Methods for making stable protein compositions
The invention relates to methods for preparing stable buffered and buffer-free protein compositions. The invention also relates to methods of purifying proteins from one or more impurities.
1 . A method for preparing a purified protein, the method comprises:
(a) subjecting a sample comprising a protein of interest to a purification process to obtain a preparation comprising the protein of interest,
(b) conducting a final ultrafiltration and diafiltration operation consisting of (i) adjusting the pH of the preparation produced by step (a) to a target pH using a pH adjusting agent prior to performing the ultrafiltration and diafiltration step, wherein the target pH is from pH 4.0 to pH 7.0, and (ii) performing the final ultrafiltration and diafiltration step in a medium that comprises a buffer, wherein the pH adjusting agent is (A) an acid selected from the group consisting of acetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzene sulfonic acid, benzoic acid, boric acid, camphorsulfonic acid, caprylic acid, formic acid, glutamic acid, hydrochloride acid, hydrobromic acid, hydroxy acids, hyaluronic acid, methanesulfonic acid, nitric acid, propionic acid, sulfuric acid, sulfonic acid, transexamic acid, and tartaric acid; (B) a base selected from the group consisting of ammonia solution, ammonium carbonate, diethanolamine, calcium hydroxide, ethanolamine, lysine, meglumine, poly-lysine, potassium hydroxide, sodium bicarbonate, sodium borate, sodium carbonate, sodium hydroxide, and triethanolamine; or (C) a buffer selected from the group consisting of acetate buffer, aspartate buffer, ascorbate buffer, borate buffer, benzoate buffer, carbonate buffer, citrate buffer, glycine buffer, HEPES buffer, MOPS buffer, MES buffer, N-(2-acetamido)iminodiacetic acid (ADA) buffer, histidine buffer, lactate buffer, phosphate buffer, succinate buffer, Tris buffer, Bis-tris buffer, and tartrate buffer; and
(c) recovering a composition comprising the protein from the final ultrafiltration and diafiltration operation, wherein the target pH is within 0.2 pH units of the pH of the composition comprising the protein recovered from the final ultrafiltration and diafiltration operation.
2 . The method of claim 1 , wherein the acid is selected from the group consisting of lactic acid, malic acid and hyaluronic acid.
3 . The method of claim 1 , wherein the target pH is from pH 4.0 to pH 6.0.
4 . The method of claim 1 , wherein the method further comprises concentrating the protein in the preparation to a target concentration.
5 . The method of claim 4 , wherein the concentrating step is carried out before the pH adjusting step.
6 . The method of claim 1 , wherein the composition obtained from the final ultrafiltration and diafiltration operation comprises the protein at a concentration of from about 40 mg/ml to about 200 mg/mL.
7 . The method of claim 1 , wherein the medium comprises a buffer at a concentration of from 2 mM to about 300 mM.
8 . The method of claim 1 , wherein the medium comprises a buffer at a concentration of from 2 mM to about 10 mM, and wherein the composition obtained from the ultrafiltration and diafiltration operation comprises the protein at a concentration of from about 1 mg/mL to about 50 mg/mL.
9 . The method of claim 1 , wherein the medium comprises a buffer at a pH outside the buffer capacity range of the buffer, and wherein the composition obtained from the ultrafiltration and diafiltration operation comprises the protein at a concentration of from about 1 mg/mL to about 50 mg/mL.
10 . The method of claim 1 , wherein the purification process comprises one or more of the following steps: centrifugation, microfiltration, TFF, virus inactivation, affinity chromatography, cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, ultrafiltration, diafiltration, SPTFF, depth filtration, and mixed-mode chromatography.
