U.S. patent application number 11/981474 was filed with the patent office on 2008-06-19 for method for the purification of albumin conjugates.
This patent application is currently assigned to ConjuChem Biotechnologies Inc.. Invention is credited to Nathalie Bousquet-Gagnon, Dominique P. Bridon, Omar Quraishi.
Application Number | 20080146783 11/981474 |
Document ID | / |
Family ID | 35196920 |
Filed Date | 2008-06-19 |
United States Patent
Application |
20080146783 |
Kind Code |
A1 |
Bousquet-Gagnon; Nathalie ;
et al. |
June 19, 2008 |
Method for the purification of albumin conjugates
Abstract
The present invention relates to a method for separating albumin
conjugate from unconjugated albumin in a solution comprising
albumin conjugate and unconjugated albumin by loading the solution
onto a hydrophobic support equilibrated in aqueous buffer having a
high salt content; applying to the support a gradient of decreasing
salt concentration; and collecting the eluted albumin
conjugate.
Inventors: |
Bousquet-Gagnon; Nathalie;
(St-Jerome, CA) ; Quraishi; Omar; (Hudson, CA)
; Bridon; Dominique P.; (San Francisco, CA) |
Correspondence
Address: |
JONES DAY
222 EAST 41ST ST
NEW YORK
NY
10017
US
|
Assignee: |
ConjuChem Biotechnologies
Inc.
|
Family ID: |
35196920 |
Appl. No.: |
11/981474 |
Filed: |
October 30, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11112277 |
Apr 22, 2005 |
7307148 |
|
|
11981474 |
|
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|
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60565228 |
Apr 23, 2004 |
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Current U.S.
Class: |
530/364 ;
530/362 |
Current CPC
Class: |
B01D 15/426 20130101;
C07K 14/765 20130101; B01D 15/166 20130101; B01D 15/327 20130101;
A61K 47/643 20170801; C07K 1/20 20130101; C07K 14/76 20130101; C07K
1/303 20130101 |
Class at
Publication: |
530/364 ;
530/362 |
International
Class: |
C07K 1/16 20060101
C07K001/16 |
Claims
1-22. (canceled)
23. A method for separating albumin conjugate from unconjugated
albumin in a solution comprising albumin conjugate and unconjugated
albumin, the method comprising: contacting said solution with a
hydrophobic interaction chromatography matrix under conditions
wherein the albumin conjugate binds to said matrix and the
unconjugated albumin does not bind to said matrix, wherein the
albumin conjugate comprises a peptide.
24. The method of claim 23, wherein said hydrophobic interaction
chromatography matrix is a column containing a hydrophobic
resin.
25. The method of claim 24, wherein said hydrophobic resin is a
bead-formed agarose-based gel filtration matrix covalently coupled
to a ligand selected from the group consisting of an octyl group, a
phenyl group and a butyl group.
26. The method of claim 25, wherein said hydrophobic resin is a
bead-formed agarose-based gel filtration matrix covalently coupled
to a butyl group.
27. The method of claim 23, wherein the hydrophobic interaction
chromatography matrix is, prior to contact with said solution,
equilibrated in aqueous buffer at a salt concentration high enough
to promote matrix-protein interactions.
28. The method of claim 27, wherein the said salt concentration is
between 500 and 3000 mM.
29. The method of claim 27, wherein said salt is selected from the
group consisting of ammonium phosphate, ammonium sulfate, magnesium
phosphate.
30. The method of claim 27, wherein said salt is ammonium
sulfate.
31. The method of claim 27, wherein the pH of said aqueous buffer
is between 3.0 and 9.0.
32. The method of claim 27, wherein the pH of said aqueous buffer
is 7.0.
33. The method of claim 27, wherein said aqueous buffer and said
hydrophobic interaction chromatography matrix are at a temperature
of between 4.degree. C. and about 25.degree. C.
34. The method of claim 27, further comprising applying a gradient
of decreasing salt concentration to said hydrophobic interaction
chromatography matrix following contact with said solution.
35. The method of claim 34, further comprising collecting the
eluted albumin conjugate.
36. The method of claim 23, wherein said albumin conjugate consists
of a peptide comprising a Michael acceptor covalently bonded to
albumin.
37. The method of claim 36, wherein said bond is between said
Michael acceptor and cysteine 34 of said albumin.
38. The method of claim 37, wherein said Michael acceptor is
maleimide-propionic acid.
39. The method of claim 36, wherein said peptide is covalently
bonded to said Michael acceptor, optionally through a linker.
40. The method of claim 39, wherein said peptide is covalently
bonded to said Michael acceptor through a linker selected from the
group consisting of (2-amino)ethoxy acetic acid (AEA),
ethylenediamine (EDA), 2-[2-(2-amino)ethoxy]ethoxy acetic acid
(AEEA), amino ethoxy ethyl amino succinic acid (AEEAS), glycine,
3-aminopropionic acid (APA), 8-aminooctanoic acid (AOA), octanoic
acid (OA), and 4-aminobenzoic acid (APhA).
41. The method of claim 39, wherein said peptide is selected from
the group consisting of glucagon like peptide 1 (GLP-1), glucagon
like peptide 2 (GLP-2), atrial natriuretic peptide (ANP), kringle 5
(K5), dynorphin, exendin-4, growth hormone releasing factor (GRF),
insulin, natriuretic peptides, enfuvirtide (T-20), T-1249, C-34,
soluble C-35 peptide EK (SC-35), peptide YY (PYY), and analogs
thereof.
42. The method of claim 39, wherein said peptide is GLP-1 (7-36)
dAla.sup.8 Lys.sup.37-CONH.sub.2.
43. The method of claim 39, wherein said peptide is Exendin-4
(1-39) Lys.sup.40-CONH.sub.2.
44. The method of claim 36, wherein said peptide comprising a
Michael acceptor is selected from the group consisting of GLP-1
(7-36) dAla.sup.8 Lys.sup.37 (.epsilon.-AEEA-MPA)-CONH.sub.2 (SEQ
ID NO:1), GRF (1-29) dAla.sup.2 Gln.sup.8 Ala.sup.15 Leu.sup.27
Lys.sup.30 (.epsilon.-MPA) CONH.sub.2 (SEQ ID NO:2), Ac-K5
Lys.sup.8 (.epsilon.-MPA)-NH.sub.2 (SEQ ID NO:3), Insulin B1-MPA
(SEQ ID NO:4), Insulin A1-MPA (SEQ ID NO:5),
MPA-AEEA-C34-CONH.sub.2 (SEQ ID NO:6), C34 (1-34) Lys.sup.35
(.epsilon.-AEEA-MPA)-CONH.sub.2 (SEQ ID NO:7), C34 (1-34)
Lys.sup.13 (.epsilon.-AEEA-MPA)-CONH.sub.2 (SEQ ID NO:8), GLP-1
(7-36) Lys.sup.37 (.epsilon.-MPA)-NH.sub.2 (SEQ ID NO:9), GLP-1
(7-36) dAla.sup.8 Lys.sup.37 (.epsilon.-MPA)-NH.sub.2 (SEQ ID
NO:10), GLP-1 (7-36) Lys.sup.26 (.epsilon.-AEEA-AEEA-MPA) (SEQ ID
NO:11), GLP-1 (7-36) Lys.sup.34 (.epsilon.-AEEA-AEEA-MPA) (SEQ ID
NO:12), Exendin-4-(1-39) Lys.sup.40 (.epsilon.-MPA)-NH.sub.2 (SEQ
ID NO:13), Exendin-4 (9-39) Lys.sup.40
(.epsilon.-AEEA-MPA)-CONH.sub.2 (SEQ ID NO:14), Dyn A (1-13)
(MPA)-NH.sub.2 (SEQ ID NO:15), MPA-AEEA-ANP (99-126)-CONH.sub.2
(SEQ ID NO:16), Dyn A (7-13) Lys.sup.13 (.epsilon.-MPA)-CONH.sub.2
(SEQ ID NO:17), acetyl-Phe-His-cyclohexylstatyl-Ile-Lys
(.epsilon.-AEEA-MPA)-CONH.sub.2 (SEQ ID NO:18), GLP-1 (7-36)
Lys.sup.23 (.epsilon.-AEEA-MPA)-CONH.sub.2 (SEQ ID NO:19), GLP-1
(7-36) Lys.sup.18 (.epsilon.-AEEA-MPA)-CONH.sub.2 (SEQ ID NO:20),
GLP-1 (7-36) Lys.sup.26 (.epsilon.-AEEA-MPA)-CONH.sub.2 (SEQ ID
NO:21), GLP-1 (7-37) Lys.sup.7 (.epsilon.-AEEA-MPA)-CONH.sub.2 (SEQ
ID NO:22), GLP-1 (7-36) Lys.sup.37
(.epsilon.-AEEA-AEEA-MPA)-CONH.sub.2 (SEQ ID NO:23), GLP-1 (7-36)
Lys.sup.37 (.epsilon.-AEEA-MPA)-CONH.sub.2 (SEQ ID NO:24),
Exendin-4-(1-39) Lys.sup.40 (.epsilon.-AEEA-MPA)-CONH.sub.2 (SEQ ID
NO:25), GLP-1 (7-36) Lys.sup.34 (.epsilon.-AEEA-MPA)-CONH.sub.2
(SEQ ID NO:26), Insulin B1-OA-MPA (SEQ ID NO:27), Insulin B29-MPA
(SEQ ID NO:28), GRF (1-29) Lys.sup.30 (.epsilon.-MPA)-CONH.sub.2
(SEQ ID NO:29), GRF (1-29) dAla.sup.2 Gln.sup.8 dArg.sup.11
Ala.sup.15 Leu.sup.27 Lys.sup.30 (.epsilon.-MPA)-CONH.sub.2 (SEQ ID
NO:30), GRF (1-29) dAla.sup.2 Lys.sup.30 (.epsilon.-MPA)-CONH.sub.2
(SEQ ID NO:31), GLP-1 (9-36) Lys.sup.37
(.epsilon.-AEEA-MPA)-CONH.sub.2 (SEQ ID NO:32), Ac-T20 (1-36)
Lys.sup.37 (.epsilon.-AEEA-MPA)-CONH.sub.2 (SEQ ID NO:33), Ac-T1249
(1-39) Lys.sup.40 (.epsilon.-AEEA-MPA)-CONH.sub.2 (SEQ ID NO:34),
3',4'-didehydro-4'-deoxy-C'-norvincaleukoblastine-AEEA-MPA, C34
(1-34) Lys.sup.13 (.epsilon.-MPA)-CONH.sub.2 (SEQ ID NO:36), C34
(1-34) Lys.sup.35 (.epsilon.-MPA)-CONH.sub.2 (SEQ ID NO:37),
MPA-C34 (1-34)-CONH.sub.2 (SEQ ID NO:38), Ac-C34 (1-34) Glu.sup.2
Lys.sup.6 Lys.sup.7 Glu.sup.9 Glu.sup.10 Lys.sup.13 Lys.sup.14
Glu.sup.16 Glu.sup.17 Lys.sup.20 Lys.sup.21 Glu.sup.23 Glu.sup.24
Lys.sup.27 Glu.sup.31 Lys.sup.34 Lys.sup.35 Lys.sup.36
(.epsilon.-AEEA-MPA)-CONH.sub.2 (SEQ ID NO:39), MPA-AEEA-C34 (1-34)
Glu.sup.2 Lys.sup.6 Lys.sup.7 Glu.sup.9 Glu.sup.10 Lys.sup.13
Lys.sup.14 Glu.sup.16 Glu.sup.17 Lys.sup.20 Lys.sup.21 Glu.sup.23
Glu.sup.24 Lys.sup.27 Glu.sup.31 Lys.sup.34 Lys.sup.35 CONH.sub.2
(SEQ ID NO:40), PYY (3-36) Lys.sup.4 (.epsilon.-OA-MPA)-CONH.sub.2
(SEQ ID NO:41), MPA-OA-PYY (3-36)-CONH.sub.2 (SEQ ID NO:42),
Insulin B29-AEES2-MPA (SEQ ID NO:43), Insulin B1-AEES2-MPA (SEQ ID
NO:44), Insulin B29-OA-MPA (SEQ ID NO:45), MPA-PYY
(3-36)-CONH.sub.2 (SEQ ID NO:46), PYY (3-36) Lys.sup.37
(.epsilon.-MPA)-CONH.sub.2 (SEQ ID NO:47), MPA-PYY
(22-36)-CONH.sub.2 (SEQ ID NO:48), Acetyl-PYY (22-36) Lys.sup.37
(.epsilon.-MPA)-CONH.sub.2 (SEQ ID NO:49), MPA-ANP
(99-126)-CONH.sub.2 (SEQ ID NO:50), MPA-EEEEP-ANP (99-126) (SEQ ID
NO:51), and GLP-2 (1-33) Gly.sup.2 Lys.sup.34
(.epsilon.-MPA)-CONH.sub.2 (SEQ ID NO:52).
45. The method of claim 23, wherein said albumin is selected from
the group consisting of serum albumin and recombinant albumin.
46. The method of claim 23, wherein said albumin is human serum
albumin.
