U.S. patent application number 15/521428 was filed with the patent office on 2017-10-19 for mixed bed ion exchange adsorber.
The applicant listed for this patent is EMD Millipore Corporation. Invention is credited to John P. Amara, Matthew T. Stone.
Application Number | 20170298091 15/521428 |
Document ID | / |
Family ID | 54293387 |
Filed Date | 2017-10-19 |
United States Patent
Application |
20170298091 |
Kind Code |
A1 |
Stone; Matthew T. ; et
al. |
October 19, 2017 |
Mixed Bed Ion Exchange Adsorber
Abstract
The present invention refers to new species of an ion exchange
adsorber which is suitable for the separation of host cell proteins
(HCPs), antibody fragments and low molecular weight substances from
solutions containing antibodies. The invention especially refers to
a process for purifying biological samples by separating
biomolecules of interest and impurities, comprising steps of
contacting a sample with said chromatography media consisting of
fibers, said fibers having imparted thereon functionality enabling
ion exchange chromatography and/or hydrophobic interaction.
Inventors: |
Stone; Matthew T.;
(Billerica, MA) ; Amara; John P.; (Billerica,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
EMD Millipore Corporation |
Billerica |
MA |
US |
|
|
Family ID: |
54293387 |
Appl. No.: |
15/521428 |
Filed: |
September 30, 2015 |
PCT Filed: |
September 30, 2015 |
PCT NO: |
PCT/US2015/053140 |
371 Date: |
April 24, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62089030 |
Dec 8, 2014 |
|
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|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01D 2311/2626 20130101;
C07K 1/18 20130101; C07K 1/36 20130101; B01D 63/02 20130101; C07K
1/20 20130101; B01D 15/3804 20130101 |
International
Class: |
C07K 1/20 20060101
C07K001/20; B01D 63/02 20060101 B01D063/02; B01D 15/38 20060101
B01D015/38; C07K 1/36 20060101 C07K001/36; C07K 1/18 20060101
C07K001/18 |
Claims
1. A process for purifying a biological sample by separating a
biomolecule of interest and impurities, comprising the steps of
contacting the sample with a chromatography media consisting of
fibers, said fibers having imparted thereon functionality enabling
ion exchange chromatography and/or hydrophobic interaction and
steps of washing either to remove unbound species or to extract
said biomolecule of interest.
2. A process according to claim 1 for purifying a sample comprising
a biomolecule of interest and impurities, comprising the steps of:
a) providing a sample, b) contacting said sample with a first
chromatography media comprising fibers, said fibers having imparted
thereon functionality enabling ion-exchange chromatography or
hydrophobic interaction chromatography, c) washing said first fiber
media to remove unbound species, d) washing said first fiber media
to extract said biomolecule of interest, e) contacting said
biomolecule of interest with a second chromatography media
comprising fibers, said fibers having imparted thereon
functionality enabling ion-exchange chromatography or hydrophobic
interaction chromatography, f) washing said second fiber media to
remove unbound species, and g) washing said second fiber media to
extract said biomolecule of interest, but with the proviso that
differently functionalized fiber media are used in steps b) and
e).
3. A process according to claim 2, wherein in step b) said sample
is contacted with a first chromatography media comprising fibers,
said fibers having imparted functionality enabling anion-exchange
chromatography, and wherein in step e) chromatography media are
used comprising fibers having functionality enabling hydrophobic
interaction chromatography.
4. A process according to claim 2, wherein in step b) said sample
is contacted with a first chromatography media comprising fibers,
said fibers having imparted functionality enabling cation-exchange
chromatography, and wherein in step e) chromatography media are
used comprising fibers having functionality enabling hydrophobic
interaction chromatography.
5. A process according to claim 2, wherein in step b)
chromatography media are used comprising fibers having
functionality enabling hydrophobic interaction chromatography, and
wherein said ion-exchange chromatography in step e) is
anion-exchange chromatography.
6. A process according to claim 2, wherein in step b)
chromatography media are used comprising fibers having
functionality enabling hydrophobic interaction chromatography, and
wherein said ion-exchange chromatography in step e) is
cation-exchange chromatography.
7. The process of claim 2, wherein said first chromatography media
in step b) is anion-exchange chromatography, and wherein said
second chromatography media in step e) is cation-exchange
chromatography.
8. The process of claim 2, wherein said first chromatography media
in step b) is cation-exchange chromatography, and wherein said
second chromatography media in step e) is anion-exchange
chromatography.
9. A process for purifying a sample comprising a biomolecule of
interest and impurities, comprising the steps: a) providing a
sample, b) contacting said sample with a mixture of a first
chromatography media comprising fibers and a second chromatography
media comprising fibers, said first chromatography media comprising
fibers having imparted thereon functionality enabling ion exchange
chromatography, said second chromatography media comprising fibers
having imparted thereon functionality enabling ion exchange
chromatography, c) washing said mixture of chromatography media to
remove unbound species, and d) washing said mixture of
chromatography media to extract said biomolecule of interest.
10. The process of claim 9, wherein said first chromatography media
is anion-exchange chromatography and said second chromatography
media is cation-exchange chromatography or vice versa.
11. A process for purifying a sample comprising a biomolecule of
interest and impurities, comprising the steps: a) providing a
sample, b) contacting said sample with a mixture of a first
chromatography media comprising fibers and a second chromatography
media comprising fibers, said first chromatography media comprising
fibers having imparted thereon functionality enabling hydrophobic
interaction chromatography, said second chromatography media
comprising fibers having imparted thereon functionality enabling
ion exchange chromatography, c) washing said mixture of
chromatography media to remove unbound species, and d) washing said
mixture of chromatography media to extract said biomolecule of
interest.
12. The process of claim 11, wherein said second chromatography
media is cation-exchange chromatography or anion-exchange
chromatography.
13. A housing comprising a packed bed of fibers; said packed bed
having a first layer and a second layer, said first layer
comprising fibers having imparted thereon functionality enabling
ion-exchange chromatography, and said second layer comprising
fibers having imparted thereon functionality enabling hydrophobic
interaction chromatography.
14. The housing of claim 13, wherein said ion-exchange
chromatography is anion-exchange chromatography or cation-exchange
chromatography.
15. A housing comprising a packed bed of fibers; said packed bed
having a first layer and a second layer, said first layer
comprising fibers having imparted thereon functionality enabling
hydrophobic interaction chromatography, and said second layer
comprising fibers having imparted thereon functionality enabling
ion-exchange chromatography.
16. The housing of claim 15, wherein said ion-exchange
chromatography is anion-exchange chromatography or cation-exchange
chromatography.
17. A housing comprising a packed bed of fibers; said packed bed
having a mixture of a first chromatography media and a second
chromatography media, said first chromatography media comprising
fibers having imparted thereon functionality enabling hydrophobic
interaction chromatography, and said second chromatography media
comprising fibers having imparted thereon functionality enabling
ion-exchange chromatography.
18. The housing of claim 17, wherein said ion-exchange
chromatography is anion-exchange chromatography or cation-exchange
chromatography.
19. A process for purifying a sample comprising a biomolecule of
interest and impurities, comprising; providing a sample, contacting
said sample with a first chromatography media comprising fibers,
said first fibers having imparted thereon functionality enabling
ion-exchange chromatography and a second chromatography media
comprising fibers, said second fibers having imparted thereon
functionality enabling ion-exchange chromatography.
20. The process of claim 19, wherein said first chromatography
media are cation-exchange fibers and said second chromatography
media are anion-exchange fibers.
21. The process of claim 19, wherein said first chromatography
media are anion-exchange fibers and said second chromatography
media are cation-exchange fibers.
22. The process of claim 19, wherein said first functionality
enables purification in a flow-through mode and said second
functionality enables purification in a flow-through mode.
23. The process of claim 19, wherein said first functionality
enables purification in a bind/elute mode and said second
functionality enables purification in a bind/elute mode.
24. The process of claim 19, wherein said first functionality
enables purification in a flow-through mode and said second
functionality enables purification in a bind/elute mode.
25. A process for purifying a sample comprising a biomolecule of
interest and impurities, comprising; providing a sample, contacting
said sample with a first chromatography media comprising fibers,
said first fibers having imparted thereon functionality enabling
ion-exchange chromatography and a second chromatography media
comprising fibers, said second fibers having imparted thereon
functionality enabling hydrophobic interaction chromatography.
26. The process of claim 25, wherein said first chromatography
media are cation-exchange fibers and said second chromatography
media are hydrophobic interaction chromatography fibers.
27. The process of claim 25, wherein said first chromatography
media are anion-exchange fibers and said second chromatography
media are hydrophobic interaction chromatography fibers.
28. The process of claim 25, wherein said first functionality
enables purification in a flow-through mode and said second
functionality enables purification in a flow-through mode.
29. The process of claim 25, wherein said first functionality
enables purification in a bind/elute mode and said second
functionality enables purification in a bind/elute mode.
