Note: Descriptions are shown in the official language in which they were submitted.
~LV5~ 3
The invention relates to modi~ied immune globulines, a
process for preparing them and pharmaceutical compositions
containing modified immune globulines~ It especially concerns
chemically modified immune globulines which may be applied via
the intravenous route.
Immune globulines prepared by fractionation from serum,
especially from human serum have the essential property to act
as antibody against antigens.
Immune globuline compositions have hitherto proved suitable
only for intramuscular administration. In the case of intra-
venous administration the recipients have shown a more or less
marked reaction with anaphylactoidal effects.
It is supposed that these secondary rea~tions are due to
the fact that the serum complement is bound by the immune glo-
buline administered. On the other hand an intravenous immuneglobuline composition is desired since it deploys its activity
faster in the organism.
It has been attempted several times to modify the immune
globulines inlsuch a way as to maintain their activity as
antibodies and to reduce the degree of complement binding to
such an extent that the modified immune globulines can be used
for intravenous administration. For example, immune globuline
molecules may be modified by enzymatic degradation, so that
the linking points for the compLement are split off, but the
rest of the molecules is capable of binding antigens~ Such
composition is administered intravenously with good success.
The reaction of immune globulines with alkylating and
acylating agents also leads to an immune globuline suitable
29 for intravenous administration. The reduction of the comple-
~9 ''
-- 2 --
: .: : - : . - ~ .
. ~ . . . . ~ . .
ment binding of immune globulines may also be obtained by N-
alkylation and benzyiation.
Furthermore, a process is known according to which immune
globulines are partly split by reduction of in~ramolecular
disulfide bonds and the sulfhydriles formed are subsequently
alkylated. In this process the original size of the molecule
is maintained.
These processes are essentially based on the modification
of the free amino groups or disulfide bonds of the immune glob-
uline moleculesO
Though these processes lead to products having rathersatisfying properties, problems are persisting to which im-
proved solutions shall be found, especially because the phy-
sical and chemical properties of the molecules are consider- ~
ably modified by the processes described~ ;
It has now been found that immune globulines in which
some carboxyl group have been modi~ied considerably change
their binding behaviour with regard to complement without loos-
ing their e~ficiency as antibodies. Such modified immune glo-
bulines which bind complement to a smaller extent or in a notdetectable degree, are suitable as medicaments for intravenous
administration.
Thus, the object of the invention are amidated immune
globulines and furthermore a process for preparing such ami-
dated immune globulines, wherein immune globulines are re-
acted with a molar excess of a primary monoamine and a carbo-
diimide in a slightly acidic to neutral aqueous solution. The
molar ratio of the amine to immune globulines expediently
29 amounts to at least 50:1 and the ratio of carbodiimide to im-
. . . - . . . ..
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. .
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~5~
mune globulines to at least 1:1.
As starting material for the reaction of the invention
there ar~ used immune globuline fractions obtained from sera,
plasmae or other body liquids or organ ~xtracts. Especially
the fractions enriched with regard to immune globuline are
used. A preferred method for preparing them is for example
the method according tv Ievy and Sober via chromatography on
DEAE cellulose. Naturally, the pure immune globulines may
also be amidated according to the invention. However, in
practice the 100% pure immune globulines do not play an im-
portant part for the time being, due to the expensive processes
of purification.
It has appeared that the complement bond of the immune
globulines provides particularly low values if the ratio of
carbodiimide to immune globulines is 5:1 to 20:1.
The reaction is generally carried out at a pH of from 3 to
7 and at a temperature in the range of from 5 to 50C.
As amines in the sense of the invention there are suitable
all primary monoamines~ i.e. compounds of the general formula
R - NH2,
wherein R is a radical which according to known conceptions
does not represent any marked antigen motive in immunology.
Examples of suitable amines are above all aliphatic amines,
methyl amine, ethyl amine and higher aliphatic amines, especially
those having up to 10 carbon atoms. In the slightly acidic
aqueous solutions applied the amines are generally present
as the corresponding ammonium cationsr According to the in-
vention the amines axe preferred which carry further functional
groups, e5pecially hydxophilic groups such as hydroxy or
acetal. Examples of such amines are ethanol amine, trishy-
droxymethyla~ino-methane or glucosamine which have a favorable
4 -
., ., , . : ,: -
, . , . , :- , . . . , : . .. ..
influence on the solubility of the reaction product in a phy-
siologically compatible aqueous medium.