47. A hydrophobic interaction chromatography matrix to which an
albumin conjugate is bound, wherein said albumin conjugate
comprises a peptide.
Description
RELATED APPLICATION
[0001] This application claims priority to U.S. provisional patent
application Ser. No. 60/565,228 filed Apr. 23, 2004, which is
expressly incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] (a) Field of the Invention
[0003] This invention relates to a method of purification for
isolating albumin conjugates from a solution comprising both
albumin conjugates and unconjugated albumin.
[0004] (b) Description of Prior Art
[0005] WO 95/10302 and WO 99/24074 describe the formation of
conjugates of albumin wherein the molecule of interest has a
reactive functionality coupled thereto that is adapted to
covalently bond to albumin, thus forming a conjugate. These
conjugates can be formed in vivo, but they can be formed in vitro
as well. The formation of the conjugate in vitro involves the
addition of a molecule coupled to a reactive functionality to a
solution of albumin. The primary end products from this reaction
are unconjugated albumin, the albumin conjugate and the unreacted
molecule coupled to the reactive functionality.
[0006] It would be highly desirable to be provided with a method
for purifying albumin conjugate from a solution comprising albumin
conjugate and unconjugated albumin.
SUMMARY OF THE INVENTION
[0007] In accordance with the present invention there is provided a
method for separating albumin conjugate from unconjugated albumin
in a solution comprising albumin conjugate and unconjugated
albumin, the method comprising:
a) loading the solution onto a hydrophobic solid support
equilibrated in aqueous buffer having a high salt content; b)
applying to the support a gradient of decreasing salt content; and
c) collecting eluted albumin conjugate.
[0008] In a preferred embodiment of the present invention, the
albumin conjugate consists of a molecule having a Michael acceptor
covalently coupled thereto which bonds to albumin, and more
preferably the bond is between the Michael acceptor and cysteine 34
of albumin.
[0009] In a more preferred embodiment of the present invention, the
Michael acceptor is a maleimide group, and more preferably, the
maleimide group is maleimide-propionic acid (MPA). The Michael
acceptor is optionally coupled to the molecule via a linker. The
linker is preferably selected in the group consisting of
hydroxyethyl motifs such as (2-amino) ethoxy acetic acid (AEA),
ethylenediamine (EDA), 2-[2-(2-amino)ethoxy)]ethoxy acetic acid
(AEEA), amino ethoxy ethyl amino succinic acid (AEEAS); one or more
alkyl chains (C1-C10) motifs such as glycine, 3-aminopropionic acid
(APA), 8-aminooctanoic acid (AOA), octanoic acid (OA),
4-aminobenzoic acid (APhA). Preferred linkers are OA, ADE, AEA,
AEEA and AEEAS. A combination of two linkers can also be used such
as, for examples, AEEA-EDA, AEEA-AEEA, AEEAS-AEEAS, and
AEA-AEEA.
[0010] In a preferred embodiment of the present invention, the
albumin is selected from the group consisting of serum albumin,
recombinant albumin and albumin from a genomic source.
[0011] In a preferred embodiment of the present invention, the
albumin is selected from the group consisting of human albumin, rat
albumin, mouse albumin, swine albumin, bovine albumin, dog albumin
and rabbit albumin, more preferable human serum albumin.
[0012] In a preferred embodiment, albumin is modified with at least
one selected from the group consisting of fatty acids, metal ions,
small molecules having high affinity to albumin, and sugars, such
as, but not limited to, glucose, lactose and mannose.
[0013] In a preferred embodiment of the present invention, the
molecule is selected from the group consisting of a peptide, DNA,
RNA, small organic molecule and a combination thereof. The peptide
has preferentially a molecular weight of at least 57 daltons. The
peptide is intended to include, but not being limited to, GLP-1,
GLP-2, ANP, K5, dynorphin, GRF, insulin, natriuretic peptides,
T-20, T-1249, C-34 and PYY. The small molecule is intended to
include, but not being limited to, vinorelbine, gemcitabine and
paclitaxel. In a more preferred embodiment of the present
invention, when the molecule is a DNA, RNA or a small organic
molecule, it is covalently attached to the albumin through an acid
sensitive covalent bond or a peptide sequence susceptible to
proteolytic cleavage, thereby allowing the separation of the
molecule from albumin and the entry of the molecule into a
cell.
[0014] In a preferred embodiment of the present invention, the
hydrophobic solid support is a column containing a hydrophobic
resin such as, but not limited to, octyl sepharose, phenyl
sepharose and butyl sepharose and more preferably butyl
sepharose.
[0015] In another embodiment of the present invention, the
hydrophobic solid support comprising a hydrophobic ligand such as
Cibacron Blue F3G-A, ether or isopropyl groups in association with
a support such as polystyrene/divinyl benzene matrix.
[0016] Substances are separated on the basis of their varying
strengths of hydrophobic interactions with hydrophobic ligands
immobilized to an uncharged matrix. This technique is usually
performed with moderately high concentrations of salts
(.apprxeq.1M) in the start buffer (salt promoted adsorption).
Elution is achieved by a linear or stepwise decrease in salt
concentration.
[0017] The type of ligand, the degree of substitution, the pH and
the type and concentration of salt used during the adsorption stage
have a profound effect on the overall performance (e.g. selectivity
and capacity) of a HIC matrix (Hydrophobic Interaction
Chromatography matrix).
[0018] The solvent is one of the most important parameters which
influence capacity and selectivity in HIC (Hydrophobic Interaction
Chromatography). In general, the adsorption process is more
selective than the desorption process. It is therefore important to
optimize the start buffer with respect to pH, type of solvent, type
of salt and concentration of salt. The addition of various
"salting-out" salts to the sample promotes ligand-protein
interactions in HIC. As the concentration of salt is increased, the
amount of bound protein increases up to the precipitation point for
the protein. Each type of salt differs in its ability to promote
hydrophobic interactions. The influence of different salts on
hydrophobic interaction follows the well-known Hofmeisters series
found below:
Hofmeisters Series
Salting-Out Effect
Anions:
[0019]
PO.sub.4.sup.3->SO.sub.4.sup.2->CH.sub.3COO.sup.->Cl.sup.--
>Br.sup.->NO.sub.3.sup.->ClO.sub.4.sup.->I.sup.->SCN.sup.-
Chaotropic Effect
Cations:
NH.sub.4.sup.+<Rb.sup.+<K.sup.+<Na.sup.+<Cs.sup.+<Li.sup.+&-
lt;Mg.sup.2+<Ba.sup.2+
[0020] Increasing the salting-out effect strengthens the
hydrophobic interactions, whereas increasing the chaotropic effect
weakens them. Therefore, ammonium sulfate exhibits a stronger
salting-out effect than sodium chloride. The most commonly used
salts for HIC are ammonium sulfate ((NH.sub.4).sub.2SO.sub.4),
sodium sulfate ((Na).sub.2SO.sub.4)), magnesium sulfate
(MgSO.sub.4), sodium chloride (NaCl), potassium chloride (KCl), and
ammonium acetate (CH.sub.3COONH.sub.4).
[0021] Protein binding to HIC adsorbents is promoted by moderate to
high concentrations of "salting-out" salts, most of which also have
a stabilizing influence on protein structure due to their
preferential exclusion from native globular proteins, i.e. the
interaction between the salt and the protein surface is
thermodynamically unfavorable. The salt concentration should be
high enough (e.g. 500-1000 mM) to promote ligand-protein
interactions yet below that which causes precipitation of the
protein in the sample. In the case of albumin, the salt
concentration should be kept below 3M (moles per liter). The
principle mechanism of salting-out consists of the salt-induced
increase of the surface tension of water (Melander and Horvath,
1977). Thus, a compact structure becomes energetically more
favorable because it corresponds to smaller protein-solution
interfacial area.
[0022] Interestingly, we found that under the same conditions (i.e.
buffer composed of SO.sub.4.sup.2-, PO.sub.4.sup.2- or
CH.sub.3COO.sup.- with any counter ion), these salts exhibit their
salting-out effect upon essentially all conjugated albumin
described herein in a manner different to non-conjugated albumin
(i.e. mercaptalbumin and albumin capped with cysteine), thus
enabling a consistent chromatographic separation between conjugated
albumin versus non-conjugated albumin. That is, we observe that
lower concentrations of salt are required to promote interactions
between ligand and conjugated albumin than between ligand and
non-conjugated albumin. This chromatographic separation is
essentially independent of (a) the sequence of albumin (e.g. human,
mouse, rat, etc.) (b) the source of albumin (i.e. plasma derived or
recombinant) (c) the molecular weight of the conjugated molecule,
(d) the position of the Michael acceptor (or maleimide group)
within the structure of the molecule, (e) the peptide sequence or
chemical structure of the molecule, and (f) the three-dimensional
structure of the conjugated molecule, e.g. linear versus loop
structure.
[0023] In a preferred embodiment of the present invention, the salt
of the aqueous buffer has a sufficient salting out effect. For
providing a sufficient salting out effect, the salt is preferably,
but not limited to, phosphate, sulfate and acetate. More
preferably, the salt is phosphate or sulfate. The selection of the
cation of the buffer is less critical and therefore, such cation
can be selected, without limitation, from the group consisting of
NH.sub.4.sup.+, Rb.sup.+, K.sup.+, Na.sup.+, Cs.sup.+, Li.sup.+,
Mg.sup.2+ and Ba.sup.2+.
[0024] The aqueous buffer is preferably ammonium phosphate,
ammonium sulfate and magnesium phosphate, and more preferably
ammonium sulfate.
[0025] In a preferred embodiment of the present invention, the
buffer pH is between 3.0 and 9.0; more preferably between 6.0 and
8.0, and even more preferably, the pH is 7.0.
[0026] In a preferred embodiment of the present invention, the
buffer and the hydrophobic solid support are at room temperature
(about 25.degree. C.) or at 4.degree. C. or in between.
[0027] Table 1 shows an example of the effect of varying salts for
purification of preformed HSA:first GLP-1 analogue conjugate from a
solution of HSA using butyl-sepharose resin (structure of the first
GLP-1 analogue is described in Example 1 below).
TABLE-US-00001 TABLE 1 Starting salt Starting salt concentration of
concentration of Salt type 750 mM 1,750 mM Ammonium phosphate Yes
yes Ammonium sulfate Yes yes Ammonium chloride No no Ammonium
iodide No no Ammonium thiocyanate No no Magnesium sulfate No yes
Magnesium phosphate* -- -- Barium sulfate* -- -- *means that the
salt is not soluble at concentrations of 1750 mM or 750 mM in 20 mM
sodium phosphate (pH 7), 5 mM caprylate Yes means that successful
resolution is achieved between the HSA:first GLP-1 analogue
conjugate and the non-conjugated HSA No means that no separation is
achieved between the HSA:first GLP-1 analogue conjugate and the
non-conjugated HSA
[0028] The term "peptide" is intended to mean an amino acid
sequence having a molecular weight of at least 57 daltons. The
peptidic sequence can be circular (loop structure) such as ANP, may
contain more than one amino acid chain such as insulin or may be
linear such as K5, dynorphin A, C-34 and GLP-1.
