{"id":{"repo_id":"mit","oai_identifier":"oai:dspace.mit.edu:1721.1/17472"},"canonical_url":"https://search.dev.ndltd.org/etd/mit/oai:dspace.mit.edu:1721.1/17472","repository":{"repo_id":"mit","name":"MIT","base_url":"https://dspace.mit.edu/oai/request"},"display":{"title":"Oxidative damage to recombinant proteins during production","abstract":"Since the introduction of recombinant human insulin nearly two decades ago, recombinant proteins have increasingly been utilized as therapeutic agents. In addition, expression of recombi­nant proteins is now a common tool used in basic research. Recombinant proteins are subject to many subtle modifications that can affect their properties; among these modifications, oxidative damage is one of the most ubiquitous. Oxidative damage, however, is only occasionally consid­ered as a \"quality concern\" since it rarely detectable using standard biochemical techniques. The production of an oxidatively-sensitive protein, a1-Antitrypsin, was investigated to ascertain the effect of fermentation parameters on the extent of oxidation. Oxidation of either of two methion­ine residues in the active site to methionine sulfoxide was sufficient for inactivation, and 50% of the antitrypsin produced under standard fermentation conditions was oxidized. Oxidative damage was linked to the dissolved oxygen concentration by experimentation and detailed modeling of the evolution and detoxification of reactive oxygen species. Under pseudo steady-state conditions, the fractional oxidation is near zero under anaerobic conditions and increases through the microaero­bic regime. At dissolved oxygen concentrations greater than 10% of air saturation, the fractional oxidation did not vary. Step changes in the dissolved oxygen concentration, designed to emulate possible time variation resulting from poor mixing or changes in gas composition, caused tran­sient increases in the fractional oxidation and enhanced proteolytic degradation. This may impli­cate oxidative stress in scale-up related protein quality and quantity limitations. In addition, oxidative damage to antitrypsin caused a 5-fold increase in the stepwise addition rate for in vitro aggregation, which suggests that oxidative damage will limit shelf stability. In addition, process simulation demonstrated that removal of oxidative variants caused a 100% increase in cost per unit when only 22% of the antitrypsin is oxidized during the fermentation step.","abstract_html":"Since the introduction of recombinant human insulin nearly two decades ago, recombinant proteins have increasingly been utilized as therapeutic agents. In addition, expression of recombi­nant proteins is now a common tool used in basic research. Recombinant proteins are subject to many subtle modifications that can affect their properties; among these modifications, oxidative damage is one of the most ubiquitous. Oxidative damage, however, is only occasionally consid­ered as a "quality concern" since it rarely detectable using standard biochemical techniques. The production of an oxidatively-sensitive protein, a1-Antitrypsin, was investigated to ascertain the effect of fermentation parameters on the extent of oxidation. Oxidation of either of two methion­ine residues in the active site to methionine sulfoxide was sufficient for inactivation, and 50% of the antitrypsin produced under standard fermentation conditions was oxidized. Oxidative damage was linked to the dissolved oxygen concentration by experimentation and detailed modeling of the evolution and detoxification of reactive oxygen species. Under pseudo steady-state conditions, the fractional oxidation is near zero under anaerobic conditions and increases through the microaero­bic regime. At dissolved oxygen concentrations greater than 10% of air saturation, the fractional oxidation did not vary. Step changes in the dissolved oxygen concentration, designed to emulate possible time variation resulting from poor mixing or changes in gas composition, caused tran­sient increases in the fractional oxidation and enhanced proteolytic degradation. This may impli­cate oxidative stress in scale-up related protein quality and quantity limitations. In addition, oxidative damage to antitrypsin caused a 5-fold increase in the stepwise addition rate for in vitro aggregation, which suggests that oxidative damage will limit shelf stability. In addition, process simulation demonstrated that removal of oxidative variants caused a 100% increase in cost per unit when only 22% of the antitrypsin is oxidized during the fermentation step.","abstract_has_math":false,"creators":["Konz, John O. (John Otto), 1971-"],"institution":"Massachusetts Institute of Technology","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":"Massachusetts Institute of Technology. Department of Chemical Engineering","school":null,"contributors":[],"advisors":["Charles L. Cooney and Jonathan King."],"committee_chairs":[],"committee_members":[],"year":1998,"date_issued":"1998","date_published":"1998","updated_at":"2026-07-22T22:21:30Z","subjects":["Chemical Engineering"],"languages":["eng"],"rights":["M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission."],"rights_urls":["http://dspace.mit.edu/handle/1721.1/7582"],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/1721.1/17472","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Charles L. Cooney and Jonathan King."]},{"key":"dc:contributor.department","label":"Department","values":["Massachusetts Institute of Technology. Department of Chemical Engineering"]},{"key":"dc:creator","label":"Author","values":["Konz, John O. 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See provided URL for inquiries about permission."]