Note: Descriptions are shown in the official language in which they were submitted.
HOE 74/B 008 - Ma 187
1063049
The present invention relates to novel compounds of
biologically active substances and water-insoluble high molecular
weight compounds, a process for their manufacture and their use
in the affinity chromatography.
In the last few years, a new technique has become more and
more important within the biochemical operating methods, the
primary feature of which consists in the utilization of the
affinity of biologically active substances bound to a carrier
to reactions that are to be carried out selectively.
By way of a specific complex formation of the carrier-
bound substance with a second compound which is present in a
mixture, it is possible to eliminate the respective substance
from the mixture and, optionally, to isol&te the said sub-
stance subsequently by deæorption.
Carrier-bound enzymes have the advantage that trans-
formations of substances can be carried out in preparative
continuous processe~ and that the reaction products are ob-
tained free from enzymes.
In the biochemical enzymatic analysis, water-insoluble
enzymes are available as reagents to be used several times.
Due to the characteristic feature of the enzymes to show an
affinity not only towards the substrates, but also towards the
specific inhibitors, the obtaining of enzyme inhlbitors by
means of the affinity chromatography on enzymes bound to a
carrier has been found to be particularly advantageous. On t~e
other hand, the bond of inhibitors to water-insoluble macro- - -
molecular resin networks permits the preparative production of ~ -
the corresponding enzymes.
29 As so-called lmmunoadsorbents, antlgens or ant~bodies are
~; - 2 - ~ -
-~. . - . : - .. . . . . . .
.. . .... ,, . . . . . ~ . . - . . ~.. ; .. : . -
.. . . .. . . . .. . . . . . . .
, . . ~ ,. . .
. - , . .
HOE 74/B 008 - Ma 187
1063~349
bound to water-insoluble macromolecular resin networks and
permit subsequently the isolation of the corresponding anti-
bodie~ and/or antigens.
By biologically active substances there are to be under-
stood, according to the description given above, natural sub-
stances and substances that have been artificislly made which
are e~fective in vivo and in vitro, substances that can be
designated, in the broadeæt sense, as enzymes, activators,
inhibitors, antigens or antibodies, vitamins, and hormones.
These biologically active subætances may be termed "effectorsn,
since they represent the active principles of the water-in-
soluble systems.
Most of the effectors bound to a carrier that have been
descr~bed 80 far are considerably more stable than the cor-
1S responding substances in solution.
As carrier substances, so-called macromolecular resin
networks, there are to be used advantageously only those sub-
stances ~hich show, besides the insolubility in aqueous systems,
an unspecific adsorption that is aæ low as possible. This
means that hydrophobic, hydrophilic and ionic interactions
between the macromolecular resin network and the reactant of
the effector have to be largely prevented. A bond with sub- -
~; stances which do not represent a reactant of the effector should --
be excluded.
The resin networks used 80 far as carriers for biologically
active substances can be æub-divided into those b~nding the
effectors by physical adsorption - this group includes
activated charcosl and glass beads -, and those whlch are bound
29 to one another with the effectors by a covalent bond.
- 3 -
. -, . .
. ~ , .
- ~ - - ,... , . - . . . .
.. . , . ~ ~ ,
,. ,: , . ; ,
~ HOE 74/B 008 - Ma 187
' 1063~)49
The latter include vinyl polymers, for example, polyacrylic
acids, polyacrylic acid amides and amino-, carboxy- or sulf-
onyl-substituted polystyrene, moreover, cellulose and its
derivatives and, finally, natural and synthetic polypeptides
~5 and proteins. Due to the balanced interaction between the
resin network and the effector, the use of carbohydrates, in
particular of cellulose, dextran, starch, agar and/or their
derivatives, as network in aqueous systems has become more and
more common, although the carboxylic groups that are frequently
contained in these natural substances are often felt to be
inconvenient, due to their unspecific reaction. Besides,
these substances show a relatively poor thermal and chemical
stability.
It has now been found that the disadvantages shown by the
carbohydrate derivatives as ~etworks in reactions depending on
affinity can be overcome if polyhydroxymethylene is used as
resin network.
The present invention provides biologically active
compounds which contain a water-insoluble poly(hydroxymethylene),
to which a biologically active substance is bound, while main- -.
taining its biological activity. In these compounds, - --.
a) the carrier network is polyhydroxymethylene,
b) the biologically active substances are substances such as
enzymes, activators, inhibitors, antigens or antibodies,
other plasmaproteins, blood group substances, phythem-
agglutinins, antibiotics, vitamins or hormones, peptides
or amino acids, or synthetically prepared efectors,
c) polyhydroxymethylene and the biologically active substances
are bound to one another by a covalent bond, direct or by
:~ - 4 - :
.. . ~ .... . .... . .. . , ,, - , .. . . . . . . . .
