Contents lists available at ScienceDirect Talanta journal homepage: www.elsevier.com/locate/talanta A Surface Acoustic Wave (SAW) biosensor method for functional quantifcation of E. coli L-asparaginase Han Yao, Cristina Soto Fernández, Xiaolong Xu, Evelien Wynendaele, Bart De Spiegeleer ∗ Drug Quality and Registration (DruQuaR) Group, Department of Pharmaceutical Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium ARTICLEINFO Keywords: Surface acoustic wave (SAW) biosensor L-asparaginase Method development and validation ABSTRACT Biosensors are rising technologies in the pharmaceutical feld 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 quantifcation of Escherichia coli L-asparaginase (E. coli L-ASNase), using polyclonal antibody (pAb) as the interaction partner immobilized on the chip. Diferent immobilization strategies of pAb were in- itiallyevaluated,resultingintheBS 3 activatedamidecouplingviaproteinGstrategyasthefnalimmobilization 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 diferent samples encom- passing diferent lots of E. coli L-ASNase, deamidated E. coli L-ASNase and dry-heated E. coli L-ASNase (80°C, 10min)indicatedthesuitabilityofthedevelopedSAWmethodtoquantify E.coli L-ASNase.WesuggestthisSAW method can be adopted as a pharmaceutical QC method. 1. Introduction L-asparaginase (L-ASNase) is a tetrameric enzyme, and has been a universal component of therapy for pediatric acute lymphoblastic leu- kaemia (ALL) since 1970's [1]. The clinically used L-ASNase prepara- tions are derived from Escherichia coli (E. coli) or from Erwinia chry- santhemi [1], with a tetrameric molecular weight (MW) of 136320Da resp.140320Da[2,3].Themechanismofanti-ALLactivityof L-ASNase is that leukemic cells cannot synthesize themselves sufcient L-aspar- agine (L-Asn) required for their protein synthesis; leukemic cells therefore have to depend upon the external L-Asn supply from the plasma and tissues [4]. The administration of L-ASNase quenches the external source of L-Asn, hence starving cancer cells [4]. For the treatment of pediatric ALL, remission induction therapy including L- ASNase, vincristine, corticosteroid and anthracycline has been the mainstay of the initial stage of the treatment [5]. In the consolidation phase of treatment, L-ASNase is also adopted in the therapy together with methotrexate, 6-mercaptopurine, vincristine and/or prednisone [5]. Three forms of L-ASNase are currently used in clinical practice, namely: (native) E. coli L-ASNase, PEGylated E. coli L-ASNase, and Er- winia L-ASNase [5]. Among these forms, E. coli L-ASNases are used for frst-line treatment of ALL, whereas Erwinia L-ASNase is used as second or third line treatment [6]. In this study, E. coli L-ASNase was in- vestigated by a biosensor based functional quantifcation method, with an anti- E. coli L-ASNase antibody used as the analytical interaction partner. Acoustic wave biosensors is a nanogram sensitive technology, em- ploying acoustic waves generated by oscillating a piezoelectric crystal quartz plate to meas. mass and viscosity changes [7]. Acoustic wave biosensors can be classifed into surface acoustic wave (SAW), bulk acoustic wave, and acoustic plate mode devices depending on the acoustic wave guiding process [8]. A surface acoustic wave is a me- chanical acoustic wave that propagates to a confned area of a piezo- electric crystal [9].LovewaveSAWsensorisarecentdevelopeddevice developed for integration in lab-on-a-chip systems. Love waves propa- gate near the surface of the piezoelectric material, the velocity and the amplitude of the wave depend on the changes occurring in the media near the surface [9]. The SAW biosensor proved to be a sensitive and efective technology for label-free detection of molecular interactions. Binding reactions are detected by measuring changes in surface wave velocity mainly caused by mass adsorption or viscosity changes on the surface layer [10]. Using SAW instruments as biosensors, antibodies or receptors are immobilized on the sensing layer to bind with analytes, usingahydrogellayeronthesensorsurface[11].Todate,awiderange https://doi.org/10.1016/j.talanta.2019.05.046 Received 8 March 2019; Received in revised form 6 May 2019; Accepted 9 May 2019 ∗ Corresponding author. E-mail address: Bart.DeSpiegeleer@UGent.be (B. De Spiegeleer). Talanta 203 (2019) 9–15 Available online 11 May 2019 0039-9140/ © 2019 Published by Elsevier B.V. T