U.S. patent application number 15/444018 was filed with the patent office on 2018-01-11 for multistep final filtration.
This patent application is currently assigned to Hoffmann-La Roche Inc.. The applicant listed for this patent is Hoffmann-La Roche Inc.. Invention is credited to Roberto Falkenstein, Klaus Schwendner.
Application Number | 20180009878 15/444018 |
Document ID | / |
Family ID | 41466840 |
Filed Date | 2018-01-11 |
United States Patent
Application |
20180009878 |
Kind Code |
A1 |
Falkenstein; Roberto ; et
al. |
January 11, 2018 |
MULTISTEP FINAL FILTRATION
Abstract
Herein is reported a method for the final filtration of
concentrated polypeptide solutions comprising the combination of
two immediately consecutive filtration steps with a first filter of
3.0 .mu.m and 0.8 .mu.m pore size and a second filter of 0.45 .mu.m
and 0.22 .mu.m pore size.
Inventors: |
Falkenstein; Roberto;
(Muenchen, DE) ; Schwendner; Klaus; (Weilheim,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hoffmann-La Roche Inc. |
Little Falls |
NJ |
US |
|
|
Assignee: |
Hoffmann-La Roche Inc.
Little Falls
NJ
|
Family ID: |
41466840 |
Appl. No.: |
15/444018 |
Filed: |
February 27, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13394766 |
Mar 7, 2012 |
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PCT/EP2010/064487 |
Sep 29, 2010 |
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15444018 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/065 20130101;
C07K 16/32 20130101; C07K 16/2866 20130101 |
International
Class: |
C07K 16/06 20060101
C07K016/06; C07K 16/32 20060101 C07K016/32; C07K 16/28 20060101
C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 1, 2009 |
EP |
09012460.3 |
Claims
1. A method for producing an immunoglobulin solution comprising a)
providing an immunoglobulin solution with a concentration of at
least 100 g/l, and b) applying the immunoglobulin solution to a
combination of a first and second filter unit, whereby the first
filter unit comprises a pre-filter with a pore size of 3.0 .mu.m
and a main-filter with a pore size of 0.8 .mu.m and the second
filter unit comprises a pre-filter with a pore size of 0.45 .mu.m
and a main-filter with a pore size of 0.22 .mu.m with a pressure of
from 0.1 to 4.0 bar, and thereby producing an immunoglobulin
solution.
2. A method for producing an immunoglobulin comprising the
following steps a) cultivating a cell comprising a nucleic acid
encoding an immunoglobulin, b) recovering the immunoglobulin from
the cell or the cultivation medium, c) purifying the immunoglobulin
with one or more chromatography steps, and providing an
immunoglobulin solution, d) optionally adding a sugar, an amino
acid and/or a detergent to the solution, e) optionally
concentrating the immunoglobulin solution to a concentration of 100
g/l or more with a method selected from diafiltration or
tangential-flow filtration, and f) applying the immunoglobulin
solution of the previous step to a combination of a first and
second filter unit, whereby the first filter unit comprises a
pre-filter with a pore size of 3.0 .mu.m and a main-filter with a
pore size of 0.8 .mu.m and the second filter unit comprises a
pre-filter with a pore size of 0.45 .mu.m and a main-filter with a
pore size of 0.22 .mu.m with a pressure of from 0.1 to 4.0 bar, and
thereby producing an immunoglobulin.
3. The method of claim 1 or 2 wherein the filter in the first and
second filter unit have about the same filter area.
4. The method of claim 1 or 2 wherein the immunoglobulin solution
has a concentration of from 100 g/l to 300 g/l.
5. The method of claim 1 or 2 wherein the immunoglobulin solution
has a volume of from 3 liter to 100 liter.
6. The method of claim 1 or 2 wherein the immunoglobulin is an
anti-IL13 receptor alpha antibody or an anti-HER2 antibody.
7. The method of claim 1 or 2 wherein the purifying is with a
protein A affinity chromatography step and at least one step
selected from cation exchange chromatography, anion exchange
chromatography, and hydrophobic interaction chromatography.
8. The method of claim 1 or 2 wherein the immunoglobulin solution
has a concentration of 160 g/l or more and the applying to the
combination of filters is by applying a pressure of 1.45 bar or
more.
9. The method of claim 1 or 2 wherein the immunoglobulin solution
comprises a sugar and a surfactant and has a concentration of 125
mg/ml or more and the applying to the combination of the filter is
by applying a pressure of 0.75 bar or less.
10. A kit comprising a first filter with a pore size of from 3.0
.mu.m to 0.8 .mu.m and a second filter with a pore size of from
0.45 .mu.m to 0.22 .mu.m.
Description
RELATED APPLICATIONS
[0001] This continuing application is a Continuation of U.S.
application Ser. No. 13/394,766, filed on Mar. 7, 2012, which is a
US national phase application of PCT/EP2010/064487 filed Sep. 29,
2010, claiming priority to European Application No. 09012460.3
filed Oct. 1, 2009, the contents of which are hereby incorporated
by reference.
FIELD OF THE INVENTION
[0002] Provided herein are methods for the final filtration of
concentrated polypeptide solutions comprising the combination of
two immediately consecutive filtration steps with a first
filtration step with a pre-filtration with a filter with a pore
size of 3.0 .mu.m and a main-filtration with a filter with a pore
size of 0.8 .mu.m and a second filtration with a pre-filtration
with a filter with a pore size of 0.45 .mu.m and with a
main-filtration with a filter with a pore size of 0.22 .mu.m.