[0029] All references herein are hereby incorporated by
reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 illustrates the purification of the conjugate
HSA:first GLP-1 analogue (SEQ ID NO:1) by a preferred embodiment of
the method of the present invention;
[0031] FIG. 2 illustrates the purification of the conjugate
HSA:first GRF analogue (SEQ ID NO:2) by a preferred embodiment of
the method of the present invention;
[0032] FIG. 3 illustrates the purification of non-conjugated HSA by
a preferred embodiment of the method of the present invention;
[0033] FIG. 4 illustrates the purification of the conjugate
rHSA:first GLP-1 analogue (SEQ ID NO:1) by a preferred embodiment
of the method of the present invention;
[0034] FIG. 5 illustrates the purification of HSA cortex by a
preferred embodiment of the method of the present invention;
[0035] FIG. 6 illustrates the purification of the conjugate HSA:K5
analogue (SEQ ID NO:3) by a preferred embodiment of the method of
the present invention;
[0036] FIG. 7 illustrates the purification of the conjugate
HSA:first insulin derivative (SEQ ID NO:4) by a preferred
embodiment of the method of the present invention;
[0037] FIG. 8 illustrates the purification of the conjugate
HSA:second insulin derivative (SEQ ID NO:5) by a preferred
embodiment of the method of the present invention;
[0038] FIG. 9 illustrates the purification of the conjugate
HSA:first C34 analogue (SEQ ID NO:6) by a preferred embodiment of
the method of the present invention;
[0039] FIG. 10 illustrates the purification of the conjugate
HSA:second C34 analogue (SEQ ID NO:7) by a preferred embodiment of
the method of the present invention;
[0040] FIG. 11 illustrates the purification of the conjugate
HSA:third C34 analogue (SEQ ID NO:8) by a preferred embodiment of
the method of the present invention;
[0041] FIG. 12 illustrates the purification of L-cysteine by a
preferred embodiment of the method of the present invention;
[0042] FIG. 13 illustrates the purification of L-cysteine:first
GLP-1 analogue (SEQ ID NO:1) by a preferred embodiment of the
method of the present invention;
[0043] FIG. 14 illustrates the purification of the conjugate
HSA:second GLP-1 analogue (SEQ ID NO:9) by a preferred embodiment
of the method of the present invention;
[0044] FIG. 15 illustrates the purification of the conjugate
HSA:third GLP-1 analogue (SEQ ID NO:10) by a preferred embodiment
of the method of the present invention;
[0045] FIG. 16 illustrates the purification of the conjugate
HSA:fourth GLP-1 analogue (SEQ ID NO:11) by a preferred embodiment
of the method of the present invention;
[0046] FIG. 17 illustrates the purification of the conjugate
HSA:fifth GLP-1 analogue (SEQ ID NO:12) by a preferred embodiment
of the method of the present invention;
[0047] FIG. 18 illustrates the purification of the conjugate
HSA:first Exendin-4 analogue (SEQ ID NO:13) by a preferred
embodiment of the method of the present invention;
[0048] FIG. 19 illustrates the purification of the conjugate
HSA:second Exendin-4 analogue (SEQ ID NO:14) by a preferred
embodiment of the method of the present invention;
[0049] FIG. 20 illustrates the purification of HSA:MPA by a
preferred embodiment of the method of the present invention;
[0050] FIG. 21 illustrates the purification of HSA by a preferred
embodiment of the method of the present invention;
[0051] FIG. 22 illustrates the purification of the conjugate
HSA:second C34 analogue (SEQ ID NO:3) by a preferred embodiment of
the method of the present invention;
[0052] FIG. 23 illustrates the purification of the conjugate
HSA:first Dynorphin A analogue (SEQ ID NO:15) by a preferred
embodiment of the method of the present invention;
[0053] FIG. 24 illustrates the purification of the conjugate
HSA:first ANP analogue (SEQ ID NO:16) by a preferred embodiment of
the method of the present invention;
[0054] FIG. 25 illustrates the purification of the conjugate
HSA:second Dynorphin A analogue (SEQ ID NO:17) by a preferred
embodiment of the method of the present invention;
[0055] FIG. 26 illustrates the purification of the conjugate
HSA:ACE inhibitor (SEQ ID NO:18) by a preferred embodiment of the
method of the present invention;
[0056] FIG. 27 illustrates the purification of the conjugate
HSA:sixth GLP-1 analogue (SEQ ID NO:19) by a preferred embodiment
of the method of the present invention;
[0057] FIG. 28 illustrates the purification of the conjugate
HSA:seventh GLP-1 analogue (SEQ ID NO:20) by a preferred embodiment
of the method of the present invention;
[0058] FIG. 29 illustrates the purification of the conjugate
HSA:eighth GLP-1 analogue (SEQ ID NO:21) by a preferred embodiment
of the method of the present invention;
[0059] FIG. 30 illustrates the purification of the conjugate
HSA:ninth GLP-1 analogue (SEQ ID NO:22) by a preferred embodiment
of the method of the present invention;
[0060] FIG. 31 illustrates the purification of the conjugate
HSA:tenth GLP-1 analogue (SEQ ID NO:23) by a preferred embodiment
of the method of the present invention;
[0061] FIG. 32 illustrates the purification of the conjugate
HSA:eleventh GLP-1 analogue (SEQ ID NO:24) by a preferred
embodiment of the method of the present invention;
[0062] FIG. 33 illustrates the purification of the conjugate
HSA:third Exendin-4 analogue (SEQ ID NO:25) by a preferred
embodiment of the method of the present invention;
[0063] FIG. 34 illustrates the purification of the conjugate
HSA:twelfth GLP-1 analogue (SEQ ID NO:26) by a preferred embodiment
of the method of the present invention;
[0064] FIG. 35 illustrates the purification of the conjugate
HSA:first insulin derivative (SEQ ID NO:4) by a preferred
embodiment of the method of the present invention;
[0065] FIG. 36 illustrates the purification of the conjugate
HSA:third insulin derivative (SEQ ID NO:27) by a preferred
embodiment of the method of the present invention;
[0066] FIG. 37 illustrates the purification of the conjugate
HSA:second insulin derivative (SEQ ID NO:5) by a preferred
embodiment of the method of the present invention;
[0067] FIG. 38 illustrates the purification of the conjugate
HSA:fourth insulin derivative (SEQ ID NO:28) by a preferred
embodiment of the method of the present invention;
[0068] FIG. 39 illustrates the purification of the conjugate
HSA:first GRF analogue (SEQ ID NO:2) by a preferred embodiment of
the method of the present invention;
[0069] FIG. 40 illustrates the purification of the conjugate
HSA:second GRF analogue (SEQ ID NO:29) by a preferred embodiment of
the method of the present invention;
[0070] FIG. 41 illustrates the purification of the conjugate
HSA:third GRF analogue (SEQ ID NO:30) by a preferred embodiment of
the method of the present invention;
[0071] FIG. 42 illustrates the purification of the conjugate
HSA:fourth GRF analogue (SEQ ID NO:31) by a preferred embodiment of
the method of the present invention;
[0072] FIG. 43 illustrates the purification of the conjugate
HSA:thirteenth GLP-1 analogue CJC 1365 (SEQ ID NO:32) by a
preferred embodiment of the method of the present invention;
[0073] FIG. 44 illustrates the purification of the conjugate HSA
lactose:first GLP-1 analogue (SEQ ID NO:1) by a preferred
embodiment of the method of the present invention;
[0074] FIG. 45 illustrates the purification of the conjugate
HSA:first T20 analogue (SEQ ID NO:33) by a preferred embodiment of
the method of the present invention;
[0075] FIG. 46 illustrates the purification of the conjugate
HSA:first T1249 analogue (SEQ ID NO:34) by a preferred embodiment
of the method of the present invention;
[0076] FIG. 47 illustrates the purification of the compound
HSA:first GLP-1 analogue (SEQ ID NO:1) by a preferred embodiment of
the method of the present invention;
[0077] FIG. 48 illustrates the purification of the compound
HSA:first C34 analogue (SEQ ID NO:6) by a preferred embodiment of
the method of the present invention;
[0078] FIG. 49 illustrates the purification of the compound
HSA:second GRF analogue (SEQ ID NO:29) by a preferred embodiment of
the method of the present invention;
[0079] FIG. 50 illustrates the purification of the conjugate
HSA:vinorelbine analogue conjugate (SEQ ID NO:35) by a preferred
embodiment of the method of the present invention;
[0080] FIG. 51 illustrates the purification of L-cysteine by a
preferred embodiment of the method of the present invention;
[0081] FIG. 52 illustrates the purification of the conjugate
L-Cysteine:vinorelbine analogue (SEQ ID NO:35) by a preferred
embodiment of the method of the present invention;
[0082] FIG. 53 illustrates the purification of the conjugate
RSA:third Exendin-4 analogue (SEQ ID NO:25) by a preferred
embodiment of the method of the present invention;
[0083] FIG. 54 illustrates the purification of the conjugate
HSA:fourth C34 analogue (SEQ ID NO:36) by a preferred embodiment of
the method of the present invention;
[0084] FIG. 55 illustrates the purification of the conjugate
HSA:fifth C34 analogue (SEQ ID NO:37) by a preferred embodiment of
the method of the present invention;
[0085] FIG. 56 illustrates the purification of the conjugate
HSA:sixth C34 analogue (SEQ ID NO:38) by a preferred embodiment of
the method of the present invention;
[0086] FIG. 57 illustrates the purification of the conjugate
HSA:seventh C34 analogue (SEQ ID NO:39) by a preferred embodiment
of the method of the present invention;
[0087] FIG. 58 illustrates the purification of the conjugate
HSA:eighth C34 analogue (SEQ ID NO:40) by a preferred embodiment of
the method of the present invention;
[0088] FIG. 59 illustrates the purification of the conjugate
HSA:first PYY analogue (SEQ ID NO:41) by a preferred embodiment of
the method of the present invention;
[0089] FIG. 60 illustrates the purification of the conjugate
HSA:second PYY analogue (SEQ ID NO:42) by a preferred embodiment of
the method of the present invention;
[0090] FIG. 61 illustrates the purification of the conjugate
HSA:fifth insulin derivative (SEQ ID NO:43) by a preferred
embodiment of the method of the present invention;
[0091] FIG. 62 illustrates the purification of the conjugate
HSA:sixth insulin derivative (SEQ ID NO:44) by a preferred
embodiment of the method of the present invention;
[0092] FIG. 63 illustrates the purification of the conjugate
HSA:seventh insulin derivative (SEQ ID NO:45) by a preferred
embodiment of the method of the present invention;
[0093] FIG. 64 illustrates the purification of the conjugate
HSA:third PYY analogue (SEQ ID NO:46) by a preferred embodiment of
the method of the present invention;
[0094] FIG. 65 illustrates the purification of the conjugate
HSA:fourth PYY analogue (SEQ ID NO:47) by a preferred embodiment of
the method of the present invention;
[0095] FIG. 66 illustrates the purification of the conjugate
HSA:fifth PYY analogue (SEQ ID NO:48) by a preferred embodiment of
the method of the present invention;
[0096] FIG. 67 illustrates the purification of the conjugate
HSA:sixth PYY analogue (SEQ ID NO:49) by a preferred embodiment of
the method of the present invention;
[0097] FIG. 68 illustrates the purification of the conjugate
HSA:second ANP analogue (SEQ ID NO:50) by a preferred embodiment of
the method of the present invention;
[0098] FIGS. 69A-B illustrates the purification of the conjugate
HSA:third ANP analogue CJC 1681 (SEQ ID NO:51) by a preferred
embodiment of the method of the present invention;
[0099] FIG. 70 illustrates the purification of the conjugate
HSA:first GLP-1 analogue (SEQ ID NO:1) by a preferred embodiment of
the method of the present invention;
[0100] FIG. 71 illustrates the purification of the conjugate
HSA:first GLP-1 analogue (SEQ ID NO:1) by a preferred embodiment of
the method of the present invention;
[0101] FIG. 72 illustrates the purification of the conjugate
HSA:first GLP-1 analogue (SEQ ID NO:1) by a preferred embodiment of
the method of the present invention;
[0102] FIG. 73 illustrates the purification of the conjugate
HSA:first GLP-1 analogue (SEQ ID NO:1) by a preferred embodiment of
the method of the present invention;
[0103] FIG. 74 illustrates the purification of the conjugate
HSA:first GLP-1 analogue (SEQ ID NO:1) by a preferred embodiment of
the method of the present invention;
[0104] FIG. 75 illustrates the purification of the conjugate
HSA:first GLP-2 analogue (SEQ ID NO:52) by a preferred embodiment
of the method of the present invention; and
[0105] FIG. 76 illustrates the purification of the conjugate
RSA:first GLP-2 analogue (SEQ ID NO:52) by a preferred embodiment
of the method of the present invention.
DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT OF THE INVENTION
[0106] In accordance with the present invention, there is provided
a method for purifying albumin conjugates from a solution
comprising albumin conjugates and unconjugated albumin.
Methods
Preparation of Control (Non-Conjugated) Human Serum Albumin (HSA)
and Preformed Albumin Conjugates
[0107] Each compound with the Michael acceptor was solubilized in
nanopure water (or in DMSO if the compound was difficult to
solubilize) at a concentration of 10 mM, then diluted to 1 mM into
a solution of HSA (25%, 250 mg/ml, Cortex-Biochem, San Leandro,
Calif.). The samples were then incubated at 37.degree. C. for 30
min. Prior to their purification, each conjugate solution was
diluted to 5% 50 mg/ml HSA in 20 mM sodium phosphate buffer (pH 7)
composed of 5 mM sodium octanoate. The initial concentration of
salt used in the elution gradient can be added to the buffer for
diluting the mixed solution. Preferably, the initial concentration
of salt is from about 750 to about 1700 mM
(NH.sub.4).sub.2SO.sub.4.
Procedure for Purification According to a Preferred Embodiment
[0108] Using an KTA purifier (Amersham Biosciences, Uppsala,
Sweden), each conjugate was loaded at a flow rate of 2.5 ml/min
onto a 50 ml column of butyl sepharose 4 fast flow resin (Amershan
Biosciences, Uppsala, Sweden) equilibrated in 20 mM sodium
phosphate buffer (pH 7) composed of 5 mM sodium octanoate and 750
mM to 1.7 M (NH.sub.4).sub.2SO.sub.4. Under these conditions, HSA
conjugates having a molecular weight addition of more than 2 kDa
relative to non-conjugated HSA adsorbed onto the hydrophobic resin
whereas essentially all non-conjugated HSA eluted within the void
volume of the column. For molecular weight additions of less than 2
kDa, a higher initial salt content may be used followed by a
stepwise gradient of decreasing salt. Each conjugate was further
purified from any free unconjugated compound by applying a
continuous or non-continuous decreasing gradient of salt (750 to 0
mM (NH.sub.4).sub.2SO.sub.4) over 4 column volumes. In a preferred
embodiment, each purified conjugate was then desalted and
concentrated by diafiltration, for instance by using Amicon.RTM.
ultra centrifugal (30 kDa) filter devices (Millipore Corporation,
Bedford, Mass.). Finally, for prolonged storage, each conjugate
solution is preferably immersed into liquid nitrogen, and
lyophilized using a Labconco freeze dry system (FreeZone.RTM.4.5),
and stored at -20.degree. C.
