PROCESS FOR PRODUCING, ISOLATING, AND PURIFYING MODIFIED RECOMBINANT PROTEINS

1 . A method for producing recombinant methionyl human granulocyte colony-stimulating factor (r-met-Hu-G-CSF) comprising:

(a) contacting cells comprising a nucleic acid encoding r-met-Hu-G-CSF with a culture medium to create a fermentation medium;

(b) fermenting the cells under fed-batch conditions causing the cells to produce r-met-Hu-G-CSF;

(c) harvesting the cells from the fermentation medium by centrifugation;

(d) lysing the cells harvested from the fermentation medium to release inclusion bodies comprising r-met-Hu-G-CSF; and

(e) storing the inclusion bodies.

2 . A method for purifying r-met-Hu-G-CSF from inclusion bodies comprising:

(a) suspending inclusion bodies comprising r-met-Hu-G-CSF in a solubilization buffer;

(b) oxidizing solubilized r-met-Hu-G-CSF to permit the r-met-Hu-G-CSF to fold and form disulfide bonds;

(c) subjecting a product of step (b) to Dowex flow-through chromatography;

(d) subjecting a product of step (c) to acid precipitation;

(e) subjecting a product of step (d) to anion exchange chromatography;

(f) subjecting a product of step (e) to cation exchange chromatography;

(g) subjecting a product of step (f) to mixed mode chromatography;

(h) concentrating a product of step (g); and

(i) exchanging r-met-Hu-G-CSF in the product of step (h) into a buffer by ultrafiltration and diafiltration.

3 . A method of producing a PEGylated and purified r-met-Hu-G-CSF comprising:

(a) contacting a r-met-Hu-G-CSF with a PEGylation reagent under suitable reaction conditions to PEGylate the r-met-Hu-G-CSF;

(b) subjecting a product of step (a) to cation exchange chromatography to remove the reaction by-products from PEGylated r-met-Hu-G-CSF;

(c) concentrating a product of step (b);

(d) exchanging the PEGylated r-met-Hu-G-CSF in a product of step (c) into a buffer by ultrafiltration and diafiltration;

(e) adding a surfactant to a product of step (d);

(f) adjusting the pH of a product of step (e) to a target value by adding HCl or NaOH;

(g) diluting a product of step (f) with additional diafiltration buffer to achieve a target PEGylated r-met-Hu-G-CSF concentration of 10.0 mg/mL; and

(h) subjecting the PEGylated r-met-Hu-G-CSF product of step (g) to 0.2-μm filtration.

4 . The method of claim 3 , further comprising, after step (h), storing the PEGylated r-met-Hu-G-CSF product at 5±3° C.

5 . The method of claim 1 , further comprising after step (e):

(f) suspending inclusion bodies comprising r-met-Hu-G-CSF in a solubilization buffer;

(g) oxidizing solubilized r-met-Hu-G-CSF to permit the r-met-Hu-G-CSF to fold and form disulfide bonds;

(h) subjecting a product of step (g) to Dowex flow-through chromatography;

(i) subjecting a product of step (h) to acid precipitation;

(j) subjecting a product of step (i) to anion exchange chromatography;

(k) subjecting a product of step (j) to cation exchange chromatography;

(l) subjecting a product of step (k) to mixed mode chromatography;

(m) concentrating a product of step (l); and

(n) exchanging r-met-Hu-G-CSF in the product of step (m) into a buffer by ultrafiltration and diafiltration.

6 . The method of 2 , further comprising after step (i):

(j) contacting a r-met-Hu-G-CSF with a PEGylation reagent under suitable reaction conditions to PEGylate the r-met-Hu-G-CSF;

(k) subjecting a product of step (j) to cation exchange chromatography to remove the reaction by-products from PEGylated r-met-Hu-G-CSF;

(l) concentrating a product of step (k);

(m) exchanging the PEGylated r-met-Hu-G-CSF in a product of step (l) into a buffer by ultrafiltration and diafiltration;

(n) adding a surfactant to a product of step (m);

(o) adjusting the pH of a product of step (n) to a target value by adding HCl or NaOH;

(p) diluting a product of step (o) with additional diafiltration buffer to achieve a target PEGylated r-met-Hu-G-CSF concentration of 10.0 mg/mL; and

(q) subjecting the PEGylated r-met-Hu-G-CSF product of step (p) to 0.2-μm filtration.

7 . The method of claim 5 , further comprising after step (n):

(o) contacting a r-met-Hu-G-CSF with a PEGylation reagent under suitable reaction conditions to PEGylate the r-met-Hu-G-CSF;

(p) subjecting a product of step (o) to cation exchange chromatography to remove the reaction by-products from PEGylated r-met-Hu-G-CSF;

(q) concentrating a product of step (p);

(r) exchanging the PEGylated r-met-Hu-G-CSF in a product of step (q) into a buffer by ultrafiltration and diafiltration;

(s) adding a surfactant to a product of step (r);

(t) adjusting the pH of a product of step (s) to a target value by adding HCl or NaOH;

(u) diluting a product of step (t) with additional diafiltration buffer to achieve a target PEGylated r-met-Hu-G-CSF concentration of 10.0 mg/mL; and

(v) subjecting the PEGylated r-met-Hu-G-CSF product of step (u) to 0.2-μm filtration.

8 . The method of claim 7 , further comprising storing the PEGylated r-met-Hu-G-CSF product at 5±3° C.