BACKGROUND OF THE INVENTION
[0003] Protein solutions with a concentration of more than 100 g/l
are prone to difficulties during the final filtration step, e.g. by
having only low transmembrane fluxes or blocking of the employed
filter by aggregates or particles formed during the formulation or
concentration process or due to added excipients resulting in an
increased viscosity of the concentrated solution.
[0004] The combination of high viscosity and increased particle or
aggregate content results often in the blocking of the pores of an
employed 0.22 .mu.m final filtration filter. As a consequence
either the filter has to be replaced during the filtration step,
i.e. before the batch is completely processed, or an increased
filter surface has to be used.
[0005] Further it has been observed that a combination of a filter
with a pore size of 0.45 .mu.m and a filter with a pore size of
0.22 .mu.m has no advantages, e.g. provided as Sartobran P
0.45/0.22 .mu.m filter. Filter with an increased pore size probable
to circumvent the before described problems are employed as
depth-filters or pre-filters but not a final filters.
[0006] In DE 4 204 444 a combination of a 1.2 .mu.m pre-filter to
remove water droplets from a gas stream prior to a 0.2 .mu.m
sterile-filtration is reported. A filter unit comprising two
filters of different pore size, whereby the filter of the smaller
pore size is flexible allowing by changing the flow direction the
filter to bend to reduce the resistance of the filter unit is
reported in U.S. Pat. No. 4,488,961. In U.S. Pat. No. 5,643,566 a
combination of a pre-filtration with a filter with a pore size of
0.45 .mu.m and a sterile-filtration with a filter of a pore size of
0.22 .mu.m is reported. A two-stage filter constructed using a
membrane with a smooth interior underlaid with a thin, flexible
porous membrane supported by a rigid screen support with a ridged
expander tube is reported in EP 0 204 836. A combination of at
least two membrane filter units of different membrane material and
different filter pore size and filter pore geometries is reported
in DE 3 818 860.
[0007] Aldington et al. (J. Chrom. B 848 (2007) 64-78) report a
scale-up of monoclonal antibody purification processes. In CS
247484 a method of preparing immunoglobulin against human
lymphocytes is reported.
SUMMARY OF THE INVENTION
[0008] It has been found that a combination of two filters each
comprising a pre-filter and a main-filter and each with a
specifically selected pore size can be used to filter highly
concentrated immunoglobulin solutions during the final packaging
step without the risk of pore blocking and the need to replace the
filter during the filtration process.
[0009] One aspect as reported herein is a method for the
preparation of an immunoglobulin solution comprising the following
steps [0010] a) providing an immunoglobulin solution with a protein
concentration of at least 100 g/l, [0011] b) filtering the
immunoglobulin solution through a combination of a first and second
filter, whereby the first filter comprises a pre-filter with a pore
size of 3.0 .mu.m and a main-filter with a pore size of 0.8 .mu.m
and the second filter comprises a pre-filter with a pore size of
0.45 .mu.m and a main-filter with a pore size of 0.22 .mu.m, and
thereby preparing an immunoglobulin solution.
[0012] Another aspect as reported herein is the use of a filter
combination as reported herein of a combination of a first and
second filter, whereby the first filter comprises a pre-filter with
a pore size of 3.0 .mu.m and a main-filter with a pore size of 0.8
.mu.m and the second filter comprises a pre-filter with a pore size
of 0.45 .mu.m and a main-filter with a pore size of 0.22 .mu.m, for
the final filtration of an immunoglobulin solution prior to active
pharmaceutical ingredient preparation.
[0013] Another aspect as reported herein is a method for producing
an immunoglobulin comprising the following steps [0014] a)
providing a cell comprising a nucleic acid encoding the
immunoglobulin, [0015] b) cultivating the cell, [0016] c)
recovering the immunoglobulin from the cell or the cultivation
medium, [0017] d) purifying the immunoglobulin with one or more
chromatography steps and providing an immunoglobulin solution, and
[0018] e) filtrating the immunoglobulin solution of step d) through
a combination of a first and second filter, whereby the first
filter comprises a pre-filter with a pore size of 3.0 .mu.m and a
main-filter with a pore size of 0.8 .mu.m and the second filter
comprises a pre-filter with a pore size of 0.45 .mu.m and a
main-filter with a pore size of 0.22 .mu.m, and thereby producing
an immunoglobulin.
[0019] A further aspect as reported herein is a kit comprising a
first filter comprising a pre-filter with a pore size of 3.0 .mu.m
and a main-filter with a pore size of 0.8 .mu.m and the second
filter comprising a pre-filter with a pore size of 0.45 .mu.m and a
main-filter with a pore size of 0.22 .mu.m.
[0020] In one embodiment the first filter has an area that is at
most twice the area of the second filter. In another embodiment the
first and second filter have about the same total filter area. In
an embodiment the immunoglobulin solution comprises a sugar, and/or
an amino acid, and/or a surfactant, and/or a salt. In a further
embodiment the immunoglobulin solution has a concentration of from
100 g/l to 300 g/l. In still another embodiment the immunoglobulin
solution has a volume of from 3 liter to 100 liter. In a further
embodiment the filtrating is with an applied pressure of from 0.1
bar to 4.0 bar. In one embodiment the immunoglobulin solution has a
concentration of 160 g/l or more and the filtrating is with an
applied pressure of 1.45 bar or more. In a further embodiment of
1.50 bar or more. In another embodiment the immunoglobulin solution
comprises a sugar and a surfactant and has a concentration of 125
mg/ml or more and the filtrating is with an applied pressure of
0.75 bar or less. In a further embodiment of 0.7 bar or less.
