U.S. patent application number 14/400389 was filed with the patent office on 2015-05-14 for purification of biological molecules.
The applicant listed for this patent is EMD Millipore Corporation. Invention is credited to William Cataldo, Christopher Gillespie, Jad Jaber, Mikhail Kozlov, Wilson Moya, Michael Phillips, Ajish Potty, Matthew T. Stone, Alex Xenopoulos.
Application Number | 20150133636 14/400389 |
Document ID | / |
Family ID | 46458134 |
Filed Date | 2015-05-14 |
United States Patent
Application |
20150133636 |
Kind Code |
A1 |
Xenopoulos; Alex ; et
al. |
May 14, 2015 |
Purification of Biological Molecules
Abstract
The present invention relates to improved processes and systems
for purification of biological molecules, where the processes can
be performed in a continuous manner.
Inventors: |
Xenopoulos; Alex;
(Billerica, MA) ; Phillips; Michael;
(Tyngsborough, MA) ; Moya; Wilson; (Concord,
MA) ; Jaber; Jad; (Sudbury, MA) ; Kozlov;
Mikhail; (Lexington, MA) ; Potty; Ajish;
(Stafford, TX) ; Stone; Matthew T.; (Cambridge,
MA) ; Cataldo; William; (Bradford, MA) ;
Gillespie; Christopher; (Shirley, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
EMD Millipore Corporation |
Billerica |
MA |
US |
|
|
Family ID: |
46458134 |
Appl. No.: |
14/400389 |
Filed: |
June 21, 2013 |
PCT Filed: |
June 21, 2013 |
PCT NO: |
PCT/US2013/046995 |
371 Date: |
November 11, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61666561 |
Jun 29, 2012 |
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61666329 |
Jun 29, 2012 |
|
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61666521 |
Jun 29, 2012 |
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Current U.S.
Class: |
530/387.1 ;
435/238; 435/297.1 |
Current CPC
Class: |
C07K 1/36 20130101; B01D
15/363 20130101; C07K 2317/14 20130101; B01D 15/362 20130101; C07K
16/00 20130101; C12M 29/04 20130101; B01D 15/3809 20130101; C12M
43/00 20130101; B01D 15/1871 20130101; B01D 15/3847 20130101; C12M
47/12 20130101; C12N 7/00 20130101; B01D 15/125 20130101 |
Class at
Publication: |
530/387.1 ;
435/238; 435/297.1 |
International
Class: |
C07K 1/36 20060101
C07K001/36; B01D 15/18 20060101 B01D015/18; C12M 1/00 20060101
C12M001/00; C07K 16/00 20060101 C07K016/00; C12N 7/00 20060101
C12N007/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 2, 2012 |
EP |
12004909.3 |
Claims
1. A process for the purification of a target molecule comprising
the steps of: a) providing a sample comprising the target molecule
and one or more impurities; b) adding at least one precipitant to
the sample and removing one or mom impurities, thereby to recover a
clarified sample; c) subjecting the clarified sample from step (b)
to a bind and elute chromatography step comprising at least two
separation units, thereby to obtain an eluate comprising the target
molecule; and d) subjecting the eluate to flow-through purification
comprising use of two or more media; wherein at least two steps are
performed concurrently for at least a portion of their duration and
wherein the process comprises only one bind and elute
chromatography step.
2. The process of claim 1, wherein the process is a continuous
process.
3. The process of claim 1, comprising a virus inactivation step
between steps (c) and (d).
4. The process of claim 3, wherein the virus inactivation step
comprises use of a virus inactivating agent selected from acid,
detergent, solvent and temperature change.
5. The process of claim 3, wherein virus inactivation step
comprises use of one or more in-line static mixers.
6. The process of claim 3, wherein virus inactivation comprises use
of one or more surge tanks.
7. The process of claim 1, wherein the target molecule is an
antibody.
8. The process of claim 7, wherein the antibody is selected from a
monoclonal antibody or a polyclonal antibody.
9. The process of claim 1, wherein the precipitant in step (b) is a
stimulus responsive polymer.
10. The process of claim 9, wherein the stimulus responsive polymer
is a modified polyallylamine polymer.
11. The process of claim 1, wherein the precipitant in step (b) is
selected from the group consisting of an acid, caprylic acid, a
flocculant and a salt.
12. The process of claim 1, wherein removing impurities in step (b)
comprises use of one or more depth filters.
13. The process of claim 1, wherein removing impurities in step (b)
comprises use of centrifugation.
14. The process of claim 1, wherein the bind and elute
chromatography step in (c) employs continuous multi-column
chromatography.
15. The process of claim 1, wherein the bind and elute
chromatography step in (c) is selected from the group consisting of
affinity chromatography, cation exchange chromatography and
mixed-mode chromatography.
16. The process of claim 1, wherein the bind and elute
chromatography step in (c) employs Protein A affinity
chromatography.
17. The process of claim 16, wherein Protein A affinity
chromatography employs a Protein A ligand coupled to a matrix
selected from the group consisting of rigid hydropbilic
polyvinylether polymer, controlled pore glass and agarose.
18. The process of claim 1, wherein the sample in step (a) is a
cell culture.
19. The process of claim 18, wherein the cell culture is provided
in a bioreactor.
20. The process of claim 19, wherein the bioreactor is a single use
bioreactor.
21. The process of claim 18, wherein the cell culture is provided
in a vessel other than a bioreactor.
22. The process of claim 1, wherein the precipitant in step (b) is
added to a bioreactor comprising a cell culture.
23. The process of claim 22, wherein the precipitant is added using
a static mixer.
24. The process of claim 1, wherein the precipitant in step (b) is
added to a vessel other than a bioreactor which comprises the
sample comprising the target molecule.
25. The process of claim 1, wherein the flow-through purification
in step (d) employs two or more media selected from activated
carbon, anion exchange chromatography media and cation exchange
chromatography media.
26. The process of claim 25, wherein the flow-through purification
in step (d) further comprises use of a virus filtration
membrane.
27. The process of claim 25, wherein the cation exchange
chromatography media is in the form of a membrane, a bead or a
fiber.
28. The process of claim 1, wherein the process comprises use of
one or more surge tanks and does not employ any pool tanks between
process steps.
29. The process of claim 1, further comprising a formulation
step.
30. The process of claim 29, wherein formulation comprises
diafiltration, concentration and sterile filtration.
31. A flow-through process for purifying a target molecule from a
Protein A eluate comprising the steps of: (a) contacting the eluate
recovered from a Protein A chromatography column with activated
carbon; (b) contacting the flow-through sample front step (a) with
an anion exchange chromatography media; and (c) contacting the
flow-through sample from step (b) with a cation exchange
cinematography media; and (d) obtaining the flow-through sample
from step (b) comprising the target molecule, wherein the eluate
flows continuously through steps (a)-(c) and wherein level of one
or more impurities in the flow-through sample in (d) is lower than
the level in the eluate in step (a).
32. The flow-through process of claim 31, further comprising
subjecting the flow-through sample from step (c) to virus
filtration.
33. The flow-through process of claim 31, further comprising use of
an in-line static mixer and/or a surge tank between steps (b) and
(c) to change pH.
34. The flow-through process of claim 31 or 32 or 33, wherein the
process employs a single skid.
35. The flow-through process of claim 31, wherein the eluate from
the Protein A chromatography column is subjected to virus
inactivation prior to contacting with activated carbon.
36. The process of claim 31, wherein steps (a)-(c) may be performed
in any order.
37. A flow-through purification process for purifying a target
molecule front a Protein A eluate comprising contacting the eluate
with two or more media selected from activated carbon, anion
exchange media, cation exchange media and virus filtration media,
wherein the flow of the eluate is continuous.
38. A system for use in a purification process comprising the
following devices: a) a bioreactor: b) a filtration device
comprising one or more depth filters: c) a single bind and elute
chromatography apparatus; and d) a flow-through purification system
comprising at least a flow-through anion exchange device, wherein
the devices in (a)-(d) are connected to be in fluid communication
with each other, such that a sample can flow continuously through
the system.
39. The system according to claim 38, wherein the bioreactor in (a)
is a single-use bioreactor.
40. The system according to claim 38, wherein the system is
enclosed in a sterile environment.
41. The system according to claim 38, wherein the bind and elute
chromatography apparatus in (c) comprises at least two separation
units, with each unit comprising the same chromatography media, and
wherein the two separation units are connected so a sample can flow
front one to the next.
42. The system according to claim 38, wherein the bind and elute
chromatography apparatus in (c) comprises three or mote separation
units having the same chromatography media, wherein the three of
more separation units are connected so that liquid can flow from
one separation unit to the next and from the last to the first
separation unit.
43. The system according to claim 38, wherein the flow-through
purification system in (d) further comprises a device selected from
an activated carbon device, a cation exchange chromatography device
and a virus filtration device.
44. The system according to claim 38, wherein the flow-through
purification system in (d) employs a single skid.
45. A process for purifying a target molecule from a sample, the
process comprising the steps of: (a) providing a bioreactor
comprising a cell culture; (b) adding a precipitant to the
bioreactor and removing one or more impurities, thereby resulting
in a clarified sample; (c) continuously transferring the clarified
sample to a Protein A affinity chromatography step, which employs
at least two separation units, thereby to obtain an eluate; (d)
continuously transferring the eluate from step (c) to a in-line
static mixer or a surge tank for mixing the eluate with one or more
virus inactivating agents; (e) continuously transferring the eluate
after step (d) into a flow-through purification operation
comprising contacting the eluate in flow-through mode with
activated carbon followed by an anion exchange chromatography media
followed by an in-line static mixer and/or a surge tank to change
pH followed by a cation exchange chromatography media followed by a
virus filtration media; and (f) formulating the flow-through sample
from step (e) at a desired concentration in a desired buffer,
wherein process steps are connected to be in fluid communication
with each other, such that a sample can flow continuously from one
process step to the next, and where at least two process steps
(b)-(f) are performed concurrently during at least a portion of
their duration.
46. A process for purifying a target molecule from a sample, the
process comprises the steps of: (a) providing a bioreactor
comprising a cell culture; (b) adding a precipitant to the
bioreactor and removing one or more impurities, thereby resulting
in a clarified sample; (c) adding one or more additives selected
from the group consisting of a salt, a detergent, a surfactant and
a polymer to the clarified sample; (d) subjecting the clarified
sample to a Protein A affinity chromatography step, which employs
at least two separation units, thereby to obtain an eluate; (e)
subjecting the eluate from step (d) to a virus inactivating agent
using an in-line static mixer or a surge tank; (f) contacting the
eluate after virus inactivation to a flow-through purification
operation comprising contacting the eluate in flow-through mode
with activated carbon followed by an anion exchange chromatography
media followed by an in-line static mixer and/or a surge tank to
change pH followed by a cation exchange chromatography media
followed by a virus filtration media; and (g) formulating the
flow-through sample from step (f) at a desired concentration in a
desired buffer, wherein the process steps are connected to be in
fluid communication with each other, such that a sample can flow
continuously from one process step to the next, and where at least
two process steps (b)-(g) are performed concurrently during at
least a portion of their duration.
47. The process of claim 46, wherein the additive is a salt.
48. The process of claim 47, wherein the salt is 0.5 M NaCl.
Description
RELATED APPLICATIONS
[0001] The present application claims the benefit of priority of
U.S. Provisional Patent Application No. 61/666,521, filing date
Jun. 29, 2012, U.S. Provisional Patent Application No. 61/666,561,
filing date Jun. 29, 2012, U.S. Provisional Patent Application No.
61/666,329, filing date Jun. 29, 2012, and European Patent
Application EP12004909.3, filing date Jul. 2, 2012, each of which
is incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
[0002] The present invention provides inventive and efficient
processes and systems for the purification of biological molecules
including therapeutic antibodies; and Fc-containing proteins.
BACKGROUND OF THE INVENTION
[0003] Efficient and economic large scale production of
biomolecules, e.g., therapeutic proteins including antibodies,
peptides or hormones, is an increasingly important consideration
for the biotechnology and pharmaceutical industries. Generally, the
purification processes are quite elaborate and expensive and
include many different steps.
[0004] Typically, proteins are produced using cell culture methods,
e.g., using either mammalian or bacterial cell lines recombinantly
engineered to produce the protein of interest. In general,
following the expression of the target protein, its separation from
one or more impurities such as, e.g., host cell proteins, media
components and nucleic acids, poses a formidable challenge. Such
separation and purification is especially important if the
therapeutic proteins are meant tor use in humans and have to be
approved by regulatory agencies, such as the Food and Drug
Administration (FDA).
[0005] Conventional processes used today for the purification of
proteins often include at least the following steps: (a) a
clarification step for the removal of cells and cellular debris,
e.g., using differential centrifugation and/or filtration; and (b)
one or more downstream chromatography steps to separate the protein
of interest from various impurities in the clarified cell culture
feed.
[0006] While the fermentation and cell culture processes can be run
either in a batch or fed-batch mode or continuously (e.g. in form
of a continuous perfusion process), the downstream purification
processes are typically run as batch processes that are often even
physically and logistically separated. Between each process step,
the sample is typically stored in a holding or pool tank or
reservoir in order to change solution conditions in order to render
it suitable for the next process step. Consequently, large vessels
are required to store the intermediate product. This leads to high
costs and very limited manufacturing flexibility and mobility.
[0007] In addition, performing a number of separate batch process
steps is labor and cost intensive as well as time consuming.
[0008] In case of monoclonal antibodies, the industry standard for
purification processes typically involves a "templated" process,
which includes several unit operations. One of the unit operations
is a purification step which employs an affinity ligand called
Protein A, isolated item Staphylococcus aureus, and which binds the
Fc-region of antibodies. Additional unit operations are usually
used in conjunction with the Protein A unit operation and most
biopharmaecutical companies employ process templates that are quite
similar in their use of the unit operations, whereas there may be
some variations in the order of the unit operations.
[0009] An exemplary templated process used in the industry today is
shown in FIG. 1. Key aspects of this template is a cell harvest
step, which typically involves use of centrifugation to remove cell
and cell debris from a cell culture broth, followed by depth
filtration. The cell harvest step is usually followed by a Protein
A affinity purification step, which is followed by virus
inactivation. Virus inactivation is typically followed by one or
more chromatographic steps, also referred to as polishing steps,
which usually include one or more of cation exchange chromatography
and/or anion, exchange chromatography and/or hydrophobic
interaction chromatography and/or mixed mode chromatography and/or
hydroxyapatite chromatography. The polishing steps are followed by
virus filtration and ultrafiltration/diafiltration, which completes
the templated process. See. e.g., Shukla et al., J. Chromatography
B., 848 (2007) 28-39; Liu et al., MAbs. 2010 Sep.-Oct. 2(5):
480-499.
[0010] Generally, the effluent of the filtration operations and the
eluate of the chromatographic operations are collected in
intermediate pool tanks and stored, often overnight, until the next
unit operation. The time needed to complete this process may be as
long as 4-7 days.
[0011] The present invention provides improved templates processes
which overcome several of the shortcomings of the templated
processes currently being used by the industry.
SUMMARY OF THE INVENTION
[0012] The present invention provides processes and systems which
provide several advantages over the typical templated processes
used in the industry today. The templated processes and systems
described herein include unit operations that are connected in a
continuous or semi-continuous manner and obviate the need for pool
tanks (also called holding tanks) between certain unit operations,
where holding tanks are typically used. Alternatively, only surge
tanks are employed.
[0013] Due to a specific combination of certain process steps, the
processes and systems described herein require fewer steps than
typical processes used in the industry and also significantly
reduce the time for the overall purification process, without
having an adverse impact on the product yield.
[0014] In one aspect according to the present invention, processes
for purifying a target molecule from a sample are provided. In some
embodiments, such a process comprises the steps of: (a) providing a
sample comprising the target molecule and one or more impurities;
(b) adding at least one precipitant to the sample and removing one
or more impurities, thereby to recover a clarified sample; (c)
subjecting the clarified sample from step (b) to a bind and elute
chromatography step comprising at least two separation units, with
each separation and comprising the same media, thereby to obtain an
eluate comprising the target molecule; and (d) subjecting the
eluate to flow-through purification comprising use of two or more
media; where at least two steps are performed concurrently for at
least a duration of their portion, and wherein the process
comprises a single bind and elute chromatography step.
[0015] In some embodiments, the flow of liquid through the process
is continuous, i.e. the process is a continuous process.
[0016] In some embodiments, the process comprises a virus
inactivation step between steps (c) and (d) above. As described
herein, the virus inactivation step comprises use of a virus
inactivation agent selected from an acid, a detergent, a solvent
and temperature change.
[0017] In some embodiments, the virus inactivation step employs the
use of one or more in-line static mixers. In other embodiments, the
virus inactivation step comprises the use of one or more surge
tanks.
[0018] In some embodiments, the target molecule is an antibody,
e.g., a monoclonal antibody or a polyclonal antibody.
[0019] In some embodiments, the precipitant employed in the
processes described herein is a stimulus responsive polymer. A
preferred stimulus responsive polymer is a modified polyallylamine
polymer, which is responsive to a phosphate stimulus.
[0020] Other exemplary precipitants include, but are not limited
to, e.g., an acid, caprylic acid, a flocculant and a salt.
[0021] In some embodiments, the removal of impurities following
addition of a precipitant employs the use of one or more depth
filter. In other embodiments, the removal of one or more impurities
employs the use of centrifugation.
[0022] Following precipitation and removal of one or more
impurities, the clarified sample is subjected to a single bind and
elute chromatography step, e.g., step (c) mentioned above, which
typically employs at least two separation units. In some
embodiments, the bind and elute chromatography step employs
continuous multi-column chromatography (CMC).
[0023] In a preferred embodiment, the bind and elute chromatography
step is an affinity chromatography step (e.g., Protein A affinity
chromatography). In other embodiments, the bind and elute
chromatography step comprises the use of a cation exchange (CEX)
bind aid elute chromatography step or a mixed mode chromatography
step.
[0024] In some embodiments, the bind and elute chromatography step
(e.g., Protein A affinity chromatography) employs the use of an
additive in the loading step, thereby resulting in reducing or
eliminating the number of intermediate wash steps that are
used.
[0025] Exemplary additives include salts, detergents, surfactants
and polymers. In some embodiments, an additive is a salt (e.g., 0.5
M NaCl)
[0026] In some embodiments, the starting sample is a cell culture.
Such a sample may be provided in a bioreactor.
[0027] In some embodiments, the sample is provided in a vessel
other than a bioreactor, e.g., it may be transferred to another
vessel from a bioreactor before subjecting it to the purification
process, as described herein.
[0028] In some embodiments, a precipitant used in step (b) above is
added directly to a bioreactor containing a cell culture. In other
embodiments, the precipitant is added to a vessel other than a
bioreactor. where the vessel contains a sample comprising a target
molecule. In some embodiments, the precipitant is added using a
static mixer.
[0029] In some preferred embodiments, the processes described
herein include a flow-through purification process operation, which
employs two or more media selected from activated carbon, anion
exchange chromatography media and cation exchange chromatography
media. In some embodiments, such a flow-through purification
operation additionally includes a virus filtration step, which
employs the use of a virus filtration membrane.
[0030] The processes described herein obviate the need to use a
pool tank between various process steps. In some embodiments, a
process according to the present invention comprises the use of one
or more surge tanks.
[0031] The processes described herein may additionally include a
formulation step. In some embodiments, such a formulation step
comprises diafiltration, concentration and sterile.
[0032] As stated above, the processes described herein include a
flow-through purification operation, which typically employs two or
more media. In some embodiments, a flow-through purification
process operation used in the processes described herein comprises
the following steps, where all steps ate performed in a
flow-through mode: (a) contacting the eluate from a Protein A
chromatography column with activated carbon; (b) contacting the
flow-through sample from step (a) with an anion exchange
chromatography media; (c) contacting the flow-through sample from
step (b) with a cation exchange chromatography media; and (d)
obtaining the flow-through sample from step (c) comprising the
target molecule, where the level of one or more impurities in the
flow-through sample after step (d) is lower than the level in the
eluate in step (a). The steps (a)-(c) described above may be
performed in any order.
[0033] To some embodiments, the flow-through purification step
further comprises a virus-filtration step, where the flow-through
sample from step (c) directly flows into a virus filtration
step.
[0034] In some embodiments, a solution change is required between
steps (b) and steps (c), where the solution change employs the use
of an in-line static mixer and/or a surge tank, to change the
pH.
[0035] In some embodiments, the entire flow-through purification
operation employs a single skid.
[0036] In some embodiments, the eluate from a Protein A
chromatography step is subjected to a virus inactivation step prior
to contacting the eluate with activated carbon.
[0037] In a particular embodiment described herein, a process for
purifying a target molecule from a sample is provided, where the
process comprises the steps of: (a) providing a bioreactor
comprising a cell culture; (b) adding a precipitant to the
bioreactor and removing one or more impurities, thereby resulting
in a clarified sample; (c) subjecting the clarified sample to a
Protein A affinity chromatography step, which employs at least two
separation units, thereby to obtain an eluate; (d) subjecting the
eluate from step (c) to a virus inactivating agent using an in-line
static mixer or a surge tank; (e) contacting the eluate after virus
inactivation to a flow-through purification operation comprising
contacting the eluate in flow-through mode with activated carbon
followed by an anion exchange chromatography media followed by an
in-line static mixer and/or a surge tank to change pH followed by a
cation exchange chromatography media followed by a virus filtration
media; and (f) formulating the flow-through sample from step (d) at
a desired concentration in a desired buffer, where the process
steps are connected to be in fluid communication with each other,
such that a sample can flow continuously from one process step to
the next, and where at least two process steps (b)-(f) are
performed concurrently during at least a portion of their
duration.
[0038] In some embodiments described herein, a process for
purifying a target molecule from a sample is provided, where the
process comprises the steps of: (a) providing a bioreactor
comprising a cell culture; (b) adding a precipitant to the
bioreactor and removing one or more impurities, thereby resulting
in a clarified sample; (c) adding one or more additives selected
from the group consisting of a salt, a detergent, a surfactant and
a polymer to the clarified sample: (d) subjecting the clarified
sample to a Protein A affinity chromatography step, which employs
at least two separation units, thereby to obtain an eluate: (e)
subjecting the eluate from step (d) to a virus inactivating agent
using an in-line static mixer or a surge tank; (f) contacting the
eluate after virus inactivation to a flow-through purification
operation comprising contacting the eluate in flow-through mode
with activated carbon followed by an anion exchange chromatography
media followed by an in-line static mixer and/or a surge tank to
change pH followed by a cation exchange chromatography media
followed by a virus filtration media: and (g) formulating the
flow-through sample from step (f) at a desired concentration in a
desired buffer, where the process steps are connected to be in
fluid communication with each other, such that a sample can flow
continuously from one process step to the next, and where at least
two process steps (b)-(g) are performed concurrently during at
least a portion of their duration.
