U.S. patent application number 13/387425 was filed with the patent office on 2014-02-13 for specific sorbent for binding proteins and peptides, and separation method using the same.
This patent application is currently assigned to INSTRACTION GMBH. The applicant listed for this patent is Markus Arendt, Klaus Gottschall, Andres Kirschfeld, Christian Meyer, Markus Weis, Martin Welter, Lothar Ziser. Invention is credited to Markus Arendt, Klaus Gottschall, Andres Kirschfeld, Christian Meyer, Markus Weis, Martin Welter, Lothar Ziser.
Application Number | 20140046023 13/387425 |
Document ID | / |
Family ID | 41503671 |
Filed Date | 2014-02-13 |
United States Patent
Application |
20140046023 |
Kind Code |
A1 |
Gottschall; Klaus ; et
al. |
February 13, 2014 |
SPECIFIC SORBENT FOR BINDING PROTEINS AND PEPTIDES, AND SEPARATION
METHOD USING THE SAME
Abstract
Sorbent comprising a solid support material, the surface of
which comprises first residues comprising a binuclear
heteroaromatic structure comprising besides carbon atoms at least
one of the heteroatoms N, O, S, and second residues comprising a
mononuclear heteroaromatic structure comprising besides carbon
atoms at least one of the heteroatoms N, O, S.
Inventors: |
Gottschall; Klaus;
(Heddesheim, DE) ; Arendt; Markus; (Hockenheim,
DE) ; Kirschfeld; Andres; (Hirschberg a.d.B, DE)
; Meyer; Christian; (Ludwigshafen, DE) ; Weis;
Markus; (Ludwigshafen, DE) ; Welter; Martin;
(Heidelberg, DE) ; Ziser; Lothar; (Altrip,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Gottschall; Klaus
Arendt; Markus
Kirschfeld; Andres
Meyer; Christian
Weis; Markus
Welter; Martin
Ziser; Lothar |
Heddesheim
Hockenheim
Hirschberg a.d.B
Ludwigshafen
Ludwigshafen
Heidelberg
Altrip |
|
DE
DE
DE
DE
DE
DE
DE |
|
|
Assignee: |
INSTRACTION GMBH
Ludwigshafen
DE
|
Family ID: |
41503671 |
Appl. No.: |
13/387425 |
Filed: |
July 28, 2010 |
PCT Filed: |
July 28, 2010 |
PCT NO: |
PCT/EP10/04628 |
371 Date: |
February 20, 2013 |
Current U.S.
Class: |
530/344 ; 502/7;
530/417 |
Current CPC
Class: |
B01J 20/3272 20130101;
B01J 20/3242 20130101; B01J 20/3255 20130101; B01J 20/3282
20130101; B01J 20/327 20130101; C07K 1/22 20130101; B01J 20/3285
20130101; B01D 15/3804 20130101; B01J 20/286 20130101 |
Class at
Publication: |
530/344 ; 502/7;
530/417 |
International
Class: |
B01J 20/286 20060101
B01J020/286; C07K 1/22 20060101 C07K001/22 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 28, 2009 |
EP |
EP09009735.3 |
Claims
1-16. (canceled)
17. A sorbent comprising a solid support material, wherein the
surface of the sorbent comprises i) a first residue comprising a
binuclear heteroaromatic structure comprising carbon atoms and at
least one heteroatom selected from N, O, and S; and ii) a second
residue comprising a mononuclear heteroaromatic structure
comprising carbon atoms and at least one heteroatom selected from
N, O, and S; wherein the first and second residues are not directly
connected with each other, and wherein the first and second
residues are separately attached to the bulk solid support material
or a polymer film supported by the solid support material as a
carrier.
18. The sorbent of claim 17, wherein the binuclear heteroaromatic
structure is a benzopyrrole (indole) structure selected from
aza-benzopyrrole structures, oxa-benzopyrrole structures and
thia-benzopyrrole structures.
19. The sorbent of claim 17, wherein the binuclear heteroaromatic
structure is a benzopyridine (quinoline or isoquinoline) structure
selected from aza-benzopyridine structures, oxa-benzopyridine
structures and thia-benzopyridine structures.
20. The sorbent of claim 17, wherein the mononuclear heteroaromatic
structure is a pyrrole structure selected from aza-pyrrole
structures, oxa-pyrrole structures, thia-pyrrole structures and
3-azapyrrole (imidazole).
21. The sorbent of claim 17, wherein the first and/or second
residue comprise(s) a covalently bound and conformationally
flexible linker of a length of from 1 to 20 atoms.
22. The sorbent of claim 17, wherein the surface of the solid
support material additionally comprises a third residue, and
optionally, a fourth residue.
23. The sorbent of claim 22, wherein the third residue comprises an
amine structure, an amide structure or a primary amine
structure.
24. The sorbent of claim 22, wherein the first, second, and third
residue are present in a molar ratio of about 1:1:2.
25. The sorbent of claim 17, wherein the surface of the solid
support material is covered with a film of a polymer comprising a
first and a second functional group, wherein the first and a second
functional group may be the same or may be different from each
other, and wherein the polymer is a carrier for the first and
second residues, and optionally, a third and a fourth residue.
26. The sorbent of claim 25, wherein the polymer exhibits
individual chains which are covalently crosslinked with each other,
and wherein the polymer is not covalently bound to the surface of
the solid support material.
27. The sorbent of claim 25, wherein the polymer is a polyamine, a
polyvinyl amine, a copolymer or a polymer blend comprising a
polyamine.
28. The sorbent of claim 25, wherein a first portion of the first
and second functional groups of the polymer is crosslinked with at
least one crosslinking reagent, and wherein a second portion of the
first and second functional groups of the crosslinked polymer is
bound to the first residue, the second residue, and, optionally, a
third and a fourth residue.
29. A method for preparing the sorbent of claim 28, comprising: (i)
providing a polymer having a first and a second functional group;
(ii) adsorbing a film of the polymer onto a surface of a solid
support material; (iii) crosslinking a first portion of the
functional groups of the adsorbed polymer with at least one
crosslinking reagent; and (iv) binding a second portion of the
functional groups of the crosslinked polymer with the first
residue, the second residue, and, optionally, the third and forth
residues.
30. A method of separating or increasing the concentration and/or
purity of a protein or a peptide from a mixture containing the
protein or the peptide, comprising: (i) contacting the mixture
which is dissolved or suspended in a first liquid with the sorbent
of claim 17 for a period of time sufficient to bind the protein or
peptide to the sorbent; (ii) optionally, rinsing the sorbent with a
second liquid; (iii) contacting the sorbent with a third liquid for
a period of time sufficient to release the bound protein or peptide
from the sorbent; (iv) separating the liquid from the sorbent to
obtain the protein or peptide; and (v) optionally, washing and/or
regenerating the sorbent with a fourth and/or fifth liquid.
31. The method of claim 30, wherein the pH of the first liquid and
the second liquid is close to the isoelectric point of the protein
or peptide.
32. The method of claim 30, wherein the pH of the first liquid is
in a range of from 5.5 to 8.5 and the pH of the third liquid is in
a range of from 3 to 6.5.
33. The method of claim 30, wherein the protein or peptide has an
isoelectric point of from 5.5 to 8.5 and a molecular weight of from
100 to 500,000 Da.
Description
FIELD OF THE INVENTION
[0001] The present patent application is related to the field of
separation technology of biomolecules, in particular to
biochromatography.
BACKGROUND OF THE INVENTION
[0002] Chromatography media for biomolecules have traditionally
been categorised according to one or more of the following possible
modes of interaction with the sample: [0003] Hydrophobic
Interaction (reversed phase) [0004] Hydrophilic Interaction (normal
phase) [0005] Cation Exchange [0006] Anion Exchange [0007] Size
Exclusion [0008] Metal Ion Chelation
[0009] Perpetual improvements in the titres of technical
fermentation processes led to an increased demand of simple,
cost-effective, and highly selective downstream purification
technologies capable of handling large protein capacities without
up-scaling the required volumes of liquid by the same factor.
Traditional stepwise application of the above chromatographic
categories to a given separation problem was accordingly mirrored
in a step-by-step, steady improvement of the product purity but
also in product losses at every stage which accumulate seriously in
the end, not to mention the operational time and cost of goods.
Introduction of affinity chromatography at an early stage into the
downstream process could be an answer to this demand since the
reduction of a consecutive series of sequential chromatography
steps into only one could thus be demonstrated many times. Affinity
chromatography is sometimes regarded as a class of its own
although, from a chemical point of view, it is based on the same
interaction modes as above, but usually on a combination of two or
more modes. The principal characteristic of affinity chromatography
is its high specificity of a pre-determined analyte which is
usually based on a known molecular recognition pair of biological
significance such as antigen--antibody, carbohydrate--lectin,
hormone--receptor, or between complementary nucleic acid strands.
Most affinity sorbents are therefore made-to-measure by the
end-user according to his particular separation task. To yield a
fully functional sorbent, the biological affinity residue is
coupled--immediately or via an optional tether allowing more
degrees of freedom in the translational and rotational motion of
the residue--by a choice of only a few standard bioconjugation
techniques to a support material which itself may be commercially
available. The shelf-life of such a sorbent is normally only short,
and it has often to be prepared on-demand.
[0010] Additionally, synthetic affinity ligands such as short
linear or cyclic synthetic peptides or peptidomimetics, but also
certain reactive dyes (mainly triazine dyes) have been found to
interact group-specifically with biomolecules. The latter are
inexpensive and easy-to-prepare low-molecular weight residues which
lack the disadvantages of the labilities and variabilities in the
tertiary structures of biopolymers. Moreover, due to their small
molecular sizes and tunable, robust activation chemistries, they
can be efficiently immobilised in a directed orientation onto solid
supports even without long tethering, whereas biopolymers under the
same conditions often suffer from lack of activity after
immobilisation due to defolding, steric hindrance, or random
orientation. In either case, the component of the sorbent which is
actively involved in the recognition process is usually only
present on the surface (often as a surface-bound monolayer) of a
supporting solid.
[0011] Apart from homogeneous solid support materials, sorbents
consisting of a 2-layered cross-sectional morphology according to
the general scheme of a bulk solid support material whose surface
is covered with a thin film of a crosslinked polymer are well-known
from the state of the art. Polymers such as heavily (radiation-)
crosslinked polybutadiene, polystyrene, polysiloxane,
poly(meth)acrylate, and polyamides have primarily been used in the
past. They have been employed primarily with the intent of creating
a dense interface which shields the surrounding medium from
unwanted interactions with the underlying part ("carrier") of the
solid support material. Such interactions may lead to unspecific or
even irreversible binding of biomolecules to the sorbent while, on
the other hand, constituents of the solid support material or its
chemical linkages to the residues may be corroded by aggressive
components of either the sample or the eluent. Polymer-coated
sorbents are basically known for applications in all
chromatographic categories as they are listed above, but in
particular for hydrophobic interaction and size exclusion. Also
known are polymer coatings which are not internally crosslinked but
grafted to the carrier material as linear or branched chains, such
as the so-called tentacle resins.
[0012] Affinity chromatography, on the other hand, has mostly been
carried out with bulk gel-phase resins. Pre-eminent gel-forming
materials are medium-crosslinked polysaccharides, polyacrylamides,
and poly(ethylene oxides). Such hydrogels ensure a biocompatible
interface which can well accommodate both the active residue and
the biological analyte interacting therewith due to their softness
(conformational flexibility, elastic modulus), large pore systems,
high polarity and high water content, as well as the absence of
reactive or denaturing chemical groups. They are able to retain
proteins in their native state, i.e. preserve their correctly
folded, three-dimensional structure, state of association, and
functional integrity. This is to a large part a consequence of the
fact that organic solvents which are often required to elute
proteins or peptides from strongly adsorbing, hydrophobic (hard)
media, can be avoided. Lack of intrinsic adsorption strength of the
support is thereby compensated by the introduction of
highly-specific, intact biological ligands as binding partners for
the separation target which are well accommodated within the
hydrogel. The mechanical resistance of these media is, however,
much weaker than that of inorganic support materials since they are
compressible under an applied pressure and do not tolerate shear
stress caused by agitation, column packing or high liquid flow
rates. Affinity sorbents that are fully compatible with robust HPLC
process conditions are therefore rare.
[0013] Only in the recent past it has been recognised that the
mechanical resistance of the stationary phase is a bulk property of
the sorbent support whereas only a thin layer at the interface
between the stationary and the mobile phases is responsible for
mass exchange and for the interaction with the biological analyte.
Therefore the concept of combining the function of a mechanically
very rigid and dimensionally stable, porous 3-dimensional core, and
a biocompatible, gel-like interface layer which carries the active
residues for binding the analyte has been brought up, and the
associated synthetic problems have been technically solved. Such
hybrid materials employ loosely crosslinked polymers of high
polarity on a base of either an inorganic oxide or a densely
crosslinked polymer of low polarity.
[0014] Methodologically, they can be prepared by applying the
polymer of high polarity onto the core material or by directly
polymerising polar monomers, precursors thereof or a prepolymer in
the presence of the core material and a crosslinker. The majority
of materials prepared according to the latter method is being
described in the literature as having either a non-pore-penetrating
or a pore-filling morphology. While non-penetrating films suffer
from restricted surface areas available for interaction with the
analyte and thus low binding capacities which only depend on the
thickness of the polymer film, pore filling films take advantage of
the full inner pore volume of the core material in the interaction
with an analyte, which usually results in good binding capacities
but slow diffusional mass transfer rates inside the pores and
exchange kinetics with the mobile phase. A polymer film covering,
but not filling completely, the interior surfaces of the core
material, would be beneficial in this respect. The best known
representative of this whole class of sorbents is the system which
consists of branched and optionally further crosslinked
polyethylene imine grafted onto a porous silica support core
material. It has been demonstrated that such sorbents can be
further derivatised but they have been commercialised only for ion
exchange and those group-specific affinity applications which
require only small standard residues.
[0015] A conceptually different approach to the production of
synthetic affinity media is the so-called molecular imprinting
technique which is based on shape and functional group
complementarity between the target substrate and polymeric cavities
formed during a polymerisation reaction which is carried out in the
presence of the target substrate and a porogen, which have to be
removed subsequently. Imprinting has been developed for a large
number of substrates including proteins and peptides, and can be
split in a covalent and a non-covalent method, as far as the
temporary fixation of the target is concerned. It is, however,
restricted to the formation of a few highly-crosslinked types of
polymers as solid support materials and has so far not found
widespread acceptance once the production scale is reached,
especially not for pharmaceutical proteins or peptides which are
under the control of a regulatory body.
[0016] The most widespread used affinity media for the purification
of immunoglobulins G (IgG) are support-bound proteins A or G, both
of which are naturally produced on the cell walls of Staphylococci,
as well as protein L, but all require rather high capital
investments for large-scale applications, which basically prevent
their use as disposables. Protein A is known to bind a particular
epitope on the constant Fc part of antibodies. It is therefore of
limited use in the purification of recombinant antibody fragments
or fusion products lacking this region. Repeated use of
protein-derived sorbents is, on the other hand, associated with the
disadvantages of protein secondary/tertiary structure and/or
chemical linkage instability towards harsh manufacturing
conditions, resulting in possible inactivation or leakages
especially during obligatory, strongly alkaline sanitisation
treatments in between chromatographic runs. In addition to an
accordingly reduced life-span there is an ongoing debate as to the
application of protein A sorbents in pharmaceutical production
since even minute amounts of leaked protein A are suspected to
cause immunological disorders in humans when products to be
purified are for in vivo pharmaceutical use. Thus, registration
approval and expected market authorisation for a regulated product
are other important factors in the decision for a technical
purification process, and therefore it has become an industry
standard that protein A chromatography must be followed by an
additional chromatography step in order to remove leached
toxicants.
[0017] Beside attempts of creating engineered variants of these
proteins with improved technical properties, as a consequence also
a few sorbents having either very short (unnatural) peptide
epitopes only or even fully synthetic residues were manufactured.
Those synthetic media useful as protein A/G/L alternatives which
are commercially available have recently been reviewed in the
January 2007 issue of Journal of Chromatography B, volume 848.
BACKGROUND ART
[0018] The usefulness of bi- and mononuclear C, N, O,
S-heteroaromatic structures, in the following exemplarily
demonstrated for the prototypical indole and imidazole structures,
as residues of biochromatography sorbents has been recognised
earlier but independently and without claiming the benefits of
their combined use. However, examples of imidazole structures are
found more often in the scientific and patent literature than
indole structures. An obvious way of introducing an imidazole
structure into a sorbent is by way of amide bond coupling with the
natural amino acid histidine, a protected form thereof, or with the
related histamine, with or without an additional linker moiety.
Histamine can be coupled to the support via its amino group by
solid-phase synthesis techniques, resulting in an imidazole
structure carrying no additional charged or dissociable groups.
With histidine, two options are feasible: coupling through amide
formation at the amino group resulting in an imidazole structure
still containing a deprotonable carboxylic group, or alternatively
through amide formation at the carboxyl group resulting in an
imidazole structure still containing a protonable amino group. All
these different possibilities have already been realised
experimentally. Analogously, indole structures can easily be
introduced as tryptophan or tryptamine residues.
[0019] Affinity chromatography on histidine sorbents was
extensively reviewed in Molecular Interactions in Bioseparations
(Ed.: T. T. Ngo), Plenum Press, New York 1993, Chapter 18 (pp.
257-275), and in Biochromatography (Ed.: M. A. Vijayalakshmi),
Taylor & Francis, London 2002, Chapter 9 (pp. 252-271). An
account of the various sorbents which have been of use in the
purification of human plasma proteins is given in these references
together with details on chromatographic parameters and proposed
mechanisms of interaction. Practical aspects of histidine affinity
chromatography were summarised by the same author in Molecular
Biotechnology 6 (1996), 347-357. Some examples with relevance for
immunoglobulin purification will be highlighted in the
following:
[0020] As an early example, in Journal of Chromatography 376
(1986), 259-267, histidine was coupled to sepharose via
epichlorohydrin or 1,4-butanediol diglycidyl ether linkages and to
epoxy-activated silica. The effect of sorbent preparation on the
chromatographic results with several protein and peptide analytes
were investigated. The sorbent was finally employed in the
semi-pilot scale purification of human placental IgG.
[0021] The purification of IgG.sub.1 and IgG.sub.2 subclasses from
human plasma on histidyl-aminohexyl-sepharose with an applied salt
gradient was described in Bioseparation 3 (1992), 47-53. In Journal
of Chromatography B 667 (1995), 57-67, and again in Journal of
Chromatography B 674 (1995), 13-21, histidine was immobilised onto
epichlorohydrin- or butanediol diglycidylether-activated
poly(ethylene vinylalcohol) hollow fibre membranes for the
subclass-selective one-step separation of IgG from untreated human
serum. The sorbent was reproducibly employed in chromatographic and
single equilibration experiments under different buffer and
temperature conditions, which allowed insights into the binding
mechanism at the Fab part of the antibody. The separation mechanism
of glycated HSA isoforms in different buffer systems on
histidyl-aminohexylsepharose was then studied in Journal of
Chromatography B 758 (2001), 163-172. The performance of histidine
immobilised on a sepharose gel and on polyethylene-vinylalcohol was
further compared to histidine-ethylenediamine bound to a monolithic
disk support in the purification of a model restriction enzyme from
a bacterial extract according to Chromatographia 65 (2007),
639-648. The same publication also reported on the adsorption of
pure IgG onto the monolithic medium and on the isolation of a
monoclonal antibody from a cell culture supernatant.
[0022] A conceptually different approach for the production of
sorbents having imidazole residues was followed by the authors of
Reactive Polymers 13 (1990), 177, and others. A polyvinylimidazole
film was prepared on a solid carrier in order to prepare a sorbent
which exhibited reversed-phase character.
[0023] In Analytical Biochemistry 201 (1992), 170-177, an agarose
sorbent with imidazole residues N-bound via a divinylsulphone
linkage was used in the fractionation of human serum and compared
to sorbents containing other aromatic or heteroaromatic residues
under a variety of mobile phase conditions. The binding was found
to be dependent on the density of residues, but the binding
patterns were always thought to contain contributions of the
sulphone moiety.
[0024] In Bioseparation 6 (1996), 165-184, a series of sorbents was
prepared wherein histidine was coupled through three different
linkers to sepharose supports. Thermodynamic, kinetic, and
stability data were presented as well as measured selectivities in
the separation of an artificial mixture of murine IgG.sub.1 with
BSA and of murine IgG.sub.1 from a cell culture filtrate. In
another approach reported in Journal of Membrane Science 207
(2002), 253-264, nylon membrane disks were covalently coated with
dextran or polyvinylalcohol layers and derivatised under
epibromohydrine activation with a hexanediamine linker and finally
with histidine. The membrane sorbents were compared to other
affinity membranes in their ability to bind human IgG.
[0025] According to Reactive & Functional Polymers 34 (1997),
103-111, retention of IgG was also observed on a sorbent prepared
from a poly(butadiene-hydroxyethylmethacrylate) support and
histidine after activation of the support with epichlorohydrin or
1,4-butanediol diglycidylether.
[0026] Chromatography with sorbents having histidine residues was
compared to other separation techniques in Applied Biochemistry and
Biotechnology 75 (1998), 93-102. Now focusing on the application of
antibody (IgG, IgM) purification, results obtained on sepharose,
nylon, nylon-polyvinyl alcohol, and silica flat membranes as well
as polyethylene vinyl alcohol hollow fiber membranes with
immobilised histidine were summarised. The recovery of antibodies
directly from sera was found to be superior to traditional capture
on protein A or protein G media in so far as a high degree of
functionality could be retained. The sorbent was discovered to have
IgG subclass-selective properties.
[0027] In Journal of Chromatography A 814 (1998), 71-81,
aminopropylimidazole, histamine, aminomethylbenzimidazole,
mercaptomethylimidazole, and 2-mercaptobenzimidazole were coupled
to activated sepharose or cellulose beads. The retention behaviour
of several model proteins on these sorbents under elution
conditions employing a salt gradient or a pH change was tested. In
another example, 2-mercapto-1-methylimidazole was coupled via a
2-hydroxypropoxy linker to sepharose as described in Journal of
Biotechnology 79 (2000), 103-115. Its suitability in the isolation
of an extracellular acid protease during work-up of a microbial
culture under pH conditions close to the pl of the protein was
tested.
[0028] The amino group of histidine was also coupled directly or
via a hexanediamine linker to an acrylate copolymer monolith, which
was then used in the separation of IgG from human serum, as
reported in Biotechnology and Bioengineering 80 (2002), 481-489.
This residue also exhibits a carboxylate group available for
interaction with the analyte.
[0029] Separation Science and Technology 37 (2002), 717-731, and
Reactive & Functional Polymers 61 (2004), 369-377, described
the partial derivatisation of poly(2-hydroxyethylmethacrylate) with
N-bound histidine residues and its repeated use in the reversible
adsorption of human IgG from plasma under various experimental
conditions. A similar crosslinked sorbent was prepared by
copolymerisation from 2-hydroxyethyl methacrylate with
2-methacrylamidohistidine according to Macromolecular Bioscience 2
(2002), 135-144, and used in the same way. Another
poly(ethyleneglycol dimethacrylate-N-methacryloyl-histidine
methylester) copolymer was prepared similarly by suspension
polymerisation on magnetic particles for use in IgG adsorption onto
a magnetically stabilised fluidised bed column, as demonstrated in
Biotechnology Progress 20 (2004), 1169-1175. Furthermore, in
Colloids and Surfaces A 301 (2007), 490-497, a non-covalent
composite comprising histidine on bentonite as a solid support
material was described and tested in an analogous way.
[0030] Tryptophanol and 2-aminobenzimidazole were, among other
residues, each independently coupled to sepharose supports as
described in Journal of Chromatography A 1016 (2003), 21-33. The
breakthrough capacities and the recoveries of BSA as a model system
for negatively charged biomolecules were measured and compared
among the different sorbents.
[0031] 2-Mercapto-5-benzimidazole sulphonic acid coupled to
cellulose beads was again described in Journal of Chromatography B
808 (2004), 25-33. IgG.sub.1 monoclonal antibodies from mouse
ascitic fluid or rat hybridoma cell culture supernatant as well as
IgG from human plasma Cohn fractions II+III were captured on the
sorbent.
[0032] International patent application WO 96/00735 (Massey
University) claims a complex formed between a resin and a protein
or peptide bound thereto, whereby the resin comprises ionisable
ligands covalently attached to a solid support material. The
experimental part and the stated adsorption/desorption mechanism
was largely identical with the following patent (see below). As an
additional example, the binding of a subtilisin variant from a
fermentation broth to 1-(3-aminopropyl)imidazole attached via amide
formation onto activated aminocaproic acid cellulose was monitored
under buffered low and high salt conditions. Related European
patent EP 783366 (Massey University) claims resins with a
relatively high density of one or more ionisable residues, and
optionally additional non-ionisable residues, bound to a support
via spacer arms. Analytes are adsorbed to the resins under
hydrophobic, low electrostatic charge conditions and desorbed at a
pH of higher electrostatic charge. 1-(3-Aminopropyl)imidazole
(optionally bound to an aminocaproic spacer),
2-(aminomethyl)benzimidazole, histamine, mercaptobenzimidazole,
2-mercapto-1-methylimidazole, and tryptamine residues were given as
examples. Either sepharose or cellulose supports were used after
their activation with epichlorohydrin, allyl glycidyl ether,
allylbromide, or carbonyl diimidazole. Acid titration data of the
different sorbent residues were presented.
[0033] International patent application WO 2006/066598 (Versamatrix
A/S) claims sorbents having covalently immobilised residues, each
of said residues containing both cationic and hydrophobic groups at
a given range of distances apart from each other. Various short
peptides or peptidomimetics were immobilised on synthetic resins
and tested for their suitability in monoclonal antibody separation
to illustrate the invention.
[0034] European Patent EP 764048 (Biosepra Inc.) claims sorbents
containing optionally substituted 5-membered heterocyclic residues
which are linked to a solid support material via a thioether
bridge. The given examples included sorbents prepared from 2-amino-
and 2-mercaptoimidazole and epoxy-activated solid supports with
hydrogel-filled pores. The 2-mercaptoimidazole sorbent was
successfully used in the purification of IgG from bovine colostrum.
International patent application WO 2004/024318 (Ciphergen
Biosystems Inc.) claims sorbents having mono- or polycyclic
heteroaromatic residues with certain anionic substituents which are
linked via a mercapto-, ether-, or amino-group to a solid support
material. As an example, 2-mercaptobenzimidazole sulphonic acid was
immobilised on cellulose beads, porous zirconia beads, and as a
dextran-based coating on silica. Antibodies were separated from
bovine serum and from milk whey by chromatography on the modified
cellulose material.
[0035] European patent EP 921855 (Upfront Chromatography A/S)
claims a method for the isolation of IgG from a solution on a
sorbent consisting of a spacer-bound, low-molecular weight residues
selected from benzimidazoles, benzothiazoles, and benzoxazoles, on
a support material. 2-Mercaptobenzimidazole on an
epichlorohydrin-activated agarose support was tested for its
alkaline stability and then used to isolate monoclonal antibodies
from an artificial culture supernatant as well as polyclonal
antibodies from various animal sera and from egg yolk. A sorbent
containing 2-mercapto-5-nitrobenzimidazole residues was also
described.
[0036] Sepharose sorbents exhibiting either tryptophanol,
2-aminobenzimidazole, or histidine residues, which were bound via
short linkers to the solid support material, were exemplarily
reported in international patent application WO 01/38227 (Amersham
Pharmacia Biotech AB), and their anion exchange properties against
four model proteins were measured. A method of binding a negatively
charged substrate from an aqueous liquid with a sorbent having
residues with simultaneously anion-exchanging and hydrophobic
properties, each residue comprising a specified, spacer-bound
arylammonium structure, is claimed. In international patent
application WO 2005/082483 (Amersham Biosciences AB), a method for
the capture of antibodies from a liquid onto a sorbent is claimed
wherein the sorbent carries residues which each comprise at least
two different structures: one cation exchange group and one C, S,
O-(hetero)aromatic ring system. Chromatography on a
2-aminobenzimidazo-sepharose sorbent under non-binding conditions
was used in the examples as a flow-through polishing step for
pre-purified humanised IgG.sub.1 obtained from a CHO cell culture.
