Asymmetrical flow field-flow fractionation for the analysis of PEG-asparaginase.

ABSTRACT

Monomethoxypolyethylene glycol L-asparaginase (PEG-ASNASE) is the PEGylated version of the enzyme L-asparaginase (ASNASE). Both are used for remission induction in acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL). The treatment control is generally carried out by performing activity assays, though methods to determine the actual enzyme rather than its activity are rare. Using asymmetrical flow field-flow fractionation (AF4) offered the chance to develop a method capable of simultaneously measuring PEG-ASNASE and PEG. A method validation was performed in accordance with FDA guidelines for PEG-ASNASE from non-biological solutions. The method unfolded a linearity of 15-750 U/mL with coefficients of correlation of r(2)>0.99. The coefficients of variation (CV) for within-run and between-run variability were 1.18-10.15% and 2.43-8.73%, respectively. Furthermore, the method was used to perform stability tests of the product Oncaspar® (PEG-ASNASE) and estimation of the molecular weight by multi-angle light scattering (MALS) of stressed samples to correlate them with the corresponding activity. The findings indicate that Oncaspar® stock solution should not be stored any longer than 24 h at room temperature and cannot be frozen in pure aqueous media. The validated method might be useful for the pharmaceutical industry and its quality control of PEG-ASNASE production.