30. The process of claim 25, wherein said first functionality
enables purification in a flow-through mode and said second
functionality enables purification in a bind/elute mode.
31. The process of claim 19, wherein said first chromatography
media and said second chromatography media are arranged in a
mixture.
32. The process of claim 19, wherein said first chromatography
media and said second chromatography media are arranged in
layers.
33. The process of claim 25, wherein said first chromatography
media and said second chromatography media are arranged in a
mixture.
34. The process of claim 25, wherein said first chromatography
media and said second chromatography media are arranged in layers.
Description
[0001] The present invention refers to new species of an ion
exchange adsorber which is suitable for the separation of host cell
proteins (HCPs), antibody fragments and low molecular weight
substances from solutions containing antibodies. The invention
especially refers to a process for purifying biological samples by
separating biomolecules of interest and impurities, comprising
steps of contacting a sample with said chromatography media
consisting of fibers, said fibers having imparted thereon
functionality enabling ion exchange chromatography and/or
hydrophobic interaction.
BACKGROUND
Purification of Monoclonal Antibodies
[0002] Since monoclonal antibodies (mAbs) are used for
pharmaceutical applications, they are required in exceptionally
high purities [A. Jungbauer, G. Carta, in: Protein Chromatography,
Process Development and Scale-Up; WILEY-VCH Verlag, Weinheim
(Germany) 2010].
[0003] In general mammalian cell cultures are employed to
manufacture the majority of therapeutic monoclonal antibodies mAb)
currently on the market. Production of these drug antibodies
typically starts in a bioreactor that contains a suspension of
Chinese Hamster Ovary (CHO) cells which secrete the antibody into
the extracellular fluid. The resulting antibodies are then
subjected to a series of processes including clarification,
filtration, and purification that removes cells, cell debris, host
cell proteins (HCP), lipids, DNA, viruses, bacteria, antibody
aggregates, etc. This series of processes is often referred to as a
downstream process (DSP).
[0004] Most commonly employed DSP includes one or two bind-elute
chromatography purification steps followed by one or two
flow-through polishing steps (FIG. 1, standard mAb purification
scheme). Typical downstream purification processes employ packed
columns filled with porous bead-based chromatography media or
membrane-based devices. These unit operations are employed in
series and each are targeted towards clearing a particular impurity
in either a flow-through polishing or a bind/elute capture
mode.
[0005] One of the primary objectives of the polishing media is to
reduce the concentration of HCP down to <10 ppm (in reference to
mAb concentration). The commercial scale purification of various
therapeutic biomolecules is currently accomplished using bead-based
chromatography resins. Biopharmaceutical manufacturers most
commonly use simple anion-exchange (AEX) chromatography media in
this flow-through polishing step. The AEX media is employed to
remove acidic HCP, DNA, endotoxins, and viruses. However, it is
often less effective in removing positively charged impurities,
such as basic HCP, product aggregates and fragments.
[0006] Monoclonal antibodies continue to gain importance as
therapeutic and diagnostic agents. The process of screening
hybridoma libraries for candidate mABs is both time consuming and
labor intensive. Once a hybridoma cell line expressing a suitable
mAB is established, a purification methodology must be developed to
produce sufficient mAB for further characterization.
[0007] A traditional method for purifying involves using Protein A
or Protein G affinity chromatography, as well as ion exchange
chromatography. The purified antibody is desalted and exchanged
into a biological buffer using dialysis. The entire process
typically requires several days to complete and can be particularly
onerous if multiple mABs are to be evaluated in parallel.
[0008] Thus, a variety of new polishing adsorbers have been
developed recently, which show greater capacities and affinities
allowing them to remove a broader range of impurities. These
adsorbers include so-called "mixed-mode" ligands, both anion (AEX)
(e.g. U.S. Pat. No. 7,714,112) and cation exchange (CEX) materials
(e.g. U.S. Pat. No. 7,385,040). However, the higher cost of these
sophisticated ligands on resins precludes their employment for
single use or in disposable processes.
[0009] In general the applied bead-based adsorbers demonstrate a
high porosity and large surface areas that provide materials with
sufficient adsorptive capacities for the batch processing of
biomolecules at production scales (e.g., 10,000 liters). From
patent literature numerous examples are known of such bead-based
media used in mixed bed stationary phases for this technical
application.
[0010] In JP 01-10178 (Asahi Chem. Ind. CO LTD., also published as
JP 2660261B2) a multifunctional module is disclosed which comprises
a combination of AEX and CEX porous hollow fiber membranes for the
purpose of removing cations, anions and fine particles within a
single device.
[0011] Bio-Rad Laboratories, Inc. has developed a multi-media
affinity column (U.S. Pat. No. 8,053,565 B) comprising a layering
of an affinity chromatography media above a second type of
chromatography media in a single column so that the second lower
chromatography media will capture any leached affinity ligands from
the upper affinity chromatography media during the process of
eluting the affinity bound protein of interest.
[0012] Promega Corp. (U.S. Pat. No. 6,270,970 A) has developed a
mixed bed solid phase for the isolation of nucleic acids from an
impure mixture. Each of the comprising two phases has the capacity
to bind and release the target nucleic acid under different
solution conditions.
[0013] In a further patent application (WO 2005/011849 A), filed by
Millipore Corp. and Ebara Corp., an electro-deionization module is
disclosed, wherein the ion-exchange means is comprised of an
assembly of a fabric of anion exchange fibers and a fabric of
cation exchange fibers which are placed in a face to face
relationship.
[0014] Mixed-bed chromatography employs two or more different
adsorbent media that are combined together in a single device. It
allows a variety of different interactions applied to be used for
analysis and purification of protein solutions within a single
device. Rassi and Horvath demonstrated that a mixed-bed column
composed of AEX-resin and CEX resin gave similar separation of
proteins as two separate columns linked in series (el Rassi, Z.;
Harvath, C.; "Tandem columns and mixed-bed columns in
high-performance liquid chromatography of proteins"; J. Chrom.
1986, 359, 255-264). Using these systems, they were able to resolve
a mixture of several different proteins that would elute
simultaneously when only a single resin was employed.
[0015] Mixed-bed chromatography has been applied to several
different applications including the analysis of proteomes, which
are particularly difficult to analyze since they are composed of a
variety of both high-abundance and low-abundance proteins
(Boschetti, E.; Righetti, P. G.; "Mixed-bed chromatography as a way
to resolve peculiar fractionation situations", J. Chomatogr. B
2011, 897, 827-835.
[0016] Separation materials, like chromatography resins typically
present a spherical structure that enables an efficient column
packing with minimal flow non-uniformities. The interstitial spaces
between the beads provide flow channels for convective transport
through the chromatography column. This enables chromatography
columns to be run with large bed depths at a high linear velocity
with a minimal pressure drop. The combination of these factors
enables chromatography resins to present the required efficiency,
high permeability, and sufficient binding capacity that are
required for the large-scale purification of biomolecules.
[0017] In bead-based chromatography, most of the available surface
area for adsorption is internal to the bead. Consequently, the
separation process is inherently slow since the rate of mass
transport is typically controlled by pore diffusion. To minimize
this diffusional resistance and concomitantly maximize dynamic
binding capacity, small diameter beads can be employed. However,
the use of small diameter beads comes at the price of increased
column pressure drop. Consequently, the optimization of preparative
chromatographic separations often involves a compromise between
efficiency/dynamic capacity (small beads favored) and column
pressure drop (large beads favored).
[0018] Chromatography media typically has a very high cost
(>$1000/L) and significant quantities are required for large
scale production columns. As a result, biopharmaceutical
manufacturers recycle chromatography resins hundreds of times. Each
of these regeneration cycles consumes substantial quantities of
media, and each step incurs additional costs associated with the
validation of each cleaning, sterilization, and column packing
operation.
[0019] As indicated above, several technologies are described in
the patent literature and marketed commercially for
biopharmaceutical separations based on functionalized fibrous media
and/or composites. Most rely on incorporating a porous gel into the
fiber matrix, the gel providing the needed surface area to gain
reasonable binding capacities. However, in such constructions, poor
uniformity in gel location and mass generally leads to poor
efficiencies (shallow breakthrough and elution fronts). In
addition, resistance to flow can be high, even for short bed
depths, a problem often aggravated by gel compression under modest
pressure loads.
[0020] Another approach taken has been the incorporation of
particulates within the fiber matrix, the particulates often are
porous and possessing a native adsorptive functionality; examples
being activated carbon and silica gel.
Object
[0021] The current downstream process (DSP) is complex and
expensive. This is why the industry is interested in the
development of new technologies to compress, simplify, and reduce
the costs of these processes. Therefore, it is an objective to
develop inexpensive disposable adsorbent media that reduces
manufacturing costs by eliminating the time and buffers required to
clean and to store the column after use. It is also an objective to
increase the performance of the adsorbent media by targeting a
wider variety of impurities.