11 . The method of claim 10 , wherein the purification process comprises
(a) a virus filtration step and one or more of the following steps: centrifugation, microfiltration, TFF, virus inactivation, affinity chromatography, cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, mixed-mode chromatography, ultrafiltration, diafiltration, SPTFF, and depth filtration, and wherein the preparation is obtained from the virus filtration step;
(b) a cation exchange chromatography step, and one or more of the following steps: centrifugation, microfiltration, TFF, virus inactivation, affinity chromatography, anion exchange chromatography, hydrophobic interaction chromatography, mixed-mode chromatography, ultrafiltration, diafiltration, SPTFF, virus filtration, and depth filtration, and wherein the preparation is obtained from the cation exchange chromatography step;
(c) an anion exchange chromatography step, and one or more of the following steps: centrifugation, microfiltration, TFF, virus inactivation, affinity chromatography, cation exchange chromatography, hydrophobic interaction chromatography, mixed-mode chromatography, ultrafiltration, diafiltration, SPTFF, virus filtration, and depth filtration, and wherein the preparation is obtained from the anion exchange chromatography step;
(d) a hydrophobic interaction chromatography step, and one or more of the following steps: centrifugation, microfiltration, TFF, virus inactivation, affinity chromatography, cation exchange chromatography, anion exchange chromatography, mixed-mode chromatography, ultrafiltration, diafiltration, SPTFF, virus filtration, and depth filtration, and wherein the preparation is obtained from the hydrophobic interaction chromatography step; or
(e) a mixed-mode chromatography step, and one or more of the following steps: centrifugation, microfiltration, TFF, virus inactivation, affinity chromatography, cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, ultrafiltration, diafiltration, SPTFF, virus filtration, and depth filtration, and wherein the preparation is obtained from the mixed-mode chromatography step.
12 . The method of claim 1 , wherein the method further comprises adding a stabilizer to the preparation.
13 . The method of claim 12 , wherein the stabilizer is one or more selected from: an amino acid, a sugar, a polyol, an anti-oxidant, a chelating agent, a lipid or lipid derivative, a salt, a polymer, an inert protein, a surfactant, and a water-miscible co-solvent.
14 . The method of claim 13 , wherein
(a) the amino acid is one or more selected from: histidine, arginine, glycine, methionine, alanine, aspartic acid, lysine hydrochloride, proline, lysine, sarcosine, gamma-aminobutyric acid, and glutamic acid;
(b) the anti-oxidant is one or more selected from: ascorbic acid, glutathione, vitamin E, and poly (ethylenimine);
(c) the sugar is one or more selected from: sucrose, trehalose, xylitol, maltose, dextrose, glucose, raffinose, and lactose;
(d) the polyol is one or more of selected from: sugar alcohol, glycerol, erythritol, caprylate, tryptophanate, and sarcosine;
(e) the polymer is one or more selected from: gelatin, hyaluronic acid, polyvinylpyrrolidone (PVP), poly (lactic-co-glycolic acid) (PLGA), polyacrylic acid (PAA), Amphipol A8-35, PAA5-25C8-40C3, polyethylene glycol (PEG), hydroxyethyl (heta) starch, sulfated polysaccharides, polyamino acids, dextran, diethylaminoethyl-dextran, hydroxypropyl-beta-cyclodextrin, sulfobutylether-beta-cyclodextrin, polyethyleneimine (PEI), and carboxymethyl cellulose;
(f) the inert protein is one or more selected from: HSA, BSA and recombinant HA;
(g) the chelating agent is one or more selected from: EDTA, DPTA, citric acid, hexaphosphate, and thioglycolic acid;
(h) the salt is one or more selected from: sodium chloride, sodium sulfate, sodium thiocyanate, potassium chloride, potassium phosphate, calcium lactate, and guanidine hydrochloride;
(i) the surfactant is one or more selected from: polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, poloxamer, PEG dodecyl ethers, and PEG tertoctylphenyl ether;
(j) the water-miscible co-solvent is one or more selected from: (DMSO), DMF, DMA and CSA; or
(j) the lipid or lipid derivative is one or more of fatty acids, phospholipids and phospholipid derivatives, DEA phosphate, DEA cetyl phosphate, oleth 10 phosphate, oleth-10, oleth-10 phosphate, DEA cetyl phosphate, mannosylglycerate, polidocanol, sulfobetaine cholate, phospholipids, and C12-15 alcohols benzoate.
