CA1063021A - Derivatives of the tetanus toxin, process for their preparation and agents containing them - Google Patents
Derivatives of the tetanus toxin, process for their preparation and agents containing themInfo
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- CA1063021A CA1063021A CA313,399A CA313399A CA1063021A CA 1063021 A CA1063021 A CA 1063021A CA 313399 A CA313399 A CA 313399A CA 1063021 A CA1063021 A CA 1063021A
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- aldehyde
- light chain
- tetanus
- tetanus toxin
- toxin
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Abstract
ABSTRACT
This invention is directed to a process for the pre-paration of a modified derivative of the light chain tetanus toxin in which the light chain of tetanus toxin free from denaturing agent is mixed with an aliphatic mono- or di-aldehyde having a chain length of from 1 to 6 carbon atoms, up to an aldehyde concentration of from 0.05 mole to 0.2 mole. The mixture is then allowed to stand for 14 to 28 days at a temperature of from 20 to 37°C, the aldehyde is eliminated and the modified derivative of the light chain is obtained. The invention is also directed to the product prepared according to this process. The products of the invention are useful in the preparation of tetanus vaccines.
This invention is directed to a process for the pre-paration of a modified derivative of the light chain tetanus toxin in which the light chain of tetanus toxin free from denaturing agent is mixed with an aliphatic mono- or di-aldehyde having a chain length of from 1 to 6 carbon atoms, up to an aldehyde concentration of from 0.05 mole to 0.2 mole. The mixture is then allowed to stand for 14 to 28 days at a temperature of from 20 to 37°C, the aldehyde is eliminated and the modified derivative of the light chain is obtained. The invention is also directed to the product prepared according to this process. The products of the invention are useful in the preparation of tetanus vaccines.
Description
HOE 74/~ 02~
10~30Zl The present lnvention relate~ to derlvatives of tetanus toxln, a proce~s for thelr preparatlon and agents contalning them.
More particularly the present invention provides derivatives of tetanus toxin, a proce~s for preparing them by modifying a partial molecule of the tetanus toxin and agents, in par-ticular tetanus vaccines, which contain one of the derivatives of tetanu~ toxin.
The common tetanus vaccine~ for the active immunization contain almost exclusively an antigen whlch has been prepared ; by the lnactivation of tetanus toxin with formaldehyde. This substance called toxoid i~ provided with a large number of antigenlc determinants, only a few of which being important, however, for the production of antibodies that protect against tetanus. The elimination of those determinants which are not - required for protection is desirable, in order to obtain i antlgenic and/or immunogenio substances, of which - due to a narrower spectrum of determinant groups - an increased speci-; ficity and an improved compatibility may be expected.
Tetanus toxin is synthesized from cells of Cl. tetani and can be obtained from the cell mass as well as from the culture filtrate. Whereas the toxin obtained in an extracellular way consists of two polypeptide chains which may be separated after having been treated with agents reducing disulfide bridges (for example, dithiothreitol (DTT), dithioerythritol ~`~ (DTE), mercaptoethanol, cysteine), under denaturing conditions, the lntracellular toxin consists of a single polypeptide chain which can be converted in known manner, by a mild trypsin (3.4.21.
29 treatment, into a product which is identical to the extra-~;~ 2 ', ~ ~ ' ' ''' ' . ' '. . . ' ' '., " , .
. 1063~)21 cellul~r toxln. The two fragments to be obtalned of thls product, whlch have been called , in this context, "llght chain" (molecular weight of about 50~000) and "heavy chain" (molecular - -welght of about 100,000)~ could only be separated, up to now, by measure~ which are carried out under denaturlng conditions, for example, in a solution containine sodium dodecyl sulfate, and the separated fragments can be kept in solutlon, according to the prior art, only by denaturing agents.
It was therefore the task of the pre~ent invention to modlfy the light chain of the tetanus toxin in a way that it could be kept in a physiologically tolerable medium without losing its antigenicity. m e light chain was to be available in particular as an es3ential immunogenic constituent of tetanus ~accine~ and was to replace the common toxoid in these substances.
It has now been found that this task can be solved by sub-~ecting the llght chain, either before or after the separation . . .
of the heavy chaln from the molecular structure of the tetanus toxin, which represents a complex of the light and heavy chains, to a slight chemical modification. By way of thi~ method, the spontaneous irreversible precipitation of the light chain, which i8 ob~eryed after the elimination o*
. denaturing agents like urea, can be avoided.
me Present invention provides a process for the pre-paratlon of a derlvatlve of the 11ght chain of the tetanus toxln, which compri~e~ treating an aqueous solutlon of the .~ .
extracellular tetanus toxin ~ith an aliphatic mono- or d~alde-29 hyde havlng a chain length of from 1 to 6 carbon atoms in an .~: . . . .