In international patent application WO 2006/043895 (GE Healthcare
Bio-Sciences AB), a method for the separation of antibodies from a
liquid is claimed which uses a sorbent having two different groups,
the first one being capable of interacting with negative charges on
an analyte and the second one being capable of a respective
non-charge interaction. The sorbent retains the impurities of the
sample while the antibodies pass through. As an example, monoclonal
antibodies were separated from protein A and host cell proteins by
chromatography on a sorbent containing 2-aminobenzimidazole
residues on an activated sepharose support.
[0037] U.S. Pat. No. 6,071,416 relates to compositions comprising
one or more N-cyclic aromatic hydrocarbon ligands the composite of
which contains at least two, and preferably four or more N-cyclic
groups bonded through an appropriate hydrophilic spacer grouping to
a solid support and to the use of such compositions in the removal
or concentration of specific ions from solutions.
[0038] WO 97/10887 relates to affinity ligands, their preparation
and attachment to matrices which may consist of solid, semi-solid,
particulate or colloidal materials, or soluble polymers, and to the
use thereof in the purification of proteinaceous materials.
OBJECTS OF THE INVENTION
[0039] One object of the invention is to provide a novel
purification method for proteins and peptides and a sorbent for
performing said method.
BRIEF SUMMARY OF THE INVENTION
[0040] The present invention is directed toward a sorbent
comprising a solid support material, the surface of which comprises
at least two different residues among which are first residues
comprising a binuclear heteroaromatic structure comprising besides
carbon atoms at least one of the heteroatoms N, O, S, and second
residues comprising a mononuclear heteroaromatic structure
comprising besides carbon atoms at least one of the heteroatoms N,
O, S. Optionally, these at least two residues are being carried by
a film of a crosslinked polymer covering said surface. Due to its
of fully synthetic origin, said sorbent is characterised by a high
physical (particularly thermal) and chemical robustness, though
still allowing the specific separation of biomolecules under gentle
physiological conditions, even from unfavourable sample matrices.
Alternative methods for the preparation of such sorbent are also
provided.
[0041] The invention also provides a method for separating or
increasing the concentration and/or purity of a protein or peptide
from a mixture containing the protein or peptide. The method
comprises contacting said mixture with a sorbent according to the
invention, to which the desired protein or peptide is bound, the
subsequent elution of said protein or peptide from the sorbent, and
optionally an intermediate rinsing step.
[0042] Disclosed are also various analytical and preparative
biochemical as well as medical applications in which the sorbent
and/or the method can be beneficially employed. Antibodies purified
according to the method are being characterised by percentages of
recovery, purity, and biological activity which are comparable to
those obtained via conventional bioaffinity separation techniques,
without suffering from the disadvantages of such techniques.
[0043] According to a general aspect, the sorbent according to the
invention comprises a solid support material, the surface of which
comprises first residues comprising a binuclear heteroaromatic
structure comprising besides carbon atoms at least one of the
heteroatoms N, O, S, and second residues comprising a mononuclear
heteroaromatic structure comprising besides carbon atoms at least
one of the heteroatoms N, O, S.
[0044] In one embodiment, the binuclear heteroaromatic structure is
a benzopyrrole (indole) structure, including all possible
aza-benzopyrrole, oxa-benzopyrrole, and thia-benzopyrrole
structures, or a benzopyridine (quinoline or isoquinoline)
structure, including all possible aza-benzopyridine,
oxa-benzopyridine, and thia-benzopyridine structures.
[0045] In one embodiment, the mononuclear heteroaromatic structure
comprises a five-membered heteroaromatic core.
[0046] In one embodiment the mononuclear heteroaromatic structure
is a pyrrole structure, including all possible aza-pyrrole,
oxa-pyrrole, and thia-pyrrole structures such as 3-azapyrrole
(imidazole).
[0047] In one embodiment, the mononuclear heteroaromatic structure
does not comprise a six-membered heteroaromatic core.
[0048] In one embodiment, the mononuclear heteroaromatic structure
comprises a six-membered heteroaromatic core under the proviso that
said six-membered heteroaromatic core is not a triazine ring or a
pyrimidine ring.
[0049] In one embodiment, the first and/or second residues comprise
a linker.
[0050] In one embodiment the first and/or second residues comprise
a covalent, conformationally flexible linker of a length of from 1
to 20 atoms.
[0051] In one embodiment the covalent, conformationally flexible
linker does not contain sulphur.
[0052] In one embodiment, the linkers comprise independently from
each other from 1 to 300 carbon atoms, e.g. 20 to 300 carbon atoms.
In said embodiment, the linker consists of or comprises
polyethylene glycol moieties.
[0053] In one embodiment, the linker does not comprise a further
heteroaromatic structure, particularly not a triazine or pyrimidine
ring.
[0054] In one embodiment further substituents are bound to the
binuclear and/or mononuclear heteroaromatic structure comprising at
least one of the heteroatoms N, O, S, respectively.
[0055] In one embodiment said further substituents are not
comprising cation-exchanging (i.e., negatively charged) groups, or
said binuclear and/or mononuclear heteroaromatic structure do not
comprise cation-exchanging (i.e., negatively charged) groups.
[0056] In one embodiment, said further substituents are not
comprising cation-exchanging groups comprising sulphate, sulfonate,
phosphate, or phosphonate groups; or said binuclear and/or
mononuclear heteroaromatic structure do not comprise
cation-exchanging groups comprising sulphate, sulfonate, phosphate,
or phosphonate groups.
[0057] In another embodiment, said further substituents are not
comprising cation-exchanging groups selected from sulphate,
sulfonate, phosphate, or phosphonate groups; or said binuclear
and/or mononuclear heteroaromatic structure do not comprise
cation-exchanging groups selected from sulphate, sulfonate,
phosphate, or phosphonate groups.
[0058] In one embodiment the first and second residues are present
in a molar ratio of from 3:2 to 2:3, preferably in a ratio of about
1:1.
[0059] In one embodiment the first residue comprises the second
residue.
[0060] In one embodiment the surface of the solid support material
additionally comprises third residues and optionally also fourth
residues.
[0061] In one embodiment the third residues comprise an amine or
amide structure, preferably a primary amine structure.
[0062] In one embodiment the first, second, and third residues are
present in a molar ratio of about 1:1:2.
[0063] In one embodiment the total density of residues amounts to
from 0.1 mol dm.sup.-3 to 1.0 mol dm.sup.-3, preferably at least
about 0.3 mol dm.sup.-3.
[0064] In one embodiment each type of residue is homogeneously and
randomly/statistically distributed on the surface of the solid
support material.
[0065] In one embodiment the solid support material consists of a
carrier the surface of which is covered with a film of a polymer
having functional groups which carries the first and second, and
optionally the third and fourth residues.
[0066] In one embodiment the polymer consists of individual chains
which are covalently crosslinked with each other, but which are not
covalently grafted or bound to the surface of the carrier.
[0067] In one embodiment the polymer chains are covalently
crosslinked with each other to an extent of from 2% to 20% based on
the number of functional groups available for crosslinking.
[0068] In one embodiment the polymer consists of individual chains
which are covalently grafted to the surface of the carrier, but not
covalently crosslinked with each other.
[0069] In one embodiment the polymer chains are covalently grafted
to the surface of the carrier via their terminal functional
groups.
[0070] In one embodiment the film of the polymer accounts for from
5% to 30%, preferably from 15% to 20%, of the total weight of the
sorbent.
[0071] In one embodiment the polymer is swellable in aqueous or
mixed aqueous-organic media.
[0072] In one embodiment the polymer is a synthetic
polyelectrolyte.
[0073] In one embodiment the crosslinking or grafting connections
of the polymer and/or the linkages of the residues are made of
amide, urethane, urea, or secondary/tertiary amine bonds.
[0074] In one embodiment the polymer is a partially derivatised
polymer selected from the group consisting of polyvinyl alcohol,
polyvinylamine, polyallylamine, polyethylene imine, polyacrylic
acid, and polymethacrylic acid, or any copolymer or polymer blend
comprising at least one of these polymers.
[0075] In one embodiment, the polymer is polyvinyl amine.
[0076] In one embodiment the solid support material or at least the
carrier is a porous material having a pore size of from 10 nm to
400 nm, or a specific surface area of from 1 m.sup.2 g.sup.-1 to
1,000 m.sup.2 g.sup.-1, or a porosity of from 30% to 80% by
volume.
[0077] In one embodiment the solid support material is a
particulate material having a particle size of from 5 .mu.m to 500
.mu.m.
[0078] In one embodiment the solid support material is a sheet- or
fibre-like material such as a membrane.
[0079] In one embodiment the material the carrier is made of is
different from the material the film of a polymer is made of.
[0080] In one embodiment the solid support material or at least the
carrier is made of a material selected from the group consisting of
generic or surface-modified polystyrene, polystyrene sulphonic
acid, polyacrylates, polymethacrylates, polyvinyl alcohol, silica,
glass, starch, cellulose, agarose, sepharose, and dextran, or
composites thereof.
[0081] In one embodiment the sorbent additionally comprises an
easily detectable tag such as an optically absorbing, an optically
emitting, a radioactive, a magnetic, or a mass- or
radiofrequency-encoding tag.
[0082] The invention also relates to a method for preparing a
sorbent, comprising: [0083] (i) providing a polymer having
functional groups; [0084] (ii) adsorbing a film of said polymer
onto the surface of a carrier; [0085] (iii) crosslinking a defined
portion of said functional groups of the adsorbed polymer with at
least one crosslinking reagent; [0086] (iv) derivatising further
defined portions of said functional groups of the crosslinked
polymer with first residues comprising a binuclear heteroaromatic
structure comprising besides carbon atoms at least one of the
heteroatoms N, O, S, and with second residues comprising a
mononuclear heteroaromatic structure comprising besides carbon
atoms at least one of the heteroatoms N, O, S, and with optional
further residues.
[0087] The invention also relates to a method for preparing a
sorbent, comprising: [0088] (i) providing a polymer having
functional groups; [0089] (ii) derivatising defined portions of
said functional groups with first residues comprising a binuclear
heteroaromatic structure comprising besides carbon atoms at least
one of the heteroatoms N, O, S, and with second residues comprising
a mononuclear heteroaromatic structure comprising besides carbon
atoms at least one of the heteroatoms N, O, S, and with optional
further residues; [0090] (iii) adsorbing a film of the derivatised
polymer onto the surface of a carrier; [0091] (iv) crosslinking a
further defined portion of said functional groups of the adsorbed
polymer with at least one crosslinking reagent.
[0092] The invention also relates to a method for preparing a
sorbent, comprising: [0093] (i) providing a polymer having
functional groups; [0094] (ii) adsorbing a film of said polymer
onto the surface of a carrier; [0095] (iii) grafting a defined
portion of said functional groups of the adsorbed polymer to said
carrier; [0096] (iv) derivatising further defined portions of said
functional groups of the grafted polymer with first residues
comprising a binuclear heteroaromatic structure comprising besides
carbon atoms at least one of the heteroatoms N, O, S, and with
second residues comprising besides carbon atoms a mononuclear
heteroaromatic structure comprising at least one of the heteroatoms
N, O, S, and with optional further residues.
[0097] The invention also relates to a method for preparing a
sorbent, comprising: [0098] (i) providing a polymer having
functional groups; [0099] (ii) derivatising defined portions of
said functional groups with first residues comprising a binuclear
heteroaromatic structure comprising besides carbon atoms at least
one of the heteroatoms N, O, S, and with second residues comprising
a mononuclear heteroaromatic structure comprising besides carbon
atoms at least one of the heteroatoms N, O, S, and with optional
further residues; [0100] (iii) adsorbing a film of the derivatised
polymer onto the surface of a carrier; [0101] (iv) grafting a
further defined portion of said functional groups of the adsorbed
polymer to said carrier.
[0102] In one embodiment of the methods for preparing a sorbent,
the polymer is soluble in aqueous or mixed aqueous-organic
media.
[0103] In one embodiment, the functional groups of the polymer are
--NH--, --NH.sub.2, --OH, --COON or --COO-- groups.
[0104] In one embodiment the polymer has a molecular weight of
between 5,000 Dalton and 50,000 Dalton.
[0105] In one embodiment the at least one crosslinking reagent is
selected from the group consisting of dicarboxylic acids, diamines,
diols, and bis-epoxides.
[0106] In one embodiment the at least one crosslinking reagent is a
linear, conformationally flexible molecule of a length of between 1
and 20 atoms.
[0107] In one embodiment the derivatisation step is carried out by
formation of amide bonds between said functional groups and said
residues.
[0108] In one embodiment the derivatisation step is carried out
stepwise with each residue.
[0109] The invention also relates to a method of separating, or
increasing the concentration and/or purity of a protein or peptide
from a mixture containing said protein or peptide, comprising:
[0110] (i) contacting said mixture being dissolved or suspended in
a first liquid with a sorbent according to the invention or with a
sorbent prepared according to a method of the invention, for a
period of time sufficient to enable said protein or peptide to
become bound to said sorbent; [0111] (ii) optionally rinsing said
sorbent with a second liquid; [0112] (iii) contacting said sorbent
with said bound protein or peptide with a third liquid for a period
of time sufficient to enable said protein or peptide to become
released from said sorbent; [0113] (iv) optionally washing and/or
regenerating the sorbent with a fourth and/or fifth liquid.
[0114] In one embodiment of the method of separating, or increasing
the concentration and/or purity of a protein or peptide, the first
liquid, the second liquid, and the third liquid are buffered
aqueous media, not containing further organic modifiers.
[0115] In one embodiment, the second liquid is the same as the
first liquid.
[0116] In one embodiment, the pH of the first and optionally the
second liquid is close to the isoelectric point pl of the target
protein or peptide.
[0117] In one embodiment, the pH of the third liquid is different,
in particular lower, than the pH of the first and optionally of the
second liquid.
[0118] In one embodiment, the pH of the first liquid is in the
range of from 5.5 to 8.5 and the pH of the third liquid is in the
range of from 3 to 6.5.
[0119] In one embodiment, the ionic strength of the third liquid is
different, in particular higher, than the ionic strength of the
first and optionally of the second liquid.
[0120] In one embodiment, the method is carried out as a
membrane-filtration technique, a solid phase extraction technique
or as a medium- to high-pressure liquid chromatography
technique.
[0121] In one embodiment, the method further comprises the
isolation of the released protein or peptide from the third liquid
subsequent to step (iii).
[0122] In one embodiment, the released protein or peptide of step
(iii) contains less than 10 ppm of leached sorbent or other
leachable substances therefrom.
[0123] In one embodiment, the method is combined with further
separation processes such as precipitation, centrifugation, drying,
(micro-/ultra-)filtration, dialysis, ion exchange, or viral
reduction treatments.
[0124] In one embodiment, said mixture containing said protein or
peptide is a crude or partially purified biosynthetic product,
obtained from a microorganism or a cell culture, or from a crop
extract.
[0125] In one embodiment, said protein or peptide has an
isoelectric point pl of from 5.5 to 8.5 and a molecular weight of
from 100 to 500,000 Da.
[0126] In one embodiment, said protein or peptide is an antibody, a
fragment thereof, oligomeric associates thereof, or an antibody- or
antibody fragment-containing fusion protein.
[0127] The invention also relates to a column for liquid
chromatography or solid phase extraction comprising a sorbent
according to the invention or a sorbent prepared according to a
method according to the invention as a stationary phase within a
tubular containment and optionally further components such as
frits, filter plates, flow distributors, seals, fittings,
screwings, valves, or other fluid handling or connection
elements.
[0128] In one embodiment, the method is further characterised by
its physical and chemical resistance against applied pressures up
to 20 bar, against applied heat up to 110.degree. C., as well as
against common sanitisation protocols, thus enabling its repetitive
use of up to 1,000 times, preferably up to 5,000 times.
[0129] The invention also relates to a collection of a plurality of
the same or different sorbents according to the invention or of
sorbents prepared according to a method according to the invention
or of columns according to the invention in the format of a
microplate or microchip array, or a multi-capillary or microfluidic
device, capable of being processed in parallel.
[0130] The invention also relates to a diagnostic or laboratory
purification kit comprising a sorbent according to the invention or
a sorbent prepared according to a method according to the invention
or a column according to the invention or a collection of sorbents
or columns according to the invention and, within the same
packaging unit, further chemical or biological reagents and/or
disposables necessary for carrying out the method according to the
invention or a different analytical, diagnostic, or laboratory
method different therefrom.
[0131] The invention also relates to the use of a sorbent according
to the invention or a sorbent prepared according to a method
according to the invention in the manufacture of a pharmaceutical
or nutritional composition comprising at least one protein or
peptide of diagnostic, therapeutic, or nutritional value.
[0132] The invention also relates to the use of a sorbent according
to the invention or a sorbent prepared according to a method
according to the invention in the removal of at least one protein
or peptide, and in the medical prevention or treatment of diseases
being caused by the presence of said at least one protein or
peptide.
[0133] The invention also relates to the use of a sorbent according
to the invention or a sorbent prepared according to a method
according to the invention in the identification, characterisation,
quantification, or laboratory purification of at least one protein
or peptide.
[0134] The invention also relates to the use of a sorbent according
to the invention or a sorbent prepared according to a method
according to the invention for the reversible immobilisation of at
least one protein or peptide and optionally testing for binding of
further chemical or biological structures to said protein or
peptide.
[0135] According to a first aspect, the invention relates to a
sorbent comprising a solid support material, the surface of which
comprises [0136] a first residue comprising a binuclear
heteroaromatic structure comprising besides carbon atoms at least
one of the heteroatoms N, O, S; and [0137] a second residue
comprising a mononuclear heteroaromatic structure comprising
besides carbon atoms at least one of the heteroatoms N, O, S;
characterized in that the first and second residue are not directly
connected with each other but are separately attached to either a
bulk solid support material itself or a polymer film supported by
it as carrier.
[0138] In one embodiment, the binuclear heteroaromatic structure is
a benzopyrrole (indole) structure, including all possible
aza-benzopyrrole, oxa-benzopyrrole, and thia-benzopyrrole
structures, or a benzopyridine (quinoline or isoquinoline)
structure, including all possible aza-benzopyridine,
oxa-benzopyridine, and thia-benzopyridine structures.
[0139] In one embodiment, the mononuclear heteroaromatic structure
is a pyrrole structure, including all possible aza-pyrrole,
oxa-pyrrole, and thia-pyrrole structures such as 3-azapyrrole
(imidazole).
[0140] In one embodiment, the first and/or second residue
comprise(s) a covalent, conformationally flexible linker of a
length of from 1 to 20 atoms.
[0141] In one embodiment, the surface of the solid support material
additionally comprises a third residue and optionally a fourth
residue.
[0142] In one embodiment, the third residue comprises an amine or
amide structure or a primary amine structure.
[0143] In one embodiment, the first, second, and third residue are
present in a molar ratio of about 1:1:2.
[0144] In one embodiment, the surface of the solid support material
is covered with a film of a polymer comprising a first and a second
functional group, which may be the same or which may be different
from each other, which in turn carry said first and second, and
optionally a third and a fourth residue.
[0145] In one embodiment, the polymer comprises or consists of
individual chains which are covalently crosslinked with each other,
but which are not covalently bound or grafted to the surface of the
carrier.
[0146] In one embodiment, the polymer is a polyamine, or a
polyvinyl amine, or a copolymer or polymer blend comprising a
polyamine.
[0147] In one embodiment, a first portion of said functional groups
of the adsorbed polymer is crosslinked with at least one
crosslinking reagent, and wherein a second portion of said
functional groups of the crosslinked polymer are bound to said
first and second, and optional further residues.
[0148] The invention also relates to a method for preparing a
sorbent according to the first aspect of the invention, comprising:
[0149] (i) providing a polymer having a first and a second
functional group; [0150] (ii) adsorbing a film of said polymer onto
the surface of a carrier; [0151] (iii) crosslinking a first portion
of said functional groups of the adsorbed polymer with at least one
crosslinking reagent; [0152] (iv) binding a second portion of said
functional groups of the crosslinked polymer with said first,
second, and optional further residues.
[0153] The invention also relates to a method of separating, or
increasing the concentration and/or purity of a protein or peptide
from a mixture containing said protein or peptide, comprising:
[0154] (i) contacting said mixture being dissolved or suspended in
a first liquid with a sorbent according to the first aspect of the
invention for a period of time sufficient to enable said protein or
peptide to become bound to said sorbent; [0155] (ii) optionally
rinsing said sorbent with a second liquid; [0156] (iii) contacting
said sorbent with said bound protein or peptide with a third liquid
for a period of time sufficient to enable said protein or peptide
to become released from said sorbent; [0157] (iv) optionally
washing and/or regenerating the sorbent with a fourth and/or fifth
liquid.
[0158] In one embodiment, the pH of the first and optionally the
second liquid is close to the isoelectric point pl of the target
protein or peptide.
[0159] In one embodiment, the pH of the first liquid is in the
range of from 5.5 to 8.5 and the pH of the third liquid is in the
range of from 3 to 6.5.
[0160] In one embodiment, said protein or peptide has an
isoelectric point pl of from 5.5 to 8.5 and a molecular weight of
from 100 to 500,000 Da.
[0161] According to a second aspect, the invention relates to a
sorbent comprising a solid support material, the surface of which
comprises [0162] a first and a second functional group, which may
be the same or different; [0163] a first residue comprising a
binuclear heteroaromatic structure comprising besides carbon atoms
at least one of the heteroatoms N, O, S; and [0164] a second
residue comprising a mononuclear heteroaromatic structure
comprising besides carbon atoms at least one of the heteroatoms N,
O, S; characterized in that the first residue is bound to the first
functional group, and the second residue is bound to the second
functional group.
[0165] According to a third aspect, the invention relates to a
sorbent comprising a solid support material, the surface of which
comprises [0166] a first and a second functional group, which may
be the same or different; [0167] a first residue comprising a
binuclear heteroaromatic structure comprising besides carbon atoms
at least one of the heteroatoms N, O, S; and [0168] a second
residue comprising a mononuclear heteroaromatic structure
comprising besides carbon atoms at least one of the heteroatoms N,
O, S; characterized in that the first residue is bound to the first
functional group, and the second residue is bound to the second
functional group; and wherein none of said functional groups is
bound to both said first residue and said second residue.
[0169] According to a fourth aspect, the invention relates to a
sorbent comprising a solid support material, the surface of which
comprises [0170] a first and a second functional group, which may
be the same or different; [0171] a first residue comprising a
binuclear heteroaromatic structure comprising besides carbon atoms
at least one of the heteroatoms N, O, S; and [0172] a second
residue comprising a mononuclear heteroaromatic structure
comprising besides carbon atoms at least one of the heteroatoms N,
O, S; characterized in that the first residue is bound to the first
functional group, and the second residue is bound to the second
functional group; under the proviso that the solid support material
does not comprise a triazine moiety or a pyrimidine moiety.
[0173] According to a fifth aspect, the invention relates to a
sorbent comprising a solid support material, the surface of which
comprises [0174] a first and a second functional group, which may
be the same or different; [0175] a first residue comprising a
binuclear heteroaromatic structure comprising besides carbon atoms
at least one of the heteroatoms N, O, S; and [0176] a second
residue comprising a mononuclear heteroaromatic structure
comprising besides carbon atoms at least one of the heteroatoms N,
O, S; characterized in that the first residue is bound to the first
functional group, and the second residue is bound to the second
functional group; under the proviso that the solid support material
does not comprise a triazine moiety or a pyrimidine moiety; and
wherein none of said functional groups is bound to both said first
residue and said second residue.
[0177] In one embodiment according to the second, third, fourth or
fifth aspect, the binuclear heteroaromatic structure is a
benzopyrrole (indole) structure, including all possible
aza-benzopyrrole, oxa-benzopyrrole, and thia-benzopyrrole
structures, or a benzopyridine (quinoline or isoquinoline)
structure, including all possible aza-benzopyridine,
oxa-benzopyridine, and thia-benzopyridine structures.
[0178] In one embodiment, the mononuclear heteroaromatic structure
is a pyrrole structure, including all possible aza-pyrrole,
oxa-pyrrole, and thia-pyrrole structures such as 3-azapyrrole
(imidazole).
[0179] In one embodiment, the first residue or the second residue
or the first and the second residue are bound to said first and
second functional group via a linker.
[0180] In one embodiment, from 5 to 95%, or from 15 to 85%, or from
25 to 75%, or from 35 to 65%, or from 40 to 60% of said first and
second functional groups are bound to said first and second
residue, and wherein said first and second residue are present in a
molar ratio of from 3:2 to 2:3.
[0181] In one embodiment, the surface of the solid support material
additionally comprises a third residue and optionally a fourth
residue.
[0182] In one embodiment, the third residue comprises an amine or
amide structure, or a primary amine structure.
[0183] In one embodiment, the first, second, and third residues are
present in a molar ratio of about 1:1:2.
[0184] In one embodiment, the surface of the solid support material
is covered with a film of a polymer comprising said first and
second functional groups which in turn carry said first and second,
and optionally a third and a fourth residue.
[0185] In one embodiment, the polymer comprises or consists of
individual chains which are covalently crosslinked with each other,
but which are not covalently grafted to the surface of the
carrier.
[0186] In one embodiment, the polymer is a partially derivatised
polyamine, or a polyvinyl amine, or a copolymer or polymer blend
comprising a polyamine.
[0187] In one embodiment, a first portion of said first and second
functional groups is crosslinked with at least one crosslinking
reagent, and wherein a second portion of said first and second
functional groups are bound to said first and second, and optional
further residues.
[0188] In one embodiment, the invention relates to a method for
preparing a sorbent according to the second, third, fourth or fifth
aspect, comprising: [0189] (i) providing a polymer having a first
and a second functional group; [0190] (ii) adsorbing a film of said
polymer onto the surface of the solid support material; [0191]
(iii) crosslinking a first portion of said functional groups of the
adsorbed polymer with at least one crosslinking reagent; [0192]
(iv) binding a second portion of said functional groups of the
crosslinked polymer with said first, second, and optional further
residues.
[0193] In one embodiment, the invention relates to a method of
separating, or increasing the concentration and/or purity of a
protein or peptide from a mixture comprising said protein or
peptide, comprising: [0194] (i) contacting said mixture being
dissolved or suspended in a first liquid with a sorbent according
to the second, third, fourth or fifth aspect of the invention, for
a period of time sufficient to enable said protein or peptide to
become bound to said sorbent; [0195] (ii) optionally rinsing said
sorbent with a second liquid; [0196] (iii) contacting said sorbent
with said bound protein or peptide with a third liquid for a period
of time sufficient to enable said protein or peptide to become
released from said sorbent; [0197] (iv) optionally washing and/or
regenerating the sorbent with a fourth and/or fifth liquid.
[0198] In one embodiment, the pH of the first and optionally the
second liquid is close to the isoelectric point pl of the protein
or peptide.
[0199] In one embodiment, the pH of the first liquid is in the
range of from 5.5 to 8.5 and the pH of the third liquid is in the
range of from 3 to 6.5.
[0200] In one embodiment, said protein or peptide has an
isoelectric point pl of from 5.5 to 8.5 and a molecular weight of
from 100 to 500,000 Da.
[0201] For the purpose of this disclosure, all embodiments as
listed for the sorbent according to the general aspect of the
invention may be combined with the sorbent according to the first,
the second, the third, the fourth, and the fifth aspect of the
invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0202] The technical problem underlying the present invention can
be stated as to provide a novel purification method for proteins
and peptides which lacks the disadvantages of the previously known
methods as they have been summarised in the foregoing sections.
This means that the method should allow to isolate the targeted
protein or peptide in a single step from the sample matrix at high
recovery without compromising its functional integrity, while
largely avoiding costly materials but still being versatile enough
to be able to adhere to standard cleaning and sanitisation
protocols of the equipment in use and thus ensuring acceptance of
the method by the respective regulatory authorities, i.e. to
provide the targeted protein or peptide in an economically feasible
way in a pharmaceutical quality.
[0203] This technical problem could now be solved by providing a
novel type of sorbent to be employed in a solid-liquid equilibrium
distribution process of the protein or peptide to be purified,
which can be distinguished from those known from the state of the
art primarily by its specific two-fold chemical derivatisation with
residues, said derivatisation being tailored to the problem of
separating the targeted proteins or peptides from their side
products, particularly from various other proteins or peptides,
with a selectivity and sensitivity that can match that of
conventional affinity media but having a composition which is
completely devoid of delicate biological material which may be
expensive to manufacture and/or degrading under harsh conditions.