[0021] In one embodiment the immunoglobulin is an anti-IL13
receptor alpha antibody or an anti-HER2 antibody. In a further
embodiment the purifying is with a protein A affinity
chromatography step and at least one step selected from cation
exchange chromatography, anion exchange chromatography, and
hydrophobic interaction chromatography.
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIG. 1 shows time course of permeate flow obtained with an
anti-HER2 antibody solution with an antibody concentration of 222
mg/ml and an applied pressure of 2.0 bar (diamonds=1.2 .mu.m pore
size filter containing combination; squares=3.0 .mu.m pore size
filter containing combination).
[0023] FIG. 2 shows time course of permeate flow obtained with an
anti-HER2 antibody solution with an antibody concentration of 125
mg/ml supplemented with about 200 mM trehalose and about 0.05%
(w/v) Tween 20 and an applied pressure of 2.0 bar (diamonds=1.2
.mu.m pore size filter containing combination; squares=3.0 .mu.m
pore size filter containing combination).
[0024] FIG. 3 shows time course of permeate flow obtained with an
anti-HER2 antibody solution with an antibody concentration of 162
mg/ml and an applied pressure of 1.8 bar (diamonds=1.2 .mu.m pore
size filter containing combination; squares=3.0 .mu.m pore size
filter containing combination).
[0025] FIG. 4 shows time course of permeate flow obtained with an
anti-IL13R.alpha. antibody solution with an antibody concentration
of 141 mg/ml supplemented with about 200 mM trehalose and about
0.2% (w/v) Poloxamer and an applied pressure of 1.6 bar
(diamonds=1.2 .mu.m pore size filter containing combination;
squares=3.0 .mu.m pore size filter containing combination).
[0026] FIG. 5 shows time course of permeate flow obtained with an
anti-HER2 antibody solution with an antibody concentration of 162
mg/ml and an applied pressure of 1.1 bar (diamonds=1.2 .mu.m pore
size filter containing combination; squares=3.0 .mu.m pore size
filter containing combination).
[0027] FIG. 6 shows time course of permeate flow obtained with an
anti-IL13R.alpha. antibody solution with an antibody concentration
of 141 mg/ml supplemented with trehalose and Poloxamer and an
applied pressure of 0.8 bar (diamonds=1.2 .mu.m pore size filter
containing combination; squares=3.0 .mu.m pore size filter
containing combination).
[0028] FIG. 7 shows time course of permeate flow obtained with an
anti-HER2 antibody solution with an antibody concentration of 125
mg/ml supplemented with trehalose and Tween 20 and an applied
pressure of 0.8 bar (diamonds=1.2 .mu.m pore size filter containing
combination; squares=3.0 .mu.m pore size filter containing
combination).
[0029] FIG. 8 shows time course of permeate flow obtained with an
anti-HER2 antibody solution with an antibody concentration of 125
mg/ml supplemented with trehalose and Tween 20 and an applied
pressure of 0.3 bar (diamonds=1.2 .mu.m pore size filter containing
combination; squares=3.0 .mu.m pore size filter containing
combination).
DETAILED DESCRIPTION OF THE INVENTION
[0030] It has been found that a combination of two filters or
filter units each comprising a pre-filter and a main-filter and
each with a specifically selected pore size can be used to filter
highly concentrated and viscous, as well as formulated
immunoglobulin solutions, i.e. comprising a sugar and a surfactant,
during the final packaging step. Especially the combination of a
first filter comprising a pre-filter an a main-filter with a pore
size of 3.0 .mu.m and 0.8 .mu.m, respectively, and a second filter
comprising a pre-filter and a main-filter with a pore size of 0.45
.mu.m and 0.22 .mu.m, respectively, is highly advantageous. With a
single filter unit of this combination it has been possible to
filtrate highly concentrated solutions containing in total e.g. 1
kg of an anti-IL-13R.alpha.1 antibody or 6 kg of an anti-HER2
antibody and to package this amounts with only minor substance
losses. In one embodiment a ratio of filer surface area to solution
volume has been determined.
[0031] In one embodiment the immunoglobulin solution comprises the
immunoglobulin and an excipient. In another embodiment the
excipient comprises one or more substances selected from sugars,
such as glucose, galactose, maltose, sucrose, trehalose and
raffinose, amino acids, such as arginine, lysine, histidine,
ornithine, isoleucine, leucine, alanine, glutamic acid, aspartic
acid, glycine, and methionine, salts, such as sodium chloride,
potassium chloride, sodium citrate, potassium citrate, sodium
phosphate, potassium phosphate, and surfactants, such as
polysorbates, and poly (oxyethylene-polyoxypropylene) polymers.
[0032] The filtrating as reported herein is used as the final
filtration step in the production of a therapeutic antibody. It can
be carried out after the required excipients, stabilizer and/or
anti-oxidants have been added to the highly concentrated antibody
solution. In one embodiment the ratio of amount of antibody in kg
to total area of the filter is of from 1000 g/m.sup.2 to 10,000
g/m.sup.2. In another embodiment the ratio is of from 1000
g/m.sup.2 to 6000 g/m.sup.2. In still another embodiment the ratio
is from 4000 g/m.sup.2 to 6000 g/m.sup.2.
