METHODS FOR PRODUCING RECOMBINANT PROTEINS
Provided herein are methods of producing a recombinant protein that include: providing a bacterium including a nucleic acid encoding a recombinant protein; and culturing the bacterium in a liquid culture medium including about 0.3 mM to about 300 mM Mg 2+ under conditions sufficient for the production and release of the recombinant protein into the culture medium.
1 . A method of producing a recombinant protein, the method comprising:
providing a recombinant bacterium comprising a nucleic acid encoding a recombinant protein; and
culturing the recombinant bacterium in a liquid culture medium comprising about 0.3 mM to about 300 mM of Mg 2+ under conditions sufficient for the production and release of the recombinant protein into the culture medium.
2 . The method of claim 1 , wherein the liquid culture medium comprises a magnesium salt.
3 . The method of claim 2 , wherein the magnesium salt is MgSO 4 .
4 . The method of claim 1 , further comprising recovering the recombinant protein from the liquid culture medium.
5 . The method of claim 4 , wherein the recovered recombinant protein is at least 95% pure.
6 . The method of claim 4 , further comprising purifying the recovered recombinant protein.
7 . The method of claim 6 , further comprising formulating the purified recombinant protein.
8 . The method of claim 7 , wherein the method does not include the performance of more than two chromatography steps.
9 . The method of claim 7 , wherein the method does not include physical or chemical disruption of the outer membrane of the recombinant bacterium.
10 . The method of claim 1 , wherein the culturing is performed using a fermentor.
11 . The method of claim 1 , wherein the culturing is batch culturing.
12 . The method of claim 1 , wherein the culturing is fed batch culturing.
13 . The method of claim 1 , wherein the culturing comprises incubating the bacterium at a rotary agitation rate of about 80 revolutions per minute (RPM) to about 1000 RPM.
14 . (canceled)
15 . The method of claim 1 , wherein the culturing is performed at about 30° C. to about 37° C.
16 . The method of claim 1 , wherein the recombinant bacterium is a Gram negative bacterium.
17 . The method of claim 1 , wherein the recombinant bacterium is selected from the group consisting of: K 12 E. coli bacterial cell, a Yersinia bacterial cell, a BL21 E. coli bacterial cell, a 60E4 E. coli , an Acinetobacter bacterial cell, a Bordella bacterial cell, a Brucella bacterial cell, a Cyanobacter bacterial cell, an Enterobacter bacterial cell, a Helicobacter bacterial cell, a Klebsiella bacterial cell, a Neisseria bacterial cell, a Pasteurella bacterial cell, a Pseudomonas bacterial cell, a Salmonella bacterial cell, and a Shigella bacterial cell.
18 . The method of claim 1 , wherein the nucleic acid encoding the recombinant protein is integrated into a chromosome of the recombinant bacterium.
19 . The method of claim 1 , wherein the nucleic acid encoding the recombinant protein is not integrated into a chromosome of the recombinant bacterium.
20 . The method of claim 1 , wherein the recombinant protein is an antibody or an antigen-binding antibody fragment.
21 . The method of claim 1 , wherein the recombinant protein is an antibody or an antigen-binding antibody fragment that specifically binds to human vascular endothelial growth factor A (VEGFA).
22 . The method of claim 21 , wherein the antigen-binding antibody fragment is ranibizumab.
23 . The method of claim 1 , wherein the nucleic acid encoding the recombinant protein is an expression vector.
24 . The method of claim 23 , wherein the nucleic acid encoding the recombinant protein comprises a sequence encoding a bacterial signal sequence.
25 . The method of claim 1 , wherein the liquid culture medium comprises about 0.4 mM to about 35 mM Mg 2+ .
26 .- 29 . (canceled)
30 . A recombinant protein produced by the method of claim 1 .
31 . A compositions comprising the recombinant protein of claim 30 .
32 . A pharmaceutical composition comprising a therapeutically effective amount of the recombinant protein of claim 30 .
33 . The pharmaceutical composition of claim 32 , wherein the recombinant protein is ranibizumab.
34 . The pharmaceutical composition of claim 33 , wherein the pharmaceutical composition comprises about 5 mg/mL to about 10 mg/mL ranibizumab.
35 . The pharmaceutical composition of claim 33 , wherein the pharmaceutical composition further comprises a tonicity agent, a buffer, a surfactant, and water for injection, and wherein the pharmaceutical composition has a pH of about 5 to about 6.
36 . The pharmaceutical composition of claim 35 , wherein the pharmaceutical composition comprises α,α-trehalose dihydrate, a histidine buffer, polysorbate 20, and water for injection, and the pharmaceutical composition has a pH of about 5 to about 6.
37 . A kit comprising the pharmaceutical composition of claim 32 .
38 . The kit of claim 37 , further comprising a sterile glass vial, wherein the pharmaceutical composition is disposed within the sterile glass vial.
39 . The kit of claim 37 , further comprising a syringe, wherein the pharmaceutical compositions is disposed within the syringe.
40 . A method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 32 .
41 . The method of claim 40 , wherein the subject has been identified or diagnosed as having wet age-related macular degeneration, diabetic macular edema, or macular edema following retinal vein occlusion.
42 . The method of claim 41 , wherein the macular edema following retinal vein occlusion is branch retinal vein occlusion.
43 . The method of claim 41 , wherein the macular edema following retinal vein occlusion is central retinal vein occlusion.