U.S. patent application number 10/723362 was filed with the patent office on 2004-04-29 for method for anion-exchange adsorption and anion-exchangers.
Invention is credited to Andersson, Mikael, Belew, Makonnen, Gustavsson, Jan, Johansson, Bo-Lennart, Maloisel, Jean-Luc.
Application Number | 20040079702 10/723362 |
Document ID | / |
Family ID | 20417786 |
Filed Date | 2004-04-29 |
United States Patent
Application |
20040079702 |
Kind Code |
A1 |
Johansson, Bo-Lennart ; et
al. |
April 29, 2004 |
Method for anion-exchange adsorption and anion-exchangers
Abstract
A method for the removal of a substance carrying a negative
charge and being present in an aqueous liquid (I). The method
comprises the steps of: (i) contacting the liquid with a matrix
carrying a plurality of ligands comprising a positively charged
structure and a hydrophobic structure, and (ii) desorbing the
substance. The characterizing feature is that (I) each of said
ligands together with a spacer has the formula:
--SP---[Ar--R.sub.1--N.sup.+(R.sub.2R.sub.3R.sub.4)] where (A)
[Ar--R.sub.1--N.sup.+(R.sub.2R.sub.3R.sub.4)] represents a ligand
a) Ar is an aromatic ring, b) R.sub.1 is [(L).sub.nR'.sub.1].sub.m
where n and m are integers selected amongst zero or 1; L is amino
nitrogen, ether oxygen or thioether sulphur; R'.sub.1 is a linker
selected among 1) hydrocarbon groups; 2) --C(.dbd.NH)--; c)
R.sub.2-4 are selected among hydrogen and alkyls; (B) SP is a
spacer providing a carbon or a heteroatom directly attached to
Ar--R.sub.1--N.sup.+(R.sub.2R.sub.3R.sub.4); (C)--- represents that
SP replaces a hydrogen in (Ar--R.sub.1--N.sup.+(R.s-
ub.2R.sub.3R.sub.4); (D)-- represents binding to the matrix; and
(II) desorption. There is also described (a) anion-exchangerrs
having high breakthrough capacities, (b) a screening method and (c)
a desalting protocol.
Inventors: |
Johansson, Bo-Lennart;
(Uppsala, SE) ; Andersson, Mikael; (Uppsala,
SE) ; Gustavsson, Jan; (Uppsala, SE) ; Belew,
Makonnen; (Uppsala, SE) ; Maloisel, Jean-Luc;
(Enebyberg, SE) |
Correspondence
Address: |
AMERSHAM BIOSCIENCES
PATENT DEPARTMENT
800 CENTENNIAL AVENUE
PISCATAWAY
NJ
08855
US
|
Family ID: |
20417786 |
Appl. No.: |
10/723362 |
Filed: |
November 26, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10723362 |
Nov 26, 2003 |
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10130958 |
Sep 16, 2002 |
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6702943 |
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10130958 |
Sep 16, 2002 |
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PCT/EP00/11605 |
Nov 22, 2000 |
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Current U.S.
Class: |
210/638 ;
210/683; 210/691 |
Current CPC
Class: |
B01J 41/09 20170101;
B01D 15/327 20130101; C02F 1/42 20130101; B01J 41/20 20130101 |
Class at
Publication: |
210/638 ;
210/683; 210/691 |
International
Class: |
B01D 011/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 22, 1999 |
SE |
9904197-2 |
Claims
1. A method for the removal of a substance carrying a negative
charge and being present in an aqueous liquid (I), said method
comprising the steps of (i) contacting the liquid with a matrix
carrying a plurality of ligands comprising a positively charged
structure (anion-exchanger) and a hydrophobic structure under
conditions permitting binding between the ligands and the
substance, and (ii) desorbing said substance from said matrix,
characterized in that (I) each of said ligand plus a spacer has the
formula: --SP---[Ar--R.sub.1--N.sup.+(R.sub.2R.sub.3R.sub.4)] where
(A) [Ar--R.sub.1--N.sup.+(R.sub.2R.sub.3R.sub.4)] represents a
ligand in which a) Ar is an aromatic ring, b) R.sub.1 is
[(L).sub.nR'.sub.1].sub.m where n and m are integers selected
amongst zero or 1; L is an amino nitrogen, an ether oxygen or a
thioether sulphur; R'.sub.1 is a bivalent linker group selected
among 1) linear, branched or cyclic hydrocarbon groups; 2)
--C(.dbd.NH)--; c) R.sub.2-4 are selected among hydrogen and lower
alkyls; (B) SP is a spacer providing a carbon, a nitrogen, a
sulphur or an oxygen directly attached to
Ar--R.sub.1--N.sup.+(R.sub.2R.s- ub.3R.sub.4); (C)--- represents
that the spacer is replacing a hydrogen in
(Ar--R.sub.1--N.sup.+(R.sub.2R.sub.3R.sub.4); (D)-- represents
binding to the matrix; and (II) desorption in step (ii) is carried
out under anion-exchange conditions when the substance is a serine
protease and in particularly when R'.sub.1=--C(.dbd.NH)--.
2. The method of claim 1, characterized in that anion-exchanger (1)
is capable of (a) binding to the substance of interest in an
aqueous reference liquid (II) under anion-exchange condition at an
ionic strength corresponding to 0.3 M NaCl and, (b) permitting a
maximal break through capacity in the pH interval 2-12 for the
substance .gtoreq.200%, such as .gtoreq.300% or .gtoreq.500% or
.gtoreq.1000%, of the maximal break through capacity in the
pH-interval 2-12 of the substance for Q-Sepharose Fast Flow
(Amersham Pharmacia Biotech, Uppsala, Sweden), said
anion-exchangers having essentially the same ligand density and
break through capacities being determined under the same
conditions.
3. The method of any of claims 1-2, characterized in that m=1 and
R'.sub.1 is a bivalent linker group selected among linear, branched
or cyclic hydrocarbon groups that may be substituted and/or have a
carbon chan that is interrupted by ether oxygen, thioether sulphur
or amino nitrogen.
4. The method according to any of claims 1-3, characterized in that
the matrix with its plurality of ligands has a pKa.ltoreq.12 and/or
is a primary or secondary nitrogen.
5. The method of any of claims 1-4, characterized in that at least
one of Ar, SP, R'.sub.1 and R.sub.2-4, comprises one or more
electron acceptor-donor atoms or groups at a distance of 1-7 atoms
from the positive nitrogen in --N.sup.+(R.sub.2R.sub.3R.sub.4),
preferably said acceptor-donor atoms or groups participating in
hydrogen-bonding, and with the proviso that for Ar this atoms or
groups are not sp.sup.2-carbons in an aromatic structure.
6. The method of any of claims 5, characterized in that said (i)
electron donor-acceptor interaction is hydrogen bonding and/or (ii)
donor atoms/groups are selected among: (a) oxygen with a free pair
of electrons, such as in hydroxy, ethers, carbonyls, and esters
(--O-- and --CO--O--) and amides, (b) sulphur with a free electron
pair, such as in thioether (--S--), (c) nitrogen with a free pair
of electron, such as in amines, amides including sulphone amides,
(d) halogen (fluorine, chlorine, bromine and iodine), and (e) sp-
and sp.sup.2-hybridised carbons; and/or (iii) acceptor groups are
selected amongst groups that consists of a electron-deficient atom
such as hydrogen and/or an electronegative atom.
7. The method of any of claims 5-6, characterized in that at least
one of said one or more hydrogen-bonding atoms is present as a
branch group in SP or as a part of the chain in SP extending from
the base matrix to the ligand.
8. The method according to any of claims 1-7, characterized in that
SP contains (a) a carbon atom with preference for a carbonyl carbon
or an sp.sup.3-hybridised carbon; or (b) a nitrogen atom with
preference for an amino or an amido nitrogen; or (c) a sulphur atom
with preference for a thioether sulphur atom; or (d) an oxygen,
with preference for an ether oxygen atom; which is directly
attached to the ligand Ar--R.sub.1--N.sup.+(R.sub.2R.sub.3R.sub.4),
with the proviso that items (b)-(d) only apply when the spacer
binds to Ar or R.sub.1.
9. The method of any of claims 1-2, characterized in that n=0, m=1,
R'.sub.1=--C(.dbd.NH)--, R.sub.2-4=hydrogen, Ar=p-C.sub.6H.sub.4--,
SP is attached to Ar via a secondary amino nitrogen, such as
--NH--.
10. The method of any of claims 1-9, characterized in that the
ionic strength during the adsorption/binding step (i) is larger or
equal with the ionic strength of 0.25 M NaCl water solution.
11. The method of any of claims 1-10, characterized in that the pH
of aqueous liquid (I) is .ltoreq.pKa+2, such as .ltoreq.pKa+1, of
the anion-exchanger or of an anion-exchanger ligand present in the
anion-exchanger.
12. The method of any of claims 1-11, characterized in that the pH
of aqueous liquid (II) is different from the pH of aqueous liquid
(I) in order to decrease the negative charge of the substance.
13. The method of any of claims 1-12, characterized in that the
polarity of aqueous liquid (II) is lower than the polarity of
aqueous liquid (I).
14. The method of any of claims 1-13, characterized in that a
structural analogue of Ar--R.sub.1--N.sup.+(R.sub.2R.sub.3R.sub.4)
is present in aqueous liquid (II) in a larger concentration than in
aqueous liquid (I).
15. An anion-exchanger (1) comprising a plurality of anion-exchange
ligands each of which is attached via a spacer to a hydrophilic
base matrix, characterized in that (a) the ligands plus their
spacers comply with the formula:
--SP---[Ar--R.sub.1--N.sup.+(R.sub.2R.sub.3R.sub.4)] where the
symbols have the same meaning as in any of claims 1-10, and (b) the
anion-exchanger (1) has a maximal breakthrough capacity in the
pH-interval 2-13 for at least one reference proteins selected
amongst ovalbumin, conalbumin, bovine serum albumin,
.beta.-lactglobulin,.alpha.-- lactalbumin, lyzozyme, IgG, soybean
trypsin inhibitor (STI) which is .gtoreq.200%, such as .gtoreq.300%
or .gtoreq.500% or .gtoreq.1000% of the maximal breakthrough
capacity in the pH-interval 2-12 obtained for a Q-exchanger
(--CH.sub.2CH(OH)CH.sub.2N.sup.+(CH.sub.3).sub.3) (anion-exchanger
2), the support matrix, degree of substitution, counter-ion and
running conditions being the same for anion-exchanger (1) and
anion-exchanger (2).
16. The anion-exchanger of claim 15, characterized in that the
relative break-through capacity is measured under anion-exchanger
condition.
17. A method for testing (screening) the appropriateness of one or
more anion-exchangers for removing a substance from a liquid, said
method comprising the steps: (a) providing a library which
comprises (i) one or more anion-exchangers to be tested (exchangers
1, 2, 3, 4 . . . n; n=an integer >0) each of which
anion-exchangers differs with respect to kind of ligand (ligands 1,
2, 3, 4, . . . n), and (ii) a reference anion-exchanger having a
reference ligand, the support matrix etc being essentially the same
in the exchangers 1, 2, 3, 4 . . . n and in the reference
anion-exchanger; (b) determining the maximal breakthrough capacity
in the pH-interval 2-12 of exchanger 1 for the substance at a
predetermined condition; (c) determining the maximal breakthrough
capacity in the pH-interval 2-12 of the reference anion-exchanger
for the substance at the same condition as in step (b); (d)
concluding with the aid of the relation between the maximal
breakthrough capacities obtained in steps (b) and (c), if
anion-exchanger 1 is appropriate to use for removing the substance;
and (e) repeating, if necessary, steps (b)-(c) for at least one of
the exchangers 2, 3, 4 . . . n.
