Rapid characterization of recombinant pharmaceutical proteins using tandem column enzymatic mapping
Abstract
An automatic tandem column enzymatic mapping method was developed for rapid characterization of pharmaceutical proteins. An immobilized enzyme column was coupled with a reversed-phase column in series and incorporated in an HPLC system. Protein samples were digested when passing through the enzyme column. The peptide fragments were transferred directly into the reversed-phase column, concentrated on the top of the column and eluted with a gradient. This mapping process was completed within 2 hours. Kinetic behaviors of immobilized trypsin columns were investigated using a variety of proteins as substrates. The degree of trypsinolysis on human growth hormone and cytochrome C as a function of incubation time was studied. The digestion reached equilibrium at around 6 minutes and half of the digestion was completed within the first 2 minutes. Efficiency of immobilized trypsin on different proteins was evaluated by comparing the peak areas of the protein samples obtained from on-column digestion with the standards. It was found to be related to the size and the number of disulfide linkage of a protein. Recombinant human growth hormone and human insulin were characterized using the tandem column peptide mapping method with the assistance of MALDI-TOF mass spectrometry. Recombinant human growth hormone was analyzed by tandem column trypsin mapping. The amino acid sequence and the location of two disulfide linkages were confirmed to be correct and disulfide mismatching was not found in the product. Possible degradants were also analyzed using this method. Methionine sulfoxide was not discovered in the original rhGH sample but detected in the sample under long-term storage. Recombinant human insulin was characterized by tandem column Glu-C proteinase mapping. The correct primary structure including the location of disulfide linkages was confirmed. In addition, disulfide scrambling which occurred in free solution Glu-C digestion on human insulin was not observed in the tandem column digestion.
Degree
Ph.D.
Advisors
Regnier, Purdue University.
Subject Area
Analytical chemistry|Chemistry|Pharmacology
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