Abstract
L-asparaginase, a therapeutic agent for the treatment of acute lymphoblastic leukemia, was evaluated for its susceptibility to cold denaturation. It was found that the enzyme derived from Erwinia chrysanthemi loses its activity when exposed to freeze-thaw cycling. When it was frozen at – 40°C and thawed, the enzyme lost 67.3% of its activity; whereas, when frozen in liquid nitrogen (–190°C), it lost almost all of its activity. Rheological studies of hetastarch showed that its viscosity dramatically increases with decreasing temperature, suggesting that at sub-zero temperatures it will create a highly viscous environment around the enzyme. It is proposed that this highly viscous environment retards the rate of conformational changes leading to losses in activity. Hetastarch solutions of various concentrations and degrees of hydroxyethylation were evaluated for their protective ability against the freeze-thaw denaturation of L-asparaginase. It was found that the cryoprotective effect of hetastarch with 0.8 degree of substitution at a concentration of 0.2% was sustained over many freeze-thaw cycles while that of the lesser substituted starch was not. The cryoprotective effect of hetastarch was compared to that of other commonly used additives such as glucose and lactose, which failed to protect the enzyme from freeze-thaw denaturation. In addition, the protective effect of a monomer of hetastarch was evaluated in order to distinguish whether the protective effect of hetastarch was due to physicochemical interactions with the individual monomer units or to its polymeric nature. The monomer showed significant cryoprotection through the first freeze-thaw cycle which was not sustained over additional freeze-thaw cycles. Thus, it appears that it is the polymeric nature of the hetastarch that is responsible for its cryoprotection of L-asparaginase. This suggests that the enzyme is protected by the highly viscous environment created by hetastarch.
- Received August 29, 1994.
- Accepted November 13, 1994.
- Copyright © Parenteral Drug Association. All rights reserved.
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