18. The method of claim 17, characterized in that the steps (b) and
(c) are carried out under anion-exchanger conditions.
19. A method for removing salt from a negatively charged substance,
preferably amphoteric, when present in a solution (liquid (I)),
which method comprises the steps of: (i) contacting liquid (I)
liquid with an anion-exchanger (1) that comprises a base matrix
carrying a plurality of ligands in which there is a positively
charged nitrogen under conditions permitting binding between the
anion-exchangerr and the substance, (ii) desorbing said substance
from said anion-exchanger by the use of a liquid (liquid (II)).
characterized in: (A) selecting anion-exchanger (1) among
anion-exchangers that are (a) capable of binding the substance of
interest in an aqueous reference liquid at an ionic strength
corresponding to 0.25 M NaCl; and (b) permitting a maximal
breakthrough capacity in the pH interval 2-12 for the substance
.gtoreq.200%, such as .gtoreq.300% or .gtoreq.500% or
.gtoreq.1000%, of the breakthrough capacity of the substance for
Q-Sepharose Fast Flow (anion-exchanger 2, Amersham Pharmacia
Biotech, Uppsala, Sweden), said anion-exchangers having essentially
the same ligand density and the breakthrough capacities being
determined under the same conditions; (B) adjusting the pH of
liquid (II) in step (ii) by the use of an acid-base pair to a value
that means a lower net positive charge on-the anion-exchanger
and/or a lower net negative or positive charge on the substance
thereby enabling elution at a lowered ionic strength compared to
liquid (I).
20. The method of claim 19, characterized in that at least one
member of the acid-base pair buffer has a vapour pressure that is
higher than the substance.
21. The method of any of claims 19-20, characterized in that the
substance in the liquid of low salt content obtained in step (ii)
is ionized in a mass spectrometer.
Description
FIELD OF INVENTION
[0001] The present invention relates a method for the removal of a
compound (=substance) carrying a negative charge from an aqueous
liquid (I). The method comprises the steps of
[0002] i) contacting the liquid with an anion-exchanger that
comprises a base matrix carrying a plurality of mixed mode
anion-exchange ligands comprising (a) a positively charged
structure and (b) a hydrophobic structure under conditions
permitting binding between the ligands and the substance, and
[0003] ii) desorbing said substance from said matrix by the use of
a liquid (II).
[0004] The invention also relates to novel anion-exchangers in
which there are anion-exchange ligands comprising both a
hydrophobic structure and a positively charged structure.
[0005] The terms "carrying a negative charge" and "negatively
charged" mean that the substance carries one or more negative
charges and/or has a negative net charge.
[0006] The terms "mixed mode anion-exchanger ligand" and "bimodal
anion-exchanger ligand", in the context of this invention, refer to
a ligand that is capable of providing at least two different, but
co-operative, sites which interact with the substance to be bound.
One of these sites gives an attractive type of charge-charge
interaction between the ligand and the substance of interest. The
second site typically gives electron acceptor-donor interaction
and/or hydrophobic interactions. Electron donor-acceptor
interactions include interactions such as hydrogen-bonding,
.pi.-.pi., charge transfer, dipole-dipole, induced dipole etc.
BACKGROUND TECHNOLOGY
[0007] The method defined above is employed in chromatographic
procedures utilizing monolithic matrices or particle matrices in
form of packed or fluidised beds, and also in batch-wise
procedures. The purpose of the procedures may be to purify a
substance carrying a negative charge, in which case the substance
is bound to the matrix, and, if necessary, further purified
subsequent to desorption from the matrix. Another purpose is to
remove an undesired substance that carries a negative charge from a
liquid. In this latter case, the liquid may be further processed
after having been contacted with the matrix in step (i). In both
cases and if so desired, the matrix may be reused after desorption
of the bound substance.
[0008] Other uses are assay procedures involving determination of
either the substance carrying the negative charge or of a substance
remaining in liquid I.
[0009] In previous anion-exchange adsorptions, the positively
charged ligands typically have comprised nitrogen structures, such
as primary, secondary, tertiary or quaternary ammonium structures.
In some instances the ligands had a dual or bimodal functionality
by comprising both a charged structure and a hydrophobic structure
which has required modifications of the desorption protocols.
[0010] Simmonds et al (Biochem. J. 157 (1976) 153-159); Burton et
al (J. Chromatog. A 814 (1998) 71-81); and Yon et al (Biochem. J.
151 (1975) 281-290) have described anion-exchanger ligands that
comprise saturated hydrocarbon groups.
[0011] Crowther et al (J. Chrom. 282 (1983) 619-628); Crowther et
al (Chromatographia 16 (1982) 349-353); Wongyai (Chromatographia
38(7/8) (1994) 485-490); Bischoff et al (J. Chrom. 270 (1983)
117-126) have described high pressure liquid chromatography of
olignucleotides and small molecules on reverse phases carrying
anion-exchanger ligands in which there is an aromatic
component.
[0012] See also Sasaki et al (J. Biochem. 86 (1979) 1537-1548) in
which a similar effect from an anion-exchanger based on a
hydrophobic matrix is discussed.
[0013] Serine proteases have been affinity adsorbed/desorbed
to/from matrices to which p-aminobenzamidine has been covalently
linked via the para amino group. See
[0014] Chang et al (J. Chem. Tech. Biotechnol. 59 (1994) 133-139)
who used an adsorption buffer in which the pH is higher and the
salt concentration is lower than in the desorption buffer;
[0015] Lee et al (J. Chromatog. A 704 (1995) 307-314) who changed
the pHs in the same manner as Chang et al but without change in
salt concentration; and
[0016] Khamlichi et al., J. Chromatog. 510 (1990) 123-132 who used
ligand analogues for desorption. The pH-values during adsorption
and desorption were the same. Desorption by only increasing the
ionic strength failed.
[0017] None of the methodologies in these three articles describe
successful desorption processes under anion-exchange
conditions.
[0018] WO 9729825 (Amersham Pharmacia Biotech AB) discloses mixed
mode anion-exchangers providing interactions based on charges and
hydrogen-bonding involving oxygen and amino nitrogen on 2-3
carbons' distance from positively charged amine nitrogen. The
publication is based on the discovery that this kind of ligands can
give anion-exchangers that require relatively high ionic strengths
for eluting bound substances.
[0019] WO 9965607 (Amersham Pharmacia Biotech AB) discloses
cation-exchangers in which there are mixed mode ligands that
require relatively high ionic strengths for eluting bound
substances.
[0020] WO 9729825 (U.S. Pat. No. 6,090,288) and WO 9965607, which
give anion and cation exchange ligands, respectively, that require
relatively high elution ionic strength are incorporated by
reference.
[0021] WO 9808603 (Upfront Chromatography) discloses separation
media of the general structure M-SP1-L where M is a support matrix
that may be hydrophilic, SP1 is a spacer and L comprises a mono- or
bicyclic homoaromatic or heteroaromatic moiety that may be
substituted (a homoaromatic moiety comprises an aromatic ring
formed only by carbon atoms). The substituents are primarily
acidic. The separation medium is suggested for the adsorption of
proteins, in particular immunoglobulins, by hydrophobic
interactions rather than ion-exchange (salt concentration up to 2
M).
[0022] WO 9600735, WO 9609116 and U.S. Pat. No. 5,652,348 (Burton
et al) disclose separation media based on hydrophobic interaction.
Adsorption and desorption are supported by increasing or
decreasing, respectively, the salt concentration of the liquid or
changing the charge on the ligand and/or the substance to be
adsorbed/desorbed by changing pH. The ligands typically comprise a
hydrophobic part that may comprise aromatic structure. Some of the
ligands may in addition also contain a chargeable structure for
permitting alteration of the hydrophobic/hydrophilic balance of the
media by a pH change. The chargeable structure may be an amine
group.
[0023] U.S. Pat. No. 5,789,578 (Burton et al) suggests to
immobilise a thiol containing ligand, such as 3-mercaptopropionic
acid, gluthathione etc, by addition of the thiol group over
carbon-carbon double bond attached to a support matrix. The
inventors in this case neither employ nor suggest the use of the
material obtained for anion-exchange adsorptions.
[0024] Dipolar adsorbents prepared by coupling sulphanilic acid
using epichlorohydrin has been described
(ligand+spacer=--CH.sub.2CHOHCH.sub.2N-
.sup.+H.sub.2C.sub.6H.sub.4SO.sub.3.sup.-) (Porat et al., J.
Chromatog. 51 (1970) 479-489; and Ohkubo et al., J. Chromatog. A,
779 (1997), 113-122). The articles do not disclose a separation
method in which the ligand is positively, and the substance to be
removed negatively, charged.
[0025] WPI Abstract Accession No. 86-312313 (=DD-A-237844, Behrend
et al) describes the use of 2,4,6-trihalo-1,3,5-triazine for
binding substances RHNR'X to carriers inter alia to cellulose. R is
hydrogen, aryl or alkyl. R' alkylene or arylene. X is carboxy,
sulphonyl, phosphate, phosphonate, boronate, etc.
THE OBJECTIVES OF THE INVENTION
[0026] The objectives of the present invention are:
[0027] a) to achieve adsorption/binding of negatively charged
substances, such as proteins, to anion-exchangers at relatively
high ionic strengths;
[0028] b) to provide anion-exchange media that can have a reduced
ligand content while retaining a sufficient capacity to bind target
substances;
[0029] c) to enable elution/desorption within broad ionic strength
intervals of substances adsorbed/bound to an anion-exchangerr;
[0030] d) to design anion-exchangers which have high breakthrough
capacities, good recovery of proteins (often 95% or higher)
etc;
[0031] e) to design anion-exchangers that can resist regeneration
and/or cleaning with alkaline and or acidic milieu without
significant loss of chromatographic properties;
[0032] f) to obviate extensive dilutions of samples of high ionic
strength that are to be used in processes requiring a lowered ionic
strength;
[0033] g) to provide simplified desalting procedures;
[0034] h) to provide a method for selecting anion-exchangers or
anion-exchange ligands that, when bound to a support matrix, are
equal or better than a conventional reference anion-exchanger in
adsorbing a negatively charged substance;
[0035] i) to provide simplified processes involving
anion-exchangers, for instance to improve productivity and/or
reduce the costs for process equipment and investments;
[0036] j) to provide anion-exchangerrs that are adapted to
preparative applications, for instance in large scale processes in
which a sample volume (=liquid (I)) larger than a litre are applied
and processed on an anion-exchangerr;
[0037] k) to provide opportunities for novel combinations of
separation principles based elution of anion-exchanger adsorbents
at high salt concentration, for instance hydrophobic interaction
adsorption after an ion exchange step.
[0038] These objectives are based on the recognition that ion
exchangers adsorbing at high salt concentrations and high ionic
strengths have benefits. This is contrary to traditional ion
exchangers which have utilized high salt concentrations and high
ionic strengths in the desorption step.
[0039] The Invention
[0040] The present inventors have discovered that ligands
containing an aromatic ring in the proximity of the positively
charged atom may provide anion-exchangers that at least partially
meet these objectives. The present inventors have also discovered
that inclusion of other atoms or groups participating in
electron-donor acceptor interactions in the proximity of the
positively charged atom in anion-exchange ligands may enhance the
strength of the interaction between the substance and the
adsorbent.
[0041] By proximity in this context is meant that the distance
between this kind of atoms or groups and the positively charged
atom is 1-7 atoms, with preference for 2, 3, 4 and 5 atoms.
[0042] Electron donor-acceptor interactions mean that an
electronegative atom with a free pair of electrons acts as a donor
and bind to an electron-deficient atom that acts as an acceptor for
the electron pair of the donor. See Karger et al., An Introduction
into Separation Science, John Wiley & Sons (1973) page 42.
