CA1056819A - Amidated immune globulines and process for preparing them - Google Patents
Amidated immune globulines and process for preparing themInfo
- Publication number
- CA1056819A CA1056819A CA235,134A CA235134A CA1056819A CA 1056819 A CA1056819 A CA 1056819A CA 235134 A CA235134 A CA 235134A CA 1056819 A CA1056819 A CA 1056819A
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- Prior art keywords
- immune globulin
- amidated
- immune
- ing
- reducing agent
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
Abstract of the disclosure:
The invention relates to modified immune globulines, a process for preparing them by treating immune globulines in a slightly acidic to neutral solution with a molar excess of a primary amine and a carbodiimide or salts thereof and phar-maceutical compositions containing modified immune globulines.
The invention relates to modified immune globulines, a process for preparing them by treating immune globulines in a slightly acidic to neutral solution with a molar excess of a primary amine and a carbodiimide or salts thereof and phar-maceutical compositions containing modified immune globulines.
Description
~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 '' : .: : - : . - ~ .
. ~ . . . . ~ . .
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-. . . - . . . ..
.. . . . . .. ..
. .
: .~, . , .' ~ : . :
:, . ..... , : .
~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 ., ., , . : ,: -, . , . , :- , . . . , : . .. ..
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 . . .
.
,' ` ~: : ~ .
: ~: : , `
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~ ' - ~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~
- 6a -- ., , :,,,. : : ~ .
~5~
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 :':.. :..
, , ` , .: " ,, , . ' ' . ' ' , ' '' ':~ ' ~s~
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
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 '' : .: : - : . - ~ .
. ~ . . . . ~ . .
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-. . . - . . . ..
.. . . . . .. ..
. .
: .~, . , .' ~ : . :
:, . ..... , : .
~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 ., ., , . : ,: -, . , . , :- , . . . , : . .. ..
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 . . .
.
,' ` ~: : ~ .
: ~: : , `
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~ ' - ~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~
- 6a -- ., , :,,,. : : ~ .
~5~
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 :':.. :..
, , ` , .: " ,, , . ' ' . ' ' , ' '' ':~ ' ~s~
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-.
: :
, .
~ 23 ,_w " ~
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 ,. . , . , , . .~ .................................... .
,,. . .. . , . , ........ ~ ., , .. , .... "
.. , . . : . ,,: . . ..
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~
:. :. . , :- . . ,
: :
, .
~ 23 ,_w " ~
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 ,. . , . , , . .~ .................................... .
,,. . .. . , . , ........ ~ ., , .. , .... "
.. , . . : . ,,: . . ..
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~
:. :. . , :- . . ,
Claims (19)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for the preparation of an amidated immune globulin in which immune globulin is reacted with a molar ex-cess of a primary monoamine and a carbodiimide or the salts of these compounds, in a slightly acidic to neutral aqueous solution.
2. A process as claimed in claim 1 in which the molar ratio of amine to immune globulin is at least 50:1 and the ratio of carbodiimide to immune globulin is at least 1:1.
3. A process as claimed in claim 1 in which the molar ratio of carbodiimide to immune globulin is 5:1 to 20:1.
4. Amidated immune globulin, whenever obtained accord-ing to a process as claimed in claim 1, claim 2 or claim 3 or by an obvious chemical equivalent thereof.
5. A process as claimed in claim 1 in which the amine is an aliphatic monoamine having 1 to 10 carbon atoms per molecule which may carry hydroxyl groups, acetal groups or hydroxyl and acetal groups.
6. A process as claimed in claim 1 in which the amine is ethanol amine, trishydroxymethylaminomethane or glucosamine.
7. A process as claimed in claim 6 in which the carbo-diimide is 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride or 1-cyclohexyl-3-(2-morpholino-ethyl)-carbodi-imide-metho-p-toluene sulfonate.
8. Amidated immune globulin, whenever obtained accord-ing to a process as claimed in claim 5, claim 6 or claim 7 or by an obvious chemical equivalent thereof.
9. A process as claimed in claim 1 in which the reaction is carried out at a pH of 3 to 7.
10. A process as claimed in claim 9 in which the reaction is carried out at a temperature of 5 to 50°C.
11. A process as claimed in claim 1 in which human immune globulin is used.
12. Amidated immune globulin, whenever obtained accord-ing to a process as claimed in claim 9, claim 10 or claim 11 or by an obvious chemical equivalent thereof.
13. A process as claimed in claim 1 in which immune globulin having at least one disulfide bond is converted into two sulfhydrile groups with the aid of a reducing agent.
14. A process as claimed in claim 13 in which the con-version of the disulfide bond into sulfhydrile bonds is carried out with a reducing agent at a slightly alkaline pH.
15. A process as claimed in claim 13 in which the con-version of the disulfide bond into the sulfhydrile bonds is 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 of immune globulin to reducing agent of 2.5 - 50:1.
with a concentration of the reducing agent of 0.01 mol/l and a molar ratio of immune globulin to reducing agent of 2.5 - 50:1.
16. Amidated immune globulin, whenever obtained accord-ing to a process as claimed in claim 13, claim 14 or claim 15 or by an obvious chemical equivalent thereof.
