title: Biochemical characterization of a new recombinant TNF receptor-hyFc fusion protein expressed in CHO cells.
abstract: The currently used Tumor Nectosis Factor (TNF)-α blockers such as infliximab, adalimumab and etanercept have Fc regions of the human IgG1 subtype have advantages in terms of in vivo half-life, however these could raise potential concerns for unwanted effector-mediated effects, such as antibody dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). To address this issue, we constructed a novel hybrid protein with decreased ADCC and CDC potentials by fusing the TNF receptor to a hybrid Fc (hyFc) containing CH2 and CH3 regions of IgG4 and highly flexible hinge regions of IgD which neither has ADCC and CDC activities. The resulting fusion protein, TNFR-hyFc, was over-expressed in CHO cells. For use as a pre-clinical material in pharmacology, PK and toxicological evaluations were carried out for biochemical characterization which was then compared with etanercept that has similarity in structure. Amino acid composition analysis and peptide mapping showed that the expressed TNFR-hyFc matched the theoretical composition derived from the DNA sequence. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) showed that TNFR-hyFc is 2.9 kDa larger than etanercept. MALDI-TOF after removal of N-glycans by PNGase treatment showed that TNFR-hyFc is 3.9 kDa larger than etanercept. Isoelectric focusing and monosaccharide analysis showed that TNFR-hyFc is slightly more acidic than etanercept. N-terminal amino acid sequencing showed that N-terminal heterogeneity is present in both TNFR-hyFc and etanercept, although the ratios are somewhat different. Glycan analysis showed that the main glycan form is bi-antennary, similar to etanercept.
authors.affiliation: Biopharmaceutical Research Labs, Research Center, Dong-A Pharmaceutical Co., Ltd., Sanggal-dong, Giheung-gu, Yongin-si, Kyungki-do 446-905, Republic of Korea. jhlee@donga.co.kr
authors.forename: Jung-Hwan
authors.initials: JH
authors.lastname: Lee
authors.forename: Jiwoo
authors.initials: J
authors.lastname: Yeo
authors.forename: Hyoung Seo
authors.initials: HS
authors.lastname: Park
authors.forename: Gong
authors.initials: G
authors.lastname: Sung
authors.forename: Sung Hee
authors.initials: SH
authors.lastname: Lee
authors.forename: Se Hwan
authors.initials: SH
authors.lastname: Yang
authors.forename: Young Chul
authors.initials: YC
authors.lastname: Sung
authors.forename: Ju-Hee
authors.initials: JH
authors.lastname: Kang
authors.forename: Chang-Shin
authors.initials: CS
authors.lastname: Park
mesh_terms: D000818:Animals; D016466:CHO Cells; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D016923:Cell Death / Q000187:drug effects; D006224:Cricetinae; D000068800:Etanercept; D006031:Glycosylation; D007141:Immunoglobulin Fc Fragments / Q000235:genetics*; D007074:Immunoglobulin G / Q000737:chemistry / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009005:Monosaccharides / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D018124:Receptors, Tumor Necrosis Factor / Q000737:chemistry / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D020539:Sequence Analysis, Protein; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000502:physiology
chemical_list: D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D009005:Monosaccharides; D010446:Peptide Fragments; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D000068800:Etanercept