U.S. patent application number 13/538649 was filed with the patent office on 2013-01-10 for depth filters for disposable biotechnological processes.
This patent application is currently assigned to EMD Millipore Corporation. Invention is credited to Kwok-Shun Cheng, Nripen Singh.
Application Number | 20130012689 13/538649 |
Document ID | / |
Family ID | 47439052 |
Filed Date | 2013-01-10 |
United States Patent
Application |
20130012689 |
Kind Code |
A1 |
Singh; Nripen ; et
al. |
January 10, 2013 |
Depth Filters For Disposable Biotechnological Processes
Abstract
A process for the primary clarification of feeds, including
chemically treated flocculated feeds, containing the target
biomolecules of interest such as mAbs, mammalian cell cultures, or
bacterial cell cultures, using a primary clarification depth
filtration device without the use of a primary clarification
centrifugation step or a primary clarification tangential flow
microfiltration step. The primary clarification depth filtration
device contains a porous depth filter having graded porous layers
of varying pore ratings. The primary clarification depth filtration
device filters fluid feeds, including chemically treated
flocculated feeds containing flocculated cellular debris and
colloidal particulates having a particle size distribution of
approximately about 0.5 .mu.m to 200 .mu.m, at a flow rate of about
10 litres/m.sup.2/hr to about 100 litres/m.sup.2/hr. Kits and
methods of using and making the same are also provided.
Inventors: |
Singh; Nripen; (Acton,
MA) ; Cheng; Kwok-Shun; (Nashua, NH) |
Assignee: |
EMD Millipore Corporation
Billerica
MA
|
Family ID: |
47439052 |
Appl. No.: |
13/538649 |
Filed: |
June 29, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61571994 |
Jul 8, 2011 |
|
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Current U.S.
Class: |
530/388.1 ;
210/500.1; 210/505; 210/656; 210/702; 210/732; 210/735; 210/767;
530/389.1; 530/412; 530/416 |
Current CPC
Class: |
B01D 2239/0428 20130101;
C07K 1/34 20130101; C02F 1/52 20130101; B01D 15/3809 20130101; C07K
1/36 20130101; B01D 2239/065 20130101; C02F 1/56 20130101; C02F
1/001 20130101; C02F 2103/343 20130101; B01D 2239/025 20130101;
B01D 15/125 20130101 |
Class at
Publication: |
530/388.1 ;
210/767; 210/702; 210/656; 210/732; 210/735; 210/500.1; 210/505;
530/389.1; 530/412; 530/416 |
International
Class: |
C07K 1/36 20060101
C07K001/36; B01D 21/01 20060101 B01D021/01; C07K 1/18 20060101
C07K001/18; C02F 1/52 20060101 C02F001/52; C07K 1/14 20060101
C07K001/14; B01D 39/04 20060101 B01D039/04; B01D 61/14 20060101
B01D061/14; B01D 15/08 20060101 B01D015/08 |
Claims
1. A process for the clarification of a feed containing a target
biomolecule of interest and a plurality of cellular debris and/or
colloidal particulates by depth filtration without the use of a
primary clarification centrifugation step or a primary
clarification tangential flow microfiltration step, the process
comprising: a) providing a depth filtration device having a porous
depth filter media; b) providing a feed containing a target
biomolecule of interest and a plurality of cellular debris and/or
colloidal particulates; c) contacting the depth filter media with
the feed; and d) separating the target biomolecule of interest from
the cellular debris and the colloidal particulates in the feed
without the use of a primary clarification centrifugation step or a
primary clarification tangential flow microfiltration step.
2. The process of claim 1, further comprising adding a chemical
flocculant to the feed in step (b), forming a chemically
flocculated feed including a plurality of flocculated cellular
debris and colloidal particulates.
3. The process of claim 1, wherein the porous depth filter media is
anisotropic, the pores have a nominal pore size rating >about 25
.mu.m, and the filter flocculated feed have >about 3% solids
resulting in a turbidity output <about 20 NTU.
4. The process of claim 1, wherein the clarification process is a
primary clarification process, and the depth filter comprises at
least 2 graded layers of non-woven fibers.
5. The process of claim 4, wherein the graded layers have a total
thickness of about 0.3 cm to about 3 cm.
6. The process of claim 1, wherein the depth filter comprises at
least 3 graded layers of non-woven fibers.
7. The process of claim 6, wherein the graded layers have a total
thickness of about 0.3 cm to about 3 cm.
8. The process of claim 1, wherein the cellular debris and the
colloidal particulates have a particle size distribution from about
0.5 .mu.m to about 200 .mu.m, and a mean particle size greater than
about 10 .mu.m.
9. The process of claim 2, wherein the depth filter media comprises
a composite of graded layers of non-woven fibers, cellulose, and
diamatoceous earth having an open nominal pore size rating
sufficient to filter the chemically flocculated feedstock.
10. The process of claim 1, wherein the target biomolecule of
interest includes monoclonal antibodies (mAbs), polyclonal
antibodies, and biotherapeutics.
11. The process of claim 1, wherein the chemical flocculant is a
polymer or an acid.
12. The process of claim 1, wherein the chemical flocculant is a
smart polymer.
13. The process of claim 12, wherein the smart polymer is a
modified polyamine.
14. The process of claim 11, wherein the acid is acetic acid.
15. The process of claim 4, wherein the non-woven fibers comprise
polypropylene, polyethylene, polyester, or nylon.
16. The process of claim 2, wherein the depth filter provides
throughputs >about 100 L/M.sup.2/hr and removes flocculated
cellular debris and colloidal particulates having a particle size
distribution of about 0.5 .mu.m to about 200 .mu.m.
17. A process according to claim 1, wherein the feed is a cell
culture located in a bioreactor and is directly loaded from the
bioreactor into the depth filtration device for primary
clarification processing.
18. A process according to claim 17, wherein the clarified cell
culture effluent is directly loaded onto a protein A bind and elute
chromatography column for chromatographic processing.
19. A process for the primary clarification of a flocculated feed
including a bimolecular species of interest and a plurality of
cellular materials by depth filtration without the use of a primary
clarification centrifugation step or a primary clarification
tangential flow microfiltration step, the process comprising: a)
providing a depth filtration device having a porous depth filter
media; b) providing a chemical flocculant; c) providing a feed
containing a target biomolecule of interest and a plurality of
cellular materials and/or colloidal particulates; d) adding the
chemical flocculant to the feed; e) forming a chemically
flocculated feed including flocculated cellular materials and/or
colloidal particulates; f) contacting the depth filter media with
the chemically flocculated feed; and g) separating the target
biomolecule of interest from the flocculated cellular materials
and/or flocculated colloidal particulates in the feed without the
use of a primary clarification centrifugation step or a primary
clarification tangential flow microfiltration clarification
step.
20. The process of claim 19, wherein the porous depth filter media
is anisotropic, the pores have a nominal pore size rating >about
25 .mu.m, and the flocculated feed has >about 3% solids
resulting in a turbidity output <about 20 NTU.
21. The process of claim 19, wherein the depth filter comprises at
least 2 graded layers of non-woven fibers.
22. The process of claim 21, wherein the graded layers have a total
thickness of about 0.3 cm to about 3 cm.
23. The process of claim 19, wherein the depth filter comprises at
least 3 graded layers of non-woven fibers.
24. The process of claim 23, wherein the graded layers have a total
thickness of about 0.3 cm to about 3 cm.
25. The process of claim 19, wherein the cellular materials and
colloidal particulates have a particle size distribution from about
0.5 .mu.m to about 200 .mu.m, and a mean particle size greater than
about 10 .mu.m.
26. The process of claim 19, wherein the depth filter media
comprises a composite of graded layers of non-woven fibers,
cellulose, and diamatoceous earth having an open nominal pore size
rating sufficient to filter the chemically flocculated feed.
27. The process of claim 19, wherein the target biomolecule of
interest includes monoclonal antibodies (mAbs), polyclonal
antibodies, and biotherapeutics.
28. The process of claim 19, wherein the chemical flocculant is a
polymer or an acid.
29. The process of claim 19, wherein the chemical flocculant is a
smart polymer.
30. The process of claim 29, wherein the smart polymer is modified
polyamine.
31. The process of claim 28, wherein the acid is acetic acid.
32. The process of claim 19, wherein the depth filter provides
throughputs >about 100 L/M.sup.2 and removes the flocculated
cellular debris and flocculated colloidal particulates.
33. The process of claim 19, wherein the feed includes cell
cultures, transgenic mammalian cell cultures, bacterial cell
cultures, nontransgenic mammalian cell cultures, tissue cultures,
microbial fermentation batches, plant extracts, biofuel, seawater
cultures, freshwater cultures, wastewater cultures, treated sewage,
untreated sewage, milk cultures, blood cultures, and combinations
thereof.
34. The process of claim 19, wherein the non-woven fibers comprise
polypropylene.
35. A process according to claim 19, wherein the feed is a cell
culture located in a bioreactor and is directly loaded from the
bioreactor into the depth filtration device for primary
clarification processing.
36. A process according to claim 35, wherein the clarified cell
culture effluent is directly loaded onto a protein A bind and elute
chromatography column for chromatographic processing.
37. A depth filtration device for the primary clarification of a
feed containing a target biomolecule of interest and a plurality of
cellular debris and/or colloidal particulates without the use of a
primary clarification centrifugation step or a primary
clarification tangential flow microfiltration step, comprising a
porous depth filter media having at least 2 graded layers of
non-woven fibers having a total thickness of about 0.3 cm to about
3 cm, and the pores have a nominal pore size rating >about 25
.mu.m.
38. The device of claim 37, wherein the media is anisotropic.
39. The device of claim 37, wherein the media comprises at least 3
graded layers of non-woven fibers.
40. The device of claim 37, wherein the media comprises a composite
of graded layers of non-woven fibers, cellulose, and diamatoceous
earth having an open nominal pore size rating sufficient to filter
the chemically flocculated feedstock.
41. The device of claim 37, wherein the non-woven fibers comprise
polypropylene, polyethylene, polyester, or nylon.
42. The device of claim 37, wherein the cellular debris and the
colloidal particulates have a particle size distribution from about
0.5 .mu.m to about 200 .mu.m, and a mean particle size greater than
about 10 .mu.m.
43. The device of claim 37, wherein the media provides throughputs
>about 100 L/M.sup.2/hr and removes flocculated cellular debris
and colloidal particulates having a particle size distribution of
about 0.5 .mu.m to about 200 .mu.m.
44. The device of claim 37, wherein the filter flocculated feed has
>about 3% solids resulting in a turbidity output <about 20
NTU when a chemical flocculant is added to the feed.
45. The device of claim 37, wherein the target biomolecule of
interest includes monoclonal antibodies (mAbs), polyclonal
antibodies, and biotherapeutics.
46. A process for purifying a target protein from a sample
comprising: (i) providing a sample comprising a target protein and
one or more impurities; (ii) adding a precipitant to the sample and
removing one of more impurities using a gradient density depth
filter, thereby to obtain a clarified sample; (iii) subjecting the
clarified sample from (ii) to a bind and elute chromatography step,
thereby to obtain an eluate; (iv) contacting the eluate from step
(iii) with one or more virus inactivating agents using one or more
in-line static mixers or one or more surge tanks; (v) subjecting
the output of the virus inactivation step to a flow-through
purification process comprising the use of two or more matrices
selected from activated carbon, anion exchange chromatography
media, cation exchange chromatography media and virus filtration
membrane; and (vi) formulating the output from step (v) at desired
concentration and buffer conditions, wherein the process is
performed continuously such that at least two or more of the
process steps are performed concurrently during at least portion of
their duration.
Description
CROSS-REFERENCED TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 61/571,994, filed on Jul. 8, 2011, the
entire contents of which are incorporated by reference herein.
DESCRIPTION OF THE INVENTION
[0002] 1. Field of the Invention
[0003] In general, the present invention relates to the primary
clarification of feeds. In certain specific embodiments, the
invention provides a primary clarification depth filtration process
of feeds, feedstreams, feedstocks, cell culture broths and the
like, which utilizes a primary clarification depth filtration
device without the use of a primary clarification centrifugation
step or primary clarification tangential flow microfiltration step.
In other embodiments, the invention provides primary clarification
depth filtration process of chemically treated feeds in which the
cell mass has been flocculated into larger aggregates.
[0004] 2. Background of the Invention
[0005] Manufacturing pharmaceutical-grade biomolecules, including
proteins such as monoclonal antibodies (mAbs), is a complex
manufacturing process comprised of multiple filtration,
centrifugation, and chromatography techniques designed to produce
high quality products for patients. The clarification of cell
culture harvests and high-solids feedstocks can be a daunting task
due to the large volumes of harvest from modern production batch
bioreactors (525,000 L) and high cell densities that often require
primary, as well as secondary clarification prior to the subsequent
chromatography operations. And as such, harvest and clarification
schemes for the production processes of cell culture harvests and
high-solids feedstocks, such as mammalian cells and mAbs, are the
product of much evolution and evaluation carried out over the last
20 years or so.
[0006] Harvest techniques for mammalian cell culture and mAbs are
now routinely expected to operate with high yields (>95%) and
minimal cell disruption. As product molecule titers have increased,
the higher cell mass and larger amounts of product create
challenges for the downstream purification steps. Higher cell
densities result in difficulties during clarification and sterile
filtration. Higher product concentrations generally result in
increased impurity load and the need for larger chromatography
installations. As such, improvements in the form of gains in
efficiency and throughput are greatly sought after.
[0007] Increasing demand and growth therapeutic mAbs have fueled
efforts towards increasing product production, quality, process
efficiency, and cost-effectiveness for production of industrial
therapeutic monoclonal antibodies. The past decade has witnessed
considerable growth in production, upstream cell culture product
titers and technical advancement in the characterization of
impurities and contaminants.
[0008] Primary clarification of feeds, feedstreams, feedstocks,
cell culture broths and the like, including high solids feeds, such
as those containing mAbs and mammalian cell culture feedstocks,
remove large amounts of biomass, particularly whole cells and other
larger cellular debris, followed by secondary clarification which
removes smaller colloidal particulates and other particles that
impair the capacity of downstream filters. Centrifugation is
typically the primary clarification step in the production
processes of mAbs and mammalian cell culture broths and
feedstocks.
