U.S. patent application number 13/588281 was filed with the patent office on 2013-02-28 for cation and anion exchange chromatography method.
This patent application is currently assigned to Hoffman-La Roche. Inc.. The applicant listed for this patent is Roberto Falkenstein, Maria Laura Magri, Michaela Mehr, Klaus Schwendner, Bernhard Spensberger. Invention is credited to Roberto Falkenstein, Maria Laura Magri, Michaela Mehr, Klaus Schwendner, Bernhard Spensberger.
Application Number | 20130053551 13/588281 |
Document ID | / |
Family ID | 46717838 |
Filed Date | 2013-02-28 |
United States Patent
Application |
20130053551 |
Kind Code |
A1 |
Falkenstein; Roberto ; et
al. |
February 28, 2013 |
CATION AND ANION EXCHANGE CHROMATOGRAPHY METHOD
Abstract
Herein is reported a method for purifying a polypeptide
comprising the steps of i) applying a solution comprising the
polypeptide to an ion exchange chromatography material, and ii)
recovering the polypeptide with a solution comprising a denaturant
and thereby purifying the polypeptide, whereby the ion exchange
chromatography material comprises a matrix of cross-linked poly
(styrene-divinylbenzene) to which ionic ligands have been attached,
and wherein the solution comprising the polypeptide applied to the
ion exchange chromatography material is free of the denaturant and
the polypeptide adsorbed to the ion exchange chromatography
material is recovered with a solution comprising a denaturant at a
constant conductivity.
Inventors: |
Falkenstein; Roberto;
(Muenchen, DE) ; Magri; Maria Laura; (Penzberg,
DE) ; Mehr; Michaela; (Uffing, DE) ;
Schwendner; Klaus; (Weilheim, DE) ; Spensberger;
Bernhard; (Eberfing, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Falkenstein; Roberto
Magri; Maria Laura
Mehr; Michaela
Schwendner; Klaus
Spensberger; Bernhard |
Muenchen
Penzberg
Uffing
Weilheim
Eberfing |
|
DE
DE
DE
DE
DE |
|
|
Assignee: |
Hoffman-La Roche. Inc.
Nutley
NJ
|
Family ID: |
46717838 |
Appl. No.: |
13/588281 |
Filed: |
August 17, 2012 |
Current U.S.
Class: |
530/416 ;
435/69.1 |
Current CPC
Class: |
C07K 1/18 20130101; B01D
15/363 20130101; B01D 15/362 20130101; C07K 14/775 20130101 |
Class at
Publication: |
530/416 ;
435/69.1 |
International
Class: |
C07K 1/18 20060101
C07K001/18; C12P 21/00 20060101 C12P021/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 25, 2011 |
EP |
11178745.3 |
Oct 4, 2011 |
EP |
11183862.9 |
Claims
1. A method for purifying a polypeptide comprising the step of:
recovering the polypeptide with a solution comprising a denaturant
from an ion exchange chromatography material at constant
conductivity and thereby purifying the polypeptide, whereby the ion
exchange chromatography material comprises a matrix of cross-linked
poly (styrene-divinylbenzene) to which ionic ligands have been
attached.
2. A method for producing a polypeptide comprising the following
steps: cultivating a prokaryotic or eukaryotic cell comprising a
nucleic acid encoding the polypeptide, recovering the polypeptide
from the cells or/and the cultivation medium, purifying the
polypeptide with an ion exchange chromatography method comprising
the following step recovering the polypeptide with a solution
comprising a denaturant from the ion exchange chromatography
material and thereby producing a polypeptide, whereby the ion
exchange chromatography material comprises a matrix of cross-linked
poly (styrene-divinylbenzene) to which ionic ligands have been
attached.
3. The method of claim 1, characterized in that the denaturant is
one denaturant and is selected from the group comprising urea,
guanidine, urea-derivatives, and guanidine-derivatives.
4. The method of claim 2, characterized in that the denaturant is
one denaturant and is selected from the group comprising urea,
guanidine, urea-derivatives, and guanidine-derivatives.
5. The method of claim 1, characterized in that the ion exchange
chromatography material is a cation exchange chromatography
material.
6. The method of claim 2, characterized in that the ion exchange
chromatography material is a cation exchange chromatography
material.
7. The method of claim 5, characterized in that the ionic ligand is
a sulfopropyl ligand or a carboxymethyl ligand.
8. The method of claim 6, characterized in that the ionic ligand is
a sulfopropyl ligand or a carboxymethyl ligand.
9. The method of claim 1, characterized in that the ion exchange
chromatography material is an anion exchange chromatography
material.
10. The method of claim 2, characterized in that the ion exchange
chromatography material is an anion exchange chromatography
material.
11. The method of claim 9, characterized in that the ionic ligand
is an ethyleneimine ligand or a quaternized ligand.
12. The method of claim 10, characterized in that the ionic ligand
is an ethyleneimine ligand or a quaternized ligand.
13. The method of claim 1, further characterized in comprising the
following steps: wherein recovering the polypeptide from the ion
exchange chromatography comprises a first ion exchange
chromatography material by applying a solution comprising a
denaturant, applying the recovered polypeptide to a second ion
exchange chromatography material, and recovering the polypeptide
from the second ion exchange chromatography material by applying a
solution comprising a denaturant and thereby obtaining or purifying
the polypeptide.
14. The method of claim 2, further characterized in comprising the
following steps: wherein recovering the polypeptide from the ion
exchange chromatography comprises a first ion exchange
chromatography material by applying a solution comprising a
denaturant, applying the recovered polypeptide to a second ion
exchange chromatography material, and recovering the polypeptide
from the second ion exchange chromatography material by applying a
solution comprising a denaturant and thereby obtaining or purifying
the polypeptide.
15. The method according to claim 13, characterized in that the
first ion exchange chromatography material is an anion exchange
chromatography material and the second ion exchange chromatography
material is a cation exchange chromatography material, or the first
ion exchange chromatography material is a cation exchange
chromatography material and the second ion exchange chromatography
material is an anion exchange chromatography material.
16. The method according to claim 14, characterized in that the
first ion exchange chromatography material is an anion exchange
chromatography material and the second ion exchange chromatography
material is a cation exchange chromatography material, or the first
ion exchange chromatography material is a cation exchange
chromatography material and the second ion exchange chromatography
material is an anion exchange chromatography material.
17. The method of claim 13, characterized in that the recovered
polypeptide is refolded prior to the applying to the second ion
exchange chromatography material.
18. The method of claim 14, characterized in that the recovered
polypeptide is refolded prior to the applying to the second ion
exchange chromatography material.
19. The method of claim 1, wherein the purifying further comprises
the following steps: applying a first solution to the ion exchange
chromatography material, applying a second solution comprising the
polypeptide to the ion exchange chromatography material, and
recovering and thereby producing or purifying the polypeptide with
a fourth solution comprising a denaturant, whereby the first
solution comprises a first buffer substance, the second solution
comprises a second buffer substance, and the fourth solution
comprises a fourth buffer substance, wherein the fourth buffer
substance comprises a denaturant.
