CA1206905A - Process for the production of capsular polyosides and capsular polyosides obtained thereby - Google Patents
Process for the production of capsular polyosides and capsular polyosides obtained therebyInfo
- Publication number
- CA1206905A CA1206905A CA000408233A CA408233A CA1206905A CA 1206905 A CA1206905 A CA 1206905A CA 000408233 A CA000408233 A CA 000408233A CA 408233 A CA408233 A CA 408233A CA 1206905 A CA1206905 A CA 1206905A
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- Prior art keywords
- capsular
- polyosides
- submitting
- retained material
- filtration
- Prior art date
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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
- A61K39/025—Enterobacteriales, e.g. Enterobacter
- A61K39/0266—Klebsiella
-
- 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
- A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
- A61K39/092—Streptococcus
-
- 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
- A61K39/102—Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
Abstract
PROCESS FOR THE PRODUCTION OF CAPSULAR POLYOSIDES, CAPSULAR POLYOSIDES OBTAINED THEREBY, AND THEIR
APPLICATION TO THE PREPARATION OF VACCINES.
ABSTRACT OF THE DISCLOSURE
The present invention relates to a process for the production of capsular polyosides from capsulated bacteriae containing same, comprising cultivating the bacteriae in a synthetic or semi-synthetic medium; at the end of the growth stage of said culture separating the microbial bodies from a supernatant phase; submitting said supernatant phase to a filtration through a membrane which retains the molecules of a molecular weight of 100,000 daltons or more, to give a capsular polyoside-rich retained material; and removing the proteins, the nucleic acids and the lipids from said retained material, to give a purified capsular polyoside.
APPLICATION TO THE PREPARATION OF VACCINES.
ABSTRACT OF THE DISCLOSURE
The present invention relates to a process for the production of capsular polyosides from capsulated bacteriae containing same, comprising cultivating the bacteriae in a synthetic or semi-synthetic medium; at the end of the growth stage of said culture separating the microbial bodies from a supernatant phase; submitting said supernatant phase to a filtration through a membrane which retains the molecules of a molecular weight of 100,000 daltons or more, to give a capsular polyoside-rich retained material; and removing the proteins, the nucleic acids and the lipids from said retained material, to give a purified capsular polyoside.
Description
Process for the production of capsular polyosides, capsular polyosides obtained thereby, and their application to the preparation of vaccines This invention relates to a ~rocess for the produc tion of capsular polyosides, to the polyosides obtained by said process, and the application of said polyosides to the preparation of vaccines.
It is known that it is possible to combat infectious diseases at two different levels: either by direct action against the pathogenic germ (antibiotics, anti-septics)~ or by an indirect action via the reinforcement of the systemic defences (vaccination, serotherapy, im-munomodulation).
The use of agents likely to exert a direct action frequently comes up against certain difficulties which may limit theix action (particularly: toxicity of the molecule and bacterial resistance).
In contrast, the reinforcement of the systemic defences via the acquisition of specific antibodies has definite advantages, particularly from the standpoint of the duration of the protection and of the specificity.
However, vaccination with whole microbial bodies is not always free from drawbacks: hypersensitization phenomena, poor tolerance or pyrogenicity are among the most fre-~uent. They are corelated to the presence of multiple antigens of the bacteria.
It is therefore ~uite naturally that searchers have specifically directed their efforts to the isola-tion of the antigen responsible for the protectiveantibodies.
In the case of capsulated bacteriae, the capsular polyosides are found to be immunogenic in humans, with little side-reactions.
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~20~9~5 A first application in humans of such capsular polyosides is the development of antimeningococcic vac-cine A and C and, quite recently, of a polyvalent vacci-ne containing the purified polyosides of fourteen sero-logic types of pneumococcus.
Several other capsulated bacteriae are involved ina variety of pathological processes, such as:
- Hemophilus influenzae, type b - Klebsiella pneumoniae, - Escherichia c _ , or - Streptococci, group B, and corresponding vaccines may be prepared from their capsular polyosides.
In European patent application 0 002 404 is descri-bed a process for the production of capsular polyosidesfrom Streptococcus pneumoniae.
According to this process, the microorganism is cul-tivated with complex culture media. Then, after inacti-vation with phenol, and without separating the microor-ganisms from the culture medium, a first addition of analcohol such as ethanol is effected, the impurities which have precipitated out are separated, and a second addition o~ alcohol is then effected to precipitate out the polyoside. The contaminants (proteins, nucleic acids) are then removed from the re-suspended polyoside, by an enzyme treatment or by treatment with a cationic surfac-tant tcitrimonium bromide), which treatments may be com-pleted by a diailtration.
In fact, this process is not generally applicable, because the specific conditions concerning each step vary with the polyoside to be separated. Thus, it is nesessary to effect pre-tests to determine the conditions of the various precipitations. In addition, due to the use of certain media, this process may cause a degrada-tion of the polyoside chains.
69~
The object of this invention is to pro~ide a moregenerally applicable, simpler and less degradint process for the production of polyosides of very high molecular weight.
Therefore, this invention relates to process for the production of capsular polyosides from capsulated bacteriae containing same,..comprising cultivating the bacteriae in a synthetic or semi-synthetic medium; at the end of the growth stage of said culture separating the microbial bodies from a supernatant phase; submitting said supernatant phase to a filtration through a membrane which retains the molecules of a molecular weight of 100,000 daltons or more, to give a capsu-lar polyoside-rich retained material, submitting said retained material to an enzyme hydrolysis, adding a butanol-chloroform mixture thereto, separating the aqueous phase, submitting said aqueous phase to a dialysis and then to a filtration through a membrane which retains the molecules having a mole-cular weight in excess of 100~000 daltons and recovering the capsular polyosides from the retained material.
In particular, the present invention provides a process for the production of capsular polyosides from capsul-ated bacteriae containing same, comprising cultivating the bacteriae in a synthetic medium containing up to 0.5 wt~ of protein hydrolysates; at the end of the growth stage of said culture separating the microbial bodies from a supernatant phase; submitting said supernatant phase to a filtration ~hrough a membrane which retains the molecules of a molecular weight of 100,000 da~tons or more, to give a capsular poly-oside-rich retained material, submitting said retained material to an enzyme hydrolysis, adding a butanol-chloroform mixture thereto, separating the aqueous phase, submitting said aqueous phase to a dialysis and then to a filtration through a mem-brane which retains the molecules having a molecular weight in excess o 100,000 daltons and recovering the capsular polyosides from the retained material.
. 1;~069(~5 The present in~ention also provides capsular polyosides produced by a process co~prising culti~ating bacteriae in a synthetic or semi-synthetic mediumi at the end of the growth stage of said culture separating the micro-bial bodies from a supernatant phase, submitting said super-natant phase to a filtration through a membrane which retains the molecules of a molecular weight of 100,000 daltons or more, to give a capsular polyoside-rich retained material, submit-ting said retained materialt~ an e~ZYmehydrolysis, adding a lG butanol~orofOrm mixture thereto, separating the aqueous phase, submitting said aqueous phase to a dialysis and then to a filtration through a membrane which retains the molecules having a molecular weight in excess of 100,000 daltons and recovering the capsular polyosides from the retained material.
The present invention further provides a vaccine comprising at least one capsular polyoside, produced by a process comprising cultivating bacteriae in a synthetic or semi-synthetic medium; at the end of the growth stage of said culture separating the microbial bodies from a super-natant phase; sumbitting said supernatant phase to a filtra-tion through a membra~e which retains the molecules of a mole-cular weight of 100,000 daltons or more, to give a capsular polyoside-rich retained material, submitting said retained nla~rial to anenzyme hydrolysis, adding a butanol-chloroform mixture thereto, separating the aqueous phase, submitting said aqueous phase to a dialysis and then to a filtration through a membrane which retains the molecules having a mole-cular weight in excess of 100~000 daltons and recovering the capsular polyosides from the retained material.
Thus, according to an essential feature of the present invention, the bacterial culture is effected in synthetic or semi-synthetic medium. By synthetic medium are meant aqueous solutions of known chemicals, of low mole-cular weight, which are present in definite amounts.
