title: Charge variant analysis of proposed biosimilar to Trastuzumab.
abstract: Trastuzumab is a humanized monoclonal antibody (mAb) employed for the treatment of HER2 Positive Breast Cancer. A HER2 overexpressing tumor cell binds to Trastuzumab and attracts immune cells which lead to induction of Antibody Dependent Cellular Cytotoxicity (ADCC) by binding to Fc receptors (CD16a or FcγRIIIa) on an effector cell, such as natural killer (NK) cells. The most commonly expressed receptor on NK cell is CD16a which binds to the Fc portion of Trastuzumab. The ligand-independent HER2-HER3 dimerization is the most potent stimulator of downstream pathways for regulation of cell growth and survival. An attempt has been made in this study to understand the impact of charge heterogeneity on the binding kinetics and potency of the monoclonal antibody. Trastuzumab has a pI range of 8.7-8.9 and is composed of mixture of acidic and basic variants beside the main peak. Ion exchange chromatography was used to isolate the acidic, basic, and main peak fractions from in-house proposed biosimilar to Trastuzumab and their activities were compared to the Innovator Trastuzumab Herclon®. Data from the mass analysis confirmed the potential modifications in both acidic and basic variant. Binding activity studies performed using Surface Plasmon Resonance (SPR) revealed that acidic variants had lesser binding to HER2 in comparison to the basic variants. Both acidic and basic variant showed no significant changes in their binding to soluble CD16a receptors. In vitro assay studies using a breast cancer cell line (BT-474) confirmed the binding potency of acidic variant to be lesser than basic variant, along with reduced anti-proliferative activity for the acidic variant of Trastuzumab. Overall, these data has provided meaningful insights to the impact of antibody charge variants on in vitro potency and CD16 binding affinity of trastuzumab.
authors.affiliation: Zumutor Biologics, Bangalore, India.
authors.forename: Pravinkumar
authors.initials: P
authors.lastname: Dakshinamurthy
authors.affiliation: Zumutor Biologics, Bangalore, India.
authors.forename: Pavithra
authors.initials: P
authors.lastname: Mukunda
authors.affiliation: Zumutor Biologics, Bangalore, India.
authors.forename: Bhargav
authors.initials: B
authors.lastname: Prasad Kodaganti
authors.affiliation: Zumutor Biologics, Bangalore, India.
authors.forename: Bharath Ravindra
authors.initials: BR
authors.lastname: Shenoy
authors.affiliation: Zumutor Biologics, Bangalore, India.
authors.forename: Bairavabalakumar
authors.initials: B
authors.lastname: Natarajan
authors.affiliation: Zumutor Biologics, Bangalore, India.
authors.forename: Amol
authors.initials: A
authors.lastname: Maliwalave
authors.affiliation: Zumutor Biologics, Bangalore, India.
authors.forename: Vivek
authors.initials: V
authors.lastname: Halan
authors.affiliation: Zumutor Biologics, Bangalore, India.
authors.forename: Sathyabalan
authors.initials: S
authors.lastname: Murugesan
authors.affiliation: Zumutor Biologics, Bangalore, India; Theramyt Novobiologics Pvt Ltd, Bangalore, India. Electronic address: sunit_maity@theramyt.com.
authors.forename: Sunit
authors.initials: S
authors.lastname: Maity
mesh_terms: D000920:Antibody-Dependent Cell Cytotoxicity / Q000187:drug effects / Q000276:immunology; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D059451:Biosimilar Pharmaceuticals / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D005260:Female; D006801:Humans; D007694:Killer Cells, Natural / Q000378:metabolism; D011485:Protein Binding; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D017452:Receptors, IgG / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D000068878:Trastuzumab / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; D059451:Biosimilar Pharmaceuticals; D018719:Erb-b2 Receptor Tyrosine Kinases; D017452:Receptors, IgG; D000068878:Trastuzumab; C496124:FCGR3A protein, human
keywords: Binding kinetics; Biosimilar; Charge variants; In vitro potency