. . .
-1063~Zl aldehyde concentratlon of from 0.01 to 0.05 mole of aldehyde at a temperature of from 1 to 20C for 5 minutes to 50 hourq, reacting the toxin before or after the sldehyde treatment in the presence of a compound reduc~ng di~ulfide bridges wlth a denaturing agent , subsequently obtaining the derivati~e of the light chain in the-presence of the denaturing agent by way of proteinchemlcal isolation processes and suitably separating it from the denaturing agent.
A preferred variant of the proces~ provide~ treatlng an aqueous solution of the tetanus toxln with an aliphatic mono-or dialdehyde having a chain length of from 1 to 6 carbon atoms, preferably formaldehyde, with an aldehyde concentration of from 0.02 mole to 0.03 mole, at a temperature of from 1 to 6C
for 2 to 20 hours, mixing the solution containing the toxin ; 15 thus modified with an agent reducing disulfide bridges, ob-taining the derivative of the light chain from the solution obtained in the presence of a denaturing agent by way of proteinchemical isolation processes and sultably separat~ng it i from the denaturing agent.
- 20 After the isolation of the derivative of the light chain of the tetanus toxin, the denaturing agent can be eliminated by way of dialysis or other comparable methods which allow the separation of low-molecular weight substances from high-~ . .
molecular weight protein, for example by gel chromatography.
~- 25 The aldehyde, too, which has been used for the reaction but ha~ not been used up, may be eliminated from the reactlon mlxtures by the same measures.
~" The derlvatlve prep~rod accordlng to the proce~ of tho 29 lnventlon does no more show, after thls treatment, tho ~: , .
, , . ,.. ,; ., .. . :
~ ` HOE 74/B 028 1063~)21 denaturlng and preclpltsting properties known from the light chaln, in phy~iologically tolerable, isotonically aqueous medla.
The derlvative no longer has the toxic property known from the tetanus toxin. It shows an antigen behavior and i~ in a positlon to lnduce antlbodle~ protecting again~t tetanus ln the human or animal organlsm.
The derlvatlve prepared accordlng to the invention is to be further purlfled by the common proteinchemical method~. In ~iew of the admittedly high toxicity of the native tetanus toxin and the fact - which 18 al~o known - that methods used for the dlssoclation of proteins only in the rarest cases lead to a 100 per cent separation of accompanying substances, in particular of protelns having a similar structure, it i8 ad-vantageous, however, to submit the product prepared accordlng to the invention to a further aldehyde treatment, which com-prises mlxing the derivative of the light chaln of the tetanus toxin with an aliphatic mono- or dialdehyde having a chain length of from 1 to 6 carbon atoms, preferably formaldehyde, up to an aldehyde concentration in the range of from 0.05 to 0.2 mole, preferably from 0.08 to 0.15 mole, and allowing it to stand for 14 to 28 days at a temperature in the range Or from 20 to 37C, elimlnating the aldehyde, for example by way of dialysis, and obtainlng the modified derivative of the light chain Or the tetanus toxin.
The additional aldehyde treatment leads to a further-stabilization of the llght chain Or the tetanus toxin and to the-elimination of the re~idual toxicity which may still be present.
29 It is obvlou~ that a stable derlvatlve of the light chaln .
~OE 74lB 028 1063~)21 may also be prepared by treatlng native tetanus toxln obtalned from culture filtrates of Cl. tetani wlth ~ubstances reduclng dlsulflde brldges, for example thlol compounds, by effecting ¦ -the separation of the light chaln from the heavy chaln under denaturing conditions, preferably by way Or chromatography ln buffer solutions containing, for example, urea, subsequently by measures which allow the separation Or low-molecular weight substances from high-molecular weight protein, by eliminating the denaturing agent suitably by gel chromatography~ and by allowing the light chain to stand immediately with an aliphatic mono- or dialdehyde having a chaln length of from 1 to 6 carbon atoms, preferably formaldehyde, wlth an aldehyde concentration of from 0.01 to 0.05 mole, for 5 minutes to 50 hours, at a temperature of irom 1 to 20C. A second aldehyde treatment in order to modify the derivative may optionally be performed.
It i8 also possible, however, to react the light chain im-mediately after its isolation with the higher amount of al-dehyde used for the modification of the derivative. However, these processes do not involve any advantage, since the yield .~ , . . .
of the derivative of the light chain prepared according to i the latter process is considerably smaller than tho one ob-tained according to the former process.