[0022] In addition, it is desirable to have a customizable
composition of ligand chemistries so that the adsorbent media can
be specifically tuned to target the particular mixture of
impurities present in a given feed. This is challenging to
accomplish with resin- and membrane-based adsorbent media.
[0023] It is also desirable to provide a combination of a high
surface area fiber with pendant adsorptive functionality for
biomolecule chromatography applications, without sacrificing bed
permeability and attainable flow rates.
[0024] In addition to this, it is also an object of the present
invention to provide an inexpensive, manufacturable and chemically
defined carrier material that can be derivatized by conventional
procedures so that it can be used both as ion exchanger material or
as a hydrophobic separator material.
SUMMARY OF THE INVENTION
[0025] The shortcomings of the prior art have been addressed by the
embodiments disclosed herein, which relate to an adsorptive media
for chromatography, particularly ion exchange chromatography and
especially a corresponding process for purifying biological samples
using this media.
[0026] The process for purifying a biological sample disclosed here
by separating a biomolecule of interest and impurities comprises
the steps of contacting the sample with a chromatography media
consisting of fibers, said fibers having imparted thereon
functionality enabling ion exchange chromatography and/or
hydrophobic interaction and comprises steps of washing either to
remove unbound species or to extract said biomolecule of
interest.
[0027] In detail, a process for purifying a sample comprising a
biomolecule of interest and impurities according to the present
invention in general comprises the steps of [0028] a) providing a
sample, [0029] b) contacting said sample with a first
chromatography media comprising fibers, said fibers having imparted
thereon functionality enabling ion-exchange chromatography or
hydrophobic interaction chromatography, [0030] c) washing said
first fiber media to remove unbound species, [0031] d) washing said
first fiber media to extract said biomolecule of interest, [0032]
e) contacting said biomolecule of interest with a second
chromatography media comprising fibers, said fibers having imparted
thereon functionality enabling ion-exchange chromatography or
hydrophobic interaction chromatography, [0033] f) washing said
second fiber media to remove unbound species, and [0034] g) washing
said second fiber media to extract said biomolecule of interest,
but with the proviso that differently functionalized fiber media
are used in steps b) and e).
[0035] In step b) the applied sample is contacted with a first
chromatography media comprising fibers, said fibers having imparted
functionality enabling anion-exchange chromatography, and wherein
in step e) chromatography media are used comprising fibers having
functionality enabling hydrophobic interaction chromatography.
However this process can also be carried out in a modified form,
wherein in step b) said sample is contacted with a first
chromatography media comprising fibers, said fibers having imparted
functionality enabling cation-exchange chromatography, and wherein
in step e) chromatography media are used comprising fibers having
functionality enabling hydrophobic interaction chromatography. In
another embodiment of this process in step b) chromatography media
are used comprising fibers having functionality enabling
hydrophobic interaction chromatography, and in step e) said
ion-exchange chromatography is anion-exchange chromatography or
cation-exchange chromatography. But it is also possible, depending
on the nature of applied biological sample that in step b)
chromatography media are used comprising fibers having
functionality enabling anion-exchange chromatography and that said
second chromatography media in step e) is cation-exchange
chromatography or vice versa.
[0036] In another embodiment of the inventive process steps are as
follows: [0037] a) providing a sample, [0038] b) contacting said
sample with a mixture of a first chromatography media comprising
fibers and a second chromatography media comprising fibers, said
first chromatography media comprising fibers having imparted
thereon functionality enabling ion exchange chromatography, said
second chromatography media comprising fibers having imparted
thereon functionality enabling ion exchange chromatography, [0039]
c) washing said mixture of chromatography media to remove unbound
species, and [0040] d) washing said mixture of chromatography media
to extract said biomolecule of interest. Here said first
chromatography media is anion-exchange chromatography and said
second chromatography media is cation-exchange chromatography or
vice versa.
[0041] Another variant of the process of the invention is [0042] a)
providing a sample, [0043] b) contacting said sample with a mixture
of a first chromatography media comprising fibers and a second
chromatography media comprising fibers, said first chromatography
media comprising fibers having imparted thereon functionality
enabling hydrophobic interaction chromatography, said second
chromatography media comprising fibers having imparted thereon
functionality enabling ion exchange chromatography, [0044] c)
washing said mixture of chromatography media to remove unbound
species, and [0045] d) washing said mixture of chromatography media
to extract said biomolecule of interest.
[0046] The second chromatography media comprising fibers having
imparted thereon functionality enabling ion exchange chromatography
may be either cation-exchange chromatography or anion-exchange
chromatography.
[0047] The present invention also relates to a housing comprising a
packed bed of fibers; said packed bed having a first layer and a
second layer, said first layer comprising fibers having imparted
thereon functionality enabling ion-exchange chromatography, and
said second layer comprising fibers having imparted thereon
functionality enabling hydrophobic interaction chromatography. Said
ion-exchange chromatography may be anion-exchange chromatography or
cation-exchange chromatography. In a special embodiment of the
housing according to the invention it comprises a packed bed of
fibers; said packed bed having a first layer and a second layer,
said first layer comprising fibers having imparted thereon
functionality enabling hydrophobic interaction chromatography, and
said second layer comprising fibers having imparted thereon
functionality enabling ion-exchange chromatography. Said
ion-exchange chromatography may be anion-exchange chromatography or
cation-exchange chromatography. In another embodiment of the
invention the housing comprises a packed bed of fibers; said packed
bed having a mixture of a first chromatography media and a second
chromatography media, said first chromatography media comprising
fibers having imparted thereon functionality enabling hydrophobic
interaction chromatography, and said second chromatography media
comprising fibers having imparted thereon functionality enabling
ion-exchange chromatography. The comprising ion-exchange
chromatography may be anion-exchange chromatography or
cation-exchange chromatography.
[0048] The inventive process for purifying a sample comprising a
biomolecule of interest and impurities may also be carried out by
contacting a sample, with a first chromatography media comprising
fibers, said first fibers having imparted thereon functionality
enabling ion-exchange chromatography and contacting it with a
second chromatography media comprising fibers, said second fibers
also having imparted thereon functionality enabling ion-exchange
chromatography. Preferably the first chromatography media are
cation-exchange fibers and said second chromatography media are
anion-exchange fibers. In another preferred embodiment of the
process said first chromatography media are anion-exchange fibers
and said second chromatography media are cation-exchange fibers.
The chromatography media in this process is selected so that said
first functionality enables purification in a flow-through mode and
said second functionality enables purification in a flow-through
mode. In another embodiment of the inventive process it is selected
so that said first functionality enables purification in a
bind/elute mode and said second functionality enables purification
in a bind/elute mode. If it proves to be advantageous for the
purification, the process can however also be modified such that
said first functionality enables purification in a flow-through
mode and said second functionality enables purification in a
bind/elute mode.
[0049] Furthermore, the inventive process for purifying a sample
comprising a biomolecule of interest and impurities may also be
carried out by contacting a sample, with a first chromatography
media comprising fibers, said first fibers having imparted thereon
functionality enabling ion-exchange chromatography and a second
chromatography media comprising fibers, said second fibers having
imparted thereon functionality enabling hydrophobic interaction
chromatography. In this embodiment of the inventive process,
preferably the first chromatography media are cation-exchange
fibers and said second chromatography media are hydrophobic
interaction chromatography fibers. Otherwise, if the biomolecule of
interest it requires said first chromatography media are
anion-exchange fibers and said second chromatography media are
hydrophobic interaction chromatography fibers. Good purification
results are achieved if said first functionality but also said
second functionality enable purification in a flow-through mode. If
necessary and advantageous the chromatography media may be chosen,
wherein said first functionality enables purification in a
bind/elute mode and said second functionality enables purification
in a bind/elute mode. In another embodiment of the inventive
process chromatography media may be chosen such that the first
functionality enables purification in a flow-through mode and said
second functionality enables purification in a bind/elute mode.
Furthermore, in a special embodiment of the inventive process said
first chromatography media and said second chromatography media is
arranged in a mixture of variously functionalized fibers, or said
first chromatography media and said second chromatography media are
arranged in layers. Corresponding embodiments of layered
chromatography media are described in more detail below.
DETAILED DESCRIPTION OF THE INVENTION
[0050] The chromatography media disclosed is derived from a shaped
or porous fiber having high surface areas. In certain embodiments,
the shaped fiber presents a fibrillated or ridged structure. An
example of the high surface area fiber in accordance with certain
embodiments is "winged" fibers, commercially available from Allasso
Industries, Inc. (Raleigh, N.C.). Suitable fibers, which may be
winged or highly porous, present a surface area of approximately 1
to 14 square meters per gram.
