Fermentative method of producing diphtheria toxin

FIELD: chemistry.

SUBSTANCE: method of producing diphtheria toxin or its mutant or fragment involves a fermentation step where the Corynebacterium diphtheriae strain is grown in a fermenter while stirring in order to maintain a homogenous culture and with limited aeration such that partial pressure of oxygen (pO2) in the culture falls to a level which is 4% lower on the bigger part of the fermentation step. Diphtheria toxin or its mutant or fragment is isolated from the culture. The invention also relates to a method of preparing a pharmaceutical composition for treating or preventing diphtheria, which includes a step for fermentative production of the toxin and mixing it with a pharmaceutically acceptable carrier after isolation.

EFFECT: use of the invention leads to high output of diphtheria toxin or mutant (for example, CRM 197), ensures high output of diphtheria toxin when the culture medium additionally contains iron or complex initial substances of varying quality.

20 cl, 6 dwg, 8 tbl, 6 ex

 

The present invention relates to the field of diphtheria antigens, in particular toxins (including mutant form of diphtheria toxin, such as CRM197), and fermentation methods of production of mixed crops such antigens.

Diphtheria toxin is a protein exotoxin produced by the bacterium Corynebacterium reagent grade. It is produced as a single polypeptide, which easily undergoes splicing to form two subunits linked by a disulfide bond, fragment a and fragment B, as the result of cleavage at residue 190, 192 or 193 (Moskaug et al., Biol. Chem. 264: 15709-15713, 1989). Fragment And is a catalytically active portion and is a NAD-dependent ADP-ribosyltransferase specific target which is a factor of protein synthesis EF-2 by which it inactivates EF-2 and stops protein synthesis in the cell.

Immunity to bacterial toxin, such as diphtheria toxin, can be acquired naturally during infection or artificially by injection detoxificating form of the toxin (toxoid) (Germanier, er, Bacterial Vaccines, Academic Press, Orlando, FI., 1984). Toxoids were traditionally obtained by chemical modification of native toxins (Lingood et al., Brit. J. Exp.Path. 44; 177, 1963), making them non-toxic while maintaining the antigenicity that protects the vaccinated animal etc is against subsequent infection natural toxin. Alternatively described several mutant of diphtheria toxin with reduced toxicity (US 4709017, US 4950740).

CRM197 is a non-toxic form of diphtheria toxin, but is immunologically indistinguishable from diphtheria toxin. CRM197 is produced .diphtheriae infected nontoxigenic phage β197tox-created by nitrosoguanidine mutagenesis of toxigenic carinifera b (carynephage b) (Uchida et al., Nature New Biology (1971) 233; 8-11). Protein CRM197 has the same molecular weight as diphtheria toxin, but differs from it in a single replacement of the base in the structural gene. This leads to the substitution of the amino acid glycine to glutamine at position 52, which makes the fragment And unable to bind NAD and therefore non-toxic (Pappenheimer 1977, Ann Rev Biochem. 46; 69-94, Rappuoli Applied and Environmental Microbiology Sept 1983, p.560-564).

Diphtheria toxoid and a mutant form with reduced toxicity, CRM197, are components of many vaccines that provide immunity against Corynebacterium diphtheriae. There are several combination vaccines that can prevent diseases caused by Bordetella pertussis, Clostridium tetani, Corynebacterium diphtheriae, and possibly hepatitis b virus and/or Haemophilus influenzae type b (see, for example, WO 93/24148 and WO 97/00697, WO 02/055105).

Diphtheria toxin and mutant forms, including CRM197, also used in vaccines as safe and effective the x media saccharides, dependent T cells. CRM197 currently used in the conjugate vaccine CRM197 with oligosaccharide Haemophilus influenzae type b (HibTitre®; Lederle Praxis Biologicals, Rochester, N.Y.).

Ways to obtain the diphtheria toxoid (DT) is well known in the art. For example, DT can be obtained by purification of the toxin from the culture of Corynebacterium diphtheriae, followed by chemical detoxification, or can be obtained by purification of recombinant or genetically detoxificating analogue of this toxin (e.g., CRM197, or other specimens, as described in US 4709017, US 5843711, US 5601827 and US 5917017). Corynebacterium diphtheriae is cultivated under aerobic conditions. Rappuoli et al., (Biotechnology February 1985, p.161-163) suggest that pO2it should be regulated at the level of 25% by aeration of the mixture of air and oxygen, which is automatically adjusted to maintain the desired pO2.

The production of significant quantities of diphtheria toxins, such as CRM197, for use in vaccines has been hampered by the small relative amount of protein. Previously this problem was solved by introducing additional copies of the gene encoding diphtheria toxin or mutant form, in Corynebacterium diphtheriae (US 4925792; US 5614382). Such methods lead to an increase in production of about three times. Ways to further improve the output of diphtheria toxin in a reproducible manner would be the useful to provide higher levels of production of these valuable antigens.

Accordingly, in the present application proposed an improved method of fermentation, including fermentation stage of growing strain of Corynebacterium diphtheriae in the medium in the fermenter under conditions of agitation sufficient to maintain a homogeneous culture, and limited aeration, so that the partial pressure of oxygen (po2in culture drops to less than 4% throughout the greater part of the fermentation stage.

This fermentation takes place in aerobic conditions, but with limited aeration, so that oxygen is used as soon as it arrives in culture, during the greater part of the fermentation, that is, after the initial phase where the density .diphtheriae is relatively low and the levels of pO2can be higher. The authors of this invention have found that culture in such conditions provides a more effective and/or consistent expression of the diphtheria toxin or mutant compared with the methods of fermentation carried out at higher pO2. The method according to the invention is more reliable than fermentation at higher oxygen levels, and ensures high output diphtheria toxin, even when the culture medium contains added iron, or when using complex source materials of varying quality.

The second is the SPECTA of the present invention, a method for manufacturing the drug, diphtheria toxin or a mutant, including fermentation method according to the invention and the selection of diphtheria toxin or a mutant of culture. Although described here diphtheria toxin and its mutants, provides that any antigen .diphtheriae can be distinguished using the method according to the invention.

The application of this method leads to higher outputs diphtheria toxin or a mutant, such as CRM197, compared with conditions in which Rho2support at the 5% level or higher, for example 20%.

In the third aspect of the present invention proposed a diphtheria toxin or a mutant, selected by the method according to the invention.

In the fourth aspect of the present invention proposed a pharmaceutical composition comprising a diphtheria toxin or a mutant according to the invention and a pharmaceutically acceptable carrier.

In an additional aspect of the present invention proposed a diphtheria toxin or a mutant for use in therapy, in particular for the treatment or prevention of bacterial disease, such as disease caused .diphtheriae.

