{"id":{"repo_id":"vt","oai_identifier":"oai:vtechworks.lib.vt.edu:10919/31318"},"canonical_url":"https://search.dev.ndltd.org/etd/vt/oai:vtechworks.lib.vt.edu:10919/31318","repository":{"repo_id":"vt","name":"Virginia Tech","base_url":"https://vtechworks.lib.vt.edu/oai/request"},"display":{"title":"Characterization of the Recombinant Human Factor VIII Expressed in the Milk of Transgenic Swine","abstract":"Factor VIII is a protein which has therapeutic applications for the treatment of Hemophilia A. Its deficiency, either qualitative or quantitative, results in Hemophilia A, a disorder affecting approximately 1 in 10,000 males. Currently, FVIII replacement therapy uses FVIII derived from plasma or cell culture. The current cost of this therapy is in excess of $150,000 per patient per year. Thus, alternative sources that are more economical are attractive. The present work focuses upon the characterization of recombinant FVIII (rFVIII) made in the milk of transgenic pigs. Two dimensional western analysis of rFVIII obtained from pig whey showed a range of FVIII species having different isoelectric points (pI) consistent with diverse glycosylation patterns. The pI of these diverse FVIII populations were accurately predicted using theoretical calculations based upon primary protein structure as variable biantennary glycosylation patterns having 0, 1, or 2 sialic acid groups present. Kinetic limitations in the adsorption of rFVIII to anion exchange media due to the nature of the complex milk environment were observed. rFVIII was purified quantitatively using batch equilibration of whey with DEAE Sepharose. This material showed proteolytic processing that was very similar to FVIII obtained from human plasma. Based upon these results, it was postulated that a dissociation of the light (A3C1C2) and heavy (A1A2B) chain due to a lack of vWF may be responsible for the low FVIII activity.","abstract_html":"Factor VIII is a protein which has therapeutic applications for the treatment of Hemophilia A. Its deficiency, either qualitative or quantitative, results in Hemophilia A, a disorder affecting approximately 1 in 10,000 males. Currently, FVIII replacement therapy uses FVIII derived from plasma or cell culture. The current cost of this therapy is in excess of $150,000 per patient per year. Thus, alternative sources that are more economical are attractive. The present work focuses upon the characterization of recombinant FVIII (rFVIII) made in the milk of transgenic pigs. Two dimensional western analysis of rFVIII obtained from pig whey showed a range of FVIII species having different isoelectric points (pI) consistent with diverse glycosylation patterns. The pI of these diverse FVIII populations were accurately predicted using theoretical calculations based upon primary protein structure as variable biantennary glycosylation patterns having 0, 1, or 2 sialic acid groups present. Kinetic limitations in the adsorption of rFVIII to anion exchange media due to the nature of the complex milk environment were observed. rFVIII was purified quantitatively using batch equilibration of whey with DEAE Sepharose. This material showed proteolytic processing that was very similar to FVIII obtained from human plasma. Based upon these results, it was postulated that a dissociation of the light (A3C1C2) and heavy (A1A2B) chain due to a lack of vWF may be responsible for the low FVIII activity.","abstract_has_math":false,"creators":["Hodges, William Anderson"],"institution":"Virginia Tech","degree_name":"Master of Science","degree_level":"masters","degree_discipline":"Chemical Engineering","degree_department":"Chemical Engineering","school":null,"contributors":[],"advisors":[],"committee_chairs":["Velander, William H."],"committee_members":["Van Cott, Kevin E.","Forsten-Williams, Kimberly","Sullivan, Joseph T."],"year":2001,"date_issued":"2001-02-19","date_published":"2001-02-19","updated_at":"2026-07-24T05:56:30Z","subjects":["batch DEAE","immunoprecipitation","Factor VIII","isoelectric focusing"],"languages":[],"rights":["In Copyright"],"rights_urls":["http://rightsstatements.org/vocab/InC/1.0/"],"identifier_entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["etd-02232001-144721"],"render_values":[{"text":"etd-02232001-144721","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/10919/31318","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.committeechair","label":"Committee Chair","values":["Velander, William H."]},{"key":"dc:contributor.committeemember","label":"Committee Member","values":["Van Cott, Kevin E.","Forsten-Williams, Kimberly","Sullivan, Joseph T."]