U.S. patent application number 15/187460 was filed with the patent office on 2016-10-13 for flocculation method.
The applicant listed for this patent is Amgen Inc.. Invention is credited to Thomas M. McNerney, Krista PETTY, Anne C. THOMAS, Xiaoyang ZHAO.
Application Number | 20160297850 15/187460 |
Document ID | / |
Family ID | 47559662 |
Filed Date | 2016-10-13 |
United States Patent
Application |
20160297850 |
Kind Code |
A1 |
McNerney; Thomas M. ; et
al. |
October 13, 2016 |
FLOCCULATION METHOD
Abstract
The present invention relates to a method for harvesting
recombinant proteins from mammalian cell culture fluid. The method
makes use of cationic polymers, non-ionic polymers and non-ionic
surfactants.
Inventors: |
McNerney; Thomas M.;
(Sammamish, WA) ; PETTY; Krista; (Newbury Park,
CA) ; THOMAS; Anne C.; (Kenmore, WA) ; ZHAO;
Xiaoyang; (Moorpark, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Amgen Inc. |
Thousand Oaks |
CA |
US |
|
|
Family ID: |
47559662 |
Appl. No.: |
15/187460 |
Filed: |
June 20, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14365496 |
Jun 13, 2014 |
9371554 |
|
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PCT/US12/69916 |
Dec 14, 2012 |
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15187460 |
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61576303 |
Dec 15, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P 21/02 20130101;
C07K 16/00 20130101; C12P 21/00 20130101; C07K 1/145 20130101 |
International
Class: |
C07K 1/14 20060101
C07K001/14; C07K 16/00 20060101 C07K016/00; C12P 21/00 20060101
C12P021/00 |
Claims
1. A mammalian cell culture harvest method comprising culturing
mammalian cells expressing a recombinant protein in a cell culture
medium for a predetermined time or until a desired cell density
and/or packed cell volume is achieved, adding a cationic polymer
and a non-ionic polymer to the cell culture medium initiating
flocculation, mixing the cell culture medium during flocculation,
allowing the flocculent to settle for a primary settle, and
recovering the primary clarified supernatant.
2. The mammalian cell culture harvest method according to claim 1,
wherein the cationic polymer is poly diallyldimethyammonium
chloride.
3. The mammalian cell culture harvest method according to claim 1,
wherein the non2-ionic polymer is selected from poly ethyleneglycol
and dextran.
4. The mammalian cell culture harvest method according to claim 1,
wherein the non-ionic polymer is selected from PEG 3,000 and PEG
6,000.
5. The mammalian cell culture harvest method according to claim 1,
further comprising adding a non-ionic surfactant to the cell
culture medium.
6. The mammalian cell culture harvest method according to claim 5,
wherein the non-ionic surfactant is Triton X-100.
7. The mammalian cell culture harvest method according to claim 1,
further comprising washing the primary settle flocculent, allowing
the washed flocculent to settle for a secondary settle, and
recovering the secondary clarified supernatant.
8. The method according to claim 1, where the cationic polymer and
the non-ionic polymer are added simultaneously.
9. The method according to claim 1, where the cationic polymer is
added first and mixed for at least 30 seconds followed by addition
of the non-ionic polymer.
10. The method according to claim 1, where the cationic polymer,
the non-ionic polymer and a non-ionic surfactant are added
simultaneously.
11. The method according to claim 1, where the cationic polymer is
added first and mixed for at least 30 seconds followed by addition
of the non-ionic polymer and a non-ionic surfactant.
12. The method according to claim 1, wherein the cationic polymer
is a polymer of diallyldimethylammonium chloride, poly
diallyldimethyammonium chloride, polyethyleneimine, polyacrylamide
or chitosan.
13. The method according to claim 1, wherein the non-ionic polymer
is polyethylene glycol or a dextran.
14. The method according to claim 5, wherein the non-ionic
surfactant is Sapoin or Triton X100.
15. The method according to claim 2, wherein the poly
diallyldimethyammonium chloride is added at a concentration of at
or about 20 to at or about 90 pg/total cell density.
16. The method according to claim 2, wherein the poly
diallyldimethyammonium chloride is added at a concentration of at
or about 25 pg/total cell density wherein the mammalian cells
originate from a diploid cell line.
17. The method according to claim 2, wherein the poly
diallyldimethyammonium chloride is added between 43 pg/total cell
density and 57 pg/total cell density wherein the mammalian cells
originate from a tetraploid cell line.
18. The method according to claim 4, wherein the concentration of
PEG 3,000 is at or about 3% to at or about 4.5%.
19. The method according to claim 4, wherein the concentration of
PEG 6,000 is at or about 2.5% to at or about 3.5%.
20. The method according to claim 6, wherein the concentration of
Triton X100 is 0.05% (w/v).
21. The method according to claim 1, wherein the mammalian cell
culture medium is between 36.degree. C. and 20.degree. C.
22. The method according to claim 1, wherein the mammalian cell
culture medium is at or above 20.degree. C.
23. The method according to claim 7, wherein the flocculent from
the primary settle is washed in a 9% sucrose solution.
Description
[0001] This application is a continuation of application Ser. No.
14/365,496, filed Jun. 13, 2014, which is a national stage
application under 35 U.S.C. .sctn.371 of International Application
No. PCT/US2012/069916, having an international filing date of Dec.
14, 2012; which claims priority to U.S. Provisional Patent
Application No. 61/576,303, filed Dec. 15, 2011, which are hereby
incorporated by reference.
FIELD OF INVENTION
[0002] The present invention relates to a method for harvesting
recombinant proteins from mammalian cell culture broth. The method
makes use of cationic polymers, non-ionic polymers and non-ionic
surfactants.
BACKGROUND OF INVENTION
[0003] Clinical manufacture of therapeutic proteins is an
expensive, large scale endeavor. Demand for greater quantities of
therapeutic recombinant proteins has driven advances in cell
culture processing which have resulted in dramatically increased
product titer. High titer cell culture processes are typically
produced by maintaining high viable cell densities over longer
culture durations. A corresponding increase in the biomass solids
(viable and non-viable cells) and the submicron cellular debris
particles are also observed. The higher burden of solids and
submicron cellular debris particles can challenge mammalian cell
culture harvest processes, making the harvest process less
effective at removing the debris without a substantial loss of
product capacity.
[0004] Cationic polymer flocculents are used for many applications
ranging from potable water purification, waste water treatment,
uses in the petroleum, mining and paper making industries,
cosmetics and medical uses, and have also been used to encapsulate
mammalian cells and enzymes and to flocculate microbial cell
cultures. However, for use in a commercial scale mammalian cell
harvest process, lengthy flocculation settling time can be
problematic, resulting in a harvest process that is time consuming
and less efficient than standard harvest practices.
[0005] There is a continuing need to improve mammalian cell culture
harvest methods, particularly commercial scale methods. Any
improvements that allow for quicker recovery times and/or greater
recovery can lead to reduced costs associated with manufacturing
protein therapeutics. The invention fulfills this need by providing
a quick and efficient method of cell culture harvest.
SUMMARY OF THE INVENTION
[0006] The present invention provides a mammalian cell culture
harvest method comprising culturing mammalian cells expressing a
recombinant protein in a cell culture medium for a predetermined
time or until a desired cell density and/or packed cell volume is
achieved, adding a cationic polymer and a non-ionic polymer to the
cell culture medium initiating flocculation, mixing the cell
culture medium during flocculation, allowing the flocculent to
settle, and recovering the clarified supernatant.
[0007] The present invention also provides a mammalian cell culture
harvest method comprising culturing mammalian cells expressing a
recombinant protein in a cell culture medium for a predetermined
time or until a desired cell density and/or packed cell volume is
achieved, adding poly diallyldimethyammonium chloride and PEG 3,000
to the cell culture medium initiating flocculation, mixing the cell
culture medium during flocculation, allowing the flocculent to
settle, and recovering the clarified supernatant.
