Contents lists available at ScienceDirect Journal of Chromatography B journal homepage: www.elsevier.com/locate/jchromb Analytical comparability study of anti-CD20 monoclonal antibodies rituximab and obinutuzumab using a stability-indicating orthogonal testing protocol: Efect of structural optimization and glycoengineering Yossra A. Trabik a , Eman M. Moenes b , Medhat A. Al-Ghobashy c,d, ⁎ , Marianne Nebsen c , Miriam F. Ayad a a Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Egypt b National Organization for Research and Control of Biologicals, Egypt c Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Egypt d Bioanalysis Research Group, School of Pharmacy, Newgiza University, Egypt ARTICLE INFO Keywords: Rituximab Obinutuzumab Glycoengineering Biosimilarity Stability ABSTRACT Glycoengineering and biosimilarity are the key factors for growing, promising and progressive approaches in monoclonal antibodies development. In this study, the physicochemical stability of anti-CD20 rituximab (RTX); originator and biosimilar was compared to its glycoengineered humanized version; obinutuzumab (OBZ). An orthogonal stability-indicating protocol using a set of validated bioanalytical techniques; size exclusion high performance liquid chromatography (SE-HPLC), reversed phase liquid chromatography (RP-HPLC), quantitative gel electrophoresis by TapeStation, receptor binding assay and dynamic light scattering (DLS) was used to in- vestigate the efect of diferent stress factors on the pattern and kinetics of degradation. SE-HPLC results sup- ported with spectral purity showed similar degradation extent with a diferent pattern of degradation between RTX and OBZ. A lower tendency to form degraded fragments and a relatively higher favorability for degradation through aggregate formation has been revealed in case of OBZ. Results were in agreement with those of DLS and receptor binding assay which showed specifcity to the intact antibodies in the presence of their degradation products. Furthermore, results were additionally confrmed through denaturing quantitative gel electrophoresis which suggested reducible covalent bonds as the mechanism for aggregates formation. RP-HPLC results showed two oxidized forms via excessive oxidation of RTX and OBZ with nearly the same degradation percent. Comparability data of RTX and OBZ using the applied methodologies showed that although glycoengineering; carried out to enhance the therapeutic and biological activity of OBZ altered the pattern of degradation but did not signifcantly afect the overall stability. Results showed also consistent stability profle between the biosi- milar and its originator RTX products. 1. Introduction Centuries ago, using antibodies as ‘magic bullets’ to cure patients was a dream for Paul Ehrlich [1,2]. In 1975 Köhler and Milstein in- vented hybridoma technology and captured the imaginations of the medical community by the development of monoclonal antibodies (mAbs) [3,4]. Since then, mAbs have become a cornerstone in the modern biotherapeutics [5]. The mAbs are monovalent antibodies produced artifcially from hybridoma cells which is a hybrid clone of antibody-producing B lymphocytes and immortal myeloma cells. They bind with high specifcity, afnity and selectivity to a unique epitope on a single antigen [6]. In 1997 the frst chimeric mAb, Rituximab (RTX) was approved for the treatment of cancer and specifcally for patients with non-Hodgkin’s lymphoma and chronic lymphocytic leukaemia [7,8]. It is a genetically engineered chimeric glycosylated protein with human IgG1 constant regions and murine light-chain and heavy-chain variable region se- quences. It is composed of two heavy chains of 451 amino acids and two light chains of 231 amino acids and has an approximate molecular weight of 145 kDa [8–10]. It has N-linked glycosyl moieties at Asn-301 in the Fc region of the heavy chain [11]. RTX has paved the way for immunotherapy biologic discovery, regulatory pathways and it has indirectly outlined how the world deals with biosimilar development in the feld of oncology. The world’s frst biosimilar for RTX was launched https://doi.org/10.1016/j.jchromb.2020.122359 Received 5 July 2020; Received in revised form 14 August 2020; Accepted 29 August 2020 ⁎ Corresponding author at: Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Egypt. E-mail address: medhat.alghobashy@cu.edu.eg (M.A. Al-Ghobashy). Journal of Chromatography B 1159 (2020) 122359 Available online 02 September 2020 1570-0232/ © 2020 Elsevier B.V. All rights reserved. T