High durability of all materials employed in the production of the
sorbent also ensures long-term reproducibility of any separation
method which uses the sorbent, which may become obvious by the
absence of drift effects in analysis results.
[0204] Of assisting help in the solution of the technical problem
given above is a layered assembly of the sorbent comprising at
least two different materials of which one is a synthetic or
biosynthetic polymer film carrying both residues and covering the
second material which serves as a solid base. This particular
assembly is characterised on the one hand by a comparatively high
weight content and high physical stability of the polymer film, but
still a rather high degree of chain flexibility resulting also in
high solvent and sample uptake capacity as well as their fast
diffusional exchange. The film is thus maintained in a homogeneous,
biocompatible, soft and gel-like state. This allows the analyte
protein or peptides to immerse with their partial or full molecular
volumes into the layer containing those active elements of the
sorbent responsible for binding and migrate either through it or
along its surface while simultaneously preventing their
denaturation. It thus ensures the creation of a
quasi-three-dimensional interaction space for the analytes and
allows multi-point contacts between epitopes distributed over the
entire protein or peptide surface and the residue-modified gel
phase. Sample components are thereby also effectively shielded by
the polymer film from unwanted interactions with the underlying
constituents of the solid support material.
[0205] With the intent to assess the entire scope of the present
invention and to render it more precisely, the meaning of a number
of terms as used within the context of the present invention
hereafter is first being defined in the subsequent paragraphs. It
has to be understood that all examples are given for illustrative
purposes only and not meant as an exclusive list of embodiments.
Persons skilled in the art will certainly recognise additional and
analogous ways of carrying out the invention without deviating from
its overall spirit. The schematic representation of FIG. 6 again
symbolises the interrelationship between a number of different
terms used herein which are related to the sorbent composition.
[0206] The term "sorbent" means any synthetic or biosynthetic
material for use as a stationary phase in a solid < > liquid
equilibrium distribution process of a sample, which exhibits
selective non-covalent binding properties as a receptor for at
least one given target protein or peptide contained in said sample,
or which is capable to distinguish in its non-covalent binding
properties between at least two given target peptides or proteins
of different constitution contained in said sample (i.e. high
absolute binding constant or high binding constant difference). It
is therefore specially designed to solve a given analytical or
preparative detection, separation, immobilisation, or (bio)chemical
conversion task which often consists of a unique combination of at
least one target protein or peptide, whose constitution may be
known, partly known, or unknown, and a sample matrix, whose
composition may similarly be known, partly known, or unknown.
[0207] As opposed to generic phases (which differentiate analytes
according to cumulative parameters which are basically averaged
over the entire analyte molecule such as electrostatic charge,
dipole moment or lipophilicity), such a sorbent binds, at least in
part, by the concept of group complementarity to at least one
domain (epitope) on the three-dimensional molecular surface of the
at least one target protein or peptide. This novel concept
therefore also reaches beyond the scope of so-called mixed-mode
sorbents which--in a traditional meaning--separate according to a
combination of two of the classical averaged effects. The sorbents
of the present invention are thus designed on the molecular level
to bind only a single protein or peptide or a group of structurally
closely related proteins or peptides with high affinity and high
individual or group selectivity out of an environment which may
contain a large spectrum of different side products.
[0208] As a "solid support material" all non-porous or preferably
porous, adsorptive media known to those skilled in the art such as
all kinds of inorganic mineral oxides like silica, alumina,
magnesia, titania, zirconia, florisil, magnetite, zeolites,
silicates (celite, kieselguhr), mica, hydroxyapatite,
fluoroapatite, metal-organic frameworks, ceramics and glasses like
controlled pore glass (CPG), metals such as aluminium, silicon,
iron, titanium, copper, silver, gold, and also graphite or
amorphous carbon, paper, (bio)polymer resins such as
polysaccharides, polyacrylamides, polystyrenes like Amberchrom.TM.
etc., whether of spherical or irregular shape, can be used for
building up the sorbent. Poly(styrene-co-divinylbenzene)
(especially poly(styrene-co-divinylbenzene) which is bulk- or
surface-sulphonated as it is used in strong cation exchange
resins), polyacrylates, polymethacrylates, polyvinyl alcohol,
silica, glass, and polysaccharides such as starch, cellulose,
cellulose esters, amylose, agarose, sepharose, mannan, xanthan and
dextran are the preferred solid support materials. The introduction
of a solid base of a minimum rigidity and hardness as an insoluble
support function provides a basis for the enlargement of the
interface between stationary and mobile phases which is the place
of interaction with the protein or peptide as the molecular basis
for the process of its partitioning between said phases, and for an
increased mechanical strength and abrasiveness, especially under
flow and/or pressurised conditions. Solid support materials
according to the invention may be of homogeneous or heterogeneous
composition, and therefore also incorporate materials which are
composites of one or more of the materials mentioned above, in
particular multi-layered composites. In this context, magnetic
particles are specifically mentioned.
[0209] In an important embodiment related hereto, the surface of
the solid support material may be covered by a polymer film. Such
an optional film is considered as a part of the solid support
material since all preparation and separation methods developed and
introduced here which rely on functional groups or residues on the
immediate surface of a unitary bulk solid support material likewise
work with respective functional groups or residues of such a
polymer overlayer. Furthermore, a meso- or macroporous topography
inherent to the bulk solid support material will often be preserved
in the coating process. If in such a resulting hybrid material the
surface polymer film has to be distinguished from all the
material(s) underneath for purposes of the invention, the latter is
summarily referred to individually as a "carrier", or, in other
words, the hybrid solid support material would comprise both the
carrier and the polymer film. In practice however, such a
distinction is often viable only if the history of the sorbent
preparation is known. The carrier as the part which provides the
rigid framework of the sorbent is analogously of solid physical
condition and may consist of any of those materials listed above as
solid support materials which can likewise be employed according to
the invention as a bulk solid support material without having a
surface polymer film on top or as a carrier for such a surface
polymer film. All characteristics, options, and restrictions as
they have been stated above except for the suitability for
adsorption of a polymer therefore apply equivalently to both terms.
A central embodiment of the invention is therefore a sorbent
wherein the solid support material consists of a carrier the
surface of which is covered with a film of a polymer having
functional groups which carries the first and second, and
optionally the third and fourth residues.
[0210] If, as preferred, a porous material is used as carrier, the
polymer film will normally cover both its external and its mostly
larger internal surface homogeneously. A "surface" thus
characterises the entire solid-liquid phase interface of the
sorbent during its preparation and application as a separation
agent, where the recognition and binding of analytes by the
residues occurs, and which is accessible to at least one dissolved
protein or peptide via (optionally pressurised) hydrodynamic flow,
convection, perfusion, diffusion, or electromigration, or
combinations of any of these. Due to possible swelling of carriers
comprising soft matter and especially of surface polymer films in
proper liquids, this is not a sharp boundary but may involve an
intermediate gel-phase layer. Surface properties of the sorbent may
be different from the bulk properties of the materials employed.
This is particularly true if two different materials are used as a
carrier and a polymer film, and if preparation methods are used
which lead to extraordinarily large specific surface areas.
[0211] "Covering" can be technically achieved by all means of
coating known to a skilled person which may either occur under
natural driving forces or be manually enforced such as spontaneous
adsorption, vapour phase deposition, polymerisation from the
liquid, gas or plasma phase, spin coating, surface condensation,
wetting, soaking, dipping, brushing, spraying, stamping,
evaporation, application of electric fields or pressure, as well as
all methods based on molecular self-assembly such as, for example,
liquid crystals, Langmuir-Blodgett- or layer-by-layer film
formation. The polymer film may thereby be coated directly as a
multilayer or as a stepwise sequence of individual monolayers on
top of each other. As long as macromolecules are concerned, single-
or multi-point-"adsorption", whether spontaneous or artificially
accelerated, is in any case considered as being the first
(incomplete) step of any coating process starting from a polymer
solution which is in physical contact with the surface of a solid.
It requires the presence of some at least weakly attractive
physical (van der Waals-) or--in case of complementary
functionalisation present on the carrier and/or the polymer--rather
specific, non-covalent chemical forces between the solid surface
and each single polymer strand and, if multilayers are adsorbed,
also between the polymers within the same and different vertically
stacked layers in order to form at least a meta-stable aggregate.
Electrostatic forces between charges of opposite sign are often
utilised for this purpose, the surface charge of the carrier
thereby being given by its zeta potential. Initial adsorption may
occur in a loose and irregular fashion which may later transform
into a larger degree of two- or three-dimensional order and/or
density. This is may be ascribed to some residual mobility of the
polymer strands on the surface as a consequence of a steady-state
equilibrium between adsorption and desorption processes at
individual surface sites and may for example be fostered by
annealing. It is usually necessary to further increase the
stability of the adsorbed aggregate by the following introduction
of covalent bonds between proximate functional groups, in addition
to a basic steric (entropic) stabilisation by physical entanglement
of the chains. For achieving still increased stabilities, the
chains of the polymer film may further be covalently grafted to the
carrier material underneath.
[0212] The external surface of the solid support material thereby
may be flat (plates, sheets, foils, disks, slides, filters,
membranes, woven or nonwoven fabrics, paper) or curved (either
concave or convex: spheres, beads, grains, (hollow) fibres, tubes,
capillaries, vials, wells in a sample tray). The pore structure of
the internal surface of the solid support material may, inter alia,
consist of regular, continuous capillary channels or of cavities of
irregular (fractal) geometry. Microscopically, it can be smooth or
rough, depending on the way of manufacture. The pore system can
either extend continuously throughout the entire solid support
material or end in (branched) cavities. The rate of a protein or
peptide's interfacial equilibration between its solvation in the
mobile phase and its retention on the surface of the stationary
phase and thus the efficiency of a continuous flow separation
system is largely determined by mass transfer via diffusion through
the pores of the solid support material and thus by its
characteristic distribution of particle and pore sizes. Pore sizes
may optionally show up as asymmetric, multimodal and/or spatially
(e.g. cross-sectionally) inhomogeneous distributions. Typical pore
sizes of porous solids suitable for use in the invention as either
full solid support materials or carriers range from 10 nm to 400 nm
and can thus be categorised as meso- or macroporous; typical
particle sizes of particulate materials range from 5 .mu.m to 500
.mu.m. Suitable solids have acceptable porosities in the range of
30% to 80% by volume and typical specific surface areas in the
range from 1 m.sup.2 g.sup.-1 to 1,000 m.sup.2 g.sup.-1.
[0213] Alternative, more recently introduced solid support
materials are the so-called monolithic chromatography media which
are cast as a single macroscopic entity of the desired (usually
rod-like) shape as opposed to classical compressible column
packings made of loose microscopic particles. Monolithic columns
can consist of silica or polymeric materials such as, for example,
polymethacrylates, and their microstructure can contain fibrous
capillaries or sintered particle agglomerates.
[0214] The term "film of a polymer" or "polymer film" means a two-
or preferably three-dimensional synthetic or biosynthetic polymer
network of at least one layer, usually between a few and a few ten
molecular layers. Such a (derivatised or underivatised) polymer
network may itself be prepared according to procedures known to a
person skilled in the art. The film of a polymer may be of a
chemically homogeneous composition, or it may be comprised of at
least two different kinds of interpenetrating polymer chains (e.g.,
polyacrylic acid and a polyamine), either irregularly entangled or
in an ordered fashion (layer-by-layer). The term "chain" generally
refers to the longest continuous main strand and also possible
branches of a polymer, along which functional groups are attached.
The term is used both to indicate the full backbone length of a
dissolved, adsorbed or grafted polymer as employed during sorbent
preparation, as well as to indicate the chain segments located
between the knots of a crosslinked polymeric mesh, since in the
latter case the full length of individual strands is hard to
identify.
[0215] "Polymers" containing at least one functional group within
their backbone or side chains are preferable since they allow an
easy derivatisation with residues at such functional groups in
homogeneous or heterogeneous media. Furthermore, many properties of
a polymer in the solid or dissolved state and also its tendency to
adsorb spontaneously onto and adhere permanently to a given solid
carrier are being determined by its functional groups.
Polyelectrolytes are specifically mentioned here. Co-polymers,
whether of alternating, statistical, or block sequence, containing
both functional and non-functional units are also realisable in
this respect. The preferred functional groups are primary and
secondary amino, hydroxyl, and carboxylic acid or ester groups.
Depending on the acidity/basicity of the surrounding medium, amino
groups may be present as protonated ammonium ions, carboxyl groups
as deprotonated carboxylate ions. If a porous or non-porous bulk
polymer is also used as the carrier of the solid support material,
it is pointed out that the film of the polymer coated thereon, as
described here, will have a different chemical composition. These
differences may result from the presence, kind, or density of the
functional groups listed below, from lower molecular weights, or
from a lower degree of crosslinking. All these parameters add to
increased hydrophilicity, solvent swellability/diffusion, and
biocompatibility, as well as to diminished unspecific adsorption on
the coated surface.
[0216] The preferred polymer film comprises at least one polymer
containing amino groups. Polyvinylamine is strongly preferred.
Other suitable polyamines may comprise polyethylene imine,
polyallylamine etc. as well as functional polymers other than those
containing amino groups, such as polyvinyl alcohol, polyvinyl
acetate, polyacrylic acid, polymethacrylic acid, their precursor
polymers such as poly(maleic anhydride), polyamides, or
polysaccharides (cellulose, dextran, pullulan etc.). If co-polymers
are employed, the preferred co-monomers are simple alkene monomers
or polar, inert monomers like vinyl pyrrolidone. Preferred
molecular weights of the polymers used range from, but are not
limited to, 5,000 Dalton to 50,000 Dalton, which is particularly
true for polyvinylamine. Polymers having a molecular weight near
the lower limit of the range given above have shown to penetrate
even narrow pores of the carrier so that solid state materials with
high surface areas and consequently with good mass transfer
kinetics, resolution and binding capacity can be used in the
sorbents of the present invention.
[0217] The polymer will be adsorbed and then crosslinked or grafted
as a thin adlayer onto the surface of a suitable carrier, either
before or after derivatisation with first and second residues. The
film content of the resulting hybrid material, including its
derivatisation with residues, may range from about 5% to 30%,
preferably from about 15% to 20% by weight, based on the total
weight of the sorbent. The exact value of the polymer content of
the fully functional sorbent will also be dependent on the degree
of derivatisation, the molecular weight of the residues, and the
specific weight of the chosen carrier. These values correspond to a
film thickness in the lower nanometer range. The coated polymer can
still retain its ability to swell or shrink, the actual film
thickness thereby being strongly dependent on the type of solvent
being used.
[0218] The degree of crosslinking of the polymer film may range
from 2% to 20% based on the number of functional groups available
for crosslinking, respectively. Particularly preferred are
crosslinkages by functional group condensation, but all other
methods known in polymer chemistry, including radical and
photochemistry, can be applied. However, crosslinking bonds can
also be formed directly between the functional groups of the
polymer(s) involved without addition of crosslinking reagents. This
is in particular possible if co-polymers or blended polymers are
employed which provide at least two different functional groups
that exhibit a latent reactivity toward each other, e.g. amine
groups and carboxylic acid groups which can form amide bonds
between each other after activation. Preferred crosslinks involve
formation of covalent C--N bonds, e.g. amide, urethane, urea or
secondary/tertiary amine bonds, and may be formed via reaction of
either activated carboxylic acids or epoxides with amines.
Crosslinks can alternatively be of non-covalent nature, making use
of ion pairing between oppositely charged functional groups or with
the help of multiply-charged counterions etc.
[0219] As used herein, the "degree of crosslinking" is given as the
maximum number of crosslinks to be formed in the crosslinking
reaction based on the total number of functional groups available
for crosslinking. If, as preferred, bifunctional reagents are used
for crosslinking, the degree of crosslinking therefore reflects the
molar ratio between the amount of crosslinking reagent, which is
submitted into the crosslinking reaction, and the number of polymer
functional groups available for crosslinking (in such case two
functional groups are required per formation of one crosslink)
whereby it is assumed that the reaction proceeds nearly
quantitatively at the ratios attempted here. In principle, it is
possible that both inter-strand and intra-strand crosslinks as well
as non-crosslinking end-terminated side chains (from partially
reacting crosslinkers) are being formed.
[0220] Conversely, the term "grafting" means a covalent anchorage
of single polymer chains to the surface of a solid carrier,
preferable formed with functional groups thereon. It would be
sufficient if each polymer strand is anchored at at least one
arbitrary position along its chain. Better stabilities of the film
can be achieved via multi-point grafting so that protruding polymer
loops are formed on the surface. The latter method, however,
reduces the three-dimensional flexibility of the polymer chains.
Single-point attachments are preferably realised through a chain
terminus so that the full elongated length of the chain along which
preferentially a plurality of functional groups/residues or only a
single one at the opposite terminus may be attached, can point
outwards away from the surface. Although the actual conformation of
the grafted polymer may be a random coil, the use of high grafting
densities on the surface and appropriate solvents can lead to
swelling and oriented self-assembling phenomena between
neighbouring chains via dispersive interactions such as in the
formation of polymer brushes which may be further stabilised by
crosslinking. Preferably, grafting is achieved via mild
condensation reactions similar to the crosslinking reactions, but
methods involving propagating free radicals, ions, or radical ions
such as oxidative or radiation-induced methods could also be
applied. The chosen method will depend on the ease, type, and
degree of functionalisation of the carrier. Grafting can be
achieved in principle via two different techniques: the first
technique uses surface-bound monomers or initiators to build up
parallel polymer chains by in situ-polymerisation from the surface,
whereas in the second technique a polymer chain is first
synthesised in its full length in a homogeneous medium, i.e. in the
absence of the surface, to which it is only subsequently grafted in
an extra step. The latter technique is preferred if a sorbent of
the invention is prepared via grafting procedures and constitutes a
methodical embodiment of the invention.
[0221] In a preferred embodiment of the present invention, the
polymer film, also if internally crosslinked by covalent bonds, is
not grafted, i.e. covalently linked, to the carrier material
underneath, i.e. it is bound thereon by physical and/or chemical
adsorption only. Accordingly, the term "binding" encompasses
physical and/or chemical adsorption. The chemical and mechanical
stability of the composite material then results from total
physical entanglement of the carrier by the crosslinked polymer
film. The thickness and density of the polymer film are still
sufficient in order to shield very polar or reactive groups on the
surface of the supporting carrier, such as phenyl or sulphonate
groups in the case of solid polystyrene sulphonate, from
accessibility which are otherwise suspected to be cleaved by
reagents or to undergo undefined, irreproducible or irreversible
interactions with the target protein or peptide or its concomitant
impurities of the mixture to be separated.
[0222] In a further embodiment, the polymer film is grafted onto
the carrier but not internally crosslinked. As a third option, the
polymer film may be internally crosslinked as well as grafted onto
the carrier. All three different resulting network morphologies of
the polymer film are depicted schematically in FIG. 2. Case A of
FIG. 2 symbolises the preferred sorbent wherein the individual
polymer chains are covalently crosslinked with each other but not
covalently grafted to the surface of the carrier. Case B represents
a sorbent wherein the individual polymer chains are covalently
grafted to the surface of the carrier but not covalently
crosslinked with each other. Case C represents a sorbent wherein
the individual polymer chains are both covalently grafted to the
surface of the carrier and covalently crosslinked with each other,
as a result of a combination of the two fixation techniques (which
may be carried out in any order).
[0223] The term "functional group" means any simple, distinct
chemical moiety belonging to an (underivatised) solid support
material or restricted to an optional polymer film on its surface,
or to a polymer during preparation of said surface via film
adsorption, which may serve as chemical attachment point or anchor
and which therefore is, at least in the swollen state of the solid
support material or a polymer film covering it, amenable to liquid
or solid phase derivatisation by chemical addition or substitution
reactions and optionally also to crosslinking. Functional groups
will therefore typically contain at least one weak bond and/or one
heteroatom, preferentially a group behaving as nucleophile or
electrophile. Less reactive functional groups may need to be
activated prior to derivatisation. They can thus both form the
structural link between the polymer strands and the residues of the
sorbent as well as forming the knots of a crosslinked network.
Opposed to residues, functional groups are primarily not designed
to interact with analytes (although it indeed cannot be rigorously
excluded that they nevertheless do interact or aid in the
separation process via repulsion of side components) but rather to
provide a surface coverage with molecularly-sized spots of defined
chemical reactivity that can be converted into the actually
interacting residues (derivatisation) or used in the formation of
covalent connections (polymer crosslinkage and grafting). The terms
"connections" or "linkages" as used herein shall cover both
directly formed covalent bonds as well as an extended series of
covalent bonds in a row via a sequence involving multiple atoms.
Other chemical moieties down to simple diatomic molecular fragments
which may be present on the sorbent or an analyte and which do not
fulfil either of these known and specified functions, are simply
named "groups".
[0224] A set of functional groups can be treated as a plurality of
separate, but identical units, and their chemical behaviour will
mainly be determined by predictable and reproducible group
properties only and to a far less extent by the materials to which
they are attached, or their exact position on these materials.
Among such functional groups are, just to mention a few, amino
groups, hydroxyl groups, thiol groups, carboxylic acid groups, or
carboxylic ester groups. Functional groups represent an integral
part of the solid support material and are thus distributed
uniformly over large areas of its surface. Suitable functional
groups often exhibit weak acid or base properties and thus give a
film-forming polymer the character of an ampholyte. Functional
groups in a polymer can either be introduced during polymerisation
from the corresponding monomers or by subsequent functional group
conversion (polymer-analogous reaction) before or after adsorption
onto the carrier. A polymer film can also contain two or more
different functional groups either if different monomers are
co-polymerised, if functional group conversion is stopped before
completion, or if different polymers are layered on top of each
other or as interpenetrating networks. The preferred functional
groups are primary and secondary amino groups. Particular
preference is given to primary amino groups.
[0225] The term "derivatisation" means any chemical reaction
capable of introducing specific residues onto the surface of a
solid support material or into a polymer used for covering said
surface during sorbent preparation in order to produce an
intermediate or fully functional sorbent, particularly by addition
to, or substitution of, its functional groups with a suitable
derivatisation reagent containing the residue or a precursor
thereof. Interconversion of a functional group into a different but
still reactive functional group shall also be covered by the term.
A "precursor" of the residue may incorporate a masked or protected
chemical moiety which can be deprotected or otherwise converted
into the final residue after or simultaneously with the formation
of a linkage with the surface or polymer in the derivatisation
step. For example, if the polymer contains primary or secondary
amino functional groups and derivatisation is made through amide
bond formation with these, additional primary or secondary amine
moieties to be contained in the residue should initially be
protected as e.g. Boc- or Fmoc-derivatives in the derivatisation
reagent. Further, if the bond to be formed during the
derivatisation reaction between a surface or polymer functional
group and a reactive center on the derivatisation reagent leads to
the formation of a new chemical moiety which plays a role in the
recognition of the target protein or peptide, the respective
residue will apparently only be fully developed after
derivatisation, and only a part or a functional modification of it
is contained as a precursor in the derivatisation reagent. In such
case, part of the precursor moiety (a leaving group) may also be
split off during the derivatisation reaction (such as a water
molecule during a condensation reaction).
[0226] Derivatisation is in each of at least one or optionally
multiple steps always being carried out on a "defined portion" of
the functional groups. This means that--taking the reactivities of
different functional groups and reagents into account--a targeted,
pre-determined percentage of each given kind of functional groups
present in the underivatised polymer or solid support material is
always being converted into functional groups derivatised with the
respective residues chosen. In order to yield homogeneously and
reproducibly derivatised sorbents, calculated appropriate amounts
of derivatisation reagents are then let to react with the polymer.
Full derivatisation (degree of derivatisation=100%) can also be
attempted, whereby the derivatisation reagent is often used in
excess, but this is not a must-have.
[0227] Since the residual materials of the sorbent as such shall
not be impaired during the derivatisation step, it is often
desirable to perform the derivatisation under mild conditions. It
may thus be necessary to either activate the functional groups or
the derivatisation reagent prior to or concomitant with the actual
bond formation step in order to maintain sufficient reactivity
under such conditions. Preferably, the derivatisation reagent is
activated. A preferred derivatisation reaction will involve a
nucleophilic polymer containing electron-rich nitrogen functional
groups such as amino groups and an electrophilic reagent containing
a leaving group attached to an electron-poor carbon such as a
carbonyl or carboxyl derivative, or vice versa. Activation can
therefore be achieved by standard techniques of solid phase or
liquid phase peptide synthesis, e.g. via activated esters.
Preferred derivatisation reactions involve the formation of amide,
urethane, urea or secondary/tertiary amine linkages with the
functional groups. Due to the asymmetry of amide and urethane
linkages with respect to the carbonyl carbon, they can be formed in
either direction from amino or carboxyl polymers, and from amino or
hydroxyl polymers, respectively.
[0228] Affinity and selectivity of the sorbent are largely
determined by a combination of two or more different residues. The
term "residue" means any distinct chemical moiety or a distinctly
identifiable, usually repeatedly occurring, arrangement of chemical
moieties of the same or different kind capable of assembling on the
nanoscopic scale (by itself or part of itself or within a cluster
of residues of the same or different kind) into a complex or a
place of high and/or selective affinity toward at least one
complementary structure or surface region of at least one protein
or peptide, as long as the affinity is stronger than a mere van der
Waals-contact with CH or CH.sub.2 repeating units of the lattice or
polymer chain on the sorbent surface. Such a place at the
solid/liquid interface is, in analogy to the description of
specific interactions involving biomacromolecules, called a
"binding site". A residue can thereby be an entirely synthetic or a
natural product or a fragment or combination thereof, but should be
amenable to chemical synthesis and/or derivatisation. It may
comprise more than one distinct chemical moiety (including
chemically unreactive moieties such as, for example, alkyl or
alkylene units which are nevertheless capable to engage in
hydrophobic or dispersive interactions).
[0229] Since two or more different residues are introduced into the
sorbent in variable ratios, a binding site will comprise two or
more, identical and different residues. The totality of residues
involved in the formation of a particular binding site is located
in close two- or three-dimensional spatial proximity of each other
and may, but does not necessarily have to, involve residues on
neighbouring surface functional groups or neighbouring repeating
units of a polymer film. Individual residues of a common binding
site may as well belong to different strands of a crosslinked or
surface-grafted polymer (the same principle applies to the
counterparts of binding exposed on the respective protein or
peptide surface). On the other hand, a particular residue can be
shared by two or more adjacent or overlapping binding sites. Due to
the random (statistical) nature of the distribution of
crosslinkages and residues onto the functional groups on the
surface or within a polymer film, a resulting distribution of
similar, but neither structurally nor energetically identical
binding sites can be formed. As a result, the sizes and affinities
of these binding sites toward the target protein or peptide may
differ to a considerable extent which has, however, in practice not
proven as a disadvantage.
[0230] "Binding" between the binding sites of the sorbent and the
target protein or peptide shall be reversible and shall therefore
take place via any form of non-covalent interaction between
complementary chemical moieties of the sorbent. Among the
prevailing non-covalent modes of binding are ionic, hydrogen
bonding, donor-acceptor charge transfer, .pi.-.pi., cation-.pi.,
dipole, coordinative, dispersive, and hydrophobic interactions, but
often mixed and non-stoichiometric forms are encountered which do
not allow to specify the individual binding mode contributions.
Thus, single, double or multiple simultaneous contacts may occur
between the binding partners which may involve the same or
different residues. Physical and entropic forces influencing the
mobility of an analyte on rough surfaces and in microscopic pores
as well as solvent-mediated interactions may add to the factors
responsible for binding. In certain instances, the resulting
complex comprising the sorbent and at least one bound protein or
peptide may be detectable or even isolable, but more often it will
be of transient character only. There is also no useful lower limit
imposed on the binding strength since such values would not only be
an intrinsic property of a given sorbent-analyte pair but also
strongly solvent-dependent. Moreover, even differential Gibbs
enthalpies as small as 1 kcal mole.sup.-1 can be still resolved by
chromatographic methods due to multiple serial equilibrations in
columns whose theoretical plate numbers can adopt values of about
10.sup.3 to 10.sup.4 per meter of chromatographic bed length. In
chromatographic applications, binding should also not be too
strong, because otherwise reversibility would be difficult to
achieve under ambient or biocompatible conditions.
