CA1153695A - S-sulfonated immunoglobulin composition having a high monomer content and a process for production thereof - Google Patents
S-sulfonated immunoglobulin composition having a high monomer content and a process for production thereofInfo
- Publication number
- CA1153695A CA1153695A CA000359215A CA359215A CA1153695A CA 1153695 A CA1153695 A CA 1153695A CA 000359215 A CA000359215 A CA 000359215A CA 359215 A CA359215 A CA 359215A CA 1153695 A CA1153695 A CA 1153695A
- Authority
- CA
- Canada
- Prior art keywords
- immunoglobulin
- sulfonated
- composition
- amino acids
- carbon atoms
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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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
- C07K16/065—Purification, fragmentation
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/827—Proteins from mammals or birds
- Y10S530/829—Blood
- Y10S530/83—Plasma; serum
- Y10S530/831—Cohn fractions
Abstract
Abstract of the Disclosure The present invention is directed to an S-sulfonated immunoglobuiin composition comprising an S-sulfonated immunoglobulin and as an aggregation preventing agent or an aggregate dissociating agent, therefor, about 1 to about 600% by weight, based on the weight of the S-sulfonated immunoglobulin of a water-soluble acid addition salt of a basic nitrogen-containing organic compound containing one or more basic nitrogen atoms in the molecule and optional-ly carboxyl groups smaller in number than the basic nitrogen atoms and having a pKb at 25°C of not more than 7.
There is also provided a process for producing an S-sulfonated immunoglobulin composition having a high monomer content, Which comprises contacting an S-sulfonated immuno-giobulin in aqueous solution with about 1 to about 600%
by weight, based on the weight of the S-sulfonated immuno-globulin, of the water-soluble acid addition salt, or reacting an immunoglobulin in aqueous solution with a compound capable of yielding a sulfite ion in water and an oxidizing agent in the presence of about 1 to about 600%
by weight, based on the immunoglobulin of the water-soluble acid addition salt.
There is also provided a process for producing an S-sulfonated immunoglobulin composition having a high monomer content, Which comprises contacting an S-sulfonated immuno-giobulin in aqueous solution with about 1 to about 600%
by weight, based on the weight of the S-sulfonated immuno-globulin, of the water-soluble acid addition salt, or reacting an immunoglobulin in aqueous solution with a compound capable of yielding a sulfite ion in water and an oxidizing agent in the presence of about 1 to about 600%
by weight, based on the immunoglobulin of the water-soluble acid addition salt.
Description
~536915 This invention relates to an S-sulfonated immunoglobulin composition having a high monomer content, and to a process for production thereof.
Immunoglobulins are of great medical signifi-cance as agents responsible for humoral immunity, and have immune activity against various pathogenic micro-organisms. Administration of immuno~lobulins can there-fore lead to the prevention and -treatment of viral in-fections such as measles and viral hepatitis ana of in-fections caused by antibiotic-resistant bacteria such as staphylococci. In such prevention and treatment, intra-venous injection is preferred to intramuscular injection in order to administer large amounts of an i~munoglohulin and cause it to produce a rapid eff~ct~ However, intra-venous administration of an immunoglobulin fractionated from huma~ plasma may cause anaphylactic side-effects involving hypotension, chill and pyrexia, dyspnaa, head-ache, etc. ~his is because aggregated immunoglobuli~
molecules in the immunoglo4ulin ~ractionated ~rom human plasma combine with complements in the blood to activate them and ~hereby liberate biologically active factors s~ch as an anaphylatoxin-like substance or a vascular-permeable factor.
~ uch aggregated immuno~lobul~ns are inherently ~ .
.
, S3~g~
contained in the immunoglobulins fractionated from human plasma, and tend to form gradually with the passage of time during their formulation into dosage forms.
Various methods have been suggested for removing or alleviating these side-effects induced by aggregated immunoglobulin molecules. For example, U.S. Patent No.
~,059,571 discloses a method for producing an intraven-ously inaectable S-sulfonated immunoglobulin having re-duced anti-complement activity while retaining various antibody activities~ which comprises reacting an immuno-globulin with sodium sulfite and sodium tetrathionate to cleave SS linkages be-tween the peptide chains of the immunoglobulin and S-sulfonate them. ~his method is con-sidered to be best among -the prior suggested methods.
S-sulfonation of an immunoglobulin does not lead to complete removal of aggregated immunoglobulin molecules. The reduced anticomplement activity of the resulting S-sulfonated immunoglobulin is presumably be-cause the aggregated molecules contained in the immuno-globulin are S-sulfonated as is the monomer.
~ he S-sulfonated immunoglobulin containing aggregated molecules has lower anticomplement activity and is safer than an immunoglobulin containing aggregated moleculRs. ~he aggregated molecules, even when S-sulfonated~ still have the abili~ to combine with com-plements. Accordingly, an S-sulfonated immunoglobulin containing a relatively large amount of aggregated molecules may possibly induce the aforesaid anaph~lactice side-effects.
An S-sulfonated immunoglobulin produced in the aforesaid manner from an immunoglobulin having a rela-tively small amount of aggregated molecules is ~ot likely to induce the aforesaid side-effects. Since, however, a commercially available immunoglobulin, for example, an immunoglobulin ob-tained by the Cohn's ethanol fractiona-ting method, contains a relatively large amount of ag-gregated molecules, an S-sulfonated immunoglobulin 9~;
obtained by directly S-sulfonating such an immunoglobulin does not have a sufficiently reduced anticomplement ac-tivity, and its safety in intravenous injection is still desired to be improved.
A method has previously been known which com-prises adding a polymeric substance such as polyethylene glycol, a salt such as ammonium sulfate, acrinol, etc.
to a solution of an immunoglobulin to precipitate the aggregated immunoglobulin molecules. It may be possible to apply this method to an S-sulfonated immunoglobulin containing aggregated molecules in an attempt to remove the aggregated molecules. Application of this ~ethod to the S-sulfonated immurloglobulin results in removal of a large amount of monomer together with the aggregated molecules. Accordingl-y, in view of the fact that immuno-globulins are obtained from valuable human blood, one cannot but hesitate to use this method in removing the aggregated molecules from an S-sulfonated immunoglobulin.
A method is also known which comprises treat-ing an immunoglobulin with an acidic aqueous solution having a pH of 4 to dissociate the aggregated immuno-globulin molecules ~see ~cta Chemica Scandinavica, vol.
22, pages 490-496 (1968)3. ~hen this method is applied to an S-sulfonated immunoglobulin, it is necessary to render the treating solution neutral after the treatment in order to avoid denaturation of the treated S-sulfona-ted immunoglobulin. In neutrality, however, the dis-sociated S-sulfonated immunoglobulin again aggregates as is the case with an immunoglobulin.
It is an object of this invention to provide an S-sulfonated immunoglobulin composition having a high monomer content.
Another object of -this inven-tion is to provide an S-sulfonated immunoglobulin composition which contains aggregated molecules only in such a low content as to provide low anticomplement activity suitable for intra-venous injection, and which can be safely used by itself " ~ ~ .
' ' :
'~ ' ~53~S
-- 4 ~
for intravenous injection.
Still another object of this invention is to provide an 15-sulfonated immunoglobulin composition having excellent storage stability which does not subst~ntially form aggregated molecules on long-term storage.
A further object of this invention is to pro~
vide an S-sulfonated immunoglobulin having a high monomer content by dissociating the aggregated molecules of the S-sulfonated immunoglobulin or preventing aggregation of the monomer of the S-sul~onated immunoglobulin.
Other objects and advantages of this invention will become apparent from the following description.
~ hese objects and advantages of this invention are achieved in accordance with this invention by an S-sulfonated immunoglobulin composition comprising an S-sulfona-ted immunoglobulin and as an aggregation prevent-ing agent or an aggregate dissociating agent therefor, about 1 to about 60~o by weight, based on the weight o~
the S-sulfonated immunoglobulin, of a water-soluble acid addition salt of a basic nitrogen-containing organic compound containing one or more basic nitrogen atoms in the molecule and optionally c~rboxyl groups smaller in number than the basic nitrogen atoms and having a pKb at 25C of not more than 70 According to another aspect, these objects and advantages are achieved in accordance with this invention by a process for producing an S-sulfonated immunoglobulin having a high monomer content, which comprises sontac-ting an S-sul~onated immunoglobulin in aqueous solution with about 1 to about 60G% by weight, based on the weight of the S-sulfonated immunoglobulin, of a water-soluble acia addition salt of a basic nitrogen-containing organic compound con-taining one or more basic nitrogen atoms in the molecule and optionally carboxyl groups smaller in number than -the basic nitrogen a-toms and having a pKb at 25C of not more than 7 thereby to prevent aggregation of the monomer of the S-sul~onated immunoglobulin, and when the ~-sulfonated immu~oglobulin contains aggregated molecules, dissociating them into monomers, and if desired, lyophilizing the resulting product.
According to still another aspect, these ob-jects and advantages are achieved in accordance withthis invention by a process for producing an S-sulfona-ted immunoglobulin having a high monomer con-tent, which comprises reacting an immunoglobulin in aqueous solution with a compound capable of yielding a sulfite ion in water and an oxidizing agen-t in the presence of about 1 to about 60~/o by weight, based on the weight of the S-sulfonated immunoglobulin9 of a water-soluble acid addi-tion salt of a basic nitrogen-containing organic compound containing one or more basic nitrogen atoms in the molec-ules and optionally carboxyl groups smaller in numberthan the basic nitrogen atoms and having a pKb at 25C
of not more than 7.
The S-sulfonated immunoglobulin used in this invention denotes a product obtained by cleaving inter-chain SS linkages of a native immunoglobulin and S-sulfona-ting them~
~ he S-sulfonated immunoglobulin can be produced, for example, by reacting an immunoglobulin composed main-ly of gamma-globulin obtained from -the serum, plasma and other body fluids or extracts of organs by a known method such as the ethanol fractionating method of Cohn et al.
[E~ G. Cohn et al., J. Am~ Chem. SocO~ 68, 459 (1946)) with, for example, sodium sulfite and sodium tetra-thionate or sodium tri-thionate by the method disclosed in UOSo Patent No. 4,059,5710 Accordingly, -the specification of U.S. Patent ~o. 4,059,571 is hereby cited as reference.
~he immunoglobulin may be used as fractionated by the aforesaid Cohn's method wi-thout purification (such an immunoglobulin usually contains at least 2~/o by weight of aggregated immunoglobulin molecules having a sedimen-tation constant of a-t least 9S). Or after fractionation, the immunoglobulin may be purified by purifying methods , , .
1~5~
known in the art, such as treatment at pH 4, .salting out, treatment with acrinol, or ion-exchange chromatograph~J
to reduce the content of aggrega~ed immunoglobulin molec-ules to varying degrees, and some-times to substantially zero.
~ he S-sulfonated immunoglobulin thus obtained may be used as such or after it is purified by known methods such as treatment at pH 4, salting out, treat-ment with acrinol or ion exchange chromatography~
~he basic nitrogen-containing organic compound used in this invention contains one or more basic nitro-gen atoms in -the molecule and has a pKb a-t 25C of not more than 7, It may contain carboxyl groups which are smaller in number -than the basic nitrogen atoms so long as the compound shows basicity.
