Process monitoring and flow ELISA techniques in biotechnology
Abstract
The first chapter of this thesis describes an automated system for rapid on-line process monitoring and harvesting of monoclonal antibody produced in a fermentor. Antibody production was monitored for the first 60 hours. When production reached a plateau, harvesting was initiated, which took 90 hours to complete. The following two chapters discuss a flow ELISA methodology for quantitation of mIgG developed in: (i) a bed packed perfusion chromatography media and (ii) a fused silica capillary. mIgG was captured by the primary antibody immobilized in either the perfusion media or the fused silica capillary; secondary antibody with ALP label was loaded and bound by mIgG; and finally, pNPP was injected and the product detected at 405nm. The lowest amount of mlgG detected was 1ng with a RSD in the range from 0.01 to 0.04. The last two chapters are devoted to the search for peptides that mimic or could be substituted for antibodies in protein purification. Candidates in this study were peptide libraries or banks and the tryptic peptides from selected antibodies. Candidates were incubated with a specific protein; size exclusion chromatography was employed to isolate the protein; then reversed phase chromatography was used to analyze the excluded SEC fraction to look for binding peptides. A few peptides were found and did show binding to the chosen protein.
Degree
Ph.D.
Advisors
Regnier, Purdue University.
Subject Area
Analytical chemistry|Biomedical research
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