Note: Descriptions are shown in the official language in which they were submitted.
~ ` ~
1336077
MA~OGRaPHY APPARATUS AND METHOD
AND MATERIAL FOR MARING THE SAME
Field of the Invention
The present invention relates to chromatography
generally, and particularly relates to the attachment of
ligands to solid supports in chromatography apparatus.
Background of the Invention -
Chromatography procedures include affinity
chromatography, ion exchange chromatography, and
hydrophobic chromatography. The use of affinity
chromatography for the purification of biological
10 macromolecules is well-established. Under optimal
conditions in the laboratory, the results of affinity
chromatography purifications can be spectacular. For
example, yields of so~ and purification factors of over
6000 have been reported for avidin, Cuatracasas, P. and
Wilchek, M., Biochem. Biophys. Res. Commun. 33, 235
(1968), and vitamin B12-binding protein, Allen, R.H., and
Majerus, P.W., ~. Biol. Chem. 247, 7702 (1972). The
success of affinity chromatography in small-scale
purifications has been harder to realize on a larger
scale partly because of the expense and difficulty
involved in chemically derivatizing solid supports with
affinity ligands. In addition, it is difficult to
achieve high flow rates with these packings, and they are
not easy to sterilize and still maintain their biological
activity. For this reason, there is a continued interest
in the development of more convenient methods of
13360~7
-2-
attaching specific ligands to solid supports.
The use of heterobifunctional ligands in affinity
chromatography has recently been proposed. See
Mattiasson, B. and Olsson, U., J. Chromatoq. 370, 21
(1986); Olsson, U. and Mattiasson, B., J. Chromatog. 370,
29 (1986) In this work, trypsin was connected to a
sepharose support by a conventional cyanogen bromide
reaction. A heterobifunctional ligand comprising
dextran, with soybean trypsin inhibitor (STI) and
Cibacron*Blue substituted thereon, was prepared, and the
heterobifunctional ligand bound to the sepharose through
the interaction of trypsin and STI. The Cibacron Blue so
bound to the sepharose was used to extract lactate
dehydrogenase from bovine heart extract. While this
procedure permits Cibacron Blue to be removed from the
sepharose*for sanitizing of the sepharose and replacement
with a different ligand, the procedure disadvantageously
employs a potentially toxic cyanogen bromide reaction,
and requires the binding of a protein, trypsin, to the
solid support. The protein is subject to denaturation
and microbial attack.
In view of the foregoing, a first object of the
present invention is to provide a chromatography
apparatus in which chromatographic functional groups are
easily and reversibly bound to a solid support.
A second object of the present invention is to
increase the capacity of the solid support for the
chromatographic functional groups bound thereto.
A third object of the present invention is to
provide new means for associating chromatographic
functional groups with the groups which reversibly bind
the chromatographic functional groups to the solid
support.
A fourth object of the present invention is to
provide a way to reduce nonspecific binding to the solid
support in a chromatography apparatus.
C * trade mark
~ 1336~7~
-3-
A fifth object of the present invention is to
provide a method of making chromatography apparatus in
which the chromatographic functional groups are
reversibly bound to the solid support, and a sixth object
of the present invention is to provide a means for
removing chromatographic functional groups reversibly
bound to the solid support.
A seventh object of the present invention is to
provide different materials as the solid support in
chromatography apparatus which achieve some or all of the
foregoing objects.
An eighth object of the present invention is to
provide ion exchange chromatography apparatus which
achieve, at least in part, the foregoing objects.
A ninth object of the present invention is to
provide hydrophobic chromatography apparatus which
achieve, at least in part, the foregoing objects.
A tenth object of the present invention is to
provide materials which can be used to produce
chromatography apparatus as described above, and
particularly materials which can be used to produce
chromatography apparatus in which nonspecific binding to
the solid support is reduced.
The foregoing objects are achieved by the apparatus,
methods, and materials disclosed below.
SummarY of the Invention
Affinity chromatography apparatus is disclosed
herein. The apparatus comprises an enclosed chamber
having an inlet opening and an outlet opening. A solid
support, such as sepharose*, is contained within the
enclosed chamber. The solid support has hydrophobic
functional groups bonded to the surface thereof and
forming a hydrophobic layer thereon. Affinity
surfactants are bound to the solid support. The affinity
surfactants comprise a polar group, a hydrophobic
functional group substituted on the polar group, and a
ligand substituted on the polar group. The hydrophobic
* Trade Mark
1336~77
--4--
functional group on the affinity surfactants are
hydrophobically adsorbed to the hydrophobic layer formed
on the solid support. The ligands are thereby bound to
the solid support and available for binding to compounds
which selectively bind to the ligands.
A first improvement to the foregoing provides a
means for increasing the capacity of the solid support
for the affinity surfactant. It has unexpectedly been
found that, when silica particles are used as the solid
support, the capacity of the solid support for the
affinity surfactants is significantly increased. A
second improvement involves the use of a polyalkoxy group
as the polar group in the affinity surfactant.
Polyalkoxy polar groups are less subject to non-specific
binding, and advantageously facilitate the construction
of affinity chromatography apparatus from which the
leakage of the ligand is low. A third improvement
provides a means for reducing non-specific binding to the
hydrophobic layer of the solid support. In this
improvement, the apparatus described above further
comprises covering surfactants bonded to the solid
support. The covering surfactants comprise a polar group
and a hydrophobic functional group substituted on the
polar group. These groups may be the same as those found
in the affinity surfactants, with the hydrophobic
functional group similarly being hydrophobically adsorbed
to the hydrophobic layer formed on the solid support.
The covering surfactant is provided in an amount
sufficient to cover the hydrophobic layer so that
non-specific binding to the hydrophobic layer during use
of the column is reduced.
A method of manufacturing an affinity chromatography
apparatus of the kind described above comprises three
steps. One step is to provide an enclosed chamber having
an inlet opening and an outlet opening, with the chamber
having a solid support contained therein. The solid
support has hydrophobic functional groups bonded to the
_5_ 133~7
surface thereof and forming a hydrophobic layer thereon.
Another step is to provide an aqueous solution containing
affinity surfactants. The affinity surfactants comprise
a polar group, a hydrophobic functional group substituted
on the polar group, and a ligand substituted on the polar
group. The final step is to pass the aqueous solution
through the enclosed chamber so that the hydrophobic
functional groups of the affinity surfactants are
hydrophobically adsorbed to the hydrophobic layer formed
on the solid support. The ligands are thereby bound to
the solid support and available for binding to compounds
which are selectively bound by the ligands. The ligands
may be removed from the apparatus by passing a polar
organic solvent through the apparatus so that the
affinity surfactants are removed from the hydrophobic
layer and taken up in the polar organic solvent. The
apparatus, free of the ligands, may then be sanitized or
sterilized for re-use.
Disclosed herein are chromatography apparatus
advantageously employing hydrophobic polymers as the
solid support. Such an apparatus comprises a solid
hydrophobic polymer support and chromatography
surfactants bonded to the polymer support. The
chromatography surfactants comprise a polar group, a
hydrophobic functional group substituted on the polar
group, and a chromatographic functional group substituted
on the polar group. The hydrophobic functional group is
hydrophobically adsorbed to the polymer support, with the
chromatographic functional group thereby being bound to
the solid support and available for binding to compounds
which are selectively bound thereby. Preferably, the
hydrophobic polymer support is contained within an
enclosed chamber having an inlet opening and an outlet
opening, though applications in which the chamber is
absent are contemplated and are explained below.