[0039] In some embodiments, the additive in step (e) is 0.5 M
NaCl.
[0040] Also provided herein are systems for use in a purification
process, as described herein. In some embodiments, a system
includes: (a) a bioreactor; (b) a filtration device comprising one
or more depth filters; (c) one bind and elute chromatography
apparatus; and (d) a flow-through purification system comprising at
least a flow-through anion exchange device, where a liquid flows
continuously through the devices in (a)-(d) during a process run,
where the devices are connected to be in fluid communication with
each other.
[0041] In some embodiments, there is a connecting line between the
various devices in the system. The devices are connected in line
such that each device in the system is in fluid communication with
devices that precede and follow the device in the system.
[0042] In some embodiments, the bioreactor used in a system
according to the present invention is a disposable or a single use
bioreactor.
[0043] In some embodiments, the system is enclosed in a sterile
environment.
[0044] In some embodiments, the bind and elute chromatography
apparatus includes at least two separation units, with each unit
comprising the same chromatography media, e.g. Protein A affinity
media. In a particular embodiment, the Protein A media comprises a
Protein A ligand coupled to a rigid hydrophilie polyvinylether
polymer matrix. In other embodiments, the Protein A ligand is
coupled to agarose or controlled pore glass. The Protein A ligand
may be based on a naturally occurring domain of Protein A from
Staphylococcus aureus or be a variant or a fragment of a naturally
occurring domain. In a particular embodiment, the Protein A ligand
is derived from the C domain of Staphylococcus aureus Protein
A.
[0045] In other embodiments, the bind and elute chromatography
apparatus includes at least three separation units. The separation
units are connected to be in fluid communication with each other,
such that a liquid can flow from one separation unit to the
next.
[0046] In yet other embodiments, the hind and elute chromatography
step employs an additive in the clarified cell culture during the
loading step where the inclusion of an additive reduces or
eliminates the need for one or more wash steps before the elution
step.
[0047] In some embodiments, a flow-through purification system
additionally comprises an activated carbon device and a cation
exchange (CEX) flow-through chromatography device. In some
embodiments, the flow-through purification system further comprises
a virus filtration device.
[0048] In some embodiments, the entire flow-through purification
system employs a single skid.
BRIEF DESCRIPTION OF THE DRAWINGS
[0049] FIG. 1 is a schematic representation of a conventional
purification process used in the industry.
[0050] FIG. 2 is a schematic representation of an exemplary
purification process, as described herein. The purification process
shown uses a bioreactor for cell culture followed by the following
process steps: clarification: Protein A bind and elute
cinematography (capture); virus inactivation; flow-through
purification; and formulation. As shown, each of the process steps
employs one or more devices used to achieve the intended result of
the process step. As shown, clarification employs precipitation and
depth filtration: Protein A bind and elute chromatography is
performed using continuous multicolumn chromatography (CMC): virus
inactivation employs two in-line static mixers: flow-through
purification employs activated carbon (AC) followed by anion
exchange (AEX) chromatography followed by a pH change using an
in-line static mixer and a surge tank followed by flow-through
cation exchange (CEX) chromatography and virus filtration; and
formulation employs a diafiltration/concentration tangential flow
filtration device followed by sterile filtration. One or more
sterile filters are also employed throughout the process.
[0051] FIG. 3 is a graph depicting the results of an experiment to
measure pressure of each depth filter (primary and secondary) and
sterile filter used during the clarification step of the process in
FIG. 2. The X-axes denote filter load (L/m.sup.2), with the top
X-axis referring to the load of the sterile filter and the bottom
X-axis referring to the load of the two depth filters; and the
Y-axis denotes the pressure in psi.
[0052] FIG. 4 is a graph depicting the results of an experiment to
measure breakthrough of HCP and MAb following depth filtration
prior to loading on the Protein A continuous multicolumn
chromatography (CMC) set up. The X-axis denotes the depth filter
load (L/m.sup.2), the left Y-axis denotes MAb concentration (mg/mL)
and the right Y-axis denotes the HCP concentration (.mu.g/mL).
[0053] FIG. 5 is a schematic depiction of the flow-through
purification process step, as further described in Example 3.
[0054] FIG. 6 is a graph depicting the results of an experiment to
measure pressure profiles after depth filter, activated carbon and
virus filtration. The Y-axis denotes pressure (psi) and the X-axis
denotes time in hours.
[0055] FIG. 7 is a graph depicting the results of an experiment to
measure HCP breakthrough alter AEX loading. The Y-axis denotes HCP
concentration (ppm) and the X-axis denotes the AEX loading
(kg/L).
[0056] FIG. 8 is a graph depicting the results of an experiment to
measure removal of MAb aggregates as a function of loading of the
virus filtration device during the flow-through purification
operation. The X-axis denotes the virus filtration loading
(kg/m.sup.2) and the Y-axis denotes percentage of MAb aggregates in
the sample after virus filtration.
[0057] FIG. 9 is a graph depicting the results of an experiment to
measure pressure profiles after activated carbon and before virus
filtration during the flow-through purification operation. The
X-axis denotes time in hours and the Y-axis denotes pressure in
psi.
[0058] FIG. 10 is a graph depicting the results of an experiment to
measure pH and conductivity profiles, where pH is measured before
activated carbon and before CEX flow-through device and the
conductivity is measured before CEX flow-through device. The left
Y-axis denotes pH, the right Y-axis denotes conductivity (mS/cm)
and the X-axis denotes time in hours.
[0059] FIG. 11 is a chromatogram for Protein A capture of untreated
clarified MAb04 using CMC which employs two separation units.
[0060] FIG. 12 is a chromatogram far Protein A capture of
smart-polymer clarified MAb04 using CMC which employs two
separation units.
[0061] FIG. 13 is a chromatogram for Protein A capture of caprylic
acid clarified MAb04 using CMC which employs two separation
units.
[0062] FIG. 14 is a graph depicting the results of an experiment to
investigate the effect of residence time on HCP removal using
activated carbon and an anion exchange chromatography (AEX) device,
as part of the flow-through purification operation. The Y-axis
denotes HCP concentration (ppm) and the X-axis denotes AEX load
(kg/L).
[0063] FIG. 15 is a graph depicting the results of an experiment to
measure the effect on pH spike after using a surge tank between the
flow-through anion exchange chromatography and cation exchange
chromatography step in a flow-through purification operation. The
X-axis denotes pH and the Y-axis denotes time in hours.
[0064] FIG. 16 is a schematic depiction of the experimental set up
used for demonstrating that running the flow-through purification
operation in a continuous manner does not have a detrimental effect
on product purity.
[0065] FIG. 17 is a graph depicting the results of an experiment to
investigate pressure profiles after virus filtration, following use
of a virus filtration device in a continuous format and in a batch
mode. The Y-axis denotes pressure in psi and the X-axis denotes
processing time in hours.
[0066] FIG. 18 is a graph depicting the results of an experiment to
investigate the effect of flow-rate on throughput of the virus
filtration device. The Y-axis denotes pressure drop (psi) and the
X-axis denotes throughput of the virus filtration device
(kg/m.sup.2).
[0067] FIG. 19 depicts a chromatogram of Lot #1712 with MAb5 at pH
5.0 and 3 minutes residence time. As depicted in FIG. 19, the
majority of the product is collected in the flow-through and this
is indicated by the relatively quick breakthrough of protein UV
trace. The strip peak size generally varies based on the conditions
and total mass loaded but it is relatively enriched with aggregate
species at 95.6%, compared to the load material which had only 5.5%
aggregates.
[0068] FIG. 20 is a graph depicting the elution (first peak between
120 to 130 ml) and regeneration (around 140 ml) peaks from the
chromatogram of Protein A purification for cell culture treated
with stimulus responsive polymer and/or NaCl. Also shown is the
control without airy treatment. The X-axis represents the volume
passed through the Protein A column and the Y-axis represents the
absorbance at 280 nm wavelength.
[0069] FIG. 21 is a bar graph depicting the HCP LRV as a function
of NaCl concentration used in the intermediate wash or the loading
step during Protein A chromatography. The X-axis represents the
NaCl concentration in Molar (M) and the Y-axis represents the HCP
LRV.
[0070] FIG. 22 is a bar graph depleting the product (MAb)
percentage recovery as a function of the NaCl concentration in
either the intermediate wash step or the loading step during
Protein A chromatography. The X-axis represents the NaCl
concentration in M and the Y-axis represents the percent MAb
recovery.
[0071] FIG. 23 is a bar graph depicting the HCP concentration in
parts per million (ppm) as a function of the additive included in
either the intermediate wash step or the loading step during
Protein A chromatography. The X-axis represents the additive
included and the Y-axis represents the HCP concentration in
ppm.
[0072] FIG. 24 is a bar graph depicting the HCP LRV as a function
of the additive included in either the intermediate wash step or
the loading step during Protein A chromatography. The X-axis
represents the additive included and the Y-axis represents the HCP
LRV.
[0073] FIG. 25 is a bar graph depicting the ratio of the additive
elution pool volume to the control elution pool volume as a
function of the additive included in either the intermediate wash
step or the loading step during Protein A chromatography. The
X-axis represents the additive included and the Y-axis represents
the ratio of the additive elution pool volume to the control
elution pool volume.
DETAILED DESCRIPTION OF THE INVENTION
[0074] The present invention provides processes and systems which
overcome several shortcomings associated with the typical templated
processes used in the industry for purification or biological
molecules such as antibodies.
[0075] As discussed above, typical templated processes for
purification of biological molecules include many different steps,
including one or more chromatographic steps, require use of holding
or pool tanks between steps as well as take several hours to days
to complete.
[0076] There have been a few efforts to move away from a typical
templated process. For example, PCT Patent Publication No. WO
2012/014183 discusses methods for protein purification in which two
or more chromatographic separation modes are combined in tandem.
Additionally, U.S. Patent Publication No. 2008/0269468 discusses
combining a continuous perfusion fermentation system with a
continuous particle removal system and a continuous purification
system, where the flow rate of the mixture through the whole
process is kept substantially constant.
[0077] Further, PCT Publication No. WO2012/051147 discusses
processes for protein purification but does not appear to describe
a continuous or a semi-continuous process.
[0078] Lastly, PCT Publication No. WO2012/078677 describes a
continuous process for manufacture of biological molecules;
however, appears to rely on the utilization of multi-valve arrays.
Further, the aforementioned PCT publication also does not teach or
suggest use of all the process steps described herein. For example,
there appears to be no teaching or suggestion of a flow-through
purification operation which includes multiple flow-through steps
including, e.g., use of a flow-through activated carbon device, a
flow-through AEX media, a flow-through CEX media and a flow-through
virus filter. In fact, PCT Publication No. WO2012/078677 does not
teach or suggest a cation exchange chromatography step performed in
a flow-through mode. Lastly, the aforementioned PCT also fails to
describe a continuous process that uses a single bind and elute
chromatography step and can be performed successfully with minimum
interventions, as per the processes described herein.
[0079] Therefore, although it appears desirable to have a
purification process which is performed in a continuous mode, it
has been difficult to achieve an efficient continuous process due
to the complexity associated with connecting several individual
unit operations to run in a continuous or even a semi-continuous
mode with minimum interventions, e.g., fewer solution adjustments
(e.g., changes in pH and/or conductivity). The present invention,
however, has been able to achieve exactly that.
[0080] The present invention also provides other advantages over
conventional processes used in the industry today, e.g., reducing
the number of process steps as well as obviating the treed to use
large pool tanks between process steps for solution adjustments. In
case of the processes and systems described herein, it is not
required to perform large volume dilutions in order to change
conductivity, thereby obviating the need to use large pool tanks
between process steps. Additionally, in some embodiments, the
processes and systems described herein employ fewer
control/monitoring equipments (also called "skids"), which are
typically associated with every single process step, compared to
conventional processes used in the art.
[0081] In some embodiments, the present invention also provides
processes which employ the inclusion of an additive during the
loading step of Protein A chromatography, resulting in the
reduction in or elimination of one or more intermediate wash steps
by going straight from the loading step to the elution step or by
reducing the number of wash steps between the loading step and the
elution step, without sacrificing product purity. While U.S. Patent
Publication No. 20130096284 discusses inclusion of an amino acid or
salt In the sample being loaded onto a Protein A chromatography
column, the foregoing publication does not appear to teach or
suggest the use of such a Protein A chromatography step in a
multi-column continuous or a semi-continuous mode, as described
herein. Instead, it discusses the Protein A step to be performed in
a batch, single column mode.
[0082] The present invention demonstrates that even upon the
elimination of or reduction in the number of wash steps performed
during the Protein A chromatography step, a reduction in the level
of impurities, e.g., HCPs, is observed, without sacrificing product
purity.
[0083] In order that the present invention may be more readily
understood, certain terms are first defined. Additional definitions
are set forth throughout the detailed description.
Definitions
[0084] Before describing the present invention in detail, it is to
be understood that this invention is not limited to specific
compositions or process steps, as such may vary. It must be noted
that, as used in this specification and the appended claims, the
singular form "a", "an" and "the" include plural referents unless
the context clearly dictates otherwise. Thus, tor example,
reference to "a ligand" includes a plurality of ligands and
reference to "an antibody" includes a plurality of antibodies and
the like.
[0085] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention is related.
[0086] The following terms are defined for purposes of the
invention as described herein.
[0087] As used herein the term "target molecule" or "target
compound" refers to any molecule, substance or compound or mixtures
thereof that is isolated, separated or purified from one or more
impurities in a sample using processes and systems described
herein. In various embodiments, the target molecule is a biological
molecule such as, e.g., a protein or a mixture of two or more
proteins. In a particular embodiment, the target molecule is an
Fc-region containing protein such as an antibody.
[0088] The term "antibody" refers to a protein which has the
ability to specifically bind to an antigen. Typically, antibodies
have a basic four-polypeptide chain structure consisting of two
heavy and two light chains, said chains being stabilized, for
example, by interchain disulfide bonds. Antibodies may be
monoclonal or polyclonal and may exist in monomeric or polymeric
form, for example, IgM antibodies which exist in pentameric form
and/or IgA antibodies which exist in monomeric, dimeric or
multimeric form. Antibodies may also include multispecific
antibodies (e.g., bispecific antibodies), and antibody fragments so
long as they retain, or are modified to comprise, a ligand-specific
binding domain. The term "fragment" refers to a part or portion of
an antibody or antibody chain comprising fewer amino acid residues
than an intact or complete antibody or antibody chain. Fragments
can be obtained via chemical or enzymatic treatment of an intact or
complete antibody or antibody chain. Fragments can also be obtained
by recombinant means. When produced recombiantly, fragments may be
expressed alone or as part of a larger protein called a fusion
protein. Exemplary fragments include Fab, Fab', F(ab')2. Fc and/or
Fv fragments.
[0089] In some embodiments, an Fc-region containing protein is a
recombinant protein which includes the Fc region of an
immunoglobulin fused to another polypeptide or a fragment thereof.
Exemplary polypeptides include, e.g., renin; a growth hormone,
including human growth hormone and bovine growth hormone; growth
hormone releasing factor; parathyroid hormone; thyroid stimulating
hormone; lipoproteins; .alpha.-1-antitrypsin; insulin
.alpha.-chain; insulin .beta.-chain; proinsulin; follicle
stimulating hormone; calcitonin; luteinizing hormone; glucagon;
clotting factors such as factor VIIIC, factor IX, tissue factor,
and von Willebrands factor; anti-clotting factors such as Protein
C; atrial natriuretic factor; lung surfactant; a plasminogen
activator, such as urokinase or human urine or tissue-type
plasminogen activator (t-PA); bombesin; thrombin; hemopoietic
growth factor; tumor necrosis factor -.alpha. and -.beta.;
enkepbalinase; RANTES (regulated on activation normally T-cell
expressed and secreted); human macrophage inflammatory protein
(MlP-1-.alpha.); a serum albumin such as human serum albumin;
Muellerian-inhibiting substance; relaxin .alpha.-chain: relaxin
.beta.-chain; prorelaxin; mouse gonadotropin-associated peptide; a
microbial protein, such as .beta.-lactamase: DNase; IgF,: a
cytotoxic T-lymphocyte associated antigen (CTLA) (e.g., CTLA-4);
inhibin: activin; vascular endothelial growth factor (VEGF);
receptors for hormones or growth factors; Protein A or D;
rheumatoid factors; a neurotrophic factor such as bone-derived
neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3,
NT-4, NT-5, or NT-6), or a nerve growth, factor such, as
NGF-.beta.; platelet-derived growth factor (PDGF); fibroblast
growth factor such as .alpha.FGF and .beta.FGF; epidermal growth
factor (EGF); transforming growth factor (TGF) such as TGF-alpha
and TGF-.beta., including TGF-.beta.1, TGF-.beta.2, TGF-.beta.3,
TGF-.beta.4, or TGF-.beta.5; insulin-like growth factor-I and -II
(IGF-I and IGF-II); des(I-3)-IGF-I (brain IGF-I), insulin-like
growth factor binding proteins (IGFBPs); CD proteins such as CD3,
CD4, CD8, CD 19 CD20, CD34, and CD40; erythropoietin;
osteoinductive factors; immuntoxins; a bone morphogenetic protein
(BMP); an interferon such as inerferon-.alpha., -.beta., and
-.gamma.; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF,
and G-CSF; interleukins (ILs), e.g., IL-1 IL-1O; superoxide
dismutase; T-cell receptors; surface membrane proteins: decay
accelerating factor: vital antigen such as, for example, a portion
of the AIDS envelope; transport proteins; homing receptors;
addressins; regulatory proteins; integrins such as CD1 1a, CD1 1b,
CD1 1c, CD 18, an ICAM, VLA-4 and VCAM; a tumor associated antigen
such as HER2, HER3 or HER4 receptor; and fragments and/or variants
of any of the above-listed polypeptides. In addition, an antibody
that may be purified using the processes described herein may bind
specifically to any of the above-listed polypeptides.
[0090] As used herein, and unless stated otherwise, the term
"sample" refers to any composition or mixture that contains a
target molecule. Samples may be derived from biological or other
sources. Biological sources include eukaryotic and prokaryotic
sources, such as plant and animal cells, tissues and organs. The
sample may also include diluents, buffers, detergents, and
contaminating species, debris and the like that are found mixed
with the target molecule. The sample may be "partially purified"
(i.e., having been subjected to one or more purification steps,
such as filtration steps) or may be obtained directly from a host
cell or organism producing the target molecule (e.g., the sample
may comprise harvested cell culture fluid). In some embodiments, a
sample is a cell culture feed.
[0091] The term "impurity" or "contaminant" as used herein, refers
to any foreign or objectionable molecule, including a biological
macromolecule such as DNA, RNA, one or more host cell proteins,
endotoxins, lipids and one or more additives which may be present
in a sample containing the target molecule that is being separated
from one or more of the foreign or objectionable molecules using a
process of the present invention. Additionally, such impurity may
include any reagent which is used in a step which may occur prior
to the method of the invention. An impurity may be soluble or
insoluble in nature.
[0092] The term "insoluble impurity," as used herein, refers to any
undesirable or objectionable entity present in a sample containing
a target molecule, where the entity is a suspended particle or a
solid. Exemplary insoluble impurities include whole cells, cell
fragments and cell debris.
[0093] The term "soluble impurity," as used herein, refers to any
undesirable or objectionable entity present in a sample containing
a target molecule, where the entity is not an insoluble impurity.
Exemplary soluble impurities include host cell proteins (HCPs),
DNA, RNA, viruses, endotoxins, cell culture media components,
lipids etc.
[0094] The terms "Chinese hamster ovary cell protein" and "CHOP"
are used interchangeably to refer to a mixture of host cell
proteins ("HCP") derived from a Chinese hamster ovary ("CHO") cell
culture. The HCP or CHOP is generally present as an impurity in a
cell culture medium or lysate (e.g., a harvested cell culture fluid
("HCCF")) comprising a target molecule such as an antibody or
immunoadhesin expressed in a CHO cell). The amount of CHOP present
in a mixture comprising a target molecule provides a measure of the
degree of purity for the target molecule. HCP or CHOP includes, but
is not limited to, a protein of interest expressed by the host
cell, such as a CHO host cell. Typically, the amount of CHOP in a
protein mixture is expressed in parts per million relative to the
amount of the target molecule in the mixture. It is understood that
where the host cell is another cell type, e.g., a mammalian cell
besides CHO, E. coli, yeast, an insect cell, or a plant cell, HCP
refers to the proteins, other than target protein, found in a
lysate of that host cell.
[0095] The term "parts per million" or "ppm" are used
interchangeably herein to refer to a measure of purity of a target
molecule purified using a process described herein. The units ppm
refer to the amount of HCP or CHOP in nanograms/milligram per
target molecule in milligrams/milliliter (i.e., (CHOP
ng/mL)/(target molecule mg/mL), where the target molecule and the
HCPs are in solution).
[0096] The terms "purifying," "purification," "separate,"
"separating," "separation," "isolated," "isolating," or
"isolation," as used herein, refer to increasing the degree of
purity of a target molecule from a sample comprising the target
molecule and one or more impurities. Typically, the degree of
purity of the target molecule is increased by removing (completely
or partially) at least one impurity from the sample.
[0097] The terms "bind and elute mode" and "bind and elute
process," as used herein, refer to a separation technique in which
at least one target molecule contained in a sample (e.g., an Fc
region containing protein) binds to a suitable resin or media
(e.g., an affinity chromatography media or a cation exchange
chromatography media) and is subsequently eluted.
[0098] The terms "flow-through process," "flow-through mode," and
"flow-through operation," as used interchangeably herein, refer to
a separation technique in which at least one target molecule (e.g.,
an Fc-region containing protein or an antibody) contained in a
biopharmaceutical preparation along with one or more impurities is
intended to flow through a material, which usually binds the one or
more impurities, where the target molecule usually does not bind
(i.e., flows through).
[0099] The term "process step" or "unit operation," as used
interchangeably herein, refers to the use of one or more methods or
devices to achieve a certain result in a purification process.
Examples of process steps or unit operations which may be employed
in the processes and systems described herein include, but are not
limited to, clarification, bind and elute chromatography, virus
inactivation, flow-through purification (including use of two or
more media selected from activated carbon, anion exchange and
cation exchange in a flow-through mode) and formulation. It is
understood that each of the process steps or unit operations may
employ more than one step or method or device to achieve the
intended result of that process step or unit operation. For
example, in some embodiments, the clarification step and/or the
flow-through purification operation, as described herein, may
employ more than one step or method or device to achieve that
process step or unit operation. In some embodiments, one or more
devices which are used to perform a process step or unit operation
are single-use devices and can be removed and/or replaced without
having to replace any other devices in the process or even having
to stop a process run.
