cleaving Agent examples (not an exhaustive list)

type Agent specificity

Enzymatic Trypsin C -terminal side of Arg and Lys

Chymotrypsin C -terminal side of hydrophobic residues (e.g.,

Leu, Met, Ala, aromatics)

Pepsin Nonspecific

Lysyl endopeptidase C -terminal side of Lys

Glutamyl endopeptidase C -terminal side of Glu and Asp

Peptidyl-Asp metallo-endopeptidase N- terminal side of Asp

Endoproteinase Asp-N N- terminal side of Asp

Clostripain C -terminal side of Arg

Chemical Cyanogen bromide C -terminal side of Met

2-Nitro-5-thio-cyano-benzoic acid N- terminal side of Cys

o -Iodosobenzoic acid C -terminal side of Trp and Tyr

Dilute acid Asp and Pro

BNPS-skatole Trp

lose its specificity, and it could become more difficult to discern differences. The
purity and activity of the cleavage agent should also be examined.
Sometimes pretreatment of either the sample or the cleavage agent is necessary.
It may also be necessary to selectively protect the sample protein to guard against
generating too many peptides, or it may be necessary to concentrate the protein or to
separate it from added substances or stabilizers that may be used in some drug for-
mulations. Sometimes, chaotropic agents (e.g., urea, guanidine, or surfactants) may
be added to unfold the protein to allow full access to cleavage sites.
The cleavage process also must be optimized, and the factors that affect the com-
pleteness and effectiveness of protein digestion are the same as those that would affect
any chemical or enzymatic reaction. Temperature, pH, time, and the amount (ratio of
enzyme/protein) of cleavage reagent used are all important factors. Generally, a tem-
perature between 25°C and 37°C is adequate for most digestions, and a pH is cho-
sen appropriate for the cleavage agent, enzyme (a pH will not denature the enzyme
before it can react with the protein), and preservation of the integrity of the sample
protein. Time can vary considerably, and if there is enough sample available, it is
advisable to perform a time course study. The amount of cleavage agent used should
be enough to accomplish a reasonably fast digestion time, but not so much that it
interferes with the chromatographic map pattern. There is a compromise between
using too much or too little of the cleavage agent, in order to avoid autodigesting the
enzyme while generating an adequate map, and overdigesting and losing the “true”
map and its information content.
Typically, a protein-to-cleavage reagent ratio between 20:1 and 200:1 is used,
and blank determinations are also made using a digestion control with all reagents
included except for the protein of interest. Immobilized enzyme reagents have also