[0051] Also disclosed herein, fibrous materials are derivatized in
a method adding surface pendant functional groups that provide
cation-exchange or anion-exchange functionalities, for example, to
the high surface area fibers. This pendant functionality is useful
for the ion-exchange chromatographic purification of biomolecules,
such as monoclonal antibodies (mAbs).
[0052] Chromatographic exchanger materials disclosed here comprise
nonwoven polymer fibers with high surface areas at least in the
range of 1-14 m.sup.2/g, which in turn comprise functional groups
at their surfaces, namely at least anion exchanging groups, cation
exchanging groups or groups with hydrophobic interaction
functionalities, and wherein the functional groups may be mixed or
as such be attached to the surface of the same fiber and whereby
fibers differently functionalized may be combined or mixed with
each other. Depending on the performed functionalization the
material of the invention is an ion exchange material which is
either an anion exchanger or a cation exchange adsorber. In another
embodiment of the invention the functionalized chromatographic
exchanger material may comprise hydrophobic interaction
functionalities either on the surfaces of the same fibers which are
already functionalized by ionic groups or on the surfaces of
separate fibers.
[0053] This means chromatographic exchanger materials of the
present invention may comprise fibers having different
functionalities or may comprise a mixture of fibers having
different functionalities. In a preferred embodiment the materials
of the present invention are blended together to present an evenly
distributed mixture of the different functional groups within the
chromatographic media. The composition of the chromatographic media
can be controlled by altering the relative amounts of the two
different types of fiber for a specific application. For instance,
a chromatographic media composed of 50% anion exchanger fibers (AEX
fibers) and 50% cation exchanger fibers (CEX fibers) could be
created for a specific application. In another instance, a
chromatographic media composed of the 25% AEX fibers and 75% CEX
fibers could be created to address the specific separation needs of
a different application.
[0054] In an alternative embodiment the materials of the present
invention have a layered structure, wherein the different layers
can be made of the same fibers or wherein each layer may have a
different functionality. Thus, layers may follow each other having
different functionalities. These layers may follow each other in a
special sequence, optionally for several times or layers with the
same functionality are repeated in direct succession for several
times to generate a desired layer thickness.
[0055] Fibers forming the chromatographic exchanger materials are
fibers with high surface areas in the range of 1-14 m.sup.2/g,
which are based either on lightweight winged fibers having eight to
32 deep channels or on highly porous fibers. The winged fibers may
be made by coextrusion forming the core features from eight to 32
deep channels between uniform, straight-edged wings. Highly porous
fibers can also be used as chromatographic exchanger materials. In
general, fibers having a length ranging from 0.5 mm to 5 cm are
used for the inventive chromatographic exchanger materials.
Preferably chromatographic exchanger materials according to the
invention are composed of fibers having a length ranging from 0.5
mm to 2.5 cm. Especially preferred are chromatographic exchanger
materials comprising fibers having a length ranging from 0.5 mm to
2 mm.
[0056] It has been found as being advantageous if the
chromatographic exchanger materials comprise fibers made of a
polymer selected from the group polystyrene, polycarbonate,
poly(ethyleneoxide), polyester, polypropylene, methyl,
methacrylate, (hydroxyethylmethacrylate,
poly(propyleneglycol)monomethacrylate, (phenyl)methacrylate,
((n-butyl)methacrylate, (n-hexyl)methacrylate, polybutylene
terephthalate (PBT), polyethylene terephthalate (PET), and
polyamide.
[0057] By means of the chromatographic exchanger materials
disclosed here new devices can be produced having significantly
improved properties in separation and purification processes of
biological fluids. Corresponding devices are therefore also subject
of the present invention. By experiments it was found, that devices
are particularly suitable comprising chromatographic exchanger
materials, wherein the fibers are made of polyamide, polybutylene
terephthalate (PBT), polyethylene terephthalate (PET) or
polypropylene (PP), especially fibers, which are derivatized with
acrylic monomers. The acrylic monomers may be selected from the
group 2-hydroxylethyl methacrylate (HEMA), acrylamide, acrylic
acid, acrylonitrile, methyl methacrylate and glycidyl methacrylate
(GMA). These acrylic monomers can be used for the derivatization as
such or in a combination thereof. Especially preferred are
corresponding devices wherein the fibers are derivatized with
glycidyl methacrylate (GMA) or with a combination of
2-hydroxylethyl methacrylate (HEMA) and glycidyl methacrylate
(GMA). According to the present invention these devices are
preferably made with such derivatized fibers which in turn are
modified by reaction with a compound comprising a functional group
or with ligands. Said functional groups are selected from amino,
carboxyl, hydroxyl, epoxy, sulfopropyl, sulfonic acid, and
sulfhydryl groups.
[0058] Since the new devices may be produced using quite
inexpensive fibers and processes, they can be made as disposable
devices.
[0059] The object of the invention is also a method for separating
a target agent from a biological sample, which comprises the steps
of: [0060] a) providing the sample containing the target agent;
[0061] b) contacting the sample with the chromatographic exchanger
material which is disclosed here; and [0062] c) allowing the target
agent to bind to the high-surface area fibers and thereby be
separated from the sample. In a following step (d) the sample
resulting from step (c) may be collected, or the sample resulting
from step (c) is retrieved in step (d) and in the next step (e) the
target agent bound to the nonwoven fibrous material is collected by
eluting through the nonwoven material an elution solution
interfering with the binding between the target agent and the
fibers so as detach the target agent from the fibers. In a special
embodiment of this method the nonwoven material is condensed into a
sheet or filled and compressed into a column.
[0063] This method is especially suitable for separation processes
wherein target agents are proteins, peptides, lipids, DNA molecule,
RNA molecule, an organic molecule, an inorganic molecule, cells,
viruses, bacteria, toxins or a prion.
[0064] The chromatography media as described above may be derived
from a shaped fiber. It has been found, that in certain
embodiments, the shaped fiber presents a fibrillated or ridged
structure. These ridges can greatly increase the surface area of
the fibers when compared to ordinary fibers. Thus, high surface
area is obtained without reducing fiber diameter, which typically
results in a significant decrease in bed permeability and a
corresponding reduction in flow rate.
[0065] An example of the high surface area fiber in accordance with
certain embodiments are "winged" fibers, commercially available
from Allasso Industries, Inc. (Raleigh, N.C.; Allasso Winged
Fiber.TM.). These fibers are made of Nylon and very lightweight and
have the same total surface area in the range of 1 to 14 square
meters per gram. These fibers comprise a shaped core polymer and a
sacrificial polymer that are coextruded through a specially
designed spinpack. The core features from eight to 32 deep channels
between uniform, straight-edged wings. The sheath polymer fills the
channels during fiber formation and is dissolved during finishing
of the final product.
[0066] As already mentioned earlier highly porous fibers are also
applicable in the present invention.
[0067] Now, it has been found, that chromatographic purification of
biomolecules, such as monoclonal antibodies (mAbs) can be processed
under simplified conditions but with improved results when a mixed
functionality of this fiber material is utilized, which includes
both ionic and hydrophobic interactions.
[0068] Thus, a method is developed wherein surface pendent
functional groups are added that provide CEX, AEX, or hydrophobic
interaction functionalities, for example, to the high surface area
fibers.
[0069] For the production of suitable functionalized high surface
area fibers not only Allasso Winged Fibers made from nylon are
applicable but also other fibers showing high surface areas in the
range of at least 1 to 14 square meters per gram may be used. For
example highly porous fibers may also be applied. Such fibers are
disclosed by S. Megelski et al. in Macromolecules 2002, 35,
8456-8466 and are spun from polystyrene, polycarbonate or
poly(ethyleneoxide). But also corresponding fibers of polyester,
polypropylene (PP), methyl methacrylate, (hydroxyethylmethacrylate,
poly(propyleneglycol)monomethacrylate, (phenyl)methacrylate,
((n-butyl)methacrylate, (n-hexyl)methacrylate), polybutylene
terephthalate (PBT), polyethylene terephthalate (PET) or polyamide
can be used for this purpose. A preferred group of fibers is made
of polyamide, polypropylene, polybutylene terephthalate (PBT) or
polyethylene terephthalate (PET).
[0070] By using these modified high surface area fibers an
alternative mode of purification may be provided, which utilizes
fiber based chromatography media with pendent AEX and CEX ligands.
In one embodiment of this invention, both the AEX fiber and the CEX
fiber media are blended and packed into a single device. This
format is advantageous in terms of its operational simplicity and
the potential for process template compression by this
approach.
[0071] In another embodiment, advantageous properties have been
found for the design of a fiber media for hydrophobic interaction
chromatography. Fiber media of this type are useful for a so-called
"tandem chromatography", where only one chromatography column is
provided and two different types of fiber chromatography media are
arranged in discrete layers within this chromatography column.
[0072] This special arrangement is suitable for an application
where a monoclonal antibody feed stream is purified by bind/elute
purification using a CEX fiber media arranged in a first layer,
followed by elution and a subsequent purification by hydrophobic
interaction chromatography with the described HIC fiber media
arranged in a second layer within the same chromatography column or
other suitable devices.
