Isolation of biologically active compounds by affinity chromatography

A method for the isolation of biologically active compounds by affinity chromatography comprising forming a sorption complex between a solvent soluble biologically active compound to be isolated and biologically active compound linked to a solid carrier by a covalent bond, said solid carrier being a hydrophillic macroporous copolymer.

This invention relates to a method of isolating biologically active 
compounds upon selective sorbents by affinity chromatography, that is, by 
selective adsorption. More particularly this invention relates to methods 
for isolating biologically active compounds such as enzymes, coenzymes, 
enzyme inhibitors, antibodies, antigens, hormones, carbohydrates, such as 
monosacharides, disacharides, polysacharides, and the like. The invention 
also relates to the selective adsorption of lipids such as triglycerides, 
fats, oils, waxes, phospholipids, glycolipids, and sterols, as well as 
carotenoids, amino acids, peptides, proteins, and the like as well as 
nucleotides nucleic acids, vitamins such as Vitamin B and the like. 
Affinity chromatography can be further applied when it is desired to 
concentrate dilute solutions of proteins in order to remove denatured 
forms thereof from refined proteins, and in the separation and resolution 
of protein and peptide components which have originated in specific 
chemical modifications. 
A number of materials have been used in the prior art as sorption complex 
carriers for biologically active components in affinity chromatography. 
Among those materials known in the art, there may be mentioned, copolymers 
of styrene and di-vinyl benzene; copolymers of acrylamide with alkylene 
bis-acrylamide; polysacharides; porous glass, etc. Many of the prior art 
materials have proved undesirable and possess many disadvantages due to 
the fact that they carry thereon linked inorganic functional groups and 
thereby, exhibit considerably non-specific and hence nondiscriminating 
sorption of the biologically active material. In other words, the prior 
art materials are neither selective nor specific enough to be of any great 
use. Other prior art materials possess unsuitable mechanical, 
hydrolytical, microbial, or thermal stabilities, as well as unstable 
distribution of pore sizes and, accordingly, have but a rather limited 
range of use. Homogeneous hydrophilic gels which are presently on the 
market, are only of use when they are moist and do not dry out. This 
latter requirement makes their storage and transport rather difficult and, 
hence, their use and availability for the above affinity chromatography 
quite unsatisfactory. 
It is known in the prior art that the most accurate and sophisticated 
separation methods operable in the biochemical arts are the processes 
based on the ability of great numbers of biologically active compounds to 
form sorption complexes with other biologically active compounds. The 
character of this complex-formation or inter-attraction is ordinarily 
quite specific to the biologically active compound being selectively 
adsorbed and is, furthermore, reversible with regard thereto, i.e., the 
adsorbed component may be released. In practive, the process works as 
follows: A component of the sorption complex sought to be insolubiliized 
is first linked to a solid carrier material by a covalent bond, that 
treated carrier is thereby brought in contact with a solution of a complex 
mixture of compounds, one of which is the biologically active compound 
sought to be adsorbed at suitable reaction conditions; only the compounds 
possessing a specific affinity for the biologically active linked 
component will be selectively adsorbed from the complex mixture onto the 
surface of the adsorption complex carrier. 
A reversal of the above process, or a dissociation of the adsorption 
complex and separation of the soluble components thereof from the 
component adsorbed or linked to the biologically active carrier surface, 
or gel, as it may be called, occurs by varying the pH, the ionic strength 
of the solvent, the temperature thereof, and the like. The aforementioned 
process is of particular application in the separation and refining of 
enzymes, enzyme inhibitors, antibodies, antigens, nucleic acids, proteins 
linked to co-enzymes, and/or vitamins, repressor proteins, proteins with 
boundary scepters of toxins or hormones, proteins containing sulf-hydro 
groups, synthetically prepared peptides, and the like, including those 
biologically active components mentioned hereinabove. 
It is an object of the instant invention to avoid one or more drawbacks of 
the prior art. 