[0008] The present invention also provides a mammalian cell culture
harvest method comprising culturing mammalian cells expressing a
recombinant protein in a cell culture medium for a predetermined
time or until a desired cell density and/or packed cell volume is
achieved, adding poly diallyldimethyammonium chloride, PEG 3,000
and Triton X-100 to the cell culture medium initiating
flocculation, mixing the cell culture medium during flocculation,
allowing the flocculent to settle, and recovering the clarified
supernatant.
[0009] The present invention also provides a mammalian cell culture
harvest method comprising culturing mammalian cells expressing a
recombinant protein in a cell culture medium for a predetermined
time or until a desired cell density and/or packed cell volume is
achieved, adding a cationic polymer and a non-ionic polymer to the
cell culture medium initiating flocculation, mixing the cell
culture medium during flocculation, allowing the flocculent to
settle for a primary settle, recovering the primary clarified
supernatant, washing the primary settle flocculent, allowing the
washed flocculent to settle for a secondary settle, and recovering
the secondary clarified supernatant.
[0010] The present invention also provides a mammalian cell culture
harvest method comprising culturing mammalian cells expressing a
recombinant protein in a cell culture medium for a predetermined
time or until a desired cell density and/or packed cell volume is
achieved, adding a cationic polymer and a non-ionic polymer to the
cell culture medium initiating flocculation, mixing the cell
culture medium during flocculation, allowing the flocculent to
settle for a primary settle, recovering the primary clarified
supernatant, washing the primary settle flocculent if product
recovery in the primary clarified supernatant is less than 80%,
allowing the washed flocculent to settle for a secondary settle,
and recovering the secondary clarified supernatant.
[0011] The present invention also provides a mammalian cell culture
harvest method comprising culturing mammalian cells expressing a
recombinant protein in a cell culture medium for a predetermined
time or until a desired cell density and/or packed cell volume is
achieved, adding poly diallyldimethyammonium chloride and PEG 3,000
to the cell culture medium initiating flocculation, mixing the cell
culture medium during flocculation, allowing the flocculent to
settle for a primary settle, recovering the primary clarified
supernatant, washing the primary settle flocculent, allowing the
washed flocculent to settle for a secondary settle, and recovering
the secondary clarified supernatant.
[0012] The present invention also provides a mammalian cell culture
harvest method comprising culturing mammalian cells expressing a
recombinant protein in a cell culture medium for a predetermined
time or until a desired cell density and/or packed cell volume is
achieved, adding poly diallyldimethyammonium chloride, PEG 3,000
and Triton X-100 to the cell culture medium initiating
flocculation, mixing the cell culture medium during flocculation,
allowing the flocculent to settle for a primary settle, recovering
the primary clarified supernatant, washing the primary settle
flocculent, allowing the washed flocculent to settle for a
secondary settle, and recovering the secondary clarified
supernatant.
[0013] In one embodiment the cationic polymer is poly
diallyldimethyammonium chloride.
[0014] In another embodiment the non-ionic polymer is selected from
poly ethylene glycol and dextran.
[0015] In another embodiment the non-ionic polymer is selected from
PEG 3,000 and PEG 6,000.
[0016] In another embodiment, the mammalian cell culture harvest
methods provided above further comprise adding a non-ionic
surfactant to the cell culture medium. In a related embodiment the
non-ionic surfactant is Triton X-100.
[0017] In another embodiment, the cationic polymer and the
non-ionic polymer are added simultaneously.
[0018] In another embodiment, the cationic polymer, the non-ionic
polymer and the non-ionic surfactant are added simultaneously.
[0019] In another embodiment, the cationic polymer is added first
and mixed for at least 30 seconds followed by addition of the
non-ionic polymer.
[0020] In another embodiment, the cationic polymer is added first
and mixed for at least 30 seconds followed by addition of the
non-ionic polymer and a non-ionic surfactant.
[0021] In another embodiment, the cationic polymer is a polymer of
diallyldimethylammonium chloride, polydiallyldimethyammonium
chloride, polyethyleneimine, polyacrylamide or chitosan.
[0022] In another embodiment, the non-ionic surfactant is Sapoin or
Triton X100.
[0023] In another embodiment, the poly diallyldimethyammonium
chloride is added at a concentration of at or about 20 to at or
about 90 pg/total cell density.
[0024] In another embodiment the poly diallyldimethyammonium
chloride is added at a concentration of at or about 25 pg/total
cell density wherein the mammalian cells originate from a diploid
cell line.
[0025] In another embodiment, the poly diallyldimethyammonium
chloride is added between 43 pg/total cell density and 57 pg/total
cell density wherein the mammalian cells originate from a
tetraploid cell line.
[0026] In another embodiment, the concentration of PEG 3,000 is at
or about 3% to at or about 4.5%.
[0027] In another embodiment, the concentration of PEG 6,000 is at
or about 2.5% to at or about 3.5%.
[0028] In another embodiment, the concentration of Triton X100 is
0.05% (w/v).
[0029] In another embodiment, the mammalian cell culture broth is
between 36.degree. C. and 20.degree. C.
[0030] In another embodiment, the mammalian cell culture broth is
at or above 20.degree. C.
[0031] In another embodiment, the flocculent from the primary
settle is washed in a 9% sucrose solution.
BRIEF DESCRIPTION OF THE FIGURES
[0032] FIG. 1 provides the structure of PDADMAC.
[0033] FIG. 2A shows the difference in settling times achieved by
PDADMAC at various molecular weights. The concentration of PDADMAC
at each molecular weight was 57 pg/Total cell density. The closed
diamond/dashed line represents PDADMAC molecular weight
100,000-200,000. The closed square with dashed line represents
PDADMAC molecular weight of 200,000-300,000. The closed triangle
and solid line represents PDADMAC molecular weight of
400,000-500,000.
[0034] FIG. 2B shows the supernatant clarity achieved when
flocculating cell culture broth with PDADMAC at various molecular
weights. From left to right the bars represent PDADMAC molecular
weight of <100,000; 100,000-200,000; 200,000-350,000; and
400,000-500,000.
[0035] FIG. 3A shows the impact of high molecular weight PDADMAC
concentration on flocculation of 15-20 .mu.m cells. The closed
diamond dashed line represents PDADMAC at 11 pg/Total cell density.
The closed square with dashed line represents PDADMAC at 18
pg/Total cell density. The closed triangle with solid line
represents PDADMAC at 25 pg/Total cell density. The closed circle
with dashed line represents PDADMAC at 39 pg/Total cell
density.
[0036] FIG. 3B shows the impact of high molecular weight PDADMAC
concentration on flocculation of 21-24 .mu.m cells. The closed
diamond dot and dashed line represents PDADMAC at 29 pg/Total cell
density. The closed square with dashed line represents PDADMAC at
43 pg/Total cell density. The closed triangle with dashed line
represents PDADMAC at 57 pg/Total cell density. The closed circle
with solid line represents PDADMAC at 71 pg/Total cell density. The
closed square with dotted line represents PDADMAC at 86 pg/Total
cell density.
[0037] FIG. 4A shows the impact of high molecular weight PDADMAC
flocculation for cell cultures that product high lactate levels.
The solid diamond with solid line represents no added lactate. The
solid square with dashed line represents lactate spiked at 3 g/L.
The solid triangle with dashed line represents lactate spiked at 6
g/L. The solid circle with dashed line represents lactate spiked at
9 g/L.
[0038] FIG. 4B shows the impact of high molecular weight PDADMAC
flocculation for cell cultures that have high cell density as
measured by packed cell volume (PCV). The solid diamond with dashed
line represents 44% PCV. The solid triangle with dashed line
represents 33% PCV. The solid square with dashed line represents
22% PCV. The solid circle with solid line represents 11% PCV.