[0073] The combination of mixed beds of fiber-based ion-exchangers
of the type as described here is found to have different unexpected
advantages. Especially, fiber media with varying ion exchange or
hydrophobic interaction chromatography ligand functionalities can
be easily arranged into layered structures, whereas with bead based
systems this format cannot be easily achieved.
[0074] Mixed-bed chromatography is a particular powerful method for
the purification therapeutic proteins derived from recombinant
cells since these sources contain a wide variety of host cell
protein (HCP) impurities. These protein impurities are particularly
challenging to remove at low concentrations when using only a
single type of chromatography media. One approach is to arrange
several different adsorbent media in a series of columns. However,
this will significantly increase the dead volume and complexity of
the downstream process. Combining the different adsorbent media
into single device allows the different impurities to be removed in
a single step.
[0075] Unexpectedly a mixed-bed chromatography device composed of
AEX functionalized fiber media and CEX functionalized fiber media
is highly efficient for the flow-through removal of residual HCP
from an antibody elution. The mixed fiber bed allows both positive
and negatively charged HCP to be removed. The relative low cost of
functionalized fibers allows them to be used as a single-use,
disposable application.
[0076] According to the present invention the layering of the
surface-modified chromatography media into discrete bands within a
housing, a single chromatography column or other suitable
chromatography device leads to improved separation and purification
results. The "layering" is accomplished by first applying a media
to the column and compressing the media to a higher density to form
the lower layer of media. A second layer can be subsequently
applied to the column and the media compressed to a higher density
to form the upper layer. Finally, the layered media can be
compressed to a third, highest density by installation of the upper
flow distribution header on the chromatography column or other
suitable chromatography device. It is possible to provide a single
column comprising a layered column comprising two types of fiber
based chromatography media with different ligands, a CEX ligand
(for example as disclosed in Example 3) and a hydrophobic
interaction chromatography ligand (for example as disclosed in
Example 6), which provides two orthogonal modes of separation. In
FIG. 2 a layered media column is disclosed for "tandem
chromatography" that provides a first layer of CEX fiber media that
is situated on top of a second layer of a HIC fiber media. This
format can be utilized for example in the purification of a
monoclonal antibody process stream by first applying the
post-protein A elution pool (mAb feed) onto the top of the layered
column.
[0077] In one embodiment, the mAb feed is at a low conductivity
(typically 3 mS/cm) and has a pH of 5 after adjustment with
appropriate buffer system (FIG. 2). This mAb feed solution is
loaded onto the column and first encounters the CEX fiber media
layer, where the mAb binds to the fiber media by an ion exchange
interaction. The fiber media is washed with an appropriate buffer
to clear any unbound HCP, DNA or other impurities. After washing,
an elution buffer of a high conductivity (30-1000) mS/cm, pH 5) is
applied to the column to elute the mAb from the CEX layer. As the
eluted mAb travels into the zone of the column occupied by the HIC
fiber media, the mAb binds to the HIC media by a hydrophobic
interaction (due to the high ionic strength of the buffer used for
elution from CEX media layer). The fiber media is again washed with
an appropriate high ionic strength buffer to clear any unbound HCP
or other impurities from the HIC fiber media portion of the column.
Finally, an elution buffer of a low ionic strength (3 mS/cm, pH 5)
is applied to the column to elute the mAb from the HIC layer. The
isolated mAb product after this step has a low conductivity and
requires only a minor pH adjustment for subsequent AEX-based
polishing operations.
[0078] FIG. 2 shows the schematic flow of the separation process
described above, which is an example of mAb purification by tandem
chromatography (CEX media and HIC) including the steps: [0079] i.
post-protein A mAb load (conductivity 3 mS, pH 5) [0080] ii. high
salt mAb elution (conductivity 30-1000 mS, pH 5), where the product
mAb is eluted from the upper CEX media layer but binds to the lower
HIC media layer. [0081] iii. low salt mAb elution (conductivity 3
mS, pH 5), where the product mAb is eluted from the lower HIC media
layer by elution with a low ionic strength eluent.
[0082] [Now, the eluted mAb is ready for a subsequent AEX polishing
operation without a need for dilution to reduce the solution
conductivity.
[0083] As mentioned above, the solution described here provides an
inexpensive separation process for mAB, comprising a disposable
format and which may be carried out at low material costs. In
addition to this, different process steps, which had to be carried
out in different columns and filtering devices under repeated
reduction of the amount of liquid and adjusting the pH value and
the conductivity, are compressed to a procedure carried out in a
single column wherein a convective transport of substrate to
binding sites takes place.
[0084] The surface functionalization of the high surface area
fibers can also be accomplished by a two step process. A suitable
functionalization process is grafting polymerization as disclosed
in WO 2012/015908 A. This functionalization begins with the
attachment of pendant allyl groups to the fiber surface, for
example to the surface of winged fibers made of Nylon6, a
polyamide. Here the fibers are treated with allyl glycidyl ether in
the presence of aqueous sodium hydroxide at 50.degree. for 12
hours. This first step of the functionalization of the fiber
surface can be carried out as disclosed in the aforementioned
patent application or under changed conditions by different
suitable monomers, like epichlorohydrin, or (meth)acrylic glycidyl
esters. [0058] The pendant allyl groups serve then as anchoring
sites on the fiber surface as attachment points for the pendant
acrylamide polymer functionality. Conditions for the solution
polymerization of acrylamide monomers are provided, and the pendant
allyl groups on the fiber surface attach to the growing polymer
chains in solution. Thus, the allyl-functionalized fibers may be
subsequently treated with an aqueous solution of
2-acrylimido-2-methyl-1-propane sulfonic acid,
N,N-dimethylacrylamide and ammonium persulfate at about 80.degree.
C. for about 4 hours. Upon heating to this temperature, persulfate
decomposition initiates a free radical polymerization of the
acrylic monomers. In this reaction a cation exchanger is received,
which comprises sulfonic acid groups. Under these conditions, the
pendant allyl groups on the fiber surface may serve as attachment
points for the pendant acrylic polymer functionality and the
acrylic polymer is covalently attached to the fiber surface.
[0085] Ceric ion redox grafting polymerizations may also be
employed for the surface modification of the high surface area
fibers. Under these conditions, the acid may be HNO.sub.3 and
Ce(VI) ions are provided by a salt like (ammonium cerium(IV)
nitrate). In this case the reaction time is much shorter and the
temperature lower.
[0086] In general the ceric ion redox grafting reaction is
processed according to Mino and Kaizerman [Mino, G., Kaizerman, S.
J. Polymer Science 31, 242-243 (1958) and J. Polymer Science 38,
393-401 (1959)], which is done in an aqueous nitric acid solution.
This reaction can be carried out with monomers, which are soluble
in aqueous solutions. If not water-soluble monomers are to be used,
the solubility can be improved by suitable solubilizers, such as
dioxane or tetrahydrofuran.
[0087] In subsequent reaction step, the surface functionalized
fibers, for example modified with poly(glycidyl methacrylate), can
be converted into an anion exchanger by simply mixing the
functionalized fibers with a solution of 50 wt % trimethylamine
(aq.) in methanol.
[0088] By appropriately adapted grafting reactions and by use of
appropriate reactants, it is possible to covalently bind polymer
chains, which are not cross-linked and which carry a variety of
functional groups, to the surfaces of the applied fibers.
[0089] With the term "functional group" are subsumed terms, such as
active, hydrolyzable, hydratable, hydrogen bond formation-causing
ionogenic (ionizable) charge-carrying (cationic or anionic) group.
Examples for such groups are --OH and/or --CO-- and/or --NH and/or
--SO.sub.3.sup.- and/or --SO.sub.4.sup.- and/or --PO.sub.4.sup.-
and/or --SO.sub.2Cl and/or --NH.sub.4.sup.+ and/or --CONH and/or
--CHO and/or --COOH and/or --COO.sup.- and/or --SH.
[0090] Thus, suitable functional groups for the anion exchange
chromatography are for example quaternary ammonium groups, like
quaternary hydroxypropyldiethylaminoethyl-, quaternary
trimethylaminoethyl-, or diethylaminoethyl groups. Depending on the
degree of ionic dissociation of the functional groups the anion
exchangers can be classified as strong, medium or weak base anion
exchanger.
[0091] In another embodiment of the invention the applied fibers
are functionalized as cation exchangers with acidic groups.
Suitable functional groups for the cation exchange chromatography
are for example sulfomethyl-, sulfopropyl-, or carboxymethyl
groups.
[0092] Groups suitable for hydrophobic interaction chromatography
are for example alkyl and aryl ligands like ether and methyl
ligands, which provide weak interactions with proteins, or butyl or
octyl ligands, phenyl, or other aryl ligands which show more
intense interactions.