[0033] 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.
[0034] The term "immunoglobulin" refers to a protein consisting of
one or more polypeptide(s) substantially encoded by immunoglobulin
genes. The recognized immunoglobulin genes include the different
constant region genes as well as the myriad immunoglobulin variable
region genes. Immunoglobulins may exist in a variety of formats,
including, for example, Fv, Fab, and F(ab).sub.2 as well as single
chains (scFv) or diabodies.
[0035] The term "complete immunoglobulin" denotes an immunoglobulin
which comprises two so called light immunoglobulin chain
polypeptides (light chain) and two so called heavy immunoglobulin
chain polypeptides (heavy chain). Each of the heavy and light
immunoglobulin chain polypeptides of a complete immunoglobulin
contains a variable domain (variable region) (generally the amino
terminal portion of the polypeptide chain) comprising binding
regions that are able to interact with an antigen. Each of the
heavy and light immunoglobulin chain polypeptides of a complete
immunoglobulin also comprises a constant region (generally the
carboxyl terminal portion). The constant region of the heavy chain
mediates the binding of the antibody i) to cells bearing a Fc gamma
receptor (Fc.gamma.R), such as phagocytic cells, or ii) to cells
bearing the neonatal Fc receptor (FcRn) also known as Brambell
receptor. It also mediates the binding to some factors including
factors of the classical complement system such as component (C1q).
The variable domain of an immunoglobulin's light or heavy chain in
turn comprises different segments, i.e. four framework regions (FR)
and three hypervariable regions (CDR).
[0036] The term "immunoglobulin fragment" denotes a polypeptide
comprising at least one domain of the variable domain of a heavy
chain, the C.sub.H1 domain, the hinge-region, the C.sub.H2 domain,
the C.sub.H3 domain, the C.sub.H4 domain of a heavy chain, the
variable domain of a light chain and/or the C.sub.L domain of a
light chain. Also comprised are derivatives and variants thereof.
For example, a variable domain, in which one or more amino acids or
amino acid regions are deleted, may be present.
[0037] The term "immunoglobulin conjugate" denotes a polypeptide
comprising at least one domain of an immunoglobulin heavy or light
chain conjugated via a peptide bond to a further polypeptide. The
further polypeptide is a non-immunoglobulin peptide, such as a
hormone, or growth receptor, or antifusogenic peptide, or
complement factor, or the like.
[0038] The term "filter" denotes both a microporous or macroporous
filter. The filter comprises a filter membrane which 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 hydroxyapatite. In one embodiment the filter membrane
of the first and second filter is made of cellulose acetate.
[0039] For the purification of recombinantly produced
immunoglobulins often a combination of different column
chromatography steps is employed. Generally a protein A affinity
chromatography is followed by one or two additional separation
steps. The final purification step is a so called "polishing step"
for the removal of trace impurities and contaminants like
aggregated immunoglobulins, residual HCP (host cell protein), DNA
(host cell nucleic acid), viruses, or endotoxins. For this
polishing step often an anion exchange material in a flow-through
mode is used.
[0040] Different methods are well established and widespread used
for protein recovery and purification, such as affinity
chromatography with microbial 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), 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) (Vijayalakshmi, M. A., Appl. Biochem. Biotech. 75
(1998) 93-102).
[0041] A first aspect as reported herein is a method for the
preparation of an immunoglobulin solution comprising [0042]
providing an immunoglobulin solution with a protein concentration
of at least 100 g/l, [0043] filtering the immunoglobulin solution
through a combination of a first and second filter unit, whereby
the first filter unit comprises a pre-filter an a main-filter with
a pore size of 3.0 .mu.m and 0.8 .mu.m, respectively, and the
second filter unit comprises a pre-filter and a main-filter with a
pore size of 0.45 .mu.m and 0.22 .mu.m, respectively, by applying
the solution to the filter combination and by applying pressure and
thereby preparing an immunoglobulin solution.
[0044] In one embodiment the protein concentration is of from 100
g/l to 300 g/l. In another embodiment the protein concentration is
of from 100 g/l up to 200 g/l. In a further embodiment the protein
concentration is of from 120 g/l to 165 g/l. In another embodiment
the immunoglobulin solution has a volume of from 3 liter to 100
liter. This solution volume is equivalent to a total mass of the
immunoglobulin of from 300 g to 50,000 g. In one embodiment the
volume is of from 3.1 liter to 80 liter. At a protein concentration
of from 120 g/l to 165 g/l this solution volume is equivalent to a
total mass of the immunoglobulin of from 370 g to 13,200 g. In one
embodiment the immunoglobulin is an anti-IL13 receptor alpha
antibody. In another embodiment the immunoglobulin is an anti-HER2
antibody.
