U.S. patent application number 12/811397 was filed with the patent office on 2011-02-10 for purification of not-glycosylated polypeptides.
Invention is credited to Roberto Falkenstein, Nicole Fuehrler, Claudia Giessel, Sybille Greithanner, Adelbert Grossmann, Friederike Hesse, Brigitte Kraemer, Marc Pompiati, Andreas Schaubmar, Birgit Weydanz.
Application Number | 20110034672 12/811397 |
Document ID | / |
Family ID | 39810157 |
Filed Date | 2011-02-10 |
United States Patent
Application |
20110034672 |
Kind Code |
A1 |
Falkenstein; Roberto ; et
al. |
February 10, 2011 |
PURIFICATION OF NOT-GLYCOSYLATED POLYPEPTIDES
Abstract
The current invention reports a method for the purification of a
not-glycosylated, heterologous polypeptide, which has been
recombinantly produced in a prokaryotic cell, wherein the method
comprises three chromatography steps of which the first
chromatography step selected from i) hydrophobic charge induction
chromatography, or ii) hydrophobic interaction chromatography, or
iii) affinity chromatography, or iv) ion exchange chromatography,
the second chromatography step is selected from i) anion exchange
chromatography, or ii) cation exchange chromatography, or iii)
hydroxylapatite chromatography, or iv) hydrophobic interaction
chromatography, and the a third chromatography step is selected
from i) hydrophobic charge induction chromatography, or ii) anion
exchange chromatography, or iii) cation exchange chromatography, or
iv) hydrophobic interaction chromatography, whereby the first
chromatography step is an affinity chromatography in case of
polypeptides capable of interacting with metal ligands, the second
chromatography step is not a hydroxylapatite chromatography step in
case of polypeptides with an isoelectric point below 6.0, and the
third chromatography step can be performed in flow-through mode
with polypeptides having a low or high isoelectric point.
Inventors: |
Falkenstein; Roberto;
(Muenchen, DE) ; Weydanz; Birgit; (Penzberg,
DE) ; Fuehrler; Nicole; (Schlehdorf, DE) ;
Giessel; Claudia; (Bad Toelz, DE) ; Greithanner;
Sybille; (Peiting, DE) ; Grossmann; Adelbert;
(Eglfing, DE) ; Hesse; Friederike; (Muenchen,
DE) ; Pompiati; Marc; (Penzberg, DE) ;
Schaubmar; Andreas; (Penzberg, DE) ; Kraemer;
Brigitte; (Penzberg, DE) |
Correspondence
Address: |
HOFFMANN-LA ROCHE INC.;PATENT LAW DEPARTMENT
340 KINGSLAND STREET
NUTLEY
NJ
07110
US
|
Family ID: |
39810157 |
Appl. No.: |
12/811397 |
Filed: |
January 15, 2009 |
PCT Filed: |
January 15, 2009 |
PCT NO: |
PCT/EP2009/000192 |
371 Date: |
July 1, 2010 |
Current U.S.
Class: |
530/351 ;
435/69.1; 435/69.51; 530/399; 530/410; 530/413; 530/415;
530/416 |
Current CPC
Class: |
C07K 1/1077 20130101;
A61K 47/60 20170801; C07K 1/22 20130101; C07K 1/36 20130101; C07K
1/16 20130101; C07K 14/56 20130101; C07K 14/65 20130101; C07K 1/20
20130101; C07K 1/18 20130101 |
Class at
Publication: |
530/351 ;
530/413; 530/416; 530/410; 435/69.1; 435/69.51; 530/399;
530/415 |
International
Class: |
C07K 1/16 20060101
C07K001/16; C07K 1/22 20060101 C07K001/22; C07K 1/18 20060101
C07K001/18; C07K 1/113 20060101 C07K001/113; C12P 21/00 20060101
C12P021/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 18, 2008 |
EP |
08000884.0 |
Claims
1. A method for the purification of a not-glycosylated,
heterologous polypeptide, which has been recombinantly produced in
an E. coli cell, wherein said not-glycosylated, heterologous
polypeptide is selected from growth factor agonists or antagonists,
or interferons or interferon variants, comprising the following
steps in the following order: a) providing a not-glycosylated,
heterologous polypeptide, which has been recombinantly produced in
an E. coli cell, b) a first chromatography step selected from the
group consisting of i) hydrophobic charge induction chromatography,
ii) hydrophobic interaction chromatography, iii) metal chelating
chromatography, or iv) ion exchange chromatography, c) a second
chromatography step selected from the group consisting of i) anion
exchange chromatography, ii) cation exchange chromatography, iii)
hydroxylapatite chromatography, iv) hydrophobic interaction
chromatography, or v) hydrophobic charge induction chromatography,
and d) a third chromatography step selected from the group
consisting of i) hydrophobic charge induction chromatography, ii)
anion exchange chromatography, iii) cation exchange chromatography,
or iv) hydrophobic interaction chromatography, whereby said first
chromatography step is an affinity chromatography or a hydrophobic
charge induction chromatography in case of polypeptides capable of
interacting with metal ligands, said second chromatography step is
not a hydroxylapatite chromatography step in case of polypeptides
with an isoelectric point below 6.0, said third chromatography step
can be performed in flow-through mode with polypeptides having a
low or high isoelectric point, and the purified non-glycosylated,
heterologous polypeptide is obtained after step d).
2.-4. (canceled)
5. The method of claim 1, characterized in that said method
comprises an additional step after step d) which is e) PEGylating
said polypeptide.
6. The method of claim 5, characterized in that said steps b) and
c) are cation exchange chromatography steps.
7. A method for the recombinant production of a not-glycosylated
heterologous polypeptide in a prokaryotic cell, characterized in
that said method comprises the following steps: a) cultivating a
prokaryotic cell comprising a nucleic acid encoding said
heterologous polypeptide under conditions suitable for the
expression of said heterologous polypeptide, wherein the
prokaryotic cell is an E. coli cell, b) recovering said
heterologous polypeptide from the culture medium or the prokaryotic
cells, and c) purifying said heterologous polypeptide with the
method of claim 1.
8. A method for the recombinant production of a not-glycosylated
heterologous polypeptide in a prokaryotic cell via inclusion
bodies, characterized in that said method comprises the following
steps: a) cultivating said prokaryotic cell comprising a nucleic
acid encoding said heterologous polypeptide under conditions
suitable for the expression of said heterologous polypeptide and
formation of inclusion bodies containing said heterologous
polypeptide, wherein the prokaryotic cell is an E. coli cell, b)
recovering said inclusion bodies from the prokaryotic cells, c)
solubilizing and renaturating said heterologous polypeptide from
said inclusion bodies, and d) purifying said heterologous
polypeptide with the method of claim 1.
9. The method of claim 1, characterized in that content of
endotoxins, and/or E. coli DNA, and/or E. coli cell proteins is
reduced in the polypeptide solution obtained after the third
chromatography step compared to the content prior to the first
chromatography step.
10. The method of claim 1, wherein the not-glycosylated,
heterologous polypeptide is IGF-1 or an IGF-1 variant, whereby the
first chromatography step is a hydrophobic charge induction
chromatography, the second chromatography step is selected from
hydroxylapatite chromatography or cation exchange chromatography,
and the third chromatography step is selected from hydrophobic
charge induction chromatography or anion exchange
chromatography.
11. The method of claim 1, wherein the not-glycosylated,
heterologous polypeptide is IFN.alpha.-2a, whereby the first
chromatography step is a hydrophobic charge induction
chromatography, the second chromatography step is an anion exchange
chromatography step, and the third chromatography step is an
hydrophobic charge induction chromatography.
12. The method of claim 1, wherein the not-glycosylated,
heterologous polypeptide is IFN.alpha.-2a, whereby the first
chromatography step is a hydrophobic interaction chromatography,
the second chromatography step is a cation exchange chromatography
step, and the third chromatography step is an hydrophobic
interaction chromatography.
13. A method for producing a not-glycosylated, PEGylated,
heterologous polypeptide, which has been recombinantly produced in
a prokaryotic cell comprising the following steps in the following
order: a) providing a not-glycosylated, heterologous polypeptide,
which has been recombinantly produced in a prokaryotic cell, b) a
first chromatography step selected from i) hydrophobic charge
induction chromatography, ii) hydrophobic interaction
chromatography, iii) affinity chromatography, or iv) ion exchange
chromatography, c) a second chromatography step selected from i)
anion exchange chromatography, ii) cation exchange chromatography,
iii) hydroxylapatite chromatography, or iv) hydrophobic interaction
chromatography, d) a third chromatography step selected from i)
hydrophobic charge induction chromatography, ii) anion exchange
chromatography, iii) cation exchange chromatography, or iv)
hydrophobic interaction chromatography, whereby said
not-glycosylated, heterologous polypeptide is PEGylated after step
d).
14. The method of claim 13, wherein the non-glycosylated PEGylated
heterologous polypeptide is IFN.alpha.-2a, whereby the first
chromatography step is selected from hydrophobic interaction
chromatography or metal affinity chromatography, the second
chromatography step is a cation exchange chromatography, and the
third chromatography step is an anion exchange chromatography and
wherein after the third chromatography step the purified
not-glycosylated and not-PEGylated IFN.alpha.-2a is PEGylated.
15. The method of claim 13, wherein the non-glycosylated PEGylated
heterologous polypeptide is PEGylated interferon, whereby the first
chromatography step is hydrophobic interaction chromatography, the
second chromatography step is a cation exchange chromatography, and
the third chromatography step is an hydrophobic charge induction
chromatography and wherein after the third chromatography step the
purified not-glycosylated and not-PEGylated IFN is PEGylated.
