CA1099635A - Atoxic, immunogenic product of tetanus toxin - Google Patents
Atoxic, immunogenic product of tetanus toxinInfo
- Publication number
- CA1099635A CA1099635A CA283,802A CA283802A CA1099635A CA 1099635 A CA1099635 A CA 1099635A CA 283802 A CA283802 A CA 283802A CA 1099635 A CA1099635 A CA 1099635A
- Authority
- CA
- Canada
- Prior art keywords
- tetanus toxin
- fragment
- atoxic
- product
- tetanus
- 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.)
- Expired
Links
- 108010055044 Tetanus Toxin Proteins 0.000 title claims abstract description 30
- 229940118376 tetanus toxin Drugs 0.000 title claims abstract description 30
- 230000002163 immunogen Effects 0.000 title claims abstract description 14
- 239000012634 fragment Substances 0.000 claims abstract description 38
- 108091005804 Peptidases Proteins 0.000 claims abstract description 8
- 102000035195 Peptidases Human genes 0.000 claims abstract description 8
- 235000019833 protease Nutrition 0.000 claims abstract description 8
- 238000000034 method Methods 0.000 claims description 18
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 6
- 230000001900 immune effect Effects 0.000 claims description 6
- 229940083575 sodium dodecyl sulfate Drugs 0.000 claims description 6
- 235000019333 sodium laurylsulphate Nutrition 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 4
- 238000001962 electrophoresis Methods 0.000 claims description 4
- 238000001502 gel electrophoresis Methods 0.000 claims description 4
- 230000036647 reaction Effects 0.000 claims description 4
- 238000001179 sorption measurement Methods 0.000 claims description 4
- 238000005194 fractionation Methods 0.000 claims description 3
- 239000002808 molecular sieve Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000004062 sedimentation Methods 0.000 claims description 3
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 claims description 3
- ZNZYKNKBJPZETN-WELNAUFTSA-N Dialdehyde 11678 Chemical compound N1C2=CC=CC=C2C2=C1[C@H](C[C@H](/C(=C/O)C(=O)OC)[C@@H](C=C)C=O)NCC2 ZNZYKNKBJPZETN-WELNAUFTSA-N 0.000 claims description 2
- 125000001931 aliphatic group Chemical group 0.000 claims description 2
- 125000004432 carbon atom Chemical group C* 0.000 claims description 2
- 238000005342 ion exchange Methods 0.000 claims description 2
- 229960005367 tetanus antitoxin Drugs 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims 1
- 229960002766 tetanus vaccines Drugs 0.000 abstract description 2
- 239000000047 product Substances 0.000 description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 14
- 239000000243 solution Substances 0.000 description 11
- 239000003795 chemical substances by application Substances 0.000 description 9
- 239000000203 mixture Substances 0.000 description 8
- 108090000526 Papain Proteins 0.000 description 7
- 239000004365 Protease Substances 0.000 description 7
- 229940055729 papain Drugs 0.000 description 7
- 235000019834 papain Nutrition 0.000 description 7
- 239000011780 sodium chloride Substances 0.000 description 7
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 6
- 206010043376 Tetanus Diseases 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 239000003053 toxin Substances 0.000 description 4
- 231100000765 toxin Toxicity 0.000 description 4
- 229960005486 vaccine Drugs 0.000 description 4
- 229920005654 Sephadex Polymers 0.000 description 3
- 239000012507 Sephadex™ Substances 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 241001077660 Molo Species 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 208000003217 Tetany Diseases 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000005227 gel permeation chromatography Methods 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 231100000636 lethal dose Toxicity 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 230000002797 proteolythic effect Effects 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- 241000237074 Centris Species 0.000 description 1
- YVGGHNCTFXOJCH-UHFFFAOYSA-N DDT Chemical compound C1=CC(Cl)=CC=C1C(C(Cl)(Cl)Cl)C1=CC=C(Cl)C=C1 YVGGHNCTFXOJCH-UHFFFAOYSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 229920001425 Diethylaminoethyl cellulose Polymers 0.000 description 1
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 1
- BRLQWZUYTZBJKN-UHFFFAOYSA-N Epichlorohydrin Chemical compound ClCC1CO1 BRLQWZUYTZBJKN-UHFFFAOYSA-N 0.000 description 1
- 102000001390 Fructose-Bisphosphate Aldolase Human genes 0.000 description 1
- 108010068561 Fructose-Bisphosphate Aldolase Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241000712079 Measles morbillivirus Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 238000007290 Pedersen coupling reaction Methods 0.000 description 1
- 201000005702 Pertussis Diseases 0.000 description 1
- 208000000474 Poliomyelitis Diseases 0.000 description 1
- 206010041415 Spastic paralysis Diseases 0.000 description 1
- 206010070863 Toxicity to various agents Diseases 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- 229940024545 aluminum hydroxide Drugs 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000003851 biochemical process Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 229940000425 combination drug Drugs 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- -1 diethylaminoethyl cellulose ion Chemical class 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229960003983 diphtheria toxoid Drugs 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N hydrochloric acid Substances Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 229940124452 immunizing agent Drugs 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 229960005030 other vaccine in atc Drugs 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 102220206201 rs1057524801 Human genes 0.000 description 1
- 235000017550 sodium carbonate Nutrition 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 229960000814 tetanus toxoid Drugs 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/08—Clostridium, e.g. Clostridium tetani
-
- 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
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
ATOXIC, IMMUNOGENIC PRODUCT OF TETANUS TOXIN
Abstract of the disclosure:
The invention provides an atoxic, immunogenic product different from the known fragment C which may be obtained from tetanus toxin by treatment with a proteinase. It also provides a tetanus vaccine containing the novel product.