[0039] FIG. 5 shows the impact of various diluents on high
molecular weight PDADMAC flocculation for a cell culture process
with high lactate levels. The solid triangle with the dotted and
dashed line represents Sucrose. The solid square with dashed line
represents cell culture media with no diluents. The solid diamond
with dashed line represents Betaine. The solid circle with dashed
line represents PEG 1,000. The solid triangle with the solid line
represents PEG 6,000. The solid triangle with the dashed line
represents Dextran 70 and Betaine.
[0040] FIG. 6 shows the impact of PDADMAC/PEG flocculation for cell
cultures with a PCV of 43.2%. The solid diamond with dashed line
represents PDADMAC only. The solid square with dashed line
represents Sucrose. The solid triangle with solid line represents
PDADMAC/PEG.
[0041] FIG. 7 shows the impact of the order of addition of PDADMAC
and PEG. PDADMAC and PEG bolus addition (1 step method) is shown by
the solid triangle with the dashed line. PDADMAC addition followed
by PEG addition (2 stem method) is shown by the solid square with
dashed line. PDADMAC alone is shown by the solid diamond with the
solid line.
[0042] FIG. 8 shows the impact of addition of Triton X-100.
PDADMAC/PEG addition is shown by the solid square with the solid
line. PDADMAC/PEG/Trition X-100 addition is shown by the solid
triangle with the dashed line.
DETAILED DESCRIPTION OF THE INVENTION
[0043] The invention provides a simple harvest flocculation
technique intended to maximize the recovery operation of high cell
mass cell culture processes. Provided is a mammalian cell culture
harvest method that makes use of cationic polymers in combination
with non-ionic polymers in the flocculation of cell culture broth.
Also provides is the use of cationic polymers in combination with
both non-ionic polymers and non-ionic surfactants.
[0044] The invention is based on the discovery that using a
non-ionic polymer or a non-ionic polymer and a non-ionic surfactant
in combination with a cationic polymer to flocculate a mammalian
cell culture broth decreased the flocculent settling time from 24
hours or longer to less than 1 hour, in some cases 15 minutes,
independent of the cell culture process density (up to 44% packed
cell volume) or lactate levels (10 g/L). The use of non-ionic
polymers also resulted in removal of high order aggregates and host
cell proteins that co-purify with the desired recombinant product.
This simple harvest method maximized the recovery operation of cell
culture processes, particularly high cell mass cell culture
commercial level processes.
[0045] Flocculation is a process whereby particles in suspension
form larger-size aggregates or clusters. In flocculation, particles
come out of suspension in the form of floc by the addition of a
flocculation agent or flocculent. Flocculents can be anionic or
cationic polymers. There are natural flocculents such as alginates
or Chitosan; mineral flocculents such as colloidal clays and
activated silica; and synthetic flocculents such as polyacrylamides
and poly diallyldimethyammonium chloride. Synthetic flocculents can
be manufactured to have specific molecular weights (based on chain
length) and molecular distribution.
[0046] Cationic polymers interact with negatively charged
particles, such as organic substances. In cell culture broth,
cationic polymers interact with negatively charged particles such
as viable and non-viable cells, cell metabolites and the cellular
debris such as nucleic acids, proteins and liposomes. Flocculation
of negatively charged compounds found in cell culture broth with
cationic polymers takes place through ionic interaction either via
the bridging of the negatively charged particles; through patch
binding of the cationic polymer that result in flocculation; or
through charge neutralization of large negatively charged particles
that result in the neutralized particle falling out of solution.
Flocs formed by cationic polymer bridging of the negatively charged
particles produced larger flocs and have increased shear
sensitivity which result in floc disruption that produce higher
levels of slower settling smaller particles. With patch or charge
neutralization, cationic polymers with high charge densities
interact with anionic patches on the particles in suspension, and
either neutralized the charge on the particle or form larger
particles that settled out of solution. Flocs formed in this manner
have a smaller floc particle volume and are less prone to
disruption due to shearing. For example, addition of poly
diallyldimethyammonium chloride (PDADMAC) to cell culture broth
flocculates negatively charged cells and cellular debris into
larger particles via an electrostatic patch mechanism (Ramsden et
al. (1998), Biotechnology Techniques, 12(8):599-603. PDADMAC also
flocculates the negatively charged submicron particles to produce a
feed stream with a significantly higher harvest filter train
throughput compared to a typical centrifuged harvest feed stream.
Flocculation via ionic interaction can be disrupted by increasing
the salt concentration or altering pH.
[0047] The addition of a cationic polymer, such as poly
diallyldimethyammonium chloride, to mammalian cell culture media
that contains or has contained cells expressing recombinant
proteins flocculates the negatively charged particles, including
cells (viable and non-viable), cell metabolites and cellular
debris. These large flocculated particles can be removed by
centrifugation or by gravity settling. The setting rate or the time
required for flocculated cells and cell debris to settle out is
dependent on the density of cells, cell debris and cell
metabolites. At low cell densities that are typical for batch cell
culture process, the flocculated material typically settles out (no
further settling) at some point over 4 hours to 24 hours, typically
around 20-24 hours. Settling rates significantly decrease with cell
culture processes that produce high cell densities of biomass
(>10% packed cell volume), submicron cellular debris, and/or
high lactate levels (>2-3 g/L). Cell culture processes that
produced high cell densities or have elevated lactate levels
require a significant amount of cell broth dilution for the floc to
settle out in 24 hours. Despite the ease of using cationic
polymers, such as PDADMAC as an alternative to traditional harvest
methods, the prolonged settling times could be less desirable on a
commercial scale.
[0048] The invention provides that flocculated particle size and
the particle size growth rate in cationic polymer flocculated
material are greatly enhanced in the presences of non-ionic
polymers and non-ionic surfactants. Floc gravity settling times of
less than 2 hours were routinely seen when PDADMAC was used in
combination with non-ionic polymers such as polyethylene glycols
and non-ionic surfactants, such as Triton X100, in spite of high
biomass/cell densities that significantly increased the
PDADMAC-only flocculation settling time. Shear forces that could
disrupt the flocs and/or reduce flocculation rates were tolerated
with the addition of non-ionic polymers and non-ionic surfactants.
Harvest recovery yields of 80-90% or greater were consistently
achieved with significantly reduced host DNA. Non-ionic polymer
addition also reduces host cell proteins and some high molecular
weight species.
[0049] As used herein, "cationic polymers" are positively charged
polymers that bind to negatively charged suspended particles.
Cationic polymers include, but are not limited to, polymers of
diallyl dimethyl ammonium chloride (DADMAC). In a preferred
embodiment, the polymerization of DADMAC forms an N-substituted
pyrrolidine structure, PDADMAC (FIG. 1). Cationic polymers also
include poly ethyleneimine (PEI), poly acrylamide (PAA) and
chitosan.
[0050] Concentrations of at or about 20 pg to at or about 90 pg of
PDADMAC per total cell density resulted in low supernatant
turbidity and good floc settling. "Total cell density" is the sum
of viable cells plus non-viable cells as measured by Trypan Blue
exclusion using a Cedex Cell Counter and Analyzer. In one
embodiment for small cell lines, such as a diploid cell line,
PDADMAC is added at or about 25 pg/total cell density. In another
embodiment, PDADMAC is added at or about 43 to at or about 57
pg/total cell density for larger cell lines, such as a tetraploid
cell line.
[0051] PDADMAC at molecular weights from 200,000-500,000 impact
flocculation performance by increasing sedimentation rate and
supernatant clarity, compared to lower molecular weight forms. In
one embodiment, the PDADMAC molecular weight is in the range of
400,000 to 500,000. In one embodiment, PDADMAC having a molecular
weight in the range of 400,000-500,000 is used at a final
concentration of 22 pg/Total cell density. In another embodiment,
PDADMAC having a molecular weight in the range of 400,000-500,000
is used at a final concentration of 25 pg/Total cell density. In
another embodiment, PDADMAC having a molecular weight in the range
of 400,000-500,000 is used at a final concentration of 45 pg/Total
cell density.
[0052] As used herein, "settling rate", "gravity settling rate" and
"flocculated packed settling rate" are used interchangeably.