16. A method for the purification of a not-glycosylated,
heterologous polypeptide, which has been recombinantly produced in
a prokaryotic cell, wherein said not-glycosylated, heterologous
polypeptide is selected from growth factor agonists or antagonists,
or interferons or interferon variants, characterized in that said
method comprises the following steps in the following order: a) a
first chromatography step selected from i) hydrophobic charge
induction chromatography, ii) hydrophobic interaction
chromatography, iii) affinity chromatography, or iv) ion exchange
chromatography, b) a second chromatography step selected from i)
anion exchange chromatography, ii) cation exchange chromatography,
iii) hydroxylapatite chromatography, iv) hydrophobic interaction
chromatography, or v) hydrophobic charge induction chromatography,
and c) a third chromatography step selected from i) hydrophobic
charge induction chromatography, ii) anion exchange chromatography,
iii) cation exchange chromatography, or iv) hydrophobic interaction
chromatography, whereby said first chromatography step is an
affinity chromatography or a hydrophobic charge induction
chromatography in case of polypeptides capable of interacting with
metal ligands, said second chromatography step is not a
hydroxylapatite chromatography step in case of polypeptides with an
isoelectric point below 6.0, said third chromatography step can be
performed in flow-through mode with polypeptides having a low or
high isoelectric point, and whereby at least two different
sequences of three chromatographic steps yield a purified
not-glycosylated, heterologous polypeptide with comparable purity.
Description
FIELD OF THE INVENTION
[0001] The current invention relates to the field of polypeptide
purification. A general method for the purification of
not-glycosylated polypeptides with a combination of three
chromatographic steps is reported.
BACKGROUND OF THE INVENTION
[0002] Proteins play an important role in today's medical
portfolio. For human application every pharmaceutical substance has
to meet distinct criteria. To ensure the safety of
biopharmaceutical agents to humans nucleic acids, viruses, and host
cell proteins, which would cause severe harm, have to be removed
especially. To meet the regulatory specification one or more
purification steps have to follow the manufacturing process. Among
other purity, throughput, and yield play an important role in
determining an appropriate purification process.
[0003] Different methods are well established and widespread used
for protein purification, such as affinity chromatography with
thiophilic ligands, Cu-chelate, or microbial proteins (e.g. protein
A or protein G affinity chromatography), ion exchange
chromatography (e.g. cation exchange, anion exchange, and
mixed-mode exchange), thiophilic adsorption, hydrophobic
interaction or aromatic adsorption chromatography, size exclusion
chromatography, and electrophoretical methods (such as gel
electrophoresis, capillary electrophoresis) (Vijayalakshmi, M. A.,
Appl. Biochem. Biotech. 75 (1998) 93-102).
[0004] Recombinant polypeptides can be produced e.g. by prokaryotic
cells such as E. coli. The recombinantly produced polypeptide
accounts for the majority of the prokaryotic cell's polypeptide
content and is often deposited as insoluble aggregate, i.e. as a so
called inclusion body, within the prokaryotic cell. For the
isolation of the recombinant polypeptide the cells have to be
disintegrated and the recombinant polypeptide contained in the
inclusion bodies has to be solubilized after the separation of the
inclusion bodies from the cell debris. For the solubilization
chaotropic reagents, such as urea or guanidinium hydrochloride, are
used. To cleave disulfide bonds reducing agents, especially under
alkaline conditions, such as dithioerythriol, dithiothreitol, or
.beta.-mercaptoethanol are added. After the solubilization of the
aggregated polypeptide the globular structure of the recombinant
polypeptide, which is essential for the biological activity, has to
be reestablished. During this so called renaturation process the
concentration of the denaturating agents is slowly reduced, e.g. by
dialysis against a suited buffer, which allows the denatured
polypeptide to refold into its biologically active structure. After
the renaturation is the recombinant polypeptide purified to a
purity acceptable for the intended use. For example, for the use as
a therapeutic protein a purity of more than 90% has to be
established. Recombinantly produced polypeptides obtained from E.
coli are normally accompanied by nucleic acids, endotoxins,
polypeptides from the producing cell, and not-renaturated
recombinant polypeptides.
[0005] With the number of different chromatographic methods
available a multitude of combinations has to be tested in order to
find a suitable purification process. In these combinations
different sequences and even different numbers of chromatographic
methods may be used. Thus, a method for determining a suitable
sequence of chromatographic steps for the purification of a
not-glycosylated polypeptide is desirable.
[0006] In WO 2007/075283 a multi step system and methods of target
molecule purification are reported. Methods for purifying compounds
comprising a protein of interest are reported in WO 2007/016250. A
process for purifying a recombinant protein including one or a few
procedural steps only is reported in WO 2006/101441. Rege et al.
(Rege, K., Biotechnol. Bioeng. 93 (2006) 618-630) report a
high-throughput process development for recombinant protein
purification. In KR 2002/080108 a process for purifying human
growth hormone from recombinant E. coli is reported.
SUMMARY OF THE INVENTION
[0007] The first aspect of the current invention is a method for
the purification of a not-glycosylated, heterologous polypeptide,
which has been recombinantly produced in a prokaryotic cell,
wherein the method comprises the following three chromatography
steps in the following order: [0008] a) a first chromatography step
selected from [0009] i) hydrophobic charge induction
chromatography, [0010] ii) hydrophobic interaction chromatography,
[0011] iii) affinity chromatography, or [0012] iv) ion exchange
chromatography, [0013] b) a second chromatography step selected
from [0014] i) anion exchange chromatography, [0015] ii) cation
exchange chromatography, [0016] iii) hydroxylapatite
chromatography, [0017] iv) hydrophobic interaction chromatography,
or [0018] v) hydrophobic charge induction chromatography, [0019] c)
a third chromatography step selected from [0020] i) hydrophobic
charge induction chromatography, [0021] ii) anion exchange
chromatography, [0022] iii) cation exchange chromatography, or
[0023] iv) hydrophobic interaction chromatography, [0024] whereby
[0025] the first chromatography step is an affinity chromatography
in case of polypeptides capable of interacting with metal ligands,
[0026] the second chromatography step is not a hydroxylapatite
chromatography step in case of polypeptides with an isoelectric
point below 6.0, [0027] the third chromatography step can be
performed in flow-through mode with polypeptides having a low or
high isoelectric point, [0028] optionally the third chromatography
step can be used for concentration of the polypeptide, [0029] and
the purified not-glycosylated, heterologous polypeptide is obtained
after step c).
[0030] The method according to the invention comprises at least
three chromatography steps, whereby for each step a chromatography
material can be selected independently of the chromatography
material selected for the previous step or for the following step,
whereby only the given provisos have to be taken into account.
Thus, the method according to the invention provides for a flexible
and exchangeable sequence of chromatography steps for the
purification of a not-glycosylated polypeptide, whereby the
obtained purity after subjecting the not-glycosylated polypeptide
to the method according to the invention is comparable
independently of the selected chromatography step sequence.
[0031] In one embodiment is the prokaryotic cell an E. coli cell.
In another embodiment is the affinity chromatography a metal
chelating chromatography. In a further embodiment comprises the
method an additional step either after step a) or after step b) or
after step c) which is d) PEGylating said polypeptide. In one
embodiment said steps a) and b) are cation exchange chromatography.
In still a further embodiment is the not-glycosylated, heterologous
polypeptide selected from growth factor agonists or antagonists, or
interferons or interferon variants.
[0032] A second aspect of the current invention is a method for the
recombinant production of a not-glycosylated heterologous
polypeptide in a prokaryotic cell, wherein the method comprises the
following steps: [0033] a) cultivating a prokaryotic cell
comprising a nucleic acid encoding a heterologous polypeptide under
conditions suitable for the expression of the heterologous
polypeptide, [0034] b) recovering the heterologous polypeptide from
the culture medium or the prokaryotic cells, [0035] c) purifying
the heterologous polypeptide with a method according to the
invention and thereby obtaining a not-glycosylated heterologous
polypeptide.
[0036] In one embodiment the methods according to the invention are
characterized in that at least two different sequences of three
chromatographic steps yield a purified not-glycosylated,
heterologous polypeptide with comparable purity. In one embodiment
the third chromatography step can be performed in flow-through mode
with polypeptides having a low, i.e. 6.0 or lower, or high, i.e.
8.0 or higher, isoelectric point.
DETAILED DESCRIPTION OF THE INVENTION
[0037] General chromatographic methods and their use are known to a
person skilled in the art. See for example, Chromatography,
5.sup.th edition, Part A: Fundamentals and Techniques, Heftmann, E.
(ed), Elsevier Science Publishing Company, New York, (1992);
Advanced Chromatographic and Electromigration Methods in
Biosciences, Deyl, Z. (ed.), Elsevier Science BV, Amsterdam, The
Netherlands, (1998); Chromatography Today, Poole, C. F., and Poole,
S. K., Elsevier Science Publishing Company, New York, (1991),
Scopes, Protein Purification: Principles and Practice (1982);
Sambrook, J., et al. (ed), Molecular Cloning: A Laboratory Manual,
Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989; Current Protocols in Molecular Biology,
Ausubel, F. M., et al. (eds)., John Wiley & Sons, Inc., New
York; or Freitag, R., Chromatographical processes in the downstream
processing of (recombinant) proteins, Meth. Biotechnol. 24 (2007)
421-453 (Animal cell biotechnology 2.sup.nd Edition).
[0038] Methods for purifying polypeptides are well established and
widespread used. They are employed either alone or in combination.