15 . The method of claim 1 , wherein the protein is any one of the following proteins: etanercept, aflibercept, adalimumab, epoetin alfa, darbepoetin alfa, filgrastim, pegfilgrastim, bevacizumab, cetuximab, infliximab, rituximab, eculizumab, trastuzumab, evolocumab, denosumab, romosozumab, erenumab, blinatumomab, and a bispecific T cell engager antibody construct.
16 . The method of claim 15 , wherein the bispecific T cell engager antibody construct is blinatumomab, anti-CD33 and anti-CD3 bispecific T cell engager antibody construct, anti-EGFRvIII and anti-CD3 bispecific T cell engager antibody construct, anti-DLL3 and anti-CD3 bispecific T cell engager antibody construct, anti-CD19 and anti-CD3 bispecific T cell engager antibody construct, anti-MSLN and anti-CD3 bispecific T cell engager antibody construct, anti-CDH19 and anti-CD3 bispecific T cell engager antibody construct, anti-FLT3 and anti-CD3 bispecific T cell engager antibody construct, anti-DLL3 and anti-CD3 bispecific T cell engager antibody construct, anti-CDH3 and anti-CD3 bispecific T cell engager antibody construct, anti-CD70 and anti-CD3 bispecific T cell engager antibody construct, anti-PSMA and anti-CD3 bispecific T cell engager antibody construct, and anti-BCMA and anti-CD3 bispecific T cell engager antibody construct.
17 . The method of claim 1 , wherein the ultrafiltration and diafiltration operation is carried out at a temperature of from about 25° C. to about 50° C.
18 . The method of claim 17 , wherein the ultrafiltration and diafiltration operation is carried out at a temperature from about 25° C. to about 40° C.
19 . The method of claim 18 , wherein the ultrafiltration and diafiltration operation is carried out at a temperature from about 30° C. to about 40° C.
20 . The method of claim 1 , wherein the composition obtained from the final ultrafiltration and diafiltration operation is more stable compared to a composition prepared by the same method without the pH adjusting step.
21 . The method of claim 1 , wherein the protein is etanercept.
22 . The method of claim 1 , wherein the protein is aflibercept.
23 . The method of claim 1 , wherein the protein is adalimumab.
24 . The method of claim 1 , wherein the target pH is pH 5.0 to pH 7.0.
25 . A method for preparing a purified protein, the method comprises:
(a) subjecting a sample comprising a protein of interest selected from etanercept, aflibercept, and adalimumab to a purification process to obtain a preparation comprising the protein of interest,
(b) conducting a final ultrafiltration and diafiltration operation consisting of (i) adjusting the pH of the preparation produced by step (a) to a target pH prior to performing the ultrafiltration and diafiltration step, and (ii) performing the final ultrafiltration and diafiltration step, and
(c) recovering a composition comprising the protein from the final ultrafiltration and diafiltration operation, wherein the target pH is within 0.2 pH units of the pH of the composition comprising the protein recovered from the final ultrafiltration and diafiltration operation.
26 . The method of claim 25 , wherein the pH is adjusted using a pH adjusting agent.
27 . The method of claim 26 , wherein the pH adjusting agent is an acid selected from the group consisting of acetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzene sulfonic acid, benzoic acid, boric acid, camphorsulfonic acid, caprylic acid, formic acid, glutamic acid, hydrochloride acid, hydrobromic acid, hydroxy acids, hyaluronic acid, methanesulfonic acid, nitric acid, phosphoric acid, propionic acid, sulfuric acid, sulfonic acid, transexamic acid, and tartaric acid.
28 . The method of claim 26 , wherein the pH adjusting agent is a base selected from the group consisting of ammonia solution, ammonium carbonate, diethanolamine, calcium hydroxide, ethanolamine, lysine, meglumine, poly-lysine, potassium hydroxide, sodium bicarbonate, sodium borate, sodium carbonate, sodium hydroxide, and triethanolamine.
29 . The method of claim 25 , wherein the target pH is from about pH 4.0 to about pH 7.0.