- 3a -,~
~69~5 By asemi-synthetic medium is meant a medium analogous to the former, but which may contain additionally a small amount (generally up to 0.5 wt%) of naturally occur-ring materials such as protein hydrolysates.
Moreover, according to the present invention, the extraction of the polyosides is effected on the super-natant of the culture after removai of the microbial bodies, and not on the o~erall culture medium.
Filtration through a membrane whose porosity is capable of retaining molecules of a molecular weight of 100,000 dal~ons or more, makes it possible to obtain rapidly a retained material comprising predominantly the polyosides, together with small amounts of proteins, nucleic acids and lipids.
B
;9~D5 Removal of the contaminants is advantageously effec-ted in the following manner: the retained material is submitted to an enzyme hydrolysis, typically with a mixture of Pronase, RNase and DNase, a butanol-chloro-form mixture (preferably a 1:1 mixture, by volume) isadded thereto, the aqueous phase is separated, the a~ueous phase is submitted to a dialysis after which the resulting material is filtered through a membrane which retains the molecules of a molecular weight of 100,000 daltons or more.
The purified polyoside may be separated, in known manner, from the retained material, by precipitation with an alcohol such as ethanol.
The following non-limiting Examples illustrate the present invention.
Pneumococcic polyoside, type 3 A freeze-dried strain of Streptococcus pneumoniae, type 3, is used as starting material.
a) Preparation of seeding materials The freeze-dried strain is added to T-broth.
Cultivation is effected for 16 hours at 37C.
5 ml of the resulting culture are mixed with 15 ml sterilized skim milk. The material is filled into ampoules in an amount of 0.5 ml/ampoule and is then freeze-dried. The freeze-dried materials are stored at +4C.
b) Pre-culture A seeding material (freeze-dried material stored in an ampoule) is added to a flask containing 200 ml of the semi-synthetic medium described below, and cultiva-tion is effected at 37C for 6 hours.
Culture medium:
Pancreatic peptone from casein ~IBF) 1500 mg Q-cystine 150 mg ~06g~
dl-Tryptophane 20 mg Q-Tyrosine 200 m~
Dipotassium phosphate 4960 mg d-G]ucose, anhydrous 1250G mg Magnesium sulfate. 7H20 500 mg Ferrous sulfate. 7H20 5 mg Zinc sulfate . 7H20 0.8 mg Manganese sulfate . 7H2O 0.3 mg Fuming hydrochloric acid RP17.8 mg d-Biotine 15 mg Nicotinic acid 1 mg Pyridoxine HCl 1 mg Riboflavine 1 mg Thiamine 1 mg Calcium pentothenate 5 mg Adenine HCl 10 mg Uracil 10 mg Choline chloride 10 mg Asparagine 100 mg Distilled water, sufficient to make 1000 ml c) Culture The culture itself is effected in a fermenter con-tainin~ 18 litres of the culture medium defined in b).
Seeding is effected in an amount of 1~ with the pre-culture.
The culture is effected at 37C, at pH 7.4, with stirring for a period of time of generally about 16 hrs, to the end of the exponential growth phase.
d) Separation of the supernatant At the end of the exponen~ial phase, the superna-tant is immediately separated by continuous centrifuga-tion at 5000 rpm. The supernatant is submitted to a sterilizing filtration through Millipore membrane 0.22 and is used immediately or is stored at -25C.
~z06~05 e) Extraction A volume of supernatant of about 20 litres is con-centrated to a volume of 2 1itres by filtration through a Millipore 10 membrane capable of retaining the mole-cules of a molecular weight of 100,000 daltons or mGre.
The xesulting retained material is filtered through a Diaflo 105 membrane after addition of 5 litres apyroge-nic steril~ distilled water to complete the separation according to the molecular weight.
Several successive diafiltrations permit a concen-tration to about 350 ml which are then lyophilized in 1 litre flasks.
All the above operations are effected at a tempera-ture of up to 8C maximum.
f) Purification The lyophilized intermediate product is dissolved in 900 ml phosphate buffer 0.01 M - pH 7.
20 mg ~Nase type 1 , 20 mg pancreatic DNase and 180 ~1 (20 units/ml) RNase Tl are added thereto. The material is incubated for 4 hrs at 37C, after which 170 mg Pronase type 9, 800 ~1 pure phenol and 2 drops toluene are added thereto.
The mixture is incubated overnight at 37C. The reaction is terminated and an equal volume (900 ml) of butanol-chloroform (1:1) is added thereto. The resulting material is stirred for 1 hour. The two phases are decanted by centrifugation at 10,000 r.p.m. for 30 mn.
The aqueous phase which contains the polyoside is dialyzed against 20 litres apyrogenic sterile distilled water for a period of time of about 60 hrs.
The dialyzate is submitted to a diafiltration through an Amicon 105 membrane. The volume of the retai-ned material is 1140 ml. 160 ml 40% sodium acetate (final concentration 5%)and 3,9 litres absolute ethanol (3 volumes) are then added.
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The precipitate is collected by centrifugation at 12,000 r.p.m. for 30 mn. Said precipitate is dissolved in apyrogenic distilled water. The resulting solution is filled in vials containing ~ ml solution each. The material is freeze-dried _ vacuo, to give 9 g of the desired product. Said product is the purified polysaccharide which contains:
- sugars 64 - proteins 0.8%
- nucleic acid <0.8%
All the above steps (apart from the incubations) are conducted between 0 and +4C.
~XAMPLE 2 Pneumococcic polyoside type 23 lS A freeze-dried strain of _treptococcus ~neumoniae type 23 is used as starting material.
The culture and the separation of the supernatant are effected under the conditions defined in Example 1 (steps a - d).
Extraction and purification of the polyoside are effected in the following manner:
e) Extraction A 15 litre volume of supernatant is concentrated to a volume of 1.5 litre under the conditions defined in Example l-e.
Three successive diafiltrations make it possible to concentrate, and then to freeze-dry, to give 1.8 g of a material of the following composition:
- sugars 60~
- proteins 19%
- nucleic acid 3.6%
f) Purification The freeze-dried intermediate product is taken up into 200 ml O.OlM phosphate buffer (pH 7). 1 mg RNase, 1 mg DNase and 20 units RNase Tl/ml are then added. The -~2~;~69(i'5 resulting material is incubated at 37C for 3 hrs, after which Pronase type 8 (180 ~g/ml), phenol(l ~l/ml) and tGluene (1 drop) are added thereto. The whole is incu-bated overnight at 37C.
Three successive extractions are effected with an equal volume (200 ml)of butanol-chloroform 1:1. The resul-ting mixture is then stirred mechanically.
The final aqueous phase is dialyzed for about 60 hrs against 4 litres apyrogenic sterile distilled water.
The dialyzate is filtered through Amicon 105 mem-brane. The material is concentrated to 50 ml. Absolute ethanol (3 volumes) and sodium acetate (final concentra-tion 5%) are added thereto and the mixture is left aside overnight at -20C.
The resulting precipitate is collected by centrifu-gation. Said precipitate is dissolved in apyrogenic ste-rile distilled water, and is then freeze-dried, to give 1050 g of a product which contains:
- sugars 75%
- proteins 1.7%
- nucleic acid 0.7~
All the above steps (except otherwise indicated) are effected between 0 and +6~C.
E~AM~E 3 Pneumococcic polyoside type 19 A freeze-dried strain of Streptococcus pneumoniae type 19 is used as starting material.
The culture and the separation of the supernatant are effected under the conditions defined in Example 1 (steps a - d).
Extraction and purification of the polyoside are effected in the following manner:
e) Extraction Using the procedure of Example l-e, a 75 litre vo-lume of supernatant is concentrated to 2 litres by three ~206~(~5 diafiltrations, to give 2.3 g having the following com-position:
- sugars 30 - proteins 15%
- nucleic acid 3.4%
f) Purification The freeze-dried intermediate product is dissolved with 150 ml O.OlM phosphate buffer (pH 7). DNase (3 mg), RNase ~3 mg) and RNase Tl (30 ,ul) are added thereto, and -the resulting material is then incubated at 37C for 4hrs, after which Pronase (62 mg), phenol (150 ,ul) and toluene (1 drop) are added. The whole is allowed to react overnight at 37C.