Such methods are, for example, affinity chromatography using thiol
ligands with complexed metal ions (e.g. with Ni(II)- and
Cu(II)-affinity material) or microbial-derived proteins (e.g.
protein A or protein G affinity chromatography), ion exchange
chromatography (e.g. cation exchange (carboxymethyl resins), anion
exchange (amino ethyl resins) and mixed-mode exchange
chromatography), thiophilic adsorption (e.g. with
beta-mercaptoethanol and other SH ligands), hydrophobic interaction
or aromatic adsorption chromatography (e.g. with phenyl-sepharose,
aza-arenophilic resins, or m-aminophenylboronic acid), size
exclusion chromatography, and preparative electrophoretic methods
(such as gel electrophoresis, capillary electrophoresis)
(Vijayalakshmi, M. A., Appl. Biochem. Biotech. 75 (1998)
93-102).
[0039] The term "buffered" as used within this application denotes
a solution in which changes of pH due to the addition or release of
acidic or basic substances is leveled by a buffer substance. Any
buffer substance resulting in such an effect can be used.
Preferably pharmaceutically acceptable buffer substances are used,
such as e.g. phosphoric acid or salts thereof, acetic acid or salts
thereof, citric acid or salts thereof, morpholine or salts thereof,
2-(N-morpholino) ethanesulfonic acid or salts thereof, histidine or
salts thereof, glycine or salts thereof, or Tris (hydroxymethyl)
aminomethane (TRIS) or salts thereof. Especially preferred are
phosphoric acid or salts thereof, or acetic acid or salts thereof,
or citric acid or salts thereof, or histidine or salts thereof.
Optionally the buffered solution may comprise an additional salt,
such as e.g. sodium chloride, sodium sulphate, potassium chloride,
potassium sulfate, sodium citrate, or potassium citrate.
[0040] The term "membrane" as used within this application denotes
both a microporous or macroporous membrane. The membrane itself is
composed of a polymeric material such as, e.g. polyethylene,
polypropylene, ethylene vinyl acetate copolymers,
polytetrafluoroethylene, polycarbonate, poly vinyl chloride,
polyamides (nylon, e.g. Zetapore.TM., N.sub.66 Posidyne.TM.),
polyesters, cellulose acetate, regenerated cellulose, cellulose
composites, polysulphones, polyethersulfones, polyarylsulphones,
polyphenylsulphones, polyacrylonitrile, polyvinylidene fluoride,
non-woven and woven fabrics (e.g. Tyvek.RTM.), fibrous material, or
of inorganic material such as zeolithe, SiO.sub.2, Al.sub.2O.sub.3,
TiO.sub.2, or hydroxylapatite.
[0041] The term "chromatography material" as used within this
application denotes on the one hand a solid material that can be
used without further modification as chromatography material, such
as hydroxylapatite or affinity chromatography material, and also
material comprising a bulk core material to which chromatographical
functional groups are attached, preferably by covalent bonds. The
bulk core material is understood to be not involved in the
chromatography process, i.e. the interaction between the
polypeptide to be separated and the chromatographical functional
groups of the chromatography material. It is merely providing a
three dimensional framework to which the chromatographical
functional groups are attached and which ensures that the solution
containing the substance to be separated can access the
chromatographical functional group. Preferably said bulk core
material is a solid phase. Thus, preferably said "chromatography
material" is a solid phase to which chromatographical functional
groups are attached, preferably by covalent bonds. Preferably said
"chromatographical functional group" is an ionizable hydrophobic
group, or a hydrophobic group, or a complex group in which
different chromatographical functional groups are combined in order
to bind only a certain type of polypeptide, or a covalently bound
charged group.
[0042] A "solid phase" denotes a non-fluid substance, and includes
particles (including microparticles and beads) made from materials
such as polymer, metal (paramagnetic, ferromagnetic particles),
glass, and ceramic; gel substances such as silica, alumina, and
polymer gels; zeolites and other porous substances. A solid phase
may be a stationary component, such as a packed chromatography
column, or may be a non-stationary component, such as beads and
microparticles. Such particles include polymer particles such as
polystyrene and poly(methylmethacrylate); gold particles such as
gold nanoparticles and gold colloids; and ceramic particles such as
silica, glass, and metal oxide particles. See for example Martin,
C. R., et al., Analytical Chemistry-News & Features, May 1
(1998) 322A-327A.
[0043] The terms "hydrophobic charge induction chromatography" or
"HCIC", which can be used interchangeably within this application,
denote a chromatography method which employs a "hydrophobic charge
induction chromatography material". A "hydrophobic charge induction
chromatography material" is a chromatography material which
comprises chromatographical function groups which can in one pH
range form hydrophobic bonds to the substance to be separated and
which are charged either positively or negatively in other pH
ranges, i.e. HCIC uses ionizable hydrophobic groups as
chromatographical functional group. Generally the polypeptide is
bound to the hydrophobic charge induction material under neutral pH
conditions and recovered afterwards by the generation of charge
repulsion by a change of the pH value. An exemplary "hydrophobic
charge induction chromatography materials" is BioSepra MEP or HEA
Hypercel (Pall Corp., USA).
[0044] The terms "hydrophobic interaction chromatography" or "HIC",
which can be used interchangeably within this application, denote a
chromatography method in which a "hydrophobic interaction
chromatography material" is employed. A "hydrophobic interaction
chromatography material" is a chromatography material to which
hydrophobic groups, such as butyl-, octyl-, or phenyl-groups, are
bound as chromatographical functional groups. The polypeptides are
separated depending on the hydrophobicity of their surface exposed
amino acid side chains, which can interact with the hydrophobic
groups of the hydrophobic interaction chromatography material. The
interactions between polypeptides and the chromatography material
can be influenced by temperature, solvent, and ionic strength of
the solvent. A temperature increase e.g. supports the interaction
between the polypeptide and the hydrophobic interaction
chromatography material as the motion of the amino acid side chains
increases and hydrophobic amino acid side chains buried inside the
polypeptide at lower temperatures become accessible. Also is the
hydrophobic interaction promoted by kosmotropic salts and decreased
by chaotropic salts. "Hydrophobic interaction chromatography
materials" are e.g. Phenylsepharose CL-4B, 6 FF, HP, Phenyl
Superose, Octylsepharose CL-4B, 4 FF, and Butylsepharose 4 FF (all
available from Amersham Pharmacia Biotech Europe GmbH, Germany),
which are obtained via glycidyl-ether coupling to the bulk
material.
[0045] The term "affinity chromatography" as used within this
application denotes a chromatography method which employs an
"affinity chromatography material". In an affinity chromatography
the polypeptides are separated based on their biological activity
or chemical structure depending of the formation of electrostatic
interactions, hydrophobic bonds, and/or hydrogen bond formation to
the chromatographical functional group. To recover the specifically
bound polypeptide from the affinity chromatography material either
a competitor ligand is added or the chromatography conditions, such
as pH value, polarity or ionic strength of the buffer are changed.
An "affinity chromatography material" is a chromatography material
which comprises a complex chromatographical functional group in
which different single chromatographical functional groups are
combined in order to bind only a certain type of polypeptide. This
chromatography material specifically binds a certain type of
polypeptide depending on the specifity of its chromatographical
functional group. Exemplary "affinity chromatographical materials"
are a "metal chelating chromatography material" such as Ni(II)-NTA
or Cu(II)-NTA containing materials, for the binding of fusion
polypeptides containing a hexahistidine tag or polypeptides with a
multitude of surface exposed histidine, cysteine, and/or
tryptophane residues, or an "antibody binding chromatography
material" such a protein A, or an "enzyme binding chromatography
material" such as chromatography materials comprising enzyme
substrate analogues, enzyme cofactors, or enzyme inhibitors as
chromatographical functional group, or a "lectin binding
chromatography material" such as chromatography materials
comprising polysaccharides, cell surface receptors, glycoproteins,
or intact cells as chromatographical functional group.
[0046] The term "metal chelating chromatography" as used within
this application denotes a chromatography method which employs a
"metal chelating chromatography material". Metal chelating
chromatography is based on the formation of chelates between a
metal ion, such as Cu(II), Ni(II) or Zn(II), which is bound to a
bulk material as chromatographical functional groups, and electron
donor groups of surface exposed amino acid side chains of
polypeptides, especially with imidazole containing side chains and
thiol group containing side chains. The chelate is formed at pH
values at which those side chains are at least partly not
protonated. The bound polypeptide is recovered from the
chromatography material by a change in the pH value, i.e. by
protonation. Exemplary "metal chelating chromatography materials"
are HiTrap Chelating HP (Amersham Pharmacia Biotec Europe GmbH,
Germany), or Fraktogel EMD (EMD Chemicals Inc, USA).