[0093] Substitution of at least one hydrogen atom with a functional
group can indirectly be made via a methylene, ethylene, propylene
or butylenes bridge or a corresponding alkoxy or aryl.
[0094] As mentioned above, the functional groups can be bound to
the surface of the applied fibers by means of suitable graft
polymers.
[0095] In general, the graft-polymerization is carried out
according to known methods in presence of suitable initiators,
which may be redox initiators such as ceric (IV) ion (ceric
ammonium nitrate: (NH.sub.4).sub.2Ce(NO.sub.3).sub.6), cerium (IV)
sulphate, ceric ammonium sulfate, iron(II)-hydrogen peroxide
(Fe.sup.2+--H.sub.2O.sub.2: Fenton reagent), cobalt (III)
acetylacetonate complex salts, Co (II)-potassium monopersulfate,
sodium sulfite-ammonium persulfate or free radical generators such
as azobisisobutyronitrile (C.sub.8H.sub.12N.sub.4: AIBN), potassium
persulfate (K.sub.2S.sub.2O.sub.8: KPS), ammonium persulfate
((NH.sub.4).sub.2S.sub.2O.sub.8: APS), and benzoyl peroxide
(C.sub.14H.sub.10O.sub.4: BPO).
[0096] For example, graft-polymerization can be processed using
acidic monomers like acrylic acid and methacrylic acid of a
suitable ceric (IV) salt. If such monomeric acids are combined,
even in a relatively low ratio, with neutral monomers such as
methyl methacrylate, methyl acrylate and acrylonitrile grafting on
a fiber takes place quite readily. The resulting graft copolymer is
a weak acidic, high-capacity, cation exchanger.
[0097] Depending on the chemical nature of the fibers employed and
the monomers used for derivatization different reactions and
mechanisms can occur and possible approaches for appropriate
surface modifications are:
[0098] atom transfer radical polymerization (ATRP)
[0099] UV-initiated free radical polymerization
[0100] thermally-initiated free radical polymerization
[0101] anionic polymerization
[0102] cationic polymerization
[0103] gamma-initiated free radical polymerization
[0104] transition metal catalyzed polymerization
[0105] reversible-addition fragmentation transfer polymerization
(RAFT)
[0106] There are various suitable monomers for carrying out the
graft-polymerization. Depending on the desired properties of the
produced material different monomers may be applied.
[0107] In order to introduce positive charges into the graft
tentacles monomers from the following group can be selected: [0108]
2-(acryloylaminoethyl)trimethylammonium chloride, [0109]
3-(acryloylamino-propyl)trimethylammonium chloride, [0110]
2-(diethylaminoethyl)acrylamide, [0111]
2-(diethylaminoethyl)methacrylamide, [0112]
2-(dimethylaminoethyl)acryl-amide, [0113]
2-(dimethylaminoethyl)methacrylamide, [0114]
3-(diethylaminopropyl)-acrylamide, [0115]
3-(diethylaminopropyl)methacrylamide, [0116]
3-(diethylamino-propyl)acrylamide, [0117]
3-(diethylaminopropyl)methacrylamide, [0118]
2-(meth-acryloylaminoethyl)trimethylammonium chloride, [0119]
3-(acryloylamino-propyl)trimethylammonium chloride, [0120]
N-(3-aminopropyl)methacrylamide hydrochloride, [0121]
[3-(Methacryloylamino)propyl]dimethyl(3-sulfopropyl)ammonium
hydroxide inner salt, [0122] 2-(dimethylamino)ethyl methacrylate,
[0123] 2-(diethylamino)ethyl methacrylate, [0124] 2-aminoethyl
methacrylate hydrochloride, [0125] 2-(diisopropylamino)ethyl
methacrylate, and [0126] 2-(tert-butylamino)ethyl methacrylate,
[0127] By using monomers selected from the following group negative
charges can be generated in the tentacles: [0128]
2-acrylamido-2-methylpropane-sulfonic acid, [0129]
2-Acrylamido-2-methyl-1-propanesulfonic acid sodium salt solution,
[0130] 2-acrylamidoethanesulfonic acid, [0131]
carboxymethylacryl-amide; carboxyethylacrylamide, [0132]
carboxypropylacrylamide, [0133] carboxy-methlymethacrylamide,
[0134] carboxyethylmethacrylamide, [0135]
carboxypropyl-methacrylamide, [0136] acrylic acid, [0137]
methacrylic acid, and [0138] 3-Sulfopropyl methacrylate potassium
salt.
[0139] By use of monomers selected from the following group on the
other hand hydrophobic groups are introduced into the generated
tentacles: [0140] N-benzyl-2-methylacrylamide, [0141]
N-isopropylmethacrylamide, [0142] N,N-dimethylmethacrylamide,
[0143] N,N-diethylmethacrylamide, [0144] methyl methacrylate,
[0145] ethyl methacrylate, [0146] hydroxyethyl methacrylate, [0147]
propyl methacrylate, [0148] n-butyl methacrylate, [0149] isobutyl
methacrylate, [0150] sec-butyl methacrylate, [0151] tert-butyl
methacrylate, [0152] hexyl methacrylate, [0153] lauryl
methacrylate, [0154] isobornyl methacrylate, [0155] benzyl
methacrylate, [0156] 1-naphthyl methacrylate, [0157] 2-naphthyl
methacrylate, [0158] 2-ethylhexyl methacrylate, [0159] cyclohexyl
methacrylate, [0160] 3,3,5-trimethylcyclohexyl methacrylate, [0161]
ferrocenylmethyl methacrylate, and [0162] phenyl methacrylate
[0163] The skilled person knows more suitable chemical compounds
other than those listed here and that can serve as monomers in this
context and which are suitable corresponding to bring positive or
negative charges into the produced polymer chains.
[0164] Suitable fibers for the production of separation materials
according to the present invention may be of any length and
diameter and are preferably cut or staple fibers or a non-woven
fabric. They need not be bonded together as an integrated structure
but can serve effectively as individual discrete entities. They may
be in the form of a continuous length such as thread or
monofilament of indeterminate length or they may be formed into
shorter individual fibers such as by chopping fibrous materials
(e.g., staple fibers) or as non-woven or woven fabrics, cutting the
continuous length fiber into individual pieces, formed by a
crystalline growth method and the like. Preferably the fibers are
made of a thermoplastic polymer, such as polypropylene, polyester,
polyethylene, polyamide, thermoplastic urethanes, polystyrenes,
co-polyesters, or liquid crystalline polymers.
[0165] Fibers which are preferably used for the purpose described
here, having a length which is much greater than the widest
dimensions of the fiber cross-section, and which do not form a
compact body. Preferably these fibers have a length in the range of
some millimeters to several centimeters. For the purpose of the
present invention, the fibers used have a length ranging from 0.5
mm to 5 cm, more suitable are corresponding fibers having a length
ranging from 0.5 mm to 2.5 cm. Particularly well suited are fibers
having a length in the range from about 0.5 mm to 2 mm.
[0166] In certain embodiments, the fiber has a cross-sectional
length of from about 1 .mu.m to about 100 .mu.m and a
cross-sectional width of from about 1 .mu.m to about 100 .mu.m. One
suitable fiber has a cross-sectional length of about 20 .mu.m and a
cross-sectional width of about 10 .mu.m. Preferably the fibers have
a cross-sectional length of about 10-20 .mu.m.
[0167] In certain embodiments, the fiber cross-section is generally
winged-shaped, with a main body region defining a substantially
longitudinal axis, and a plurality of projections extending
radially outwardly from the main body region. The projections form
an array of co-linear channels that extend along the length of the
fiber, typically 20-30 such channels per fiber. In certain
embodiments, the length of the projections is shorter than the
length of the main body region. In certain embodiments, the fiber
cross-section is generally winged-shaped, with a middle region
comprising a longitudinal axis that runs down the center of the
fiber and having a plurality of projections that extend from the
middle region. In certain embodiments, a plurality of the
projections extends generally radially from the middle region. As a
result of this configuration, a plurality of channels is defined by
the projections. Suitable channel widths between projections range
from about 200 to about 1000 nanometers. Suitable fibers are
disclosed in U.S. Patent Publication No. 2008/0105612, the
disclosure of which is incorporated herein by reference.
[0168] Fibers with surface areas ranging from at least 1 m.sup.2/g
to about 14 m.sup.2/g or even more are suitable. Furthermore,
preferably suitable are fibers having a surface area of at least 20
m.sup.2/g, also more preferably a surface area of at least 25
m.sup.2/g, and also further preferably a surface area of at least
30 m.sup.2/g. This applies for winged fibers as well as for highly
porous fibers. Thus, preferably fibers are used having surface
areas in this range, but also fibers having much higher surface
areas may be applied for the preparation of separation materials
according to the invention.
