Peptide mapping of therapeutic monoclonal antibodies: improvements for increased speed and fewer artifacts.
Peptide mapping is a widely utilized technique to characterize monoclonal antibodies for the purpose of product identity and is becoming increasing important as a stability indicating assay. Many conventional peptide-mapping methods are extremely time consuming and yield a map that is wrought with processing artifact peaks such as deamidation, carbamylation, and missed cleavages. Therefore, this work examines the many common individual sample preparation steps of the peptide-mapping procedure for monoclonal antibodies including the steps of denaturing, reduction, sample cleanup, digestion, and HPLC solvent selection. Improvements in each of these steps are demonstrated that greatly help to reduce artifacts and also allow for reduction of overall sample preparation time. After evaluating the many different parameters for increased speed and fewer artifacts, the sample preparation was reduced from days to hours and the resulting peptide map is nearly free of sample or background artifacts. Therefore, this peptide map procedure and optimization scheme is an excellent tool to further examine real sample changes in a shorter amount of time.
Dick
Lawrence W
LW
Analytical and Formulation Development Department, Medarex, Inc., 519 Route 173 West, Bloomsbury, NJ 08804, USA.
Mahon
David
D
Qiu
Difei
D
Cheng
Kuang-Chuan
KC
D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism / Q000627:therapeutic use; D016477:Artifacts; D002851:Chromatography, High Pressure Liquid; D003641:Deamination; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010449:Peptide Mapping / Q000379:methods*; D011489:Protein Denaturation; D019163:Reducing Agents; D012997:Solvents / Q000737:chemistry; D013696:Temperature; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism; D014508:Urea / Q000737:chemistry
D000911:Antibodies, Monoclonal; D019163:Reducing Agents; D012997:Solvents; D014508:Urea; D014357:Trypsin