Examples of LC/EMS Analysis
[0109] Following purification, 1 .mu.l of each conjugate sample is
preferably injected onto LC/EMS system. The HSA:first GLP-1
analogue (SEQ ID NO:1) conjugate was confirmed by detection of a
species of highest abundance with a total mass of 70 160 Da which
corresponds to the mass of mercaptalbumin (66 448 Da) where
cysteine 34 is in the free thiol form, plus the mass of only one
molecule of the first GLP-1 analogue (3 719.9 Da). The structure of
the first GLP-1 analogue (SEQ ID NO:1) is described in Example 1
below. This is illustrated in Table 2.
TABLE-US-00002 TABLE 2 Molecular Absolute Relative Component Weight
Abundance Abundance A 70160.58 321970 100.00 B 65862.95 70008 21.74
C 64545.45 62888 19.53 D 70320.04 41167 12.79 E 61287.67 16842 5.23
F 60623.81 16522 5.13 G 58090.04 12473 3.87
[0110] The HSA:first GRF analogue (SEQ ID NO:2) conjugate was
confirmed by detection of a species of highest abundance with a
total mass of 70 086 Da which corresponds to the mass of
mercaptalbumin (66 448 Da) where cysteine 34 is in the free thiol
form, plus the mass of only one molecule of the first GRF analogue
(3648.2 Da). The structure of the first GRF analogue (SEQ ID NO:2)
is described in Example 2 below. This is illustrated in Table
3.
TABLE-US-00003 TABLE 3 Molecular Absolute Relative Component Weight
Abundance Abundance A 70086.06 279413 100.00 B 63214.84 53333 19.09
C 62148.17 38582 13.81 D 70247.98 34870 12.48 E 56795.96 10523 3.77
F 62695.49 9813 3.51
[0111] The following examples illustrate several compounds having a
maleimide group as Michael acceptor that have been conjugated to
albumin and purified in accordance with the method of the present
invention.
[0112] The following examples are for the purpose of illustrating
the present invention and not of limiting its scope.
[0113] In the following examples, the gradient numbers refer to the
following gradient details, where CV means a column volume of 50
ml.
[0114] Gradient #1: Linear 750-0 mM (NH.sub.4).sub.2SO.sub.4, over
4 CV, flow rate of 2.5 ml/min.
[0115] Gradient #2: Step gradient 1.75M-1.2M
(NH.sub.4).sub.2SO.sub.4 over 0.5 CV, followed by 1.2M-875 mM
(NH.sub.4).sub.2SO.sub.4 over 5 CV, and finally 875 mM-0 mM
(NH.sub.4).sub.2SO.sub.4 over 0.5 CV flow rate of 2.5 ml/min.
[0116] Gradient #3: Linear 900-0 mM (NH.sub.4).sub.2SO.sub.4 over 4
CV, flow rate of 2.5 ml/min.
[0117] Gradient #4: Step gradient 1.5M-1.1M
(NH.sub.4).sub.2SO.sub.4 over 0.5 CV, followed by 1.1M-375 mM
(NH.sub.4).sub.2SO.sub.4 over 6 CV, and finally 375 mM-0 mM
(NH.sub.4).sub.2SO.sub.4 over 0.5 CV, flow rate of 2.5 ml/min.
[0118] Gradient #5: Linear 750-0 mM (NH.sub.4).sub.2SO.sub.4 over 2
CV, flow rate of 2.5 ml/min.
[0119] Gradient #6: Step gradient 1.75M-0M (NH.sub.4).sub.2SO.sub.4
over 6 CV, flow rate of 2.5 ml/min.
[0120] Gradient #7: Linear 750-0 mM (NH.sub.4).sub.2SO.sub.4 over 6
CV, flow rate of 2.5 ml/min.
Example 1
Purification of HSA:First GLP-1 Analogue (SEQ ID NO:1)
Conjugate
[0121] The first GLP-1 analogue is GLP-1 (7-36) dAla.sup.8
Lys.sup.37 (.epsilon.-AEEA-MPA)-CONH.sub.2 and has the following
sequence:
TABLE-US-00004
H(dA)EGTFTSDVSSYLEGQAAKEFIAWLVKGRK(AEEA-MPA)-CONH.sub.2
[0122] The purification of a conjugate made from reacting 1 ml 25%
250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM first
GLP-1 analogue diluted into 9 ml of buffer made of 20 mM sodium
phosphate buffer pH 7.0, 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4, was performed on a column of Butyl
sepharose using the gradient #1 described above. In FIG. 1 the
purified conjugate fraction elutes during the gradient of
decreasing (NH.sub.4).sub.2SO.sub.4 concentration as fraction B
(F8-F9), whereas non-conjugated albumin elutes within the void
volume of the column (fraction A). The conjugate fraction was
concentrated with Ultrafree.TM. filter 30 kDa and analyzed using
LC-EMS.
Example 2
Purification of HSA:First GRF Analogue (SEQ ID NO:2) Conjugate
[0123] The first GRF analogue is GRF (1-29) dAla.sup.2 Gln.sup.8
Ala.sup.15 Leu.sup.27 Lys.sup.30 (.epsilon.-MPA) CONH.sub.2 and has
the following sequence:
TABLE-US-00005 YaDAIFTQSYRKVLAQLSARKLLQDILSRK(MPA)-CONH.sub.2
[0124] The purification of a conjugate made from reacting 1 ml 25%
250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM first
GRF analogue diluted into 9 ml of 20 mM sodium phosphate buffer (pH
7.0), 5 mM sodium caprylate and 750 mM (NH.sub.4).sub.2SO.sub.4,
was performed on a column of Butyl sepharose using the gradient #1
described above. In FIG. 2 the purified conjugate fraction appears
in fraction B (F6-F7) whereas non-conjugated albumin elutes within
the void volume of the column (fraction A). The conjugate fraction
was concentrated with Ultrafree.TM. filter 30 kDa and analyzed
using LC-EMS.
Example 3
Purification of Non-Conjugated HSA 1 ml
[0125] The purification of 1 ml 25% 250 mg/ml non-conjugated HSA
(Cortex-Biochem, San Leandro, Calif.) diluted into 9 ml of buffer
(pH 7.0) made of 20 mM sodium phosphate buffer, 5 mM sodium
caprylate and 750 mM (NH.sub.4).sub.2SO.sub.4, was performed on a
column of Butyl sepharose using the gradient #1 described above.
Essentially all albumin molecules elute within the void volume and
no protein species is observed at 280 nm during
(NH.sub.4).sub.2SO.sub.4 gradient. FIG. 3 illustrates the
separation curve obtained.
Example 4
Purification of rHSA:First GLP-1 Analogue (SEQ ID NO:1)
Conjugate
[0126] The first GLP-1 analogue is GLP-1 (7-36) dAla.sup.8
Lys.sup.37 (.epsilon.-AEEA-MPA)-CONH.sub.2 and his sequence is
shown in Example 1.
[0127] The purification of a conjugate made from reacting 5 ml 5%
rHSA (recombinant HSA new century culture grade) with 200 .mu.M
first GLP-1 analogue diluted into 5 ml of a buffer made of 20 mM
sodium phosphate buffer, 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using the gradient #1 described above. In FIG. 4 the
purified conjugate fraction appears in fraction B (F7-F8-F9).
Example 5
Purification of HSA 10 ml
[0128] The purification of 10 ml 25% 250 mg/ml HSA (Cortex-Biochem,
San Leandro, Calif.) diluted into 40 ml of a buffer made of 20 mM
sodium phosphate buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using the gradient #1 described above. Essentially all
albumin molecules elute within a void volume and no protein species
is observed at 280 nm during (NH.sub.4).sub.2SO.sub.4 gradient.
FIG. 5 illustrates the separation curve obtained.
Example 6
Purification of HSA:K5 Analogue (SEQ ID NO:3) Conjugate
[0129] The K5 analogue is Ac-K5 Lys.sup.8 (.epsilon.-MPA)-NH.sub.2
and has the following sequence:
##STR00001##
[0130] The purification of a conjugate made from reacting 4 ml 25%
250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM K5
analogue diluted into 16 ml of 20 mM sodium phosphate buffer (pH
7.0), 5 mM sodium caprylate and 750 mM (NH.sub.4).sub.2SO.sub.4 was
performed on a column of Butyl sepharose using the gradient #1
described above. In FIG. 6 the purified conjugate fraction appears
in fraction A with albumin and in fraction B (F6-F7-F8).
Example 7
Purification of HSA:First Insulin Derivative (SEQ ID NO:4)
Conjugate
[0131] The first insulin derivative is human insulin with MPA on
position B1 and is represented in FIG. 1 below.
[0132] The purification of a conjugate made from reacting 1 ml 25%
250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM first
insulin derivative diluted into 9 ml of 20 mM sodium phosphate
buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using the gradient #1 described above. In FIG. 7 the
purified conjugate fraction appears in fraction B (F6-F7-F8).
Example 8
Purification of HSA:Second Insulin Derivative (SEQ ID NO:5)
Conjugate
[0133] The second insulin derivative is human insulin with MPA on
position A1 and is represented in FIG. 1 shown above in Example
7.
[0134] The purification of a conjugate made from reacting 1 ml 25%
250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM
second insulin derivative diluted into 9 ml of 20 mM sodium
phosphate buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 8 the purified
conjugate fraction appears in fraction B (F6-F7-F8).
Example 9
Purification of HSA:First C34 Analogue (SEQ ID NO:6) Conjugate
[0135] The first C34 analogue is MPA-AEEA-C34-CONH.sub.2 and has
the following sequence:
##STR00002##
[0136] The purification of a conjugate made from reacting 5 ml 25%
250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM first
C34 analogue diluted into 20 ml of 20 mM sodium phosphate buffer
(pH 7.0), 5 mM sodium caprylate and 750 mM (NH.sub.4).sub.2SO.sub.4
was performed on a column of Butyl sepharose using gradient #1
described above. In FIG. 9 the purified conjugate fraction appears
in fraction F2.
Example 10
Purification of HSA:Second C34 Analogue (SEQ ID NO:7) Conjugate
[0137] The second C34 analogue is C34 (1-34) Lys.sup.35
(.epsilon.-AEEA-MPA)-CONH.sub.2 and has the following
structure:
##STR00003##
[0138] The purification of a conjugate made from reacting 5 ml 25%
250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM
second C34 analogue diluted into 20 ml of 20 mM sodium phosphate
buffer, 5 mM sodium caprylate and 750 mM (NH.sub.4).sub.2SO.sub.4
was performed on a column of Butyl sepharose using gradient #1
described above. In FIG. 10 the purified conjugate fraction appears
in fraction F2.
Example 11
Purification of HSA:Third C34 Analogue (SEQ ID NO:8) Conjugate
[0139] The third C34 analogue is C34 (1-34) Lys.sup.13
(.epsilon.-AEEA-MPA)-CONH.sub.2 and has the following
structure:
##STR00004##
[0140] The purification of a conjugate made from reacting 5 ml 25%
250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM third
C34 analogue diluted into 20 ml of 20 mM sodium phosphate buffer
(pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4, was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 11 the
purified conjugate fraction appears in fraction F2.
Example 12
Purification of l-Cysteine
[0141] The purification of 121 mg of 1-cysteine in 2 ml of a buffer
made of 20 mM sodium phosphate, 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4, was performed on a column of Butyl
sepharose using the gradient #5 described above. FIG. 12
illustrates the separation curve obtained, where L-cysteine elutes
within the void volume of the column (F3).
Example 13
Purification of L-cysteine:First GLP-1 Analogue (SEQ ID NO:1)
Conjugate
[0142] The first GLP-1 analogue is GLP-1 (7-36) dAla.sup.8
Lys.sup.37 (.epsilon.-AEEA-MPA)-CONH.sub.2 and his sequence is
shown above in Example 1.
[0143] The purification of a conjugate made from reacting 121 mg
L-cysteine with 36.36 mg first GLP-1 analogue diluted into 2 ml of
20 mM sodium phosphate buffer (pH 7.0), 5 mM sodium caprylate and
750 mM (NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #5 described above. FIG. 13 illustrates
the separation curve obtained where the excess L-cysteine elutes in
F3 (column void volume) and the L-Cysteine:first GLP-1 analogue
conjugate elutes in 0 mM (NH.sub.4).sub.2SO.sub.4.
Example 14
Purification of HSA:Second GLP-1 Analogue (SEQ ID NO:9)
Conjugate
[0144] The second GLP-1 analogue is GLP-1 (7-36) Lys.sup.37
(.epsilon.-MPA)-NH.sub.2 and has the following sequence:
TABLE-US-00006 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRK(.epsilon.-MPA)
[0145] The purification of a conjugate made from reacting 2.5 ml
25% 250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM
second GLP-1 analogue diluted into 10 ml of 20 mM sodium phosphate
buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #5 described above. In FIG. 14 the
purified conjugate fraction appears in fraction F2.