As is well known, pKb used herein is a dissoci-ation index of a basic compolmd which is defined by the following formula pKb - - log K
wherein K=~BH )/~B)o~H ~ in which ~B) is the concentration of the basic compound (i.e. the basic nitrogen-containing organic compound), ~X+) is the hydrogen ion concentration, and ~BH ) is the concentration of a conjugated acid.
Examples of suitable basic nitrogen-containing organic compounds which can be used in this inven-tion include lower alkylamines, ~- or 6-membered heterocyclic compounds having 1 to 3 nitrogen atoms9 guanidines op-tionally substituted by lower alkyl groups, lower alkyl-or aryl-amidines, basic amino acids, esters or amides of neutral amino acids at the carboxyl group, and amine derivatives of glucose. ~hese basic nitrogen-con~aining organic compounds can be used either singly or in com-bination with each other.
~S36 Specific examples of the lower alkylamines are primary lower alkylamines such as meth~lamine, ethylamine, propylamine and butylamine, secondary lower alkylamines such as dimeth~lamine, diethylamine, dipropylamine and dibutylamine and tertiary lower alkylamines such as tri-methylamine, triethylamine, tripropylamine and tributyl-amine. Preferably, the lower alkyl group in the lower alkylamines has l to 4 carbon atoms.
Specific examples of the 5- or 6-membered heterocyclic compounds having l to 3 nitrogen atoms include pyrrolidine, piperidine, imidazole, p~razole and triazole, ~hese compounds may be substituted with an alkyl group having 1 -to 4 carbon atoms (such as 2-methylimidazole).
~pecific examples of the optionally substituted guanidies are guanidine and methylguanidine. Preferably, the substituent alkyl group has l to 4 carbon atoms.
Specific examples of the amidines are Cl - C4 alkyl amidines such as methylamidine, and benzamidines optionally substituted with a Cl - CL~ alkyl group, such as methylbenzamidineO
Examples of the basic amino acids are lysine, ornithine, arginine, hydro~ylysine and histidine.
Speclfic examples of the esters or amides of neutral amino acids include esters formed between neutral amino acids such as glycine, alanine9 valine, leucine, isoleucine, cysteine, cystine and methionine and alcohols, for example aliphatic alcohols having l to 4 carbon atoms such as methanol, ethanol, n-propanol and n-butanol, and amides formed between these neu-tral amino acids and primary or secondary amines having a Cl - C4 alkyl group or ammonia, such as glycinamide, alaninamide and leucin-amide.
~he amine derivatives of glucose are, for example, compounds resulting from substitution of an amino group for at leas-t one of the hydroxyl groups of glucose, such as D-glucosamine.
!
, , ': ' ' ' ~ he basic nitrogen-containing organic compound is used in the form of a water-soluble acid addition salt in this invention. The water-soluble acid addition salt can be favorably prepared by using a mineral acid such as hydrochloric acid, hydrobromic acid, phosphoric acid or sulfuric acid or an organic carboxylic acid such as acetic acid. ~he mineral acid salts are preferred~
Especially preferred are the mineral acid salts, above all hydrochlorides, of arginine, guanidine, leucinamide, imidazole, 2-methylimidazole and D-glucosamineO
~ he S-sulfonated immunoglobulin composition of this invention contains about 1 to about 60~/o by weight, preferably 10 to 600% by weight, more preferably 20 to 40~/0 by weight, based on the S-sulfonated i~munoglobulin, of the water-soluble acid addition salt of the basic nitrogen-containing organic compound. If the amount of the water-soluble acid addition salt is less than about 1% by weight, the effect of this compound to dissociate the aggregated S-sulfonated immunoglobulin molecules and prevent re-aggregation of the molecular chains of the S-sulfonated immunoglobulin becomes so small as to deviate from the range intended by the present invention. If, on the other hand, i-t exceeds about 60~/o by weight, the intended effect can be obtained but economic or opera-tional disadvantages arise.
~ he S-sulfonated immunoglobulin composition of this invention contains the water-soluble acid addition salt of the basic nitrogen-containing organic compound as an aggregation preventing agent or as an aggregate dissociating agent for the S-sulfonated immunoglobulin.
The water-soluble acid addition salt serves to dissociate the aggregated S-sulfonated immunoglobulin molecules and prevent aggregation of the molecules of the S-sulfonated immunoglobulinO
~he essence of this invention lies in the pro-vision of an S-sulfonated immunoglobulin composition which has a low content of aggregated molecules and ' .
~5;~S9~i therefore a high monomer conten-tO
Since the S-sulfonated immunoglobulin composi-tion provided by this invention has a low aggregate con-tent and a very low anticomplement activity, i-t encom-passes a composition which is intravenously injectablewhile containing a water-soluble acid addition salt of a basic nitrogen-containing organic compound such as L-arginine hydrochloride. For example, a composi-tion in accordance with this invention comprlsing an S-sulfonated immunoglobulin and abou-t 10 to about 100% by weight, based on the S-sulfonated immunoglobulin, of ~-arginine hydrochloride is preferably used for this purpose.
The S-sulfonated immunoglobulin composition provided by -this invention also encompasses a composition which cannot be used by itself for intravenous injection and which can be regarded as an intermediate composition for the production of an intravenously injectable ~-sulfonated i~munoglobulin preparation~
This in-termediate composition in accordance with this invention can be formed into an S-sulfonated immunoglobulin prepara-tion suitable for intravenous injection by removing the water-soluble acid addition salt of a basic nitrogen-containing organic compound therein by dialysis, etc~ in a step as close as possible to the final step of lyophilization in the drug formula-ting process, and then lyophilizing the residue.
Such an S-sulfonated immunoglobulin composition can be provided by contacting an S-sulfonated imm~noglo-bulin containing aggregated molecules with the water~
soluble acid addition salt; or contacting a purified S-sulfonated immunoglobulin substantially free from aggre-gatea molecules wi-th the water-soluble acid addition salt; or preparing an S-sulfonated immunoglobulin from an immunoglo~ulin containing aggregated molecules in the presence of the water-soluble acid addition salt.
~ hus7 according to this invention, there is first provided a process for producing an S-sulfonated ' ~ ;
.
~3~
imrnunoglobulin composi.tion having a high monomer content, which comprises contacting an S-sulfonated immunoglobulin containing aggregated molecules in aqueous solution with about 1 to about 600% by weight, based on the S-sulfonated immunoglobulin, of at least one water-soluble acid addi-tion salt of a basic nitrogen~containing organic compound con-taining one or more basic nitrogen atoms in the mole-cule and op-tionally carboxyl groups smaller in number than the basic nitrogen atoms and having a pKb at 2~C
of not more than 7 thereby -to prevent aggregation of the monomer of the S-sulfonated imrnunoglobulin and to dis-sociate the aggregated molecules into monomers, and then if desired, lyophilizing the resulting productO
~econdly, there is pro~ided a process for producing an ~sulfonated immunoglobulin composition having a high monomer content, which comprises contacting a purified S-sulfonated immunoglobulin substantially free from aggregated molecules with the sarne amount as above of at lea:st one aforesaid water-soluble acid addi-tion salt in aqueous solution thereby to prevent aggregation of the monomer of the S-sulfonated immunoglobulin, and then if desired, lyophilizing the resulting product.
Thirdly, the inven-tion provides a process for producing an S-sulfonated immunoglobulin composition having a high monomer content, which comprises reacting an immunoglobulin in aqueous solution with a compound capable of yielding a sulfite ion in water and an oxidi-zing agent in the presence of about 1 to about 600% by weight, based on the weight of -the immunoglobulin, of at least one aforesaid water-soluble acid addition salt~
In the first and second processes of this in-vention, the S-sulfona-ted immunoglobulin and the water-soluble acid addition salt of a basic nitrogen-containing organic compound are contacted with each other in aqueous solutionO The con-tacting is effected at a temperature of about 0 to about 50C, preferably about 0 to about 30C.
Desirably, the pH of the aqueous solution during contac-' ~ ~
-369~i ting is about 5 to about 8O
The greatest characteristic of -the process of this invention is that as seen in the second process as soon as the aggregated S-sulfonated immunoglobulin mole-cules in the starting S-sulfonated immunoglobulin are contacted with the water-soluble acid addition sal-t under the aforesaid con-tacting conditions, dissociation of the aggregated molecules begins, and within a period of as short as l hour, dissociation of the aggrega-ted S-sul-fonated immunoglobulin molecules can be substantiallyachieved and thereby an S-sulfonated immunoglobulin having a high monomer content can be provided.
A second feature of the process of this inven-tion is that as typically expressed by the second process even when the contacting is continued for a period longer than the aforesaid time, substantial dissociation of the aggregated S-sulfonated immunoglobulin molecules is retained over a long period of time.
~he first and second processes in accoraance with this invention can be practiced by directly adding the water-soluble acid addition salt to a solution of the S-sulfonated immunoglobulin, or by mixing a solution of the S-sulfonated immunoglobulin wi-th a solution of the water-soluble acid addition salt.
In the latter-mentioned procedure, the water-soluble acid addition salt may be produced in situ by preparing an aqueous solution of the basic nitrogen-containing organic compound and adding an acid such as hydrochloric acid to the aqueous solution to adaust its pX to about 5 to abou-t 8.
According -to the process of this invention, the resulting aqueous solution of S-sulfonated immunoglobulin having a high monomer content, if required, can be lyo-philized by a method known ~ se to provide the composi-tion of -this invention in the form of a lyophilized solidO
Pre~erably, the composition of this invention provided in the form of a lyophilized solid is an intravenously j ~5;~
inaectable ccmposition comprising a non-toxic water-soluble acid addition salt capable of ~eing used in intravenous injectionO Such an intravenously injectable composition is dissolved in sterilized water or physio-logical saline to form an intravenous injecting prepara-tion.
Preferably, the composi-tion of this invention provided in ~he form of a lyophilized solid contains about 1 to about 10~/o by weight, based on the weight of the immunoglobulin, of a nontoxic water-soluble acid addition salt such as ~-arginine hydrochloride.
The sulfonation reaction in the third process of this invention is carried out in water The pH of the reaction system during the reaction is preferably in the range of 5.0 to 9Ø ~he reaction temperature is from 0 to 50C, preferably from lO to 45C~ At a temperature of more than 50C, the immunoglobulin molecules are undesir-ably susceptible -to denaturation. At a temperature of less than 0C, the reaction proceeds too slowly to be commercially feasible. The reaction is continued until almost all of the interchain SS linkages of the immuno-globulin are cleaved and S-sulfonated. ~he reaction time, which varies depending upon the amounts of the reagents, the reaction temperature, e~tc , is generally from 0~5 to 24 hours.
Suitable oxidizing agents are those whic~ have low reactivity with a sulfite ion generated from the other reagent. E~amples of the oxidizing agents are com-pounds capable of forming a polythionate ion having 3 to 6 sulfur atoms, such as a trithionate ion, a tetrathionate ion, a pentathionate ion or a hexathionate ion, in water (e.g., the sodium, potassium or ammonium salt of a poly-thionic acid having 3 to 6 sulfur atoms); compounds capable of forming A cupric ion in water (e.g., cupric chloride, cupric bromide or cupric sulfate); compounds capable of forming an iodobenzoate ion in water (e.g., the sodium, potassium or ammonium sal~ of iodobenzoic ,, acid); and molecular oxygen-containing gases such as water (in this case, the gases desirably contain a catalytic amount of cysteine or 2-mercaptoet~ylamine).