' ' ''"'; ' "'' .' , ' " '."' ' ''' ' " '. '' ', ' ~' ' ' .', ' .' ~ " ' . ' , ' '
HOE 74/B 008 - Ma 187
1~63049
way of an intermediate member, a so-called "spacern.
Polyhydroxymethylene is a synthetic polymer with a con-
tinuous carbon chain. Each carbon atom carries a hydrogen
atom and a hydroxyl group. Its preparation and its properties
have been de~cribed, inter alia, by N.D. Field, J.R. Schaefgen,
J.Polymer Sci., vol. 58, 533-543 (1962) and G. Smets, K.Hayashi,
J. Polymer Sci., vol.29, 257-274 (1958). It can be prepared by
way of basic or acid hydrolysis from polyvinylene carbonate.
In a broader sense, the polyhydroxymethylene is to be under-
stood as a derivative of polyvinylene carbonate. The bond of
; the effectoræ to the polyhydroxymethylene can be obtained, on
one hand, by the measure used for the covalent bond of the
effectors with the hydroxyl group, and on the other hand~ by
reacting the polyvinylene carbonate by way of the equally kno~m
am~nolytic reaction of a cyclocarbonte ring of the polyvinylene
carbonate with a prlmary amine, for example, with hexamethylene
diamine, to obtain a polyvinylene carbonate substituted by a
~pacer or effector over a urethane bond, and by subsequently
saponifying the remaining cyclo-carbonate groups of said poly-
vinylene carbonate to give hydroxyl groups:
._ -CH---C~ ~ CH - c~L CH - CH ~
l l ~ H2 l l OH 1 saponi-
~C ~ --> ~ucf I fication
~ ~ o o Jn 1 ~H
polyvinylene carbonate
H - ~ -~H-~
OH OH OH O
. _ 1l-1 C = o
NH
'
-
.. ,,, ... ;
\ HOE 74/B 008 - Ma 187
1063049
It is especially advantageous for the application of
the products if the biologically active effectors are not
reacted directly with the polyhydroxymethylene, but are linked
~ to the network by a covalent bond by way of an intermediate member
termed "arm" in the affinity chromatography or known by the
term of "spacer" in British literature.
The spacer substances are in most cases hydrocarbon
structures of a length of about 1 - 2 nm. ~wo functional end
groups of the spacer permit in each case the reaction with the
network as well as with the effector; in the reaction with the
network, polyhydroxymethylene as well as polyvinylene carbonate
may be used under appropriate conditions.
The present invention also provides a process for the
covalent binding of the biologically active substances to the
carrier.
To reach this goal, use can be made of a series of
generally known reactions:
The binding of the effector to the network is effected in
a particularly simple way a~fter an activation of the hydroxyl
groups of the polyhydroxymethylene by means of cyanohalides,
advantageously by bromocyanogen, and a subsequent reaction of
the biologically active effectors containing amino groups by
way of these activated groups.
Another process - which is a variant of the azide method
described by Curtius - comprises reacting polyhydroxymethylene,
instead of polysaccharides used by Curtiusj;with a halogeno-
carboxylic acid, such as chloroacetic acid, in an alkaline medium,
esterifying the corresponding acid with aloohol, subsff~ntly
oonver~ the ester into the hydrazide, and finally binding the resulting
- 6 -
HOE 74/B 008 - Ma 187
1063~49
azide with the amino group of the effector protein to the
polyhydroxymethylene network.
Another possibility of binding the effector to a poly-
hydroxymethylene network consists in the acylation of the
hydroxyl groups w1th bromo-acetylbromide, followed by the
alkylation of the amino group of the effector.
Similar reactions are obtained, for example, by reacting
the hydroxyl groups of the carrier with reactive triazines;
in this çase, a part of the reactive groups of the triaz1ne
is reacted with the polyhydroxymethylene compound, another
part reacts-with amino groups of the effector.
Diazotizable aromati~ amines which may be linked, on one
hand, to the hydroxyl groups of the carrier by way of a further
reactive group, permit on the other hand the coupling with
appropriate activated amino acids, for example, the tyrosine
or histidine radicals of the protein effector.
Vinylsulfone derivatives containing arylamino groups and
sulfuric acid semi-esteræ of B-hydroxyethylsulfones can be
reacted with the hydroxyl groups of the carrier. They make the
binding of the effector possible according to the above-
mentioned diazotizing reaction.