Example 15
Purification of HSA:Third GLP-1 Analogue (SEQ ID NO:10)
Conjugate
[0146] The third GLP-1 analogue is GLP-1 (7-36) dAla.sup.8
Lys.sup.37 (.epsilon.-MPA)-NH.sub.2 and has the following
sequence:
TABLE-US-00007
H(dA)EGTFTSDVSSYLEGQAAKEFIAWLVKGRK(MPA)-CONH.sub.2
[0147] The purification of a conjugate made from reacting 2.5 ml
25% 250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM
third GLP-1 analogue diluted into 10 ml of 20 mM sodium phosphate
buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #5 described above. In FIG. 15 the
purified conjugate fraction appears in fraction F2.
Example 16
Purification of HSA:Fourth GLP-1 Analogue (SEQ ID NO:11)
Conjugate
[0148] The fourth GLP-1 analogue is GLP-1 (7-36) Lys.sup.26
(.epsilon.-AEEA-AEEA-MPA) and has the following sequence:
TABLE-US-00008 HAEGTFTSDVSSYLEGQMK(.epsilon.-AEEA-AEEA-MPA)
EFIAWLVKGR
[0149] The purification of a conjugate made from reacting 2.5 ml
25% 250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM
fourth GLP-1 analogue diluted into 10 ml of 20 mM sodium phosphate
buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 16 the
purified conjugate fraction appears in fraction F2.
Example 17
Purification of HSA:Fifth GLP-1 Analogue (SEQ ID NO:12)
Conjugate
[0150] The fifth GLP-1 analogue is GLP-1 (7-36) Lys.sup.34
(.epsilon.-AEEA-AEEA-MPA) and has the following sequence:
TABLE-US-00009
HAEGTFTSDVSSYLEGQAAKEFIAWLVK(.epsilon.-AEEA-AEEA-MPA)GR
[0151] The purification of a conjugate made from reacting 2.5 ml
25% 250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM
fifth GLP-1 analogue diluted into 10 ml of 20 mM sodium phosphate
buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 17 the
purified conjugate fraction appears in fraction F2.
Example 18
Purification of HSA:First Exendin-4 Analogue (SEQ ID NO:13)
Conjugate
[0152] The first exendin-4 analogue is Exendin-4-(1-39) Lys.sup.40
(.epsilon.-MPA)-NH.sub.2 and has the following sequence:
TABLE-US-00010
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSK(.epsilon.-MPA)-
CONH.sub.2
[0153] The purification of a conjugate made from reacting 1 ml 25%
250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM first
Exendin-4 analogue diluted into 9 ml of 20 mM sodium phosphate
buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 18 the
purified conjugate fraction appears in fraction F2.
Example 19
Purification of HSA:Second Exendin-4 Analogue (SEQ ID NO:14)
Conjugate
[0154] The second Exendin-4 analogue is Exendin-4 (9-39) Lys.sup.40
(.epsilon.-AEEA-MPA)-CONH.sub.2 and has the following sequence:
TABLE-US-00011
DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSK(AEEA-MPA)-CONH.sub.2
[0155] The purification of a conjugate made from reacting 3.5 ml
25% HSA cortex with 1 mM second Exendin-4 analogue diluted into
21.5 ml of 20 mM sodium phosphate buffer (pH 7.0), 5 mM sodium
caprylate and 750 mM (NH.sub.4).sub.2SO.sub.4 was performed on a
column of Butyl sepharose using gradient #1 described above. In
FIG. 19 the purified conjugate fraction appears in fraction F2.
Example 20
Purification of HSA:MPA
[0156] The purification of a conjugate made from reacting 1 ml 25%
250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 2 mM MPA
diluted into 9 ml of 20 mM sodium phosphate buffer (pH 7.0), 5 mM
sodium caprylate and 1750 mM (NH.sub.4).sub.2SO.sub.4 was performed
on a column of Butyl sepharose using gradient #2 described above.
In FIG. 20 the fraction of mercaptalbumin is in fraction A (F5) and
capped albumin is in fraction B (F7-F8). The conjugate fraction was
concentrated with Amicon.TM. filter 30 kDa.
Example 21
Purification of HSA
[0157] The purification of 1 ml 25% 250 mg/ml HSA (Cortex-Biochem,
San Leandro, Calif.) diluted into 9 ml of 20 mM sodium phosphate
buffer (pH 7.0), 5 mM sodium caprylate and 1750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using the gradient #2 described above. When using
gradient #2, unlike gradients #1 and #5, both conjugated albumin
and non-conjugated albumin adsorbs onto the hydrophobic resin
during sample loading. FIG. 21 illustrates the separation curve
obtained where F4 and F5 are enriched in mercaptalbumin and F6, F7
and F8 are enriched in capped albumin.
Example 22
Purification of HSA:Second C34 Analogue (SEQ ID NO:3) Conjugate
[0158] The second C34 analogue is C34 (1-34) Lys.sup.35
(.epsilon.-AEEA-MPA)-CONH.sub.2 and his structure is shown in
Example 10.
[0159] The purification of a conjugate made from reacting 1 ml 25%
250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM
second C34 analogue diluted into 9 ml of a buffer made of 20 mM
sodium phosphate buffer (pH 7.0), 5 mM sodium caprylate and 1750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #2 described above. In FIG. 22
mercaptalbumin appears in fraction A (F5) and capped albumin and
the purified conjugated is in fraction B (F7-F8).
Example 23
Purification of HSA:First Dynorphin A Analogue (SEQ ID NO:15)
Conjugate
[0160] The first Dynorphin A analogue is Dyn A (1-13)
(MPA)-NH.sub.2 and has the following sequence:
TABLE-US-00012 YGGFLRRIRPKLK(MPA)-CONH.sub.2.
[0161] The purification of a conjugate made from reacting 1 ml 25%
250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM first
Dynorphin A analogue diluted into 9 ml of 20 mM sodium phosphate
buffer (pH 7.0), 5 mM sodium caprylate and 1750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #2 described above. In FIG. 23 the
purified conjugate fraction appears in fraction A (F11-F12)
Example 24
Purification of HSA:First ANP Analogue (SEQ ID NO:16) Conjugate
[0162] The first ANP analogue is MPA-AEEA-ANP (99-126)-CONH.sub.2
and has the following structure:
##STR00005##
[0163] The purification of a conjugate made from reacting 1 ml 25%
250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM first
ANP analogue diluted into 9 ml of 20 mM sodium phosphate buffer (pH
7.0), 5 mM sodium caprylate and 1750 mM (NH.sub.4).sub.2SO.sub.4
was performed on a column of Butyl sepharose using gradient #2
described above. In FIG. 24 the purified conjugate fraction appears
in fraction A (F14).
Example 25
Purification of HSA:Second Dynorphin A Analogue (SEQ ID NO:17)
Conjugate
[0164] The second Dynorphin A analogue is Dyn A (7-13) Lys.sup.13
(.epsilon.-MPA)-CONH.sub.2 and has the following sequence:
TABLE-US-00013 RIRPKLK(MPA)-CONH.sub.2
[0165] The purification of a conjugate made from reacting 1 ml 25%
250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM
second Dynorphin A analogue diluted into 9 ml of 20 mM sodium
phosphate buffer (pH 7.0), 5 mM sodium caprylate and 1750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #2 described above. In FIG. 25 the
purified conjugate fraction appears in fraction A (F9).
Example 26
Purification of HSA:ACE Inhibitor (SEQ ID NO:18) Conjugate
[0166] The ACE inhibitor used in this example is
acetyl-Phe-His-cyclohexylstatyl-Ile-Lys
(.epsilon.-AEEA-MPA)-CONH.sub.2 and has the following sequence:
##STR00006##
[0167] The purification of a conjugate made from reacting 1 ml 25%
250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM ACE
inhibitor diluted into 9 ml of 20 mM sodium phosphate buffer (pH
7.0), 5 mM sodium caprylate and 1750 mM (NH.sub.4).sub.2SO.sub.4
was performed on a column of Butyl sepharose using gradient #2
described above. In FIG. 26 the purified conjugate fraction appears
in fraction A (F14).
Example 27
Purification of HSA:Sixth GLP-1 Analogue (SEQ ID NO:19)
Conjugate
[0168] The sixth GLP-1 analogue is GLP-1 (7-36) Lys.sup.23
(.epsilon.-AEEA-MPA)-CONH.sub.2 and has the following sequence:
TABLE-US-00014
HAEGTFTSDVSSYLEGK(AEEA-MPA)AAKEFIAWLVKGR-CONH.sub.2
[0169] The purification of a conjugate made from reacting 3 ml 25%
250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM sixth
GLP-1 analogue diluted into 22 ml of 20 mM sodium phosphate buffer
(pH 7.0), 5 mM sodium caprylate and 1750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 27 the
purified conjugate fraction appears in fraction F2.
Example 28
Purification of HSA:Seventh GLP-1 Analogue (SEQ ID NO:20)
Conjugate
[0170] The seventh GLP-1 analogue is GLP-1 (7-36) Lys.sup.18
(.epsilon.-AEEA-MPA)-CONH.sub.2 and has the following sequence:
TABLE-US-00015
HAEGTFTSDVSK(AEEA-MPA)YLEGQAAKEFIAWLVKGR-CONH.sub.2
[0171] The purification of a conjugate made from reacting 3 ml 25%
250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM
seventh GLP-1 analogue diluted into 22 ml of 20 mM sodium phosphate
buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 28 the
purified conjugate fraction appears in fraction F2.
Example 29
Purification of HSA:Eighth GLP-1 Analogue (SEQ ID NO:21)
Conjugate
[0172] The eighth GLP-1 analogue is GLP-1 (7-36) Lys.sup.26
(.epsilon.-AEEA-MPA)-CONH.sub.2 and has the following sequence:
TABLE-US-00016
HAEGTFTSDVSSYLEGQAAK(AEEA-MPA)EFIAWLVKGR-CONH.sub.2
[0173] The purification of a conjugate made from reacting 2.5 ml
25% 250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM
eighth GLP-1 analogue diluted into 22.5 ml of 20 mM sodium
phosphate buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 29 the
purified conjugate fraction appears in fraction F2.
Example 30
Purification of HSA:Ninth GLP-1 Analogue (SEQ ID NO:22)
Conjugate
[0174] The ninth GLP-1 analogue is GLP-1 (7-37) Lys.sup.27
(.epsilon.-AEEA-MPA)-CONH.sub.2 and has the following sequence:
TABLE-US-00017
HAEGTFTSDVSSYLEGQAAKK(AEEA-MPA)FIAWLVKGR-CONH.sub.2
[0175] The purification of a conjugate made from reacting 3 ml 25%
250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM ninth
GLP-1 analogue diluted into 22 ml of 20 mM sodium phosphate buffer
(pH 7.0), 5 mM sodium caprylate and 750 mM (NH.sub.4).sub.2SO.sub.4
was performed on a column of Butyl sepharose using gradient #1
described above. In FIG. 30 the purified conjugate fraction appears
in fraction F2.
Example 31
Purification of HSA:Tenth GLP-1 Analogue (SEQ ID NO:23)
Conjugate
[0176] The tenth GLP-1 analogue is GLP-1 (7-36) Lys.sup.37
(.epsilon.-AEEA-AEEA-MPA)-CONH.sub.2 and has the following
sequence:
TABLE-US-00018 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRK-AEEA-AEEA-MPA-
CONH.sub.2
[0177] The purification of a conjugate made from reacting 2.5 ml
25% 250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM
tenth GLP-1 analogue diluted into 22.5 ml of 20 mM sodium phosphate
buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 31 the
purified conjugate fraction appears in fraction F2.
Example 32
Purification of HSA:Eleventh GLP-1 Analogue (SEQ ID NO:24)
Conjugate
[0178] The eleventh GLP-1 analogue is GLP-1 (7-36) Lys.sup.37
(.epsilon.-AEEA-MPA)-CONH.sub.2 and has the following sequence:
TABLE-US-00019
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRK(AEEA-MPA)-CONH.sub.2
[0179] The purification of a conjugate made from reacting 2.5 ml
25% 250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM
eleventh GLP-1 analogue diluted into 22.5 ml of 20 mM sodium
phosphate buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 32 the
purified conjugate fraction appears in fraction F2.
Example 33
Purification of HSA:Third Exendin-4 Analogue (SEQ ID NO:25)
Conjugate
[0180] The third Exendin-4 analogue is Exendin-4-(1-39) Lys.sup.40
(.epsilon.-AEEA-MPA)-CONH.sub.2 and has the following sequence:
TABLE-US-00020
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSK(.epsilon.-AEEA-
MPA)-CONH.sub.2
[0181] The purification of a conjugate made from reacting 2.5 ml
25% 250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM
third Exendin-4 analogue diluted into 22.5 ml of 20 mM sodium
phosphate buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 33 the
purified conjugate fraction appears in fraction F2.
