U.S. patent application number 12/988707 was filed with the patent office on 2011-02-24 for chromatography medium.
Invention is credited to Jan Bergstrom, Gunnar Glad, Bo-Lennart Johansson, Jean-Luc Maloisel, Nils Norrman, Tobias E. Soderman.
Application Number | 20110045574 12/988707 |
Document ID | / |
Family ID | 41217058 |
Filed Date | 2011-02-24 |
United States Patent
Application |
20110045574 |
Kind Code |
A1 |
Bergstrom; Jan ; et
al. |
February 24, 2011 |
CHROMATOGRAPHY MEDIUM
Abstract
The present invention is within the field of chromatography.
More precisely, it relates to a novel chromatography medium, namely
a hydrophobic medium provided with different lids excluding
molecules over a certain size due to the porosity of the
hydrophobic medium and/or the porosity of the lid. The invention
also relates to use of the separation medium for purification of
large molecules, which do not enter the separation medium, as well
as small molecules, which enter the separation medium and are
eluted from there.
Inventors: |
Bergstrom; Jan; (Balinge,
SE) ; Glad; Gunnar; (Uppsala, SE) ; Johansson;
Bo-Lennart; (Uppsala, SE) ; Maloisel; Jean-Luc;
(Uppsala, SE) ; Norrman; Nils; (Uppsala, SE)
; Soderman; Tobias E.; (Uppsala, SE) |
Correspondence
Address: |
GE HEALTHCARE BIO-SCIENCES CORP.;PATENT DEPARTMENT
101 CARNEGIE CENTER
PRINCETON
NJ
08540
US
|
Family ID: |
41217058 |
Appl. No.: |
12/988707 |
Filed: |
April 21, 2009 |
PCT Filed: |
April 21, 2009 |
PCT NO: |
PCT/SE2009/050406 |
371 Date: |
October 20, 2010 |
Current U.S.
Class: |
435/239 ;
252/62.51R; 435/252.1; 435/320.1; 502/400; 502/402; 530/387.1;
530/412; 536/25.4; 554/175 |
Current CPC
Class: |
B01D 15/327 20130101;
B01J 20/28009 20130101; C12N 7/00 20130101; C07K 16/00 20130101;
B01J 20/287 20130101; B01J 39/26 20130101; B01J 20/3293 20130101;
G01N 1/405 20130101; B01J 20/285 20130101; C12N 2760/16151
20130101; B01J 20/3212 20130101; C07K 1/20 20130101 |
Class at
Publication: |
435/239 ;
252/62.51R; 435/252.1; 435/320.1; 502/400; 502/402; 530/387.1;
530/412; 536/25.4; 554/175 |
International
Class: |
C12N 7/02 20060101
C12N007/02; H01F 1/00 20060101 H01F001/00; C12N 1/20 20060101
C12N001/20; C12N 15/63 20060101 C12N015/63; B01J 20/00 20060101
B01J020/00; B01J 20/26 20060101 B01J020/26; C07K 16/00 20060101
C07K016/00; C07K 1/14 20060101 C07K001/14; C07H 21/00 20060101
C07H021/00; C11B 3/00 20060101 C11B003/00 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 22, 2008 |
SE |
0800923-5 |
Claims
1. A separation medium comprising a porous, hydrophobic core
entity; and a porous hydrophilic lid covering the whole outer part
of the core entity, wherein the lid only allows molecules under a
certain size to penetrate and interact with the internal part of
the core entity of the separation medium.
2. The separation medium of claim 1, wherein the hydrophobic core
entity only allows molecules under a certain size to penetrate and
interact with the internal part of the core entity of the
separation medium.
3. The separation medium of claim 1, wherein the hydrophobic core
entity is hydrophobic per se and is based on a hydrophobic
polymer.
4. The separation medium of claim 1, wherein the hydrophobic core
entity is hydrophilic per se and based on a hydrophilic polymer,
functionalized with hydrophobic interaction ligands.
5. The separation medium of claim 4, wherein the hydrophobic
interaction ligands comprise aliphatic or aromatic
hydrocarbons.
6. The separation medium of claim 1, wherein the hydrophobic core
entity allows adsorption of low molecular weight molecules and/or
small size molecules at low ionic strength 0-1M in the pH interval
2-11.
7. The separation medium of claim 4, wherein the core entity
comprises further ligands besides said hydrophobic ligands which
may be located on and/or associated with the core entity and/or on
the hydrophobic ligands.
8. The separation medium of claim 7, wherein said further ligands
are electrostatic interaction ligands.
9. The separation medium of claim 1, wherein the core entity has a
pore size preventing molecules over a certain size from entering
the pores.
10. The separation medium of claim 1, wherein the hydrophilic lid
comprises ligands that reversibly bind the molecules within the
sample.
11. The separation medium of claim 4, wherein the core entity and
lid are made of agarose and the hydrophobic ligands are
C.sub.4-C.sub.16 ligands.
12. The separation medium of claim 1, wherein a hydrogel is
provided in the lid to fill the pores and thereby further decrease
and adjust the pore size to prevent high molecular weight molecules
from entering the pores.
13. The separation medium of claim 1, wherein magnetic particles
are incorporated into the core entities.
14. The separation medium of claim 1, wherein the lid and/or core
entity are functionalized by layer activation.
15. The separation medium of claim 1, which is mixed with
chromatography media, wherein the separation media comprises up to
10% of the total media volume.
16. The separation medium of claim 15, wherein the separation
medium comprises octyl ligands and the chromatography media is a
cation exchange media.
17. A method for separation comprising the step of adsorbing
molecules under a certain size from a sample, onto the separation
medium of claim 1, at low ionic strength above or equal to 0M, at
pH 2-11.
18. The method for separation of claim 17, wherein the certain size
corresponds to the size range represented by
molecules/organisms/particles selected from cells, cell particles,
bacteria, virus, virus like particles, plasmids, antibodies,
lipids, proteins, peptides or nucleic acid.
19. The method for separation of claim 17, wherein the target
molecule is a small molecule or particle <60 000D.
20. The method for separation of claim 17, wherein the target
molecule is a molecule or particle <1 000 kD.
21. The method for separation of claim 17, wherein the target
molecule is a large molecule/organism/particle, such as a cell,
cell particles, bacteria, virus, virus like particles, plasmids,
antibodies, proteins, which is not adsorbed on the separation
medium but obtained in the flow-through.