[0231] As the sorbents of the present invention are concerned, a
residue may be connected to a functional group on the surface of a
solid support material, including an optional polymer film covering
said surface, and, if so, comprises the entire partial structure
pointing away from the surface from the point of attachment at the
functional group, or at least that part of it which occurs in an
identical manner on different functional groups. Not necessarily
has the entire residue to engage directly in the binding of the
target protein or peptide. The residue may as well contain such
atoms or moieties which only have the purpose of separating or
connecting the actually binding structures from/with each other or
to provide a geometrically suitable framework for the binding site
in order to present the binding structures to the target. Optional
spacer, branching or other linker units between the functional
groups on the solid support material, especially on an optional
polymer film on its surface, and the actually binding structures
are thus formally assigned to be part of each residue to which they
make at least one connection. The connection can usually be
achieved via at least one special derivatisation process of the
functional groups, in a stochastic (ubiquitous) or selective
manner, prior or subsequent to the application of an optional
polymer film onto the carrier medium, in a homogeneous or
heterogeneous fashion. Accordingly, a solution or thin film of the
polymer may be reacted with pre-synthesised derivatisation
reagents, which already contain the residues or precursors
thereof.
[0232] However, if the functional groups, or structural parts of
them, are converted by derivatisation with residues or precursors
thereof into moieties of a different kind, or are then forming an
integral chemical unit with additional atoms of said residue (e.g.,
the nitrogen atom in the conversion of an --NH.sub.2 functional
group into a --NHCO--R residue), they may as well be regarded as
having basically lost their character as functional groups and
instead be regarded as a structural moiety belonging to said
residue.
[0233] If residues of the same or different kind are attached
individually to functional groups of the solid support material
either directly or via a covalent, conformationally flexible
linker, it is assumed that they adapt to their complementary
counterparts on the target protein or peptide surface
independently, the driving force being the minimisation of the
overall Gibbs enthalpy. It is therefore not necessary for the
purpose of the present invention that the residues of the binding
site are organised in the correct three-dimensional orientation for
optimum binding of a given protein or peptide epitope (as for
example in a natural antibody); they only need to be able to assume
such an orientation through exploration of their conformational
space (substrate-induced fit). In many cases, especially if
differential binding is strived for, two or more, different or
overlapping epitopes of the target protein or peptide may be
recognised by the same sorbent.
[0234] While the term "residue", which refers to the overall unit
which is pointing away from the sorbent surface and repeated many
times identically or similarly thereon with the intent to engage in
analyte binding, is as such functionally defined, such a residue
may consist on the molecular-structural level of one or more
distinguishable, but within themselves contiguous subunits, into
which it may--just formally--be fragmented, so-called "structures".
This term is being used throughout the invention in its broadest
possible meaning. Although somewhat arbitrary, the division of a
residue into different structures should follow the principle of
chemical likeness and intuition, whereby molecular moieties or
fragments should be meaningfully grouped together according to
common structural and/or physical properties. The functions
associated with different structures belonging to the same residue
may thereby likewise be different: some structures (C, N, O,
S-heteroaromatics, in particular) may be related to analyte binding
while others are not. In view of myriads of possibilities of
realising sorbents according to the invention due to small
structural changes of the residues, on such a basis the essential
parts of a residue can be separated from the non-essential parts.
To those optional structures not primarily involved in analyte
binding, "linkers" are belonging which are short molecular (often
simple hydrocarbon) tethers, optionally comprising functionalities
or unsaturated valencies at one or both ends for making the
necessary connections, and forming the ties between the actually
binding structures and adjacent structures and/or the sorbent
surface. It would thus for example be possible to employ several
different residues in a sorbent of the invention which all comprise
a binuclear C, N, O, S-heteroaromatic structure but linkers of
different kind, length, or connectivity and optional or missing
further structures. Such a group of residues could then be
distinguished on the molecular level but they would functionally
altogether qualify as "first residues" within the meaning of the
invention. The use of linkers will be discussed in more detail
farther below.
[0235] The structures of the residues responsible for the target
recognition involve "heteroaromatic structures", which are in a
narrower sense those principal structural parts of the residues
with which the interaction contact with a protein or peptide
analyte takes place. The expression "heteroaromatic" within this
context means a ring or fused ring system showing the aromatic
characteristics of a continuously delocalised, closed-loop
.pi.-electron system (as indicated for example by an anisotropic
magnetic shielding in the NMR spectrum) and consisting only of
carbons and at least one endocyclic heteroatom taken from the group
comprising nitrogen, oxygen, and sulphur. Combinations of
heteroatoms of the same or different kind within the same or
different rings are possible, as long as a stable structure results
wherein the usual valencies of the heteroatoms are not
exceeded.
[0236] According to common nomenclature, "binuclear" denotes a
fused bicyclic ring system consisting of two aromatic rings of any
size each which are sharing two adjacent ring atoms (carbons in
nearly all cases), the covalent bond between them (i.e., a
[n.m.0]-bridge of zero atoms length) and a common conjugated
.pi.-electron system, whereas "mononuclear" denotes an isolated
monocyclic ring of any size. Further aromatic, heteroaromatic,
aliphatic or heteroaliphatic rings or ring systems may nevertheless
be attached to both heteroaromatic structures as substituents via
immediate single bond connections to at least one ring atom,
optionally via spacer units. Extended ring fusion (two-point
substituent attachment) is, however, only possible with aliphatic
or heteroaliphatic rings, such that the heteroaromatic
.pi.-electron system is not extended into the full length of the
additional ring, and ring systems of higher nuclearity are avoided.
The number, combination, and distribution of heteroatoms as well as
their formal .pi.-bond order (which may be fractional) are thereby
irrelevant with respect to the classification as heteroaromatic;
the only prerequisite is that the at least one heteroatom is
positioned within a ring and shares at least one .pi. electron of
the common conjugated ring system. Due to the multiple individual
possibilities of fragmenting a residue into structures, it should
be sufficient within the context used here if at least one viable
first residue and second residue fragmentation leads to a binuclear
and mononuclear C, N, O, S-heteroaromatic structure, respectively.
Preferred heteroaromatic structures comprise at least one, more
preferably exact one five-membered ring.
[0237] The term "C, N, O, S-heteroaromatic structure" as used
herein means a binuclear respectively a mononuclear heteroaromatic
structure comprising besides carbon atoms at least one of the
heteroatoms N, O, S.
[0238] In one embodiment, the heteroaromatic structure comprises at
least one N atom and at least one additional heteroatom N, O, or S.
In one such embodiment, the heteroaromatic structure comprises two
N atoms. Such a heteroaromatic structure is e.g. imidazole,
[0239] "Substituents" are organic radicals (except hydrogen) which
are considered as optional parts of the heteroaromatic structures
and are thus thought to engage also in analyte binding. They are
resembling the exocyclic parts of these heteroaromatic structures
to whose cores, i.e. the binuclear or mononuclear heteroaromatic
ring-forming atoms, they are covalently bound via at least one
covalent bond without comprising said ring-forming atoms. Except
for the case where several heteroaromatic structures are connected
with each other via direct covalent linkages, such substituents by
themselves will usually only exhibit weak and unselective
interactions with the target protein or peptide and may rather be
used to tailor the hydrophobic/hydrophilic properties of the
residues. They would not be able to fulfil the object of the
invention satisfactorily if they were bound to the sorbent surface
in the absence of any C, N, O, S-heteroaromatic ring.
[0240] In the prior art, sorbents showing high affinities and
selectivities are predominantly known from solid support materials
to which antibodies or other high-molecular weight receptors of
biological origin are affixed. Such antibodies first have to be
raised specifically against the target antigen in a biological
process involving living organisms, or the target protein or
peptide must be reversibly conjugated to an antigen or to one
component of only few previously known natural affinity pairs. The
sorbents of the present invention can be distinguished from those
by the fact that their residues are accessible by chemical
synthesis, by a low molecular weight and high chemical stability.
However, they may as well be implemented as stationary phases in
all types of affinity chromatographic methods.
[0241] The terms "protein" and "peptide" represent poly- and
oligoaminoacids, respectively, as chemically, biosynthetically or
bioanalytically distinctly identifiable entities which can be of
synthetic or biological origin (regardless of their possible
occurrence in nature), of linear or branched, homo- or heteromeric
sequences, and upon which no minimum or maximum sequence length or
molecular weight limit is imposed. A minimum requirement is that
they should be composed of at least two amino acids which are
connected via at least one amide bond, which would, for example,
correspond to a dipeptide. The presence of non-proteinogenic or
completely unnatural amino acids, .beta.-amino acids, N-alkyl amino
acids, additional peptidomimetic units etc., which are all still
capable of forming peptidic bonds, should not be detrimental. Small
(oligo)peptides can often be prepared synthetically via stepwise or
convergent methods; the term peptide shall in such case
additionally encompass obtainable structures formed via unusual
connectivities such as, for example, depsipeptides or peptoids.
Larger proteins typically possess a defined three-dimensional
structure which may adopt numerous of different shapes such as, for
example, globular (albumin) or filamentous/fibrous (actin,
collagen) shapes; they may be soluble in the cytosol,
membrane-bound, part of the extracellular matrix, or can be
presented on the surface of a cell. Due to the tiny amounts of
proteins that can be handled with modern molecular biological
methods, their primary amino acid sequence does not need to be
known in order to identify them; sometimes it is not even known
whether they are present as a homogeneous composition. Proteins or
peptides bound to the surface of a (colloidally) dispersed carrier
(nano)particle such as, for example, a virus, a quantum dot, or a
latex sphere, are usually required to be cleaved off first in order
to expose also the otherwise shielded parts of their entire
molecular surface for interaction with the sorbent before they can
be employed in the separation method of the invention.
[0242] The above terms include on the one hand non-covalent peptide
aggregates as well as homo- and heteromultimeric proteins, but on
the other hand also functional or non-functional subunits of a full
protein such as the products of enzymatic digests or disulphide
bond reductions, but also de-novo designed mini-proteins such as
Affibodies.TM., Anticalins.TM., Nanobodies.TM., or other
artificially reconstituted active sites. Metal ions or complexes
may be contained in proteins, usually in their active sites.
Analyte proteins can be modified by in-vivo posttranslational
modifications, such as phosphorylation, sulphatisation,
glycosylation, glucuronidation, or ubiquitinylation. Conjugation
with glycosides and lipids results in glyco- and lipoproteins,
respectively, consisting of additional structural units beyond just
amino acids. Protein modifications whose up- or downregulation can
serve as markers for certain pathological states of the organism in
which they are produced are thereby generally of utmost importance.
Similarly, in-vitro biochemical modifications of a surface as well
as an active or allosteric site of a protein include the formation
of reversible or irreversible complexes with substrate agonists or
antagonists as well as all kinds of protective group chemistry of
amino, carboxyl, and side chain functions. Proteins or peptides can
further be chemically or biochemically tagged (e.g. oligohistidine
sequence-tags, conjugated dyes or radioactive labels) or fused with
another (carrier) protein with the aim of enhanced expression,
solubility, excretion, detection or separation of the protein or
peptide, whereby the point of conjugation might be cleavable, but
they can also lack part of their native sequence such as, for
example, a membrane anchoring tail.
[0243] If one of the terms "target protein", "target peptide", or
simply "target" is used, the particular protein or peptide, or
multitude of proteins or peptides (usually related by structure,
classification, synthesis, or origin), is meant for which the
sorbent with its specific residues is designed. This is normally
the analyte or component of the feed mixture showing the highest
affinity for the sorbent. The target protein or peptide may be
distinguished from its potential proteinacious side products not
only by its amino acid sequence (down to single-point sequence
mutations or deletions and including those resulting from
alternative splicing or SNP variants during gene transcription) but
by its full secondary and tertiary structure elements which include
the presence of differently folded (native, unfolded or misfolded)
states. The target protein or peptide often is, but does not
necessarily need to be the main component of the feed mixture (by
weight or molarity), not even the main peptidic component.
Regardless of its abundance within the mixture, the target often
is, but does not necessarily be a valuable mixture component or the
particular substance required to be purified, the latter possibly
being contained in the flow-through fraction. Since many proteins
or peptides have demonstrable toxic properties, in health- or
environment-oriented applications predominantly the target can also
be such a toxic or otherwise unwanted properties exhibiting protein
or peptide in a mixture from which it has to be depleted. It could
also be that the target is not a major product but a minor side
product of a manufacturing process which is required to be
separated or removed from the remainder of the mixture, whereby the
concentration or purity of another mixture component--usually the
principal product--which itself may or may not be a protein or
peptide, is increased. Pointing towards the multi-step blood plasma
fractionation process, many consecutive fractionations may be
necessary to rectify a whole bunch of different proteins or
peptides being simultaneously present in the feed mixture, whereby
the flow-through of a particular stage of fractionation may be
adsorbed at the next stage, or vice versa.
[0244] The collectivity of all solutes within the mixture to be
separated--including the target--which are capable of at least weak
interactions with the sorbent of the invention under suitable
conditions is termed as "analytes". Most analytes will be proteins
or peptides, because these are the analytes the sorbent is designed
for, but under certain circumstances it is possible that small,
non-peptidic molecules may belong to this group. Closely related
analytes may form together a synthetic or biosynthetic library, for
example one derived from a tryptic digest, a phage display library
or an expression product of a randomised cDNA library which has
been appropriately transcribed in vivo or in vitro. The affinity of
the sorbent, however, usually drops rapidly for analytes having
structures deviating from the target group, and approaches zero if
they are structurally unrelated to the target(s).
[0245] Preferred proteins or peptides will have an isoelectric
point pl of from 5.5 to 8.5 and their molecular weight can range
from 100 to 500,000 Da. These pl values will approximately match
the acidity pK.sub.a of at least one of the heteroaromatic
structures incorporated in at least one of the first and second
residues. The particularly preferred target proteins of the sorbent
of the invention are "antibodies" or mixtures thereof, a term which
shall also include fragments (light and heavy chains, Fab and Fc
regions, Sc variable regions, etc.) of antibodies, artificial
molecular constructs from such fragments (diabodies, triabodies),
oligomeric associates of antibodies, as well as antibody- or
antibody fragment-containing fusion proteins, or other types of
conjugates such as those containing detectable tags like
glutathione or GFP which may also be chemically linked with each
other. It may be a polyclonal or a monoclonal antibody. Among the
immunoglobulins (Ig, .gamma.-globulin) in general, the antibody may
belong to any of the isotypes IgA, IgD, IgE, IgG, or IgM, each of
which can in turn be divided into several subclasses. The antibody
can be of human or other mammalian (typical: murine or rodent
(mouse, rat, rabbit, hamster, guinea pig), goat, sheep, dog, pig,
bovine, horse) origin. The preferred antibodies are human or
humanised (chimeric) antibodies. Their idiotypes can be directed
against all types of antigens (other antibodies or biological
substances, small molecules).
[0246] A "mixture containing a protein or peptide" means a mixture
that can be of various origin. There is no severe limitation of the
present invention as to the source from which the mixture has been
obtained. The only requirement is that it contains at least one
protein or peptide which would qualify as an analyte for which the
sorbent of the present invention exhibits at least weak receptor
properties. The mixture may thereby contain two or more different
proteins or peptides which are either intended to be separated
collectively from the remainder of the mixture (i.e., all of them
are separation targets) or to be separated from each other (i.e.,
only one or a few of them are separation targets). The structural
motifs (epitopes) of the at least two proteins or peptides within
the mixture which are recognised by the residues of the sorbent may
both be identical, similar or partially identical, or different. It
is assumed that the latter cases will lead in many instances to
different types of interaction with the sorbent, and thus to larger
differences in binding strength, provided that the at least two
proteins or peptides are of comparable molecular weight and contain
about the same number of recognisable epitopes.
[0247] If the protein or peptide is a naturally occurring or
recombinantly produced substance, it may be obtained from fresh or
dry extracts of liquid or solid biological material such as
animals, plants, microbes, or viruses (including breeded or
transgenic species which overproduce the product), extracts from
cell cultures or cell culture media, microbial (bacterial or
fungal) or enzymatic fermentation broths, commercial feedstocks, or
any combination thereof. Alternatively, the mixture containing the
protein or peptide can be the raw product of a chemical synthesis
or partial synthesis. This especially includes standard solution
and solid-phase peptide synthetic methods, performed either
manually or in an automated fashion.
[0248] As typical for any purification technique and especially any
chromatographic technique, the exact conditions to be used are not
only dependent on the constitution of the target protein or peptide
but on that of the sample matrix as well. The "matrix" is a term in
use for the collectivity of all active and non-active constituents
of the mixture, with the exception of the target(s) but including
the medium in which they are dissipated. This is because not the
absolute physical or chemical properties of the target protein or
peptide are commonly utilised in a separation process but rather
the differences of said properties between the target protein or
peptide and all or a few specific matrix components. Usually, the
composition of the matrix is at most only partially known (both
qualitatively and quantitatively) since one single analysis method
is often not able to detect all constituents, at least not with
equal sensitivity. Intermediate products obtained at different
process stages during the downstream isolation and purification of
a chemical or biological material represent different matrices
within the meaning used in the context of the present invention.
The entire mixture (target and matrix combined) to be tested for
its adsorption behaviour on the sorbent is in an analytical context
often also termed the "sample".
[0249] Prior to treatment with the sorbent of the invention, raw
chemical or biological materials can be partially purified via
further pre-processing by any combination of further
non-destructive unit operations, in particular traditional
separation processes which may comprise filtration (including
micro- or ultrafiltration), dialysis and electrodialysis, washing,
precipitation, centrifugation, ion exchange, gel filtration,
dissolution, evaporation, crystallisation, drying, grinding, any
way of viral reduction treatment, and also conventional
chromatography (either chromatography on sorbents of low
specificity or conventional affinity chromatography with biological
residues) in order to remove as much waste material as feasible
(e.g., insoluble matter and the majority of proteins, nucleic
acids, carbohydrates, lipids, and inorganics in case of biological
material, leaving only the valuable substances), harmful or
aggressive substances or those substances which are suspected to
possibly deteriorate the sorbent or diminish its separation
ability, from the chemical or biological material, thereby
increasing the concentration of the target prior to contacting it
with the sorbent. Within this context, LC/LC-coupling techniques
are referenced to. Dry mixtures such as freeze-dried or lyophilised
material need to be taken up in a suitable feed solvent before they
are treated with the sorbent. It is desirable that the dissolved
mixture is homogeneous and free of suspended or colloidal
particles. Similarly, the separation method of the invention can
also be combined subsequently with one or more steps of the kind
given above.
[0250] Many proteins or peptides are already manufactured on an
industrial scale and have found applications in medicine, nutrition
(e.g. dietary supplements), cosmetics, or agriculture. A
large-scale production of most of them can until now economically
and within a reasonable timeframe only be achieved by extraction of
biomass, i.e. biological material obtained for example from
medicinal plants, microbial fermentations using prokaryotic or
eukaryotic microorganisms, or cell cultures of higher organisms up
to insect or mammalian cells (e.g. the frequently used CHO, NS0,
BHK, or the immortalized HeLa cells). In summary, frequent sources
of mixtures according to the invention are therefore biosynthetic
products, such as those obtained from a microorganism or a cell
culture, or from a crop extract.
[0251] Microbial fermentations include submerged or floating
cultures of bacterial or fungal (e.g. yeast) strains. Products can
be extracted from whole organism harvests or from separated parts
such as the mycelium and/or the corresponding culture medium
supernatant into which they may be secreted. Semi-synthetic
procedures include both downstream chemical modifications of
natural products or intermediates and the biotransformation of
synthetic feedstocks. In all cases, side products often comprise
protein isoforms, truncated forms and accumulated intermediates or
follow-up products along the biosynthetic pathways leading to the
targeted protein or peptide. These may additionally be accompanied
by ubiquitously secreted antibiotics, endotoxins, mycotoxins,
pyrogens, promoters or inhibitors of cell proliferation, protease
inhibitors, defoaming agents, residuals of incompletely digested
nutrients, products of partial degradation, as well as
high-molecular weight and partially insoluble components (e.g. cell
debris) as they may result from final-stage cell lysis of the
producing organism. Cell lysates often further increase the
complexity of the mixture due to the release of additional
substance classes like nucleic acids and a vast number of so-called
host cell proteins into the extractable medium.
[0252] The term "separation" with relevance for the separation
method of the invention includes all kinds of segregating or
splitting a mixture into its parts, particularly dividing one or
more structurally different components, which are molecularly
dissolved in a liquid, and spreading them into different liquid
fractions. One outstanding component of the mixture is always the
target protein or peptide which should experience a separation from
at least one other mixture component. It thereby does not matter
whether the target is separated in one fraction and the
collectivity of side products separated in one other (common)
fraction, or if each individual mixture component is separated in
its own fraction from any other component, or if the method results
in anything located in between these extremes. It is sufficient if
in at least one liquid fraction obtained after performing the
method an enrichment of at least one dissolved protein or peptide
already present in the original (feed) mixture is observed.
Separated side products do not necessarily need to be recovered as
separate liquid fractions; they may also stay bound to the sorbent
for being discarded as such, for example. It would not be unusual
if the separation process remains incomplete which would turn into
yield losses in the fractions containing the desired product of
value. Sharp fractionation which avoids overlapping elution bands
would increase the quality of separation (i.e. purity) at the cost
of further yield losses.
[0253] The terms "concentration" and "purity" relate to the given
or achievable fractional content of the respective substance in the
mixture, whereby the term concentration is referring to solutions
with inclusion of the amount of solvent in the total reference
amount of mixture, whereas the term purity refers to (sometimes
hypothetical) dry mixtures without giving consideration to solvents
(including residual water). Most often they are stated as either
weight or molar fractions (weight/weight, weight/volume,
moles/moles, moles/volume). A higher purity can thus be attained at
the cost of a higher dilution (i.e. lower concentration) or vice
versa, depending on the more important end to be achieved in a
particular system. The measure for determining the actual values of
these indicators as used herein is by HPLC peak area, whereby it
has to be noted that every quantification method except for weight
shows a certain bias for well-detectable mixture components versus
badly-detectable mixture components, and may also yield non-linear
calibration curves. Insoluble material, for example, is not
quantifiable by HPLC. Depending on its origin, the way of its
isolation and pre-processing, the mixture may typically contain the
targeted protein(s) or peptide(s) in a (combined) purity of from 1%
to 99%, preferably of at least 10%, more preferably of at least
50%, the remainder being side products or compounds which are
structurally and functionally unrelated to the target such as
residual solvents, reagents etc. Depending on the actual
purification task, the separation method of the invention can
therefore be used both as an initial capturing or isolation step
out of very dilute or crude mixtures, or as a final polishing step
of an already pre-purified mixture containing an almost pure target
protein or peptide. The number of side products and other
constituents of the mixture may range from one (e.g. a single-point
sequence mutation or deletion) to an essentially infinite number
(e.g. untreated physiological samples). The kind of side products
is as well dependent on the source of the raw material and prior
processing.
[0254] The term "contacting" refers to any appropriate treatment of
the initial (feed) mixture being present in a liquid (mobile) phase
with the sorbent as the solid (stationary) phase by establishing
physical contact between the phases both on the phenomenological
(wetting) as well as on the molecular (surface or pore diffusion)
scale. Contacts formed should be intense enough to enable possibly
all molecularly dissipated components of the mixture, but at least
the target protein or peptide, to reach all external and optional
internal sorbent surfaces where residues are located and then to
interact with them. Contact formation can occur under static or
(plug, laminar, turbulent etc.) flow conditions, e.g. over a fixed
or fluidised (expanded) bed of sorbent particles. Since the mixture
will be dissolved in a first liquid (feed liquid or adsorption
liquid), this will be a heterogeneous process and contact formation
may macroscopically be accelerated via stirring or shaking of the
resulting suspension, although there is no time limit for
terminating this operational step unless the establishment of a
steady-state binding equilibrium of the target protein or peptide
and optionally of the side products to the sorbent would be
approaching.
[0255] As used herein, the term "liquid" refers to any solvent
(including water as the most important one) or mixture of solvents
which possess at least weak solubilising properties for one or more
components of the mixture to be separated. Liquids of different
composition may be employed for treatment of the sorbent in the
different steps of the method, since in each step the respective
liquid employed therein has to fulfil a particular task which it
should enable, such as target adsorption (binding), target
desorption (release), or sorbent cleaning. Within a chromatographic
environment, a liquid which enables a dynamic equilibrium exchange
of one or more components of the mixture with the sorbent is often
also termed as a mobile phase. Since chromatographic separations on
the sorbent of the present invention are predominantly dependent on
both strongly polar and hydrophobic interactions, a broad variety
of liquid compositions having differentiating solvation
capabilities for individual mixture components can be used,
depending on which type of interaction should be favoured. To
further modulate the strength of any or all of these interactions
over the time course of any given step of the separation method, it
may sometimes also be advisable to gradually change the composition
of the liquid used within said step, e.g. via gradient mixing.
Therefore, the composition of a liquid dedicated to fulfil a
specified task does not need to be constant over the full time
lapse of the process step in which it is employed. The specific
solubility of the target protein or peptide has also to be taken
into account when choosing suitable adsorption and elution liquids.
Proteins, except for those which are membrane-bound, normally
require the use of liquids of high aqueous content, if they have to
be conserved in their native states and aggregation has to be
prevented. Many proteins or peptides tolerate also low to moderate
percentages of dimethyl sulphoxide, dimethyl formamide,
acetonitrile, or the lower alcohols and glycols. Since the sorbents
of the invention are chemically resistant to almost all protic and
aprotic organic solvents, especially if the bulk solid support
material contained in the carrier is shielded by a surface polymer
film being the only material in direct contact with the liquids,
preference is further given to those predominantly polar liquids
which facilitate swelling of the sorbent or at least said optional
polymer film located thereon. The exact polarity of a compatible
liquid mixture can thereby be easily fine-tuned by way of its
composition.
[0256] Furthermore, to such liquids or liquid mixtures small
amounts of auxiliary substances such as--preferably
volatile--acids, bases, or buffers may favourably be added, thus
enabling to switch between different solvation capabilities via
adjustment of the pH of the applied liquid (or, in partially
organic eluents, the apparent pH) and thereby the degree of
protonation and/or deprotonation of selected or all analytes and/or
of selected or all residues of the sorbent. Useful substances in
this respect are, for example, formic acid, acetic acid,
trifluoroacetic acid, and their salts. The addition of high
concentrations of inert, organic or inorganic salts can also be
useful to modify the ionic strength of a liquid and thus to
selectively break ion pairs between analytes and the sorbent via
competitive interactions. However, in preparative applications such
non-volatile salt additives are difficult to remove later on from
the recovered eluate if the target protein or peptide is intended
to be further purified by crystallisation.
[0257] It may under certain circumstances be advantageous to use
further organic modifiers together with the sorbent in the
resolution of protein or peptide mixtures, which are acting by a
mechanism reaching beyond a pure adjustment of liquid pH or ionic
strength. As "modifiers" small molecules or macromolecules or
mixtures thereof are summed up which are not liquids with solvating
properties by themselves but which may be dissolved or suspended in
small amounts in one or more of the various liquids employed in the
separation method of the invention either to help or prevent the
solubilisation/elution of certain components of the mixture to be
separated during the particular step of the method, or for a number
of secondary (technological) reasons, such as, for example,
long-term stabilisation and storage of solvents, prevention of
sorbent biofouling, preservation of analytes from chemical or
biological degradation or from coagulation, enhanced solvent
miscibility, sorbent swelling, improved analyte detection, breaking
of water structure, controlled protein unfolding or refolding etc.,
depending on the individual separation problem. Special examples of
organic modifiers are ion-pairing reagents, surfactants
(detergents) and chaotropic reagents.
[0258] "Rinsing", "washing", and "regenerating" are different
expressions used for better distinguishing the stepwise treatment
of the same sorbent with different kinds of liquids. The liquids
are thereby rather differentiated by the tasks they perform than by
their composition. The actual procedure of treatment may thereby be
very similar and sometimes only differs by the decision to be made
whether the liquid has to be further refined, fractionated,
recollected, or discarded based on the substances dissolved therein
after the treatment. Rinsing is directed to a treatment with a
liquid that ideally solubilises and releases from the sorbent any
mixture component except for the target which may have been
unspecifically bound by the sorbent. Washing is directed to a
treatment that is intended to solubilise and release from the
sorbent all residually bound mixture components, even those which
may be stronger binding than the target. Regenerating is directed
to the use of liquids which are capable to remove traces of the
washing liquid and to restore the ideal physical and chemical
properties of the clean sorbent for use in the adsorption step at
the beginning of the next run of the method.