Ion exchange chromatography apparatus are disclosed
herein. Such an apparatus comprises an enclosed chamber
` ~ 1336Q77
having an inlet opening and an outlet opening, a solid
hydrophobic support contained within the enclosed
chamber, and ion exchange surfactants bonded to the solid
support. The ion exchange surfactants comprise a polar
group, a hydrophobic functional group substituted on the
polar group, and an ionogenic group substituted on the
polar group. The hydrophobic functional group is
hydrophobically adsorbed to the solid support, the
ionogenic groups thereby being bound to the solid support
and available for binding to compounds which are bound
thereby.
Hydrophobic chromatography apparatus are also
disclosed herein. Such an apparatus comprises an
enclosed chamber having an inlet opening and an outlet
opening, a solid hydrophobic support contained within the
enclosed chamber, and hydrophobic surfactants bonded to
the solid support. The hydrophobic surfactants comprise
a polar group, a first hydrophobic functional group
substituted on the polar group, and a second hydrophobic
functional group substituted on the polar group. The
first hydrophobic functional group is hydrophobically
adsorbed to the solid support. The second hydrophobic
functional group has a partition coefficient not greater
than (and preferably less than) the first hydrophobic
functional group. In this apparatus, the second
hydrophobic functional group is bound to the solid
support and available for bonding to compounds which will
adsorb thereto.
Disclosed herein are solutions usefùl for making
chromatography apparatus of the type described herein.
The solution, preferably an aqueous solution, contains
chromatography surfactants. The chromatography
surfactants comprise a polar group, a hydrophobic
functional group substituted on the polar group, and a
chromatographic functional group substituted on the polar
group. The polar group is a polyalkoxy group having at
least two alkoxy groups, with the alkoxy groups selected
~7~ 1336077
from the class consisting of ethoxy and isopropoxy.
Preferably, the polar group, the hydrophobic group and
the chromatographic group are selected so that the
solution is a single phase solution.
Also disclosed herein are solutions, useful for
making chromatography apparatus as described herein, in
which nonspecific binding to the solid support is
reduced. These solutions, also preferably aqueous
solutions, comprise chromatographic surfactants and
covering surfactants in combination in a liquid solvent.
The chromatographic surfactants comprise a polar group, a
hydrophobic functional group substituted on the polar
group, and a chromatographic functional group substituted
on the polar group. The covering surfactants comprise a
polar group and a hydrophobic functional group
substituted on the polar group. The covering
surfactants, when bound to a solid support in a
chromatography apparatus, serve to inhibit nonspecific
binding to the support during use of the apparatus.
Preferably, the polar group and hydrophobic group of the
covering surfactants and the polar group, the hydrophobic
group, and chromatographic group of the chromatography
surfactants are selected so that the solution is a single
phase solution.
Brief Description of the Drawinqs
Figure l shows the elution profile of a
cholinesterase/bovine serum albumin mixture from an
affinity chromatography apparatus of the present
invention; and
Figure 2 shows the elution profile of cholinesterase
in partially purified human serum sample from an affinity
chromatography apparatus of the present invention.
1336077
-8-
Detailed Description of the Invention
The term "chromatography surfactant," as used
herein, refers to affinity surfactants, ion exchange
surfactants, and hydrophobic surfactants. Each of these
latter terms is explained below.
The term "chromatographic functional group," as used
herein, refers to ligands, ionogenic groups, and
hydrophobic functional groups. Each of these latter
terms is explained below.
The term "solid hydrophobic support," as used
herein, refers to all hydrophobic solid supports,
including, for example, (a) hydrophobic agarose,
sepharose, and cellulose, (b) hydrophobic silica, and (c)
hydrophobic polymers.
Enclosed chambers used for practicing the present
invention are typically columns of the type used in
affinity chromatography and high performance affinity
chromatography, ion exchange chromatography, and
hydrophobic chromatography. The solid support contained
within the column may be any conventional solid support,
such as agarose, sepharose*, or cellulose, but among these
silica particle supports are preferred. The silica
particles may, for example, be those porous silica
particles conventionally employed in liquid
chromatography, having pores ranging in width from 60 to
4000 Angstroms, but may also be non-porous particles.
The hydrophobic functional groups bound to the solid
supports may be benzyl groups or linear or branched alkyl
groups having from 2 to 50 carbon atoms and any degree of
unsaturation. Preferably, the hydrophobic groups are
linear alkyl groups having from 8 to 50 carbon atoms.
Exemplary alkyl groups are octyl, decyl, dodecyl,
tetradecyl, hexadecyl, octadecyl, and eicosyl.
Hydrophobic functional groups may be covalently
bonded to solid supports by conventional techniques.
Such solid supports are commercially available as reverse
phase packings. As noted above, superior results are
* Trade Mark
'C
. <
1336077
g
obtained when silica particles are used as the solid
support. To achieve these superior results, the silica
particles should have bonded thereto at least 100
micromoles, more preferably at least 200 micromoles, and
most preferably at least 400 micromoles, of the
hydrophobic functional group per milliliter of silica
particles.
Affinity surfactants of the present invention have
the general formula A-B-L, where A is a hydrophobic
functional group, B is a polar group, and L is a ligand
which specifically binds to a target molecule. The
hydrophobic functional group may be any of the
hydrophobic functional groups noted with respect to
silica particles above, with the same class preferred for
silica particles being preferred for the affinity
surfactants.
The polar group may be any hydrophilic spacer arm
conventionally used in affinity chromatography and having
a substitution site at each end. See qenerally C. Lowe
and P. Dean, Affinity ChromatographY, 218-220 (1974).
Preferably, however, the polar group is a polyalkoxy
group having at least 2 alkoxy groups selected from the
class consisting of ethoxy and isopropoxy. Broadly
speaking, the polyalkoxy group will have not more than 50
alkoxy groups. Preferably, however, the polyalkoxy group
will have from 5 to 30 alkoxy groups therein. A ligand
can be substituted for the hydroxyl group at one end of
the polyalkoxy group and a hydrophobic functional group
substituted for the hydroxyl group at the other end of
the polyalkoxy group by known procedures. Minor
substitutions to polyalkoxy groups which do not
substantially affect the polarity of the polyalkoxy
groups produce equivalent compounds for purposes of this
invention. Fluorinated polyalkoxy groups are exemplary
of such equivalent compounds.