Examples of the compound capable of yielding a sulfite ion in water include sulfurous acid salts such as sodium sulfite, potassium sulfite and ammonium sulfite;
bisulfites such as sodium bisulfite, potassium bisulfite and ammonium bisulfite; and pysobisulfites such as - sodium pyrobisulfite, potassium pyrobisulfite and am-monium pyrobisulfite.
~ he amount of the compound capable of yielding a sulfite ion in water is at least 2 moles, preferably at least lO moles, per mole of the interchain SS linkage of the immunoglobulin to be cleaved. ~he amount of the oxidizing agent is at least 1 mole, preferably at least
Immunoglobulins are of great medical signifi-cance as agents responsible for humoral immunity, and have immune activity against various pathogenic micro-organisms. Administration of immuno~lobulins can there-fore lead to the prevention and -treatment of viral in-fections such as measles and viral hepatitis ana of in-fections caused by antibiotic-resistant bacteria such as staphylococci. In such prevention and treatment, intra-venous injection is preferred to intramuscular injection in order to administer large amounts of an i~munoglohulin and cause it to produce a rapid eff~ct~ However, intra-venous administration of an immunoglobulin fractionated from huma~ plasma may cause anaphylactic side-effects involving hypotension, chill and pyrexia, dyspnaa, head-ache, etc. ~his is because aggregated immunoglobuli~
molecules in the immunoglo4ulin ~ractionated ~rom human plasma combine with complements in the blood to activate them and ~hereby liberate biologically active factors s~ch as an anaphylatoxin-like substance or a vascular-permeable factor.
~ uch aggregated immuno~lobul~ns are inherently ~ .
.
, S3~g~
contained in the immunoglobulins fractionated from human plasma, and tend to form gradually with the passage of time during their formulation into dosage forms.
Various methods have been suggested for removing or alleviating these side-effects induced by aggregated immunoglobulin molecules. For example, U.S. Patent No.
~,059,571 discloses a method for producing an intraven-ously inaectable S-sulfonated immunoglobulin having re-duced anti-complement activity while retaining various antibody activities~ which comprises reacting an immuno-globulin with sodium sulfite and sodium tetrathionate to cleave SS linkages be-tween the peptide chains of the immunoglobulin and S-sulfonate them. ~his method is con-sidered to be best among -the prior suggested methods.
S-sulfonation of an immunoglobulin does not lead to complete removal of aggregated immunoglobulin molecules. The reduced anticomplement activity of the resulting S-sulfonated immunoglobulin is presumably be-cause the aggregated molecules contained in the immuno-globulin are S-sulfonated as is the monomer.
~ he S-sulfonated immunoglobulin containing aggregated molecules has lower anticomplement activity and is safer than an immunoglobulin containing aggregated moleculRs. ~he aggregated molecules, even when S-sulfonated~ still have the abili~ to combine with com-plements. Accordingly, an S-sulfonated immunoglobulin containing a relatively large amount of aggregated molecules may possibly induce the aforesaid anaph~lactice side-effects.
An S-sulfonated immunoglobulin produced in the aforesaid manner from an immunoglobulin having a rela-tively small amount of aggregated molecules is ~ot likely to induce the aforesaid side-effects. Since, however, a commercially available immunoglobulin, for example, an immunoglobulin ob-tained by the Cohn's ethanol fractiona-ting method, contains a relatively large amount of ag-gregated molecules, an S-sulfonated immunoglobulin 9~;
obtained by directly S-sulfonating such an immunoglobulin does not have a sufficiently reduced anticomplement ac-tivity, and its safety in intravenous injection is still desired to be improved.
A method has previously been known which com-prises adding a polymeric substance such as polyethylene glycol, a salt such as ammonium sulfate, acrinol, etc.
to a solution of an immunoglobulin to precipitate the aggregated immunoglobulin molecules. It may be possible to apply this method to an S-sulfonated immunoglobulin containing aggregated molecules in an attempt to remove the aggregated molecules. Application of this ~ethod to the S-sulfonated immurloglobulin results in removal of a large amount of monomer together with the aggregated molecules. Accordingl-y, in view of the fact that immuno-globulins are obtained from valuable human blood, one cannot but hesitate to use this method in removing the aggregated molecules from an S-sulfonated immunoglobulin.
A method is also known which comprises treat-ing an immunoglobulin with an acidic aqueous solution having a pH of 4 to dissociate the aggregated immuno-globulin molecules ~see ~cta Chemica Scandinavica, vol.
22, pages 490-496 (1968)3. ~hen this method is applied to an S-sulfonated immunoglobulin, it is necessary to render the treating solution neutral after the treatment in order to avoid denaturation of the treated S-sulfona-ted immunoglobulin. In neutrality, however, the dis-sociated S-sulfonated immunoglobulin again aggregates as is the case with an immunoglobulin.
It is an object of this invention to provide an S-sulfonated immunoglobulin composition having a high monomer content.
Another object of -this inven-tion is to provide an S-sulfonated immunoglobulin composition which contains aggregated molecules only in such a low content as to provide low anticomplement activity suitable for intra-venous injection, and which can be safely used by itself " ~ ~ .
' ' :
'~ ' ~53~S
-- 4 ~
for intravenous injection.
Still another object of this invention is to provide an 15-sulfonated immunoglobulin composition having excellent storage stability which does not subst~ntially form aggregated molecules on long-term storage.
A further object of this invention is to pro~
vide an S-sulfonated immunoglobulin having a high monomer content by dissociating the aggregated molecules of the S-sulfonated immunoglobulin or preventing aggregation of the monomer of the S-sul~onated immunoglobulin.
Other objects and advantages of this invention will become apparent from the following description.
~ hese objects and advantages of this invention are achieved in accordance with this invention by an S-sulfonated immunoglobulin composition comprising an S-sulfona-ted immunoglobulin and as an aggregation prevent-ing agent or an aggregate dissociating agent therefor, about 1 to about 60~o by weight, based on the weight o~
the S-sulfonated immunoglobulin, of a water-soluble acid addition salt of a basic nitrogen-containing organic compound containing one or more basic nitrogen atoms in the molecule and optionally c~rboxyl groups smaller in number than the basic nitrogen atoms and having a pKb at 25C of not more than 70 According to another aspect, these objects and advantages are achieved in accordance with this invention by a process for producing an S-sulfonated immunoglobulin having a high monomer content, which comprises sontac-ting an S-sul~onated immunoglobulin in aqueous solution with about 1 to about 60G% by weight, based on the weight of the S-sulfonated immunoglobulin, of a water-soluble acia addition salt of a basic nitrogen-containing organic compound con-taining one or more basic nitrogen atoms in the molecule and optionally carboxyl groups smaller in number than -the basic nitrogen a-toms and having a pKb at 25C of not more than 7 thereby to prevent aggregation of the monomer of the S-sul~onated immunoglobulin, and when the ~-sulfonated immu~oglobulin contains aggregated molecules, dissociating them into monomers, and if desired, lyophilizing the resulting product.
According to still another aspect, these ob-jects and advantages are achieved in accordance withthis invention by a process for producing an S-sulfona-ted immunoglobulin having a high monomer con-tent, which comprises reacting an immunoglobulin in aqueous solution with a compound capable of yielding a sulfite ion in water and an oxidizing agen-t in the presence of about 1 to about 60~/o by weight, based on the weight of the S-sulfonated immunoglobulin9 of a water-soluble acid addi-tion salt of a basic nitrogen-containing organic compound containing one or more basic nitrogen atoms in the molec-ules and optionally carboxyl groups smaller in numberthan the basic nitrogen atoms and having a pKb at 25C
of not more than 7.
The S-sulfonated immunoglobulin used in this invention denotes a product obtained by cleaving inter-chain SS linkages of a native immunoglobulin and S-sulfona-ting them~
~ he S-sulfonated immunoglobulin can be produced, for example, by reacting an immunoglobulin composed main-ly of gamma-globulin obtained from -the serum, plasma and other body fluids or extracts of organs by a known method such as the ethanol fractionating method of Cohn et al.
[E~ G. Cohn et al., J. Am~ Chem. SocO~ 68, 459 (1946)) with, for example, sodium sulfite and sodium tetra-thionate or sodium tri-thionate by the method disclosed in UOSo Patent No. 4,059,5710 Accordingly, -the specification of U.S. Patent ~o. 4,059,571 is hereby cited as reference.
~he immunoglobulin may be used as fractionated by the aforesaid Cohn's method wi-thout purification (such an immunoglobulin usually contains at least 2~/o by weight of aggregated immunoglobulin molecules having a sedimen-tation constant of a-t least 9S). Or after fractionation, the immunoglobulin may be purified by purifying methods , , .
1~5~
known in the art, such as treatment at pH 4, .salting out, treatment with acrinol, or ion-exchange chromatograph~J
to reduce the content of aggrega~ed immunoglobulin molec-ules to varying degrees, and some-times to substantially zero.
~ he S-sulfonated immunoglobulin thus obtained may be used as such or after it is purified by known methods such as treatment at pH 4, salting out, treat-ment with acrinol or ion exchange chromatography~
~he basic nitrogen-containing organic compound used in this invention contains one or more basic nitro-gen atoms in -the molecule and has a pKb a-t 25C of not more than 7, It may contain carboxyl groups which are smaller in number -than the basic nitrogen atoms so long as the compound shows basicity.
As is well known, pKb used herein is a dissoci-ation index of a basic compolmd which is defined by the following formula pKb - - log K
wherein K=~BH )/~B)o~H ~ in which ~B) is the concentration of the basic compound (i.e. the basic nitrogen-containing organic compound), ~X+) is the hydrogen ion concentration, and ~BH ) is the concentration of a conjugated acid.
Examples of suitable basic nitrogen-containing organic compounds which can be used in this inven-tion include lower alkylamines, ~- or 6-membered heterocyclic compounds having 1 to 3 nitrogen atoms9 guanidines op-tionally substituted by lower alkyl groups, lower alkyl-or aryl-amidines, basic amino acids, esters or amides of neutral amino acids at the carboxyl group, and amine derivatives of glucose. ~hese basic nitrogen-con~aining organic compounds can be used either singly or in com-bination with each other.
~S36 Specific examples of the lower alkylamines are primary lower alkylamines such as meth~lamine, ethylamine, propylamine and butylamine, secondary lower alkylamines such as dimeth~lamine, diethylamine, dipropylamine and dibutylamine and tertiary lower alkylamines such as tri-methylamine, triethylamine, tripropylamine and tributyl-amine. Preferably, the lower alkyl group in the lower alkylamines has l to 4 carbon atoms.
Specific examples of the 5- or 6-membered heterocyclic compounds having l to 3 nitrogen atoms include pyrrolidine, piperidine, imidazole, p~razole and triazole, ~hese compounds may be substituted with an alkyl group having 1 -to 4 carbon atoms (such as 2-methylimidazole).
~pecific examples of the optionally substituted guanidies are guanidine and methylguanidine. Preferably, the substituent alkyl group has l to 4 carbon atoms.
Specific examples of the amidines are Cl - C4 alkyl amidines such as methylamidine, and benzamidines optionally substituted with a Cl - CL~ alkyl group, such as methylbenzamidineO
Examples of the basic amino acids are lysine, ornithine, arginine, hydro~ylysine and histidine.