Abstract of the disclosure:
The invention provides an atoxic, immunogenic product different from the known fragment C which may be obtained from tetanus toxin by treatment with a proteinase. It also provides a tetanus vaccine containing the novel product.
Description
~9~3S HOE 76/B Q16 The present invention relates to an atoxic, immunogenic product of tetanus toxin.
The invention provides an atoxic, immunogenic product which ma~ be obtained from ~etanus toxin by means of a proteinase.
It also provides a tetanus vaccine containing the novel product.
As has already been known, tetanus toxin is highly toxic towards mammals and also humans and must be detox~cated, in order to be used as an immunizing agent. It has been known to detoxicate the tetanus toxin by treating it with formaldehyde.
It has also been known that an atoxic, immunogenic product can be obtained by a treatment of tetanus toxin ~ith a proteinase.
This atoxic, immunogenic product is labelled as fragment C of the tetanus toxin in scientific literature.
It was a surprising fact which could not have been ~oreseen that in the treatment o~ tetanus toxin with a ~roteinase, pre-ferably with papain, a further substance having especially advantageous properties is formed which can be characterized chemically In a definite manner and which may be isolated ac-cording to known biochemical processes.
The particularly advantageous properties of the product ~res er~,~af~o~ of ~h c termed fragment B include the absence of toxi~city and the~im-munogenicity of the same. These properties make the product suitable as a component of vaccines againSt tetanus~
The present ~nvention therefore provides an atoxic, immuno-genic product which may be obtained from tetanus tox~n by treat-ing the same with a proteinase, preferably with papain, which product IS characterized by the follow~ng parameters:
1. Sedimentation constant S20W 5~66+0~34 ,9 2, Immunologic reaction with tetanus antitoxin:
~ 2 -.
1(39~63s ., parti`~lly identical wi.th.that of tetanus toxi~n, 3, Immunologic reaction with fragment C:
not ident~cal.
4. Molecular weight~ determined by a gel electrophoresi~s in sodium dodecylsulfate, of 95 000 - 5 000.
5. Cleavable by reduction into a sub-unit having a molecular weight of 45~0Q + 2 50Q and a sub-unit immunoiogically ,i:dentical with the known derivat~ve of the l~ght chains of the tetanus toxin, having a molecular weight of 48 0Qn +
The invention provides an atoxic, immunogenic product which ma~ be obtained from ~etanus toxin by means of a proteinase.
It also provides a tetanus vaccine containing the novel product.
As has already been known, tetanus toxin is highly toxic towards mammals and also humans and must be detox~cated, in order to be used as an immunizing agent. It has been known to detoxicate the tetanus toxin by treating it with formaldehyde.
It has also been known that an atoxic, immunogenic product can be obtained by a treatment of tetanus toxin ~ith a proteinase.
This atoxic, immunogenic product is labelled as fragment C of the tetanus toxin in scientific literature.
It was a surprising fact which could not have been ~oreseen that in the treatment o~ tetanus toxin with a ~roteinase, pre-ferably with papain, a further substance having especially advantageous properties is formed which can be characterized chemically In a definite manner and which may be isolated ac-cording to known biochemical processes.
The particularly advantageous properties of the product ~res er~,~af~o~ of ~h c termed fragment B include the absence of toxi~city and the~im-munogenicity of the same. These properties make the product suitable as a component of vaccines againSt tetanus~
The present ~nvention therefore provides an atoxic, immuno-genic product which may be obtained from tetanus tox~n by treat-ing the same with a proteinase, preferably with papain, which product IS characterized by the follow~ng parameters:
1. Sedimentation constant S20W 5~66+0~34 ,9 2, Immunologic reaction with tetanus antitoxin:
~ 2 -.