In another aspect of the present invention proposed the use of diphtheria toxin or a mutant according to the invention in the manufacture of a medicinal product for the treatment or prevention of bacterial diseases, particularly diseases caused .diphthriae.

In another aspect of the present invention, a method for prevention or treatment of bacterial infections, particularly infections caused by .diphtheriae, including the introduction to the patient the pharmaceutical composition according to the invention.

Description of graphic materials

Figure 1 - graphs showing the profile of the oxygen saturation and its use in determining KLa fermentation. Panel a shows the dynamics of oxygen saturation after transfer from nitrogen to oxygen. Panel B shows a graph of ln(100-ro2against time, which enables to estimate KLa by determining the gradient of the line.

Figure 2 is an overview diagram of fermentation method for .diphtheriae.

Figure 3 is a chart showing typical growth kinetics of the culture .diphtheriae. Line with circular markers shows the optical density (OD) at 650 nm after different periods of cultivation. The line with diamond markers shows the pH of the culture.

Figure 4 - polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE) supernatant culture. Lane 1 - molecular weight markers, lane 2 - 1 g standard CRM197, band 3 - 0.5 μg of standard CRM197, band 4 0.25 microgram standard CRM197, lines 5 - 11 - supernatant from fermentati .diphtheriae. Gel a shows supernatant from CDT082 in the band 5, CDT198 in lanes 6-8 (line 6 - supernatant was removed at the time of 22.5 hours, line 7 in oment 24 hours and the line 8 - at the time 28 hours), CDT199 in the bands 9, 10 and 11 (lane 9 supernatant was removed at the time of 22 hours and 45 minutes, line 10, in the moment of 24 hours and 45 minutes and the line 11 - serial after filtration and filtration). Gel B shows supernatant from CDT082 in line 5, from CDT205 in lines 6-9 (line 7 - after 21 hours 43 minutes fermentation, line 8 - after 23 hours of fermentation, line 9 - after 24 hours of fermentation) and from CDT206 in lines 10-13 (line 10 after 22 hours and 10 min of fermentation, line 11 - after 23 hours 49 minutes fermentation, line 12 - 24 hours after 30 min of fermentation, line 13 after filtration and filtration).

5 is a Graph showing KLa 150-liter fermenter at different mixing speeds under conditions of aeration 23 litres per minute.

Detailed description of the invention

The authors of the present invention assume that the terms "include", "contain" and "contains" in each case can be substituted, respectively, the terms "comprising", "comprise" and "comprises".

One aspect of the present invention is a method of fermentation, including fermentation stage of growing strain of Corynebacterium diphtheriae in the medium in the fermenter under conditions of agitation sufficient to maintain a homogeneous culture (e.g., sufficient to achieve the time of mixing is less 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 second), and limited aeration, so that pO2 in the culture falls to less than 5%, 4%, 3%, 1% or 0.5% over the greater part of the fermentation stage. In the preferred embodiment of the Rho2drops to levels close to zero, preferably throughout the greater part of the fermentation stage.

For example, Rho2in the culture falls to less than 5%, 4%, 3%, 1% or 0.5% of the time when Corynebacterium diphtheriae grown to a density sufficient for the consumption of mostly oxygen, as oxygen enters the culture (latent period, for example, from at least 1, 2, 3, 4, 5 or 6 hours after the start of the fermentation)before fermentation, when the concentration of Rho2increases again, close to the end of the fermentation stage (for example, through 16, 18, 20, 22 or 24 hours after a latent period).

Fermentation usually ends, and the culture is harvested when Rho2increases to levels that exceed the terms of the limited aeration. It should be noted that under different conditions of inoculation, for example, when the fermenter inoculant a considerably large number of culture .diphtheriae, limited aeration begin shortly after the beginning of fermentation (for example, through 1, 5, 10, 20, 30, 40 or 60 minutes after the start of fermentation).

100%Rho2represents the amount of oxygen that is present when the medium (in the absence of a culture saturated with oxygen PEFC the ozonation compressed air through this Wednesday at 34.5°C and a pressure of 0.5 bar (50 kPa). For a 150-liter fermenter speed aeration and stirring speed should be set to 23 liter/min and 240 rpm, whereas for a 20-liter fermenter speed aeration and stirring speed should be set to 3 liter/min and 300 rpm, Rho2can be defined as the amount of oxygen present in fully aerated fermentation medium before inoculation.

Homogeneous culture is a culture in which the bacteria are evenly dispersed throughout the fermenter, so that at least 3, 4, 5, 6, 7, 8, 9 or 10% of the bacteria present in the top 10% of culture medium.

Fermentation stage is defined as the stage at which Corynebacterium diphtheriae grown in the fermenter. Fermentation stage starts from the moment of introduction into the fermenter preculture and ends when the conditions of limited aeration, described here, Rho2eventually rises to a level above 10%. Fermentation stage usually lasts for more than 12, 14, 16, 18, 20 or 24 hours, for example from 16 to 40 hours or, for example, from 22 to 28 hours.

Mixing is carried out by mixing the culture in the fermenter, but it can be accomplished in any other suitable way, for example by shaking, using fibromites and/or by ozonation gas. Mixing is up to is enough to get the time of mixing culture less than 20, 15, 10, 8, 7, 6, 5, 4, 3, 2 or 1 seconds.

The mixing time of culture can be measured in a glass fermenter. It represents the time elapsed after the introduction of colored water solution to this colored aqueous solution was uniformly dispersed throughout the culture medium.

The fermenter is any installation for industrial production of bacterial cultures. However, this term does not include culture flasks, which are usually used for growing bacteria on a smaller scale.

A large part of the fermentation stage is defined as the time in excess of 50%, 60%, 70%, 80% or 90% of the total duration of the fermentation stage. The fermentation is usually carried out under conditions of limited aeration during 12, 14, 16, 18, 20, 21, 22, 23, 24, 25, 26 or 28 hours.

The term "limited aeration" describes the conditions of aeration, which allow .diphtheriae to use aerobic respiration and yet limit the amount of available oxygen, so after increasing the density of the culture (for example, after at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 hours of fermentation) oxygen is consumed very quickly after entering the culture, so Rho2is less than 5, 4, 3, 2, 1 or 0.5%. It should be noted that by increasing the number of culture used for inoculation of the fermenter, the terms of the limited Aer the tion could be achieved very quickly after inoculation (for example, after 1, 5, 10, 20 or 30 minutes after the start of fermentation).

Such conditions of limited aeration lead to stable expression of a toxin such as diphtheria toxin or its mutants.