[0009] mAb manufacturers have invested a great deal of time and
effort increasing the product titer of a feedstock. However, while
higher titers increase cell culture productivity, it also produces
feedstocks with larger amounts of biomass and cell debris content.
Feeds containing such larger amounts of biomass and cell debris can
produce high turbidity centrate after centrifugation. High
turbidity centrates often reduce the throughput of the secondary
clarification depth filter and the subsequent sterile filter used
downstream of the centrifuge. The reduced throughput causes a range
of problems from increased process cost to deviations in process
procedures due to plugging of filters and long processing delays.
Finally, the need for primary clarification using a centrifuge
requires extensive, validated cleaning procedures between runs to
attempt to reduce the risk of cross contamination between batches
and therapeutic molecular species.
[0010] This is particularly problematic at pilot or clinical scale
biotherapeutic production where it is desirable to process multiple
products in a relatively short time. The centrifuge cleaning
procedures slow down the pilot plant's ability to change over to
the production of a different biomolecule and greatly increase the
risk of cross contamination between production runs. In addition,
centrifugation cannot efficiently remove all particulates and
cellular debris from these feedstocks in the primary clarification
step, hence the need for the secondary clarification step utilizing
depth filtration after the centrifugation step, but prior to the
subsequent chromatographic steps.
[0011] Alternatively, successive filtration runs have proven useful
in removing different-sized cell and cellular debris from
feedstocks, but typically the volumetric throughputs limit the
application to smaller volumes (<1000 L) where the filter
installation has a reasonable size. The use of filtration greatly
reduces the risk of cross contamination and eliminates the need for
cleaning and cleaning validation between runs due the disposable
nature of filtration devices. Unfortunately, the low throughput
requires a large number of filter units which can reduce filtration
yields because each successive step results in the loss of a
portion of the feed solution through hold-up volumes of the filter
device and equipment.
[0012] In order to further enhance clarification performance,
throughput and downstream filtration operations, the flocculation
of a cell culture harvests have been used. Flocculants are a class
of materials that can aggregate and agglutinate fine particles from
a solution, resulting in their settling from the liquid phase and a
reduction in solution turbidity.
[0013] Flocculation can be accomplished in a variety of ways
including polymer treatment, chemical treatment (changes in pH) or
the addition of a surfactant. Precipitation using flocculants can
be used to selectively remove impurities while leaving the protein
product in the solution. However, flocculants have not been widely
used in the clarification of mAbs, mammalian cells, and other
bimolecular cellular materials of interest feedstocks.
[0014] Flocculation of cell culture harvests by chemicals require
the use of either acids, such as acetic acid, or polymers such as
chitosan, polysaccharides, polyamines or polyacrylamides.
Flocculation has been used as an alternative separation technology
to enhance centrifuge clarification throughput and centrate
quality, thereby improving the downstream filtration operations.
While chemical flocculation is quite effective in agglomerating
cellular debris and cellular contaminants (host cell proteins and
DNA), the resulting flocculated suspension is generally not easily
separable by ordinary filtration methods without the use of a
centrifuge prior to filtration.
[0015] Flocculants precipitate cells, cell debris and proteins
because of the interaction between the charges on the proteins and
charges on the polymer (e.g. polyelectrolytes), creating insoluble
complexes, and subsequent bridging of insoluble complexes either by
residual charge interaction or through hydrophobic patches on the
complexes to form larger clusters. In order to remove these large
clusters, a centrifuging step or tangential flow microfiltration is
the primary mode of clarification followed by the secondary
clarification step whereby depth filtration is widely used in the
clarification of cell culture broth prior to the capture
chromatography step. Since centrifugation cannot deliver a
particle-free centrate, depth filter (secondary depth filtration)
and sterile filter need to be installed further downstream.
[0016] Tangential flow microfiltration (also called cross-flow
microfiltration) competes with centrifugation for the harvest and
clarification of mAbs and therapeutic products from mammalian cell
culture. One advantage this technique offers is the creation of a
particle-free harvest stream that requires minimal additional
filtration. However, tangential flow microfiltration membranes used
for cell culture harvests are often plagued with the problem of
membrane fouling (i.e., irrecoverable declines in membrane flux)
and typically require strict complex operating condition followed
by a thorough cleaning regimen (as is also the case with a
centrifuge) for the membranes after each use. The use of optimized
membrane chemistry, with more hydrophilic tangential flow
microfiltration membranes generally being somewhat less susceptible
to significant fouling, to address this issue.
[0017] Traditionally, flocculation is generally used to agglomerate
non-deformable solid particles. For example, dilute suspensions of
submicron sized clay or titanium dioxide particles, which are very
difficult to filter because of their small particle size, can be
chemically flocculated and easily separated easily by, because the
size of these submicron particles size greatly increases by the
formation of agglomerated flocs., which settle more quickly, and
thereby filter faster because of large flow channels inside the
cake.
[0018] However, when chemical flocculation is applied to mAb
feedstocks or other biomolecule/cellular feedstocks, the resulting
agglomerate is unique and quite unlike the non-deformable solid
particles of earth materials and metal oxides because of the nature
of the biological properties of these bimolecular materials. Most
solid non-deformable particles such as earth materials or metal
oxides have a density much higher than water. Therefore, once these
small particles are flocculated, their particle size greatly
increases, and the resulting flocs quickly settle (i.e., in
minutes) by gravity. In contrast, cells, mAbs and other biomolecule
species are made of amino acids and water, and have a density very
close to the density of water. Therefore, flocculated cells and
other biomolecules don't readily settle and often take a number of
hours before settling occurs.
[0019] Another problem is the relatively low density of the
flocculated cell mass which, typically form a fluffy mass that
occupies significant part of the feed volume rather than forming a
compacted cake. Also, because of the biological origin of the
particles, the flocs are fragile and tend to break down easily
under pressure.
[0020] For this reason, most conventional solid-liquid separation
methods while useful for solid particle systems, fail in
flocculated cell masses such as mAb feedstocks.
[0021] Particle retention is believed to involve both size
exclusion and adsorption through hydrophobic, ionic and other
interactions. Fouling mechanisms may include pore blockage, cake
formation and/or pore constriction. Depth filters are advantageous
because they are very effective in removing contaminants and come
in disposable formats thereby eliminating validation and
contamination issues related to re-usable hardware installations,
such as those encountered when using a centrifuge. However, depth
filters are currently unable to handle the high solids feedstreams
that are typical of high titer mAb processes, such that depth
filters are therefore often used after centrifuging. The high
particulate load and high turbidity present in unclarified cell
culture supernatant adds challenges to the primary clarification by
depth filtration alone.
[0022] However, depth filters are currently unable to handle the
primary clarification of high-solids feedstreams, and often must be
used after centrifugation or tangential flow microfiltration. The
high particulate load and high turbidity present in unclarified
cell culture supernatant adds challenges to primary clarification
by depth filtration alone. Currently, the limited throughput
results in large installations of depth filters for primary
clarification which results in yield losses due to the large hold
up volume and scale-up issues as discussed above.
[0023] In addition, mAb feedstocks are challenging feed streams to
clarify and filter because of the presence of a higher biomass
content, and result in a high turbidity centrate after
centrifugation. Because of the need to remove large amounts of
biomass, the high turbidity centrates shorten the life of the depth
filter for clarification downstream. A need exists improve the
clarification of mAbs thereby resulting in higher throughputs.
[0024] In light of the above primary clarification processes which
rely on the use of a primary clarification centrifugation step or a
primary clarification microfiltration step followed by a secondary
clarification step which relies on depth filtration media to remove
larger particles, a need exists for a disposable, reasonably
reliable, and not inordinately expensive to implement, primary
clarification process that does not use a primary clarification
centrifugation or microfiltration step followed by an additional
secondary clarification step.
SUMMARY OF THE INVENTION
[0025] In response to the above needs and problems associated with
the primary clarification processes of feeds, feedstreams,
feedstocks, cell culture broths and the like, the present invention
overcomes the challenges by using a primary clarification depth
filtration process which utilizes a primary clarification depth
filtration device without the use a primary clarification
centrifugation step or primary clarification tangential flow
microfiltration step.
[0026] The present invention encompasses a process for the primary
clarification, by depth filtration, of feeds, feedstreams,
feedstocks, cell culture broths and the like, containing a target
biomolecule of interest and a plurality of cellular debris and
colloidal particulates without the use of a primary clarification
centrifugation step or a primary clarification tangential flow
microfiltration step, the process comprising:
[0027] a) providing a depth filtration device having a porous depth
filter media;
[0028] b) providing a feed stream containing a target biomolecule
of interest and a plurality of cellular debris and particulates,
wherein the cellular debris and particulates have a particle size
distribution of about 0.5 .mu.m to about 200 .mu.m;
[0029] c) contacting the porous depth filter media with the feed
stream, such that the depth filter media is capable of filtering
cellular debris and particulates having a particle size
distribution of about 0.5 .mu.m to about 200 .mu.m at a flow rate
of about 10 litres/m.sup.2/hr to about 100 litres/m.sup.2/hr;
and
[0030] d) separating the target biomolecule of interest from the
cellular debris and particulates without the use of a primary
clarification centrifugation step or a primary clarification
tangential flow microfiltration step.
[0031] The present invention further encompasses a process for the
primary clarification by depth filtration of a flocculated feed
containing therein a target biomolecule of interest or
biotherapeutic of interest and flocculated cellular debris,
materials, and colloidal particulates using a primary clarification
depth filtration device without the use of a primary clarification
centrifugation step or a primary clarification tangential flow
microfiltration step, the process comprising:
[0032] a) providing a depth filtration device containing a porous
depth filter media;
[0033] b) providing a chemical flocculant;
[0034] c) providing a feed containing a target biomolecule of
interest and a plurality of cellular material, debris and colloidal
particulates;
[0035] d) combining the chemical flocculant to the feed;
[0036] e) forming chemically flocculated cellular materials, debris
and colloidal particulates in the feed, and optionally chemically
flocculating the target biomolecule of interest;
[0037] f) contacting the porous depth filter media with the feed
containing the chemically flocculated cellular materials, debris
and colloidal particulates; and
[0038] g) separating the flocculated bimolecular species of
interest and the plurality of flocculated cellular material without
the use of a centrifugation clarification step or a tangential flow
microfiltration clarification step.
[0039] The present invention is directed towards the primary
clarification of feed using depth filtration devices without the
use of a primary clarification centrifugation step or primary
clarification tangential flow microfiltration step. The depth
filtration devices are able to filter high solids 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 litres/m.sup.2/hr to
about 100 litres/m.sup.2/hr until the TMP reaches 20 psi. The
primary clarification depth filter media taught herein include
graded porous layers of varying pore ratings.
[0040] One preferred application of the primary clarification
porous anisotropic depth filters media provided herein is the
primary clarification of chemically treated flocculated high solids
feeds containing a bimolecular species or biotherapeutic of
interest, and a plurality of flocculated cellular debris and
flocculated colloidal particulates.
[0041] In certain embodiments, the invention provides a process for
using a depth filtration device having a porous filter media in the
primary clarification of flocculated feeds containing mAbs,
mammalian cell cultures, plant cell cultures, bacteria cell
cultures, insect cell cultures, and other bimolecular cellular
materials and cultures of interest, by efficiently separating
flocculated aggregated cellular masses and debris from the
biomolecular species of interest without the use of a primary
clarification centrifugation step or a primary clarification
tangential flow microfiltration step by using a fiber based porous
depth filter media capable of performing depth filtration of a high
volume of feedstock containing very large particles without the
unintended effect of cake filtration.
[0042] In certain embodiments, the invention provides a depth
filtration device including a porous depth filter media having
multiple graded layers for use in primary clarification and the
removal of aggregated cellular biomass, including flocculated
cellular debris and colloidal particulates with a size larger than
about 10 microns (.mu.m) or smaller particles with the use of a
flocculating agent.
[0043] In still other embodiments, the invention provides a depth
filtration device including a porous depth filter media having open
graded layers for use in primary clarification depth filtration
that enables the depth filtration of cellular debris and colloidal
particulates having particle sizes varying from 0.5 .mu.m to 200
.mu.m, thereby improving the throughput for the unclarified
feedstreams without the unintended effect of cake filtration.
[0044] In certain embodiments, the present invention provides a
depth filtration media:
[0045] a) having large pores for the large flocs of cellular debris
to penetrate without the unintended effect of cake filtration or
the formation of floc bridging inside the pores of the filter;
[0046] b) with very high depth to spread out the cell masses to
prevent internal floc bridging, which would lead to internal cake
filtration inside the media, external to or internal to the media
in order to avoid concentration of the pressure drop which could
cause floc breakdown;
[0047] c) having an anisotropic depth filter layer, i.e. with a
gradual reduction in pore size that matches the population of the
floc size in the feedstock. For certain feedstocks with significant
amount of fine flocs such as those produced from acid flocculation
instead of polymers, the depth filter layer includes a composite
media having a combination of felt material, DE and chopped fibers
for fine removal is needed;
[0048] d) for the primary clarification of bimolecular species of
interest with mean particle sizes greater than 10 um, typical of
flocculant and chemically treated feed streams where the depth
filter includes graded layers of non-woven fibers with open nominal
pore size ratings capable of filtering flocculated feed streams
with high amount of solids;
[0049] e) of composite graded layers of non-woven fibers and
cellulose/diamatoceous earth with open nominal pore size ratings
capable of filtering polymer flocculant treated feed streams with
high amount of solids;
[0050] f) having good retentive properties for the polymer
flocculant (eg. smart polymer (SmP), chitosan etc) treated feeds
despite the greater permeability;
[0051] g) having good retentive properties for the polymer
flocculant (eg. acid precipitation, caprylic acid etc) treated
feeds despite the greater permeability;
[0052] h) used in the process for primary clarification of cell and
cell debris containing cultures using a depth filter comprising
graded layers of non-woven fibers; and
[0053] i) used in the process for primary clarification of
flocculated cell and cell debris containing cultures using a depth
filter comprising graded layers of non-woven fibers.