20. The method of claim 2, wherein the purifying further comprises
the following steps: applying a first solution to the ion exchange
chromatography material, applying a second solution comprising the
polypeptide to the ion exchange chromatography material, and
recovering and thereby producing or purifying the polypeptide with
a fourth solution comprising a denaturant, whereby the first
solution comprises a first buffer substance, the second solution
comprises a second buffer substance, and the fourth solution
comprises a fourth buffer substance, wherein the fourth buffer
substance comprises a denaturant.
21. The method according to claim 19, characterized in that after
applying the second solution and prior to applying the fourth
solution the following step is added: applying a third solution to
the ion exchange chromatography material, whereby the third
solution comprises a third buffer substance.
22. The method of claim 1, characterized in that the polypeptide
has an amino acid sequence selected from the group of amino acid
sequences of SEQ ID NO: 01, SEQ ID NO: 02, SEQ ID NO: 03, and SEQ
ID NO: 04.
23. The method of claim 2, characterized in that the polypeptide
has an amino acid sequence selected from the group of amino acid
sequences of SEQ ID NO: 01, SEQ ID NO: 02, SEQ ID NO: 03, and SEQ
ID NO: 04.
24. The method of claim 1, wherein the denaturant is a urea or a
urea-derivate.
25. The method of claim 2, wherein the denaturant is a urea or a
urea-derivate.
Description
RELATED APPLICATION
[0001] This application claims the benefit of European Patent
Application No. EP 11178745.3 filed Aug. 25, 2011, and European
Patent Application No. 11183862.9 filed Oct. 4, 2011, which is
fully incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Proteins play an important role in today's medical
portfolio. Expression systems for the production of recombinant
polypeptides are well-known in the state of the art. Polypeptides
for use in pharmaceutical applications are mainly produced in
prokaryotic cells, such as E. coli, and mammalian cells such as CHO
cells, NS0 cells, Sp2/0 cells, COS cells, HEK cells, BHK cells,
PER.C6.RTM. cells, and the like.
[0003] For human application every pharmaceutical substance has to
meet distinct criteria. To ensure the safety of biopharmaceutical
agents to humans, for example, nucleic acids, viruses, and host
cell proteins, which would cause severe harm, have to be removed.
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.
[0004] Different methods are well established and widespread used
for protein purification, such as affinity chromatography with
microbial proteins (e.g. protein A or protein G affinity
chromatography), ion exchange chromatography (e.g. cation exchange
(sulfopropyl or carboxymethyl resins), anion exchange (amino ethyl
resins) and mixed-mode ion exchange), 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), metal chelate affinity chromatography (e.g. with Ni(II)- and
Cu(II)-affinity material), size exclusion chromatography, and
electrophoretical methods (such as gel electrophoresis, capillary
electrophoresis) (see e.g. Vijayalakshmi, M. A., Appl. Biochem.
Biotech. 75 (1998) 93-102). Necina, R., et al. (Biotechnol. Bioeng.
60 (1998) 689-698) reported the capture of human monoclonal
antibodies directly from cell culture supernatants by ion exchange
media exhibiting high charge density. In WO 89/05157 a method is
reported for the purification of product immunoglobulins by
directly subjecting the cell culture medium to a cation exchange
treatment. A one-step purification of monoclonal IgG antibodies
from mouse ascites is described by Danielsson, A., et al., J.
Immun. Meth. 115 (1988) 79-88. A method for purifying a polypeptide
by ion exchange chromatography is reported in WO 2004/024866 in
which a gradient wash is used to resolve a polypeptide of interest
from one or more contaminants. In EP 0 530 447 a process for
purifying IgG monoclonal antibodies by a combination of three
chromatographic steps is reported. Wang, et al. (Wang, H., et al.,
Peptides 26 (2005) 1213-1218) reports the purification of hTFF3
expressed in E. coli by a two-step cation exchange
chromatography.
[0005] In WO 2010/063717 polypeptide purification is reported.
Protein purification and identification is reported in WO
2008/002235. Cole, D. R., reports the chromatography of insulin in
urea-containing buffer (J. Biol. Chem. 235 (1960) 2294-2299. In
U.S. Pat. No. 4,129,560 a process for the purification of high
molecular weight peptides using non-ionic detergents is reported. A
method for the preparation of growth hormone and antagonist thereof
having lower levels of isoform impurities thereof is reported in WO
2004/031213. In U.S. Pat. No. 6,451,987 ion exchange chromatography
of proteins and peptides is reported. A process for purifying
insulin and insulin so prepared is reported in EP 0 013 826.
[0006] Herein is reported an ion exchange chromatography method for
the purification of polypeptides by elution of the polypeptide from
the ion chromatography material with a solution comprising a
denaturant.
SUMMARY OF THE INVENTION
[0007] It has been found that a polypeptide can be recovered from
an ion exchange chromatography material (cation and/or anion
exchange chromatography material) with a solution comprising a
denaturant/chaotropic agent, whereby during the recovering of the
polypeptide from the ion exchange chromatography material the
conductivity of the applied solutions is maintained constant, i.e.
the conductivity is kept constant.
[0008] One aspect as reported herein is a method for obtaining or
purifying a polypeptide by ion exchange chromatography in
bind-and-elute mode comprising the following step: [0009]
recovering the polypeptide from the ion exchange chromatography
material by applying a solution comprising a denaturant and thereby
obtaining or purifying the polypeptide, whereby the ion exchange
chromatography material comprises a matrix of cross-linked poly
(styrene-divinylbenzene) to which ionic ligands have been
attached.
[0010] In one embodiment the method comprises the following steps:
[0011] recovering the polypeptide from a first ion exchange
chromatography material by applying a solution comprising a
denaturant, [0012] applying the recovered polypeptide to a second
ion exchange chromatography material, and [0013] recovering the
polypeptide from the second ion exchange chromatography material by
applying a solution comprising a denaturant and thereby obtaining
or purifying the polypeptide.
[0014] In one embodiment the first ion exchange chromatography
material is an anion exchange chromatography material and the
second ion exchange chromatography material is a cation exchange
chromatography material.
[0015] In one embodiment the first ion exchange chromatography
material is a cation exchange chromatography material and the
second ion exchange chromatography material is an anion exchange
chromatography material.
[0016] In one embodiment the denaturant is urea or a
urea-derivative.
[0017] In one embodiment the denaturant is a mixture of two or
three denaturants.
[0018] In one embodiment the solution applied in the recovering has
a constant conductivity.
[0019] In one embodiment the solution applied in the wash step has
a constant conductivity.
[0020] In one embodiment the solution applied in the recovering has
a constant pH-value.
[0021] In one embodiment the solution applied in the wash step has
a constant pH-value.
[0022] In one embodiment the method comprises the following steps:
[0023] applying a solution comprising the polypeptide in native
form to an ion exchange chromatography material, and [0024]
recovering the polypeptide from the ion exchange chromatography
material by applying a solution comprising a denaturant and thereby
obtaining or purifying the polypeptide.
[0025] In one embodiment the ion exchange chromatography material
is a cation exchange chromatography material. In one embodiment the
ligand is sulfopropyl or carboxymethyl.
[0026] In one embodiment the ion exchange chromatography material
is an anion exchange chromatography material. In one embodiment the
ligand is poly ethyleneimine or quaternized ethyleneimine.
