The icIEF instrument (Maurice) was obtained from ProteinSimple, Inc

The icIEF instrument (Maurice) was obtained from ProteinSimple, Inc. were comprehensively characterized. Despite the consistency of purity, drifts in N-glycan profile were observed, which resulted in the variation of antibody-dependent cellular cytotoxicity (ADCC) activity. In detail, four parametric curves of ADCC activity of the reference product were unparalleled, and the maximum response value was highly related to the content of %afucose than half-maximal effective concentration (EC50). Lorcaserin As ADCC is a potential critical quality attribute of Perjeta?, the glycosylation of Perjeta? and its biosimilars should be tightly monitored and controlled. 1. Introduction Biosimilars are biological products that are highly similar to the previously authorized reference products, and they have no clinically meaningful differences. The US Food and Drug Administration (FDA), the European Medicines Agency (EMA), the World Health Organization (WHO), and other local drug regulatory authorities, including China National Medical Products Administration (NMPA), have published a number of guidelines for the development of biosimilars, which could be used to further clarify clinical and nonclinical evidences [1C3]. Minor changes in the manufacturing process may cause drifts in the quality of biological products. Therefore, to ensure the consistency between processes and products, a robust quality management system is urgently required [4, 5]. Evaluating the similarity of structural and functional properties is crucial to indicate whether the existing differences can influence clinical outcomes. In order to ensure that the full spectrum of product variability could be accurately captured, manufacturers should gather multiple batches of reference products throughout the biosimilar development period [6]. Considering the inherent heterogeneity among protein products and the expected lot-to-lot differences derived from manufacturing processes, the FDA guideline has recommended that manufacturers should collect at least 10 lots of reference products[1]. Pertuzumab, a full-length humanized recombinant IgG1(receptor IIIa binding and glycosylation [7]. Perjeta? was approved by the United States FDA (2012), EMA (2013), and NMPA (2018) in combination with trastuzumab and chemotherapy for the treatment of patients with HER2-positive metastatic breast cancer. For the development of biosimilars of pertuzumab, 13 lots of China-resourced Perjeta? with an expiration date from August 2020 to October 2021 were collected intensively for the characterization of physicochemical and functional attributes. The state-of-the-art and orthogonal methods were chosen to analyze the size, charge, and unpaired cysteine variants of pertuzumab. As reported previously, an unpaired cysteine variant of Cys23/Cys88 in one or both light chains of pertuzumab could be identified, which was proved to be related to the antiproliferation effect, and it was estimated to have a reduced potency of ~50% compared with the native Fab [8]. N-linked glycosylation of the crystallizable fragment (Fc) of the immunoglobulin class G (IgG) is a nicein-125kDa well-known attribute that is significantly associated with Fc-mediated antibody effector functions, such as ADCC and complement-dependent cytotoxicity (CDC). Afucosylation content dramatically enhances the ADCC activity by increasing the binding affinity between the antibody and Fcreceptor IIIa [7, 9, 10]. The drifts in N-glycan-related quality attributes have been extensively discussed in previous studies (e.g., trastuzumab) [4, 11, 12]. However, no previous research has demonstrated that the quality attributes of pertuzumab Lorcaserin have drifted. In the present study, we found that the size, charge, unpaired cysteine variants, and bioactivity were highly consistent among all batches of pertuzumab, while two sequential drifts of glycosylation were observed, and the same drifts were also detected in Fc= 13)2.614.180.982.0NASD (= 13) (%)30.818.411.714.0 Open in a separate window The CellTiter-Glo Luminescent and Bio-Glo Luciferase Assay Reagent Lorcaserin was obtained from Promega (Madison, WI, USA). Goat anti-human IgG- (Fc specific) peroxidase antibody, sodium dodecyl sulfate (SDS), n-ethyl maleimide (NEM), sodium cyanoborohydride, 2-aminobenzamide (2-AB), and 3,3,5,5-tetramethylbenzidine (TMB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). HER2 antigen was purchased from Sino Biological Co., Ltd. (Beijing, China). The PNGase F kit was obtained from New England Biolabs (Ipswich, MA, USA). The Measure-iT? Thiol Assay kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). FabALACTICA (IgdE) was obtained from Genovis (Lund, Sweden). TSKgel FcR-IIIA-NPR column was obtained from Tosoh Bioscience (Tokyo, Japan). 2.2. Cell Lines.