[0259] "Immobilisation" means a process of eliminating or
substantially retarding the long-range lateral and/or vertical
mobility of a protein or peptide on the surface of a sorbent which
may otherwise be caused by either statistical, diffusional
migration (Brown's motion) or directed physical or chemical forces
(e.g. osmotic pressure, shear flow). The macroscopic two- or
three-dimensional position of an immobilised protein or peptide on
the adsorptive part of a surface can therefore be regarded as being
fixed on a short time scale. Inevitable small fluctuations in the
order of nanometres around the centre of immobilisation such as
conformational changes, molecular rotations or oscillations,
hopping between adjacent binding sites, or any translational motion
within the combined radii of the protein or peptide itself and the
residue to which it is bound as well as an (optionally polymeric)
tether applied for fixation of the respective binding site residue
to the surface, still remain unaffected. Slow release of the bound
protein or peptide by crossing the binding surface layer on a large
time scale may as well be a desired property.
[0260] In a central embodiment defining a composition of matter,
the present invention is directed toward the target-specific design
of a novel sorbent. Solid support materials having functional
groups have been used for subsequent surface derivatisation,
yielding a two- or three-dimensional arrangement of multiple
residues suitable for multivalent and/or multifunctional spatial
interaction with the target protein or peptide included therein.
After testing various residues, it was particularly found that
two-fold derivatisation of portions of the functional groups of the
solid support material with both binuclear and mononuclear
heteroaromatic residues results in a superior performance in the
subsequent chromatographic separation of proteins or peptides from
each other or from their side products if the sorbent comprising
said heteroaromatic residues is used as stationary phase.
[0261] A general aspect of the invention can therefore be described
with a sorbent comprising a solid support material, the surface of
which comprises first residues comprising a binuclear
heteroaromatic structure comprising besides carbon atoms at least
one of the heteroatoms N, O, S, and second residues comprising a
mononuclear heteroaromatic structure comprising besides carbon
atoms at least one of the heteroatoms N, O, S.
[0262] More specific aspects of the invention may be described with
sorbents according to the first, second, third, fourth and fifth
aspect as specified in the section "Brief Summary of the
Invention".
[0263] The term "wherein none of said functional groups comprises
both said first residue and said second residue" as used for the
description of the sorbents according to the third and fifth aspect
means that less than 5% of the available functional groups of the
surface of the carrier, preferably less than 1%, more preferred
less than 0.1%, still more preferred none of the functional groups,
carry both a first and a second residue.
[0264] In a specific embodiment, it is not detectable by common
analytical methods such as spectroscopic methods that a functional
group carries a first and a second residue.
[0265] Said solid support material of the sorbent can be chosen
from the group comprising polystyrene, polystyrene sulphonic acid,
polyacrylates, polymethacrylates, polyvinyl alcohol, silica, glass,
starch, cellulose, agarose, sepharose, and dextran, or any
composites thereof. The solid support material may belong to the
class of generic bulk or further surface-modified materials, e.g.
to introduce surface functional groups or to increase aqueous
wettability.
[0266] In a special embodiment, the sorbent may also comprise an
easily detectable tag, such as an optically absorbing, an optically
emitting, a radioactive, or a mass- or radiofrequency-encoding tag.
The tag may be used to identify a particular sorbent with its
individual combination of residues even in sorbent mixtures or to
facilitate the detection of protein or peptide binding. The tag can
be incorporated into the core of the solid support material, or
alternatively together with the residues onto its surface.
[0267] To the C, N, O, S-heteroaromatic structures, as they were
mentioned above, especially belong some which are frequently
occurring in chemical structures of small organic molecules, such
as those of the list depicted in FIGS. 3 and 4. Heteroaromatic
structures containing at least one nitrogen atom within at least
one ring are preferred in both residue types. The ring cores of
typical heteroaromatic structures will predominantly be assembled
from one or more moieties of the --NH--, C.dbd.N, --O-- or --S--
type in addition to C--C or C.dbd.C moieties. 5- and 6-membered
ring cores are strictly preferred. Fine-tuning of the specific
affinity of the sorbent for a given particular protein or peptide
is attained via careful selection of the respective heterocyclic
cores and substituents, the molar ratio of first and second
residues, and the introduction of optional further residues.
Therefore it will become clear that the full variability cannot be
exhaustively dealt with; instead the conceptual framework for
building up a sorbent according to the existing demands will be
given.
[0268] For simplicity, only one mesomeric formula is shown for each
structure in FIGS. 3 and 4. Moreover, for the purpose of the
present invention it is sufficient within the meaning of the term
"heteroaromatic" if at least one reasonable mesomeric or tautomeric
formula of heteroaromatic character of such a structure exists even
if there are additional non-heteroaromatic formulae possible. The
connection between the ring system and the remainder of the
residue, and thus eventually the solid support material, can be
made via any of the ring atoms, including free valencies at the
heteroatoms, as attachment points.
[0269] Some of the heteroaromatic structures shown, especially
those containing one or more weakly basic ring nitrogens, are
ionogenic which means that an electronically neutral atom or any
group containing it can, under the conditions of the separation to
be performed (i.e., usually mild or ambient conditions that do not
affect the structural integrity of sorbent or analytes), be
reversibly converted, (e.g. by protonation or deprotonation) into a
cation or anion which is either stable under ambient conditions or
in equilibrium with the uncharged form. More specifically, the C,
N, O, S-heteroaromatic residues of the sorbent will, at least in
part, be amenable to protonation and thus, at least in part, be
present in their protonated form. Although the charged forms are
not explicitly shown in the figures, the equilibrium can actually
reside almost entirely on either side under the given conditions,
and there can still be a measurable mutual interconversion between
the charged and the uncharged form. Protonation depends on the
environmental pH but is also prevalent in most aprotic organic
solvents and makes it difficult to distinguish whether both forms
or only one of them is responsible for the affinity exhibited by
the sorbent. The exact degree of protonation of each residue will
depend on its basicity, the concentration and kind of acid present,
on the mobile phase used and on the way of pre-conditioning of the
sorbent.
[0270] Depending on the particular separation task, it may thus be
advantageous to either treat the mixture to be separated with a
sorbent which exhibits residues which have been conditioned to be
predominantly in the uncharged state or predominantly in the
charged state, or which may even change the state of charge one or
more times during the separation (e.g. by buffer exchange as known
from weak ion exchangers). Conditions under which ionogenic
heteroaromatic structures of the sorbent are partially ionised are
also possible, as can easily be imagined if a separation is
performed in an environment whose pH approaches the pK value of the
respective heteroaromatic structure. It might also be necessary to
manufacture or store the sorbent in an uncharged state while
performing the separation in a charged state, or vice versa.
[0271] A pre-conditioning of the sorbent involving an aqueous
buffer system of an about neutral pH is preferred, especially if
further (third) residues comprising an amine structure (see below)
are present. Such treatment will establish a uniform distribution
of counterions belonging to each sort of ammonium structure or
other ionogenic residue. The strength of hydrogen bonding exhibited
by the residues towards an analyte is also influenced by the nature
of the counterions which are expected to stay within the
surrounding solvate shell and to form ion pairs with protonated
residues, their basicity and/or their hard vs. soft polarisability
behaviour.
[0272] Further substituents can be bound to the binuclear and/or to
the mononuclear C, N, O, S-heteroaromatic structures, respectively,
and can be chosen for both kinds of structures independently. As
shown in FIG. 5 for exemplary structures, wherein the substituents
R.sup.1, . . . , R.sup.n independently represent an electron pair,
hydrogen (H), an organic radical, or a surface linkage. Without
wishing to be confined to a particular ring geometry or
substitution pattern, suitable substituents of C, N, O,
S-heteroaromatic structures may especially comprise those which are
composed of one or more of the following simple organic radicals:
C.sub.1-C.sub.20 linear or branched alkyl, alkenyl, alkinyl,
cycloalkyl, cycloalkenyl, aryl, arylalkyl, arylalkenyl,
arylalkinyl, alkyloxy, alkenyloxy, alkinyloxy, cycloalkyloxy,
aryloxy, arylalkyloxy, alkylthiyl, alkenylthiyl, alkinylthiyl,
cycloalkylthiyl, arylthiyl, arylalkylthiyl, halogenalkyl,
halogenalkenyl, halogenalkinyl, halogencycloalkyl, halogenaryl,
halogenarylalkyl, halogenalkyloxy, halogenaryloxy,
halogenarylalkyloxy, halogenalkylthiyl, halogenarylthiyl, or
halogenarylalkylthiyl. In particular, substituents may contain at
least one further binuclear or mononuclear C, N, O,
S-heteroaromatic structure or heteroatoms bound in any other way,
and especially one taken from the list depicted in FIGS. 3 and 4.
Also, two or more of the substituents R.sup.1, . . . , R.sup.n may
be connected to each other by an aliphatic linkage of any kind and
length, but especially via any of the aforementioned organic
radicals or a combination thereof, so as to form a polycyclic ring
structure. Condensation into multinuclear C, N, O, S-heteroaromatic
structures (number of fused aromatic rings 3) is, however,
excluded, as are substituents comprising permanent
cation-exchanging groups.
[0273] Preferred binuclear structures are benzopyrrole (indole)
structures, including all possible aza-benzopyrrole,
oxa-benzopyrrole, and thia-benzopyrrole structures, or
benzopyridine (quinoline or isoquinoline) structures, including all
possible aza-benzopyridine structures. Preferred mononuclear
structures are pyrrole structures, including all possible
aza-pyrrole, oxa-pyrrole, and thia-pyrrole structures. Here,
3-azapyrrole (imidazole) structures are particularly preferred.
[0274] The fully synthetic sorbents of the present invention have
to be distinguished further from conventional affinity media in
which the surface-bound residues are themselves often proteins or
peptides or parts thereof and which closely mimic known biological
ligand-receptor interactions. In general, such media suffer from
the disadvantages stated in the beginning. Since the natural amino
acids tryptophan and histidine also contain binuclear or
mononuclear C, N-heteroaromatic structures in their side chains,
respectively, any polypeptide residue or group-protected variant
thereof, which could easily be built up by sequential solid-phase
synthesis techniques, would theoretically qualify as a residue
according to the present invention. It has thus to be made clear
that neither tryptophan nor histidine, their esters or carbamates,
or any peptides comprising either of them as residues are within
the scope of the invention. This is, however, not true for any
synthetic and predominantly non-peptidic structure which may
contain a tryptophan- or histidine-related building block.
[0275] Roughly equal degrees of derivatisation with each residue
are preferred, i.e. the first and second residues will be present
in a molar ratio of about 1:1, in a broader sense at least of from
3:2 to 2:3. The sum of the degrees of derivatisation for first and
second residues combined is preferably kept close to at least 50%
(based on the number of functional groups available for
derivatisation) in order to promote the formation of multivalent
interaction sites of mixed composition while still keeping the
binding capacity of the sorbent for the target protein or peptide
high. Both first and second residues may then be present, for
example, at degrees of derivatisation of close to 25% each.
Preferred third residues (see below) comprise amine or amide, more
preferably primary amine structures. In such case, the first,
second, and third residues are present in a molar ratio of about
1:1:2, respectively. Relative deviations of ca. 10% around these
values are tolerable.
[0276] In one embodiment, from 5 to 95% of the functional groups
are linked to said binuclear and mononuclear heteroaromatic
structures, preferably from 15 to 85%, more preferred from 25 to
75%, still more preferred from 35 to 65%, further preferred from 40
to 60%. Since the ratio of first to second residues may be freely
selected, it is possible to optimally adjust within said given
ranges a sorbent to a specific separation problem, e.g. the
separation of a protein or peptide from a mixture comprising said
peptide or protein, or to adjust the sorbent to the optimal
increase of the concentration and/or purity of a peptide or protein
from a mixture comprising said peptide or protein. This variability
renders particularly useful the sorbent or the sorbents according
to the invention for the mentioned separation or increase and/or
purity problems.
[0277] Accordingly, in one embodiment, from 5 to 95% of the
functional groups are linked to said binuclear and mononuclear
heteroaromatic structures, preferably from 15 to 85%, more
preferred from 25 to 75%, still more preferred from 35 to 65%,
further preferred from 40 to 60%; wherein the first and second
residues are present in a molar ratio of from 3:2 to 2:3.
[0278] The type of residue attachment can be any variant of a
covalent bond (homo- or heteroatomic, variable bond order) and may
either be made directly with functional groups on the surface of
the solid support material or on an optional polymer film covering
said surface, whether attached to its backbone or to its pendant
linear or branched side chains or optionally coupled via the
termini of bifunctional linkers. In addition to the heteroaromatic
structures which are the designated parts of the first and second
residues to interact selectively with the target protein or
peptide, these residues may thus also comprise covalent linkers.
Such bifunctional linkers are intentionally not shown in the
figures due to their large possible variability in length and
chemical composition but they are known from standard solid phase
synthesis or bioconjugation methods (e.g. succinyl); the most
simple bifunctional linker would be an alkylene chain of a
predetermined number of from 1 to about 20 atoms. Best suited
linkers are conformationally flexible ones. The preferred covalent
linkages which connect the entire residues to a polymer film will
again be made of amide, urethane, urea, or secondary/tertiary amine
bonds.
[0279] Within this context, it has to be mentioned that especially
long alkylene chains or polyethylene glycol moieties used as
linkers could exert additional, largely unspecific hydrophobic
forces on said proteins or peptides in superposition or
amplification of the primary effect of the heteroaromatics.
Previously described linkers containing sulphur, however, which are
easily synthesised and connected to activated surfaces, are not
within the focus of the present invention since it is well known
that sulphur atoms or sulphur-containing groups interact well with
corresponding groups of the same kind on the molecular surface of
an analyte, a fact that may be able to introduce special
selectivities on its own which could possibly interfere with the
binding mechanism of the sorbents presented here.
[0280] It can therefore not be excluded that possible additional
chemical structures formed between such an optional linker and the
functional groups of the solid support material and/or the
heteroaromatic structures by way of their attachment are also
accessible to various analytes and may thus aid in the selective
retention of the target protein or peptide. The only practical
limitations with regard to the chemical composition of the
additional structural entities placed between the heteroaromatic
structure as part of the respective residue and the surface of the
solid support material are imposed by the requirement of chemical
stability and compatibility with the conditions applied during the
manufacture, storage, and use of the sorbent. Therefore it is also
possible that the respective residue is incorporated via a
specified attachment point as a sub-structure into a scaffold of
higher complexity (including polymers) which may comprise
additional residues of the same and/or different kind.
[0281] The residues can be coupled directly to the surface of a
bulk solid support material, in particular by forming covalent
bonds with functional groups on said surface. For example, the
method of choice for coupling residues to the surface silanol
groups of silica is performed with the help of chlorosilane- or
alkoxysilane-terminated linkers whereas coupling to the hydroxyl
groups of carbohydrate supports can be achieved through a variety
of methods such as the classic cyanogen bromide activation. These
methods are sufficiently known to those skilled in the art.
[0282] In a preferred embodiment, however, the bulk solid support
material represents only a carrier the immediate surface of which
is covered with a film of a polymer having functional groups, said
polymer in turn carrying pendant first and second and optionally
further residues. Thus a thin interlayer is formed which moves the
macroscopic shape-defining and the analyte-interacting parts of the
sorbent apart from each other but does not significantly change the
overall underlying surface topology and is therefore being
considered as a part of that surface. The residues can be attached
to said polymer functional groups which will turn the employed base
polymer into an at least partially derivatised co-polymer. Suitable
polymers having functional groups are for example polyvinyl
alcohol, polyvinylamine, polyallylamine, polyethylene imine,
polyacrylic acid, polymethacrylic acid, and any copolymer or
polymer blend comprising at least one of these polymers. Especially
if the solid support material consists of a bulk polymeric material
as a carrier whose surface is further covered with a film of a
polymer, but also if non-polymeric carriers are used, the material
the carrier is made of can be different from the material the film
of a polymer is made of. Such difference can manifest itself for
example in a different monomer composition, polymerisation regio-
or stereochemistry, stereoregularity (tacticity), molecular weight
distribution, degree of crosslinking, or combinations thereof.
[0283] The exact thickness of the polymer film and also the
separation kinetics and capacity of the sorbent are thereby
dependent on the state of swelling of the polymer, which itself
will always be a function of the mobile phase composition, and can
thus vary under different external conditions. For separations of
proteins and peptides carried out in aqueous or mixed
aqueous-organic media, it is preferable if the polymer is swellable
in such media. This is accomplished most easily if the polymer is a
synthetic polyelectrolyte. As explained above, the charge character
of any possible ionogenic residues also influences the swellability
of the polymer film to a certain degree which is again
solvent-dependent. The term "aqueous" is used herein to describe
liquids which contain more than 50% by volume of water, the
remainder being other water-miscible solvents or additives such as
inorganic or organic buffers, salts etc.
[0284] Such a morphology is designed to maintain unusually high
mass transfer rates between mobile and stationary phases via pore
diffusion. The linear or branched polymer itself has to be durably
fixed to the surface of the rigid and firm carrier in order for the
polymer film to withstand the conditions of the separation process
for which it is made and stay in position throughout the entire
process. The fixation can either be performed by internal
crosslinkage of the individual polymer strands resulting in the
formation of a continuous polymer network, or by grafting of
individual polymer strands at one or more positions along the chain
to the carrier solid. Crosslinkage as well as grafting can easily
be achieved between the same or different functional groups of the
polymer, or between the functional groups present anywhere in the
polymer and those present on the surface of the uncoated carrier,
respectively. The preferred crosslinking or grafting connections of
the polymer will be made of amide, urethane, urea, or
secondary/tertiary amine bonds. The terminal functional groups of
the individual polymer strands are best used for grafting, which
will result in an end-on configuration giving the highest chain
flexibility.
[0285] Though a combination of both techniques would certainly be
feasible, usually one of them is sufficient. The preferred way of
fixation is crosslinkage (without grafting). The polymer chains may
thereby be covalently crosslinked with each other to an extent of
from 1% to 20% based on the number of functional groups available
for crosslinking.
[0286] Additional supplementary residues could thus in principle
result from the introduction of crosslinks into the polymer film if
the crosslinking reagents contain chemical structures that are
suited to interact with one or more analytes. Since the degree of
crosslinking of the polymer is preferably held at a comparably low
percentage, their contributions are believed to be rather
negligible. The same is thought to be true for the contribution of
additional amide (e.g. formamide) or urethane groups which may be
remaining in a variable amount, but usually less than 1%, as a
result from the synthesis of polymer films containing amino
functional groups via incomplete hydrolysis reactions, resulting in
statistical amine/amide or amine/urethane copolymers.
[0287] It is nevertheless possible to derivatise a solid support
material with two or more different first residues and/or two or
more different second residues, according to the definition of
their respective partial structures. These may then differ from
each other in their heteroaromatic structures or in their ways of
linking these structures to the surface of the solid support
material, or both. In a preferred embodiment, the total number of
first residues and total number of second residues (or their degree
of derivatisation equivalents, respectively) will be about equal in
order to realise the maximum number of mixed-composition binding
sites comprising all different first and second residues under the
provision of a random (statistical) spatial residue
distribution.
[0288] Usually and in a preferred embodiment, the first and second
residues are not connected directly with each other but are
separately attached to either a bulk solid support material itself
or a polymer film supported by it as a carrier. Both the binuclear
and mononuclear C, N, O, S-heteroaromatic structures are two
distinguishable entities, each possessing its own linker to a bulk
solid support material or polymer surface, which shall--within this
embodiment--constitute the shortest connection path between them.
Such a form makes the second residue easily separable by structure
from the first residue. Accordingly, the binuclear heteroaromatic
structure and the mononuclear heteroaromatic structure are not
linked to the surface of the support material via the one and same
functional group.
[0289] On the other hand, two or more residues of the same or
different kind can also be connected directly with each other
through covalent bonds not involving the backbone of a polymer film
or in any other way the surface of the solid support material. In
such case, the boundaries between the individual residues begin to
blur and are becoming arbitrary since they may only be left
meaningful if the derivatisation history of the sorbent (i.e., the
sequence and kind of derivatisation steps) is known. As is
exemplarily shown in the schematic representations A-H of FIG. 1,
two pendant functional groups on the surface of the solid support
material can be derivatised with two different residues in many
different ways (the long horizontal wiggly line here denotes a part
of the surface which may itself contain further residues).
[0290] In addition to the case mentioned above of an equal
distribution wherein each individual functional group carries one
residue (in formulae A or B), they can also, for example, be
aligned sequentially in a row (in formulae C, D) or in parallel (in
formulae E, G) onto the same functional group. Such configurations
can experimentally be achieved, inter alia, in that a residue
contains itself a functional group which is the same as the polymer
or surface functional group or different therefrom, and which may,
after derivatisation of the polymer or surface functional group
with said first residue, be derivatised itself (optionally after
deprotection and/or activation) with the second residue (case C),
or in that one functional group is derivatised at least twofold (in
a single step or in a number of consecutive steps, such that a
common functional group (case G) or a linker having a branched
structure (case E) is shared by both residues (an appropriate
example would be a two- or threefold alkylation of a primary amino
group to yield a tertiary amino or quaternary ammonium moiety). The
resulting configurations C, D, E, F could, however, also be
achieved via an alternate path in which the surface or the polymer
having functional groups is derivatised with a single
derivatisation reagent already carrying both first and second
residues in the correct mutual arrangement. More complex mutual
arrangements of both residues such as macro- or polycyclic ring
systems (cases F and H) are also imaginable, of course. In all
cases except for A, the first portion of functional groups which is
derivatised with the first residue always equals the second portion
of functional groups which is derivatised with the second
residue.
[0291] All situations described above can under a unified view also
be regarded as borderline cases of a more general situation in
which the individual residues are arranged in a hierarchical order.
According to this interpretation and as a special embodiment of the
invention, the first (larger) residue comprises the second
(smaller) residue (or vice versa) in addition to its binuclear C,
N, O, S-heteroaromatic structure, whereby the larger residue is
chosen such that it directly derivatises a functional group of the
solid support material, as is shown in the general representation I
of FIG. 1. In such case, all atoms and chemical moieties of the
second residue are also contained in the first residue. This means
that there is only one, or two different, distinguishable residues,
each of which comprising all covalently connected structural
elements including both the binuclear and the mononuclear C, N, O,
S-heteroaromatic structures. Within such a given hierarchical
order, basically any type of connection and geometry (linear,
branched, cyclic, etc.) can be realised to attach the atoms and
structural entities of the second residue to those of the remainder
of the first residue which is not overlapping with the second
residue. Under such circumstances, the splitting of the structural
hierarchy into two different residues, however, would rather be a
matter of formality; at the borderline the chemical properties will
be unique to the overall sum of structures as an intergral chemical
unit. Multiple arrangements are thereby possible regarding the
mutual orientation of the structural subunits of the two residues,
with the exception that both first and second residues should not
share a common, single heteroaromatic ring system.
[0292] If one of the given examples is re-examined in view of this
general representation, such a configuration can, inter alia, be
realised in that the two different heteroaromatic structures share
a common linker or a part thereof through which they are attached
to the surface of the solid support material itself. The two
heteroaromatic structures can thereby be arranged linearly on the
same branch or on different branches, if the linker has a branched
structure. The entire residue, i.e. the largest possible, uniform
structural unit (including a possible linker terminating in the
surface functional groups and all other substructures connected
therewith), would then--just formally--be attributed to the first
residue, while the second residue would--formally, again--in such a
configuration only comprise the respective mononuclear
heteroaromatic structure and possibly its immediate connective
elements with the remainder of the overall (first) residue.
[0293] It has now surprisingly been found that any sorbent
possessing a combination of the two structural features described
above allows the easy recovery of a number of proteins or peptides,
in certain instances with a purity higher than 98%, or with a final
concentration of each impurity below 1% in a single step starting
from only partially purified mixtures. Pharmaceutical grades can
thus be obtained without laborious or cumbersome procedures. The
concentration of proteins or peptides in crude materials such as
those directly resulting from manufacturing on the industrial
scale, can be enriched to high levels in a single step, too.
Applicable titres may range from about 1% to about 90% in the
mixture. The recovered yields of said steps are thereby at least as
high as those of conventional purification methods and can approach
values of 95%. The markedly good performance of a sorbent
comprising residues of both kinds of the two different
heteroaromatic structures for the given object of the invention is
even more surprising since it could be shown that sorbents
comprising residues of closely related structures or only one kind
of the two heteroaromatic structures necessary showed only a
moderate separation efficiency at most.
[0294] Without wishing to be bound to a theory, the high
performance of this particular sorbent compared to a sorbent coated
with a film of a simple, underivatised polymeric amine can be
attributed to the presence of additional and structurally novel
multivalent binding sites. The structures responsible for the
creation of such novel binding sites can predominantly be
attributed to the partial statistical modification of the polymer.
Among those structures particularly to be noted is the potential
presence of extended, either electron-rich or electron-poor
.pi.-systems and/or conjugated systems of weak basicity within said
binding sites. The underlying interaction mode is thought to both
involve interactions belonging to the group of polar/dipolar ones
like electrostatic forces, charge transfer and hydrogen bonding, as
well as those belonging to the group of apolar ones like
hydrophobic interaction and .pi.-stacking. The heteroatoms of the
.pi. system are expected to be the potential sites of dipole forces
and hydrogen bonding, whether through the electronic .pi. system
itself or through an extra electron lone pair. However, without
having performed investigations into the actually operating
mechanism in a given separation and the exact kind of the partial
contribution of each residue to the over-all binding strength, a
definite conclusion cannot be drawn in advance for any such
structure, partly because hydrogen bonding forming competition with
solvent molecules may also complicate the case. Steric factors may
additionally contribute to the selectivity of the designed sorbent.
At least, pure ionic contributions from the first or second
residues are unlikely or can even be excluded.
[0295] Moreover, after testing a large number of differently
derivatised sorbents, it was strikingly found that the presence of
a third residue in addition to derivatisation of first and second
portions of functional groups on the surface of the solid support
material with first and second residues yielded even superior
results in view of the given separation object of the invention.
The solid support material may thus be further derivatised with a
supplemental third, fourth, and fifth residue, and so forth. A
sorbent comprising a solid support material, the surface of which
in addition to first and second residues, as described above, also
comprises a third residue, is therefore a further embodiment of the
present invention. Binuclear or mononuclear C, N, O,
S-heteroaromatic structures are being excluded as structural
building blocks of the third and each further residues. Apart from
this exclusion, all options regarding possible structural
relationships between two residues, as exemplarily set forth in
FIG. 1 for the first and second residue, analogously apply to the
mutual relationships between the third and the first, the third and
the second, as well as between any additional residues. Each
additional residue of a different kind promotes the sorbent's
potential of creating very specific binding sites for a given
protein or peptide and to distinguish it from closely related side
products. Each category of residues should, however, be present at
a degree of derivatisation of at least about 20% since
significantly lower degrees of derivatisation are in most cases
negligible for statistical reasons. For the majority of
applications, it is therefore sufficient to keep the number of
residue categories.ltoreq.5 at about equal degrees of
derivatisation. Regardless of the number and mutual ratio of
different residues, each type of residue should still be
homogeneously and randomly (statistically) distributed on the
surface of the solid support material.
[0296] Whereas said first portion of functional groups may thereby
comprise said second portion of functional groups, or may be
different therefrom, the third residue may also arise from
incomplete derivatisation of the surface functional groups of the
solid support material with portions of first and second residues.
Depending on the reagents and synthetic conditions used, the
derivatisation reactions often remain incomplete. Therefore, a
certain number of underivatised functional groups generic to the
surface of the solid support material including the optional base
polymer covering it (i.e., those incorporated into at least one of
its corresponding monomers or repeating units) to be derivatised
may survive intentionally or for technical reasons. These may still
be accessible to various analytes, can act as supplementary part of
a binding site, assist in binding the target protein or peptide,
and thus add to the separation ability of the sorbent. This means
that a third (leftover) portion of said functional groups itself
may represent a kind of said third residues. In the present
invention, it is preferred to employ solid support materials
covered with a polyamine film, particularly a polyvinylamine film.