Fall Rho2before approaching to zero is achieved by the speed of aeration and mixing, which are such that oxygen is introduced into the culture used by a culture to breath soon after its introduction in this culture, so despite the aeration of the culture Rho2read the oxygen sensor as zero or close to zero.

During the fermentation stage is ro2starts with a higher level for specific facilities agitation speed and aeration. This is due to the fact that at the beginning of the fermentation stage, the density of bacteria in the culture is low and increases during the fermentation stage. Usually requires a period of time (for example, up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 hours), before pO2will fall to less than 5%. Since then pO2remains at less than 5, 4, 3, 2, 1 or 0.5%, preferably at a level close to zero, until close to the end of the fermentation stage, for example, to collect the culture of the fermenter.

Perhaps fermentation stage is carried out at constant volume ratio of macroprolactinoma (KLa) throughout the fermentation stage. Alternatively, the fermentation stage is carried out at one or more than one KLa, so limited aeration is achieved when the values of KLa, persisting for the most part (for example, 50%, 60%, 70%, 80%, 90%, 95%) fermentation stage.

KLa is a measure of the rate at which oxygen enters the culture. The higher KLa, the greater the rate at which oxygen is introduced into the culture. Several factors, including the amount and composition of the medium, mixing, aeration, pressure, temperature, position, and characteristics of the moving parts of the fermenter, will affect the KLa specific fermentation stage.

Usually oxygen is introduced into a fermentation culture by ozonation of compressed air through the culture. When air is introduced into the culture, there are different concentrations of oxygen, the flow rate should be adapted accordingly. For example, when the culture is injected source of 100%oxygen, then the flow rate must be correspondingly lower. When culture is injected gas containing less oxygen than air, then you can use a higher flow velocity.

KLa can be measured using the method described in Example 1. This method consists of installing the fermenter conditions volume of medium, temperature, pressure, mixing and aeration, which should be measured KLa, Tkacheva the e gas by the replacement of air with gaseous nitrogen, gas pumping by restoring the aeration air and measuring the speed at which Rho2returns to its stationary level.

ln(100-pO2)=-KLa.T+C

When plotting ln(100-pO2from the time the gradient (or slope) of the line represents the KLa.

The value of KLa fermentation stage is influenced by a number of factors, including the degree of mixing culture and the rate of aeration of the culture. Constant KLa can be maintained, for example, by reducing the mixing of culture and the increase in the rate of aeration or Vice versa. However, in one embodiment as the mixing of cultures, and the rate of aeration during the fermentation stage are constant.

Fermentation stage is carried out, for example, when KLa 10-200 h-1, 10-150 h-1, 10-100 h-1, 10-80 h-1, 10-50 h-1, 10-40 h-1, 10-30 h-1, 20-150 h-1, 20-100 h-1, 20-50 h-1, 20-60 h-1, 20-80 h-1, 20-30 h-1, 20-40 h-1, 30-60 h-1, 60-80 h-1, 60-150 h-1or 60-200 h-1.

The value of KLa method of fermentation according to the invention may vary depending on the size of the fermentation culture. For crops 10-30 liters can be used KLa 10-30 h-1, 15-30, 20-30 or 22-28 h-1. For crops 30-250 liters can be used KLa or 30-60 40-50 h-1. For crops 250-800 liters can be used KLa 30-50, 40-50, 40-60, 0-60, 30-80 or 60-150 h-1. For crops 800-3000 liters can be used KLa 30-50, 40-50, 40-60, 30-60, 30-80, 60-150 or 60-200 h-1.

For the size of the fermentation culture 10-30 liters KLa 10-30 h-1for example, is achieved by using air flow or speed aeration 1-5 liter/min and the stirring speed of 200-400 rpm, for example, the speed of aeration 2-4 liter/min and the stirring speed is about 250-350/min

For fermentation culture 30-250 liters KLa 30-60 h-1is achieved, for example, when using the air flow rate 15-25 l/min and the stirring speed of 150 to 250 rpm, for example, when using the air flow rate 20-25 liter/min and the stirring speed of 200-250 rpm, for example, when using the air flow rate of 15-20 litres/min and the stirring speed of 200-250 rpm

the pH of the culture .diphtheriae environment CY during the fermentation stage depends on the conditions of aeration and mixing of the culture (Nikolajewski et al., J. Biological Standardization, 1982, 10; 109-114). At the beginning of the fermentation stage pH CY is 7.4. In the case of low aeration or KLa, the pH drops to values around 5. In the case of increased aeration pH increases until a value of about 8.5. In one embodiment of the present invention .diphtheriae cultivate in the environment CY or in the environment SOC (Sambrook J et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), or in a similar environment. the pH from 7.0 to 7.8 m which may be maintained in the fermenter intensity aeration, possibly without the need of adding acid or base.

The method according to the invention can be used with any strain of Corynebacterium diphtheriae. Such strains can produce diphtheria toxin wild type, fused proteins, including diphtheria toxin or fragment (e.g., proteins, described in US 5863891), or mutant forms, or fragments of diphtheria toxin, preferably those that have low toxicity. Examples of such mutant toxins are CRM176, CRM197, CRM228, CRM45 (Uchida et al., J. Biol. Chem. 218; 3838-3844, 1973); CRM9, CRM45, CRM102, CRM103 and CRM107, and other mutations described Nicholls and Co. in Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc. 1992; deletion or mutation of Glu-148 Asp, Gln or Ser and/or Ala-158 on Gly and other mutations disclosed in US 4709017 or US 4950740; mutation of at least one or more than one residue Lys 516, Lys 526, Phe 530 and/or Lys 534, and other mutations disclosed in US 5917017 or US 6455673; or a fragment disclosed in US 5843711. In one embodiment the strain .diphtheriae produces CRM197.

In one embodiment of the methods according to the invention using the following strains .diphtheriae: ATSC, ATSC, ATSC, ATSC, ATSC, ATSC, ATSC, ATSC, ATSC, ATSC, ATSC, ATSC, ATSC, ATSC, ATSC, ATSC or ATSC.

Environment for use in this invention may contain one or more than one of the following components: 5-20 g/l, 10-16 g/l or 10 g/l Kazimirovich acids is whether the hydrolysate of casein, 5-20 g/l, 7-15 g/l or 9-12 g/l soy peptone and/or 10-40 g/l, 14-32 g/l or 18-22 g/l yeast extract.