Illustrative examples of donor atoms/groups are:
[0043] (a) oxygen with a free pair of electrons, such as in
hydroxy, ethers, carbonyls, and esters (--O-- and --CO--O--) and
amides,
[0044] (b) sulphur with a free electron pair, such as in thioethers
(--S--),
[0045] (c) nitrogen with a free pair of electron, such as in
amines, amides including sulphone amides], cyano,
[0046] (d) halo (fluorine, chlorine, bromine and iodine), and
[0047] (e) sp- and sp.sup.2-hybridised carbons.
[0048] Typical acceptor atoms/groups are electron deficient atoms
or groups, such as metal ions, cyano, nitrogen in nitro etc, and
include a hydrogen bound to an electronegative atom such as HO-- in
hydroxy and carboxy, --NH-- in amides and amines, HS-- in thiol
etc.
[0049] The first aspect of the invention is thus a method for the
removal of a substance that carries a negative charge and is
present in an aqueous liquid (I). The method comprises step (i) and
step (ii) as defined above. The main characterizing features are
that
[0050] the ligands with a spacer have the formula:
--SP---[Ar--R.sub.1--N.sup.+(R.sub.2R.sub.3R.sub.4)]
[0051] desorption in step (ii) is performed under anion-exchange
conditions when R.sub.1 is --C(.dbd.NH)-- and the substance is a
serine protease. For desorption of other substances see under the
heading "Desorption" below.
[0052] Anion-exchange ligands as contemplated in the context of the
present invention typically have molecular weights <1000, such
as <700 daltons excluding the molecular weight contribution of
halogens that may be present.
[0053] The Anion-Exchanger
[0054] In the formula
--SP---[Ar--R.sub.1--N.sup.+(R.sub.2R.sub.3R.sub.4)]- :
[0055] [Ar--R.sub.1--N.sup.+(R.sub.2R.sub.3R.sub.4)] represents a
ligand structure
[0056] SP is a spacer that attaches the ligand
[Ar--R.sub.1--N.sup.+(R.sub- .2R.sub.3R.sub.4)] to the base
matrix.
[0057] --- represents that the spacer replaces a hydrogen in
[Ar--R.sub.1--N.sup.+(R.sub.2R.sub.3R.sub.4)].
[0058] -- represents a link to the base matrix.
[0059] Henceforth the terms lower hydrocarbon group and lower alkyl
(including lower alkylene) mean C.sub.1-10, such as C.sub.1-6,
saturated hydrocarbon groups that optionally are substituted and
have carbon chains as discussed below for R.sub.2-4. See below.
[0060] The preferred positively charged structure
--N.sup.+(R.sub.2R.sub.3- R.sub.4) in the ligand has a pKa value
that is below 12.0, such as below 10.5. This typically means that
the ligand is a primary or secondary ammonium group. For
measurement of pKa see under the heading "Adsorption" below.
[0061] In the ligand
[Ar--R.sub.1--N.sup.+(R.sub.2R.sub.3R.sub.4)]:
[0062] a) Ar is an aromatic ring structure,
[0063] b) R.sub.1 is [(L).sub.nR'.sub.1].sub.m where
[0064] n and m are integers selected amongst 0 or 1, with
preference for (a) m=0 or (b) n=0 when m=1;
[0065] L is an amino nitrogen, an ether oxygen or a thioether
sulphur;
[0066] R'.sub.1 is a bivalent linker group selected among
[0067] 1) linear, branched or cyclic hydrocarbon groups;
[0068] 2) --C(.dbd.NH)--;
[0069] c) R.sub.2, R.sub.3, and R.sub.4 are selected among hydrogen
and lower alkyls.
[0070] The positive charge on the nitrogen may be more or less
delocalised to atoms or groups in Ar--R.sub.1 and/or R.sub.2-4. For
R'.sub.1 equals --C(.dbd.NH)-- (m=1), one, two or three of
R.sub.2-4 are preferably hydrogen.
[0071] The Group Ar
[0072] The aromatic ring structure Ar may comprise one or more
aromatic rings, for instance a phenyl, a biphenyl or a naphthyl
structure and other aromatic ring systems that comprise fused rings
or bicyclic structures. Aromatic rings may be heterocyclic, i.e.
contain one or more nitrogen, oxygen or sulphur atoms. The ring may
have further substituents in addition to R.sub.1 and a possible
spacer. These other substituents may contain an electron donor or
acceptor atom or group, for instance enabling hydrogen-bonding.
[0073] Illustrative Ar-groups are: hydoxyphenyl (2-, 3- and 4-),
2-benzimadozolyl, methylthioxyphenyl (2-, 3- and 4-), 3-indolyl,
2-hydroxy-5-nitrophenyl, aminophenyl (2-, 3- and 4-),
4-(2-aminoethyl)phenyl, 3,4-dihydroxyphenyl, 4-nitrophenyl,
3-trifluoromethylphenyl, 4-imidazolyl, 4-aminopyridine,
6-aminopyrimidyl, 2-thienyl, 2,4,5-triaminophenyl,
4-aminotriazinyl-, 4-sulphoneamidophenyl etc.
[0074] The Group R'.sub.1
[0075] For m=1, R'.sub.1 is a bivalent hydrocarbon group or
--C(.dbd.NH)--. In preferred bivalent hydrocarbon groups there are
typically one or more atoms or groups participating in
hydrogen-bonding or other electron acceptor-donor interactions as
defined above. Thus R'.sub.1 in form of a hydrocarbon group may be
substituted with
[0076] a) one or more primary ammonium groups (--N.sup.+H.sub.3) in
which one or more of the hydrogens may be replaced with lower
alkyl, and/or
[0077] b) one or more hydroxy (--OH) in which the hydrogen may be
replaced with a lower alkyl, and/or
[0078] the carbon chain in the hydrocarbon group may be interrupted
at one or more positions by thioether sulphur, ether oxygen or
amine nitrogen
[0079] The preferred hydrocarbon chains between Ar and
--N.sup.+(R.sub.2R.sub.3R.sub.4) have a length of 1-20 atoms (in
R'.sub.1).
[0080] Typical R'.sub.1s are selected amongst --CH.sub.2--,
--CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH(CH.sub.3)--, --CH.sub.2OCH.sub.2--, --C(.dbd.O)--,
--C(.dbd.NH)--,
--CH.sub.2N.sup.+(C.sub.2H.sub.5).sub.2CH.sub.2CH.sub.2--- ,
--CH.sub.2CH.sub.2(OCH.sub.2CH.sub.2--).sub.n' (where n' is an
integer larger than 1, such as .ltoreq.100 for instance .ltoreq.25
or .ltoreq.10) etc. For cases in which n=m=1, R'.sub.1 is
preferably a bivalent hydrocarbon group, such as (a) --CH.sub.2--,
(b) --CH.sub.2CH.sub.2-- possibly substituted with a hydroxy and/or
a hydroxy lower alkyl or lower alkyl (for instance hydroxy methyl
or methyl, respectively) at one or two of its carbon atoms, (c)
--(CH.sub.2).sub.n"SCH.sub.2).sub.n'" (where n" and n'"
independently are integers 1-3, such as 1 and 2, respectively), and
(d) --CH.sub.2N.sup.+(C.sub.2H.sub.5).sub.2CH.sub.2CH.sub.2--, (e)
--CH.sub.2CH.sub.2(OCH.sub.2CH.sub.2--).sub.n' (where n' is 1, 2 or
3).
[0081] The Group R.sub.2-4
[0082] In a preferred case two or three of R.sub.2-4 are hydrogen
if the spacer is attached to R'.sub.1 or Ar, and three if the
spacer is attached to the nitrogen. Thus, the anion-exchanger
ligands are preferably primary or secondary amine/ammonium
groups
[0083] R.sub.2-4 alkyl groups may contain hydroge n-bonding atoms
as defined above. Thus, a hydrogen in R.sub.2-4 may be replaced at
one or more positions with --OR".sub.1 and/or
--N.sup.+(R'.sub.2R'.sub.3R'.sub.4- ), in which R'.sub.2-4 and
R".sub.1 are hydrogen or lower alkyl. In addition, a carbon chain
in R.sub.2-4 may be interrupted at one or more positions with ether
oxygen, thioether sulphur or amino nitrogen.
[0084] One or more of R.sub.2-4 may be a bivalent alkylene forming
a 5- or 6-membered ring by having one end attached to the nitrogen
and the other replacing a hydrogen in R.sub.1 or one of the
remaining R.sub.2, R.sub.3, and R.sub.4.
[0085] If not hydrogen, one or more of R.sub.2-4, R'.sub.2-4 and
R".sub.1 groups are often selected amongst lower alkyl groups
having 1-3 carbon atoms.
[0086] In variants of R.sub.2-4, R'.sub.1-4 and R".sub.1, each
sp.sup.3-hybridised carbon carries at most one atom selected from
amino nitrogen, thioether sulphur, and ether and hydroxy
oxygens.
[0087] The Spacer (SP)
[0088] The spacer (SP) starts at the base matrix and extends (a) to
the nitrogen in --N.sup.+(R.sub.2R.sub.3R.sub.4) by replacing one
of R.sub.2-4, or (b) to the chain of atoms connecting
--N.sup.+(R.sub.2R.sub.3R.sub.4) with Ar by replacing a hydrogen in
R.sub.1, or (c) to an aromatic ring in Ar by replacing a hydrogen
in Ar. SP always replaces a hydrogen in the ligand
[Ar--R.sub.1--N.sup.+(R.sub.2- R.sub.3R.sub.4)]. It is thus
presumed that if the spacer binds directly to the nitrogen or the
sulphur atom in the ligand, then the replaced group R.sub.2-5 has
been hydrogen.
[0089] The spacer as such is conventional as in traditional ion
exchangers and may thus comprise linear, branched, cyclic
saturated, unsaturated and aromatic hydrocarbon groups (e.g. with
up to 1-20, such as 1-10 carbon atoms). As discussed above for
R.sub.1-5, hydrocarbon groups may carry hydroxy groups, alkoxy and
aryloxy and the corresponding thio analogues, and/or amino groups.
Carbon chains may at one or more positions be interrupted by amino
nitrogen, ether oxygen, thioether sulphur as discussed above for
R.sub.1-4. There may also be carbonyl groups, such as in amides and
ketones, and other groups having the comparable stability against
hydrolysis. At most one atom selected from oxygen, sulphur and
nitrogen is preferably bound to one and the same
sp.sup.3-hybridised carbon atom.
[0090] SP may provide one or more electron donor or acceptor atoms
or groups enhancing binding of the substance to the anion-exchanger
as discussed above, for instance by participating in
hydrogen-bonding. These atoms or groups may (a) be part of or
attached directly to the chain of atoms in the spacer extending
from the base matrix to the ligand or (b) be part of a branch group
attached to this chain. A branch group in this context is a group
which
[0091] is attached directly to the chain of atoms referred to in
the preceding paragraph, and
[0092] comprises an atom or group participating in electron-donor
acceptor interaction, such as hydrogen-bonding.
[0093] In a preferred variant, the part of SP binding directly to
Ar--R.sub.1--N.sup.+(R.sub.2R.sub.3R.sub.4) is:
[0094] a carbon with preference for a carbonyl carbon or an
sp.sup.3-hybridised carbon; or
[0095] a nitrogen with preference for an amino or amido nitrogen;
or
[0096] a sulphur with preference for a thioether sulphur; or
[0097] an oxygen, with preference for an ether oxygen;
[0098] with the proviso that SP is attached to a carbon in
[Ar--R.sub.1--N.sup.+(R.sub.2R.sub.3R.sub.4)] for (b)-(d).