Accordingly, the preferred functional groups are primary and
optionally secondary amino structures which may therefore be
regarded as supplemental third residues. It could also be shown
that fractions of derivatised functional groups which were
apparently too high led to a decrease in the selectivity for the
given separation object. This fact may be taken as an indication
that novel multifunctional binding sites are thus created within
the sorbent comprising both first and second residues and
underivatised functional groups in close spatial proximity.
[0297] By way of such a designed tertiary derivatisation or an
incomplete primary and secondary derivatisation, the selectivity
for a given protein or peptide can in many cases be further
increased, and as an accompanying practical benefit an optional
polymer film covering the surface of the solid support material is
often observed to gain additional chemical stability and better
solvent compatibility or swelling properties, depending on the
relative polarities of the first and second residues and the
functional groups involved. Nevertheless, it can be stated that the
first and second residues are the most essential residues for
achieving the underlying separation object in terms of specificity
since a film of a completely underivatised polymer like crosslinked
polyvinylamine, which exhibits only backbone primary amino
functional groups to the analytes, does not achieve the separation
object of the present invention satisfactorily. Ideally, the total
density of residues (including underivatised functional groups
acting as supplementary residues) amounts to from 0.1 mol dm.sup.-3
to 1.0 mol dm.sup.-3, but preferably to at least about 0.3 mol
dm.sup.-3.
[0298] On the other hand, underivatised reactive functional groups
of the solid support material or of an optional polymer film
thereon, more specifically amino groups, may still exhibit
considerable reversible or irreversible reactivity towards the
target or possible reactive side products of the mixture to be
separated which may lead to firm capture of those substances--even
if they are present in low concentrations only--and, after repeated
use, to a slow deterioration of the sorbent and loss of binding
capacity. In order to avoid such unwanted interactions, it is
common practice in the preparation of chromatographic stationary
phases to render such residual functional groups inactive via final
end-capping of said groups. Thus, additional (third or fourth)
residues may be created here via at least partial conversion of
originally free functional groups into structurally different
end-capped functional groups. End-capping may in this way be
regarded as a special case of a derivatisation reaction
establishing an improved compatibility of the
solid/liquid-interface with the demands of the respective analyte,
matrix, and mobile phase but which can hardly create additional
binding strength and thus no additional selectivity. Partial or
full end-capping of residual functional groups may nevertheless
eventually turn out to be favourable in terms of long-term process
stability despite the additional effort in stationary phase
preparation.
[0299] Preferably, end-capping of nucleophilic functional groups
such as amino groups is achieved through reactions which reduce
functional group nucleophilicity. End-capping groups are designed
to be of simple molecular structure so as to exhibit no interaction
or at least only non-covalent and non-specific interactions of low
strength with a broad range of analytes and to not alter the
overall polarity of the stationary phase significantly. It is
conceivable, however, that they may assist at high degrees of
derivatisation the first and second residues in multivalent
interactions with the substrate. Despite the possibility of more
than two-fold mixed tertiary derivatisations on the sorbent due to
incomplete or mixed endcapping, it has turned out that it is
preferable to aim at either a uniform endcapping (i.e. to a
degree>95%), or no end-capping at all, throughout the sorbent.
Depending on the structure of the end-capping groups, they thus may
or may not potentially act in the role of tertiary residues, if
treated formally.
[0300] In a first methodical embodiment, the present invention is
directed to methods for preparing sorbents of the invention having
the characteristics as presented above. They will result in that
special class of sorbents wherein the solid support material
consists of a carrier the surface of which is covered with a film
of a polymer having functional groups which carries the residues.
The special characteristics of this preferred class of sorbents
have also been extensively outlined above. Now, said preparation
methods comprise at least the steps of: [0301] (i) providing a
polymer having functional groups; [0302] (a) adsorbing a film of
the polymer onto the surface of a carrier ("adsorption step");
[0303] (b-I) crosslinking a defined portion of the functional
groups of the adsorbed polymer with at least one crosslinking
reagent ("crosslinkage step"); [0304] or: [0305] (b-II) grafting a
defined portion of the functional groups of the adsorbed polymer to
the carrier ("grafting step"); [0306] (c) derivatising defined
portions of the functional groups of the polymer with first
residues comprising a binuclear C, N, O, S-heteroaromatic structure
and with second residues comprising a mononuclear C, N, O,
S-heteroaromatic structure, and with optional further residues
("derivatisation step").
[0307] Several variations concerning the detailed layout of the
above preparation method are conceivable. First, steps (b-I) and
(b-II), crosslinkage and grafting, respectively, are considered as
equivalent alternatives, and either one of these steps is
sufficient to carry out the method in order to build up a sorbent
according to the invention which will show the characteristics
described further above. Both alternatives serve as means to fulfil
the task of a durable fixation of the adsorbed polymer onto the
carrier under the conditions of further processing and use of the
sorbent, even if treated with strongly solubilising solvents. This
is achieved by either forming a continuous network of additional
covalent bonds between all polymer strands and thus physically
entangling the carrier (crosslinkage) or by forming covalent bonds
between each single polymer strand and the carrier (grafting). Of
course, both alternative processes for fixation can also be
combined within the method, either concurrently into a single step
or subsequently as two distinguishable subordinate steps, without
suffering from disadvantages for the stability of the sorbent.
[0308] Secondly, further variations are possible concerning the
relative temporal order of the derivatisation step (c) in relation
to the adsorption step (a). It is thus conceivable to first
derivatise a polymer in homogeneous solution with the residues and
then adsorb a film of the derivatised polymer already containing
the residues onto a suitable carrier. Such a procedure will require
to investigate and optimise the experimental conditions of the
coating step for each differently derivatised polymer. The
preferred variant is therefore rather to first adsorb an
underivatised polymer onto the carrier as will be carried out
within the adsorption step (a) parallel or prior to the
derivatisation step (c), in order to obtain a thin homogeneous
layer.
[0309] The crosslinkage step (b-I) or the grafting step (b-II),
respectively, will in any case immediately follow the adsorption
step (a) since, once crosslinked, the polymer would be difficult to
be adsorbed as a film. A further boundary condition is that step
(i) will always be the first step of the sequence. Taken together,
the following four combinations of said two independent variations
of steps (choice of step b-I or b-II combined with relative order
of steps (a) and (c)) are possible:
[0310] 1.sup.st Method: Method for preparing a sorbent according to
the invention, comprising, in the following order:
[0311] (i) providing a polymer having functional groups;
[0312] (ii) adsorption step (a);
[0313] (iii) crosslinkage step (b-I);
[0314] (iv) derivatisation step (c).
[0315] 2.sup.nd Method: Method for preparing a sorbent according to
the invention, comprising, in the following order:
[0316] (i) providing a polymer having functional groups;
[0317] (ii) derivatisation step (c);
[0318] (iii) adsorption step (a);
[0319] (iv) crosslinkage step (b-I).
[0320] 3.sup.rd Method: Method for preparing a sorbent according to
the invention, comprising, in the following order:
[0321] (i) providing a polymer having functional groups;
[0322] (ii) adsorption step (a);
[0323] (iii) grafting step (b-II);
[0324] (iv) derivatisation step (c).
[0325] 4.sup.th Method: Method for preparing a sorbent according to
the invention, comprising, in the following order:
[0326] (i) providing a polymer having functional groups;
[0327] (ii) derivatisation step (c);
[0328] (iii) adsorption step (a);
[0329] (iv) grafting step (b-II).
[0330] Each step of the sequences is meant to be carried out with
the polymer in its state as resulting from completion of the
immediately preceding step, i.e. a derivatisation step following a
crosslinkage or grafting step will be carried out with the already
crosslinked or grafted polymer, whereas a derivatisation step
preceding an adsorption step will be carried out with the free,
non-adsorbed polymer. If a defined portion of the functional groups
of the polymer is reacted in a particular step and a similar
portion has already been reacted in a preceding step, it is meant
that the defined portion in that particular step will be taken from
the totality of those functional groups that are leftover from the
preceding steps and have not been reacted previously (with the
exemption of bi- or multivalent functional groups). While all four
methods will in principle yield comparable results, the first
method is preferred for its practical simplicity.
[0331] In a further variation which has not explicitly been
mentioned so far, a first portion of the functional groups of the
polymer could be derivatised in solution, the partially derivatised
polymer then adorbed, and a second portion of the same or different
functional groups as before on the thus adsorbed polymer
derivatised with the same or different residues as before. Or
functional groups of the polymer could first be converted into
different functional groups or residue precursors by solution
derivatisation, which would then, after adsorption, be converted
into the final residues. The most reasonable order in which
individual residues are introduced by such a mixed combination of
preparation steps will thereby strongly depend on the particular
kind of carrier material and the easiness of adsorption of a
particular, partially derivatised polymer on the carrier.
[0332] Intra- and intermolecular crosslinking of the layer will
form a stable two- or preferably three-dimensional polymer network
and prevent its desorption from the enwrapped carrier medium.
Although crosslinking can be achieved according to all procedures
known as state of the art, also incorporating unselective methods
based on the generation of radical species anywhere on the polymer
chains such as electrochemical, light- or (ionising)
radiation-induced methods, the crosslinking step will preferably be
carried out only between the functional groups of the polymer using
crosslinking reagents which for example are to designed to undergo
condensation reactions with said functional groups. Linear,
conformationally flexible molecules, such as
.alpha.,.omega.-bifunctional condensation reagents, of a length of
between 1 and 20 atoms are preferred for crosslinking. Also, two or
more crosslinking reagents of different length and/or different
reactivity and/or different chain rigidity can be employed,
preferably in consecutive steps. Crosslinking will not be carried
out in an exhaustive manner which would lead to a rigid material,
but always to a predetermined extent only, i.e. with a defined
portion of polymer functional groups, which is easily controllable
via the stoichiometric fraction of added crosslinking reagent(s) in
relation to available polymer functional groups. Suitable
crosslinking reagents in this respect comprise dicarboxylic acids,
diamines, diols, and bis-epoxides, for example
1,10-decanedicarboxylic acid or ethyleneglycol diglycidylether
(EGDGE). 4,4'-Biphenyldicarboxylic acid is useful as a rigid
crosslinker.
[0333] Crosslinking reagents are preferentially chosen to react
specifically with the functional groups of the polymer but neither
with the template nor with the underlying carrier material such as
to accomplish stable crosslinks within the polymer film only but
not between the polymer film and the carrier surface. Anyway,
establishing additional crosslinks of the latter type in a moderate
number would certainly not alter the properties of the sorbent
significantly.
[0334] If additional capping groups are desired, they are usually
introduced last in the process (after the last derivatisation with
a specific residue) if prior derivatisation has been incomplete.
End-capping can in principle be carried out analogously to the
specific derivatisation steps described above. However, activation
methods leading to highly reactive reagents are usual in capping
reactions since they are required to react with those functional
groups which have proven to be the least reactive ones during the
prior derivatisation steps. Preferred are acyl anhydrides and acyl
chlorides, particularly those of acetic acid, or isocyanates and
isothiocyanates, or epoxides. Also, two or more different
end-capping reagents or reagents comprising two or more different
capping groups such as, for example, mixed anhydrides can be
employed. It can also be imagined to use other typical alkylation
reagents having good leaving groups such as methyl iodide, dimethyl
sulphate, or diazomethane. Other suitable end-capping methods both
for polymeric and non-polymeric stationary phases as known from the
prior art can analogously be used. Usually, an exhaustive
end-capping of as many residual functional groups as possible is
desired although the process can also be managed to stop at
essentially any arbitrary degree of capping, if required.
[0335] It is also possible to temporarily derivatise functional
groups of the polymer film or substituents of the residue with
protecting groups. Said functional groups or substituents can thus
be protected during the introduction of one or more further sets of
residues from sometimes undesired reactions with the respective
derivatisation reagents which may otherwise lead to uncontrollable
accumulation of residues or higher-order substitution patterns such
as branching. Once the additional set of residues has been put in
place, the protecting groups are usually removed again.
[0336] The preferred functional groups of the polymer to be
adsorbed as a film onto the surface of a carrier are primary or
secondary amino groups, hydroxyl groups, and carboxylic acid or
carboxylic ester groups. These groups are easily derivatisable,
biocompatible, and increase the water solubility of the polymer. It
is thus also preferred to employ polymers in the method which are
soluble in aqueous or mixed aqueous-organic media because the
adsorption step is preferably carried out from such media onto the
carrier material suspended therein. Although the adsorption step
itself can in principle be carried out stepwise using different
polymers in each step, it is preferentially carried out with a
single type of polymer (i.e., polymers having the same type of
functional groups, or functional groups bearing charges of the same
prefix) only. Particularly preferred are polymers having a
molecular weight of between 5,000 Dalton and 50,000 Dalton.
[0337] In general, all further preferred embodiments as outlined
above with respect to the composition and properties of the sorbent
of the invention also apply to the methods of its preparation and
the materials to be employed in said method in an analogous way and
thus do not need to be repeated within this context.
[0338] The anchor group, i.e. the site of activation of the
derivatisation reagent used in the derivatisation step may be close
to the binding site to be formed or at a short or long distance
remote from it, basically depending on structural, functional, or
synthetic requirements, i.e. it may incorporate a spacer group
between the structures forming the binding site and the activation
site. Such spacer can be either rigid or flexible and of variable
length, whereupon a longer spacer group often transforms into
increased conformational flexibility which may sometimes be
required by the complex between the binding site of the sorbent and
the target protein or peptide in order to adopt a favourable
geometry. Spacers can either be coupled first with the
corresponding heteroaromatic structures in separate (possibly
homogeneous) reactions and the formed conjugates, which resemble
the full residues, then, after optional deprotection, coupled with
the polymer, or spacers can be coupled to the polymer first and the
formed conjugates then, after optional deprotection, coupled with
the corresponding heteroaromatic structures to form the full
residues. The two coupling reactions may thereby be of the same or
different kind. In general, if a polymer containing primary amino
functional groups is used as the film-forming polymer, the nitrogen
atom of the functional amino group can directly be incorporated
into the residue.
[0339] Preferred derivatisation reagents comprise amines, epoxides,
carboxylic acids or esters, and iso(thio)cyanates, resulting in the
formation of amide, urethane, or urea linkages with the preferred
polymer functional groups. For structural, stability, and
convenience reasons, it is most preferred if the derivatisation
step is carried out by formation of amide bonds between the
functional groups and the residues, i.e. either between an
amino-containing polymer and a carboxyl-terminated derivatisation
reagent or between a carboxyl-containing polymer and an
amino-terminated derivatisation reagent. In conjunction with amino
polymers, particularly preferred derivatisation reagents are
activated carboxylic acid derivatives.
[0340] If chemical activation is necessary prior to derivatisation,
it can be carried out in an extra step upstream of the
derivatisation step or concurrently with the derivatisation step.
Either the polymer functional groups or, preferably, the
derivatisation reagent can be activated. Activation of a carboxyl
group, for example, can be achieved by standard techniques of solid
phase peptide synthesis, e.g. via activated esters such as OBt
(benzotriazolyloxy) or ONB (norbornendicarboximidyloxy) esters.
Hydroxyl groups can be treated analogously. In an economic and thus
particularly preferred embodiment, the activation will be performed
in situ during the derivatisation step with the help of methods
also known from peptide chemistry, i.e. as a one-pot reaction in
which a steady-state concentration of the activated species is
being produced, but not isolated.
[0341] Both residues can be introduced into the polymer in a single
derivatisation step. Optionally, a single derivatisation reagent is
used here which already comprises both residues (or precursors
thereof, respectively) or which comprises the first residue which
comprises the second residue (or vice versa). Or at least two
different derivatisation reagents are employed as a mixture, each
of which comprising at least one but different residue. The
derivatisation step can alternatively be carried out stepwise with
each residue. Then the derivatisation reagent employed in the first
derivatisation step comprises the first residue and the
derivatisation reagent employed in the second derivatisation step
comprises the second residue, or vice versa.
[0342] In one variation of the preparation method, steps (iii) and
(iv) can be carried out as a single step. This embodiment takes
into account that in a derivatisation reaction of the functional
groups of the polymer both the first and the second residue can
easily be introduced simultaneously. This can either be achieved in
a way that a mixture of at least two derivatisation reagents is
used, the first of which comprising the first residue, and the
second one comprising the second residue. Although a random,
irregular distribution of the two residues along the polymer
backbone will then result, the derivatised polymer can be
characterised by a statistical ratio of first and second residues
which will basically be determined by the relative amounts and
reactivities of the at least two derivatisation reagents.
Alternatively, it is feasible to use only one derivatisation
reagent if this derivatisation reagent already comprises both the
first and the second residue (or if the first residue comprises the
second residue, or vice versa). Naturally, both residues will then
be present in the resulting derivatised polymer in a 1:1 ratio and
in a pre-defined mutual regio- and stereochemistry. Instead of two
fully developed residues it is also possible that at least one
residue is present in the derivatisation reagent as a
precursor.
[0343] Within the scope of the same variation of the preparation
method of the present invention, configurations can be realised in
which a mixture of derivatisation reagents is used, each of which
comprising both the first and the second residue. In particular, in
such a mixture a partial structure of the first residue (or
precursor thereof) can be varied among the derivatisation reagents
whereas the second residue (or precursor thereof) may be kept
identical, or vice versa. Very particularly, derivatisation
reagents can be combined with each other in pursuit of the
preparation method, a defined amount of which contains both first
and second residues whereas another defined amount may contain only
first or only second residues. The resulting product would then
exhibit one residue in excess over the other if reagent amounts and
reactivities are otherwise comparable. In such way, inter alia,
tailor-made, but still homogeneous and random (statistical)
distributions of first and second residues among the functional
groups of the polymer can be achieved.
[0344] If additional third, fourth, . . . etc. residues are to be
introduced into the polymer, the derivatisation step can optionally
be repeated step-wise multiple times employing further residues
comprising a desired structural motif accordingly. Economically
feasible are up to about four repetition steps. Preferably, each
derivatisation step is always carried out to roughly the same
degree of derivatisation, the degree for each residue thereby
accounting for about 25%.
[0345] The sorbent of the present invention can predominantly be
applied to the purification of mixtures containing proteins or
peptides. In a second methodical embodiment, the present invention
is therefore directed to a method of separating, or increasing the
concentration and/or purity of one or more proteins or peptides
from a mixture containing said protein or peptide and optional side
products using a target-specifically designed sorbent as described
above. The method comprises at least the steps of: [0346] (i)
contacting said mixture being dissolved or suspended in a first
liquid with a sorbent of the invention for a period of time
sufficient to enable said protein or peptide to become bound to
said sorbent; [0347] (iii) contacting said sorbent with said bound
protein or peptide with a third liquid for a period of time
sufficient to enable said protein or peptide to become released
from said sorbent.
[0348] In a first variation of the above method, a separate rinsing
step with a second (wash) liquid that ideally does not
significantly disrupt the non-covalent bonds between the sorbent
residues and the protein or peptide to be purified or otherwise
acts to release said sorbent-bound protein or peptide can be
included between step (i) and step (iii). Depending on the kind and
number of side products and further constituents contained in the
mixture, such a change of liquids during the separation process can
sometimes increase separation efficiency. The second liquid will
mostly have low elution strength and will elute unspecifically. The
method then comprises the optional intermediate step of: [0349]
(ii) rinsing said sorbent with a second liquid;
[0350] After contacting the mixture of the target protein or
peptide and side products with the sorbent in step (i), the sorbent
with the protein or peptide adsorbed to it can also be separated
again from the remaining mixture contained in the first liquid
before it is then rinsed with the second liquid in step (ii). The
remaining mixture may itself be recollected if it contains valuable
side products. The latter variation can also be used as a capturing
means for very dilute feedstocks and also be a feasible way to
remove potential side products in a rapid batch process which are
suspected to interfere with a subsequent, full and more
sophisticated chromatographic separation. Among such possible side
products are those which may lead to a slow deterioration of the
sorbent by irreversible physical or chemical adsorption and thus to
shortened column durability.
[0351] In a special but important case in practice, the second
liquid can be chosen identical to the first (feed, adsorption)
liquid. This means that the sorbent is rinsed in step (ii) with the
same liquid as the one from which the target protein or peptide is
adsorbed when it is applied as a mixture to the sorbent in step
(i). This is often possible since the first liquid is usually
chosen such that it has only medium-to-poor solubilising properties
for the target protein or peptide because an efficient adsorption
will only be possible if the interaction enthalpy between the
target protein or peptide and the liquid is smaller than between
the target protein or peptide and the sorbent. If, on the other
hand, this liquid has good solubilising properties for the side
products which are supposed to be eluted from the sorbent in step
(ii), it can also be applied for rinsing the sorbent while the
target protein or peptide will still adhere to it without being
simultaneously released.
[0352] Similarly, the second liquid can be chosen identical to the
third (desorption, elution) liquid. If the solubilising properties
of the third liquid for the target protein or peptide and the side
products are different to a degree large enough while their
adsorption enthalpies on the sorbent are comparable, the same
liquid can be used for rinsing the sorbent. This essentially means
that step (ii) and step (iii) of the method can under these
circumstances be combined into one step. In a continuous flow
system, the better solubilised side products will then be rinsed
off first, followed by the released target protein or peptide in a
later eluted fraction of the same liquid. Of course, this sequence
may again be followed by additional fractions of the third liquid
containing further, less solubilised and therefore slower eluted
side products.
[0353] Even all three liquids may be identical. However, even if
two or three liquids are chosen identical, they may still be
applied to the sorbent at different flow rates in different steps
of the method. Volumetric flow rates in chromatography are in
general a function of the applied pressure regime, the column
dimensions, and the liquid viscosity. Corresponding one-dimensional
velocities of the mobile phase in HPLC are typically in the order
of about 1-5 mm s.sup.-1. The numeration first, second, third, . .
. liquid thus serves to define the relative sequence of applying
liquids that fulfil different tasks, but is not meant to define
necessarily particular compositions of the respective liquids.
Instead of exchanging the kind of liquid or its applied flow-rate
discretely or stepwise (i.e., as a step-gradient), other continuous
gradient shapes, in particular linear gradients, may be used to
switch slowly between the different liquids and/or flow rates. This
requires the installation of a mechanism to gradually mix
increasing fractions of the succeeding liquid into the preceding
liquid, respectively.
[0354] In one embodiment of the present invention, the third liquid
will differ from the first and optionally also from the second
liquid in its pH. In a particular embodiment, the pH of the third
liquid is lower than the pH of the first and optionally of the
second liquid. Still more preferred, the pH of the first and
optionally the second liquid is close to (i.e.: within .+-.1 unit
approximately matches) the isoelectric point pl of the target
protein or peptide, whereas the third liquid has a pH which is
largely different therefrom, at least by ca. 2 pH units, and in
particular lower. The pH of the first liquid may favourably be in
the range of from 5.5 to 8.5 whereas the resulting pH of the third
liquid would be in the range of from 3 to 6.5. This embodiment
deals with the case that the enthalpy of binding between the
sorbent and the target protein or peptide is dominated to a
significant part by electrostatic or other polar interactions
(dipole forces, hydrogen bonds) involving one or more ionisable
residues (e.g. amino groups or nitrogen-containing heteroaromatics)
on either binding partner. In particular, hydrophobic and polar
interactions are expected to be dominant close to neutral pH,
whereas ionic repulsion is expected to partially replace the
attracting polar forces of the same, hitherto uncharged residues,
when approaching either extreme of the pH spectrum (e.g., between
protonated nitrogens at low pH). This effect can considerably
weaken the enthalpy of binding and, as a result, release the bound
protein or peptide from the sorbent. In the opposite way, an
attractive ionic interaction can also be weakened upon loss of a
point charge of either binding partner as a result of a pH shift.
Of particular importance are in this respect nitrogen-containing
heteroaromatics as residues on the sorbent since these are capable
of exhibiting both hydrophobic as well as polar/ionic interactions.
The attractiveness of such heteroaromatic residues for use in the
separation method of the invention results from the fact that their
binding behaviour can be switched at pH values which closely
resemble physiological conditions, whereby the exact pH range of
switching is dependent on the isoelectric point of the specific
residue and may thus be fine-tuned by its structure and the
relative composition of the sorbent which contains at least two
different residues of such kind. On the other hand, the pH
dependency of the enthalpy of binding should be less pronounced
with regard to interactions between the sorbent and at least one
side product to be separated off. This can for example be due to
different isoelectric points of target and side products or to
quantitatively different relative contributions of
hydrophobic/polar vs. electrostatic interactions.
[0355] In a further embodiment of the present invention, the third
liquid will differ from the first and optionally also from the
second liquid in its ionic strength. In a particular embodiment,
the ionic strength of the third liquid is higher than the ionic
strength of the first and optionally the second liquid. This
embodiment deals with the case that the enthalpy of binding between
the sorbent and the target protein or peptide is dominated to a
significant part by electrostatic interactions under participation
of one or more ionic or ionisable residues, whereas such
participation is different, in particular less pronounced, in the
electrostatic interaction between the sorbent and at least one side
product to be separated off. On the other hand, hydrophobic
contributions to the enthalpy of binding will be strengthened upon
an increase in ionic strength, if all other parameters are kept
constant. Preferably, the adsorption step (i) of the separation
method is performed under low-salt conditions (0-0.2 M sodium
chloride) in the first liquid, whereas the release step (iii) can
be performed at up to 1 M sodium chloride in the third liquid.
Although the sorbent of the invention tolerates high-salt
conditions very well, it is under most circumstances neither
necessary nor advisable to add high salt concentrations to the
third liquid of step (iii) in order to desorb sorbent-bound
proteins or peptides. Instead, the affinity of the sorbent for many
proteins or peptides can be largely invariable with changes in salt
concentration. Therefore salt gradients may not be effective merely
by themselves to release adsorbed proteins or peptides, but they
can be efficient in combination with assistive pH gradients.
[0356] Release of the target protein or peptide from the sorbent
can thus be accomplished via increasing the solvation strength of
the third liquid for the target as compared to the first and second
liquids. It can alternatively be accomplished via displacement of
the target protein or peptide from the binding sites of the sorbent
with a displacement reagent which is dissolved in the third liquid.
The displacement effect (preferable binding of the displacement
reagent by the sorbent rather than of the competing target) can
either be achieved if the displacement reagent is present in molar
excess over the target protein or peptide or if the displacement
reagent's binding strength toward the sorbent is even higher than
that of the target protein or peptide. The displacement reagent may
itself be a protein or peptide having similar properties as the
target, or a fragment thereof, but also a small synthetic molecule
with high affinity for C, N, O, S-heteroaromatic residues. It may
also itself be a binuclear or mononuclear C, N, O, S-heteroaromatic
molecule; displacement would then rather occur as a consequence of
saturating the target protein or peptide's sorbent-interacting
groups with soluble heteroaromatics in preference over the
sorbent's residues. Excess displacement reagent must, however, be
removed later from the eluate in order to isolate the target
protein or peptide in pure form.
[0357] Another eluent change, after the target protein or peptide
has become completely released from the sorbent, can similarly be
useful in terms of economics in order to accelerate a
chromatographic run at the expense of chromatographic resolution,
or if other valuable products are eluted behind.
[0358] In a second variation, the method is augmented by the
optional final step of: [0359] (iv) washing and/or regenerating the
sorbent with a fourth and/or fifth liquid; which is introduced
after step (iii).
[0360] Here, as a fourth (cleaning) liquid a liquid is used which
will mostly have very high elution strength, may contain additives
of the above-mentioned kind, and elute unspecifically. If the
sorbent is used in the form of a chromatographic column, the fourth
liquid may be applied at high volumetric flow rates in the normal
or reverse direction since its task is to clean the sorbent and
permanently remove any build-up of residual, strongly adsorbing or
otherwise interfering chemical or biological impurities, especially
particulate matter, in order to prevent gradual fouling, clogging,
or capacity reduction of the column. For medical hygiene and
safety, typical sanitisation or sterilisation protocols (e.g.,
alkaline (1.0 M sodium hydroxide), acidic (0.4 M acetic acid),
oxidative (hypochlorite) and/or heat treatment) to eliminate
microbial contamination can also be applied to the sorbent at this
point.