A particularly stable ether bond is obtained in the
reaction of the hydroxyl groups of the polyhydroxymethylene
with epoxides which do not form ions and which contain at leaæt
2 reactive groups, such aæ the epihalohydrines or the poly-
epoxides, for example, epichlorhydrin or bisepoxide.
Besides the processes mentioned above as examples for
the preparation of the covalent bond between the carrier net-
29 work and the protein effector or other effectors, there may be
. - . - . - . - . . ., .. , ~
-
,. . .
.
.
1063049
mentioned further methods which lead to the reaction of the
hydroxyl groups of the polyhydroxymethylene on one hand, and
to the reaction of determined groups of the effector on the
other hand, thus forming a covalent bond between the two
reactants, such as the know reaction with complex-forming
metal compounds, for example, titanium compounds.
Besides by its above-mentioned chemical and thermal
stability, the polyhydroxymethylene is marked by advantageous
processing properties, thus becoming superior to the carrier
networks on the basis of natural carbohydrates that had been
considered optimal up to this time. Polyhydroxymethylene can
be manufactured, for example, in the form of fibers, filaments,
films or spherical particles or, perferably, powdered material,
so that the most appropriate form may be chosen, depending on
the purpose of application of the effector to be bound to it.
Another advantage of the polyhydroxymethylene as com-
pared against the carrier materials according to the state of
the art is to be seen in the fact that the surface which is
accessible for the bond of the effectors and/or the spacers
can be controlled during production.
According to the analogous reaction mechanisms as they
have been described for the bond of the effector with the poly-
hydroxymethylene network, the spacer substances can also be
bound to the network, for example if the spacers carry an
amino group as one of the functional groups. The introduction
of a spacer also makes it possible, however, to introduce
~ additional reaction possibilities for the bond of the effector.
- In the case that the spacer bound to the network carries a-~ free carboxyl group, this carboxyl group can be activated, for
- 8 -
_, - - ~ . , ~ - .
- : , . . : . , , , , -~ . , .: : .,
. ' . . .. , : ; ., : :, ~
.
HOE 74/B 008 - Ma 187
.
1063~49
example, by carbodiimide compounds and can then form an amide
bond with amino groups of the effector. Carboxyl groups also
permit the formation of amide bonds by means of the isoxazolium
salts introduced by Woodward.
If the spacer bound to the network carries an available
free amino group, the introduction of arylamino groups in the
extension of the spacer is possible, by means of vinylsulfone
derivatives containing arylamino groups or sulfuric acid esters
of B-hydroxyethylsulfones, said arylamino groups subsequently
being diazotized according to known methods and then bond to
correspondingly reactive groups of the effector, for example:
, . . . ..
r
. n
0 NH- (CH2) 6-NH-CH2-CH2 s 2 ~ -NH2 .
... . ...
The biologically active compounds of the invention are
suitable for most application processes that have so far been
known for other effectors bound to hydrop~ilic water-insoluble
carriers. This means that enzymes can be made water-insolub~e.
The insoluble enzymes are more and more used to deterDine sub-
~trates in automatic analyzers and as so-called enzyme elec-
trodes. Due to the increased stability, a series of carrier-
bound enzymes is suitable for the implementation of enzymatic
reactions on an industrial scale.
Carrier-bound biologically active substances are used on
a large scale in the afflnity chromatography, due to their
properties as specific adsorbents. By means of carrier-bo~d
29 natural or synthetic enzyme inhibitors, a high degree Or purity
_ 9 _
.. . . .. . . . .. .. . .. . .. . ..
. . , ~ . , . . - , . . ~ . -
- . . , . .. ~ . -
. . . .. , . . . , . - - - . .. . . . . .. . . . .. . .
: . . . , ~ . .~, ,
, : . . ~ . - -
HOE 74/B 008 - Ma 187
1~63~49
is obtained for enzymes, whereas the enzymes have been found
to be extremely suitable as effectors particularly for the
production of natural enzyme inhibitors from raw extracts.
Carrier-bound water-insoluble antigens are used for the iso- -
lation of the corresponding antibodies, which are thus obtained
free from other serum constituents and free from other antigens.
By means of affinity chromatography it is also possible to
isolate those antibodies that cannot be precipitated, as well as
those that exclude precipitation due to their low concentration
- 10 in the serum, and also to determine them quantitatively.
The Examples given below in order to illustrate the in-
vention show that a carrier prepared according to a single
process is suitable for the bond of many different effectors
and that the basic structure of polyhydroxymethylene can be
;~ 15 made suitable, by an appropriate substitution, for the bond of
any desired effector.
Moreover, the Examples show how the effectors bound to
the network can be used.
. \
'' \'
~1 . ' \ .
\
.' ' , \
\_
~ - 10 -
.