Settling rates can be determined by methods known in the art and
described herein. For example, gravity settling is at 1 g. Settling
rates are determined by taking the floc volume divided by the total
volume measured in a 0.5 L or 1 L glass graduated cylinder. Total
volume is the volume of the cell broth with all
flocculating/settling agents included.
[0053] "Settling time" is the time it takes the floc to have
settled. Settling time is achieved when the floc settling rate is
less than or equal to 1% per hour. Settling times of as little as
15 minutes for PDADMAC flocculation in combination with dosing of a
non-ionic polymer or non-ionic polymers in combination with
non-ionic surfactants are described herein. The rapid settling time
occurred in spite of the high biomass/cell densities that
significantly increase the PDADMAC-only flocculation settling time.
Shear forces that disrupt the flocs and/or reduce flocculation
rates are tolerated with the addition of non-ionic polymers or
non-ionic surfactants.
[0054] Supernatant clarity is independent of the settling rate, but
is dependent on the cationic polymer dosing level. Other factors
such as temperature, cell culture fluid density and viscosity had
little impact on settling rate or supernatant clarity. PDADMAC
dosing, in particular, is a function of the cell volume, the total
density of cells (viable and non-viable cells), and the
concentration of submicron cellular debris particles.
[0055] As used herein, "non-ionic polymer" refers to hydrophilic
polymers that increase interactions between molecules, enhancing
precipitation. Non-ionic polymers include, but are not limited to,
polyethylene glycols (PEG) maltodextran, starches, methyl
celluloses, and dextrans.
[0056] Increased settling rates were achieved when PEG or dextrans
were added to mammalian cell culture media simultaneously with or
subsequent to the addition of a cationic polymer flocculent.
Product recovery was dependent on the non-ionic polymer
concentration, PEG molecular weight, order of addition of the
non-ionic polymer and PDADMAC (simultaneously or PDADMAC first
followed by the non-ionic polymer) and cell culture duration or
debris level in the cell culture broth.
[0057] PEG 3,000 is useful in the range of at or about 3 to at or
about 4.5% (w/v). PEG 6,000 is useful in the range of at or about
2.5% to at or about 3.5% (w/v). In one embodiment PEG 3,000 is used
at a final concentration of 3% (w/v). In another embodiment, PEG
3,000 is used at a final concentration of 15% (w/v). In another
embodiment, PEG 3,000 is used at a final concentration of 25%
(w/v).
[0058] As used herein, "non-ionic surfactant" refers to organic
compounds that are amphiphilic, meaning they contain both
hydrophobic groups and hydrophilic groups and includes, but is not
limited to, Sapoin and Triton X100 In one embodiment, Triton X-100
is used at a final concentration of 0.05% (w/v).
[0059] The non-ionic polymer can be added alone or in combination
with a non-ionic surfactant. Either can be added simultaneously
with the cationic polymer or subsequent to addition of cationic
polymer. The non-ionic polymer and surfactant can both be added
quickly with an addition time of 1 minute or less.
[0060] Once the flocculent has settled (primary settling), the
clarified supernatant can be harvested. To increase recombinant
product recovery, the floc can be washed or resuspended to remove
any residual recombinant product. Suitable wash diluents include
sucrose, PEG, cell culture media and buffer saline solution. In one
embodiment, the wash diluent is 9% sucrose. The floc and wash
diluent is mixed for <1 minute to 60 minutes and allowed to
settle for .about.1 hour to 24 hours. Once floc has settled
(secondary settling) the clarified secondary supernatant is
harvested. The supernatants from the primary and secondary settling
can be combined or purified separately.
[0061] Clarified supernatant can be harvested by removing the
supernatant by pumping or decanting followed by filtration through
a depth filter containing diatomaceous earth and followed by a
0.2.mu. cut off membrane filter or just a 0.2.mu. cutoff
filter.
[0062] Cationic polymer clearance can be monitored by methods known
in the art, such as assays for monitoring mammalian cell toxicity;
assays for determining the inhibition of DNA or RNA transcription
by DNA polymerase or reverse transcription, and by assays that
determine protein translation of mRNA; and by methods described
herein. For example, PDADMAC clearance from the recombinant protein
purification process intermediates can be monitored by inhibition
of DNA amplification in using quantitative polymerase chain
reaction (QPCR).
[0063] The cell culture broth can be used directly from the
bioreactor or it can be cooled prior to flocculation. In a
preferred embodiment the temperature range for the cell culture
broth is from at or about 36.degree. C. to at or about 20.degree.
C. In another embodiment, the cell culture broth is cooled to at or
about 20.degree. C.
[0064] The present invention provides a method of harvesting
recombinant proteins from mammalian cell cultures. The typical
methods used in commercial processes for the production of
recombinant proteins by mammalian cell culture include batch
culture, fed-batch and perfusion culture. Batch culture, a
discontinuous method where cells are grown in a fixed volume of
culture media for a short period of time followed by a full
harvest. Harvest typically occurs at the point when the maximum
cell density is achieved (typically 5-10.times.10.sup.6 cells/mL.
Fed-batch culture provides bolus or continuous media feeds to
replenish those media components that have been consumed. Since
fed-batch cultures receive additional nutrients throughout the run,
they have the potential to achieve higher cell densities (>10 to
30.times.10.sup.6 cells/ml) and increased product titers, when
compared to the batch method. With perfusion methods, typical large
scale commercial cell culture strategies strive to reach high cell
densities, 60-90(+).times.10.sup.6 cells/mL where almost a fifty to
over one-half of the reactor volume is biomass. With perfusion
culture, extreme cell densities of >1.times.10.sup.8 cells/mL
have been achieved and even higher densities are predicted.
[0065] As used herein "peptide," "polypeptide" and "protein" are
used interchangeably throughout and refer to a molecule comprising
two or more amino acid residues joined to each other by peptide
bonds. Peptides, polypeptides and proteins are also inclusive of
modifications including, but not limited to, glycosylation, lipid
attachment, sulfation, gamma-carboxylation of glutamic acid
residues, hydroxylation and ADP-ribosylation. Polypeptides can be
of scientific or commercial interest, including protein-based
drugs. Polypeptides include, among other things, antibodies, fusion
proteins, and cytokines. Peptides, polypeptides and proteins are
produced by recombinant animal cell lines using cell culture
methods and may be referred to as "recombinant peptide",
"recombinant polypeptide" and "recombinant protein". The expressed
protein(s) may be produced intracellularly or secreted into the
culture medium from which it can be recovered and/or collected.
[0066] Examples of polypeptides that can be harvested using the
methods of the invention include proteins comprising amino acid
sequences identical to or substantially similar to all or part of
one of the following proteins: tumor necrosis factor (TNF), flt3
ligand (WO 94/28391), erythropoeitin, thrombopoeitin, calcitonin,
IL-2, angiopoietin-2 (Maisonpierre et al. (1997), Science
277(5322): 55-60), ligand for receptor activator of NF-kappa B
(RANKL, WO 01/36637), tumor necrosis factor (TNF)-related
apoptosis-inducing ligand (TRAIL, WO 97/01633), thymic
stroma-derived lymphopoietin, granulocyte colony stimulating
factor, granulocyte-macrophage colony stimulating factor (GM-CSF,
Australian Patent No. 588819), mast cell growth factor, stem cell
growth factor (U.S. Pat. No. 6,204,363), epidermal growth factor,
keratinocyte growth factor, megakaryote growth and development
factor, RANTES, human fibrinogen-like 2 protein (FGL2; NCBI
accession no. NM_00682; Ruegg and Pytela (1995), Gene 160:257-62)
growth hormone, insulin, insulinotropin, insulin-like growth
factors, parathyroid hormone, interferons including
.alpha.-interferons, .gamma.-interferon, and consensus interferons
(U.S. Pat. Nos. 4,695,623 and 4,897,471), nerve growth factor,
brain-derived neurotrophic factor, synaptotagmin-like proteins (SLP
1-5), neurotrophin-3, glucagon, interleukins, colony stimulating
factors, lymphotoxin-.beta., leukemia inhibitory factor, and
oncostatin-M. Descriptions of proteins that can be produced
according to the inventive methods may be found in, for example,
Human Cytokines: Handbook for Basic and Clinical Research, all
volumes (Aggarwal and Gutterman, eds. Blackwell Sciences,
Cambridge, Mass., 1998); Growth Factors: A Practical Approach
(McKay and Leigh, eds., Oxford University Press Inc., New York,
1993); and The Cytokine Handbook, Vols. 1 and 2 (Thompson and Lotze
eds., Academic Press, San Diego, Calif., 2003).