[0361] The fifth (reconditioning) liquid is used to condition the
sorbent, its degree of swelling, and the solvation of its attached
residues after prior treatment with aggressive or strongly
solvating liquids such that the original state of the sorbent is
restored and constant, equilibrated conditions are installed at the
beginning of each separation run. Apart from the removal of traces
of elution or cleaning liquids, counterions of ionic residues, if
present, will thereby also be replaced to their original uniform
distribution in order to maintain constant acid/base properties of
the sorbent. The fifth liquid can be identical to the first or
second liquid, and will usually be applied at the same flow rate.
It is also possible to switch from a quick and simple
wash/regeneration program after each run to a more sophisticated
procedure after every fifth, tenth etc. run, for example, depending
on the actual load of those contaminants which are critical to
reach the attempted product quality specifications.
[0362] The preferred way of carrying out the separation method is
as a medium-to-high pressure liquid chromatography technique. Due
to its operational simplicity, and by way of either of the variety
of variations cited above, the method may also be used
discontinuously in the manner of a batch purification as with the
affinity (membrane-) filtration or solid phase extraction
techniques or continuously as with the simulated moving-bed (SMB)
technique. All variations may also be combined with one
another.
[0363] The strong chemical stability as well as static and dynamic
binding capacity of the sorbent (up to ca. 0.3 I feed load or ca.
20 g protein or peptide per litre of sorbent, respectively, are
possible) allows large degrees of freedom in the independent
variability of all five liquids used in the method. Also strongly
solvating eluent systems not compatible with conventional affinity
chromatography are now accessible so that there is plenty of room
to optimise the liquids for properties such as solubilising power,
low cost, low toxicity, and low waste production. A system of
liquids compatible with the implementation of the method basically
comprises any liquid or mixture of liquids which possesses at least
weak solubilising properties for the substrate of the separation
method, i.e. in particular a protein or peptide, and preferably
also for the side products--the latter being of particular
importance for the second liquid. Since chromatographic separations
on the sorbent of the present invention will usually be carried out
under biocompatibility restrictions, buffered aqueous media are
often used as first, second, and third liquids. Organic modifiers
other than buffers or metal salts which are essential to preserve
the protein function (e.g. detergents, chaotropic additives,
antioxidants, antifoams) could hypothetically also be added to the
liquids, but in order to retain the highest possible biological
activitiy of the protein or peptide to be purified, these reagents
are best be avoided completely. Small amounts of volatile organic
acids may be added though prior or subsequent to the actual
separation process for reasons of enhancing the detectability of
certain analytes.
[0364] If further additives are being used nevertheless, they
usually have to be removed later on, i.e. after completion of the
method, from the liquid containing the target protein or peptide
obtained in step (iii), especially if it is required to obtain said
protein or peptide in crystalline form. To achieve this purpose, a
broad range of such potentially additional steps is well known to
those skilled in the art. In order to remove additives, the method
of the invention may therefore as well be combined subsequently
with any other type of common separation processes.
[0365] Though it would be feasible to apply virtually any organic
or aqueous liquid or liquid mixture including supercritical fluids
to the sorbent of the present invention, preference is given to
those polar liquids which facilitate swelling of a polymer film, if
present on the surface of the solid support material. The exact
polarity of a liquid mixture can be easily fine-tuned by way of its
composition.
[0366] Since the adsorbed target protein or peptide (and also side
products) are often not released instantaneously (like an on-off
state) in step (iii) of the method but rather slowly and gradually,
step (iii) itself can favourably be carried out stepwise, i.e. as a
fractionation, for an increased resolution of the overall
separation process. Two or more fixed-volume fractions of either
the same or different size are then collected manually or
automatically of the third liquid after the sorbent has been
contacted with it for a sufficient time. Then step (iii) is
repeated and the sorbent is again contacted with fresh third liquid
(of a modified composition, if necessary) until all bound target
protein or peptide has been released. A continuous supply of the
third liquid is also realisable during the collection of fractions.
Purity and recovery of the released target protein or peptide in
each fraction is subsequently determined, and only those fractions
which meet the pre-set acceptance specifications in terms of
quality and/or economy are further processed while all other
fractions may either be discarded or recycled into the
feedstock.
[0367] Frontal as well as zonal elution techniques can be employed.
The best performance and productivity are often achieved with
gradient elution, especially with increasing content of polar
organic solvents (lower alcohols, acetonitrile, acetone) to the
second and/or third liquids. However, if used in process
chromatography or within a manufacturing environment in general,
isocratic elution or simple gradient shapes such as step gradients
might be preferred for operational simplicity and technical
robustness. pH and salt gradients can also be successfully
implemented. Depending on the particular residues of the sorbent,
pH values in the range between 1 and 14 for short durations, and
between 2 and 13 for continuous operation, are possible, as far as
the chemical stability of the sorbent is concerned. The respective
optimum liquid compositions will also depend on the actual degree
of derivatisation of the sorbent and has to be determined
experimentally from case to case.
[0368] What makes the method clearly distinguishable from
conventional ion exchange sorption in that it can also and
particularly be applied to separation tasks in which the protein or
peptide does not contain any net ionic charge, i.e. if the pH of
the solubilising medium resembles closely its isoelectric point.
Although anionic charges may add to the binding strength toward the
sorbent of the present invention due to its potential content of
protonable nitrogen-containing residues, their presence is not
obligatory for a successful completion of the method. The same
holds true for the side products and other components of the
mixture. The extent to which charged interactions are able to
affect the sorption or separation of a compound on a sorbent is
also determined by the dipolar character and salt concentration of
the surrounding medium. What has been explained above for opposite
charges of sorbent and analytes is also true for charges of the
same prefix which may lead in some cases to repellence and
exclusion from the sorbent instead of an additional attraction.
[0369] The method may also additionally comprise the isolation of
the protein or peptide, subsequent to step (iii), from at least one
fraction of the third liquid into which it has there been released.
In preparative applications, it is possible to isolate the protein
or peptide in concentrated or even neat form from a solution in the
third liquid for the purpose of characterisation and/or subsequent
treatment. In the easiest way, it can be recovered from the liquid
of step (iii) by gentle methods of solvent evaporation (including
freeze drying, lyophilisation). Solvent evaporation would, however,
also enrich possibly contained substances of low vapour pressure
stemming from the third liquid. Such substances may comprise
additives such as buffer salts or stabilising agents, or
contaminants such as higher boiling solvent homologues and/or
degradation products which are usually contained in trace amounts
in solvents of commercially available qualities. Due to the high
physical and chemical stability of the sorbent, however,
practically no leaching from the stationary phase will occur during
steps (ii)-(iv), so that the released protein or peptide of step
(iii) will typically contain less than 10 ppm of leached sorbent or
other leachable substances therefrom (i.e. its constituents
(polymer, residues), or decomposition products).
[0370] A preferable method of isolation consists of a
crystallisation step of the third liquid containing the purified
protein or peptide or said evaporated residue, if necessary, after
re-dissolution. During such a crystallisation step, which may for
example be induced by changing the temperature and/or the
composition of the liquid, even higher degrees of purification can
be achieved since contaminants of low vapour pressure are usually
kept in solution and are thus easily separated from the targeted
product crystals. After drying, the crystals are often ready for
use in compounding and formulation processes. If dry storage is
unwanted or impossible, it may alternatively be necessary to
perform a transfer of the purified product into a solution of
differing composition, i.e. the third liquid would be exchanged
against a storage liquid by standard operations like dialysis, ion
exchange etc.
[0371] As usual in chromatography, the method and an associated
apparatus on which it is run may also favourably be supplemented by
a suitable detection technique which allows for qualitative,
semi-quantitative or quantitative measurement of the concentration
of the target protein or peptide and/or side products or other
components of the mixture in the eluate for sharp and fine
fractionation. Preferred detection methods involve on-line
flow-cell detectors of physical or spectroscopic properties such as
refractometers, polarimeters, conductometers, ultraviolet/visible
absorbance or fluorescence spectrometers, infrared spectrometers,
mass spectrometers, and nuclear magnetic resonance spectrometers.
An online pre- or post-column derivatisation or degradation unit
may also be added to the system in order to convert all or specific
components of the mixture to be separated into derivatives or
fragments with improved detectability, or to accelerate or delay
their elution. A universal non-destructive detection method for
proteins or peptides is UV absorbance at a wavelength of 280
nm.
[0372] On the large scale, the sorbent and thus also the separation
method of the present invention employing the sorbent can
beneficially be used in the manufacture of a pharmaceutical or
nutritional composition for human or veterinary use (e.g. an
antiserum or vaccine), if such composition comprises at least one
protein or peptide of diagnostic, therapeutic, or nutritional value
which can be bound by the sorbent. The benefit of the present
invention mainly arises from the fact that such applications often
require purities of the valuable active ingredient in the range of
>99% or even>99.9% which are realisable by conventional
methods only under lengthy and costly procedures, which may even
render some applications prohibitive from an economic
viewpoint.
[0373] On the small scale, they can alternatively be used in the
identification, characterisation, quantification, or laboratory
purification of the at least one protein or peptide. For this
purpose, which is related to qualitative and quantitative analysis,
the separation method is likely to be complemented by a specific
biological assay or by a spectroscopic method, e.g. using
hyphenated techniques, but can also be accomplished by comparison
of retention volumes with pure, authentic samples or peptide
standards. In microscale formats, they may be interesting for
proteomic applications, i.e. the simultaneous identification or
quantification of the expression levels and modifications of a
plurality of different proteins in a cell or in an organism.
[0374] As part of a medical device, they can also be used in the
removal of at least one protein or peptide from a biological fluid,
which includes the medical prevention or treatment of diseases
being caused by the presence of said at least one protein or
peptide in said biological fluid. The device may be applied as a
kind of detoxification or decontamination unit in all cases in
which a patient has already taken up or is about to take up harmful
or infectious proteins or peptides, as they are for example
secreted by pathogens, but also in those cases in which the body of
the patient itself has produced such harmful or infectious
proteins, as it is often the case in autoimmune diseases. Potential
sources of uptake include food, water, air, contact with infected
persons, blood transfusions etc. In a specialised application, the
medical device may be constructed as an apheresis or plasmapheresis
unit. Such a device will predominantly be operated ex-vivo or
in-vitro, but construction as a miniaturised, implantable device
also appears to be within imagination. A biological fluid of the
patient could (either continuously or batch-wise) be taken from the
patient, depleted from the contaminant via treatment with the
sorbent, and then returned to the patient. Biological fluids from
external sources (other humans, animals) could also be treated with
the sorbent to reduce the risk of transmission of infectious
diseases before the fluids or parts thereof or compositions
manufactured therefrom are administered to a patient in need
thereof. In such case, the separation method of the invention would
be used to diminish the concentration/purity of the target protein
or peptide in the <value> fraction (thereby increasing the
purity of the proteins or peptides of value therein), whereas it
would be enriched in the <waste> fraction.
[0375] Finally, they can be used for the immobilisation of at least
one protein or peptide on the sorbent. Due to the non-covalent
nature of the interactions between the sorbent and the targeted
protein or peptide, such immobilisation will be reversible. This
may be a potential advantage in applications such as the
preparation of filterable reagents or catalysts, the surface-bound
culture of cells, in drug delivery devices (e.g. drug eluting or
healing stents), or in drug discovery screenings. In the latter
case, the separation method of the invention can be complemented by
a method of testing for binding of further chemical or biological
structures to the immobilised protein or peptide. The detection of
such secondary binding can then serve as a first indication of a
possible physiological effect of either binding partner. If a
polymer coating is used, the immobilised protein or peptide may
become physically entrapped by the surrounding gel-forming medium
and will thus additionally experience an environment of high
biocompatibility. Expressed differently, a non-covalent, isolable
complex formed between a sorbent as described herein and at least
one protein or peptide is thus also embodied within the present
invention. Such a complex containing an antibody as the preferred
protein of the invention may be used in immunosorption
techniques.
[0376] A further object of the present invention which can
immediately be derived from the explanations given above is a
pre-packed column, comprising a sorbent of the present invention
within a tubular containment. Such a column can be used as
stationary phase of a fixed, desired size (length.times.diameter)
in liquid chromatography or solid phase extraction applications.
Beside the tubular containment, such a column can optionally
comprise further components such as frits, filter plates, flow
distributors, seals, fittings, screwings, valves, or other fluid
handling or connection elements, which are known from the state of
the art. The sorbent may be packed either as a slurry under
gravitational or centrifugal force, under externally applied
hydrodynamic pressure, or under additional axial compression by a
piston into the column, and made commercially available in such a
pre-packed format. For the added convenience of the user, a more
reproducible packing can thus be assured and stationary phases can
easily be stored if not in use and quickly be exchanged within a
chromatographic system. The material the containment is made of
(chemically and biologically inert materials such as stainless
steel, borosilicate glass, plastics like PEEK etc.) is typically
chosen such that the high stability of the sorbent itself is not
sacrificed, which means that the entire column should ideally be
characterised by a physical and chemical resistance against applied
pressures up to 20 bar or against applied heat up to 110.degree. C.
as well as against common sanitisation protocols including
autoclavability. Under favourable circumstances, this will enable a
repetitive use of the column of up to 1,000 times, preferably up to
5,000 times, and add to overall process economy. However, it can
also be a disposable or incinerable unit. Another option is to
design only the immediate tubular housing of the sorbent cheap and
disposable and to place it inside a second, outer housing made of
long-lived and durable materials which also contains all re-usable,
supplementary components (cartridge design).
[0377] A column can be part of a full chromatography system. Apart
from the detection system described above, other pertinent
components of a chromatography system include pumps, flow
regulators, liquid reservoirs, degassers, injection ports, column
switching valves, pressure and flow meters, temperature-controlled
chambers, outlet collection trays (carousels), and robotic
fractionators.
[0378] A further object of the present invention is a collection
(or "library") of a plurality of the same or different sorbents of
the present invention either as loose materials (of granular or
block (monolithic) design) or as pre-packed columns, cartridges
(see above), or membranes, whereby the individual sorbents may be
the same or different. A collection of different sorbents may for
example be used in an initial screening campaign for suitable
sorbents that are planned to be used in a more sophisticated
preparative chromatographic setup afterwards, whereas a collection
of the same sorbents may for example be used in multiple medical
diagnostic tests of large numbers of samples having similar
matrixes, or in quasi-continuous process monitoring. The advantage
of such a collection is its ability to be processed in parallel,
either in a manual or in a automated fashion. Such parallel
processing allows--beside time savings due to higher sample
throughput as compared to serial processing--to compare different
sorbents or other process parameters also under standardised or at
least identical (reproducible) conditions. This advantage can
especially be exploited if the individual members of the collection
are arranged in a standardised and positionally addressable format,
preferably a two-dimensional rectangular grid compatible with
robotic workstations, such as a microplate array or a microchip
array, or as a multi-capillary or microfluidic device. As far as
the readout of miniaturised formats is regarded, reference is made
again to proteomics technologies.
[0379] All intermediate products beginning with the
crosslinkage/grafting step of the preparation methods described
above are sufficiently stable to be stored for future usage. Such
product can then be split into several subsets upon which the
derivatisation step is performed with individual derivatisation
reagents. In such way a library of different sorbents (i.e.
sorbents derivatised with different residues or combinations
thereof or at different residue ratios or different degrees of
derivatisation) can be formed on demand. If the derivatisation step
is carried out in parallel on the entirety of subsets, it is
feasible to form such a library in a very short time in order to
perform an initial screening search of the best sorbent for a given
application which would allow to respond rapidly to changing
separation objects. Apart from different derivatisations, different
solid support materials, including the possibility of different
polymer films, carriers and/or activation chemistries, may also be
applied in the formation of the sorbent library.
[0380] Random or targeted library screening is a means which may
sometimes complement or even replace rational sorbent design. It is
used especially in those cases where the relative importance of
contributions from different residues on the sorbent and/or their
counterparts on the target protein or peptide are non-obvious, if
structural information is scarce, or if additional tight
boundaries, e.g. concerning the choice of compatible liquid phases,
apply. The screening of such a library toward a given separation
object can be carried out in such a way that one or more parameters
that characterise the performance of a particular sorbent
(affinity, selectivity, capacity, recovery, stability etc.) are
measured either consecutively or in parallel with the full library
or one or more subsets thereof. The most prominent characteristics
are affinity- and selectivity-related thermodynamic and kinetic
parameters regarding the formation of complexes between the sorbent
and protein or peptide targets. A pre-selection of sorbents
suitable for incorporation into the library could be performed with
computational methods.
[0381] A viable screening method would for example consist of
treating a mixture containing at least one protein or peptide as
well as side products and/or other components with the respective
sorbents of the present invention under suitable batch conditions
and measure the individual equilibrium Gibbs enthalpies of complex
formation between the sorbents and the targeted protein or peptide.
An alternative method would consist of measuring the differential
Gibbs enthalpies between the formation of complexes of the sorbent
with the targeted protein or peptide on one hand and those with
appropriately chosen side products on the other hand. Measurements
can directly be carried out with the help of all thermodynamic
and/or kinetic methods known to the person skilled in the art such
as, e.g., calorimetry. Measurements can also be made indirectly
with the help of chromatographic runs under the process-like
conditions of the envisaged application on the transient formation
of such complexes, whereby the obtained results may need to be
corrected for eluent contributions. In a chromatographic
environment, k' and .alpha. values may serve at first approximation
as indicators of the Gibbs enthalpy or differential Gibbs enthalpy,
respectively.
[0382] A further object of the present invention is a diagnostic or
laboratory purification kit. which comprises beside a sorbent of
the invention (or a collection of sorbents, or a column containing
the sorbent), within the same packaging unit, a set of further (or
even all) chemical or biological reagents and/or disposables
necessary for carrying out the separation method of the invention
or a different analytical, diagnostic, or laboratory method in
which said sorbent can be employed. Such a pre-packed collection of
materials in the right number, amount, or concentration is intended
to increase the convenience of the user if standardised
experimental protocols have to be followed when the separation
method is carried out, and especially if the sorbent or column is
used as a disposable device. Said protocol can be incorporated
together with safety data sheets etc. into the directions for use
which can optionally accompany the kit.
FIGURE CAPTIONS
[0383] FIG. 1: Different individual configurations A-H and one
general representation I resulting from derivatisation of two
adjacent surface functional groups (FG) with one first and one
second residue.
[0384] FIG. 2: Different schematic morphologies A-C of a solid
support material consisting of a carrier the surface of which is
covered with a film of a polymer (here exemplified for a
non-porous, particulate carrier depicted as a grey sphere; not
drawn to scale).
[0385] FIG. 3: Choice of possible first residues comprising a
binuclear C, N, O, S-heteroaromatic structure composed of fused 5-
and 6-membered rings and one or two heteroatoms contained
therein.
[0386] FIG. 4: Choice of possible second residues comprising a
mononuclear C, N, O, S-heteroaromatic structure composed of 5- or
6-membered rings and one or two heteroatoms contained therein.
[0387] FIG. 5: General representation of exemplary first and second
residues composed of 5- and 6-membered rings. R.sup.1 . . .
R.sup.8=electron pair, H, organic radical or surface linkage;
X.sup.1 . . . X.sup.6=C or N; Y.sup.1 . . . Y.sup.2.dbd.N, O,
S.
[0388] FIG. 6: Symbolic representation (not drawn to scale) of
terms used to characterise the analyte-interacting surface of the
sorbent. Not all items depicted are necessary to carry out the
invention.
[0389] FIG. 7: Structures of first and second residues used in this
study (functional groups provided by a surface polymer film are
printed in bold).
[0390] FIG. 8: Stepwise pH-elution of immunoglobulin G from sorbent
no. ND 10003 for the loading capacity determination of Example
4.
[0391] FIG. 9: Analytical chromatograms of a protein test mixture
containing catalase and of its individual components on sorbent no.
ND 08037 of Example 5.
[0392] FIG. 10: Analytical chromatograms of a protein test mixture
containing catalase and of its individual components on sorbent no.
ND 070002 of Example 5.
[0393] FIG. 11: Analytical chromatograms of a protein test mixture
containing catalase and of its individual components on sorbent no.
ND 06380 of Example 5.
[0394] FIG. 12: Analytical chromatography of a protein test mixture
containing conalbumin and of its individual components under the
influence of high salt concentrations on sorbent no. ND 08236 of
Example 6.
[0395] FIG. 13: Analytical chromatography of a protein test mixture
containing conalbumin and of its individual components under the
influence of high salt concentrations on sorbent no. ND 07200 of
Example 6.
[0396] FIG. 14: Analytical chromatography of a protein test mixture
containing conalbumin and of its individual components under the
influence of high salt concentrations on sorbent no. ND 07122 of
Example 6.
[0397] FIG. 15: Analytical chromatography of a protein test mixture
containing conalbumin and of its individual components under the
influence of high salt concentrations on sorbent no. ND 06386 of
Example 6.
[0398] FIG. 16: UV-Chromatograms of the semi-preparative isolation
of conalbumin from a mixture of further proteins on sorbents no. ND
07200 (a) and PRC 10014 (b) according to Example 7 (the dotted
lines symbolise the course of the applied pH gradients; sections
I-VII: time windows of collected fractions).
EXAMPLES
General
[0399] HPLC systems from Dionex (formerly Gynkotek) consist of a
four channel low-pressure gradient pump (LPG 580, LPG 680 or LPG
3400), auto sampler (Gina 50, ASI-100 or WPS-300), six-channel
column switching valves (Besta), column oven and a diode-array UV
detector (UVD 170U, UVD 340S or VWD 3400).
[0400] All sorbents employed were based on the same porous
spherical carrier of sulphonated polystyrene-divinylbenzene
copolymer (35 .mu.m mean particle diameter, 1000 .ANG. mean pore
diameter) covered with a film of crosslinked polyvinylamine which
is derivatised in a randomly distributed fashion with residues as
given in the respective experimental description. The binuclear and
mononuclear heteroaraomatic structures employed as first and second
residues are shown in the formula of FIG. 7. Both five- and
six-membered heterocyclic rings containing one or two nitrogen
atoms are represented therein. For all chromatographic experiments
the sorbents were used in standard stainless steel HPLC columns of
33.5.times.4 mm actual bed size, if not stated otherwise. Columns
were packed by flow sedimentation of water-methanol (1:1)
suspensions under a pressure of 20 bar.
[0401] The following Table 1 lists the proteins that were employed
in the various experiments. The given choice spans a broad variety
ranging from medium-sized peptides to large multimeric protein
complexes, with different functions such as catalysis, transport,
immune response, and signalling/regulation being represented, too.
Isoelectric point data were taken from the literature and were
reproduced by isoelectric focussing. Protein purities were
additionally checked by gel permeation chromatography. All other
reagents used were of standard laboratory grade quality.
TABLE-US-00001 TABLE 1 List of poteins used in this study Molecular
Isoelectric Protein Source Distributor CAS No. Weight Point Alcohol
Bakers yeast Sigma 9031-72-5 141 kDa 5.4-5.8 Dehydrogenase Catalase
Bovine liver Sigma 9001-05-2 250 kDa 5.4 Catalase Murine liver
Sigma 9001-05-2 250 kDa 6.7 .alpha.-Chymo- Bovine pancreas Sigma
9035-75-0 26 kDa 9.5 trypsinogen A Conalbumin Chicken egg white
Sigma 1391-06-6 77 kDa 6.7 Cytochrome C Bovine heart Sigma
9007-43-6 12 kDa 10.5 Hemoglobin Human Sigma 9008-02-0 64 kDa 7.4
Hexokinase Bakers yeast Sigma 9001-51-8 54 kDa 4.5-5.0
Immunoglobulin G Human plasma Octapharma 89957-37-9 144 kDa 6.4
(Gammanorm .RTM.) Insulin Recombinant Bioton 9004-10-8 6 kDa 5.3
human Lysozyme Chicken egg white Fluka 12650-88-3 14 kDa 8.9 Pepsin
Porcine gastric Sigma 9001-75-6 36 kDa 2.9 mucosa Proteinase K
Tritirachium album Sigma 39450-01-6 29 kDa 8.9 Pyruvate Kinase
Rabbit muscle Sigma 9001-59-6 237 kDa 7.6
Example 1
Synthesis of Specific Sorbents Having Indole and Imidazole
Containing Residues in Different Ratios
[0402] Commercial polystyrene-divinylbenzene copolymer spherical
resin beads (Rohm & Haas Company: Amberchrom.TM.) were first
excessively sulphonated in concentrated sulphuric acid, then
commercial polyvinylamine-polyvinylformamide copolymer solution
(BASF: Lupamin.RTM.) was adsorbed onto the porous beads and lightly
chemically crosslinked with a bis-epoxide. To this underivatised
intermediate, which contained ca. 0.35-0.45 mmol/ml free amino
groups and was pre-swollen in dimethyl formamide, in situ-activated
3-indolylpropionic acid and 4-imidazolylacrylic acid were
successively and independently coupled to the amino groups via a
standard solid phase amide coupling protocol in a slight excess
over the predetermined amount corresponding to the targeted degrees
of derivatisation. The sorbents were washed free of excessive
reagents and dried until constant weight was achieved. Degrees of
derivatisation were determined after each derivatisation step via
solid phase titration with aqueous toluenesulphonic acid as
differences of the proton binding capacities of the residual amino
groups. According to this general procedure, the sorbents listed in
Table 2 were prepared.
TABLE-US-00002 TABLE 2 Compositions of selected exemplary sorbents
prepared (accuracy ca. .+-. 2%; if acetyl is present as third
residue, the difference between the combined degrees of
substitution and 100% usually equals the content of acetyl groups;
if no third ligand is present, the difference between the combined
degrees of substitution and 100% equals the content of residual
amino groups) 3-Indolylpropionyl 4-Imidazolylacryl [%
Derivatisation] [% Derivatisation] 3rd Residue (optional) 12 21 --
14 9 Acetyl 14 31 Acetyl, Succinic Amide (12%), N-Imidazolinone-
ethyloxycarbonyl (15%) 18 32 -- 20 6 Acetyl 20 24 Acetyl, Succinic
Amide (26%), Succinic Amide (26%) + Acetyl, Succinic Amide (26%) +
Succinic Acid 21 17 Acetyl 22 13 Acetyl 22 30 -- 23 37 -- 24 25
Acetyl, Succinic Acid (6%, 23%) 25 30 -- 26 19 Acetyl, Succinic
Amide (21%), N-Imidazolinone- ethyloxycarbonyl (10%, 19%),
2-Hydroxyacetyl (8%, 12%) 27 14 Acetyl 28 27 Acetyl 28 37 -- 29 33
Acetyl 38 24 Acetyl 38 60 Acetyl 43 24 -- 43 27 Acetyl 43 32 Acetyl
47 11 -- 55 19 Acetyl
Example 2
Screening of a Collection of Sorbents of the Invention for their
Binding Properties Towards Different Proteins
[0403] Experimental:
[0404] Solutions of the pure proteins conalbumin, hemoglobin,
catalase (from murine liver) and pyruvate kinase, all having
isoelectric points between 6 and 8, in 25 mM phosphate buffer pH
7.4 were prepared in concentrations of ca. 15-18 mg/ml (pyruvate
kinase: 5 mg/ml). Analytical amounts of 30 .mu.l (hemoglobin: 125
.mu.l) of these solutions were independently injected onto
different HPLC columns containing the sorbents which had been
derivatised with one or more individual residues comprising
heteroaromatic structures in the respective degrees as listed in
the tables below. A step gradient was employed starting with a 25
mM phosphate buffer pH 7.4 binding step for 40 min at 0.25 mL/min,
followed by a 50 mM acetate buffer pH 3.5 elution step for 20 min
at 0.5 ml/min and a final acidic purge with 1 M aqueous acetic acid
(pH 2.25) for another 20 min at the same flow rate. Reconditioning
with the binding buffer for 20 min at a flow of 1 mL/min restored
the original conditions of the sorbent for the following run.
Yields of eluted protein were determined independently for each
elution step via integration of the UV absorbance signal at 230 nm,
280 nm, or 500 nm wavelength. Total protein recoveries were
determined through quantitative comparison with additional bypass
runs (no column installed).