22. The method for separation of claim 17, wherein the target
molecule is a large molecule/organism/particle, such as a cell,
cell particles, bacteria, virus, virus like particles, plasmids,
antibodies, proteins, which is adsorbed on the lid.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a filing under 35 U.S.C. .sctn.371 and
claims priority to international patent application number
PCT/SE2009/050406 filed Apr. 21, 2009, published on Oct. 29, 2009
as WO 2009/131526, which claims priority to application number
0800923-5 filed in Sweden on Apr. 22, 2008.
FIELD OF THE INVENTION
[0002] The present invention is within the field of chromatography.
More precisely, it relates to a novel chromatography medium, namely
a hydrophobic medium provided with different lids excluding
molecules over a certain size due to the porosity of the
hydrophobic medium and/or the porosity of the lid.
[0003] Within biotechnology, the chromatographic methods suggested
up to date are based on different modes of interaction with a
target. Thus, for example, in ion-exchange chromatography, the
functional groups are permanently bonded ionic groups with their
counter ions of opposite charge, while in hydrophobic interaction
chromatography (HIC), the interaction between the stationary phase
and the component to be separated is based on hydrophobicity.
[0004] Chromatography based on hydrophobic interaction is generally
divided in to two types named HIC and RPC.
[0005] HIC refers to hydrophobic interaction chromatography of
proteins using very hydrophilic matrixes to which hydrophobic
ligands are immobilized to a very low degree in order to avoid
denaturation of separated proteins. In order to adsorb proteins to
be separated it is necessary to use high concentrations of an
inorganic salt in water. Desorption is usually achieved by
gradually lowering the ionic strength in an elution step.
[0006] RPC refers to chromatography using matrixes usually silica
based functionlized to a very high degree with hydrophobic non
polar molecules/ligands usually aliphatic carbon chains containing
4 to 18 carbon atoms. Adsorption is normally achieved without the
need of added salt. Desorption or elution is most often done by
gradually increasing the content of an organic solvent in the
elution solution.
[0007] RPC media with 18 carbon chains binds proteins strongly. The
proteins are also denaturated to different degree during the
binding process. In order to elute the proteins it is necessary to
use extraordinary conditions. However RPC media with shorter chains
(4 Carbons) may be used together with water/acetonitrile based
eluents containing additives such as triflouracetic acid for
protein separations.
[0008] The stationary phase, also known as the separation matrix,
comprises a support, which is commonly a plurality of essentially
spherical particles, and ligands coupled to the support. In most
separation matrices, the support is porous to allow a larger amount
of ligand and consequently more bound target compound in each
particle. The support is most often a natural or synthetic polymer
and the spherical particles may be produced in a number of
different ways. Silica and glass beads are also used. Natural
polymers often used for this purpose are the polysaccharides
dextran and agarose.
[0009] For separation of biomolecules, by chromatographic and
batch-wise procedures, the porosity of the matrix (beads,
monoliths, packed membranes etc) is very important. Advantages of
polymeric media are that it is easy to vary pore size over broad
ranges and their high chemical stability e.g. tolerance of high
pH:values. A general rule, which is accepted throughout the
literature, is to use media with large pore sizes for large
molecules. Mass transfer in these pores is a result of diffusion
processes and not of convection.
[0010] In WO2009/099375, a method is described based on the
Spinning Disc technology (Walton and Prewett (Walton, W. H.;
Prewett, W. C. (1949) Proc. Phys. Soc. B. 62, 341-350) to produce
agarose beads having a selected porosity to exclude large
molecules.
[0011] Humanized monoclonal antibodies (mAbs) hold significant
promise as biopharmaceuticals. One of the most challenges faced in
the purification of mAbs is their separation from host cell
proteins (HCPs) in the cell culture media. A wide variety of mAbs
isolation technique is available today that include affinity
chromatography on Protein A, ion exchange chromatography and
hydrophobic interaction chromatography (HIC). All these techniques
involve the use of a mobile phase (adsorption buffer) that must be
adjusted to accomplish interaction between mAb-molecules and the
ligands. For example, in HIC high amounts of salt must be added to
the mobile phase and in case of ion exchange chromatography the pH
of the mobile phase must be adjusted so the mAbs are oppositely
charged compared to the ion exchange ligand. Furthermore, when the
mAbs are adsorbed to the ligand the mobile phase must be adjusted
to accomplish desorption of the mAbs.
[0012] It would be desirable to obtain a chromatographic medium
which has a pore size distribution and surface properties that
prevents large molecules, such as mAbs, from entering the beads and
which internally can adsorb proteins and other peptides or
biomolecules independently of buffer conditions. Ion exchangers
aimed for separation of biomolecules are able to adsorb most
charged biomolecules at low ionic strength but when the ionic
strength is increased over a certain level these media loose the
ability to adsorb the sample molecules.
SUMMARY OF THE INVENTION
[0013] The present invention provides a medium having such a high
hydrophobicity in the core entity that proteins can interact or be
adsorbed both at very low and high ionic strengths in a broad pH
range.
[0014] In a first aspect, the invention relates to a separation
medium comprising a porous, hydrophobic core entity; and a porous,
hydrophilic lid covering the whole exterior of the core entity,
wherein the lid only allows molecules under a certain size to
penetrate and interact with the internal part of the core entity of
the separation medium.
[0015] In a preferred embodiment, preferably also the hydrophobic
core entity only allows molecules under a certain size to penetrate
and interact with the internal part of the core entity of the
separation medium.
[0016] The porous hydrophilic lid is a porous outer layer or
jacket/shell surrounding the core entity.
[0017] The separation medium is preferably provided with a highly
hydrophobic core in order to bind proteins and other hydrophobic
molecules independently of the properties of the sample and of
running conditions such as ionic strength and pH, in case of
chromatography, or the supernatant, in case of a batch-wise
procedure. The separation medium is preferably bead-shaped but may
also be a membrane.
[0018] The size exclusion property of the medium is determined by
the porosity of one or both of its porous constituents. The certain
size referred to above is below the size that excludes the target
molecules/organism/particles such as cells, cell particles, virus,
virus like particles, plasmids, any type of antibodies, lipids,
proteins, peptides and nucleic acids. This medium will efficiently
separate low size molecules from larger molecules of the type
mentioned above.
[0019] The hydrophobic core entity may be hydrophobic per se and
may be based on a hydrophobic polymer. For example,
styren/ethylstyren/DVB, vinylethers and acrylates containing
hydrophobic substituents as well as fluoroalkane-containing
polymers.