~, ' ' ~ . .. .
' .': , . ' .
:
- ' ~ , :
- ~ HOE 74/B 008 - Ma 187
1063~49
E X A M P L E ls
Pol~hydrox~meth~lene-tetanu~ toxoid compound
Preparation of the network from (~ -amino-n-hexyl~-substituted
pol~h~droxymethvlene: -
20 Grams of polyhydroxymethylene suspended in 900 ml of
water were mixed with a solution of 20 g Or BrCN in 50 ml of
--dimethylformamide and were maintained at a pH ~alue of 11.5
for 6 minutes at an internal temperature Or 15C by 810wly
adding dropwise, while stirring, 2 n sodium hydroxide solu-
tion. Subsequently the mixture was suction-filtered i~nediate-
ly, was washed thoroughly with ice water and was squeezed to
remo~e the remaining water. The polyhydroxymethylenc thus
activated, which was still moist with water, was added to
a well-stirred solution of 20 g of hexamethylene diamine in
500 ml of H20, which was maintained at room temperature and
was ad~usted with concentrated HCl to a pH value of 8.5,
the mixture was then made up with water to a total ~olume of
1 liter, while the pH value was constantly ad~usted between
8.5 and 9.0, and was stirred at room temperature for another
5 hours. SubsequQntly the reaction mixture was suction-fil-
tered and was washed thoroughly with water. A dried sample
of the polyhydroxymethylene substituted by (~ -amino-n-he~yl)
group~ had a nitrogen content of 1.9~. By means of Sanger~
reagent for the detection of primary free amino groups, pro-
ducts we~e ob$iained which had a ~trong yellow color shade.
Preparation of the carrier-bound effector
Effeotors Tetanus antigen
10 Grams of the carrier of EY.ample 1 were suspended in
- 29 200 ml of a 0.5 molar sodium pho~phate ~olution ha~ing a pH
'- - 11 -
." ' '
. ~ .: : i . : . ~ . . - , :
: .,............ ; . . , : . ,. , ~ -. : .. ~ ' .~ : . :
. ; i , . ; ~.
HOE 74/B 008 - Ma 187
_,~
1063~49
value of 10. The suspension wag heated, while ~tirring, at
50 C and was mixed with 5 g Or l-aminobenzene-4-B-hydro~yethyl-
sulrone-sulfuric acid-semi-ester. After ~he pH value had
been corrected to 10 by means Or 2 molar NaOH, the reaction
mixture was- stirred for 1 hour at 50C. Subsequently the
mixture was suction-filtered by means of a strainer and was
washed with 2 1 of water, 2 1 Or acetone and 2 1 of 0.2 molar
HCl. ~or the diazotizing Or the arylamine groups introduced
into the polyhydroxymethylene, the filter residue was su~-
pended in 200 ml of 0.2 molar HCl, was cooled to 2C and
was mixed, while stirring, with 1 molar NaN02 solution until
a slight exces~ Or nitrite could be detected by means of
KJ-starch paper. After 10 mimlteJ the reaction mixture was
suction-filtered by way of a stra$ner, and the filter residue
was washed with 1 1 Or an aqueous 0.01 molar amidosulfonic
acid Or a temperature of ~C and then with 200 ml Or 0.2 mo-
lar sodium phosphate of 2C and a pH ~alue of 7.5. For the
coupling, the.product containing diazonium groups was sus-
pended in 150 ml Or a solution of 2 g Or tetanus toxoide with
a 1260 Lf/mg Or N in 0.2 molar sodium phosphate Or a p~ value
of 7.5 and a temperature Or 2C, and was stlrred for 20
hour~ at 5C. Those portions of the toxo~de that had not been
bound were washed out with 1 1 of 1 molar NaCl solution on a
trainer.
; 25 AD~lication Or the compound of polvhydroxvmethylene-tetanus
toxoide
Preparation of tetanu~ antitoxin
10 Grams of the product described in Example 1 were ou~-
29 pended in 150 ml Or o- 15 molar NaCl solution~ with the addi-
:~ ,. .
~- ~ tion of M/15 sodium pho~phate, ("PBS") of a pH ~alue Or 7.2,
~` _ 12 -
.; ~ ; ' .
". ' ~' ' ' .
~OE 74/~ 0~8 - Ma 187
1063049
and wero introduced into a column of a diameter of 3 cm and a
height of 10 cm. 20 Millimeters-of tetanu~ horse serum with
1650 IU of tetanus antitoxin/ml were introduced into the co-
lumn which was subsequently washed with 500 ml of PBS, pH
value 7.2. The elution of the antibodies was effected with
0.5 molar glycine/HCl buffer, pH value of 2.5. After neu-
tralization with 2 molar NaOH and subsequent centrifuging,
the eluate contained a total of 240 mg of protein and 12.300
IU of ~ ~*~in as was found in a protection test with mice.