[0054] In certain embodiments, the invention provides a depth
filtration media prefilter for depth filters having a nominal pore
size <about 25 .mu.m comprising graded layers of non-woven
fibers.
[0055] In still other embodiments, the invention provides a depth
filtration media prefilter for depth filters with a nominal pore
size <25 about .mu.m comprising at least three graded layers of
non-woven fibers.
[0056] In still other embodiments, the invention provides a depth
filtration media including a depth filter including at least two
layers of graded non-woven fibers with a nominal pore size rating
>about 25 .mu.m capable of filtering flocculated feed streams
with >about 3% solids.
[0057] In still other embodiments, the invention provides a depth
filtration media including a depth filter including at least two
layers graded non-woven fibers with a nominal pore size rating
>about 25 .mu.m capable of filtering polymer flocculated feed
streams with >about 3% solids.
[0058] In still other embodiments, the invention provides a depth
filtration media including a depth filters comprising of at least
two layers of graded non-woven fibers with a nominal pore size
rating >about 25 .mu.m capable of filtering polymer flocculated
feed streams with >about 3% solids resulting in a turbidity
output <20 NTU.
[0059] To overcome the present challenges associated with the depth
filters, the present invention is directed towards the development
of a primary clarification depth filter media and process of using
the same which can filter high solid containing fluid streams
having a particle size distribution of approximately 0.5 microns to
200 microns at a flow rate of about 10 litres/m.sup.2/hr (10 LMH)
to 100 litres/m.sup.2/hr (100 LMH) until the TMP reaches 20 psi.
The primary clarification depth filter includes graded layers
having various pore ratings with the application in primary
clarification for polymer and chemically treated flocculated
feeds.
[0060] The present invention is directed towards depth filters for
disposable primary clarification processes. The use of open graded
layers for depth filtration enables the filtration of feeds
containing large clusters with the potential to eliminate
centrifugation, and enables the filtration of higher solids having
particle sizes varying from about 0.5 microns to about 200 microns,
thereby improving the throughput for these unclarified
feedstreams.
[0061] Additional features and advantages of the invention will be
set forth in the detailed description and claims, which follows.
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. It is to be understood that the foregoing
general description and the following detailed description, the
claims, as well as the appended drawings are exemplary and
explanatory only, and are intended to provide an explanation of
various embodiments of the present teachings. The specific
embodiments described herein are offered by way of example only and
are not meant to be limiting in any way.
BRIEF DESCRIPTION OF THE DRAWINGS
[0062] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate the presently
contemplated embodiments of the invention and, together with the
description, serve to explain the principles of the invention.
[0063] FIGS. 1A, 1B, 1C, 1D, 1E, and 1F depict different schematic
embodiments of examples of primary clarification depth filters
according to the invention, wherein FIG. 1A (eight layers), FIG. 1C
(seven layers) and FIG. 1E (eight layers) depict primary
clarification depth filters for use with polymer flocculant (smart
polymer) treated feeds, and FIGS. 1B, 1D and 1F depict primary
clarification depth filters having at least eight layers for use
with chemically treated feeds (acid treatment); and
[0064] FIG. 2 is a schematic representation of an exemplary primary
clarification depth filter purification process, as described
herein. The purification process shown uses a bioreactor for cell
culture followed by the following process steps: primary
clarification depth filtration; Protein A bind and elute
chromatography (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 a graded
clarification depth filtration as taught herein and depicted in
FIGS. 1A to 1F; 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.
DESCRIPTION OF THE EMBODIMENTS
[0065] All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entirety to the same extent as if each
individual publication, patent or patent application was
specifically and individually indicated to be incorporated by
reference.
[0066] For the purposes of this specification and appended claims,
unless otherwise indicated, all numbers expressing quantities of
ingredients, percentages or proportions of materials, reaction
conditions, and other numerical values used in the specification
and claims, are to be understood as being modified in all instances
by the term "about" whether or not explicitly indicated. The term
"about" generally refers to a range of numbers that one would
consider equivalent to the recited value (i.e., having the same
function or result). In many instances, the term "about" may
include numbers that are rounded to the nearest significant
figure.
[0067] Accordingly, unless indicated to the contrary, the numerical
parameters set forth in the following specification and attached
claims are approximations that may vary depending upon the desired
properties sought to be obtained by the present invention. At the
very least, and not as an attempt to limit the application of the
doctrine of equivalents to the scope of the claims, each numerical
parameter should at least be construed in light of the number of
reported significant digits and by applying ordinary rounding
techniques.
[0068] Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the invention are approximations,
the numerical values set forth in the specific examples are
reported as precisely as possible. Any numerical value, however,
inherently contains certain errors necessarily resulting from the
standard deviation found in their respective testing measurements.
Moreover, all ranges disclosed herein are to be understood to
encompass all subranges subsumed therein. For example, a range of
"1 to 10" includes any and all subranges between (and including)
the minimum value of 1 and the maximum value of 10, that is, any
and all subranges having a minimum value of equal to or greater
than 1 and a maximum value of equal to or less than 10, e.g., 5.5
to 10.
[0069] Before describing the present invention in further detail, a
number of terms will be defined. Use of these terms does not limit
the scope of the invention but only serve to facilitate the
description of the invention.
[0070] 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. The
following terms are defined for purposes of the invention as
described herein.
[0071] As used herein, the singular forms "a," "an," and "the"
include plural referents unless the context clearly dictates
otherwise.
[0072] 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 which
involves organisms or biochemically active substances derived from
such organisms. Such a process may be either 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.
[0073] The term "cell culture," refers to cells grown in
suspension, roller bottles, flasks and the like, as well as the
components of the suspension itself, including but not limited to
cells, cell debris, cellular contaminants, colloidal particles,
biomolecules, HCP, host cell proteins (HCP) and DNA, mAbs,
flocculants. Large scale approaches, such as bioreactors, including
adherent cells growing attached to microcarriers in stirred
fermentors, are also encompassed by the term "cell culture."
Moreover, it is possible to not only to culture contact-dependent
cells, but also to use the suspension culture techniques in the
methods of the claimed invention. Exemplary microcarriers include,
for example, dextran, collagen, plastic, gelatin and cellulose and
others as described in Butler, Spier & Griffiths, Animal cell
Biotechnology 3:283-303 (1988). Porous carriers, such as, for
example, Cytoline.RTM. or Cytopore.RTM., as well as dextran-based
carriers, such as DEAE-dextran (Cytodex 1.RTM.), quaternary
amine-coated dextran (Cytodex 2.RTM.) or gelatin-based carriers,
such as gelatin-coated dextran (Cytodex 3.RTM.) may also be used.
Cell culture procedures for both large and small-scale production
of proteins are encompassed by the present invention. Procedures
including, but not limited to, a fluidized bed bioreactor, hollow
fiber bioreactor, roller bottle culture, or stirred tank bioreactor
system may be used, with or without microcarriers, and operated
alternatively in a batch, fed-batch, or perfusion mode.
[0074] The terms "cell culture medium," and "culture medium" refer
to a nutrient solution used for growing animal cells, e.g.,
mammalian cells. Such a nutrient solution generally includes
various factors necessary for cell attachment, growth, and
maintenance of the cellular environment. For example, a typical
nutrient solution may include a basal media formulation, various
supplements depending on the cell type and, occasionally,
antibiotics. In some embodiments, a nutrient solution may include
at least one component from one or more of the following
categories: 1) an energy source, usually in the form of a
carbohydrate such as glucose; 2) all essential amino acids, and
usually the basic set of twenty amino acids plus cystine; 3)
vitamins and/or other organic compounds required at low
concentrations; 4) free fatty acids; and 5) trace elements, where
trace elements are defined as inorganic compounds or naturally
occurring elements that are typically required at very low
concentrations, usually in the micromolar range. The nutrient
solution may optionally be supplemented with one or more components
from any of the following categories: 1) hormones and other growth
factors as, for example, insulin, transferrin, and epidermal growth
factor; 2) salts and buffers as, for example, calcium, magnesium,
and phosphate; 3) nucleosides and bases such as, for example,
adenosine and thymidine, hypoxanthine; and 4) protein and tissue
hydrolysates. In general, any suitable cell culture medium may be
used. The medium may be comprised of serum, e.g. fetal bovine
serum, calf serum or the like. Alternatively, the medium may be
serum free, animal free, or protein free.
[0075] The term "cell culture additive," as used herein, refers to
a molecule (e.g., a non-protein additive), which is added to a cell
culture process in order to facilitate or improve the cell culture
or fermentation process. In some embodiments according to the
present invention, a stimulus responsive polymer, as described
herein, binds and precipitates one or more cell culture additives.
Exemplary cell culture additives include anti-foam agents,
antibiotics, dyes and nutrients.
[0076] The terms "Chinese hamster ovary cell protein" and "CHOP,"
as used interchangeably herein, refer to a mixture of host cell
proteins ("HOP") 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
containing a protein or polypeptide of interest (e.g., an antibody
or immunoadhesin expressed in a CHO cell). Generally, the amount of
CHOP present in a mixture comprising a protein of interest provides
a measure of the degree of purity for the protein of interest.
Typically, the amount of CHOP in a protein mixture is expressed in
parts per million relative to the amount of the protein of interest
in the mixture. It is understood that where the host cell is
another mammalian cell type, an E. coli, a yeast cell, an insect
cell, or a plant cell, HCP refers to the proteins, other than
target protein, found in a lysate of the host cell.
[0077] The terms "contaminant," "impurity," and "debris," as used
interchangeably herein, refer to any foreign or objectionable
material, including a biological macromolecule such as a DNA, an
RNA, one or more host cell proteins (HCPs or CHOPs), endotoxins,
viruses, lipids and one or more additives which may be present in a
sample containing a protein or polypeptide of interest (e.g., an
antibody) being separated from one or more of the foreign or
objectionable molecules using a stimulus responsive polymer
according to the present invention. In some embodiments, a stimulus
responsive polymer described herein binds and precipitates a
protein or polypeptide of interest from a sample containing the
protein or polypeptide of interest and one or more impurities. In
other embodiments, a stimulus responsive polymer described herein
binds and precipitates one or more impurities, thereby to separate
the polypeptide or protein of interest from one or more
impurities.
[0078] 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 step 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 step in a process or system 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 the 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 to be purified.
[0079] 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.
[0080] As used herein the term "depth filter" (e.g.,
gradient-density depth filter) achieves filtration within the depth
of the filter material. A common class of such filters are those
that comprise a random matrix of fibers bonded (or otherwise
fixed), to form a complex, tortuous maze of flow channels. Particle
separation in these filters generally results from entrapment by or
adsorption to, the fiber matrix. The most frequently used depth
filter media for bioprocessing of cell culture broths and other
feedstocks consists of cellulose fibers, a filter aid such as DE,
and a positively charged resin binder. Depth filter media, unlike
absolute filters, retain particles throughout the porous media
allowing for retention of particles both larger and smaller than
the pore size. Particle retention is thought to involve both size
exclusion and adsorption through hydrophobic, ionic and other
interactions. The fouling mechanism may include pore blockage, cake
formation and/or pore constriction. Depth filters are advantageous
because they remove contaminants and also come in disposable
formats thereby eliminating the validation issues.
[0081] 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
matrix. One example of an affinity chromatography matrix is a
ProteinA matrix. An affinity chromatography matrix 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 matrices are matrices carrying protein A
ligands like Protein A SEPHAROSE.TM. or PROSEP.RTM.-A. In the
processes and systems described herein, an affinity chromatography
step may be used as the bind and elute chromatography step in the
entire purification process.
[0082] 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 mixt 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
interacts 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.
[0083] "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 "CIEX") or can predominately bind the
impurities while the target molecule "flows through" the column
(cation exchange flow through chromatography FT-CIEX). 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.
[0084] The term "ion exchange matrix" refers to a matrix 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 the 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).
[0085] The term "anion exchange matrix" is used herein to refer to
a matrix which is positively charged, e.g. having one or more
positively charged ligands, such as quaternary amino groups,
attached thereto. Commercially available anion exchange resins
include DEAE cellulose, QAE SEPHADEX.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).
[0086] The term "cation exchange matrix" refers to a matrix which
is negatively charged, and which has free cations for exchange with
cations in an aqueous solution contacted with the solid phase of
the matrix. A negatively charged ligand attached to the solid phase
to form the cation exchange matrix or resin may, for example, be a
carboxylate or sulfonate. Commercially available cation exchange
matrices include carboxy-methyl-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).
[0087] 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.
[0088] 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.
[0089] 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.
[0090] 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 where 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 not 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 step,
in which case, during the performance of a process step including
multiple steps, the sample flows continuously through the multiple
steps that are necessary to perform the process step. One example
of such a process step described herein is the flow through
purification step which includes multiple steps that are performed
in a continuous manner, e.g., flow-through activated carbon
followed by flow-through AEX media followed by flow-through CEX
media followed by flow-through virus filtration.
[0091] In some embodiments, a depth filter, as described herein, is
used for clarification, following which the clarified cell culture
can continuously flow onto the next step in the purification
process, e.g., a bind and elute chromatography step (e.g., Protein
A affinity chromatography).
[0092] The term "semi-continuous process," as used herein, refers
to a generally continuous process for purifying a target molecule,
where input of the fluid material in any single process step or the
output is discontinuous or intermittent. 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 are "semi-continuous" in
nature, in that they 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.
[0093] 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 in direct fluid
communication with each other, such that fluid material
continuously flows through the process step 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 step, e.g., when a process step requires several
steps to be performed in order to achieve the intended result of
the process step. One such example is the flow-through purification
process step, as described herein, which may include several steps
to be performed in a flow-through mode, e.g., activated carbon;
anion exchange chromatography, cation exchange chromatography and
virus filtration.
[0094] The term "fluid communication," as used herein, refers to
the flow of fluid material between two process steps or flow of
fluid material between steps of a process step, 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.
[0095] The terms "purifying," "purification," "separate,"
"separating," "separation," "isolate," "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.
