Date of Award

2006

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Pharmaceutical Sciences

First Advisor

Thomas Needham

Abstract

Purpose: To identify factors affecting the concentration loss during the manufacturing process of Beta Domain Deleted Recombinant Human Factor VIII (BDDrFVIII SQ)

Methods: Lyophilization cycles were stopped after each stage to characterize the protein through the cycle. Three different types of glass tubing vials; untreated, silicon dioxide coated, and siliconized were evaluated. Two formulations of BDDrFVIII SQ were evaluated to determine the effects of a concentrated formula. Factor VIII concentration (μg/ml) was determined using an anion exchange high performance liquid chromatography (HPLC) assay, with ultraviolet (UV) and fluorescence detection.

Results: The freezing and annealing stages were found be critical aspects of the lyophilization cycle. Comparison of the three types of glass vials demonstrated that treated vials (siliconized or silicon dioxide) yielded greater protein concentrations. The formulation experiment indicated that a concentrated formula (2X) is superior to the standard formulation for protein recovery.

Conclusions: The current freezing cycle produces an ice crystal structure that damages the protein and may increase susceptibility to denaturation and aggregation upon reconstitution. Treated vials showed a significant benefit over untreated glass vials in the recovery of protein, but did not ameliorate the problem entirely. This indicates that the protein loss is only partially due to protein-surface interactions resulting in adsorption. Through fortification of the formula and decreasing the amount of water, there is less protein lost during lyophilization and reconstitution.

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