[0047] The term "ion exchange chromatography" as used within this
application denotes a chromatography method which employs an "ion
exchange chromatography material". The term "ion exchange
chromatography material" encompasses depending whether a cation is
exchanged in a "cation exchange chromatography" a "cation exchange
chromatography material" or an anion is exchanged in an "anion
exchange chromatography" an "anion exchange chromatography
material". The term "ion exchange chromatography material" as used
within this application denotes an immobile high molecular weight
solid phase that carries covalently bound charged groups as
chromatographical functional groups. For overall charge neutrality
not covalently bound counter ions are associated therewith. The
"ion exchange chromatography material" has the ability to exchange
its not covalently bound counter ions for similarly charged ions of
the surrounding solution. Depending on the charge of its
exchangeable counter ions the "ion exchange chromatography
material" is referred to as "cation exchange chromatography
material" or as "anion exchange chromatography material". Further
depending on the nature of the charged group the "ion exchange
chromatography material" is referred to as e.g. in the case of
cation exchange chromatography materials with sulfonic acid groups
(S), or carboxymethyl groups (CM). Depending on the chemical nature
of the charged group the "ion exchange chromatography material" can
additionally be classified as strong or weak ion exchange
chromatography material, depending on the strength of the
covalently bound charged substituent. For example, strong cation
exchange chromatography materials have a sulfonic acid group as
chromatographical functional group and weak cation exchange
chromatography materials have a carboxylic acid group as
chromatographical functional group. "Cation exchange chromatography
materials", for example, are available under different names from a
multitude of companies such as e.g. Bio-Rex, Macro-Prep CM
(available from Biorad Laboratories, Hercules, Calif., USA), weak
cation exchanger WCX 2 (available from Ciphergen, Fremont, Calif.,
USA), Dowex.RTM. MAC-3 (available from Dow chemical company--liquid
separations, Midland, Mich., USA), Mustang C (available from Pall
Corporation, East Hills, N.Y., USA), Cellulose CM-23, CM-32, CM-52,
hyper-D, and partisphere (available from Whatman plc, Brentford,
UK), Amberlite.RTM. IRC 76, IRC 747, IRC 748, GT 73 (available from
Tosoh Bioscience GmbH, Stuttgart, Germany), CM 1500, CM 3000
(available from BioChrom Labs, Terre Haute, Ind., USA), and
CM-Sepharose.TM. Fast Flow (available from GE Healthcare--Amersham
Biosciences Europe GmbH, Freiburg, Germany).
[0048] The term "hydroxylapatite chromatography" as used within
this application denotes a chromatography method that employs a
certain form of calcium phosphate as chromatography material.
Exemplary hydroxylapatite chromatography materials are Bio-Gel HT,
Bio-Gel HTP, Macro-Prep Ceramic (available from Biorad
Laboratories), Hydroxylapatite Type I, Type II, HA Ultrogel (Sigma
Aldrich Chemical Corp., USA), Hydroxylapatite Fast Flow and High
Resolution (Calbiochem), or TSK gel HA-1000 (Tosoh Haas Corp.,
USA)
[0049] A "polypeptide" is a polymer of amino acid residues joined
by peptide bonds, whether produced naturally or synthetically.
Polypeptides of less than about 20 amino acid residues are referred
to as "peptides." A "protein" is a molecule comprising one or more
polypeptide chains whereof at least one comprises 100 or more amino
acid residues. Polypeptides and protein may also comprise non-amino
acid components, such as carbohydrate groups. Carbohydrate groups
and other non-amino acid components may be added by the cell in
which the polypeptide or protein is produced, and will vary with
the type of cell. Polypeptides and proteins are defined herein in
terms of their amino acid backbone structures; substituents such as
carbohydrate groups are generally not specified, but may be present
nonetheless.
[0050] The terms "antibody" and "immunoglobulin", which can be used
interchangeably within this application, denote a molecule
generally comprising two light chains and two heavy chains. Each of
the heavy and light chains comprises a variable region (generally
the amino terminal portion of the chain) which contains specific
binding regions (CDR, complementary determining region) which
interacts with the antigen. Each of the heavy and light chains also
comprises a constant region (generally, the carboxyl terminal
portion of the chains) which may mediate the binding of the
immunoglobulin to host tissues or factors including various cells
of the immune system, some phagocytic cells and a first component
(Clq) of the classical complement system. Typically, the light and
heavy chains of an immunoglobulin are complete chains, each
consisting essentially of a variable region and a complete constant
region. Generally a light chain comprises a light chain variable
domain, a hinge region, and a light chain constant domain, whereas
a heavy chain comprises a heavy chain variable domain, a hinge
region, and a heavy chain constant domain consisting of a C.sub.H1
domain, a C.sub.H2 domain, a C.sub.H3 domain, and optionally a
C.sub.H4 domain. Antibodies may exist in a variety of forms,
including, for example, Fv, Fab, and F(ab).sub.2 as well as single
chains (e.g. Huston, J. S., et al., Proc. Natl. Acad. Sci. USA 85
(1988) 5879-5883; Bird et al., Science 242 (1988) 423-426; and, in
general, Hood et al., Immunology, Benjamin N.Y., 2nd edition (1984)
and Hunkapiller and Hood, Nature 323 (1986) 15-16). Depending on
the amino acid sequence of the constant region of the heavy chain
are immunoglobulins assigned to different classes: IgA, IgD, IgE,
IgG, and IgM. Some of these classes are further divided into
subclasses (isotypes), i.e. IgG in IgG 1, IgG2, IgG3, and IgG4, or
IgA in IgA1 and IgA2. According to the immunoglobulin class to
which an immunoglobulin belongs the heavy chain constant regions of
immunoglobulins are called .alpha. (IgA), .delta. (IgD), .epsilon.
(IgE), .gamma. (IgG), and .mu. (IgM), respectively.
[0051] The term "bind-and-elute mode" and grammatical equivalents
thereof as used in the current invention denotes an operation mode
of a chromatography method, in which a solution containing a
substance of interest is brought in contact with a stationary
phase, preferably a solid phase, whereby the substance of interest
binds to the stationary phase. As a result the substance of
interest is retained on the stationary phase whereas substances not
of interest are removed with the flow-through or the supernatant.
The substance of interest is afterwards eluted from the stationary
phase in a second step and thereby recovered from the stationary
phase with an elution solution. This does not necessarily denote
that 100% of the substances not of interest are removed but
essentially 100% of the substances not of interest are removed,
i.e. at least 50% of the substances not of interest are removed,
preferably at least 75% of the substances not of interest are
removed, preferably at least 90% of the substances not of interest
are removed, preferably more than 95% of the substances not of
interest are removed.
[0052] The term "flow-through mode" and grammatical equivalents
thereof as used within the current invention denotes an operation
mode of a chromatography method, in which a solution containing a
substance of interest is brought in contact with a stationary
phase, preferably a solid phase, whereby the substance of interest
does not bind to that stationary phase. As a result the substance
of interest is obtained either in the flow-through or the
supernatant. Substances not of interest, which were also present in
the solution, bind to the stationary phase and are removed from the
solution. This does not necessarily denote that 100% of the
substances not of interest are removed from the solution but
essentially 100% of the substances not of interest are removed,
i.e. at least 50% of the substances not of interest are removed
from the solution, preferably at least 75% of the substances not of
interest are removed from the solution, preferably at least 90% of
the substances not of interest are removed from the solution,
preferably more than 95% of the substances not of interest are
removed from the solution.
[0053] The terms "continuous elution" and "continuous elution
method", which are used interchangeably within this application,
denote a chromatography method wherein e.g. the concentration of a
substance causing elution, i.e. the dissolution of a bound
substance from a chromatography material, is raised or lowered
continuously, i.e. the concentration is changed by a sequence of
small steps each not bigger than a change of 2%, preferably of 1%,
of the concentration of the substance causing elution. In this
"continuous elution" one or more conditions, for example the pH,
the ionic strength, concentration of a salt, and/or the flow of a
chromatography method, may be changed linearly, or changed
exponentially, or changed asymptotically. Preferably the change is
linear.
[0054] The terms "step elution" and "step elution method", which
are used interchangeably within this application, denote a
chromatography method wherein e.g. the concentration of a substance
causing elution, i.e. the dissolution of a bound substance from a
chromatography material, is raised or lowered at once, i.e.
directly from one value/level to the next value/level. In this
"step elution" one or more conditions, for example the pH, the
ionic strength, concentration of a salt, and/or the flow of a
chromatography method, is/are changed all at once from a first,
e.g. starting, value to a second, e.g. final, value. The change in
the step is bigger than a change of 5%, preferably of 10%, of the
concentration of the substance causing elution. "Step elution"
denotes that the conditions are changed incrementally, i.e.
stepwise, in contrast to a linear change. In the "step elution
method" is after each increase a new fraction collected. After each
increase the conditions are maintained till the next step in the
elution method.
[0055] An "isolated polypeptide" is a polypeptide that is
essentially free from contaminating cellular components, such as
carbohydrate, lipid, or other proteinaceous impurities associated
with the polypeptide in nature. Typically, a preparation of an
isolated polypeptide contains the polypeptide in a highly purified
form, i.e. at least about 80% pure, at least about 90% pure, at
least about 95% pure, greater than 95% pure, or greater than 99%
pure. One way to show that a particular protein preparation
contains an isolated polypeptide is by the appearance of a single
band following sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis of the protein preparation and Coomassie Brilliant
Blue staining of the gel. However, the term "isolated" does not
exclude the presence of the same polypeptide in alternative
physical forms, such as dimers, derivatized forms, not correctly
folded forms, not correctly disulfide bridged forms, or scrambled
forms.
[0056] "Heterologous DNA" or "heterologous polypeptide" refers to a
DNA molecule or a polypeptide, or a population of DNA molecules or
a population of polypeptides that do not exist naturally within a
given cell. DNA molecules heterologous to a particular cell may
contain DNA derived from the cell's species (i.e. endogenous DNA)
so long as that cell's DNA is combined with non-cell's DNA (i.e.
exogenous DNA). For example, a DNA molecule containing a non-cell's
DNA segment encoding a polypeptide operably linked to a cell's DNA
segment comprising a promoter is considered to be a heterologous
DNA molecule. Conversely, a heterologous DNA molecule can comprise
an endogenous structural gene operably linked with an exogenous
promoter. A peptide or polypeptide encoded by a non-cell's DNA
molecule is a "heterologous" peptide or polypeptide.
[0057] It has now surprisingly been found that with the method
according to the invention the purification of not-glycosylated
polypeptides, which have been produced recombinantly by a
prokaryotic cell, can be performed. It has been found that only a
small defined number of a maximum of three chromatography steps is
required and also that only a defined number of different
chromatography methods have to be tested in order to establish a
chromatographic purification process with allows the purification
of a not-glycosylated, recombinantly produced polypeptide to a
purity that allows the use of said not-glycosylated, recombinantly
produced polypeptide for therapeutic purposes.