It is generally understood that the macroporous hydrophilic carrier 
(hydrogel) is a copolymer defined as a material with large inner surface 
of the pores measurable in the dry state and having physically stable 
pores, e.g., channells even in the contact with the solvent. This property 
gives the carriers a considerable mechanical strength and binding capacity 
in contrast to homogeneous xerogels mentioned in the prior art. This 
property makes the most important difference to all materials in the prior 
art. 
It is another object of the invention to provide for a method for the 
isolation of biologically active compounds by affinity chromatography. 
It is still another object of the instant invention to provide for a method 
of carrying out affinity chromatography upon selective adsorbents. 
It is a further object of the instant invention to carry out the above 
process by forming an adsorption complex between the compound which is 
desired to be isolated and the biologically active compound linked to a 
solid carrier. 
It is still another object of the instant invention to carry out the 
process by using as solid carrier, a hydrogel carrier. 
These and other objects of the invention will become apparent as the 
description proceeds. 
Broadly speaking, the instant invention relates to a method for the 
isolation of biologically active compounds by affinity chromatography by 
taking advantage of the formation of sorption complexes between a soluble 
compound (biologically active) which is desired to be isolated and a 
biologically active compound (which need not be the same compound) which 
is linked to a solid hydrogel carrier by a covalent bond therebetween. 
The carriers contemplated by the instant invention are the hydrophilic 
macroporous copolymers of hydrophilic monomers, such as: 
hydroxyloweralkylacrylates, i.e., methyl, ethyl, propyl, and through 
octyl; hydroxyloweralkylmethacrylates; oligo- or polyglycol acrylates; 
oligo- or polyglycol methacrylates; acrylonitriles; methacrylonitriles; 
aminoloweralkylacrylates (aminoloweralkylmethacrylates); acrylic or 
methacrylic acid and methylolacrylamide, polymerized with a cross-linking 
agent, such as: di-vinyl or polyvinyl monomers, such as, alkylene 
di-acrylates; alkylene dimethacrylates, oligo- or polyglycoldiacrylates, 
oligo- and polyglycoldimethacrylates; alkyl triacrylates and 
trimethacrylates; glycoltriacrylates or trimethacrylates; alkyl 
polyacrylates or polymethacrylates; glycolpolyacrylates or 
polymethacrylates, alkylene-bis-acrylamides, alkylene-bis-methacrylamides, 
or di-vinyl benzenes. The hydrophilic gels referred to above are employed 
in carrying out the instant invention by virtue of their capacity to carry 
the chemically linked biologically active compounds therein in a gel known 
as the first phase, which phase is brought into contact with a solution 
containing at least the compound which is desired to be separated 
therefrom. Components which are operative to form the sorption complex are 
then selectively sorbed and form a covalent link with the modified gel 
under given conditions in this first phase. In a second phase, the 
biologically active compounds which had been sorbed are desorbed and 
separated from the gel by changing the environment thereof, i.e., by 
changing the physical or physicochemical conditions, without, however, 
causing any damage to the activity of either the still linked if any, of 
the desorbed components. The cycle of the specific sorption and 
physico-chemical desorption may be repeated as long as the biological 
activity of the compound linked to the gel lasts. 
The gel material is based essentially upon hydrophilic esters of acrylic 
and methacrylic acids and can advantageously be used in the form of their 
discrete particles, preferably globular particles, or in the form of 
blocks, membranes, films, fibers, tapes, moving belts, or in any physical 
form so long as the gel material is operative to provide a necessary area 
of contact with the mixture containing the biologically active material 
sought to be separated therefrom. 
It is noted that acrylamide and methacrylamide are unsuitable as the 
primary monomer because the resultant polymer possess unsuitable 
mechanical, hydrolytic or thermal stability, as well as an inconsistant 
distribution of pore size and a lower molecular weight exclusion limit. 
Similar difficulties are encountered with methylene-bis-acrylamide or 
methacrylamide except that as cross-linking agents the 
alkylene-bis-acrylamides yield a lower hydrolytic stability but are 
otherwise acceptable. Cross-linking agents are always used in minor 
amounts in order to obtain insolubility or to control the swellng capacity 
and do not markedly affect the chemical properties of the resultant 
polymers. 