1(39~63s ., parti`~lly identical wi.th.that of tetanus toxi~n, 3, Immunologic reaction with fragment C:
not ident~cal.
4. Molecular weight~ determined by a gel electrophoresi~s in sodium dodecylsulfate, of 95 000 - 5 000.
5. Cleavable by reduction into a sub-unit having a molecular weight of 45~0Q + 2 50Q and a sub-unit immunoiogically ,i:dentical with the known derivat~ve of the l~ght chains of the tetanus toxin, having a molecular weight of 48 0Qn +
2 50Q (each time determined in the sodium dodecyl-sulfate electrophoresis~.
The variation$ shown in the parameters are due to the limits of error within the methods of determinati.on~
The sedimentation constant was determined in an aqueous ~5 soluti,on of 0.2 molar NaCl, 0.02 molar Na2HPO4~ Q.03 molar NaH2PO4 at a pH of 6,8 according to T~ Svedberg and K~O. Peder-sen, "The Ultra~centri:fuge", The Clarendon Pres$J Oxford, ~940, in an overlayer cell of an analytical ultracentrifuge, with re,ference to V~nograd, Proc.Acad.,~ci.. USA, 49, 902 (1963~. The overlayering action of the test solution containing the product of the invention was effected on a ~ molar NaCl solution having a pH value of 7.Q.
The immunologic reactivity was examined while using anti-sera, which were obtained by the immunisation of rabbits with tetanus toxoid or the fragment B of the invention, Use was made of the known double diffusion technique. By means of this technique it can be shown that the formerly described fragment C IS clearl~ distin~ui$hed from the fragment B of the invention.
`~9 ~s. could further be shown, fragment B can be dlssociated into .
1(~9963S
two fragments, one of which shaws the same immunologic behavior as the derivative of the light chain described in Canadian Patent No.
1,066,621, issued Novenber 20, 1979.
The gel electrophoresis in a 7 % polyacrylamide gel with the S use of sodium dodecylsulfate as a camponent of the electrophoresis buffer has been carried out according to K. Weber and M. Osborn, J.Biol.Chem., Vol. 244, 4406 - 4412 (1969). Prior to carrying out the gel electro-phoresis, the samples to be analyzed were mixed in an aqueous solution with sodium dodecylsulfate up to a concentration of 1 % and were kept for 1 minute in boiling water. m e calculation of the molecular weight is effected by a ca~parison with standard substances for determining the molecular weight (Aldolase and Katalase which may be obtained by Messrs.
Boehringer, Mbnnheim, No. of Cat. 15 575 - "Eichproteine Grosse II").
The calculation method has been indicated in the above-cited paper by Weber and Osborn.
Ihe navel atoxic, immunogenic product labelled fragment B
of the tetanus toxm may be obtained according tothe process described in Canadian Patent No. 1,038,293 issued September 12, 1978, by treating tetanus tox m with a proteinase. According to a preferred enbodlment, fragment B is obtained by the treatment of tetanus toxin, which may be produced fram culture filtrates of clostrium Tetani by way of ion exchange chramatography, with papain, preferably in the form of a substance being bound to a carrier, in the presence of a reducing agent.
In this process it has proved to be advantageous to choose an incubation temperature which is higher than usual, i.e. between 30 and 60 C, preferably between 45 and 55 C. By this measure it is possible to increase the yield of the product of ~9963S
the invention. Following the proteolytic action on the tetanus toxin, the enzyme bound to a carrier is eliminated from the re-action mixture, suitably by way of centrifuging, and the remain-ing solution is subjected to a fractionation through a molecular sieve. In this process the use of dextran cross-linked with epichlorhydrin has proved to be advantageous, for example Sephadex(R) G 100 of Firm Pharmacia, Uppsala or Ultrogel(R) of LKB, Bromma or bio-gel p(R) of Bio-Rad Laboratories, Richmond, Calif In case there is used a soluble enzyme preparation, the separation of the enzyme is effected in the process of frac-tionation through the molecular sieve.
Once fragment B has been obtained in this manner, an anti-serum may be prepared with the same by the immunization of test ~5 animals with fragments B, and by means of said antiserum it is then possible to obtain an immuno-adsorbing agent in common manner. This agent serves in its turn to extract fragment B in a particularly pure form from the incubation mixture of said fermentation solution.
It is also possible to eliminate fragment C or tetanus toxin from a mixture with fragment B by immuno-adsorptive measures.
