Studies On The Preparation, Purification And Identification Of N-terminal Site-Specific Mono-PEGylation Of L-Asparaginase

The L-asparaginase (L-ASP) is one kind of white or light yellow freeze-dry dust, smell-less and tasteless. It is composed of homeotype tetramer and its relative molecular weight is 133000±5000. The main function is to hydrolyze aspartamic acid in blood serum to aspartic acid and the ammonia. L-ASP in clinical is mainly used to treat the acute lymphocyte leukemia (abbreviation AL). And the aspartamic acid is a must for cell protein synthetic and proliferation. Normal cells can synthesize aspartamic acid, but tumor cells of acute leukemia etc do not have that function. Thus when L-ASP causes the aspartamic acid sudden missing, the tumor cell cannot obtain enough aspartamic acid from the blood, and cannot synthesize either. Then massive tumor cells will be destructed because the protein synthesis is blocked and the proliferation is suppressed. L-ASP can also disturb the process of DNA and RNA synthesis in cells, possibly by affecting the G1 multiplication cycle of cells. In clinical practice, reorganized L-ASP also has short half-life, strong immunogenicity and poisonous side effect as other reorganized products. Because its half-life is short, it must be injected every day to maintain effective blood concentration. As a result, long-term and frequent injection increases the treatment cost, brings serious pain and inconvenience to patients, reduces the compliance and aggravate patients'economical burden.In order to overcome the clinical limit of L-ASP, this research chose the single methoxy polyethylene glycol propylaldehyde (mPEG-ALD) to carry out localized modification on the N-terminal of the laevo-rotatory asparaginase. By increasing its molecular weight, the kidney elimination rate was reduced and in vivo half-life was enhanced; Because of stereo barrier effect of coupling PEG three-dimensional, the trypsin degradation was slowed down and its immunogenicity was also greatly reduced simultaneously; and because of the PEG biological good compatibility and lengthening of half-life, the injection amount of L-ASP was reduced, which correspondingly reduced its poisonous side effect.The detailed contents of research were described as the following:1 This research established one optimized PEG-L-ASP preparation system by screening proper mole ratio, reaction temperature, reaction time, pH value of the modification reaction of PEG. The optimized preparation condition was: mPEG was 1:10;temperature was 37 centigrade; time was 6 hours; pH value was 5; enzyme solution...