[0020] Alternatively, the hydrophobic core entity is hydrophilic
per se and is based on a hydrophilic polymer, functionalized with
hydrophobic interaction ligands. The hydrophobic interaction
ligands may comprise aliphatic hydrocarbons, such as C1-C30 alkyl,
preferably C4-C16 alkyl, and/or aromatic hydrocarbons, such as
phenyl, antracene, naphtalene. Independent of ligand, the ligand
density should always be optimized for adsorption at low ionic
strength and in the purpose of obtaining the desired highly
hydrophobic nature of the medium. Preferably, the ligand density
should be above normal HIC-levels, i.e. >90 .mu.mole/ml core
entity.
[0021] The hydrophobic core entity allows adsorption of the
molecules (with the lowest molecular weight and/or smallest size)
in the sample that are able to penetrate the lid at low ionic
strength, such as 0-1 M, preferably 0-0.4 M, most preferably 0-0.1
M, and in the pH interval 2-11.
[0022] In one embodiment, the core entity comprises further ligands
besides said hydrophobic ligands which may be located on and/or
associated with the core entity and/or on the hydrophobic ligands.
The further ligands may be hydrophobic and may contain a non
dominating charged group. Preferably, said further ligands are
electrostatic interaction ligands, i.e. a positive and/or a
negative charge ligand. For example, these further ligands may be
octylamine ligands, provided with a positive charge, which
increases the binding of small negatively charged molecules to the
core entity. The amount of further added charged ligands should be
adjusted so that they do not interfere too much with the
hydrophobic interaction to the main hydrophobic ligand.
[0023] In a preferred embodiment, the core entity and lid are made
of agarose and the hydrophobic ligands are hydrocarbon ligands
comprising 4-16 carbons, preferably octyl ligands.
[0024] Preferably, the core entity has a pore size preventing
molecules over a certain size from entering the pores.
Alternatively, or in addition thereto, a polymeric hydrogel e.g.
dextran is provided in the lid to fill the pores and thereby
further decrease and adjust the pore size to prevent high molecular
weight molecules from entering the pores.
[0025] In a further embodiment, the hydrophilic lid comprises any
type of chromatography ligands that reversibly bind the molecules
within the sample with the highest molecular weight(s) and/or
largest size(s). Some of the small molecules will probably also
bind to the lid but during the elution step they most probably will
penetrate the lid and bind to the core.
[0026] For certain applications, such as proteomic analysis,
magnetic particles may be incorporated into the core entities of
the separation medium.
[0027] The invention also relates to a separation medium which
comprises the lid bead medium described above in mixture with
conventional chromatography media, such as HIC, IE or affinity
media. Preferably the lid bead medium comprises up to 10% of the
total media volume. In a preferred embodiment of this mixed media,
the lid bead medium comprises octyl ligands and the chromatography
media comprises a cation exchange media. Benefits of this mixed
media are increased resolution between large and small
molecules.
[0028] In a second aspect, the invention relates to use of the
separation medium as described above, for adsorbing molecules under
a certain size from a sample at low ionic strength at pH 2-11,
preferably in one single step. The procedure may be used to purify
molecules over or under said size. Thus this aspect involves a
method of using the separation medium comprising a step of
adsorbing molecules and optional further step(s).
[0029] The pore size referred to above varies with, and is
determined by, the size of the target molecule in the specific
application. This certain size range preferably corresponds to the
size range of molecules/organisms/particles selected from cells,
cell particles, bacteria, virus, virus like particles, plasmids,
antibodies, lipids, proteins, peptides, nucleic acid. The sample
may be, for example, a culture supernatant and the high molecular
weight molecule may be a monoclonal antibody. The separation medium
will be very useful for addition at cell harvest to rapidly remove
undesired enzymes, like proteases, peptidases and nucleases, such
as trypsin, chymotrypsin, DNase and RNase. The separation medium
may be used in chromatographic or batch mode. Any column or batch
format may be used. For some applications, such as purification of
biopharmaceuticals for clinical phase I and II studies, disposable
columns in RTP (ready to process) format are preferred.
[0030] In an alternative embodiment, the adsorbed low molecular
weight molecules, such as below 60 000 D, may be the desired
molecules, such as biomarkers or drug markers, which are eluted,
for example by decreased polarity in the eluent, from the core
entity and further analysed. Another example is when the target
molecule is adsorbed in the core entity but not used for further
analysis, such as for lipid removal.
[0031] When the target molecule is a large
molecule/organism/particle, such as a cell, cell particles,
bacteria, virus, virus like particles, plasmids, antibodies,
proteins, it may obtained in the flow-through and thus not adsorbed
by the medium.
[0032] Alternatively the target molecule is a large
molecule/organism/particle, such as a cell, cell particles,
bacteria, virus, virus like particles, plasmids, antibodies,
proteins, which is adsorbed on the lid. The target molecule is then
eluted by a technique appropriate for the chosen absorption mode
(e.g. ion exchange: salt and/or a pH gradient, IMAC: imidazol
gradient, boronate: sugar or pH gradient, HIC: lowering the salt
concentration).
[0033] The preferred use of the separation medium according to the
invention is for purification of monoclonal antibodies. Another
preferred use is wherein the target molecule is a molecule or
particle <1 000 kD. This embodiment is suitable for purification
of virus, such as influenza virus, but also for other large
molecules such as IgM antibodies.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 is an illustration of three different bead designs
that have been tested. FIG. 1A: SEPHAROSE.TM. 20 Fast Flow (20%
agarose particle) and Spinning disc and FIG. 1B: lid-dextran
SEPHAROSE.TM. 6 Fast Flow. IgG=monoclonal antibodies; P=proteins
with molecular weight less than ca 70 000.
DETAILED DESCRIPTION OF THE INVENTION
[0035] The present invention relates to a chromatographic medium
which has both hydrophilic and hydrophobic regions in the matrix
and may be based on a matrix which is hydrophobic per se, or on a
hydrophilic matrix with hydrophobic ligands, and is provided with a
hydrophilic outer layer. The medium may also be based on a
hydrophilic matrix internally functionalized with hydrophobic
ligands (e.g. a hydrophobic internal core in a bead).
[0036] Example of Synthetic Path Ways:
[0037] Hydrophobic per se start material e.g. DVB beads covered by
a hydrophilic and porous layer.
[0038] Per se hydrophilic start material fully functionalized with
hydrophobic functional groups and then covered by a hydrophilic and
porous layer. Layer activation of a hydrophilic matrix and
internally couple hydrophobic ligands (FIG. 1).
[0039] Layer activation of a hydrophobic matrix and make a
hydrophilic functionalization of the outer parts.