Similarly, it is noted that di-vinyl benzene is acceptable as a 
cross-linking agent, but styrene-di-vinyl benzene is hydrophobic and 
possesses considerable non-specificity and hence results in the 
non-discriminatory sorption of biologically active materials and is 
unacceptable. 
Preparation of the hydrogel copolymers useful as the carrier is carried out 
in accordance with polymerization procedures well known in the art as is 
the formation of the physical form thereof ultimately employed. 
It is to be understood, that throughout the disclosure herein, the method 
is described either by using a biologically active compound bound in the 
gel, which compound corresponds to that sought to be separated, or by 
using a different biologically active compound. It is not necessary that 
both compounds be the same; however, the ability of one biologically 
active compound to separate either a like compound or a dissimilar one is 
dependent upon the following: The isolation makes use of the ability of 
biologically active substances to form specific reversible complexes with 
other active substances. E.g., enzymes form specific complexes with their 
inhibitors, antibodies with antigens, toxins with antitoxins, receptors 
with hormones, etc. If one component of the specific complex is bound to a 
solid carrier, it is possible to adsorb the other component from the 
solution. 
The isolated biologically active compounds are prevented from denaturation 
by stabilizing their tertiary structure employing ligand links to the 
active centers thereof. The entire process is relatively rapid and enables 
one to obtain highly active products in only one operation. Considerable 
mechanical and hydrolytical stabilities of the hydrophilic macroporous 
carriers permit their use in the most convenient form for a given 
application in production. 
The above separation technique may be carried out either discontinuously by 
a batch method or continuously in repeated cycles, such as in a column or 
the like. In the continuous process, the gel carrier may be moved as a 
continuous medium, such as a belt or tape, along a conveyor mechanism and 
the solutions to be acted upon are stationary with respect thereto or a 
container holding the solution may move in the opposite direction. 
A great advantage of the separation by affinity chromatography performed in 
the above described manner is one rapid separation of the isolated 
biologically active compounds from inhibitors and destructive 
contaminants. 
All methods suitable for carrying out affinity chromatography as are known 
in the art are incorporated herewith by reference. 
The physical apparatus and/or technique employed in the actual separation 
method (i.e., discontinously or batch) is not critical to the invention 
and accordingly any of the well known procedures are operative. 
The instant invention, broadly includes the provision of a method for the 
isolation of biologically active compounds by affinity chromatography 
comprising forming a sorption complex between a solvent soluble 
biologically active compound to be isolated and a biologically active 
compound linked to a solid carrier by a covalent bond, said solid carrier 
being a hydrophilic macroporous copolymer derived from hydrophilic 
monomers selected from the group consisting of hydroxyalkyl acrylates, 
hydroxyalkyl methacrylates; oligo- and polyglycol acrylates, oligo- and 
polyglycol methacrylates; acrylonitrile, methacrylonitrile; aminoalkyl 
acrylates, aminoalkyl methacrylates; acrylic acid, methacrylic acid or 
methylolacrylamide and cross-linked by copolymerization with divinyl or 
polyvinyl monomers selected from the group consisting of alkylene 
diacrylates, alkylene dimethacrylates, oligo- and polyglycol diacrylates, 
oligo- and polyglycol dimethacrylates, alkyl tri- and polyacrylates, alkyl 
tri- and polymethacrylates, glycol tri- and poly- acrylates, glycol tri- 
and polymethacrylates, alkylenebis acrylamides, alkylenebismethacrylamides 
and divinylbenzene. 
In a broader aspect of the invention, it is seen that the copolymer can 
comprise a hydrophilic monomer selected from the group consisting of 
hydroxy alkyl acrylates and methacrylates, polyglycol acrylates and 
methacrylates, acrylonitrile and methacrylonitrile, aminoalkyl acrylates 
and methacrylates, acrylic and methacrylic acid or methylolacrylamide 
copolymerized with another member of said group as well as derivatives of 
acrylic and methacrylic acid such as substituted amides, alkyl esters and 
anhydrides or copolymerized with a minor amount of the above noted 
polyolefinic cross-linking agents. 
