کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
5832173 1122593 2015 12 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Mammalian cell transient expression, non-affinity purification, and characterization of human recombinant IGFBP7, an IGF-1 targeting therapeutic protein
موضوعات مرتبط
علوم زیستی و بیوفناوری ایمنی شناسی و میکروب شناسی ایمونولوژی
پیش نمایش صفحه اول مقاله
Mammalian cell transient expression, non-affinity purification, and characterization of human recombinant IGFBP7, an IGF-1 targeting therapeutic protein
چکیده انگلیسی


- Successfully expressed and purified the mammalian rhIGFBP7, an IGF-1 targeted inhibition protein, using transient gene expression and conventional non-affinity column chromatography purification method.
- High quality material IGFBP7 was generated for further pre-clinical drug development studies.
- Transient gene expression technology through optimization of vectors is practical to be used for production of recombinant non-antibody protein products for biopharmaceutical development.

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 ~ 50 mg/L was selected for production at 2 L 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 ~ 50 mg/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.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: International Immunopharmacology - Volume 29, Issue 2, December 2015, Pages 476-487
نویسندگان
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