Insulin-fc fusions and methods of use
An insulin and fusion protein technology, applied in the field of insulin-Fc fusion protein composition, can solve problems such as inability to normalize blood sugar levels
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Embodiment approach 1
[0308] Embodiment 1: Synthesis and method for preparing insulin-Fc fusion protein in HEK293 cells
[0309] Insulin-Fc fusion protein was synthesized as follows. The gene sequence of interest was constructed using proprietary software (LakePharma, Belmont, CA) and cloned into a high-expression mammalian vector. HEK293 cells were seeded in shake flasks 24 hours before transfection and cultured in serum-free chemically defined medium. A DNA expression construct encoding the insulin-Fc fusion protein of interest was transiently transfected into 2L of HEK293 cell suspension using Syd Labs (Natick, MA) standard operating procedures for transient transfection. After 20 h, count cells to determine viability and live cell count, and pass (Pall Forté Bio LLC, Fremont, CA) to measure titers. Additional readouts were performed throughout the transient transfection production run. Cultures were harvested on or after day 5.
[0310] As shown in Table 1, an exemplary insulin-Fc fus...
Embodiment approach 2
[0312] Embodiment 2: Synthesis and method of making insulin-Fc fusion protein in CHO cells
[0313] The CHO cell line was originally derived from CHO-K1 (LakePharma, Belmont, CA), and the endogenous glutamine synthetase (GS) gene was knocked out by recombinant techniques using methods known in the art. Stable expression DNA vectors were designed and optimized for CHO expression and GS selection and integrated into high expression mammalian vectors (LakePharma, Belmont, CA). Confirm the sequence of each completed construct before starting scale-up experiments. Place the suspended CHO cells in humidified 5% CO 2 in a chemically defined medium (CD OptiCHO TM ; Invitrogen, Carlsbad, CA). No serum or other products of animal origin were used to grow CHO cells.
[0314] Grow in CD OptiCHO during exponential growth phase TM medium, use 80 μg DNA System (MaxCyte, Inc., Gaithersburg, MD) transfected approximately 8,000 suspension-adapted CHO cells by electroporation to create ...
Embodiment approach 3
[0318] Embodiment 3: Purification of insulin-Fc fusion protein
[0319]Purification of the insulin-Fc fusion protein was performed as follows. Conditioned medium supernatants containing secreted insulin-Fc fusion protein were harvested from transiently or stably transfected HEK or CHO production runs and clarified by centrifugation. The supernatant containing the desired insulin-Fc fusion protein was run on a protein A column and eluted using a low pH gradient. Then, the eluted desired protein fractions were pooled and buffer exchanged into 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0 buffer. A 0.2 μm membrane filter was used for the final filtration step. Calculate the final protein concentration from the solution optical density at 280 nm. Further optional purification is carried out by ion exchange chromatography, gel filtration chromatography or other methods, if necessary. The titer results (mg / L) obtained after protein A column purification are shown in figure ...
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