Example 34
Purification of HSA:Twelfth GLP-1 Analogue (SEQ ID NO:26)
Conjugate
[0182] The twelfth GLP-1 analogue is GLP-1 (7-36) Lys.sup.34
(.epsilon.-AEEA-MPA)-CONH.sub.2 and has the following sequence:
TABLE-US-00021
HAEGTFTSDVSSYLEGQAAKEFIAWLVK(.epsilon.-AEEA-MPA)GR-CONH.sub.2
[0183] The purification of a conjugate made from reacting 2.5 ml
25% 250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM
twelfth GLP-1 analogue diluted into 22.5 ml of 20 mM sodium
phosphate buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 34 the
purified conjugate fraction appears in fraction F2.
Example 35
Purification of HSA:First Insulin Derivative (SEQ ID NO:4)
Conjugate
[0184] The first insulin derivative is human insulin with MPA on
position B1 and his structure is detailed in Example 7.
[0185] The purification of a conjugate made from reacting 2.5 ml
25% 250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM
first insulin derivative diluted into 22.5 ml of 20 mM sodium
phosphate buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 35 the
purified conjugate fraction appears in fraction F2.
Example 36
Purification of HSA:Third Insulin Derivative (SEQ ID NO:27)
Conjugate
[0186] The third insulin derivative is human insulin with OA-MPA on
position B1 and is represented in FIG. 1 shown above in Example
7.
[0187] The purification of a conjugate made from reacting 4 ml 25%
250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM third
insulin derivative diluted into 21 ml of 20 mM sodium phosphate
buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 36 the
purified conjugate fraction appears in fraction F2.
Example 37
Purification of HSA:Second Insulin Derivative (SEQ ID NO:5)
Conjugate
[0188] The second insulin derivative is human insulin with MPA on
position A1 and is represented in FIG. 1 shown above in Example
7.
[0189] The purification of a conjugate made from reacting 3 ml 25%
250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM
second insulin derivative diluted into 22 ml of 20 mM sodium
phosphate buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 37 the
purified conjugate fraction appears in fraction F2.
Example 38
Purification of HSA:Fourth Insulin Derivative (SEQ ID NO:28)
Conjugate
[0190] The fourth insulin derivative is human insulin with MPA on
position B29 and is represented in FIG. 1 shown above in Example
7.
[0191] The purification of a conjugate made from reacting 3 ml 25%
250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM
fourth insulin derivative diluted into 22 ml of 20 mM sodium
phosphate buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 38 the
purified conjugate fraction appears in fraction F2.
Example 39
Purification of HSA:First GRF Analogue (SEQ ID NO:2) Conjugate
[0192] The first GRF analogue is GRF (1-29) dAla.sup.2 Gln.sup.8
Ala.sup.15 Leu.sup.27 Lys.sup.30 (.epsilon.-MPA) CONH.sub.2 and his
sequence is shown in Example 2.
[0193] The purification of a conjugate made from reacting 3.7 ml
25% 250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM
first GRF analogue diluted into 22 ml of 20 mM sodium phosphate
buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 39 the
purified conjugate fraction appears in fraction F2.
Example 40
Purification of HSA:Second GRF Analogue (SEQ ID NO:29)
Conjugate
[0194] The second GRF analogue is GRF(1-29) Lys.sup.30
(.epsilon.-MPA)-CONH.sub.2 and has the following sequence:
TABLE-US-00022 YADAIFTNSYRKVLGQLSARKLLQDIMSRK(MPA)-CONH.sub.2
[0195] The purification of a conjugate made from reacting 2.5 ml
25% 250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM
second GRF analogue diluted into 22.5 ml of 20 mM sodium phosphate
buffer (pH 7.0), 5 mM sodium caprylate and 900 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #3 described above. In FIG. 40 the
purified conjugate fraction appears in fraction F2.
Example 41
Purification of HSA:Third GRF Analogue (SEQ ID NO:30) Conjugate
[0196] The third GRF analogue is GRF (1-29) dAla.sup.2 Gln.sup.8
dArg.sup.11 Ala.sup.15 Leu.sup.27 Lys.sup.30
(.epsilon.-MPA)-CONH.sub.2 and has the following sequence:
TABLE-US-00023 YaDAIFTQSYrKVLAQLSARKLLQDILSRK(MPA)-CONH.sub.2
[0197] The purification of a conjugate made from reacting 2.5 ml
25% 250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM
third GRF analogue diluted into 22.5 ml of 20 mM sodium phosphate
buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #3 described above. In FIG. 41 the
purified conjugate fraction appears in fraction F2.
Example 42
Purification of HSA:Fourth GRF Analogue (SEQ ID NO:31)
Conjugate
[0198] The fourth GRF analogue is GRF (1-29) dAla.sup.2 Lys.sup.30
(.epsilon.-MPA)-CONH.sub.2 and has the following sequence:
TABLE-US-00024 YaDAIFTNSYRKVLGQLSARKLLQDIMSRK(MPA)-CONH.sub.2
[0199] The purification of a conjugate made from reacting 2.5 ml
25% HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM fourth GRF
analogue diluted in 22.5 ml of 20 mM sodium phosphate buffer (pH
7.0), 5 mM sodium caprylate and 900 mM (NH.sub.4).sub.2SO.sub.4 was
performed on a column of Butyl sepharose using gradient #3
described above. In FIG. 42 the purified conjugate fraction appears
in fraction F2.
Example 43
Purification of HSA:Thirteenth GLP-1 Analogue CJC 1365 (SEQ ID
NO:32) Conjugate
[0200] The thirteenth GLP-1 analogue is GLP-1 (9-36) Lys.sup.37
(.epsilon.-AEEA-MPA)-CONH.sub.2 and has the following sequence:
TABLE-US-00025
EGTFTSDVSSYLEGQAAKEFIAWLVKGRK(.epsilon.-AEEA-MPA)-CONH.sub.2
[0201] The purification of a conjugate made from reacting 3.5 ml
25% HSA (Cortex-Biochem, San Leandro, Calif.) and 1 mM thirteenth
GLP-1 analogue diluted in 21.5 ml of 20 mM sodium phosphate buffer
(pH 7.0), 5 mM sodium caprylate and 750 mM (NH.sub.4).sub.2SO.sub.4
was performed on a column of Butyl sepharose using gradient #1
described above. In FIG. 43 the purified conjugate fraction appears
in fraction F2.
Example 44
Purification of HSA Lactose:First GLP-1 Analogue (SEQ ID NO:1)
Conjugate
[0202] The first GLP-1 analogue is GLP-1 (7-36) dAla.sup.8
Lys.sup.37 (.epsilon.-AEEA-MPA)-CONH.sub.2 and his sequence is
shown above in Example 1.
[0203] The purification of a conjugate made from reacting 4 ml 25%
lactosaminated albumin (HSA pre-incubated with excess lactose at
37.degree. C., pH 7.0) with 200 .mu.M first GLP-1 analogue in 4 ml
of a buffer made of 20 mM sodium phosphate, 5 mM sodium caprylate
and 750 mM (NH.sub.4).sub.2SO.sub.4, (pH 7.0) was performed on a
column of Butyl sepharose using gradient #1 described above. In
FIG. 44 the purified lactosaminated conjugate fraction appears in
fraction F2.
Example 45
Purification of HSA:First T20 Analogue (SEQ ID NO:33) Conjugate
[0204] The first T20 analogue is Ac-T20 (1-36) Lys.sup.37
(.epsilon.-AEEA-MPA)-CONH.sub.2 and ahs the following sequence:
TABLE-US-00026 Ac-YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFK(AEEA-
MPA)-CONH.sub.2
[0205] The purification of a conjugate made from reacting 2.5 ml
25% HSA with 1 mM first T20 analogue in 10 ml of 20 mM sodium
phosphate buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 45 the
purified conjugate fraction appears in fraction F2.
Example 46
Purification of HSA:First T1249 Analogue (SEQ ID NO:34)
Conjugate
[0206] The first T1249 analogue is Ac-T1249 (1-39) Lys.sup.40
(.epsilon.-AEEA-MPA)-CONH.sub.2 and has the following sequence:
TABLE-US-00027 Ac-WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWFK(AEEA-
MPA)-CONH.sub.2
[0207] The purification of a conjugate made from reacting 2 ml 25%
HSA and 1 mM first T1249 analogue in 10.5 ml of 20 mM sodium
phosphate buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 46 the
purified conjugate fraction appears in fraction F4.
Example 47
Purification of a HSA:First GLP-1 Analogue (SEQ ID NO:1)
[0208] The first GLP-1 analogue is GLP-1 (7-36) dAla.sup.8
Lys.sup.37 (.epsilon.-AEEA-MPA)-CONH.sub.2 and his sequence is
shown in Example 1.
[0209] The purification of 114.45 mg of the preformed conjugate of
the first GLP-1 analogue in 12.5 ml of 20 mM sodium phosphate
buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #5 described above. FIG. 47 illustrates
the separation curve obtained with the conjugate found in fraction
F2.
Example 48
Purification of a HSA:First C34 Analogue (SEQ ID NO:6)
[0210] The first C34 analogue is MPA-AEEA-C34-CONH.sub.2 and his
sequence is shown above in Example 9.
[0211] The purification of 114.45 mg of the preformed conjugate of
the first C34 analogue in 12.5 ml of 20 mM sodium phosphate buffer
(pH 7.0), 5 mM sodium caprylate and 750 mM (NH.sub.4).sub.2SO.sub.4
was performed on a column of Butyl sepharose using gradient #5
described above. FIG. 48 illustrates the separation curve obtained
with the conjugate found in fraction F2.
Example 49
Purification of a HSA:Second GRF Analogue (SEQ ID NO:29)
[0212] The second GRF analogue is GRF(1-29) Lys.sup.30
(.epsilon.-MPA)-CONH.sub.2 and his sequence is shown above in
Example 40.
[0213] The purification of 125.53 mg of the preformed conjugate of
the second GRF analogue in 12.5 ml of 20 mM sodium phosphate buffer
(pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4, pH 7.0 was performed on a column of Butyl
sepharose using gradient #5 described above. FIG. 49 illustrates
the separation curve obtained with the conjugate found in fraction
F2.
Example 50
Purification of HSA:Vinorelbine Analogue Conjugate (SEQ ID
NO:35)
[0214] The vinorelbine analogue is a molecule of vinorelbine with
AEEA-MPA coupled thereto as illustrated in the following
structure:
##STR00007##
[0215] The purification of a conjugate made from 2.5 ml 25% HSA and
1 mM vinorelbine analogue in 22.5 ml of a buffer made of 20 mM
sodium phosphate buffer, 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4, pH 7.0 was performed on a column of Butyl
sepharose using gradient #4 described above. In FIG. 50 the
purified conjugate fraction appears in fraction F2. The conjugate
fraction was concentrated with Amicon.TM. filter 30 kDa.
Example 51
Purification of L-Cysteine
[0216] The purification of 2.5 ml 40 mM L-cysteine in 22.5 ml of 20
mM sodium phosphate buffer (pH 7.0), 5 mM sodium caprylate and 1500
mM (NH.sub.4).sub.2SO.sub.4, was performed on a column of Butyl
sepharose using gradient #4 described above. FIG. 51 illustrates
the separation curve obtained with L-cysteine eluting within the
void volume of the column (fraction F3).
Example 52
Purification of L-Cysteine:Vinorelbine Analogue (SEQ ID NO:35)
Conjugate
[0217] The vinorelbine analogue is a molecule of vinorelbine with
AEEA-MPA coupled thereto as illustrated in the structure shown in
Example 50.
[0218] The purification of a conjugate made from reacting 2.5 ml 40
mM L-cysteine with 1 mM vinorelbine analogue in 22.5 ml of 20 mM
sodium phosphate buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #4 described above. FIG. 52 illustrates
the separation curve obtained with the L-cysteine conjugate eluting
within fractions F8, F9 and F10.
Example 53
Purification of RSA:Third Exendin-4 Analogue (SEQ ID NO:25)
Conjugate
[0219] The third Exendin-4 analogue is Exendin-4-(1-39) Lys.sup.40
(.epsilon.-AEEA-MPA)-CONH.sub.2 and his sequence shown in Example
33.
[0220] The purification of a conjugate made from reacting 11 ml 5%
RSA (rat serum albumin) with 200 .mu.M third Exendin-4 analogue in
11 ml of 20 mM sodium phosphate buffer (pH 7.0), 5 mM sodium
caprylate and 750 mM (NH.sub.4).sub.2SO.sub.4, was performed on a
column of Butyl sepharose using gradient #1 described above. In
FIG. 53 the purified conjugate fraction appears in fraction F2.
Example 54
Purification of HSA:Fourth C34 Analogue (SEQ ID NO:36)
Conjugate
[0221] The fourth C34 analogue is C34 (1-34) Lys.sup.13
(.epsilon.-MPA)-CONH.sub.2 and has the following sequence:
TABLE-US-00028
WMEWDREINNYTK(MPA)LIHSLIEESQNQQEKNEQELL-CONH.sub.2
[0222] The purification of a conjugate made from reacting 2 ml 25%
HSA with 1 mM fourth C34 analogue in 13 ml of 20 mM sodium
phosphate buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 54 the
purified conjugate fraction appears in fraction F2.