},{"key":"dc:rights.uri","label":"Rights URI","values":["http://dspace.mit.edu/handle/1721.1/7582"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/1721.1/17472"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 1998.","Includes bibliographical references (p. 209-222)."]},{"key":"dc:description.abstract","label":"Abstract","values":["Since the introduction of recombinant human insulin nearly two decades ago, recombinant proteins have increasingly been utilized as therapeutic agents. In addition, expression of recombi­nant proteins is now a common tool used in basic research. Recombinant proteins are subject to many subtle modifications that can affect their properties; among these modifications, oxidative damage is one of the most ubiquitous. Oxidative damage, however, is only occasionally consid­ered as a \"quality concern\" since it rarely detectable using standard biochemical techniques. The production of an oxidatively-sensitive protein, a1-Antitrypsin, was investigated to ascertain the effect of fermentation parameters on the extent of oxidation. Oxidation of either of two methion­ine residues in the active site to methionine sulfoxide was sufficient for inactivation, and 50% of the antitrypsin produced under standard fermentation conditions was oxidized. Oxidative damage was linked to the dissolved oxygen concentration by experimentation and detailed modeling of the evolution and detoxification of reactive oxygen species. Under pseudo steady-state conditions, the fractional oxidation is near zero under anaerobic conditions and increases through the microaero­bic regime. At dissolved oxygen concentrations greater than 10% of air saturation, the fractional oxidation did not vary. Step changes in the dissolved oxygen concentration, designed to emulate possible time variation resulting from poor mixing or changes in gas composition, caused tran­sient increases in the fractional oxidation and enhanced proteolytic degradation. This may impli­cate oxidative stress in scale-up related protein quality and quantity limitations. In addition, oxidative damage to antitrypsin caused a 5-fold increase in the stepwise addition rate for in vitro aggregation, which suggests that oxidative damage will limit shelf stability. In addition, process simulation demonstrated that removal of oxidative variants caused a 100% increase in cost per unit when only 22% of the antitrypsin is oxidized during the fermentation step."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Ph.D."]},{"key":"dc:format.mimetype","label":"Dc Format Mimetype","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Oxidative damage to recombinant proteins during production"]}]}],"canonical_facts":{"dc:contributor.advisor":["Charles L. Cooney and Jonathan King."],"dc:contributor.department":["Massachusetts Institute of Technology. Department of Chemical Engineering"],"dc:creator":["Konz, John O. (John Otto), 1971-"],"dc:date.accessioned":["2005-06-02T15:22:35Z"],"dc:date.available":["2005-06-02T15:22:35Z"],"dc:date.issued":["1998"],"dc:description":["Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 1998.","Includes bibliographical references (p. 209-222)."],"dc:description.abstract":["Since the introduction of recombinant human insulin nearly two decades ago, recombinant proteins have increasingly been utilized as therapeutic agents. In addition, expression of recombi­nant proteins is now a common tool used in basic research. Recombinant proteins are subject to many subtle modifications that can affect their properties; among these modifications, oxidative damage is one of the most ubiquitous. Oxidative damage, however, is only occasionally consid­ered as a \"quality concern\" since it rarely detectable using standard biochemical techniques. The production of an oxidatively-sensitive protein, a1-Antitrypsin, was investigated to ascertain the effect of fermentation parameters on the extent of oxidation. Oxidation of either of two methion­ine residues in the active site to methionine sulfoxide was sufficient for inactivation, and 50% of the antitrypsin produced under standard fermentation conditions was oxidized. Oxidative damage was linked to the dissolved oxygen concentration by experimentation and detailed modeling of the evolution and detoxification of reactive oxygen species. Under pseudo steady-state conditions, the fractional oxidation is near zero under anaerobic conditions and increases through the microaero­bic regime. At dissolved oxygen concentrations greater than 10% of air saturation, the fractional oxidation did not vary. Step changes in the dissolved oxygen concentration, designed to emulate possible time variation resulting from poor mixing or changes in gas composition, caused tran­sient increases in the fractional oxidation and enhanced proteolytic degradation. This may impli­cate oxidative stress in scale-up related protein quality and quantity limitations. In addition, oxidative damage to antitrypsin caused a 5-fold increase in the stepwise addition rate for in vitro aggregation, which suggests that oxidative damage will limit shelf stability. In addition, process simulation demonstrated that removal of oxidative variants caused a 100% increase in cost per unit when only 22% of the antitrypsin is oxidized during the fermentation step."],"dc:description.degree":["Ph.D."],"dc:format.mimetype":["application/pdf"],"dc:identifier.uri":["http://hdl.handle.net/1721.1/17472"],"dc:language.iso":["eng"],"dc:publisher":["Massachusetts Institute of Technology"],"dc:rights":["M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission."],"dc:rights.uri":["http://dspace.mit.edu/handle/1721.1/7582"],"dc:subject":["Chemical Engineering"],"dc:title":["Oxidative damage to recombinant proteins during production"],"dc:type":["Thesis"]},"updated_at":"2026-07-22T22:21:30Z"}