[0096] 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 effected, such
that the undesirable impurities can be more easily separated from
the soluble target molecule. Precipitation by phase change can be
effected 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.
[0097] In some embodiments described herein, clarification employs
the addition of a precipitant to a sample containing a target
molecule and one or more impurities followed by depth filtration.
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 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.
[0098] 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.
[0099] There are many ways known to those skilled in the art of
removing the precipitated material, such as filtration or settling
or any combinations thereof.
[0100] The term "settling," as used herein, refers 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.
[0101] As used herein the term "smart polymer" (SmP), (also known
as stimuli-responsive polymers or intelligent polymers or Affinity
Macro Ligands (AML)), as used herein means a group of polymers that
are biologically, chemically, or physically responsive to an
external stimulus such as to changes in environmental conditions
such pH, temperature, light, ionic strength, radiation, voltage,
external pressure, solvent composition, or other stimulus. Smart
polymers respond with large property changes to small physical or
chemical stimuli, and can reversibly change their physical or
chemical properties in response to these environmental stimuli (Roy
and Gupta, 2003; Kopecek, 2007). Smart polymers can take many
forms; they may be dissolved in an aqueous solution, adsorbed or
grafted on aqueous-solid interfaces, or cross-linked to form
hydrogels [Hoffman J Controlled Release (1987) 6:297-305; Hoffman
Intelligent polymers. In: Park K, ed. Controlled drug delivery.
Washington: ACS Publications, (1997) 485-98; Hoffman Intelligent
polymers in medicine and biotechnology. Artif Organs (1995)
19:458-467]. Typically, when the polymer's critical response is
stimulated, the smart polymer in solution will show a sudden onset
of turbidity as it phase-separates; the surface-adsorbed or grafted
smart polymer will collapse, converting the interface from
hydrophilic to hydrophobic; and the smart polymer (cross-linked in
the form of a hydrogel) will exhibit a sharp collapse and release
much of its swelling solution. Smart polymers may be physically
mixed with, or chemically conjugated to, biomolecules to yield a
large family of polymer-biomolecule systems that can respond to
biological as well as to physical and chemical stimuli.
Biomolecules that may be polymer-conjugated include proteins and
oligopeptides, sugars and polysaccharides, single- and
double-stranded oligonucleotides and DNA plasmids, simple lipids
and phospholipids, and a wide spectrum of recognition ligands and
synthetic drug molecules. A number of structural parameters control
the ability of smart polymers to specifically precipitate proteins
of interest; smart polymers should contain reactive groups for
ligand coupling; not interact strongly with the impurities; make
the ligand available for interaction with the target protein; and
form compact precipitates.
[0102] As used herein the phrase "high solids" containing feed,
means a feed having approximately >7% solids, while the phrase
"low solid" containing feeds would be approximately 0.1%-7%
solids.
[0103] 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 according to the present invention. Accordingly, the
present invention provides novel polymers which are responsive to a
stimulus and which stimulus results in a change in the solubility
of the polymer. Examples of stimuli to which one or more polymers
described 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.
[0104] The term "polymer" as used herein, refers to a molecule
formed by covalent linkage of two or more monomer units. These
monomer units can be synthetic or occur in nature. The polymers
formed by the repeating units can be linear or branched. Examples
of polymers include, but are not limited to, polyethylene glycol,
polypropylene glycol, polyethylene, polyallylamine,
polyvinylalcohol, polystyrene and copolymers (e.g.
polystyrene-co-polypyridine, polyacrylic acid-co-methyl
methacrylate, pluronics, PF68 etc). In some embodiments according
to the present invention, polymers comprise a polytelectrolyte
backbone.
[0105] 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 bind 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 IgGI. An
interaction compliant with such value for the binding constant is
termed "high affinity binding" in the present context. In some
embodiments, such functional derivative or variant of Protein A
comprises 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.
[0106] 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.
[0107] 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.
[0108] In some embodiments, Protein A derivatives of variants are
attached via multi-point attachment to suitable a chromatography
matrix.
[0109] 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
matrix. One example of an affinity chromatography matrix is a
ProteinA matrix. An affinity chromatography matrix 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 matrices are matrices carrying protein A
ligands like Protein A SEPHAROSE.TM. or PROSEP.RTM.-A. In the
processes and systems described herein, an affinity chromatography
step may be used as the bind and elute chromatography step in the
entire purification process.
[0110] The term "stimulus responsive polymer," as used herein, is a
polymer 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.
[0111] The term "flocculation," as used herein, refers to the
addition of a flocculant, such as a polymer or chemically treated
(e.g., acid treatment) described herein, to a solution in order to
remove one or more suspended insoluble or soluble impurities. The
polymer must be added to the solution at a concentration which
allows for spontaneous formation of insoluble aggregates which can
be removed from solution via typical solid-liquid separation
methods.
[0112] The term "composition," "solution" or "sample," as used
herein, refers to a mixture of a target molecule or a desired
product to be purified using one or more stimulus responsive
polymers or chemically treated (e.g., acid treatment) described
herein along with one or more undesirable entities or impurities.
In some embodiments, the sample comprises feedstock or cell culture
media into which a target molecule or a desired product is
secreted. In some embodiments, the sample comprises a target
molecule (e.g., a therapeutic protein or an antibody) along with
one or more impurities (e.g., host cell proteins, DNA, RNA, lipids,
cell culture additives, cells and cellular debris). In some
embodiments, the sample comprises a target molecule of interest
which is secreted into the cell culture media.
[0113] In some embodiments, a sample from which a target molecule
is to be purified using one or more stimulus responsive polymers or
chemically treated (e.g., acid treatment) described herein is
"partially purified" prior to contacting the sample with a stimulus
responsive polymer. Partial purification may be accomplished, for
example, by subjecting the sample to one or more purification
steps, such as, e.g., one or more non-affinity chromatography
steps. The target molecule may be separated from one or more
undesirable entities or impurities either by precipitating the one
or more impurities or by precipitating the target molecule.
[0114] The term "precipitate," precipitating" or "precipitation,"
as used herein, refers to the alteration of a bound (e.g., in a
complex with a biomolecule of interest) or unbound polymer or other
soluble species from an aqueous and/or soluble state to a
non-aqueous and/or insoluble state.
[0115] The term "biomolecule of interest," as used herein, can be a
desired target molecule such as, for example, a desired product or
polypeptide of interest (e.g., an antibody), or it can be an
undesirable entity, which needs to be removed from a sample
containing the desired target molecule. Such undesirable entities
include but are not limited to, for example, one or more impurities
selected from host cell protein, DNA, RNA, protein aggregates, cell
culture additives, viruses, endotoxins, whole cells and cellular
debris. In addition, the biomolecule of interest may also be bound
and precipitated by a stimulus responsive polymer or chemically
treated (e.g., acid treatment) as described herein.
[0116] The terms "target molecule", "target biomolecule", "desired
target molecule" and "desired target biomolecule," as used
interchangeable herein, generally refer to a polypeptide or product
of interest, which is desired to be purified or separated from one
or more undesirable entities, e.g., one or more impurities, which
may be present in a sample containing the polypeptide or product of
interest.
[0117] The terms "protein of interest," "target polypeptide,"
"polypeptide of interest," and "target protein," as used
interchangeably herein, generally refer to a therapeutic protein or
polypeptide, including but not limited to, an antibody that is to
be purified using a stimulus responsive polymer according to the
present invention.
[0118] As used herein interchangeably, the term "polypeptide" or
"protein," generally refers to peptides and proteins having more
than about ten amino acids. In some embodiments, a stimulus
responsive polymer described herein is used to separate a protein
or polypeptide from one or more undesirable entities present in a
sample along with the protein or polypeptide. In some embodiments,
the one or more entities are one or more impurities which may be
present in a sample along with the protein or polypeptide being
purified. As discussed, above, in some embodiments, a stimulus
responsive polymer described herein specifically binds and
precipitates a protein or polypeptide of interest upon the addition
of a stimulus to the sample. In other embodiments, a stimulus
responsive polymer described herein binds to and precipitates an
entity other than the protein or polypeptide of interest such as,
for example, host cell proteins, DNA, viruses, whole cells,
cellular debris and cell culture additives, upon the addition of a
stimulus.
[0119] In some embodiments, a protein or polypeptide being purified
using a stimulus responsive polymer described herein is a mammalian
protein, e.g., a therapeutic protein or a protein which may be used
in therapy. Exemplary proteins include, but are not limited to, for
example, 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 A-chain; insulin B-chain; proinsulin;
follicle stimulating hormone; calcitonin; luteinizing hormone;
glucagon; clotting factors; anti-clotting factors; atrial
natriuretic factor; lung surfactant; a plasminogen activator;
bombesin; thrombin; hemopoietic growth factor; tumor necrosis
factor-alpha and -beta; enkephalinase; RANTES (regulated on
activation normally T-cell expressed and secreted); human
macrophage inflammatory protein (MIP-1-alpha); a serum albumin such
as human serum albumin; Muellerian-inhibiting substance; relaxin
A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated
peptide; a microbial protein, such as beta-lactamase; Dnase; IgE; a
cytotoxic T-lymphocyte associated antigen (CTLA), such as CTLA-4;
inhibin; activin; vascular endothelial growth factor (VEGF);
receptors for hormones or growth factors; Protein A or D;
rheumatoid factors; a neurotrophic factor, 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; epidermal growth factor (EGF); transforming growth
factor (TGF); insulin-like growth factor-I and -II (IGF-I and
IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor
binding proteins (IGFBPs); CD proteins; erythropoietin;
osteoinductive factors; immunotoxins; a bone morphogenetic protein
(BMP); an interferon; interleukins (IIs), e.g., IL-1 to IL-10;
superoxide dismutase; T-cell receptors; surface membrane proteins;
decay accelerating factor; viral antigen such as, for example, a
portion of the AIDS envelope; transport proteins; homing receptors;
addressins; regulatory proteins; integrins; a tumor associated
antigen such as HER2, HER3 or HER4 receptor; and fragments and/or
variants of any of the above-listed polypeptides.
[0120] Further, in some embodiments, a protein or polypeptide
purified using a smart polymer according to the present invention
is an antibody, functional fragment or variant thereof. In some
embodiments, a protein of interest is a recombinant protein
containing an Fc region of an immunoglobulin.
[0121] The term "immunoglobulin," "Ig" or "antibody" (used
interchangeably herein) refers to a protein having a basic
four-polypeptide chain structure consisting of two heavy and two
light chains, said chains being stabilized, for example, by
interchain disulfide bonds, which has the ability to specifically
bind antigen. The term "single-chain immunoglobulin" or
"single-chain antibody" (used interchangeably herein) refers to a
protein having a two-polypeptide chain structure consisting of a
heavy and a light chain, said chains being stabilized, for example,
by interchain peptide linkers, which has the ability to
specifically bind antigen. The term "domain" refers to a globular
region of a heavy or light chain polypeptide comprising peptide
loops (e.g., comprising 3 to 4 peptide loops) stabilized, for
example, by 3-pleated sheet and/or intrachain disulfide bond.
Domains are further referred to herein as "constant" or "variable",
based on the relative lack of sequence variation within the domains
of various class members in the case of a "constant" domain, or the
significant variation within the domains of various class members
in the case of a "variable" domain. Antibody or polypeptide
"domains" are often referred to interchangeably in the art as
antibody or polypeptide "regions". The "constant" domains of
antibody light chains are referred to interchangeably as "light
chain constant regions", "light chain constant domains", "CL"
regions or "CL" domains. The "constant" domains of antibody heavy
chains are referred to interchangeably as "heavy chain constant
regions", "heavy chain constant domains", "CH" regions or "CH"
domains. The "variable" domains of antibody light chains are
referred to interchangeably as "light chain variable regions",
"light chain variable domains", "VL" regions or "VL" domains. The
"variable" domains of antibody heavy chains are referred to
interchangeably as "heavy chain variable regions", "heavy chain
variable domains", "VH" regions or "VH" domains.
[0122] Immunoglobulins or 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.
Immunoglobulins or 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 recombinantly, 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. Exemplary fusion proteins include Fc fusion
proteins.
[0123] Generally, an immunoglobulin or antibody is directed against
an "antigen" of interest. Preferably, the antigen is a biologically
important polypeptide and administration of the antibody to a
mammal suffering from a disease or disorder can result in a
therapeutic benefit in that mammal. However, antibodies directed
against nonpolypeptide antigens (such as tumor-associated
glycolipid antigens; see U.S. Pat. No. 5,091,178) are also
contemplated. Where the antigen is a polypeptide, it may be a
transmembrane molecule (e.g. receptor) or a ligand such as a growth
factor.
[0124] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to conventional
(polyclonal) antibody preparations which typically include
different antibodies directed against different determinants
(epitopes), each monoclonal antibody is directed against a single
determinant on the antigen. The modifier "monoclonal" indicates the
character of the antibody as being obtained from a substantially
homogeneous population of antibodies, and is not to be construed as
requiring production of the antibody by any particular method. For
example, the monoclonal antibodies to be used in accordance with
the present invention may be made by the hybridoma method first
described by Kohler et al., Nature 256:495 (1975), or may be made
by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).
"Monoclonal antibodies" may also be isolated from phage antibody
libraries using the techniques described in Clackson et al., Nature
352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597
(1991), for example.
[0125] Monoclonal antibodies may further include "chimeric"
antibodies (immunoglobulins) in which a portion of the heavy and/or
light chain is identical with or homologous to corresponding
sequences in antibodies derived from a particular species or
belonging to a particular antibody class or subclass, while the
remainder of the chain(s) is identical with or homologous to
corresponding sequences in antibodies derived from another species
or belonging to another antibody class or subclass, as well as
fragments of such antibodies, so long as they exhibit the desired
biological activity (U.S. Pat. No. 4,816,567; and Morrison et al.,
Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
[0126] "Framework" or "FR" residues are those variable domain
residues other than the hypervariable region residues as herein
defined.