[0027] In one embodiment the polypeptide is an antibody, or an
antibody fragment, or a fusion polypeptide comprising at least one
antibody domain.
[0028] In one embodiment the polypeptide is a
tetranectin-apolipoprotein A-I fusion protein. In one embodiment
the tetranectin-apolipoprotein A-I fusion protein has an amino acid
sequence of SEQ ID NO: 01, or SEQ ID NO: 02, or SEQ ID NO: 03, or
SEQ ID NO: 04.
[0029] One aspect as reported herein is a method for producing a
polypeptide comprising the following steps: [0030] cultivating a
prokaryotic or eukaryotic cell comprising a nucleic acid encoding
the polypeptide, [0031] recovering the polypeptide from the cells
or/and the cultivation medium, [0032] if the polypeptide is
recovered in the form of inclusion bodies solubilizing and/or
re-folding the polypeptide, [0033] purifying the polypeptide with
an ion exchange chromatography method as reported herein and
thereby producing the polypeptide.
DESCRIPTION OF THE FIGURES
[0034] FIG. 1 Chromatogram of a purification of
tetranectin-apolipoprotein A-I fusion protein of SEQ ID NO: 01 on
an anion exchange chromatography column with sodium chloride
conductivity gradient.
[0035] FIG. 2 Chromatogram of a purification of
tetranectin-apolipoprotein A-I fusion protein of SEQ ID NO: 01 on
an anion exchange chromatography column with urea gradient at
constant conductivity and constant pH-value.
[0036] FIG. 3 Chromatogram of a purification of
tetranectin-apolipoprotein A-I fusion protein of SEQ ID NO: 02 on
an anion exchange chromatography column with urea gradient at
constant conductivity and constant pH-value.
[0037] FIG. 4 Chromatogram of a purification of
tetranectin-apolipoprotein A-I fusion protein of SEQ ID NO: 01 on
an anion exchange chromatography column with urea wash, isopropanol
wash and guanidinium hydrochloride gradient elution.
[0038] FIG. 5 Chromatogram of a purification of
tetranectin-apolipoprotein A-I fusion protein of SEQ ID NO: 01 on
an anion exchange chromatography column with urea wash, guanidinium
hydrochloride wash and sodium chloride gradient elution.
[0039] FIG. 6 Chromatogram of a purification of
tetranectin-apolipoprotein A-I fusion protein of SEQ ID NO: 02 on
an anion exchange chromatography column with urea wash, guanidinium
hydrochloride wash and sodium chloride gradient elution.
[0040] FIG. 7 Chromatogram of a purification of
tetranectin-apolipoprotein A-I fusion protein of SEQ ID NO: 01 on a
cation exchange chromatography column with urea gradient at
constant conductivity and constant pH-value.
[0041] FIG. 8 Chromatogram of a purification of anti-TSLP receptor
antibody on an anion exchange chromatography column with Tris
buffer wash and urea gradient elution.
DETAILED DESCRIPTION OF THE INVENTION
[0042] Herein is reported a scalable ion exchange chromatography
method operated in bind-and-elute mode for the purification of
polypeptides wherein the recovering of the polypeptide from the ion
exchange chromatography material is with a solution comprising a
denaturant, wherein the conductivity of the solution used in the
recovering step is maintained constant.
[0043] The terms "applying to" and grammatical equivalents thereof
denote a partial step of a purification method in which a solution
containing a substance of interest to be purified is brought in
contact with a stationary phase. This denotes that a) the solution
is added to a chromatographic device in which the stationary phase
is located, or b) that a stationary phase is added to the solution
comprising the substance of interest. In case a) the solution
containing the substance of interest to be purified passes through
the stationary phase allowing for an interaction between the
stationary phase and the substances in solution. Depending on the
conditions, such as e.g. pH, conductivity, salt concentration,
temperature, and/or flow rate, some substances of the solution are
bound to the stationary phase and, thus, are removed from the
solution. Other substances remain in solution. The substances
remaining in solution can be found in the flow-through. The
"flow-through" denotes the solution obtained after the passage of
the chromatographic device irrespective of its origin. It can
either be the applied solution containing the substance of interest
or the buffer, which is used to flush the column or which is used
to cause the elution of one or more substances bound to the
stationary phase.
[0044] In one embodiment the chromatographic device is a column, or
a cassette. The substance of interest can be recovered from the
solution after the purification step by methods familiar to a
person of skill in the art, such as e.g. precipitation, salting
out, ultrafiltration, diafiltration, lyophilization, affinity
chromatography, or solvent volume reduction to obtain the substance
of interest in purified or even substantially homogeneous form. In
case b) the stationary phase is added, e.g. as a solid, to the
solution containing the substance of interest to be purified
allowing for an interaction between the stationary phase and the
substances in solution. After the interaction the stationary phase
is removed, e.g. by filtration, and the substance of interest is
either bound to the stationary phase and removed therewith from the
solution or the substance of interest is not bound to the
stationary phase and remains in the solution.
[0045] 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. In one
embodiment the buffer substance is selected from phosphoric acid or
salts thereof, acetic acid or salts thereof, citric acid or salts
thereof, morpholine, 2-(N-morpholino) ethanesulfonic acid or salts
thereof, imidazole or salts thereof, histidine or salts thereof,
glycine or salts thereof, or tris (hydroxymethyl) aminomethane
(TRIS) or salts thereof. In one embodiment the buffer substance is
selected from imidazole or salt thereof or histidine or salts
thereof. Optionally the buffered solution may also comprise an
additional inorganic salt. In one embodiment the inorganic salt is
selected from sodium chloride, sodium sulphate, potassium chloride,
potassium sulfate, sodium citrate, and potassium citrate.
[0046] A "polypeptide" is a polymer consisting of amino acids
joined by peptide bonds, whether produced naturally or
synthetically. Polypeptides of less than about 20 amino acid
residues may be referred to as "peptides", whereas molecules
consisting of two or more polypeptides or comprising one
polypeptide of more than 100 amino acid residues may be referred to
as "proteins". A polypeptide may also comprise non-amino acid
components, such as carbohydrate groups, metal ions, or carboxylic
acid esters. The non-amino acid components may be added by the
cell, in which the polypeptide is expressed, and may vary with the
type of cell. Polypeptides are defined herein in terms of their
amino acid backbone structure or the nucleic acid encoding the
same. Additions such as carbohydrate groups are generally not
specified, but may be present nonetheless.
[0047] The term "antibody" denotes a protein that comprises at
least two light polypeptide chains and two heavy polypeptide
chains. Each of the heavy and light polypeptide chains contains a
variable region (generally the amino terminal portion of the
polypeptide chain) which contains a binding domain for interaction
with the antigen. Each of the heavy and light polypeptide chains
also comprises a constant region (generally the carboxyl terminal
portion) which may mediate the binding of the antibody to host
tissues or factors including various cells of the immune system,
some phagocytic cells and a first component (C1q) of the classical
complement system. Typically, the light and heavy polypeptide
chains are complete chains, each consisting essentially of a
variable region, i.e. V.sub.L or V.sub.H, and a complete constant
region, i.e. of C.sub.L in case of a light polypeptide chain or of
C.sub.H1, C.sub.H2, C.sub.H3, and optionally C.sub.H4 in case of a
heavy polypeptide chain. The variable regions of the antibody
according to the invention can be grafted to constant regions of
other isotypes. For example, a polynucleotide encoding the variable
region of a heavy chain of the 1-isotype can be grafted to
polynucleotide encoding the constant region of another heavy chain
class (or subclass).