Deproteination is effected with an equal volume (3 successive extractions) of chl.oroform-butanol 1:1.
The aqueous phase is dialyzed for 60 hrs against 4 litres ap~Troyenic sterile distilled water. The dialyzate is submitted to a diafiltration through Amicon XM 100 membrane.
The retained material is precipitated with 3 volu-mes ethanol containing 5% sodium acetate.
After overnight storage in a freezer, the precipi-tate is dissolved in .apyrogenic steril.edistilled water, and freeze-dried, to give 730 mg of product which con-tains - sugars 73%
- proteins 0.2%
- nucleic acid 0.5%.
Here, again, all the above steps (aside from the incubations) are effected.between 0 and +4C.
Pneumococcic polyoside type 1 A freeze-dried strain of Streptococcus pneumoniae type 1 is used as starting material. The culture and the separation of the supernatant are effected under the ~lZ0~9Q~
conditions defined in Example 1~
Extraction and purification of the polyoside are effected in the following manner:
e) ~xtraction 100 Litres of the culture supernatant are submitted to a diafiltration through Pellicon* cassettes (Milli-pore 105 membrane~. The volume of the final retained material is 3 litres which are freeze-dried in flasks, to give 6.1 g of product which contains:
- galacturonic acid 46.5%
- proteins 7.7%
f) Purification The resulting product is dissolved in 800 ml apyro-genic sterile distilled water. To the solution are added - 16 mg RNase - 16 mg DNase - 160 ~1 RNase Tl.
The material is incubated at 37C for 4 hrs, after which phenol (300 ,ul), toluene (2 drops) and Pronase type 8 (150 mg) are added thereto and the whole is then incubated overnight at 37C. The proteins are extrac-ted with an equal volume of chloroform-butanol (1:1)~
The aqueous phase is dialyzed for 60 hrs. Diafiltration is effected through Amicon~XM 100 membrane The volume of the final retained material is 150 ml. The solution is precipitated with 3 volumes cold ethanol containing 5~ sodium acetate.
The plug is taken up into 250 ml apyrogenic sterile distilled water and freeze-dried in flasks in vacuo, to give 3.7 g purified polyoside which contains:
- galacturonic acid 55.5% (which corresponds to 85% sugars) - proteins 1%
The diffusion coefficient in the KD gel of the polyoside is 0.05. Therefore, the mclecular weight is ~'~ * Trade mark ~2(g~g~
at least 2 x 10 .
Polyoside from K. neumoniae,type 2 A strain of Klebsiella pneumoniae type 2 is used as starting material.
The culture and the separation of the supernatant are effected under the conditions defined in Example 1 (steps a - _), but using as culture medium the following synthetic culture medium:
Trisodium citrate 0.85 g Ammonium sulfate 0.17 g Magnesium sulfate 0.17 g Glutamic acid 0.17 g d-Glucose 16.70 g lS Dipotassium phosphate 10 g Monopotassium phosphate 6.66 g Distilled water, sufficient, to make 1000 ml final pH : 6.8 Extraction and purification of the polyoside are effected in the following manner:
e) Extraction 90 litres of the culture supernatant are used. Dia-filtration is effected through Pellicon cassettes (2 cassettes, 105 membrane). After concentration to 1.5 litre, the material is washed 3 times with 10 litres water. The final retained material is 2.6 litres. The material is freeze-dried in flas]cs ,to give 6.1 g of product which contains:
- sugars 50%
- proteins 15%
f) Purification The resulting product is dissolved in 350 ml water.
There are added thereto:
- 7 ml 0.5M phosphate buffer, pH 7.0 - 7 mg RNase ~20691~5 - 7 m~ DNase - 70 ,ul RNase Tl (prior filtration of the enzymes through 0.22 ~).
The whole is incubated at 37C for 4 hrs, after which protease type 8 (70 mg), phenol (350 ~1) and to-luene (1 drop) are added thereto, and the mixture is incubated overnight at 37C. The reaction is terminated and deproteination is completed by 3 extractions with butanol-chloroform (1:1). The a~ueous phase is dialyzed for 60 hrs against apyrogenic sterile distilled water.
Diafiltration is effected through Amicon XM 100 membra-ne. The volume of the final retained material is 150 ml.
The solution is precipitated with 3 volumes cold absolu-te ethanol containing 5% sodium acetate. The precipitate is collected by centrifugation, dissolved in 250 ml water and then freeze-dried in flasks,ln vacuo. The pure polyoside thus obtained contains:
- sugars 76~ (Hexoses/uronic acid = 3:1) - proteins 3.8%
The KD of the polyoside obtained is 0.1. Thus, the molecular weiyht is in excess of 107.
Polyoside from H. influenza,type b A freeze-dried strain of Hemophilus lnfluenzae type b is used as starting material.
The culture and the separation of the supernatant are effected under the conditions de~ined in Example 1 (steps a - d), but using as culture medium the following semi-synthetic culture medium:
Proteose peptone 5 g Sodium chloride 3.5 g d-Glucose ) 4 g Monopotassium phosphate 1.3 g Dipotassium phosphate 3.5 g NAD (nicotine adenosine dinucleotide) 0.001 g , ; , ~ 2~ 5 Globular extract 50 ml Distilled water, sufficient, to make lOOO ml Extraction and purification of the polyoside are effected in the following manner:
e) Extraction 30 Litres of the culture supernatant are used. Dia-filtration is effected through Millipore 100,000 membra-ne (2 cassettes ). The first concentration gives a volume of 1 litre. The material is washed with 3 x 10 litres sterile distilled water. It is concentrated to 1.3 litre and lyophilized in flasks, to give 3.9 g of a product which contains:
- sugars 53%
- proteins 13~
The product thus obtained is dissolved in 200 ml water.
After filtration through 0.22 ~, the following materials are added:
4.5 ml 0.5M phosphate buffer, pH 7 4 mg RNase 4 mg DNase 40 ~1 RNase T1 The whole is incubated at 37C for 4 hrs, after which protease type 8 (65 mg), phenol (200 ~ul) and to-luene (2 drops) are added and the resulting material is incubated overnight at 37C. The reaction is terminated with 3 extractions with butanol-chloroform (1:1). The aqueous phase is collected by centrifugation at 12,000 r.p.m. at +4C for 30 minutes. It is dialyzed for 60 hrs against apyrogenic sterile dist-lled water (frequent changes of the dialysis medium). Concentration is effec-ted through Amicon XM 100 membrane ( 2 washings x lO0 ml). The volume of the diafiltrate is 100 ml. The dia-filtrate is precipitated with 3 volumes cold absolute ethanol containing 5% sodium acetate. The precipitate, ' ,;
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collected by centrifugation, is taken up into 100 ml apyrogenic sterile distilled water. The solution is freeze-dried in flasks, in vacuo, to give 1.3 g pure polyoside which contains:
- sugars (PRP) 63%
- proteins 2~
he ratio of the polyoside components is the following:
Ribose: Ribitol: Phosphorus : 0.9 : 1 : 0.98 (theore-tical ratio: 1 : 1 : 1).
The KD is close to 0.1, which corresponds to a mole-cular weight of about 107.
The purified immunogenic capsular polyosides obtai-ned according to the present invention may be used for the preparation of vaccines intended to be administered to humans and to animals for preventive (or curative) purposes. The vaccines may be prepared by incorporation of the purified capsular polyosides obtained according to the present invention in a physiologically acceptable liquid carrier such a physiologic solution or water for injection.
For this purpose, one or - preferably - more puri-fied capsular polyosides obtained according to the pre-sent invention and from differen~ serologic types may be incorporated. In addition, polyvalent vaccines may be prepared by incorporation of two or more purified capsu-lar polyosides obtained from different germs.
The results of tests which demonstrate the immuno-genic activity of the polyosides produced according to the present invention are given hereinafter.
a) Titration of the serum antibodies in mice ~) Male Swis5 mice of 20 g body weight were used in groups of 10 animals each:
1 reference group was not given any treatment.
1 group was treated by the I.P. route at the rate of several doses of the polyoside of Example 1 per animal.