[0099] Typical structures in SP that are attached directly to
[Ar--R.sub.1--N.sup.+(R.sub.2R.sub.3R.sub.4)] are: --CH.sub.2--,
--CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH(CH.sub.3)--, --C(CH.sub.3).sub.2--,
--C(CH.sub.2CH.sub.3).su- b.2--, --C(OCH.sub.3).sub.2--,
--CH.sub.2OCH.sub.2--, --CH.sub.2SCH.sub.2--,
--CH.sub.2NHCH.sub.2--, --CH.sub.2O--, --CH.sub.2CH.sub.2O--,
--CH.sub.2S--, --CH.sub.2CH.sub.2S--, --CH.sub.2NH--, --CONH--,
--NHCO--, --CONH(CH.sub.2).sub.2SCH.sub.2--,
--NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CONH--,
--CH.sub.2CH.sub.2NH--,
--CH.sub.2CH(OH)CH.sub.2OCH.sub.2CH(OH)CH.sub.2O-- -, (the right
valence binds to Ar--R.sub.1--N.sup.+(R.sub.2R.sub.3R.sub.4)- ).
The remaining part of the spacer may be of the same kind as in
traditional ion-exchangers.
[0100] The spacer may be introduced according to conventional
covalent coupling methodologies including also techniques to be
developed in the future. Illustrative coupling chemistries involve
epichlorohydrin, epibromohydrin, allyl-glycidylether, bis-epoxides
such as butanedioldiglycidylether, halogen-substituted aliphatic
substances such as di-chloro-propanol, divinyl sulfone,
carbonyldiimidazole, aldehydes such as glutaric dialdehyde,
quinones, cyanogen bromide, periodates such as sodium-meta
periodate, carbodiimides, chloro-triazines, sulfonyl chlorides such
as tosyl chlorides and tresyl chlorides, N-hydroxy succinimides,
oxazolones, maleimides, 2-fluoro-1-methylpyridinium
toluene-4-sulfonates, pyridyl disulfides and hydrazides.
[0101] The Base Matrix
[0102] The base matrix is based on organic and/or inorganic
material.
[0103] The base matrix is preferably hydrophilic and in the form of
a polymer, which is insoluble and more or less swellable in water.
Hydrophobic polymers that have been derivatized to become
hydrophilic are included in this definition. 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 polymerization of
monomers exhibiting groups which can be converted to OH, or by
hydrophilization of the final polymer, e.g. by adsorption of
suitable compounds, such as hydrophilic polymers.
[0104] Suitable inorganic materials to be used in base matrices are
silica, zirconium oxide, graphite, tantalum oxide etc.
[0105] Preferred matrices lack groups that are unstable against
hyrolysis, such as silan, ester, amide groups and groups present in
silica as such. This in particular applies with respect to groups
that are in direct contact with the liquids used.
[0106] The matrix may be porous or non-porous. This means that the
matrix may be fully or partially permeable (porous) or completely
impermeable to the substance to be removed (non-porous), i.e. the
matrix should have a Kav in the interval of 0.40-0.95 for
substances to be removed. This does not exclude that Kav may be
lower, for instance down to 0.10 or even lower for certain
matrices, for instance having extenders. See for instance WO
9833572 (Amersham Pharmacia Biotech AB).
[0107] In a particularly interesting embodiment of the present
invention, the 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.
[0108] An interesting form of matrices has densities higher or
lower than the liquid. 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 9218237 (Amersham Pharmacia Biotech AB) and WO
9200799 (Kem-En-Tek).
[0109] The term hydrophilic matrix means that the accessible
surface of the matrix is hydrophilic in the sense that aqueous
liquids are able to penetrate the matrix. 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, ester,
ether of lower alkyls (such as (--CH.sub.2CH.sub.2O--).sub.nH where
n is an integer).
[0110] The level of anion-exchange ligands in the anion-exchangers
used in the invention is usually selected in the interval of
0.001-4 mmol/ml matrix, such as 0.002-0.5 mmol/ml matrix, with
preference for 0.005-0.3 mmol/ml matrix. Possible and preferred
ranges are among others determined by the kind of matrix, ligand,
substance to be removed etc. Thus, the level of anion-exchange
ligands is usually within the range of 0.01-0.3 with preference for
0.01-0.1 mmol/ml for agarose based matrices. For dextran based
matrices the interval is typically 0.01-0.6 mmol/ml matrix with
subrange being 0.01-0.2 mmol/ml matrix. In the certain variants,
for instance when R.sub.1 is --C(.dbd.NH)--, the level of the mixed
mode ligand is often at the lower half part of these intervals. In
these variants of the invention the levels of anion-exchange ligand
thus are smaller than 0.150 mmol per ml matrix and/or smaller than
1 mmol per gram dry weight of matrix. The expression "mmol per ml
matrix" refers to fully sedimented matrices saturated with water.
The capacity range refers to the capacity of the matrix in fully
protonated form to bind chloride ions. It includes a possible
contribution also from positively charged groups other than the
nitrogen in the ligand [Ar--R.sub.1--N.sup.+(R.sub.-
2R.sub.3R.sub.4)], for instance in the spacer or in any of the
groups R.sub.2-4, R'.sub.1-4 and R".sub.1.
[0111] Stability of the Novel Anion-Exchangers
[0112] The inventive anion-echangers/anion-exchange ligands should
resist the conditions typically applied in processes comprising
anion-exchange absorptions. As a general rule, this means that an
anion-exchanger according to the invention should be able to resist
0.1 or 1 M NaOH in water for at least 10 hours with essentially no
reduction in total ion binding capacity. By "essentially no
reduction in total ion binding capacity" is contemplated that the
total ion binding capacity is reduced at most by 10%. In structural
terms this means that the anion-exchange ligand in preferred
variants should only contain structures selected among pure
hydrocarbon groups (including homoaromatic and heteroaromatic
structures), thioether and ether groups, disulphide groups, hydroxy
groups, sulphoxide or sulphone groups, carboxamide groups, sulphone
amide groups, acetal and ketal groups and groups of similar
hydrolytic stability.
[0113] Selection of anion-exchanger to be used for removal of a
particular substance. In the preferred variants of the inventive
method the anion-exchanger is selected among anion-exchangers that
adsorb the particular substance at relatively high ionic strengths.
The anion-exchanger (l) used should thus be capable of:
[0114] (a) binding the substance of interest in an aqueous
reference liquid (II) at an ionic strength corresponding to 0.25 M
NaCl, and
[0115] (b) permitting a maximal break through capacity somewhere in
the pH interval 2-12 for the substance .gtoreq.200%, such as
.gtoreq.300% or .gtoreq.500% or .gtoreq.1000%, of the break through
capacity of the substance for a conventional anion-exchanger (2)
(reference anion-exchanger).
[0116] Primarily these percentage figures apply to measurements
made during anion-exchanger conditions. The reference liquid
typically consists of a buffered aqueous liquid and the
substance.
[0117] An indirect way of finding this kind of anion-exchangers is
to screen for anion-exchangers that have an increased maximal
elution ionic strength for the substance (carrying the negative
charge) compared to the elution ion strength required for the same
substance on a conventional anion-exchanger. Thus, the
anion-exchanger may be selected among those requiring more than
125%, such as more than 140% or more than 200%, of the elution
ionic strength required for a standard anion-exchanger at the
particular conditions applied for a selected substance to be
removed. See WO 9729825 (Amersham Pharmacia Biotech AB)
[0118] The comparisons above refer to measurements performed under
essentially the same conditions for anion-exchangerr (1) and (2),
i.e. essentially the same support matrix (support material, bead
size, pore sizes, pore volume, packing procedure etc), pH,
temperature, solvent composition, number of charged ligand having
the formula given above etc. The breakthrough capacities are
measured at the same relative concentration of the substance in the
flow through (for instance c/c.sub.0=10%, for c/c.sub.0 see the
experimental part). The spacer and coupling chemistry may differ.
Certain kinds of coupling chemistries may lead to cross-linking of
the support matrix resulting in a more rigid matrix. In this case
the flow conditions at which the comparison is made is selected at
a level where the matrix is essentially non-compressed.
[0119] As a reference ion-exchanger, the commercially available
anion-exchanger Q-Sepharose Fast Flow (Amersham Pharmacia Biotech,
Uppsala, Sweden) was selected in the context of the present
invention. This anion-exchanger is a strong anion-exchanger whose
ligand and spacer arm structure are:
--O--CH.sub.2CHOHCH.sub.2OCH.sub.2CHOHCH.sub.2N.sup.+(CH.sub.3).sub.3.
[0120] Its chloride ion capacity is 0.18-0.25 mmol/ml gel. The base
matrix is epichlorohydrin cross-linked agarose in beaded form. The
beads have diameters in the interval 45-165 .mu.m. The exclusion
limit for globular proteins is 4.times.10.sup.6.
[0121] Best Mode
[0122] Based on experimental results achieved at the priority date
the best ligand was considered to have n=0, m=1,
R'.sub.1--N.sup.+H.sub.3=--C- (.dbd.N.sup.+H.sub.2)NH.sub.2, and
Ar=p-C.sub.6H.sub.4--. The formula
--C(.dbd.N.sup.+H.sub.2)NH.sub.2, is only a representation of a
group in which the charge in reality is delocalised over the
--N--C--N-- grouping and a hydrogen is binding accordingly. SP was
attached to Ar via the nitrogen atom of an amido group. See further
the experimental part for information about the best mode for
spacer and base matrix.
[0123] During the priority year the experimental support has been
extended to a large number of various ligands. The best mode varies
with substance of interest and is apparent from the experimental
part in which the best ligands discovered so far are given.
[0124] Adsorption/Desorption
[0125] The adsorption and/or desorption steps may be carried out as
a chromatographic procedure with the anion-exchange matrix in a
monolithic form or as particles in the form of a packed or a
fluidised bed. For particulate matrices, these steps may be carried
out in a batch-wise mode with the particles being more or less
completely dispersed in the liquid (e.g. in a fluidised/expanded
bed).
[0126] The liquids used in steps (i) and (ii) are aqueous, i.e.
water, possibly mixed with a water-miscible solvent.
[0127] Adsorption
[0128] During adsorption, a liquid sample containing the negatively
charged substance is contacted with the anion-exchanger defined
above under conditions permitting adsorption (binding), preferably
by anion-exchange. In other words the substance is at least
partially negative and the ligand at least partially positive.
[0129] By anion-exchanger is contemplated that the substance to be
removed carries a negative charge and the anion-exchangerr is
positively charged (=anion-exchanger conditions). For an amphoteric
substance that is present in an aqueous liquid this means a
pH.gtoreq.pl-0.5, preferably pH.gtoreq.pl.
[0130] In the preferred variants, weak anion-exchangers (preferably
present as a primary or secondary amine group in the
anion-exchangerr) are buffered to a pH within the interval
.ltoreq.pKa+2, preferably .ltoreq.pKa+1. The lower limit can extend
down to at least pH=1 or 2 and is primarily determined by the
stability of the anion-exchanger in acidic milieu and by the
isoelectric point (pl) and stability of the substance to be
removed. The pKa-value of an anion-exchanger is taken as the pH at
which 50% of its titratable groups are neutralized.
[0131] The ionic strength (measured as salt concentration or
conductivity) is typically below the elution ionic strength for the
particular combination of ion-exchanger, substance to be bound,
temperature and pH, solvent composition etc. One of the benefits of
the invention is that by using the mixed mode anion-exchangers
defined above, it will be possible to carry out adsorption/binding
also at elevated ionic strengths compared to what normally has been
done for conventional ion-exchangers (reference anion-exchangerrs).