As starting material, use is made of tetanus toxin whicb ; has been obtalned from culture filtrates of Cl. tetani in known manner (extracellular toxin). As an alternative there may be used a tetanus toxin prepared according to known processes which was obtained by extracting the Cl. tetani `~ bacteria (intracellular toxin). In this caso~ however, a trypsin ~9 (3-4 21;4)treatment to be carried out according to a known .. . .
" " ' ~ ; ' ', " ' ' ' ' . ' . -' '` .
. .. : ~ ., ,. , , .......... . : .
HOE 74/~ 028 method is required before or after the reaction with aldehyde.
If the light chain is not to be reacted with the aldehyde until after the separation from the heavy chain, it is clear that a trypsin (3.4.21.4) treatment is to be effected, in the case of intracellular toxin, before the reaction with aldehyde, as the proteolytic step represents a pre-requisite for the isolation of the light chain from this starting material. the tetanus toxin solution used as starting material contains the toxin suitably in a concentration of from 0.5 to 10 mg/ml.
As compounds reducing disulfide bridges, preference is given to thiol compounds. For this purpose there may be mentioned, for example, cysteine, mercapto-ethanol or dithio-erythritol, preferably however, dithiothreitol. These com-pounds are added in amounts which cause a concentration of the compounds reducing disulfide bridges in the reaction mixture of from 0.05 to 0.3 mole.
By denaturing agents in the sense of the present process there are to be understood chemical compounds which help to dissociate protein molecules to obtain sub-units, in particular by dissolving hydrogen bonds. Known agents to dissolve hydrogen bonds are, for example, urea or guanidine.hydrochloride.
Urea has proved to be effective preferably in a concentration - of from 4 to 6 moles, whereas guanidine hydrochloride is par-- - ticularly effective in a concentration of from 2 to 4 moles.
It is not possible to isolate a modified light chain from the convention~l tetanus toxoid, since in this case the use of con~iderably higher amounts of formaldehyde has resulted in a covalent cross-linking between the light and the heavy chains.
~ .
~ ~- 29 However, there is no reason not to react any solution con-. ~ , .
.~ , , . , , . . - . - , ~ HOE 74/~ 028 1063~)Zl taining the light chain which has not been denatured, with aldehydes, in order to obtain derivatives having the advan-tageous properties with regard to stabllity and to antigenlcity.
Be~ide~ the above-mentioned proce~ variants, derivatives of the tetanus toxln are also a sub~ect of this ~nvention, whlch are to be characterized by parameters resulting from their preparation according to the pre~ent process .
Finally, the present invention provides agents containing the tetanus toxin derivatives of the inventlon, in particular tetanus vaccines for the prophylaxis again~t the tetanus disease or for the preparation of tetanuæ antisera to be u~ed for thera-peutical or diagnostic purposes, but also diagnostic prepara--~ tlon~ contalnlng the tètanus toxin derivatlves or antisera ob-tained from them. In order to increase the solution stability of the product prepared according to the invention, lt may be advantageous to add com-pounds used for the stabilization of proteln solutions, such as amino aclds or carbohydrates, to the physiologically tolerable aqueous medium, in which the above product-ls dis-solved.
; - It i8 recommended, for the protection against microbial contamination, to add antimicrobial sUbstances~ such as sodium timerfonate, to the solutions which are ready for use. For ` the preparation of a polyvalent vaccin~, the tetanus toxln }
derlvatives may be mixed wlth other antlgens and/or toxoids in known manner.
~ The suitabillty Or the products as vacclnes has been proved -~ in the following by te~ts which certlfy a good potency of the 29 product.
:
HOE 74/B 028_ 1063~Zl Potency test a) In vitro In order to determine the protective potency of the determinant groups which are pre~ent on the light chain, the followlng test was carried out:
A tetanus antitoxin serum of equine origin was ab-sorbed with increasing amounts of the product according to Example 1. By way of examining the supernatants with the aid of the immunological double dlffusion technique, the equivalence zone, in which the entire antibodies directed against the derivative of the light chain were precipitated, was determined. The evaluation of the anti- -bodies in the animal test showed that up to 40 % of the protecting antibodies from the antiserum of the product of the invention could be absorbed.
. .
b) In vivo Groups of 10 Guinea pigs each were given suboutaneously 1 ml (20 ~g) of a suspension of the product prepared ac-cordlng to Example 1, adsorbed on 0.2 ~ Al(OH)3 gel.