E X A M P L E 2: -
Pol~hvdroxymethylene-aminobenzamidine compound
- Preparation of the network from (~ -carbox~-n-pentyl~ subst~-
tuted_pol~h~droxv~eth~lene-
20 Grams of polyhydroxymethylene activated with BrCN
according to Example 1 were added to an aqueous solution of
25 g Or ~-amino-caproic acid that had been adjusted to a pH
value Or 8.5 by mean~ of diluted sodium hydroxide solution,
and the total volume of which had been made up to 1 liter.
The reaction mi~ture was then stirred for another 5 hours at
room temperature and a pH ~alue of 8.5 and was suction-fil-
tered, then the product was wa~hed thoroughly with ~ater.
dried sample of the polyhydroxymethylene substituted by (~ -
carboxy-n-pentyl) groupq had a nitrogen content of o.86~.
Preparation of the carrier-bound effector
Efrector: Aminobenzamidine
2 Grams of a polyhydroxymethylene derivati~e prepared
aocording to ExamplQ 2 were ~uspended in 25 ml of 0.1 molar
morpholino-ethane-sulfonic acid and wero mixed with 1 ~ of
29 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide-HCl. By ad-
- 13 -
.
' ~ , . ' . ',
, ' ' , ,'
~ , . ' . .
NOE 74/8 008 - Ma 187
. 1063049
ding 2 molar HCl while controlling the pH value, *he pH wa~
adjusted to 4.8. 1 Gram of m-aminobenzamidine was added to
the reaction mixture while stirring. The su~pension was
stirred for 1 hour while correcting the pH value, and the
adsorbent was subsequently introduced into a chromatography
column ha~ing a diameter Or 1 cm. The adsorbent was then
rinsed with 200 ml of 0.05 molar trishydroxymethylaminomethane-
HCl-buffer solution, pH of 8.0, with an addition of 0.5 molar
KCl (tris-KCl).
Application of the compound of polyhvdroxvmethylene-aminobenz-
amidine
Preparation of thrombln ~C 3 ~ ~/s)
(~C3 y~/5~
100 Milligrams of bovine raw thrombin~ were dissolved in
5 ml of tris-KCl~ and were introduced into the column which
had been pr0pared as has been described a~ove. The column
was subsequently rinsod with 200 ml of tris-KCl buffer solution.
The thrombin bound to the insolubilized m-aminob~nzamidine
was separated from this compound with 100 ml Or a solution of
0.05 molar m-aminobenzamidine in tris-KCl buffer.
The fractions containing protein were combined and were
pas~ed over a column of l x 30 cm of sephadex G-25 into a tris-
KCl buffer solution. In the first peak of the column eluate
- the peak containing the protein - 78% of the initial activity
- could be detected by determining the activity according to
Chase and Shaw, ~iochem. 8, 1969, page 2212.
:
_ 14 -
.
r . - . . - . ' ' '. ' ' ~ ' ' ' ' '.' . ;' .
, ' , ~ .. . . ,; , ,.
,., ' ; ' ". "; ' '
'' ' '~ " ' ' ~ ; ' '' ,
' ~ ' , ' ' ' , ' . '
..
~ HOE 74/~ 008 - Ma 187
1063~49
E X A M P L E 3s
Pol~hydroxymethylene-popsin compound
Preparation of the net*ork from (~ -amino-n-hexyl)-substituted
polyh~droxymethylenes
. 8.0 Grams of polyvinylene carbonate dissolved and repreci-
pitated from dimethylformamide and methanol, which product
- had been prepared according to N.D. Field, J.R. Schaefgen,
J. Polymer Sci., vol. 58, p. 534 (1962), were suspended at
room temperature in a solution of 7.5 g of hexame~yle~e d~m~ in
180 ml of methanol and were stirred for ~8 houri at room tem-
perature, then suction-filtered, washed with methanol and . :
suspended in a solution of 10 g Or sodium methylate in 300 ml
of ~ethanol of 96~ strength for 4 days at room temperature,
and were then washed with methanol and vory thoroughly with
watër. A dried sample of the polyhydro~ymethylene adsorbent
substituted by primary amino groups over a spacer of 6 CH2
groups had a nitrogen content of 5.7%.