17. A process as claimed in claim 13 in which the re-ducing agent is dithioerythritol or dithiothreitol.
18. A process as claimed in claim 15 in which the re-ducing agent is dithioerythritol or dithiothreitol.
19. Amidated immune globulin, whenever obtained accord-ing to a process as claimed in claim 17 or claim 18 or by an obvious chemical equivalent thereof.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE2442655A DE2442655C3 (en) | 1974-09-06 | 1974-09-06 | Amidated immunoglobulins and their use for intravenous administration |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1056819A true CA1056819A (en) | 1979-06-19 |
Family
ID=5925019
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA235,134A Expired CA1056819A (en) | 1974-09-06 | 1975-09-05 | Amidated immune globulines and process for preparing them |
Country Status (21)
| Country | Link |
|---|---|
| JP (1) | JPS5161623A (en) |
| AR (1) | AR212909A1 (en) |
| AT (1) | AT342763B (en) |
| AU (1) | AU499871B2 (en) |
| BE (1) | BE833186A (en) |
| BR (1) | BR7505721A (en) |
| CA (1) | CA1056819A (en) |
| CH (1) | CH615194A5 (en) |
| DE (1) | DE2442655C3 (en) |
| DK (1) | DK399175A (en) |
| EG (1) | EG11813A (en) |
| FR (1) | FR2283695A1 (en) |
| GB (1) | GB1517026A (en) |
| IE (1) | IE41671B1 (en) |
| IL (1) | IL48049A (en) |
| IT (1) | IT1048485B (en) |
| LU (1) | LU73328A1 (en) |
| NL (1) | NL7510289A (en) |
| NZ (1) | NZ178580A (en) |
| SE (1) | SE403704B (en) |
| ZA (1) | ZA755687B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5004697A (en) * | 1987-08-17 | 1991-04-02 | Univ. Of Ca | Cationized antibodies for delivery through the blood-brain barrier |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3704282A (en) * | 1971-04-09 | 1972-11-28 | Sidney Spector | Catecholamine antigens and antibodies specific therefor |
-
1974
- 1974-09-06 DE DE2442655A patent/DE2442655C3/en not_active Expired
-
1975
- 1975-09-01 NL NL7510289A patent/NL7510289A/en not_active Application Discontinuation
- 1975-09-04 CH CH1147275A patent/CH615194A5/en not_active IP Right Cessation
- 1975-09-04 GB GB36451/75A patent/GB1517026A/en not_active Expired
- 1975-09-04 LU LU73328A patent/LU73328A1/xx unknown
- 1975-09-04 IT IT26918/75A patent/IT1048485B/en active
- 1975-09-04 AU AU84536/75A patent/AU499871B2/en not_active Expired
- 1975-09-04 IL IL48049A patent/IL48049A/en unknown
- 1975-09-04 NZ NZ178580A patent/NZ178580A/en unknown
- 1975-09-05 IE IE1944/75A patent/IE41671B1/en unknown
- 1975-09-05 FR FR7527354A patent/FR2283695A1/en active Granted
- 1975-09-05 CA CA235,134A patent/CA1056819A/en not_active Expired
- 1975-09-05 BR BR7505721*A patent/BR7505721A/en unknown
- 1975-09-05 DK DK399175A patent/DK399175A/en not_active Application Discontinuation
- 1975-09-05 SE SE7509898A patent/SE403704B/en unknown
- 1975-09-05 AT AT689475A patent/AT342763B/en not_active IP Right Cessation
- 1975-09-05 ZA ZA00755687A patent/ZA755687B/en unknown
- 1975-09-06 JP JP50107581A patent/JPS5161623A/ja active Pending
- 1975-09-06 EG EG521/75A patent/EG11813A/en active
- 1975-09-08 BE BE159842A patent/BE833186A/en unknown
- 1975-09-14 AR AR260263A patent/AR212909A1/en active
Also Published As
| Publication number | Publication date |
|---|---|
| FR2283695B1 (en) | 1978-09-22 |
| DE2442655A1 (en) | 1976-03-18 |
| IL48049A (en) | 1978-08-31 |
| SE7509898L (en) | 1976-03-08 |
| ZA755687B (en) | 1976-08-25 |
| FR2283695A1 (en) | 1976-04-02 |
| CH615194A5 (en) | 1980-01-15 |
| DE2442655B2 (en) | 1979-02-01 |
| AR212909A1 (en) | 1978-11-15 |
| SE403704B (en) | 1978-09-04 |
| IE41671L (en) | 1976-03-06 |
| LU73328A1 (en) | 1977-05-11 |
| BE833186A (en) | 1976-03-08 |
| IT1048485B (en) | 1980-11-20 |
| DK399175A (en) | 1976-03-07 |
| NL7510289A (en) | 1976-03-09 |
| DE2442655C3 (en) | 1979-09-20 |
| AT342763B (en) | 1978-04-25 |
| JPS5161623A (en) | 1976-05-28 |
| NZ178580A (en) | 1978-03-06 |
| GB1517026A (en) | 1978-07-05 |
| BR7505721A (en) | 1976-08-03 |
| AU8453675A (en) | 1977-03-10 |
| EG11813A (en) | 1978-09-30 |
| IE41671B1 (en) | 1980-02-27 |
| IL48049A0 (en) | 1975-11-25 |
| ATA689475A (en) | 1977-08-15 |
| AU499871B2 (en) | 1979-05-03 |
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