After 4 weeko , the animals were poisoned with tetanus ~` toxin, corresponding to 10~ minimum lethal doses (dlm =
dosls letalls minima). All Or the animals survived the ~ poisoning.
i 2 Rabbit~ were immunized twice in an interval of 14 days with the product of the invention according to ; Example 3 (160~ug of tetanus toxin derivative suspended in 1 ml of complete Freund's ad~uvant per application and animal). Three day~ after the second in~ection, blood test~
29 wero performed. The evaluation of the tetanus antitoxin : _ 9 _ ~ . .
.' . '' . . ' . ~ ~ ~ .'. , ` ' '' : :
- . ' . . , . ' '' ''' , , - : ' ' , 1063~21 tlters in the serum was 100 IU/ml and/or 50 IU/ml.
The following Examples serve to illu~trate the invention.
E X A M P L E 1:
Tetanus toxin obtained from the culture filtrate of Cl.
tetani (40,000 floculation units (= Lf units) corresponding to about 100 mg of protein) was dissolved in 100 ml of 0.1 molar phosphate buffer having a pH of 7.8 and was mixed wlth form-aldehyde up to a final concentration of 0.03 mole of aldehyde;
the solution was then allowed to stand for 16 hours at 4C.
Subsequently the ~olution was dialyzed again~t isotonic phos-phate-buffered sodium hydroxide solUtion (PBS) having a pH value of 7.4,was concentrated ~ ~xut 20 mlJand 150 mg of dithiothreitol and urea were added up to a final concentration of 6 moles/l.
After having stood for 30 minutes at room temperature, the solution was introduced into a column (4.5 x 100 cm) of Sephadex(R) -G 150 ), which was equilibrated with 6 moles of urea in 0.1 molar trishydroxy~ethylaminomethane-HCl buffer (Tris) having a pH value of 8.0, and with an addition of dithio-threitol of 0.001 mole. The elution was performed with the equilibration mixture. Upon chromatography, two protein peaks were found by measuring the W absorption at 280 nm.
The materlal which corresponded to the flrst peak was dlscarded.
The second peak contalned the derivatlve of the llght chain of the tetanus toxln. The solutlon of the isolated derivatlve of the light chaln was dialyzed against 0.1 molar phosphate buffer and was mixed, in a protein concentration of 100/ug of protein/ml, with formaldehyde up to a final concentration of 29 )Trade mark of Messrs. Pharmacia, Uppsala .. . .
.. . ... .. . ..
. .
.. . . . .
. - ~ . ; . , . ~
.
1063~21 0.1 mole of aldehyde and was then allowed to stand for three weeks at room temperature. After a final dialysis against 0.15 molar NaCl in order to eliminate the free formaldehyde, the modified derivative of the light chain was obtained.
After concentration on an ultrafilter it could be processed into a vaccine in known manner. In this process, glycin or lysin could be used as stabilizers. As an adjuvant, use was made of a suspension of AltO~)3. Instead of formaldehyde, there could also be used - calculated on the molar aldehyde amount - glutardialdehyde, propionaldehyde or butyraldehyde, either in both steps or only in one step.
E X A M P L E 2: -Tetanus toxin from cell extracts was mixed in a protein concentration of 0.1 % in 0.1 molar phosphate buffer having a pH value of 7.8, with 100 /ug of trypsin (3.4.21.4) and was allcwed to stand for 60 minutes at room temperature. Subsequently trypsin inhibitor obtained from bovine lungs (150 /ug) was added, and the product was reacted according to Example 1 with formaldehyde, was dialyzed, reduced with dithiothreitol and subjected to chromatography in urea solution.
E X A M P L E 3:
Tetanus toxin (20 ml, 0.5% protein) in 0.1 molar Tris having a pH value of 8.0 was reacted with dithioerythrite (150 mg) and guanidine hydrochloride up to a final concen-tration of 4 moles/l, and was then subjected to chromatographyin a manner analogous to that of Example 1. The second peak was pooled, the volume was adjusted to 400 ml, and the guani- -dine hydrochloride was eluted, together with the low-molecular weight components of the reaction mixture, by way of chromato-. - 11 -. ! ~ ~ . ' ' . , . , ., . ' ~ .
. ,, ,, ... . . . . .. . ~
' "" . : ' ', '' ' ' ' ~ .
1063~Zl I
graphy on a column with Sephadex(R)-G 25 (column contents 2000 ml) with 0.1 molar pho~phate buffer having a pH value of 7.8. The light chain waR then mixed with formaldehyde up to , a flnal concentration of 0.1 molar aldehyde and was allowed to stand for 3 weeks at room temperature. The processing into a vaccine wa~ effected according to Example 1.
This application is a division of Serial No. 240,888 filed December 2, 1975. . .
:,. , , , .
,,- . ~.................. . . . ..