Preparation of the carrier-bound effector
B Effectors Pep8in ~C 3~
20 Gram~ of (~ -amino-n-hexyl)-substituted polyhydroxy-
methylene prepared according to Example 3 were suspended in
400 ml of 0.2 molar sodium phosphate, pH ~alue of 10. 40 Hilli-
liter~ of a glutardialdehyde solution o~ 25% strength were
added to the suspension, while stirring. After having been
25. stirred for 2 hours at room temperature, the reaction mi~ture --
wa~ filtered off by means Or a strainer, and the filter resi-
due was washed with 4 liter~ of 0.2 molar sodium acetate buffer
solution, pH value of 4.0, Subsequently the reaction product
. ~C3~-4-~)
29 was introduced into 500 ml Or a solution Or 5 g.of pepsi~ with
. - 15 -
.
.:
,,. . .. -, , . : : , .: . . - ,, ,: . .
~OE 74~B 008 - Ma 187
1063049
a total of 500.000 units, determined according to Anson, ~n
0.2 molar sodium acetate, pH value of 4.0, and was then
~tirred for 15 hours at 6C. The product obtained was then
washed on a ~trainer with 5 liters of 0.1 molar sodium acetate/
acetic acid, pH value of 4.0, and an addition Or 1 molar
NaCl.
A~plication of th~ compound polyhvdroxymethylene-pePsin
Proteolytic separation of immuno~lobulins
.
The adsorbent of the present Examplè wa~ suspended in 0.1
molar acetate buffsr solution, pH value of 4.0, and the acti-
vity was dermined according to Anson. The su~pension contained
~ EC ~ 4 /)
B a total of 21.000 units of pepsin~in a bound state which could
be detected by mean of thi~ te~t. For the proteolytic ~epara-
tion of human immunoglobulin G, the pepsin adsorbent ~ ~
filtered off and the residue was suspended in a ~olution of
2.5% strength of 30 g of human IgG in a physiological NaCl
solution, pH value of 4.1. The separation mixture was ~tirred
~or 24 hour~ at 37C. After elimination of the carrier-bound
pepsin by filtration the filtrate was added to 1250 ml of
~aturated ammonium sulf~te solution. The precipitate obtained
in this process was then removed by centrifuging; it contained
;~ the ~(ab)2 ~ragment of the immunoglobulin with the antibody
activity of the latter. The residue liquid was re~ected.
,
E X A M P_L E 4s
~re~aration of the com~ound Or ~olvhvdroxvmeth~lene-o~amic acid
10 Grams Or polyhydroxymethylene were activated with
BrCN a~ has been described in Example 1 and were reacted with
5 g o~ tyramin dlssolved in 150 ml o~ 0.1 molar 30dium~ydro-
29 geno-carbonate, at a temperature of 5C for 60 minutes, while
- 16 -
"
.. . , ... ~ . . . ... ..
: . , .:, . . :
- - . '
.. . . . . . .. . . . .
. , ., .:
~ HOE 74/B 008 - Ma 187
1063049
stirring. Those portion~ of the tyr~min which had not been
bound were suction-filtered by mean~ of a strainer, and the
filter residue was washed with 1 1 of 0.1 molar sodiumpho~-
phate buffer solution having a pH value of 7.0 and a tempera-
ture of 5C. The filter residue was suspended in 150 ml of
a solution of 1.0 g of diazotized p-aminophenyloxamic acid,
the solution having been cooled to 5C, and the mixture was
stirred for 15 hours at a temperature of 5C. The adsorbent
wa~ washed on a s*rainer with 1 1 of 0.05 molar sodium ace-
tate-acetic acid buffer solution, pH value of 5.5, with an
addition of 2 mil~les of CaC12 and 0.2 mM of EDTA,
~pl~cation of_the comnound of nolvhvdroxvmethvlene-oxamic acid
~ . . .
o~ r ~-~r~ 3~l~
The adsorbent according to E~ample 4 was resuspended in
15 the above-mentioned acetate buffer solution and was introduced
into a chromatography column having a diameter Or 2 cm. 1 Li-
ter of a culture filtrate of Vibrio Cholerae with 800 IU of
~3-Z~I-I~)
neuraminida~/ml were passed through this column,- the filtra-
te having been adJusted beforehand with 2 molar acetic acid
to a pH ~alue Or 5.5 and having been ~ixed with CaC12 and
EDTA up to a concentration Or 2 millimoles and 0.2 milli-
mole~ respec*ively. Tho~e proteins which had not been
bound were ~ashed out by means Or 500 ml of acotate buffer
solution.
The elution o f the neuraminidaoe adsorbed to the carrier-
bound inhibitor was effected by washing the column with 0.1
molar sodium-~ydrogeno-carbonate solution and collecting the
fraction~ containing active neuraminidase. They contain ~6
29 f the ~ctivity used.
1? :
.
