Characterization of the Recombinant Human Factor VIII Expressed in the Milk of Transgenic Swine. William Anderson Hodges

Transcription

1 Characterization of the Recombinant Human Factor VIII Expressed in the Milk of Transgenic Swine William Anderson Hodges Thesis submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Master of Science in Chemical Engineering APPROVED: William Velander, Chair Kimberly Forsten Kevin Van Cott Joseph Sullivan February 9, 2001 Blacksburg, Virginia Keywords: Factor VIII; immunoprecipitation, isoelectric focusing; batch DEAE

2 Characterization of the Recombinant Human Factor VIII Expressed in the Milk of Transgenic Swine William Anderson Hodges (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.

3 Table of Contents Chapter 1. An Introduction to Factor VIII and its Inherent Instabilities... 1 Factor VIII Production in Cell Culture... 2 Factor VIII Production in Transgenic Animals... 4 Chapter 2. Immunoprecipitation Assays Introduction Methods Results Discussion Chapter 3. Isoelectric Focusing Introduction Methods Results Discussion Chapter 4. Batch-mode DEAE Adsorption Introduction Methods Results Discussion Chapter 5. Conclusions and Future Work Vita... 81

4 List of Illustrations Chapter 1 Figure 1a. Western Analysis of FVIII Chains Obtained Human Plasma... 7 Figure 1b. Thrombin Activation of WT-FVIII vwf Complex... 8 Figure 2. Inactivation Resistant Factor VIII (IR8) in CHO Cell... 9 Figure 3. Hypothesized rhfviii Inactivation in Transgenic Milk Figure 4. Factor VIII Activity Levels Obtained in Cell Culture Chapter 2 Figure 1. Rocket Figure 2. Rocket Figure 3. Rocket Figure 4. Rocket Figure 5. Rocket Figure 6. Rocket Figure 7. Rocket Figure 8. Rocket Figure 9. Rocket Figure 10. Rocket Figure 11. Coupled Rocket performed with Red Cross FVIII and Cedarlane Sheep anti-fviii antibody Figure 12. SIA Figure 13. SIA Figure 14. Crossed Plates Figure 15. Crossed Plates Figure 16. Standard Curve Using Western... 32

5 Chapter 3 Figure 1. Primary Structure of Biantennary glycan from porcine lactoferrin Figure 2. Expected pi range for Factor FVIII Fragments Based upon Primary Structure and Glycosylation Figure 3. Isoelectric Focusing Unit Figure 4. Two-dimensional Western of hfviii obtained from American Red Cross Anti-hemophilic Factor Figure 5. Two-dimensional Western of rhfviii obtained from Transgenic Swine Milk Figure 6. Protein Concentration and ph gradient for Azocasein / Hemoglobin IEF Run Figure 7. Slot Blot Performed on Fractions from DEAE Column Performed on Milk from Pig 22-5 Figure % Bio-Rad Tris-Glycine Gel: Reduced Anti-FVIII Western On DEAE Column Fractions Figure 9. ph Profile of IEF on Wash 2 from DEAE Column Figure % Bio-Rad Tris-Glycine Gel: Reduced Anti-FVIII Western On Wash 1 IEF Fractions Figure 11. IEF Run on Elute 1 from DEAE Column Figure % Novex Tris-Glycine Gel: Reduced Anti-FVIII Western On 250 mm Elute IEF Fractions Figure 13. ph Profile of IEF Run Figure 14. Protein Concentration of IEF Supernatent Samples Figure % Tris-Glycine gel: Reduced Anti-FVIII Western on IEF Supernatent Samples Figure % Tris-Glycine gel: Reduced Anti-FVIII Western on IEF Pellet samples Chapter 4 Figure 1. Non-reduced 4-12% Bis-Tris Gel Analysis of DEAE Purification of hfviii

6 Figure 2. Non-reduced 4-12% Bis-Tris Western Analysis of DEAE Purification of hfviii Figure 3. Western Analysis of Column-mode DEAE treatment of pig milk containing rhfviii Figure 4. Reduced 4-12% Bis-Tris Gel Analysis of Batch DEAE Processing of Transgenic Milk Figure 5. Reduced 4-12% Bis-Tris Western Analysis of Batch DEAE Processing of Transgenic Milk Figure 6. Non-reduced 4-12% Bis-Tris Gel Analysis of Zinc Acetate Precipition of DEAE column fractions Figure 7. Reduced 4-12% Bis-Tris Western Analysis of Zinc Acetate Precipitation of DEAE column fractions Figure 8. Standard Curve for APTT Test Performed on DEAE Column Fractions Chapter 5 Figure 1. Theoretical pi of IR8 Fragments... 79