Speclfic examples of the esters or amides of neutral amino acids include esters formed between neutral amino acids such as glycine, alanine9 valine, leucine, isoleucine, cysteine, cystine and methionine and alcohols, for example aliphatic alcohols having l to 4 carbon atoms such as methanol, ethanol, n-propanol and n-butanol, and amides formed between these neu-tral amino acids and primary or secondary amines having a Cl - C4 alkyl group or ammonia, such as glycinamide, alaninamide and leucin-amide.
~he amine derivatives of glucose are, for example, compounds resulting from substitution of an amino group for at leas-t one of the hydroxyl groups of glucose, such as D-glucosamine.
!
, , ': ' ' ' ~ he basic nitrogen-containing organic compound is used in the form of a water-soluble acid addition salt in this invention. The water-soluble acid addition salt can be favorably prepared by using a mineral acid such as hydrochloric acid, hydrobromic acid, phosphoric acid or sulfuric acid or an organic carboxylic acid such as acetic acid. ~he mineral acid salts are preferred~
Especially preferred are the mineral acid salts, above all hydrochlorides, of arginine, guanidine, leucinamide, imidazole, 2-methylimidazole and D-glucosamineO
~ he S-sulfonated immunoglobulin composition of this invention contains about 1 to about 60~/o by weight, preferably 10 to 600% by weight, more preferably 20 to 40~/0 by weight, based on the S-sulfonated i~munoglobulin, of the water-soluble acid addition salt of the basic nitrogen-containing organic compound. If the amount of the water-soluble acid addition salt is less than about 1% by weight, the effect of this compound to dissociate the aggregated S-sulfonated immunoglobulin molecules and prevent re-aggregation of the molecular chains of the S-sulfonated immunoglobulin becomes so small as to deviate from the range intended by the present invention. If, on the other hand, i-t exceeds about 60~/o by weight, the intended effect can be obtained but economic or opera-tional disadvantages arise.
~ he S-sulfonated immunoglobulin composition of this invention contains the water-soluble acid addition salt of the basic nitrogen-containing organic compound as an aggregation preventing agent or as an aggregate dissociating agent for the S-sulfonated immunoglobulin.
The water-soluble acid addition salt serves to dissociate the aggregated S-sulfonated immunoglobulin molecules and prevent aggregation of the molecules of the S-sulfonated immunoglobulinO
~he essence of this invention lies in the pro-vision of an S-sulfonated immunoglobulin composition which has a low content of aggregated molecules and ' .
~5;~S9~i therefore a high monomer conten-tO
Since the S-sulfonated immunoglobulin composi-tion provided by this invention has a low aggregate con-tent and a very low anticomplement activity, i-t encom-passes a composition which is intravenously injectablewhile containing a water-soluble acid addition salt of a basic nitrogen-containing organic compound such as L-arginine hydrochloride. For example, a composi-tion in accordance with this invention comprlsing an S-sulfonated immunoglobulin and abou-t 10 to about 100% by weight, based on the S-sulfonated immunoglobulin, of ~-arginine hydrochloride is preferably used for this purpose.
The S-sulfonated immunoglobulin composition provided by -this invention also encompasses a composition which cannot be used by itself for intravenous injection and which can be regarded as an intermediate composition for the production of an intravenously injectable ~-sulfonated i~munoglobulin preparation~
This in-termediate composition in accordance with this invention can be formed into an S-sulfonated immunoglobulin prepara-tion suitable for intravenous injection by removing the water-soluble acid addition salt of a basic nitrogen-containing organic compound therein by dialysis, etc~ in a step as close as possible to the final step of lyophilization in the drug formula-ting process, and then lyophilizing the residue.
Such an S-sulfonated immunoglobulin composition can be provided by contacting an S-sulfonated imm~noglo-bulin containing aggregated molecules with the water~
soluble acid addition salt; or contacting a purified S-sulfonated immunoglobulin substantially free from aggre-gatea molecules wi-th the water-soluble acid addition salt; or preparing an S-sulfonated immunoglobulin from an immunoglo~ulin containing aggregated molecules in the presence of the water-soluble acid addition salt.
~ hus7 according to this invention, there is first provided a process for producing an S-sulfonated ' ~ ;
.
~3~
imrnunoglobulin composi.tion having a high monomer content, which comprises contacting an S-sulfonated immunoglobulin containing aggregated molecules in aqueous solution with about 1 to about 600% by weight, based on the S-sulfonated immunoglobulin, of at least one water-soluble acid addi-tion salt of a basic nitrogen~containing organic compound con-taining one or more basic nitrogen atoms in the mole-cule and op-tionally carboxyl groups smaller in number than the basic nitrogen atoms and having a pKb at 2~C
of not more than 7 thereby -to prevent aggregation of the monomer of the S-sulfonated imrnunoglobulin and to dis-sociate the aggregated molecules into monomers, and then if desired, lyophilizing the resulting productO
~econdly, there is pro~ided a process for producing an ~sulfonated immunoglobulin composition having a high monomer content, which comprises contacting a purified S-sulfonated immunoglobulin substantially free from aggregated molecules with the sarne amount as above of at lea:st one aforesaid water-soluble acid addi-tion salt in aqueous solution thereby to prevent aggregation of the monomer of the S-sulfonated immunoglobulin, and then if desired, lyophilizing the resulting product.
Thirdly, the inven-tion provides a process for producing an S-sulfonated immunoglobulin composition having a high monomer content, which comprises reacting an immunoglobulin in aqueous solution with a compound capable of yielding a sulfite ion in water and an oxidi-zing agent in the presence of about 1 to about 600% by weight, based on the weight of -the immunoglobulin, of at least one aforesaid water-soluble acid addition salt~
In the first and second processes of this in-vention, the S-sulfona-ted immunoglobulin and the water-soluble acid addition salt of a basic nitrogen-containing organic compound are contacted with each other in aqueous solutionO The con-tacting is effected at a temperature of about 0 to about 50C, preferably about 0 to about 30C.
Desirably, the pH of the aqueous solution during contac-' ~ ~
-369~i ting is about 5 to about 8O
The greatest characteristic of -the process of this invention is that as seen in the second process as soon as the aggregated S-sulfonated immunoglobulin mole-cules in the starting S-sulfonated immunoglobulin are contacted with the water-soluble acid addition sal-t under the aforesaid con-tacting conditions, dissociation of the aggregated molecules begins, and within a period of as short as l hour, dissociation of the aggrega-ted S-sul-fonated immunoglobulin molecules can be substantiallyachieved and thereby an S-sulfonated immunoglobulin having a high monomer content can be provided.
A second feature of the process of this inven-tion is that as typically expressed by the second process even when the contacting is continued for a period longer than the aforesaid time, substantial dissociation of the aggregated S-sulfonated immunoglobulin molecules is retained over a long period of time.
~he first and second processes in accoraance with this invention can be practiced by directly adding the water-soluble acid addition salt to a solution of the S-sulfonated immunoglobulin, or by mixing a solution of the S-sulfonated immunoglobulin wi-th a solution of the water-soluble acid addition salt.
In the latter-mentioned procedure, the water-soluble acid addition salt may be produced in situ by preparing an aqueous solution of the basic nitrogen-containing organic compound and adding an acid such as hydrochloric acid to the aqueous solution to adaust its pX to about 5 to abou-t 8.
According -to the process of this invention, the resulting aqueous solution of S-sulfonated immunoglobulin having a high monomer content, if required, can be lyo-philized by a method known ~ se to provide the composi-tion of -this invention in the form of a lyophilized solidO
Pre~erably, the composition of this invention provided in the form of a lyophilized solid is an intravenously j ~5;~
inaectable ccmposition comprising a non-toxic water-soluble acid addition salt capable of ~eing used in intravenous injectionO Such an intravenously injectable composition is dissolved in sterilized water or physio-logical saline to form an intravenous injecting prepara-tion.
Preferably, the composi-tion of this invention provided in ~he form of a lyophilized solid contains about 1 to about 10~/o by weight, based on the weight of the immunoglobulin, of a nontoxic water-soluble acid addition salt such as ~-arginine hydrochloride.
The sulfonation reaction in the third process of this invention is carried out in water The pH of the reaction system during the reaction is preferably in the range of 5.0 to 9Ø ~he reaction temperature is from 0 to 50C, preferably from lO to 45C~ At a temperature of more than 50C, the immunoglobulin molecules are undesir-ably susceptible -to denaturation. At a temperature of less than 0C, the reaction proceeds too slowly to be commercially feasible. The reaction is continued until almost all of the interchain SS linkages of the immuno-globulin are cleaved and S-sulfonated. ~he reaction time, which varies depending upon the amounts of the reagents, the reaction temperature, e~tc , is generally from 0~5 to 24 hours.
Suitable oxidizing agents are those whic~ have low reactivity with a sulfite ion generated from the other reagent. E~amples of the oxidizing agents are com-pounds capable of forming a polythionate ion having 3 to 6 sulfur atoms, such as a trithionate ion, a tetrathionate ion, a pentathionate ion or a hexathionate ion, in water (e.g., the sodium, potassium or ammonium salt of a poly-thionic acid having 3 to 6 sulfur atoms); compounds capable of forming A cupric ion in water (e.g., cupric chloride, cupric bromide or cupric sulfate); compounds capable of forming an iodobenzoate ion in water (e.g., the sodium, potassium or ammonium sal~ of iodobenzoic ,, acid); and molecular oxygen-containing gases such as water (in this case, the gases desirably contain a catalytic amount of cysteine or 2-mercaptoet~ylamine).
Examples of the compound capable of yielding a sulfite ion in water include sulfurous acid salts such as sodium sulfite, potassium sulfite and ammonium sulfite;
bisulfites such as sodium bisulfite, potassium bisulfite and ammonium bisulfite; and pysobisulfites such as - sodium pyrobisulfite, potassium pyrobisulfite and am-monium pyrobisulfite.
~ he amount of the compound capable of yielding a sulfite ion in water is at least 2 moles, preferably at least lO moles, per mole of the interchain SS linkage of the immunoglobulin to be cleaved. ~he amount of the oxidizing agent is at least 1 mole, preferably at least
2 mole, per mole of the interchain S~ linkage of the immunoglobulin to be cleaved~
~ he water-soluble acid addition sal-t of a basic nitrogen-containing organic compound to be present in the reaction system may be added to -the reaction system as such a salt or as a basic nitrogen-containing organic compound. I~hen it is added as a basic nitrogen-contain-ing compound, it reacts with an acid such as sulfurous acid present in the reaction system -to change to its water-soluble acid addition salt.
The basic nitrogen-containing organic compound or its water-soluble acid addition salt is added prior to the sulfonation reaction to the reaction system in an amount of about 1 to about 600% by weight, preferably lO
to 600% by weight, more preferably 20 to 400/0 by weight, based on the immunoglobulin. When the amount of the basic nitrogen-containing organic compound is less than 1% by weight, the effect of the invention cannot be sufficiently obtained. If it exceeds 600% by weight, the effect of this inven-tion can be obtained, bu-t economical or opera-tional disadvantages ariseO ~he temperature at which the basic nitrogen containing organic compound is added is ,:
-~;36~S
from 0 to 50C, preferably from 0 to 30C.
According to the third process, dissociation of the aggrega-ted molecules of the resulting S-sulfonated immunGglobulin in the reaction system proceeds simultane-cusly with the S-sulfona-tion reaction of the immunoglo-bulin.