: ~
10~30Zl The present lnvention relate~ to derlvatives of tetanus toxln, a proce~s for thelr preparatlon and agents contalning them.
More particularly the present invention provides derivatives of tetanus toxin, a proce~s for preparing them by modifying a partial molecule of the tetanus toxin and agents, in par-ticular tetanus vaccines, which contain one of the derivatives of tetanu~ toxin.
The common tetanus vaccine~ for the active immunization contain almost exclusively an antigen whlch has been prepared ; by the lnactivation of tetanus toxin with formaldehyde. This substance called toxoid i~ provided with a large number of antigenlc determinants, only a few of which being important, however, for the production of antibodies that protect against tetanus. The elimination of those determinants which are not - required for protection is desirable, in order to obtain i antlgenic and/or immunogenio substances, of which - due to a narrower spectrum of determinant groups - an increased speci-; ficity and an improved compatibility may be expected.
Tetanus toxin is synthesized from cells of Cl. tetani and can be obtained from the cell mass as well as from the culture filtrate. Whereas the toxin obtained in an extracellular way consists of two polypeptide chains which may be separated after having been treated with agents reducing disulfide bridges (for example, dithiothreitol (DTT), dithioerythritol ~`~ (DTE), mercaptoethanol, cysteine), under denaturing conditions, the lntracellular toxin consists of a single polypeptide chain which can be converted in known manner, by a mild trypsin (3.4.21.
29 treatment, into a product which is identical to the extra-~;~ 2 ', ~ ~ ' ' ''' ' . ' '. . . ' ' '., " , .
. 1063~)21 cellul~r toxln. The two fragments to be obtalned of thls product, whlch have been called , in this context, "llght chain" (molecular weight of about 50~000) and "heavy chain" (molecular - -welght of about 100,000)~ could only be separated, up to now, by measure~ which are carried out under denaturlng conditions, for example, in a solution containine sodium dodecyl sulfate, and the separated fragments can be kept in solutlon, according to the prior art, only by denaturing agents.
It was therefore the task of the pre~ent invention to modlfy the light chain of the tetanus toxin in a way that it could be kept in a physiologically tolerable medium without losing its antigenicity. m e light chain was to be available in particular as an es3ential immunogenic constituent of tetanus ~accine~ and was to replace the common toxoid in these substances.
It has now been found that this task can be solved by sub-~ecting the llght chain, either before or after the separation . . .
of the heavy chaln from the molecular structure of the tetanus toxin, which represents a complex of the light and heavy chains, to a slight chemical modification. By way of thi~ method, the spontaneous irreversible precipitation of the light chain, which i8 ob~eryed after the elimination o*
. denaturing agents like urea, can be avoided.
me Present invention provides a process for the pre-paratlon of a derlvatlve of the 11ght chain of the tetanus toxln, which compri~e~ treating an aqueous solutlon of the .~ .
extracellular tetanus toxin ~ith an aliphatic mono- or d~alde-29 hyde havlng a chain length of from 1 to 6 carbon atoms in an .~: . . . .
. . .
-1063~Zl aldehyde concentratlon of from 0.01 to 0.05 mole of aldehyde at a temperature of from 1 to 20C for 5 minutes to 50 hourq, reacting the toxin before or after the sldehyde treatment in the presence of a compound reduc~ng di~ulfide bridges wlth a denaturing agent , subsequently obtaining the derivati~e of the light chain in the-presence of the denaturing agent by way of proteinchemlcal isolation processes and suitably separating it from the denaturing agent.
A preferred variant of the proces~ provide~ treatlng an aqueous solution of the tetanus toxln with an aliphatic mono-or dialdehyde having a chain length of from 1 to 6 carbon atoms, preferably formaldehyde, with an aldehyde concentration of from 0.02 mole to 0.03 mole, at a temperature of from 1 to 6C
for 2 to 20 hours, mixing the solution containing the toxin ; 15 thus modified with an agent reducing disulfide bridges, ob-taining the derivative of the light chain from the solution obtained in the presence of a denaturing agent by way of proteinchemical isolation processes and sultably separat~ng it i from the denaturing agent.
- 20 After the isolation of the derivative of the light chain of the tetanus toxin, the denaturing agent can be eliminated by way of dialysis or other comparable methods which allow the separation of low-molecular weight substances from high-~ . .
molecular weight protein, for example by gel chromatography.
~- 25 The aldehyde, too, which has been used for the reaction but ha~ not been used up, may be eliminated from the reactlon mlxtures by the same measures.
~" The derlvatlve prep~rod accordlng to the proce~ of tho 29 lnventlon does no more show, after thls treatment, tho ~: , .