Initially, the hydrogel-sorption complex is formed by first preparing a 
hydrogel (as above defined) in accordance with copolymerization condition 
and techniques well known in the art. The thus prepared hydrogel is 
thereafter brought into contact with a solution containing at least, in 
soluble form, the biologically active material sought to be later 
isolated. The biologically active component may be present in the solution 
in amounts of mg to kg, in dependence on its solubility and on the 
capacity of the gel. The solvent for the biologically active compound may 
be any one of the following aqueous electrolytes and organic water- 
miscible solvents. The hydrogel is brought into contact with the liquid 
solution of the biologically active compound at temperatures of 0.degree. 
to 50.degree.C, preferably 0.degree. to 25.degree.C, under such conditions 
that there occurs an intimate contact between the two and thus the 
production of a bond therebetween (the first phase). Ordinarily the 
biologically active compound will first be placed in a buffer solution, 1 
to 10 parts compounds to 1 to 100 parts buffer solution; thereby creating 
a buffered solution of pH 2 to 11, preferably 3 to 10. The two buffered 
solutions are then allowed to contact and thereafter the sorption complex 
is formed. Suitable buffer materials include HC1 and other mineral acids 
as well as solutions of electrolytes and organic water-miscible solvents. 
The contact times between the buffered solutions, each containing its 
operative agent will ordinarily be for 1/2 an hour to 100 hours, 
preferably 1 hour to 8 hours, though the contact time is not critical. 
The thus sorbed biologically active material may be desorbed or dissociated 
from the hydrogel carrier by a technique know as the second phase. The 
second phase or desorption is carried out by bringing about a change in 
the physico-chemical environment of the hydrogel material containing 
thereon the biologically active material. Broadly speaking the 
dissociation may be accomplished by varying the pH of the environment, 
generally downward, such as from 1 to 11, preferably 1 to 10. The pH 
variance is accomplished by adding such amount parts acid donator to the 
solution which is necessary to achieve to necessary value pH. 
The biologically active compound may also be dissociated by changing the 
ionic strength of the solvent by adding thereto 1 to 60 parts per 100 
weight parts solvent, an electrolyte, or organic water-miscible solvent. 
The dissociation may also be accomplished by varying the temperature of 
the environment, such as from 0.degree. to 60.degree.C, preferably 
10.degree. to 50.degree.C for a period of time to effect destruction of 
the sorption complex, ideally that period of time will vary from 2 minutes 
to 10 hours. Such dissociationing carried out by admixing moving streams 
of the hydrogel carrier and the solvent solution.

The above described invention can be further defined and illustrated by way 
of the following Examples which are given by way of illustration only and 
are not to be interpreted as limiting. All parts, proportions, and ratios 
therein as wall as in the appended claims, are by weight unless indicated 
otherwise. 
EXAMPLE 1 
Isolation of highly active chymotrypsin by affinity chromatography on a 
hydroxyethyl methacrylate gel with covalently bound tryspin inhibitor was 
carried out as follows: 0.02 weight parts of crystalline chymotrypsin was 
dissolved in 1 weight part of 0.05M Tris-HC1 buffer solution of pH 8.0. 
The solution was supplied to a column (10 .times. 80 mm) containing the 
hydroxyethyl methacrylate gel, which had been prepared by a suspension 
copolymerization of 2-hydroxyethyl methacrylate with ethylene 
dimethacrylate in the presence of an inert solvent, the resulting 
copolymer had a molecular weight exclusion limit of 300,000. Pancreatic 
trypsin inhibitor was covalently linked to the gel which was then 
equilibrated with 0.05M Tris-HCl buffer solution having a pH of 8.0. After 
the sample had soaked into the column, the column was eluted with the same 
buffer solution using a flow rate of 300 ml per hour and fractions thereby 
obtained were collected at ten minute intervals. As soon as the fractions 
did not contain any compound absorbing in the ultraviolet region of the 
spectrum (at 280 nm), the elution with 0.05M Tris-HCl buffer solution was 
stopped and the gel column was further eluted with about 0.1M acetic acid 
solution having a pH of 3. The dissociation of the sorption complex 
occurred at the change of pH of the elution agent and chymotrypsin was 
eluted from the column which exhibited high activity after lyophilization. 