By matching the anion-exchanger with the substance to be removed,
the adsorption may be carried out at an ionic strength that is
higher than when using the reference ion-exchanger (measured at the
same pH and otherwise the same conditions). Depending on the
anion-exchanger used the ionic strength may be more than 25% higher
such as more than 40% higher. Some combinations of anion-exchanger
and substance to be removed may permit adsorption at more than 100%
higher ionic strength than when using the corresponding reference
ion-exchanger according to above.
[0132] In absolute figures the discussion in the preceding
paragraph means that adsorption according to the present invention
may be performed at ionic strengths above or below 15 or 20 mS/cm.
The ionic strength may exceed 30 mS/cm and in some cases even
exceed 40 mS/cm. Useful ionic strengths often correspond to NaCl
concentrations (pure water) .gtoreq.0.1 M, such as .gtoreq.0.3 M or
even .gtoreq.0.5 M. The conductivity/ionic strength to be used will
depend on the ligand used, its density on the matrix, the substance
to be bound, its concentration etc.
[0133] Depending on the anion-exchanger selected, breakthrough
capacities .gtoreq.200%, such as .gtoreq.300% or .gtoreq.500% and
even .gtoreq.1000% of the breakthrough capacity obtained for a
particular substance with the reference anion-exchanger may be
accomplished (the same conditions as discussed before).
[0134] Desorption
[0135] Desorption may be carried out according to established
protocols. Preferably the desorption process comprises at least one
of the following procedures:
[0136] (A) Increasing the salt concentration (ionic strength),
[0137] (B) Increasing pH in order to lower the positive charge on
the ligands,
[0138] (C) Decreasing pH for decreasing a negative charge or for
reversing the charge on the substance bound to the matrix,
[0139] (D) Adding a ligand analogue or an agent (e.g. a solvent)
that reduces the polarity of the aqueous liquid (I).
[0140] According to the invention serine proteases are only
desorbed under anion-exchange conditions if R'.sub.1 is
--C(.dbd.NH)--.
[0141] The conditions provided by (A)-(D) may be used in
combination or alone. The proper choice will depend on the
particular combination of
[0142] (a) substance to be desorbed,
[0143] (b) anion-exchanger (ligand, kind of matrix, spacer and
ligand density), and
[0144] (c) various variables of aqueous liquid II (composition,
polarity, temperature, pH etc).
[0145] Replacing aqueous liquid I (adsorption buffer) with aqueous
liquid II (desorption buffer), thus means that at least one
variable such as temperature, pH, polarity, ionic strength, content
of soluble ligand analogue etc shall be changed while maintaining
the other conditions unchanged so that desorption can take
place.
[0146] In the simplest cases this means:
[0147] (a) an increase in ionic strength and/or
[0148] (b) a decrease in pH for reducing the negative charge of the
substance to be desorbed,
[0149] when changing from aqueous liquid I to aqueous liquid II.
Alternative (a) includes a decreased, a constant or an increased
pH. Alternative (b) includes a decreased, an increased or a
constant ionic strength.
[0150] In chromatographic and/or batch procedures the matrix with
the substance to be desorbed is present in a column or other
suitable vessel in contact with the adsorption liquid (aqueous
liquid I). The conditions provided by the liquid are then changed
as described above until the desired substance is eluted from the
matrix. After adsorption, a typical desorption process means that
the ionic strength is increased compared to that used during
adsorption and in many cases correspond to at least 0.4 M NaCl,
such as at 0.6 M NaCl, if pH or any of the other variables except
ionic strength are not changed. The actual values will depend on
the various factors discussed above.
[0151] The requirement for using an increased ionic strength for
desorption may be less strict depending on the conditions provided
by aqueous liquid II. See below.
[0152] The change in conditions can be accomplished in one or more
steps (step-wise gradient) or continuously (continuous gradient).
The various variables of the liquid in contact with the matrix may
be changed one by one or in combination.
[0153] Typical salts to be used for changing the ionic strength are
selected among chlorides, phosphates, sulphates etc of alkali
metals or ammonium ions).
[0154] Typical buffer components to be used for changing pH are
preferably selected amongst acid-base pairs in which the buffering
component can not bind to the ligand, i.e. piperazine,
1,3-diaminopropane, ethanolamine etc. A decrease in pH in step (ii)
will reduce the negative charge of the substance to be desorbed,
assist desorption and thus also reduce the ionic strength needed
for release from the matrix.
[0155] Depending on the pKa of the ligand used and the pl of the
substance to be released, an increase in pH may result in the
release of the substance or increase its binding to the
ion-exchange matrix.
[0156] Desorption may also be assisted by adjusting the polarity of
liquid (II) to a value lower than the polarity of the adsorption
liquid (I). This may be accomplished by including a water-miscible
and/or less hydrophilic organic solvent in liquid II. Examples of
such solvents are acetone, methanol, ethanol, propanols, butanols,
dimethyl sulfoxide, dimethyl formamide, acrylonitrile etc. A
decrease in polarity of aqueous liquid II (compared to aqueous
liquid I) is likely to assist in desorption and thus also reduce
the ionic strength needed for release of the substance from the
matrix.
[0157] Desorption may also be assisted by including a soluble
structural analogue of the ligand
[Ar--R.sub.1--N.sup.+(R.sub.2R.sub.3R.sub.4)]. Its concentration in
liquid (II) should be larger than its concentration in aqueous
liquid (I). A "structural analogue of the ligand" or "ligand
analogue" is a substance that has a structural similarity with the
ligand and in soluble form inhibits binding between the ligand
attached to the matrix and the substance to be removed.
[0158] Recovery
[0159] In a sub-aspect the present inventive method enables high
recoveries of an adsorbed substance, for instance recoveries above
60% such as above 80% or above 90%. Recovery is the amount of the
desorbed substance compared to the amount of the substance applied
to an anion-exchanger in the adsorption/binding step. In many
instances, the recovery can exceed even 95% or be essentially
quantitative. Typically the amount of the substance applied to an
anion-exchanger is in the interval of 10-80%, such as 20-60%, of
the total binding capacity of the anion-exchanger for the
substance.
[0160] The Substance to be Removed From the Liquid (I).
[0161] Removal of a substance according to the invention is
primarily carried out in order to purify the substance or some
other substance that is present in liquid (I).
[0162] The present invention is primarily intended for large
molecular weight substances that have several structural units that
can interact with mixed mode ligands defined above. Appropriate
substances typically have a molecular weight that is above 1000
dalton and/or are bio-organic and/or polymeric. The number of net
negatively charged groups per molecule is typically one or more.
Preferably the charge of the substances is dependent on pH (i.e.
the substance is amphoteric). Among biomolecules those having
polypeptide structure, nucleic acid structure, lipid structure, and
carbohydrate structure are normally possible to remove from a
liquid according to the invention (provided they have, or can be
provided with, a negative charge). In principle the invention is
applicable also to other biomolecules and organic substances
provided they meet the structural demands given above.
[0163] The substance may be dissolved in the aqueous medium or be
in the form of small bio-particles, for instance of colloidal
dimensions. Illustrative examples of bio-particles are viruses,
cells (including bacteria and other unicellular organisms) and cell
aggregates and parts of cells including cell organelles.
[0164] It is believed that the invention in particular will be
applicable to aqueous liquids that are derived from biological
fluids comprising a substance of interest together with high
concentration of salts. The novel anion-exchangers are likely to be
extremely useful in desalting, e.g. by enabling adsorption at high
ionic strength and desorption at a lowered ionic strength by first
changing the pH to reduce the positive charge of the adsorbed
substance.
[0165] Typical liquids of high ionic strength that contain a target
substances of interest are fermentation broths/liquids, for
instance from the culturing of cells, and liquids derived
therefrom. The cells may originate from a vertebrate, such as a
mammal, or an invertebrate (for instance cultured insect cells such
as cells from butterflies and/or their larvae), or a microbe (e.g.
cultured fungi, bacteria, yeast etc). Included are also plant cells
and other kinds of living cells, preferably cultured.
[0166] In case aqueous liquid (I) containing the substance to be
removed contains particulate matter then it may be beneficial to
utilize fluidised particulate support matrices carrying the novel
anion-exchange ligands together with an upward flow. Aqueous
liquids (I) of this type may originate from (a) a fermentation
broth/liquid from the culture of cells, (b) a liquid containing
lysed cells, (c) a liquid containing cell and/or tissue
homogenates, and (d) pastes obtained from cells.
[0167] The Second Aspect of the Invention
[0168] This aspect comprises an anion-exchanger (1) comprising a
plurality of anion-exchange ligands attached to a hydrophilic base
matrix. The ligands plus spacer comply with the formula:
--SP---[Ar--R.sub.1--N.sup.+(R.sub.2R.sub.3R.sub.4)]
[0169] where the symbols have the same meaning as previously.
[0170] The characteristic feature is that the anion-exchanger (1)
has a maximal breakthrough capacity somewhere in the pH-interval
2-12 for at least one of the reference proteins: ovalbumin,
conalbumin, bovine serum albumin,
.beta.-lactglobulin,.alpha.-lactalbumin, lyzozyme, IgG, soybean
trypsin inhibitor (STI) which is .gtoreq.200%, such as .gtoreq.300%
or .gtoreq.500% or .gtoreq.1000% of the corresponding maximal
breakthrough capacity obtained for a Q-exchanger
(--CH.sub.2CH(OH)CH.sub.2N.sup.+(CH.s- ub.3).sub.3 (anion-exchanger
2). The same support matrix, degree of substitution, counter-ion
etc are essentially the same in the same sense as discussed above.
The preferred reference anion-exchangerr is Q Sepharose Fast Flow
as discussed above. The running conditions for determining
breakthrough capacities of anion-exchanger (1) and anion-exchanger
(2) are essentially the same as discussed elsewhere in this
text.
[0171] Breakthrough capacities are determined under the
anion-exchanger conditions defined under the heading "Selection of
anion-exchangerr to be used for the removal of a particular
substance". The relative breakthrough capacity for each reference
substance is in the typical case determined separately by using an
aqueous liquid consisting of a buffer and the reference substance
for which breakthrough capacity is to be determined.
[0172] The various embodiments and their preferences are the same
as above. Thus in preferred variants at least one of SP, R'.sub.1
and R.sub.2-4 typically comprises an electron acceptor-donor atom
or group, as defined above for the first aspect of the invention,
for instance participating in hydrogen-bonding. The electron
donor-acceptor group or atom may for instance be present in a
branch in the spacer (SP).
[0173] The Third Aspect of the Invention
[0174] This aspect is a method for testing (screening) the
appropriateness of one or more anion-exchangers for removing a
substance from a liquid. The method comprises the steps of:
[0175] (a) providing a library which comprises
[0176] (i) one or more anion-exchangers to be tested (test
anion-exchangerrs, exchangers 1, 2, 3, 4 . . . n; n=an integer
>0) each of which anion-exchangers differs with respect to kind
of ligand (ligands 1, 2, 3, 4 . . . n), and
[0177] (ii) a reference anion-exchanger having a reference ligand,
the support matrix, counter-ion etc being essentially the same in
the exchangers 1, 2, 3, 4 . . . n and in the reference
anion-exchanger;
[0178] (b) determining the maximal breakthrough capacity somewhere
in the pH-interval 2-12 of exchanger 1 for the substance at a
predetermined condition;
[0179] (c) determining the maximal breakthrough capacity in the
pH-interval 2-12 of the reference anion-exchanger for the substance
at the same condition as in step (b);
[0180] (d) concluding with the aid of the relation between the
maximal breakthrough capacities obtained in steps (b) and (c), if
anion-exchanger (1) is appropriate to use for removing the
substance; and
[0181] (e) repeating, if necessary, steps (b)-(c) for at least one
of the exchangers 2, 3, 4 . . . n.