It is known that the iron content in the growth environment can affect the growth .diphtheriae and influence the production of the toxin (see WO 00/50449). Iron is essential for bacterial growth, however, it was shown that iron in high concentrations inhibits the production of toxin. For the process according to the invention the iron content in the environment has a lower bound of 10, 50, 75, 100, 120 or 150 billion-1and the upper bound 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000 or 5000 billion-1. For example, the concentration of iron in the environment are: 50-1000 billion-1, 100-1000 billion-1, 200-1000 billion-1, 400-1000 billion-1, 500-1500 billion-1, 700-1300 billion-1, 50-2000 billion-1, 100-2000 billion-1, 200-2000 billion-1, 400-2000 billion-1, 700-2000 billion-1, 50-3000 billion-1, 100-3000 billion-1, 200-3000 billion-1, 400-3000 billion-1, 700-3000 billion-1, 1000-3000 billion-1, 1500-3000 billion-1, 1700-3000 billion-1, 50-4000 billion-1, 100-4000 billion-1, 200-4000 billion-1, 400-4000 billion-1, 700-4000 billion-1, 1000-4000 billion-1, 1500-4000 billion-1, 1700-4000 billion-1or 2000-4000 billion-1. The iron may be in the form of Fe2+and/or Fe3+.

In one embodiment of the method according to the invention is sufficiently resistant to the presence in the environment of iron salts, so h is about before application does not require processing environment for removal of iron.

Fermentation phase is at a temperature suitable for culture .diphtheriae, for example at 25-45°C, 25-40°C, 30-38°C or 34-35°C.

Fermentation stage are susceptible to formation of large amounts of foam. To control foaming in the fermenter may add defoamer. Perhaps in the fermenter using a sensor foam or mechanical protivovospalitel, for example, along with antifoam.

The second aspect of the present invention is a method of manufacturing the drug, antigen, such as diphtheria toxin or a mutant or fragment, including the implementation phase of the fermentation method according to the invention, as described above, and selection of antigen, such as diphtheria toxin or a mutant or fragment from the culture.

The third aspect of the present invention is a diphtheria toxin or a mutant or fragment (e.g., CRM197)allocated by the method according to the invention.

The toxicity of diphtheria toxin possibly reduced through chemical processing, including the processing of cross-linking reagents for the formation of toxoid. Links to the toxin include toxoids.

An additional aspect of the present invention is a pharmaceutical composition comprising diphtheria toxin or mutant (e.g., CRM197) or the slice image is ateneu and a pharmaceutically acceptable carrier.

The pharmaceutical composition according to the invention may further comprises additional antigens in combination vaccines. In one embodiment the antigen(s), subject to Association with diphtheria toxin, mutant or fragment, as described above, include one or more than one tetanus toxoid, whole cell pertussis antigen (Pw), acellular pertussis antigen (RA) (as described below), the surface antigen of hepatitis b virus, hepatitis a virus, polysaccharides or oligosaccharides Haemophilus influenzae b, neisseriaceae (for example, N. meningitidis) polysaccharides or oligosaccharides, proteins of N. meningitidis serotype In may as part of outer membrane vesicles, polysaccharides or oligosaccharides pneumococcal pneumococcal proteins or any of the antigens listed below. Bacterial polysaccharides may be conjugated to a protein carrier. Diphtheria toxin or toxoid or mutant of diphtheria toxin, such as CRM197, or its fragments, for example, obtained using the method according to the invention, can be used as protein carriers. However, you can also use other carrier proteins such as tetanus toxoid, the fragment of tetanus toxoid, pneumolysin, Protein D (US 6342224). This pharmaceutical composition may contain a variety of polysaccharides or oligosaccharides to yugirovannykh with different protein-carriers.

Diphtheria toxin or a mutant, such as CRM197, or its fragment, obtained using the method according to the invention, can be prepared in the form of the drug with capsular polysaccharides or oligosaccharides derived from one or more than one of Neisseria meningitidis, Haemophilus influenzae b, Streptococcus pneumoniae, Streptococcus group a, Streptococcus group b, Staphylococcus aureus or Staphylococcus epidermidis. For example, the pharmaceutical or immunogenic composition can contain capsular polysaccharides originating from one or more than one serogroup a, C, W-135 and Y of Neisseria meningitidis. For example, serogroup a and C; a and W, and Y; and W, and Y, W and Y; a, C, and W; a, C and Y; A, W and Y; C, W and Y, or a, C, W and Y can be prepared in the form of the drug with CRM197. In another example, this immunogenic composition comprises capsular polysaccharides derived from Streptococcus pneumoniae. Pneumococcal capsular polysaccharide antigens are preferably selected from serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F (most preferably from serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F). Another example includes polyribosylribitol (PRP) capsular polysaccharides or oligosaccharides) Haemophilus influenzae type b. Additional example contains capsular polysaccharides Type 5, Type 8, 336, poly-N-acetylglucosamine (PNAG or deacetylation form dPNAG) Staphylococcus aureus. Additional primerselect capsular polysaccharides of Type I, Type II, Type III or PIA Staphylococcus epidermidis. Additional example contains capsular polysaccharides of Type Ia, Type Ic, Type II or Type III Streptococcus group C. Additional sample contains capsular polysaccharides of Streptococcus group a may optionally contain at least one protein M and, more preferably, many types of M protein

Bacterial polysaccharides for use in this invention can be full-size, representing a purified native polysaccharides. Alternatively, these polysaccharides can have a size of 2-20 times 2-5 times 5-10 times 10 to 15 times or 15-20 times smaller for easier handling. Oligosaccharides typically contain from 2 to 20 repeating units.

Such capsular polysaccharides can be unconjugated or conjugated to a protein carrier such as tetanus toxoid, the fragment of tetanus toxoid, diphtheria toxoid or CRM197 (both, for example, obtained by the method according to the invention), pneumolysin or protein D (US 6342224). Tetanus toxin, diphtheria toxin and pneumolysin detoxify or genetic mutations and/or chemical treatment.

Polysaccharide or oligosaccharide conjugate can be obtained by any known technique combinations. For example, the polysaccharide can be obtained by the combination through a thioester bond. This way it is Nyugati based on activation of the polysaccharide tetrafluoroborate 1-cyano-4-dimethylaminopyridine (CDAP) with the formation of sanatoga ether. The activated polysaccharide, thus, it is possible to get a combination, either directly or through spacer elements group with the amino group on the protein carrier. Perhaps tianity ether are combined with hexanediamine, and amino derivatives of polysaccharide kongugiruut with protein carrier using the chemistry getreligion, including the formation of thioester linkages. Such conjugates are described in published PCT application WO 93/15760 Uniformed Services University.

These conjugates can also be obtained by direct reductive amination, as described in US 4365170 (Jennings) and US 4673574 (Anderson). Other methods are described in EP-0161188, EP-208375 and ER-0477508.

An additional method involves the combination of activated bromine cyan polysaccharides, derivatizing the adipic acid hydrazide (ADH), with protein carrier by carbodiimide condensation (C. Chu et al., Infect. Immunity, (1983) 245; 256).