[0405] Results:
[0406] The screening results are listed in Tables 3-6 (the sum of
the absolute yields of all three elution steps thereby equals the
total recovery) which allow the direct comparison of different
first and second residues as well as the effect of either missing
residue. While derivatisation with second residues only usually
resulted in a diminished binding capacity of the sorbent (higher
breakthrough), derivatisation with first residues only resulted in
binding which often proved to be too strong for practical
(chromatographic) purposes. Reversible and reproducible binding can
only be assured if the target protein is recovered during a
moderately acidic buffered elution step (here: pH 3.5). If, on the
other hand, the target protein can only be released from the
sorbent in a strongly acidic purge step, it may suffer from
denaturation, thus making the sorbent useless for preparative
purposes. Irreversible binding was sometimes also indicated by low
total recoveries which may imply that even the aqueous acid used in
the purge step does not have sufficient eluotropic strength to
release the full amount of protein from the sorbent. As a general
trend, the maximum overall performance was thus achieved only if
both ligands were present on the sorbent. The optimum composition
of the sorbent, however, was also to a certain extent dependent on
the target protein. Depending on the individual synthetic
capabilities, degrees of derivatisation with both first and second
residues could be varied over a wide range. Moreover, a measurable
performance response could be observed even upon minor variations
of the degrees of derivatisation if the kind of residues remained
unchanged. No reversible retention was observed on the reference
sorbents included in the study which contained neither a first nor
a second residue. Derivatisation with acetyl or succinic acid
groups as third ligands delivered rather adverse properties. Under
the same conditions as applied here, basic proteins such as
proteinase K, cytochrome C, or lysozyme as well as acidic proteins
such as pepsin or insulin were shown to break through at pH 7.4 in
a high percentage on the majority of stationary phases or to
(partially) elute at pH 3.5. An exception was given by the protein
hexokinase which was found in all three fractions including the
acid purge step. Total recoveries were often unacceptably low with
these proteins (data not shown). A parallel experiment performed
with hemoglobin under the addition of 150 mM sodium chloride to
both binding and elution buffers did not alter the raw data
significantly. An additional high salt elution step (1 M NaCl) at
the binding pH 7.4 prior to lowering the pH of the eluent led to
further elution (12%) only from the sorbent derivatised with 30%
residues comprising an indole structure (i.e., lacking the second
residue).
TABLE-US-00003 TABLE 3 Phase screening with the target protein
conalbumin Second Third Yield Yield Yield First Residue Residue
Residue Breakthrough Elution Purge Total Structure Structure
Structure pH 7.4 pH 3.5 pH 2.25 Recovery Benzimidazole Imidazole --
0% 100% 0% 63% (13%) (10%) Indole Imidazole -- <1% 94% 6% 98%
(ca. 15%) (ca. 25%) Indole Imidazole -- 0% 86% 14% 84% (25%) (20%)
Indole Imidazole -- 0% 64% 36% 91% (26%) (26%) Quinoline Imidazole
-- 2% 84% 14% 97% (ca. 40%) (ca. 30%) Benzimidazole Imidazole -- 0%
100% 0% 59% (ca. 50%) (ca. 30%) Indole Imidazole Acetyl 52% 43% 5%
88% (25%) (21%) (54%) Indole Imidazole Succinic 87% 0% 13% 81%
(26%) (24%) Acid (ca. 50%) Indole -- -- 0% 100% 0% 81% (ca. 25%)
Indole -- -- 0% 17% 83% 86% (30%) Quinoline -- -- 0% 100% 0% 83%
(ca. 40%) Indole -- -- 1% 13% 86% 69% (ca. 50%) Benzimidazole -- --
0% 100% 0% 72% (ca. 50%) Benzimidazole -- -- <1% 92% 8% 97% (ca.
50%) -- Pyridine -- 58% 42% 0% 61% (24%) -- Imidazole -- 29% 71% 0%
89% (30%) -- Imidazole -- 0% 100% 0% 59% (62%) -- Pyridine Acetyl
91% 9% 0% 103% (ca. 20%) + (ca. 50%) Imidazole (ca. 30%) -- -- --
42% 5% 53% 75% -- -- -- 36% 0% 64% 91% -- -- Acetyl 100% 0% 0% 100%
(ca. 100%) -- -- Succinic 100% 0% 0% 95% Acid (ca. 100%)
TABLE-US-00004 TABLE 4 Phase screening with the target protein
hemoglobin Second Third Yield Yield Yield First Residue Residue
Residue Breakthrough Elution Purge Total Structure Structure
Structure pH 7.4 pH 3.5 pH 2.25 Recovery Benzimidazole Imidazole --
0% 100% 0% 105% (13%) (10%) Indole Imidazole -- 4% 95% 1% 88% (ca.
15%) (ca. 25%) Quinoline Imidazole -- 57% 43% 0% 92% (19%) (30%)
Indole Imidazole -- 3% 97% 2% 89% (25%) (20%) Indole Imidazole --
9% 89% 0% 59% (26%) (26%) Benzimidazole Imidazole -- 0% 100% <1%
91% (ca. 50%) (ca. 30%) Indole Imidazole Acetyl 75% <1% 25% 93%
(25%) (21%) (54%) Indole Imidazole Succinic 60% 40% 0% 77% (26%)
(24%) Acid (ca. 50%) Indole -- -- 0% 96% 4% 66% (ca. 25%) Indole --
-- 0% 96% 4% 73% (30%) Quinoline -- -- 58% 41% 1% 67% (ca. 40%)
Indole -- -- 3% 90% 7% 56% (ca. 50%) Benzimidazole -- -- <1% 99%
1% 88% (ca. 50%) Benzimidazole -- -- 70% 30% 0% 74% (ca. 50%)
Quinoline -- Acetyl 100% 0% 0% 82% (ca. 40%) (ca. 60%) -- Imidazole
-- 84% 16% 0% 83% (30%) -- Imidazole -- 81% 19% 0% 79% (62%) -- --
-- 89% 10% 2% 91% -- -- -- 85% 2% 13% 78% -- -- Acetyl 100% 0% 0%
91% (ca. 100%)
TABLE-US-00005 TABLE 5 Phase screening with the target protein
catalase from murine liver (n.d. = not determined) Second Third
Yield Yield Yield First Residue Residue Residue Breakthrough
Elution Purge Total Structure Structure Structure pH 7.4 pH 3.5 pH
2.25 Recovery Benzimidazole Imidazole -- 2% 98% n.d. 86% (13%)
(10%) Quinoline Imidazole -- 3% 97% n.d. 85% (19%) (30%) Indole
Imidazole -- 4% 96% n.d. 45% (25%) (20%) Indole Imidazole -- 4% 75%
n.d. 45% (26%) (26%) Benzimidazole Imidazole -- 2% 98% n.d. 82%
(ca. 50%) (ca. 30%) Indole Imidazole Acetyl 6% 94% n.d. 90% (25%)
(21%) (54%) Indole Imidazole Succinic 72% 28% n.d. 86% (26%) (24%)
Acid (ca. 50%) Indole -- -- 29% 71% n.d. 25% (ca. 25%) Indole -- --
27% 73% n.d. 27% (30%) -- -- -- 63% 37% n.d. 49% -- -- Acetyl 100%
0% n.d. 84% (ca. 100%)
TABLE-US-00006 TABLE 6 Phase screening with the target protein
pyruvate kinase (n.d. = not determined) Second Third Yield Yield
Yield First Residue Residue Residue Breakthrough Elution Purge
Total Structure Structure Structure pH 7.4 pH 3.5 pH 2.25 Recovery
Benzimidazole Imidazole -- <1% 100% n.d. 55% (13%) (10%) Indole
Imidazole -- 0% 100% 0% 61% (ca. 15%) (ca. 25%) Quinoline Imidazole
-- 0% 100% n.d. 55% (19%) (30%) Indole Imidazole -- 0% 100% n.d.
31% (25%) (20%) Indole Imidazole -- 53% 47% 0% 13% (ca. 25%) (ca.
25%) Indole Imidazole -- 0% 0% n.d. 0% (26%) (26%) Benzimidazole
Imidazole -- 0% 100% n.d. 44% (ca. 50%) (ca. 30%) Indole Imidazole
Acetyl 31% 69% n.d. 87% (25%) (21%) (54%) Indole Imidazole Succinic
100% 0% n.d. 58% (26%) (24%) Acid (ca. 50%) Indole -- -- 0% 0% 0%
0% (ca. 25%) Indole -- -- 0% 0% n.d. 0% (30%) Quinoline -- -- 0%
100% 0% 56% (ca. 40%) Benzimidazole -- -- 0% 0% 0% 0% (ca. 50%)
Benzimidazole -- -- 0% 100% 0% 45% (ca. 50%) Quinoline -- Acetyl
100% 0% 0% 93% (ca. 40%) (ca. 60%) -- -- -- 100% 0% n.d. 36% -- --
Acetyl 100% 0% n.d. 71% (ca. 100%)
Example 3
Determination of Loading Capacities of Sorbents Having Indole and
Imidazole Containing Residues for Neutral and Weakly Acidic
Proteins
[0407] Experimental:
[0408] The three proteins hemoglobin, conalbumin, and catalase
(from bovine liver) were each dissolved at a concentration of 32
mg/ml in 25 mM phosphate buffer pH 7.4. Increasing volumes (15 . .
. 250 .mu.l) of these stock solutions corresponding to absolute
amounts of 0.5 . . . 8 mg protein, or loadings of 1.2 . . . 19.0 mg
protein per ml stationary phase (equals 0.2 . . . 3.6%
weight/weight), respectively, were injected stepwise onto HPLC
columns containing the sorbents which had been derivatised to
varying degrees with 3-indolylpropionyl and 4-imidazolylacryl
residues as shown below.
[0409] ND 08240: 25% 3-indolylpropionyl+20% 4-imidazolylacryl
residues
[0410] ND 08236: 14% 3-indolylpropionyl+29% 4-imidazolylacryl
residues
[0411] ND 08037: 26% 3-indolylpropionyl+26% 4-imidazolylacryl
residues
[0412] A step gradient was employed starting with a 25 mM phosphate
buffer pH 7.4 binding step for 40 min at 0.25 mL/min, followed by a
50 mM acetate buffer pH 3.5 elution step for 20 min at 0.5 ml/min
and a final acidic purge with 1 M aqueous acetic acid (pH 2.25) for
another 20 min at the same flow rate. Yields of eluted protein were
determined independently for each step via integration of the UV
absorbance signal at 280 nm or 450 nm wavelength. The total protein
recoveries were checked through quantitative comparison with
additional bypass runs (no column installed).
[0413] Results:
[0414] The elution behaviours of the three proteins at increased
loadings on the different sorbents are being summarised in Table 7.
ND 08240: Breakthrough at pH 7.4 ranged from 4.3% to 52% over the
measured range of 4.8-19.0 mg/ml load for the neutral protein
hemoglobin. The maximum loading capacity was determined to be 4.8
mg/ml. Higher loading led to a rapid rise of the breakthrough
portion. The remainder of the protein was eluted at pH 3.5 over the
entire range of loading. ND 08236: Breakthrough at pH 7.4 ranged
from 0.32% to 47% over the measured range of 1.2-19.0 mg/ml load
for the neutral protein conalbumin. The maximum loading capacity
was determined to be 7.13 mg/ml. Higher loading led to a rapid rise
of the breakthrough portion. The remainder of the protein was
eluted at pH 3.5 over the entire range of loading. ND 08037:
Breakthrough at pH 7.4 ranged from 2.1% to 10.4% over the measured
range of 1.2-19 mg/ml load for the weakly acidic protein catalase.
The maximum loading capacity was determined to be 11.88 mg/ml.
Higher loading led only to a slow rise of the breakthrough portion;
the remainder of the protein, however, was divided among both
acidic elution steps, with the portion at pH 2.25 decreasing from
56% at 1.19 mg/ml load to 16% at 19.0 mg/ml load.
TABLE-US-00007 TABLE 7 Yields after adsorption and stepwise
desorption for three sorbent-protein combinations at their
respective maximum loading capacities Maximum Yield Yield Yield
Sorbent Load Breakthrough Elution Purge No. Protein [mg/ml] pH 7.4
pH 3.5 pH 2.25 ND 08240 Hemoglobin 4.75 4% 92% 2% ND 08236
Conalbumin 7.13 3% 95% 2% ND 08037 Catalase 11.88 5% 82% 13%
Example 4
Determination of the Loading Capacity of Immunoglobulin G on a
Sorbent Having Indole and Imidazole Containing Residues
[0415] Experimental:
[0416] A solution of 10 mg/ml immunoglobulin G (IgG) was prepared
by diluting 303 .mu.l commercial Gammanorm.RTM. pharmaceutical
product with 100 mM phosphate buffer pH 7.4 in a 5 ml volumetric
flask. 842 .mu.l of this stock solution corresponding to an
absolute amount of 8.58 mg IgG, or a loading of 20.4 mg IgG per ml
stationary phase, were injected onto a HPLC column containing
sorbent no. ND 10003 which had previously been equilibrated with
ca. 70 column volumes of 100 mM phosphate buffer pH 7.4. This
sorbent had been derivatised with 22% 3-indolylpropionyl and 30%
4-imidazolylacryl residues. During injection, a 100 mM phosphate
binding buffer pH 7.4 was applied at a flow rate of 0.1 mL/min
which was maintained for 90 min, followed by a stepwise change to
100 mM acetate buffer pH 4.3+50 mM sodium chloride elution buffer
for 20 min at 0.5 mL/min, an acidic purge pH 2.6 with 0.5 M aqueous
acetic acid for another 20 min at the same flow, an intermediate
dilution with 100 mM phosphate buffer pH 7.4 for 10 min at 1
ml/min, and a final cleaning with 1 M aqueous sodium hydroxide
solution for 12 min at the same flow. A corresponding chromatogram
is shown in FIG. 8. Three fractions resembling the eluents of
different pH (pH 7.4, pH 4.3, pH 2.6) were collected and analysed
off-line for their IgG contents photometrically at 280 nm. For
assessment of the 100% reference value, a bypass run was performed
with the column being replaced by a piece of empty tubing but under
otherwise identical conditions.
[0417] Results:
[0418] IgG could be found in all fractions, with the majority being
reversibly bound at pH 7.4 and desorbed at pH 4.3. The yields of
IgG in the three analysable fractions were measured as 9%
(breakthrough pH 7.4), 81% (elution pH 4.3), and 12% (purge pH
2.6). Total protein recovery over all three fractions therefore
amounted to a sum of ca. 102%. The reversible loading capacity (pH
4.3 fraction) of sorbent no. ND 10003 could thus be determined as
16.5 mg IgG per ml stationary phase, the total loading capacity
(injected amount less pH 7.4 breakthrough) as 18.5 mg IgG per ml
stationary phase. Since the sorbent was repeatedly used under the
same experimental protocol, the stability of the sorbent against
cleaning/sanitisation with 1 M NaOH is also notable.
Example 5
Analytical Separation of Catalase on Different Sorbents of the
Invention
[0419] Experimental:
[0420] A mixture of the five proteins catalase (from bovine liver),
hexokinase, pepsin, cytochrome C, and .alpha.-chymotrypsinogen A
(each in an amount of 0.48 mg dissolved in 25 mM phosphate buffer
pH 7.4) was injected onto different HPLC columns containing the
sorbents which had been derivatised with first and/or second
residues as listed below.
[0421] ND 08037: 26% 3-indolylpropionyl+26% 4-imidazolylacryl
residues
[0422] ND 070002: 50% benzimidazole-5-carbonyl+30%
4-imidazolylacryl residues
[0423] ND 06380: 32% benzimidazole-5-carbonyl residues
[0424] The eluent was applied as a gradient starting from 25 mM
phosphate buffer pH 7.4 for 10 min at 0.25 ml/min, followed by a
first linear pH change to 0.1 M aqueous acetic acid (pH 2.86)
within 50 min at 0.5 ml/min and a second linear pH change to 1 M
aqueous acetic acid (pH 2.25) within an additional 50 min at the
same flow which level was finally held for 20 min. The eluate
composition was monitored at absorbance wavelengths of 225 nm, 280
nm, and 290 nm. For comparison, each single protein was injected
also as a pure solution. Chromatographic runs on the different
sorbents are depicted in FIGS. 9-11.
[0425] Results:
[0426] The chromatogram of the mixture matches the superposition of
the single protein runs. Elution of the full injected amount of the
targeted protein catalase was retarded on all three sorbents. As
expected, the acidic proteins hexokinase and pepsin were also
electrostatically bound to the sorbent but with low capacities
(5-25% breakthrough for hexokinase; 35-50% breakthrough for
pepsin). The consistently low overall recoveries of pepsin suggest
that the bound fraction of this protein cannot be released from the
sorbent again even at the lowest pH values applied. Among the basic
proteins, cytochrome C was entirely found in the breakthrough
fraction whereas of .alpha.-chymotrypsinogen A about 25-35% were
breaking through and the remainder was retained. The relative order
of elution along the pH-gradient was in all cases found as (1)
.alpha.-chymotrypsinogen A--(2) catalase--(3) hexokinase with
peak-to-peak differences ranging between 3 and 9 min. Although the
eluted peaks were always overlapping, for qualitative analytical
purposes catalase was sufficiently resolved from both proteins
during the applied gradient while separation from cytochrome C and
pepsin was actually complete. Catalase could thus be detected in
the simultaneous presence of both acidic and basic proteins with
the help of a sorbent of the invention. When comparing sorbent no.
ND 070002 with ND 06380, it becomes also evident that the
additional mononuclear heterocyclic substituent present in ND
070002 effects a general increase in retention as well as a reduced
overlap of catalase and .alpha.-chymotrypsinogen A peaks.
TABLE-US-00008 TABLE 8 Retention times [min] of three proteins on
three sorbents in the elution gradient of Example 5 Sorbent
.alpha.-Chymo- No. trypsinogen A Catalase Hexokinase ND 08037 25.8
29.2 38.3 ND 070002 28.9 33.9 41.8 ND 06380 24.4 27.0 32.3
Example 6
Effect of Added Salt on the Retention of Proteins on Different
Sorbents of the Invention
[0427] Experimental:
[0428] A mixture of the five proteins conalbumin, hexokinase,
pepsin, cytochrome C, and .alpha.-chymotrypsinogen A (each in an
amount of 0.48 mg dissolved in 25 mM phosphate buffer pH 7.4) was
injected onto different HPLC columns containing the sorbents which
had been derivatised with residues as indicated in the
corresponding tables below. The eluent was applied as a gradient
starting from 25 mM phosphate buffer pH 7.4 for 10 min at 0.25
ml/min, followed by a linear salt increase from 0 to 1 M sodium
chloride at constant pH within 50 min at 0.5 mL/min which level was
then held for 20 min at constant flow. A final purging step with 1
M aqueous acetic acid for 10 min at 0.5 ml/min concluded the
elution sequence. The eluate composition was monitored at
absorbance wavelengths of 225 nm, 280 nm, and 290 nm. Given yields
were based on these signals after integration. Protein recoveries
were calculated here by summing up the yields of each elution step.
For comparison, each protein was injected also as a pure solution
(see FIGS. 12-15).
[0429] Results:
[0430] The retention data of all five proteins are listed in Tables
9-12. Sorbents no. ND 08236 and ND 06386 did neither show any
notable influence on the binding strength towards the targeted
conalbumin nor towards the more acidic proteins hexokinase and
pepsin. In accordance with the intended protocol, these proteins
could only be eluted from the sorbents after a downward pH jump
with an acidic eluent. Only a small percentage of the basic protein
.alpha.-chymotrypsinogen A (which is not supposed to be bound
electrostatically) was eluted at an increased salt concentration.
On the other hand, the quinolyl-4-carbonyl containing sorbents no.
ND 07200 and ND 07122 exhibited a moderate salt effect on the
retention of both conalbumin and .alpha.-chymotrypsinogen A. This
effect was more pronounced in ND 07122 than in ND 07200 in terms of
yield losses during salt application, thereby confirming the
beneficial effect of the additional mononuclear heteroaromatic
ligand on the binding strength in ND 07200. These combined findings
can be regarded as an indication that the binding mechanism of the
sorbents differs from a simple ion exchange. As a consequence,
proteins may also be captured on the sorbents from (unclarified)
feed solutions containing high concentrations of pH-neutral salts.
Contrariwise, the salt concentration of the eluent can be used as
an additional parameter to adjust protein selectivity in
separations of more complex mixtures.
TABLE-US-00009 TABLE 9 Retention data with added salt on sorbent
no. ND 08236 (14% 3- indolylpropionyl + 29% 4-imidazolylacryl
residues) Retention Yield Yield Yield Time [min] Break- Salt Acidic
Total Protein Salt Elution through Elution Purge Recovery
Conalbumin -- -- -- 68% 68% .alpha.-Chymo- -- 53% -- 43% 96%
trypsinogen A Cytochrome C -- 88% -- -- 88% Hexokinase -- 9% -- 47%
56% Pepsin -- 31% -- -- 31%
TABLE-US-00010 TABLE 10 Retention data with added salt on sorbent
no. ND 07200 (19% quinoline- 4-carbonyl + 30% 4-imidazolylacryl
residues) Retention Yield Yield Yield Time [min] Break- Salt Acidic
Total Protein Salt Elution through Elution Purge Recovery
Conalbumin 25.1 -- 26% 26% 52% .alpha.-Chymo- 21.3 48% 18% 3% 69%
trypsinogen A Cytochrome C -- 88% -- -- 88% Hexokinase -- 17% --
21% 37% Pepsin -- 32% -- -- 32%
TABLE-US-00011 TABLE 11 Retention data with added salt on sorbent
no. ND 07122 (19% quinoline- 4-carbonyl residues) Retention Yield
Yield Yield Time [min] Break- Salt Acidic Total Protein Salt
Elution through Elution Purge Recovery Conalbumin 30.6 -- 56% 32%
88% .alpha.-Chymo- 19.9 45% 42% 3% 91% trypsinogen A Cytochrome C
-- 83% -- -- 83% Hexokinase -- 14% -- 32% 46% Pepsin -- 33% -- --
33%
TABLE-US-00012 TABLE 12 Retention data with added salt on sorbent
no. ND 06386 (13% benzimidazolyl-2-thioacetyl residues) Retention
Yield Yield Yield Time [min] Break- Salt Acidic Total Protein Salt
Elution through Elution Purge Recovery Conalbumin -- -- -- 78% 78%
.alpha.-Chymo- 10.5 61% 11% 28% 100% trypsinogen A Cytochrome C --
78% -- -- 78% Hexokinase -- 10% -- 38% 48% Pepsin -- 28% -- --
28%
Example 7
Semi-Preparative Separation of Conalbumin from a Mixture of
Proteins on Sorbents Having Imidazole and Either Indole or
Quinoline Containing Residues
[0431] Experimental:
[0432] A mixture of the five proteins conalbumin, hexokinase,
pepsin, cytochrome C, and .alpha.-chymotrypsinogen A (each-in an
amount of 0.48 mg dissolved in 25 mM phosphate buffer pH 7.4) was
injected onto two different HPLC columns containing sorbent no. ND
07200 (33.5.times.4 mm) or PRC 10014 (250.times.4 mm; 0.96 mg
loaded). The sorbents had either been derivatised with 19%
quinoline-4-carbonyl and 30% 4-imidazolylacryl residues (ND 07200)
or with 22% 3-indolylpropionyl and 28% 4-imidazolylacryl residues
(PRC 10014). The eluent was applied as a gradient starting from 25
mM phosphate buffer pH 7.4 for 10 min (PRC 10014: 40 min) at 0.25
ml/min, followed by a linear buffer change to 50 mM acetate buffer
pH 3.5 within 50 min at 0.5 ml/min which level was finally held for
20 min (PRC 10014: 50 min) at constant flow rate. The eluate
composition was monitored at an absorbance wavelength of 225 nm
(FIG. 16 a/b) and collected in seven fractions. After concentration
to a volume of 0.2 ml through centrifugal membrane filtration the
protein content of each fraction was qualitatively analysed by
SDS-PAGE (exemplarily shown in FIG. 17 corresponding to the
fractionation on ND 07200). For purposes of comparison, analytical
runs of all five individual proteins on sorbent no. ND 07200 are
depicted in FIG. 18.
[0433] Results:
[0434] ND 07200: Fraction IV (31:30-40:00 min; 4.25 ml) contained
the majority of conalbumin together with some residual
.alpha.-chymotrypsinogen A but not contaminated by any further
protein. Cytochrome C was found in fraction I,
.alpha.-chymotrypsinogen A in fractions II and III, and hexokinase
in fractions V and VI. Minor amounts of conalbumin were also
detected in fractions II, III, and V. Although still incomplete,
the purification ability of the chosen combination of stationary
and mobile phases was acceptable when considering the short column
dimension and coarse fractionation. PRC 10014: Fraction V
(98:00-109:00 min; 5.50 ml) contained the majority of conalbumin
together with residual .alpha.-chymotrypsinogen A and hexokinase
from which it had been partly separated. Cytochrome C was found in
fraction I, .alpha.-chymotrypsinogen A in fractions IV and VI, and
hexokinase in fractions V-VII. Minor amounts of conalbumin were
also detected in fractions IV and VI.
Example 8
Analytical and Semi-Preparative Separation of Hemoglobin from a
Mixture of Proteins on a Sorbent Having Benzimidazole and Imidazole
Containing Residues
[0435] Experimental:
[0436] A mixture of the five proteins hemoglobin, hexokinase,
pepsin, cytochrome C, and .alpha.-chymotrypsinogen A (each in an
amount of 0.48 mg dissolved in 25 mM phosphate buffer pH 7.4) was
injected onto a HPLC column containing sorbent no. ND 07003 which
had been derivatised with 13% benzimidazolyl-2-thioacetyl and 10%
4-imidazolylacryl residues. The eluent was applied as a gradient
starting from 25 mM phosphate buffer pH 7.4 for 10 min at 0.25
ml/min, followed by a linear buffer change to 50 mM acetate buffer
pH 3.5 within 50 min at 0.5 ml/min which level was finally held for
20 min at constant flow rate. The eluate composition was monitored
at absorbance wavelengths of 225 nm (FIG. 19), 280 nm, 290 nm, and
500 nm and collected in seven fractions. After concentration to a
volume of 0.2 ml through centrifugal membrane filtration the
protein content of each fraction was qualitatively analysed by
SDS-PAGE (FIG. 20). For purposes of comparison, parallel analytical
runs of all five individual proteins (hemoglobin: 1.92 mg injected
amount) on sorbent no. ND 07003 are plotted in FIG. 21 together
with the measured pH values of collected eluent at the end of the
column. Analytical runs on the analogous reference sorbent no. ND
06386, which was lacking the imidazolylacryl residue, are added in
FIG. 22.
[0437] Results:
[0438] Some key data of this experiment are summarised in Table 13.
The overlay of the analytical chromatograms obtained with sorbent
no. ND 07003 demonstrate a reasonably good separation efficiency of
the stationary phase. Hemoglobin could thus be detected in the
simultaneous presence of both acidic and basic proteins with a
minimum retention time difference of 7.5 min and recovered with
100% (reference sorbent ND 06386: 86%). Furthermore, the
chromatogram of the mixture matches the superposition of the single
protein runs. As expected, the acidic proteins hexokinase and
pepsin were also electrostatically bound to the sorbent but with
low capacities (i.e., partial breakthrough). Most difficult problem
to be solved turned out to be the separation of hemoglobin from
hexokinase due to the broad elution behaviour of the latter. The
consistently low overall recoveries of pepsin suggest that the
bound fraction of this protein cannot be released from the sorbent
again even at the lowest pH values applied. In contrast,
.alpha.-chymotrypsinogen A and hemoglobin could not be separated on
sorbent no. ND 06386 (retention times of 35.1 and 35.7 min) if the
same gradient is used. In accordance with the analytical runs, the
fraction IV of the semi-preparative run (31:30-40:00 min; 4.25 ml)
contained the majority of the hemoglobin together with some
residual .alpha.-chymotrypsinogen A but not contaminated by any
further protein. The other proteins of the initial mixture were
found in fraction I (cytochrome C) and fractions V and VI
(hexokinase, .alpha.-chymotrypsinogen A). Traces of hemoglobin were
also carried into the latter two fractions.
TABLE-US-00013 TABLE 13 Retention data of the test mixture of
Example 8 on sorbent no. ND 07003. Retention Time [min] Yield Yield
Buffered Approximate Break- Buffered Total Protein Elution Elution
pH through Elution Recovery Hemoglobin 25.5 6.8 4% 96% 100%
.alpha.-Chymo- 33.0 5.6 36% 13% 49% trypsinogen A Cytochrome -- --
99% -- 99% C Hexokinase 49.2 4.5 12% 48% 61% Pepsin -- -- 34% --
34%
* * * * *