[0182] In case the degree of substitution varies between the
reference anion-exchangerr and the individual anion-exchangerrs to
be tested this should be accounted for when step (d) is carried
out. This in particular applies if the variation in degree of
substitution is large for instance with a factor greater than 3, 5
or 10 for anion-exchangerrs 1, 2 . . . n.
[0183] In particular it is believed that in case the maximal
breakthrough capacity determined for a test anion-exchanger/ligand
is larger than for the reference anion-exchanger/ligand then the
test anion-exchanger/ligand will have advantages over the reference
anion-exchanger/ligand. This conclusion will be more pronounced in
case the maximal breakthrough capacity determined for the test
anion-exchanger/ligand is .gtoreq.200%, such as .gtoreq.300% or
.gtoreq.500% or .gtoreq.1000% of the breakthrough capacity of the
reference anion-exchanger/ligand.
[0184] This screening method is in particular adapted for screening
libraries in which at least one of the anion-exchangers 1-n are as
defined
[0185] (a) in the first and second aspects of the present
invention,
[0186] (b) in U.S. Pat. No. 6,090,288 (Amersham Pharmacia Biotech
AB), and
[0187] (c) in International Patent Application filed in parallel
with this application and based on SE 9904197-2 (A method for
anion-exchange adsorption and thioether anion-exchangerrs).
[0188] The method also applies to cases where the anion-exchangerrs
1-n also include one or more conventional anion-exchangerrs and
anion-exchangerrs in which a positively charged nitrogen is part of
an aromatic ring, such as pyridine, pyrrole, imidazole etc.
[0189] Two or more of the anion-exchangers 1-n may be tested in the
method in parallel or in sequence.
[0190] The reference anion-exchanger may have a ligand that is
defined in the first and/or second aspects of the invention.
[0191] Selection of running conditions and reference
anion-exchanger can be done as outlined for the first and second
aspects of the invention. Steps (b) and/or (c) may be performed at
an ionic strength corresponding to the ionic strength of a water
solution that consists of 0.1 M NaCl or more, preferably
.gtoreq.0.25 M NaCl. Step (b) and (c) may or may not be carried out
under anion-exchanger conditions.
[0192] In the third aspect of the invention, tabulated or
predetermined breakthrough capacities for the reference
anion-exchanger may be used. Thus the method also encompasses
measurements carried out at different times and/or by different
individuals or by machines, including using tabulated values from
outside sources for the reference anion-exchanger or the reference
anion-exchange ligand.
[0193] An anion-exchanger ligand found by this screening method can
often be used in an inventive manner in the method of the invention
for removal of a substance from a liquid as defined above.
[0194] An innovative method for lowering the salt concentration in
liquids which contain proteins and the like.
[0195] This method is primarily intended for removing salt
(desalting) from a liquid (I) containing a negatively charged
substance as defined above, preferably amphoteric. Henceforth the
term desalting of a liquid will encompass any kind of ionic
strength reduction by removing charged substances from the liquid.
For the largest advantages to be accomplished the ionic strength of
liquid (I) is above the ionic strength of an aqueous solution of
0.1 M NaCl or 0.15 M NaCl or 0.2 M NaCl or 0.5 M NaCl.
[0196] The method comprises the steps of:
[0197] (i) contacting a liquid (I) with an anion-exchanger (1) that
comprises a base matrix carrying a plurality of ligands in which
there is a positively charged nitrogen under conditions permitting
binding between the anion-exchangerr and the substance,
[0198] (ii) desorbing said substance from said anion-exchanger by
the use of a liquid (liquid (I)).
[0199] The method is characterized in:
[0200] (A) selecting anion-exchanger (1) among anion-exchangers
that are
[0201] (a) capable of binding the substance of interest in an
aqueous reference liquid at an ionic strength corresponding to 0.1
M NaCl, preferably 0.25 M NaCl; and
[0202] (b) permitting a maximal breakthrough capacity in the pH
interval 2-12 for the substance which is .gtoreq.100%, such as
.gtoreq.125% or .gtoreq.200% or .gtoreq.300% or .gtoreq.500% or
.gtoreq.1000%, of the breakthrough capacity of the substance for
Q-Sepharose Fast Flow (anion-exchanger 2, Amersham Pharmacia
Biotech, Uppsala, Sweden),
[0203] said anion-exchangers (1) and (2) having essentially the
same ligand density, and the breakthrough capacities being
determined under the essentially same conditions.
[0204] B. adjusting the pH of liquid (II) in step (ii) by the use
of a buffering acid-base pair to a pH value that means a lower net
positive charge or zero charge or a net negative charge (if
possible) on the anion-exchanger and/or a lower net negative, zero
or net positive charge on the substance thereby, if necessary by
combining with a lowered ionic strength and/or a lowered polarity
compared to liquid (I) and/or by including a neutral structural
analogue.
[0205] The anion-exchanging ligand may be a primary, a secondary, a
tertiary, a quaternary ammonium group, or an amidinium group.
Heteroaromatic groups in which there is a nitrogen atom in the
aromatic ring are included in the term tertiary ammonium groups. In
the same fashion, N-alkylated forms of such heteroaromatic groups
are included in quaternary ammonium groups. The pKa of the ligands
are found in the interval from 4 and upwards.
[0206] The anion-exchangerrs and the ligands to be used may be of
the mixed mode kind as described
[0207] (a) herein or
[0208] (b) in International Patent Application filed in parallel
with this application and based on SE 9904197-2 (A method for
anion-exchange adsorption and thioether anion-exchangerrs), or
[0209] (c) in U.S. Pat. No. 6,090,288 (Amersham Pharmacia Biotech
AB).
[0210] U.S. Pat. No. 6,090,288 corresponds to WO-A-9729825
(Amersham Pharmacia Biotech) and is together with the patent
application under item (b) hereby incorporated by reference. U.S.
Pat. No. 6,090,288 discloses mixed mode anion-exchangers having
enhanced binding to substances based on charge interaction and
hydrogen-bonding involving oxygen and amino nitrogen on 2-3
carbons' distance from positively charged amine nitrogen. The
anion-exchangerrs under item (b) have ligands in which there is a
thioether function in the proxiimity of a positively charged atom
as also defined for the instant invention.
[0211] Step (ii) as defined above means that the elution (step
(ii)) will require a significantly lowered ionic strength compared
to the ionic strength of liquid (I) in step (i). The substance can
thus be eluted in concentrated form in a solution of low
concentration of salt, e.g .ltoreq.100 mM or even .ltoreq.10 mM.
Typically the appropriate pH in step (ii) shall be below pl+2 or
below pl+1, with preference for pH<pl, of the desired substance
to be desorbed. Depending on the particular ligands involved, it
may also be preferred to adjust pH.ltoreq.pKa of the
ligands/anion-exchangers used.
[0212] pH may be adjusted between steps (i) and (ii) by the use of
a buffering acid-base pair in which the acid and/or the base is
volatile and/or uncharged. This will mean that the buffer
components can be removed simply by evaporation after step (ii).
Volatile buffer components typically have a vapour pressure that is
.gtoreq.1 mm Hg, such as .gtoreq.10 mm Hg, at 25.degree. C. General
rules regarding selection of buffer components are given above.
[0213] Elution by changing the pH may be supported by including an
organic water-miscible solvent in liquid (II), preferably a
volatile solvent with a vapour pressure .gtoreq.10 mm Hg, such as
.gtoreq.10 mm Hg, at 25.degree. C. Structural analogues should be
neutral and preferably volatile as defined for a solvent.
[0214] By the term salt in this context is meant a compound that,
when dissolved in either liquid (I) or liquid (II), forms
positively charged moieties and negatively charged moieties. Each
negatively charged moiety carries a low number of negative charges
per molecule, for instance one, two or three, and is preferably
non-polymeric. The positively charged moiety doesn't have any such
restrictions. If the principle of this aspect is applied to
desalting with cation exchangers the demands on the cation in the
salt will correspond to those for the anion when anion-exchangerrs
are used.
[0215] This innovative aspect is particularly useful for liquids
containing the above-mentioned substances which cannot be desalted
by existing methods, such as membrane filtration, dialysis, gel
filtration etc, or for liquids that are handled in processes
requiring low salt concentration or absence of salt, for instance
affinity adsorption, mass spectrometry, etc. Thus it will be
advantageous to desalting according to the invention a solution
containing a desired substance before some kind of analyses is made
on the substance as such.
[0216] This aspect of the invention also embraces the analogous
aspect in which a cation exchanger is used. The cation exchanger
may be as defined in SE 0002688-0 filed Jul. 17, 2000 (Adsorption
method and ligands), WO 996507 (Amersham Pharmacia Biotech AB)
etc.
[0217] The invention will now be illustrated with patent examples.
The invention is further defined in the appending claims.
EXPERIMENTAL PART
[0218] Part I: Synthesis of Anion-Exchangerrs
[0219] General:
[0220] Volumes of matrix refer to sedimented bed volume. Weights of
matrix given in gram refer to the suction dried weight. It is
understood that these matrices are still water solvated material.
For reactions on a large scale, stirring refers to the use of a
suspended, motor-driven stirrer since the use of a magnet bar
stirrer will to damage the beads. Small-scale reactions (up to 20
ml) were performed in closed vials on a shaking-table.
Determination of the functionality and the extent of allylation,
epoxidation, or the degree of substitution of ion exchanger groups
on the beads were made using conventional methods. Elementary
analyses of the gels were also performed especially for analysing
of the sulphur content.
[0221] A typical example for preparing the anion-exchangerrs
described above is exemplified below using Sepharose 6 Fast Flow
(Amersham Pharmacia Biotech, Uppsala, Sweden) as the base
matrix.
[0222] 1. Introduction of Allyl Groups on the Matrix:
[0223] In a typical procedure allylation was carried out using
allyl glycidyl ether, but note that the introduction of allyl
groups on the solid support can as well be easily achieved with
using allyl bromide. 80 g of Sepharose 6 Fast Flow was mixed with
0.5 g of NaBH.sub.4, 13 g of Na.sub.2SO.sub.4 and 40 ml of 50%
aqueous solution of NaOH. The mixture was stirred for 1 hour at
50.degree. C. After addition of 100 ml of allylglycidyl ether the
temperature of the suspension was maintained at 50.degree. C. and
stirred for 18 hours. The mixture was filtered and the gel washed
successively with 500 ml distilled water, 500 ml ethanol, 200 ml
distilled water, 200 ml 0.2 M acetic acid and 500 ml distilled
water.
[0224] Titration gave a degree of substitution of 0.3 mmol of
allyl/ml of settled gel. It was possible to obtain a degree of
substitution of 0.45 mmol of allyl/ml gel by starting from a gel
that had been drained (100 ml gel to 75 ml gel).
[0225] 2. Introduction of Amines Groups on the Matrix:
[0226] In a typical procedure the amines groups were introduced on
the matrix directly via the nitrogen atom of the amine groups or
via the sulphur atom in thiol containing derivatives. Derivatives
containing other reactive nucleophilic groups such as phenol for
example can as well be used. Coupling to the matrix was realised in
preference via bromination of the allyl group and nucleophilic
substitution under basic conditions. In some cases and for
thiol-containing derivatives radical addition to the allyl was as
well performed. In the case where the attachment point to the gel
was achieved via other nucleophilic groups than the amine, the
amine group can be introduced as a protected form and a
deprotection step is then necessary.
[0227] Amine groups can also be introduced by other conventional
methods, for example reductive amination.