[0058] Therefore, the current invention provides in a first aspect
a method for purifying a not-glycosylated, heterologous
polypeptide, which has been recombinantly produced in a prokaryotic
cell, comprising the following steps in the following order: [0059]
a) a first chromatography step selected from [0060] i) hydrophobic
charge induction chromatography, [0061] ii) hydrophobic interaction
chromatography, [0062] iii) affinity chromatography, or [0063] iv)
ion exchange chromatography, [0064] b) a second chromatography step
selected from [0065] i) anion exchange chromatography, [0066] ii)
cation exchange chromatography, [0067] iii) hydroxylapatite
chromatography, or [0068] iv) hydrophobic interaction
chromatography, or [0069] v) hydrophobic charge induction
chromatography, [0070] c) a third chromatography step selected from
[0071] i) hydrophobic charge induction chromatography, [0072] ii)
anion exchange chromatography, [0073] iii) cation exchange
chromatography, or [0074] iv) hydrophobic interaction
chromatography.
[0075] In one embodiment is the purified not-glycosylated,
heterologous polypeptide obtained after step c) of the method
according to the invention. Due to the different characteristics of
different polypeptides, which are depending on its physical
properties, such as e.g. the isoelectric point (Ip) or the
distribution of surface exposed amino acid residues, not all
chromatography methods are suited for all polypeptides. Therefore
the following provisos apply to the method according to the
invention: [0076] the first chromatography step is an affinity
chromatography or a hydrophobic charge induction chromatography in
case of polypeptides capable of interacting with metal ligands,
[0077] the second chromatography step is not a hydroxylapatite
chromatography step in case of polypeptides with an isoelectric
point below 6.0, [0078] the third chromatography step can be
performed in flow-through mode with polypeptides having a low or
high isoelectric point, [0079] optionally the third chromatography
step can be used to concentrate the polypeptide solution.
[0080] The method according to the current invention will be
exemplified in the following. These examples are only presented to
exemplify the method according to the current invention but not to
restrict the scope of the invention, which is presented in the
appended claims.
IGF-1 Agonist
[0081] A first exemplary polypeptide is an IGF-1 agonist as
reported e.g. in WO 2006/066891.
[0082] For the purification of the IGF-1 agonist a sequence of
three chromatography steps according to the method according to the
invention have been performed. The sequence comprises the
chromatography steps: [0083] 1) hydrophobic charge induction
chromatography (a-i), [0084] 2) hydroxylapatite chromatography
(b-iii), and [0085] 3) hydrophobic charge induction chromatography
(c-i).
[0086] This sequence fulfills the provisos for the method according
to the invention as the polypeptide has a hexahistidine tag and an
isoelectric point above 6.0.
[0087] The starting material had a purity of 50% (determined by
HPLC) of the IGF-1 agonist. After performing the purification
method according to the invention with the chromatography steps as
outlined above a purity of more than 97% (determined by HPLC) has
been obtained. All three chromatography steps have been performed
in a bind-and-elute mode.
[0088] To show the versatility of the method according to the
invention the IGF-1 agonist has also been purified with a different
sequence of chromatography steps according to the method according
to the invention which are: [0089] 1) hydrophobic interaction
chromatography (a-ii), [0090] 2) cation exchange chromatography
(b-ii), and [0091] 3) anion exchange chromatography (c-ii).
[0092] After performing the purification method according to the
invention with the chromatography steps as outlined above a purity
of 97% (determined by HPLC) has been obtained. The first and second
chromatography steps have been performed in a bind-and-elute mode
and the third chromatography step has been performed in
flow-through-mode.
[0093] Thus, it has surprisingly been found that with different
sequences of three chromatography steps the same molecule can be
purified to a similar purity. Furthermore has been found that the
final chromatography step can be performed in different elution
modes, i.e. in a bind-and-elute mode or in a flow-through mode.
Also has been found that the different chromatography steps can be
performed either as step elution or as continuous elution.
[0094] Thus, another aspect of the current invention is a method
for the purification of a polypeptide, especially of IGF-1 or an
IGF-1 variant as reported in WO 2006/066891, comprising a sequence
of three successive chromatography steps whereby the first
chromatography step is a hydrophobic charge induction
chromatography, the second chromatography step is selected from
hydroxylapatite chromatography or cation exchange chromatography,
and the third chromatography step is selected from hydrophobic
charge induction chromatography or anion exchange
chromatography.
Interferon
[0095] A second exemplary polypeptide is interferon alpha-2a
(IFN.alpha.-2a) as reported e.g. in EP 0 043 980.
[0096] For the purification of the IFN.alpha.-2a a sequence of
three chromatography steps according to the method according to the
invention have been performed. The sequence comprises the
chromatography steps: [0097] 1) hydrophobic charge induction
chromatography (a-i), [0098] 2) anion exchange chromatography
(b-ii), and [0099] 3) hydrophobic interaction chromatography
(c-iv).
[0100] This sequence fulfills the provisos for the method according
to the invention as the recombinantly produced IFN.alpha.-2a has no
tag for the interaction with a metal chelating chromatography
material and has an isoelectric point above 6.0.
[0101] The starting material had a purity of 49% (determined by
HPLC). After performing the purification method according to the
invention with the chromatography steps as outlined above a purity
of more than 99% (determined by HPLC) has been obtained. The
chromatography steps have been performed in a bind-and-elute
mode.
[0102] Thus, another aspect of the current invention is a method
for the purification of IFN.alpha.-2a comprising a sequence of
three successive chromatography steps, whereby the first
chromatography step is a hydrophobic charge induction
chromatography step, the second chromatography step is an anion
exchange chromatography step, and the third chromatography step is
an hydrophobic charge induction chromatography step.
[0103] The IFN.alpha.-2a has also been purified for comparison with
a different sequence of chromatography steps: [0104] 1) hydrophobic
interaction chromatography (a-ii), [0105] 2) cation exchange
chromatography (b-ii), and [0106] 3) hydrophobic interaction
chromatography (c-iv).
[0107] After performing the purification method with the
chromatography steps as outlined above a purity of more than 97%
(determined by HPLC) has been obtained.
[0108] Thus, another aspect of the current invention is a method
for the purification of IFN.alpha.-2a comprising a sequence of
three successive chromatography steps, whereby the first
chromatography step is a hydrophobic interaction chromatography,
the second chromatography step is a cation exchange chromatography
step, and the third chromatography step is an hydrophobic
interaction chromatography.
PEGylated Interferon
[0109] The method according to the invention is not only applicable
to not-glycosylated, recombinantly produced polypeptides, it is
further more also suitable for the production of PEGylated,
not-glycosylated polypeptides. For exemplary PEGylated interferon
see e.g. EP 0 809 996.
[0110] Thus, another aspect of the current invention is a method
for producing a not-glycosylated, PEGylated, heterologous
polypeptide, which has been recombinantly produced in a prokaryotic
cell comprising the following steps in the following order: [0111]
a) providing a not-glycosylated, heterologous polypeptide, which
has been recombinantly produced in a prokaryotic cell, [0112] b) a
first chromatography step selected from [0113] i) hydrophobic
charge induction chromatography, [0114] ii) hydrophobic interaction
chromatography, [0115] iii) affinity chromatography, or [0116] iv)
ion exchange chromatography, [0117] c) a second chromatography step
selected from [0118] i) anion exchange chromatography, [0119] ii)
cation exchange chromatography, [0120] iii) hydroxylapatite
chromatography, or [0121] iv) hydrophobic interaction
chromatography, [0122] d) a third chromatography step selected from
[0123] i) hydrophobic charge induction chromatography, [0124] ii)
anion exchange chromatography, [0125] iii) cation exchange
chromatography, or [0126] iv) hydrophobic interaction
chromatography, whereby said not-glycosylated, heterologous
polypeptide is obtained after PEGylation after step d).
[0127] Due to the different characteristics of different
polypeptides, which are depending on its physical properties, such
as e.g. the isoelectric point (Ip) or the distribution of surface
exposed amino acid residues, not all chromatography methods are
suited for all polypeptides. Therefore the following provisos apply
to the method according to the invention: [0128] the first
chromatography step is an affinity chromatography or a hydrophobic
charge induction chromatography in case of polypeptides capable of
interacting with metal ligands, [0129] the second chromatography
step is not a hydroxylapatite chromatography step in case of
polypeptides with an isoelectric point below 6.0, [0130] the third
chromatography step can be performed in flow-through mode with
polypeptides having a low or high isoelectric point.
[0131] An exemplary PEGylated IFN is reported in EP 0 809 996.
[0132] For the production of the PEGylated IFN a sequence of three
chromatography steps according to the method according to the
invention have been performed. The sequences comprises the
chromatography steps: [0133] 1) hydrophobic interaction
chromatography (b-ii), [0134] 2) cation exchange chromatography
(c-ii), and [0135] 3) anion exchange chromatography (d-ii), and
after step 3) the purified not-glycosylated and not-PEGylated IFN
is PEGylated.
[0136] The starting material had a purity of 58% (determined by
HPLC). After performing the purification method according to the
invention with the chromatography steps as outlined above a purity
of more than 90% (determined by HPLC) has been obtained. All the
chromatography steps have been performed in a bind-and-elute
mode.
[0137] To show the versatility of the production method according
to the invention also with PEGylated polypeptides the IFN has also
been purified prior to PEGylation with a further sequence of
chromatography steps according to the method according to the
invention: [0138] 1) metal affinity chromatography (b-iii), [0139]
2) cation exchange chromatography (c-ii), and [0140] 3) anion
exchange chromatography (d-ii), and after step 3) the purified
not-glycosylated and not-PEGylated IFN is PEGylated.