Ligands which will bind to any of a variety of
target molecules can be employed in the affinity
133~Q7~
--10--
surfactants of the present invention. Exemplary of such
ligands, and the target molecules bound thereby, are the
following: Biotin and Avidin; Monoclonal Antibodies and
Inhibin; Procainamide and Cholinesterase; N-methyl
Acridinium and Acetylcholinesterase; P-aminobenzamidine
and Trypsin; P-aminophenol-beta-D-thiogalacto-pyranoside
and Beta-Galactosidase; Chitin and Lysozyme; Methotrexate
and Dihydrofolate Reductase; NAD and Alcohol
Dehydrogenase; Sulfanilamide and Carbonic Anhydrase; DNA
lo and DNA Polymerase; DNA and cDNA; DNA and RNA; cDNA and
Genetically Engineered Plasmids; Oxidized Glutathione and
Glutathione Reductase; P-aminobenzamidine and Urokinase;
Monoclonal Antibodies and Insulin; Trypsin and Soybean
Trypsin Inhibitor; N6-aminocaproyl-3',5'-cAMP and Protein
Kinase; Pepstatin and Renin; 4-Chlorobenzylamine and
Thrombin; Monoclonal Antibodies and Interferon;
N-(4-amino phenyl) Oxamic Acid and Influenza Virus;
Prealbumin and Retinal-binding Protein; Neurophysin and
Vasopressin; Lysine and Plasminogen; Heparin and
Antithrombin; Cycloheptaamylose and Human Serum Amylase;
Cortisol and Transcortin; PyridoxaI-5-phosphate and
Glutamate-pyruvate transaminase; Chelating Agents and
Metal Ions; Chelating Agent-Cu and Superoxide Dismutase;
Chelating Agent-Zn and Human Fibrinogen; Coenzyme A and
Succinic Thiokinase; Flavin and Luciferase; Pyridoxal
Phosphate and Tyrosine Aminotransferase; Porphyrin and
Haemopexin; Lysine and Ribosomal RNA; Polyuridine and
mRNA; Concanavalin A and Immunoglobulins;
3-phospho-3-hydroxypropionate and Enolase; D-malate and
Fumarate Hydratase; Atropine or Cobratoxin and
Cholinergic Receptors; 6-Aminopenicillanic acid and
D-Alanine Carboxypeptidase; Plant Lectins and Epidermal
Growth Factor Receptors; Alprenolol and Epinephrine
Receptors; Growth Hormone and Prolactin Receptors;
Insulin and Insulin Receptors; Estradiol or
Diethylstilbestrol and Estrogen Receptors; Dexamethasone
and Glucocorticoid Receptors; Hydroxycholecalciferol and
-11- 1336077
Vitamin D Receptors; Virus Monoclonal Antibodies and
Blood Viruses; and Monoclonal Antibodies and
BacteriophagesO Suitable chelating agents for practicing
the present invention include ethylenediaminetetraacetic
acid (EDTA) and other compounds containing the
iminodiacetic acid group, phosphonoacetic acid (Hz03P-
CH2COOH), pyrophosphate (such as dibasic pyrophosphate
hexahydrate), dibasic orthophosphate, crown ethers such
as dicylohexano-18-crown-6, cyclodextrins, and cryptands.
In overview, suitable ligands include, but are not
limited to, antibodies, nucleic acids, antitoxins,
peptides, chelating agents, enzyme inhibitors, receptor
agonists, and receptor antagonists. The term
"antibody", as used herein, means immunoglobulins such as
IgA, IgG, IgM, IgD, and IgE, whether monoclonal or
polyclonal in origin.
Aqueous solutions containing the affinity
surfactants described above are used to make the
chromatography apparatus described above. In preparing
these solutions, the polar group, the hydrophobic group
and the ligand are preferably selected so that the
affinity surfactants are soluble in the aqueous solution.
The aqueous solution will thus be a single phase aqueous
solution.
We have found solid hydrophobic polymer supports
particularly advantageous for practicing the present
invention. With such supports, the need for further
covering the support with hydrophobic functional groups
is eliminated. Such solid hydrophobic polymers comprise
a matt of intertwined hydrophobic polymer chains, the
chains having molecular weights of from about 10,000
daltons to about lO,000,000 daltons. The polymer may
optionally be porous. Suitable polymer materials
include, for example, polyethylene, polypropylene,
polyether sulfone, polystyrene, polydivinylbenzene,
polytetrafluoroethylene, polymethyl methacrylate,
polydimethyl siloxane, and blends thereof. Copolymers of
-12- 13 3 6077
hydrophobic and hydrophilic polymers which are
sufficiently hydrophobic to bind surfactants are intended
as encompassed by the term "hydrophobic polymer" herein.
Porous polypropylene, such as that available from the
AKZO company under the trade name ACCUREL~, is
particularly preferred. The polymer support may be in
any form, including, for example, particles, beads,
cards, sheets, fibers, hollow fibers, and semipermeable
membranes. The polymer supports, except for cards, are
lo preferably contained within an enclosed chamber having an
inlet and an outlet. Polymer cards with chromatographic
surfactants (preferably affinity surfactants) bound
thereto are useful as a diagnostic or analytic tool which
need not be contained within an enclosed chamber.
Ion exchange surfactants of the present invention
preferably have the general formula A-B-I, where A and B
are as given in connection with the affinity surfactants
described above and I is an ionogenic group. The
ionogenic group is selected from the class consisting of
cation and anion. Suitable anions are, for example,
-S03, -CH2s03 ~ -C2H4so3 ~ -C3H6S03, _po32, -Coo-, and
-CH2Coo-. Suitable cations include quaternary amines and
sulfonium ions, with the quaternary amines preferred.
Exemplary quaternary amines are -CH2N (CH3) 3,
-CHzN (CH3)2C2H40H, -C2H4N (C2Hs)3~ -C2H4N (C2H5)2cH2cH(OH)cH3
-C2H4NHC(NH2)N H2, -C5H4N CH3, -C2H4N H(C2Hs)2~ -C2H4N H3~
-(C2H4N H2)nC2H4N H3 wherein n is from one to ten, -N HR2
wherein R is H or a linear or branched alkyl group having
from one to ten carbon atoms and any degree of
unsaturation, -CH2C6H4N H3, and -N+C5H5.
Hydrophobic chromatography surfactants of the
present invention preferably have the general formula A-
B-H, where A and B are the same as given in connection
with the affinity surfactants described above and H is a
second hydrophobic group (A being the first hydrophobic
group). H has a partition coefficient not greater than,
and preferably less than, A~ The term "partition
-13- 133~077
coefficient," as herein used, means the amount of
material which goes into the oil phase (n-decane) over
the water phase of a two phase solution containing equal
parts by weight of each liquid. Preferably, the group
"H" is either phenyl or a linear or branched saturated
alkyl group having from two to four carbon atoms. It is
also preferred that the group "A" be a linear or branched
alkyl group having at least six carbon atoms and any
degree of unsaturation.
Covering surfactants have the general formula A-B,
with A and B being the same as given in connection with
the affinity surfactants described above. Covering
surfactants may be included in apparatus of the present
invention in quantities sufficient to partially or
completely cover the surface of the support, so long as
nonspecific binding of the support is, thereby, at least
partially reduced.
Solutions containing chromatography surfactants,
either alone or in combination with covering surfactants,
are preferably single phase solutions. It is also
preferred that liquid solvents employed in such solutions
be aqueous solvents, though solutions of alcohol (e.g.,
methanol, ethanol, isopropanol), or other organic
solvents containing no water can also be used.
Surfactants can also be solubilized in an aqueous
solution by adding an alcohol, such as methanol, ethanol,
or isopropanol or other organic solvent, thereto. Hence
the term "aqueous solution" as used herein is intended to
include mixed aqueous solutions, such as water and
organic solvent solutions, and the term "water soluble,
when applied to surfactants herein, is intended to
encompass surfactants soluble in mixed aqueous solutions
which would not be soluble in water alone.
The present invention is explained in greater detail
in the following examples. These examples are provided
to illustrate the present invention, and are not to be
taken as limiting thereof.
~ 1 3 ~ 7
-14-
EXAMP~E8 1-3
Preparation of Af~inity Surfactants
E. coli beta-galactosidase, chromatographically
purified, was obtained from Worthington Biochemicals.