For this purpose, an antiserum against the known fragment C is at first prepared. By means of this antiserum, an immuno-ad-sorbing agent can be obtained in known manner. Said agent is suitable in its turn to eliminate the remainder of fragment C
and of tetanus toxin from fragment B, since ragment C as well as tetanus toxin react with the anti-fragment C-serum, however, fragment B does not~
~, 29 Other processes of isolation, as they are known to the man 1~9963S
skilled in the art for separating proteins having a different electric charge, are also successful. They include above all electrophoretic processes as well as processes of ion exchange chromatography.
The pure fragment B from tetanus toxin which may be obtain-ed in this manner proves to be non toxic as compared against the tetanus toxin. Whereas 30 x 106 min.lethal doses are detected per mg of the tetanus toxin, fragment B has 5 min.lethal doses per mg. With regard to the toxic symptoms, fragment B is seen to be completely different from tetanus toxin; it does not show the symptoms characterized by spastic paralysis. In spite of the reduction of the toxicity to 1:6x 106 it is advantageous to treat fragment B with an aldehyde, as has been described in the following.
For this process the product obtained by means of protei-nases is treated after its isolation with a protein concentra-tion of about 1 mg of protein per ml or less in a buffer solu-tion having a pH value of from 6.0 to 8.5, preferably 7.8, and ~ /~5 ~e~ tre a molarity in the range of from 0.01 to 0.2 molo with 0.015 to ~o/e~ rer/~r~
0.3 molo of an aliphatic mono- or dialdehyde of ~ to 6 carbon atoms, preferably formaldehyde, during 14 to 28 days at a tem-perature in the range of from 20 to 37 C. If desired, the product may be subjected to a dialysis of 10 to 20 hours, pre-ferably 15 hours, against a physiologically acceptable solution, such as 0.15 molar sodium chloride, and/or it may be filtered under sterile conditions. For preparing the vaccine, the pro-duct is suitably also mixed with an adjuvant, for example, alu-minum hydroxide. By dilution with a physiologically acceptable 29 solution, such as 0.1$ molar sodium chloride solution, the con-' ,' ' - ' ', .
1~399635 centration is adjusted to the desired antigen content.
These vaccines may be used by themselves, but also in com-bination with other vaccines. ~ For combination there are sui-table, above all, diphtheria toxoid, pertussis immunogenic agents, poliomyelitis viruses, or measles viruses. By way of adsorption tests it can be shown that about one half of all protecting antibodies directed against tetanus toxin are direct-ed against fragment B.
The following Examples serve to further illustrate the present invention.
E X A M P L E 1:
~f~
t~ Clostrium-tetani are fermented in a latham medium. The toxin formed in this process is adsorbed at diethylaminoethyl cellulose ion exchangers and is elu_ted by a gradiently rising molarity of a phosphate buffer having a pH value of 7 from 0.01 mole to 0.4 mole. The fractions containing the toxin are again separated by chromatography in a column which is filled with Sephadex(R) G 100, and the fractions containing the toxin are extracted and combined.
2.5 Grams of tetanus toxin in a solution having a final concentration of 15 mg of tetanus toxin/ml of a 0~1 molar phos-phate buffer of a pH of 6.5 containing 0.01 mole of Na2EDTA
and 0.001 mole of cystein-HCl are mixed with 40 mg of papain.
The papain contains 30 units of enzymatic activity/mg of sub-stance. At first, the mixture is maintained for 1 hour at 45 C, and thereafter for another 2 hours at 55 C.
For the separation of the tetanus toxin there may be used preferably also papain bound to a carrier.
SO ~9/~/
29 For th;s purpose, a solution of 500 ml' of papain,dialyzed - 7 ~
1~9963S
against ~r0.1 molar Na2CO3 buffer of a pH of 10.0 is combined with 50 mg/m~ of an agarose gel activated with cyanobromide and the mixture is maintained for 24 hours at 4 C. The reac-tion product washed several times with a solution containing 4 moles of urea and 0.5 mole of NaCl per liter is used in a manner analogous to that of the soluble enzyme for the proteo-lytic separation of the tetanus toxin.
Upon cooling of the mixture, the volume is concentrated by means of an ultrafilter, and the mixture is then introduced into a column of a length of 10 x 100 cm filled with Sephadex(R) G 100. The elution is effected with a 0.1 molar trishydroxy-methylaminomethane-hydrochloric acid buffer having a pH value of 8.0 and containing 1 mole of NaCl.
During the elution, the adsorption in the range of the wave length of 280 nm is measured continuously. The fractions showing an adsorption in this range are collected separately.
There are formed 4 fractions, the first of which representing a double peak.