[0100] As used herein, the term "system" generally refers to the
physical form of the whole purification process, which includes two
or more devices to perform the process steps or unit operations, as
described herein. In some embodiments, the system is enclosed in a
sterile environment.
[0101] As used herein, the term "separation unit" refers to an
equipment or apparatus, which can be used in a bind and elute
chromatographic separation or a flow-through step or a filtration
step. For example, a separation unit can be a chromatography column
or a chromatography cartridge which is filled with a sorbent matrix
or a chromatographic device that contains a media that has
appropriate functionality. In some embodiments according to the
processes and systems described herein, a single bind and elute
chromatography step is used in the purification process which
employs two or more separation units. In a preferred embodiment,
the two or more separation units include the same media.
[0102] In various embodiments, the processes and systems described
herein obviate the need to necessarily use pool tanks, thereby
significantly reducing the overall time to run a purification
process as well as the overall physical footprint occupied by the
system. Accordingly, in various embodiments according to the
present invention, the output from one process step (or unit
operation) is the input for the nest step (or unit operation) in
the process and flows directly and continuously into the next
process step (or unit operation), without the need for collecting
the entire output from a process step.
[0103] As used herein, the term "pool tank" refers to any
container, vessel, reservoir, tank or bag, which is generally used
between process steps and has a size/volume to enable collection of
the entire volume of output from a process step. Pool tanks may be
used far holding or storing or manipulating solution conditions of
the entire volume of output from a process step. In various
embodiments according to the present invention, the processes and
systems described herein obviate the need to use one or more pool
tanks.
[0104] In some embodiments, the processes and systems described
herein may use one or more surge tanks throughout a purification
process.
[0105] The term "surge tank" as used herein, refers to any
container or vessel or bag, which is used between process steps or
within a process step (e.g., when a single process operation
comprises more than one step); where the output from one step flows
through the surge tank onto the next step. Accordingly, a surge
tank is different from a pool tank, in that it is not intended to
hold or collect the entire volume of output from a step; but
instead enables continuous flow of output from one step to the
next. In some embodiments, the volume of a surge tank used between
two process steps or within a process operation (e.g., flow-through
purification operation) described herein, is no more than 25% of
the entire volume of the output from the process step. In another
embodiment, the volume of a surge tank is no more than 10% of the
entire volume of she output from a process step. In some other
embodiments, the volume of a surge tank is less than 35%, or less
than 30%, or less than 25%, or less than 20%, or less than 15%, or
less than 10% of the entire volume of a cell culture in a
bioreactor, which constitutes the starting material from which a
target molecule is purified.
[0106] The term "continuous process," as used herein, refers to a
process for purifying a target molecule, which includes two or more
process steps (or unit operations), such that the output from one
process step flows directly into the next process step in the
process, without interruption and/or without the need to collect
the entire volume of the output from a process step before
performing the next process step. In a preferred embodiment, two or
more process steps can be performed concurrently for at least a
portion of their duration. In other words, in case of a continuous
process, as described herein, it is nor necessary to complete a
process step before the next process step is started, but a portion
of the sample is always moving through the process steps. The term
"continuous process" also applies to steps within a process
operation, in which case, during the performance of a process
operation Including multiple steps, the sample flows continuously
through the multiple steps that are necessary to perform the
process operation. One example of such a process operation
described herein is the flow through purification operation which
includes multiple steps that are performed in a continuous manner
and employs two or more of flow-through activated carbon,
flow-through AEX media, flow-through CEX media, and flow-through
virus filtration. In one embodiment, the flow through purification
operation is carried out in the order: activated carbon followed by
AEX media followed by CEX media followed by virus filtration.
However, it is understood that activated carbon, AEX media and CEX
media may be used in any order. Accordingly, in some embodiments,
AEX is followed by activated carbon followed by CEX media; or
alternatively, CEX is followed by activated carbon followed by AEX
media. In yet other embodiments, activated carbon is followed by
CEX media followed by AEX media. In still other embodiments, AEX
media is followed by CEX media followed by activated carbon: or
alternatively, CEX media is followed by AEX media followed by
activated carbon.
[0107] Continuous processes, as described herein, also include
processes where the input of the fluid material in any single
process step or the output is discontinuous or intermittent. Such
processes may also be referred to as "semi-continuous" processes.
For example, in some embodiments according to the present
invention, the input in a process step (e.g., a bind and elute
chromatography step) may be loaded continuously; however, the
output may be collected intermittently, where the other process
steps in the purification process are continuous. Accordingly, in
some embodiments, the processes and systems described herein
include at least one unit operation which is operated in an
intermittent matter, whereas the other unit operations in the
process or system may be operated in a continuous manner.
[0108] The term "connected process" refers to a process for
purifying a target molecule, where the process comprises two or
more process steps (or unit operations), which are connected to be
in direct fluid communication with each other, such that fluid
material continuously flows through the process steps in the
process and is in simultaneous contact with two or more process
steps during the normal operation of the process. It is understood
that at times, at least one process step in the process may be
temporarily isolated from the other process steps by a barrier such
as a valve in the closed position. This temporary isolation of
individual process steps may be necessary, for example, during
start up or shut down of the process or during removal/replacement
of individual unit operations. The term "connected process" also
applies to steps within a process operation which am connected to
be in fluid communication with each other, e.g., when a process
operation requires several steps to be performed in order to
achieve the intended result of the operation (e.g., the
flow-through purification operation used in the methods described
herein).
[0109] The term "fluid communication," as used herein, refers to
the flow of fluid material between two process steps or flow of
fluid material between process steps of a process operation, where
the process steps are connected by any suitable means (e.g., a
connecting line or surge tank), thereby to enable the flow of fluid
from one process step to another process step. In some embodiments,
a connecting line between two unit operations may be interrupted by
one or more valves to control the flow of fluid through the
connecting line. A connecting line may be in the form of a tube, a
hose, a pipe, a channel or some other means that enables flow of
liquid between two process steps.
[0110] The terms "clarify," "clarification," and "clarification
step," as used herein, refers to a process step for removing
suspended particles and or colloids, thereby to reduce turbidity,
of a target molecule containing solution, as measured in NTU
(nephelometric turbidity units). Clarification can be achieved by a
variety of means, including centrifugation or filtration.
Centrifugation could be done in a batch or continuous mode, while
filtration could be done in a normal flow (e.g., depth filtration)
or tangential flow mode. In processes used in the industry today,
centrifugation is typically followed by depth filtration intended
to remove insoluble impurities, which may not have been removed by
centrifugation. Furthermore, methods for enhancing clarification
efficiency can be used, e.g. precipitation. Precipitation of
impurities can be performed by various means such as by
flocculation, pH adjustment (acid precipitation), temperature
shirts, phase change due to stimulus-responsive polymers or small
molecules, or any combinations of these methods. In some
embodiments described herein, clarification involves any
combinations of two or more of centrifugation, filtration, depth
filtration and precipitation. In some embodiments, the processes
and systems described herein obviate the need for
centrifugation.
[0111] The term "precipitate," precipitating" or "precipitation" as
used herein, refers to process used in clarification, in which the
properties of the undesirable impurities are modified such that
they can be more easily separated from the soluble target molecule.
This is typically accomplished by forming large aggregate particles
and/or insoluble complexes containing the undesirable impurities.
These particles have properties (e.g. density or size) such that
they can be more easily separated from the liquid phase that
contains the soluble target molecule, such as by filtration or
centrifugation. In some cases, a phase change is caused, such that
the undesirable impurities can be more easily separated from the
soluble target molecule. Precipitation by phase change can be
achieved by the addition of a precipitating agent, such as a
polymer or a small molecule. In a particular embodiment, the
precipitant is a stimulus responsive polymer, also referred to as a
smart polymer. In some embodiments described herein, the
precipitant or precipitating agent is a flocculant. Flocculation,
as used herein, is one way of performing precipitation where the
performance typically depends on the flocculant concentration used
("dose dependent"). Typical flocculating agents are
polyelectrolytes, such as polycations, that complex with oppositely
charged impurities.
[0112] In some embodiments described herein, clarification employs
the addition of a precipitant to a sample containing a target
molecule and one or more impurities. In some cases, a change in
solution conditions (such as temperature, pH, salinity) may be used
to initiate the precipitation, such as in the case of stimulus
responsive polymers. The precipitated material which contains the
one or more impurities as well as the precipitating agent is
subsequently removed thereby recovering the target molecule in the
liquid phase, where the liquid is then typically subjected to
further process steps in order to further purify the target
molecule.
[0113] Precipitation may be performed directly in a bioreactor
containing a cell culture expressing a target molecule to be
purified, where a precipitant is added directly to the bioreactor.
Alternatively, the precipitant may be added to the cell culture,
which typically contains the target molecule, in a separate
vessel.
[0114] In some embodiments, the precipitant is added using a static
mixer. In case the precipitant is a stimulus responsive polymer,
both the polymer and the stimulus to which it is responsive, may be
added using a static mixer.
[0115] There are many ways known to those skilled in the art of
removing the precipitated material, such as centrifugation,
filtration or settling or any combinations thereof.
[0116] The term "settlings," as used herein, raters to a
sedimentation process in which the precipitated material migrates
to the bottom of a vessel under the influence of gravitational
forces. Settling can be followed by decanting or filtering of the
liquid phase or supernatant.
[0117] The term "stimulus" or "stimuli," as used interchangeably
herein, is meant to refer to a physical or chemical change in the
environment which results in a response by a stimulus responsive
polymer. Accordingly, such polymers are responsive to a stimulus
and the stimulus results in a change in the solubility of the
polymer. Examples of stimuli to which one or more polymers used
herein are responsive, include, but are not limited to, e.g.,
changes in temperature, changes in conductivity and/or changes in
pH. In some embodiments, a stimulus comprises addition of a
complexing agent or a complex forming salt to a sample. In various
embodiments, a stimulus is generally added after the addition of a
polymer to a sample. Although, the stimulus may also be added
during or before addition of a polymer to a sample.
[0118] The term "stimulus responsive polymer" as used herein,
refers to a polymer or copolymer which exhibits a change in a
physical and/or chemical property after the addition of a stimulus.
A typical stimulus response is a change in the polymer's
solubility. For example, the polymer poly(N-isopropylacrylamide) is
water soluble at temperatures below about 35.degree. C., but become
insoluble in water at temperatures of about 35.degree. C. In a
particular embodiment, a stimulus responsive polymer is a modified
polyallylamine (PAA) polymer which is responsive to a multivalent
ion stimulus (e.g. phosphate stimulus). Further details regarding
this polymer can be found, e.g., in U.S. Publication No.
20110313066, incorporated by reference herein in its entirety.
[0119] In some embodiments, a cell culture is subjected to a depth
filter to remove one or more impurities.
[0120] The terms "depth filter" or "depth filtration" as used
herein refer to a filter that is capable of retaining particulate
matter throughout the filter medium, rather than just on the filter
surface. In some embodiments described herein, one or more depth
fibers are used in the clarification process step.
[0121] In some embodiments, clarification results in the removal of
soluble and/or insoluble impurities in a sample which may later on
result in the fouling of a filter or device used in a process step
in a purification process, thereby making the overall purification
process more economical.
[0122] In various embodiments described herein, one or more
chromatography steps are included in a protein purification
process.
[0123] The term "chromatography" refers to any kind of technique
which separates an analyte of interest (e.g. a target molecule)
from other molecules present in a mixture through differential
adsorption onto a media. Usually, the target molecule is separated
from other molecules as a result of differences in rates at which
the individual molecules of the mixture migrate through a
stationary medium under the influence of a moving phase, or in bind
and elute processes.
[0124] The term "matrix," as used herein, refers to any kind of
particulate sorbent, bead, resin or other solid phase (e.g., a
membrane, non-woven, monolith, etc.) which usually has a functional
group or ligand attached to it. A matrix having a ligand or
functional group attached to it is referred to as "media," which in
a separation process, acts as the adsorbent to separate a target
molecule (e.g., an Fc region containing protein such as an
immunoglobubn) from other molecules present in a mixture (e.g., one
or more impurities), or alternatively, acts as a sieve to separate
molecules based on size (e.g., in ease of a virus filtration
membrane).
[0125] Examples of materials for forming the matrix include
polysaccharides (such as agarose and cellulose); and other
mechanically stable substances such as silica (e.g. controlled pore
glass), poly(styrenedivinyl)benzene. polyacrylamide, ceramic
particles and derivatives of any of the above. In a particular
embodiment, a rigid hydrophilic polyvinylether polymer is used as a
matrix.
[0126] Certain media may not contain ligands. Examples of media
that may be used in the processes described herein that do not
contain a ligand include, best are not limited to, activated
carbon, hydroxyapatite, silica, etc.
[0127] The term "ligand," as used herein, refers to a functional
group that is attached to a matrix and that determines the binding
properties of the media. Examples of "ligands" include, but are not
limited to, ion exchange groups, hydrophobic interaction groups,
hydrophilic interaction groups, thiophilic interactions groups,
metal affinity groups, affinity groups, bioaffinity groups, and
mixed mode groups (combinations of the aforementioned). Other
exemplary ligands which may be used include, but are not limited
to, strong cation exchange groups, such as sulphopropyl, sulfonic
acid; strong anion exchange groups, such as trimethylammonium
chloride; weak cation exchange groups, such as carboxylic acid;
weak anion exchange groups, such as N.sub.5N diethylamino or DEAE;
hydrophobic interaction groups, such as phenyl, butyl, propyl,
hexyl; and affinity groups, such as Protein A, Protein G, and
Protein L. In a particular embodiment, the ligand that is used in
the processes and systems described herein includes one or more
Protein A domains or a functional variant or fragment thereof as
described in U.S. Patent Publication Nos. 201002218442 and
20130046056, both incorporated by reference herein, which relate to
ligands based either on wild-type multimeric forms of B, Z or C
domains or on multimeric variants of Protein A domains (e.g., B, Z
or C domain pentamers). The ligands described therein also exhibit
reduced Fab binding.
[0128] The term "affinity chromatography" refers to a protein
separation technique in which a target molecule (e.g., an Fc region
containing protein of interest or antibody) specifically binds to a
ligand which is specific for the target molecule. Such a ligand is
generally covalently attached to a suitable chromatography matrix
material and is accessible to the target molecule in solution as
the solution contacts the chromatography media. In a particular
embodiment, the ligand is Protein A or a functional variant
thereof, immobilized onto a rigid hydrophilic polyvinylether
polymer matrix. The target molecule generally retains its specific
binding affinity for the ligand during the chromatographic steps,
while other solutes and/or proteins in the mixture do not bind
appreciably or specifically to the ligand. Binding of the target
molecule to the immobilized ligand allows contaminating proteins
and impurities to be passed through the chromatography matrix while
the target molecule remains specifically bound to the immobilized
ligand on the solid phase material. The specifically bound target
molecule is then removed in its active form from the immobilized
ligand under suitable conditions (e.g., low pH, high pH, high salt,
competing ligand etc.), and passed through the chromatographic
column with the elution buffer, substantially free of the
contaminating proteins and impurities that were earlier allowed to
pass through the column. It is understood that any suitable ligand
may be used for purifying its respective specific binding protein,
e.g. an antibody.
[0129] In some embodiments according to the present invention.
Protein A is used as a ligand for purifying an Fc region containing
target protein. The conditions for elution from the ligand (e.g.,
based on Protein A) of the target molecule (e.g., an Fc-region
containing protein) can be readily determined by one of ordinary
skill in the art. In some embodiments, Protein G or Protein L or a
functional variant thereof may be used as a ligand. In some
embodiments, a process which employs a ligand such as Protein A,
uses a pH range of 5-9 for binding to an Fc-region containing
protein, followed by washing or re-equilibrating the ligand/target
molecule conjugate, which is then followed by elution with a buffer
having pH about or below 4 which contains at least one salt.
[0130] The terms "Protein A" and "Prot A" are used interchangeably
herein and encompasses Protein A recovered from a native source
thereof. Protein A produced synthetically (e.g., by peptide
synthesis or by recombinant techniques), and variants thereof which
retain the ability to hind proteins which have a CH.sub.2/CH.sub.3
region, such as an Fc region. Protein A can be purchased
commercially from Repligen, GE or Fermatech. Protein A is generally
immobilized on a chromatography matrix. A functional derivative,
fragment or variant of Protein A used in the methods and systems
according to the present invention may be characterized by a
binding constant of at least K=10.sup.8 M, and preferably
K=10.sup.9 M, for the Fc region of mouse IgG2a or human IgG1. An
interaction compliant with such value for the binding constant is
termed "high affinity binding" in the present context. In some
embodiments, such functional derivatives or variants of Protein A
comprise at least part of a functional IgG binding domain of
wild-type Protein A, selected from the natural domains E, D, A, B,
C or engineered mutants thereof, which have retained IgG binding
functionality.
[0131] Also, Protein A derivatives or variants engineered to allow
a single-point attachment to a solid support may also be used in
the affinity chromatography step in the claimed methods.
[0132] Single point attachment generally means that the protein
moiety is attached via a single covalent bond to a chromatographic
support material of the Protein A affinity chromatography. Such
single-point attachment may also occur by use of suitably reactive
residues which are placed at an exposed amino acid position, namely
in a loop, close to the N- or C-terminus or elsewhere on the outer
circumference of the protein fold. Suitable reactive groups are
e.g. sulfhydryl or amino functions.
[0133] In some embodiments, Protein A derivatives of variants are
attached via multipoint attachment to suitable a chromatography
matrix.
[0134] The term "affinity chromatography matrix," as used herein,
refers to a chromatography matrix which carries ligands suitable
for affinity chromatography. Typically the ligand (e.g., Protein A
or a functional variant or fragment thereof) is covalently attached
to a chromatography matrix material and is accessible to the target
molecule in solution as the solution contacts the chromatography
media. One example of an affinity chromatography media is a Protein
A media. An affinity chromatography media typically binds the
target molecules with high specificity based on a lock/key
mechanism such as antigen/antibody or enzyme/receptor binding.
Examples of affinity media carrying Protein A ligands include
Protein A SEPHAROSE.TM. and PROSEP.RTM.-A. In the processes and
systems described herein, an affinity chromatography step may be
used as the single bind and elute chromatography step in the entire
purification process. In a particular embodiment, a Protein A based
ligand is attached to a rigid hydrophilie polyvinylether polymer
matrix. In other embodiments, such a ligand is attached to agarose
or to controlled pore glass.
[0135] The terms "ion-exchange" and "ion-exchange chromatography,"
as used herein, refer to the chromatographic process in which a
solute or analyte of interest (e.g., a target molecule being
purified) in a mixture, interacts with a charged compound linked
(such as by covalent attachment) to a solid phase ion exchange
material, such that the solute or analyte of interest internets
non-specifically with the charged compound more or less than solute
impurities or contaminants in the mixture. The contaminating
solutes in the mixture elute from a column of the ion exchange
material faster or slower than the solute of interest or are bound
to or excluded from the resin relative to the solute of
interest.
[0136] "Ion-exchange chromatography" specifically includes cation
exchange, anion exchange, and mixed mode ion exchange
chromatography. For example, cation exchange chromatography can
bind the target molecule (e.g., an Fc region containing target
protein) followed by elution (e.g., using cation exchange bind and
elute chromatography or "CEX") or can predominately bind the
impurities while the target molecule "flows through" the column
(cation exchange flow through chromatography FT-CEX).
[0137] Anion exchange chromatography can bind the target molecule
(e.g., an Fc region containing target protein) followed by elution
or can predominately bind the impurities while the target molecule
"flows through" the column, also referred to as negative
chromatography. In some embodiments and as demonstrated in the
Examples set forth herein, the anion exchange chromatography step
is performed in a flow through mode.
[0138] The term "ion exchange media" refers to a media that is
negatively charged (i.e., a cation exchange media) or positively
charged (i.e., an anion exchange media). The charge may be provided
by attaching one or more charged ligands to a matrix, e.g., by
covalent linkage. Alternatively, or in addition, the charge may be
an inherent property of the matrix (e.g., as is the case of silica,
which has an overall negative charge).
[0139] The term "anion exchange media" is used herein to refer to a
media which is positively charged, e.g. having one or more
positively charged ligands, such as quaternary amino groups,
attached to a matrix. Commercially available anion exchange media
include DEAE cellulose, QAE SEPBADEX.TM. and FAST Q SEPHAROSE.TM.
(GE Healthcare). Other exemplary materials that may be used in the
processes and systems described herein are Fractogel.RTM. EMD TMAE,
Fractogel.RTM. EMD TMAE highcap. Eshmuno.RTM. Q and Fractogel.RTM.
EMD DEAE (EMD Millipore).
[0140] The term "cation exchange media" refers to a media which is
negatively charged, and which has free cations for exchange with
cations in an aqueous solution contacted with the solid phase of
the media. A negatively charged ligand attached to the solid phase
to form the cation exchange media may, for example, be a
carboxylate or sulfonate. Commercially available cation exchange
media include carboxy-mtehyl-cellulose, sulphopropyl (SP)
immobilized on agarose (e.g., SP-SEPHAROSE FAST FLOW.TM. or
SP-SEPHAROSE HIGH PERFORMANCE.TM., from GE Healthcare) and
sulphonyl immobilized on agarose (e.g. S-SEPHAROSE FAST FLOW.TM.
from GE Healthcare). Preferred is Fractogel.RTM. EMD SO.sub.3,
Fractogel.RTM. EMD SE Highcap, Eshmuno.RTM. S and Fractogel.RTM.
EMD COO (EMD Millipore).
[0141] The term "mixed-mode chromatography" or "multi-modal
chromatography," as used herein, refers to a process employing a
chromatography stationary phase that carries at least two distinct
types of functional groups, each capable of interacting with a
molecule of interest. Mixed-mode chromatography generally employs a
ligand with more than one mode of interaction with a target protein
and/or imparities. The ligand typically includes at least two
different but cooperative sites which interact with the substance
to be bound. For example, one of these sites may have a
charge-charge type interaction with the substance of interest,
whereas the other site may have an electron acceptor-donor type
interaction and/or hydrophobic and/or hydrophilic interactions with
the substance of interest. Electron donor-acceptor interaction
types include hydrogen-bonding, .pi.-.pi., cation-.pi., charge
transfer, dipole-dipole and induced dipole interactions. Generally,
based on the differences of the sum of interactions, a target
protein and one or more impurities may be separated under a range
of conditions.
[0142] The term "mixed mode ion exchange media" or "mixed mode
media" refers to a media which is covalently modified with cationic
and/or anionic and hydrophobic moieties. A commercially available
mixed mode ion exchange media is BAKERBOND ABX.TM. (J. T. Baker,
Phillipsburg, N.J.) containing weak cation exchange groups, a low
concentration of anion exchange groups, and hydrophobic ligands
attached to a silica gel solid phase support matrix. Mixed mode
cation exchange materials typically have cation exchange and
hydrophobic moieties. Suitable mixed mode cation exchange materials
are Capto.RTM. MMC (GE Healthcare) and Eshmuno.RTM. HCX (EMD
Millipore).