[0127] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies which contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which
hypervariable region residues of the recipient are replaced by
hypervariable region residues from a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, and capacity. In some instances, Fv
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues which are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FR
regions are those of a human immunoglobulin sequence.
[0128] The humanized antibody optionally also will comprise at
least a portion of an immunoglobulin constant region (Fc),
typically that of a human immunoglobulin. For further details, see
Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature
332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596
(1992).
[0129] In some embodiments, an antibody which is separated or
purified using a stimulus responsive polymer according to the
present invention is a therapeutic antibody. Exemplary therapeutic
antibodies include, for example, trastuzumab (HERCEPTIN.TM.,
Genentech, Inc., Carter et al (1992) Proc. Natl. Acad. Sci. USA,
89:4285-4289; U.S. Pat. No. 5,725,856); anti-CD20 antibodies such
as chimeric anti-CD20 "C2B8" U.S. Pat. No. 5,736,137; anti-IgE
(Presta et al (1993) J. Immunol. 151:2623-2632; WO 95/19181);
anti-CD18 (U.S. Pat. No. 5,622,700; WO 97/26912); anti-IgE,
including E25, E26 and E27 (U.S. Pat. No. 5,714,338; U.S. Pat. No.
5,091,313; WO 93/04173; U.S. Pat. No. 5,714,338); anti-Apo-2
receptor antibody (WO 98/51793); anti-TNF-alpha antibodies
including cA2 (REMICADE.TM.), CDP571 and MAK-195 (U.S. Pat. No.
5,672,347; Lorenz et al (1996) J. Immunol. 156(4):1646-1653;
Dhainaut et al (1995) Crit. Care Med. 23(9):1461-1469); anti-Tissue
Factor (TF) (EP 0 420 937 B1); anti-CD4 antibodies such as the
cM-7412 antibody (Choy et al (1996) Arthritis Rheum 39(1):52-56);
anti-Fc receptor antibodies such as the M22 antibody directed
against Fc gamma RI as in Graziano et al (1995) J. Immunol.
155(10):4996-5002; anti-GpIIb/IIIa antibodies; anti-RSV antibodies
such as MEDI-493 (SYNAGIS.TM.); anti-CMV antibodies such as
PROTOVIR.TM.); anti-HIV antibodies such as PRO542; anti-hepatitis
antibodies such as the anti-Hep B antibody OSTAVIR.TM.); anti-CA
125 antibody OvaRex; anti-idiotypic GD3 epitope antibody BEC2;
anti-alpha v beta3 antibody VITAXIN.TM.; anti-human renal cell
carcinoma antibody such as ch-G250; ING-1; anti-human 17-1A
antibody (3622W94); and anti-human leukocyte antigen (HLA)
antibodies such as Smart ID10 and the anti-HLA DR antibody Oncolym
(Lym-1).
[0130] The terms "isolating," "purifying" and "separating," are
used interchangeably herein, in the context of purifying a target
molecule (e.g., a polypeptide or protein of interest) from a
composition or sample comprising the target molecule and one or
more impurities, using a stimulus responsive polymer described
herein. In some embodiments, the degree of purity of the target
molecule in a sample is increased by removing (completely or
partially) one or more impurities from the sample by using a
stimulus responsive polymer, as described herein. In another
embodiment, the degree of purity of the target molecule in a sample
is increased by precipitating the target molecule away from one or
more impurities in the sample.
[0131] In some embodiments, a purification process additionally
employs one or more "chromatography steps." Typically, these steps
may be carried out, if necessary, after the separation of a target
molecule from one or more undesired entities using a stimulus
responsive polymer according to the present invention.
[0132] In some embodiments, a "purification step" to isolate,
separate or purify a polypeptide or protein of interest using a
stimulus responsive polymer described herein, may be part of an
overall purification process resulting in a "homogeneous" or "pure"
composition or sample, which term is used herein to refer to a
composition or sample comprising less than 100 ppm HCP in a
composition comprising the protein of interest, alternatively less
than 90 ppm, less than 80 ppm, less than 70 ppm, less than 60 ppm,
less than 50 ppm, less than 40 ppm, less than 30 ppm, less than 20
ppm, less than 10 ppm, less than 5 ppm, or less than 3 ppm of HCP.
As used herein "primary clarification" includes the removal of
aggregated cellular biomass, including flocculated cellular debris
and colloidal particulates with a size larger than about 10 microns
(.mu.m) or smaller particles with the use of a flocculating
agent.
[0133] The terms "clarify", "clarification", "clarification step,"
as used herein, generally refers to one or more steps used
initially in the purification of biomolecules. The clarification
step generally comprises removal of cells and/or cellular debris
using one or more steps including any of the following alone or
various combinations thereof, e.g., clarification depth filtration,
precipitation, flocculation and settling. In some embodiments, the
present invention provides an improvement over the conventional
clarification step commonly used in various purification schemes.
Clarification steps generally involve the removal of one or more
undesirable entities and 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 insoluble
components 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. 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 shifts, phase change due to
stimulus-responsive polymers or small molecules, or any
combinations of these methods.
[0134] The term "chromatography," as used herein, refers to any
kind of technique which separates an analyte of interest (e.g., a
target molecule) from other molecules present in a mixture.
Usually, the analyte of interest 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.
[0135] The term "chromatography resin" or "chromatography media"
are used interchangeably herein and refer to any kind of phase
(e.g., a solid phase) which separates an analyte of interest (e.g.,
a target molecule) from other molecules present in a mixture.
Usually, the analyte of interest is separated from other molecules
as a result of differences in rates at which the individual
molecules of the mixture migrate through a stationary solid phase
under the influence of a moving phase, or in bind and elute
processes. Examples of various types of chromatography media
include, for example, cation exchange resins, affinity resins,
anion exchange resins, anion exchange membranes, hydrophobic
interaction resins and ion exchange monoliths.
[0136] The term "capture step" as used herein, generally refers to
a method used for binding a target molecule with a stimulus
responsive polymer or a chromatography resin, which results in a
solid phase containing a precipitate of the target molecule and the
polymer or resin. Typically, the target molecule is subsequently
recovered using an elution step, which removes the target molecule
from the solid phase, thereby resulting in the separation of the
target molecule from one or more impurities. In various
embodiments, the capture step can be conducted using a
chromatography media, such as a resin, membrane or monolith, or a
polymer, such as a stimulus responsive polymer, polyelectrolyte or
polymer which binds the target molecule.
[0137] The term "salt," as used herein, refers to a compound formed
by the interaction of an acid and a base. Various salts which may
be used in various buffers employed in the methods described herein
include, but are not limited to, acetate (e.g. sodium acetate),
citrate (e.g., sodium citrate), chloride (e.g., sodium chloride),
sulphate (e.g., sodium sulphate), or a potassium salt.
[0138] The term "solvent," as used herein, generally refers to a
liquid substance capable of dissolving or dispersing one or more
other substances to provide a solution. Solvents include aqueous
and organic solvents, where useful organic solvents include a
non-polar solvent, ethanol, methanol, isopropanol, acetonitrile,
hexylene glycol, propylene glycol, and 2,2-thiodiglycol.
[0139] The term "parts per million" or "ppm," as used
interchangeably herein, refers to a measure of purity of a desired
target molecule (e.g., a target protein or antibody) purified using
a stimulus responsive polymer described herein. Accordingly, this
measure can be used either to gauge the amount of a target molecule
present after the purification process or to gauge the amount of an
undesired entity. In some embodiments, the units "ppm" are used
herein to refer to the amount of an impurity in a solution, e.g.,
HCP or CHOP, in nanograms/milliliter of protein of interest in
milligrams/milliliter (i.e., CHOP ppm=(CHOP ng/ml)/(protein of
interest mg/ml). When the proteins are dried (e.g., by
lyophilization), ppm refers to (CHOP ng)/(protein of interest
mg)).
[0140] The term "pl" or "isoelectric point" of a polypeptide, as
used interchangeably herein, refers to the pH at which the
polypeptide's positive charge balances its negative charge. pl can
be calculated from the net charge of the amino acid residues or
sialic acid residues of attached carbohydrates of the polypeptide
or can be determined by isoelectric focusing.
[0141] 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.
[0142] 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).
[0143] 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 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 step, 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.
[0144] As used herein the term "pore size" and "nominal pore size"
refers to the pore size which retains the majority of the
particulate at 60-98% of the rated pore size.
[0145] As used herein the term "throughput" means the volume
filtered through a filter.
[0146] As used herein, the term "system" generally refers to the
physical form of the whole purification process, which includes two
or more process steps or unit operations, as described herein. In
some embodiments, the system is enclosed in a sterile
environment.
[0147] In the present invention, the use of open graded layers
allows the larger particles to penetrate and become captured within
the depth of the filters, rather than collecting on the surface
(Refer to Examples 2A and 2B).
[0148] The advantage is higher throughput, and retention of large
solids (0.5 microns to about 200 microns) while eliminating the
problem of cake formation. The use of open pores in the primary
clarification filters provides these depth filters with the linear
increase in pressure with the solid retention with no significant
increase in the pressure and hence resulting in high throughputs.
The structural dimension of the filter in combination with the
optimization of layers (pore sizes and thickness) gives exceptional
filtration properties which can retain high amount of solids.
[0149] In the present invention, the use of open graded layers
allows the larger flocculated 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 (Refer
to Examples 9 (A-E) and 11 (A-J)). The primary clarification depth
filter provided herein are arranged such that the "open" top layers
constitute the prefiltration zone of the depth filters in order to
capture larger flocculated particles, while the bottom layers
constitute the polishing zone which captures the smaller residual
aggregated flocculated particles. One advantage with the primary
clarification depth filter having this type of arrangement is
higher throughput, and the retention of larger flocculated solids,
while also eliminating the problem of cake formation. The use of
such open pores in the primary clarification filter taught herein
provides a linear increase in pressure with the solids retention,
with no significant increase in the pressure, and hence resulting
in higher, more desirable throughputs.
[0150] Examples of primary clarification depth filters according to
the invention are depicted in FIGS. 1A, 1B, 1C, 1D, 1E and 1F.
[0151] FIG. 1C depicts a primary clarification depth filters having
at least seven layers, and is used when the cell-culture feeds are
treated with a polymer flocculant (e.g., smart polymer or
traditional flocculant).
[0152] FIGS. 1A and 1E depict primary clarification depth filters
having at least eight layers, and are each used when the
cell-culture feeds are treated with a polymer flocculant (e.g.,
smart polymer or traditional flocculant).
[0153] FIGS. 1B, 1D, and 1F depict primary clarification depth
filters having at least eight layers, and are each used when the
cell-culture feeds are chemically treated (e.g., acid
treatment).
[0154] The primary clarification depth filter depicted in FIG. 1A
shows a primary clarification depth filter used when the
cell-culture feeds are treated with a polymer flocculant (e.g.,
smart polymer) having two (upper) layers with a nominal pore size
of about 100 microns of a non woven such as polypropylene about 0.4
cm thick, having two more layers with a nominal pore size of about
50 microns of a non woven such as polypropylene about 0.4 cm thick,
having two additional layers with a nominal pore size of about 25
microns of a non woven such as polypropylene about 0.4 cm thick,
followed by a single layer about 0.35 cm thick of a material such
as cellulose (CE25) for example, and another single layer about
0.35 cm thick of a material such as diatomaceous earth (DE40) for
example.
[0155] The primary clarification depth filter depicted in FIG. 1B
shows a primary clarification depth filter used when the
cell-culture feeds are chemically treated (e.g., acid treatment)
having two (upper) layers with a nominal pore size of about 25
microns of a non woven such as polypropylene about 0.4 cm thick,
having two more layers with a nominal pore size of about 10 microns
of a non woven such as polypropylene about 0.4 cm thick, having two
additional layers with a nominal pore size of about 5 microns of a
non woven such as polypropylene about 0.4 cm thick, followed by a
single layer about 0.35 cm thick of a material such as cellulose
(CE25) for example, and followed by another single of layer about
0.35 cm thick of a material such as diatomaceous earth (DE40) for
example. Either the cellulose or diatomaceous earth layer can be
selected as the lowest (bottom) layer.
[0156] The primary clarification depth filter depicted in FIG. 1C
shows a primary clarification depth filter used when the
cell-culture feeds are treated with a polymer flocculant (e.g.,
smart polymer) having two (upper) layers with a nominal pore size
of about 100 microns comprising a non woven such as polypropylene
about 0.4 cm thick, having two more layers with a nominal pore size
of about 100 microns of a non woven such as polypropylene about 0.4
cm thick, having two additional layers with a nominal pore size of
about 100 microns comprising a non woven such as polypropylene
about 0.4 cm thick, followed by a single layer (bottom) about 8
microns thick of a non woven such as polypropylene about 0.2 cm
thick.
[0157] The primary clarification depth filter depicted in FIG. 1D
shows a primary clarification depth filter used when the
cell-culture feeds are chemically treated (e.g., acid treatment)
having two (upper) layers with a nominal pore size of about 50
microns comprising a non woven such as polypropylene about 0.4 cm
thick, having two additional layers with a nominal pore size of
about 25 microns of a non woven such as polypropylene about 0.4 cm
thick, having two more layers with a nominal pore size of about 10
microns of a non woven such as polypropylene about 0.4 cm thick,
followed by a single layer about 0.35 cm thick of a material such
as cellulose (CE25) for example, and followed by another single of
layer about 0.35 cm thick of a material such as diatomaceous earth
(DE40) for example. Either the cellulose or diatomaceous earth
layer can be selected as the lowest (bottom) layer.
[0158] The primary clarification depth filter depicted in FIG. 1E
shows a primary clarification depth filter used when the
cell-culture feeds are treated with a polymer flocculant (e.g.,
smart polymer) having two (upper) layers with a nominal pore size
of about 100 microns comprising a non woven such as polypropylene
about 0.4 cm thick, having two more layers with a nominal pore size
of about 50 microns of a non woven such as polypropylene about 0.4
cm thick, having two additional layers with a nominal pore size of
about 25 microns comprising a non woven such as polypropylene about
0.4 cm thick, followed by a layer about 0.35 cm thick of a material
such as cellulose (CE25) for example, and followed by another
single of layer about 0.35 cm thick of a material such as
diatomaceous earth (DE40) for example.