In the S-sulfonated immunoglobulin formed in tke reaction system, the H chain-H chain and H chain-L
chain of the immunoglobulin are mostly cleaved, but the three-dimensional structure of the immunoglobulin is substantially retained by a non-covalent bond such as hydrogen bond between the chainsO
~ he S-sulfonated immunogLobulin is generally separated from the reaction system by using a purifying method such as dialysis, salting out or column chromato-graphy. For example, by dial~zing -the resulting reaction solution ~ith a physiological saline, a solution of -the fin~l product in physiological saline is obtained.
As can be appreciated from the foregoing, the process of this invention includes the following embodi-ments so long as the aggregated S-sulfona-ted immunoglo-bulin molecules can be substantially dissociated and aggregation of the S-sulfonated immunoglobulin molecules is substantially inhibited.
1) A process comprising contacting an S-sulfonated immunoglobulin (obtained by S-sulfonating by, for example, the method described in UOSo Pa-tent ~o. 4,059,571 an immunoglobulin containing at least about 20% by weight of aggregated molecules and prepared by the Cohn's ethanol fractionating method) with the water-soluble ~cid addi-tion salt in aqueous solution. If the S-sulfonated immunoglobulin is provided as a lyophilized product, it is used as an aqueous solution. ~he lyophilized S-sul-folated immunoglobulin contains aggregated molecules.
However, a solid mix-ture of such a solid S~sulfonated immunoglobulin and a predetermined amoun-t of the water-soluble acid addition salt should be understood as ~S~9S
constituting part of the S-sulfonated immunoglobulin composition of this invention because by converting it into an aqueous solution, subst~ntial dissociation of the aggregated S_sulfonated immunoglobulin molecules can be achieved~
2) A process comprising contacting an S-sulfona-ted immunoglobulin (obtained by S-sulfonating by, for ex&mple, the method described in UOSO Patent NoO ~,0597571, an immunoglobulin treated with an acid by the method of Hanson et al. ~Acta Chemica Scandinavica, vol. 22, pages 490 - 496 (1968)~)with the water-soluble acid ad-dition salt in aqueous solution. When the immunoglobulin treated with an acid by the method of Hanson et alO is adjusted to a pH near neutrality, the immunoglobulin molecules again aggregateO Moreover, when the immuno-globulin is maintained at a p~ of about 4 for a long period of time, it undergoes denatura-tion. In -this case, it is desirable to use an immunoglobulin which is acid-treated at a time as close as possible to the performance of -the sulfonation reactionO
~ he water-soluble acid addition sal-t of a basic nitrogen-containing organic compound to be present in the reaction system may be added to -the reaction system as such a salt or as a basic nitrogen-containing organic compound. I~hen it is added as a basic nitrogen-contain-ing compound, it reacts with an acid such as sulfurous acid present in the reaction system -to change to its water-soluble acid addition salt.
The basic nitrogen-containing organic compound or its water-soluble acid addition salt is added prior to the sulfonation reaction to the reaction system in an amount of about 1 to about 600% by weight, preferably lO
to 600% by weight, more preferably 20 to 400/0 by weight, based on the immunoglobulin. When the amount of the basic nitrogen-containing organic compound is less than 1% by weight, the effect of the invention cannot be sufficiently obtained. If it exceeds 600% by weight, the effect of this inven-tion can be obtained, bu-t economical or opera-tional disadvantages ariseO ~he temperature at which the basic nitrogen containing organic compound is added is ,:
-~;36~S
from 0 to 50C, preferably from 0 to 30C.
According to the third process, dissociation of the aggrega-ted molecules of the resulting S-sulfonated immunGglobulin in the reaction system proceeds simultane-cusly with the S-sulfona-tion reaction of the immunoglo-bulin.
In the S-sulfonated immunoglobulin formed in tke reaction system, the H chain-H chain and H chain-L
chain of the immunoglobulin are mostly cleaved, but the three-dimensional structure of the immunoglobulin is substantially retained by a non-covalent bond such as hydrogen bond between the chainsO
~ he S-sulfonated immunogLobulin is generally separated from the reaction system by using a purifying method such as dialysis, salting out or column chromato-graphy. For example, by dial~zing -the resulting reaction solution ~ith a physiological saline, a solution of -the fin~l product in physiological saline is obtained.
As can be appreciated from the foregoing, the process of this invention includes the following embodi-ments so long as the aggregated S-sulfona-ted immunoglo-bulin molecules can be substantially dissociated and aggregation of the S-sulfonated immunoglobulin molecules is substantially inhibited.
1) A process comprising contacting an S-sulfonated immunoglobulin (obtained by S-sulfonating by, for example, the method described in UOSo Pa-tent ~o. 4,059,571 an immunoglobulin containing at least about 20% by weight of aggregated molecules and prepared by the Cohn's ethanol fractionating method) with the water-soluble ~cid addi-tion salt in aqueous solution. If the S-sulfonated immunoglobulin is provided as a lyophilized product, it is used as an aqueous solution. ~he lyophilized S-sul-folated immunoglobulin contains aggregated molecules.
However, a solid mix-ture of such a solid S~sulfonated immunoglobulin and a predetermined amoun-t of the water-soluble acid addition salt should be understood as ~S~9S
constituting part of the S-sulfonated immunoglobulin composition of this invention because by converting it into an aqueous solution, subst~ntial dissociation of the aggregated S_sulfonated immunoglobulin molecules can be achieved~
2) A process comprising contacting an S-sulfona-ted immunoglobulin (obtained by S-sulfonating by, for ex&mple, the method described in UOSO Patent NoO ~,0597571, an immunoglobulin treated with an acid by the method of Hanson et al. ~Acta Chemica Scandinavica, vol. 22, pages 490 - 496 (1968)~)with the water-soluble acid ad-dition salt in aqueous solution. When the immunoglobulin treated with an acid by the method of Hanson et alO is adjusted to a pH near neutrality, the immunoglobulin molecules again aggregateO Moreover, when the immuno-globulin is maintained at a p~ of about 4 for a long period of time, it undergoes denatura-tion. In -this case, it is desirable to use an immunoglobulin which is acid-treated at a time as close as possible to the performance of -the sulfonation reactionO
3) A process comprising contacting ~ purified S-sulfonated immunoglobulin substantially free from ag-gregated molecules with the water-soluble acid addition salt in aqueous solution~
4) A process comprising S-sulfonating in accord-ance with the method described in UOSO Patent No.
4,059,571 an immunoglobulin obtained by the Cohnls ethanol fractionating method or an immunoglobulin acid-treated by the method of ~anson et al. in the presence of the wa-ter-soluble acid addition salt, whereby the S-sulfonated immunoglobulin formed in the S-sulfonation reaction system is contacted with the water-soluble acid addition salt~
rrhe present invention provides the following t~pes of composition.
1) An S-sulfona-ted immunoglobulin composition being substantially free from aggregated molecules and - ~LS3695 being in the form of an aqueous solution capable of be-ing directly used for intravenous inaection.
2) An S-sulfonated immunoglobulin composition in the form of a lyophilized solid which does not substan-tially contain aggregated molecules and can be used asan intravenous injecting preparation by being formed into an aqueous solutionO
3) An S-sulfona-ted immunoglobulin composition which does not substantially contain aggregated molec-ules but is in the form of an aqueous solution whichcannot be directly used as an intravenously injectable prepara-tion~ An intravenously injectable S-sulfonated immunoglobulin preparation can be prepared by removing the water-soluble acid addition salt from this composi-tion not suitable for intravenous injection.
As can be appreciated from the detaileddescription above, the process of this invention makes it possible to dissociate the aggregated molecules of an S-sulfonated immunoglobulin, and to prevent aggrega-tion of -the S-sulfonated immunoglobulin molecules.
Accordingly, the invention also brings about the excel-lent advantage that aggregated molecules can be utilized as dissociated S-sulfonated immunoglobUlinS which can be intravenously injected.
~he following ~xamples and Comparative E~amples illustrate the present invention in detail~ All percen-tages in these examples are by weightO
In these examples, measurement of the monomer content and the anticomplement activity of an S-sulfonated immurloglobulin and sodium dodecylsulfate (SDS) disc elec-trophoresis were performed by the following methods.
Monom r content of S-sulfonated immuno~lobulin ~ he content of monomers (sedimentation constant 7S; molecular weight about 160,000) is determined by sub-aecting 0.3 ml of a 5% aqueous solution of an S-sulfo~a-ted immunoglobulin to gel-filtration analysis. Sepharose C~-6B (Pharmacia CoO) is used as a gel, and a col~n ~ ffaG~e /~/C
l~S3 having a diamete~ of 1.5 cm and a length of ~0 cm is usedO The rate o~ flow of -the solution is 0.17 ml/min.
Anticom~l_ment activit-~
A 1% ~-sulfonated immunoglobulin (GVB ) solu-tion (5 ml) con-taining guinea pig serum, 20 CH50/mlO is incubated at 37C for an hour, and the consumed comple-ment is measured by the method described in Kabat ~
Ma~er, "Experimental Immunochemistry", page 225, 1961.
The an-ticomplement activity levels are indicated by the percentage of consumption to 20 CH50/ml.
SDS disc electroPhoresis The resulting S-sulfonated immunoglobulin is subjec-ted to SDS disc electrophoresis by the method of Weber and Osborne ~J. Biol. Chem. 244, 4406 (19~9)~ to determine the amount of the unreacted immunoglobulin.
Examples l to_10 and Com~ ve Examples l to_4 A solution of 7~5 g of sodium tetrathionate in 50 ml of a OolM phosphate buffer containing sodium chloride and having a p~ of 7~2 and a solution of 12.3 g of sodium sulfite in 100 ml of a O.lM phosphate bufler COlltaining sodium chloride and having a pH of 7.2 ~ere added to 300 ml of a l~/o solu-tion of human immunoglobulin (fraction II obtained b~ the method of Cohn's ethanol fr~ctionating method), and they were reacted at 42C for 405 hours. After the reaction, the reaction mixture was cooled with ice, and dialyzed against a O.~/o a~ueous solution of sodium chloride to obtain a solution of an S-sulfonated immunoglobulin.
A predetermined amount of each of the additive shown in Table 1 (wa-ter-soluble acid addition salts of basic nitrogen-containing organic compounds having a pKb of not more than 7 in accordance with this invention or comparative compounds) was mixed with 10 ml of a 5% solu-tion of the resulting S-sulfonated immunoglobulin. One hour after -the addition and on standing at 4C for 3 weeks, the monomer content and anticomplement activity of -the S-sulfonated immunoglobulin were measuredO The results are shown in Table 1.
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It is seen from ~able 1 that the S~sulfonated immunoglobulin compositions obtained by adding water-soluble salts of basic nitrogen-containing organic compounds having a pKb at 25C of not more than ?
(Examples 1 to 10) have a higher monomer content and a much lower anticomplement activity than the comparative compositions (Comparative Examples 1 to 4) because the aggregated S-sulfonated immunoglobulin molecules are dissociated, and that this state is subs-tantially re-tained even ~fter a lapse of 3 weeks.Exam~le 11 A 10% solu-tion of the same S-sulfonated immuno-globulin as used in Example 1 was purified by salting out with sodium sulfate, and one gram of ~-arginine hydro-chloride was added to 10 ml of a 5% solution of thepurified S-sulfonated immunoglobulin. ~he mixture was allowed to stand at 4C for 1 week. ~he resulting com-position had a monomer content of 930~/O and an anticom-plement activity of 3~5. When ~-arginine hydrochloride was not added, the S-sulfonated immunoglobulin showed an anticomplemen-t ac-tivity of 17.6 and a monomer content of 7805% after standing for 1 week at 4C.