[0143] Mixed mode anion exchange materials typically have anion
exchange and hydrophobic moieties. Suitable mixed mode anion
exchange materials are Capto.RTM. Adhere (GE Healthcare).
[0144] The term "hydrophobic interaction chromatography" or "HIC,"
as used herein, refers to a process for separating molecules based
on their hydrophobicity. i.e., their ability to adsorb to
hydrophobic surfaces from aqueous solutions. HIC is usually
differentiated from the Reverse Phase (RP) chromatography by
specially designed HIC resins that typically have a lower
hydrophobicity, or density of hydrophobic ligands compared to RP
resins.
[0145] HIC chromatography typically relies on the differences in
hydrophobic groups on the surface of solute molecules. These
hydrophobic groups tend to bind to hydrophobic groups on the
surface of an insoluble matrix. Because HIC employs a more polar,
less denaturing environment than reversed phase liquid
chromatography, it is becoming increasing popular for protein
purification, often in combination with ion exchange or gel
filtration chromatography.
[0146] The term "break-through," as used herein, refers to the
point of time during the loading of a sample containing a target
molecule onto a packed chromatography column or separation unit,
when the target molecule first appears in the output from the
column or separation unit. In other words, the term "break-through"
is the point of time when loss of target molecule begins.
[0147] A "buffer" is a solution that resists changes in pH by the
action of its acid-base conjugate components. Various buffers which
can be employed depending, for example, on the desired pH of the
buffer, are described in: Buffers, A Guide for the Preparation and
Use of Buffers in Biological Systems, Grueffroy, D., ed. Calbiochem
Corporation (1975). Non-limiting examples of buffers include MES,
MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate,
and ammonium buffers, as well as combinations of these.
[0148] When "loading" a sample onto a device or a column or a
separation unit containing a suitable media, a buffer is used to
load the sample comprising the target molecule and one or more
imparities onto the device or column or separation unit. In the
bind and elute mode, the buffer has a conductivity and/or pH such
that the target molecule is bound to media, while ideally all the
imparities are not bound and flow through the column. Whereas, in a
flow-through mode, a buffer is used to load the sample comprising
the target molecule and one or more impurities onto a column or
device or separation unit, wherein the buffer has a conductivity
and/or pH such that the target molecule is not bound to the media
and flows through while ideally all or most of the impurities bind
to the media.
[0149] The term "additive" as used herein, refers to any agent
which is added to a sample containing a target protein prior to
loading of the sample onto a chromatography matrix or during the
loading step, where the addition of the agent eliminates one or
more wash steps or reduces the number of wash step which are
otherwise designed for impurity removal, to be used subsequent to
the loading step and before the elution of the target protein. A
single agent may be added to a sample prior to or during the
loading or the number of agents may be more than one. Exemplary
additives include, but are not limited to, salts, polymers,
surfactants or detergents, solvents, chaotropic agents and any
combinations thereof. In a particular embodiment, such an additive
is sodium chloride salt.
[0150] In a particular embodiment, a static mixer is used for
contacting the output from the clarification step with an additive,
where the use of a static mixer significantly reduces the time,
thus allowing for a simplified connection of the clarification step
to the protein A chromatography step.
[0151] The term "re-equilibrating" refers to the use of a buffer to
re-condition the media prior to loading the target molecule. The
same buffer used for loading may be used for re-equilibrating.
[0152] The term "wash" or "washing" a chromatography media refers
to passing an appropriate liquid, e.g., a buffer, through or over
the media. Typically washing is used to remove weakly bound
contaminants front the media prior to eluting the target molecule
and/or to remove non-bound or weakly bound target molecule after
loading. In some embodiments, the wash buffer is different from the
loading buffer. In other embodiments, the wash buffer and the
loading buffer are the same. In a particular embodiment, a wash
step is eliminated or the number of wash steps is reduced in a
purification process by altering the conditions of the sample
load.
[0153] In some embodiments, the wash steps that are used in the
processes described herein employ a buffer having a conductivity of
20 mS/cm or less, and accordingly, are different from, the buffers
that are typically used for impurity removal, as those typically
have a conductivity greater than 20 mS/cm.
[0154] The term "conductivity" refers to the ability of an aqueous
solution to conduct an electric current between two electrodes. In
solution, the current flows by ion transport. Therefore, with an
increasing amount of ions present in the aqueous solution, the
solution will hove a higher conductivity. The unit of measurement
for conductivity is milliSeimens per centimeter (mS/cm or mS), and
can be measured using a commercially available conductivity meter
(e.g., sold by Orion). The conductivity of a solution may be
altered by changing the concentration of ions therein. For example,
the concentration of a buffering agent and/or concentration of a
salt (e.g. NaCl or KCl) in the solution may be altered in order to
achieve the desired conductivity. In some embodiments, the salt
concentration of the various buffers is modified to achieve the
desired conductivity. In some embodiments, in processes where one
or more additives are added to a sample load, if one or more wash
steps are subsequently used, such wash steps employ a buffer with a
conductivity of about 20 mS/cm or less.
[0155] The term "elute" or "eluting" or "elution" refers to removal
of a molecule (e.g., a polypeptide of interest or an impurity)
front a chromatography media by using or altering certain solution
conditions, whereby the buffer (referred to as an "elution buffer")
competes with the molecule of interest for the ligand sites on the
chromatography resin. A non-limiting example is to elute a molecule
from an ion exchange resin by altering the ionic strength of the
buffer surrounding the ion exchange material such that the buffer
competes with the molecule for the charged sites on the ion
exchange material.
[0156] In some embodiments, the elution buffer has a low pH (e.g.,
having a pH in the range of about 2 to about 5, or from about 3 to
about 4) and which disrupts the interactions between ligand (e.g.,
Protein A) and the target protein. Exemplary elution buffers
include phosphate, acetate, citrate and ammonium buffers, as well
as combinations or these. In some embodiments, an elution buffer
may be used which has a high pH (e.g., pH of about 9 or higher).
Elution buffers may also contain additional compounds, e.g.,
MgCl.sub.2 (2 mM) for facilitating elution.
[0157] In case virus inactivation (VI) is desired, a virus
inactivation buffer may be used to inactivate certain viruses prior
to during the target molecule. In such instances, typically, the
virus inactivation buffer differs from loading buffer since it may
contain detergent/detergents or have different properties
(pH/conductivity/salts and their amounts). In some embodiments,
virus inactivation is performed before the bind and elute
chromatography step. In some embodiments, virus inactivation is
performed after during or after elution from a bind and elute
chromatography step. In some embodiments, virus inactivation is
performed in-line using a static mixer. In other embodiments, virus
inactivation employs use of one or more surge tanks.
[0158] The term "bioreactor," as used herein, refers to any
manufactured or engineered device or system that supports a
biologically active environment. In some instances, a bioreactor is
a vessel in which a cell culture process is carried out. Such a
process may either be aerobic or anaerobic. Commonly used
bioreactors are typically cylindrical, ranging in size from liters
to cubic meters, and are often made of stainless steel. In some
embodiments described herein, a bioreactor is made of a material
other than steel and is disposable or single-use. It is
contemplated that the total volume of a bioreactor may be any
volume ranging from 100 mL, to up to 10,000 Liters or more,
depending on a particular process. In some embodiments according to
the processes and systems described herein, the bioreactor is
connected to a unit operation such as a depth filter. In some
embodiments described herein, a bioreactor is used for both cell
culturing as well as for precipitation, where a precipitant may be
added directly to a bioreactor, thereby to precipitate one or more
impurities.
[0159] The term "active carbon" or "activated carbon," as used
interchangeably herein, refers to a carbonaceous material which has
been subjected to a process to enhance its pore structure.
Activated carbons are porous solids with very high surface areas.
They can be derived from a variety of sources including coal, wood,
coconut husk, nutshells, and peat. Activated carbon can be produced
from these materials using physical activation involving heating
under a controlled atmosphere or chemical activation using strong
acids, bases, or oxidants. The activation processes produce a
porous structure with high surface areas that give activated carbon
high capacities for impurity removal. Activation processes can be
modified to control the acidity of the surface. In some embodiments
described herein, activated carbon is used in a flow through
purification step, which typically follows a bind and elute
chromatography step or a virus inactivation step which in turn
follows the bind and elute chromatography step. In some
embodiments, activated carbon is incorporated within a cellulose
media, e.g., in a column or some other suitable device.
[0160] The term "static mixer" refers to a device for mixing two
fluid materials, typically liquids. The device generally consists
of mixer elements contained in a cylindrical (tube) housing. The
overall system design incorporates a method for delivering two
streams of fluids into the static mixer. As the streams move
through the mixer, the non-moving elements continuously blend the
materials. Complete mixing depends on many variables including the
properties of the fluids, inner diameter of the tube, number of
mixer elements and their design etc. In some embodiments described
herein, one or more static mixers are used throughout the
purification process or system. In a particular embodiment, a
static mixer is used for contacting the output from the bind and
elute chromatography step with a virus inactivating agent (e.g., an
acid or any other suitable virus inactivating agent), where the use
of a static mixer significantly reduces the time, which would
otherwise be needed to accomplish effective virus inactivation.
Process According to the Present Invention
[0161] As discussed above, the present invention provides novel and
improved processes for purification of target molecules from a
sample (e.g., a cell culture feed) containing a target molecule and
one or more impurities. The processes described herein are a vast
improvement over existing methods used in the art, in that they
reduce the overall time frame required for a process run (12-24
hours relative to several days); include fewer steps relative to
most conventional processes; reduce the overall physical footprint
of a process by virtue of having fewer unit operations and are
easier to execute than a conventional process. Additionally, in
some embodiments, processes according to the present invention
employ devices that may be disposable.
[0162] The processes according to the present invention include
several process steps or unit operations which are intended to
achieve a desired result and where the process steps (or unit
operations) are connected such that to be in fluid communication
with each other and further that two or more process steps can be
performed concurrently for at least a part of the duration of each
process step. In other words, a user does not have to wait for a
process step to be completed before executing the next process step
in the process, but a user can start a process run such that the
liquid sample containing the target molecule flows through the
process steps continuously or semi-continuously, resulting in a
purified target molecule. Accordingly, the sample containing the
target molecule is typically in contact with mare than one process
step or unit operation in the process at any given time.
[0163] Each process step (or unit operation) may involve the use of
one or more devices or methods to accomplish the process step.
[0164] The processes described herein are different from
conventional processes used in the industry, in that they obviate
the need to use pool tanks for holding, diluting, manipulating and
sometimes storing the output from a process step before the output
is subjected to the next process step. In contrast, the processes
described herein enable any manipulation of the sample in-line
(e.g., using a static mixer) or employ the use of surge tanks
(which am usually not more than 10% or 20% or 25% of total volume
of the output from a process step) between process steps or
sometimes within a process operation (e.g., when a process
operation employs more than one method or device), thereby
significantly reducing the overall time to perform the process as
well as the physical footprint of the overall system for performing
the process. In a preferred embodiment, processes described herein
use no pool tanks but only surge tanks having a volume of less than
25%, preferably less than 10% of the volume of the output front the
preceding step.
[0165] The processes described herein include at least three
process steps-clarification, bind and elute chromatography and
flow-through purification. Typically, clarification is the first
step followed by bind and elute chromatography followed by
flow-through purification operation. The processes may include
additional process steps including, but not limited to, virus
inactivation and formulation. An important aspect of the processes
described herein is that regardless of the number of steps, the
process includes only one bind and elute chromatography step.
[0166] The various process steps are performed in a continuous or a
semi-continuous manner, as described herein. Following are examples
of process steps which may be used in a continuous or
semi-continuous process, as described herein. It is understood that
any combinations of the process steps shown below can be used. In
other words, any process step under Step 1 in the Table 1 below may
be combined with any process step under Step 2 and/or any process
step under Step 3 and so forth. It is also understood that
additional process steps, which are described elsewhere in the
specification may be combined with or used instead of one or more
of the process steps described in Table 1 below.
TABLE-US-00001 TABLE I Step 4 Step 2 Step 3 Flow- Step 1 Bind and
Elute Virus though Step 5 Clarification Chromatography Inactivation
purification Formulation Precipitation Continuous/semi- Virus Flow
Diafiltration in a vessel continuous bind inactivation through and
followed by and elute Protein in a surge AEX media concentration
depth A tank with or followed by filtration chromatography without
sterile virus filtration filtration Precipitation Simulated moving
Virus Flow Concentration in a vessel bed bind and elute
inactivation through followed by followed by Protein A using a AEX
media sterile centrifugation chromatography static mixer and CEX
filtration media with or without virus filtration Precipitation
Continuous/ Flow Diafiltration in a vessel semi-continuous through
followed by followed by Cation exchange activated sterile settling
and bind and elute carbon filtration microfiltration chromatography
media and of the AEX media supernatant with, or without virus
filtration Precipitation Continuous/semi- Flow in a bioreactor
continuous Mixed through followed by mode bind and activated depth
elute carbon filtration chromatography media and AEX media and CEX
media with or without virus filtration Precipitation in a
bioreactor followed by centrifugation Precipitation in a bioreactor
followed by settling and microfiltration of the supernatant
[0167] The various process steps (or unit operations) are described
in more detail infra.
[0168] The starting material for the purification process is
usually a sample containing a target molecule being purified.
Typically, a cell culture producing the target molecule is used.
However, samples other than cell cultures may also be used.
Exemplary samples include, but are not limited to, transgenic
mammalian cell cultures, non-transgenic mammalian cell cultures,
bacterial cell cultures, tissue cultures, microbial fermentation
batches, plant extracts, biofuels, seawater cultures, freshwater
cultures, wastewater cultures, treated sewage, untreated sewage,
milk, blood, and combinations thereof. Generally, the samples
contain various impurities in addition to the target molecule. Such
impurities include media components, cells, cell debris, nucleic
acids, host cell proteins, viruses, endotoxins, etc.
Clarification
[0169] One of the first process steps (or unit operations) in the
processes and systems described herein is typically clarification.
Clarification is intended to separate one or more soluble and/or
insoluble imparities from the target molecule. In some embodiments,
insoluble impurities like cells and cellular debris are removed
from the sample resulting in a clarified fluid containing the
target molecule in solution as well as other soluble impurities.
Clarification is typically performed prior to a step involving
capture of the desired target molecule. Another key aspect of
clarification is the removal of soluble and/or insoluble impurities
in a sample which may later on result in the fouling of a sterile
filter in a purification process, thereby making the overall
purification process more economical.
[0170] As used in the industry today, clarification generally
comprises removal of cells and/or cellular debris and typically
involves centrifugation as the first step, followed by depth
filtration. See, e.g., Shukla et al., J. Chromatography B, 848
(2007): 28-39; Liu et al., MAbs. 2(5): 480-499 (2010).
[0171] In some preferred embodiments described herein,
clarification obviates the need to use centrifugation.
[0172] For example, in some embodiments, where the starting volume
of the cell culture sample in a bioreactor is less than 2000
liters, or less than 1000 liters or less than 500 liters, the cell
culture sample may be subjected to depth filtration alone or to
settling and depth filtration, without the need for
centrifugation.
[0173] In some preferred embodiments, use of precipitation before
depth filtration increases throughput and therefore, the amount of
sample volume which may be processed without the need bar
centrifugation is also increased. In other words, in some
instances, if 1000 liters of a sample can be processed by depth
filtration alone, by combining that with precipitation, a user may
be able to process almost twice that amount, i.e., 2000 liters.
[0174] Depth filters are typically used to remove one or more
insoluble impurities. Depth filters are filters that use a porous
filtration medium to retain particles throughout the medium, rather
than just on the surface of the medium.
[0175] In some preferred embodiments, a depth filter is used, for
clarification, which is capable of filtering cellular debris and
particulate matter having a particle sixe distribution of about 0.5
.mu.m to about 200 .mu.m at a flow rate of about 10
liters/m.sup.2/hr to about 100 liters/m.sup.2/hr.
[0176] It has been found that especially good results in the
primary removal of particulate impurities can be achieved if the
porous depth filter is anisotropic (i.e. with a gradual reduction
in pore size). In some preferred embodiments, the pores have a
nominal pore size rating>about 25 .mu.m. In some preferred
embodiments, the depth filter comprises at least 2 graded layers of
non-woven fibers, wherein the graded layers have a total thickness
of about 0.3 cm to about 3 cm.
[0177] In some embodiments, the depth filters are configured in a
device which is able to filter high solid feeds containing
particles having a particle size distribution of approximately 0.5
.mu.m to 200 .mu.m at a flow rate of about 10 liters/m.sup.2/hr to
about 100 liters/m.sup.2/hr until the transmembrane pressure (TMP)
reaches 20 psi.
[0178] In some embodiments, depth filters comprise a composite of
graded layers of non-woven fibers, cellulose, and diatomaceous
earth. The non-woven fibers comprise polypropylene, polyethylene,
polyester, nylon or mixtures thereof.
[0179] Exemplary depth fibers and methods of use thereof may be
found in U.S. Patent Publication No. 20130012689, incorporated by
reference herein, which are particularly useful for filtering
samples containing particles have a size distribution of about 0.5
.mu.m to 200 .mu.m. Accordingly, in some embodiments, depth filters
used in the clarification step include open graded layers, allowing
the larger particles in the feed stream to penetrate into the depth
of the filter, and become captured within the pores of the filter
rather than collect on the surface. The open top layers of the
graded depth filters enable capturing of larger particles, while
the bottom layers enable capturing the smaller residual aggregated
particles. Various advantages of the graded depth filters include a
higher throughput retention of larger solids and eliminating the
problem of cake formation.
[0180] As discussed above, in some embodiments, clarification
includes the use of depth filtration following precipitation.
Precipitation may employ acid precipitation, use of a stimulus
responsive polymer, flocculation or settling and any other suitable
means/agent for achieving precipitation. Accordingly, in some
embodiments, a precipitant, e.g., a stimulus responsive polymer, is
added to a sample to precipitate one or more soluble and/or
insoluble impurities prior to depth filtration.
[0181] Other means of precipitation include, but are not limited
to, use of short-chain fatty acids such as caprylic acid, use of
flocculants, changing solution conditions (e.g., temperature, pH,
salinity) and acid precipitation. For example, it has been reported
that in mildly acidic conditions, the addition of short-chain fatty
acids such as caprylic acid typically precipitates non IgG proteins
while IgG is not precipitated.
[0182] Flocculation, as used herein, is one way of performing
precipitation where the precipitation typically depends on the
flocculant concentration used (i.e., is "close dependent"). Typical
flocculating agents are polyelectrolytes, such as polycations, that
complex with oppositely charged impurities.
[0183] Flocculants generally precipitate cells, cell debris and
proteins because of the interaction between the charges on the
cells/proteins and charges on the polymer (e.g. polyelectrolytes),
thereby creating insoluble complexes.
[0184] The use of polyelectrolyte polymers in flocculation to
purify proteins is well established in the art (see, e.g.,
international PCT Patent Application No. WO2008/091740,
incorporated by reference herein). Precipitation by flocculants can
be accomplished with a wide range of polymers, with the only
required general characteristic being the polymer must have some
level of interaction with a species of interest (e.g., a target
molecule or an impurity). Exemplary flocculants include polymers
such as chitosan and polysaccharides.
[0185] Flocculation may also be achieved by chemical treatment
resulting in changes in pH or by addition of a surfactant.
[0186] There are many ways known to those skilled in the art of
removing the precipitated material, such as centrifugation, depth
filtration, filtration or settling or any combinations thereof.
Settling can be followed by decanting or filtering of the liquid
phase or supernatant.
[0187] In some preferred embodiments, stimulus responsive polymers
are used for precipitating one or more impurities. Examples of such
stimulus responsive polymers can be found, e.g., in U.S.
Publication Nos., 20090036651, 20100267933 and 20110313066; each of
which is incorporated by reference herein in its entirety. Stimulus
responsive polymers are generally soluble in an aqueous based
solvent under a certain set of process conditions such as pH
temperature and/or salt concentration and are rendered insoluble
upon a change in one or more of such conditions and subsequently
precipitate out. Exemplary stimulus responsive polymers include,
but are not limited to, polyallylamine, polyallylamine modified
with a benzyl group or polyvinylamine and polyvinylamine modified
with a benzyl group, where the stimulus is phosphate or
citrate.
[0188] In some embodiments, a stimulus responsive polymer is
continuously added using a static mixer, in other embodiments, both
the polymer as well as the stimulus to which it is responsive are
added using a static mixer.
[0189] In some embodiments, small molecules are used for
precipitating one or more impurities, especially insoluble
impurities.
[0190] In some embodiments, small molecules used in the processes
described herein are non-polar and cationic, e.g., as described in
PCT Publication No. WO2013028334, incorporated by reference herein.
Exemplary small molecules that may be used for clarification
include, but are not limited to, monoalkyltrimethyl ammonium salt
(non-limiting examples include cetyltrimethylammonium bromide or
chloride, tetradecyltrimethylammonium bromide or chloride,
alkyltrimethyl ammonium chloride, alkylaryltrimethyl ammonium
chloride, dodecyltrimethylammonium bromide or chloride,
dodecyldimethyl-2-phenoxyethylammonium bromide, hexadecylamine
chloride or bromide, dodecyl amine or chloride, and
cetyldimethylethyl, ammonium bromide or chloride), a
monoalkyldimethylbenzyl ammonium salt (non-limiting examples
include alkyldimethylbenxyl ammonium chloride and benzethonium
chloride), a dialkyldimethyl ammonium salt (non-limiting examples
include domiphen bromide, didecyldimethyl ammonium halides (bromide
and chloride salts) and octyldodecyldimethyl ammonium chloride or
bromide), a heteroaromatic ammonium salt (non-limiting examples
include cetylpyridium halides (chloride or bromide salts) and
hexadecylpyridinium bromide or chloride, cis-isomer
1-[3-chloroallyl]-3,5,7-triaza-1-azoniaadamantane,
alkyl-isoquinolinium bromide, and alkyldimethylnaphthylmethyl
ammonium chloride), a polysubstituted quaternary ammonium salt,
(non-limiting examples include alkyldimethylbenzyl ammonium
saccharinate and alkyldimethylethylbenzyl ammonium
cyclohexylsulfamate), and a bis-quaternary ammonium salt
(non-limiting examples include
1,10-bis(2-methyl-4-amminoquinolinium chloride)-decane, 1,6-Bis
{1-methyl-3-(2,2,6-trimethyl cyclohexyl)-propyldimethyl ammonium
chloride hexane or triclobisonium chloride, and the bis-quat
referred to as CDQ by Buckman Brochures).
[0191] In a particular preferred embodiment, the small molecule is
benzethonium chloride (BZC).