[0040] The invention also provides a method of separating large
molecular weight molecules, such as antibodies, from other
components of a liquid, which requires less time and process steps
than the prior art methods. This is achieved by a method wherein
the liquid comprising the desired high molecular weight molecules,
such as antibodies, is contacted with the chromatographic medium,
and substantially pure high molecular weight molecules are
recovered in non-binding or reversible binding mode. Another
advantage is that low molecular weight molecules, such as host cell
proteins, adsorb to the internal parts of the medium even at very
low ionic strengths.
[0041] The support matrix and core entity of the chromatographic
medium can be based on organic or inorganic material. It is
preferably in the form of an organic polymer, which is insoluble
but may be more or less swellable in water. Suitable polymers are
polyhydroxy polymers, e.g. based on polysaccharides, such as
agarose, dextran, cellulose, starch, pullulan, etc. and completely
synthetic polymers, such as polyacrylic amide, polymethacrylic
amide, poly (hydroxyalkylvinyl ethers), poly(hydroxyalkylacrylates)
and polymethacrylates (e.g. polyglycidylmethacrylate),
polyvinylalcohols and polymers based on styrenes and
divinylbenzenes, and copolymers in which two or more of the
monomers corresponding to the above-mentioned polymers are
included. Polymers, which are soluble in water, may be derivatized
to become insoluble, e.g. by cross-linking and by coupling to an
insoluble body via adsorption or covalent binding. Hydrophilic
groups can be introduced on hydrophobic polymers (e.g. on
copolymers of monovinyl and divinylbenzenes) by graft
polymerization of monomers exhibiting groups which can be converted
to OH, or by hydrophilization of the final polymer, e.g. by binding
or adsorption of suitable compounds, such as hydrophilic polymers
and other hydrophilic compounds. Reactions including epoxides or
reactive halogenids may be used.
[0042] Suitable inorganic materials to be used in support matrices
are silica, glass, zirconium oxide, graphite, tantalum oxide
etc.
[0043] Preferred support matrices lack groups that are unstable
against hydrolysis, such as silanol, ester, amide groups and groups
present in silica as such.
[0044] In a particularly interesting embodiment of the present
invention, the support matrix is in the form of irregular or
spherical particles with sizes in the range of 1-1000 .mu.m,
preferably 5-50 .mu.m for high performance applications and 50-300
.mu.m for preparative purposes.
[0045] An interesting form of support matrix has densities higher
or lower than the liquid sample or buffer solutions to be used,
such as fermentation feeds. This kind of matrices is especially
applicable in large-scale operations for fluidised or expanded bed
chromatography as well as for different batch wise procedures, e.g.
in stirred tanks. Fluidised and expanded bed procedures are
described in WO 92/18237 and WO 92/00799. The most practical use of
these matrices has been to combine particles/beads with a density
higher than the density of a fluidising liquid with an upward flow.
This kind of support matrices in expanded bed mode is particularly
beneficial in case the sample solution contains particulate and
sticky materials.
[0046] The term hydrophilic support matrix in practice means that
the accessible surface of the matrix is hydrophilic and protein
friendly, i.e. that the surface not irreversibly adsorbs and
denaturates proteins. Typically the accessible surfaces on a
hydrophilic base matrix expose a plurality of polar groups for
instance comprising oxygen and/or nitrogen atoms. Examples of such
polar groups are hydroxyl, amino, carboxy, sulphonate (S and SP
ligands) ester, ether of lower alkyls (such as
(--CH.sub.2CH.sub.2O--).sub.nH where n is an integer 2, 3, 4 and
higher).
[0047] A hydrophilic surface coat, possible in the form of
hydrophilic extenders belongs conceptually to the support
matrix.
[0048] In ordinary/established HIC media an increase in polarity by
addition of salts to the mobile phase is required to accomplish
binding of proteins. The bound proteins are subsequently released
from the matrix by lowering the concentration of salt. Thus, a
disadvantage of this procedure is the necessity to add salt to the
raw material, as this may cause problems and a consequently
increased cost to the large-scale user. Adsorption of proteins to
ordinary HIC-media is usually performed with a salt concentration
between 0.75 and 4 M (Jansson, J.-C. and Ryden, L., Protein
Purification--Principles, High Resolution Methods and Applications,
VCH, Weinheim, pp. 220-221).
[0049] This invention relates to a medium which maximizes the
interaction with molecules small enough to penetrate into the
hydrophobic core of the separation matrix, such as HCPs, and
minimizes the interaction with the largest proteins in the samples,
such as MAb-molecules and MAb-dimers in a MAb purification process.
Furthermore, one embodiment of the invention enables that mobile
phases with no extra addition of salt can be used and that no
elution buffer (desorption buffer) is needed to recover the desired
high molecular weight molecules or organisms for embodiments with
neutral or almost neutral lids (non-binding lids).
[0050] Three different approaches for the design of beads aimed for
capture of host cell proteins but not large monoclonal antibodies
have been tested. In one approach agarose beads based on high
contents of agarose (20%) was designed to obtain a bead with a
porosity that excludes large proteins (monoclonal antibodies) (FIG.
1A). In a further approach, a prototype based on SEPHAROSE.TM. 6
Fast Flow and a gel filtration lid was produced. The lid was
obtained by coupling dextran to the outer segment of the beads. The
pore sizes obtained in the dextran filled segment is designed to
prevent large molecules, such as mAbs, to enter the core of the
beads (FIG. 1B). The third approach is based on the spinning disc
media described in WO2009/099375. Also in this case the beads were
designed to exclude large proteins (FIG. 1A) by the introduction of
a neutral outer segment achieved as described by commonly owned
layer activation patents EP 0 966 488 B1 (Process for introducing a
functionality.)
[0051] During the attachment of the hydrophobic ligands to the
different prototypes a synthesis procedure was used to prevent
ligands to be coupled to the surface of the beads and in that way
also eliminate adsorption due to hydrophobic interaction of
monoclonal antibodies to the outer part of the beads. The
prototypes are named lid-OH core-octyl media meaning that the octyl
ligands only are attached in the core of the beads. Furthermore,
the amount of ligands in the core of the beads is adjusted so that
host cell proteins are adsorbed using adsorption buffers with very
low ionic strengths adjusted to any pH relevant for chromatography
of biomolecules.
EXAMPLES
[0052] The present examples are presented herein for illustrative
purpose only, and should not be constructed to limit the invention
as defined by the appended claims.