[0141] After performing the purification method according to the
invention with the chromatography steps as outlined above a purity
of more than 90% (determined by HPLC) has been obtained. All the
chromatography steps have been performed in a bind-and-elute
mode.
[0142] Thus, another aspect of the current invention is a method
for the production of a PEGylated IFN.alpha.-2a comprising a
sequence of three successive chromatography steps whereby the first
chromatography step is selected from hydrophobic interaction
chromatography or metal affinity chromatography, the second
chromatography step is a cation exchange chromatography, and the
third chromatography step is an anion exchange chromatography and
wherein after the third chromatography step the purified
not-glycosylated and not-PEGylated IFN is PEGylated.
[0143] The production of PEGylated IFN has also been performed for
comparison with a different sequence of three chromatography steps:
[0144] 1) hydrophobic interaction chromatography (b-ii), [0145] 2)
cation exchange chromatography (c-ii), and [0146] 3) hydrophobic
charge induction chromatography (d-i), and after step 3) the
purified not-glycosylated and not-PEGylated IFN is PEGylated.
[0147] After performing the purification method with the
chromatography steps as outlined above a purity of 89% (determined
by HPLC) has been obtained.
[0148] Thus, another aspect of the current invention is a method
for the production of PEGylated interferon, especially
IFN.alpha.-2a, comprising a sequence of three successive
chromatography steps whereby the first chromatography step is
hydrophobic interaction chromatography, the second chromatography
step is a cation exchange chromatography, and the third
chromatography step is an hydrophobic charge induction
chromatography and wherein after the third chromatography step the
purified not-glycosylated and not-PEGylated IFN is PEGylated.
[0149] Escherichia, Salmonella, Streptomyces or Bacillus are, for
example, suitable as prokaryotic host organisms. In one embodiment
is the prokaryotic cell an E. coli cell. Preferably the E. coli
cell is an E. coli XL1-blue cell, or an E. coli BL21(DE3) cell, or
an E. coli K-12 cell. In another embodiment is the
not-glycosylated, heterologous polypeptide selected from growth
factor agonists or antagonists, or interferons or interferon
variants.
[0150] Another aspect of the current invention is a method for the
recombinant production of a not-glycosylated heterologous
polypeptide in a prokaryotic cell, characterized in that said
method comprises the following steps: [0151] a) cultivating a
prokaryotic cell comprising a nucleic acid encoding said
heterologous polypeptide under conditions suitable for the
expression of said heterologous polypeptide, [0152] b) recovering
said heterologous polypeptide from the culture medium or the
prokaryotic cells, [0153] c) purifying said heterologous
polypeptide with a method comprising the following steps in the
following order: [0154] .alpha.) a first chromatography step
selected from [0155] i) hydrophobic charge induction
chromatography, [0156] ii) hydrophobic interaction chromatography,
[0157] iii) affinity chromatography, or [0158] iv) ion exchange
chromatography, [0159] .beta.) a second chromatography step
selected from [0160] i) anion exchange chromatography, [0161] ii)
cation exchange chromatography, [0162] iii) hydroxylapatite
chromatography, or [0163] iv) hydrophobic interaction
chromatography, or [0164] v) hydrophobic charge induction
chromatography, [0165] .gamma.) a third chromatography step
selected from [0166] i) hydrophobic charge induction
chromatography, [0167] ii) anion exchange chromatography, [0168]
iii) cation exchange chromatography, or [0169] iv) hydrophobic
interaction chromatography.
[0170] In one embodiment is the not-glycosylated heterologous
polypeptide obtained after step c). Due to the different
characteristics of different polypeptides, which are depending on
its physical properties, such as e.g. the isoelectric point (Ip) or
the distribution of surface exposed amino acid residues, not all
chromatography methods are suited for all polypeptides. Therefore
the following provisos apply to the method according to the
invention: [0171] the first chromatography step is an affinity
chromatography or a hydrophobic charge induction chromatography in
case of polypeptides capable of interacting with metal ligands,
[0172] the second chromatography step is not a hydroxylapatite
chromatography step in case of polypeptides with an isoelectric
point below 6.0, [0173] the third chromatography step can be
performed in flow-through mode with polypeptides having a low or
high isoelectric point.
[0174] The term "under conditions suitable" as used within this
application denotes conditions which are used for the cultivation
of a cell expressing a polypeptide and which are known to or can
easily be determined by a person skilled in the art. It is known to
a person skilled in the art that these conditions may vary
depending on the type of cell cultivated and type of polypeptide
expressed. In general the cell is cultivated at a temperature, e.g.
between 20.degree. C. and 40.degree. C., and for a period of time
sufficient to allow effective production, e.g. for of from 4 to 28
days.
[0175] In one embodiment said chromatographic steps are performed
in bind and elute mode. The term "bind and elute mode" as used in
the current invention denotes an operation mode of a purification
method, in which a solution containing a substance of interest to
be purified is brought in contact with a stationary phase,
preferably a solid phase, whereby the substance of interest binds
to the stationary phase. As a result the substance of interest is
retained on the stationary phase whereas substances not of interest
are removed with the flow-through or the supernatant. The substance
of interest is afterwards optionally after a washing step eluted
from the stationary phase in a second step and thereby recovered
from the stationary phase with an elution solution.
[0176] The term "PEGylating" means the formation of a covalent
linkage of a (polyethylene) glycol residue at the N-terminus of the
polypeptide and/or an internal lysine residue. PEGylation of
proteins is widely known in the state of the art and reviewed by,
for example, Veronese, F. M., Biomaterials 22 (2001) 405-417. PEG
can be linked using different functional groups and polyethylene
glycols with different molecular weight, linear and branched PEGs
as well as different linking groups (see also Francis, G. E., et
al., Int. J. Hematol. 68 (1998) 1-18; Delgado, C., et al., Crit.
Rev. Ther. Drug Carrier Systems 9 (1992) 249-304). Activated PEG
derivatives are known in the art and are described in, for example,
Morpurgo, M., et al., J. Bioconjug. Chem. 7 (1996) 363-368, for
PEG-vinylsulfone. Linear chain and branched chain PEG species are
suitable for the preparation of the PEGylated fragments. Examples
of reactive PEG reagents are iodo-acetyl-methoxy-PEG, or
methoxy-PEG-vinylsulfone.
[0177] In one embodiment of the methods according to the current
invention is the content of endotoxins, and/or E. coli DNA, and/or
E. coli cell proteins reduced in the polypeptide solution obtained
after the third chromatography step compared to the content prior
to the first chromatography step.
[0178] In another embodiment is the method according to the
invention a method for the recombinant production of a
not-glycosylated heterologous polypeptide in a prokaryotic cell via
inclusion bodies, whereby the method comprises the following steps:
[0179] a) cultivating a prokaryotic cell comprising a nucleic acid
encoding said heterologous polypeptide under conditions suitable
for the expression of said heterologous polypeptide and formation
of inclusion bodies containing said heterologous polypeptide,
[0180] b) recovering said inclusion bodies from the prokaryotic
cells, [0181] c) solubilizing and renaturating said heterologous
polypeptide from said inclusion bodies, [0182] d) purifying said
heterologous polypeptide with a method according to the first
aspect of the current invention.
[0183] In one embodiment is the not-glycosylated heterologous
polypeptide obtained after step d). Inclusion bodies are found in
the cytoplasm and contain the expressed polypeptide in an
aggregated form insoluble in water. Usually, such proteins of
inclusion bodies are in a denatured form (e.g., randomly linked
disulfide bridges). These inclusion bodies are separated from other
cell components, for example by centrifugation after cell lysis.
According to the invention, the inclusion bodies are washed under
denaturing conditions. Such denaturing agents are well known in the
state of the art and are, for example, highly concentrated
solutions of guanidinium hydrochloride (e.g. about 6 mol/l) or urea
(e.g. about 8 mol/l). The denaturing agent is preferably used as a
buffered solution. After washing, the inclusion bodies are
solubilized.
[0184] The term "PEGylation" means a covalent linkage of a poly
(ethylene glycol) residue at the N-terminus of the polypeptide
and/or an internal lysine residue. PEGylation of proteins is widely
known in the state of the art and reviewed by, for example,
Veronese, F. M., Biomaterials 22 (2001) 405-417. PEG can be linked
using different functional groups and polyethylene glycols with
different molecular weight, linear and branched PEGs as well as
different linking groups (see also Francis, G. E., et al., Int. J.
Hematol. 68 (1998) 1-18; Delgado, C., et al., Crit. Rev. Ther. Drug
Carrier Systems 9 (1992) 249-304). PEGylation can be performed in
aqueous solution with PEGylation reagents as described, for
example, in WO 00/44785, in one embodiment by using NHS-activated
linear or branched PEG molecules of a molecular weight between 5
kDa and 40 kDa. PEGylation can also be performed at the solid phase
according to Lu, Y., et al., Reactive Polymers 22 (1994) 221-229.
Not randomly, N-terminally PEGylated polypeptide can also be
produced according to WO 94/01451.
[0185] Activated PEG derivatives are known in the art and are
described in, for example, Morpurgo, M., et al., J. Bioconjug.