[0192] In some embodiments, clarification is perforated directly in
a bioreactor. In other words, a precipitant, e.g., a stimulus
responsive polymer, may be added directly to a bioreactor
containing a culture of cells expressing a target molecule, thereby
precipitating the cells and cell debris, and where the target
molecule remains in the liquid phase obtained as a result of the
precipitation. In some preferred embodiments, the liquid phase is
further subjected to depth filtration. The liquid phase may also be
subjected to centrifugation, filtration, settling, or combinations
thereof.
[0193] In other embodiments, a stimulus responsive polymer is added
to a vessel which contains the cell culture and is separate from a
bioreactor. Therefore, as used herein, the term "vessel," refers to
a container separate from a bioreactor which is used for culturing
cells.
[0194] In some embodiments, a stimulus responsive polymer is added
in a sample before centrifugation, and centrifugation is followed
by depth filtration. In such a process, the size/volume of the
depth filter which may be required following centrifugation is
smaller than what is required in the absence of stimulus responsive
polymer.
[0195] In some embodiments, a clarified cell culture feed is
further subjected to a charged fluorocarbon composition (CFC), to
further remove host cell proteins (HCPs), as described in PCT
Application No. PCT/US2013/32768 (internal ref. no. MCA-1303PCT),
filed Mar. 18, 2013, which describes a CFC-modified membrane for
removal of HCPs, CFC-modified membranes can also be used after
other process steps in the purification process, e.g., following
Protein A bind and elute chromatography step or following
flow-through purification process step or following the
anion-exchange chromatography step, which is part of the
flow-through purification process step.
[0196] The clarified sample is typically subjected to a bind and
elute chromatography step.
Bind and Elute Chromatography
[0197] In various embodiments described herein, the processes and
systems include only a single bind and elute chromatography process
step for capture, which typically follows clarification. Bind and
elute chromatography is intended to bind the target molecule,
whereas the one or more impurities flow through (also referred to
as the "capture step"). The bound target molecule is subsequently
eluted and the eluate or output from the bind and elute
chromatography step may be subjected to further purification
steps.
[0198] Bind and elute chromatography may employ a single separation
unit or two or three or more separation units.
[0199] In various embodiments described herein, bind and elute
chromatography that is used is affinity bind and elute
chromatography or cation exchange bind and elute chromatography or
mixed mode bind and elute chromatography. Typically, bind and elute
chromatography employs the use of a media which is intended to bind
the target molecule.
[0200] In some preferred embodiments, the bind and elute
chromatography is an affinity chromatography. Suitable
chromatography media, that may be used for affinity chromatography
include, but are not limited to, media having Protein A, Protein G
or Protein I, functional groups (e.g., ProSep.RTM. High Capacity
(EMD Millipore), ProSep.RTM. Ultra Plus (EMD Millipore). Poros.RTM.
MabCapture.TM. A (Life Technologies), AbSolute.RTM. (NovaSep),
Protein A Ceramic HyperD.RTM. (Pall Corporation), Toyopearl
AF-rProtein A-650F (Tosoh), MabSelect.RTM. Sure (GE Healthcare)).
Suitable media are usually packed in a chromatography column or
device.
[0201] In a particular embodiment, the affinity chromatography
media includes a Protein A based ligand coupled to a hydrophilic
rigid polyvinylether polymer matrix.
[0202] In some embodiments according to the present invention, the
bind and elute chromatography process employs continuous
multi-column chromatography, also referred to as CMC.
[0203] In continuous chromatography, several identical columns are
typically connected in an arrangement that allows columns to be
operated in series and/or in parallel, depending on the method
requirements. Thus, all columns can be run simultaneously or may
overlap intermittently in their operation. Each column is typically
loaded, eluted, and regenerated several times during a process run.
Compared to conventional chromatography, where a single
chromatography cycle is based on several consecutive steps, such as
loading, washing, elution and regeneration, in case of continuous
chromatography based on multiple identical. columns, all these
steps may occur on different columns. Accordingly, continuous
chromatography operation may result in a better utilization of
chromatography resin and reduced buffer requirements, which
benefits process economy.
[0204] Continuous bind and elute chromatography also includes
simulated moving bed (SMB) chromatography.
[0205] In some preferred embodiments, bind and elute chromatography
employs CMC which uses two separation units. In some other
preferred embodiments, bind and elute chromatography employs CMC
which uses two or three or more units. In case of CMC, the loading
of a sample is usually continuous; however, the elution is
intermittent or discontinuous (i.e., CMC is semi-continuous in
nature).
[0206] In some preferred embodiments, CMC employs three separation
units, each containing the same chromatography media, and where the
separation units are connected such that liquid can flow from one
separation unit to the next separation unit and from the last to
the first separation unit, where the sample is loaded onto the
first separation unit at a pH and conductivity which enables
binding of the target molecule to the separation unit and where at
least part of duration of loading time overlaps with the loading of
the consecutive separation unit, where the two separation units are
in fluid communication, such that to enable any target molecules
that do not bind to the first separation unit being loaded to bind
to the next separation unit.
[0207] Different separation units can be at different stages of the
process at any given time; i.e., while one separation unit is being
loaded, the next separation unit could be subjected to washing,
eluting, re-equilibration etc. Also, while the first separation
unit is being subjected to the washing, eluting, re-equilibrating
steps, the consecutive separation unit is subjected to the loading
step and so forth, such that the sample flows continuously through
the separation units and has a velocity above 800 cm/h and that the
chromatography media of the separation units comprises particles
with a diameter between 40 and 200 .mu.m and with pore diameters in
the range between 50 nm and 200 nm.
[0208] In some embodiments, each separation unit includes an
affinity chromatography media such as, e.g., Protein A based media.
In other embodiments, each separation unit includes an ion exchange
media (e.g., a cation exchange chromatography media) or a
mixed-mode chromatography media.
[0209] Exemplary continuous chromatography processes which may be
used in the bind and elute chromatography process step, as
described herein, can be found, e.g., in European Patent
Application Nos. EP11008021.5 and EP 12002828.7, both incorporated
by reference herein.
[0210] In some embodiments, the separation units are connected in a
circular manner, also referred to as a simulated moving bed. For
example, in certain instances, at least three separation units are
connected in a circle and the loading of the sample is shifted
sequentially from one separation unit to the next, e.g., as
described in European Patent No. 2040811, incorporated by reference
herein.
[0211] It has been found that running bind and elute chromatography
in a semi-continuous or continuous mode enables using a reduced
volume of an affinity media, by up to 90% of the volume used in a
conventional process. Further, separation units with a reduced
diameter, between one third and one fifth compared to a batch
process, can be used. The separation units can be re-used multiple
times within the processing of a particular batch of a target
molecule, e.g., during the batch production of a target molecule
which is a therapeutic candidate.
[0212] In some embodiments, a separation unit that is being loaded
with a sample is in fluid communication with another separation
unit over the entire duration of the loading time.
[0213] In other embodiments, the separation unit that is being
loaded is in fluid communication with another separation unit for
only part of the duration of the loading time. In some embodiments,
two separation units are in fluid communication only for a second
half of the duration of the loading time.
[0214] In a batch chromatography mode, typically loading of a
separation unit (e.g., a chromatography column) is stopped, prior
to an excess of target molecule saturating the separation unit. In
contrast, in case of a CMC bind and elute chromatography process
step, as used in the processes and systems described herein, the
loading of a separation unit does not have to be stopped as target
molecules that do not bind to one separation unit move on to the
next separation unit because of fluid communication between the two
separation units, where the outlet of one separation unit is
connected with the inlet of a second separation unit and so forth.
It is understood that a person skilled in the art can readily
determine when during the loading step, the amount of a target
molecule that is not bound, to the separation unit that is being
loaded is sufficiently high, such that the outlet of the separation
unit being loaded needs to be connected to the inlet of another
separation unit. It has been found that this embodiment is
especially effective if the separation units comprise media having
a particle diameter between 40 and 200 .mu.m and pore diameter
ranging from 50 to 200 nm. With such media, the loading feed can be
run continuously at a velocity above 800 cm/h. Further details can
be found in EP12002828.7, filed, on Apr. 23, 2012. In some
embodiments, the outlet of the separation unit or the separation
units that are being washed is in a fluid communication with the
previous separation unit so that target molecules removed by said
washing are not lost but loaded onto the previous separation
unit.
[0215] It has been found that the level of impurities (e.g., HCPs)
that end up in the elation pool containing rite target molecule can
be significantly reduced with use of certain additives to the
sample load during bind and elute chromatography. In fact, addition
of certain additives to the sample prior to loading or during the
loading of the sample may obviate the need to use specific wash
steps typically designed to enhance impurity clearance. In other
words, the number of wash steps that are typically used is reduced
by inclusion of certain additives prior to loading or during the
loading of the sample.
[0216] In the context of continuous chromatography, a Protein A
column that has completed the loading step and is moved to
subsequent zones is required to complete all necessary steps within
a time frame expected such that the column will be ready to accept
fresh loading solution, e.g., as described herein, can be found,
e.g., in European Patent Application Nos. EP11008021.5 and
EP12002828.7, both incorporated by reference herein. The time that
is required to complete all necessary steps depends on the number
of steps or zones that the column must go through to be ready for
loading again. By reducing or eliminating steps, such as
intermediate washing, the application of continuous chromatography
for higher titers (target protein concentrations) is enabled where
the loading phase is expected to be shorter as well as simplifies
the timing required for all titer conditions during continuous
chromatography.
[0217] Exemplary additives which may be employed to reduce or
eliminate one or more intermediate wash steps include, hut are not
limited to, salts, polymers, surfactants or detergents, solvents,
chaotropic agents and any combinations thereof. A "salt" is a
compound formed by the interaction of an acid and a base. Examples
of salts include any and all chloride salts, sulfate salts,
phosphate salts, acetate and/or citrate salts, e.g., sodium
chloride, ammonium sulfate, ammonium chloride, potassium chloride,
sodium acetate. In a particular embodiment, the salt is NaCl (e.g.,
added to a final concentration of 0.5 M NaCl). The term
"hydrophobic salt" refers to a specific salt type with a
hydrophobic component such as, alkylamines; tetramethylammonium
chloride (TMAC), tetraethylammonium chloride (TEAC),
tetrapropylammonium chloride and tetrabutylammonium chloride. As
used herein, a "polymer" is a molecule formed by covalent linkage
of two or more monomers, where the monomers are not amino acid
residues. Examples of polymers include polyethylene glycol (PEG),
propylene glycol, and copolymers of ethylene and propylene glycol
(e.g., Pluronics, PF68, etc). In a particular embodiment, the
polymer is PEG.
[0218] The term "detergent" refers to surfactants, both ionic and
nonionic, such as polysorbates (e.g., polysorbates 20 or 80);
poloxamers, (e.g., poloxamer 188); Triton; sodium dodecyl sulfate
(SDS); sodium lauryl sulfate; sodium octyl glycoside; lauryl-,
myristyl-,, linoleyl-, or steatyl-sulfobetaine; (see U.S. Pat. No.
6,870,034B2 for more detergents). In a particular embodiment, the
detergent is a polysorbate, such as polysorbate 20 (Tween 20).
[0219] The terms "solvent" refers to a liquid substance capable of
dissolving or dispensing one or more other substances to provide a
solution. In some embodiments, the solvent is an organic, non-polar
solvent such as ethanol, methanol, isopropanol, acetonitrile,
hexylene glycol, propylene glycol and 2,2-thiodiglycol. The term
"chaotropic salt" refers to a salts that is known to disrupt the
intermolecular water structure. An example of such a salt is urea
and guamdimum HCl.
[0220] In some embodiments, the one or more additives are mixed
continuously with a clarified cell culture using one or more static
mixers. Accordingly, in some embodiments, a clarified cell culture
sample continuously flows to the Protein A chromatography step in a
protein purification process, where one or more additives, as
described herein, are continuously mixed, with the clarified cell
culture prior to loading onto a Protein A chromatography
matrix.
Virus Inactivation
[0221] In some embodiments according to the processes and systems
described herein, bind and elute chromatography is followed by
virus inactivation (VI). It is understood that virus inactivation
may not necessarily be performed but is considered optional.
[0222] Preferably, the output or eluate from bind and elute
chromatography is subjected to virus inactivation. Viral
inactivation renders viruses inactive, or unable to infect, which
is important, especially in case the target molecule is intended
for therapeutic use.
[0223] Many viruses contain lipid or protein coats that can be
inactivated by chemical alteration. Rather than simply rendering
the virus inactive, some viral inactivation processes are able to
denature the virus completely. Methods to inactivate viruses are
well known to a person skilled in the art. Some of the more widely
used virus inactivation processes include, e.g., use of one or more
of the following: solvent/detergent inactivation (e.g. with Triton
X 100); pasteurization (heating); acidic pH inactivation; and
ultraviolet (UV) inactivation. It is possible to combine two or
more of these processes; e.g., perform acidic pH inactivation at
elevated temperature.
[0224] In order to ensure complete and effective virus
inactivation, virus inactivation is often performed over an
extended period of time with constant agitation to ensure proper
mixing of a virus inactivation agent with the sample. For example,
in many large scale processes used in the industry today, an output
or eluate from a capture step is collected in a pool tank and
subjected to virus inactivation over an extended period of time
(e.g., >1 to 2 hours, often followed by overnight storage).
[0225] In various embodiments described herein, the time required
for virus inactivation is significantly reduced by performing virus
inactivation in-line or by employing a surge tank instead of a pool
tank for this step.
[0226] Examples of virus inactivation techniques that can be used
in the processes described herein can be found, e.g., in PCT Patent
Application No PCT/US2013/45677 (Internal ref. no. P12/098PCT).
[0227] In some preferred embodiments, virus inactivation employs
use of acidic pH, where the output from the bind and elute
chromatography step is subjected to in-line exposure to acidic pH
for virus inactivation. The pH used for virus inactivation is
typically less than 5.0, or preferably between 3.0 and 4.0. In some
embodiments, the pH is about 3.6 or lower. The duration of time
used for virus inactivation using an in-line method can range from
10 minutes or less, 5 minutes or less, 3 minutes or less, 2 minutes
or less, to about 1 minute or less. In ease of a surge tank the
time requited for inactivation is typically less than 1 hour, or
preferably less than 30 minutes.
[0228] In some embodiments described herein, a suitable virus
inactivation agent is introduced in-line into a tube or connecting
fine between bind and elute chromatography and the next unit
operation in the process (e.g., flow through purification), where
preferably, the tube or connecting line contains a static mixer
which ensures proper mixing of the output from the bind and elute
chromatography process step with the virus inactivation agent,
before the output goes on to the next unit operation. Typically,
the output from the bind and elute chromatography flows through the
static mixer at a certain flow rate, which ensures a minimum
contact time with the virus inactivation agent. The contact time
can be adjusted by using static mixers of a certain length and/or
diameter.
[0229] In some embodiments, a base or a suitable buffer is
additionally introduced into the tube or connecting line after
exposure to an acid for a duration of time, thereby to bring the pH
of the sample to a suitable pH for the next step, where the pH is
not detrimental to the target molecule. Accordingly, in some
preferred embodiments, both exposure to a low pH as well as that to
a basic buffer is achieved in-line with mixing via a static
mixer.
[0230] In some embodiments, instead of an in-line static mixer, or
in addition to an in-line static mixer, a surge tank is used for
treating use output from bind and elute chromatography with a virus
inactivation agent, where the volume of the surge tank is not more
than 25% of the total volume of the output from bind and elute
chromatography or not more than 1.5% or not more than 10% of volume
of the output from bind and elute chromatography. Because the
volume of the surge tank is significantly less than the volume of a
typical pool tank, more efficient mixing of the sample with the
virus inactivation agent can be achieved.
[0231] In some embodiments, virus inactivation can be achieved by
changing the pH of the elation buffer during bind and elute
chromatography, rather than having to change pH of the output from
bind and elute chromatography.
[0232] In some embodiments described herein, the sample is
subjected to a flow-through purification process, following virus
inactivation. In some embodiments, a filtration step may be
included after virus inactivation and before flow-through
purification. Such a step may be desirable, especially in cases
where turbidity of the sample is observed following virus
inactivation. Such a nitration step may employ a microporous filter
or a depth filter.
[0233] Although, in processes where virus inactivation step is
optional, the output from bind and elute chromatography may be
directly subjected to flow-through purification.
[0234] In various embodiments described herein, the output from
bind and elute chromatography is subjected to a flow-through
purification operation either directly, or following virus
inactivation. In some embodiments, flow-through purification
operation used in the processes and systems described herein
employs two or more process steps or devices or methods for
achieving flow-through purification, which is intended to remove
one or more impurities present in the output from bind and elute
chromatography, with or without virus inactivation.
[0235] In some preferred embodiments, flow through publication
operation, as described herein, includes one or more of the
following steps performed in a flow-through mode; activated carbon;
anion exchange chromatography; cation exchange chromatography,
mixed mode chromatography, hydrophobic interaction chromatography
and virus filtration, or combinations thereof. In some embodiments,
one or more valves, in-line static mixers and/or surge tanks may be
used between two or more-of these steps, in order to change
solution conditions.
[0236] The various steps, one or more of which may be used, to
achieve flow-through purification, are described in more detail
infra.
[0237] As described herein, in some embodiments, in some preferred
embodiments, flow-through purification employs at least one
flow-through anion exchange chromatography (AEX) step, where one or
more impurities still remaining in the sample containing the target
molecule bind the anion exchange chromatography media, whereas the
target molecule flows through.
[0238] In some embodiments, flow-through mixed-mode chromatography
or flow-through hydrophobic interaction chromatography may be used
instead, or in addition to flow-through anion-exchange
chromatography.
[0239] Exemplary anion exchange media which may be employed, for
AEX chromatography, include, but are not limited to, such as those
based on quaternary ammonium ions, as well as weak anion
exchangers, such as those based on primary, secondary, and tertiary
amine. Additional examples of suitable anion exchange media are Q
Sepharose.RTM. available from GE Healthcare Bio-Sciences AB,
Fractogel TMAE and Eshmuno Q available from EMD Chemicals,
Mustang.RTM. Q available from Pall Corp., Sariobind.RTM. Q
available from Sartorius Stedim, and ChromaSorb.TM. devices
available from EMD Millipore.
[0240] The media can be in the form of particles, membranes, porous
materials or monolithic materials, in preferred embodiments, media
are membrane based matrices, also called membrane absorbers. The
membrane adsorber is preferably a porous membrane sheet made by
phase separation methods well known in the art. See, for example,
Zeman L J, Zydney A L, Microfiltration and Ultrafiltration:
Principles and Applications, New York: Marcel Dekker, 1996. Hollow
fiber and tubular membranes are also acceptable matrices. The
membrane absorbers typically have a bed height of 0.5 to 5 mm.
[0241] Membranes can be manufactured from a broad range of
polymeric materials known in the art, including polyolefins, such
as polyethylene and polypropylene, polyvinylidene fluoride,
polyamide, polytetrafluoroethylene, cellulosics, polysulfone,
polyacrylonitrile, etc.
[0242] In order to impart anion exchange properties, the surface of
the membranes is usually modified by coating, grafting, adsorption,
and plasma-initiated modification with suitable monomers and/or
polymers.
[0243] In some embodiments, the anion exchange media that is used
for flow-through anion exchange is a membrane based media having a
surface coated with a crosslinked polymer having attached primary
amine groups such as a polyallylamine or a protonated
polyallylamine.
[0244] Additional suitable media can be found in, e.g., U.S. Pat.
No. 8,137,561, incorporated by reference herein, which describes
porous chromatographic or adsorptive media having a porous,
polymeric coating formed on a porous, self-supporting substrate and
anionic exchangers including such media as well as use methods of
purifying a target molecule using such media. Such media are
particularly suited for the robust removal of low-level impurities
from manufactured target molecules, such as monoclonal antibodies,
in a manner that integrates well into existing downstream
purification processes. Typical impurities include DNA endotoxin,
HCP and viruses. Such media function well at high salt
concentration and high conductivity (high affinity), effectively
removing viruses even under such conditions. High binding capacity
without sacrificing device permeability is achieved. Indeed,
depending on the coating properties, nucleic acid binding
capacities of greater than about 5 mg/mL, or greater than about 25
mg/mL, or greater than about 35-40 mg/mL, may be achieved. The
amount of the anion exchange adsorber is much less than that used
for a comparable bead-based process.
[0245] In some embodiments, the membranes having an anion exchange
functionality are encapsulated in a suitable multi-layer device
providing uniform flow through the entire stack of the membrane.
The devices can be disposable or reusable, and can either be
preassembled by the membrane manufacturer or assembled by the end
user. Device housing materials include thermoplastic resins, such
as polypropylene, polyethylene, polysulfone, polyvinylidene
fluoride, and the like; thermoset resins such as acrylics,
silicones and epoxy resins; and metals such as stainless steel. The
membrane can either be permanently bonded to the device housing,
such as by using an adhesive or thermal bonding, or held in place
by compression and carefully placed gaskets.
[0246] In some preferred embodiments, the anion-exchange adsorber
device is used at the solution pH value that is at least 0.5-1.0
units below the isoelectric point of the target protein. The
preferred pH range of anion-exchange adsorber device is from about
6 to about 8. Suitable range of salt concentration is between 0 and
500 mM, more preferably between 10 and 200 mM.
[0247] In some embodiments, flow-through purification may employ
additional steps. For example, in a preferred embodiment one or
more additional flow-through steps are used in addition to
anion-exchange chromatography (AEX). The additional flow-through
steps include, e.g., mixed-mode chromatography, cation exchange
chromatography, hydrophobic interaction chromatography, activated
carbon, size exclusion or combinations thereof.
[0248] Additional steps which may be included in flow-through
purification include, e.g., use of activated carbon prior to
anion-exchange chromatography or after anion-exchange
chromatography (and/or one or more of mixed mode and HIC). It some
embodiments, activated carbon is incorporated into a cellulose
media, e.g., in a column or a device. Alternatively, activated
carbon can be combined with an anion-exchange media (e.g., in a
column or a cartridge), thereby to bather remove one or more
impurities from a sample containing a target molecule. The column
or cartridge may also be deposable, e.g., Millistak.RTM. Pod. The
media can be in the form of particles, membranes, fibrous porous
materials or monolithic materials. In case of activated carbon, it
can be impregnated into a porous material, e.g., a porous fibrous
material.
[0249] It has been found that allow through activated carbon step
prior to the flow-through anion exchange chromatography is
especially suitable for the removal of host cell proteins and
leached Protein A. It is also capable of removing a significant
amount of potential impurities from cell culture media, such as
hormones, surfactants, antibiotics, and autifoam compounds. In
addition, it has been found that an activated carbon containing
device reduces the level of turbidity in the sample, for example
generated during pH increase of Protein A elution fractions.