.
~2~)69C 5 The titration ofthe hemagglutinating antibodies was effected according to the techniques described in the literature, using the red blood corpuscle of sheep sensitized by the polyoside by means of chromium chlo-ride.
The results reported in Table I demonstrate anincrease of the serum level of hemagglutinating antibo-dies with respect to the reference animals, together with a conventional immunitary paralysis at high do-sages.
~ ) - Analogous tests were conducted in mice with the polyoside of Example 5 (polyoside from Klebsiella pneumoniae, type 2).
The results obtained are given in Table II.
b) Titration of serum antibodies in rats.
The same study was conducted with male Sprague Dawley rats of 400-450 g body weight.
The groups of 10 animals were administered an I.P.
injection of 2 and 20 ~g polvoside from Streptococcus ~neumoniae, type 3, per animal.
Titration of thehemagglutinating antibodies was effected on the 4th, 6th and 14th day after administra-tion.
The results obtained are given in Table III.
Consistent with the literature, the cerum antibody level increases gradually to reach a maximum level on the 6th day.
c) Specific protection ~- Groups of 10 male Swi.ss strain mice were im-munized with the polyoside from Streptococcus pneumoniae type 1, obtained according to the procedure of Example 4, by the sub-cutaneous route (two injections at a one week interval); dosages injected: 0.47 - 4.7 - 47 and 470 ug/kg.
On the 10th day following the 2nd immunization, lZ069(35 ,____.~_____ .____. __________________~____1____1________~
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the animals were infected with the virulent pneumococcus(~ype 1), by the sub-cutaneous route (1250 germs/animal).
After ten days observation, the death rate was as follows:
- untreated animals 100%
- treated animals (0.47 ~g/kg) 30%
- treated animals (4.7 ~g/kg) 10%
- treated animals (47 ug/kg) 30%
- treated animals (470 Jug/kg) 40%
10 ~- Groups of 10 male Swiss mice were immunized with the KP2 polyoside by the sub-cutaneous route (2 injec-tions at a weekly interval; dosages injected: 53 - 533 -5332 ~g/kg).
On the 8th day after the last treatment, Klebsiella pneumoniae type 2 strain was injected to the animals at a rate of 650 germs/animal. After 10 days observation, the death rate was found to be as follows:
- untreated animals 100 - treated animals (53 ~g/kg) o%
- treated animals (533 /ug/kg) 0%
- treated animals (5333 ~g/kg) 0%
The polyosides produced according to the present invention are typically useful for the preparation of the following vaccines:
a) vaccines for the preventive and curative treat-ment of acute and chronic respiratory diseases, compri-sing one of the purified polyosides corresponding to each of the following germs:
Streptococcus E_eumoniae _ mophilus _nfluenzae, type b Klebsiella pneumoniae Escherichia coli For example:
lZ069V5 1) mixture of polyosides from S. pneumoniae types 1, 6, 14, 23 . . . . . . . . . . . . . . . 50 ~ug polyoside from K. pneumoniae, type 2 . . 20 jug polyoside from H. _nfluenzae, type b . . 20 ,ug final volume: 0.5 ml physiological solution
It is known that it is possible to combat infectious diseases at two different levels: either by direct action against the pathogenic germ (antibiotics, anti-septics)~ or by an indirect action via the reinforcement of the systemic defences (vaccination, serotherapy, im-munomodulation).
The use of agents likely to exert a direct action frequently comes up against certain difficulties which may limit theix action (particularly: toxicity of the molecule and bacterial resistance).
In contrast, the reinforcement of the systemic defences via the acquisition of specific antibodies has definite advantages, particularly from the standpoint of the duration of the protection and of the specificity.
However, vaccination with whole microbial bodies is not always free from drawbacks: hypersensitization phenomena, poor tolerance or pyrogenicity are among the most fre-~uent. They are corelated to the presence of multiple antigens of the bacteria.
It is therefore ~uite naturally that searchers have specifically directed their efforts to the isola-tion of the antigen responsible for the protectiveantibodies.
In the case of capsulated bacteriae, the capsular polyosides are found to be immunogenic in humans, with little side-reactions.
3~$
~20~9~5 A first application in humans of such capsular polyosides is the development of antimeningococcic vac-cine A and C and, quite recently, of a polyvalent vacci-ne containing the purified polyosides of fourteen sero-logic types of pneumococcus.
Several other capsulated bacteriae are involved ina variety of pathological processes, such as:
- Hemophilus influenzae, type b - Klebsiella pneumoniae, - Escherichia c _ , or - Streptococci, group B, and corresponding vaccines may be prepared from their capsular polyosides.
In European patent application 0 002 404 is descri-bed a process for the production of capsular polyosidesfrom Streptococcus pneumoniae.
According to this process, the microorganism is cul-tivated with complex culture media. Then, after inacti-vation with phenol, and without separating the microor-ganisms from the culture medium, a first addition of analcohol such as ethanol is effected, the impurities which have precipitated out are separated, and a second addition o~ alcohol is then effected to precipitate out the polyoside. The contaminants (proteins, nucleic acids) are then removed from the re-suspended polyoside, by an enzyme treatment or by treatment with a cationic surfac-tant tcitrimonium bromide), which treatments may be com-pleted by a diailtration.
In fact, this process is not generally applicable, because the specific conditions concerning each step vary with the polyoside to be separated. Thus, it is nesessary to effect pre-tests to determine the conditions of the various precipitations. In addition, due to the use of certain media, this process may cause a degrada-tion of the polyoside chains.
69~
The object of this invention is to pro~ide a moregenerally applicable, simpler and less degradint process for the production of polyosides of very high molecular weight.
Therefore, this invention relates to process for the production of capsular polyosides from capsulated bacteriae containing same,..comprising cultivating the bacteriae in a synthetic or semi-synthetic medium; at the end of the growth stage of said culture separating the microbial bodies from a supernatant phase; submitting said supernatant phase to a filtration through a membrane which retains the molecules of a molecular weight of 100,000 daltons or more, to give a capsu-lar polyoside-rich retained material, submitting said retained material to an enzyme hydrolysis, adding a butanol-chloroform mixture thereto, separating the aqueous phase, submitting said aqueous phase to a dialysis and then to a filtration through a membrane which retains the molecules having a mole-cular weight in excess of 100~000 daltons and recovering the capsular polyosides from the retained material.
In particular, the present invention provides a process for the production of capsular polyosides from capsul-ated bacteriae containing same, comprising cultivating the bacteriae in a synthetic medium containing up to 0.5 wt~ of protein hydrolysates; at the end of the growth stage of said culture separating the microbial bodies from a supernatant phase; submitting said supernatant phase to a filtration ~hrough a membrane which retains the molecules of a molecular weight of 100,000 da~tons or more, to give a capsular poly-oside-rich retained material, submitting said retained material to an enzyme hydrolysis, adding a butanol-chloroform mixture thereto, separating the aqueous phase, submitting said aqueous phase to a dialysis and then to a filtration through a mem-brane which retains the molecules having a molecular weight in excess o 100,000 daltons and recovering the capsular polyosides from the retained material.
. 1;~069(~5 The present in~ention also provides capsular polyosides produced by a process co~prising culti~ating bacteriae in a synthetic or semi-synthetic mediumi at the end of the growth stage of said culture separating the micro-bial bodies from a supernatant phase, submitting said super-natant phase to a filtration through a membrane which retains the molecules of a molecular weight of 100,000 daltons or more, to give a capsular polyoside-rich retained material, submit-ting said retained materialt~ an e~ZYmehydrolysis, adding a lG butanol~orofOrm mixture thereto, separating the aqueous phase, submitting said aqueous phase to a dialysis and then to a filtration through a membrane which retains the molecules having a molecular weight in excess of 100,000 daltons and recovering the capsular polyosides from the retained material.
