PURIFICATION OF PROTEINS
The present invention relates to a method for purifying proteins, which proteins are produced by homologous or heterologous expression in a ciliate host, from harvested cell culture fluid (HCCF) which was not chromatographically purified. The method comprises the steps of incubating a harvested cell culture fluid with a kosmotropic agent, efflorescing protein by formation of crystals, and, optionally, harvesting effloresced protein.
1 . A method for purifying proteins, which proteins are produced by homologous or heterologous expression in a ciliate host, from harvested cell culture fluid (HCCF) which was not chromatographically purified,
the method comprising the steps of
a) incubating a harvested cell culture fluid with a kosmotropic agent,
b) efflorescing protein by formation of crystals, and
c) optionally, harvesting effloresced protein,
wherein the method does not comprise a step of crosslinking the proteins before or after efflorescing.
2 . The method according to claim 1 , wherein the protein is a glycosylated protein.
3 . The method according to claim 1 or 2 , wherein the kosmotropic agent is at least one of
ammonium sulfate,
sodium dihydrogen phosphate, and/or
polyethylene glycol
4 . The method according to any one of the aforementioned claims , wherein the harvested cell culture fluid comprises between ≥3 and ≤200 g/l of protein.
5 . The method according to any one of the aforementioned claims , wherein for efflorescing the protein,
a pH of between ≥5 and ≤8 is established in the medium, and/or
a temperature of between ≥10 and ≤40° C. is established.
6 . The method according to any one of the aforementioned claims , wherein, for the incubation of the harvested cell culture fluid, a kosmotropic salt concentration is established in the range of between ≥50 and ≤2500 mM.
7 . The method according to any one of the aforementioned claims , wherein, for the incubation of the harvested cell culture fluid, a polyalcohol concentration is established in the range of between ≥2 and ≤25% w/v.
8 . The method according to any one of the aforementioned claims , wherein the incubation of the harvested cell culture fluid with the kosmotropic agent is carried out over a period of between ≥10 min and ≤36 hrs.
9 . The method according to any one of the aforementioned claims , wherein a pre-harvest cell culture fluid is subjected to one or more filtration and/or centrifugation steps prior to step a), to obtain the harvested cell culture fluid.
10 . The method according to any one of the aforementioned claims , wherein the ciliate host is Tetrahymena sp.
11 . The method according to any one of the aforementioned claims , wherein the homologous or heterologous expression is under control of
a metallothionein gene (MTT1) inducible promoter, and/or
a heat inducible promoter.
12 . The method according to any one of the aforementioned claims , wherein the protein is an enzyme.
13 . The method according to claim 12 wherein the protein is at least one selected from the group consisting of:
lipase
protease
amylase
glutenase
glutamine-specific cysteine protease
prolyl endopeptidases
saccharase, and/or
lactase.
14 . The method according to any one of the aforementioned claims , wherein the enzyme is
a) Tetrahymena lipase according to any one of SEQ ID NOs 1-3 or 8-10,
b) Tetrahymena amylase according to SEQ ID NOs 4 or 11,
c) Tetrahymena protease according to any of SEQ ID NOs 5-7, 12-14 or 15-17, or
d) a variant of any one of the above enzymes having
(i) at least 90% amino acid sequence identity therewith, wherein said variant retains enzyme functionality, and/or
(ii) between 1 and 3 amino acid residues removed or added at the N-terminus and/or the C terminus
15 . The method according to any one of claims 1-14 , the method further comprising the step of solubilizing the effloresced protein.
16 . A pharmaceutical intermediate which has been obtained with a method according to any one of claims 1-15 .
17 . The pharmaceutical intermediate according to claim 16 , which comprises a solubilized form of the effloresced protein or the effloresced protein per se.
18 . A purified protein that has been obtained with a method according to any one of claims 1-15 .
19 . A purified, crystallized protein or pharmaceutical intermediate which comprises a ciliate-type glycosylation pattern.
20 . The intermediate according to any one of claim 16 or 17 or protein according to any one of claim 18 or 19 , which is or comprises at least one of
a) Tetrahymena lipase according to any one of SEQ ID NOs 1-3 or 8-10,
b) Tetrahymena amylase according to SEQ ID NOs 4 or 11,
c) Tetrahymena protease according to any of SEQ ID NOs 5-7, 12-14 or 15-17, or
d) a variant of any one of the above enzymes having
(i) at least 90% amino acid sequence identity therewith, wherein said variant retains enzyme functionality, and/or
(ii) between 1 and 3 amino acid residues removed or added at the N-terminus and/or the C terminus
21 . The intermediate according to any one of claim 16, 17 or 20 or the protein according to any one of claims 18-20 , which is provided in crystalline form.
22 . The intermediate according to any one of claim 16, 17, 20 or 21 or the protein according to any one of claims 18-21 , which has a purity grade of ≥80%.
23 . The intermediate according to any one of claims 16, 17, or 20-22 or the protein according to any one of claims 18-22 , wherein the protein crystals have a length of between ≥2 and ≤1000 μM.
24 . A pharmaceutic preparation comprising the intermediate according to any one of claims 16, 17, or 20-23 or the protein according to any one of claims 18-22 , and optionally further one or more further pharmaceutically acceptable excipients.
25 . The pharmaceutic preparation according to claim 24 , wherein the protein is an enzyme.
26 . The pharmaceutic preparation according to claim 25 , wherein the enzyme is at least one selected from the group consisting of
lipase
protease
amylase
glutenase
glutamine-specific cysteine protease
prolyl endopeptidases
saccharase, and/or
lactase.
27 . The pharmaceutic preparation according to claim 26 , wherein the enzyme is or comprises
a) Tetrahymena lipase according to any one of SEQ ID NOs 1-3 or 8-10,
b) Tetrahymena amylase according to SEQ ID NOs 4 or 11,
c) Tetrahymena protease according to any of SEQ ID NOs 5-7, 12-14 or 15-17, or
d) a variant of any one of the above enzymes having
(i) at least 90% amino acid sequence identity therewith, wherein said variant retains enzyme functionality, and/or
(ii) between 1 and 3 amino acid residues removed or added at the N-terminus and/or the C terminus
28 . The protein according to any one of claims 18-22 , or the pharmaceutic preparation according to any one of claims 26-27 (for the manufacture of a medicament) for use in the treatment of a human or animal subject
being diagnosed for,
suffering from or
being at risk of developing
a disorder caused by enzyme deficiency or dysfunction, or for the prevention of such condition.
29 . A method for treating or preventing a disorder caused by enzyme deficiency or dysfunction, which method comprises administration, to a human or animal subject, the enzyme according to any one of claims 18-22 , or the pharmaceutic preparation according to any one of claims 26-27 , in a therapeutically sufficient dose.
30 . The protein for use according to claim 28 or the method according to claim 29 , wherein the disorder caused by enzyme deficiency or dysfunction is at least one selected from the group consisting of
a lipid digestion deficiency and/or a digestive disorder
disorder caused by enzyme deficiency or dysfunction.
pancreatic exocrine insufficiency can have the following causes:
chronic pancreatitis
cystic fibrosis
main pancreatic duct obstruction
pancreatic resection
gastric resection
short bowel syndrome
hereditary hemochromatosis
celiac disease
Zollinger-Ellison syndrome
celiac sprue
sucrose intolerance or genetic sucrase-isomaltase deficiency (GSID), and/or
lactose intolerance
31 . A dosage unit comprising the protein according to any one of claims 18-22 , or the pharmaceutic preparation according to any one of claims 26-27 .