.. ,. . "..., . ~ .. ;., .,.... ...., , -
... .. . ... . . .. . . ., .... . . . , . ~ . ~
.. . . . . . , . ., . . . . . .. .. .. - . .
.
, . . . : . - . ~.
HOE 74/B 008 - Ma 187
-- 1063049
E X A M P L E 5:
a)Reaction of polyhydroxymethylene with epichlorhydrin
50 Grams of polyhydroxymethylene were suspended in 1 1
of 2n NaOH, were mixed with 250 ml of epichlorhydrin and
were then stirred for 2 hour~ at a temperature in the range
Or from 55 to 60C. The pH value of the suspension was re-
duced after a short time to 10 - 11. Ihis pH ~alue wa
maintained for another hour by adding NaOH. After a reaction
time of 2 hours, the solid matter was suction-flltered, and
was washed with water, acetone and finally again with water.
. .
b) 50 Grams Or hexamethylene-diamine were dissol~ed in 1.5 1
of water and were mixed with HCl up to a pH value of 10. The
poly~ydro~ymethylene activated under 5a) was added to the
solution and was stirred for 6 hours at a temperature in the
- range of from 50 to 55 C. Then the product was 6uction-fil-
tered and was washed with water until it was free from hexa-
methylene-diamine.
¢) The product obtained under Example 5b) was stirred with
50 g of 1-aminobenzene-4-B-hydroxyethyl-sulfone-sulfuric acid
e~ter for 1 hour at 55 C and a pH ~alue o~ 10. Subsequently
th0 ~olid matter was filtered ofr, and was then washed with
water, acetone and again with water.
d) 10 Grams 0r the product obtained ac¢ording to 5 c) were
; - washed on a strainer with 200 ml Or 0.1 n HCl and were after-
25- wards sus~ended in 300 ml Or 0.5 n HCl. At a temperature in
the range Or ~rom 0 to 4C, the suspension was mixed, while
Jtirring, with 0.1 n NaN0~ solution, until a Rlight excess ~-
-~ of nitrite could be detected in the diazotl~ing ~ith KJ-starch
29 pap~r. Af ter 10 minutes, the reaction mixturq was suction-
_ 18 -
'
.. . . . , .. , .: .. ,, - , , . . . ~ : , -
,
HOE 74/B 008 - Ma 187
1063049
filtered by way of a ~trainer, and the residue was washed
with ice water and subsequently with 0.15 molar sodium phos-
phate buffer solution having a pH value of 7.5 and a tempera-
ture of from 0 to 4C.
e) 0.75 Grams of albumin were dissolved in 250 ml of phos-
phate buffer solution, pH value of 7.5, were then cooled to
4C, and the produot prepared under 5d) was added. The sus-
pension was stirred at 4C for 20 hours, was then filtered
off, and the so~id matter was washed with 1 molar NaCl and
a phosphatebuffered solution Or sod-i-um chloride (P~S) (aque-
ous NaCl solution of 0.9% strength with a content of 1/15 mo-
les of Na2}3P04-KH2P04 buffer of a pH value of 7.2).
The filtrate and the washing lye~ were tested for albumin
according to the method o~ the radia~ imm-~odif~usion. 75 Mil-
~5 ligrams o~ albumin w~e-bound to 1 g of the carrier thus pre-
- pared.
- r) 2.4 Grams of IgM were dissolved in 250 ml of phosphate
bufrer solution, pH of 7.5, and were ~ooled to 4C. 10 Grams
o~ the product diazotized according to 5d) were added, and
the suspension was stirred at 4C *or 20 hours. A~ter filtra-
tion the solid-matter was wsshed wlth 1 molar NaCl solution
and wlth PBS.
1 Gram of the carrier prepared according to 5 f) was able
to bind 240 mg of IgM.
~ ~ A ~ L 6 s
a) A~ has bcen described in Example 5 a)b?, 10 g of poly-
hydroxy~thyle~e were activated wi~h epichlorhydrin, were
reacted with hexamothylel1e-diamiIla and wore worked up.
; 29 b) 10 Grams o~ the carrier thus prepared were`succinoylated
- 19 ~
:
;,. . -
,
. .
HOE 74/B 008 - Ma 187
. .
1063~49
for 4 hours at 10 C and a pH ~alue of 6, with 5 g of succinic
acid a~ydride suspended in 200 ml of water. The pH value
was adjusted with 2 n NaOH. After washing the solid matter
with water, the product was Rtirred at 5C for 30 minutes,
at a pH value of 5, with 2.5 g of N-cyclohexyl-N'-(-(N-methyl-
morpholino)-ethyl)~carbodiimide-p-toluene-sulfonate, was
filtered off and was washed fast with ice water.
c) 1 Gram of IgG was dissolved in 250 ml of a phosphate
buffer solution, pH value of 7.5, and wa~ stirred for 24
hours at 4C with the carrier prepared under 6 f). After
filtration, the product wa~ washed out with 1 molar ~odium
chlor~de and with PBS.