Chem. 7 (1996) 363-368, for PEG-vinylsulfone. Linear chain and
branched chain PEG species are suitable for the preparation of the
PEGylated fragments. Examples of reactive PEG reagents are
iodo-acetyl-methoxy-PEG, or methoxy-PEG-vinylsulfone (m is
preferably an integer from about 450 to about 900 and R is a
C.sub.1- to C.sub.6-alkyl, linear or branched, having one to six
carbon atoms such as methyl, ethyl, isopropyl, etc. whereby in one
embodiment R=methyl):
##STR00001##
[0186] The use of these iodo-activated substances is known in the
art and described e.g. by Hermanson, G. T., in Bioconjugate
Techniques, Academic Press, San Diego (1996) p. 147-148.
[0187] In one embodiment is the PEG species an activated PEG ester,
e.g., N-hydroxysuccinimidyl propionate, or N-hydroxysuccinimidyl
butanoate, or N-hydroxysuccinimides such as PEG-NHS (Monfardini,
C., et al., Bioconjugate Chem. 6 (1995) 62-69). In one embodiment
the activated N-hydroxysuccinimide ester is
##STR00002##
using alkoxy-PEG-N-hydroxysuccinimide, such as
methoxy-PEG-N-hydroxysuccinimide (MW 30000; Shearwater Polymers,
Inc.), wherein R and m are as defined above. In one embodiment the
PEG species is the N-hydroxysuccinimidyl ester of methoxy poly
(ethylene glycol)-butyric acid. The term "alkoxy" refers to an
alkyl ether group in which the term `alkyl` means a straight-chain
or branched-chain alkyl group containing a maximum of four carbon
atoms, such as methoxy, ethoxy, n-propoxy and the like, preferably
methoxy.
[0188] One aspect of the invention is a method for the purification
of a not-glycosylated, heterologous polypeptide, which has been
recombinantly produced in a prokaryotic cell, characterized in that
said method comprises the following steps in the following order:
[0189] a) a first chromatography step selected from [0190] i)
hydrophobic charge induction chromatography, [0191] ii) hydrophobic
interaction chromatography, [0192] iii) affinity chromatography, or
[0193] iv) ion exchange chromatography, [0194] b) a second
chromatography step selected from [0195] i) anion exchange
chromatography, [0196] ii) cation exchange chromatography, [0197]
iii) hydroxylapatite chromatography, or [0198] iv) hydrophobic
interaction chromatography, or [0199] v) hydrophobic charge
induction chromatography, [0200] c) a third chromatography step
selected from [0201] i) hydrophobic charge induction
chromatography, [0202] ii) anion exchange chromatography, [0203]
iii) cation exchange chromatography, or [0204] iv) hydrophobic
interaction chromatography, [0205] whereby [0206] said first
chromatography step is an affinity chromatography or a hydrophobic
charge induction chromatography in case of polypeptides capable of
interacting with metal ligands, [0207] said second chromatography
step is not a hydroxylapatite chromatography step in case of
polypeptides with an isoelectric point below 6.0, [0208] said third
chromatography step can be performed in flow-through mode with
polypeptides having a low or high isoelectric point, [0209] and
with the proviso that the combination of three chromatographic
steps is not [0210] affinity chromatography, ion exchange
chromatography and hydrophobic interaction chromatography, [0211]
hydrophobic interaction chromatography, cation exchange
chromatography and anion exchange chromatography, [0212] cation
exchange chromatography, anion exchange chromatography and
hydrophobic interaction chromatography, [0213] cation exchange
chromatography, hydrophobic interaction chromatography and cation
exchange chromatography, [0214] anion exchange chromatography,
hydrophobic interaction chromatography and hydrophobic interaction
chromatography.
[0215] In one embodiment of the method according to the invention
is the not-glycosylated heterologous polypeptide obtained after the
third chromatography step.
[0216] The term "comparable" as used within this application
denotes that two results are within 10% of each other. For example,
a purity of 90% and a purity of 95% are comparable as 95% is within
10% of a purity of 90% (90%+10% of 90%=90%+9%=99%).
[0217] The following examples, references, and figures are provided
to aid the understanding of the present invention, the true scope
of which is set forth in the appended claims. It is understood that
modifications can be made in the procedures set forth without
departing from the spirit of the invention.
DESCRIPTION OF THE FIGURES
[0218] FIG. 1 Reversed Phase HPLC chromatogram of the IGF-1 agonist
before (a) and after (b) the first HCIC.
[0219] FIG. 2 Reversed Phase HPLC chromatogram of the IGF-1 agonist
before (a) and after (b) the hydroxylapatite chromatography
step.
[0220] FIG. 3 Reversed Phase HPLC chromatogram of the IGF-1 agonist
before (a) and after (b) the second HCIC.
[0221] FIG. 4 Reversed Phase HPLC chromatogram of the IGF-1 agonist
before (a) and after (b) the HIC.
[0222] FIG. 5 Reversed Phase HPLC chromatogram of the IGF-1 agonist
before (a) and after (b) the cation exchange chromatography
step.
[0223] FIG. 6 Reversed Phase HPLC chromatogram of the IGF-1 agonist
before (a) and after (b) the anion exchange chromatography
step.
EXPERIMENTAL PART
Material and Methods
[0224] If not otherwise indicated have the different chromatography
methods been performed according to the chromatography material
manufacturer's manual.
Recombinant DNA Techniques:
[0225] Standard methods were used to manipulate DNA as described in
Sambrook, J., et al., Molecular cloning: A laboratory manual; Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The
molecular biological reagents were used according to the
manufacturer's instructions.
Protein Determination:
[0226] Protein concentration was determined by determining the
optical density (OD) at 280 nm, with a reference wavelength of 320
nm, using the molar extinction coefficient calculated on the basis
of the amino acid sequence.
Size-Exclusion-HPLC:
[0227] The chromatography was conducted with a Tosoh Haas TSK 3000
SWXL column on an ASI-100 HPLC system (Dionex, Idstein, Germany).
The elution peaks were monitored at 280 nm by a UV diode array
detector (Dionex). After dissolution of the concentrated samples to
1 mg/ml the column was washed with a buffer consisting of 200 mM
potassium dihydrogen phosphate and 250 mM potassium chloride pH 7.0
until a stable baseline was achieved. The analyzing runs were
performed under isocratic conditions using a flow rate of 0.5
ml/min. over 30 minutes at room temperature. The chromatograms were
integrated manually with Chromeleon (Dionex, Idstein, Germany).
Reversed Phase HPLC(RP-HPLC):
[0228] The purity is analyzed by RP-HPLC. The assay is performed on
a Poroshell column using an acetonitrile/aqueous TFA gradient. The
elution profile is monitored as UV absorbance at 215 nm. The
percentages of the eluted substances are calculated based upon the
total peak area of the eluted proteins.
DNA-Threshold-System:
[0229] see e.g. Merrick, H., and Hawlitschek, G., Biotech Forum
Europe 9 (1992) 398-403
Host Cell Protein Determination:
[0230] The walls of the wells of a micro titer plate are coated
with a mixture of serum albumin and Streptavidin. A goat derived
polyclonal antibody against HCP is bound to the walls of the wells
of the micro titer plate. After a washing step different wells of
the micro titer plate are incubated with a HCP calibration sequence
of different concentrations and sample solution. After the
incubation not bound sample material is removed by washing with
buffer solution. For the detection the wells are incubated with an
antibody peroxidase conjugate to detect bound host cell protein.
The fixed peroxidase activity is detected by incubation with ABTS
and detection at 405 nm.
General Method for the Isolation, Solubilization and Renaturation
of Inclusion Bodies:
[0231] In addition to the method performed in the cited literature
can the preparation of inclusion bodies e.g. be performed according
the method by Rudolph et al. (Rudolph et al., Folding Proteins, In:
T. E. Creighton (ed.): Protein function: A Practical Approach, 57
(1996)). The inclusion bodies were stored at -70.degree. C.
Solubilization of the inclusion bodies can likewise be performed
according the method by Rudolph et al. (Rudolph et al., Folding
Proteins, In: T. E. Creighton (ed.): Protein function: A Practical
Approach, 57 (1996)).
Example 1
Purification of an IGF-1 Agonist
[0232] The polypeptide was expressed in E. coli. The polypeptide is
first applied to a HCIC column, then to a hydroxylapatite column
and finally to a second HCIC column.
[0233] The chromatographic conditions were as follows:
1.sup.st Column:
[0234] Resin: HCIC with MEP-Hypercel (Pall Corporation, USA) as
single step elution [0235] Loading: 10 mg polypeptide per ml of
column volume [0236] Buffer A: 25 mM tris(hydroxymethyl)amino
methane buffer, adjusted to pH 9.0 [0237] Buffer B: 10 mM sodium
acetate buffer, adjusted to pH 5.0
[0238] The solution containing the IGF-1 agonist was applied in a
first step to a column containing a hydrophobic charge induction
chromatography material (MEP-Hypercel from Pall Corporation).
[0239] In FIG. 1 the reversed phase chromatogram of the IGF-1
agonist before and after HCIC is presented.
2.sup.nd Column:
[0240] Resin: Hydroxylapatite chromatography (Biorad Laboratories,
USA) [0241] Loading: 6.5 mg polypeptide per ml of column volume
[0242] Buffer A: 5 mM potassium phosphate buffer, adjusted to pH
6.5 [0243] Buffer B: 10 mM MES buffer supplemented with 1 M sodium
chloride, adjusted to pH 6.5
[0244] FIG. 2 presents the reversed phase chromatogram before and
after the hydroxylapatite chromatography step.
3.sup.rd Column:
[0245] Resin: HCIC with HEA-Hypercel (Pall Corporation, USA) [0246]
Loading: 20 mg polypeptide per ml of column volume [0247] Buffer A:
20 mM sodium acetate buffer, adjusted to pH 4.0
[0248] FIG. 3 presents the reversed phase chromatogram before and
after the second HCIC step.
