title: Rapid online proteolytic mapping of PEGylated rhGH for identity confirmation, quantitation of methionine oxidation and quantitation of UnPEGylated N-terminus using HPLC with UV detection.
abstract: Proteolytic mapping is a widely used tool in the BioPharmaceutical Industry for the analysis of post-translation modifications as well as confirmation of protein identity by comparison to a well-characterized reference standard. This manuscript presents an integrated chromatographic approach which provides the ability to rapidly digest and analyze a PEGylated rhGH for methionine oxidation, identity confirmation and free (unPEGylated) N-terminal peptide by RP-HPLC using UV detection at 280 nm. This approach utilizes an online procedure in which the digestion step is integrated to the RP-HPLC analysis via an external column switching valve. A Poroszyme Trypsin cartridge is used in the digestion step, followed by delivery of the digested sample plug through a sample loop to an orthogonal RP-HPLC column for separation and quantitation of the resulting tryptic peptides. Oxidation of the methionine (met14) in the T2 tryptic fragment was quantified with a sensitivity of approximately 1.0% (peak area percent relative to parent T2). The RP-HPLC profile obtained with the integrated system was nearly identical to that obtained via traditional methods (e.g. batch digestion followed by RP-HPLC analysis). The integrated technique, however, represents a 10-fold reduction in total analysis time when compared to the optimized batch digestion procedure. In addition, the identity of the PEGylated rhGH compound could be confirmed as well as the percentage of free N-terminus in a single injection.
authors.affiliation: Pfizer Global Biologics Pharmaceutical Sciences, Analytical Research and Development, St. Louis Laboratories, 700 Chesterfield Parkway N, St. Louis, MO 63017, USA. jeffrey.m.schneiderheinze@pfizer.com
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Schneiderheinze
authors.forename: Zara
authors.initials: Z
authors.lastname: Walden
authors.forename: Rob
authors.initials: R
authors.lastname: Dufield
authors.forename: Charles
authors.initials: C
authors.lastname: Demarest
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019382:Human Growth Hormone / Q000737:chemistry* / Q000378:metabolism*; D006868:Hydrolysis; D020407:Internet; D008715:Methionine / Q000032:analysis* / Q000378:metabolism*; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry*; D040901:Proteomics / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
chemical_list: D010446:Peptide Fragments; D011994:Recombinant Proteins; D019382:Human Growth Hormone; D011092:Polyethylene Glycols; D008715:Methionine