In the course of a repeated chromatography carried out under the same conditions, the two peaks are separated, and finally the second peak is isolated. This latter peak con-tains the atoxic, immunogenic product of the invention, the fragment B from tetanus toxin.
The fragment B of the invention can be obtained in a particularly simple manner, if an antiserum directed against the known fragment C (10 ml with 1 000 IU/ml~ is bound in common manner to agarose activated by BrCN, and a preparation of fragment B ~hich has been partially purified by gel chroma-29 tography is subsequently mixed with such an immuno-adsorbing ~099635 agent. After 60 minutes of stirring, the gel is separated to-gether with the impurities bound to it, and fragment B is ob-tained by another gel chromatography.
It is also possible to neutralize the impurities by adding anti-fragment C-antiserum (IgG fraction) and to separate the f ~
_Lu~ *L}~ complex compounds from fragment B by way of gel chromatography.
Finally, fragment B of the invention may also be obtained if an immuno-adsorbing agent for fragment B is prepared via an antiserum, with the aid of a fragment B once prepared. For this purpose, known processes may be applied. By means of the immu-no-adsorbing agent, fragment B is adsorbed selectively from the mixture with other reaction products from the proteinase treatment of tetanus toxin, whereupon it may be eluted selecti-vely from the immuno-adsorbing agent.
E X A M P L E 2:
Fragment B is diluted with 0.~ molar phosphate buffer solution of a pH of 6.5 to 200 ~g of protein per ml, mixed with 0.06 % formaldehyde and allowed to stand at 37 C for 21 days.
Thereafter, the solution is dialyzed for 16 hours against several t~mes its volume of 0.15 molar sodium chloride solu-tion and is then processed in common manner into a vaccine.
_ g .~
.
The variation$ shown in the parameters are due to the limits of error within the methods of determinati.on~
The sedimentation constant was determined in an aqueous ~5 soluti,on of 0.2 molar NaCl, 0.02 molar Na2HPO4~ Q.03 molar NaH2PO4 at a pH of 6,8 according to T~ Svedberg and K~O. Peder-sen, "The Ultra~centri:fuge", The Clarendon Pres$J Oxford, ~940, in an overlayer cell of an analytical ultracentrifuge, with re,ference to V~nograd, Proc.Acad.,~ci.. USA, 49, 902 (1963~. The overlayering action of the test solution containing the product of the invention was effected on a ~ molar NaCl solution having a pH value of 7.Q.
The immunologic reactivity was examined while using anti-sera, which were obtained by the immunisation of rabbits with tetanus toxoid or the fragment B of the invention, Use was made of the known double diffusion technique. By means of this technique it can be shown that the formerly described fragment C IS clearl~ distin~ui$hed from the fragment B of the invention.
`~9 ~s. could further be shown, fragment B can be dlssociated into .
1(~9963S
two fragments, one of which shaws the same immunologic behavior as the derivative of the light chain described in Canadian Patent No.
1,066,621, issued Novenber 20, 1979.
The gel electrophoresis in a 7 % polyacrylamide gel with the S use of sodium dodecylsulfate as a camponent of the electrophoresis buffer has been carried out according to K. Weber and M. Osborn, J.Biol.Chem., Vol. 244, 4406 - 4412 (1969). Prior to carrying out the gel electro-phoresis, the samples to be analyzed were mixed in an aqueous solution with sodium dodecylsulfate up to a concentration of 1 % and were kept for 1 minute in boiling water. m e calculation of the molecular weight is effected by a ca~parison with standard substances for determining the molecular weight (Aldolase and Katalase which may be obtained by Messrs.
Boehringer, Mbnnheim, No. of Cat. 15 575 - "Eichproteine Grosse II").
The calculation method has been indicated in the above-cited paper by Weber and Osborn.
Ihe navel atoxic, immunogenic product labelled fragment B
of the tetanus toxm may be obtained according tothe process described in Canadian Patent No. 1,038,293 issued September 12, 1978, by treating tetanus tox m with a proteinase. According to a preferred enbodlment, fragment B is obtained by the treatment of tetanus toxin, which may be produced fram culture filtrates of clostrium Tetani by way of ion exchange chramatography, with papain, preferably in the form of a substance being bound to a carrier, in the presence of a reducing agent.
In this process it has proved to be advantageous to choose an incubation temperature which is higher than usual, i.e. between 30 and 60 C, preferably between 45 and 55 C. By this measure it is possible to increase the yield of the product of ~9963S
the invention. Following the proteolytic action on the tetanus toxin, the enzyme bound to a carrier is eliminated from the re-action mixture, suitably by way of centrifuging, and the remain-ing solution is subjected to a fractionation through a molecular sieve. In this process the use of dextran cross-linked with epichlorhydrin has proved to be advantageous, for example Sephadex(R) G 100 of Firm Pharmacia, Uppsala or Ultrogel(R) of LKB, Bromma or bio-gel p(R) of Bio-Rad Laboratories, Richmond, Calif In case there is used a soluble enzyme preparation, the separation of the enzyme is effected in the process of frac-tionation through the molecular sieve.