[0048] 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, R. E., et al., Science 242 (1988) 423-426; and, in
general, Hood, et al., Immunology, Benjamin N.Y., 2nd edition, The
Benjamin/Cummings Publishing Company, Inc. (1984), and Hunkapiller,
T. and Hood, L., Nature 323 (1986) 15-16). In one embodiment the
antibody is selected from monoclonal antibody, isolated heavy or
light chain, or heavy or light chains only consisting of constant
regions as well as fragments thereof.
[0049] The term "constant" denotes that a certain value is
maintained at a level with a relative change of at most 10%. In one
embodiment the conductivity of the solution in the recovering step
is maintained constant with a change of at most +/-10%. In one
embodiment the conductivity of the solution in the recovering step
is maintained constant with a change of at most +/-5%. In one
embodiment the conductivity of the solution in the recovering step
is maintained constant with a change of at most +/-2%.
[0050] The term "bind-and-elute mode" denotes a way to perform a
chromatography purification method. Herein a solution containing a
polypeptide of interest to be purified is applied to a stationary
phase, particularly a solid phase, whereby the polypeptide of
interest interacts with the stationary phase and is retained
thereon. Substances not of interest are removed with the
flow-through or the supernatant, respectively. The polypeptide of
interest is afterwards recovered from the stationary phase in a
second step by applying an elution solution.
[0051] The term "inclusion body" denotes a dense intracellular mass
of aggregated polypeptide of interest, which constitutes a
significant portion of the total cell protein, including all cell
components of a prokaryotic cell.
[0052] The term "denaturized" denotes forms of polypeptides wherein
these have a secondary, tertiary, and/or quaternary structure that
is not the native one. The polypeptide in this non-native form may
be soluble but concomitantly in a biologically inactive
conformation. Or the polypeptide may be insoluble and in a
biologically inactive conformation with e.g. mismatched or unformed
disulfide bonds. This insoluble polypeptide can be, but need not
be, contained in inclusion bodies.
[0053] The term "refolded" refers to a polypeptide obtained from a
denaturized form. Typically, the goal of refolding is to produce a
protein having a higher level of activity than the protein would
have if produced without a refolding step. A folded protein
molecule is most stable in the conformation that has the least free
energy. Most water soluble proteins fold in a way that most of the
hydrophobic amino acids are in the interior part of the molecule,
away from water. The weak bonds that hold a protein together can be
disrupted by a number of treatments that cause a polypeptide to
unfold, i.e. to denaturize. A folded protein is the product of
several types of interactions between the amino acids themselves
and their environment, including ionic bonds, Van der Waals
interactions, hydrogen bonds, disulfide bonds and covalent
bonds.
[0054] The terms "denatured" or "denaturized" as used herein refer
to a polypeptide in which ionic and covalent bonds and Van der
Waals interactions which exist in the molecule in its native or
refolded state are disrupted. Denaturation of a polypeptide can be
accomplished, for example, by treatment with 8 M urea, reducing
agents such as mercaptoethanol, heat, pH, temperature and other
chemicals. Reagents such as 8 M urea disrupt both the hydrogen
bonds and the hydrophobic bonds, and if mercaptoethanol is also
added, the disulfide bridges (S--S) which are formed between
cysteines are reduced to two --S--H groups. Refolding of
polypeptides which contain disulfide linkages in their native or
refolded state may also involve the oxidation of the --S--H groups
present on cysteine residues for the protein to reform the
disulfide bonds.
[0055] The term "chaotropic agent" or "denaturant", which can be
used interchangeably, denotes a compound that distorts the
three-dimensional structure of a polypeptide. This process is also
called denaturation. The chaotropic agent distorts/disrupts
interactions by non-covalent forces such as hydrogen bonds, or van
der Waals forces. In one embodiment the chaotropic agent is
selected from the group comprising butanol, ethanol, 1- and
2-propanol, guanidinium chloride, magnesium chloride, sodium
dodecyl/sodium lauryl sulfate, urea, and thiourea.
[0056] The term "in native form" denotes the form of a polypeptide
wherein it has a secondary, tertiary, and/or quaternary structure
in which the polypeptide has his biological activity.
[0057] The term "ion exchange chromatography material" denotes an
immobile high molecular weight matrix that carries covalently bound
charged substituents. For overall charge neutrality not covalently
bound counter ions are bound to the charged substituents by ionic
interaction. The "ion exchange chromatography material" has the
ability to exchange its not covalently bound counter ions for
similarly charged binding partners or 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". Depending on the nature of the charged
group (substituent) the "ion exchange chromatography material" is
referred to as, e.g. in the case of cation exchange materials,
sulfonic acid or sulfopropyl resin (S), or carboxymethyl resin
(CM). Depending on the chemical nature of the charged
group/substituent the "ion exchange chromatography material" can
additionally be classified as strong or weak ion exchange material,
depending on the strength of the covalently bound charged
substituent. For example, strong cation exchange materials have a
sulfonic acid group, such as a sulfopropyl group, as charged
substituent, weak cation exchange materials have a carboxylic acid
group, such as a carboxymethyl group, as charged substituent.
Strong anion exchange materials have a quarternary ammonium group,
and weak anion exchange materials have a diethylaminoethyl group as
charged substituent.
[0058] Generally, the position of an ion exchange chromatography
step is variable in a multi-step purification sequence of a
polypeptide.
[0059] 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).
[0060] The purification process of immunoglobulins in general
comprises a multistep chromatographic part. In the first step
non-immunoglobulin polypeptides and proteins are separated from the
immunoglobulin fraction by an affinity chromatography, e.g. with
protein A. Afterwards an ion exchange chromatography can be
performed. Finally a third chromatographic step can be performed to
separate immunoglobulin monomers from multimers and fragments of
the same class. Sometimes the amount of aggregates is high (5% or
more) and it is not possible to separate them efficiently in the
third purification step necessitating further purification
steps.
[0061] It has been found that a polypeptide can be recovered from
an ion exchange chromatography material, which comprises a matrix
of cross-linked poly (styrene-divinylbenzene) to which ionic
ligands have been attached, with a solution comprising a
denaturant, whereby the conductivity of the solution is kept
constant during the recovering. This finding was very surprising as
generally an increase in ionic strength is used to recover
polypeptides from ion exchange chromatography materials. At the
same time this chromatography material has sufficient binding
capacity for industrial production scale separations.
[0062] Therefore, one aspect as reported herein is a method for
obtaining or purifying a polypeptide comprising the following step:
[0063] recovering the polypeptide from an ion exchange
chromatography material by applying a solution comprising a
denaturant and thereby obtaining or purifying the polypeptide,
whereby the ion exchange chromatography material comprises a matrix
of cross-linked poly (styrene-divinylbenzene) to which ionic
ligands have been attached.