Since the process is carried out under conditions under
which the antibody activity of the immune globuline must not
be adversely affected, it is expedient to carry out the re~
action in known manner in the presence of a carbodiimide as
activator. As carbodiimide there are suitable all representa-
tives of this class of compounds which are capable of having
an activating effect on the formation of peptide bonds. Ex-
amples for such carbodiimides are the 1-ethyl-3-(3-dimethyl-
aminopropyl)-carbodiimide-hydrochloride (EDC) or the l-cyclo-
hexyl-3-(2-morpholinoethyl)-carbodiimide-metho-p-toluene-sul-
fonate (CMC). In the same way as the amines the carbodiimides
are present as salts in the slishtly acidic aqueous solutions
applied. The carbodiimides are generally used as salt since
they are more stable and easier to handle in this formO On
principle, the free carbodiimides may also be used; when being
dissolved in the a~ueous soluti~on they pass to the salt form.
The testing of the complement bond may ~e carried out ac-
cording to A. Nowotny, sasic Exercices in Immunochemistry,page 160 et seq~ (1969).
The process of the invention for preparing amidated immune
globulines may also be carried out with immune globulines in
which disulfide groups present are reduced to sulfhydrile
groups. The products amidated after the reduction have the
same ad~antageous properties.
For the reduction of disulfide bonds it is for example
particularly advantage~us to use dithiothreitol or dithioery-
thritol (Cleland's reagent). ~he disulfide bonds may also be
~9 reduced according to
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HOE 74/B 023
a known process (see siochem. J. 99, P8, 1966) with reducing
agents such as 2-mercapto-ethanol or mercaptoethyl amine while
using high concentrations of the reducing agent.
The conversion of the disulfide bond into sulfhydrile
bonds may be carried out with a reducing agent at a slightly
alkaline pH with a concentration of the reducing agent of 0.01
mol/l and a molar ratio o~ immune globulin to reducing agent
of 2.5 - 50~
The reducing agents described by Cleland having
the following formulae
H H H H
HS - C - C - C - C - SH
H OH OH H
H H OH H
HS - C - C - C - C - S~
.
~ OH H H
may be used for the reduction of the immune globulines.
Amidated immune globulines having a sufficient re-
ductlon of the complement bond may be administered by intra-
venousroute. They may be treated with physiologically com-
patible solvents to obtain the corresponding compositions.
Medicaments containing amidated immune globulines may be made
available in a liquid or freeze-dried form.
The following Examples illustrate the invention.
E X A M P L E _ 1:
Preparation of the starting material.
22.1 Liters of human serum obtained from spontaneous-
1~ '
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- ~5~ HOE 74/B 023
ly coagulated blood were passed for salt formation over a
column equilibrated with 0.0175 molar sodium phosphate, pH
6.4, filled with Sephadex G-2 ~ "med.ium" (registered trade
mark of Messr. Pharmacia for cross-linked dextran). With a
passage photometer the absorption was measured in the column
t~
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,
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The first peak formed by the serum proteins was collected
separately from the following low molecular portions and pass-
ed over a aolumn rinsed with the above-mentioned phosphate
buffer, of 15 kg of DEAE - cellulose with l mol equivalents/g
of exchanger capacity. The column was rinsed afterwards with
1~5 column volumes o~ buffer. The immune globulines being in
the passage of the column were precipitated by addition of
solid ammonium sulfate up to a concentration of 2.2 moles per
liter. Most part of the supernatent liquid was siphoned off
after standing for 24 hours, the rest was eliminated by centri-
fugation at 5200 g. The residue of the centrifuge was freed
from ammonium sulfate by dialysis against 0.1 molar NaCl solu-
tion. The volume of the dialysed immune globuline solution
was filled up with a NaCl solution to 2000 ml. It contained
155 g of protein on the whole.