Example 1
Preparation of Octyl Media Based on SEPHAROSE.TM. 20 Fast Flow (A),
SEPHAROSE.TM. 6 Fast Flow (B), and Spinning Disc Media (C)
[0053] Volumes of matrix refer to settled bed volume and weights of
matrix given in gram refer to suction dry weight. For large scale
reaction stirring is referring to a suspended, motor-driven stirrer
since the use of magnet bar stirrer is prompt to damage the beads.
Small-scale reactions (up to 20 mL of gel) were performed in closed
vials and stirring refers to the use of a shaking table.
[0054] Conventional methods were used for the analysis of the
functionality and the determination of the degree of allylation,
epoxidation, or the degree of substitution of ion exchanger groups
on the beads.
A: Preparation of Lid-OH core-octyl SEPHAROSE.TM. 20 Fast Flow
A1: Preparation of SEPHAROSE.TM. 20 Fast Flow
[0055] Agarose (50 g) was dissolved in water (250 g) by heating at
95.degree. C. for approximately 10 hours. The solution was added to
toluene (375 mL) and ethyl cellulose (35 g) in an emulsification
vessel, the temperature was kept at 70.degree. C. The
emulsification vessel was equipped with a blade stirrer. The speed
of the stirrer was increased step by step from 150 rpm to 340 rpm,
maintaining the temperature at 70.degree. C. When the agarose
particles were judged by a microscope to have a desired size the
speed was decreased to 150 rpm. Thereafter the emulsion was cooled
and the beads were allowed to gel. The beads were washed with
ethanol and water.
[0056] Water was added to 250 mL beads to a total weight of 310 g.
To this suspension 38 g Na.sub.2SO.sub.4, 3.0 mL 50% NaOH and 0.25
g NaBH.sub.4 was added and the temperature was increased to
47.degree. C. Epichlorohydrine (31.4 mL) and sodium hydroxide
solution (21.6 mL) were then added with pumps during 6 h. After the
addition was completed the reaction continues over night at
47.degree. C. The slurry was then cooled and neutralized to pH 5-6
with 60% acetic acid. Finally the gel was carefully washed on a
glass filter with distilled water. The beads were thereafter sieved
on 43 .mu.m and 166 .mu.m cloths.
A2: Preparation of an OH-lid Allyl SEPHAROSE.TM. 20 Fast Flow
[0057] Allyl activated SEPHAROSE.TM. 20 Fast Flow. 50 g of drained
SEPHAROSE.TM. 20 Fast Flow (SEPHAROSE.TM. 6 Fast Flow, GE
Healthcare, Uppsala, Sweden) was mixed with 15 mL of 50% NaOH
solution, 6 g of Na.sub.2SO.sub.4 and 0.1 g of NaBH.sub.4. The
mixture was stirred at 50.degree. C. for 1 h, followed by addition
of 30 mL allyl glycidyl ether (AGE). The reaction slurry was
stirred at 50.degree. C. for 17 h. Then the gel was washed on a
glass filter with distilled water, ethanol and finally with
distilled water again.
[0058] Partial bromination and NaOH treatment (preparation of
OH-lid). 10 mL of drained allylated SEPHAROSE.TM. 20 Fast Flow, was
stirred in 5 mL of distilled water and 0.4 g sodium acetate. 125
.mu.L of bromine, dissolved in a capped vial with 10 mL distilled
water was added and the stirring was continued for 5 minutes. After
washing with water, the drained gel was stirred with 7.5 mL of 2 M
NaOH at 50.degree. C. for 18 h, followed by washing with water.
Remaining allyl content in the core of the beads was measured by
titration; 0.37 mmol/mL.
A3: Core Coupling
[0059] Coupling of octyl in the core of the beads. 4.5 mL of OH-lid
SEPHAROSE.TM. 20 Fast Flow (see above), is fully brominated and
washed. Drained gel was mixed with a solution of 1.25 mL
octanethiol in 4.5 mL 2 M NaOH and 1 mL of ethanol, followed by
stirring at 50.degree. C. for 16 h. After washing with water,
ethanol and water, the gel was stored in 20% ethanol.
B: Preparation of Lid-Dextran Core-Octyl SEPHAROSE.TM. 6 Fast
Flow
B1: Preparation of a Lid-dextran Allyl SEPHAROSE.TM. 6 Fast
Flow
[0060] Allylation of SEPHAROSE.TM. 6 Fast Flow. SEPHAROSE.TM. 6
Fast Flow was allylated according to above and the allyl content
was determined by titration to 259 .mu.mol/mL.
[0061] Partial bromination. Allylated SEPHAROSE.TM. 6 Fast Flow
(100 mL) was weighed into a flask and 900 mL of distilled water and
1.3 g sodium sulphate was added. 0.3 equivalents of bromine, 400
.mu.L, were then added with a pipette during vigorous stirring.
After approximately 5 minutes (when the bromine was consumed) the
gel was washed with distilled water on a glass filter.
[0062] Dextran coupling. 51 mL of the partially brominated gel was
transferred to a flask, and a solution of 30 g Dextran AB in 125 mL
of distilled water was added. After stirring for 30 minutes, 10 g
of NaOH and 0.5 g NaBH.sub.4 were added and the slurry was heated
to 50.degree. C. and left stirring over night. After approximately
18 hours the pH was adjusted to approximately 7 with acetic acid
(60% solution). The gel was then washed with distilled water on a
glass filter.
B2: Core Coupling
[0063] Coupling of octyl in the core of the beads. 10 mL of drained
gel (Dextran coupled SEPHAROSE.TM. 6 Fast Flow, see above) were put
with distilled water into a beaker and vigorous overhead stirring
was begun. Bromine was added until the slurry had a remaining
deeply orange/yellow colour. After 10 minutes of stirring sodium
formiate (approximately 1.5 g) was added until the slurry was
completely discoloured. The gel was then washed with distilled
water on a glass filter. Drained brominated gel was weighed into a
flask and 10 mL of distilled water and 2.3 mL of octanethiol was
added. The pH was then adjusted to approximately 12.5 with NaOH
(50%-solution). The mixture was then left stirring in 50.degree. C.
over night. After 20 hours the gel was washed with distilled water
and ethanol.
C: Preparation of Lid-OH Core-Octyl Spinning Disc Medium
C1: Preparation of Spinning Disc Medium
[0064] 6% agarose solutions were used as starting material.
Temperature, cooling rate, viscosity, speed and agarose flow rate
(to the disc) were investigated with respect to the porosity
response.