In the specific examples diphtheria toxin or a fragment or mutant (e.g., CRM197) kongugiruut with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 additional antigens pharmaceutical composition. In one embodiment it kongugiruut with the polysaccharide(s) component(s), for example, bacterial polysaccharides, including polysaccharides listed above.

Pharmaceutical or immunogenic composition according to the invention also may contain additional the additional protein components. It may make in the form of the drug antigens, providing protection against one or more than one infection caused by tetanus bacteria Bordetella pertussis. The pertussis component may constitute killed C. whole cell pertussis (Pw), or acellular pertussis component (RA), which contains at least one antigen (preferably two or all three) of RT, FHA and 69 kDa of pertactin. Some other acellular pertussis preparations also contain agglutinogen, such as Fim2 and Fim3, and these vaccines are also being considered for use in this invention. Typically, the antigen conferring protection against tetanus is tetanus toxoid, which is either chemically inactivated toxins (for example, after treatment with formaldehyde), or inactivated by the introduction of one or more than one point mutation(s).

Pharmaceutical or immunogenic composition according to the invention may contain pneumococcal protein antigens, for example those pneumococcal proteins that are exposed on the outer surface of the pneumococcus (which can be recognized by the immune system of the host during at least part of the life cycle of the pneumococcus), or represent proteins that are secreted or released by the pneumococcus. For example, the protein can the t to be a toxin, adhesin, two-carrier signal or lipoprotein of Streptococcus pneumoniae, or fragments thereof. Examples of such proteins include: pneumolysin (preferably detoxified enduring chemical treatment or mutation) [Mitchell et al. Nucleic Acids Res. 1990 Jul; 18(13): 4010 "Comparison of pneumolysin genes and proteins from Streptococcus pneumoniae types 1 and 2.", Mitchell et al. Biochim Biophys Acta 1989 Jan 23; 1007(1): 67-72 "Expression of the pneumolysin gene in Escherichia coli: rapid purification and biological properties", WO 96/05859 (A. Cyanamid), WO 90/06951 (Paton et al.), WO 99/03884 (NAVA)]; PspA and its variants with a deletion of the transmembrane (US 5804193 - Briles et al.); PspC and its variants with a deletion of the transmembrane (WO 97/09994 - Briles et al.); PsaA and its variants with a deletion of the transmembrane (Wegge & Paton, Infect Immun 1996 Dec; 64(12):5255-62 "Sequence heterogeneity of PsaA, a 37-kilodalton putative adhesion essential for virulence of Streptococcus pneumoniae"); pneumococcal proteins that bind choline, and their variants with a deletion of the transmembrane; b and its variants with a deletion of the transmembrane (WO 97/41151; WO 99/51266); glyceraldehyde-3-phosphate dehydrogenase (Infect. Immun. 1996 64:3544); HSP70 (WO 96/40928); PcpA (Sanchez-Beato et al. FEMS Environ Lett 1998, 164:207-14); M-like protein (EP 0837130) and adhesin 18627 (EP 0834568), but are not limited to them. Additional pneumococcal protein antigens for inclusion in immunogenic composition represent the antigens disclosed in WO 98/18931, WO 98/18930 and PCT/US 99/30390.

Examples neisserial proteins destined for inclusion in immunogenic composition according to the invention include TbpA (WO 93/06861; EP 586266; WO 92/0367; US 5912336), b (WO 93/06861; EP 586266), Hsf (WO 99/31132), NspA (WO 96/29412), Nar (PCT/EP 99/02766), PorA, PorB, OMP85 (also known as D15) (WO 00/23595), PilQ (PCT/EP 99/03603), PldA (PCT/EP 99/06718), FrpB (WO 96/31618, see SEQ ID NO:38), FrpA or FrpC or conservative, common to both, of at least 30, 50, 100, 500, 750 amino acids (WO 92/01460), LbpA and/or LbpB (PCT/EP 98/05117; Schryvers et al. Med. Environ. 1999 32: 1117), FhaB (WO 98/02547 SEQ ID NO:38), HasR (PCT/EP 99/05989), lipo02 (PCT/EP 99/08315), MltA (WO 99/57280) and ctrA (PCT/EP 00/00135). Neisserial proteins may add as purified proteins or as part of the preparation of the outer membrane.

Pharmaceutical or immunogenic composition according to the invention may contain one or more than one antigen, which can protect the host against Netherweave Haemophilus influenzae, RSV and/or one or more than one antigen, which can protect the host against influenza virus.

Examples of protein antigens Netherweave .influenzae include protein fimbrin (US 5766608) and fused proteins containing peptides derived from it (for example, merger LB1) (US 5843464 Ohio State Reserch Foundation), OMP26, P6, protein D, TbpA, TbpB, Hia, Hmw1, Hmw2, Nar and D15.

Examples of antigens of influenza virus include the whole live or inactivated virus, split influenza virus, grown in eggs or MDCK cells, or Vero cells, or entire virosome influenza (as described by R. Gluck, Vaccine, 1992, 10, 915-920)or purified or recombinant proteins, such as proteins, NP, NA, or M, or combined the promotion.

Examples of antigens of RSV (respiratory syncytial virus) include the F glycoprotein, glycoprotein G, HN protein, protein M or its derivatives.

You should take into account that the antigenic composition according to this invention may contain one or more than one capsular polysaccharide from one bacterial species. Antigenic compositions can also contain capsular polysaccharides originating from one or more than one species of bacteria.

An additional aspect of the present invention includes immunogenic compositions or vaccines containing diphtheria toxin, fragment or mutant (e.g., CRM197)obtained by the methods according to the invention, and a pharmaceutically acceptable carrier.

Immunogenic composition or vaccine may contain a number of adjuvant sufficient to enhance the immune response to the immunogen. Suitable adjuvants include aluminum salts, a mixture of squalene (SAF-1), muramylpeptide, saponin derivatives, preparations of cell walls of mycobacteria, monophosphorylated And derived microway acids, surface-active substances on the basis of non-ionic block copolymers, Quil A, subunit In the cholera toxin, polyphosphazene and derivatives, and immunostimulating complexes (ISCOMs)such as the complexes described by Takahashi et al. (1990) Nature 344:873-875, but are not limited to them. For veterinary use and for the floor is to be placed antibodies in animals can be used mitogenic components of the adjuvant's adjuvant.

As with all immunogenic compositions or vaccines, immunological effective amount of immunogenic determined empirically. Factors that should be considered include immunogenicity, whether or not the immunogen to form complexes or covalently contact adjuvant or protein carrier or other carrier, route of administration and the number of immunizing doses to be administered. Such factors are known in the field of vaccines, and such definitions without undue experimentation certainly is within the skills of immunologists.