Example 55
Purification of HSA:Fifth C34 Analogue (SEQ ID NO:37) Conjugate
[0223] The fifth C34 analogue is C34 (1-34) Lys.sup.35
(.epsilon.-MPA)-CONH.sub.2 and has the following sequence:
TABLE-US-00029
WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLK(MPA)-CONH.sub.2
[0224] The purification of a conjugate made from 2 ml 25% HSA and 1
mM fifth C34 analogue in 13 ml of a buffer made of 20 mM sodium
phosphate buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4, was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 55 the
purified conjugate fraction appears in fraction F2.
Example 56
Purification of HSA:Sixth C34 Analogue (SEQ ID NO:38) Conjugate
[0225] The sixth C34 analogue MPA-C34 (1-34)-CONH.sub.2 and has the
following sequence:
TABLE-US-00030
MPA-WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL-CONH.sub.2
[0226] The purification of a conjugate made from reacting 2 ml 25%
HSA and 1 mM sixth C34 analogue in 13 ml of 20 mM sodium phosphate
buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 56 the
purified conjugate fraction appears in fraction F2.
Example 57
Purification of HSA:Seventh C34 Analogue (SEQ ID NO:39)
Conjugate
[0227] The seventh C34 analogue is Ac-C34 (1-34) Glu.sup.2
Lys.sup.6 Lys.sup.7 Glu.sup.9 Glu.sup.10 Lys.sup.13 Lys.sup.14
Glu.sup.16 Glu.sup.17 Lys.sup.20 Lys.sup.21 Glu.sup.23 Glu.sup.24
Lys.sup.27 Glu.sup.31 Lys.sup.34 Lys.sup.35 Lys.sup.36
(.epsilon.-AEEA-MPA)-CONH.sub.2 and has the following sequence:
TABLE-US-00031 Ac-WEEWOKKIEEYTKKIEELIKKSEEQQKKNEEELKKK(AEEA-MPA)-
CONH.sub.2
[0228] The purification of a conjugate made from reacting 2 ml 25%
HSA with 1 mM seventh C34 analogue in 13 ml 20 mM sodium phosphate
buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4, was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 57 the
purified conjugate fraction appears in fraction F2.
Example 58
Purification of HSA:Eighth C34 Analogue (SEQ ID NO:40)
Conjugate
[0229] The eighth C34 analogue is MPA-AEEA-C34 (1-34) Glu.sup.2
Lys.sup.6 Lys.sup.7 Glu.sup.9 Glu.sup.10 Lys.sup.13 Lys.sup.14
Glu.sup.16 Glu.sup.17 Lys.sup.20 Lys.sup.21 Glu.sup.23 Glu.sup.24
Lys.sup.27 Glu.sup.31 Lys.sup.34 Lys.sup.35-CONH.sub.2 and has the
following sequence:
TABLE-US-00032
MPA-AEEA-WEEWDKKIEEYTKKIEELIKKSEEQQKKNEEELKK-CONH.sub.2
[0230] The purification of a conjugate made from reacting 2 ml 25%
HSA with 1 mM eighth C34 analogue in 13 ml of 20 mM sodium
phosphate buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4, was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 58 the
purified conjugate fraction appears in fraction F2.
Example 59
Purification of HSA:First PYY Analogue (SEQ ID NO:41) Conjugate
[0231] The first PYY analogue is PYY (3-36) Lys.sup.4
(.epsilon.-OA-MPA)-CONH.sub.2 and has the following structure:
##STR00008##
[0232] The purification of a conjugate made from reacting 1.5 ml
25% HSA with 1 mM first PYY analogue in 6 ml of 20 mM sodium
phosphate buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4, was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 59 the
purified conjugate fraction appears in fraction F2.
Example 60
Purification of HSA:Second PYY Analogue (SEQ ID NO:42)
Conjugate
[0233] The second PYY analogue is MPA-OA-PYY (3-36)-CONH.sub.2 and
has the following sequence:
##STR00009##
[0234] The purification of a conjugate made from reacting 1.5 ml
25% HSA with 1 mM second PYY analogue in 6 ml of 20 mM sodium
phosphate buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4, was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 60 the
purified conjugate fraction appears in fraction F2.
Example 61
Purification of HSA:Fifth Insulin Derivative (SEQ ID NO:43)
Conjugate
[0235] The fifth insulin derivative is human insulin with
AEEAS-AEEAS-MPA on position B29 and is represented in FIG. 1 shown
above in Example 7.
[0236] The purification of a conjugate made from reacting 2 ml 25%
HSA with 1 mM fifth insulin derivative in 15 ml of 20 mM sodium
phosphate buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 61 the
purified conjugate fraction appears in fraction F2.
Example 62
Purification of HSA:Sixth Insulin Derivative (SEQ ID NO:44)
Conjugate
[0237] The sixth insulin derivative is human insulin with
AEEAS-AEEAS-MPA on position B1 and is represented in FIG. 1 shown
above in Example 7.
[0238] The purification of a conjugate made from reacting 2.5 ml
25% HSA with 1 mM sixth insulin derivative in 15 ml of 20 mM sodium
phosphate buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 62 the
purified conjugate fraction appears in fraction F2.
Example 63
Purification of HSA:Seventh Insulin Derivative (SEQ ID NO:45)
Conjugate
[0239] The seventh insulin derivative is human insulin with OA-MPA
on position B29 and is represented in FIG. 1 shown above in Example
7.
[0240] The purification of a conjugate made from reacting 2 ml 25%
HSA with 1 mM seventh insulin derivative in 15 ml of 20 mM sodium
phosphate buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 63 the
purified conjugate fraction appears in fraction F2.
Example 64
Purification of HSA:Third PYY Analogue (SEQ ID NO:46) Conjugate
[0241] The third PYY analogue is MPA-PYY (3-36)-CONH.sub.2 and has
the following sequence:
TABLE-US-00033
MPA-NH-IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY-CONH.sub.2
[0242] The purification of a conjugate made from reacting 3 ml 25%
HSA with 1 mM third PYY analogue in 18 ml of 20 mM sodium phosphate
buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 64 the
purified conjugate fraction appears in fraction F2.
Example 65
Purification of HSA:Fourth PYY Analogue (SEQ ID NO:47)
Conjugate
[0243] The fourth PYY analogue is PYY (3-36) Lys.sup.37
(.epsilon.-MPA)-CONH.sub.2 and has the following sequence:
TABLE-US-00034
IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRYK(MPA)-CONH.sub.2
[0244] The purification of a conjugate made from reacting 3 ml 25%
HSA with 1 mM fourth PYY analogue in 18 ml of 20 mM sodium
phosphate buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #1 described above. In FIG. 65 the
purified conjugate fraction appears in fraction F2.
Example 66
Purification of HSA:Fifth PYY Analogue (SEQ ID NO:48) Conjugate
[0245] The fifth PYY analogue is MPA-PYY (22-36)-CONH.sub.2 and has
the following sequence: (MPA)-ASLRHYLNLVTRQRY-CONH.sub.2.
[0246] The purification of a conjugate made from reacting 6 ml 25%
HSA with 1 mM fifth PYY analogue in 36 ml of 20 mM sodium phosphate
buffer (pH 7.0), 5 mM sodium caprylate and 900 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #3 described above. In FIG. 66 the
purified conjugate fraction appears in fraction F2.
Example 67
Purification of HSA:Sixth PYY Analogue (SEQ ID NO:49) Conjugate
[0247] The sixth PYY analogue is Acetyl-PYY (22-36) Lys.sup.37
(.epsilon.-MPA)-CONH.sub.2 and has the following sequence:
Ac-ASLRHYLNLVTRQRYK(MPA)-CONH.sub.2.
[0248] The purification of a conjugate made from reacting 6 ml 25%
HSA with 1 mM sixth PYY analogue in 36 ml of 20 mM sodium phosphate
buffer (pH 7.0), 5 mM sodium caprylate and 900 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #3 described above. In FIG. 67 the
purified conjugate fraction appears in fraction F2.
Example 68
Purification of HSA:Second ANP Analogue (SEQ ID NO:50)
Conjugate
[0249] The second ANP analogue is MPA-ANP (99-126)-CONH.sub.2 and
has the following structure:
##STR00010##
[0250] The purification of a conjugate made from reacting 1 ml 25%
HSA with 1 mM second ANP analogue in 14 ml of 20 mM sodium
phosphate buffer (pH 7.0), 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4 was performed on a column of Butyl
sepharose using gradient #3 described above. In FIG. 68 the
purified conjugate fraction appears in fraction F2.
Example 69
Purification of HSA:Third ANP Analogue (SEQ ID NO:51) Conjugate
[0251] The third ANP analogue is ANP (99-126) having reacted with
MAL-dPEG.sub.4.TM. (Quanta Biodesign, Powell, Ohio, USA) coupled to
Ser.sup.99. The resulting ANP analogue is MPA-EEEEP-ANP (99-126)
where EEEEP is ethoxy ethoxy ethoxy ethoxy propionic acid; and its
sequence is the following:
##STR00011##
The purification of a conjugate made from reacting 1 ml 25% HSA
with 1 mM CJC 1681 in 14 ml of 20 mM sodium phosphate buffer (pH
7.0), 5 mM sodium caprylate and 900 mM (NH.sub.4).sub.2SO.sub.4 was
performed on a column of Butyl sepharose using gradient #3
described above. In FIGS. 69A and 69B the purified conjugate
fraction appears in fraction F2.
Example 70
Purification of HSA:First GLP-1 Analogue (SEQ ID NO:1)
Conjugate
[0252] The first GLP-1 analogue is GLP-1 (7-36) dAla.sup.8
Lys.sup.37 (.epsilon.-AEEA-MPA)-CONH.sub.2 and his sequence is
shown above in Example 1.
[0253] The purification of a conjugate made from reacting 1 ml 25%
250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM first
GLP-1 analogue diluted into 9 ml of buffer made of 20 mM sodium
phosphate buffer pH 7.0, 5 mM sodium caprylate and 1.75M
(NH.sub.4).sub.2SO.sub.4, was performed on a column of Butyl
sepharose using the gradient #6 described above. In FIG. 70 the
purified conjugate fraction appears in fraction B.
Example 71
Purification of HSA:First GLP-1 Analogue (SEQ ID NO:1)
Conjugate
[0254] The first GLP-1 analogue is GLP-1 (7-36) dAla.sup.8
Lys.sup.37 (.epsilon.-AEEA-MPA)-CONH.sub.2 and his sequence is
shown above in Example 1.
[0255] The purification of a conjugate made from reacting 1 ml 25%
250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM first
GLP-1 analogue diluted into 9 ml of buffer made of 20 mM sodium
phosphate buffer pH 7.0, 5 mM sodium caprylate and 1.75M magnesium
sulfate, was performed on a column of Butyl sepharose using the
gradient #6 described above. In FIG. 71 the purified conjugate
fraction appears in fraction F2.
Example 72
Purification of HSA:First GLP-1 Analogue (SEQ ID NO:1)
Conjugate
[0256] The first GLP-1 analogue is GLP-1 (7-36) dAla.sup.8
Lys.sup.37 (.epsilon.-AEEA-MPA)-CONH.sub.2 and his sequence is
shown above in Example 1.
[0257] Example with 750 mM ammonium sulfate The purification of a
conjugate made from reacting 1 ml 25% 250 mg/ml HSA
(Cortex-Biochem, San Leandro, Calif.) with 1 mM first GLP-1
analogue diluted into 9 ml of buffer made of 20 mM sodium phosphate
buffer pH 7.0, 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4, was performed on a column of Butyl
sepharose using the gradient #1 described above. In FIG. 72 the
purified conjugate fraction appears in fraction F2.
Example 73
Purification of HSA:First GLP-1 Analogue (SEQ ID NO:1)
Conjugate
[0258] The first GLP-1 analogue is GLP-1 (7-36) dAla.sup.8
Lys.sup.37 (.epsilon.-AEEA-MPA)-CONH.sub.2 and his sequence is
shown above in Example 1.
[0259] Example with 1.75M ammonium phosphate The purification of a
conjugate made from reacting 1 ml 25% 250 mg/ml HSA
(Cortex-Biochem, San Leandro, Calif.) with 1 mM first GLP-1
analogue diluted into 9 ml of buffer made of 20 mM sodium phosphate
buffer pH 7.0, 5 mM sodium caprylate and 1.75M ammonium phosphate,
was performed on a column of Butyl sepharose using the gradient #6
described above. In FIG. 73 the purified conjugate fraction appears
in fraction B.
Example 74
Purification of HSA:First GLP-1 Analogue (SEQ ID NO:1)
Conjugate
[0260] The first GLP-1 analogue is GLP-1 (7-36) dAla.sup.8
Lys.sup.37 (.epsilon.-AEEA-MPA)-CONH.sub.2 and his sequence is
shown above in Example 1.