30 . The method of claim 29 , wherein the target pH is from about pH 4.0 to about pH 6.0.
31 . The method of claim 29 , wherein the target pH is pH 5.0 to pH 7.0.
32 . The method of claim 25 , wherein the method further comprises concentrating the protein in the preparation to a target concentration.
33 . The method of claim 32 , wherein the concentrating step is carried out before the pH adjusting step.
34 . The method of claim 25 , wherein the composition obtained from the final ultrafiltration and diafiltration operation comprises the protein at a concentration of from about 40 mg/ml to about 200 mg/mL.
35 . The method of claim 25 , wherein the purification process comprises one or more of the following steps: centrifugation, microfiltration, TFF, virus inactivation, affinity chromatography, cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, ultrafiltration, diafiltration, SPTFF, depth filtration, and mixed-mode chromatography.
36 . The method of claim 25 , wherein the method further comprises adding a stabilizer to the preparation.
37 . The method of claim 36 , wherein the stabilizer is one or more selected from: an amino acid, a sugar, a polyol, an anti-oxidant, a chelating agent, a lipid or lipid derivative, a salt, a polymer, an inert protein, a surfactant, and a water-miscible co-solvent.
38 . The method of claim 37 , wherein
(a) the amino acid is one or more selected from: histidine, arginine, glycine, methionine, alanine, aspartic acid, lysine hydrochloride, proline, lysine, sarcosine, gamma-aminobutyric acid, and glutamic acid;
(b) the anti-oxidant is one or more selected from: ascorbic acid, glutathione, vitamin E, and poly (ethylenimine);
(c) the sugar is one or more selected from: sucrose, trehalose, xylitol, maltose, dextrose, glucose, raffinose, and lactose;
(d) the polyol is one or more of selected from: sugar alcohol, glycerol, erythritol, caprylate, tryptophanate, and sarcosine;
(e) the polymer is one or more selected from: gelatin, hyaluronic acid, polyvinylpyrrolidone (PVP), poly (lactic-co-glycolic acid) (PLGA), polyacrylic acid (PAA), Amphipol A8-35, PAA5-25C8-40C3, polyethylene glycol (PEG), hydroxyethyl (heta) starch, sulfated polysaccharides, polyamino acids, dextran, diethylaminoethyl-dextran, hydroxypropyl-beta-cyclodextrin, sulfobutylether-beta-cyclodextrin, polyethyleneimine (PEI), and carboxymethyl cellulose;
(f) the inert protein is one or more selected from: HSA, BSA and recombinant HA;
(g) the chelating agent is one or more selected from: EDTA, DPTA, citric acid, hexaphosphate, and thioglycolic acid;
(h) the salt is one or more selected from: sodium chloride, sodium sulfate, sodium thiocyanate, potassium chloride, potassium phosphate, calcium lactate, and guanidine hydrochloride;
(i) the surfactant is one or more selected from: polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, poloxamer, PEG dodecyl ethers, and PEG tertoctylphenyl ether;
(j) the water-miscible co-solvent is one or more selected from: (DMSO), DMF, DMA and CSA; or
(j) the lipid or lipid derivative is one or more of fatty acids, phospholipids and phospholipid derivatives, DEA phosphate, DEA cetyl phosphate, oleth 10 phosphate, oleth-10, oleth-10 phosphate, DEA cetyl phosphate, mannosylglycerate, polidocanol, sulfobetaine cholate, phospholipids, and C12-15 alcohols benzoate.
39 . The method of claim 25 , wherein the ultrafiltration and diafiltration operation is carried out at a temperature of from about 25° C. to about 50° C.
40 . The method of claim 25 , wherein the final ultrafiltration and diafiltration operation is carried out using a medium that comprises essentially no buffer.
41 . The method of claim 40 , wherein the protein is etanercept or aflibercept.
42 . The method of claim 25 , wherein the final ultrafiltration and diafiltration operation is carried out in a medium that comprises a buffer.
43 . The method of claim 42 , wherein the protein is adalimumab.