[0064] As a denaturant is used for the recovery of the bound
polypeptide the solution comprising the polypeptide which is
applied to the ion exchange chromatography material is free of
denaturants. The polypeptide retained on the ion exchange
chromatography material is recovered with a solution comprising a
denaturant such as urea or a urea-derivative and a constant
conductivity.
[0065] The method as reported herein is, thus, operated in
bind-and-elute mode, i.e. the polypeptide is first bound to the ion
exchange chromatography material and thereafter, in a further step,
recovered from the ion exchange chromatography material.
Intermittent wash steps can be included in the methods as reported
herein.
[0066] In these wash steps the applied solution(s) is (are)
substantially free of a denaturant. The term "substantially free of
a denaturant" denotes that a denaturant can be present in the
applied (wash) solution but at a concentration that is below the
concentration required for the recovery of the polypeptide from the
ion exchange material.
[0067] In the method as reported herein all solutions are free of,
i.e. do not contain, a denaturant except for the solution for
recovering the polypeptide from the ion exchange chromatography
material. In one embodiment the solution comprising the denaturant
is an aqueous solution. In a further embodiment the solution
comprising the denaturant does not comprise, i.e. it is free of, an
organic solvent and/or an aliphatic alcohol. In a further
embodiment the solution comprising the denaturant is consisting of
water, the denaturant, a buffer substance, and optionally one or
two or three inorganic salts.
[0068] The term "denaturant" or "chaotropic agent", which can be
used interchangeably within this application, denotes compounds
that transfer a polypeptide from its native form in a non-native,
i.e. denatured, form. Denaturants are generally chaotropic agents.
Exemplary denaturants are urea and urea-derivatives, guanidine and
guanidine-derivatives, tetraalkyl ammonium salts, long chain
sulfonic acid esters, and lithium perchlorate.
[0069] The addition of urea, to be more precise, the change of the
concentration of urea does not affect the conductivity of a
solution, i.e. the conductivity of a solution remains constant upon
the addition or change of the concentration of urea.
[0070] In one embodiment the denaturant is urea or a
urea-derivative.
[0071] In one embodiment the denaturant is urea. In one embodiment
the urea has a concentration of from 4 mol/l to 9 mol/l.
[0072] In one embodiment the denaturant is thiourea. In one
embodiment the thiourea has a concentration of from 1.5 mol/l to 3
mol/l.
[0073] In one embodiment the denaturant is a mixture of two or
three denaturants. In one embodiment the denaturant is a mixture of
urea and thiourea. In one embodiment the denaturant is a mixture of
urea and a guanidinium salt.
[0074] In one embodiment of the aspects as reported herein the
method for purifying or obtaining a polypeptide comprises the
following steps: [0075] applying a first solution to the ion
exchange chromatography material to produce a conditioned ion
exchange chromatography material, [0076] applying a second solution
comprising the polypeptide to the conditioned ion exchange
chromatography material, [0077] optionally applying a third
solution to the ion exchange chromatography material, [0078]
recovering and thereby purifying or obtaining the polypeptide with
a fourth solution comprising a denaturant from the ion exchange
chromatography material.
[0079] The first and second solutions are substantially free of a
denaturant. The third solution is substantially free of a
denaturant.
[0080] Polypeptides can be produced recombinantly in eukaryotic and
prokaryotic cells, such as CHO cells, HEK cells and E. coli. If the
polypeptide is produced in prokaryotic cells it is generally
obtained in the form of insoluble inclusion bodies. The inclusion
bodies can easily be recovered from the prokaryotic cell and the
cultivation medium. The polypeptide obtained in insoluble form in
the inclusion bodies has to be solubilized before purification
and/or re-folding procedure can be carried out.
[0081] Thus, a second aspect as reported herein is a method for
producing a polypeptide comprising the following steps: [0082]
cultivating a prokaryotic or eukaryotic cell comprising a nucleic
acid encoding the polypeptide, [0083] recovering the polypeptide
from the prokaryotic or eukaryotic cells or/and the cultivation
medium, [0084] optionally if the polypeptide is recovered in form
of inclusion bodies solubilizing and/or re-folding the polypeptide,
[0085] purifying the polypeptide with an ion exchange
chromatography method as reported herein and thereby producing a
polypeptide.
[0086] In one embodiment the ion exchange chromatography method
comprises the following steps: [0087] applying a first solution to
the ion exchange chromatography material to produce a conditioned
ion exchange chromatography material, [0088] applying a second
solution comprising the polypeptide to the conditioned ion exchange
chromatography material, [0089] optionally applying a third
solution (wash step) to the ion exchange chromatography material,
[0090] recovering and thereby producing the polypeptide with a
fourth solution comprising one or more denaturants from the ion
exchange chromatography material,
[0091] whereby the first to third solutions are free of
denaturants.
[0092] In the following different embodiments of all the aspects as
reported before are presented.
[0093] In one embodiment the first solution comprises a first
buffer substance, the second solution comprises a second buffer
substance, the third solution comprises a third buffer substance,
and the fourth solution comprises a fourth buffer substance,
whereby the fourth buffer substance comprises one or more
denaturants.
[0094] In one embodiment the second buffer substance and the third
buffer substance and the fourth buffer substance are all different
buffer substances.
[0095] In one embodiment the first solution and/or the second
solution and/or the third solution is/are free of a denaturant. In
one embodiment the third solution is substantially free of a
denaturant.
[0096] In one embodiment the applying of the first solution is for
3 to 20 column volumes.
[0097] In one embodiment the applying of the first solution is for
3 to 10 column volumes. In one embodiment the applying of the
second solution is for 1 to 10 column volumes.
[0098] In one embodiment the applying of the third solution is for
1 to 10 column volumes.
[0099] The ion exchange chromatography material is in the first
step conditioned with a buffered solution. This solution is free
of, i.e. does not comprise, a denaturant. The buffer substance of
the conditioning, first buffer solution can be the same or
different from the buffer substance of the second solution
comprising the polypeptide.
[0100] Thereafter a second solution comprising the polypeptide is
applied to the conditioned ion exchange chromatography material. In
this step the polypeptide is retained on the ion exchange
chromatography material. This solution does not comprise a
denaturant. The buffer substance of the loading, i.e. second,
buffer solution can be the same or different from the buffer
substance of the third solution.
[0101] After the loading of the ion exchange chromatography
material with the polypeptide optionally a washing, i.e. third,
solution can be applied to the loaded ion exchange chromatography
material. This solution is substantially free of a denaturant.
[0102] Finally for recovering the polypeptide from the ion exchange
chromatography material a recovering, i.e. fourth, solution
comprising one or more denaturants is applied to the chromatography
material.
[0103] In one embodiment the method for purifying or obtaining a
polypeptide is a column chromatography method.
[0104] In one embodiment the conductivity of the solution in the
recovering step is constant.
[0105] In one embodiment the pH-value of the solution in the
recovering step is constant.
[0106] The volume applied to the ion exchange chromatography
material in the different steps is independently of each other of
from 3 to 20 column volumes, in one embodiment of from 4 to 10
column volumes.
[0107] In one embodiment the ion exchange chromatography material
is made of polystyrene divinyl benzene derivatized with functional
groups. In one embodiment the anion exchange chromatography
material is a polystyrene divinyl benzene derivatized with
quaternized poly ethyleneimine functional groups. In one embodiment
the cation exchange chromatography material is a polystyrene
divinyl benzene derivatized with sulfopropyl functional groups.