[0065] The Spinning Disc apparatus was manufactured by ABB
Industriservice according to given specification (see below):
Steel quality: SS 2343-02 (for all specified items) Polymeric
materials: PTFE, polycarbonate (dome protection), EPDM (Ethylene
Propylene Diene Monomer), silicon rubber. Dome height: 900 mm
Capture basin diameter: 2400 mm including water drain channel
Capture basin slope: 3.degree. Number of discs: 6 Disc diameter:
200 mm General disc thickness: maximum 5.2 mm Disc thickness at
edges: 12.4 mm including 135.degree. slope Upper pressure
compensation chamber diameter (for liquid agarose solution): 73 mm
Upper pressure compensation chamber height: 6 mm Number of
distribution needles: 6 Inner diameter of needles: 0.7 mm
[0066] The agarose solution was fed to six discs via needles. By
using six discs instead of one, there is an increase in capacity.
The agarose flow was the same to each of the six discs. This means
that the bead size originating from each disc is the same. The
speed range of the discs was adjusted within 3001-3010 rpm and the
relative humidity in the dome was 100%. If the relative humidity is
less than 100% there is a risk that water will be evaporated from
the agarose drops.
[0067] Allylated, 6% agarose solutions adjusted to 69.7.degree. C.
and a viscosity within 397-421 mPas were used to feed the spinning
discs. The flow rate of the agarose solution to the discs was
adjusted to 170 mL/min. The capture water temperature was
20.1.degree. C.
[0068] The porosity of the spinning disc beads after cross-linking
with epichlorohydrin is presented in Table 1. The porosity was
estimated with different dextrans and the void volume was obtained
with blue dextran 2000. The spinning disc prototype was produced to
obtain a porosity that not allows immunoglobulins to penetrate the
beads. This means that molecules with a molecular weight larger
than ca. 150 000 g/mol should not diffuse into the beads.
TABLE-US-00001 TABLE 1 The K.sub.av-value of five different dextran
standards for the spinning disc prototype Mw Dx K.sub.av 9890 0.655
43500 0.382 66700 0.276 123600 0.021 196300 0.013 .sup.1 The
K.sub.av-value vas calculated as: (V.sub.R - V.sub.O )/(V.sub.C -
V.sub.O ) where V.sub.R = retention volume of dextran standards,
V.sub.O = void volume and V.sub.C = geometric volume of the
column.
[0069] The particle size was 190 .mu.m.+-.5 .mu.m. The spinning
disc prototype was used to produce media for capture of proteins
with a molecular weight less than approximately 70 000 g/mol while
larger molecules such as immunoglobulins (human IgG) should not be
able to diffuse into the beads and interact with the ligands in the
core of the beads.
C2: Preparation of an OH-lid-Allyl Spinning Disc Medium
[0070] Allyl activated spinning disc medium. Spinning disc medium
was washed with distilled water on a glass filter. The gel, 25 mL,
was drained on the filter and weighed into a 3-necked round
bottomed flask. NaOH (20 mL, 50%-solution) was added and mechanical
stirring started. Sodium borohydride, 0.1 g, and sodium sulphate,
2.9 g, were added to the flask and the slurry heated to 50.degree.
C. on a water bath. After approximately one hour 27.5 mL of allyl
glycidyl ether was added. The slurry was then left under vigorously
stirring over night. After about 20 hours the slurry was
transferred to a glass filter and the pH adjusted to around 7 with
acetic acid (60%). The gel was then washed with distilled water
(.times.4), ethanol (.times.4) and distilled water (.times.4). The
allyl content was then determined by titration to 321
.mu.mol/mL.
[0071] Partial bromination and NaOH treatment (preparation of
OH-lid). Allylated gel, 11.6 mL, was weighed into a flask and 90 mL
of distilled water and 1 g sodium sulphate was added. 0.3
equivalents of bromine, 57 .mu.L, were then added with a pipette
during vigorous stirring. After approximately 5 minutes (when the
bromine was consumed) the gel was washed with distilled water on a
glass filter. 5 mL of the partially brominated gel was transferred
to a flask with water solution. NaOH (50%-solution) was then added
to pH>13 and the slurry were heated to 50.degree. C. and left
stirring over night. After approximately 18 hours the pH was
adjusted to approximately 7 with acetic acid (60% solution). The
gel was then washed with distilled water on a glass filter.
C3: Core Coupling
[0072] Coupling of octyl in the core of the beads. 5 mL of drained
OH-lid spinning disc medium was put with distilled water into a
beaker and vigorous overhead stirring was begun. Bromine was added
until the slurry had a remaining deeply orange/yellow colour. After
10 minutes of stirring sodium formiate (approximately 1.5 g) was
added until the slurry was completely discoloured. The gel was then
washed with distilled water on a glass filter.
[0073] Drained brominated gel was weighed into a flask and 10 mL of
distilled water and 2 mL of octanethiol was added. The pH was then
adjusted to approximately 12.5 with NaOH (50%-solution). The
mixture was then left stirring in 50.degree. C. over night. After
20 hours the gel was washed with distilled water and ethanol.
Example 2
Chromatographic Evaluation of the Three Prototypes Based on Octyl
Ligands in the Core of the Beads
[0074] The three different octyl media to be investigated
(Prototypes: lid-OH core-octyl SEPHAROSE.TM. 20 Fast Flow, lid-OH
core-octyl spinning disc and lid-dextran octyl core SEPHAROSE.TM.
Fast Flow), with respect to breakthrough capacity, were packed in
HR 5/5 columns and the sample solution was pumped at a flow rate of
0.3 or 1.0 mL/min through the column after equilibration with
buffer solution. The breakthrough capacity was evaluated at 10% of
the maximum UV detector signal (280 nm). The maximum UV signal was
estimated by pumping the test solution directly into the detector.
The breakthrough capacity at 10% of absorbance maximum (Q.sub.b10%)
was calculated according to the formula:
Q.sub.b10%=(T.sub.R10%-T.sub.RD).times.C/V.sub.c
where T.sub.R10% is the retention time (min) at 10% of absorbance
maximum, T.sub.RD the void volume time in the system (min), C the
concentration of the sample (4 mg protein/mL) and V.sub.C the
column volume (mL). The adsorption buffer used at breakthrough
capacity measurements was 25 mM TRIS (pH 8.0) or 50 mM acetate (pH
4.75).
[0075] Prototype lid-OH core-octyl SEPHAROSE.TM. 20 Fast Flow was
also tested with a clarified feed in flow-through mode. The feed
(32 mL) was pumped through a HR 5/5 column packed with the
prototype and the flow-through fraction (36 mL) was analysed with
respect of the amount of host cell proteins (HCP) and the amount of
monoclonal antibody recovered. The adsorption buffer used at these
experiments was 25 mM phosphate buffer adjusted to pH 7.2.