The present invention further provides a vaccine comprising at least one capsular polyoside, produced by a process comprising cultivating bacteriae in a synthetic or semi-synthetic medium; at the end of the growth stage of said culture separating the microbial bodies from a super-natant phase; sumbitting said supernatant phase to a filtra-tion through a membra~e which retains the molecules of a mole-cular weight of 100,000 daltons or more, to give a capsular polyoside-rich retained material, submitting said retained nla~rial to anenzyme hydrolysis, adding a butanol-chloroform mixture thereto, separating the aqueous phase, submitting said aqueous phase to a dialysis and then to a filtration through a membrane which retains the molecules having a mole-cular weight in excess of 100~000 daltons and recovering the capsular polyosides from the retained material.
Thus, according to an essential feature of the present invention, the bacterial culture is effected in synthetic or semi-synthetic medium. By synthetic medium are meant aqueous solutions of known chemicals, of low mole-cular weight, which are present in definite amounts.
- 3a -,~
~69~5 By asemi-synthetic medium is meant a medium analogous to the former, but which may contain additionally a small amount (generally up to 0.5 wt%) of naturally occur-ring materials such as protein hydrolysates.
Moreover, according to the present invention, the extraction of the polyosides is effected on the super-natant of the culture after removai of the microbial bodies, and not on the o~erall culture medium.
Filtration through a membrane whose porosity is capable of retaining molecules of a molecular weight of 100,000 dal~ons or more, makes it possible to obtain rapidly a retained material comprising predominantly the polyosides, together with small amounts of proteins, nucleic acids and lipids.
B
;9~D5 Removal of the contaminants is advantageously effec-ted in the following manner: the retained material is submitted to an enzyme hydrolysis, typically with a mixture of Pronase, RNase and DNase, a butanol-chloro-form mixture (preferably a 1:1 mixture, by volume) isadded thereto, the aqueous phase is separated, the a~ueous phase is submitted to a dialysis after which the resulting material is filtered through a membrane which retains the molecules of a molecular weight of 100,000 daltons or more.
The purified polyoside may be separated, in known manner, from the retained material, by precipitation with an alcohol such as ethanol.
The following non-limiting Examples illustrate the present invention.
Pneumococcic polyoside, type 3 A freeze-dried strain of Streptococcus pneumoniae, type 3, is used as starting material.
a) Preparation of seeding materials The freeze-dried strain is added to T-broth.
Cultivation is effected for 16 hours at 37C.
5 ml of the resulting culture are mixed with 15 ml sterilized skim milk. The material is filled into ampoules in an amount of 0.5 ml/ampoule and is then freeze-dried. The freeze-dried materials are stored at +4C.
b) Pre-culture A seeding material (freeze-dried material stored in an ampoule) is added to a flask containing 200 ml of the semi-synthetic medium described below, and cultiva-tion is effected at 37C for 6 hours.
Culture medium:
Pancreatic peptone from casein ~IBF) 1500 mg Q-cystine 150 mg ~06g~
dl-Tryptophane 20 mg Q-Tyrosine 200 m~
Dipotassium phosphate 4960 mg d-G]ucose, anhydrous 1250G mg Magnesium sulfate. 7H20 500 mg Ferrous sulfate. 7H20 5 mg Zinc sulfate . 7H20 0.8 mg Manganese sulfate . 7H2O 0.3 mg Fuming hydrochloric acid RP17.8 mg d-Biotine 15 mg Nicotinic acid 1 mg Pyridoxine HCl 1 mg Riboflavine 1 mg Thiamine 1 mg Calcium pentothenate 5 mg Adenine HCl 10 mg Uracil 10 mg Choline chloride 10 mg Asparagine 100 mg Distilled water, sufficient to make 1000 ml c) Culture The culture itself is effected in a fermenter con-tainin~ 18 litres of the culture medium defined in b).
Seeding is effected in an amount of 1~ with the pre-culture.
The culture is effected at 37C, at pH 7.4, with stirring for a period of time of generally about 16 hrs, to the end of the exponential growth phase.
d) Separation of the supernatant At the end of the exponen~ial phase, the superna-tant is immediately separated by continuous centrifuga-tion at 5000 rpm. The supernatant is submitted to a sterilizing filtration through Millipore membrane 0.22 and is used immediately or is stored at -25C.
~z06~05 e) Extraction A volume of supernatant of about 20 litres is con-centrated to a volume of 2 1itres by filtration through a Millipore 10 membrane capable of retaining the mole-cules of a molecular weight of 100,000 daltons or mGre.
The xesulting retained material is filtered through a Diaflo 105 membrane after addition of 5 litres apyroge-nic steril~ distilled water to complete the separation according to the molecular weight.
Several successive diafiltrations permit a concen-tration to about 350 ml which are then lyophilized in 1 litre flasks.
All the above operations are effected at a tempera-ture of up to 8C maximum.
f) Purification The lyophilized intermediate product is dissolved in 900 ml phosphate buffer 0.01 M - pH 7.
20 mg ~Nase type 1 , 20 mg pancreatic DNase and 180 ~1 (20 units/ml) RNase Tl are added thereto. The material is incubated for 4 hrs at 37C, after which 170 mg Pronase type 9, 800 ~1 pure phenol and 2 drops toluene are added thereto.
The mixture is incubated overnight at 37C. The reaction is terminated and an equal volume (900 ml) of butanol-chloroform (1:1) is added thereto. The resulting material is stirred for 1 hour. The two phases are decanted by centrifugation at 10,000 r.p.m. for 30 mn.
The aqueous phase which contains the polyoside is dialyzed against 20 litres apyrogenic sterile distilled water for a period of time of about 60 hrs.
The dialyzate is submitted to a diafiltration through an Amicon 105 membrane. The volume of the retai-ned material is 1140 ml. 160 ml 40% sodium acetate (final concentration 5%)and 3,9 litres absolute ethanol (3 volumes) are then added.
~ZQ6!~0~
The precipitate is collected by centrifugation at 12,000 r.p.m. for 30 mn. Said precipitate is dissolved in apyrogenic distilled water. The resulting solution is filled in vials containing ~ ml solution each. The material is freeze-dried _ vacuo, to give 9 g of the desired product. Said product is the purified polysaccharide which contains:
- sugars 64 - proteins 0.8%
- nucleic acid <0.8%
All the above steps (apart from the incubations) are conducted between 0 and +4C.
~XAMPLE 2 Pneumococcic polyoside type 23 lS A freeze-dried strain of _treptococcus ~neumoniae type 23 is used as starting material.
The culture and the separation of the supernatant are effected under the conditions defined in Example 1 (steps a - d).
Extraction and purification of the polyoside are effected in the following manner:
e) Extraction A 15 litre volume of supernatant is concentrated to a volume of 1.5 litre under the conditions defined in Example l-e.
Three successive diafiltrations make it possible to concentrate, and then to freeze-dry, to give 1.8 g of a material of the following composition:
- sugars 60~
- proteins 19%
- nucleic acid 3.6%
f) Purification The freeze-dried intermediate product is taken up into 200 ml O.OlM phosphate buffer (pH 7). 1 mg RNase, 1 mg DNase and 20 units RNase Tl/ml are then added. The -~2~;~69(i'5 resulting material is incubated at 37C for 3 hrs, after which Pronase type 8 (180 ~g/ml), phenol(l ~l/ml) and tGluene (1 drop) are added thereto. The whole is incu-bated overnight at 37C.
Three successive extractions are effected with an equal volume (200 ml)of butanol-chloroform 1:1. The resul-ting mixture is then stirred mechanically.
The final aqueous phase is dialyzed for about 60 hrs against 4 litres apyrogenic sterile distilled water.
The dialyzate is filtered through Amicon 105 mem-brane. The material is concentrated to 50 ml. Absolute ethanol (3 volumes) and sodium acetate (final concentra-tion 5%) are added thereto and the mixture is left aside overnight at -20C.
The resulting precipitate is collected by centrifu-gation. Said precipitate is dissolved in apyrogenic ste-rile distilled water, and is then freeze-dried, to give 1050 g of a product which contains:
- sugars 75%
- proteins 1.7%
- nucleic acid 0.7~
All the above steps (except otherwise indicated) are effected between 0 and +6~C.
E~AM~E 3 Pneumococcic polyoside type 19 A freeze-dried strain of Streptococcus pneumoniae type 19 is used as starting material.
The culture and the separation of the supernatant are effected under the conditions defined in Example 1 (steps a - d).
