Asparaginase (ASNASE), an enzyme catalyzing the deamidation of asparagine, is therapeutically used for the treatment of pediatric acute lymphoblastic leukemia (ALL). Current administration protocols are exclusively using bolus injection. No information is available about other dosages, like slow intravenous administration, thus hampering the full clinical exploitation of the drug. Preliminary observation indicates inconsistent issue of incompatibility with the infusion solution, which seems dependent of i.a. the source and batch of asparaginase used. Therefore, a selective pharmaceutical characterization is urgently required, so that the right choices and use of ASNASE can be defined, extending the clinical use.
We present our current results of the development and evaluation of (1) the enzymatic activity by determination of the formed ammonia by the Nessler reaction (see Figure 1) and (2) the amide-bond characterization (secondary/tertiary structures) by Fourier Transform Infrared Spectroscopy (FT-IR) and Circular Dichroism (CD). Different methods were selected for the ASNASE activity determination; whereof the Nessler assay was withheld for optimization. Using Design of Experiments (DOE) with four variables, i.e. CKI/CHgI2, CNaOH/CHgI2, CHgI2 final and reaction time in a D-optimal onion design space, a maximum enzyme activity response could be determined. For the characterization of the primary structure, peptide-mapping LC-MS method was adopted. The ASNASE amide-bond (secondary/tertiary structures) was characterized by FT-IR (see Figure 2) and CD. The secondary structure elements of the ASNASE are quantified by FT-IR, combined with mathematical deconvolution of the different amide peaks (mainly amide I, II and III bands). Using CD, the melting temperature (Tm) in relation with the enzyme stability is examined. Denaturation of the β-sheet is observed in a temperature range of 60-63°C, while the α-helix denaturated in 63-65°C