Example 12 A 10% solution of the same S-sulfonated immuno-25 globulin as used in Example 1 was purified wi-th acrinol~ -One gram of I~arginine hydrochloride was added to 10 ml of a ~/0 solution of the purified S-sulfonated immunoglo-bulin, and the mixture was allowed to stand at 4C for one week. The composition showed an anticomplement activity of 207 and a monomer content of 91~/o~ When ~-arginine hydrochloride was not added, the S-sulfonated immunoglobulin showed an anticomplement activity of 15.1 and a monomer content of 770 5% on s-tanding at 4C for 1 week.
35 Exa~les 1~ to 17 and ~
~ en milliliters of a 10% solution of the same S-sulfonated i~nunoglobulin as used in Example 1 was ~l~iL5~895 dialyzed with a OolM acetate buffer h~ving a pH of 4, and treated with an acid in accordance with the method of Hanson et al. ~see Acta Chemica Scandinavica, vol. 22~
pages 490 - 496 (1968)~. ~he treated S-sulfonated immuno-globulin was then dialyzed agains-t a 0005M phosphate buffer containing 0.5M sodium chloride (pH 7.0) to neu-tralize it and to obtain 12.5 ml of a solution of the S-sulfonated imrnunoglobulin (concentration 7.6%).
A predetermined amount of each of the additive (water-soluble sal-ts of basic nitrogen-containing organic compounds having a pKb at 25C of not more than 7 in accordance with -this inven-tion and comparative additives) shown in ~able 2 was added to 10 ml of a 5/0 solution of the acid-treated S-sulfonated imrnunoglobulin. One hour after the addition and on standing at 4C for one week, the monomer con-tent and anticomplement activity of the acid--treated S-sul~onated immlunoglobulin were measured.
~he results are shown in ~able 2.
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~o a)~r~ . ... ... .
~1 ~4 I ~1 ~ co (`~I o rl O U~ J
rl a) .,1 ~rl ~I r-l ~1 ~I r-l O ~ ~
3: ~5 o c) C~, C) 0 U~ __ h h o ~o ~ ~ U~ o U~ o ~ U~
0~ .. . o o o o C' O ~D ~ [~ ~ O
~0 G CO CO (~ CO CO 1~ L~ CO
h ~1~ O O O ~~1 ~9 ~1) 0 ~V O~rl o ~ o o I ~ ~ ~ ~ O O O O
0 rl F~
,-1 ~ o ~
~ Z C) 0 o h ~ ~ ~
a) ~ a,~ . . . O O O O O
-~ ~ Lr\ ~ O ~D CO U~ ~ ~
o ~ ~`' o~ a ~ ----~
a ~ ~u~
~ ~~ ~ u~ u~
0 o ~ ~ o o E~ ~ ~ O O ~ ~ O O ~1 ~
- --r~
h ~c1 o .~ o ~
r~5 h c~
h a~ a r~ r~ ~ ~
p~ ~r l ~rl ~1 ~ o ~1 (1) 02 r--I
S:l o,si a) a~
~r-J ~rl V C) $~
~ O O U~ ~ O ~ r-l O
h ~ r-l h O ~
0 .4 ~ r-l ~D rc I rl ~r^l I I ~? ~0 r--l r--l )~ Q i ~ Q~i ~ ~ C~) h-l~ ~ ~ '~
O O ~ D ~I r~ r-l r--l r~
~4 ~ 0h ~^ o o ~ X ~ X X ~C X~ ~d ~
0 X ~ ~S C~l r.7l r~] r.~ rSl r~ V V V
X ~1 0 X V
~ ~ V
-. _,_ -~3LS36~5 I-t is seen from '~able 2 that in the S-sulfona-ted immunoglobulin compositions obtained by adding the water-soluble salts of basic nitrogen-con-taining organic compounds having a pKb of not more than 7 at 25C (Ex-amples 13 to 17) ~ the formation of aggreg^ated moleculesis inhibited, and their anticomplement activities are maintained low, over a longer period of time than in the case of the comp~r~tive compositions~
Exam~le 18 ~-arginine hydrochloride (1.5 g) was added to 30 ml of a l~/o solution of human immunoglobulin (fraction II ob-tained by the Cohn's ethanol frac-tionating method;
monomer content 76.2%; an-ticomplement activity less than 9~; antibody value diphteria 2.0 units/ml). ~o the re-sulting solution were added a solution of 0.5 g of sodium tetrathionate in 2 ml of a OolM phosphate buffer containing sodium chloride (pH 7.2) and a solution of 0 7 82 g of sodium sulfite in 8 ml of a O.lM phosphate buffer containing sodium chloride (pH 7.2). ~hus, the i~nunoglobulin was S-sulfonated at 37C for 4~5 hours~
After the reaction, the reaction mixture was cooled with ice, and dialyzed against a 0.9~ aqueous solution of sodium chloride to obtain 42 ml of a 6. 5% solution o~ the S-sulfonated immu~oglobulin. ~he resulting S-sulfonated 25 immunoglobulin had the following properties.
Monomer content:
85.6 %
Anticomplement activity:
9..0 %
~0 Amount of immunoglobulin (H2~2):
0.8% by SDS disc electrophoresis Antibody value:
diphteria 2. 0 units/mlO
~e~
By operating under the same conditions as in Example 18 except that I-arginine hydrochloride was not added, 40 ml of a 6.7% solution of the S-sulfonated
4,059,571 an immunoglobulin obtained by the Cohnls ethanol fractionating method or an immunoglobulin acid-treated by the method of ~anson et al. in the presence of the wa-ter-soluble acid addition salt, whereby the S-sulfonated immunoglobulin formed in the S-sulfonation reaction system is contacted with the water-soluble acid addition salt~
rrhe present invention provides the following t~pes of composition.
1) An S-sulfona-ted immunoglobulin composition being substantially free from aggregated molecules and - ~LS3695 being in the form of an aqueous solution capable of be-ing directly used for intravenous inaection.
2) An S-sulfonated immunoglobulin composition in the form of a lyophilized solid which does not substan-tially contain aggregated molecules and can be used asan intravenous injecting preparation by being formed into an aqueous solutionO
3) An S-sulfona-ted immunoglobulin composition which does not substantially contain aggregated molec-ules but is in the form of an aqueous solution whichcannot be directly used as an intravenously injectable prepara-tion~ An intravenously injectable S-sulfonated immunoglobulin preparation can be prepared by removing the water-soluble acid addition salt from this composi-tion not suitable for intravenous injection.
As can be appreciated from the detaileddescription above, the process of this invention makes it possible to dissociate the aggregated molecules of an S-sulfonated immunoglobulin, and to prevent aggrega-tion of -the S-sulfonated immunoglobulin molecules.
Accordingly, the invention also brings about the excel-lent advantage that aggregated molecules can be utilized as dissociated S-sulfonated immunoglobUlinS which can be intravenously injected.
~he following ~xamples and Comparative E~amples illustrate the present invention in detail~ All percen-tages in these examples are by weightO
In these examples, measurement of the monomer content and the anticomplement activity of an S-sulfonated immurloglobulin and sodium dodecylsulfate (SDS) disc elec-trophoresis were performed by the following methods.
Monom r content of S-sulfonated immuno~lobulin ~ he content of monomers (sedimentation constant 7S; molecular weight about 160,000) is determined by sub-aecting 0.3 ml of a 5% aqueous solution of an S-sulfo~a-ted immunoglobulin to gel-filtration analysis. Sepharose C~-6B (Pharmacia CoO) is used as a gel, and a col~n ~ ffaG~e /~/C
l~S3 having a diamete~ of 1.5 cm and a length of ~0 cm is usedO The rate o~ flow of -the solution is 0.17 ml/min.
Anticom~l_ment activit-~
A 1% ~-sulfonated immunoglobulin (GVB ) solu-tion (5 ml) con-taining guinea pig serum, 20 CH50/mlO is incubated at 37C for an hour, and the consumed comple-ment is measured by the method described in Kabat ~
Ma~er, "Experimental Immunochemistry", page 225, 1961.
The an-ticomplement activity levels are indicated by the percentage of consumption to 20 CH50/ml.
SDS disc electroPhoresis The resulting S-sulfonated immunoglobulin is subjec-ted to SDS disc electrophoresis by the method of Weber and Osborne ~J. Biol. Chem. 244, 4406 (19~9)~ to determine the amount of the unreacted immunoglobulin.
Examples l to_10 and Com~ ve Examples l to_4 A solution of 7~5 g of sodium tetrathionate in 50 ml of a OolM phosphate buffer containing sodium chloride and having a p~ of 7~2 and a solution of 12.3 g of sodium sulfite in 100 ml of a O.lM phosphate bufler COlltaining sodium chloride and having a pH of 7.2 ~ere added to 300 ml of a l~/o solu-tion of human immunoglobulin (fraction II obtained b~ the method of Cohn's ethanol fr~ctionating method), and they were reacted at 42C for 405 hours. After the reaction, the reaction mixture was cooled with ice, and dialyzed against a O.~/o a~ueous solution of sodium chloride to obtain a solution of an S-sulfonated immunoglobulin.
A predetermined amount of each of the additive shown in Table 1 (wa-ter-soluble acid addition salts of basic nitrogen-containing organic compounds having a pKb of not more than 7 in accordance with this invention or comparative compounds) was mixed with 10 ml of a 5% solu-tion of the resulting S-sulfonated immunoglobulin. One hour after -the addition and on standing at 4C for 3 weeks, the monomer content and anticomplement activity of -the S-sulfonated immunoglobulin were measuredO The results are shown in Table 1.
, . . .
.
:
~1~3~5 ~ ~ O ~ O r~ o U~ o ~
~ ~ O O ~ O 1~ 9 ~ I ~ > Ci~ O C`~ C' OO ~
~; a.) ~ ' r~ I r~
0 C:) r~ ~
U~ ~
h h o a ci~ ~ ~ u~ ~ ~ ~ ~ ~ ~ ~O
o ~ . . . . o . . o o C' O ~D O ~1 U~ C' CO ~ O r~ o~ U\
~o o c~ co cs~ a) ~ ~ co G~ CO ~ C' ~ C~ C~
_ ~ P`
h ~ ~ ~ ~ ~U r~ ~D ~ N~ (~J ~ ) ~ (~J o r~
a) ~rl o ~ ~ o ~ o o ~ o ~ I ~1 ~ cO (~ D O Ci` ~
-1 ~ ~ I
~ OC) ~ ~ C~ ~
o . . I
h a~ ~ a~, 0O ~ ~ ~ C~ ~ Lr\ ~ r~
~1 o ~ - . ~ O
O ~ ~\ 00 r-l LJ'~ O r~l ~D ~ ~ O ~ ~9 0 ) ~ c~ CO a~ c~ co c~ OE~ cO 00 00 ~ C' C' C~) C--1 ~ - -- .
~ ~ ~ L~
r-l ;~ bl01 11~
3 o o r~ r-l r~l r I r-l O r-l r~ O r~ l 0 __ __ __ ____.__ C' O CO C' Lr~.