P-aminophenyl-beta-D-thiogalactopyranoside (APGP) was
purchased from Calbiochem. O-nitrophenyl-beta-D-
galactopyranoside (ONPG), bovine trypsin (twice
recrystallized), DL-benzoylarginine-p-nitroanilide
(DL-BAPNA), p-aminobenzamidine (PAB), cholinesterase
(affinity purified from horse serum), procainamide,
dithiobisnitrobenzoate, and butyrylthiocholine were
obtained from Sigma Chemicals Co. Tresyl chloride
(2,2,2-trifluoroethanesulfonyl chloride) was obtained
from Fluka and ethyl-(dimethyl-aminopropyl)carbodiimide
(EDC) from Aldrich. The surfactant Sandopan JA36 was
obtained from Sandoz Chemicals and the surfactant
octaethylene glycol n-hexadecyl ether (C16E8) was obtained
from Nikkol Chemicals Co. (Japan). The structures of
these surfactants is as shown below:
20(a) Sandopan JA36
O
CH3--(CH2) 12----(C2H4o) n~ (CH2) 3 C OH
CH3- (CH2) 12-- (C2H40) n~ (CHz)3-OH (46%)
1 < n < 100, n = 19
25(b) C16Es
CH3- (CH2) 15-- (C2H4o) a~OH
The Attachment of APGP to JA36 was performed using a
water-soluble carbodiimide synthesis. 100 mg of APGP
(0.35 mmoles) were dissolved in 150 ml of distilled water
and cooled to 5 degrees Centigrade. 500 mg of EDC (2.5
mmoles) were added to the solution and dissolved. 340 mg
~ Trade Mark
, ...
133~0~7
-15-
of JA36 (0.34 mmoles) were then added dropwise and the pH
adjusted to 4.25 with dilute HCl (0.1 N). The pH was
maintained between 4.25 and 5.5 during the reaction by
addition of either dilute HCl or NaOH. The course of the
reaction was monitored by a quantitative determination of
residual free amine using the ophthaldialdehyde (OPA)
test. See Chen, R.F. et al., Biochim. Biophys. Acta 576,
440 (197~); Rowlett, R. and Murphy, J., AnalYtical
Biochemistry 112, 163 (1981); Sredos, V.-J. et al.,
AnalYtical Biochemistry 101, 188 (1980). The reaction
proceeded for 60 hours. The product JA36-APGP was
purified by repeated foam fractionation of the reaction
mixture and the final product was positively identified
by 5134C nmr spectroscopy and by ultraviolet absorption.
The specific inhibitor of trypsin, PAB, was also
attached to JA36 by a similar EDC-catalyzed reaction.
485 mg of JA36 (0.43 mmoles) was dissolved in distilled
water and cooled to 5 degrees Centigrade. 400 mg of EDC
(2 mmoles) was then dissolved by dropwise addition with
stirring. The pH was adjusted to 4.5 with dilute HCl
and, after 15-20 minutes, 250 mg of PAB (1.2 mmoles) were
added to the mixture. The reaction mixture was
maintained at a pH of between 4.5 and 6.0 by addition of
dilute HCl or NaOH. The reaction was followed by
monitoring the shift in the absorbance maximum of the
ultraviolet spectrum (lambdamax of PAB = 290 nm, lambdamax
of JA36-PAB = 270 nm). The reaction proceeded for 72
hours. The product JA36-PAB was purified by repeated
foam fractionation of the reaction mixture and was
positively identified by 13C nmr.
Pyridinium, the specific inhibitor of
cholinesterase, was attached to C16E8 by tresylation of
the surfactant followed by nucleophilic substitution with
the inhibitor. See qenerallY Nilsson, K. and Mosbach,
K., Methods in EnzymologY 104, 56 (1984). Originally we
carried out this procedure for the purpose of linking
C~6E8 to procainamide, using pyridine as an organic base
13360~7
,
-16-
for the reaction. The reaction which we originally
believed to produce C16E8-procainamide, we now know to
have produced C16E8-pyridinium. Both pyridinium and
procainamide are, however, specific inhibitors of
cholinesterase.
To attach C16E8 to pyridinium, 600 mg of C16E8 (1.0
mmoles) is dissolved in 10 ml of dichloromethane and the
solution cooled to 4 Centigrade. 400 microliters of
pyridine and 400 microliters of tresyl chloride is added
to the surfactant solution. The reaction proceeds for
1.5 hours with stirring, whereupon an additional 400
microliters of pyridine is added. The solution is then
stirred at ambient temperature for 16 hours. The
reaction mixture is then dried under vacuum in a rotary
1~ evaporator and redissolved in 50 ml of distilled water.
The product C16E8-Procainamide is purified by preparative
reverse phase HPLC. The chromatographic system consists
of a Waters Associates system controller and data module,
a U6K injector, two Model 6000A pumps, and a Model 440
UV-visible absorbance detector. Column effluent is
monitored at 254 and 280 nm. The aqueous solution
containing the product is applied to a stainless steel
column (2.5 x 25 cm) packed with Davisil octadecyl-bonded
silica gel (300 Angstrom pore size, 30-40 micron particle
size). The flowrate is maintained at 2.0 ml/min and the
solvent was 0.1% trifluoroacetic acid (TFA) in water
(solvent A). The eluate has no detectable surface
activity (as gauged by its inability to foam) and it is
deduced that the surfactant is completely retained by the
column. The surfactant is then eluted with a 120 minute
linear gradient from solvent A to 100% solvent B
(methanol:iso-propanol:acetonitrile:TFA, 6:3:1:0.1 by
volume). The flowrate during elution is maintained at
3.0 ml/min and 10 ml fractions were collected and
redissolved in water to gauge surface activity.
Pyridine has an absorbance maximum at 256 nm.
Several peaks are eluted from the column having
1336077
-17-
significant absorbance at 259 nm. One of these peaks
elutes with a retention time of 127.5 minutes and is
surface active (as judged by its ability to foam in
aqueous solution). None of the other peaks which
absorbed at 259 nm are surface active. The surface
active material significantly inhibits the enzymatic
activity of horse cholinesterase and is, therefore,
judged the desired product. An additional surface active
peak elutes at 85 minutes but has no significant
absorbance at wavelengths higher than 220 nm and is
judged to be unreacted C16E8. The purity of the final
C16E8-pyridinium is determined by reverse phase HPLC on a
Perkin-Elmer octadecyl-bonded silica cartridge column.
Sample aliquots eluted isocratically (95~ acetonitrile,
5% water) at the rate of 1.0 milliliters per minute give
single, symmetrical peaks with uv detection at both 260
and 210 nm.
The molar extinction coefficient of C16E8-pyridinium
at 259 nm is 3118 M-1 cm~1 (by dry weight). The product
final yield is 71.9%. The modified surfactant in
solution has a K; toward horse serum cholinesterase of
5.14 x 10-7 M as determined by the method of Dixon.
Unmodified surfactant does not have any effect on the
activity of horse cholinesterase even at concentrations
as high as 10-3 M.
EXAMPLE 4
AdsorPtion of Affinity Surfactant
to Silica Particles
A high performance affinity chromatographic (HPAC)
column was prepared from an Upchurch Scientific HPLC
pre-column (2 cm length, 2 mm inside diameter) packed
with 0.021 g of Davisil*octadecyl-bonded silica (400
Angstrom pore size, 30-40 micron particle size). A
dilute (10 micromolar) solution of C16E8-PYridiniUm was
applied to the column and the eluate was monitored at 259
* Trade Mark
_ _,r
1336077
-18-
nm, the maximum in the absorbance spectrum of the
surfactant-inhibitor. The solution was continuously
applied until the absorbance at 259 nm was constant. The
capacity of the column was determined in a separate
experiment in which O.l gms of packing was equilibrated
with a O.l micromolar C16E8-pyridinium solution by
recirculation through the packing on a glass frit until
the absorbance at 259 nm was constant. Based on the
decrease in absorbance and the extinction coefficient, a
capacity of 0.302 micromoles/mg of packing was computed.