A: Sample
[0076] The samples used for breakthrough measurements were human
immunoglobulin (IgG, Gammanorm), ovalbumin and lysozyme. The
proteins were dissolved in the adsorption buffers at a
concentration of 4 mg/mL and only one protein at a time was applied
into the column
[0077] The monoclonal antibody (mAb) was an IgG1 (based on IEF its
pI is in the range 7.5 to 8.4), expressed in NS0 cells. The
filtered non-purified cell culture supernatant was used as sample.
The concentration of the mAb in the sample was 1.3 mg/mL and 32 mL
of the sample were applied to the column (HR 5/5 column packed with
prototype lid-OH core-octyl SEPHAROSE.TM. 20 Fast Flow). The growth
medium used at production of the monoclonal antibody was DMEM
(Gibco) and 10% fetal calf serum (FCS, Gibco).
B: Instrumental
Apparatus
[0078] LC System: AKTAexplorer 10.times.T or equal
Software: UNICORN.TM.
Column: HR 5/5
Instrument Parameters
[0079] Flow rate: 0.3, 0.5 or 1.0 mL/min Detector cell: 10 mm
Wavelength: 280 nm
B1: UNICORN.TM. Method
[0080] The main method used at breakthrough experiments is depicted
below:
0.00 Base CV 1.00 {mL} #Column volume {mL} Any
0.00 Block Start Conditions
[0081] 0.00 Base SameAsMain
[0082] 0.00 Wave length 280 {nm} 254 {nm} 215 {nm}
[0083] 0.00 AvaragingTime 2.56 {sec}
[0084] 0.00 Alarm Pressure Enable 3.00 {MPa} 0.00 {MPa}
[0085] 0.00 End Block
0.00 Block Column Position
0.00 Block Equilibration
[0086] 0.00 Base SameAsMain
[0087] 0.00 PumpAInlet A1
[0088] 0.00 BufferValveA1 A11
[0089] 0.00 Flow 0.3 {mL/min}
[0090] 1.00 Set Mark ( )# column name
[0091] 3.9 AutoZeroUV
[0092] 5.0 #Equilibration volume End Block
0.00 Block Sample Loading
[0093] 0.00 Base volume
[0094] 0.00 Flow (1)#flow rate {mL/min}
[0095] 0.00 Set Mark ( )# sample
[0096] 0.00 InjectionValve Inject
[0097] 0.00 Watch UV Greater Than (100) #20 percent maxabs {mAu}
END BLOCK
[0098] 49.00 InjectionValve Load
[0099] 49.00 End Block
0.00 Block Column Wash
[0100] 0.00 Base SameAsMain
[0101] 0.00 InjectionValve Load
[0102] 0.00 Watch Off UV
[0103] 0.00 PumpAInlet A1
[0104] 0.00 BufferValveA1 A11
[0105] 0.00 Watch UV Less Than (20) #5 percent {mAu} END BLOCK
[0106] 20.00 End Block
0.00 Block Gradient Elution
[0107] 0.00 Base SameAsMain
[0108] 0.00 PumpBInlet B1
[0109] 0.00 Gradient 100 {% B} 2.00 {base}
[0110] 0.00 Flow 0.30 {mL/min}
[0111] 10.00 Gradient 0.00 {% B} 0.00 {base}
[0112] 10.00 End Block
Block Reequilibration
[0113] 0.00 End Method
C: Host Cell Protein (HCP) Analysis
[0114] Samples for ELISA were pre-treated by addition of 10% 2.0 M
Tris, 1% BSA, 0.5% TWEEN.TM. 20, pH 8.0 (50 .mu.L BSA solution to
450 .mu.L sample). The samples were diluted in "Sample Diluent
Buffer" (catalogue number F223A, Cygnus Technologies) and analyzed
by a NS/0 HCP ELISA kit (catalogue number F220, Cygnus
Technologies) using the "High sensitivity protocol" specified in
the kit. The spectrophotometer VERSAMAX.TM. and the software
SOFTMAX.RTM. Pro, both from Molecular Devices, was used for reading
and evaluation of the plates.
D: Results
[0115] D1: Lid-OH core-octyl SEPHAROSE.TM. 20 Fast Flow
[0116] Lid-OH core-octyl SEPHAROSE.TM. 20 Fast Flow is based on
SEPHAROSE.TM. 20 Fast Flow with octyl as core ligand. The base
matrix SEPHAROSE.TM. 20 Fast Flow is designed with high content of
agarose in order to obtain a porosity that prevents monoclonal
antibodies to penetrate the beads. In this case SEPHAROSE.TM. 20
Fast Flow was activated with a high degree of allyl groups (0.37
mmol/mL) meaning that high ligand content was obtained in the core
of the beads.
[0117] The breakthrough capacity of Lysozyme and IgG of the
prototype based on SEPHAROSE.TM. 20 Fast Flow (Lid-OH core-octyl
SEPHAROSE.TM. 20 Fast Flow) was tested. As adsorption buffer 25 mM
TRIS (pH=8.0) was used and 1.0 M NaOH+30% isopropanol was used as
desorption buffer and the flow rate was adjusted to 1.0 mL/min The
prototypes were packed in HR 5/5 columns
[0118] As a result of the test, IgG was not adsorbed (Q.sub.b10=0)
while lysozyme had a breakthrough capacity (Q.sub.b10) of about 10
mg/mL. It can be noted that these results were obtained with 25 mM
TRIS (pH=8.0) as mobile phase and therefore clearly shows that no
extra salt addition is necessary to accomplish adsorption of small
proteins.
[0119] This type of media is mainly aimed to be used for only one
cycle. However it is possible to elute adsorbed host cell proteins.
To verify this adsorbed lysozyme was eluted by using 1.0 M NaOH+30%
isopropanol as desorption buffer.
D2: Lid-Dextran Core-Octyl SEPHAROSE.TM. 6 Fast Flow
[0120] This prototype is based on SEPHAROSE.TM. 6 Fast Flow that
has a porosity that makes it possible for IgG to diffuse into the
matrix. Therefore, according to FIG. 1 the pore sizes in the outer
part of the beads have been reduced by attaching dextran and in
that way prevent IgG to diffuse into the beads. According to Table
2 the breakthrough capacity of IgG was 0 mg/mL which clearly proves
that IgG not diffuse into the beads. However, a relatively high
breakthrough capacity was observed for ovalbumin (16 mg/mL). The
molecular weight of ovalbumin and IgG is approximately 43 000 and
150 000 g/mol, respectively. This means that the dextran lid has a
high "size-selectivity" and can allow ovalbumin to diffuse into the
beads while IgG is prevented to interact with the core ligands.
