Note: Descriptions are shown in the official language in which they were submitted.
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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-
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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
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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
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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
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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
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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-
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~ HOE 74/~ 028
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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.
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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
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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
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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-
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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. . .
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