Extraction and purification of the polyoside are effected in the following manner:
e) Extraction Using the procedure of Example l-e, a 75 litre vo-lume of supernatant is concentrated to 2 litres by three ~206~(~5 diafiltrations, to give 2.3 g having the following com-position:
- sugars 30 - proteins 15%
- nucleic acid 3.4%
f) Purification The freeze-dried intermediate product is dissolved with 150 ml O.OlM phosphate buffer (pH 7). DNase (3 mg), RNase ~3 mg) and RNase Tl (30 ,ul) are added thereto, and -the resulting material is then incubated at 37C for 4hrs, after which Pronase (62 mg), phenol (150 ,ul) and toluene (1 drop) are added. The whole is allowed to react overnight at 37C.
Deproteination is effected with an equal volume (3 successive extractions) of chl.oroform-butanol 1:1.
The aqueous phase is dialyzed for 60 hrs against 4 litres ap~Troyenic sterile distilled water. The dialyzate is submitted to a diafiltration through Amicon XM 100 membrane.
The retained material is precipitated with 3 volu-mes ethanol containing 5% sodium acetate.
After overnight storage in a freezer, the precipi-tate is dissolved in .apyrogenic steril.edistilled water, and freeze-dried, to give 730 mg of product which con-tains - sugars 73%
- proteins 0.2%
- nucleic acid 0.5%.
Here, again, all the above steps (aside from the incubations) are effected.between 0 and +4C.
Pneumococcic polyoside type 1 A freeze-dried strain of Streptococcus pneumoniae type 1 is used as starting material. The culture and the separation of the supernatant are effected under the ~lZ0~9Q~
conditions defined in Example 1~
Extraction and purification of the polyoside are effected in the following manner:
e) ~xtraction 100 Litres of the culture supernatant are submitted to a diafiltration through Pellicon* cassettes (Milli-pore 105 membrane~. The volume of the final retained material is 3 litres which are freeze-dried in flasks, to give 6.1 g of product which contains:
- galacturonic acid 46.5%
- proteins 7.7%
f) Purification The resulting product is dissolved in 800 ml apyro-genic sterile distilled water. To the solution are added - 16 mg RNase - 16 mg DNase - 160 ~1 RNase Tl.
The material is incubated at 37C for 4 hrs, after which phenol (300 ,ul), toluene (2 drops) and Pronase type 8 (150 mg) are added thereto and the whole is then incubated overnight at 37C. The proteins are extrac-ted with an equal volume of chloroform-butanol (1:1)~
The aqueous phase is dialyzed for 60 hrs. Diafiltration is effected through Amicon~XM 100 membrane The volume of the final retained material is 150 ml. The solution is precipitated with 3 volumes cold ethanol containing 5~ sodium acetate.
The plug is taken up into 250 ml apyrogenic sterile distilled water and freeze-dried in flasks in vacuo, to give 3.7 g purified polyoside which contains:
- galacturonic acid 55.5% (which corresponds to 85% sugars) - proteins 1%
The diffusion coefficient in the KD gel of the polyoside is 0.05. Therefore, the mclecular weight is ~'~ * Trade mark ~2(g~g~
at least 2 x 10 .
Polyoside from K. neumoniae,type 2 A strain of Klebsiella pneumoniae type 2 is used as starting material.
The culture and the separation of the supernatant are effected under the conditions defined in Example 1 (steps a - _), but using as culture medium the following synthetic culture medium:
Trisodium citrate 0.85 g Ammonium sulfate 0.17 g Magnesium sulfate 0.17 g Glutamic acid 0.17 g d-Glucose 16.70 g lS Dipotassium phosphate 10 g Monopotassium phosphate 6.66 g Distilled water, sufficient, to make 1000 ml final pH : 6.8 Extraction and purification of the polyoside are effected in the following manner:
e) Extraction 90 litres of the culture supernatant are used. Dia-filtration is effected through Pellicon cassettes (2 cassettes, 105 membrane). After concentration to 1.5 litre, the material is washed 3 times with 10 litres water. The final retained material is 2.6 litres. The material is freeze-dried in flas]cs ,to give 6.1 g of product which contains:
- sugars 50%
- proteins 15%
f) Purification The resulting product is dissolved in 350 ml water.
There are added thereto:
- 7 ml 0.5M phosphate buffer, pH 7.0 - 7 mg RNase ~20691~5 - 7 m~ DNase - 70 ,ul RNase Tl (prior filtration of the enzymes through 0.22 ~).
The whole is incubated at 37C for 4 hrs, after which protease type 8 (70 mg), phenol (350 ~1) and to-luene (1 drop) are added thereto, and the mixture is incubated overnight at 37C. The reaction is terminated and deproteination is completed by 3 extractions with butanol-chloroform (1:1). The a~ueous phase is dialyzed for 60 hrs against apyrogenic sterile distilled water.
Diafiltration is effected through Amicon XM 100 membra-ne. The volume of the final retained material is 150 ml.
The solution is precipitated with 3 volumes cold absolu-te ethanol containing 5% sodium acetate. The precipitate is collected by centrifugation, dissolved in 250 ml water and then freeze-dried in flasks,ln vacuo. The pure polyoside thus obtained contains:
- sugars 76~ (Hexoses/uronic acid = 3:1) - proteins 3.8%
The KD of the polyoside obtained is 0.1. Thus, the molecular weiyht is in excess of 107.
Polyoside from H. influenza,type b A freeze-dried strain of Hemophilus lnfluenzae type b is used as starting material.
The culture and the separation of the supernatant are effected under the conditions de~ined in Example 1 (steps a - d), but using as culture medium the following semi-synthetic culture medium:
Proteose peptone 5 g Sodium chloride 3.5 g d-Glucose ) 4 g Monopotassium phosphate 1.3 g Dipotassium phosphate 3.5 g NAD (nicotine adenosine dinucleotide) 0.001 g , ; , ~ 2~ 5 Globular extract 50 ml Distilled water, sufficient, to make lOOO ml Extraction and purification of the polyoside are effected in the following manner:
e) Extraction 30 Litres of the culture supernatant are used. Dia-filtration is effected through Millipore 100,000 membra-ne (2 cassettes ). The first concentration gives a volume of 1 litre. The material is washed with 3 x 10 litres sterile distilled water. It is concentrated to 1.3 litre and lyophilized in flasks, to give 3.9 g of a product which contains:
- sugars 53%
- proteins 13~
The product thus obtained is dissolved in 200 ml water.
After filtration through 0.22 ~, the following materials are added:
4.5 ml 0.5M phosphate buffer, pH 7 4 mg RNase 4 mg DNase 40 ~1 RNase T1 The whole is incubated at 37C for 4 hrs, after which protease type 8 (65 mg), phenol (200 ~ul) and to-luene (2 drops) are added and the resulting material is incubated overnight at 37C. The reaction is terminated with 3 extractions with butanol-chloroform (1:1). The aqueous phase is collected by centrifugation at 12,000 r.p.m. at +4C for 30 minutes. It is dialyzed for 60 hrs against apyrogenic sterile dist-lled water (frequent changes of the dialysis medium). Concentration is effec-ted through Amicon XM 100 membrane ( 2 washings x lO0 ml). The volume of the diafiltrate is 100 ml. The dia-filtrate is precipitated with 3 volumes cold absolute ethanol containing 5% sodium acetate. The precipitate, ' ,;
~z~6~
collected by centrifugation, is taken up into 100 ml apyrogenic sterile distilled water. The solution is freeze-dried in flasks, in vacuo, to give 1.3 g pure polyoside which contains:
- sugars (PRP) 63%
- proteins 2~
he ratio of the polyoside components is the following:
Ribose: Ribitol: Phosphorus : 0.9 : 1 : 0.98 (theore-tical ratio: 1 : 1 : 1).
The KD is close to 0.1, which corresponds to a mole-cular weight of about 107.
The purified immunogenic capsular polyosides obtai-ned according to the present invention may be used for the preparation of vaccines intended to be administered to humans and to animals for preventive (or curative) purposes. The vaccines may be prepared by incorporation of the purified capsular polyosides obtained according to the present invention in a physiologically acceptable liquid carrier such a physiologic solution or water for injection.