[0045] Another aspect as reported herein is a method for producing
an immunoglobulin comprises the following steps [0046] cultivating
a cell comprising a nucleic acid encoding the immunoglobulin,
[0047] recovering the immunoglobulin from the cell or the
cultivation medium, [0048] purifying the immunoglobulin with one or
more chromatography steps, and providing a purified immunoglobulin
solution, and [0049] filtrating the purified immunoglobulin
solution through a combination of filters as reported herein, i.e.
a combination of a first and second filter unit, whereby the first
filter unit comprises a pre-filter with a pore size of 3.0 .mu.m
and a main-filter with a pore size of 0.8 .mu.m, respectively, and
the second filter unit comprises a pre-filter with a pore size of
0.45 .mu.m and a main-filter with a pore size of 0.22 .mu.m,
respectively, by applying the solution to the filter combination
and by applying pressure.
[0050] In one embodiment the cell is a prokaryotic cell or a
eukaryotic cell. In one embodiment in which the cell is a
prokaryotic cell the cell is selected from E. coli cells, or
bacillus cells. In one embodiment in which the cell is a eukaryotic
cell the cell is selected from mammalian cells, in a special
embodiment from CHO cells, BHK cells, HEK cells, Per.C6.RTM. cells
and hybridoma cells. In one embodiment the cell is a mammalian cell
selected from CHO-K1 and CHO DG44. In one embodiment the
cultivating is at a temperature of from 20.degree. C. to 40.degree.
C., and for a period of from 4 to 28 days. In one embodiment the
purifying is with a protein A affinity chromatography step and at
least one step selected from cation exchange chromatography, anion
exchange chromatography, and hydrophobic interaction
chromatography.
[0051] It has been found that a combination of a first filter unit
comprising a pre-filter an a main-filter with a pore size of 3.0
.mu.m and 0.8 .mu.m, respectively, and a second filter unit
comprising a pre-filter and a main-filter with a pore size of 0.45
.mu.m and 0.22 .mu.m, respectively, is advantageous for processing
(filtrating) highly concentrated immunoglobulin solution by
allowing the filtration of a complete batch of a concentrated
immunoglobulin solution without the need to replace the filter.
[0052] It has further been found that in the filter combination it
is advantageous that each of the two filters employed in the units
as well as the filter combination has approximately the same filter
area, i.e. within two times the area of the smallest filter.
[0053] It has further been found that depending on the components
of the solution beside the immunoglobulin different pressure and
concentration ranges provide for advantageous processes.
[0054] If the solution is a concentrated immunoglobulin solution
with a concentration of 160 g/l or more, i.e. 165 g/l or 170 g/l,
to which no sugar or surfactant has been added then the method is
operated in one embodiment with an applied pressure of 1.45 bar or
more, in another of 1.5 bar or more. If the solution is a
concentrated immunoglobulin solution with a concentration of 125
g/l or more, i.e. 130 g/l or 135 g/l, to which at least a sugar and
a surfactant have been added then the method is operated in an
embodiment with an applied pressure of 0.75 bar or less, in another
embodiment of 0.7 bar or less.
[0055] Another aspect as reported herein is a kit comprising a
first filter unit comprising a pre-filter and a main-filter with a
pore size of 3.0 .mu.m and 0.8 .mu.m, respectively, and a second
filter unit comprising a pre-filter and a main-filter with a pore
size of 0.45 .mu.m and 0.22 .mu.m, respectively. Another aspect as
reported herein is the use of a filter comprising a first filter
unit comprising a pre-filter and a main-filter with a pore size of
3.0 .mu.m and 0.8 .mu.m, respectively, and a second filter unit
comprising a pre-filter and a main-filter with a pore size of 0.45
.mu.m and 0.22 .mu.m, respectively for the filtration of a
concentrated immunoglobulin solution with a protein concentration
of at least 100 g/l.
[0056] The following examples and referenced 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
[0057] Material and Methods
[0058] Antibody
[0059] An exemplary antibody is an immunoglobulin against the IL13
receptor .alpha.1 protein (anti-IL13R.alpha.1 antibody) e.g. as
reported in SEQ ID NO: 01 to 12 of WO 2006/072564 (incorporated
herein by reference).
[0060] Another exemplary immunoglobulin is an anti-HER2 antibody
reported in WO 92/022653, WO 99/057134, WO 97/04801, U.S. Pat. No.
5,677,171 and U.S. Pat. No. 5,821,337 (incorporated herein by
reference).
[0061] Filter
[0062] Herein among others a Sartobran P 0.45 .mu.m+0.2 .mu.m
filter cartridge and a Sartoclean CA 3.0 .mu.m+0.8 .mu.m filter
cartridge have been exemplarily employed. Both filter cartridges
are available from Sartorius AG, Gottingen, Germany.
[0063] Analytical Methods
[0064] Size Exclusion Chromatography: [0065] resin: TSK 3000
(Tosohaas) [0066] column: 300.times.7.8 mm [0067] flow rate: 0.5
ml/min [0068] buffer: 200 mM potassium phosphate containing 250 mM
potassium chloride, adjusted to pH 7.0 [0069] wavelength: 280 nm
[0070] DNA-threshold-system: see e.g. Merrick, H., and Hawlitschek,
G., Biotech Forum Europe 9 (1992) 398-403 [0071] Protein A ELISA:
The wells of a micro titer plate are coated with a polyclonal
anti-protein A-IgG derived from chicken. After binding non-reacted
antibody is removed by washing with sample buffer. For protein A
binding a defined sample volume is added to the wells. The protein
A present in the sample is bound by the chicken antibody and
retained in the wells of the plate. After the incubation the sample
solution is removed and the wells are washed. For detection are
added subsequently a chicken derived polyclonal anti-protein
A-IgG-biotin conjugate and a Streptavidin peroxidase conjugate.