[0261] Example with 750 mM ammonium phosphate The purification of a
conjugate made from reacting 1 ml 25% 250 mg/ml HSA
(Cortex-Biochem, San Leandro, Calif.) with 1 mM first GLP-1
analogue diluted into 9 ml of buffer made of 20 mM sodium phosphate
buffer pH 7.0, 5 mM sodium caprylate and 750 mM ammonium phosphate,
was performed on a column of Butyl sepharose using the gradient #1
described above. In FIG. 74 the purified conjugate fraction appears
in fraction F2.
Example 75
Purification of HSA:First GLP-2 Analogue (SEQ ID NO:52)
Conjugate
[0262] The first GLP-2 analogue is GLP-2 (1-33) Gly.sup.2
Lys.sup.34 (.epsilon.-MPA)-CONH.sub.2 and has the following
sequence:
TABLE-US-00035
HGDGSFSDEMNTILDNLAARDFINWLIQTKITDK(MPA)-CONH.sub.2
[0263] The purification of a conjugate made from reacting 2 ml 25%
250 mg/ml HSA (Cortex-Biochem, San Leandro, Calif.) with 1 mM first
GLP-2 analogue diluted into 14 ml of buffer made of 20 mM sodium
phosphate buffer pH 7.0, 5 mM sodium caprylate and 750 mM
(NH.sub.4).sub.2SO.sub.4, was performed on a column of Butyl
sepharose using the gradient #1 described above. In FIG. 75 the
purified conjugate fraction appears in fraction F2.
Example 76
Purification of RSA:First GLP-2 Analogue (SEQ ID NO:52)
Conjugate
[0264] The first GLP-2 analogue is GLP-2 (1-33) Gly.sup.2
Lys.sup.34 (.epsilon.-MPA)-CONH.sub.2 and his sequence is shown in
Example 75.
[0265] The purification of a conjugate made from reacting 9 ml 25%
250 mg/ml RSA (rat serum albumin) with 1 mM first GLP-2 analogue
diluted into 14 ml of buffer made of 20 mM sodium phosphate buffer
pH 7.0, 5 mM sodium caprylate and 750 mM (NH.sub.4).sub.2SO.sub.4,
was performed on a column of Butyl sepharose using the gradient #1
described above. In FIG. 76 the purified conjugate fraction appears
in fraction F2.
[0266] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth, and as follows in the scope of the appended
claims.
Sequence CWU 1
1
53131PRTArtificial SequenceGLP-1 Analogue 1His Xaa Glu Gly Thr Phe
Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys
Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Xaa 20 25
30230PRTArtificial SequenceGRF Analogue 2Tyr Xaa Asp Ala Ile Phe
Thr Gln Ser Tyr Arg Lys Val Leu Ala Gln 1 5 10 15Leu Ser Ala Arg
Lys Leu Leu Gln Asp Ile Leu Ser Arg Xaa 20 25 3038PRTArtificial
SequenceK5 Analogue 3Pro Arg Lys Leu Tyr Asp Tyr Xaa 1 5430PRTHomo
sapiensMOD_RES1Xaa is Phe linked to MPA 4Xaa Val Asn Gln His Leu
Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 10 15Leu Val Cys Gly
Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr 20 25 30521PRTHomo
sapiensMOD_RES1Xaa is Gly linked to MPA 5Xaa Ile Val Glu Gln Cys
Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu 1 5 10 15Glu Asn Tyr Cys
Asn 20634PRTArtificial SequenceC34 Analogue 6Xaa Met Glu Trp Asp
Arg Glu Ile Asn Asn Tyr Thr Ser Leu Ile His 1 5 10 15Ser Leu Ile
Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu 20 25 30Leu
Leu735PRTArtificial SequenceC34 Analogue 7Trp Met Glu Trp Asp Arg
Glu Ile Asn Asn Tyr Thr Ser Leu Ile His 1 5 10 15Ser Leu Ile Glu
Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu 20 25 30Leu Leu Xaa
35834PRTArtificial SequenceC34 Analogue 8Trp Met Glu Trp Asp Arg
Glu Ile Asn Asn Tyr Thr Xaa Leu Ile His 1 5 10 15Ser Leu Ile Glu
Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu 20 25 30Leu
Leu931PRTArtificial SequenceGLP-1 Analogue 9His Ala Glu Gly Thr Phe
Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys
Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Xaa 20 25
301031PRTArtificial SequenceGLP-1 Analogue 10His Xaa Glu Gly Thr
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala
Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Xaa 20 25
301130PRTArtificial SequenceGLP-1 Analogue 11His Ala Glu Gly Thr
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala
Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25
301230PRTArtificial SequenceGLP-1 Analogue 12His Ala Glu Gly Thr
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala
Lys Glu Phe Ile Ala Trp Leu Val Xaa Gly Arg 20 25
301340PRTArtificial SequenceExendin-4 Analogue 13His Gly Glu Gly
Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15Glu Ala
Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser
Gly Ala Pro Pro Pro Ser Xaa 35 401432PRTArtificial
SequenceExendin-4 Analogue 14Asp Leu Ser Lys Gln Met Glu Glu Glu
Ala Val Arg Leu Phe Ile Glu 1 5 10 15Trp Leu Lys Asn Gly Gly Pro
Ser Ser Gly Ala Pro Pro Pro Ser Xaa 20 25 301513PRTArtificial
SequenceDynorphin A Analogue 15Tyr Gly Gly Phe Leu Arg Arg Ile Arg
Pro Lys Leu Xaa 1 5 101628PRTArtificial SequenceANP Analogue 16Xaa
Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly 1 5 10
15Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr 20
25177PRTArtificial SequenceDynorphin A Analogue 17Arg Ile Arg Pro
Lys Leu Xaa 1 5184PRTArtificial SequenceACE Inhibitor 18Phe Xaa Ile
Xaa 11930PRTArtificial SequenceGLP-1 Analogue 19His Ala Glu Gly Thr
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Xaa Ala Ala
Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25
302030PRTArtificial SequenceGLP-1 Analogue 20His Ala Glu Gly Thr
Phe Thr Ser Asp Val Ser Xaa Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala
Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25
302130PRTArtificial SequenceGLP-1 Analogue 21His Ala Glu Gly Thr
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala
Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25
302230PRTArtificial SequenceGLP-1 Analogue 22His Ala Glu Gly Thr
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala
Lys Xaa Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25
302331PRTArtificial SequenceGLP-1 Analogue 23His Ala Glu Gly Thr
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala
Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Xaa 20 25
302431PRTArtificial SequenceGLP-1 Analogue 24His Ala Glu Gly Thr
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala
Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Xaa 20 25
302540PRTArtificial SequenceExendin-4 Analogue 25His Gly Glu Gly
Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15Glu Ala
Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser
Gly Ala Pro Pro Pro Ser Xaa 35 402631PRTArtificial SequenceGLP-1
Analogue 26His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Xaa
Gly Arg Lys 20 25 302730PRTHomo sapiensMOD_RES1Xaa is Phe linked to
OA-MPA 27Xaa Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala
Leu Tyr 1 5 10 15Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro
Lys Thr 20 25 302830PRTHomo sapiensMOD_RES29Xaa is Lys linked to
MPA 28Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu
Tyr 1 5 10 15Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Xaa
Thr 20 25 302930PRTArtificial SequenceGRF Analogue 29Tyr Ala Asp
Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln 1 5 10 15Leu
Ser Ala Arg Lys Leu Leu Gln Asp Ile Met Ser Arg Xaa 20 25
303030PRTArtificial SequenceGRF Analogue 30Tyr Xaa Asp Ala Ile Phe
Thr Gln Ser Tyr Xaa Lys Val Leu Arg Gln 1 5 10 15Leu Ser Ala Arg
Lys Leu Leu Gln Asp Ile Leu Ser Arg Xaa 20 25 303130PRTArtificial
SequenceGRF Analogue 31Tyr Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg
Lys Val Leu Gly Gln 1 5 10 15Leu Ser Ala Arg Lys Leu Leu Gln Asp
Ile Met Ser Arg Xaa 20 25 303229PRTArtificial SequenceGLP-1
Analogue 32Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
Gln Ala 1 5 10 15Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
Xaa 20 253337PRTArtificial SequenceT20 Analogue 33Tyr Thr Ser Leu
Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln 1 5 10 15Glu Lys
Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu 20 25 30Trp
Asn Trp Phe Xaa 353440PRTArtificial SequenceT1249 Analogue 34Trp
Gln Glu Trp Glu Gln Lys Ile Thr Ala Leu Leu Glu Gln Ala Gln 1 5 10
15Ile Gln Gln Glu Lys Asn Glu Tyr Glu Leu Gln Lys Leu Asp Lys Trp
20 25 30Ala Ser Leu Trp Glu Trp Phe Xaa 35 403521PRTHomo sapiens
35Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu 1
5 10 15Glu Asn Tyr Cys Asn 203634PRTArtificial SequenceC34 Analogue
36Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Xaa Leu Ile His 1
5 10 15Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln
Glu 20 25 30Leu Leu3735PRTArtificial SequenceC34 Analogue 37Trp Met
Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu Ile His 1 5 10
15Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu
20 25 30Leu Leu Xaa 353834PRTArtificial SequenceC34 Analogue 38Xaa
Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Leu Ile His 1 5 10
15Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu
20 25 30Leu Leu3936PRTArtificial SequenceC34 Analogue 39Trp Glu Glu
Trp Asp Lys Lys Ile Glu Glu Tyr Thr Lys Lys Ile Glu 1 5 10 15Glu
Leu Ile Lys Lys Ser Glu Glu Gln Gln Lys Lys Asn Glu Glu Glu 20 25
30Leu Lys Lys Xaa 354035PRTArtificial SequenceC34 Analogue 40Xaa
Glu Glu Trp Asp Lys Lys Ile Glu Glu Tyr Thr Lys Lys Ile Glu 1 5 10
15Glu Leu Ile Lys Lys Ser Glu Glu Gln Gln Lys Lys Asn Glu Glu Glu
20 25 30Leu Lys Lys 354134PRTArtificial SequencePYY Analogue 41Ile
Xaa Pro Glu Ala Pro Gly Glu Asp Arg Ser Pro Glu Glu Leu Asn 1 5 10
15Arg Tyr Tyr Lys Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln
20 25 30Arg Tyr4234PRTArtificial SequencePYY Analogue 42Xaa Lys Pro
Glu Ala Pro Gly Glu Asp Arg Ser Pro Glu Glu Leu Asn 1 5 10 15Arg
Tyr Tyr Lys Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln 20 25
30Arg Tyr4330PRTHomo sapiensMOD_RES29Xaa is Lys linked to
AEEAS-AEEAS-MPA 43Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val
Glu Ala Leu Tyr 1 5 10 15Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr
Thr Pro Xaa Thr 20 25 304430PRTHomo sapiensMOD_RES1Xaa is Phe
linked to AEEAS-AEEAS-MPA 44Xaa Val Asn Gln His Leu Cys Gly Ser His
Leu Val Glu Ala Leu Tyr 1 5 10 15Leu Val Cys Gly Glu Arg Gly Phe
Phe Tyr Thr Pro Lys Thr 20 25 304530PRTHomo sapiensMOD_RES29Xaa is
Lys linked to OA-MPA 45Phe Val Asn Gln His Leu Cys Gly Ser His Leu
Val Glu Ala Leu Tyr 1 5 10 15Leu Val Cys Gly Glu Arg Gly Phe Phe
Tyr Thr Pro Xaa Thr 20 25 304634PRTArtificial SequencePYY Analogue
46Xaa Lys Pro Glu Ala Pro Gly Glu Asp Arg Ser Pro Glu Glu Leu Asn 1
5 10 15Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg
Gln 20 25 30Arg Tyr4735PRTArtificial SequencePYY Analogue 47Ile Lys
Pro Glu Ala Pro Gly Glu Asp Arg Ser Pro Glu Glu Leu Asn 1 5 10
15Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln
20 25 30Arg Tyr Xaa 354815PRTArtificial SequencePYY Analogue 48Xaa
Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln Arg Tyr 1 5 10
154916PRTArtificial SequencePYY Analogue 49Arg Ser Leu Arg His Tyr
Leu Asn Leu Val Thr Arg Gln Arg Tyr Xaa 1 5 10 155028PRTArtificial
SequenceANP Analogue 50Xaa Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg
Met Asp Arg Ile Gly 1 5 10 15Ala Gln Ser Gly Leu Gly Cys Asn Ser
Phe Arg Tyr 20 255128PRTArtificial SequenceANP Analogue 51Xaa Leu
Arg Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly 1 5 10
15Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr 20
255234PRTArtificial SequenceGLP-2 Analogue 52His Gly Asp Gly Ser
Phe Ser Asp Glu Met Asn Thr Ile Leu Asp Asn 1 5 10 15Leu Ala Ala
Arg Asp Phe Ile Asn Trp Leu Ile Gln Thr Lys Ile Thr 20 25 30Asp
Xaa5330PRTHomo sapiens 53Phe Val Asn Gln His Leu Cys Gly Ser His
Leu Val Glu Ala Leu Tyr 1 5 10 15Leu Val Cys Gly Glu Arg Gly Phe
Phe Tyr Thr Pro Lys Thr 20 25 30
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