,D U~
o ~1 rl r~ r~1r~ ~ O ~
__ ~ ~ a~
~ rl r~ rl rl o rl I S-l I I h ~ r-l h o o O c O
rJ ~1 0 h ,I h h ~1 ~ c~ r1 ~ .~i ~ ~ ,5~ a) rd A P~ c~ h P~ v r~
h c~ ~ o ,S:I .5:1 o o q~ ~ o ~ h N
~l ~ h ~ ~ O o ~ 0 o P~ ~ ~ ~ P~ ~ ~ k., ~
tl ~ r 5::1 r .d ~ -rl ~ 0 a) td) 0 a) rl O
rl (I) ~ O ~ r~ O c~ G) rl r-l r-l ~ rl ~ rl ~rd O ~ O i~ G) a~ ~
~1 r! C.) rl r~ rl V rl N ~ c~ rl bf) o o U~ ~ h rl ~ $-1 ~ h 0 ~ O rl h ~ ~ ~ a~ O ~i G) O r~ O ~ tD h Q) v v ::~
0 4 ~ r~ r-l r-l 0 rl r~ Dr~ rl ~ ~ ~ r ~ $ ~ v ~ ~hl ~ d ~ ~ ~ ~ ~o ~ r~ ~1 _ _ - _ h ~ O r~ <\J r~ ~
h ~l (~J ~\ ~ u~ ~D C~ co C~ ~ ~ , ~ o Q~ Q~ ~ ~ o ~ ~ o o ~ ~ 1 r~ i 1 0 pl V
~ - v ~-~ - - -- ~
'1~3~5 - 19 ~
It is seen from ~able 1 that the S~sulfonated immunoglobulin compositions obtained by adding water-soluble salts of basic nitrogen-containing organic compounds having a pKb at 25C of not more than ?
(Examples 1 to 10) have a higher monomer content and a much lower anticomplement activity than the comparative compositions (Comparative Examples 1 to 4) because the aggregated S-sulfonated immunoglobulin molecules are dissociated, and that this state is subs-tantially re-tained even ~fter a lapse of 3 weeks.Exam~le 11 A 10% solu-tion of the same S-sulfonated immuno-globulin as used in Example 1 was purified by salting out with sodium sulfate, and one gram of ~-arginine hydro-chloride was added to 10 ml of a 5% solution of thepurified S-sulfonated immunoglobulin. ~he mixture was allowed to stand at 4C for 1 week. ~he resulting com-position had a monomer content of 930~/O and an anticom-plement activity of 3~5. When ~-arginine hydrochloride was not added, the S-sulfonated immunoglobulin showed an anticomplemen-t ac-tivity of 17.6 and a monomer content of 7805% after standing for 1 week at 4C.
Example 12 A 10% solution of the same S-sulfonated immuno-25 globulin as used in Example 1 was purified wi-th acrinol~ -One gram of I~arginine hydrochloride was added to 10 ml of a ~/0 solution of the purified S-sulfonated immunoglo-bulin, and the mixture was allowed to stand at 4C for one week. The composition showed an anticomplement activity of 207 and a monomer content of 91~/o~ When ~-arginine hydrochloride was not added, the S-sulfonated immunoglobulin showed an anticomplement activity of 15.1 and a monomer content of 770 5% on s-tanding at 4C for 1 week.
35 Exa~les 1~ to 17 and ~
~ en milliliters of a 10% solution of the same S-sulfonated i~nunoglobulin as used in Example 1 was ~l~iL5~895 dialyzed with a OolM acetate buffer h~ving a pH of 4, and treated with an acid in accordance with the method of Hanson et al. ~see Acta Chemica Scandinavica, vol. 22~
pages 490 - 496 (1968)~. ~he treated S-sulfonated immuno-globulin was then dialyzed agains-t a 0005M phosphate buffer containing 0.5M sodium chloride (pH 7.0) to neu-tralize it and to obtain 12.5 ml of a solution of the S-sulfonated imrnunoglobulin (concentration 7.6%).
A predetermined amount of each of the additive (water-soluble sal-ts of basic nitrogen-containing organic compounds having a pKb at 25C of not more than 7 in accordance with -this inven-tion and comparative additives) shown in ~able 2 was added to 10 ml of a 5/0 solution of the acid-treated S-sulfonated imrnunoglobulin. One hour after the addition and on standing at 4C for one week, the monomer con-tent and anticomplement activity of the acid--treated S-sul~onated immlunoglobulin were measured.
~he results are shown in ~able 2.
i , ~ .
s .
~,, ~
O ~ ~D O r~ ~ ~ cO
~o a)~r~ . ... ... .
~1 ~4 I ~1 ~ co (`~I o rl O U~ J
rl a) .,1 ~rl ~I r-l ~1 ~I r-l O ~ ~
3: ~5 o c) C~, C) 0 U~ __ h h o ~o ~ ~ U~ o U~ o ~ U~
0~ .. . o o o o C' O ~D ~ [~ ~ O
~0 G CO CO (~ CO CO 1~ L~ CO
h ~1~ O O O ~~1 ~9 ~1) 0 ~V O~rl o ~ o o I ~ ~ ~ ~ O O O O
0 rl F~
,-1 ~ o ~
~ Z C) 0 o h ~ ~ ~
a) ~ a,~ . . . O O O O O
-~ ~ Lr\ ~ O ~D CO U~ ~ ~
o ~ ~`' o~ a ~ ----~
a ~ ~u~
~ ~~ ~ u~ u~
0 o ~ ~ o o E~ ~ ~ O O ~ ~ O O ~1 ~
- --r~
h ~c1 o .~ o ~
r~5 h c~
h a~ a r~ r~ ~ ~
p~ ~r l ~rl ~1 ~ o ~1 (1) 02 r--I
S:l o,si a) a~
~r-J ~rl V C) $~
~ O O U~ ~ O ~ r-l O
h ~ r-l h O ~
0 .4 ~ r-l ~D rc I rl ~r^l I I ~? ~0 r--l r--l )~ Q i ~ Q~i ~ ~ C~) h-l~ ~ ~ '~
O O ~ D ~I r~ r-l r--l r~
~4 ~ 0h ~^ o o ~ X ~ X X ~C X~ ~d ~
0 X ~ ~S C~l r.7l r~] r.~ rSl r~ V V V
X ~1 0 X V
~ ~ V
-. _,_ -~3LS36~5 I-t is seen from '~able 2 that in the S-sulfona-ted immunoglobulin compositions obtained by adding the water-soluble salts of basic nitrogen-con-taining organic compounds having a pKb of not more than 7 at 25C (Ex-amples 13 to 17) ~ the formation of aggreg^ated moleculesis inhibited, and their anticomplement activities are maintained low, over a longer period of time than in the case of the comp~r~tive compositions~
Exam~le 18 ~-arginine hydrochloride (1.5 g) was added to 30 ml of a l~/o solution of human immunoglobulin (fraction II ob-tained by the Cohn's ethanol frac-tionating method;
monomer content 76.2%; an-ticomplement activity less than 9~; antibody value diphteria 2.0 units/ml). ~o the re-sulting solution were added a solution of 0.5 g of sodium tetrathionate in 2 ml of a OolM phosphate buffer containing sodium chloride (pH 7.2) and a solution of 0 7 82 g of sodium sulfite in 8 ml of a O.lM phosphate buffer containing sodium chloride (pH 7.2). ~hus, the i~nunoglobulin was S-sulfonated at 37C for 4~5 hours~
After the reaction, the reaction mixture was cooled with ice, and dialyzed against a 0.9~ aqueous solution of sodium chloride to obtain 42 ml of a 6. 5% solution o~ the S-sulfonated immu~oglobulin. ~he resulting S-sulfonated 25 immunoglobulin had the following properties.
Monomer content:
85.6 %
Anticomplement activity:
9..0 %
~0 Amount of immunoglobulin (H2~2):
0.8% by SDS disc electrophoresis Antibody value:
diphteria 2. 0 units/mlO
~e~
By operating under the same conditions as in Example 18 except that I-arginine hydrochloride was not added, 40 ml of a 6.7% solution of the S-sulfonated
5~1695 - 2~ -immunoglobulin was obtainedO ~he resulting S-sulfonated immunoglobulin had a monomer content of 7?.1/~ and an anticomplement activity of 28Ø
Example 19 ~y operating in the same way as in Example 18 except that 1.5 g of ~-ornithine hy~rochloride was used instead oP I-arginine hydrochloride, 40 ml o~ a 6.4%
solution of S-sulfonated immunoglobulin was obtained.
~he resulting S-sulfonated immunoglobulin had a monomer content of 84.1%.
Example 20 By operating in ~the same way as in Example 18 except that 1.5 g of ~-lysine hydrochloride was used irstead of ~-arginine hydrochloride, 41 ml of a 6.6%
solution of S-sulfonated immunoglobulin was obtained.
~he resulting S~sulfonated immunoglobulin had a monomer content of 83. 2%.
Example 21 By operating in the same way as in Example 18 20 except that l-leucinamide hydrochloride was used instead of I-arginine hydrochloride~ 43 ml of a 6.3% solution of S-sulfonated i~munoglobulin was obtainedO ~he resulting S-sulfonated immunoglobulin had a monomer content of 85. 3%.
2 5 Example 2?
By operating in the same way as in Example 18 except that D-glucosa~ine hydrochloride was used instead of ~-arginine hydrochloride, 42 ml of a 606% solution of S-sulfonated immunoglobulin was obtained~ ~he resulting S-sulfonated immunoglobulin had a monomer content of 83.9~/o.
ExamPle ?3 L-arginine hydrochloride (1.5 g) was added to 30 ml of a l~/o solution of human immunoglobulin (~raction 35 II obtained by the Cohn's ethanol fractiona-ting method).
~o the resulting solution were added a solution of 0.4 g of sodium trithionate in 2 ml of a O.lM phosphate buffer ., , , ~53~9S
- 24 _ containing sodium chloride (pH 7.2) and a solution of 0.82 g of sodium sulfite in 8 ml of a OolI1 phosphate buffer containing sodium chloride (pH 7.2)~ ~he im~uno-globulin was thus S-s~lfonated at 37C for 4.5 hours.
After the reaction, the reaction mixture was cooled with ice, and dialyzed agains-t a 0.9~ aqueous solution of sodium chloride to a obtain 45 ml of a 6.0%
solution of an S-sulfonated immunoglobulin. ~he result-ing S-sulfonated immunoglobulin had a monomer content of 86.2% and an anticomplement activity of 8.7%o ', ~
~ . , : - :
Example 19 ~y operating in the same way as in Example 18 except that 1.5 g of ~-ornithine hy~rochloride was used instead oP I-arginine hydrochloride, 40 ml o~ a 6.4%
solution of S-sulfonated immunoglobulin was obtained.
~he resulting S-sulfonated immunoglobulin had a monomer content of 84.1%.
Example 20 By operating in ~the same way as in Example 18 except that 1.5 g of ~-lysine hydrochloride was used irstead of ~-arginine hydrochloride, 41 ml of a 6.6%
solution of S-sulfonated immunoglobulin was obtained.
~he resulting S~sulfonated immunoglobulin had a monomer content of 83. 2%.