[0108] The methods as reported herein are exemplified in the
following with a tetranectin-apolipoprotein A-I fusion protein as
reported in WO 2012/028526 and an anti-TSLP receptor antibody as
reported in WO 2012/007495.
[0109] The following examples 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.
Example 1
Material and Methods
[0110] If not otherwise indicated the different chromatography
methods have been performed according to the chromatography
material manufacturer's manual.
Recombinant DNA Techniques:
[0111] 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:
[0112] 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:
[0113] 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 min. at room temperature. The chromatograms were
integrated manually with Chromeleon (Dionex, Idstein, Germany).
Reversed Phase HPLC(RP-HPLC):
[0114] The purity is analyzed by RP-HPLC. The assay is performed on
a Phenomenex C18 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:
[0115] See e.g. Merrick, H., and Hawlitschek, G., Biotech Forum
Europe 9 (1992) 398-403.
Host Cell Protein Determination:
[0116] 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.
DNA Determination:
[0117] Biotin was bound to a microtiter plate. A reaction mixture
of streptavidin, single-stranded DNA and biotinylated
single-stranded DNA binding protein was added. The binding protein
was able to bind DNA and was biotinylated. In this manner it was
possible to specifically remove the DNA from the sample mixture.
The streptavidin bound the biotin on the microtiter plate as well
as the biotin which was coupled to the single-stranded DNA binding
protein. A DNA-specific antibody which was coupled to urease was
added to this total complex. Addition of urea resulted in a
hydrolysis of the urea which caused a local change in the pH. This
change can be detected as an altered surface potential. The change
in the surface potential was proportional to the amount of bound
DNA. Single stranded DNA was obtained by proteinase K digestion and
denaturation with SDS.
General Method for the Isolation, Solubilization and Re-Folding of
Polypeptide from Inclusion Bodies:
[0118] 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, R., et al., Folding
Proteins, In: Creighton, T.E., (ed.): Protein function: A Practical
Approach, Oxford University Press (1997) 57-99). 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, R., et al., Folding Proteins, In:
Creighton, T.E., (ed.): Protein function: A Practical Approach,
Oxford University Press (1997) 57-99).
Example 2
Comparative Example
Purification of Tetranectin-Apolipoprotein A-I Fusion Protein of
SEQ ID NO: 01 on an Anion Exchange Chromatography Column with
Sodium Chloride Conductivity Gradient
[0119] resin: POROS.RTM. HQ [0120] load: 443 mg polypeptide [0121]
column load: 30 mg/ml [0122] elution method: linear gradient 0 M to
1 M sodium chloride
Result:
[0123] As can be seen from FIG. 1 the fusion protein cannot be
obtained in a defined peak. The analytical results are shown in the
following Table.
TABLE-US-00001 TABLE c (fusion DNA ECP LAL protein) yield [pg/mg]
[ng/ml] [EU/ml] [mg/ml] [%] applied 1210000 4844100 21845 2.4
solution flow through <495458 35500 827 1.2 wash <37337 79950
549 1.6 peak 1 <2999 482490 21856 2.0 44.7 peak 2 <26786
79850 19573 0.2 3.3 post peak 23981818 35000 17476 0.1
Example 3
Purification of Tetranectin-Apolipoprotein A-I Fusion Protein of
SEQ ID NO: 01 on an Anion Exchange Chromatography Column with Urea
Gradient at Constant Conductivity and Constant pH-Value
[0124] resin: POROS.RTM. HQ [0125] load: 366 mg polypeptide [0126]
column load: 24.8 mg/ml [0127] elution method: linear gradient 0 M
to 6 M urea
Result:
[0128] As can be seen from FIG. 2 the fusion protein can be
obtained in a defined peak. The analytical results are shown in the
following Table.
TABLE-US-00002 TABLE c (fusion DNA ECP LAL protein) yield [pg/mg]
[ng/ml] [EU/ml] [mg/ml] [%] applied 989196 732700 9099 3.3 solution
flow through <60000 11933 53 0 wash <2400000 8153 140 0.03
peak 1 <28860 4999 44 2.1 56.2 peak 2 <62959 3424 20 1.0 9.4
post peak <78431 83300 2089 0.8
Example 4
Purification of Tetranectin-Apolipoprotein A-I Fusion Protein of
SEQ ID NO: 02 on an Anion Exchange Chromatography Column with Urea
Gradient at Constant Conductivity and Constant pH-Value
[0129] resin: POROS.RTM. HQ [0130] elution method: linear gradient
0 M to 6 M urea
Result:
[0131] As can be seen from FIG. 3 the fusion protein can be
obtained in a defined peak. The analytical results are shown in the
following Table.
TABLE-US-00003 TABLE c (fusion DNA ECP LAL protein) yield [pg/mg]
[ng/ml] [EU/ml] [mg/ml] [%] applied 8025 247340 1565 2.3 solution
peak 1 <2.2 137 <3 2.3 54.0 post peak 11144 24420 198 1.2
Example 5
Comparative Example
Purification of Tetranectin-Apolipoprotein A-I Fusion Protein of
SEQ ID NO: 01 on an Anion Exchange Chromatography Column with Urea
Wash, Isopropanol Wash and Guanidinium Hydrochloride Gradient
Elution
[0132] resin: Q-Sepharose.RTM. FF (GE Healthcare) [0133] load: 281
mg polypeptide [0134] column load: 15 mg/ml [0135] equilibration:
30 mM potassium phosphate buffer pH 8.0; 5.94 mS/cm [0136] urea
wash: 6 M urea solution pH 8.0; 435 .mu.S/cm [0137] 2-propanol
wash: 20% (v/v) 2-propanol [0138] elution solution: 6 M guanidinium
hydrochloride pH 8.0; LF=278 mS/cm [0139] wash steps: wash with 5
column volumes 6 M urea solution; wash with 5 column volumes 20%
2-propanol [0140] elution method: linear gradient 0 M to 6 M
guanidinium hydrochloride in 10 column volumes
Result:
[0141] As can be seen from FIG. 4 the fusion protein cannot be
obtained during the wash steps with the urea solution and the
2-propanol solution. Elution can only be effected by using the
guanidinium hydrochloride solution.
Example 6
Comparative Example
Purification of Tetranectin-Apolipoprotein A-I Fusion Protein of
SEQ ID NO: 01 on an Anion Exchange Chromatography Column with Urea
Wash, Guanidinium Hydrochloride Wash and Sodium Chloride Gradient
Elution
[0142] resin: Q-Sepharose.RTM. FF (GE Healthcare) [0143] load: 280
mg polypeptide [0144] column load: 20 mg/ml [0145] equilibration:
30 mM potassium phosphate buffer pH 8.0; 5.9 mS/cm [0146] urea
wash: 6 M urea solution pH 8.0; 435 .mu.S/cm [0147] guanidinium
hydrochloride solution: [0148] 0.1 M guanidinium hydrochloride pH
8.0 [0149] elution solution: 1 M sodium chloride in 50 mM potassium
phosphate buffer pH 8.0; 91.7 mS/cm [0150] wash steps: wash with 5
column volumes 6 M urea solution; wash with 5 column volumes 0.1 M
guanidinium hydrochloride solution [0151] elution method: linear
gradient 0 M to 1 M sodium chloride in 10 column volumes
Result:
[0152] As can be seen from FIG. 5 that in each of the wash steps
only a minor fraction of the fusion protein can be obtained. The
analytical results are shown in the following Table.