Amidation of the immune globuline.
lO00 ml of the immune globuline solution obtained accord-
ing to the process described above were dialysed for 24 hours
while stirring against lO00 ml of l molar tris-hydroxymethyl-
aminomethane-~"tris")-HCl bu~fer; pH 5.4, transferred to a
glas~ vessel and mixed while stirring with 0.96 l-ethyl-3-(3-
dimethylaminopropyl)-carboxydiimide~HCl. The batch was stirred
for 2 hours at room temperature.
The immune globuline amidated with tris-hydroxymethyl-
amino-methane was passed over a column containing 8 liters of
Sephadex G-25 ~ which had been previously rinsed with a so-
lution having 0.15 molar NaCl and 0.3 molar glycine as well as
a pH value of 7.3. The optical density of the column eluate
29 was measured at 280 nm with a passage photometer. The portion
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of the eluate containing the clmidated immune globuline was
combined and concentrated with an ultra filter to a content
of protein of 5%.
The following Table shows a comparison of starting immune
5 globuline to amidated immune globuline with regard to comple-
ment bond and antibody activity:
T A B L E 1:
Antibody specifit~
Complement German meas- diphtheritis tetanus
bond 1) les titer I~ / ml IU / ml
starting
immune 22% 1:1024 ~0.5 ~1.0 >1 ~2
globuline
amidated
immune 0% 1:1024 ~0.5 ~1.0 > 1 ~2
globuline
1) the evaluation of the comp:Lement bond was effected accord-
iny.:to NOWOTNY, Aol Basic Exercices in Immunochemistry;
Spxinger Verlag, 1969, page 160.
E X A M P L E 2:
~ , . .
1000 ml of immune globuline solution obtained according to
Example 1, containing 75.5 g of protein were mixed with 890 mI
of a solution containing 52.5 g of glucosamine-HCl and the pH
value was adjusted to 6.0 with 2 molar NaOH. With stirring
2.06.g of 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)car~odiimide--
metho-p toluene sulfonate were added to the solution and stir-
ring of the batch was continuedO The reaction temperature was
29 25C. The transer of the amidated immune globuline into a so-
.
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~ 23
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lution containing 0.15 mole per liter NaCl a~d ~ ole per
liter of glycine ol pH 7.3 was carried out in the manner des
cribed in Example 1, but with a col~u~l containing 10 li-ters
o~ Sephadex G-25 ~ The concentration to 5% of protein was
also eff'ected as described in Example 1. The results of the
evaluation of the amidated i~mune globuline obtained accord~
ing to Example 2 were opposed to those of the ~tarting globu~
line in Table 2.
T A B ~ E 2:
ntibod~ specif~
Complement German meas~ diphtherilis tetanus
bond les~titer IU / ml IU / ml . .
~_~ , . . .
starting
immune 22~ 1:102~ ~0.5 ~100 ~1 ~2
globuline
amida-ted
immune 1.5% 1:1024 ~0.5 C1.0 ~1 ~2
- ~- -- ' .
n _. _ P L ~ 9:
Reduction and amidation of the immune globulines.
The amidation o~ the i~mune globulines described iil Ex-
ample 1 and 2 may be carried out in the same way with reduced
immune globulines. The reduction was carried ou-t as follows:
A ~olution o~ 3.3 g of immune globuline in 330 ml Q~ 0. 15
molar NaCl solution was adjusted to p~ 8.2 with -tris(hydroxy-
methyl)-ami~lomethane (Tris). To the imm~le globuline ~olution
15,~ mg o~ dithioerythrite (DTE) dlssol~ed in 2 ml o~ water
were added~ After 60 minutes 20 ml of a tris-HCl solution
29 containing 5 g of Tris~ of pH 1.0, were added while stirrixlg,
g
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aç~ 02-~
The pH value of the mixture was adjusted to 5.0 with HCl and
amidated as described in Example 1 after addi-tion of 82.2 mg
of N-ethyl-N' (3 dimethylaminopropyl)-carbodiimide HCl.
The reduction of -the immune glcbulines may be carried out
instead wi-th dithioery-thrite with dithiothreitol (DTT) under
the same test co~ditions~
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:. :. . , :- . . ,