For this purpose, one or - preferably - more puri-fied capsular polyosides obtained according to the pre-sent invention and from differen~ serologic types may be incorporated. In addition, polyvalent vaccines may be prepared by incorporation of two or more purified capsu-lar polyosides obtained from different germs.
The results of tests which demonstrate the immuno-genic activity of the polyosides produced according to the present invention are given hereinafter.
a) Titration of the serum antibodies in mice ~) Male Swis5 mice of 20 g body weight were used in groups of 10 animals each:
1 reference group was not given any treatment.
1 group was treated by the I.P. route at the rate of several doses of the polyoside of Example 1 per animal.
.
~2~)69C 5 The titration ofthe hemagglutinating antibodies was effected according to the techniques described in the literature, using the red blood corpuscle of sheep sensitized by the polyoside by means of chromium chlo-ride.
The results reported in Table I demonstrate anincrease of the serum level of hemagglutinating antibo-dies with respect to the reference animals, together with a conventional immunitary paralysis at high do-sages.
~ ) - Analogous tests were conducted in mice with the polyoside of Example 5 (polyoside from Klebsiella pneumoniae, type 2).
The results obtained are given in Table II.
b) Titration of serum antibodies in rats.
The same study was conducted with male Sprague Dawley rats of 400-450 g body weight.
The groups of 10 animals were administered an I.P.
injection of 2 and 20 ~g polvoside from Streptococcus ~neumoniae, type 3, per animal.
Titration of thehemagglutinating antibodies was effected on the 4th, 6th and 14th day after administra-tion.
The results obtained are given in Table III.
Consistent with the literature, the cerum antibody level increases gradually to reach a maximum level on the 6th day.
c) Specific protection ~- Groups of 10 male Swi.ss strain mice were im-munized with the polyoside from Streptococcus pneumoniae type 1, obtained according to the procedure of Example 4, by the sub-cutaneous route (two injections at a one week interval); dosages injected: 0.47 - 4.7 - 47 and 470 ug/kg.
On the 10th day following the 2nd immunization, lZ069(35 ,____.~_____ .____. __________________~____1____1________~
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the animals were infected with the virulent pneumococcus(~ype 1), by the sub-cutaneous route (1250 germs/animal).
After ten days observation, the death rate was as follows:
- untreated animals 100%
- treated animals (0.47 ~g/kg) 30%
- treated animals (4.7 ~g/kg) 10%
- treated animals (47 ug/kg) 30%
- treated animals (470 Jug/kg) 40%
10 ~- Groups of 10 male Swiss mice were immunized with the KP2 polyoside by the sub-cutaneous route (2 injec-tions at a weekly interval; dosages injected: 53 - 533 -5332 ~g/kg).
On the 8th day after the last treatment, Klebsiella pneumoniae type 2 strain was injected to the animals at a rate of 650 germs/animal. After 10 days observation, the death rate was found to be as follows:
- untreated animals 100 - treated animals (53 ~g/kg) o%
- treated animals (533 /ug/kg) 0%
- treated animals (5333 ~g/kg) 0%
The polyosides produced according to the present invention are typically useful for the preparation of the following vaccines:
a) vaccines for the preventive and curative treat-ment of acute and chronic respiratory diseases, compri-sing one of the purified polyosides corresponding to each of the following germs:
Streptococcus E_eumoniae _ mophilus _nfluenzae, type b Klebsiella pneumoniae Escherichia coli For example:
lZ069V5 1) mixture of polyosides from S. pneumoniae types 1, 6, 14, 23 . . . . . . . . . . . . . . . 50 ~ug polyoside from K. pneumoniae, type 2 . . 20 jug polyoside from H. _nfluenzae, type b . . 20 ,ug final volume: 0.5 ml physiological solution
2) mixture of polyosides from S. pneumoniae types 1, 6, 14, 23 . . . . . . . . . . . . . . . 25 ,ug polyoside from K.pneumoniae, type 2 . . 10 ~g polyoside from H.influenzae, type b . . 10 ,ug b) Antipneumococcic vaccine comprising the purified poly-osides from the following types of Streptococcus pneu-moniae:
1, 3, 4, 6A, 7F, 8, 9N, 12F, 14, 18L, l9F, 23, 2, llA, 15F.
For example:
- mixture comprising 50 ~g active material in a volume of 0.5 ml physiological solution, - mixture comprising 25 ,ug active material in a volume of 0.5 ml physiological solution.
1, 3, 4, 6A, 7F, 8, 9N, 12F, 14, 18L, l9F, 23, 2, llA, 15F.
For example:
- mixture comprising 50 ~g active material in a volume of 0.5 ml physiological solution, - mixture comprising 25 ,ug active material in a volume of 0.5 ml physiological solution.
Claims (8)
1. Process for the production of capsular poly-osides from capsulated bacteriae containing same, comprising cultivating the bacteriae in a synthetic or semi-synthetic medium, at the end of the growth stage of said culture separat-ing the microbial bodies from a supernatant phase; submitting said supernatant phase to a filtration through a membrane which retains the molecules of a molecular weight of 100,000 daltons or more, to give a capsular polyoside-rich retained material, submitting said retained material to an enzyme hydro-lysis, adding a butanol-chloroform mixture thereto, separating the aqueous phase, submitting said aqueous phase to a dialysis and then to a filtration through a membrane which retains the molecules having a molecular weight in excess of 100,000 dal-tons and recovering the capsular polyosides from the retained material.
2. Process for the production of capsular polyosides from capsulated bacteriae containing same, comprising cultivating the bacteriae in a synthetic medium containing up to 0.5 wt% of protein hydrolysates; at the end of the growth stage of said culture separating the micro-bial bodies from a supernatant phase; submitting said super-natant phase to a filtration through a membrane which retains the molecules of a molecular weight of 100,000 daltons or more to give a capsular polyoside-rich retained material, submitting said retained material to an enzyme hydrolysis, adding a butanol-chloroform mixture thereto, separating the aqueous phase, submitting said aqueous phase to a dialysis and then to a filtration through a membrane which retains the molecules having a molecular weight in excess of 100,000 daltons and recovering the capsular polyosides from the retained material.
3. A process as claimed in claim 1 for the production of capsular polyosides from Streptococcus pneu-moniae, using as culture medium a medium having substantially the following composition Pancreatic peptone from casein (IBF) 1500 mg ?-cystine 150 mg dl-Tryptophane 20 mg ?-Tyrosine 200 mg Dipotassium phosphate 4960 mg d-Glucose, anhydrous 12500 mg Magnesium sulfate. 7H2O 500 mg Ferrous sulfate. 7H2O 5 mg Zinc sulfate. 7H2O 0.8 mg Manganese sulfate. 7H2O 0.3 mg Fuming hydrochloric acid RP 17.8 mg d-Biotine 15 mg Nicotinic acid 1 mg Pyridoxine HCl 1 mg Riboflavine 1 mg Thiamine 1 mg Calcium pentothenate 5 mg Adenine HCl 10 mg Uracil 10 mg Choline chloride 10 mg Asparagine 100 mg Distilled water, sufficient to make 1000 ml
4. A process as claimed in claim 1 for the production of capsular polyosides from Klebsiella pneumoniae, using as culture medium a medium having substantially the following composition Trisodium citrate 0.85 g Ammonium sulfate 0.17 g Magnesium sulfate 0.17 g Glutamic acid 0.17 g d-Glucose 16.70 g Dipotassium phosphate 10 g Monopotassium phosphate 6.66 g Distilled water, sufficient to make 1000 ml
5. A process as claimed in claim 1 for the pre-paration of capsular polyosides from Hemophilus influenzae, using as culture medium a medium having substantially the following composition Proteose peptone 5 g Sodium chloride 3.5 g d-Glucose 4 g Monopotassium phosphate 1.3 g Dipotassium phosphate 3.5 g NAD (nicotine adenosine dinucleotide) 0.001g Globular extract 50 ml Distilled water, sufficient to make 1000 ml
6. Capsular polyosides whenever produced by a process comprising cultivating bacteriae in a synthetic or semi-synthetic medium; at the end of the growth stage of said culture separating the microbial bodies from a supernatant phase; submitting said supernatant phase to a filtration through a membrane which retains the molecules of a molecular weight of 100,000 daltons or more, to give a capsular poly-oside-rich retained material, submitting said retained material to an enzyme hydrolysis, adding a butanol-chloroform mixture thereto, separating the aqueous phase, submitting said aqueous phase to a dialysis and then to a filtration through a mem-brane which retains the molecules having a molecular weight in excess of 100,000 daltons and recovering the capsular poly-osides from the retained material.