Once fragment B has been obtained in this manner, an anti-serum may be prepared with the same by the immunization of test ~5 animals with fragments B, and by means of said antiserum it is then possible to obtain an immuno-adsorbing agent in common manner. This agent serves in its turn to extract fragment B in a particularly pure form from the incubation mixture of said fermentation solution.
It is also possible to eliminate fragment C or tetanus toxin from a mixture with fragment B by immuno-adsorptive measures.
For this purpose, an antiserum against the known fragment C is at first prepared. By means of this antiserum, an immuno-ad-sorbing agent can be obtained in known manner. Said agent is suitable in its turn to eliminate the remainder of fragment C
and of tetanus toxin from fragment B, since ragment C as well as tetanus toxin react with the anti-fragment C-serum, however, fragment B does not~
~, 29 Other processes of isolation, as they are known to the man 1~9963S
skilled in the art for separating proteins having a different electric charge, are also successful. They include above all electrophoretic processes as well as processes of ion exchange chromatography.
The pure fragment B from tetanus toxin which may be obtain-ed in this manner proves to be non toxic as compared against the tetanus toxin. Whereas 30 x 106 min.lethal doses are detected per mg of the tetanus toxin, fragment B has 5 min.lethal doses per mg. With regard to the toxic symptoms, fragment B is seen to be completely different from tetanus toxin; it does not show the symptoms characterized by spastic paralysis. In spite of the reduction of the toxicity to 1:6x 106 it is advantageous to treat fragment B with an aldehyde, as has been described in the following.
For this process the product obtained by means of protei-nases is treated after its isolation with a protein concentra-tion of about 1 mg of protein per ml or less in a buffer solu-tion having a pH value of from 6.0 to 8.5, preferably 7.8, and ~ /~5 ~e~ tre a molarity in the range of from 0.01 to 0.2 molo with 0.015 to ~o/e~ rer/~r~
0.3 molo of an aliphatic mono- or dialdehyde of ~ to 6 carbon atoms, preferably formaldehyde, during 14 to 28 days at a tem-perature in the range of from 20 to 37 C. If desired, the product may be subjected to a dialysis of 10 to 20 hours, pre-ferably 15 hours, against a physiologically acceptable solution, such as 0.15 molar sodium chloride, and/or it may be filtered under sterile conditions. For preparing the vaccine, the pro-duct is suitably also mixed with an adjuvant, for example, alu-minum hydroxide. By dilution with a physiologically acceptable 29 solution, such as 0.1$ molar sodium chloride solution, the con-' ,' ' - ' ', .
1~399635 centration is adjusted to the desired antigen content.
These vaccines may be used by themselves, but also in com-bination with other vaccines. ~ For combination there are sui-table, above all, diphtheria toxoid, pertussis immunogenic agents, poliomyelitis viruses, or measles viruses. By way of adsorption tests it can be shown that about one half of all protecting antibodies directed against tetanus toxin are direct-ed against fragment B.
The following Examples serve to further illustrate the present invention.
E X A M P L E 1:
~f~
t~ Clostrium-tetani are fermented in a latham medium. The toxin formed in this process is adsorbed at diethylaminoethyl cellulose ion exchangers and is elu_ted by a gradiently rising molarity of a phosphate buffer having a pH value of 7 from 0.01 mole to 0.4 mole. The fractions containing the toxin are again separated by chromatography in a column which is filled with Sephadex(R) G 100, and the fractions containing the toxin are extracted and combined.
2.5 Grams of tetanus toxin in a solution having a final concentration of 15 mg of tetanus toxin/ml of a 0~1 molar phos-phate buffer of a pH of 6.5 containing 0.01 mole of Na2EDTA
and 0.001 mole of cystein-HCl are mixed with 40 mg of papain.
The papain contains 30 units of enzymatic activity/mg of sub-stance. At first, the mixture is maintained for 1 hour at 45 C, and thereafter for another 2 hours at 55 C.
For the separation of the tetanus toxin there may be used preferably also papain bound to a carrier.