[0159] The primary clarification depth filter depicted in FIG. 1F
shows a primary clarification depth filter used when the
cell-culture feeds are chemically treated (e.g., acid treatment)
having two (upper) layers with a nominal pore size of about 35
microns comprising a non woven such as polypropylene about 0.4 cm
thick, having two more layers with a nominal pore size of about 15
microns of a non woven such as polypropylene about 0.4 cm thick,
having two additional layers with a nominal pore size of about 10
microns comprising a non woven such as polypropylene about 0.4 cm
thick, followed by a single layer about 0.35 cm thick of a material
such as cellulose (CE25) for example, and followed by another
single of layer 0.35 cm thick of a material such as diatomaceous
earth (DE40) for example. Either the cellulose or diatomaceous
earth layer can be selected as the lowest (bottom) layer.
[0160] The structural dimension of the primary clarification depth
filter provided herein in combination with the optimization of the
pore sizes and/or thickness of layer of the primary clarification
depth filter results in highly advantageous filtration properties
which also retain high amounts of solids. Since depth filters
achieve filtration through the depth of media via a combination of
various mechanisms, the column volume of feed (V.sub.f) versus
column volume of media (V.sub.m) shows the effectiveness of
different filters.
[0161] In addition, various feeds have different amounts of solids
which result in the highly variable performance of the depth
filters, hence (V.sub.f) versus (V.sub.m)) value gives a better
estimate of the actual "efficiency" of filters.
[0162] Another important parameter, K, is used to describe the
filter efficiency while normalizing for the solid content of the
feedstock. The parameter K allows for filtration of feeds with
different solids content to be effectively compared.
[0163] The K parameter is actually function of three measurable
parameters, volume throughput (TP), collection efficiency (.eta.),
and initial concentration of solids (C.sub.i). as shown in Equation
1.
K=[TP].times.[.eta.].times.[C.sub.i].times.100 (1)
[0164] Where volume throughput (TP) is given by volume of feed
filtered (V.sub.f) divided by volume of media (V.sub.m), collection
efficiency (q) is given by volume of solids captured (V.sub.sc)
divided by volume of solids in the feed (V.sub.s), and initial
concentration of solids (C.sub.i) is given by volume of solids in
the feed (V.sub.s) divided by volume of feed filtered (V.sub.f) as
given in Equation 2.
K = [ V f V m ] .times. [ V sc V s ] .times. [ V s V f ] .times.
100 ( 2 ) ##EQU00001##
[0165] The following examples will demonstrate the usefulness of
Equations (1) and (2) and the K parameter in determining and
comparing the efficiency of particle depth filters when used with
particular feedstocks.
[0166] The following examples are provided so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make the compositions of the invention and
how to practice the methods of the invention and are not intended
to limit the scope of what the inventor regards as his invention.
Efforts have been made to insure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.), but some experimental
errors and deviation should be accounted for. Unless indicated
otherwise, temperature is in degrees C., chemical reactions were
performed at atmospheric pressure or transmembrane pressure, as
indicated, the term "ambient temperature" refers to approximately
25.degree. C. and "ambient pressure" refers to atmospheric
pressure.
Comparative Examples
[0167] Settling often fails because of long settling time (hours).
Disposing large volume of unconsolidated cell floc mass causes low
mAb yield as a significant fraction of the mAb is trapped in the
cell mass. (Refer to Example 5)
[0168] Cake filtration fails because the cell mass breaks down
under pressure. In other words, cell debris break apart from the
polymer under stress and flow through as high turbidity in the
filtrate (refer to Example 6).
[0169] Depth filtration fails because the flocs, particularly those
from polymer systems, are quite large, in the order of 100 microns.
Instead of penetrating into the media, which is needed for depth
filtration, large flocs soon pile up on top of the surface of the
depth filter media, reducing the depth filter media into an
inefficient cake filter. (refer to Example 7)
[0170] Dynamic filtration such as tangential filtration or vibrated
filter media have been tried with little success because the shear
stress needed to reduce cake formation break up the floes back into
individual particles, nullifying the benefits of flocculation.
(refer to Example 8)
[0171] The invention will be further clarified by the following
examples which are intended to be exemplary of the invention.
EXAMPLES
Example 1
Preparation of Unclarified Non-Expressing Cell Culture Fluid
(CCF)
[0172] In a representative experiment, cells derived from an
expressing Chinese Hamster Ovary (CHO) cell line were grown in a 10
L bioreactor (New Brunswick Scientific, Edison, N.J.) to a density
of 10.times.10.sup.6 cells/mL and harvested at 80% viability.
Monoclonal antibody (mAb) titer was determined to be 0.8 g/L. The
level of host cell proteins (HCP) was found to be 200,000 ng/mL
using an ELISA assay (Cygnus Technologies, Southport, N.C., #3G
ELISA). The pH of the unclarified cell culture was pH 6.9.
Example 2
Preparation of Multivalent Ion Stimulus Sensitive Polymer
[0173] 10 g of polyallylamine (PAA) (Nittobo Medical Co., Ltd.,
Tokyo, Japan 150 kD; 40% wt/wt) is placed in a 100 mL round bottom
flask and a solution of 3.34 g of sodium hydroxide (1.2 Eq. per
monomer) in 25 mL H.sub.2O is added at room temperature under
magnetic stirring and in small amounts. Benzyl chloride (2.30 g,
2.09 mL) is then added, stirred for few minutes at room temperature
and then heated to 60.degree. C. overnight for 17 hours. The
reaction is then cooled to room temperature and solvent is removed
resulting in polymer precipitation. The precipitated polymer is
washed with water and stirred in 1M aqueous AcOH solution (40 ml)
until complete solubilization is achieved. The solution is then
diluted with H.sub.2O to a final volume of 400 ml (1% polymer
solution), potassium dibasic phosphate (K.sub.2HPO.sub.4) (3.48 g)
is added under stirring and pH of the solution is adjusted to pH
6.8 to precipitate the modified polymer. The polymer is collected
by filtration over a fritted funnel and finally dried overnight in
a vacuum oven overnight at 50.degree. C. to 60.degree. C. The
polymer was then redissolved in 1M acetic acid to generate a 10% wt
polymer concentrate solution.
Example 3
Smart Polymer (SmP) Treatment of CHO-S Feed
[0174] In order to flocculate the cell culture with SmP, a 500 ml
sample of cell culture broth from Example 1 was added to a 1000 ml
media bottle. While stirring, a sample of polymer concentrate from,
Example 2 to the desired polymer dose (wt %), typically 0.2%. The
solution was allowed to mix for 15 minutes.
Example 4
Acid Treatment of CHO-S Feed
[0175] In order to flocculate the cell culture with addition of
acid to reduce pH, a 500 ml sample of cell culture broth from
Example 1 was added to a 1000 ml media bottle. While stirring,
concentrated acetic acid was added dropwise until the desired pH
was achieved. The target pH was pH 4.8-5.0 unless otherwise noted.
The solution was allowed to mix for 15 minutes.
Example 5
Settling Studies for the Smart Polymer (SmP) Treated CHO-S Feed
[0176] Settling experiments were conducted at different settling
times to determine effectiveness of using density differences to
perform the solid liquid separation of SmP treated feeds. 500 ml of
cell culture broth was prepared according to Example 3.
[0177] The SmP treated feed was allowed to settle for about 0.5 to
6 hours, and samples of the supernatant were taken and measured for
turbidity and volume of solids. The turbidity was measured using a
HACH Model #2100P turbiditimeter. Table 1 shows the settling
studies on SmP treated CHO-S feed for times varying from 0.5 to 6
hours.
[0178] It was observed that the settling time was >about 180
minutes to reach the equilibrium turbidity of <about 20 NTU for
the SmP treated CHO-S feed, and about 120 minutes to reach the
equilibrium turbidity of <about 20 NTU for the SmP treated
CHO-DH44 feed.
[0179] When the smallest polymer dose (0.05%) was used, the
incomplete flocculation lead to increased turbidity (>about 350
NTU) even after 12 hours settling time for the SmP treated CHO-S
feed. In addition, the settling times of 3 hours has a large volume
of unconsolidated cell mass (about 30% to 40%) for SmP treated
CHO-S feed and 20% for the SmP treated CHO-DH44 feed which could
apparently resulted in low mAb yield (about 60% to 80%) as
significant amount of mAb is trapped in the unconsolidated
mass.
TABLE-US-00001 TABLE 1 Settling studies for the smart polymer
treated CHO-S feed. Turbidity Turbidity Turbidity Turbidity Time
(0.05%) (0.2%) (0.4%) (0.6%) (min) (NTU) (NTU) (NTU) (NTU) 0 451
549 601 666 60 128 26 81 112 120 101 16 54 90 180 98 10 38 68
Example 6
Cake Filtration for the Smart Polymer (SmP) Treated DG44 and CHO-S
Feeds
[0180] Diamatoceous earth (DE) media was used to determine the
effectiveness of the cake filtration. First, DE was packed to a
depth of least about 4 cm in the Buchner funnel after which we
passed the SmP treated feed from Example 3 by applying vacuum
through it. During filtration through the diatomaceous earth media,
CHO-S cells formed a film of filter cake on the 20 .mu.m sieve. It
was observed that the filter cake impeded the outward flow of
filtrate resulting in a throughput <about 10 L/m.sup.2. In
addition, it was observed that the cell mass broke apart from the
polymer under stress resulting in a high turbid filtrate (about 200
NTU to 300 NTU) which can significantly impact the secondary
filtration operations.
Example 7
Depth Filtration for the Smart Polymer (SmP) Treated CHO-S Feeds
Using Commercially Available Primary Clarification Filters (D0HC
and F0HC)
[0181] Filtration experiments were performed with smart polymer
(SmP) treated CHO-S feed from Example 3 and acid treated feed from
Example 4 to determine the throughput of commercially available
MilliStak.RTM. D0HC depth filters. The D0HC filters were flushed
with water according to the user instructions. Feed was applied to
the depth filters using a peristaltic pump at a flow rate of about
100 L/m.sup.2/hr. However, depth filters were unable to handle
high-solids feedstreams. It is believed that depth filtration
primarily failed because the aggregated cells were larger and
instead of penetrating into the media, which is needed for depth
filtration, larger particulates build up on the top of the depth
filter media surface, reducing the depth filter media into an
inefficient cake filter. D0HC had a throughput of about 20
L/m.sup.2 for the SmP treated feeds and F0HC had a throughput of
about 5 L/m.sup.2 for the acid treated feeds. The filter cake
formation from the larger floc particles is largely due to the
tightness of filter which reduced the throughput of filters
significantly.
Example 8
Dynamic Filtration for the Smart Polymer (SmP) Treated Feed
[0182] Dynamic filtration experiments using tangential flow
Pellicon.RTM. 3 (0.11 m.sup.2) filtration devices (available from
Millipore Corporation, Billerica, Mass. USA) was performed to
determine the effectiveness of solid liquid separation of SmP
treated feed. The filtration devices contained microfiltration
membranes (0.45 .mu.m) constructed of a polyvinyldifluoride (PVDF)
membrane. SmP treated cell culture harvest was loaded at 50
L/m.sup.2/h until the TMP reached 15 psi. An instantaneous plugging
of the Pellicon.RTM. 3 devices was observed, resulting in a
throughput <5 L/m.sup.2. Poor yield was observed due to the
material loss created by the rapid plugging and system and device
hold-up volume.
Example 9A
Depth Filters for the Removal of Aggregated and Large Biomolecule
Particulates
[0183] A depth filtration device 10 was assembled using seven (7)
layers of non-woven fibers (polypropylene) having a total thickness
of all the layers of 1.6 cm. The layers are arranged in the depth
filtration device from most open nominal pore size 200 .mu.m (2
layers) followed by nominal 50 .mu.m (2 layers), nominal 40 .mu.m
(2 layers) to a single nominal 8 .mu.m layer (see FIG. 1).
[0184] The individual properties of the seven (7) layers of (needle
felt) non-woven fibers (Rosedale Products, Inc., Ann Arbor, Mich.)
are shown in Table 3 (2 layers.times.200 .mu.m, 2 layers.times.50
.mu.m, 2 layers.times.40 .mu.m, and 1 layers.times.8 .mu.m). After
assembling the stack of layers, polypropylene hose barb end caps
were added to the top and bottom and the entire assembly overmolded
into a single, integral Mini Cap filtration device (available from
Millipore Corporation, Billerica, Mass. USA). The depth filtration
device was tested for water permeability resulting in a value of
0.45 L/min at 4 psi.
TABLE-US-00002 TABLE 3 Characterization of physical properties of
non-woven felt material from Rosedale Products, Inc. and Midwest
Filtration Company Av. Basis Water Flow Nom. Pore Pore Weight Rate
Supplier Layer (.mu.m) (.mu.m) (g/m.sup.2) (gallons/min) Rosedale
Needle Punch 200 100 425 555 Rosedale Needle Punch 100 85 380 529
Rosedale Needle Punch 50 70 309 524 Rosedale Needle Punch 40 70 347
514 Rosedale Needle Punch 30 60 320 523 Rosedale Needle Punch 25 50
266 492 Rosedale Needle Punch 20 40 413 330 Rosedale Needle Punch
10 35 396 360 Rosedale Melt Blown 8 8 288 264 Midwest UniPro 760 PP
.gtoreq.200 110 260 552 (Needle Punch) Midwest Needle Punch 200 80
336 500 Midwest Needle Punch 100 70 369 497 Midwest Needle Punch 50
65 335 476 Midwest Needle Punch 25 50 390 436 Midwest Needle Punch
10 35 390 410 Midwest Needle Punch 5 31 368 400 Midwest Needle
Punch 1 30 486 350 Midwest UniPro 530 MM .ltoreq.1 15 187 338 (Melt
Blown)
Example 9B
Depth Filters for the Removal of Aggregated and Large Biomolecule
Particulates
[0185] The graded depth filter of Example 9B consists of graded
non-woven fibers, having a depth of 1.6 cm, and are capable of
filtering an acid flocculated feed stream comprising particles in
the range of about 0.5 .mu.m to about 200 .mu.m. The graded depth
filter consists of seven (7) layers of non-woven fibers from
Rosedale Products, Inc., Ann Arbor, Mich. (2 layers.times.200
.mu.m, 2 layers.times.100 .mu.m, 2 layers.times.50 .mu.m, and 1
layer.times.8 .mu.m). After assembling the stack of layers,
polypropylene hose barb end caps were added to the top and bottom
and the entire assembly overmolded into a single, integral Mini Cap
filtration device (available from Millipore Corporation, Billerica,
Mass. USA). The device was tested for water permeability resulting
in a value of 0.55 L/min at 4 psi.