The column was then washed with lO0 ml of 50 mM tris-HCl
buffer containing 0.1 M NaCl at pH 8Ø This 0.1 M NaCl
Tris-HCl buffer was also the solution used to apply
protein mixtures to the column in subsequent examples.
Upon saturation of the packing with the affinity
surfactant, the binding was so strong in aqueous
solutions that no observable leakage of the
surfactant-inhibitor from the packing was detected during
elution, even in the presence of 3 M guanidine
hydrochloride. From the measured extinction coefficient
of the C16E8-pyridinium, as noted above, the specific
adsorption of the affinity surfactant to the reverse
phase material was determined to be 0.302 micromoles/mg
of packing. Since the density of the octadecyl-bonded
silica particles is about 1.5 gms/ml, the average ligand
loading is roughly 400 micromoles/ml of packing. This is
an extraordinarily high capacity when compared to the
typical ligand concentrations in ordinary affinity
columns.
EXAMPLE 5
Separation of Purified Cholinesterase
From Bovine Serum Albumin
The ability of the column prepared in Example 4
above to bind cholinesterase preferentially was first
demonstrated by separating a mixture of horse serum
133~7
--19--
cholinesterase and bovine serum albumin (0.70 mg/ml total
protein concentration, 1:4 w/w). The specific activity
of this solution was 22.1 units/mg. one unit of activity
is deined as the amount of enzyme hydrolyzing 1.0
micromole of butyrylthiocholine per minute at 25 degrees
Centigrade and a pH of 8Ø The column was equilibrated
with 0.05 M Tris-HCl buffer, pH 8.0, 0.1 M NaCl at the
rate of 1. 0 ml/min. When a 100 microliter aliquot of the
test protein mixture was applied to the column, all of
lo the cholinesterase was retained. Over 90% of the
cholinestrase activity was recovered upon application of
a sharp 2-minute linear gradient to 0.05 M Tris-HCl, pH
9.0, 1.0 M NaCl. The specific activity of the purified
eluate was 250 units/mg, corresponding to a ll-fold
purification.
Figure l shows the HPAC elution profile of a 100
microliter aliquot of the test cholinesterase-BSA
mixture. Repeated injections of 100 microliter aliquots
resulted in cholinesterase peaks of similar area and
purity. Doubling the iniection size resulted in doubling
of the cholinesterase peak area. Figure 1 shows the
cholinesterase peak to be sharp and well-defined. Yet,
as already mentioned, reverse phase analysis of the
purified product indicated the presence of contaminants.
A plausible explanation for this apparent discrepancy is
that, due to the small si~e of the column (2 cm length)
and the relatively high gradient flow rate, peak
resolution was negligible. Modification of the column
length, flow rate, or gradient time should allow for the
resolution of the contaminant peaks from the main
cholinesterase peak.
EXAMPLE8 6-7
Separation of Cholinesterase from Serum
The HPAC column described in Example 4 was then used
in the purification of horse and human serum
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cholinesterase~ Non-sterile, non-filtered grade horse
serum was obtained from Pel-Freeze Biologicals. Human
serum was donated by one of the inventors. In this case,
a sample of whole blood was centrifuged at 3000 RPM for
15 minutes. The supernatant was removed and the buffy
coat discarded~ The specific activity of cholinesterase
in the horse serum was 0.054 units/mg and that in the
human serum 0.066 units/mg.
Application of 200 microliter aliquots of both horse
and human serum to the column resulted, in each case, in
the elution of a broad and large protein peak. In both
cases, application of the same 2-minute gradient to 1.0 M
NaCl resulted in elution of a sharp peak containing
cholinesterase activity. These peaks had the same
retention times as the cholinesterase peaks obtained with
the enzyme test mixture of cholinesterase-BSA. With the
horse serum, the amount of cholinesterase recovered
corresponded to 84.2% of the activity applied and with
the human serum, the activity yield was 80.7%. The
specific activities of the purified cholinesterase from
horse and human serum were 15.1 and 5.18 units/mg,
respectively. These values correspond to 280 and 79-fold
enrichments of cholinesterase. Reverse phase HPLC
analysis showed that the gradient-induced eluate from
horse serum was 2.2% cholinesterase while that obtained
from the human serum 0.95% pure enzyme.
Figure 2 shows the HPAC elution profile for the
purification of the human serum cholinesterase. As with
the enzyme test mixture, even though the gradient-induced
eluate appears to be a sharp peak, the reverse phase HPLC
analysis described above reveals that the cholinesterase
in this peak is only 0.95% pure. Again, the resolution
in the small test column was insufficient to separate
components eluting at the beginning of the gradient and
those at the end of the gradient. This is not surprising
after one considers that a column this short will allow
significant mixing, especially with such a sharp
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gradient. The product purity could undoubtedly be
greatly enhanced by applying a longer gradient, by using
a longer column, or by eluting the enzyme with a specific
inhibitor such as choline chloride.
An analysis of Figure 2 also shows that the initial
peak, corresponding to the non-binding serum proteins, is
much smaller than what one would expect based on the
original protein content of the serum. The reason for
this is that a significant fraction of the proteins that
do not interact with the immobilized ligand are
irreversibly retained by the column in aqueous solution.
This was established from binding studies of proteins
other than cholinesterase with octadecyl reverse phase
packing. However, there is a finite number of these
binding sites, and when they are filled, the column only
retains molecules interacting specifically with the
immobilized inhibitor.
The irreversible, non-specific binding of proteins
to the HPAC column does not appear to interfere with the
specific interaction of cholinesterase with the column.
The ethylene oxide groups between the hydrophobic tail
and the procainamide moiety serve as a long (ca. 20-30
Angstroms) spacer arm which effectively places the
inhibitor in free solution. Since the spacer arm is so
long and contains no ionizable groups, the possibility of
non-specific ionic binding to the immobilized surfactant,
relative to conventional affinity chromatography, is
greatly reduced.
BXAMPLE 8
Removal of AffinitY Surfactant
From Solid SuPport
The affinity surfactant is easily removed from the
column described in Example 4 above by passing a 6:4
Volume/Volume mixture of methanol and isopropanol through
the column. The packing can then be sterilized and new
-22- 1336077
affinity surfactants -- either the same as or different
from those removed -- adsorbed to the packing by the
procedures described herein.
The invention described above may be used for a
broad variety of separation procedures, and embodied in a
variety of different apparatus and compositions.
Accordingly, the foregoing discussion is to be considered
illustrative, and not limiting, with the scope of the
present invention being defined by the following claims.
Equivalents of the claims are to be included therein.
EXAMPLE 9
Binding of Surfactants to PolYProPylene Support
A 2.5 x 10-3 Molar aqueous solution of pentaethylene
glycol mono-n-dodecYl ether (C12E5) is circulated over a
hydrophobic polypropylene membrane in an enclosed chamber
until the transmembrane flux for the a~ueous solution
increases to a noticeably improved steady state. After
this treatment, the chamber is emptied, dried, and
aspirated with air for 1.5 hours. After this treatment,
the transmembrane flux for water is measured, and is
found to be essentially the same as the steady state
transmembrane flux for the aqueous C12E5 solution. After
this, a .lM Tris-HCl buffer solution at pH 8 is
continuously circulated across the membrane for 24 hours,
during which time the transmembrane pressure remains
constant. These procedures show that the surfactant
remains adsorbed to the membrane while water, air, and
buffer solution are circulated across the membrane.