SO ~9/~/
29 For th;s purpose, a solution of 500 ml' of papain,dialyzed - 7 ~
1~9963S
against ~r0.1 molar Na2CO3 buffer of a pH of 10.0 is combined with 50 mg/m~ of an agarose gel activated with cyanobromide and the mixture is maintained for 24 hours at 4 C. The reac-tion product washed several times with a solution containing 4 moles of urea and 0.5 mole of NaCl per liter is used in a manner analogous to that of the soluble enzyme for the proteo-lytic separation of the tetanus toxin.
Upon cooling of the mixture, the volume is concentrated by means of an ultrafilter, and the mixture is then introduced into a column of a length of 10 x 100 cm filled with Sephadex(R) G 100. The elution is effected with a 0.1 molar trishydroxy-methylaminomethane-hydrochloric acid buffer having a pH value of 8.0 and containing 1 mole of NaCl.
During the elution, the adsorption in the range of the wave length of 280 nm is measured continuously. The fractions showing an adsorption in this range are collected separately.
There are formed 4 fractions, the first of which representing a double peak.
In the course of a repeated chromatography carried out under the same conditions, the two peaks are separated, and finally the second peak is isolated. This latter peak con-tains the atoxic, immunogenic product of the invention, the fragment B from tetanus toxin.
The fragment B of the invention can be obtained in a particularly simple manner, if an antiserum directed against the known fragment C (10 ml with 1 000 IU/ml~ is bound in common manner to agarose activated by BrCN, and a preparation of fragment B ~hich has been partially purified by gel chroma-29 tography is subsequently mixed with such an immuno-adsorbing ~099635 agent. After 60 minutes of stirring, the gel is separated to-gether with the impurities bound to it, and fragment B is ob-tained by another gel chromatography.
It is also possible to neutralize the impurities by adding anti-fragment C-antiserum (IgG fraction) and to separate the f ~
_Lu~ *L}~ complex compounds from fragment B by way of gel chromatography.
Finally, fragment B of the invention may also be obtained if an immuno-adsorbing agent for fragment B is prepared via an antiserum, with the aid of a fragment B once prepared. For this purpose, known processes may be applied. By means of the immu-no-adsorbing agent, fragment B is adsorbed selectively from the mixture with other reaction products from the proteinase treatment of tetanus toxin, whereupon it may be eluted selecti-vely from the immuno-adsorbing agent.
E X A M P L E 2:
Fragment B is diluted with 0.~ molar phosphate buffer solution of a pH of 6.5 to 200 ~g of protein per ml, mixed with 0.06 % formaldehyde and allowed to stand at 37 C for 21 days.
Thereafter, the solution is dialyzed for 16 hours against several t~mes its volume of 0.15 molar sodium chloride solu-tion and is then processed in common manner into a vaccine.
_ g .~
.
Claims (4)
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for the preparation of an atoxic, immunogenic product from tetanus toxin, which is characterized by the following parameters:
a. Sedimentation constant S?0W 5.66 ? 0.34 b. Immunologic reaction with tetanus antitoxin:
partially identical with that of tetanus toxin.
c. Immunologic reaction with fragment C:
not identical.
d. Molecular weight, determined by a gel electrophoresis in sodium dodecylsulfate, of 95,000 ? 5,000.
e. Cleavable by reduction into a sub-unit having a molecular weight of 45,000 ? 2,500 and a sub-unit immunologically identical with the known derivative of the weight of 48,000 ? 2,500 (each time determined in the sodium dodecylsulfate electrophoresis).
in which the product obtained by treating tetanus toxin with a proteinase is extracted by a fractionation through a molecular sieve, by an immuno-adsorption process, by an electrophoresis, by ion exchange chromotography or by a combination of these extraction processes.
a. Sedimentation constant S?0W 5.66 ? 0.34 b. Immunologic reaction with tetanus antitoxin:
partially identical with that of tetanus toxin.
c. Immunologic reaction with fragment C:
not identical.
d. Molecular weight, determined by a gel electrophoresis in sodium dodecylsulfate, of 95,000 ? 5,000.
e. Cleavable by reduction into a sub-unit having a molecular weight of 45,000 ? 2,500 and a sub-unit immunologically identical with the known derivative of the weight of 48,000 ? 2,500 (each time determined in the sodium dodecylsulfate electrophoresis).
in which the product obtained by treating tetanus toxin with a proteinase is extracted by a fractionation through a molecular sieve, by an immuno-adsorption process, by an electrophoresis, by ion exchange chromotography or by a combination of these extraction processes.
2. An atoxic, immunogenic product from tetanus toxin, as defined in claim 1, whenever obtained according to a process as claimed in claim 1 or by an obvious chemical equivalent thereof.
3. A process as claimed in claim 1 in which the product is treated at a pH value of from 6.0 to 8.5 with from 0.015 to 0.3 mole of an aliphatic mono- or dialdehyde with 1 to 6 carbon atoms for 14 to 28 days at a temperature in the range of from 20 to 37°C.