Its proteolytic activity to hemoglobin and esterase activity to 
acetylyrosine ethyl ester were measured at a pH of 8; both were related to 
1 mg of chymotrypsin. The chymotrypsin concentration was determined 
photometrically from the absorbance of chymotrypsin solution in 1mM HCl at 
280 nm. 
EXAMPLE 2 
The isolation of a highly active trypsin by affinity chromatography on a 
hydroxyethyl methacrylate gel with covalently bound trypsin inhibitor was 
carred out using the same procedure as is described in Example 1, with the 
exception that the copolymeric gel carrier had a molecular weight 
exclusion limit of 350,000 and that the esterase activity of the enzymatic 
preparation refined by the affinity chromatography (trypsin) was 
determined by means of benzoylarginine ethyl ester. 
EXAMPLE 3 
This example describes the isolation of antibodies of insulin by means of 
the antigen (insulin) covalently bound to an ethylene glycol acrylate gel 
having a molecular weight exclusion limit of 300,000. Antiinsulin serum (2 
weight parts) in 0.1M sodium barbiturate buffer having a pH of 8.8 
containing 3% of albumin was supplied to the column (8 .times. 40mm) 
packed with ethylene glycol acrylate - ethylene diacrylate copolymer, 
which copolymer contained therein, covalently bound insulin. The gel was 
equilibrated by washing with 50 weight parts of 0.5M phosphate buffer 
having a pH of 8.0 containing 0.8M NaCl. After the sample had soaked into 
the gel, the column was eluted with the same buffer solution. Pure insulin 
antibodies were eluted from the column using an acid solution (3N HCl) 
similarly as described in Example 1. 
EXAMPLE 4 
This example describes the isolation of papain SH-proteinase by affinity 
chromatography on a hydroxyethyl methacrylate gel (molecular weight 
exclusion limit of 300,000) with covalently bound p-aminophenyl mercury 
acetate. The swollen gel above, was activated by cyanogen bromide (50 
weight parts) and suspended into 20 weight parts of 10% aqueous 
dimethylsufoxide. A solution of 1 weight part of p-aminophenyl mercury 
acetate in 20 weight parts of dimethylsulfoxide was slowly added to the 
suspension. The suspension was then stirred with a magnetic stirrer at 
4.degree.C for 24 hours, heated to 30.degree.C and the gel was decanted 5 
times with 150 weight parts of 20% aqueous solution of dimethylsulfoxide. 
The gel suspension was finally used for packing a column and the gel was 
eluted with 500 weight parts of 20% aqueous dimethylsulfoxide at a flow 
rate of 10 ml/hr. The perfectly washed gel obtained in this way was packed 
into a column 10 .times. 60 mm and treated with 0.5% solution of papain in 
a standard buffer (0.5 weight % of butanol, 10 weight % of 
dimethylsulfoxide, 0.1M KC1, 0.5M sodium acetate, pH 5.0) adapted to 1mM 
ethylenediamine tetraacetic acid and 10mM Na.sub.2 SO.sub.3, as long as 
the absorbance A.sub.280 of the solution eluted from the column was equal 
to that of the solution fed into the column. The column was then washed 
with the standard buffer till the eluate absorbed at a wave length 280nm. 
The active papain sorbed at the gel was released with the standard buffer 
solution adapted to 0.5mM HgCl.sub.2. A highly active papain was obtained 
after desalting of the eluate on Sephadex G25 or after dialysis. 