The active agent in the pharmaceutical composition or vaccine according to the invention can be present in different concentrations. Usually the minimum concentration of a substance is the amount necessary to achieve its target application, while the maximum concentration is the maximum amount that will remain in solution or suspended in homogeneous initial mixture. For example, the minimum amount of therapeutic agent preferably is a number that will give one a therapeutically effective dosage. For biologically active substances minimum concentration represents the amount needed for biological activity is rastvorenii, and the maximum concentration is located at the point, which may not be supported homogeneous suspension. In the case of roller units this number represents the number for a single therapeutic applications. Usually expected that each dose will contain 1-100 µg protein antigen, preferably 5-50 μg, and most preferably 5-25 μg. The preferred dose of bacterial polysaccharides represent 10-20 mcg, 10-5 µg, 5-2,5 μg or 2.5 to 1 µg. The preferred amount of the substance varies from substance to substance, but it can easily be determined by a qualified specialist in this field.

The vaccine preparations of the present invention can be used to protect or treat a mammal (for example, patients who are human, prone to infection, through the introduction of specified vaccines via the system path or through mucous membranes. This introduction may include injection via the intramuscular, intraperitoneal, intradermal or subcutaneous route; or introduction through the mucous membrane in the mouth/digestive tract, respiratory tract, urinary ways. Despite the fact that the vaccine according to the invention can be introduced in the form of a single dose, its components can also be introduced together at the same time or at different times (for example, if the vaccine is rootstown polysaccharides, they can be introduced separately at the same time or within 1-2 weeks after the introduction of a combination of bacterial protein for optimal coordination of immune responses in relation to each other). Apart from one route of administration can be used two different ways of introduction. For example, viral antigens can enter the ID (intradermal), whereas bacterial proteins can enter IM (intramuscularly) or IN (intranasal). If polysaccharides are present, they can be administered IM (or ID), and bacterial proteins, you can type IN (or ID). In addition, the vaccine according to the invention it is possible to enter IM doses for the primary immunization and IN repeated doses of immunization.

Obtaining vaccines in General are described in Vaccine Design (The subunit and adjuvant approach" (eds M.F. Powell & Newman M.J.) (1995) Plenum Press New York). Encapsulation in liposomes described Fullerton, U.S. patent 4235877.

Another aspect of the present invention is a method of manufacturing a pharmaceutical composition, comprising a step for diphtheria toxin or fragment or mutant (e.g., CRM197) using the method of fermentation according to the invention and combining it with a pharmaceutically acceptable carrier and you can add any additional antigens mentioned above.

Such a method may optionally include the phase conjugation of diphtheria toxin or fragment or mutant (for example the EP, CRM197) with one or more than one additional component of the pharmaceutical composition, preferably a bacterial polysaccharides or oligosaccharides, as described above.

An additional aspect of the present invention is the application of diphtheria toxin or fragment or mutant (e.g., CRM197) according to the invention in the manufacture of a medicinal product for the treatment or prevention of bacterial diseases, particularly diseases caused .diphtheriae.

Another aspect of the present invention is a method of prevention or treatment of bacterial infections, particularly infections caused by .diphtheriae, including the introduction of the patient pharmaceutical compositions, immunogenic compositions or vaccines according to the invention.

All references or patent applications cited in this application are included in it by reference.

This invention illustrated by the accompanying examples. The examples below are carried out using standard techniques that are well known and are common for professionals in the art, except in those cases where it is described in detail otherwise. These examples are illustrative, but not limiting the invention.

EXAMPLES

Example 1: Measurement of KLa

For measuring KLa enzyme is operating stage fermenter was filled with the desired volume of water, used the settings fermentation (e.g., temperature, pressure, agitation and aeration) and left the system to achieve steady state. The sensor pO2calibrated at 100%.

Aeration quickly translated into gaseous nitrogen, maintaining the same flow rate. The value of pO2tracked until then, until it fell to less than 5%. When he reached this point, aeration quickly translated into air, maintaining the same flow rate. Level pO2tracked and recorded in a few moments time in the form of a percentage of the original stationary 100%level.

KLa was calculated by plotting log(100-ro2) against time. The angular coefficient of the linear part of the graph corresponds to - KLa. Usually consider only the data from 20%to 80%Rho2.

Results

Indicators Rho2at different points in time are shown in table 1.

Table 1
Time (seconds)Time (hours)pO2(%)ln(100-pO2)
2000,056204,382026635
2100,058of 21.94,357990057
2200,061234,343805422
2300,064254,317488114
2400,067274,290459441
2500,069294,262679877
2600,072324,219507705

These results were plotted on a graph as shown in figure 1, and the KLa was determined from the angular coefficient of the line shown in figure 1,B.

Example 2: Fermentation of the strain ADS 39255 .diphtheriae scale 150 liters

The bacterium used in obtaining CRM197 (giving a cross-reaction material), is a mutant strain of Corynebacterium diphtheriae, obtained by processing nitrosoguanidine according to method A. Pappenheimer (Nature New Biol. 233:8-11, 1971). He expresses detoxified enduring diphtheria toxin (AA 52: mutation, which is a substitution of glycine for glutamic acid). He the floor of the EN of the American collection tissue cultures (ATS) and has room 39255. A General description of how the fermentation is shown in figure 2.

Work seed material containing 1.1×1010CFU/ml (colony forming units/ml), were removed from the freezer (-70°C) and thawed at room temperature. Immediately after thawing vessel was shaken in vibromaster, and 250 μl of inoculum was collected 1-ml syringe with a needle.

This volume were injected with 100 ml of sterile physiological solution (0.9%). The flask was shaken. Two ml of the suspension was collected 2-ml syringe with needle and used for inoculation of 3-liter Erlenmeyer flask containing 500 ml of medium as described in US 4925792. This flask was incubated at 34.5°C±0,5°C at the stirring speed of 250 rpm until the optical density (650 nm) did not reach 4,0-6,0 (after 16-19 h incubation).

150-liter fermenter was sterilized and 100 l of culture medium is aseptically transferred into the fermenter. The bottle of acid was filled with 500 ml of N3RHO425% (vol./vol.); the pH of this environment source amounted to about 7.4, and it is not regulated.

The fermenter was prepared the day before inoculation and supported him in the ready state at a temperature of 34.5°C, a pressure of 0.5 bar (50 kPa), the air flow 23 N L/min in the free space above the medium and the stirring speed of 50 rpm for 16-20 h before inoculation.