EXAMPLE 10
SYnthesis of 2-(n-hexadecylheptaethoxY)acetic Acid
C~6E8 (1 gram) was dissolved in approximately 80
milliliters of deionized water. Potassium permanganate
(355 milligrams) was added to the solution, together with
-23- 1336077
three drops of l M sodium hydroxide. The mixture was
stirred overnight at room temperature. The excess
permanganate was reduced by addition of sodium bisulfite
and the resulting manganese dioxide precipitate removed
by vacuum filtration. The filtrate was then acidified
with concentrated hydrochloric acid and the precipitated
C16E7-CH2-COOH was removed by centrifugation. 13C-NMR
analysis confirmed the presence of carbonyl carbon group
in the product and showed characteristic peaks for the
surfactant. Isocratic reverse phase HPLC showed a single
peak for the product.
EXAMPLE ll
Synthesis of l-(n-hexadecYloctaethoxy)ethylenediamine
C~6E8 (0.5 gram) was dissolved in 20 milliliters of
dimethylsulfoxide and the solution cooled to 5 degrees C.
Tresyl chloride (0.37 milliliter, 4 mmol) and O.l
milliliter of triethylamine were added to the solution
with stirring. The solution was stirred for l.5 hours at
room temperature, after which l.25 milliliters of
ethylene diamine (20 mmol) was added to the reaction
mixture. The reaction was allowed to proceed for 24
hours at which point the solvent was distilled under
vacuum. The mixture was redissolved in 50 milliliters of
water.
The aminated surfactant was purified by reverse
phase HPLC using a stainless steel column (2.5 x 25
centimeters) packed with Davisiltm octadecyl-bonded
silica. The column was washed with 250 milliliters of
0.1% trifluoroacetic acid (TFA) in water (solvent A) at
the rate of 2.0 milliliters per minute, prior to sample
application. The surfactant was completely retained by
the column in solvent A, since the column eluate had no
detectable surface activity (as gauged by its inability
to foam in an aqueous solution). The surfactant was
eluted with solvent B (methanol: iso-propanol:
133~077
-24-
acetonitrile: TFA, 6:3:1:0.01 by volume). The absorbance
of the column effluent was monitored at 220 nm. The
flowrate during elution was kept at 5.0 milliliters per
minute at 7.5 milliliter fractions were collected and
redissolved in water to gauge surface activity. Three
purification steps with different linear gradient
combinations were required to obtain homogeneous
material. The purified material gave a positive test for
amines using sodium 2,4,6-trinitrobenzene sulphonate.
See J. Inman and H. Dintzis, Biochemistry 8, 4074 (1969).
EXAMPL~ 12
Synthesis of N-(n-hexadecyloctaethoxy)PYridinium
C16E8-Pyridinium was synthesized by tresylation of
C16E8 followed by reaction with pyridine and purification
by reverse phase HPLC as described in Bxamples 1-3 above.
EXAMPL~S 13-15
Preparation of test columns
Test columns consisted of Upchurch Scientific
precolumns (internal dimensions: 2 x 20 millimeters)
packed with 24 + 1 milligram of Davisiltm octadecyl-bonded
wide-pore silica (30-40 micrometer particle size, 300 A
pore diameter). The freshly packed columns were
initially washed with several hundred column volumes of
methanol at 1.0 milliliters per minutes, followed by a
similar volume of deionized water. Adsorption of the
surfactants described in Examples 10-12 above to the
packing is done by recirculating 10-4 M aqueous solutions
of the appropriate surfactant through the columns at the
rate of 1.0 milliliters per minute for a period of no
less than four hours. For this purpose a Perkin-Elmer
series lo isocratic HPLC pump is used. A m~i rum value
of 0.37 micromoles of surfactant per milligram of packing
is obtained from adsorption studies with C16E8-Pyridinium.
-25- 1336077
This value compares well with the .6 micromoles of
surfactant per milligram of packing found by Bischoff et
al., J. of Chrom. 257, 305 (1983).
The columns were then rinsed with water followed by
several hundred column volumes of 50 mM Tris-HCl buffer,
pH 8.0 (Buffer A). Unless otherwise stated, the columns
were equilibrated with Buffer A prior to protein
application. Proteins were eluted with various linear
gradients to 50 mM Tris-HCl buffer, pH 8.0, 1.0 M NaCl
(Buffer B). The absorbance of the effluent was monitored
at 280 nm. The surfactant was removed in preparation for
reverse phase mode of operation by rinsing the column
with acetonitrile at 1 milliliter per minute overnight.
BXAMPLE5 16-17
Preparation of Test Solutions
Protein determinations: Standard protein solutions
were prepared by dissolving a dry weight amount of each
test protein in Buffer A. The concentration of these
solutions were as follows: bovine serum albumin, 1.2
milligrams per milliliter; lysozyme, 3.0 milligrams per
milliliter; trypsin, 2.9 milligrams per milliliter;
cholinesterase, 1.5 milligrams per milliliter. The
detector response was calibrated using these standard
solutions by directly connecting the outlet of the
injector to the inlet of the detector. The average of
the integrated peak areas for multiple injections of each
of the standard solutions was used to calibrate the
detector at 280 nm.
The activity of cholinesterase and carboxylesterase
solutions was determined by the method of Ellman et al.,
Biochem. Pharmacol. 7, 88 (1961). In the case of
cholinesterase, butyrylthiocholine was used as substrate,
and phenylthiobutyrate was used as substrate for
determination of carboxylesterase activities. One unit
of enzymatic activity corresponded to the hydrolysis of
133~077
-26-
one micromole per minute of a l mM substrate solution at
25 degrees Celsius in 0.05 M Tris-HCl buffer, pH 8Ø
Preparation of clarified liver homogenate. Frozen
beef liver (446 grams) was homogenized in 500 milliliters
of 50 mM Tris-HCl buffer, pH 8.0 (Buffer A) at 5 degrees
Celsius. The homogenate was allowed to stand for 15
minutes and then diluted with an additional 500
milliliters of Buffer A. A fraction of the homogenate
(500 milliliters) was saved and the remainder filtered
through six layers of cheese cloth, followed by a course
glass-sintered funnel. The ~iltrate was centrifuged for
30 minutes at 4000 RPM at 5 degrees Celsius. The
supernatant (630 milliliters) was saved.
Ammonium sulfate (123.5 grams) was slowly added to
the clarified homogenate at 5 degrees Celsius. After
allowing the solution to stand for 0.5 hours at ~ degrees
Celsius, the solution was centrifuged at 4000 RPM for one
hour and the supernatant (470 milliliters) was dialyzed
against 6 liters of Buffer A for 6 hours and then against
12 liters of Buffer A for 12 hours at 5 degrees Celsius.
A fraction of the dialyzate (180 milliliters) was
ultrafiltered using an Amicon (lO0,000 MW cutoff) YM100
membrane at 5 degrees Celsius. Most of the
phenylthiobutyrate activity (98.0%~ remained in the
retentate. The retentate was used for the purification
experiment.
EXAMPLE 18
Cation-Exchange Chromatography of LysozYme and BSA
The chromatographic system used consisted o~ a
Perkin-Elmer series 410 quaternary gradient elution pump
and a Perkin-Elmer series LC-95 UV/visible
spectrophotometric detector. Data was acquired in part
with a Perkin-Elmer series 3600 data station and in part
with a MacIntosh Se digital computer equipped with the
Dynamaxtm Software Data Acquisition package (Rainin Co.).
l~36~7
~ . -
-27-
The cation-exchange characteristics of the test
octadecyl-bonded column adsorbed with C16E7-CH2-COOH
described in Examples 13-15 above were studied initially
using lysozyme and BSA. Lysozyme, with an isoelectric
point (pI) of 11.0 should be positively charged in the
equilibriating buffer at a pH of 8Ø BSA, on the other
hand, has a reported pI value of 4.9 and should therefore
bear a negative charge at a pH of 8.