[0169] Suitable fibers include porous fibers, such as those
described in U.S. provisional application 62/044,630, the
disclosure of which is incorporated herein by reference.
[0170] Porous fibers may have high surface areas, but it is
essential that the pores are of sizes that allow to be
functionalized by graft polymerization, whereby, however, the
effectiveness and accessibility is not restricted in later
separation processes. Furthermore, it is important that these
porous fibers in application having necessary stability, so that
they are suitable for use in compressed chromatography column.
[0171] In certain embodiments, the fibers can readily be packed
under compression into a device or container with appropriate ports
and dimensions so that no packing is required by the user as the
product arrives ready for service. The fibers also can be used in a
pre-formed bed format such as nonwoven sheetstock material created
by a spunbond (continuous filament) or wet-laid (cut fiber)
process, common in the nonwovens industry. Suitable preformed fiber
formats include sheets, mats, webs, monoliths, etc.
[0172] The shaped fiber media offers certain advantages over porous
chromatographic beads by nature of its morphology. Typically in
bead-based chromatography, the rate limiting step in the separation
process is penetration of the adsorbate (solute) into the depths of
porous beads as controlled by diffusion; for macromolecules such as
proteins, this diffusional transport can be relatively slow. For
the high surface area fibers disclosed herein, the binding sites
are mainly exposed on the exterior of the fibers and therefore are
easily accessed by adsorbate molecules in the flow stream. The
rapid transport offered by this approach allows for short residence
time (high flow velocity), thereby enabling rapid cycling of the
media by means such as simulated moving bed systems. As speed of
processing is a critical parameter in the production of biologies,
fiber-based chromatographic media as described herein has
particular process advantages over conventional bead-based
media.
[0173] A suitable column packing density of between about 0.1-0.4
g/ml, preferably about 0.32 g/ml, at a bed height of 1-5 cm will
provide sufficient flow uniformity for acceptable performance in a
chromatographic evaluation. The surface functionalized fiber media
of the embodiments disclosed herein show a high permeability in a
packed bed format.
[0174] A particular advantage of this new surface functionalized
fiber media described here, is its versatility. Depending on the
used fibers and on their derivatization, separation and
purification of a variety of target molecules is possible, for
example proteins, especially protein A, peptides, lipids, DNA
molecule, RNA molecule, organic molecules, inorganic molecule,
cells, viruses, bacteria, toxins or a prion. Especially in
separation processes that are applied to biological fluids, the
properties of the materials described herein prove to be
particularly advantageous and effective.
Abbreviations:
[0175] AEX anion-exchange chromatography media [0176] aq. aqueous
[0177] ArIS-ArIR two-component system, which is a regulator of
virulence gene expression in Staphylococcus aureus [0178] CEX
cation-exchange chromatography media [0179] CHO Chinese Hamster
Ovary [0180] CV column volume [0181] DI deionized water [0182] DNA
deoxyribonucleic acid [0183] DSP downstream process [0184] Fab
fragment antigen-binding (Fab fragment) is a region on an antibody
that binds to antigens. [0185] Fc region (Fragment, crystallizable)
region, which is composed of two heavy chains that contribute two
or three constant domains depending on the class of the antibody
[0186] GMA glycidyl methacrylate [0187] HCP host cell protein
[0188] HIC hydrophobic interaction chromatography [0189] IgG
Immunoglobulin G (IgG), or gamma globulin, the antibodies
(immunoglobulins) of class G, [0190] LRV "log removal value" The
term refers to the log (base 10) of the ratio of the mass of
impurity in the load of a purification step to the mass of impurity
in the product pool. [0191] mAb monoclonal antibody [0192]
Q-functionalization surface functionalization with quaternary
ammonium (Q)
FIGURES
[0193] FIG. 1: A standard mAb purification scheme is shown, which
employs DSP including a bind-elute chromatography purification step
followed by a flow-through polishing step.
[0194] FIG. 2: A schematic flow scheme of the separation process is
shown of a mAb purification by tandem chromatography (CEX media and
HIC) including the steps: [0195] i. post-protein A mAb load
(conductivity 3 mS, pH 5) [0196] ii. high salt mAb elution
(conductivity 30-100 mS, pH 5), where the product mAb is eluted
from the upper CEX media layer but binds to the lower HIC media
layer and [0197] iii. low salt mAb elution (conductivity 3 mS, pH
5), where the product mAb is eluted from the lower HIC media layer
by elution with a low ionic strength eluent
[0198] FIG. 3: shows HCP clearance from the mAb04 Protein A feed
after flow through purification in the form of bar charts, where
the described media from example 5 are used (AEX fiber, CEX fiber,
and a blend of both AEX and CEX media).
[0199] FIG. 4: Modified surface of the winged nylon fiber media
with a selection of reactive methacrylic monomers
((hydroxyethyl)methacrylate,
poly(propyleneglycol)-monomethacrylate, (phenyl)methacrylate,
(n-butyl)methacrylate, (n-hexyl)methacrylate)) in presence of 0.4 M
cerium(IV) ammonium nitrate.
TABLES
[0200] Table 1: Characteristics of columns packed with either AEX
fibers, CEX fibers or a blend of both AEX and CEX fibers in view of
bed depth and column volume, pressure and flowrate, permeability
and velocity.
[0201] Table 2: Flow-through purification data for mAb04 using AEX
fiber media, CEX fiber media and blended AEX/CEX fiber media
columns in view of the characteristics, flow through, loading of
mAb on fibers, recovery of mAb, HCP concentration and LRV of
HCP.
[0202] The present description enables the person skilled in the
art to apply the invention comprehensively. Even without further
comments, it is therefore assumed that a person skilled in the art
will be able to utilise the above description in the broadest
scope.
[0203] If anything is unclear, it goes without saying that the
publications and patent literature cited should be consulted.
Accordingly, these documents are regarded as part of the disclosure
content of the present description.
[0204] For better understanding and in order to illustrate the
invention, examples are given below which are within the scope of
protection of the present invention. These examples also serve to
illustrate possible variants. Owing to the general validity of the
inventive principle described, however, the examples are not
suitable for reducing the scope of protection of the present
application to these alone.
[0205] Furthermore, it goes without saying to the person skilled in
the art that, both in the examples given and also in the remainder
of the description, the component amounts present in the
compositions always only add up to 100% by weight or mol %, based
on the composition as a whole, and cannot exceed this, even if
higher values could arise from the percent ranges indicated. Unless
indicated otherwise, % data are % by weight or mol %, with the
exception of ratios, which are shown in volume data, such as, for
example, eluents, for the preparation of which solvents in certain
volume ratios are used in a mixture.
[0206] The temperatures given in the examples and the description
as well as in the claims are always in .degree. C.
EXAMPLES
Example 1
Graft Polymerization of Un-Modified Nylon Fibers
[0207] 10 g Allasso nylon fibers and water (466 ml) are added into
a 500 ml bottle. 14 ml 1M HNO.sub.3 (14.4 mmol) are added to the
reaction mixture, followed by the addition of 1.2 ml of a 0.4 M
ammonium cerium(IV) nitrate solution in 1M HNO.sub.3 (0,480 mmol).
The reaction mixture is agitated for 15 minutes. 3.39 g Glycidyl
methacrylate (GMA, 24 mmol) are added. Now the agitated reaction
mixture is heated to 35.degree. C. for 1 hour. After cooling down
to room temperature, the solids are washed with DI water
(3.times.300 ml) and the damp material is used immediately in the
following step.
Example 2
Q-Functionalization of Epoxy-Functionalized Fibers (AEX Fiber
Media)
[0208] The damp GMA functionalized fibers from example 1 are added
into a 2 L bottle together with water (500 ml) and a solution of 50
wt % trimethylamine (aq.) in methanol (500 ml). The mixture is
agitated for 18 hours at room temperature. Then the fiber solids
are subsequently washed with a solution of 0.2 M ascorbic acid in
0.5 M sulphuric acid (3.times.400 ml), DI water (3.times.400 ml),
1M sodium hydroxide solution (3.times.400 ml), DI water
(3.times.400 ml) and ethanol (1.times.400 ml). Subsequently, the
material is placed in an oven to dry at 40.degree. C. for 48
hours.
Yield: 11.74 g of a white fibrous solid
Example 3
Graft Polymerization of Un-Modified Nylon Fibers (CEX Fiber
Media)
[0209] 10 g Allasso nylon fibers and water (460 ml) are added into
a 1000 ml bottle. 29.8 ml 1M HNO.sub.3 solution (30 mmol) are added
to the reaction mixture, followed by the addition of a solution
7.46 ml of a 0.4 M ammonium cerium(IV) nitrate solution in 1M
HNO.sub.3 (3.00 mmol). The reaction mixture is agitated for 15
minutes. Then 61.5 g 3-sulfopropylmethacrylate potassium salt
(3-SPMA, 250 mmol) are added and the resulting agitated reaction
mixture is heated to 35.degree. C. for 18 hours. After cooling to
room temperature, the fiber solids from each bottle are washed with
DI water (3.times.300 ml), 0.2 M ascorbic acid in 0.5 M sulphuric
acid (3.times.300 ml), DI water (3.times.300 ml), 1M sodium
hydroxide solution (3.times.300 ml), DI water 3.times.300 ml) and
ethanol (1.times.300 ml). The prepared material is then placed in
an oven to dry at 40.degree. C.