[0250] Further details about carbonaceous materials, activated
carbon and their use in flow-through purification processes can be
found in PCT Publication No. WO2013/028330, which is hereby
incorporated by reference.
[0251] As discussed above, the flow-through purification operation
used in the processes and systems described herein may include more
than one flow-through step.
[0252] In preferred embodiments, flow-through purification further
includes one or more additional flow-through steps, e.g., for
aggregate removal and virus filtration. In some embodiments, the
sample is passed through an adsorptive depth filter, or a charged
or modified microporous layer or layers in a normal flow filtration
mode of operation, for aggregate removal. Examples of flow-through
steps which may be used for aggregate removal can be found in,
e.g., U.S. Pat. Nos. 7,118,675 and 7,465,397, incorporated by
reference herein. Accordingly, in some embodiments, a two-step
filtration process for removing protein aggregates and viral
particles may be used, wherein a sample is first filtered through
one or more layers of absorptive depth filters, charged or surface
modified porous membranes, or a small bed of chromatography media
to produce a protein aggregate-free sample. This may be followed by
the use of an ultrafiltration membrane for virus filtration, as
described in more detail below. Ultrafiltration membranes used tor
virus filtration are typically referred to as nanofiltration
membranes.
[0253] In some embodiments, an additional flow-through step employs
a cation exchange chromatography (CEX) media. Further details about
cation exchange flow through devices and their use to flow-through
purification processes can be found in U.S. patent application Ser.
No. 13/783,941 (internal ref no. MCA-1423), incorporated by
reference herein. Accordingly, in some embodiments, a cation
exchange chromatography media that is used after the anion exchange
chromatography step employs a solid support containing one or more
cation exchange binding groups at a density of 1 to 30 mM. Such
solid supports are able to bind protein aggregates relative to
monomers at a selectively greater than about 10.
[0254] In some embodiments, a negatively charged filtration medium
may be used tor removal of protein aggregates, e.g., comprising a
porous substrate coated with a negatively charged polymerized
cross-linked acrylamidoalkyl coating, polymerized in situ on the
surface of the substrate upon exposure to an electron beam and in
the absence of a chemical polymerization free-radical initiator,
where the coating is formed from a polyrmerizable acrylamidoalkyl
monomer having one or more negatively charged pendant groups and an
acrylamido cross-linking agent. Additional details concerning such
filtration media can be found, e.g., in PCT Publication No.
WO2010/098867, incorporated by reference herein.
[0255] The use of a flow-through cation-exchange step (CEX) may
necessitate a reduction of solution pH to increase affinity and
capacity for impurities, such as antibody aggregates. Such pH
reduction can be performed by a simple in-line addition of suitable
solution containing acid, via a three-way valve, a T-style
connector, a static mixer, or other suitable devices well known in
the art. In addition, a small surge vessel can be employed to
provide additional mixing and access for sampling. The volume of
the surge vessel, which can be in the form of a bag, a container,
or a tank, is usually considerably smaller that the volume of the
fluid processed with flow-through setup, for example not more than
10% of the volume of the fluid.
[0256] In some embodiments, the cation exchange media removes
protein aggregates and/or acts as a pre-filter for a
virus-filtration membrane, typically used after cation exchange
chromatography.
[0257] In another embodiment, protein aggregates can be removed
using a composite filter material that comprises a calcium
phosphate salt. Suitable calcium phosphate salts are dicalcium
phosphate anhydrous, dicalcium phosphate dehydrate, tricalcium
phosphate and tetracalcium phosphate. In another embodiment, the
calcium phosphate salt is hydroxyapatite. The solution conditions
are typically adjusted prior to loading the sample on such device,
in particular concentrations of phosphate ion and the ionic
strength. Further details about the removal of protein aggregates
using a composite filler material that comprises a calcium
phosphate salt in flow-through mode can be found in WO2011156073
A1, which is incorporated by reference herein.
[0258] The entire flow-through purification operation (including
the anion exchange chromatography step and one or more additional
steps, as described herein), are performed continuously without the
use of a pool tank between flow-through process steps.
[0259] In some embodiments, the flow-through purification process
additionally includes virus filtration. However, virus filtration
is optional and may not necessarily always be used.
[0260] In some embodiments, virus filtration involves filtration
based on size exclusion, also referred to as sieving.
[0261] For virus removal, the sample is typically passed through an
ultrafiltration fiber that retains the viruses while the target
molecule passes through. According to IUPAC, ultrafiltration is a
"pressure-driven membrane-based separation process in which
particles and dissolved macromolecules smaller than 0.1 .mu.m and
larger than about 2 nm are rejected," (IUPAC, "Terminology for
membranes and membrane processes" published in Pure Appl. Chem.,
1996, 68, 1479). The ultrafiltration membranes used in this step
are usually specifically designed to remove viruses. In contrast to
ultrafiltration membranes used for protein concentration and buffer
exchange, these membranes are usually not characterized by the
molecular weight cut-offs, but rather by typical retention of viral
particles. Viral retention is expressed in log reduction value
(LRV), which is simply a Log.sub.10 of the ratio of viral particles
in feed and filtrate in a standardized test. Use of viral
filtration in purification processes can be found in, e.g.,
Meltzer, T., and Jornitz, M,. eds., "Filtration and Purification in
the Biopharmaceutical Industry", 2nd ed., Informa Healthcare, 2008,
Chapter 20.
[0262] Virus-retentive membranes can be manufactured in the form of
a flat sheet, such as Viresolve.RTM. Pro from EMD Millipore
Corporation, Ultipor.RTM. VP Grade DV20 from Pall Corporation,
Virosart.RTM. CPV from Sartorius Stedim Biotech, or in the form of
hollow fiber, such as Planova.andgate. 20N from Asahi Kasei Medical
Co. They can be single-layer or multi-layer products, and can be
manufactured by one of many membrane production processes known in
the art. A particularly beneficial combination of throughput
retention can be achieved for an asymmetric, composite
virus-retentive membrane, as described in U.S. Publication No.
20120076934 A1, incorporated by reference herein.
[0263] In a particular embodiment the flow-through purification
operation involves at least an activated carbon step, an anion
exchange chromatography step, a cation exchange chromatography step
and a virus filtration step.
[0264] Following virus filtration, the sample containing the target
molecule may be subjected to one or more additional
formulation/concentration steps.
Additional Process Steps
[0265] As discussed above, in some embodiments, the sample is
subjected to one or more additional process steps following virus
filtration.
[0266] In some embodiments, the one or more additional steps
include formulation, which may employ diafiltration/concentration
followed by sterile filtration.
[0267] In some embodiments, following virus filtration, the sample
containing target molecule is subjected to diafiltration, which
typically employs the use of an ultrafiltration membrane in a
Tangential Flow Filtration (TFF) mode. In case of Tangential Flow
Filtration (TFF), the fluid is pumped tangentially along the
surface of the filter medium. An applied pressure serves to force a
portion of the fluid through the fiber medium to the filtrate
side.
[0268] Diafiltration results in the replacement of the fluid which
contains the target molecule with the desired buffer for
formulation of the target molecule. Diafiltration is typically
followed by a step to concentrate the target molecule, performed
using the same membrane.
[0269] In another embodiment, single-pass tangential flow
filtration (SPTFF) can be used for concentration/diafiltration. A
SPIFF module includes multiple ultrafiltration devices connected in
series. The target protein is sufficiently concentrated/diafiltered
after a single pass through the SPTFF module without the need for a
retentate loop and pump, enabling continuous operation. More
information can be found in the presentation entitled "Single pass
TFF" by Herb Lutz et al., presented at the American Chemical
Society conference in the spring of 2011.
[0270] Following diafiltration/concentration, the sample is
subjected to a sterile filtration step for storage or any other
use.
[0271] Sterile filtration is typically carried out using Normal
Flow Filtration (NFF), where the direction of the fluid stream is
perpendicular to the fitter medium (e.g. a membrane) coder an
applied pressure.
Systems According to the Present Invention
[0272] The present invention also provides systems for purifying a
target molecule, wherein the systems include two or more unit
operations connected to be in fluid communication with each other,
such that to perform a process for purifying a target molecule in a
continuous or semi-continuous manner. Each unit operation may
employ one or more devices to achieve the intended purpose of that
unit operation. Accordingly, in some embodiments, the systems
described herein, include several devices which are connected to
enable the purification process to be run in a continuous or
semi-continuous manner.
[0273] Without wishing to be bound by theory, it is contemplated
that a system can be enclosed in a closed sterile environment,
thereby to perform the whole purification process in a sterile
manner.
[0274] In various embodiments, the very first device in a system is
a bioreactor containing the starting material, e.g., culturing
cells expressing a protein to be purified. The bioreactor can be
any type of bioreactor like a batch or a fed batch bioreactor or a
continuous bioreactor like a continuous perfusion fermentation
bioreactor. The bioreactor can be made of any suitable material and
can be of any size. Typical materials are stainless steel or
plastic. In a preferred embodiment, the bioreactor is a disposable
bioreactor, e.g. in form of a flexible, collapsible bag, designed
for single-use.
[0275] Clarification may be performed directly in the bioreactor,
or alternatively, the bioreactor can simply be used for culturing
the cells, and clarification is performed in a different vessel. In
yet another embodiment, the cell culture is simply flowed through a
depth filtration device in order to remove one or more impurities.
Accordingly, in some preferred embodiments, the bioreactor is in
fluid communication with a device for performing depth
filtration.
[0276] The device for performing clarification (e.g., a depth
filtration device) is generally connected to be in fluid
communication with a device for performing capture using a bind and
elute chromatography (e.g., a continuous multi-column
chromatography device comprising two or more separation units). In
some embodiments, the device for bind and elute chromatography is
connected to be in fluid communication with a unit operation for
performing flow-through purification, which may include more than
one device/step. In score embodiments, an in-line static mixer or a
surge tank is included between the device for bind and elute
chromatography and the first device used for flow-through
purification.
[0277] In some embodiments, the flow-through purification operation
includes more than one device, e.g., an activated carbon device
followed, by a AEX chromatography device followed by an in-line
static mixer and/or a surge tank for changing pH, followed by a CEX
chromatography device followed by a virus filtration device. The
devices could generally be in any suitable format, e.g., a column
or a cartridge.
[0278] The last unit operations in the system may include one or
more devices for achieving formulation, which includes
diafiltration/concentration and sterile filtration.
[0279] Typically, each device includes at least one inlet and at
least one outlet, thereby to enable the output from one device to
be in fluid communication with the inlet of a consecutive device in
the system.
[0280] In most processes and systems used in the industry today,
each device used in a purification process employs a process
equipment unit, also referred to as a "skid," which typically
includes the necessary pumps, valves, sensors and device holders.
Typically, at least one skid is associated with each device. In
some of the embodiments described herein, the number of skids used
throughout the purification process is reduced. For example, in
some embodiments, only one skid is used to perform the entire
flow-through purification operation, which may include multiple
devices, e.g., activated carbon device, anion exchange
chromatography device, cation exchange chromatography device and
virus filtration device, along with any equipment needed for
solution condition changes. Accordingly, in some embodiments, a
single skid may be used for all of the foregoing steps in
flow-through purification.
[0281] In some embodiments, fluid communication between the various
devices is continuous; in that the fluid flows directly through all
the devices without interruptions. In other embodiments, one or
more valves, sensors, detectors, surge tanks and equipment for any
in-line solution changes may be included between the various
devices, thereby to temporarily interrupt the flow of fluid through
the system, if necessary, for example, to replace/remove a
particular device.
[0282] In some embodiments, one or more surge tanks are included
between the various devices. In some embodiments, not more than 3
and not more than 2 surge tanks are present in the entire system.
The surge tanks located between different devices have no more than
25%, and preferably no more than 10% of the entire volume of the
output from the first of the two devices.
[0283] In some preferred embodiments, the systems described herein
include one or more static mixers for buffer exchange and/or
in-line dilution.
[0284] In some embodiments, a system further includes one or more
sensors and/or probes for controlling and/or monitoring one or more
process parameters inside the system, for example, temperature,
pressure, pH conductivity, dissolved oxygen (DO), dissolved carbon
dioxide (DCO.sub.2), mixing rate, flow rate, product parameters.
The sensor may also be an optical sensor in some cases.
[0285] In some embodiments, process control may be achieved in ways
which do not compromise the sterility of the system.
[0286] In some embodiments, sensors and/or probes may be connected,
to a sensor electronics module, the output of which can be sent to
a terminal board and/or a relay box. The results of the sensing
operations may be input into a computer-implemented control system
(e.g., a computers for calculation and control of various
parameters (e.g., temperature and weight/volume measurements,
purity) and for display and user interface. Such a control system
may also include a combination of electronic, mechanical, and/or
pneumatic systems to control process parameters. It should be
appreciated that the control system may perform other functions and
the invention is not limited to having any particular function or
set of functions.
[0287] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application, as well as the Figures, are
incorporated herein by reference.
EXAMPLES
Example 1
Process for Purifying a Monoclonal Antibody
[0288] In this representative example, the purification of a
monoclocal antibody is achieved using a purification process in a
continuous manner, where various unit operations are connected in a
manner to operate continuously. An exemplary process is depleted in
FIG. 2.
[0289] The representative example described below includes the
following steps performed in the sequence listed: clarification
using depth filtration; use of one or more in-line static mixers to
change solution conditions: Protein A bind and elute chromatography
using continuous multicolumn chromatography which employs two
separation units; pH adjustment of the output using, one or more
static mixers; and flow-through purification which employs depth
filtration followed by activated carbon followed by anion exchange
chromatography followed by pH adjustment using a static mixer
followed by cation exchange chromatography followed by virus
filtration.
[0290] In this example a CHO-based monoclonal antibody (MAb05) is
produced in a fed batch bioreactor. Approximately 5.5 L of cell
culture processed with a 0.054 m.sup.2 D0HC (EMD Millipore) primary
depth biter then further clarified with a 0.054 m.sup.2 X0HC (EMD
Millipore) secondary depth filter where both are processed at a 10
LMH flux making the loading approximately 100 L/m.sup.2.
[0291] The effluent from the depth filtration is contacted with a 5
M NaCl solution at a 1:10 ratio that is then mixed through a static
mixer followed by a sterile filter. The pressure is monitored prior
to each depth filter and after the sterile filter (FIG. 3).
Following the static mixer, the solution is passed through a SHC
sterile filter (EMD Millipore) to a final loading of 3200 L/m2. The
effluent from the sterile filter is directed to a surge tank that
is monitored with a load cell to determine the amount filtered. One
mL samples are collected just prior to each load cycle on Protein A
continuous multi-column chromatography (CMC) (FIG. 4). After
approximately 70 mL of cell culture is processed and collected in a
surge tank, the clarified solution is simultaneously loaded into
the next step for Protein A capture.
[0292] Protein A capture consists of two Protein A columns running
on a modified Akta Explorer 100. The Protein A columns have 10 mL
of ProSep Ultra Plus Protein A media packed into 1.6 cm ID
Vantage-L (EMD Millipore) chromatography columns to bed heights of
10.25 and 10.85 cm. The columns are equilibrated with 1l X PBS, 0.5
M NaCl for 5 column volumes (all column volumes are based on the
smallest column). Throughout the run, the loading flow rate is set
so as to have a loading residence time of .about.1 minute. During
the initial loading both columns are placed in series, where the
effluent of the primary column is loaded directly onto the
secondary column until a specific load volume is reached. After a
specific loading volume is passed over the columns, the feed is
stopped and 2 column volumes (CVs) of the equilibration buffer is
passed through the primary column to the secondary column. The
primary column is then positioned to undergo washing, elution,
cleaning and reequilibration, while the secondary column is loaded
as the primary column. Following the reequilibration of the first
column, the column is then moved to the secondary position to
reside in series with the now primary column. This series of events
is repeated with each column taking the primary position after the
original primary position column is loaded to a set volume. The
first column is loaded a total of three times and the second column
is loaded twice. The elutions from each column are collected info a
beaker with mixing, using a UV trigger to control the start and end
time of the elution.
[0293] After the first two elutions are collected and pooled, the
solution is pumped out into a surge tank and mixed with a 2 M
solution of tris and processed through two static mixers to
increase the pH to pH 8.0, where the pH of the resulting solution
is measured immediately after the static mixers. The pH adjusted
solution is then processed through an AlHC depth filter (EMD
Millipore) followed by a 1 cm ID Omnifit column packed with
activated carbon. The effluent from the activated carbon column is
then continuously (lowed through an anion exchange chromatography
device (e.g., ChromaSorb.TM.) (EMD Millipore) to a loading of 4 kg
of MAb/L of ChromaSorb.TM.. The effluent from the ChromaSorb.TM.
anion exchanger is then mixed with 1 M acetic acid, then processed
through a static mixer to lower the pH to pH 5.5. The pH-adjusted
solution from the static mixer is then flowed through a cation
exchange chromatography device used as a prefilter, followed by
virus filtration using the Viresolve.RTM. Pro membrane (EMD
Millipore). The effluent from the virus filter is directed to a
pool tank and sampled.
[0294] This purification process provides final solution that meets
all purification targets, specifically HCP <1 ppm, aggregates
<1% with a mAb05 recovery >60% for the overall process.
Example 2
Process for Purifying a Monoclonal Antibody
[0295] In this representative example, the purification of a
monoclocal antibody is achieved using a purification process, where
various unit operations are connected in the sequence described
below.
[0296] The representative example described below includes the
following steps performed in the sequence listed: clarification
using stimulus responsive polymer following centrifugation;
contacting the supernatant with salt; Protein A bind and elute
chromatography using continuous multicolumn chromatography which
employs two separation units; pH adjustment of the output using one
or more static mixers; and flow-through purification which employs
depth filtration followed by activated carbon followed by anion
exchange chromatography followed by pH adjustment using a static
mixer followed by cation exchange chromatography followed by virus
filtration.
[0297] In this example, a CHO-based monoclonal antibody (MAb05) is
produced in a fed-batch bioreactor. A total of 7 liters of cell
culture is contacted with a solution of a stimulus responsive
polymer (modified polyallylamine; responsive to salt addition) to a
final polymer concentration of 0.2% v/v. The cell culture is mixed
with the stimulus responsive polymer solution for approximately 10
minutes. About 175 mL of of 2 M K.sub.2HPO.sub.4 solution is added
and the cell culture is mixed for an additional 10 minutes. The pH
is then raised to 7.0 by adding 2 M tris base and mixing for 15
minutes. The solution is then centrifuged in 2 L aliquots at
4.500.times.g for 10 minutes and the supernatant is decanted and
retained. The solids are disposed off. The cell culture supernatant
is pooled and then mixed with 5 M NaCl at a 1:10 ratio in a batch
mode with continuous stirring. The final conductivity of the
solution is measured at this point and is at 55.+-.5 mS/cm. The
resulting solution is sterile filtered through a 0.22 .mu.m Express
SHC filter (EMD Millipore). The sterile filtered solution is the
loading material for the Protein A chromatography.
[0298] The Protein A capture step consists of two Protein A columns
running on a modified Akta Explorer 100. The Protein A columns have
10 mL of ProSep Ultra Plus Protein A media packed into 1.6 cm ID
Vantage-L (EMD Millipore) chromatography column to bed heights of
10.25 and 10.85 cm. The columns are equilibrated with 1.times. TBS.
0.5 M NaCl for 5 column volumes, CVs (all column volumes are baaed
on the smallest column). Throughout the run, the loading flow rate
is set so as to have a loading residence time of about one
minute.
[0299] During the initial loading, both columns are placed in
series, where the effluent of the primary column is loaded directly
onto the secondary column until a specific load volume is reached.
After a specific loading volume is passed over the columns, the
feed is stopped and two CVs of the equilibration buffer is passed
through the primary column to the secondary column. The primary
column is then positioned to undergo washing, elution cleaning and
reequilibration, while the secondary column is loaded as the
primary column. Following the reequilibration of the first column,
that column is then moved to the secondary position to reside in
series with the now primary column. This series of events is
repeated with each column taking the primary position after the
original primary position column is loaded to a set volume. Each
column is loaded a total of seven times. The elutions from each
column are collected with a fraction collector, using a UV trigger
to control the start time of the elution and collected to a
constant volume of approximately 3.5 CVs.
[0300] Flow-through purification comprises of six main steps; depth
filter; activated carbon; anion exchange chromatography; in-line pH
adjustment; cation exchange chromatography; and virus
filtration.
[0301] FIG. 5 illustrates the order in which these steps are
connected. The necessary pumps and sensors, e.g., sensors for
pressure, conductivity and UV are also shown in the schematic.
[0302] All devices are individually wetted at a different station,
and then assembled as shown in FIG. 5. The devices are wetted and
pre-treated according to the manufacturer's protocol or as
described herein. Briefly, the depth filter (AlHC) is flushed with
100 L/m.sup.2 of water followed by 5 volumes of equilibration
buffer 1 (EB1: Protein A elution buffer adjusted to pH 7.5 with 1 M
Tris-base, pH 11), 10 mL of activated carbon is packed into a 2.5
cm ID Omnifit column. The column is flushed with 10 CVs water, and
then equilibrated with EB1 until the pH is stabilized to pH 7.5,
1.2 mL of anion exchange membrane (7 layers) is stacked into a 47
mm diameter Swinex device. The device is wetted with water at 12.5
CV/min for at least 10 min, followed by 5 device volumes (DVs) of
EB1. A disposable helical static mixer (Koflo Corporation, Cary,
Ill.) with 12 elements is used to perform in-line pH adjustments. A
3-layer cation-exchange chromatography device (0.12 mL membrane
volume) is wetted with 10 DVs water, followed by 5 DVs of
equilibration buffer 2 (EB2: EB1 adjusted to pH 5.0 using 1 M
acetic acid). The device is further treated with 5 DVs of EB2+1 M
NaCl, and then equilibrated with 5 DV EB2. A 3.1 cm.sup.2
Viresolve.RTM. Pro virus filtration device is wetted with water
pressurized at 30 psi for at least 10 minutes. The flow rate is
then monitored every minute until the flow rate remains constant
for 3 consecutive minutes. After all the devices are wetted and
equilibrated, they are connected as shown in FIG. 5.
[0303] EB1 is run through the entire system until all pressure
readings and pH readings are stabilized. Following equilibration,
the feed (i.e., Protein A eluate adjusted to pH 7.5) is subjected
to flow-through purification. During the run, samples are collected
before the surge tank and alter Viresolve.RTM. Pro to monitor MAb
concentration and impurity levels (e.g., HCP, DNA, leached Protein
A and aggregates). After the feed is processed, the system is
flushed with 3 device volumes of EB1 to recover any MAb still
remaining in the various devices as well as the connecting lines
between devices.