85 Milli~rams of IgG were bound in a covalent bond to 1 g
of the carrier.
E X A M P L E 7s
a) 20 Grams of the product prepared under 5 a) were stirred
for 6 hours, at a temperature in the range of from 50 to 55 C
and at a pH value of 10, with 20 g of aminocaproic acid. Sub-
~equently the product was suctlon-filtered and washed with
water-
b) 10 Grams of the product prepared'under 7 a) were mixed
with 2.5 g Or N-cyclohesyl-N'-(N-methylmorpholino)-ethyl)-carb- -
,
odiimide-p-toluene-sulfonate, and after 5 minutes, 2 g of
~ N-hydroxysuccinimide were added which had a pH value of 5.
- 25 After having been stirred for 5 hours at 24C, the ~olid
matt-r wao filtered off and was washed with water.
c) 0.5 Gram of albumin were dissolYed in 200 ml of phosphate
buffer solution and were stirred for 24 hours, at a tempera-
29 ture of 4C, with the activated carrier propared under ~ b).
.
- 20 -
- . ,: . ,
HOE ?4/B 00~ - Ma 187
-- 1063049
After filtration, the product was washed out with 1 molar
sodium chloride and with PBS.
- 50 Milligrams of albumin were bound in a covalent bond
to 1 g of the carrier.
5 E X A M P L E 8:
10 Gram6 of polyhydroxymethylene were activated with
epichlorhydrin as has been described ill Example 5 a) b), were
reacted wlth hexaJmethylene-diamine and were worked up.
a) 10 Grams of the carrier thus prepared were suspended in
1Q- 100 ml of water and were reacted with 500 ml of glutardialde-
hyd~ of 25% strength. After having been stir~cd for 1 hour~
the solid matter was flltered off and was washed with water
and/or PBS.
b) 0.5 Gr~m of albumin were dissolved in 200 ml of a phos-
p'.late buffer solution and were stirred for 20 hours at 4C
~ith the carrier prepared under 8 a). After filtration, ths
product was washed with 1 molar sodium chloride and with PBS.
E X A M P L E 9s
D~rect reaction of polyhydrox~ethylene activated with
20 - epichlorhydrins - -
a) 10 Grams of polyhydroxymethylene were activated with 50
ml of epichlorhydrin as has been described in Example 5 a).
After filtration, the product was washed fast with water,
acetone~water, and finally with PBS.
b) 0.5 Gram of albumin were dissolved in 200 ml of PBS and
were stirred for 60 hours at 4C with the product prepared
under 9 a). After filtration, the carrier-bound protein
was washed with 1 molar sodium chlorlde and with PBS.
1 Gram of the carrier thus prepared was able to bind 45
mg of albumin. Ik . . ` .
~ . - 2~ -
, ~- .
- ; . . . . ....
.. , . . . . - . , . . .: .
':~ ''. `.' ~- ........ ~:
'' . ;'' ' :
HOE 74/B 008 - ~a 187
.
1063049
E X A M P L E 10 -
10 Grams of water-insoluble polyhydroxymethylene were
stirred at a pH value of 9.5 (0.15 molar sodium bicarbonate/
. NaOH buffer) for 3 days-- at 25C with 30 g of
diethylene glycol-diglycidylether; the product was suction-
filtered, was washed thoroughly, was added to 0.5 g of human
albumen in 200 ml of 0.15 molar phosphate buffer having a pH
value of 8, then the reaction mixture was stirred for 60 hours
at 10C. After filtration, the carrier-bound protein was
washed with 1 molar sodium chloride solution and with "PBS".
- 1 Gram of the carrier thus prepared contained 35 mg of
bound albumen.
E X A M P L E 11:
10 Grams of polyhydroxymethylene were reacted with 30 g
f glycidyl-tris-(glycidylether), as it has been described in
Example 10, were introduced into 0.5 g of immunoglobulin G
(IgG) in 200 ml of 0.15 molar phosphate buffer having a pH
value of 7.5, and the reaction mixture was stirred for 60 hours
at 4C. After filtration, the product was washed with 1 molar
sodium chloride solution and with "PBS".
1 Gram of the protein resin thus prepared contained 50 mg
of bound IgG.
.
' .
. . _ 21 a - -
,
.,~ , , ~ . . . . ... .. .. . ,5,-
, ,
