High-yield deblocking of amino termini of recombinant immunoglobulins with pyroglutamate aminopeptidase.
For larger proteins, efficient deblocking prior to Edman sequencing is especially important to obtain quality, extended sequencing data which is limited by the stepwise accumulation of background from the random acid hydrolysis of the protein. Therefore, any portion that remains blocked contributes to the undesirable background. We report an optimized procedure for the removal of pyroglutamate (pGlu) by pyroglutamate aminopeptidase (PGAP) and demonstrate its use for the quantitative deblocking of several humanized recombinant antibodies (rIgGs). The rIgGs with blocked heavy chain provided an advantageous system in which removal of pGlu from the heavy chain was determined as a ratio of the deblocked heavy chain to the light chain in the first cycle of sequencing; i.e., the light chain was used as an internal standard. The reaction temperature, reaction time, enzyme-to-substrate ratio, denaturation, and reduction/carboxymethylation prior to digestion, and different commercial enzymes were evaluated. The optimized procedure involves reduction/carboxymethylation in guanidine buffer, buffer exchange by gel-permeation chromatography, and overnight PGAP digestion at 37 degrees C. Five different rIgGs, including one with blocked heavy and light chains, were deblocked in nearly quantitative yields using this procedure.
Mozdzanowski
J
J
Bioanalytical Sciences, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA.
Bongers
J
J
Anumula
K
K
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D007145:Immunoglobulin kappa-Chains / Q000096:biosynthesis; D007146:Immunoglobulin lambda-Chains / Q000096:biosynthesis; D007202:Indicators and Reagents; D008099:Liver / Q000201:enzymology; D010449:Peptide Mapping; D019711:Pyrococcus / Q000201:enzymology; D011751:Pyroglutamyl-Peptidase I; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013552:Swine; D014162:Transfection / Q000379:methods
D007074:Immunoglobulin G; D007147:Immunoglobulin Light Chains; D007145:Immunoglobulin kappa-Chains; D007146:Immunoglobulin lambda-Chains; D007202:Indicators and Reagents; D011994:Recombinant Proteins; D011751:Pyroglutamyl-Peptidase I