Before inoculation in the fermenter was set out in the conditions of cultivation: the temperature of 34.5°C, pressure 0.5 bar (50 kPa), the air flow 23 N L/min, injected into the environment, and the mixing speed 240 rpm Stirring with a speed of 240 rpm gives the ring rate of 1.76 m/s and theoretical mixing time of 3.9 seconds. The level of dissolved oxygen is not regulated, and only monitored system, foam control was included; the pH was allowed to reach 7.8 and then maintained at this level by the addition of N3RHO4. Before inoculation sensor Rho2set on 100%.

The mixing speed was set at 240 rpm, which corresponds to the peripheral speed of 1.76 m/C. the mixing Speed 240 rpm combined with the speed of aeration 23 l/min, which resulted in KLa (20-80%, water 30°C, pressure of 0.5 bar (50 kPa)), defined at 42 h-1(figure 5).

The fermenter was inoculable port for inoculation, using 400 ml of seed culture described above.

The fermentation is continued until, until you have met both of the following conditions: it has been 20 hours of fermentation, and the level of dissolved oxygen increased to 10%. The total duration of fermentation usually ranged from 22 to 28 hours

At the end of fermentation set temperature was changed to 20°C, off regulation of pH, air flow is translated into the free space above the environment in order to limit the foaming, the ICI is it foam control off, other parameters did not change.

The microfiltration system was connected to the fermenter, and microfiltration was started when the temperature of the suspension was reached 21°C. the Microfiltration was carried out in two phases: concentration and diafiltration. During phase concentration parameters were as follows: the pressure inside of 0.6 bar (60 kPa), the pressure outside is ~0.1 bar (10 kPa), the speed of the filtrate was maintained constant at 2 l/min using a calibrated peristaltic pump (the pressure of the filtrate was approximately 0.3 bar (30 kPa). This suspension was concentrated, until it occurred one of the following events: either the pressure at the inlet port reached 0.9 bar (90 kPa), or received 75 liters of filtrate.

During phase diafiltration used the following parameters: the internal pressure was maintained at a level that was achieved in the late stage of concentration (maximum of 0.9 bar (90 kPa)); water was added at the rate of 2 liter/min; the total quantity of water added was 3 volume ultraconnected.

Ultraconnected was filtered on a 0.22 μm membrane and stored at +4°C. the Stability of CRM197 in such suspensions tested after up to 4 days in storage or at 4°C or at room temperature (+20°C<CT<23°C). No decomposition and no differences were not observed on the basis of quantitatively the estimates by the method of ELISA or SDS-PAGE. Test to confirm the absence of growth was carried out on agar VAV, incubarea at 36°C.

Results

Two fermentation scale 150 l was carried out using the Protocol fermentation described above (CDT 199 and CDT 206). Conditions for preculture and culture are shown in tables 2 and 3, and outputs CRM197 shown in table 4. Figure 3 shows a graph of a typical kinetics of the growth of preculture. When OD (650 nm) ranged from 4.0 to 6.0, culture obviously were in exponential growth phase, and the pH is only slightly changed.

Table 2
Conditions for preculture
no cultureThe duration of the cycle preculture (h:min)OP650precultureThe final pH value
CDT19916:445,887,30
CDT20617:534,037,25

Table 3
Conditions for crops
no cultureThe duration of fermentation (h:min)Used 25%N3RHO4(g)The final OD value650The total required amount of antifoam (g)Evaluation of SOME (BAC./ml)
CDT19924:45217,21024,9 e
CDT20624:31213,9119no data

Table 4
Assessment CRM197
no cultureDensitometry of the SDS-PAGE (mg/l)ELISA (mg/l)
CDT19989/106138
CDT20692/83116

During fermentation observed different phases.

The first phase was characterized by the decrease of the level of the solution is aqueous oxygen before reaching 0% (duration approx 6-7 hrs). During this phase, the pH remains stable or slightly reduced (approximately 0.1 unit pH).

During the second phase, the pH increases and reaches a plateau at about a pH of 7.8. At this level began regulation of pH, however, often under these conditions the fermentation acid no need to add. During this plateau pH, an increase in the level of dissolved oxygen is more than 0% with a subsequent drop to 0%.

The third phase was characterized by the decrease of pH to approximately 7.4.

Finally, from 22 to 24 h of fermentation observed an increase in Rho2. This is the signal for the collection.

Gases to be pumped out of the fermenter, were analyzed on a mass spectrometer. Observed typical profile products CO2.

Two presents the fermentation was continued after receiving the signal collection to assess the kinetics products CRM197. The authors of this invention have observed that the level of CRM197 is not incremented after receiving the signal on the collection, but had an increase in pricing. In order to limit the consumption of antifoam, it is preferable to stop the fermentation at the time of signal collection. However, CRM197, apparently, is not affected by the excess foam, and will not be decomposition.

Microfiltration

Data shown for microfiltration CDT206.

74,3 l of filtrate was collected when the pressure n the inlet reached 0.9 bar (90 kPa). The pressure on the output hole on the entire stage of concentration ranged from 0.15 to 0.10 bar (15 to 10 kPa). The pressure of the filtrate at all stages of concentration was 0.3-0.4 bar (30 to 40 kPa)and the concentration step lasted 36 minutes.

For phase diafiltration was gradually added 75 l of water (2 l/min), whereas the filtrate was extracted with the same flow rate. Pressure at inlet was 0.9 bar (90 kPa) at the beginning and fell to 0.7 bar (70 kPa) at the end of diafiltration. The pressure on the exit hole was stable at 0.1 bar (10 kPa). The duration of the stage of diafiltrate was 39 minutes

Quantitative assessment of CRM197

The level of expression of CRM197 in conditions of low aeration was usually 2-4 times higher than the expression level that was achieved in terms of strong aeration, when Rho2was maintained at 5% or higher throughout the fermentation.

Stained SDS-PAGE of supernatant culture

The gels SDS-PAGE (figure 4) was divided supernatant culture under reducing conditions, so it is possible to define any band decomposition (after cutting can be defined two subunits, respectively, 35 and 23 kDa). They then painted Kumasi blue.

Results

Gels of samples from two fermentati, colored Kumasi, shown in figure 4,And (CDT 199) and 4 In (CDT 206). CRM197 was manifested in idamay molecular weight (theoretical molecular weight: 58,4 kDa). CRM197 was not decomposed, as in samples taken after signal the end of fermentation (figure 4,lanes 9 and 10), was not observed changes in the electrophoretic pattern on the gel. The number of CRM197 was not affected by the filtration and final filtration on 0.22 μm, since the strips 9 and 11, figure 4,And shows an equivalent amount CRM197.

Temperature can have a negative impact on the stability of the CRM (figure 4,B, lane 12). This sample was selected when the sampling valve was warm, and the number of CRM197, present in the sample decreases.