[0228] 2.1. Activation of Allyl Sepharose Via Bromination:
[0229] Bromine was added to a stirred suspension of 100 ml of allyl
activated Sepharose 6 Fast Flow (0.4 mmol allyl groups/ml drained
gel), followed by 4 g of AcONa and 100 ml of distilled water, until
a persistent yellow colour was obtained. Sodium formiate was then
added till the suspension was fully decolourised. The reaction
mixture was filtered and the gel washed with 500 ml of distilled
water. A suitable aliquot of the activated gel was then transferred
to a reaction vessel and coupled with the appropriate ligand
according to the following procedures.
[0230] 2.1.a. Coupling of Octopamine to Sepharose 6 Fast Flow.
[0231] 5.5 g of bromine activated gel (0.4 mmol allyl groups/ml
drained gel) were transferred to a reaction vessel containing a
solution of octopamine (2 g) in water (4 ml) that has been adjusted
to pH 11.5 by addition of a 50% aqueous solution of NaOH. The
mixture was stirred for 17 hours at 50.degree. C. The suspension
was filtered and the gel was washed successively with 3.times.10 ml
of distilled water, 3.times.10 ml EtOH, 3.times.10 ml aqueous 0.5 M
HCl and finally 3.times.10 ml of distilled water. The degree of
substitution was 0.10 mmol amine group/ml of gel.
[0232] 2.1.b. Coupling of 2-Amino-4-(trifluoromethyl)-benzenethiol
to Sepharose 6 Fast Flow.
[0233] 6 g of bromine activated gel (0.4 mmol allyl groups/ml
drained gel) were transferred to a reaction vessel containing a
solution of 2-Amino-4-(trifluoro methyl)-benzenethiol (2.5 g) in
water/DMF (2:1, 4 ml) that has been adjusted to pH 11.5 by addition
of a 50% aqueous solution of NaOH. The reaction was stirred for 18
hours at 60.degree. C. The suspension was filtered and the gel was
successively washed with 3.times.10 ml of distilled water,
3.times.10 ml EtOH, 3.times.10 ml aqueous 0.5 M HCl and finally
with 3.times.10 ml. The degree of substitution was 0.07 mmol amine
group/ml of gel.
[0234] 2.1.c. Coupling of 2-(Boc-amino) ethanethiol to Sepharose 6
Fast Flow.
[0235] A 30 g quantity of bromine activated gel (0.4 mmol allyl
groups/ml drained gel) was transferred to a reaction vessel
containing a solution of 2-(Boc-amino) ethanethiol-l (7.35 g) in
water/DMSO (1:3, 40 ml). The pH was adjusted to pH 11 with 1 M
NaOH. The reaction was stirred for 16 hours at 50.degree. C. After
filtration of the reaction mixture the gel was successively washed
with 3.times.50 ml of distilled water, 3.times.50 ml DMSO,
3.times.50 ml of distilled water and finally with 3.times.50 ml of
EtOH.
[0236] 2.1.d. Coupling of Cysteamine to Amino Ethane Thiol Derived
Sepharose 6 Fast Flow.
[0237] The Boc protected amino ethanethiol gel (6 ml) (from 2.1.c)
was treated with a 10% solution of trifluoroacetic acid in
CH.sub.2Cl.sub.2 (60 ml) for 2 hours at room temperature. The
suspension was filtered and the gel was washed successively with
3.times.10 ml CH.sub.2Cl.sub.2, 3.times.10 ml EtOH, and 3.times.10
ml of distilled water. The degree of substitution was 0.29 mmol
amine group/ml of gel.
[0238] 2.2. Direct Coupling to the Allyl Group.
[0239] 2.2.a. Cysteamine Derived Sepharose 6 Fast Flow.
[0240] A solution of cysteamine (4.7 g) in MeOH (15 ml) was added
to a slurry of 10 ml of allyl activated Sepharose 6 Fast Flow (0.4
mmol allyl groups/ml drained gel) in MeOH (40 ml). The reaction
mixture was left under UV irradiation and stirring at 40.degree. C.
for 16 hours. The reaction mixture was filtered and the gel was
successively washed with 3.times.10 ml MeOH, 3.times.10 ml
distilled water, 3.times.10 ml 0.5 M HCl and finally 3.times.10 ml
of distilled water. The degree of substitution was 0.34 mmol amine
group/ml of gel.
[0241] 3. Coupling to Cysteamine Sepharose 6 Fast Flow:
[0242] 3.1. Boc-L-Phenylalanine Derived Cysteamine Sepharose.
[0243] A solution of Boc-L-Phenylalanine N-hydroxysuccinimide ester
(0.44 g, 1.2 mmol) in DMF (2 ml) was added to a mixture of
cysteamine Sepharose (4 ml, 0.2 mmol amine group/ml gel) and
N,N-diisopropylethylamine (1 mmol) in DMF (5 ml). The reaction was
allowed to continue for 18 hours at room temperature. The reaction
mixture was filtered and the gel was washed successively with
3.times.10 ml DMF, 3.times.10 ml acetone, and finally 3.times.10 ml
of distilled water. Residual amine groups were calculated to be
0.033 mmol amine group/ml of gel after titration.
[0244] 3.2. L-Phenylalanine Derived Cysteamine Sepharose.
[0245] The Boc protected L-phenylalanine derived cysteamine gel (3
ml) (from 3.1 or 2.1.d) was treated with a 10% solution of
trifluoroacetic acid in CH.sub.2Cl.sub.2 (4 ml) for 2 hours at room
temperature. The reaction mixture was filtered and the gel was
washed successively with 3.times.10 ml CH.sub.2Cl.sub.2, 3.times.10
ml acetone, and 3.times.10 ml of distilled water. The degree of
substitution of the product was 0.19 mmol amine group/ml of
gel.
[0246] 3.3. Fmoc-L-Tyrosine Derived Cysteamine Sepharose.
[0247] A solution of Fmoc-L-tyrosine N-hydroxysuccinimide ester
(1.1 mmol) in DMF (3 ml) was added to a slurry of cysteamine
Sepharose (3.3 ml, 0.3 mmol amine group/ml gel) in DMF (5 ml). The
mixture was stirred for 18 hours at room temperature. The
suspension was filtered and the gel was washed with 3.times.10 ml
DMF.
[0248] 3.4. L-Tyrosine Derived Cysteamine Sepharose:
[0249] The Fmoc protected L-tyrosine derived cysteamine gel (3.3
ml) (from 3.3) was treated with a 10% solution of
1,8-diazabicyclo[5,4,0]-undec-7-e- ne in DMF (10 ml) for 18 hours
at room temperature. The reaction mixture was filtered and the gel
was washed successively with 3.times.10 ml DMF, 3.times.10 ml
acetone, and 3.times.10 ml of distilled water. The degree of
substitution was 0,28 mmol amine group/ml of gel.
[0250] Part II. Chromatography
[0251] To verify that the ligands suggested in this invention
adsorb proteins at higher ionic strengths than the reference
anion-exchanger, breakthrough capacities of bovine serum albumin
(BSA) was determined. The new "high-salt" anion-exchange ligands
attached to Sepharose Fast Flow were compared to Q Sepharose Fast
Flow in this study. Furthermore, the elution conductivity of three
proteins, namely conalbumin (Con A), lactalbumin (Lactalb) and
soybean trypsin inhibitor (STI), was also determined for all
anion-exchangers. This function test was used to verify retardation
at high salt conditions for other proteins as well. Four of the
"high-salt" anion-exchanger ligands with high breakthrough
capacities were also tested with respect of recovery of the protein
(BSA) applied.
[0252] A. Breakthrough Capacity (Qb10%) at High Salt Condition
[0253] The Qb10%-value was evaluated at relatively high
concentration of salt (0.25 M NaCl) relative to the reference
anion-exchanger Q Sepharose Fast Flow that was operated under
identical conditions. The Qb10%-values for the different
anion-exchangers were determined using the method of frontal
analysis described below.
EXPERIMENTAL
[0254] I. Buffer and Sample Solutions
[0255] The sample solution was BSA dissolved in 20 mM piperazin
(pH=6.0) with 0.25 M NaCl added. The concentration of BSA was 4
mg/ml. Buffer and sample solutions were filtered through 0.45 .mu.m
Millipore Millex HA filters before use.
[0256] II. Chromatographic System
[0257] All experiments were performed at room temperature using
Akta Explorer 100 chromatography system (Amersham Pharmacia Biotech
AB, Uppsala, Sweden) equipped with Unicorn 3.1 software. Samples
were applied to the column via a 150 ml superloop. A flow rate of 1
ml/min (ca 300 cm/h) was used throughout. The effluents were
monitored continuously by absorbance measurements at 280 nm using a
10 mm flow cell.
[0258] III. Frontal Analysis
[0259] Each prototype anion-exchanger was packed in a HR 5/5 column
(packed bed volume=1) and equilibrated with the piperazine buffer
(20 mM piperazin, pH=6.0, with 0.25 M NaCl). The breakthrough
capacity (Q.sub.b) 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 UV detector. The breakthrough
capacity was calculated from the retention volume at 10% height of
the maximum signal after correction of the dead volume.
[0260] A column equilibrated with the piperazine buffer was
continuously fed (via a 150 ml superloop) with the sample solution
at a flow rate of 1 ml/min (i.e. ca. 300 cm/h). The application of
sample was continued until the A.sub.280 of the effluent reached a
level of 10% of A.sub.280 of the sample solution. On the basis of
data so obtained (i.e. volume of the packed gel bed (V.sub.c), its
void volume, flow rate and concentration of BSA to the column), the
breakthrough capacity of the gel (Qb 10%) can be calculated. The
results obtained have formed the basis for screening a large number
of "high salt ligand" candidates and the results will be presented
below.
[0261] IV. Evaluation
[0262] The breakthrough capacity at a level of 10% of absorbance
maximum of the BSA sample solution (QbBSA) was calculated with the
formula:
QbBSA=(T.sub.R10%-T.sub.RD).times.C/Vc
[0263] where:
[0264] T.sub.R10%=retention time at 10% of absorbance maximum
(min)
[0265] T.sub.RD=Dead time in the system (min)
[0266] C=Concentration of BSA (4 mg/mL)
[0267] V.sub.C=Column volume (mL)
[0268] B. Function Test
[0269] The anion-exchange media were packed in 1.0 ml HR 5/5
columns and equilibrated with 20 column volumes of the A-buffer (20
mM phosphate buffer; pH 6.8). 50 .mu.l of a protein mixture (6
mg/ml Con A, 4 mg/ml Lactalbumin and 6 mg/ml STI) were applied to
the column and eluted with a linear gradient (gradient volume=20
column volumes) to 100% of the B-buffer (A-buffer plus 2.0 M NaCl).
The flow rate was adjusted to 0.3 ml/min (100 cm/h). All
experiments were performed at room temperature using kta Explorer
100 chromatography system equipped with Unicorn 3.1 software.
[0270] C. Recovery of BSA Bound to "High Salt" Anion-Exchanger
Ligands
[0271] Details concerning type of column, packed bed volume,
buffers, protein solution, flow rate and type of apparatus are
outlined above. To a column equilibrated with piperazine buffer (20
mM piperazine, pH=6.0, with 0.25 M NaCl) was applied a solution of
BSA from a 50 ml super loop until an amount corresponding to 30% of
its breakthrough capacity was applied. The column was then washed
with 2 bed volumes of the equilibrium buffer and the bound BSA was
eluted with the appropiate de-sorption buffer. In case of ligands
(Tyrosine) and (2-Aminobenzimidazole) adsorbed BSA were eluted with
a piperazinee buffer (20 mM piperazine, pH=6.0, with 2.0 M NaCl).
In addition, adsorbed BSA on ligands (Octopamine) and (Tyrosinol)
were eluted with a TRIS buffer (0.2 M TRISS, pH=9.0, with 2 M
NaCl).