TABLE-US-00004 TABLE c (fusion DNA ECP LAL protein) yield [pg/mg]
[ng/ml] [EU/ml] [mg/ml] [%] applied 1210000 4844100 21845 4.0
solution flow through 88000 379270 4572 n.d. urea wash 69700 22220
286 n.d. guanidinium 2330 30810 1229 n.d. wash peak 1 117 308650
23484 4.3 37.7 peak 2 19040 139645 6827 1.3 13.3 n.d. = not
determined
Example 7
Comparative Example
Purification of Tetranectin-Apolipoprotein A-I Fusion Protein of
SEQ ID NO: 02 on an Anion Exchange Chromatography Column with Urea
Wash, Guanidinium Hydrochloride Wash and Sodium Chloride Gradient
Elution
[0153] resin: Q-Sepharose.RTM. FF (GE Healthcare) [0154] load: 239
mg polypeptide [0155] column load: 15 mg/ml [0156] equilibration:
30 mM potassium phosphate buffer pH 8.0; 5.9 mS/cm [0157] urea
wash: 6 M urea solution pH 8.0 [0158] guanidinium hydrochloride
solution: [0159] 0.1 M guanidinium hydrochloride pH 8.0 [0160]
elution solution: 0.35 M sodium chloride in 20 mM potassium
phosphate buffer pH 8.0 [0161] wash steps: wash with 4 column
volumes 6 M urea solution; [0162] wash with 4 column volumes 0.1 M
guanidinium hydrochloride solution [0163] elution method: step
elution with 0.35 M sodium chloride for 7 column volumes
Result:
[0164] As can be seen from FIG. 6 in each of the wash steps only a
minor fraction of the fusion protein can be obtained. The
analytical results of three runs are shown in the following
Table.
TABLE-US-00005 TABLE c (fusion DNA ECP LAL protein) yield run
[pg/mg] [ng/ml] [EU/ml] [mg/ml] [%] 1 applied 2280 477300 28115 2.6
2 solution 2280 477300 28115 2.6 3 2833 108250 216531 1.2 1 peak 1
n.d. n.d. n.d. n.d. 9.3 2 n.d. n.d. n.d. n.d. 16.1 3 n.d. n.d. n.d.
n.d. 11.1 1 peak 2 99 38550 263 0.8 31.7 2 25 45200 670 1.0 35.1 3
25 31920 38 0.9 37.1 1 post peak 66929 29520 22195 0.3 9.4 2 23265
35900 928 0.3 12.7 3 15619 11510 45 0.5 23.1 n.d. = not
determined
Example 8a
Purification of Tetranectin-Apolipoprotein A-I Fusion Protein of
SEQ ID NO: 01 on a Cation Exchange Chromatography Column with Urea
Gradient at Constant Conductivity and Constant pH-Value
[0165] resin: POROS.RTM. HS [0166] load: 5.58 g polypeptide [0167]
wash 1: 50 mM sodium formiate, adjusted to pH 3.0 [0168] wash 2: 1
M sodium chloride, 30 mM potassium phosphate buffer, adjusted to pH
8.0 [0169] wash 3: 30 mM potassium phosphate buffer, adjusted to pH
8.0 [0170] elution solution: 6 M urea in 10 mM potassium phosphate
buffer pH 8.0 [0171] elution method: wash 1 for 3 column volumes,
[0172] wash 2 for 20 column volumes, [0173] wash 3 for 5 column
volumes, [0174] linear gradient 0 M to 6 M urea in 10 column
volumes
Result:
[0175] As can be seen from FIG. 7 the fusion protein can be
obtained in a defined peak. The analytical results are shown in the
following Table.
TABLE-US-00006 TABLE c (fusion DNA ECP LAL protein) yield [pg/mg]
[ng/ml] [EU/ml] [mg/ml] [%] applied 1933426 >99661 1712 3.3
solution recovered 428784 145887 458 2.7 64.4 solution
Example 8b
Purification of Tetranectin-Apolipoprotein A-I Fusion Protein of
SEQ ID NO: 01 on a Cation Exchange Chromatography Material Followed
by an Anion Exchange Chromatography Column with Urea Gradient at
Constant Conductivity and Constant pH-Value
[0176] resin: POROS.RTM. HQ [0177] load: 3.19 g polypeptide
obtained in example 8a (see above)
Result:
[0178] The analytical results of the second chromatography step are
shown in the following Table.
TABLE-US-00007 TABLE c (fusion DNA ECP LAL protein) yield [pg/mg]
[ng/ml] [EU/ml] [mg/ml] [%] applied 354545 81055 94 0.55 solution
recovered 6 203 6 4.3 82.8 solution
Example 9
Purification of Anti-TSLP Receptor Antibody on an Anion Exchange
Chromatography Column with Tris Buffer Wash and Urea Gradient
Elution
[0179] resin: POROS.RTM. HQ [0180] load: 189 mg polypeptide [0181]
Tris wash: 5 mM Tris buffer with 10 mM sodium chloride pH 8.4; 4
mS/cm [0182] elution solution: 5 mM Tris buffer with 10 mM sodium
chloride and 6 M urea pH 8.4; 4 mS/cm [0183] wash step: wash with 3
column volumes Tris buffer solution [0184] elution method: linear
gradient 0 M to 6 M urea in 30 column volumes
Result:
[0185] As can be seen from FIG. 8 the antibody can be obtained.