},{"key":"dc:contributor.department","label":"Department","values":["Chemical Engineering"]},{"key":"dc:creator","label":"Author","values":["Hodges, William Anderson"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2014-03-14T20:32:05Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2014-03-14T20:32:05Z","2002-02-28"]},{"key":"dc:date.issued","label":"Date","values":["2001-02-19"]},{"key":"dc:publisher","label":"Institution","values":["Virginia Tech"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Chemical Engineering"]},{"key":"thesis:degree_level","label":"Degree Level","values":["masters"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Master of Science"]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["Virginia Polytechnic Institute and State University"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["batch DEAE","immunoprecipitation","Factor VIII","isoelectric focusing"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:rights","label":"Dc Rights","values":["In Copyright"]},{"key":"dc:rights.uri","label":"Rights URI","values":["http://rightsstatements.org/vocab/InC/1.0/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["etd-02232001-144721"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/10919/31318"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Factor VIII is a protein which has therapeutic applications for the treatment of Hemophilia A. Its deficiency, either qualitative or quantitative, results in Hemophilia A, a disorder affecting approximately 1 in 10,000 males. Currently, FVIII replacement therapy uses FVIII derived from plasma or cell culture. The current cost of this therapy is in excess of $150,000 per patient per year. Thus, alternative sources that are more economical are attractive. The present work focuses upon the characterization of recombinant FVIII (rFVIII) made in the milk of transgenic pigs. Two dimensional western analysis of rFVIII obtained from pig whey showed a range of FVIII species having different isoelectric points (pI) consistent with diverse glycosylation patterns. The pI of these diverse FVIII populations were accurately predicted using theoretical calculations based upon primary protein structure as variable biantennary glycosylation patterns having 0, 1, or 2 sialic acid groups present. Kinetic limitations in the adsorption of rFVIII to anion exchange media due to the nature of the complex milk environment were observed. rFVIII was purified quantitatively using batch equilibration of whey with DEAE Sepharose. This material showed proteolytic processing that was very similar to FVIII obtained from human plasma. Based upon these results, it was postulated that a dissociation of the light (A3C1C2) and heavy (A1A2B) chain due to a lack of vWF may be responsible for the low FVIII activity."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Master of Science"]},{"key":"dc:title","label":"Title","values":["Characterization of the Recombinant Human Factor VIII Expressed in the Milk of Transgenic Swine"]}]}],"canonical_facts":{"dc:contributor.committeechair":["Velander, William H."],"dc:contributor.committeemember":["Van Cott, Kevin E.","Forsten-Williams, Kimberly","Sullivan, Joseph T."],"dc:contributor.department":["Chemical Engineering"],"dc:creator":["Hodges, William Anderson"],"dc:date.accessioned":["2014-03-14T20:32:05Z"],"dc:date.available":["2014-03-14T20:32:05Z","2002-02-28"],"dc:date.issued":["2001-02-19"],"dc:description.abstract":["Factor VIII is a protein which has therapeutic applications for the treatment of Hemophilia A. Its deficiency, either qualitative or quantitative, results in Hemophilia A, a disorder affecting approximately 1 in 10,000 males. Currently, FVIII replacement therapy uses FVIII derived from plasma or cell culture. The current cost of this therapy is in excess of $150,000 per patient per year. Thus, alternative sources that are more economical are attractive. The present work focuses upon the characterization of recombinant FVIII (rFVIII) made in the milk of transgenic pigs. Two dimensional western analysis of rFVIII obtained from pig whey showed a range of FVIII species having different isoelectric points (pI) consistent with diverse glycosylation patterns. The pI of these diverse FVIII populations were accurately predicted using theoretical calculations based upon primary protein structure as variable biantennary glycosylation patterns having 0, 1, or 2 sialic acid groups present. Kinetic limitations in the adsorption of rFVIII to anion exchange media due to the nature of the complex milk environment were observed. rFVIII was purified quantitatively using batch equilibration of whey with DEAE Sepharose. This material showed proteolytic processing that was very similar to FVIII obtained from human plasma. Based upon these results, it was postulated that a dissociation of the light (A3C1C2) and heavy (A1A2B) chain due to a lack of vWF may be responsible for the low FVIII activity."],"dc:description.degree":["Master of Science"],"dc:identifier.other":["etd-02232001-144721"],"dc:identifier.uri":["http://hdl.handle.net/10919/31318"],"dc:publisher":["Virginia Tech"],"dc:rights":["In Copyright"],"dc:rights.uri":["http://rightsstatements.org/vocab/InC/1.0/"],"dc:subject":["batch DEAE","immunoprecipitation","Factor VIII","isoelectric focusing"],"dc:title":["Characterization of the Recombinant Human Factor VIII Expressed in the Milk of Transgenic Swine"],"dc:type":["Thesis"],"thesis:degree_discipline":["Chemical Engineering"],"thesis:degree_level":["masters"],"thesis:degree_name":["Master of Science"],"thesis:institution_name":["Virginia Polytechnic Institute and State University"]},"updated_at":"2026-07-24T05:56:30Z"}