TABLE-US-00001 Purity determined Column Yield [%] by HPLC [%] Start
49.2 HCIC step 15.7 86.6 Hydroxylapatite 62.5 97.0 chromatography
step HCIC step 94.2 97.7
Example 2
Purification of IGF-1 Agonist--Comparative Example to Example 1
[0249] The polypeptide is first applied to a HIC column, followed
by a cation exchange chromatography and finally to an anion
exchange chromatography operated in flow-through mode.
1.sup.st Column:
[0250] Resin: HIC with Super Butyl Toyopearl (Tosoh Haas Corp.,
USA) [0251] Loading: 5 mg polypeptide per ml of column volume
[0252] Buffer A: 50 mM potassium phosphate buffer supplemented with
1 M potassium chloride, adjusted to pH 8.0 [0253] Buffer B:
2-propanol 5-10% (w/v), adjusted to pH 4.0
[0254] Elution was performed with a linear gradient over 30 column
volumes from 0% (v/v) to 100% (v/v) of buffer B.
[0255] FIG. 4 presents the reversed phase chromatogram before and
after the HIC step.
2.sup.nd Column:
[0256] Resin: Cation exchange chromatography with CM-Sepharose FF
(GE-Healthcare., USA) [0257] Loading: 4.1 mg polypeptide per ml of
column volume [0258] Buffer A: 50 mM acetic acid, adjusted to pH
5.8 [0259] Buffer B: 100 mM tris(hydroxymethyl)amino methane buffer
supplemented with 1 M sodium chloride, adjusted to pH 9.5
[0260] Elution was performed as follows: change to 15% (v/v) buffer
B at the start, maintaining 15% (v/v) buffer B for 5 column
volumes, afterwards a linear gradient to 55% (v/v) buffer B over 20
column volumes, and finally maintaining 55% (v/v) buffer B for 10
column volumes.
[0261] FIG. 5 presents the reversed phase chromatogram before and
after the cation exchange chromatography step.
3.sup.rd Column:
[0262] Resin: Anion exchange chromatography with Q-Sepharose in
flow-through mode (GE-Healthcare., USA) [0263] Loading: 20 mg
polypeptide per ml of column volume [0264] Buffer A: 25 mM
tris(hydroxymethyl)amino methane buffer, adjusted to pH 9.5 [0265]
Buffer B: 10 mM acetic acid (pH 3.6)
[0266] FIG. 6 presents the reversed phase chromatogram before and
after the anion exchange chromatography step.
TABLE-US-00002 Purity determined Column Yield [%] by HPLC [%] Start
49.6 HIC step 16.7 approx. 90 Cation exchange 14.2 90.1
chromatography step Anion exchange 93.8 97.0 chromatography
step
Example 3
Purification of Interferon
[0267] The polypeptide is first applied to a HIC column, then to an
anion exchange column and finally to a cation exchange column.
[0268] The chromatographic conditions were as follows:
1.sup.st Column:
[0269] Resin: HIC with Butyl Sepharose (GE-Healthcare, USA) as
single step elution [0270] Loading: 8 mg polypeptide per ml of
column volume [0271] Buffer A: 20 mM potassium phosphate buffer,
adjusted to pH 8.0
2.sup.nd Column:
[0271] [0272] Resin: Anion exchange chromatography with Q-Sepharose
FF [0273] (GE-Healthcare, USA) [0274] Loading: 1.5 mg polypeptide
per ml of column volume [0275] Buffer A: 30 mM ammonium acetate,
adjusted to pH 5.9 [0276] Buffer B: 1.8 mM ammonium acetate,
adjusted to pH 3.5
[0277] Elution was performed as follows: change to 15% (v/v) buffer
B at the start, maintaining 15% (v/v) buffer B for 3 column
volumes, and afterwards a linear gradient to 90% (v/v) buffer B
over 37.5 column volumes.
3.sup.rd Column:
[0278] Resin: Cation exchange chromatography with SP-Sepharose as
single step elution [0279] Loading: 2.84 mg polypeptide per ml of
column volume [0280] Buffer A: 50 mM borate buffer supplemented
with 250 mM sodium chloride, adjusted to pH 9.0
TABLE-US-00003 [0280] Purity determined Column Yield [%] by HPLC
[%] Start 48.8 HIC step 13.4 69.3 Anion exchange 55.0 97.5
chromatography step Cation exchange 89.5 Approx. 100 chromatography
step
Example 4
Purification of Interferon--Comparative Example to Example 3
[0281] The polypeptide is first applied to a HIC column, then to a
cation exchange column and finally to an anion exchange column.
[0282] The chromatographic conditions were as follows:
1.sup.st Column:
[0283] Resin: HIC with Butyl Sepharose (GE-Healthcare, USA) [0284]
Loading: 8 mg polypeptide per ml of column volume [0285] Buffer A:
20 mM potassium phosphate buffer supplemented with 2 m potas-sium
chloride, adjusted to pH 8.0 [0286] Buffer B: 20 mM potassium
phosphate buffer, adjusted to pH 8.0
2.sup.nd Column:
[0286] [0287] Resin: Cation exchange chromatography with CM
Toyopearl [0288] (Tosoh Hass Corp., USA) [0289] Loading: 5 mg
polypeptide per ml of column volume [0290] Buffer A: equilibration:
75 mM sodium acetate, adjusted to pH 4.0 wash: 15 mM sodium
acetate, adjusted to pH 5.5 [0291] Buffer B: 30 mM sodium acetate,
adjusted to pH 7.0
3.sup.rd Column:
[0291] [0292] Resin: Anion exchange chromatography with Q-Sepharose
[0293] Loading: 3 mg polypeptide per ml of column volume [0294]
Buffer A: 30 mM ammonium acetate buffer, adjusted to pH 6.8 [0295]
Buffer B: 1) 25 mM ammonium acetate, adjusted to pH 6.5 [0296] 2)
1.8 mM ammonium acetate supplemented with 3 mM acetic acid,
adjusted to pH 4.5
TABLE-US-00004 [0296] Purity determined Column Yield [%] by HPLC
[%] Start 58 HIC step 2.3 71.6 Cation exchange 28.3 88.3
chromatography step Anion exchange 95.3 93.2 chromatography
step
Example 5
Purification of Interferon--Comparative Example to Examples 3 and
4
[0297] The polypeptide is first applied to a metal chelating
column, then to a cation exchange column and finally to an anion
exchange column.
[0298] The chromatographic conditions were as follows:
1.sup.st Column:
[0299] Resin: Copper chelating Sepharose (GE-Healthcare, USA) as
single step elution [0300] Loading: 51 mg polypeptide per ml of
column volume [0301] Buffer A: equilibration: 300 mM guanidinium
hydrochloride supplemented with 150 mM sodium chloride and 20 mM
sodium phosphate buffer, adjusted to pH 6.45 wash: 50 mM acetic
acid supplemented with 100 mM sodium chloride, adjusted to pH 4.95
[0302] Buffer B: 50 mM acetic acid supplemented with 100 mM sodium
chloride, adjusted to pH 3.9
2.sup.nd Column:
[0302] [0303] Resin: Cation exchange chromatography with CM
Toyopearl [0304] (Tosoh Hass Corp., USA) as single step elution
[0305] Loading: 5 mg polypeptide per ml of column volume [0306]
Buffer A: equilibration: 75 mM sodium acetate, adjusted to pH 4.0
wash: 15 mM sodium acetate, adjusted to pH 5.5 [0307] Buffer B: 30
mM sodium acetate, adjusted to pH 7.0
3.sup.rd Column:
[0308] Resin: Anion exchange chromatography with Q-Sepharose
Loading: 3 mg polypeptide per ml of column volume Buffer A: 30 mM
ammonium acetate buffer, adjusted to pH 6.8 Buffer B: 1) 25 mM
ammonium acetate, adjusted to pH 6.5 [0309] 2) 1.8 mM ammonium
acetate supplemented with 3 mM acetic acid, adjusted to pH 4.5
[0310] Elution was performed as follows: change to 10% (v/v) buffer
B at the start, maintaining 15% (v/v) buffer B for 3 column
volumes, and afterwards a linear gradient to 90% (v/v) buffer B
over 27.5 column volumes.
TABLE-US-00005 Purity determined Column Yield [%] by HPLC [%] Start
47.3 Metal chelating 3.2 59.8 chromatography step Cation exchange
28.3 88.3 chromatography step Anion exchange 95.3 93.2
chromatography step
Sequence CWU 1
1
21105PRTHomo sapiensMOD_RES(27)..(27)Any naturally occurring amino
acid 1Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln
Phe1 5 10 15Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Xaa Pro Thr Gly
Tyr Gly 20 25 30Ser Ser Ser Arg Xaa Ala Pro Gln Thr Gly Ile Val Asp
Glu Cys Cys 35 40 45Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr
Cys Ala Pro Leu 50 55 60Xaa Pro Ala Xaa Ser Ala Arg Ser Val Arg Ala
Gln Arg His Thr Asp65 70 75 80Met Pro Lys Thr Gln Lys Glu Val His
Leu Lys Asn Ala Ser Arg Gly 85 90 95Ser Ala Gly Asn Lys Asn Tyr Arg
Met 100 1052165PRTHomo sapiens 2Cys Asp Leu Pro Gln Thr His Ser Leu
Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala Gln Met Arg Lys Ile
Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30Arg His Asp Phe Gly Phe Pro
Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys Ala Glu Thr Ile Pro
Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60Asn Leu Phe Ser Thr
Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65 70 75 80Leu Asp Lys
Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90 95Ala Cys
Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys 100 105
110Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu
115 120 125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val
Val Arg 130 135 140Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn
Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu 165
* * * * *