7. Capsular polyosides as claimed in claim 6, obtained from bacteria selected from the group consisting of Streptococcus pneumoniae, Hemophilus influenzae, Klebsiella pneumoniae and Escherichia coli.
8. Capsular polyosides as claimed in claim 6, obtained from Streptococcus pneumoniae.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR8114842A FR2510606A1 (en) | 1981-07-30 | 1981-07-30 | PROCESS FOR OBTAINING CAPSULAR POLYOSIDES, CAPSULAR POLYOSIDES THUS OBTAINED AND THEIR APPLICATION TO THE PREPARATION OF VACCINES |
| FR8114842 | 1981-07-30 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1206905A true CA1206905A (en) | 1986-07-02 |
Family
ID=9261011
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000408233A Expired CA1206905A (en) | 1981-07-30 | 1982-07-28 | Process for the production of capsular polyosides and capsular polyosides obtained thereby |
Country Status (11)
| Country | Link |
|---|---|
| EP (1) | EP0071515B1 (en) |
| AT (1) | ATE16244T1 (en) |
| CA (1) | CA1206905A (en) |
| DD (1) | DD202623A5 (en) |
| DE (1) | DE3267160D1 (en) |
| DK (1) | DK327182A (en) |
| ES (1) | ES514496A0 (en) |
| FR (1) | FR2510606A1 (en) |
| IE (1) | IE53582B1 (en) |
| NO (1) | NO159394C (en) |
| PT (1) | PT75301B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008129559A2 (en) | 2007-04-23 | 2008-10-30 | Serum Institute Of India Ltd | Antigenic polysaccharides and process for their preparation |
| WO2011148382A1 (en) | 2010-05-28 | 2011-12-01 | Biological E Limited | An improved process for the purification of capsular polysaccharides of haemophilus influenza - b, neisseria meningitis such as serotypes a, c, y and w-135, and other similar related capsular polysaccharides produced from both gram negative and gram positive microorganisms using aluminium phosphate with alcohol. |
| WO2024062494A1 (en) | 2022-09-19 | 2024-03-28 | Biological E Limited | Method for the purification of capsular polysaccharides |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ATE72401T1 (en) * | 1986-03-27 | 1992-02-15 | Schweiz Serum & Impfinst | KLEBSIELLA CAPSULE POLYSACCHARID VACCINE. |
| US6426074B1 (en) * | 1997-03-19 | 2002-07-30 | The Brigham And Women's Hospital Inc. | Group B Streptococcus vaccine |
| JP4856345B2 (en) * | 2000-03-16 | 2012-01-18 | ザ・チルドレンズ・ホスピタル・オブ・フィラデルフィア | Controlled production of Pneumococcus capsular polysaccharide |
| NZ511680A (en) | 2001-05-14 | 2004-07-30 | Univ Waikato | Method for preparing nucleic acid or DNA samples and a DNA extraction process using thermophilic proteases |
| GB0522303D0 (en) * | 2005-11-01 | 2005-12-07 | Chiron Srl | Culture method |
| WO2011151841A1 (en) * | 2010-05-31 | 2011-12-08 | Panacea Biotec Limited | Fermentation process for streptococcus pneumoniae |
| US11951165B2 (en) | 2016-12-30 | 2024-04-09 | Vaxcyte, Inc. | Conjugated vaccine carrier proteins |
| MX2019007910A (en) | 2016-12-30 | 2019-12-05 | Sutrovax Inc | Polypeptide-antigen conjugates with non-natural amino acids. |
| WO2020010016A1 (en) | 2018-07-04 | 2020-01-09 | Sutrovax, Inc. | Self-adjuvanted immunogenic conjugates |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US2166963A (en) * | 1936-11-19 | 1939-07-25 | Sharp & Dohme Inc | Antigenic polysaccharide complex |
| US2528972A (en) * | 1947-05-15 | 1950-11-07 | Western Reserve University | Prophylactic toxoid compound and method of making same |
| FR2407262A1 (en) * | 1977-10-27 | 1979-05-25 | Cassenne Lab Sa | NEW POLYSACCHARIDES MICROBIAL BODY EXTRACTS OF HAEMOPHILUS INFLUENZAE, PROCESS FOR THE PREPARATION AND APPLICATION AS MEDICINAL PRODUCTS OF THESE NEW PRODUCTS |
| CA1115210A (en) * | 1977-11-28 | 1981-12-29 | Dennis J. Carlo | Pneumococcal vaccine |
| US4264764A (en) * | 1979-01-12 | 1981-04-28 | Merck & Co., Inc. | Meningitis vaccine |
| US4221906A (en) * | 1979-03-19 | 1980-09-09 | American Cyanamid Company | Stabilization of pneumococcal polysaccharides |
| US4287173A (en) * | 1979-09-24 | 1981-09-01 | Merck & Co., Inc. | Vaccine for dental caries |
| US4307080A (en) * | 1979-09-24 | 1981-12-22 | Merck & Co., Inc. | Meningitis vaccine |
-
1981
- 1981-07-30 FR FR8114842A patent/FR2510606A1/en active Granted
-
1982
- 1982-07-15 IE IE1705/82A patent/IE53582B1/en unknown
- 1982-07-19 EP EP82401343A patent/EP0071515B1/en not_active Expired
- 1982-07-19 AT AT82401343T patent/ATE16244T1/en not_active IP Right Cessation
- 1982-07-19 DE DE8282401343T patent/DE3267160D1/en not_active Expired
- 1982-07-21 DK DK327182A patent/DK327182A/en not_active Application Discontinuation
- 1982-07-23 PT PT75301A patent/PT75301B/en not_active IP Right Cessation
- 1982-07-28 CA CA000408233A patent/CA1206905A/en not_active Expired
- 1982-07-28 NO NO822592A patent/NO159394C/en unknown
- 1982-07-29 DD DD82242065A patent/DD202623A5/en unknown
- 1982-07-29 ES ES514496A patent/ES514496A0/en active Granted
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008129559A2 (en) | 2007-04-23 | 2008-10-30 | Serum Institute Of India Ltd | Antigenic polysaccharides and process for their preparation |
| WO2011148382A1 (en) | 2010-05-28 | 2011-12-01 | Biological E Limited | An improved process for the purification of capsular polysaccharides of haemophilus influenza - b, neisseria meningitis such as serotypes a, c, y and w-135, and other similar related capsular polysaccharides produced from both gram negative and gram positive microorganisms using aluminium phosphate with alcohol. |
| WO2024062494A1 (en) | 2022-09-19 | 2024-03-28 | Biological E Limited | Method for the purification of capsular polysaccharides |
Also Published As
| Publication number | Publication date |
|---|---|
| ES8305827A1 (en) | 1983-04-16 |
| DK327182A (en) | 1983-01-31 |
| NO822592L (en) | 1983-01-31 |
| FR2510606B1 (en) | 1984-10-26 |
| PT75301B (en) | 1984-07-31 |
| PT75301A (en) | 1982-08-01 |
| ATE16244T1 (en) | 1985-11-15 |
| IE53582B1 (en) | 1988-12-21 |
| FR2510606A1 (en) | 1983-02-04 |
| NO159394B (en) | 1988-09-12 |
| ES514496A0 (en) | 1983-04-16 |
| DE3267160D1 (en) | 1985-12-05 |
| IE821705L (en) | 1983-01-30 |
| DD202623A5 (en) | 1983-09-28 |
| NO159394C (en) | 1988-12-21 |
| EP0071515A1 (en) | 1983-02-09 |
| EP0071515B1 (en) | 1985-10-30 |
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