A Surface Acoustic Wave (SAW) biosensor method for functional quantification of E. colil-asparaginase.

Biosensors are rising technologies in the pharmaceutical field for medicine discovery, development and Quality Control (QC) stages. Surface acoustic wave (SAW) biosensor employs acoustic waves generated by oscillating a piezoelectric crystal quartz plate to meas. mass and viscosity, and allows to detect and quantify binding events between the analyte and an immobilized interacting ligand. We present here a SAW biosensor based approach for the functional quantification of Escherichia colil-asparaginase (E. colil-ASNase), using polyclonal antibody (pAb) as the interaction partner immobilized on the chip. Different immobilization strategies of pAb were initially evaluated, resulting in the BS3 activated amide coupling via protein G strategy as the final immobilization method. The method was validated by evaluating the selectivity, linearity, as well as accuracy (a recovery of 102.4%) and precision (RSD of 8.5%). The application of the validated method on different samples encompassing different lots of E. colil-ASNase, deamidated E. colil-ASNase and dry-heated E. colil-ASNase (80 °C, 10 min) indicated the suitability of the developed SAW method to quantify E. colil-ASNase. We suggest this SAW method can be adopted as a pharmaceutical QC method.

Development of a capillary zone electrophoresis method to quantify E. colil-asparaginase and its acidic variants.

A capillary zone electrophoresis (CZE) method with UV detection was developed for the quantification of the E.colil-asparaginase (l-ASNase) and its acidic variants. During the initial method development, a variety of experimental conditions were screened. Subsequently, a Design of Experiments (DoE) was used to optimize the pH and concentration of the selected background electrolyte (BGE) containing both TRIS and boric acid. Optimization was performed taking into account both the separation efficiency of l-ASNase and its acidic variants as well as overall method robustness. A repeatable separation between E.colil-ASNase and its acidic variants was achieved on a bare fused silica capillary in combination with a BGE consisting of both 400 mM TRIS and boric acid. The method was validated for linearity, accuracy, precision, LOD, LOQ and robustness. The recovery for l-ASNase was 97.9-104.4% with a precision RSD of 1.5-3.2%, while the recovery of impurities was 92.1-109.8% with a RSD of 1.7-4.6%. The quantification limit was 1.9% (m/m). Moreover, the CZE-UV method was applied to determine the degradation rate in the presence of ammonium bicarbonate, confirming the suitability of the method. The degraded, partially charged l-ASNase was evaluated for its in-vitro enzymatic activity showing an insignificant different enzyme activity compared to the unmodified sample.