title: Mammalian cell transient expression, non-affinity purification, and characterization of human recombinant IGFBP7, an IGF-1 targeting therapeutic protein.
abstract: Targeted inhibiting insulin-like growth factor 1 is an effective approach for cancer therapy. Insulin-like growth factor binding protein 7 (IGFBP7) is considered as a potential therapeutic protein. However, producing high quality of such non-IgG proteins in mammalian cells is still a challenge in biopharmaceutical development. Here, we report a rapid production process by using transient gene transfection in HEK 293E cells. A set of constructs combining several expression promoters, leader sequences, and 5' un-translated regions were generated and optimized, from which the best vector with expression level at ~50mg/L was selected for production at 2L cell culture scale. Comparison study in downstream purification methods led to development of a scalable, non-affinity chromatography strategy through Super Q, Fast Flow Q, and Heparin columns. The product was characterized in purity (99%), isoelectric point, molecule weight, glycosylation, and stability by using SEC-HPLC, SDS-PAGE, isoelectric focusing and mass spectrometry. The highly purified product shows IGF-1 binding activity and inhibits IGF-1-induced cell proliferation. This process not only provides a remarkable high expression at ~50mg/L and pure glycosylated mammalian rhIGFBP7, also highlights that transient gene expression technology is practical to be used for production and early development of recombinant non-IgG therapeutic proteins.
authors.affiliation: Biopharmaceutical Development Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, United States.
authors.forename: Man-Shiow
authors.initials: MS
authors.lastname: Jiang
authors.affiliation: Biopharmaceutical Development Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, United States.
authors.forename: Xiaoyi
authors.initials: X
authors.lastname: Yang
authors.affiliation: Protein Expression Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, United States.
authors.forename: Dominic
authors.initials: D
authors.lastname: Esposito
authors.affiliation: Biopharmaceutical Development Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, United States.
authors.forename: Earl
authors.initials: E
authors.lastname: Nelson
authors.affiliation: Biopharmaceutical Development Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, United States.
authors.forename: Jinhui
authors.initials: J
authors.lastname: Yuan
authors.affiliation: Protein Expression Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, United States.
authors.forename: Ralph F
authors.initials: RF
authors.lastname: Hopkins
authors.affiliation: Biopharmaceutical Development Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, United States.
authors.forename: Trevor
authors.initials: T
authors.lastname: Broadt
authors.affiliation: Protein Characterization Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, United States.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Xiao
authors.affiliation: Protein Characterization Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, United States.
authors.forename: Simona
authors.initials: S
authors.lastname: Colantonio
authors.affiliation: Protein Characterization Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, United States.
authors.forename: DaRue A
authors.initials: DA
authors.lastname: Prieto
authors.affiliation: Biological Resources Branch, National Cancer Institute at Frederick, Frederick, MD 21702, United States.
authors.forename: Anthony R
authors.initials: AR
authors.lastname: Welch
authors.affiliation: Biological Resources Branch, National Cancer Institute at Frederick, Frederick, MD 21702, United States.
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Creekmore
authors.affiliation: Biopharmaceutical Development Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, United States.
authors.forename: George
authors.initials: G
authors.lastname: Mitra
authors.affiliation: Biopharmaceutical Development Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, United States. Electronic address: jianweiz@sjtu.edu.cn.
authors.forename: Jianwei
authors.initials: J
authors.lastname: Zhu
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D000595:Amino Acid Sequence; D049109:Cell Proliferation / Q000187:drug effects; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D018969:Insulin-Like Growth Factor Binding Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000187:drug effects*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000494:pharmacology; D014162:Transfection
chemical_list: D020121:5' Untranslated Regions; D018969:Insulin-Like Growth Factor Binding Proteins; D011994:Recombinant Proteins; C102529:insulin-like growth factor binding protein-related protein 1; D007334:Insulin-Like Growth Factor I
keywords: IGFBP7; Protein expression; Protein purification; Transient gene expression