Title

Investigation of hetastarch as a cryoprotectant for L-asparaginase

Date of Completion

January 1998

Keywords

Chemistry, Analytical|Chemistry, Pharmaceutical|Chemistry, Physical|Health Sciences, Pharmacy

Degree

Ph.D.

Abstract

With recent advances in recombinant DNA technology, the stability of therapeutic proteins has been receiving renewed interest. The objective of this research was to investigate the effects of hetastarch on the freeze/thaw stability of l-asparaginase. Preliminary rheological studies of hetastarch showed that its viscosity dramatically increases with decreasing temperature. This led to the evaluation of its solutions for their protective ability against freeze/thaw denaturation of l-asparaginase. In contrast, glucose, lactose and 2-hydroxyethyl glucose, the monomer of hetastarch, showed very little protective effect. ^ The cause(s) for the loss of activity upon exposure to consecutive freeze/thaw cycles was determined by characterizing the protein using multiple techniques. Circular dichroism studies showed a decrease of α-helical structure with a concomitant increase in β-sheet and random coil suggesting alterations in secondary structure leading to unfolding. The elution profiles obtained from SEC studies indicated the formation of multiple species during the process of freezing and thawing. SDS-PAGE studies showed bands corresponding to 1–3 kDa and 32 kDa suggesting that some of the species are fragments and shortened monomers resulting from the cleavage of monomers. The molecular weight distribution obtained using SEC-linked light scattering indicated a substantial fraction of aggregate corresponding to 300 kDa. From these findings, a model for the degradation of l-asparaginase was developed. ^ The fragments found on SEC and PAGE were separated by reversed phase HPLC and sequenced using Edman chemistry and mass spectrometry. MALDI-TOF mass analysis identified a fragment with a mass of 1425 Da. ES/MS/MS identified two more fragments with masses of 616 Da and 1666 Da. The sequences of all the identified fragments indicate that they are the result of cleavage at the C-terminal end of l-asparaginase. While not directly involved in the enzyme's active site, the possible effects of the fragmentation on the loss of asparaginase activity is discussed in the dissertation. ^ The kinetics of cold denaturation of l-asparaginase in the absence and presence of hetastarch was investigated. It is clear that hetastarch slows the rate of cold denaturation. A model was developed to explain the kinetics data. ^