[0067] Additionally the methods of the invention would be useful to
harvest proteins comprising all or part of the amino acid sequence
of a receptor for any of the above-mentioned proteins, an
antagonist to such a receptor or any of the above-mentioned
proteins, and/or proteins substantially similar to such receptors
or antagonists. These receptors and antagonists include: both forms
of tumor necrosis factor receptor (TNFR, referred to as p55 and
p75, U.S. Pat. No. 5,395,760 and U.S. Pat. No. 5,610,279),
Interleukin-1 (IL-1) receptors (types I and II; EP Patent No.
0460846, U.S. Pat. No. 4,968,607, and U.S. Pat. No. 5,767,064),
IL-1 receptor antagonists (U.S. Pat. No. 6,337,072), IL-1
antagonists or inhibitors (U.S. Pat. Nos. 5,981,713, 6,096,728, and
5,075,222) IL-2 receptors, IL-4 receptors (EP Patent No. 0 367 566
and U.S. Pat. No. 5,856,296), IL-15 receptors, IL-17 receptors,
IL-18 receptors, Fc receptors, granulocyte-macrophage colony
stimulating factor receptor, granulocyte colony stimulating factor
receptor, receptors for oncostatin-M and leukemia inhibitory
factor, receptor activator of NF-kappa B (RANK, WO 01/36637 and
U.S. Pat. No. 6,271,349), osteoprotegerin (U.S. Pat. No.
6,015,938), receptors for TRAIL (including TRAIL receptors 1, 2, 3,
and 4), and receptors that comprise death domains, such as Fas or
Apoptosis-Inducing Receptor (AIR).
[0068] Other proteins that can be harvested using the invention
include proteins comprising all or part of the amino acid sequences
of differentiation antigens (referred to as CD proteins) or their
ligands or proteins substantially similar to either of these. Such
antigens are disclosed in Leukocyte Typing VI (Proceedings of the
VIth International Workshop and Conference, Kishimoto, Kikutani et
al., eds., Kobe, Japan, 1996). Similar CD proteins are disclosed in
subsequent workshops. Examples of such antigens include CD22, CD27,
CD30, CD39, CD40, and ligands thereto (CD27 ligand, CD30 ligand,
etc.). Several of the CD antigens are members of the TNF receptor
family, which also includes 41BB and OX40. The ligands are often
members of the TNF family, as are 41BB ligand and OX40 ligand.
[0069] Enzymatically active proteins or their ligands can also be
harvested using the invention. Examples include proteins comprising
all or part of one of the following proteins or their ligands or a
protein substantially similar to one of these: a disintegrin and
metalloproteinase domain family members including TNF-alpha
Converting Enzyme, various kinases, glucocerebrosidase, superoxide
dismutase, tissue plasminogen activator, Factor VIII, Factor IX,
apolipoprotein E, apolipoprotein A-I, globins, an IL-2 antagonist,
alpha-1 antitrypsin, ligands for any of the above-mentioned
enzymes, and numerous other enzymes and their ligands.
[0070] The term "antibody" includes reference to both glycosylated
and non-glycosylated immunoglobulins of any isotype or subclass or
to an antigen-binding region thereof that competes with the intact
antibody for specific binding, unless otherwise specified,
including human, humanized, chimeric, multi-specific, monoclonal,
polyclonal, and oligomers or antigen binding fragments thereof.
Also included are proteins having an antigen binding fragment or
region such as Fab, Fab', F(ab').sub.2, Fv, diabodies, Fd, dAb,
maxibodies, single chain antibody molecules, complementarity
determing region (CDR) fragments, scFv, diabodies, triabodies,
tetrabodies and polypeptides that contain at least a portion of an
immunoglobulin that is sufficient to confer specific antigen
binding to a target polypeptide. The term "antibody" is inclusive
of, but not limited to, those that are prepared, expressed, created
or isolated by recombinant means, such as antibodies isolated from
a host cell transfected to express the antibody.
[0071] Examples of antibodies include, but are not limited to,
those that recognize any one or a combination of proteins
including, but not limited to, the above-mentioned proteins and/or
the following antigens: CD2, CD3, CD4, CD8, CD11a, CD14, CD18,
CD20, CD22, CD23, CD25, CD33, CD40, CD44, CD52, CD80 (B7.1), CD86
(B7.2), CD147, IL-1.alpha., IL-1.beta., IL-2, IL-3, IL-7, IL-4,
IL-5, IL-8, IL-10, IL-2 receptor, IL-4 receptor, IL-6 receptor,
IL-13 receptor, IL-18 receptor subunits, FGL2, PDGF-.beta. and
analogs thereof (see U.S. Pat. Nos. 5,272,064 and 5,149,792), VEGF,
TGF, TGF-.beta.2, TGF-.beta.1, EGF receptor (see U.S. Pat. No.
6,235,883) VEGF receptor, hepatocyte growth factor, osteoprotegerin
ligand, interferon gamma, B lymphocyte stimulator (BlyS, also known
as BAFF, THANK, TALL-1, and zTNF4; see Do and Chen-Kiang (2002),
Cytokine Growth Factor Rev. 13(1): 19-25), C5 complement, IgE,
tumor antigen CAl25, tumor antigen MUC1, PEM antigen, LCG (which is
a gene product that is expressed in association with lung cancer),
HER-2, HER-3, a tumor-associated glycoprotein TAG-72, the SK-1
antigen, tumor-associated epitopes that are present in elevated
levels in the sera of patients with colon and/or pancreatic cancer,
cancer-associated epitopes or proteins expressed on breast, colon,
squamous cell, prostate, pancreatic, lung, and/or kidney cancer
cells and/or on melanoma, glioma, or neuroblastoma cells, the
necrotic core of a tumor, integrin alpha 4 beta 7, the integrin
VLA-4, B2 integrins, TRAIL receptors 1, 2, 3, and 4, RANK, RANK
ligand, TNF-.alpha., the adhesion molecule VAP-1, epithelial cell
adhesion molecule (EpCAM), intercellular adhesion molecule-3
(ICAM-3), leukointegrin adhesin, the platelet glycoprotein gp
IIb/IIIa, cardiac myosin heavy chain, parathyroid hormone, rNAPc2
(which is an inhibitor of factor VIIa-tissue factor), MHC I,
carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), tumor
necrosis factor (TNF), CTLA-4 (which is a cytotoxic T
lymphocyte-associated antigen), Fc-.gamma.-1 receptor, HLA-DR 10
beta, HLA-DR antigen, sclerostin, L-selectin, Respiratory Syncitial
Virus, human immunodeficiency virus (HIV), hepatitis B virus (HBV),
Streptococcus mutans, and Staphlycoccus aureus. Specific examples
of known antibodies which can be produced using the methods of the
invention include but are not limited to adalimumab, bevacizumab,
infliximab, abciximab, alemtuzumab, bapineuzumab, basiliximab,
belimumab, briakinumab, canakinumab, certolizumab pegol, cetuximab,
conatumumab, denosumab, eculizumab, gemtuzumab ozogamicin,
golimumab, ibritumomab tiuxetan, labetuzumab, mapatumumab,
matuzumab, mepolizumab, motavizumab, muromonab-CD3, natalizumab,
nimotuzumab, ofatumumab, omalizumab, oregovomab, palivizumab,
panitumumab, pemtumomab, pertuzumab, ranibizumab, rituximab,
rovelizumab, tocilizumab, tositumomab, trastuzumab, ustekinumab,
vedolizomab, zalutumumab, and zanolimumab.