After a further washing step substrate solution is added resulting
in the formation of a colored reaction product. The intensity of
the color is proportional to the protein A content of the sample.
After a defined time the reaction is stopped and the absorbance is
measured. [0072] Host cell protein (HCP) ELISA: 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.
Example 2
[0073] Filtration of an Anti-HER2 Antibody with a Single Filter of
0.45 .mu.m and 0.22 .mu.m Pore Size
[0074] In this example it is shown that a highly concentrated
immunoglobulin solution cannot be filtered with a single sterile
filter with a pore size of 0.45 .mu.m (pre-filter) and 0.22 .mu.m
(main-filter) without blocking of the pores of the filter with a
loading of more than 2,460 g protein per square meter of filter
area.
[0075] In this example a single filter with a pore size of 0.45
.mu.m and 0.22 .mu.m and a total filter area of 0.2 square meters
has been employed.
TABLE-US-00001 TABLE 1 Solutions employed in the single filter
filtration. solution No. 1 2 3 4 5 protein mass 473 491 496 501 542
[g] volume [l] 3.940 4.200 4.134 4.139 4.516 loading 2,365 2,455
2,480 2,505 2,710 [g/m.sup.2]
[0076] The concentrated immunoglobulin solutions were filtered
through the single filter with the parameters as shown in Table
2.
TABLE-US-00002 TABLE 2 Process parameters. solution No. 1 2 3 4 5
volume flow 1.97 2.1 Drop to 0 Drop to 0 Drop to 0 [l/h] due to
pore due to pore due to pore blocking blocking blocking mass flow
237 246 Drop to 0 Drop to 0 Drop to 0 [g/h] due to pore due to pore
due to pore blocking blocking blocking
[0077] For solutions No. 3 to 5 the pores of the single filter were
blocked prior to the complete filtration of the batch volume. To
complete the filtration the blocked filter had to be changed
resulting in additional time required and loss of product.
TABLE-US-00003 TABLE 3 Results of the filtration. solution No. 1 2
3 4 5 protein mass 2,365 2,455 960 968 1,440 passing the filter
[g/m.sup.2] volume passing 3.940 4.200 1.600 1.600 2.400 the filter
[l] pore blocking of NO NO YES YES YES the filter
Example 3
[0078] Filtration of an Anti-HER2 Antibody with a Combination of a
First Filter with a Pore Size of 3.0 .mu.m and 0.8 .mu.m and a
Second Filter with a Pore Size of 0.45 .mu.m and 0.22 .mu.m
[0079] In this example it is shown that a highly concentrated
immunoglobulin solution can be filtered with a combination of two
filters with a pore size of 3.0 .mu.m (pre-filter) and 0.8 .mu.m
(main-filter) and of 0.45 .mu.m (pre-filter) and 0.22 .mu.m
(main-filter) without blocking of the pores of the filter
independent from the loading of protein per square meter of total
filter area.
[0080] In this example a combined filter with a first filter unit
with a pore size of 3.0 .mu.m and 0.8 .mu.m, respectively, and a
second filter unit with a pore size of 0.45 .mu.m and 0.22 .mu.m,
respectively, and a filter area each of 0.6 square meters has been
employed.
TABLE-US-00004 TABLE 4 Solutions employed in the combined filter
filtration. solution No. 6 7 8 9 10 protein mass 5,217 5,191 5,356
6,151 5,580 [g] volume [l] 42.070 42.201 43.542 48.055 44.998
loading 4,347.5 4,325.8 4,463.3 5,125.8 4,650.0 [g/m.sup.2]
[0081] The concentrated immunoglobulin solutions were filtered
through the combination of the two filters with the parameters as
shown in Table 5.
TABLE-US-00005 TABLE 5 Process parameters. solution No. 6 7 8 9 10
volume flow [l/h] 38.95 42.20 43.54 33.02 45.00 mass flow [g/h]
4830 5191 5356 4226 5580
[0082] For none of the solutions No. 6 to 10 the pores of the
combined filters were blocked prior to the complete filtration of
the batch volume.
TABLE-US-00006 TABLE 6 Results of the filtration. solution No. 6 7
8 9 10 protein mass 4,347.5 4,325.8 4,463.3 5,125.8 4,650.0 passing
the filter [g/m.sup.2] volume 42.070 42.201 43.542 48.055 44.998
passing the filter [l] pore NO NO NO NO NO blocking of the
filter
Example 4
[0083] Filtration of an Anti-IL13R.alpha. Antibody with a Filter
Combination of a Filter with 3.0 .mu.m and 0.8 .mu.m Pore Size and
a Filter with 0.45 .mu.m and 0.22 .mu.m Pore Size and Both Filters
with Different Filter Areas
[0084] In this example it is shown that a conditioned protein A
eluate can be filtered with a combination of two filters but the
flow has to be reduced if the filter area does not match between
the two filters.
[0085] In this example a filter unit with a pore size of 3.0 .mu.m
(pre-filter) and 0.8 .mu.m (main-filter) with a filter area of 1.8
square meters and a filter unit with a pore size of 0.45 .mu.m
(pre-filter) and 0.22 .mu.m (main-filter) with a filter area of 0.6
square meters has been employed.