Example 9C
Depth Filters for the Removal of Aggregated and Large Biomolecule
Particulates
[0186] The graded depth filter of Example 9C consists of graded
non-woven fibers, have a depth of 1.6 cm, and are capable of
filtering an acid flocculated feed stream comprising particles in
the range of about 0.5 .mu.m to about 200 .mu.m. The graded depth
filter consists of seven (7) layers of non-woven fibers from
Rosedale Products, Inc., Ann Arbor, Mich. (3 layers.times.200
.mu.m, 3 layers.times.100 .mu.m and 1 layer.times.8 .mu.m). The
graded depth filter provided herein was assembled in a 23 cm.sup.2
of an integral Mini Cap filtration device (available from Millipore
Corporation, Billerica, Mass. USA) and tested for water
permeability resulting in a value of 0.55 L/min at 4 psi.
Example 9D
Depth Filters for the Removal of Aggregated and Large Biomolecule
Particulates
[0187] The graded depth filter of Example 9D consists of graded
non-woven fibers, have a depth of 1.6 cm, and are capable of
filtering an acid flocculated feed stream comprising particles in
the range of 0.5 .mu.m to 100 .mu.m. The graded depth filter
consists of seven (7) layers of non-woven fibers from Rosedale
Products, Inc., Ann Arbor, Mich. (2 layers.times.100 .mu.m, 2
layers.times.50 .mu.m, 2 layers.times.40 .mu.m and 1 layer.times.8
.mu.m). After assembling the stack of layers, polypropylene hose
barb end caps were added to the top and bottom and the entire
assembly overmolded into a single, integral Mini Cap filtration
device (available from Millipore Corporation, Billerica, Mass.
USA). filtration device. The device was tested for water
permeability resulting in a value of 0.5 L/min at 4 psi.
Example 9E
Depth Filters for the Removal of Aggregated and Large Biomolecule
Particulates
[0188] The graded depth filter of Example 9E consists of a graded
non-woven fibers, have a depth of 1.6 cm, and are capable of
filtering a polymer flocculated feed stream comprising particles in
the range of about 0.5 .mu.m to about 200 .mu.m. The graded depth
filter consists of seven (7) layers of non-woven fibers from
Midwest Filtration Company, Cincinnati, Ohio (2
layers.times.UniPro.RTM. 760 PP, 2 layers.times.100 .mu.m, 2
layers.times.50 .mu.m and 1 layer.times.UniPro.RTM. 530 MM). The
graded depth filter feeds consists of seven (7) layers of non-woven
fibers from Midwest Filtration Company, Cincinnati, Ohio (2
layers.times.UniPro.RTM. 760 PP, 2 layers.times.100 .mu.m, 2
layers.times.50 .mu.m and 1 layer.times.UniPro.RTM. 530 MM). The
graded depth filter provided herein was assembled in a 23 cm.sup.2
integral Mini Cap filtration device (available from Millipore
Corporation, Billerica, Mass. USA) and tested for water
permeability resulting in a value of 0.65 L/min at 4 psi.
Example 10
Filtration Performance of Depth Filters Removal of Aggregated and
Large Biomolecule Particulates
[0189] Filter devices from Examples 9A-9E were tested for
filtration performance using the following method. The depth
filters were run with untreated and SmP treated unclarified feed
after flushing out with the Milli-Q water with the TMP across each
filter monitored by pressure transducers. The unclarified cell
culture harvest was treated with 0.2 wt % smart polymer (SmP) dose
(wt %) and stirred for 15 minutes. The depth filters were first
flushed with .gtoreq.about 50 L of Milli-Q water for each square
meter of filter area at 600 L/m.sup.2/h to wet the filter media and
flush out extractables. Untreated and SmP treated unclarified
harvest were loaded at 100 L/m.sup.2/h until the TMP across any one
filter reached 20 psig.
[0190] Table 4 compares the filter throughput of two Millistak.RTM.
filters (X0HC and D0HC) with Primary clarification depth filter for
the filtration of the feed described in Example 3. (0.2% (w/v)
smart polymer (SmP) treated feed). X0HC and D0HC gave a throughput
of 10 L/m.sup.2 and 44 L/m.sup.2 whereas throughput of primary
clarification depth filter was 325 L/m.sup.2. Filtrate turbidity in
all the cases was <about 5 NTU as shown in Table 4. In terms of
column volume of filtrate by column volume of media, X0HC and D0HC
gave a throughput of 1.5 V.sub.f/V.sub.m and 6 V.sub.f/V.sub.m
whereas throughput of primary clarification depth filter was 16.5
CV.sub.f/CV.sub.m. Example 9A performed the best with the volume
throughput of 16.5 V.sub.f/V.sub.m (325 L/m.sup.2) with a K
efficiency of 84%. From this comparison, it is evident that Example
9A filter composed of layers described in the present claim is
capable of removing large amount of solids during clarification of
unclarified cell harvests.
TABLE-US-00003 TABLE 4 Comparison of the Primary Clarification (PC)
Depth Filter described in Example 9A for the filtration throughput
of SMP treated feed with 0.2% (w/v). Dose (%) PCV Turbidity TP TP K
Feed Treatment Filter Type (w/v) (%) (NTU) (L/m.sup.2)
(V.sub.f/V.sub.m) (%) CHO-S Untreated X0HC NA 3.8 5 10 2 8 CHO-S
Untreated D0HC NA 3.8 45 44 6.5 26 CHO-S SMP treated X0HC 0.2 4.0 2
8 1.5 8 CHO-S SMP treated D0HC 0.2 4.0 2 39 6 24 CHO-S SMP treated
PC (Ex. 9A) 0.2 4.0 6 325 16.5 66
[0191] The present invention has a significant advantage in terms
of linear differential pressure growth. In the case of X0HC and
D0HC the fluid pressure was lesser and constant at the start but
suddenly increased exponentially thereby reaching its limit. One
possible explanation consistent with the observed pressure response
is the rapid formation of a cake layer on the surface of the
filter. In the case of primary clarification depth filters, the
pressure increase is close to linear, with no significant abrupt
increase in pressure. This result is consistent with particulates
being trapped throughout the depth of the filter avoiding the cake
formation. A large increase in filter volumetric throughput is also
observed, which again is consistent of depth filtration instead of
the cake filtration observed in the commercial X0HC and D0HC
filters.
[0192] Table 5 compares the filter throughput of primary
clarification depth filters described in Examples 9B-9E in terms of
column volume of feed versus column volume of media.
[0193] The graded depth filter described in the Example 9B gave the
volume throughput of 32 V.sub.f/V.sub.m (640 L/m.sup.2) with a K
efficiency of 90% for SmP treated CHO-DG44 feed and volume
throughput of 22 V.sub.f/V.sub.m (430 L/m.sup.2) with a K
efficiency of 90% for SmP treated CHO-S feed.
[0194] In another Example 9C, the graded depth filter resulted in
the volume throughput of 33 V.sub.f/V.sub.m (660 L/m.sup.2) with a
Kman efficiency of 94% for SmP treated CHO-DG44 feed and volume
throughput of 22 V.sub.f/V.sub.m (435 L/m.sup.2) with a K
efficiency of 90% for SmP treated CHO-S feed.
[0195] The graded depth filter described in the Example 9D resulted
in a volume throughput of 29 V.sub.f/V.sub.m (580 L/m.sup.2) with a
K efficiency of 81% for SmP treated CHO-DG44 feed and volume
throughput of 20 V.sub.f/V.sub.m (390 L/m.sup.2) with a K
efficiency of 81% for SmP treated CHO-S feed.
[0196] In yet another Example 9E, the graded depth filter described
in the present claim resulted in a volume throughput of 33
V.sub.f/V.sub.m (650 L/m.sup.2) with a K efficiency of 92% for SmP
treated CHO-S feed.
[0197] From this comparison, it is evident that Example 9A-9E,
filter composed of layers described in the claims is capable of
removing large amount of solids during clarification of unclarified
cell harvests.
TABLE-US-00004 TABLE 5 Comparison of the Primary Clarification (PC)
Depth Filter described in Example 9B-9E for the filtration
throughput of SMP treated feed with 0.2% (w/v). Dose (%) PCV
Turbidity TP TP K Feed Treatment Filter Type (w/v) (%) (NTU)
(L/m.sup.2) (V.sub.f/V.sub.m) (%) CHO- SMP PC (Ex. 9B) 0.2 2.8
<5 640 30 90 DG44 treated CHO-S SMP PC (Ex. 9B) 0.2 4.0 <5
430 22 90 treated CHO- SMP PC (Ex. 9C) 0.2 2.9 <5 660 33 94 DG44
treated CHO-S SMP PC (Ex. 9C) 0.2 4.0 <5 435 22 90 treated CHO-
SMP PC (Ex. 9D) 0.2 2.9 <5 580 29 81 DG44 treated CHO-S SMP PC
(Ex. 9D) 0.2 4.0 <10 390 20 80 treated CHO-S SMP PC (Ex. 9E) 0.2
3.0 <5 650 33 92 treated
Example 11A
Depth Filters for the Removal of Aggregated and Small Biomolecule
Particulates
[0198] Chemically treated feeds (e.g., acid treatment) has the
capability to increase the average particle size from <about 5
.mu.m to >about 20 .mu.m. In addition, the acid treated feeds
gives a broad particle size distribution. In response to the need
for separation of this broad range of particles, a combination of
open graded non-woven layers and tighter (CE and DE) provides the
effective depth filtration. A depth filtration device was assembled
using eight (8) layers of non-woven fibers (polypropylene) having a
total thickness of all the layers of 2.0 cm. The layers are
arranged in the filtration device 50, FIG. 1, with the open nominal
pore size 200 .mu.m (2 layers), followed by nominal 50 .mu.m (2
layers), nominal 40 .mu.m (2 layers), followed by a layer of
Cellulose (CE25), and a layer of diatomaceous earth (DE40). The
individual properties of the six (6) layers of (needle punched)
non-woven fibers (Rosedale Products, Inc., Ann Arbor, Mich.) are
shown in Table 3 (2.times.200 .mu.m, 2.times.50 .mu.m, and
2.times.40 .mu.m). After assembling the stack of layers,
polypropylene hose barb end caps were added to the top and bottom
and the entire assembly overmolded into a single, integral Mini Cap
filtration device (available from Millipore Corporation, Billerica,
Mass. USA). The device was tested for water permeability resulting
in a value of 0.40 L/min at 4 psi.
Example 11B
Depth Filters for the Removal of Aggregated and Small Biomolecule
Particulates
[0199] The graded depth filter of Example 11B consists of graded
non-woven fibers, have a depth of 2 cm, and are capable of
filtering an acid flocculated feed stream comprising particles in
the range of about 0.5 .mu.m to about 100 .mu.m. The graded depth
filter consists of six (6) layers of non-woven fibers (2
layers.times.30 .mu.m, 2 layers.times.25 .mu.m, 2 layers.times.20
.mu.m) from Rosedale Products, Inc., Ann Arbor, Mich., followed by
a layer of cellulose (CE25), and a layer of diatomaceous earth
(DE40). After assembling the stack of layers, polypropylene hose
barb end caps were added to the top and bottom and the entire
assembly overmolded into a single, integral Mini Cap filtration
device (available from Millipore Corporation, Billerica, Mass.
USA). The filtration device was tested for water permeability
resulting in a value of 0.15 L/min at 4 psi.
Example 11C
Depth Filters for the Removal of Aggregated and Small Biomolecule
Particulates
[0200] The graded depth filter of Example 11B consists of graded
non-woven fibers, has a depth of 2 cm, and is capable of filtering
an acid flocculated feed stream comprising particles in the range
of about 0.5 .mu.m to about 100 .mu.m. The graded depth filter
feeds comprises of six (6) layers of non-woven fibers (2
layers.times.25 .mu.m, 2 layers.times.20 .mu.m, 2 layers.times.10
.mu.m) from Rosedale Products, Inc., Ann Arbor, Mich., followed by
a layer of cellulose (CE25), and a layer of diatomaceous earth
(DE40). After assembling the stack of layers, polypropylene hose
barb end caps were added to the top and bottom and the entire
assembly overmolded into a single, integral Mini Cap filtration
device (available from Millipore Corporation, Billerica, Mass.
USA). The device was tested for water permeability resulting in a
value of 0.15 L/min at 4 psi.
Example 11D
Depth Filters for the Removal of Aggregated and Small Biomolecule
Particulates
[0201] The graded depth filter of Example 11D consists of graded
non-woven fibers, have a depth of 1.6 cm, and are capable of
filtering an acid flocculated feed stream comprising particles in
the range of about 0.5 .mu.m to about 100 .mu.m. The graded depth
filter feeds comprises of four (4) layers of non-woven fibers
(2.times.20 .mu.m, 2.times.10 .mu.m) from Rosedale Products, Inc.,
Ann Arbor, Mich. and cellulose (CE 25)/diatomaceous earth (DE 60).
