Process for the production of preformed conjugates of albumin and a therapeutic agent
The present invention provides processes for the production of preformed albumin conjugates. In particular, the invention provides processes for the in-vitro conjugation of a therapeutic compound to recombinant albumin, wherein a therapeutic compound comprising a reactive group is contacted to recombinant albumin in solution to form a conjugate. The processes provide for conjugation to albumin species of increasing homogeneity. The resulting conjugate is purified by chromatography, in particular hydrophobic interaction chromatography comprising phenyl sepharose and butyl sepharose chromatography.
1 . A process for the preparation of a conjugate, said conjugate comprising albumin covalently linked to a compound, the process comprising purifying the conjugate by a first hydrophobic interaction chromatography followed by a second hydrophobic interaction chromatography.
2 . The process of claim 1 , wherein the first hydrophobic interaction chromatography is phenyl sepharose chromatography.
3 . The process of claim 1 , wherein the second hydrophobic interaction chromatography is butyl sepharose chromatography.
4 . The process of claim 3 , wherein the butyl sepharose chromatography comprises:
a. equilibrating butyl sepharose resin in 750 mM ammonium sulfate;
b. contacting the butyl sepharose resin with a solution comprising the conjugate; and
c. applying a decreasing salt gradient from 750-0 mM ammonium sulfate to separate monomeric conjugated albumin species from non-monomeric albumin species.
5 . The process of claim 1 , wherein the first hydrophobic interaction chromatography is different than the second hydrophobic interaction chromatography.
6 . The process of claim 1 , further comprising the step of further purifying the conjugate by ultrafiltration.
7 . The process of claim 1 , further comprising the step of further purifying the conjugate by a method selected from ion exchange chromatography, affinity chromatography, and size exclusion chromatography.
8 . The process of claim 1 , wherein the conjugate is formed in a solution by contacting albumin contained in the solution with a compound, said compound comprising a reactive group, under reaction conditions wherein the reactive group is capable of covalently binding cysteine 34 thiol of the albumin to form a conjugate.
9 . The process of claim 8 wherein the solution comprises a culture medium of a host organism secreting recombinant albumin therein.
10 . The process of claim 9 , wherein the culture medium is separated from the host organism prior to contacting the albumin with the compound.
11 . The process of claim 8 , wherein the solution is a lysate of a host organism producing recombinant albumin.
12 . The process of claim 8 , wherein the solution comprises recombinant albumin purified by hydrophobic interaction chromatography.
13 . The process of claim 8 , wherein the albumin is mercaptalbumin-enriched albumin.
14 . The process of claim 13 , wherein mercaptalbumin is enriched by contacting the albumin with thioglycolic acid.
15 . The process of claim 13 , wherein mercaptalbumin is enriched by contacting the albumin with dithiothreitol.
16 . The process of claim 8 , wherein the albumin is deglycated albumin.
17 . The process of claim 8 , wherein the albumin is deglycated albumin enriched for mercaptalbumin.
18 . The process of claim 16 or 17 , wherein the albumin is deglycated by aminophenylboronic acid agarose affinity chromatography.
19 . The process of claim 16 or 17 , wherein the albumin is deglycated by concanavalin A sepharose affinity chromatography.
20 . The process of claim 8 , wherein said reaction conditions comprise a reaction temperature of 20° to 25° C.
21 . The process of claim 8 , wherein said reaction conditions comprise a reaction time of at least 30 minutes.
22 . The process of claim 8 , wherein said reaction conditions comprise a final molar ratio of the compound to recombinant albumin of 0.1:1 to 1:1.
23 . The process of claim 8 , wherein said reaction conditions comprise a final molar ratio of the compound to albumin of 0.5:1 to 0.9:1.
24 . The process of claim 8 , wherein said reaction conditions comprise a final molar ratio of the compound to albumin of 0.7:1.
25 . The process of claim 1 , wherein the compound comprises an amino acid, a peptide, a protein, an organic molecule, RNA, or DNA.
26 . The process of claim 1 , wherein the compound is less than 30 kDa.
27 . The process claim 1 , wherein the compound is insulin, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), peptide YY (PYY), growth hormone releasing factor (GRF), glucagon-like peptide-1 (GLP-1), exendin-3, or exendin-4.
28 . The process of claim 1 , wherein the compound is GLP-1.
29 . The process of claim 1 , wherein the compound is exendin-3.
30 . The process of claim 1 , wherein the compound is exendin-4.
31 . The process of claim 1 , wherein the compound comprises a reactive group.
32 . The process of 31, wherein the reactive group is a Michael acceptor, a succinimidyl-containing group, a maleimido-containing group or an electrophilic thiol acceptor.
33 . The process of claim 31 , wherein the reactive group is a maleimido-containing group.
34 . The process of claim 31 , wherein the reactive group is maleimid-propionic acid (MPA).
35 . The process of claim 31 , wherein the reactive group is a cysteine residue.
36 . The process of claim 8 , wherein the albumin is recombinant serum albumin.
