Characterization of the Recombinant Human Factor VIII Expressed in the Milk of Transgenic Swine

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.

Name thesis:degree_name

Master of Science

Level thesis:degree_level

masters

Discipline thesis:degree_discipline

Chemical Engineering

Department dc:contributor.department

Chemical Engineering

Grantor dc:publisher

Virginia Tech

Year dc:date.issued

2001

Author dc:creator

• Hodges, William Anderson

Chair dc:contributor.committeechair

• Velander, William H.

Committee members dc:contributor.committeemember

• Van Cott, Kevin E.
• Forsten-Williams, Kimberly
• Sullivan, Joseph T.

• batch DEAE
• immunoprecipitation
• Factor VIII
• isoelectric focusing

Statement dc:rights

• In Copyright

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Dc Identifier Other

etd-02232001-144721

OAI identifier oai:identifier

oai:vtechworks.lib.vt.edu:10919/31318