After assembling the stack of layers, polypropylene hose barb end
caps were added to the top and bottom and the entire assembly
overmolded into a single, integral Mini Cap filtration device
(available from Millipore Corporation, Billerica, Mass. USA). The
device was tested for water permeability resulting in a value of
0.3 L/min at 4 psi.
Example 11E
Depth Filters for the Removal of Aggregated and Small Biomolecule
Particulates
[0202] The graded depth filter of Example 11E consists of graded
non-woven fibers, have a depth of 2 cm, and are capable of
filtering an acid flocculated feed stream comprising particles in
the range of about 0.5 .mu.m to about 100 .mu.m. The graded depth
filter consists of six (6) layers of non-woven fibers from Rosedale
Products, Inc., Ann Arbor, Mich. (2 layers.times.25 .mu.m, 2
layers.times.20 .mu.m, 2 layers.times.10 .mu.m), followed by a
layer of cellulose (CE25), and a layer of diatomaceous earth
(DE40). After assembling the stack of layers, polypropylene hose
barb end caps were added to the top and bottom and the entire
assembly overmolded into a single, integral Mini Cap filtration
device (available from Millipore Corporation, Billerica, Mass.
USA). The device was tested for water permeability resulting in a
value of 0.2 L/min at 4 psi.
Example 11F
Depth Filters for the Removal of Aggregated and Small Biomolecule
Particulates
[0203] The graded depth filter of Example 11F consists of graded
non-woven fibers, have a depth of 1.6 cm, and are capable of
filtering an acid flocculated feed stream comprising particles in
the range of about 0.5 .mu.m to about 100 .mu.m. The graded depth
filter consists of four (4) layers of non-woven fibers from
Rosedale Products, Inc., Ann Arbor, Mich. (2 layers.times.20 .mu.m,
2 layers.times.10 .mu.m), followed by a layer of cellulose (CE25),
and a layer of diatomaceous earth (DE40). After assembling the
stack of layers, polypropylene hose barb end caps were added to the
top and bottom and the entire assembly overmolded into a single,
integral Mini Cap filtration device (available from Millipore
Corporation, Billerica, Mass. USA). The device was tested for water
permeability resulting in a value of 0.3 L/min at 4 psi.
Example 11G
Depth Filters for the Removal of Aggregated and Small Biomolecule
Particulates
[0204] The graded depth filter of Example 11G consists of graded
non-woven fibers, have a depth of 1.6 cm, and are capable of
filtering an acid flocculated feed stream comprising particles in
the range of about 0.5 .mu.m to about 100 .mu.m. The graded depth
filter consists of six (6) layers of non-woven fibers from Midwest
Filtration Company, Cincinnati, Ohio (2 layers.times.50 .mu.m, 2
layers.times.25 .mu.m, 2 layers.times.10 .mu.m) followed by a layer
of cellulose (CE25), and a layer of diatomaceous earth (DE40).
After assembling the stack of layers, polypropylene hose barb end
caps were added to the top and bottom and the entire assembly
overmolded into a single, integral Mini Cap filtration device
(available from Millipore Corporation, Billerica, Mass. USA). The
device was tested for water permeability resulting in a value of
0.35 L/min at 4 psi.
Example 11H
Depth Filters for the Removal of Aggregated and Small Biomolecule
Particulates
[0205] The graded depth filter of Example 11H consists of graded
non-woven fibers, have a depth of 2 cm, and are capable of
filtering an acid flocculated feed stream comprising particles in
the range of about 0.5 .mu.m to about 100 .mu.m. The graded depth
filter consists of six (6) layers of non-woven fibers from Midwest
Filtration Company, Cincinnati, Ohio (2.times.25 .mu.m, 2.times.10
.mu.m, 2.times.5 .mu.m) followed by a layer of cellulose (CE25),
and a layer of diatomaceous earth (DE40). After assembling the
stack of layers, polypropylene hose barb end caps were added to the
top and bottom and the entire assembly overmolded into a single,
integral Mini Cap filtration device (available from Millipore
Corporation, Billerica, Mass. USA). The device was tested for water
permeability resulting in a value of 0.4 L/min at 4 psi.
Example 11I
Depth Filters for the Removal of Aggregated and Small Biomolecule
Particulates
[0206] The graded depth filter of Example 11I consists of graded
non-woven fibers, have a depth of 2 cm, and are capable of
filtering an acid flocculated feed streams comprising particles in
the range of about 0.5 .mu.m to about 100 .mu.m. The graded depth
filter comprises six (6) layers of non-woven fibers from Midwest
Filtration Company, Cincinnati, Ohio (2.times.10 .mu.m, 2.times.5
.mu.m, 2.times.1 .mu.m), followed by a layer of cellulose (CE25),
and a layer of diatomaceous earth (DE40). After assembling the
stack of layers, polypropylene hose barb end caps were added to the
top and bottom and the entire assembly overmolded into a single,
integral Mini Cap filtration device (available from Millipore
Corporation, Billerica, Mass. USA). The device was tested for water
permeability resulting in a value of 0.4 L/min at 4 psi.
Example 12
Filtration Performance of Depth Filters Removal of Aggregated and
Small Biomolecule Particulates
[0207] Filter devices from Examples 11A-11I were tested for
filtration performance using the following method. The unclarified
cell culture harvest was treated with 1M glacial acetic acid to
adjust the pH to 4.8 and stirred for 30 minutes. Depth filters were
run with untreated and acid treated unclarified feed after flushing
out with the Milli-Q water with the TMP across each filter
monitored by pressure transducers. The depth filters were first
flushed with .gtoreq.about 50 L of Milli-Q water for each square
meter of filter area at 600 L/m.sup.2/h to wet the filter media and
flush out extractables. Untreated and acid precipitated unclarified
harvest were loaded at 100 L/m.sup.2/h until the TMP across any one
filter reached 20 psig.
[0208] Table 6 compares the filter throughput of two Millistak.RTM.
filters (X0HC and DOHC) with primary clarification depth filter for
the acid treated feed. X0HC and D0HC gave a throughput of 5
L/m.sup.2 and 20 L/m.sup.2 whereas throughput of primary
clarification depth filter was 210 L/m.sup.2.
TABLE-US-00005 TABLE 6 Comparison of the Acid Precipitation Primary
Clarification (APPC) Depth Filter for the filtration throughput for
acid treated feed (pH = 4.8). PCV Turbidity TP TP K Feed Treatment
Filter Type pH (%) (NTU) (L/m.sup.2) (V.sub.f/V.sub.m) (%) CHO-S
Untreated X0HC 6.9 3.8 5 10 2 8 CHO-S Untreated D0HC 6.9 3.8 45 44
6.5 26 CHO-S Acid X0HC 4.8 3.9 1 5 1.5 6 treated CHO-S Acid D0HC
4.8 3.9 3 20 4 16 treated CHO-S Acid APPC (Ex. 11A) 4.8 3.9 25 210
9 36 treated
[0209] Table 2 compares the filter throughput of two Millistak.RTM.
filters (X0HC and D0HC) with acid precipitated primary
clarification depth filter for the acid treated feed in terms of
column volume of feed versus column volume of media. X0HC and D0HC
gave a throughput of 1.5 V.sub.f/V.sub.m (K=6) and 4
V.sub.f/V.sub.m ((K=16) whereas throughput of primary clarification
depth filter was 9 V.sub.f/CV.sub.m (K=36). From this comparison,
it is evident that Example 9A filter composed of layers described
in the present claim is capable of removing large amount of solids
during clarification of unclarified cell harvests.
TABLE-US-00006 TABLE 2 Settling studies for the smart polymer
treated CHO-DG44 feed. Turbidity Turbidity Turbidity Turbidity Time
(0.05%) (0.2%) (0.4%) (0.6%) (min) (NTU) (NTU) (NTU) (NTU) 0
>1000 >1000 >1000 >1000 60 365 33 50 84 120 344 26 31
68 360 325 17 21 49
[0210] Table 7 compares the filter throughput of primary
clarification depth filters described in Examples 11B-11I in terms
of column volume of feed versus column volume of media. From this
comparison, it is evident that in Examples 11A-11I, filters
composed of layers as provided herein are capable of removing large
amount of solids during clarification of unclarified cell
harvests.
TABLE-US-00007 TABLE 7 Comparison of the Acid Precipitation Primary
Clarification (APPC) Depth Filter for the filtration throughput for
acid treated feed (pH = 4.8). PCV Turbidity TP TP K Feed Treatment
Filter Type pH (%) (NTU) (L/m.sup.2) (V.sub.f/V.sub.m) (%) CHO-DG44
Acid treated APPC (Ex. 11B) 4.8 2.8 <10 554 27 77 CHO-S Acid
treated APPC (Ex. 11B) 4.8 4.0 <10 347 18 72 CHO-DG44 Acid
treated APPC (Ex. 11C) 4.8 2.8 <10 660 30 84 CHO-S Acid treated
APPC (Ex. 11C) 4.8 4.0 <10 391 20 80 CHO-DG44 Acid treated APPC
(Ex. 11D) 4.8 2.8 <10 580 29 81 CHO-S Acid treated APPC (Ex.
11E) 4.8 4.0 <10 425 22 87 CHO-S Acid treated APPC (Ex. 11F) 4.8
4.0 <10 395 20 81 CHO-S Acid treated APPC (Ex. 11G) 4.8 12.0
<10 122 6.1 73 CHO-S Acid treated APPC (Ex. 11H) 4.8 12.0 <10
132 6.9 82 CHO-S Acid treated APPC (Ex. 11I) 4.8 12.0 <10 140
7.2 86
Example 13
Depth Filters for the Removal of Aggregated and Small Colloidal
Particulates in the Range of 0.1 .mu.m to 200 .mu.m
[0211] The graded depth filter of Example 13 consists of graded
non-woven fibers, have a depth of 2 cm, and are capable of
filtering an acid flocculated feed stream comprising particles in
the range of about 0.1 .mu.m to about 200 .mu.m. The graded depth
filter consists of six (6) layers of non-woven fibers from Midwest
Filtration Company, Cincinnati, Ohio (2 layers.times.25 .mu.m, 2
layers.times.10 .mu.m, 2 layers.times.5 .mu.m) followed by
commercially available cellulose (CE 25)/diatomaceous earth (DE
40), and IM75. The graded depth filter provided herein were
assembled in a 23 cm.sup.2 integral Mini Cap filtration device
(available from Millipore Corporation, Billerica, Mass. USA) and
tested for water permeability resulting in a value of 0.25 L/min at
4 psi. Next, acid precipitated unclarified harvest was loaded at
100 L/m.sup.2/h until the TMP across any one filter reached 20
psig. Filtration performance was compared against the control
graded filter consisting of six (6) layers of non-woven fibers from
Midwest Filtration Company, Cincinnati, Ohio (2 layers.times.25
.mu.m, 2 layers.times.10 .mu.m, 2 layers.times.5 .mu.m) followed by
a layer of cellulose (CE25), and a layer of diatomaceous earth
(DE40). The filter described in this example resulted in a
throughput of 11 V.sub.f/V.sub.m (K=66) whereas throughput of
control graded depth filter was 12 V.sub.f/CV.sub.m (K=72).
However, the graded depth filter described in the example resulted
in a turbidity of 1 NTU as compared to 4 NTU for control graded
filter. From this comparison, it is evident that Example 13 filter
composed of layers as provided herein are capable of removing
smaller colloidal particulates in addition to cells and cell debris
during clarification which can potentially lead to removal of
secondary clarification filters in the process.
[0212] Another major benefit for the customer is improved
high-solids feedstock clarification economics. As previously noted,
in the clarification process applications for many high-solids
feedstock, centrifuges and/or tangential flow microfiltration are
used as the primary clarification step upstream from the secondary
clarification which typically includes a depth filter. By
incorporating the depth filter into the primary clarification
process in the manner described herein, the preceding (upstream
clarification step) and subsequent (downstream clarification step)
use of a centrifugation step and/or tangential flow microfiltration
step are eliminated. Furthermore, less down time would be
anticipated to be spent in cleaning, checking and replacing the
centrifuge (s) and/or tangential flow microfiltration
membranes.
Example 14
Clarification Depth Filtration Device and System for Purifying a
Target Molecule
[0213] FIG. 5 is a schematic representation of an exemplary
clarification depth filtration device purification process
incorporated into a system for purifying a target molecule, wherein
the system includes two or more unit operations connected in fluid
communication with each other, in order 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.
[0214] Without wishing to be bound by theory, it is contemplated
that a system can be enclosed in a closed sterile environment, so
as to perform the entire purification process in a sterile
manner.
[0215] In various embodiments, the very first device in such 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 particular embodiment, the bioreactor is a
disposable bioreactor, e.g. in form of a flexible, collapsible bag,
designed for single-use.
[0216] 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
clarification depth filtration device as taught herein in order to
remove one or more impurities. Accordingly, in some embodiments,
the bioreactor is in fluid communication with a device for
performing depth filtration.
[0217] The clarification depth filtration device as taught herein
is in fluid communication with a device for performing capture
using a bind and elute chromatography (e.g., a continuous
multi-column chromatography device). In some embodiments, the
device for bind and elute chromatography is connected in fluid
communication with a unit operation for performing flow-through
purification, which may include more than one device/step. In some
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.
[0218] In some embodiments, the flow-through purification process
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.
[0219] The last unit operations in the system may include one or
more devices for achieving formulation, which includes
diafiltration/concentration and sterile filtration.
[0220] 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.
[0221] 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 process, 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 the
flow-through purification process.
[0222] 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 unit operation.
[0223] 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.
[0224] 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.
[0225] In some embodiments, process control may be achieved in ways
which do not compromise the sterility of the system.
[0226] 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 computer) 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.
[0227] The disclosure set forth above may encompass multiple
distinct inventions with independent utility. Although each of
these inventions has been disclosed in its preferred form(s), the
specific embodiments thereof as disclosed and illustrated herein
are not to be considered in a limiting sense, because numerous
variations are possible. The subject matter of the inventions
includes all novel and nonobvious combinations and subcombinations
of the various elements, features, functions, and/or properties
disclosed herein. The following claims particularly point out
certain combinations and subcombinations regarded as novel and
nonobvious. Inventions embodied in other combinations and
subcombinations of features, functions, elements, and/or properties
may be claimed in applications claiming priority from this or a
related application. Such claims, whether directed to a different
invention or to the same invention, and whether broader, narrower,
equal, or different in scope to the original claims, also are
regarded as included within the subject matter of the inventions
taught herein.
* * * * *