[0072] The invention can also be used to harvest recombinant fusion
proteins comprising, for example, any of the above-mentioned
proteins. For example, recombinant fusion proteins comprising one
of the above-mentioned proteins plus a multimerization domain, such
as a leucine zipper, a coiled coil, an Fc portion of an
immunoglobulin, or a substantially similar protein, can be produced
using the methods of the invention. See e.g. WO94/10308; Lovejoy et
al. (1993), Science 259:1288-1293; Harbury et al. (1993), Science
262:1401-05; Harbury et al. (1994), Nature 371:80-83; Hakansson et
al. (1999), Structure 7:255-64. Specifically included among such
recombinant fusion proteins are proteins in which a portion of a
receptor is fused to an Fc portion of an antibody such as
etanercept (a p75 TNFR:Fc), and belatacept (CTLA4:Fc).
[0073] For the purposes of this invention, cell culture medium is a
media suitable for growth of animal cells, such as mammalian cells,
in in vitro cell culture. Cell culture media formulations are well
known in the art. Typically, cell culture media are comprised of
buffers, salts, carbohydrates, amino acids, vitamins and trace
essential elements. The cell culture medium may or may not contain
serum, peptone, and/or proteins. Various tissue culture media,
including serum-free and defined culture media, are commercially
available, for example, any one or a combination of the following
cell culture media can be used: RPMI-1640 Medium, RPMI-1641 Medium,
Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium
Eagle, F-12K Medium, Ham's F12 Medium, Iscove's Modified Dulbecco's
Medium, McCoy's 5A Medium, Leibovitz's L-15 Medium, and serum-free
media such as EX-CELL.TM. 300 Series (JRH Biosciences, Lenexa,
Kans.), among others. Cell culture media may be supplemented with
additional or increased concentrations of components such as amino
acids, salts, sugars, vitamins, hormones, growth factors, buffers,
antibiotics, lipids, trace elements and the like, depending on the
requirements of the cells to be cultured and/or the desired cell
culture parameters.
[0074] Cell culture media may be serum-free, protein-free, and/or
peptone-free. "Serum-free" applies to a cell culture medium that
does not contain animal sera, such as fetal bovine serum.
"Protein-free" applies to cell culture media free from exogenously
added protein, such as transferrin, protein growth factors IGF-1,
or insulin. Protein-free media may or may not contain peptones.
"Peptone-free" applies to cell culture media which contains no
exogenous protein hydrolysates such as animal and/or plant protein
hydrolysates. Cell culture broth or like terminology refers to the
cell culture media that contains, among other things, viable and
non-viable mammalian cells, cell metabolites and cellular debris
such as nucleic acids, proteins and liposomes.
[0075] By cell culture or "culture" is meant the growth and
propagation of cells outside of a multicellular organism or tissue.
Suitable culture conditions for mammalian cells are known in the
art. See e.g. Animal cell culture: A Practical Approach, D.
Rickwood, ed., Oxford University Press, New York (1992). Mammalian
cells may be cultured in suspension or while attached to a solid
substrate. Fluidized bed bioreactors, hollow fiber bioreactors,
roller bottles, shake flasks, or stirred tank bioreactors, with or
without microcarriers, and operated in a batch, fed batch,
continuous, semi-continuous, or perfusion mode are available for
mammalian cell culture.
[0076] Mammalian cells, such as CHO cells, may be cultured in small
scale cultures, such as for example, in 100 ml to large scale cell
cultures, such as systems that have culture sizes in the thousand
and tens of thousands mls, for clinical and commercial
manufacturing of protein therapeutics.
[0077] The cell lines (also referred to as "host cells") are
genetically engineered to express a polypeptide of commercial or
scientific interest. Cell lines are typically derived from a
lineage arising from a primary culture that can be maintained in
culture for an unlimited time. Genetically engineering the cell
line involves transfecting, transforming or transducing the cells
with a recombinant polynucleotide molecule, and/or otherwise
altering (e.g., by homologous recombination and gene activation or
fusion of a recombinant cell with a non-recombinant cell) so as to
cause the host cell to express a desired recombinant polypeptide.
Methods and vectors for genetically engineering cells and/or cell
lines to express a polypeptide of interest are well known to those
of skill in the art; for example, various techniques are
illustrated in Current Protocols in Molecular Biology, Ausubel et
al., eds. (Wiley & Sons, New York, 1988, and quarterly
updates); Sambrook et al., Molecular Cloning: A Laboratory Manual
(Cold Spring Laboratory Press, 1989); Kaufman, R. J., Large Scale
Mammalian Cell Culture, 1990, pp. 15-69.
[0078] A wide variety of mammalian cell lines suitable for growth
in culture are available from the American Type Culture Collection
(Manassas, Va.) and commercial vendors. Examples of mammalian cell
lines commonly used in the industry include VERO, BHK, HeLa, CV1
(including Cos), MDCK, 293, 3T3, myeloma cell lines (e.g., NSO,
NS1), PC12, WI38 cells, and Chinese hamster ovary (CHO) cells. CHO
cells are widely used for the production of complex recombinant
proteins, e.g. cytokines, clotting factors, and antibodies (Brasel
et al. (1996), Blood 88:2004-2012; Kaufman et al. (1988), J. Biol
Chem 263:6352-6362; McKinnon et al. (1991), J Mol Endocrinol
6:231-239; Wood et al. (1990), J. Immunol. 145:3011-3016). The
dihydrofolate reductase (DHFR)-deficient mutant cell lines (Urlaub
et al. (1980), Proc Natl Acad Sci USA 77: 4216-4220), DXB11 and
DG-44, are desirable CHO host cell lines because the efficient DHFR
selectable and amplifiable gene expression system allows high level
recombinant protein expression in these cells (Kaufman R. J.
(1990), Meth Enzymol 185:537-566). In addition, these cells are
easy to manipulate as adherent or suspension cultures and exhibit
relatively good genetic stability. CHO cells and proteins
recombinantly expressed in them have been extensively characterized
and have been approved for use in clinical commercial manufacturing
by regulatory agencies.
[0079] While the terminology used in this application is standard
within the art, definitions of certain terms are provided herein to
assure clarity and definiteness to the meaning of the claims.
Units, prefixes, and symbols may be denoted in their SI accepted
form. Numeric ranges recited herein are inclusive of the numbers
defining the range and include and are supportive of each integer
within the defined range. Unless otherwise noted, the terms "a" or
"an" are to be construed as meaning "at least one of". The section
headings used herein are for organizational purposes only and are
not to be construed as limiting the subject matter described. The
methods and techniques described herein are generally performed
according to conventional methods well known in the art and as
described in various general and more specific references that are
cited and discussed throughout the present specification unless
otherwise indicated. See, e.g., Sambrook et al. Molecular Cloning:
A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (2001) and Ausubel et al., Current
Protocols in Molecular Biology, Greene Publishing Associates
(1992), and Harlow and Lane Antibodies: A Laboratory Manual Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990).
All documents, or portions of documents, cited in this application,
including but not limited to patents, patent applications,
articles, books, and treatises, are hereby expressly incorporated
by reference.
[0080] The present invention is not to be limited in scope by the
specific embodiments described herein that are intended as single
illustrations of individual aspects of the invention, and
functionally equivalent methods and components are within the scope
of the invention. Indeed, various modifications of the invention,
in addition to those shown and described herein will become
apparent to those skilled in the art from the foregoing description
and accompanying drawings. Such modifications are intended to fall
within the scope of the appended claims.
EXAMPLES
Example 1
[0081] This experiment compares different molecular weight
preparations of diallyldimethylammonium chloride (PDADMAC) for
flocculating mammalian cell culture broth and comparing their
settling times.