[0304] FIG. 6 depicts the pressure readings after depth filter,
activated carbon, and Viresolve.RTM. Pro in flow-through
purification. Generally, an increase in pressure denotes fouling of
filter columns. Notably, the activated carbon column remains fairly
protected from any precipitate due the depth biter used before the
activated carbon. The Viresolve.RTM. Pro pressure rises slowly with
time, but is well below the operating maximum limit (50 psi).
[0305] The HCP breakthrough as a function of time, as measured
after the anion exchange chromatography device is below the target
of 10 ppm. The final HCP in the Viresolve.RTM. Pro pool is <1
ppm (Table I). The average leached Protein A in the elution
fractions is 32 ppm. The leached Protein A in the Viresolve.RTM.
Pro pool is 4 ppm. The aggregates are reduced from 1% to 0.4%.
[0306] The results from the experiment are summarized in Table II
below.
TABLE-US-00002 TABLE II Property measured Results MAb Yield (%)
following flow-through 97.8% purification, where the baseline of
100% is amount after Protein A CMC Average HCP measured, alter
Protein A CMC 172 .fwdarw. 1.75 elution relative to that measured
after virus filtration (ppm) Average aggregates after Protein A CMC
1 .fwdarw. 0.4% elution relative to aggregates after virus
filtration (%) Average leached Protein A after Protein A 32
.fwdarw. 4 CMC elution relative to leached Protein A after virus
filtration (ppm) Virus Filtration throughput (kg/m.sup.2) >6.1
Dilution factor of MAb as measured by ratio 1.15x of MAb
concentration after Protein A CMC and after virus filtration
Example 3
Flow-Through Purification Process Following Batch Protein A
Chromatography
[0307] In this representative experiment, a monoclonal antibody
solution previously purified by batch protein A is further purified
using flow-through purification to meet final purity and yield
targets. This is done by performing the following steps in a
flow-through manner: activated carbon; anion exchange
chromatography; in-line pH change; cation exchange chromatography
and virus filtration.
[0308] The set-up, equilibration and run is similar to Example 2
except for some minor modifications. The starting material is a
protein A eluate from a batch protein A process. Specifically, the
MAb feed processed for this run is 102 mL of 135 mg/mL MAb05 at a
flow rate of 0.6 mL/min. A depth filter is not used in this study
as the feed is filtered through a sterile 0.22 .mu.m filter prior
to performing the flow-through purification. A 2.5 mL activated
carbon column is used which corresponds to a loading of 0.55 kg/L.
Two anion exchange chromatography devices (0.2 and 0.12 mL) are
connected in series to get a loading of 4.3 kg/L. Two 1.2 mL cation
exchange chromatography devices (7 layers of the membrane on each
device) are connected in parallel to handle aggregates. The MAb
loading on the cation exchange chromatography devices is about 570
mg/mL. A 3.1 cm.sup.2 Viresolve.RTM. Pro device is used for virus
filtration.
[0309] The HCP breakthrough as a function of loading after anion
exchange chromatography device is below the target of 10 ppm (FIG.
7). The final HCP in the Viresolve.RTM. Pro pool is <1 ppm
(Table 2). The aggregates are reduced from 5% to 1.1% by the cation
exchange chromatography device (FIG. 8).
[0310] The results from the experiment are summarized in Table III
below.
TABLE-US-00003 TABLE III Property measured Results MAb Yield (%)
following flow-through purification. 92% where the baseline of 100%
is amount after Protein A batch chromatography Average HCP measured
after Protein A batch 591 .fwdarw. 0.61 chromatography elution
relative to that measured after virus filtration (ppm) Average
aggregates after Protein A batch ~5 .fwdarw. 1.1% chromatography
elution relative to aggregates after virus filtration (%) Virus
Filtration throughput (kg/m.sup.2) >3.7 Dilution factor of MAb
as measured by ratio of MAb 1.25x concentration after Protein A
batch chromatography and after virus filtration
[0311] FIG. 9 shows the pressure readings before activated carbon
and Viresolve.RTM. Pro. Generally, increased pressure implies the
filters are getting fouled. In this case, there is a modest (about
5 psi) increase in pressure in case of activated carbon. The
Viresolve.RTM. Pro pressure rises slowly with time, but is well
below the operating maximum limit (50 psi).
[0312] As shown in FIG. 10, the pH after adjustment remains at the
target set-point of pH 4.9 except during start-up. The pH spikes
can be dampened by using a surge tank after the in-line pH
adjustment and before pumping to the cation exchange chromatography
device.
Example 4
Clarification Connected to Protein A Chromatography
[0313] In this representative example, clarification is connected
to Protein A chromatography in a continuous manner.
[0314] In this experiment, the flow-rate that is used for depth
filtration is determined by the residence time used for Protein A
chromatography, which follows depth filtration. The flow-rate used
in this representative example is slower than that used in
conventional depth filtration, resulting in a higher HCP removal in
the output recovered after Protein A chromatography.
[0315] A monoclonal antibody (MAb04) cell culture feed is obtained
and split into three equal portions. The first portion (sample #1
in Table IV) is clarified using D0HC and X0HC Millistak+.RTM.
primary and secondary depth filters (EMD Millipore) at a filter
area ratio of 1:1 and a flow rate of 100 Liters/m.sup.2/hour (LMH),
which is a typical flux used in standard clarification processes.
The effluent is tested for MAb concentration and HCP amount. The
second portion of the cell culture feed (sample #2) is also
clarified with the same type and ratio of filters but at allow rate
of 10 LMH. This flow rate is based on a six minute residence time
of the Protein A chromatography column, which follows
clarification. In both cases, the same amount of material is
processed, corresponding to a throughput of about 100 L/m.sup.2,
and the two samples are treated in the same manner.
[0316] Both clarified cell culture fluids are subjected to Protein
A bind and elute chromatography, resulting in samples #3 and #4 in
Table IV, where the clarification and Protein A chromatography are
performed separately (i.e., are not connected). Sample #4, relating
to slower flow-rate, is lost and therefore, not able to be
analyzed.
[0317] The third portion of cell culture feed (sample #5) is
processed through an assembly that has the effluent of the two
depth filters continuously loaded onto a Protein A chromatography
column (i.e., where clarification and Protein A chromatography are
connected). Same chromatographic conditions as above are used. All
protein A eluates are tested for MAb concentration and HCP amount.
In case of sample #5, the six minute residence time of the Protein
A chromatography determines the flow-rate for clarification of
about 10 LMH.
[0318] All results are shown in table IV. The results indicate that
the HCP levels are 2.times. lower after slow clarification (i.e.,
sample #2 relative to sample #1). A direct comparison of the
corresponding samples following Protein A chromatography is not
reported due to the sample loss (i.e., sample #4). A comparison of
results relating to sample #5 relative to sample #3 suggests that
performing clarification and Protein A chromatography in a
continuous and connected manner provides a 8.times. improvement in
MAb purify compared to the purity when clarification and Protein A
chromatography are run separately. Comparing the estimated LRV
values documents the difference.
TABLE-US-00004 TABLE IV HCP # Sample MAb (g/L) (ng/mL) HCP (ppm)
LRV 1 100 LMH clarified 1.19 (80%) 256,894 221,426 2 10 LMH
clarified 1.01 (69%) 118,114 117,421 3 100 LMH clarified and 9.03
7,553 837 2.4 Protein A purified 4 10 LMH clarified and not tested
Protein A purified 5 10 LMH clarified and 7.94 808 102 (3.1)
Protein A purified in a connected manner
Example 5
Clarification using Stimulus Responsive Polymer
[0319] In this representative experiment, clarification is
performed using a stimulus responsive polymer using two different
processes.
[0320] In one process, a stimulus responsive polymer is added
directly to a bioreactor (which may be a single use or disposable
bioreactor) containing a cell culture expressing a target molecule.
In a second process, a cell culture is pumped out of a bioreactor
and contacted with a stimulus responsive polymer using one or more
in-line static mixers.
[0321] In case of the process relating to performing clarification
directly in a bioreactor, 60 mL of 10 wt % stimulus responsive
polymer is added into a 3 L disposable bioreactor containing a cell
culture and mixed for at least 5 minutes, 75 mL of 2 M
K.sub.2HPO.sub.4 is added into the bioreactor and mixed for at
least 5 minutes, 2 M Tris base is added into the bioreactor while
mixing in order to increase pH to between 7-7.3 (approximately
50-100 mL). The solution is allowed to mix for at least 1 minute
and then pumped out of the bioreactor and loaded directly onto a
depth filter at a rate of 100 LMH to remove the precipitate.
[0322] In case of the process relating to the use of an in-line
static mixer, cell culture is pumped out of bioreactor at a rate of
93.5 LMH to a valve or connector where it is contacted with a
stimulus responsive polymer stream flowing at a rate of 1.9 LMH.
The combined stream then flows into an inline static mixer sized
appropriately to provide efficient mixing. The stream then flows
into a second valve or connector where it is contacted with a
stimulus for the polymer flowing at a rate of 2.3 LMH. The combined
stream flows into a second static mixer sized appropriately to
provide efficient mixing. The stream then flows into a third valve
or connector where it is contacted with a 2 M Tris base stream
flowing at an approximate rate of 2.3 LMH (flow of tris is adjusted
to maintain a pH of 7-7.3 of the combined stream). The combined
stream flows into a third static mixer that is sized appropriately
to provide efficient mixing. This stream then is loaded directly on
one or more depth filters in order to remove the precipitate.
[0323] It is noted that different feeds may be more sensitive to pH
or may interact with a stimulus responsive polymer differently.
Yields can be maximized by having the ability to treat feeds either
in bioreactor, inline or a combination of the two may be used.
[0324] Use of a stimulus responsive polymer, as described herein,
results in a better performance in the bind and elute
chromatography process step (e.g., Protein A chromatography step),
which follows the clarification step. Additionally, it is observed
that the method described in this representative example results in
an increase number of chromatographic cycles of the next bind and
elute chromatography step, relative to clarification schemes that
do not involve use of a stimulus responsive polymer. Lastly, the
eluate obtained subsequent to the bind and elute chromatographic
step appears to exhibit less turbidity generation upon pH change,
when a stimulus responsive polymer is used upstream of the bind and
elute chromatography step.
Example 6
Effect of Clarification using Precipitation on Elution Performance
of Protein A Chromatography
[0325] In this representative experiment, the effect of the type of
clarification performed on a CHO-based cell culture producing MAb04
on elution performance of Protein A chromatography is
investigated.
[0326] A single batch of cell culture is split evenly into three
aliquots. One aliquot is subjected to clarification using caprylic
acid; another aliquot is subjected to clarification using a
stimulus responsive polymer (i.e., modified polyallylamine); and
the third aliquot is left untreated.
[0327] Following precipitation with the caprylic acid or stimulus
responsive polymer, the solids are removed using centrifugation.
The untreated cell culture is also centrifuged alter mixing for the
same amount of time as the two treated cultures. All are sterile
filtered prior to use.
[0328] For each clarified solution, the conductivity of the
solutions is measured and adjusted with 5 M NaCl until the
conductivity reaches about 54 mS/cm. The average concentration of
added NaCl is approximately 0.5 M for all solutions. The higher
conductivity cell culture solutions are sterile filtered prior to
loading on to separate Protein A chromatography columns.
[0329] In order to perform Protein A chromatography for each feed
solution, three columns are packed with ProSep Ultra Plus media
with one column having 4 mL of packed media and the other two both
having 4.24 mL of packed media in 10 mm inner diameter OmniFit
columns. The column bed heights are 5.1 and 5.4 cm for the 4 mL and
4.24 mL columns, respectively.
[0330] All chromatography experiments are performed on an Akta
Explorer 100 (caprylic and stimulus responsive polymer treated) or
an Akta Avant 25 (untreated). Prior to the first loading, each
column is washed with at least five column volumes (CVs) of 0.15 M
phosphoric acid. All chromatography runs are performed using the
same basic procedure. The columns are equilibrated with 5 CVs of
1.times. TBS+0.5 M NaCl, followed by loading to .about.30 g of
MAb04 per liter of packed media. The loading solution is flushed
out with 2 CVs of equilibration buffer followed by a 4 CV wash with
25 mM Tris pH 7.0. Following washing of the column, the product
(MAb04) is elated from the column with 5 CVs of 25 mM glycine HCl,
25 mM acetic acid pH 2.5. The elution is collected using the
system's fraction collector with collection starting using a UV
trigger and collected for a constant volume of 4 CVs. The column is
cleaned with 4 CVs of 0.15 M phosphoric acid followed by a
reequilibration step of 10 CVs with equilibration buffer.
[0331] The Protein A purification of the different clarified
samples is performed for twelve (untreated) and nine (capryilic
acid and stimulus responsive polymer treated) successive
cycles.
[0332] FIGS. 11, 12 and 13 depict the overlaid chromatograms for
all experiments, in each case displaying the load, elution and
cleaning peaks in sequence. It is evident that the elution peak
without stimulus responsive polymer treatment has visible and
significant trailing as compared to the elution peaks from the
stimulus responsive polymer treated cell culture, suggesting a less
efficient elution when stimulus responsive polymer was not
used.
[0333] Additionally, the absorbance of the loading solution is
noticeably lower for the stimulus responsive polymer treated cell
culture compared to the untreated cell culture, where the
absorbance is reduced by 0.4-0.5 absorbance units (AU), suggesting
a lower impurity challenge to the column.
[0334] After elution, the pH is raised to 5.0 for both sets of
samples, then further raised to pH 7.5. At pH 5.0 there is no
visible turbidity of the solutions. However, at pH 7.5 all elution
samples exhibit increased levels of turbidity with significantly
higher levels (99-644 NTU) for the untreated samples, while the
stimulus responsive polymer treated elution pool turbidity ranges
from 69.5 to 151 NTU, which is significantly lower relative to the
untreated samples.
Example 7
Simultaneous Removal of Soluble and Insoluble Impurities from
Affinity Capture Eluate using Activated Carbon
[0335] In this representative experiment, it was demonstrated that
activated carbon, when packed with cellulose media, was capable of
removing both insoluble (i.e., particulates) as well as soluble
impurities from an eluate from the Protein A bind and elute
chromatography step (i.e., capture step).
[0336] In many conventional processes used in the industry today, a
depth filter is often used following the Protein A affinity capture
step to remove insoluble impurities (i.e., particulates) before the
next step, which typically is a cation exchange bind and elute
chromatography step.
[0337] In the processes described herein, the use of a depth filter
following the Protein A bind and elute chromatography is obviated.
Notably, by using activated carbon following the Protein A bind and
elute chromatography step, not only is the need for the cation
exchange bind and elute chromatography step obviated, but also is
the need to use a depth filter. This offers many advantages
including, e.g., reducing the overall cost, process time as well as
the overall physical footprint due to elimination of steps that are
typically used.
[0338] As demonstrated herein, use of activated carbon leads to
simultaneous removal of both soluble impurities (e.g., HCPs) as
well as insoluble impurities (e.g., particulates).
[0339] A cell culture of monoclonal antibody MAb04 is subjected to
Protein A affinity chromatography, and the pH of the elution pool
is adjusted from pH 4.8 to pH 7.5, with dropwise addition of 1.0 M
Tris base, in order to change solution conditions suitable for the
next step in the process. However, raising the pH of the solution
increases the turbidity, which in this case is measured to be 28.7
NTU. This solution is referred to below as the MAb04 Protein A
eluate.
[0340] A circular section of a sheet of activated carbon-cellulose
media 5/8 inch in diameter and 5 mm in thickness is cut and
carefully loaded into 1.5 mm diameter Omnifit.RTM. Chromatography
Columns (SKU: 006BCC-25-15-AF, Diba Industries, Inc. Danbury, Conn.
06810 USA) to result in a column volume of 0.89 mL. The column is
flushed with 25 mM Tris pH 7. About 40 mL of the turbid MAb04
Protein A eluate is passed through the column at a flow rate of
0.20 mL/min, resulting in a residence time of 4.5 minutes. Four 10
mL fractions are collected. Each individual fraction as well as a
combined pool composed of all four fractions is evaluated for
turbidity and analyzed for the concentrations of HCP and MAb. HCP
analysis is performed using a commercially available ELISA kit from
Cygnus Technologies, Southport N.C. USA, catalog number F550,
following kit manufacturers protocol. The MAb concentration is
measured using an Agilent HPLC system equipped with a Poros.RTM. A
Protein A analytical column. The results are summarized in Table
V.
[0341] The results show that activated carbon is unexpectedly
effective for the simultaneous removal of both the insoluble
impurities (i.e., particulate matter) as well as the soluble
impurities (i.e., HCPs) front the Protein A eluate. The turbidity
of the Protein A eluate is reduced front 28.7 NTU to 9.9 NTU, while
the concentration of HCP is reduced from 758 ppm to 104 ppm.
[0342] This result demonstrates that activated carbon, when packed
with cellulose media, can be used for removing both soluble as well
as insoluble impurities.
TABLE-US-00005 TABLE V activated volume carbon loaded loading
Turbidity MAb HCP HCP HCP (mL) (kg/L) (NTU) (mg/mL) (ng/mL) (ppm)
LRV control -- 28.7 10.38 7,872 758 -- 10 0.13 8.8 9.76 130 13 1.77
20 0.25 10.3 10.53 850 81 0.97 30 0.38 10.3 10.55 1,660 157 0.68 40
0.50 10.6 10.38 2,333 225 0.53 40 (pool) 0.50 9.9 10.59 1,098 104
0.86
Example 8
Effect of Residence Time on Impurity Removal by Activated
Carbon
[0343] In this representative experiment, it was demonstrated that
when activated carbon is used in a continuous process, as described
herein, it results in a greater impurity removal relative to when
it is not used in a continuous manner. Notably, when activated
carbon is employed in a continuous process, as described herein,
where it is usually in fluid communication with the Protein A bind
and elute chromatography step upstream and with an anion exchange
chromatography media downstream, the sample flows through the
activated, carbon at a flow-rate which is slower (i.e., having a
longer residence time) relative to when activated carbon is used
separately as a stand alone operation.
[0344] In this example, Protein A-purified MAb04 eluate is further
subjected to a flow-through purification step which employs
activated carbon (AC) and an anion exchange chromatography membrane
device (e.g., a ChromaSorb.TM. device) configured in series, at
four different flow rates. Antibody concentration in the feed is
determined to be 7.5 g/L; HCP concentration is determined to be 296
ppm. The experiment is performed at pH 7.0. Activated Carbon, grade
Nuchar HD, is obtained from Mead West Vaco. It is packed in a glass
Omnifit column to bed volume of 0.8 mL. An anion exchange
chromatography device with membrane volume 0.08 mL is connected in
series to the AC column. The flow rates are chosen such that the
residence time (RT) on AC is 1, 2, 4 or 10 mins. The MAb loading on
AC and the anion exchange chromatography device is held constant at
0.5 kg/L and 5 kg/L, respectively, for the 4 different runs (i.e.,
having the four different residence times stated above).
[0345] Samples from the breakthrough of the anion exchange
chromatography device are collected and analyzed for MAb and HCP
concentrations. The breakthrough of HCP as a function of MAb
loading on the anion exchange chromatography device at the 4
different residence times on AC mentioned above, is shown in FIG.
14.
[0346] As demonstrated in FIG. 14, lower flow rates on AC (i.e.,
longer residence times), provide better purify at same MAb
loadings. Alternatively, the same purify can be achieved with a
smaller volume of AC and the anion exchange chromatography device
at a slower flow rate. For example, the target purity of .about.1
ppm HCP can be achieved using 2 kg/L loading on the anion exchange
chromatography device (and 0.2 kg/L on AC), operating at 1 minute
residence time. Notably, the same purity can be achieved while
operating at a longer residence time of 10 mins (i.e., slower flow
rate) with a significantly higher loading of 5 kg/L on the anion
exchange chromatography device (and 0.5 kg/L on AC). This finding
highlights a potential economic advantage in using less consumable
purification material to achieve the same purity when the flow rate
is reduced.
Example 9
Advantage of using a Surge Tank in the Flow-Through Purification
Process Step
[0347] In this representative experiment, one or more advantages of
using a surge tank in the flow-through purification process step,
as described herein, are demonstrated.
[0348] Typically, cation exchange flow-through chromatography
requires the sample to be at a pH of about 5. Accordingly, the pH
of the sample has to be changed from about neutral pH to about pH
5.0, as it flows front the anion exchange chromatography step to
the cation exchange chromatography step, when performing
flow-through purification.
[0349] While the change in pH can be achieved by using an in-line
static mixer, this example demonstrates that it is advantageous to
additionally use a surge tank. Accordingly, the flow of the sample
is as follows: anion exchange chromatography step to an in-line
static mixer to a surge tank to the cation exchange chromatography
step.
[0350] It is observed that if only art in-line static miner is used
for changing pH conditions between the anion exchange and the
cation-exchange flow-through chromatography steps, a sudden
increase (i.e., a spike) is seen, as measured using a pH probe. It
is understood that the pH spike is observed due to chemical
differences in the composition of the sample and that of the buffer
being added to change the pH. This pH spike is undesirable as it
results in the sample being processed for a duration of time at a
higher than is required for optimal results. This example
demonstrates that this pH spike can be reduced or eliminated by the
use of a surge tank after the in-line static mixer and before the
sample contacts the cation exchange chromatography step, as shown
in FIG. 15.
[0351] As shown in FIG. 15, a pH spike of about pH 6.5 is observed
without the use of a surge tank. However, when a surge tank is
used, as described herein, the pH falls to below pH 5.3, which is
closer to the desirable pH for the subsequent cation exchange
chromatography step.
Example 10
Running the Flow-Through Purification Process Step in a Continuous
Manner is not Detrimental to Product Purity
[0352] In this representative experiment, it is demonstrated that
running a flow-through purification process in a continuous manner
is not detrimental to product purity. In other words, by comparing
the product purity from a flow-through purification process step
run in a continuous manner to one where the individual steps are
performed separately, it is shown that there is no detrimental
effect on the product purity.
[0353] This example demonstrates that by connecting activated
carbon and an anion exchange chromatography device (e.g.,
ChromaSorb.TM.) in series to a cation exchange chromatography
device, which acts as a virus prefilter and a virus filtration
device, and operating the entire flow-through purification process
step continuously results in similar capacity of the virus filter,
compared to when the activated carbon and the anion exchange
chromatography device are decoupled from the cation exchange
chromatography device and the virus filtration device.
[0354] The experimental set-up for this experiment is shown in FIG.
16. Option 1 in FIG. 16 refers to the continuous process, where the
sample from the surge tank (present after anion exchange
chromatography device) is fed directly into a cation-exchange
chromatography device followed by a virus filtration device. Option
2 in FIG. 16 refers to a batch process where sample is pooled after
activated carbon and the anion exchange chromatography device, and
after a duration of time, it is processed through through a cation
exchange chromatography device and the virus filtration device.