37 . The process of claim 8 , wherein the albumin is recombinant human serum albumin.
38 . The process of claim 8 , wherein the albumin is fused to a peptide.
39 . The process of claim 38 , wherein the peptide is glucagon-like peptide 1, exendin 3, or exendin-4.
40 . The process of claim 1 , wherein the conjugate is according to the following:
wherein the protein is albumin and X is S of Cysteine 34.
41 . The process of claim 1 , wherein the conjugate is according to the following:
wherein the protein is albumin and X is S of Cysteine 34.
42 . The process of claim 8 , wherein the albumin is produced by a host organism.
43 . The process of claim 42 , wherein the host is a yeast strain transformed to express recombinant albumin.
44 . The process of claim 43 , wherein the yeast is selected from the group comprising Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces lactis, Arxula adeninivorans , and Hansenula polymorpha.
45 . The process of claim 42 , wherein the host is a bacterium transformed to express recombinant albumin.
46 . The process of claim 45 , wherein the bacterium is Escherichia coli.
47 . The process of any claim 42 , wherein the host is a transgenic plant expressing recombinant albumin.
48 . The process of claim 42 , wherein the host is a transgenic animal expressing recombinant albumin.
49 . The process of claim 8 , wherein the recombinant albumin is produced by a mammalian cell transformed with a vector encoding albumin, or a variant or derivative thereof.
50 . A process for the preparation of a conjugate, the conjugate comprising recombinant albumin and a compound having less than 30 kDa that is selected from the group consisting of an amino acid, a peptide, a protein, an organic molecule, RNA and DNA, where the compound is modified by coupling a reactive group thereto and the conjugate is formed by the reaction of the modified compound and recombinant albumin, the process comprising the steps of:
a. producing recombinant albumin by culturing a host organism in a culture media, such that the recombinant albumin is secreted in the culture media;
b. concurrently with step (a), adding the modified compound to the culture media and allowing the modified compound to react with recombinant albumin secreted in the culture media; and
c. purifying the conjugate resulting from the reaction of step (b).
51 . A process for the preparation of a conjugate, the conjugate comprising recombinant albumin and a compound having less than 30 kDA that is selected from the group consisting of an amino acid, a peptide, a protein, an organic molecule, RNA, and DNA, where the compound is modified by coupling a reactive group thereto and the conjugate is formed by the reaction of the modified compound and recombinant albumin, the process comprising the steps of:
a. producing recombinant albumin by culturing a host organism in a culture media, such that the recombinant albumin is secreted in the culture media;
b. collecting the culture media containing the recombinant albumin;
c. adding the modified compound to the collected culture media obtained at step (b) and allowing the modified compound to react with recombinant albumin; and
d. purifying the conjugate resulting from the reaction of step (b).
52 . A process for the preparation of a conjugate, the conjugate comprising recombinant albumin and a compound having less than 30 kDA that is selected from the group consisting of an amino acid, a peptide, a protein, an organic molecule, RNA, and DNA, where the compound is modified by coupling a reactive group thereto and the conjugate is formed by the reaction of the modified compound and recombinant albumin, the process comprising the steps of:
a. producing recombinant albumin by culturing a host organism in a culture media, such that the recombinant albumin is secreted in the culture media;
b. purifying the secreted recombinant albumin;
c. adding the modified compound recombinant albumin purified at step (b) and allowing the modified compound to react with recombinant albumin; and
d. purifying the conjugate resulting from the reaction of step (c).
53 . A process according to claim 52 , wherein the purified recombinant albumin of step (b) comprises capped albumin and mercaptalbumin, and the process further comprises a step of enrichment of mercaptalbumin prior to the reaction with the modified compound of step (c).
54 . A process according to claim 50 , 51 , or 52 , wherein the host organism is a yeast.
55 . A process for the preparation of a conjugate, the conjugate comprising recombinant albumin and a compound having less than 30 kDA, that is selected from the group consisting of an amino acid, a protein, an organic molecule, RNA, and DNA, where the compound is modified by coupling a reactive group thereto and the conjugate is formed by the reaction of the modified compound and recombinant albumin, the process comprising the steps of:
a. producing recombinant albumin by culturing a host organism in a culture media, such that recombinant albumin is stored intracellularly;
b. physically separating recombinant albumin from the cell of the host organism;
c. adding the modified compound to the recombinant albumin obtained in step (b) and allowing the modified compound to react with recombinant albumin; and
d. purifying the conjugate resulting from the reaction of step (b).
56 . A process according to claim 50 , having an additional step (b-1) of purification of the recombinant albumin obtained in step (b) prior to its reaction with the modified compound of step (c).
57 . A process according to claim 56 , wherein the recombinant albumin obtained by the purification step (b-1) comprises capped albumin and mercaptalbumin, and the process further comprises a step (b-2) of enrichment of mercaptalbumin, and the process further comprises a step (b-2) of enrichment of mercaptalbumin prior to the reaction with the modified compound of step (c).
58 . A process according to claim 55 , 56 , or 57 wherein the host organism is a bacteria.