Standard solutions of lysozyme (3.0 milligrams of
protein per milliliter) and BSA (1.2 milligrams of
protein per milliliter) were prepared in Buffer A as
described in Examples 16-17 above. Aliquots of 20
microliters of these solutions were applied to the C16E7-
CH2-COOH test column. Adsorbed proteins were eluted with
a 20 minute linear gradient to Buffer B (50 mM HCl, pH
8.0, 1.0 M NaCl). Proteins that were not retained by the
test column would elute in approximately 0.30 minute
whereas retained proteins eluted after about 8.5 minutes
after application of the gradient to Buffer B. The
amount of protein in each eluted peak was determined from
the integrated peak areas and calibration injections as
described above. High peak efficiencies and recoveries
were not observed initially when freshly adsorbed columns
were used to analyze the test proteins. Optimum and
consistent column behavior was obtained only after the
column had been treated with several injections (20
microliters) of the lysozyme standard solution. After
this treatment, typical analyses of the test proteins
gave the following results.
In the case of lysozyme 106% of the applied protein
was calculated to be present in the retained peak. This
value was based on the integrated peak area and the
calibration value obtained as described above. When BSA
was applied to the column 102% of the applied protein was
calculated to be present in the unretained peak. In both
cases there was almost no loss of material in the column
due to irreversible hydrophobic adsorption. When
1336077
-28-
mixtures of lysozyme and BSA were applied to the test
column (30 micrograms of lysozyme and 12 micrograms of
BSA per injection) there was near complete resolution of
the two proteins. Figure 3 shows the elution profiles
for the analyses of BSA, lysozyme, and the BSA/lysozyme
mixture. Recoveries of greater than 100% were obtained
because of a positive bias present in the integration of
broad peaks which had some degree of tailing, as with the
elution of retained peaks, versus very narrow peaks, as
obtained in the calibration procedure with no column.
The magnitude of this bias was not higher than 15% and
depended on the shape of the particular peak.
EXAMPLE 19
Cation Exchange Chromatography of TrYpsin
This example was conducted in accordance with
Example 18 above. The activity of trypsin solutions were
determined by a method similar to that described by
Erlanger et al., Arch. Biochem. BioPhys. 95, 271 (1961),
using BAPNA as substrate. An aliquot (0.5 milliliter) of
the trypsin solution to be assayed was diluted in 1.5
milliliters of 0.05 M Tris-HCl buffer, pH 8.15,
containing 0.02 M CaCl2 in a 3.5 milliliter disposable
polystyrene cuvette. A 100 microliter aliquot of a lOZ M
solution of BAPNA in dimethylsulfoxide was then added to
this solution. The rate of change in absorbance at 388
nm at 25 degrees Celsius was then measured in a Schimadzu
UV 160 spectrophotometer. One unit of activity was
defined as the amount of enzyme causing an absorbance
change of 0.1 per minute under these conditions.
A solution of trypsin (2.9 milligrams of protein per
milliliter) was prepared in Buffer A and several activity
assays were made over the course of three days to
determine its stability at 4 degrees Celsius. No
significant loss in tryptic activity was observed, and
therefore the solution was assumed to be stable for
1336077
-29-
several days at this temperature. Aliquots of 20 and 100
microliters were applied to the C16E7-CH2-COOH test column
as described for the analysis of lysozyme and BSA samples
in Example 18 above. In this case the eluted peaks were
collected and assayed for enzymatic activity.
Initially, operation of the column was done at room
temperature. At this temperature, when 58 micrograms of
trypsin (2.56 units) were injected into the column under
initial conditions of 100% Buffer A, an unretained peak
was eluted at 0.22 minutes which contained no tryptic
activity. In the retained peak, only 24% of the
enzymatic activity was recovered. Larger injections of
trypsin, also at room temperature (290 micrograms, 11.0
units), resulted in the same low recovery of enzymatic
activity. In the latter cases, 37% of the total injected
activity was recovered, of which 35% was eluted in the
void volume and 65% was eluted during the course of the
gradient.
In an effort to improve overall recovery, the
gradient to 100% Buffer B was shortened to 5 minutes and
an ice bath was used to cool the outside of the column to
4 degrees Celsius. When 290 micrograms of trypsin were
applied to the column under these conditions, 13% of the
activity was found in the unretained peak and 86% of the
activity was found in the peak which eluted upon
application of the gradient, with an overall activity
recovery of 99%. During the experiments with this
column, no loss in performance was observed even after
85,000 column volumes of buffer had circulated through
the column. Bischoff et al., Anal. Biochem. 151, 526
(1985); J. of Chrom. 296, 329 (1984); J. of Chrom. 257,
305 (1983), found that their columns also continued to
perform well after several thousand column volumes of
use.
~30- i336 077
EXANPhE 20
Reverse Phase Chromatography of Test Proteins
The C16E7-CH2-COOH test column used as a cation-
exchange support in Examples 18-l9 above was washed with
acetonitrile to remove the adsorbed surfactant. This
column was then used in the reverse phase chromatography
of lysozyme, BSA, and trypsin. All the proteins were
retained by the column under initial conditions of 0.1%
trifluoroacetic acid in water. A 5 minute linear
gradient from initial conditions to 0.1% trifluoroacetic
acid in acetonitrile was used to elute the proteins.
Lysozyme eluted at 8.84 minutes whereas BSA eluted at
9.07 minutes. The chromatogram of trypsin showed three
peaks with retention times of 7.77, 8.16, and 8.85
minutes. The relative peak areas were 46.8%, 13.7%, and
39.5%, respectively. The column was then equilibrated
with Buffer A and a lOO microliter injection of the
lysozyme stock solution was made. No protein eluted upon
application of a gradient to lOO~ Buffer B, indicating
that the stationary phase of hydrophobic and that no
residual ion-exchange capacity remained. The
multiplicity of peaks in the chromatogram of trypsin can
be explained by different molecular species of the same
enzyme (i.e., isozymes).
The column was again adsorbed with C16E7-CHz-COOH
using the same procedure done initially. The column was
equilibrated with several hundred column volumes of
Buffer A and used for the analysis of lysozyme, BSA, and
trypsin as before. The protein retention behavior was
similar to that described previously.
EXAMPLE 21
Anion-Exchanqe Chromatoqraphy with N-(n-
hexadecyloctaethoxY)Pyridinium
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The anion exchange properties of the C16E8-Pyridinium
test column prepared in Examples 13-15 above were
evaluated with BSA and lysozyme. The same stock protein
solutions used in the evaluation of the C16E7-CH2-COOH test
column were also used for these experiments. The column
was initially conditioned with multiple injections of BSA
standard solution as previously described. Injection of
a 25 microliter aliquot of BSA resulted in the adsorption
of all the protein and a calculated recovery of 113% of
the protein upon applying a two minute gradient to Buffer
B. Application of a 25 microliter aliquot of the
lysozyme standard solution resulted in the elution of
110% of the applied protein unretained.