Sequence CWU 1
1
41284PRTArtificial SequenceTetranectin-apolipoprotein A-I (1) 1Pro
Ile Val Asn Ala Lys Lys Asp Val Val Asn Thr Lys Met Phe Glu 1 5 10
15 Glu Leu Lys Ser Arg Leu Asp Thr Leu Ala Gln Glu Val Ala Leu Leu
20 25 30 Lys Glu Gln Gln Ala Leu Gln Thr Val Asp Glu Pro Pro Gln
Ser Pro 35 40 45 Trp Asp Arg Val Lys Asp Leu Ala Thr Val Tyr Val
Asp Val Leu Lys 50 55 60 Asp Ser Gly Arg Asp Tyr Val Ser Gln Phe
Glu Gly Ser Ala Leu Gly 65 70 75 80 Lys Gln Leu Asn Leu Lys Leu Leu
Asp Asn Trp Asp Ser Val Thr Ser 85 90 95 Thr Phe Ser Lys Leu Arg
Glu Gln Leu Gly Pro Val Thr Gln Glu Phe 100 105 110 Trp Asp Asn Leu
Glu Lys Glu Thr Glu Gly Leu Arg Gln Glu Met Ser 115 120 125 Lys Asp
Leu Glu Glu Val Lys Ala Lys Val Gln Pro Tyr Leu Asp Asp 130 135 140
Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys Val 145
150 155 160 Glu Pro Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln Lys
Leu His 165 170 175 Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu Glu
Met Arg Asp Arg 180 185 190 Ala Arg Ala His Val Asp Ala Leu Arg Thr
His Leu Ala Pro Tyr Ser 195 200 205 Asp Glu Leu Arg Gln Arg Leu Ala
Ala Arg Leu Glu Ala Leu Lys Glu 210 215 220 Asn Gly Gly Ala Arg Leu
Ala Glu Tyr His Ala Lys Ala Thr Glu His 225 230 235 240 Leu Ser Thr
Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu Arg 245 250 255 Gln
Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser Phe Leu Ser 260 265
270 Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr Gln 275 280
2283PRTArtificial SequenceTetranectin-apolipoprotein A-I with
N-terminal His-tag 2Ile Val Asn Ala Lys Lys Asp Val Val Asn Thr Lys
Met Phe Glu Glu 1 5 10 15 Leu Lys Ser Arg Leu Asp Thr Leu Ala Gln
Glu Val Ala Leu Leu Lys 20 25 30 Glu Gln Gln Ala Leu Gln Thr Val
Asp Glu Pro Pro Gln Ser Pro Trp 35 40 45 Asp Arg Val Lys Asp Leu
Ala Thr Val Tyr Val Asp Val Leu Lys Asp 50 55 60 Ser Gly Arg Asp
Tyr Val Ser Gln Phe Glu Gly Ser Ala Leu Gly Lys 65 70 75 80 Gln Leu
Asn Leu Lys Leu Leu Asp Asn Trp Asp Ser Val Thr Ser Thr 85 90 95
Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro Val Thr Gln Glu Phe Trp 100
105 110 Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln Glu Met Ser
Lys 115 120 125 Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro Tyr Leu
Asp Asp Phe 130 135 140 Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr
Arg Gln Lys Val Glu 145 150 155 160 Pro Leu Arg Ala Glu Leu Gln Glu
Gly Ala Arg Gln Lys Leu His Glu 165 170 175 Leu Gln Glu Lys Leu Ser
Pro Leu Gly Glu Glu Met Arg Asp Arg Ala 180 185 190 Arg Ala His Val
Asp Ala Leu Arg Thr His Leu Ala Pro Tyr Ser Asp 195 200 205 Glu Leu
Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys Glu Asn 210 215 220
Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu His Leu 225
230 235 240 Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu
Arg Gln 245 250 255 Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser
Phe Leu Ser Ala 260 265 270 Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
Gln 275 280 3285PRTArtificial SequenceTetranectin-apolipoprotein
A-I (2) 3Ala Pro Ile Val Asn Ala Lys Lys Asp Val Val Asn Thr Lys
Met Phe 1 5 10 15 Glu Glu Leu Lys Ser Arg Leu Asp Thr Leu Ala Gln
Glu Val Ala Leu 20 25 30 Leu Lys Glu Gln Gln Ala Leu Gln Thr Val
Asp Glu Pro Pro Gln Ser 35 40 45 Pro Trp Asp Arg Val Lys Asp Leu
Ala Thr Val Tyr Val Asp Val Leu 50 55 60 Lys Asp Ser Gly Arg Asp
Tyr Val Ser Gln Phe Glu Gly Ser Ala Leu 65 70 75 80 Gly Lys Gln Leu
Asn Leu Lys Leu Leu Asp Asn Trp Asp Ser Val Thr 85 90 95 Ser Thr
Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro Val Thr Gln Glu 100 105 110
Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu Gly Leu Arg Gln Glu Met 115
120 125 Ser Lys Asp Leu Glu Glu Val Lys Ala Lys Val Gln Pro Tyr Leu
Asp 130 135 140 Asp Phe Gln Lys Lys Trp Gln Glu Glu Met Glu Leu Tyr
Arg Gln Lys 145 150 155 160 Val Glu Pro Leu Arg Ala Glu Leu Gln Glu
Gly Ala Arg Gln Lys Leu 165 170 175 His Glu Leu Gln Glu Lys Leu Ser
Pro Leu Gly Glu Glu Met Arg Asp 180 185 190 Arg Ala Arg Ala His Val
Asp Ala Leu Arg Thr His Leu Ala Pro Tyr 195 200 205 Ser Asp Glu Leu
Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala Leu Lys 210 215 220 Glu Asn
Gly Gly Ala Arg Leu Ala Glu Tyr His Ala Lys Ala Thr Glu 225 230 235
240 His Leu Ser Thr Leu Ser Glu Lys Ala Lys Pro Ala Leu Glu Asp Leu
245 250 255 Arg Gln Gly Leu Leu Pro Val Leu Glu Ser Phe Lys Val Ser
Phe Leu 260 265 270 Ser Ala Leu Glu Glu Tyr Thr Lys Lys Leu Asn Thr
Gln 275 280 285 4285PRTArtificial
SequenceTetranectin-apolipoprotein A-I 4Xaa Pro Ile Val Asn Ala Lys
Lys Asp Val Val Asn Thr Lys Met Phe 1 5 10 15 Glu Glu Leu Lys Ser
Arg Leu Asp Thr Leu Ala Gln Glu Val Ala Leu 20 25 30 Leu Lys Glu
Gln Gln Ala Leu Gln Thr Val Asp Glu Pro Pro Gln Ser 35 40 45 Pro
Trp Asp Arg Val Lys Asp Leu Ala Thr Val Tyr Val Asp Val Leu 50 55
60 Lys Asp Ser Gly Arg Asp Tyr Val Ser Gln Phe Glu Gly Ser Ala Leu
65 70 75 80 Gly Lys Gln Leu Asn Leu Lys Leu Leu Asp Asn Trp Asp Ser
Val Thr 85 90 95 Ser Thr Phe Ser Lys Leu Arg Glu Gln Leu Gly Pro
Val Thr Gln Glu 100 105 110 Phe Trp Asp Asn Leu Glu Lys Glu Thr Glu
Gly Leu Arg Gln Glu Met 115 120 125 Ser Lys Asp Leu Glu Glu Val Lys
Ala Lys Val Gln Pro Tyr Leu Asp 130 135 140 Asp Phe Gln Lys Lys Trp
Gln Glu Glu Met Glu Leu Tyr Arg Gln Lys 145 150 155 160 Val Glu Pro
Leu Arg Ala Glu Leu Gln Glu Gly Ala Arg Gln Lys Leu 165 170 175 His
Glu Leu Gln Glu Lys Leu Ser Pro Leu Gly Glu Glu Met Arg Asp 180 185
190 Arg Ala Arg Ala His Val Asp Ala Leu Arg Thr His Leu Ala Pro Tyr
195 200 205 Ser Asp Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala
Leu Lys 210 215 220 Glu Asn Gly Gly Ala Arg Leu Ala Glu Tyr His Ala
Lys Ala Thr Glu 225 230 235 240 His Leu Ser Thr Leu Ser Glu Lys Ala
Lys Pro Ala Leu Glu Asp Leu 245 250 255 Arg Gln Gly Leu Leu Pro Val
Leu Glu Ser Phe Lys Val Ser Phe Leu 260 265 270 Ser Ala Leu Glu Glu
Tyr Thr Lys Lys Leu Asn Thr Gln 275 280 285
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