EXAMPLE 5 
Chymotrypsin inhibitor was isolated from potatoes by means of affinity 
chromatography on a hydroxyethyl methacrylate gel which gel was 
crosslinked by ethylene dimethacrylate and carried covalently linked 
chymotrypsin. Potatoes (1500 weight parts) were homogenized in a 
high-speed blender and extracted with 200 weight parts of a mixture of 
0.9% NaCl and 0.03% Na.sub.2 SO.sub.3 solutions mixed in a ratio 1:1. The 
homogenized mixture was kept for 2 hours in a refrigerator at a 
temperature of +4.degree.C and then was centrifuged. The supernatant was 
filtered through a 0.5 cm thick layer of kieselguhr and lyophilized. The 
crude light brown extract was obtained in the yield of 33 weight parts. 
This crude lyophilized extract (from potatoes 12 weight parts) was 
dissolved in 75 weight parts of 0.2M Tris-HCl buffer solution of pH 8.0 
and after the whole amount had been dissolved the solution was adjusted 
with 1M NaOH to a pH of 8. The sample was again filtered through the 
kieselguhr layer and introduced into a column (14 .times. 455 mm) packed 
with a hydroxyethyl methacrylate gel carrying covalently linked 
chymotrypsin and equilibrated with 0.2M Tris-HCl buffer to pH 8. After the 
sample had soaked into the column, the column was eluted with 0.2M 
Tris-HCl at pH 8.0 by a flow rate 3.5 ml per hour and fractions were 
collected each hour. When the fractions indicated that they did not any 
longer contain any protein, the column was eluted with 0.2M KCl-HCl 
colution of pH 2.0. By changing pH in the column the inhibitor was eluted 
in a narrow zone. The inhibition activity of the isolated inhibitor 
increased 13 times by the single operation. 
EXAMPLE 6 
The hen ovoinhibitor was isolated from a crude ovomucoid by means of the 
affinity chromatography on a diethylene glycol acrylate gel with 
covalently linked trypsin. The crude hen ovomucoid was prepared from white 
of eggs by means of trichloroacetic acid and acetone (according to 
Lineweater and Murray [J. Biol. Chem. 171,565 c1947]) and the chymotryptic 
ovoinhibitor was isolated by the process analogous to that described in 
Example 5. The inhibition activity of the ovoinhibitor increased 30 times 
by this single operation. 
EXAMPLE 7 
The affinity chromatography of a commercial trypsin inhibitor from soybeans 
was carried out on a column of the hydroxyethyl methacrylate gel having 
the molecular weight exclusion limit 300,000 carrying covalently linked 
trypsin by a procedure described in Example 5. 
It is thus seen that in the present biologically active compounds are 
isolated by affinity chromatography. In the system of the invention, a 
sorption complex is formed between a solvent soluble biologically active 
compound to be isolated and a biologically active compound chemically 
linked by a covalent bond to a solid, or hydrogel carrier. The carrier is 
a hydrophilic macroporous copolymer as heretofore described. 
The isolation makes use of the ability of biologically active substances to 
form specific complexes with other substances, e.g., enzymes form specific 
complexes with their inhibitors, antibodies with antigens, toxins with 
antitoxins, receptors with hormones, etc. If one component of the specific 
complex is bound to a solid carrier, it is possible to adsorb the other 
component from the solution. 
A prime advantage of the separation by affinity chromatography performed in 
the above described manner is the rapid separation of the isolated 
biologically active compounds from inhibitors and destructive 
contaminants. 
The sorption complex system has increased porosity, mechanical and 
hydrolytic properties as compared to systems in which a biologically 
active compound is linked to a material such as polyacrylamides, glass, 
agarose or sepharose. 
The low degree of porosity of the otherwise essentially desirable solid 
carriers render them relatively ineffective as adsorbents for purification 
of enzymes of even low molecular weight. Synthetic polyacrylamide gels 
also possess many desirable features, but the porosity however, is 
diminished during the chemical modifications required for attachment of 
ligands and in this respect the polyacrylamide beads are inferior to those 
of agarose. While agarose has been recommended, experience with the use of 
this material explicitly shows that the mechanical and hydrolytical 
properties are unsatisfactory for the use in technological methods. 
The use of carriers on the basis of macroporous methacrylate gels 
significantly speeds up the process due to the good flow properties of the 
column and the quick adjustment of the carrier-substrate balance.