Preparation of Remedial Substances.
Year: 1894

page_text: Date of Application, 1st Feb., 1894
Complete Specification Left, 31st Oct., 1894-Accepted, 26th Jan., 1895
PROVISIONAL SPECIFICATION.
Preparation of Remedial Substances.
I, AUGUST ZIMMERMANN, of 6 and 7 Cross Lane, St. Mary-at-Hill, in the
City of London, Merchant, (a communication to me from abroad by Hans Aronson,
of 11 Fasanenstrasse, Charlottenburg, in the Empire of Germany, Doctor of
Medicine,) do hereby declare the nature of this invention to be as follows:-
Behring has shown in the course of his investigations that substances are found
in the blood serum of animals rendered by artificial means immune to a certain
disease, which substances are able to protect animals otherwise susceptible to this
disease, and, if employed in large quantities, even to cure animals already suffering
from the specified disease. Ehrlich made the same observation in regard to milk.
It has been attempted to isolate these active principles from the blood serum or
from the whey in a solid form.
According to published investigations (Fizzoni and Gattani, Emmerich and
Tsuboi, Ehrlich and Brieger, Buchner) only the ordinary albuminoid precipitants
employed in physiological chemistry, such as alcohol and the sulphates of sodium,
ammonium and magnesium, have been up to the present used for this purpose.
These methods however only effect a preservation but no practically useful
concentration of the immunising principles of the blood-serum, which is of primary
consideration for therapeutical purposes, because of its large percentage of
immunising substances.
The substances, the so-called anti-toxines, obtained in solid form by these
processes are at the most only ten times as strong as the mother material, the
blood serum.
As these substances in therapeutical use must be subcutaneously
injected and solutions cannot be prepared more concentrated than 10 per cent., it
follows that the volume of injected liquid is not less than would have been injected
had the blood serum been employed direct.
By means of the method now to be described not only preservation but also a
nuch greater concentration of the active principles, even from blood serum, is
attained. The basis for the elaboration of this method was afforded by some
observations made during an investigation into the filtration of liquids infected
with bacteria through colloidal alumina. It was found that if strongly poisonous
broth cultures of tetanus or diphtheria microbes were filtered through a layer of
freshly precipitated aluminium hydroxide, the filtrate is not only sterile "but also
nearly non-poisonous. The latter effect is directly opposite to that obtained by
filtration through cones of burnt alumina or infusorial earth. The colloidal, that
is to say freshly precipitated, alumina retains not only the bacteria but also the
poisonous substances produced by them in the solution. By filtration of the serum
of immune animals through colloidal alumina it was found that the active principles
are likewise taken up by the alumina and that the filtrate is almost inactive.
In the method for the preparation of immunising substances forming the subject
of this invention a different principle is adopted to that in methods hitherto
employed, which depend upon the direct precipitation of albuminoids. The
therapeutically active albuminoids are combined with inorganic precipitates,
extract the active principles from these precipitates by shaking with a solvent and
obtain them in solid form either by concentration of the solution or by their
precipitation by means of the ordinary albuminoid precipitants. This method is
based upon the experience gained in experiments on the immunity of animals from
địnhtheria.

page_text: 1. It was discovered that if a precipitate of a metallic hydroxide, for instance,
aluminium hydroxide, iron hydroxide, or of an insoluble metallic salt, for instance
calcium phosphate, is produced in a dilute aqueous solution of the blood serum of
an immune animal, the active principle is found in the inorganic precipitate, whilst
the solution is practically free from the active principle, though other albuminoid
bodies are still largely contained in the solution.
The same result can be obtained, though less practically by filtering through
the freshly precipitated hydroxide or salt, or by shaking with the same.
2. It was further discovered that the active principles can be extracted from the
precipitate by shaking with solvents, especially with dilute solutions of alkalies,
ammonia or organic bases. The active principles are readily brought into solution
by this treatment; a greater part of the other worthless albuminoid substances
remain in the precipitate.
3. The active principles are comparatively stable substances, so that the faintly
alkaline solutions may be evaporated in vacuum. In this way the active principles
are obtained in solid form. As already mentioned, instead of evaporation, however,
these active principles can be thrown out of the solution by the ordinary albuminoid
precipitating agents.
Stable products in solid form can be thus prepared which are about one hundred
times as active as the mother material, the blood serum. At the same time about
85 per cent. albuminoid substances which are worthless ballast, are removed and the
volume of liquid required for subcutaneous injection is reduced to one-tenth the
original volume of the serum, as 10 per cent. solutions of the finished product can
be prepared without difficulty. The yield of active principle amounts to about
80 per cent. Further it has been determined that poisonous, non-immunising
substances (toxines), which at first pass into the blood when the injections of
bacteria cultures are made in the process of rendering animals immune, are also
got rid of, and that by careful application of the method to filtered poisonous broth
cultures of diphtheria germs, a comparatively non-poisonous but at the same time
immunising substance can be produced.
The products so obtained are white, soluble in water, more soluble in dilute
alkalies, and chemically cannot be distinguished from ordinary albuminoid
substances, but are only distinguished physiologically by their activity against the
infectious disease towards which the animal was rendered immune whose serum
afforded the material for their preparation.
For the preparation of these immunising substances the blood serum of animals
that are naturally immune, and into whose systems large quantities of virulent
bacteria cultures or of the poison produced by the bacteria had been previously
introduced, can also be employed. This has been proved in face of the contrary
observations of others first of all in the case of diphtheria, after Waillard had
found in tetanus that the immunising substances were also produced in the blood
of naturally immune animals, when they had been treated in the above manner.
For the preparation of immunising substances from serum one proceeds for
example, in the following way :—
100 c.c. blood serum (taken from an animal rendered immune) is diluted with
100 c.c. distilled water and 30 c.c. 10 per cent. aluminium sulphate solution added.
To the mixture about 4 c.c. 20 per cent. solution of ammonia is added with constant
stirring. The precipitate produced is removed by filtration and washed with a
moderate quantity of distilled water, then shaken in an agitating machine for
24 hours with 75 c.c. distilled water containing a small quantity of ammonia
(about 0.8 gramme NH, per litre).
The mass is then filtered and the slightly turbid filtrate evaporated to dryness in
vacuum at the lowest possible temperature. About 0.8 gramme of a white organic
substance is thus obtained, which exhibits all the reactions of albuminoid bodies,
and which by experiment on animals is found to possess about hundred times the
activity of the blood serum used for its preparation. If, for instance, it had been
necessary to inject 01 c.c. of the serum serving as mother material in order to