TABLE-US-00002 TABLE 2 Breakthrough capacity (Q.sub.b10) of IgG and
ovalbumin for two different prototypes packed in HR 5/5 columns.
Q.sub.b10 IgG.sup.1 Q.sub.b10 Ovalbumin.sup.1 Prototype (mg/mL)
(mg/mL) Lid-dextran octyl-core 0 16 SEPHAROSE .TM. 6 Fast Flow
Lid-OH octyl-core 0.6 14 spinning disc .sup.1As adsorption buffer
was 50 mM acetate buffer (pH = 4.75) used and 1.0 M NaOH + 30%
isopropanol was used as desorption buffer and the flow rate was
adjusted to 1.0 mL/min
D3: Lid-OH Core-Octyl Spinning Disc
[0121] Breakthrough measurements. Lid-OH core-octyl Spinning disc
is based on a Spinning disc beads with octyl as core ligand. The
Spinning disc medium was designed to obtain a porosity that
prevents monoclonal antibodies to penetrate the beads. The spinning
disc prototype was activated with a high degree of allyl groups
(0.32 mmol/mL) meaning that high ligand content was obtained in the
core of the beads. Furthermore, no ligands were coupled in the
outer part of the beads (see the preparation of the beads).
According to Table 1 the IgG breakthrough capacity was very low
(0.6 mg/mL) while the capacity of ovalbumin was 23 times higher.
These results also show that the porosity of the spinning disc
prototype means that very small amounts of IgG are able to diffuse
into the core of the beads.
[0122] MAb feed application. Clarified feed that had not been run
on any other chromatography column was applied to an HR 5/5 column
packed with prototype lid-OH core-octyl spinning disc. The column
was run in flow-through mode and the flow-through fraction was
sampled and analysed with respect of the recovery of the mAb and
the amount of host cell proteins (HCP).
[0123] 32 mL of mAb (1.3 mg mAb/mL) was applied to a HR 5/5 column
packed with the prototype lid-OH core-octyl spinning disc. As
adsorption buffer was 25 mM Na-phosphate (pH=7.2) used and 1.0 M
NaOH+30% isopropanol was used as desorption buffer. The flow-rate
was adjusted to 0.5 mL/min and 36 mL of the flow-through fraction
was sampled.
[0124] In Table 3 are the results from recovery and HCP
measurements presented. The HCP content was reduced by more than
50% and the recovery of the mAb was 89%.
TABLE-US-00003 TABLE 3 Yield and HCP reduction obtained in the
experiment described in Example 3. Yield HCP Sample (%) (.mu.g/mL)
Start material 100 791 Flow-through fraction.sup.1 89 341 .sup.136
mL were sampled (flow-through fraction) and 32 mL of the mAb
solution were applied to the column (see the experimental
section).
Example 3
Purification of Influenza Virus
[0125] When producing influenza virus at large scale aiming at
influenza vaccines it is important to reduce the levels of protein
and DNA in the final preparation.
[0126] The particles of the present invention are well suited for
the purification of viruses since viral particles are significantly
larger in size than most of the contaminants.
[0127] This is illustrated in the following example.
Analysis Methods
Virus Concentration
[0128] The DotBlot HA assay was used according to a standard
protocol.
Dna Concentration
[0129] The PICOGREEN.RTM. DNA assay was used according to the
manufacturers instruction (available from Invitrogen).
Protein Concentration
[0130] The Bradford protein assay was used according to the
manufacturers instruction. (Available from Bio-Rad)
Agarose Gel Electrophoresis for Analysis of Molecular Weight
Distribution in DNA Sample.
[0131] An E-GEL.RTM. Agarose Gel 0.8% (Invitrogen) precast gel was
used according to the manufacturers instructions
[0132] The DNA ladder used was 1 kb Plus DNA marker
(Invitrogen)
Sample
[0133] An influenza virus sample produced in-house was used in the
study. The virus was propagated in MDCK cells until lysis occurred.
Influenza virus strain A/H1N1/Solomon Islands was used. After
lysis, the material was concentrated .about.10.times. in an
ultrafiltration (UF) step and another .about.2.times. in a
diafiltration (DF) step (Hollow Fiber Cartridge 500 kDa). The
diafiltration buffer was 50 mM Tris-HCl, 150 mM NaCl pH 7.3 and the
sample was frozen until used.
Chromatography Method and Results
[0134] Column: 2 mL TRICORN.TM. 5/100 column packed with particles
of the present invention with 7 .mu.m thick neutral outer layer and
octyl amine as ligand in the interior. The particles in this
example are agarose particles.
[0135] 10 mL of influenza virus sample was applied at a flow rate
of 75 cm/h. The flow-through fraction was collected.
[0136] The starting material and flow-through fraction was analysed
for virus concentration, DNA concentration and protein
concentration. The virus recovery, DNA depletion and protein
depletion was calculated. The results are shown in Table 4 and
Table 5.
TABLE-US-00004 TABLE 4 Analysis results Amount of Amount of Amount
of Sample virus [.mu.g HA] DNA [.mu.g] protein [.mu.g] Start
Material 600 555 4300 Flow-through 582 233 780 fraction
TABLE-US-00005 TABLE 5 Virus recovery, protein and DNA depletion
Virus recovery DNA depletion Protein depletion [%] [%] [%] 97 42
80
[0137] The results show that good protein depletion is obtained
which is the main function of this purification step. The DNA
depletion is lower, and this can be explained by the presence of
high molecular weight DNA in this particular sample.
[0138] DNA with molecular weight up to around 500 base pairs are
efficiently removed while the larger DNA cannot enter the pore
structure and bind to the positively charged octyl amine ligands
situated in the inner part of the particles.
[0139] A second purification step such as a conventional anion
exchange step could remove the larger DNA in this case.
[0140] Alternatively the sample can be treated with a nuclease such
as BENZONASE.RTM. to reduce the molecular weight to well below 500
base pairs and in that way obtain almost complete DNA and protein
depletion when the influenza virus sample is passed through a
column packed with particles of the present invention.
[0141] It is apparent that many modifications and variations of the
invention as hereinabove set forth may be made without departing
from the spirit and scope thereof. The specific embodiments
described are given by way of example only, and the invention is
limited only by the terms of the appended claims.
* * * * *