TABLE-US-00007 TABLE 7 Solutions employed in the combined filter
filtration. solution No. 11 12 13 14 15 protein mass [g] 1,169.0
1,299.6 1,154.4 1,220.4 1,284.7 volume [l] 71.4 76.0 74.0 67.8 70.2
loading [g/m.sup.2] 487.1 541.5 481.0 508.5 535.3
[0086] The concentrated immunoglobulin solutions were filtered
through the combined filter with the parameters as shown in Table
8.
TABLE-US-00008 TABLE 8 Process parameters. solution No. 11 12 13 14
15 volume flow Drop to 0 22 13 12 98 [l/h] due to pore blocking
mass flow Drop to 0 376 203 216 1793 [g/h] due to pore blocking
[0087] For solution No. 11 the pores of the combined filter were
blocked prior to the complete filtration of the batch volume. To
complete the filtration the blocked filter had to be changed
resulting in additional time required and loss of product.
TABLE-US-00009 TABLE 9 Results of the filtration. solution No. 1 2
3 4 5 protein mass 347.9 541.5 481.0 508.5 535.3 passing the filter
[g/m.sup.2] volume passing 51.0 76.0 74.0 67.8 70.2 the filter [l]
pore blocking of YES NO NO NO NO the filter
[0088] In order to prevent filter blocking as in the experiment
with solution No. 11 the flow through the membrane had to be
reduced in experiments with solutions No. 12 to 14. In experiment
with solution No. 15 the protein A eluate has been decanted
resulting in a loss of protein.
Example 5
[0089] Filtration of an Anti-IL13R.alpha. Antibody with a Filter
Combination of a Filter with 3.0 .mu.m and 0.8 .mu.m Pore Size and
a Filter with 0.45 .mu.m and 0.22 .mu.m Pore Size and Both Filters
Each with the Same Filter Area
[0090] In this example it is shown that a conditioned protein A
eluate can be filtered with a combination of two filters without a
reduction of the flow if the filter area does match between the two
filters.
[0091] In this example the filter unit with a pore size of 3.0
.mu.m and 0.8 .mu.m has a filter area of 0.2 square meters and the
filter unit with a pore size of 0.45 .mu.m and 0.22 .mu.m has a
filter area of 0.2 square meters.
TABLE-US-00010 TABLE 10 Solutions employed in the combined filter
filtration. solution No. 16 17 18 19 20 protein mass [g] 495 634
825 861 956 volume [l] 3.5 4.14 5.5 5.6 6.3 loading [g/m.sup.2]
1,237.5 1,585.0 2,062.5 2,152.5 2,390
[0092] For none of the solutions No. 16 to 20 the pores of the
combined filters were blocked prior to the complete filtration of
the batch volume.
TABLE-US-00011 TABLE 11 Results of the filtration. solution No. 16
17 18 19 20 Protein mass 1,237.5 1,585.0 2,062.5 2,152.5 2,390
passing the filter [g/m.sup.2] Volume passing 3.5 4.14 5.5 5.6 6.3
the filter [l] Pore blocking of NO NO NO NO NO the filter
Example 6
[0093] Filtration of Different Antibody Solutions with Different
Filter Combinations with Different Protein Concentrations,
Different Compounds in Solution and Different Applied Pressures
[0094] Solutions comprising either an anti-IL13R.alpha. antibody or
an anti-HER2 antibody were filtered with a filter combination
employing different filter area and filter pore size as well as
different excipients and applied pressure.
[0095] The used filter combinations are listed in Table 12. In the
following the denotation `A1`, `A2`, B1', and B2' will be used
therefore.
TABLE-US-00012 TABLE 12 Filter combinations filter 1 filter 2
filter 3 filter 4 com- pore size/ pore size/ pore size/ pore size/
bination diameter diameter diameter diameter A1 1.2 .mu.m/ 0.8
.mu.m/26 mm 0.45 .mu.m/26 mm 0.2 .mu.m/26 mm 26 mm A2 1.2 .mu.m/
0.8 .mu.m/26 mm 0.45 .mu.m/26 mm 0.2 .mu.m/26 mm 47 mm B1 3.0
.mu.m/ 0.8 .mu.m/26 mm 0.45 .mu.m/26 mm 0.2 .mu.m/26 mm 26 mm B2
3.0 .mu.m/ 0.8 .mu.m/26 mm 0.45 .mu.m/26 mm 0.2 .mu.m/26 mm 47
mm
[0096] In the following Tables 13 to 20 and in corresponding FIGS.
1 to 8 the results obtained with different filter combinations,
different antibody solutions and different filtering conditions are
presented.