page_text: protect an animal against the specific infectious disease to which the first animal
was immune, it would only be necessary to use 0.001 gramme of the product
obtained by the method described.
By a repetition of the extraction of the alumina with slightly ammoniacal water
a further quantity (about 0.4 gramme) of a less active product is obtained.
Dated this First day of February 1894.
Preparation of Remedial Substances.
I, AUGUST ZIMMERMANN, of 6 and 7 Cross Lane, St. Mary-at-Hill, in the
City of London, Merchant, (a communication to me from abroad by Hans Aronson,
of 11 Fasanenstrasse, Charlottenburg, in the Empire of Germany, Doctor of
Medicine,) do hereby declare the nature of this invention and in what manner
the same is to be performed to be particularly described and ascertained in and
by the following statement :-
Behring has shown in the course of his investigations that substances are found
in the blood serum of animals rendered by artifical means immune to a certain
disease, which substances are able to protect animals otherwise susceptible to this
disease, and, if employed in large quantities, even to cure animals already suffering
from the specified disease. Ehrlich made the same observation in regard to milk.
It has been attempted to isolate these active principles from the blood serum or
from the whey in a solid form.
According to published investigations (Fizzoni and Gattani, Emmerich and
Tsuboi, Ehrlich and Brieger, Buchner) only the ordinary albuminoid precipitants
employed in physiological chemistry, such as alcohol and the sulphates of sodium,
ammonium and magnesium, have been up to the present used for this purpose.
These methods however only effect a preservation but no practically useful
concentration of the immunising principles of the blood-serum, which is of primary
consideration for therapeutical purposes, because of its large percentage of
immunising substances.
The substances, the so-called anti-toxines, obtained in solid form by these
processes are at the most only ten times as strong as the mother material, the
blood serum.
As these substances in therapeutical use must be subcutaneously
injected and solutions cannot be prepared more concentrated than 10 per cent., it
follows that the volume of injected liquid is not less than would have been injected
had the blood serum been employed direct.
By means of the method now to be described not only preservation but also a
much greater concentration of the active principles, even from blood serum, is
attained. The basis for the elaboration of this method was afforded by some
observations made during an investigation into the filtration of liquids infected
with bacteria through colloidal alumina. It was found that if strongly poisonous
broth cultures of tetanus or diphtheria microbes were filtered through a layer of
freshly precipitated aluminium hydroxide, the filtrate is not only sterile but also
nearly non-poisonous. The latter effect is directly opposite to that obtained by
filtration through cones of burnt alumina or infusorial earth. The colloidal, that
is to say freshly precipitated, alumina retains not only the bacteria but also the
poisonous substances produced by them in the solution. By filtration of the serum
of immune animals through colloidal alumina it was found that the active principles
are likewise taken up by the alumina and that the filtrate is almost inactive.
In the method for the preparation of immunising substances forming the subject.
of this invention a different principle is adopted to that in methods hitherto
employed, which depend upon the direct precipitation of albuminoids. The