Yield: 11.38 g of a white fibrous solid
Example 4
Blended Ion-Exchange Media Column Packing
[0210] 0.35 g of a slurry of the described fiber media (see Table
1) in 25 mM Tris pH 8 is added into a 6.6 mm ID Omnifit column. The
fiber media is compressed to a bed depth of 3.0 cm (1.03 ml column
volume, 0.35 g/ml fiber packing density). Fiber bed permeability is
assessed by flowing 25 mM Tris pH 8 buffer through the column at a
flow rate of 2.0 ml/min and measuring the column pressure drop by
means of an electric pressure transducer. Fiber bed permeability
values are also provided in Table 1.
TABLE-US-00001 TABLE 1 Characteristics of columns packed with
either AEX fibers, CEX fibers or a blend of both AEX and CEX fibers
Pressure, Permeability Bed depth, [PSI] [mDarcy] Media type, [cm]
flowrate velocity Column type amount [g] CV [ml] [ml/min] [cm/h]
AEX column AEX fibers, 3.0 cm, 23.5 PSI 185 mDa, example 2 1.03 ml
2.0 ml/min 350 cm/h 0.35 g CEX column CEX fibers, 3.0 cm, 20.0 PSI
269 mDa, example 3, 1.03 ml 2.5 ml/min 440 cm/h 0.35 g AEX and CEX
AEX fibers, 3.0 cm, 28.0 PSI 144 mDa blended column example 2, 1.03
ml 1.9 ml/min 330 cm/h 0.18 g CEX fibers, example 3, 0.18 g
Example 5
[0211] Comparison of HCP Removal from mAb04 Protein a Elution with
AEX Fiber, CEX Fiber, and a Blend of Both AEX and CEX Media
[0212] A cell culture of mAb04 was clarified and then captured at a
concentration of 7.2. mg/ml using Protein A column chromatography.
The pH of the mAb04 Protein A elution was then adjusted to pH 5
with Tris base for storage and then filtered through a Stericup-GP
0.22 .mu.m Millipore ExpressPLUS membrane (1 L, catalogue number:
SCGPU02RE, Millipore Corp. Billerica, Mass., 01821, USA). The pH of
the solution was adjusted to pH 7.0 with Tris base just prior to
use. The resulting solution was then filtered through a Stericup-GP
0.22 mm Millipore Express PLUS membrane (1 L, catalogue number
SCGPU02RE, Millipore Corp. Billerica, Mass., 01821, USA).
[0213] Three columns containing functionalized fibers were prepared
as described in example 4. The first 1 ml column consisted of AEX
fibers functionalized with quaternary ammonium ligands from example
2 (lot ID # JA7654-163B). The second 1 ml column consisted of CEX
fibers functionalized with sulfonate ligands from example 3 (lot
ID# JA7654-131). The third 1 ml column consisted of a blend of
equal quantities of AEX fibers functionalized with quaternary
ammonium ligands from example 2 (JA7654-163B) and CEX fibers
functionalized with sulfonate ligands from example 3 (JA7654-131),
see example 4. The three columns are equilibrated with a buffer
solution (25 mM Tris at pH 7).
[0214] 120 ml of a Protein A elution pool is passed through each
column at a flow rate of 0.33 ml/min giving a residence time of 3
min in each fiber packed column. Three 40 ml fractions are
collected from each column. Pooled samples representing the elution
pool composition after 80 ml and 120 ml, which have passed through
the column, are submitted for analysis. The solutions are analyzed
for host cell protein (HCP) and IgG concentration. HCP analysis is
performed using a commercially available ELISA kit from Cygnus
Technologies, Southport, N.C., USA, catalogue number F550,
following kit manufacturer's protocol. IgG concentration is
measured using an Agilent HPLC system equipped with a Poros.RTM. A
Protein A analytical column. Results are summarized in Table 3 and
FIG. 3.
[0215] The results of the experiment show that combining the mixed
bed column containing the AEX and CEX fiber gave greater HCP
removal with a log removal value (LRV) of approximately 1.6 LRV.
This greatly exceeds the approximately 0.9 LRV of HCP observed for
the column containing only the AEX fibers and the approximately 0.6
LRV of HCP observed for the column that contained only the CEX
fibers. The greater amount of HCP removed by the column with a
blend of both fibers is likely due to the fact that it has two
different ligands which are able to adsorb HCP with different
characteristics. The AEX functionalized fibers are able to bind to
impurities that have negatively charged regions on their surfaces,
which is typical for proteins with lower isoelectric points. The
CEX functionalized fibers are able to bind impurities that have
positively charged regions on their surface, which is typical of
proteins with higher isoelectric points.
TABLE-US-00002 TABLE 2 Flow-through purification data for mAb04
using AEX fiber media, CEX fiber media and blended AEX/CEX fiber
media columns Loading Flow of mAb through on fibers mAb mAb HCP HCP
LRV of train [kg/l] [g/l] recovery [ng/ml] [ppm] HCP untrea-ted --
7.20 -- 1876 261 -- AEX fibers 0.54 7.15 99% 223 31 0.92 AEX fibers
0.82 7.16 99% 242 34 0.89 CEX fibers 0.54 6.67 93% 476 71 0.57 CEX
fibers 0.82 6.82 95% 494 72 0.56 blend of 0.54 6.97 97% 38 5 1.68
AEX and CEX fibers blend of 0.82 7.03 98% 47 7 1.59 AEX and CEX
fibers
FIG. 3: shows HCP clearance from the mAb04 Protein A feed after
flow through purification using the described media from example
5.
Example 6
[0216] Fiber Media with Hydrophobic Interaction Chromatography
Ligand
[0217] The hydrophobic interaction chromatography media described
in the text above can be prepared by using the fiber surface
modification procedures described in examples 1 and 3 and a
methacrylate monomer or other polymerizable functionality selected
from a group comprising [0218] methyl methacrylate, [0219]
(hydroxyethylmethacrylate, [0220]
poly(propyleneglycol)monomethacrylate, [0221] (phenyl)methacrylate,
[0222] ((n-butyl)methacrylate, [0223] (n-hexyl)methacrylate).
[0224] The ceric ion redox polymerization procedure described in
examples 1 and 3 can be used to directly modify the surface of the
Allasso nylon fiber media with reactive methacrylic monomers
disclosed in this example. (see FIG. 4) After grafting
polymerization and suitable washing procedures (also described in
examples 1 and 3), the fiber media now displays an appropriate
hydrophobic ligand functionality for hydrophobic interaction
chromatography (HIC). The media is now ready to be loaded into a
chromatography column or other device for the tandem chromatography
application described in the text above.
FIG. 4: Selection of HIC ligands suitable for fiber media of the
present invention and process for attachment to the surface of the
Allasso nylon fiber media. i: Allasso fiber media surface
modification using a methacrylate monomer selected from the group
comprising: (hydroxyethyl)methacrylate,
poly(propyleneglycol)monoethacrylate, (phenyl)methacrylate,
(n-butyl)methacrylate, (n-hexyl)methacrylate), Allasso nylon fiber,
0.4 M cerium(IV) ammonium nitrate, nitric acid, water 35.degree.
C., 18 hrs according to the surface modification procedure employed
in examples 1 and 3.
Example 7
[0225] Fiber Media Modified with Poly(Hydroxyethylmethacrylate)
Ligand for Hydrophobic Interaction Chromatography
[0226] Hydroxyethylmethacrylate (HEMA, 1.69 g, 13 mmol) and water
(232.5 ml) are added into a 500 ml bottle. Then 5.00 g of Allasso
nylon fibers (Winged Fiber.TM.), are added to this solution. 1M
HNO.sub.3 solution (7.21 ml, 7.2 mmol) are added to this reaction
mixture, followed by the addition of a 0.4 M solution of ammonium
cerium(IV) nitrate in 1 M HNO.sub.3 (0,601 ml, 0,240 mmol). The
reaction mixture is heated to 35.degree. C. for 1 hour. After
cooling to room temperature, the solids are washed with a solution
of 0.2 M ascorbic acid in 0.5 M sulphuric acid (3.times.100 ml), DI
water (3.times.100 ml) 1 M sodium hydroxide solution (3.times.100
ml) DI water (3.times.100 ml) and ethanol (1.times.100 ml). The
material is placed in an oven to dry at 40.degree. C. for 12
hours.
Yield: 5.58 g as a white fibrous solid
* * * * *