[0082] CHO cells expressing a recombinant monoclonal antibody were
grown in 2,000 L bioreactors in a fed batch culture for 15 days.
Cell culture broth was cooled to 10.degree. C. prior to testing. A
series of spin flasks were set up with 1 L of cell culture broth in
each flask. PDADMAC was supplied as 20% (w/v) liquid (Sigma
Aldrich, St. Louis, Mo.) and a working stock solution used in all
these experiments was prepared by diluting with purified water to
10% (w/v). PDADMAC, at molecular weights of 100,000-200,000;
200,000-350,000; and 400,000-500,000 was added to each flask to a
final concentration between 29 and 86 pg PDADMAC per Total cell
density. The PDADMAC solutions were added continuously for about 1
minute and incubated for 15 minutes, with stirring at 70-80 rpm at
10.degree. C. The floc was allowed to settle at ambient
temperature. This material was used for the settling time
determination.
[0083] A second fed batch culture was grown in a 1,000 L disposable
reactor for 15 days. The cell culture broth was maintained at
.about.36.degree. C. A series of spin flasks were set up with 1 L
of cell culture broth in each flask. PDADMAC at molecular weights
of <100,000, 100,000-200,000; 200,000-350,000; and
400,000-500,000 was added to each flask to a final concentration
between 25 and 76 pg PDADMAC per Total cell density. The PDADMAC
solutions were added continuously for about 1 minute and incubated
for 15 minutes, with stirring at 70-80 rpm at .about.36.degree. C.
The floc was allowed to settle at ambient temperature. This
material was used for the turbidity determination.
[0084] Total cell density was determined by adding the total number
of viable cells to the total non-viable cells as measured by Trypan
Blue exclusion using a Cedex Cell Counter and Analyzer (Roche
Innovatis AG, Indianapolis, Ind.). The flocculated solutions were
then transferred into 1 L glass graduated cylinders to determine
flocculated packed settling rate. Readings were taken at 15 minute
intervals for 90 minutes and the relative flocculation volume was
calculated as Settled Floc Volume/Total Volume.
[0085] The supernatant was removed from the settled flocculated
cell mass by decanting followed by 0.2.mu. filter. Turbidity was
measured using a 2100P turbidimeter (Hach, Loveland, Colo.).
[0086] FIGS. 2A and 2B show flocculation with PDADMAC having an
average molecular weight greater than 200,000, but less than
500,000 results in an optimum settling time and clarity compared to
PDADMAC with an average molecular weight less than 200,000.
Example 2
[0087] This experiment compares the amount of PDADMAC necessary for
flocculating mammalian cell culture broth expressing a recombinant
antibody from a small cell line, such as a diploid cell, and a
large cell line, such as a tetraploid cell line.
[0088] Diploid and tetraploid cell lines were grown as described
above. A series of spin flasks were set up with 1 L of cell culture
broth in each flask from each of the diploid and tetraploid
cultures. In this experiment and all the following experiments,
unless otherwise noted, PDADMAC having an average molecular weight
of 400,000 to 500,000 is used. PDADMAC was added to each flask to a
final concentration as shown in Table 2. Total cell density was
determined as described above.
TABLE-US-00001 TABLE 2 Final concentrations of PDADMAC for diploid
and tetraploid cultures Cell Type Final PDADMAC Concentration 15-20
.mu.m 11 pg per Total cell density cells, such as a 18 pg per Total
cell density diploid cell 25 pg per Total cell density 30 pg per
Total cell density 21-24 .mu.m 29 pg per Total cell density cells,
such as a 43 pg per Total cell density tetraploid cell 57 pg per
Total cell density 71 pg per Total cell density 86 pg per Total
cell density
[0089] The PDADMAC solutions were added continuously and stirred as
described above. The floc was allowed to settle at ambient
temperature.
[0090] The flocculated solutions were then transferred into 1 L
glass graduated cylinders to determine flocculated packed settling
rate. Readings were taken at 15 minute intervals for 90 to 120
minutes and the relative flocculation volume was calculated as
described above.
[0091] FIG. 3A shows flocculation with PDADMAC at a concentration
of 25 pg per Total cell had the fastest settling time.
[0092] FIG. 3B shows flocculation with PDADMAC at a concentration
of 57 pg per Total cell had the fastest settling time.
Example 3
[0093] This experiment looks at the impact of PDADMAC flocculation
for cell cultures that produce high lactate levels and/or have high
cell densities.
[0094] CHO cells expressing a recombinant monoclonal antibody were
grown in 1,000 L disposable bioreactor for 14 days. The cell
culture broth was at ambient temperature. Four spin flasks were set
up with 1 L of cell culture broth in each flask. Prior to adding
the PDADMAC, each flask was spiked with either 3 g/L, 6 g/L or 9
g/L Na DL-Lactate, at 60% (w/w) (Sigma Aldrich, St. Louis, Mo.), or
a no lactate as a control. PDADMAC was then added to each flask to
a final concentration of 25 pg/Total cell density (PDADMAC stock as
described above). Total cell density was determined as described
above.
[0095] PDADMAC was added continuously at ambient temperature and
stirred as described above. The floc was then allowed to settle at
ambient temperature.
[0096] The flocculated solutions were then transferred into 1 L
glass graduated cylinders to determine flocculated packed settling
rate. Readings were taken at various intervals for 1500 minutes and
the relative flocculation volume was calculated as described
above.
[0097] A second batch of CHO cell cultures were grown in a 1,000 L
disposable bioreactor, in a perfusion culture for 20 days. Cell
broth was cooled to ambient temperature. Prior to PDADMAC addition,
the cell broth was diluted 25%, 50% and 75% with cell culture
medium. Cell broth at a packed cell volume of 44% was not diluted
prior to PDADMAC addition. The final concentration of PDADMAC was
25 to 26 pg/Total cell density. The PDADMAC addition rate was
.about.1 minute, with stirring and settling at ambient temperature
as described above.
[0098] The flocculated solutions were then transferred into 1 L
glass graduated cylinders to determine flocculated packed settling
rate. Readings were taken at various intervals for 1400 minutes and
the relative flocculation volume was calculated as described
above.
[0099] FIGS. 4A and 4B show that settling rates significantly
decrease with cell culture processes that have high lactate levels
(>2-3 g/L) and/or produce high cell densities of biomass
(>10% packed cell volume).
Example 4
[0100] This experiment compares the settling times of different
diluents used in combination with PDADMAC.
[0101] CHO cells expressing a recombinant monoclonal antibody were
grown in a 1,000 L disposable bioreactor in a perfusion culture for
18 days. Cell broth from Day 16 was delivered for testing at
36.degree. C. and was cooled down to ambient temperature for 2.5
hours prior to flocculation. Addition and settling was performed at
ambient. Prior to PDADMAC addition, the cell broth was diluted to
67% with the various dilutents. Lactate level was 5 g/L. PDADMAC
addition rate .about.1 minute with an incubation period of 15
minutes at 75-85 rpm. PDADMAC stock solution was at 10% (w/v) from
the original stock solution of 20 (w/v) with purified water. Final
concentration of PDADMAC was 25 pg/Total cell density. The floc was
allowed to settle at ambient temperature.
[0102] A series of diluents was prepared; the concentrations are
shown in Table 3.
TABLE-US-00002 TABLE 3 Final concentrations of various diluents
Compound Final Concentration Sucrose 90 g/L (EMD, Philadelphia, PA)
Betaine 32 g/L (Sigma Aldrich, St. Louis, MO) PEG 1,000 146 g/L
(Sigma Aldrich, St. Louis, MO) PEG 6,000 260 g/L (Sigma Aldrich,
St. Louis, MO) Dextran 70/Betaine Dextran 100 g/L (Sigma Aldrich,
St. Louis, MO) Betaine 30 g/L Cell culture medium Control
[0103] The flocculated solutions were then transferred into 1 L
glass graduated cylinders to determine flocculated packed settling
rate. Readings were taken at various intervals for 1600 minutes and
the relative flocculation volume was calculated as described
above.