, , . ,.. ,; ., .. . :
~ ` HOE 74/B 028 1063~)21 denaturlng and preclpltsting properties known from the light chaln, in phy~iologically tolerable, isotonically aqueous medla.
The derlvative no longer has the toxic property known from the tetanus toxin. It shows an antigen behavior and i~ in a positlon to lnduce antlbodle~ protecting again~t tetanus ln the human or animal organlsm.
The derlvatlve prepared accordlng to the invention is to be further purlfled by the common proteinchemical method~. In ~iew of the admittedly high toxicity of the native tetanus toxin and the fact - which 18 al~o known - that methods used for the dlssoclation of proteins only in the rarest cases lead to a 100 per cent separation of accompanying substances, in particular of protelns having a similar structure, it i8 ad-vantageous, however, to submit the product prepared accordlng to the invention to a further aldehyde treatment, which com-prises mlxing the derivative of the light chaln of the tetanus toxin with an aliphatic mono- or dialdehyde having a chain length of from 1 to 6 carbon atoms, preferably formaldehyde, up to an aldehyde concentration in the range of from 0.05 to 0.2 mole, preferably from 0.08 to 0.15 mole, and allowing it to stand for 14 to 28 days at a temperature in the range Or from 20 to 37C, elimlnating the aldehyde, for example by way of dialysis, and obtainlng the modified derivative of the light chain Or the tetanus toxin.
The additional aldehyde treatment leads to a further-stabilization of the llght chain Or the tetanus toxin and to the-elimination of the re~idual toxicity which may still be present.
29 It is obvlou~ that a stable derlvatlve of the light chaln .
~OE 74lB 028 1063~)21 may also be prepared by treatlng native tetanus toxln obtalned from culture filtrates of Cl. tetani wlth ~ubstances reduclng dlsulflde brldges, for example thlol compounds, by effecting ¦ -the separation of the light chaln from the heavy chaln under denaturing conditions, preferably by way Or chromatography ln buffer solutions containing, for example, urea, subsequently by measures which allow the separation Or low-molecular weight substances from high-molecular weight protein, by eliminating the denaturing agent suitably by gel chromatography~ and by allowing the light chain to stand immediately with an aliphatic mono- or dialdehyde having a chaln length of from 1 to 6 carbon atoms, preferably formaldehyde, wlth an aldehyde concentration of from 0.01 to 0.05 mole, for 5 minutes to 50 hours, at a temperature of irom 1 to 20C. A second aldehyde treatment in order to modify the derivative may optionally be performed.
It i8 also possible, however, to react the light chain im-mediately after its isolation with the higher amount of al-dehyde used for the modification of the derivative. However, these processes do not involve any advantage, since the yield .~ , . . .
of the derivative of the light chain prepared according to i the latter process is considerably smaller than tho one ob-tained according to the former process.
As starting material, use is made of tetanus toxin whicb ; has been obtalned from culture filtrates of Cl. tetani in known manner (extracellular toxin). As an alternative there may be used a tetanus toxin prepared according to known processes which was obtained by extracting the Cl. tetani `~ bacteria (intracellular toxin). In this caso~ however, a trypsin ~9 (3-4 21;4)treatment to be carried out according to a known .. . .
" " ' ~ ; ' ', " ' ' ' ' . ' . -' '` .
. .. : ~ ., ,. , , .......... . : .
HOE 74/~ 028 method is required before or after the reaction with aldehyde.
If the light chain is not to be reacted with the aldehyde until after the separation from the heavy chain, it is clear that a trypsin (3.4.21.4) treatment is to be effected, in the case of intracellular toxin, before the reaction with aldehyde, as the proteolytic step represents a pre-requisite for the isolation of the light chain from this starting material. the tetanus toxin solution used as starting material contains the toxin suitably in a concentration of from 0.5 to 10 mg/ml.
As compounds reducing disulfide bridges, preference is given to thiol compounds. For this purpose there may be mentioned, for example, cysteine, mercapto-ethanol or dithio-erythritol, preferably however, dithiothreitol. These com-pounds are added in amounts which cause a concentration of the compounds reducing disulfide bridges in the reaction mixture of from 0.05 to 0.3 mole.
By denaturing agents in the sense of the present process there are to be understood chemical compounds which help to dissociate protein molecules to obtain sub-units, in particular by dissolving hydrogen bonds. Known agents to dissolve hydrogen bonds are, for example, urea or guanidine.hydrochloride.
Urea has proved to be effective preferably in a concentration - of from 4 to 6 moles, whereas guanidine hydrochloride is par-- - ticularly effective in a concentration of from 2 to 4 moles.