4. An atoxic, immunogenic product, whenever obtained according to a process as claimed in claim 3 or by an obvious chemical equivalent thereof
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP2634584.6 | 1976-07-31 | ||
| DE2634584A DE2634584C3 (en) | 1976-07-31 | 1976-07-31 | Atoxic, immunogenic product from tetanus toxin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1099635A true CA1099635A (en) | 1981-04-21 |
Family
ID=5984461
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA283,802A Expired CA1099635A (en) | 1976-07-31 | 1977-07-29 | Atoxic, immunogenic product of tetanus toxin |
Country Status (18)
| Country | Link |
|---|---|
| JP (1) | JPS5326319A (en) |
| AT (1) | AT362498B (en) |
| AU (1) | AU516191B2 (en) |
| BE (1) | BE857373A (en) |
| CA (1) | CA1099635A (en) |
| DE (1) | DE2634584C3 (en) |
| DK (1) | DK342577A (en) |
| EG (1) | EG12732A (en) |
| FI (1) | FI58500C (en) |
| FR (1) | FR2360315A1 (en) |
| GB (1) | GB1570958A (en) |
| IE (1) | IE45539B1 (en) |
| IL (1) | IL52617A (en) |
| IT (1) | IT1085660B (en) |
| LU (1) | LU77874A1 (en) |
| NL (1) | NL7708279A (en) |
| NO (1) | NO772704L (en) |
| SE (1) | SE7708693L (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB8516442D0 (en) * | 1985-06-28 | 1985-07-31 | Wellcome Found | Cloned antigen |
-
1976
- 1976-07-31 DE DE2634584A patent/DE2634584C3/en not_active Expired
-
1977
- 1977-07-26 NL NL7708279A patent/NL7708279A/en not_active Application Discontinuation
- 1977-07-27 EG EG446/77A patent/EG12732A/en active
- 1977-07-27 AU AU27380/77A patent/AU516191B2/en not_active Expired
- 1977-07-28 FI FI772305A patent/FI58500C/en not_active IP Right Cessation
- 1977-07-28 SE SE7708693A patent/SE7708693L/en not_active Application Discontinuation
- 1977-07-29 AT AT563477A patent/AT362498B/en not_active IP Right Cessation
- 1977-07-29 NO NO772704A patent/NO772704L/en unknown
- 1977-07-29 IL IL52617A patent/IL52617A/en unknown
- 1977-07-29 LU LU77874A patent/LU77874A1/xx unknown
- 1977-07-29 IT IT26354/77A patent/IT1085660B/en active
- 1977-07-29 CA CA283,802A patent/CA1099635A/en not_active Expired
- 1977-07-29 IE IE1590/77A patent/IE45539B1/en unknown
- 1977-07-29 JP JP9213677A patent/JPS5326319A/en active Pending
- 1977-07-29 GB GB31968/77A patent/GB1570958A/en not_active Expired
- 1977-07-29 DK DK342577A patent/DK342577A/en not_active Application Discontinuation
- 1977-08-01 FR FR7723600A patent/FR2360315A1/en active Granted
- 1977-08-01 BE BE179821A patent/BE857373A/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| FI772305A (en) | 1978-02-01 |
| JPS5326319A (en) | 1978-03-11 |
| FR2360315A1 (en) | 1978-03-03 |
| AT362498B (en) | 1981-05-25 |
| EG12732A (en) | 1979-09-30 |
| FI58500B (en) | 1980-10-31 |
| AU516191B2 (en) | 1981-05-21 |
| DE2634584B2 (en) | 1979-03-22 |
| AU2738077A (en) | 1979-02-01 |
| NO772704L (en) | 1978-02-01 |
| DE2634584C3 (en) | 1979-11-08 |
| LU77874A1 (en) | 1978-02-02 |
| IT1085660B (en) | 1985-05-28 |
| DK342577A (en) | 1978-02-01 |
| DE2634584A1 (en) | 1978-02-02 |
| NL7708279A (en) | 1978-02-02 |
| IE45539L (en) | 1978-01-31 |
| FR2360315B1 (en) | 1980-04-11 |
| BE857373A (en) | 1978-02-01 |
| IL52617A (en) | 1980-12-31 |
| FI58500C (en) | 1981-02-10 |
| IE45539B1 (en) | 1982-09-22 |
| GB1570958A (en) | 1980-07-09 |
| ATA563477A (en) | 1980-10-15 |
| IL52617A0 (en) | 1977-10-31 |
| SE7708693L (en) | 1978-02-01 |
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