[0104] As shown in FIG. 5, the combination of PDADMAC and PEG 6,000
has the fastest settling time. The combination of PDADMAC with PEG
6,000, PEG1,000 or Dextran 70/Betaine had improved settling rates
compared to PDADMAC alone. The addition of non-ionic polymers
significantly increased the floc settling rate. The combination of
PDADMAC with either PEG or Dextran also decreased settling time
compared to PDADMAC alone, independent of lactate levels in the
culture.
Example 5
[0105] This experiment looks at the impact of the combination of
PDADMAC and PEG 3,000 on settling times for cell cultures with high
cell densities.
[0106] CHO cells expressing a recombinant monoclonal antibody were
grown in 80 L bioreactor in a perfusion culture for 20 days. Cell
culture broth was cooled to ambient temperature prior to testing.
Prior to PDADMAC addition, the cell broth was diluted to 10% with
the 36% (w/v) Sucrose or 25% (w/v) PEG 3,000 (both in purified
water). Final cell broth sucrose concentration was 3.6% (w/v) and
the final PEG 3,000 concentration was 2.5% (w/v). PDADMAC addition
rate .about.1 minute with a incubation period of 15 minutes at
75-85 rpm. PDADMAC stock solution was at 10% (w/v) from the
original stock solution of 20 (w/v) with purified water. Final
concentration of PDADMAC was 22 pg/Total cell density for PEG 3,000
and 25 pg/Total cell density for Sucrose and control or undiluted
cell broth. Control or undiluted cell broth PCV was 48%. The
diluted cell broth PCV=(control PCV.times.dilution
factor)=43.2%.
[0107] The flocculated solutions were then transferred into 1 L
glass graduated cylinders to determine flocculated packed settling
rate. Readings were taken at various intervals for 240 minutes and
the relative flocculation volume was calculated as described
above.
[0108] As shown in FIG. 6, the combination of PDADMAC and PEG 3,000
resulted in more rapid settling than PDADMAC alone. As seen in
Example 3, the settling rate for PDADMAC alone significantly
decreased with an increase in cell density. The combination of PEG
3,000 with PDADMAC decreased the flocculent settling time over that
of PDADMAC alone, independent of the cell culture process density
(in this case 43% packed cell volume).
Example 6
[0109] This experiment looks at the impact of the timing of the PEG
addition with PDADMAC on settling times.
[0110] CHO cells expressing a recombinant monoclonal antibody were
grown in 80 L bioreactor in a 19 day perfusion cell culture
process. Cell culture broth was cooled to 21.degree. C. Three spin
flasks were set up with 1 L of cell culture broth in each flask. To
one flask PDADMAC was added at a concentration of 45 pg/Total cell
density (molecular weight 400,000-500,000). To another flask
PDADMAC 45 pg/Total cell density and PEG 3,000, 15% (w/v) were
added as a bolus addition (the 1-Step Method). To the third flask,
PDADMAC was added at a concentration of 45 pg/Total cell density
and PEG 3,000 at a final concentration of 15% (w/v) was added
following the PDADMAC addition (the 2-Step Method). PDADMAC
addition rate was .about.1 minute. PDADMAC/PEG addition rate was
.about.5 minutes. All additions were at ambient temperature. All
flasks were incubated for 15 minutes at 75-85 rpm. The floc was
allowed to settle at ambient temperature.
[0111] The flocculated solutions were then transferred into 1 L
glass graduated cylinders to determine flocculated packed settling
rate. Readings were taken at various intervals for 240 minutes and
the relative flocculation volume was calculated as described
above.
[0112] As shown in FIG. 7, the combination of PDADMAC and PEG 3,000
either added simultaneously or sequentially decreased the
flocculent settling time over that of PDADMAC alone.
[0113] A large scale culture was next prepared. CHO cells
expressing a recombinant monoclonal antibody were grown in an 80 L
bioreactor in a perfusion culture for 19 days. Cell culture broth
was cooled to 21.degree. C. PDADMAC at a concentration of 45
pg/Total cell density and PEG 3,000 at a final concentration of 15%
(w/v) were added simultaneously at ambient temperature, addition
rate was 21 minutes, followed by 5 minute incubation at 100 rpm.
The floc was allowed to settle at ambient temperature.
[0114] Once the flocculent has settled (primary settling), the
clarified supernatant was be harvested by pumping the fluid from
the bioreactor followed by filtration through a depth filter
containing diatomaceous earth followed by a 0.2.mu. cut off
membrane filter.
[0115] The floc was washed in an equal volume of 9% sucrose
solution to remove any residual recombinant protein and allowed to
settle for 16 hours. Once floc settled (secondary settling) the
clarified secondary supernatant was harvested as described
above.
[0116] Clarified harvested cell culture supernatants from the above
flocculations (small scale and large scale) were purified using
Protein A chromatography followed by a product quality
determination. The Protein A eluate was not neutralized prior to
product quality determination. Protein A eluate product quality
attributes measured were molecular variants as measured by SEC,
host cell proteins as determined by ELISA.
[0117] The Protein A purified material was then passed over a CEX
column at pH 7.5.
TABLE-US-00003 TABLE 4 CEX @ pH 7.5 SEC CHOP Post- Harvest HMW
Monomer LMW (ppm) Pre Main Shoulder Post Control 11.6% 87.2% 1.2%
9,602 8.7% 82.9% 6.8% 1.6% Small Scale 9.2% 89.6% 1.2% 3,950 8.2%
83.5% 6.8% 1.6% 2 Step Method Small Scale 9.0% 89.8% 1.2% 3,602
8.2% 83.5% 6.8% 1.5% 1 Step Method Large Scale 9.6% 89.2% 1.2%
5,056 8.1% 83.5% 7.0% 1.4% Primary Settle Large Scale 11.7% 86.9%
1.5% 11,268 8.4% 83.8% 6.4% 1.5% Secondary Settle with Sucrose
Wash
[0118] Product quality is similar between the control and the
primary flocculate harvest for both scales. The PDADMAC/PEG primary
harvest tends to remove the higher order aggregate which is
reflected by the low HMW levels in the Protein A pool. A reduction
in the host cell protein level for PDADMAC/PEG harvest was
observed. The resuspension with sucrose resulted in slightly higher
levels of CHOP and HMW compared to the primary PDADMAC/PEG harvest
and is believed to be the resolubization of these impurities.
Example 7
[0119] This experiment looks at the impact on settling time by
adding a surfactant along with PDADMAC and PEG.
[0120] CHO cells expressing a recombinant monoclonal antibody were
grown in an 80 L bioreactor in a perfusion cell culture for 15
days. Cell broth from day 14 was cooled to 30.degree. C. for
testing. Two spin flasks were set up with 1 L of cell culture broth
in each flask. To one flask PDADMAC and PEG 3,000 were added
simultaneously, PDADMAC was added at a concentration of 25 pg/Total
cell density (molecular weight 400,000-500,000) and PEG 3,000 at a
final concentration of 3% (w/v). To the other flask Triton X-100 at
a final concentration of 0.05% (v/v) was added in addition to
PDADMAC and PEG at the above concentrations. (Triton X-100 stock
solution was at 10% (v/v) from an original stock solution of 20%
(v/v), Sigma Aldrich, St. Louis, Mo.) The three components were
added simultaneously. The addition rate was .about.1 minute, with
incubation for 15 minutes at 75-85 rpm. All flasks were spun as
described in earlier examples. The floc was allowed to settle at
ambient temperature.
[0121] The flocculated solutions were then transferred into 1 L
glass graduated cylinders to determine flocculated packed settling
rate. Readings were taken at various intervals for 240 minutes and
the relative flocculation volume was calculated as described
above.
[0122] As shown in FIG. 8, the addition of Triton X-100 along with
PDADMAC and PEG 3,000, decreased the flocculent settling time over
that of PDADMAC and PEG 3,000 alone.
* * * * *