It is not possible to isolate a modified light chain from the convention~l tetanus toxoid, since in this case the use of con~iderably higher amounts of formaldehyde has resulted in a covalent cross-linking between the light and the heavy chains.
~ .
~ ~- 29 However, there is no reason not to react any solution con-. ~ , .
.~ , , . , , . . - . - , ~ HOE 74/~ 028 1063~)Zl taining the light chain which has not been denatured, with aldehydes, in order to obtain derivatives having the advan-tageous properties with regard to stabllity and to antigenlcity.
Be~ide~ the above-mentioned proce~ variants, derivatives of the tetanus toxln are also a sub~ect of this ~nvention, whlch are to be characterized by parameters resulting from their preparation according to the pre~ent process .
Finally, the present invention provides agents containing the tetanus toxin derivatives of the inventlon, in particular tetanus vaccines for the prophylaxis again~t the tetanus disease or for the preparation of tetanuæ antisera to be u~ed for thera-peutical or diagnostic purposes, but also diagnostic prepara--~ tlon~ contalnlng the tètanus toxin derivatlves or antisera ob-tained from them. In order to increase the solution stability of the product prepared according to the invention, lt may be advantageous to add com-pounds used for the stabilization of proteln solutions, such as amino aclds or carbohydrates, to the physiologically tolerable aqueous medium, in which the above product-ls dis-solved.
; - It i8 recommended, for the protection against microbial contamination, to add antimicrobial sUbstances~ such as sodium timerfonate, to the solutions which are ready for use. For ` the preparation of a polyvalent vaccin~, the tetanus toxln }
derlvatives may be mixed wlth other antlgens and/or toxoids in known manner.
~ The suitabillty Or the products as vacclnes has been proved -~ in the following by te~ts which certlfy a good potency of the 29 product.
:
HOE 74/B 028_ 1063~Zl Potency test a) In vitro In order to determine the protective potency of the determinant groups which are pre~ent on the light chain, the followlng test was carried out:
A tetanus antitoxin serum of equine origin was ab-sorbed with increasing amounts of the product according to Example 1. By way of examining the supernatants with the aid of the immunological double dlffusion technique, the equivalence zone, in which the entire antibodies directed against the derivative of the light chain were precipitated, was determined. The evaluation of the anti- -bodies in the animal test showed that up to 40 % of the protecting antibodies from the antiserum of the product of the invention could be absorbed.
. .
b) In vivo Groups of 10 Guinea pigs each were given suboutaneously 1 ml (20 ~g) of a suspension of the product prepared ac-cordlng to Example 1, adsorbed on 0.2 ~ Al(OH)3 gel.
After 4 weeko , the animals were poisoned with tetanus ~` toxin, corresponding to 10~ minimum lethal doses (dlm =
dosls letalls minima). All Or the animals survived the ~ poisoning.
i 2 Rabbit~ were immunized twice in an interval of 14 days with the product of the invention according to ; Example 3 (160~ug of tetanus toxin derivative suspended in 1 ml of complete Freund's ad~uvant per application and animal). Three day~ after the second in~ection, blood test~
29 wero performed. The evaluation of the tetanus antitoxin : _ 9 _ ~ . .
.' . '' . . ' . ~ ~ ~ .'. , ` ' '' : :
- . ' . . , . ' '' ''' , , - : ' ' , 1063~21 tlters in the serum was 100 IU/ml and/or 50 IU/ml.
The following Examples serve to illu~trate the invention.
E X A M P L E 1:
Tetanus toxin obtained from the culture filtrate of Cl.
tetani (40,000 floculation units (= Lf units) corresponding to about 100 mg of protein) was dissolved in 100 ml of 0.1 molar phosphate buffer having a pH of 7.8 and was mixed wlth form-aldehyde up to a final concentration of 0.03 mole of aldehyde;
the solution was then allowed to stand for 16 hours at 4C.
Subsequently the ~olution was dialyzed again~t isotonic phos-phate-buffered sodium hydroxide solUtion (PBS) having a pH value of 7.4,was concentrated ~ ~xut 20 mlJand 150 mg of dithiothreitol and urea were added up to a final concentration of 6 moles/l.
After having stood for 30 minutes at room temperature, the solution was introduced into a column (4.5 x 100 cm) of Sephadex(R) -G 150 ), which was equilibrated with 6 moles of urea in 0.1 molar trishydroxy~ethylaminomethane-HCl buffer (Tris) having a pH value of 8.0, and with an addition of dithio-threitol of 0.001 mole. The elution was performed with the equilibration mixture. Upon chromatography, two protein peaks were found by measuring the W absorption at 280 nm.