TABLE-US-00013 TABLE 13 Results obtained with an anti-HER2 antibody
solution with an antibody concentration of 222 mg/ml and an applied
pressure of 2.0 bar. filtration flow filtration duration [ml/
duration flow combination [min] min] Combination [min] [ml/min] A1
1 3.7 B1 1 3.4 2 3.5 2 3.2 3 3.2 3 3.1 4 3.0 4 3.0 5 2.9 5 2.8 6
2.6 6 2.9 7 2.5 7 2.8 8 2.3 8 2.7 9 2.0 9 2.7 10 2.0 10 2.7 11 1.7
11 2.7 12 1.6 12 2.5 13 1.5 13 2.6 14 1.3 14 2.5 15 1.2 15 2.5
TABLE-US-00014 TABLE 14 Results obtained with an anti-HER2 antibody
solution with an antibody concentration of 125 mg/ml supplemented
with about 200 mM trehalose and about 0.05% (w/v) Tween 20 and an
applied pressure of 2.0 bar. filtration flow filtration duration
[ml/ duration flow combination [min] min] Combination [min]
[ml/min] A1 1 22.4 B1 1 20.1 2 20.2 2 17.7 3 18.3 3 15.5 4 16.8 4
13.8 5 15.9 5 12.2 6 14.3 6 11.1 7 13.1 7 10.0 8 12.3 8 8.7 9 11.3
9 8.1 10 10.3 10 7.0 11 9.7 11 6.6 12 9.2 12 5.8 13 8.4 13 5.2 14
8.1 14 4.8 15 7.4 15 4.3
TABLE-US-00015 TABLE 15 Results obtained with an anti-HER2 antibody
solution with an antibody concentration of 162 mg/ml and an applied
pressure of 1.8 bar. filtration flow filtration duration [ml/
duration flow combination [min] min] Combination [min] [ml/min] A2
1 7.6 B2 1 8.1 2 6.5 2 6.9 3 6.1 3 6.4 4 5.7 4 6.2 5 5.4 5 5.9 6
5.1 6 5.6 7 5.2 7 5.5 8 5.0 8 5.3 9 4.9 9 5.2 10 4.7 10 5.1 11 4.8
11 5.0 12 4.8 12 4.8 13 4.6 13 4.9 14 4.7 14 4.6 15 4.6 15 4.6
TABLE-US-00016 TABLE 16 Results obtained with an anti-IL13R.alpha.
antibody solution with an antibody concentration of 141 mg/ml
supplemented with about 200 mM trehalose and about 0.2% (w/v)
Poloxamer and an applied pressure of 1.6 bar. filtration flow
filtration duration [ml/ duration flow combination [min] min]
Combination [min] [ml/min] A2 1 15.6 B2 1 13.2 2 9.4 2 8.1 3 7.0 3
5.5 4 5.5 4 4.1 5 4.6 5 3.3 6 3.8 6 2.6 7 3.3 7 2.3 8 2.9 8 1.9 9
2.5 9 1.6 10 2.2 10 1.5 11 1.5 11 1.2 12 0.5 12 1.2 13 0.3 13 1.0
14 0.3 14 0.9 15 0.3 15 0.8
TABLE-US-00017 TABLE 17 Results obtained with an anti-HER2 antibody
solution with an antibody concentration of 162 mg/ml and an applied
pressure of 1.1 bar. filtration flow filtration duration [ml/
duration flow combination [min] min] Combination [min] [ml/min] A1
1 4.4 B1 1 4.3 2 4.0 2 4.0 3 3.6 3 3.5 4 3.5 4 3.0 5 3.3 5 3.0 6
3.2 6 3.0 7 3.2 7 2.9 8 3.1 8 2.8 9 3.1 9 2.8 10 2.9 10 2.7 11 3.0
11 2.6 12 2.9 12 2.8 13 2.8 13 2.5 14 2.8 14 2.6 15 2.8 15 2.5
TABLE-US-00018 TABLE 18 Results obtained with an anti-IL13R.alpha.
antibody solution with an antibody concentration of 141 mg/ml
supplemented with trehalose and Poloxamer and an applied pressure
of 0.8 bar. filtration flow filtration duration [ml/ duration flow
combination [min] min] Combination [min] [ml/min] A2 1 7.6 B2 1 8.1
2 5.0 2 5.5 3 3.7 3 4.2 4 2.9 4 3.1 5 2.5 5 2.6 6 2.1 6 2.2 7 1.8 7
1.8 8 1.5 8 1.5 9 1.4 9 1.4 10 1.2 10 1.2 11 1.1 11 1.1 12 1.0 12
1.0 13 0.9 13 0.8 14 0.8 14 0.8 15 0.8 15 0.8
TABLE-US-00019 TABLE 19 Results obtained with an anti-HER2 antibody
solution with an antibody concentration of 125 mg/ml supplemented
with trehalose and Tween 20 and an applied pressure of 0.8 bar.
filtration flow filtration duration [ml/ duration flow combination
[min] min] Combination [min] [ml/min] A1 1 9.3 B1 1 9.7 2 8.7 2 8.8
3 8.1 3 8.4 4 7.9 4 8.0 5 7.7 5 7.4 6 7.2 6 7.0 7 7.1 7 6.4 8 6.6 8
6.1 9 6.2 9 5.7 10 6.0 10 5.4 11 5.6 11 5.0 12 5.3 12 4.6 13 5.0 13
4.5 14 4.8 14 4.1 15 4.5 15 3.3
TABLE-US-00020 TABLE 20 Results obtained with an anti-HER2 antibody
solution with an antibody concentration of 125 mg/ml supplemented
with trehalose and Tween 20 and an applied pressure of 0.3 bar.
filtration flow filtration duration [ml/ duration flow combination
[min] min] Combination [min] [ml/min] A1 1 3.9 B1 1 3.7 2 3.2 2 4.8
3 3.0 3 4.6 4 2.7 4 3.8 5 2.6 5 4.0 6 2.3 6 3.8 7 2.1 7 3.8 8 2.0 8
3.7 9 1.8 9 3.6 10 1.5 10 3.6 11 1.4 11 3.5 12 1.3 12 3.5 13 1.2 13
3.3 14 1.1 14 3.3 15 1.1 15 3.2
* * * * *