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prior to crystallization. Gels 3 and 4 are two preparations of the 
crystallized enzyne. These gels were loaded with 100 micrograms of protein 
each. The crystallized asparaginase was non-pyrogenic in rabbits at a dose 
of 5000 I.U./kg. This is equivalent to about 17 mg/kg. 
The marked increase in specific activity across crystallization which 
amounted to about a three-fold purification suggested the possibility that 
the crystallized enzyme might offer some therapeutic advantage over 
amorphous preparations of asparaginase that were then being evaluated 
clinically. Purification of the enzyme by crystallization rather than by 
chromatography also appeared to be a favorable factor for the scale-up to 
commercial production. Accordingly, acute toxicity studies were conducted 
on the material appearing in gels 2 and 3, that is, the material before and 
after crystallization. The specific activities of these particular 
preparations were 135 and 275 I.U./mg respectively, approximately a two-fold 
difference in purity. This comparative acute toxicity study was performed 
at the suggestion of the Division of Biological Standards which was the FDA 
bureau responsible for regulatory aspects of asparaginase at that time. 
Four species were used in the acute toxicity study: mouse, rabbit, dog and 
morkey. There were no significant differences in acute toxicity of the two 
preparations, except that the cruder material induced a weak in vitro 
hemolysis of canine erythrocytes. The LD Q in both the dog and the monkey 
was greater than 16,000 I.U./kg body weight; this is equivalent to more than 
120 mg protein/kg in the case of the cruder preparation. 
[ 462 ] 