Example 21 By operating in the same way as in Example 18 20 except that l-leucinamide hydrochloride was used instead of I-arginine hydrochloride~ 43 ml of a 6.3% solution of S-sulfonated i~munoglobulin was obtainedO ~he resulting S-sulfonated immunoglobulin had a monomer content of 85. 3%.
2 5 Example 2?
By operating in the same way as in Example 18 except that D-glucosa~ine hydrochloride was used instead of ~-arginine hydrochloride, 42 ml of a 606% solution of S-sulfonated immunoglobulin was obtained~ ~he resulting S-sulfonated immunoglobulin had a monomer content of 83.9~/o.
ExamPle ?3 L-arginine hydrochloride (1.5 g) was added to 30 ml of a l~/o solution of human immunoglobulin (~raction 35 II obtained by the Cohn's ethanol fractiona-ting method).
~o the resulting solution were added a solution of 0.4 g of sodium trithionate in 2 ml of a O.lM phosphate buffer ., , , ~53~9S
- 24 _ containing sodium chloride (pH 7.2) and a solution of 0.82 g of sodium sulfite in 8 ml of a OolI1 phosphate buffer containing sodium chloride (pH 7.2)~ ~he im~uno-globulin was thus S-s~lfonated at 37C for 4.5 hours.
After the reaction, the reaction mixture was cooled with ice, and dialyzed agains-t a 0.9~ aqueous solution of sodium chloride to a obtain 45 ml of a 6.0%
solution of an S-sulfonated immunoglobulin. ~he result-ing S-sulfonated immunoglobulin had a monomer content of 86.2% and an anticomplement activity of 8.7%o ', ~
~ . , : - :
Claims (29)
- WHAT WE CLAIM IS:
l. An S-sulfonated immunoglobulin composition com-prising an S-sulfonated immunoglobulin and as an aggrega-tion preventing agent or an aggregate dissociating agent therefor, about 1 to about 600 % by weight, based on the weight of the S-sulfonated immunoglobulin, of a water-soluble acid addition salt of a basic nitrogen-containing organic compound containing one or more basic nitrogen atoms in the molecule and optionally carboxyl groups smaller in number than the basic nitrogen atoms and having a pKb at 25°C of not more than 7. - 2. The composition of claim 1 wherein said basic nitrogen-containing organic compound is at least one member selected from the group consisting of lower alkylamines, 5- or 6-membered heterocyclic compounds having 1 to 3 nitrogen atoms, guanidines optionally substituted by a lower alkyl group, lower alkyl- or aryl-amidines, basic amino acids, esters or arnides of neutral amino acids at the carboxyl group, and amine derivatives of glucose.
- 3. The composition of claim 2 wherein the lower alkylamines have 1 to 4 carbon atoms in the alkyl group.
- 4. The composition of claim 2 wherein the hetero-cyclic compounds are optionally substituted by a lower alkyl group having 1 to 4 carbon atoms.
- 5. The composition of claim 2 wherein the guanidines are guanidine or guanidine derivatives resulting from sub-stitution by a lower alkyl group having 1 to 4 carbon atoms.
- 6. The composition of claim 2 wherein the amidines are lower alkylamidines with 1 to 4 carbon atoms, or benzamidine optionally substituted by a lower alkyl group having 1 to 4 carbon atoms.
- 7. The composition of claim 2 wherein the basic amino acids are lysine, ornithine and arginine.
- 8. The composition of claim 2 wherein the esters of neutral amino acids are esters of the neutral amino acids with lower alcohols having 1 to 4 carbon atoms.
- 9. The composition of claim 2 wherein the amides of neutral amino acids are amides of the neutral amino acids with primary or secondary amines having a Cl - C4 alkyl group or with ammonia.
- 10. The composition of claim 2 wherein the amine derivatives of glucose result from substitution of an amino group for at least one of the hydroxyl groups of glucose.
- 11. The composition of claim 1 which is in the form of an aqueous solution.
- 12. The composition of claim 1 which is in the form of a solid.
- 13. A process for producing an S-sulfonated immunoglobulin composition having a high monomer content, which comprises contacting an S-sulfonated immunoglobulin in aqueous solution with about 1 to about 600% by weight, based on the weight of the S-sulfonated immunoglobulin, of a water-soluble acid addition salt of a basic nitrogen-containing organic compound containing one or more basic nitrogen atoms in the molecule and optionally carboxyl groups smaller in number than the basic nitrogen atoms and having a pKb at 25°C of not more than 7 thereby to prevent aggregation of the monomer of the S-sulfonated immunoglobulin, and when the S-sulfonated immunoglobulin contains aggregated molecules, dissociating the aggregated molecules into monomers, and then if required, lyophilizing the resulting product.
- 14. A process according to claim 13, wherein said basic nitrogen-containing organic compound is at least one member selected from the group consisting of lower alkylamines, 5- or 6-membered heterocyclic compounds having 1 to 3 nitrogen atoms, guanidines optionally substituted by a lower alkyl group,lower alkyl- or aryl-amidines, basic amino acids, esters or amides of neutral amino acids at the carboxyl group, and amine derivatives of glucose.
- 15. A process according to claim 14, wherein the lower alkylamines have 1 to 4 carbon atoms in the alkyl group.
- 16. A process according to claim 14, wherein the heterocyclic compounds are optionally substituted by a lower alkyl group having 1 to 4 carbon atoms.
- 17. A process according to claim 14, wherein the guanidines are guanidine or guanidine derivatives resulting from substitution by a lower alkyl group having 1 to 4 carbon atoms.
- 18. A process according to claim 14, wherein the amidines are lower alkylamidines with 1 to 4 carbon atoms, or benzamidine optionally substituted by a lower alkyl group having 1 to 4 carbon atoms.
- 19. A process according to claim 14, wherein the basic amino acids are lysine, ornithine and arginine.
- 20. A process according to claim 14, wherein the esters of neutral amino acids are esters of the neutral amino acids with lower alcohols having 1 to 4 carbon atoms.
- 21. A process according to claim 14, wherein the amides of neutral amino acids are amides of the neutral amino aGids with primary or secondary amines having a Cl - C4 alkyl group or with ammonia.
- 22. A process according to claim 14, wherein the amine derivatives of glucose result from substitution o an amino group for at least one of the hydroxyl groups of glucose.
- 23. A process according to claim 13, wherein said resulting product is lyophilized to form a solid.
- 24. A process according to claim 23, further comprising dissolving said solid in sterile water or physiological saline to form an aqueous solution.
- 25. The process of claim 13 wherein said contacting is carried out at a temperature of about 0°C to about 50°C.
- 26. The process of claim 13 wherein said contacting is carried out at a pH of from about 5 to about 8.
- 27. A process for producing an S-sulfonated immunoglobulin composition having a high monomer content, which comprises reacting an immunoglobulin in aqueous solution with a compound capable of yielding a sulfite ion in water and an oxidizing agent in the presence of about 1 to about 600% by weight, based on the immunoglobulin, of a water-soluble acid addition salt of a basic pH of about 5 to about 8.
- 28. The process of claim 27 wherin said reaction is carried out at a temperature of about 0°C to about 50°C.
- 29. The process of claim 27 wherein said reaction is carried out at a pH of about 5 to about 8.
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP109,644/79 | 1979-08-30 | ||
| JP109,645/79 | 1979-08-30 | ||
| JP10964579A JPS5634633A (en) | 1979-08-30 | 1979-08-30 | Preparation of s-sulfonated immunoglobulin rich in monomer |
| JP10964479A JPS5634632A (en) | 1979-08-30 | 1979-08-30 | S-sulfonated immunoglobulin composition |
| JP132,786/79 | 1979-10-17 | ||
| JP13278679A JPS5657718A (en) | 1979-10-17 | 1979-10-17 | Preparation of s-sulfonated immunoglobulin containing large amount of monomer |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1153695A true CA1153695A (en) | 1983-09-13 |
Family
ID=27311524
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000359215A Expired CA1153695A (en) | 1979-08-30 | 1980-08-28 | S-sulfonated immunoglobulin composition having a high monomer content and a process for production thereof |
Country Status (4)
| Country | Link |
|---|---|
| US (1) | US4360457A (en) |
| EP (1) | EP0025321B1 (en) |
| CA (1) | CA1153695A (en) |
| DE (1) | DE3067150D1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4470925A (en) * | 1982-05-05 | 1984-09-11 | E. I. Du Pont De Nemours And Company | Immunoglobulin half-molecules and process for producing hybrid antibodies |
| US4479895A (en) * | 1982-05-05 | 1984-10-30 | E. I. Du Pont De Nemours And Company | Immunoglobulin half-molecules and process for producing hybrid antibodies |
| US4597966A (en) * | 1985-01-09 | 1986-07-01 | Ortho Diagnostic Systems, Inc. | Histidine stabilized immunoglobulin and method of preparation |
| GB9418092D0 (en) * | 1994-09-08 | 1994-10-26 | Red Cross Found Cent Lab Blood | Organic compounds |
| WO2002013860A1 (en) * | 2000-08-11 | 2002-02-21 | Chugai Seiyaku Kabushiki Kaisha | Stabilized antibody-containing preparations |
| AU2003219958B2 (en) | 2002-02-27 | 2006-01-05 | Immunex Corporation | Polypeptide formulation |
| US7425618B2 (en) * | 2002-06-14 | 2008-09-16 | Medimmune, Inc. | Stabilized anti-respiratory syncytial virus (RSV) antibody formulations |
| US7132100B2 (en) | 2002-06-14 | 2006-11-07 | Medimmune, Inc. | Stabilized liquid anti-RSV antibody formulations |
| US20040002451A1 (en) | 2002-06-20 | 2004-01-01 | Bruce Kerwin | Compositions of pegylated soluble tumor necrosis factor receptors and methods of preparing |
| US20040208869A1 (en) * | 2003-01-30 | 2004-10-21 | Medimmune, Inc. | Uses of anti-integrin alphanubeta3 antibody formulations |
| JP2008543839A (en) | 2005-06-14 | 2008-12-04 | アムジェン インコーポレーテッド | Self-buffering protein formulation |
| EA201990998A1 (en) | 2016-10-21 | 2019-11-29 | PHARMACEUTICAL COMPOSITIONS AND METHODS FOR PRODUCING THEM |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2560155C2 (en) * | 1974-03-08 | 1984-04-26 | Teijin Ltd., Osaka | Human immunoglobulin derivative |
| US4118379A (en) * | 1974-09-06 | 1978-10-03 | Behringwerke Aktiengesellschaft | Amidated immune globulins and process for preparing them |
| DE2658334B2 (en) * | 1976-12-23 | 1980-06-04 | Behringwerke Ag, 3550 Marburg | Process for the production of immunoglobulin preparations with reduced complement binding and a pharmaceutical containing the same |
-
1980
- 1980-08-28 CA CA000359215A patent/CA1153695A/en not_active Expired
- 1980-08-28 US US06/182,053 patent/US4360457A/en not_active Expired - Lifetime
- 1980-08-29 DE DE8080303004T patent/DE3067150D1/en not_active Expired
- 1980-08-29 EP EP80303004A patent/EP0025321B1/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| EP0025321A3 (en) | 1981-12-23 |
| EP0025321A2 (en) | 1981-03-18 |
| DE3067150D1 (en) | 1984-04-26 |
| US4360457A (en) | 1982-11-23 |
| EP0025321B1 (en) | 1984-03-21 |
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