[0355] In this experiment, the starting sample is the Protein A
bind and elute chromatography eluate, winch has an HCP
concentration of 250 ppm. It is observed that after activated
carbon and anion exchange chromatography device, the HCP levels is
reduced to about 4 ppm.
[0356] In ease of the batch process (Option 2), the final HCP
concentration following virus filtration is observed to be around 1
ppm; whereas in case of the continuous process (Option 1), the HCP
concentration following virus filtration was about 2 ppm, both of
which are towards the lower end of what can be quantified using
methods known in the art and those described herein, e.g., assays
described in Example 7.
[0357] This result implies that performing all the steps in the
flow-through purification process step in a continuous manner
results in a product purity, which is comparable to when one or
more steps are performed as a stand alone operation.
[0358] Additionally, it is noted that the the pressure profiles for
the two processes are also very similar, as shown in FIG. 17.
Example 11
Effect of Residence Time an Performance of Virus Filtration
Membrane
[0359] In this representative experiment, the effect of residence
time on performance of the virus filtration is investigated. It is
observed that a lower flow rate through the cation exchange
chromatography step and the virus filtration step during the flow
through purification process step, results in a higher throughput
of the virus filter.
[0360] A three-layer cation exchange chromatography device, as
described in U.S. patent application Ser. No. 13/783,941 (internal
ref. no. MCA-1423), having membrane area 3.1 cm.sup.2 and membrane
volume 0.12 mL, is connected in a series to a virus filtration
device, having a membrane area of 3.1 cm.sup.2. About 3 mg/mL of a
polyclonal human IgG (Seracare) in 20 mM sodium acetate, pH 5.0
buffer, is processed through the two connected devices. The
experiment is performed at two separate flow-rates, 100 and 200
LMH. A 0.22 .mu.m sterile filter is placed between cation exchange
chromatography device and the virus filtration device.
[0361] A pressure sensor is used for measuring the pressure across
the assembly at the different flow rates. Normally, a pressure of
about 50 psi is an indication of fouling or plugging of the virus
filtration membrane. As shown in FIG. 18, when the experiment is
performed at a lower flow-rate (i.e., 100 LMH), more sample volume
can be processed through the virus filtration membrane (i.e.,
higher throughput) relative to when the sample is processed at a
higher flow-rate (i.e., 200 LMH). This could be attributed to
longer residence time of the sample in the cation exchange
chromatography device, which may result in an improvement in
binding of high molecular weight IgG aggregates, thereby presenting
early plugging of the virus filter.
Example 12
Removal of Aggregates at Various Residence times from a MAb Feed
using a Strong Cation-Exchange (CEX) Resin Modified with an
AMPS/DMAM Grafted Copolymer
[0362] In a 250 mL glass jar, 64 ml wet cake of Toyopearl HW75-F
chromatography resin was added. Next, 115 g of 5 M sodium
hydroxide, 18.75 g of sodium sulfate, and 4 mL of allyl glycidyl
ether (AGE) were added to the jar containing the resin. The jar was
then placed in a hybridizer at 50.degree. C. overnight, with
rotation at medium speed. The next day, the main was filter drained
in a sintered glass filter assembly (EMD Millipore Corporation,
Billerica, Mass.) and the wet cake was washed with methanol and
then rinsed with deionzed water. In a glass vial, 10 mL wet cake of
the AGE activated resin was added. To the glass vial, 0.2 g of
Ammonium persulfate, 0.3 g AMPS, 1.2 g DMAM, and 48 g of deionizer
water were added and the vial was heated to 60.degree. C. for 16
hours. The next day, the resin was filter drained in a sintered
glass filter assembly (EMD Millipore Corporation, Billerica, Mass.)
and the wet cake was washed with a solution of methanol and
deionized water and the resin was labeled as Lot #1712.
[0363] The resin, labeled as Lot #1712, was packed in no
Omnifit.RTM. Chromatography Column with an internal diameter of 6.6
mm to a bed height of 3 cm resulting in about 1 mL packed resin
bed. An AKTA Explorer 100 (chromatography system) was equipped and
equilibrated with buffers appropriate to screen these columns for
flow-through chromatography. The chromatography columns containing
the resin sample were loaded onto the chromatography system with
equilibration buffer. The feedstock was an IgG1 (MAb5) feedstock
that was purified using ProSep.RTM. Ultra Plus Affinity
Chromatography Media, and was adjusted to pH 5.0 with 2 M Tris
Base. The final concentration of the Protein A pool was diluted to
4 mg/mL, contained 5.5% aggregated product, and a conductivity of
about 3.2 mS/cm. The resin was loaded at a residence time of 1.3,
or 6 minutes and to a load density of 144 mg/mL. The strip peak
fraction for the 3 minute residence time contained 95.6% aggregates
indicating a high level of selectivity for aggregated species. The
results are depleted in Table VI below.
[0364] Table VI depicts retention of monomer and aggregates for Lot
#1712 with MAb5 at pH 5.0 at 6.3, or 1 minute residence time. As
shown in Table VI, on average, the monomeric species can be
collected at concentrations close to the feed concentration
relatively early compared to the aggregated species for all
residence times tested, which suggests that selectivity is
relatively insensitive to flow rates.
TABLE-US-00006 TABLE VI Average of 6, 3, or 1 Minute Cumulative
Residence 6 Minutes 3 Minutes 1 Minute Flow- Protein Time Residence
Residence Residence through Load % Protein Time Time Time
Collection Density in flow- % Aggre- % Aggre- % Aggre- Fraction #
(mg/mL) through gates gates gates 1 16 13.5 0.0% 0.0% 0.0% 2 32
94.3 0.0% 0.0% 0.0% 3 48 94.4 0.0% 0.0% 0.0% 4 64 95.2 0.0% 0.0%
0.0% 5 80 98.3 0.5% 0.0% 0.0% 6 96 100.0 0.7% 0.3% 0.0% 7 112 99.3
1.1% 0.9% 2.1% 8 128 100.0 2.3% 1.6% 2.8% 9 144 100.0 3.1% 3.6%
4.8%
[0365] As depicted in FIG. 19, the majority of the product is
collected in the flow-through and this is indicated by the
relatively quick breakthrough of protein UV trace. The strip peak
size generally varies based on the conditions and total mass loaded
but it is relatively enriched with aggregate species at 95.6%,
compared to the load material which had only 5.5% aggregates.
Example 13
Removal of Aggregates from a MAb Feed using Cation-Exchange (CEX)
Winged Fibers Modified with an AMPS/DMAM Grafted Copolymer
[0366] In this representative experiment, cation-exchange winged
fibers were used as the solid support.
[0367] In a 1 L glass jar, 20 g of dry Nylon multi-lobed, or
winged, fibers were combined with 400 g of 4 M sodium hydroxide, 24
g of sodium sulfate, and 100 mL of allyl glycidyl ether (AGE). The
jar was then placed in a hybridizer at 50.degree. C. overnight
rotating at medium speed. The following day, the fibers were
filtered in a sintered glass filter assembly and the fibers were
then washed with methanol and rinsed with Milli-Q water. A day
later, the fibers were washed with water, followed by methanol, and
then water again, suctioned to a dry cake and dried in vacuum oven
at 50.degree. C. for 1 day. The resulting sample was labeled Sample
#1635. In three separate glass vials, 2 grams dry cake of Sample
#1635. AGE activated fibers, were weighed out and added to a glass
vial for additional modification by grafting. To the glass vial,
ammonium persulfate, AMPS, DMAM, and deionized water were added in
amounts specified in Table VII and the vial was heated to
60.degree. C. for 16 hours with continuous rotation. The following
day, the fiber samples were filtered in a sintered glass filter
assembly and the wet cake was washed with a solution of deionized
water. The vials containing the fibers were labeled as Lot #1635-1,
1635-2, and 1635-5. Next, Lot #1635-5 was titrated for small ion
capacity, which was found to be about 28 .mu.mol/mL. It was then
assumed that samples #1635-1 and #1635-2 also had small ion
capacity less than 28 .mu.mol/mL.
TABLE-US-00007 TABLE VII Ingredients #1635-1 #1635-2 #1635-5 Fibers
(g) 2.0 2.0 2.0 Ammonium 0.18 0.18 0.18 persulfate (g) AMPS (g)
0.48 0.60 0.72 DMAM (g) 0.48 0.60 0.72 Water (g) 28.86 28.62
28.38
[0368] The resulting modified winged fibers. Lot #1635-1, #1635-2,
#1635-5 were packed in an Omnifit.RTM. Chromatography Column with
an internal diameter of 6.6 mm to a bed height of 3 cm resulting in
about 1 mL packed fiber bed. An AKTA Explorer 100 (chromatography
system) was equipped and equilibrated with buffers appropriate to
screen these columns for flow-through chromatography. The
chromatography columns containing the winged fiber samples were
loaded onto the chromatography system with equilibration buffer.
The feedstock was an IgG1 (MAb5) feedstock that was purified using
protein A affinity chromatography, and was adjusted to pH 5.0 with
2 M Tris Base. The final concentration of the protein A pool was 4
mg/mL and contained 5.5% aggregated or HMW product. This is the
same feedstock as used in Example 12. The columns packed with fiber
Lot #1635-1 and Lot #1635-2 were loaded to a mass loading of 64
mg/mL and the column packed with fiber Lot 1635-5 was loaded to a
mass loading of 80 mg/mL. The results are depicted In Table VIII
below.
TABLE-US-00008 TABLE VIII Cumulative protein Fiber Lot #1635-1
Fiber Lot #1635-2 Fiber Lot #1635-5 load Monomer Dimers LMW Monomer
Dimers LMW Monomer Dimers LMW mg/mL (%) (%) (%) (%) (%) (%) (%) (%)
(%) 8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 16 85.8 0.0 14.0 86.0 0.0
15.6 1.7 0.0 10.2 24 84.3 0.0 15.6 85.6 0.0 14.8 51.8 0.0 13.4 32
83.2 0.6 16.0 83.0 0.0 17.1 85.6 0.0 15.1 40 79.4 2.4 17.7 81.1 0.0
14.9 86.5 0.0 13.9 48 79.6 4.4 15.8 82.5 1.5 15.9 83.3 0.0 15.8 56
82.4 4.4 12.8 79.4 2.1 18.3 87.1 0.0 13.1 64 81.1 4.5 14.2 83.1 3.1
14.7 85.4 2.3 13.6 72 N/A* N/A N/A N/A N/A N/A 85.7 4.0 13.6 80 N/A
N/A N/A N/A N/A N/A 85.1 5.1 13.8 Strip 38.0 63.5 0.0 54.4 35.0
11.0 51.3 43.0 5.6 Peak *N/A = Not applicable
Example 14
The Combined Effect of Stimulus Responsive Polymer Clarification
and Addition of NaCl to the Clarified Cell Culture on the Protein A
Elution Pool Purity
[0369] In this representative experiment a MAb04 cell culture fluid
is clarified using either depth filtration or a precipitation step,
specifically using a stimulus responsive polymer (i.e., modified
polyallylamine). The resulting clarified solutions are either
loaded on to a column containing ProSep.RTM. Ultra Plus resin or
have NaCl added to a final concentration of 0.5 M NaCl prior to
loading onto the column. In the absence of the NaCl addition, the
column is equilibrated with 1.times. TBS prior to loading, whereas
with NaCl, the equilibration buffer is 1.times. TBS, 0.5 M NaCl.
All columns are loaded to approximately 30 g of MAb04 per liter of
resin and then washed with equilibration buffer for 2 CVs followed
by a 3 CV wash with 25 mM Tris pH 7.0. The bound MAb04 is eluted
with the 25 mM glycine HCl, 25 mM acetic acid pH 2.5 solution and
then cleaned with 5 CVs of 0.15 M phosphoric acid pH 1.6 before
being reequilibrated with the appropriate equilibration buffer.
[0370] FIG. 20 displays an overlay of the elution and cleaning
peaks, where the elution peak generated from the stimulus
responsive polymer treated cell culture displays a sharper tail and
a reduced cleaning peak. In the presence of NaCl during the load
with the stimulus responsive polymer treated cell culture, the
cleaning peak is further reduced, thereby indicating a lower level
of strongly bound impurities on the resin.
Example 15
Comparison of the Effect of Different NaCl Concentrations During
Intermediate Washing Steps with the Effect of 0.5 M NaCl Present in
the Loading Solution
[0371] In this representative experiment, the impact of different
NaCl concentrations during the intermediate washing steps on the
monoclonal antibody (MAb) elution pool purity achieved by Protein A
chromatography is directly compared to the impact of having 0.5 M
NaCl in the sample being loaded onto a Protein A chromatography
column.
[0372] A 5 mL column of ProSep.RTM. Ultra Plus Protein A media is
acquired as a pre-packed column. All chromatography runs are
performed using an Akta Explorer chromatography system with a flow
rate of 1.7 mL/min (.about.3 minute residence time) for all steps.
The same column is used for all experiments. For the first set of
experiments, where NaCl is present in the intermediate wash, the
chromatography step employed a 5 column volume equilibration with
1.times. TBS followed by loading of 300 mL of clarified cell
culture containing a target protein (referred to as MAb05) at a
concentration of approximately 0.57 g/L. Following the loading of
cell culture, the column is flushed with 10 mL of equilibration
buffer to remove any unbound product, impurities and other cell
culture components. The column is then washed with 5 column volumes
of 25 mM tris, pH 7.0 that also includes 0.5 M NaCl. The column is
subsequently washed with 5 column volumes of 25 mM Tris, pH 7.0
without NaCl. The product protein (MAb05) is eluted from the column
over 5 column volumes using a buffer containing 25 mM Acetic acid
and 25 mM Glycine HCl at pH 2.5. The column is subsequently cleaned
with 5 column volumes of 0.15 M Phosphoric acid followed by a 10
column volume regeneration step using the equilibration buffer.
Equivalent runs are performed where the NaCl concentration during
the first wash is varied between 0, 1.5 and 2 M.
[0373] The next experiment is performed using the same column with
the following changes to the protocol. The cell culture sample
being loaded is mixed with a volume of 5 M NaCl such that the final
NaCl concentration in the clarified cell culture is 0.5 M NaCl. The
equilibration buffer is modified to include 0.5 M NaCl in the
1.times. TBS solution. The column is loaded with 333 mL of
clarified cell culture with 0.5 M NaCl present to maintain a
constant mass loading of the target protein (MAb05). Intermediate
washing is performed as previously described where a total of 10
column volumes of 25 mM Tris, pH 7.0 is used throughout. The
elution and cleaning steps and buffers remain identical as
described above. Table IX provides an abbreviated summary of the
steps performed for each experiment, identifying the buffers and
volumes used for each step where a potential change is made.
TABLE-US-00009 TABLE IX Equilibration Intermediate Sample buffer
(25 Loading wash buffer 1 (25 Intermediate wash number mL) volume
mL) buffer 2 (25 mL) 1 1 X TBS 300 mL 25 mM Tris pH 7 25 mM Tris pH
7 2 1 X TBS 300 mL 25 mM Tris, 0.5M 25 mM Tris pH 7 NaCl pH 7 3 1 X
TBS 300 mL 25 mM Tris, 1.5M 25 mM Tris pH 7 NaCl pH 7 4 1 X TBS 300
mL 25 mM Tris, 2M 25 mM Tris pH 7 NaCl pH 7 5 1 X TBS, 333 mL 25 mM
Tris pH 7 25 mM Tris pH 7 0.5M NaCl
[0374] Samples of the loading cultures and the elution pools are
measured for MAb05 concentration and HCP concentration. FIG. 21
shows the HCP LRV as a function of the NaCl concentration used in
the intermediate wash or used in the loading step. The Figure
illustrates the improved level of HCP removal (purification) when
0.5 M NaCl is present in the equilibration buffer and clarified
cell culture during the loading phase compared to the addition of
NaCl to the intermediate washing steps, at varying concentrations.
The results also provide the measured HCP concentrations in parts
per million (ppm) based on the ng of HCP per mg of MAb05, shown as
number within the corresponding bar. Further, FIG. 22 shows the %
MAb05 recovered in the elution pool as compared to the mass loaded,
where it is clearly observed that with 2 M NaCl present during the
intermediate wash step, a significant loss of product is
realized.
Example 16
Comparison of the Product Purification Achieved Based on the
Protein A Purification Step during which an Additive is
Included
[0375] In this representative experiment, a direct comparison is
made between the purification achieved by Protein A chromatography
with an additive present in the intermediate wash only to the
purification achieved with an additive in the equilibration buffer
and cell culture samples that are loaded.
[0376] A 5 mL column of ProSep.RTM. Ultra Plus Protein A media is
acquired as a pre-packed column. Ah chromatography runs are
performed using an Akta Avant chromatography system with a flow
rate of 1.7 mL/min (.about.3 minute residence time) for ah steps.
Use same column is used for all experiments. For all washing
experiments, the method described in Example 15 above applies where
NaCl is replaced with a specific additive at a defined
concentration. The additives and concentrations used are provided
in Table X.
TABLE-US-00010 TABLE X Measured conductivity of solutions (mS/cm)
MAb05 Wash cell buffer (25 culture + mM Tris + additive Final TBS +
additive - (0.016 - Stock working additive 1.2 - Tris cell Additive
Concen- concen- (160.7 - buffer culture identity tration tration
TBS alone) alone) alone) NaCl 5M 0.5M 53.4 50.0 31.9 Urea 8M 0.5M
13.4 1.96 8.29 Ammonium 3M 0.5M 78.0 69.2 47.6 Sulfate Tween-20
100% 0.5% 14.0 1.97 15.6 Plutonic F- 10% 0.5% 13.3 1.89 15.4 68 PEG
400 100% 10% 9.89 1.44 4.66 PEG 8000 30% 5% 1.53 10.9 TMAC 5M 0.5M
48.1 37.9 47.1 Hexylene 100% 5% 12.0 1.69 5.84 glycol
[0377] FIG. 23 shows the concentration of HCP remaining in the
product elution pool for each additive used whether it is present
in the first intermediate wash step or in the equilibration buffer
and cell culture sample. The addition of different salts (NaCl
Ammonium Sulfate or TMAC) shows the lowest HCP levels, when the
salts are present in the loading phase.
[0378] FIG. 24 shows the LRV of HCP (relative to the loading HCP
concentration) as a function of the additive used and the
purification step during which the additive is used. This figure
illustrates, again, that salts are most effective at reducing the
HCP concentrations, i.e., increasing the HCP LRV. As demonstrated,
the presence of the additive in the loading solutions shows
improved impurity clearance when compared to the same additive
present only in the intermediate wash. Tables XI and XII summarize
the numerical results illustrated in FIGS. 23 and 24 with Table XI
showing data when the additive is present in the loading step and
Table XII showing data when the additive is present only during the
intermediate wash.
TABLE-US-00011 TABLE XI [HCP] in cell [HCP] in [MAb05] culture
elution in elution MAb05 % load pool HCP Additive pool (g/L)
Recovery (ppm) (ppm) LRV None 13.26 93.1 220350 1226 2.25 NaCl
13.49 92.0 217850 106.8 3.31 Ammonium 13.64 94.9 279871 124.6 3.35
Sulfate TMAC 15.44 99.9 218200 49.5 3.64 Urea 12.83 95.6 166382
3133 1.73 Tween-20 13.01 94.2 222464 2371 1.97 Pluronic F- 13.28
87.5 232195 2140 2.04 68 PEG 400 14.15 76.2 341212 252.0 3.13 PEG
8000 14.18 173.8 374388 4475 1.92 Hexylene 13.73 99.0 230147 2053
2.05 Glycol
TABLE-US-00012 TABLE XII [HCP] in cell [HCP] in [MAb05] culture
elution in elution MAb05 % load pool HCP Additive pool (g/L)
Recovery (ppm) (ppm) LRV None 13.26 93.1 220350 1226 2.25 NaCl
13.04 103.9 232274 350.0 2.82 Ammonium 13.11 98.5 208246 279.8 2.87
Sulfate TMAC 15.20 83.9 241755 85.28 3.45 Urea 12.93 90.1 189352
3211 1.77 Tween-20 12.79 91.7 227974 3032 1.88 Pluronic F- 13.76
103.5 243325 2034 2.08 68 PEG 400 14.47 112.4 236376 915.0 2.41 PPG
8000 14.40 101.9 251847 2204 2.06 Hexylene 14.00 75.3 244069 1738
2.15 Glycol
[0379] FIG. 25 illustrates an example of the relative elation pool
volume depending on the additive identity and the chromatography
step during which the additive is included. The Figure shows that
when the ratio of the additive elution pool volume to the control
elation pool volume (i.e., where no additives are present) is
greater than 100%, the additive elation pool volume exceeds the
control elution volume. Conversely, the values less than 100%
indicate a decrease in the additive elution pool volume relative to
tire control elution pool volume. This Figure further demonstrates
the impact of the additives on the elution pool volume. A value
less than 100% is preferred. Combining the information provided in
FIGS. 23, 24 and 25 with the numerical data in Tables XI and XII,
the order of the best performing conditions are TMAC in
load>TMAC in wash>Ammonium Sulfate in load>NaCl. in
load>Ammonium sulfate in wash. The order provided here is based
on the HCP LRV as of primary importance followed by product
recovery. If HCP concentration in ppm is of primary importance, the
order changes slightly to TMAC in load>TMAC in wash>NaCl in
load>Ammonium Sulfate in load>Ammonium sulfate in wash.
[0380] The specification is most thoroughly understood in light of
the teachings of the references cited within the specification
which are hereby incorporated by reference. The embodiments within
the specification provide an illustration of embodiments in this
invention and should not be construed, to limit its scope. The
skilled artisan readily recognizes that many other embodiments are
encompassed by this invention. All publications and inventions are
incorporated by reference in their entirety. To the extent that the
material incorporated by reference contradicts or is inconsistent
with the present specification, the present specification will
supercede any such material. The citation of any references herein
is not an admission that such references are prior art to the
present invention.
[0381] Unless otherwise indicated, ail numbers expressing
quantities of ingredients, cell culture, treatment conditions, and
so forth used in the specification, including claims, are to be
understood as being modified in all instances by the term "about."
Accordingly, unless otherwise indicated to the contrary, the
numerical parameters are approximations and may vary depending upon
the desired properties sought to be obtained by the present
invention. Unless otherwise indicated, the term "at least"
preceding a series of elements is to be understood to refer to
every element in the series. Those skilled in the art will
recognize, or be able to ascertain using no more than routine
experimentation, many equivalents to the specific embodiments of
the invention described herein. Such equivalents are intended to be
encompassed by the following claims.
[0382] Many modifications and variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. The specific embodiments
described herein are offered by way of example only and are not
meant to be limiting in any way. It is intended that the
specification and examples be considered as exemplary only, with a
true scope and spirit of the invention being indicated by the
following claims.
* * * * *