A solution of C16E8-Pyridinium (10-4M) was again
recirculated through the column for four hours at the
rate of 1.0 milliliter per minute. This was done to
investigate the effect of re-equilibration with
surfactant on column performance. The interaction of
bovine liver carboxylesterase (CE) with the readsorbed
column was studied with a solution of purified enzyme
having an enzymatic activity of 29.1 phenylthiobutyrate
units per milliliter and a protein concentration of 1.72
milligrams per milliliter. Initially, a 25 microliter
aliquot of the CE solution was applied to the C16E8-
Pyridinium test column. This resulted in all the proteinand activity present in the aliquot being retained by the
column. Upon application of a 2.0 minute gradient to
Buffer B, 90.8% of the applied protein eluted. However,
no enzymatic activity was recovered. The column was then
treated with a large injection (200 microliters) of the
CE stock solution. After this treatment, analysis of a
25 microliter aliquot of the CE solution resulted in
86.7% of the applied protein being retained. The
remaining protein was not retained, and contained no
enzymatic activity. Upon elution with Buffer B all the
adsorbed protein was recovered and this fraction
contained all the activity units present in the original
1336077
-32-
25 microliter aliquot. Therefore, the 13% of protein
that was unretained by the column contained no CE and
were probably positively-charged contaminants in the
original CE sample.
The C16E8-Pyridinium test column was then used to
purify the carboxylesterase present in the clarified
liver homogenate prepared in Examples 16-17 above. The
liver homogenate had an activity of 14.1
phenylthiobutyrate units per milliliter and a protein
concentration of 21.1 milligrams per milliliter. Protein
concentration was determined, in this case, by measuring
the absorbance at 280 nm of the protein solutions and
assuming an extinction coefficient of 1.0 (milligram per
milliliter)~1. Therefore, the specific activity of the
homogenate was 0.67 units per milligram of protein.
Injection of a 50 microliter aliquot of homogenate under
initial conditions of 100% Buffer A resulted in two
unretained and poorly-resolved peaks which corresponded
to 57% of the applied protein as determined from the
integrated peak areas. The material under these peaks
contained 0.20 units of activity which corresponded to
28% of the applied activity. Upon application of a two
minute linear gradient to Buffer B, two peaks eluted and
these contained 43% of the applied protein. The first
peak (8.32 minute retention time) contained 0.40 units of
activity (56%), whereas the second peak (8.87 minute
retention time) had no activity. This indicated an
overall activity recovery of 85%. The specific activity
of the purified CE was calculated to be 1.25 units per
milligram of protein, corresponding to a purification
factor of 1.9. The reason that a small number of peaks
was observed for the analysis of a complex protein
mixture, such as a liver homogenate, is that due to the
small size of the column relative to the particle
diameter and the steep gradient used, the column had very
few theoretical plates. Improvements in peak resolution
were not addressed since the objective was to study the
~ _33- 133~7~
retention characteristics of the modified supports and,
for such purposes, the simple retention behavior of the
test columns used was adequate.
EXAMPLE 22
Anion Exchanqe with l-(n-hexadecyloctaethoxy)
ethylenediamine
The anion-exchange characteristics of the C16E7-
CH2CH2NHCH2CH2NH2 test column prepared in Examples 13-15
above were initially tested using BSA and lysozyme. In
this case, however, the pH of Buffers A and B was
adjusted to 7.4 in an effort to minimize the amount of
silica degradation which occurred at a pH of 8.0 during
the studies using C16E8-Pyridinium test columns and which
resulted in excessively high pressure drops across the
columns. As mentioned above, this degradation was not
observed in the C16E7-CH2-COOH column. This observation
can be attributed to a localized lowering of the pH
brought about by the concentration of carboxylate groups
of the derivatized surfactant. Use of pH 7.4 buffers
resulted in greatly improved column stability in the
anion-exchange systems.
As in the case of C16E8-Pyridinium, BSA was retained
by the column whereas lysozyme was not. Initially, 25
microliter aliquots of the standard BSA solution were
injected into the column. This resulted in poor protein
recovery (<30%) and considerable peak tailing. When
larger injections sizes were used (>100 microliters),
peak recoveries were much improved and consistent. Peak
efficiencies were also improved and symmetrical peaks
were observed.
In the case of lysozyme an in~ection of 100
microliters resulted in the elution of an unretained peak
at 0.2 minutes. The integrated peak area indicated 106%
recovery. No protein was eluted from the column with a
two minute gradient to Buffer B. In the case of BSA (200
1336077
-
-34-
microliter injection), an unretained peak was observed
(0.36 minutes) corresponding to 40% of the applied
sample. This was possibly due to exceeding the column
capacity. A peak eluted at 8.3 minutes upon application
of the gradient to Buffer B. The area of this peak
corresponded to 50% of the applied protein. Therefore,
the overall protein recovery was 90%.
Application of a 50 microliter aliquot of affinity-
purified serum cholinesterase (1.90 units) resulted in
the elution of an initial peak (0.27 minutes) containing
no enzymatic activity and corresponding to 16.2% of the
applied protein. Upon application of the gradient to
Buffer B, a peak eluted (8.3 minute retention time)
containing 1.95 units of activity (103% activity
recovery) and corresponding to 83.7% of the applied
protein. When horse serum (100 microliters, 5.77 units
per milliliter) was applied to the column over 30% of the
applied protein was retained by the column, but all the
cholinesterase activity applied was found in the
unretained fraction.
EXAMPLE 23
Ion Exchange ChromatograPhy with Surfactants
Bound to a HYdroPhobic Polymer Support
High density polypropylene porous particles
purchased under the trade name of ACCUREL~ were
hydrophilyzed with C16E7CH2COOH and used in the Ion-
Exchange purification of bovine heart cytochrome C. A
glass column (8.5 x 2.5 cm) was packed with about 2.63
grams of ACCUREL~ powder in an aqueous suspension. The
particles had been washed with methanol, deionized water,
and finally had been allowed to equilibrate for four
hours in 100 milliliters of a 0.05 M solution of
C16E7CH2COOH. The column was equilibrated with 50 ml of 10
mM Tris-acetate buffer pH 6.9 prior to use.
1336077
-35-
A clarified bovine heart homogenate was prepared by
homogenizing 114 grams of beef heart tissue in a 0.3%
aluminum sulfate solution. The homogenate was filtered
through five layers of cheesecloth and the pH of the
filtrate adjusted to 8.6 with ammonium hydroxide. The
solution was finally filtered through a coarse glass-
fritted funnel. A five milliliter aliquot of this
homogenate was applied to the surfactant-modified
ACCUREL~ column. The cytochrome C content of the bovine
heart homogenate was effectively retained by the column.
The column was washed with several column volumes of
equilibrating buffer. The cytochrome C was then eluted
with a 1.0 M NaCl solution in equilibrating buffer.
Approximately 80% of the applied cytochrome C was
recovered and this corresponded to a thirty-fold
purification.
The extent of hydrophilization of ACCUREL~ was
determined using the parent surfactant, namely C16E8. A
small glass column was packed with 126.3 milligrams of
ACCUREL~. The column was washed with 20 milliliters of
methanol, followed by 20 milliliters of deionized water.
The column was then treated with 146 milliliters of a 10 3
M solution of C16E8. The ligand loading was estimated to
be 0.96 ~moles per milligram of packing. The column was
then equilibrated with 20 milliliters of 10 mM Tris-
acetate, pH 7.5. Treatment of the column with several
five milliliter aliquots of a purified bovine cytochrome
C solution (0.8 milligrams per milliliter) resulted, in
each case, in the recovery of 99% of the applied protein.
The foregoing examples are for illustrative purposes
only, and are not to be taken as restricting the scope of
this invention. Applicants' invention is defined by the
following claims, with equivalents of the claims to be
included therein.