[0272] Results
[0273] The results obtained for breakthrough capacities for a
series of representative "high salt" anion-exchanger ligands are
summarised in Table 1 and the structures of the ligands are
depicted in section part III. The degree of ligand substitution on
the majority of these new anion-exchangers was ca. 0.05-0.3 mmol/ml
packed gel. As a reference anion-exchanger, the commercially
available Q Sepharose Fast Flow was used. The ligand density is in
the same range as the new series of anion-exchangers. The results
indicate the following trends.
[0274] 1. The new anion-exchange ligands have much higher elution
conductivity for all three proteins compared to the reference
anion-exchangerr Q Sepharose Fast Flow (Table 1).
[0275] 2. The new anion-exchange ligands have also a much higher
breakthrough capacity for BSA (QbBSA) compared to Q Sepharose Fast
Flow. The ligand that gave the highest Qb-value corresponds to an
increase of 4300% relative the reference anion-exchanger. Of the
presented ligands (Table 1), the one that gave the lowest Qb-value
corresponds to a 500% increase compared to Q Sepharose Fast
Flow.
[0276] 3. All good anion-exchange ligands are primary or secondary
amines or both primary and secondary amines. No good ligand based
on a quartenary amine has been found.
[0277] 4. The recovery data from good anion-exchange ligands (7, 5,
27 and 4) show that adsorbed BSA can be eluted by a salt step (7
and 5) and/or a combined pH and salt step (2 and 4) with recoveries
larger than 80% (see Part III).
1TABLE 1 Elution conductivity at pH 6 for three proteins and
breakthrough capacity of BSA (pH 6 and 0.25 M NaCl) on different
anion-exchangers. Break- through Elution conductivity Ligand
Capacity Lactalbumi density QbBSA ConA ne STI Ligand mmol/ml
(mg/ml) (mS/cm) (mS/cm) (mS/cm) Q Sepharose Fast Flow 0.21 1 12 20
30 1. Thiomicamine 0.13 43 ne ne ne 2. Tyrosinol 0.13 39 132 ne ne
3. Tryptophanol 0.15 37 ne ne ne 4. Octopamine 0.10 37 ne ne ne 5.
2-Aminobenz- 0.17 34 46 117 ne imidazole 6. Phenylalanine/ 0.20 33
ne ne ne cysteamine 7. Tyrosine 0.30 31 45 68 121 8. 2-Amino-3-
0.17 31 ne ne ne phenyl propanol 9. a-(1-amino ethyl)-4- 0.09 29 79
88 ne hydroxy benzyl alcohol 10. 2-Amino-4- na 27 ne ne ne
nitrophenol 11. 2-(4-amino-phenyl) 0.20 26 99 ne ne ethyl amine 12.
Noradrenaline 0.08 25 ne ne ne 13. Benzylcysteine/ na 25 ne ne ne
cysteamine 14. 2-Amino-1-(4- 0.09 23 ne ne ne nitrophenyl)
1,3-propanediol 15. 2-Amino-1-phenyl- 0.09 22 24 81 ne
1,3-propanediol 16. 2-Amino-4- 0.01 21 ne ne ne (trifluoromethyl)-
benzenethiol 17. 3,4-Dihydroxy 0.11 18 46 65 110 benzylamine 18.
p-Aminobenz- 0.10 17 43 62 ne amidine 19. Boc-His(Boc)-Osu 0.30 17
21 39 78 20. 4,6-Diamino-2- 0.10 14 ne ne ne mercapto pyrimidine
21. na 14 29 54 78 Treophenylserine/ cysteamine 22. Tyrosinol 0.07
13 ne ne ne 23. 4-Aminothio phenol 0.05 13 ne ne ne 24.
Thienylserine/ na 12 27 50 72 cysteamine 25. 1,2,4,5-Tetra 0.10 9
71 ne ne aminobenzene 26. 4-Amino-1,3,5- 0.07 9 na na na
triazine-2-thiol 27. Sulfanilamide 0.05 8 ne 63 ne 28.
4-Aminophenol 0.05 5 ne 40 ne ne = not eluted, na = not
analysed
[0278] Part III. Ligands and Recovery.
[0279] The best ligands tested was derived from the following
compounds: 1
[0280] 1. Thiomicamine: Ar=4-methylthioxyphenyl,
R'.sub.1=--CH(OH)CH(CH.su- b.2OH)--, n=0, m=1, SP ends with
--CH.sub.2CH(OH)CH.sub.2O-- (coupling at the amino group). 2
[0281] 2. Tyrosinol: Ar=4-hydroxyphenyl, n=0, m=1,
R'.sub.1=--CH.sub.2CH(C- H.sub.2OH)--, SP ends with
--CH.sub.2CH(OH)CH.sub.2O-- (coupling at the amino group). Recovery
80% 3
[0282] 3. Tryptophanol: Ar=3-indolyl, n=0, m=1,
R'.sub.1=--CH.sub.2CH(CH.s- ub.2OH)--, SP ends with
--CH.sub.2CH(OH)CH.sub.2O-- (coupling at the amino group). 4
[0283] 4. Octopamine: Ar=4-hydroxyphenyl, n=0, m=1,
R'.sub.1=--CH(OH)CH.sub.2--, SP ends with
--CH.sub.2CH(OH)CH.sub.2O-- (coupling at the amino group). Recovery
80% 5
[0284] 5. 2-aminobenzimidazole: Ar=2-benzimidazolyl, m=0, SP ends
with --CH.sub.2CH(OH)--CH.sub.2O-- (coupling at the amino group).
Recovery 82% 6
[0285] 6. Phenylalanine/cysteamine: Ar=phenyl, n=0, m=1,
R'.sub.1=--CH.sub.2CH<, SP ends with
--CONHCH.sub.2CH.sub.2SCH.sub.27. 7
[0286] 7. Tyrosine: Ar=4-hydroxyphenyl, n=0, m=1,
R'.sub.1=--CH.sub.2CH<- ;, SP ends with --CONHCH.sub.2--
(coupling at the carboxy group). Recovery=98% 8
[0287] 8. 2-Amino-3-phenylpropanol: Ar=phenyl, n=0, m=1,
R'.sub.1=--CH.sub.2CH(CH.sub.2OH)--, SP ends with
--CH.sub.2CH(OH)CH.sub.- 2O-- (coupling at the amino group). 9
[0288] 9. Alpha-(1-aminoethyl)-4-hydroxybenzyl alcohol:
Ar=4-hydroxyphenyl, n=0, m=1, R'.sub.1=--CH(OH)CH(CH.sub.3)--, SP
ends with --CH.sub.2CH(OH)CH.sub.2O-- (coupling at the amino
group). 10
[0289] 10. 2-Amino-4-nitrophenol: Ar=2-hydroxy-5-nitrophenyl, m=0,
SP ends with --CH.sub.2CH(OH)CH.sub.2O-- (coupling at the amino
group). 11
[0290] 11. 2-(4-aminophenyl) ethylamine:
[0291] Ar=4-(2-aminoethyl)phenyl, m=0, SP ends with
--CH.sub.2CH(OH)CH.sub.2O-- (coupling at aromatic amino group).
[0292] Ar=4-aminophenyl, n=0, m=1, R'.sub.1=--CH.sub.2CH.sub.2--,
SP ends with --CH.sub.2CH(OH)CH.sub.2O-(coupling at aliphatic amino
group) 12
[0293] 12. Noradrenaline: Ar=3,4-dihydroxyphenyl, n=0, m=1,
R'.sub.1=--CH(OH)CH.sub.2--, SP ends with
--CH.sub.2CH(OH)CH.sub.2O-- (coupling at the amino group). 13
[0294] 13. Benzylcysteine/cysteamine: Ar=phenyl, n=0, m=1,
R'.sub.1=--CH.sub.2CH.sub.2S CH.sub.2CH<, SP ends with
--CONHCH.sub.2CH.sub.2SCH.sub.2--. 14
[0295] 14. 2-Amino-1(4-nitrophenyl)-1,3-propanediol:
Ar=4-notrophenyl, n=0, m=1, R'.sub.1=--CH(OH)CH(CH.sub.2OH)--, SP
ends with --CH.sub.2CH(OH)CH.sub.2O-- (coupling at the aminogroup).
15
[0296] 15. 2-Amino-1-phenyl-1,3-propanediol: Ar=phenyl, n=0, m=1,
R'.sub.1=--CH.sub.2CH(CH.sub.2OH)--, SP attaches at amino and ends
with --CH.sub.2CH(OH)CH.sub.2O-- (coupling at the amino group).
16
[0297] 16. 2-Amino-4-(trifluoromethyl)-benzenethiol:
Ar=5-trifluoromethylphen-1,2-diyl, m=0,
R'.sub.1=--CH.sub.2CH(CH.sub.2OH)- --, SP attaches at Ar and ends
with --SCH.sub.2(coupling at the mercapto group) 17
[0298] 17. 3,4-Dihydroxybenzylamine: Ar=3,4-dihydroxyphenyl, n=0,
m=1, R'.sub.1=--CH.sub.2--, SP attaches at amino and ends with
--CH.sub.2CH(OH)CH.sub.2O-- (coupling at the amino group). 18
[0299] 19. Boc-HIS(Boc)-Osu: Ar=4-imidazolyl, n=0, m=1,
R'.sub.1=--CH.sub.2CH<, SP ends with --CONHCH.sub.2CH--. 19
[0300] 20. 4, 6-diamino-2-mercaptopyrimidine:
Ar=4-aminopyrimid-2,6-yl or 6-aminopyrimid-2,4-diyl, m=0, SP
attaches at Ar and ends with --SCH.sub.2-- (coupling at the
mercapto group). 20
[0301] 21. Treophenylserine/cysteamine: Ar=phenyl, n=0, m=1,
R'.sub.1=--CH(OH)CH<, SP attaches at R'.sub.1 and ends with
--CONHCH.sub.2CH.sub.2SCH.sub.2--.
[0302] QbBSA=14 mg/ml 21
[0303] 22. Tyrosinol: Ar=4-hydroxyphenyl, n=0, m=1,
R'.sub.1=--CH.sub.2CH(CH.sub.2OH)--, SP attaches at amino and ends
with --CH.sub.2CH(OH)CH.sub.2O-- (coupling at the amino group).
22
[0304] 23. 4-aminothiophenol: Ar=phenyl, m=0, SP attaches at Ar and
ends with --SCH.sub.2(coupling at the amino group). 23
[0305] 24. Thienylserine/cysteamine: Ar=2-thienyl, n=0, m=1,
R'.sub.1=--CH(OH)CH<, SP attaches at R'.sub.1 and ends with
--CONHCH.sub.2CH.sub.2SCH.sub.2--. 24
[0306] 25. 1,2,4,5-tetraminobenzene: Ar=2,4,5-triaminophen-1-yl,
m=0, SP attaches at aryl and ends with --NHCH.sub.2CH(OH)CH.sub.2--
(coupling at an amino group). 25
[0307] 26. 4-amino-1,3,5-triazine-2-thiol:
Ar=1,3,5-triazin-2,6-diyl, m=0, SP attaches at Ar and ends with
--SCH.sub.2-- (coupling at the mercapto group). 26
[0308] 27. Sulfanilamide: Ar=4-sulphonamidophenyl, m=0, SP attaches
at amino and ends with --NHCH.sub.2CH(OH)CH.sub.2-- (coupling at
the amino group) 27
[0309] 28. 4-aminophenol: Ar=4-hydroxyphenyl, m=0, SP attaches at
amino and ends with --NHCH.sub.2CH(OH)CH.sub.2-- (coupling at the
amino group). Ligand density: low
* * * * *