The materlal which corresponded to the flrst peak was dlscarded.
The second peak contalned the derivatlve of the llght chain of the tetanus toxln. The solutlon of the isolated derivatlve of the light chaln was dialyzed against 0.1 molar phosphate buffer and was mixed, in a protein concentration of 100/ug of protein/ml, with formaldehyde up to a final concentration of 29 )Trade mark of Messrs. Pharmacia, Uppsala .. . .
.. . ... .. . ..
. .
.. . . . .
. - ~ . ; . , . ~
.
1063~21 0.1 mole of aldehyde and was then allowed to stand for three weeks at room temperature. After a final dialysis against 0.15 molar NaCl in order to eliminate the free formaldehyde, the modified derivative of the light chain was obtained.
After concentration on an ultrafilter it could be processed into a vaccine in known manner. In this process, glycin or lysin could be used as stabilizers. As an adjuvant, use was made of a suspension of AltO~)3. Instead of formaldehyde, there could also be used - calculated on the molar aldehyde amount - glutardialdehyde, propionaldehyde or butyraldehyde, either in both steps or only in one step.
E X A M P L E 2: -Tetanus toxin from cell extracts was mixed in a protein concentration of 0.1 % in 0.1 molar phosphate buffer having a pH value of 7.8, with 100 /ug of trypsin (3.4.21.4) and was allcwed to stand for 60 minutes at room temperature. Subsequently trypsin inhibitor obtained from bovine lungs (150 /ug) was added, and the product was reacted according to Example 1 with formaldehyde, was dialyzed, reduced with dithiothreitol and subjected to chromatography in urea solution.
E X A M P L E 3:
Tetanus toxin (20 ml, 0.5% protein) in 0.1 molar Tris having a pH value of 8.0 was reacted with dithioerythrite (150 mg) and guanidine hydrochloride up to a final concen-tration of 4 moles/l, and was then subjected to chromatographyin a manner analogous to that of Example 1. The second peak was pooled, the volume was adjusted to 400 ml, and the guani- -dine hydrochloride was eluted, together with the low-molecular weight components of the reaction mixture, by way of chromato-. - 11 -. ! ~ ~ . ' ' . , . , ., . ' ~ .
. ,, ,, ... . . . . .. . ~
' "" . : ' ', '' ' ' ' ~ .
1063~Zl I
graphy on a column with Sephadex(R)-G 25 (column contents 2000 ml) with 0.1 molar pho~phate buffer having a pH value of 7.8. The light chain waR then mixed with formaldehyde up to , a flnal concentration of 0.1 molar aldehyde and was allowed to stand for 3 weeks at room temperature. The processing into a vaccine wa~ effected according to Example 1.
This application is a division of Serial No. 240,888 filed December 2, 1975. . .
:,. , , , .
,,- . ~.................. . . . ..
: ~
Claims (3)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for the preparation of a modified derivative of the light chain of tetanus toxin in which the light chain of tetanus toxin free from denaturing agent is mixed with an aliphatic mono- or di-aldehyde having a chain length of from 1 to 6 carbon atoms, up to an aldehyde concentration of from 0.05 mole to 0.2 mole, the mixture is allowed to stand for 14 to 28 days at a temperature of from 20 to 37°C, the aldehyde is eliminated and the modified derivative of the light chain is obtained.
2. A process as claimed in claim 1, in which the aldehyde is formaldehyde.
3. A modified derivative of the light chain of tetanus toxin, whenever obtained according to a process as claimed in claim 1 or claim 2 or by an obvious chemical equivalent thereof.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA313,399A CA1063021A (en) | 1974-12-03 | 1978-10-13 | Derivatives of the tetanus toxin, process for their preparation and agents containing them |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE2457047A DE2457047C3 (en) | 1974-12-03 | 1974-12-03 | Process for the preparation of a derivative of the light chain of tetanus toxin, its use for tetanus prophylaxis |
| CA240,888A CA1066621A (en) | 1974-12-03 | 1975-12-02 | Dialdehyde treatment of tetanus toxin and derivative obtained |
| CA313,399A CA1063021A (en) | 1974-12-03 | 1978-10-13 | Derivatives of the tetanus toxin, process for their preparation and agents containing them |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1063021A true CA1063021A (en) | 1979-09-25 |
Family
ID=27164225
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA313,399A Expired CA1063021A (en) | 1974-12-03 | 1978-10-13 | Derivatives of the tetanus toxin, process for their preparation and agents containing them |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1063021A (en) |
-
1978
- 1978-10-13 CA CA313,399A patent/CA1063021A/en not_active Expired
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