page_text: thereapeutically active albuminoids are combined with inorganic precipitates,
extract the active principles from these precipitates by shaking with a solvent and
obtain them in solid form either by concentration of the solution or by their
precipitation by means of the ordinary albuminoid precipitants. This method is
based upon the experience gained in experiments on the immunity of animals from
diphtheria.
1. It was discovered that if a precipitate of a metallic hydroxide, for instance,
aluminium hydroxide, iron hydroxide, or of an insoluble metallic salt, for instance
calcium phosphate, is produced in a dilute aqueous solution of the blood serum of
an immune animal, the active principle is found in the inorganic precipitate, whilst
the solution is practically free from the active principle, though other albuminoid
bodies are still largely contained in the solution.
The same result can be obtained, though less practically by filtering through
the freshly precipitated hydroxide or salt, or by shaking with the same.
2. It was further discovered that the active principles can be extracted from
the precipitate by shaking with solvents, especially with dilute solutions of alkalies,
ammonia or organic bases. The active principles are readily brought into solution
by this treatment; a greater part of the other worthless albuminoid substances
remain in the precipitate.
3. The active principles are comparatively stable substances, so that the faintly
alkaline solutions may be evaporated in vacuum. In this way the active principles
are obtained in solid form. As already mentioned, instead of evaporation, however,
these active principles can be thrown out of the solution by the ordinary albuminoid
precipitating agents.
Stable products in solid form can be thus prepared which are about one hundred
times as active as the mother material, the blood serum. At the same time about
85 per cent. albumenoid substances which are worthless ballast, are removed and
the volume of liquid required for subcutaneous injection is reduced to one-tenth
the original volume of the serum, as 10 per cent. solutions of the finished product
can be prepared without difficulty. The yield of active principle amounts to about
80 per cent. Further it has been determined that. poisonous, non-immunising
substances (toxines), which at first pass into the blood when the injections of
bacteria cultures are made in the process of rendering animals immune, are also got
rid of, and that by careful application of the method to filtered poisonous broth
cultures of diphtheria germs, a comparatively non-poisonous but at the same time
immunising substance can be produced.
The products so obtained are white, soluble in water, more soluble in dilute
alkalies, and chemically cannot be distinguished from ordinary albuminoid substances,
but are only distinguished physiologically by their activity against the infectious
disease towards which the animal was rendered immune whose serum afforded the
material for their preparation.
For the preparation of these immunising substances the blood serum of animals
that are naturally immune, and into whose systems large quantities of virulent
bacteria cultures or of the poison produced by the bacteria had been previously
introduced, can also be employed. This has been proved in face of the contrary
observations of others first of all in the case of diphtheria, after Waillard had
found in tetanus that the immunising substances were also produced in the blood of
naturally immune animals, when they had been treated in the above manner.
For the preparation of immunising substances from serum one proceeds for
example, in the following way:-
100 c.c. blood serum (taken from an animal rendered immune) is diluted with 100 c.c.
distilled water and 30 c.c. 10 per cent. aluminium sulphate solution added.
To the
mixture about 4 c.c. 20 per cent. solution of ammonia is added with constant
stirring. The precipitate produced is removed by filtration and washed with a
moderate quantity of distilled water, then shaken in an agitating machine for
24 hours with 75 c.c. distilled water containing a small quantity of ammonia
(about 0.8 gramme NH, per litre).

page_text: The mass is then filtered and the slightly turbid filtrate evaporated to dryness in
vacuum at the lowest possible temperature. About 0.8 gramme of a white organic
substance is thus obtained, which exhibits all the reactions of albuminoid bodies,
and which by experiment on animals is found to possess about hundred times the
activity of the blood serum used for its preparation. If, for instance, it had been
necessary to inject 0.1 c.c. of the serum serving as mother material in order to
protect an animal against the specific infectious disease to which the first animal
was immune, it would only be necessary to use 0.001 gramme of the product
obtained by the method described.
By a repetition of the extraction of the alumina with slightly ammoniacal water
a further quantity (about 0-4 gramme) of a less active product is obtained.
Having now particularly described and ascertained the nature of my said inven-
tion and in what manner the same is to be performed I declare that I wish it to be
understood that the invention extends not only to the blood serum but also to the
whey and other tissue liquors of animals rendered immune or immune by nature
and that what I claim is :-
1. Isolating the immunising principles from the other albuminoids substantially
as described.
2. As new articles of manufacture the immunising principles in an isolated form
substantially as described.
3. The process of isolating the immunising principles from the other albuminoids
by combining them with an inorganic precipitate and then extracting them from
the precipitate substantially as described.
4. The process of isolating the inmunising principles from the other albuminoids
by filtration through a metallic hydroxide or salt and then extracting them from
the filtering medium substantially as described.
5. In the processes referred to in the preceding claims separating the immunising
principles from the precipitates by shaking with solvents substantially as described.
6. In the processes referred to in the preceding claims obtaining the immunising
principles in a solid form by evaporation in vacuo substantially as described.
Dated this 29th day of October 1894.
AUG. ZIMMERMANN.