Example 3: Fermentation of the strain ADS 39255 .diphtheriae in 20-liter scale

For fermentation .diphtheriae used a method similar to the method described in example 2, except that used a 20-liter fermenter. The culture was stirred at a speed of 300 rpm, and the air flow was set at 3 liters/minute. During fermentation did not require the addition of an acid or base, as throughout the fermentation pH remained near neutral values.

This culture grew to a final OD (650 nm) 18,3. Found that out CRM197 is 103 mg/liter in the evaluation by densitometry on the stained gel.

Example 4: Fermentation of the strain ADS 39255 .diphtheriae at different scales

Fermentation is a method of growing .diphtheriae under conditions of constant KLa can be adapted deprimente in fermenters other sizes and different designs. Good output production CRM197 was reached in accordance with the terms of the size of the fermenter, air flow and mixing speeds listed in table 5. Three fermentation 150-liter scale were carried out in fermenters of various designs.

Table 5
Scale (l)Air flow (l/min)Stirring speed (rpm)KLa (h-1)
20330022-28
15023240~42
15023185~50
15017200~40

As shown in table 5, it was important the value of KLa, and not the specific choice of terms of air flow and the speed of mixing. Thus, a weaker air flow could be offset by the higher mixing rate, which led to similar KLa, and yet achieved harasiewicz CRM197. The stirring speed should be sufficient to obtain a homogeneous suspension, and aeration limit to maintain low Rho2. N.B. Different fermenters used for 20-liter fermentati. Different geometric shapes of different fermenters led to the interval of values of KLa, shown in table 5.

However, conditions with different KLa were optimal for scale fermentation. Therefore, for fermentation in 20-liter fermenter lower KLa 22-28 h-1was the best.

Conditions with optimal KLa are conditions that provide limited aeration, so Rho2in culture drops to low levels. The person skilled in the art should be able to easily identify such conditions for the particular size and geometric shape of the tank.

Example 5: Effect of iron concentration on the yield of fermentati at different Rho2

A number of fermentati strain ADS 39255 .diphtheriae was performed in a 20-liter scale, following the method described in Example 3, so that during the greater part of the fermentation pO2was low or was at a prescribed constant level of 5% pO2. The number of present Fe3+ranged from the absence of added Fe3+to add 250 billion-1Fe3+, add 500 billion-1Fe3+and adding the s 500 billion -1Fe2+. Output CRM197 at the end of fermentation was measured by densitometry of the SDS-PAGE gel. This method gives results approximately 20% lower than the results achieved ELISA.

Results

Table 6
Fermentation conditionOutput CRM197 (mg/liter)
medium with 5%pO2without the addition of Fe3+38
medium with 5%Rho2with the addition of 250 billion-1Fe3+14
medium with 5%Rho2with the addition of 500 billion-1Fe3+18
medium with 5%Rho2with the addition of 500 billion-1Fe2+13
Low Rho2constant KLa, the medium without addition of Fe3+100
Low Rho2constant KLa, the environment adding 250 billion-1Fe3+88
Low Rho2constant KLa, the environment adding 500 billion-1Fe3+ 97

As shown in table 6, when .diphtheriae fermented with 5%Rho2the addition of iron leads to lower output. However, when the level of Rho2reduced, and the fermentation is carried out under conditions of low Rho2at constant KLa, as described in example 3, achieved higher outputs, and output is not affected by the addition of Fe3+.

Example 6: Effect of iron concentration on the yield of DT or CRM197 under conditions of low aeration

The range of concentration of iron, which does not affect the expression of CRM197, were measured in the microplate. Microplates imitate the conditions of limited aeration, by way of fermentation according to the invention.

Cultivation in microplates was performed in the same medium, which was used when fermentation described above (in an environment similar to the environment CY). Fe3+added from the original solution of FeCl3·6N2About ion concentration of Fe3+1 g/L.

Wells of microtiter tablets filled environment and inoculable at a concentration of 8 E5 bacteria/ml.

The microtiter plates were incubated at 34.5°C with agitation of 250 rpm/min for 46 h in the case, as Corynebacterium diphtheriae expressing CRM197, and Corynebacterium diphtheriae expressing a diphtheria toxin.

These samples were filtered through 0.22 μm filter.

Expression was measured by way of dense is hometree on SDS-PAGE (Criterion XT 4-12% bis tris from BioRad) with staining of Kumasi blue (Gelcode blue dye from Pierce). The benchmark used for the quantitative evaluation was a mutant diphtheria toxin CRM from List Biological Laboratories INC. introduced in the gel at different concentrations.

Results

Table 7 shows the expression of CRM197 with different concentrations of iron for .diphtheriae grown under conditions of limited aeration in microtiter wells. Expression of CRM197 was slightly sensitive to repression Fe3+and it was not significantly influenced by the addition of 1 million-1or 2 million-1Fe3+. Only when 3 million-1indeed has been a significant decrease in the expression CRM197. Even with this level of Fe3+expression CRM197 still accounted for 79% of the expression level, which was achieved without the addition of Fe3+.

Table 7
Corynebacterium diphtheriae, expessialy CRM197
Added Fe3+(m-1)The optical density 46 hpHCRM (%)
018,17,66100
200 billion-117,97,7296
300 billion-116,9to 7.7797
400 billion-116,97,8106
500 billion-117,27,83109
600 billion-117,77,81112
700 billion-117,2to 7.77109
800 billion-117,2to 7.77103
900 billion-116,57,79101
1 million-116,9of 7.7596
2 million-1177,7988
3 million-116,9 7,8379

Outputs CRM197 expressed as a percentage of output, which is achieved without adding additional Fe3+.

Results expression of DT for .diphtheriae grown under specified conditions in microtiter wells, shown in table 8. Products DT under conditions of limited aeration was also slightly sensitive to repression Fe3+. Expression of DT was increased with increasing concentration of Fe3+with a maximum DT product, which was achieved at 700 billion-1. The expression DT started to fall only when the concentration of Fe3+increased to 3 million-1.

Table 8
Corynebacterium diphtheriae expressing a diphtheria toxin
Added Fe3+(m-1)The optical density 46 hpHDT (%)
04,168,66100
200 billion-14,728,42106
300 billion-1 4,6of 8.47107
400 billion-1a 4.98,51125
500 billion-14,228,59155
600 billion-14,488,51148
700 billion-14,048,63167
800 billion-14,28charged 8.52164
900 billion-14,588,62168
1 million-14,488,64166
2 million-1the 4.78,68176
3 million-14,28,68141

Outputs DT expressed the AK percentage of output, which is achieved without adding additional Fe3+.

1. The manufacturing method of preparation of diphtheria toxin or a mutant or fragment, including the method of fermentation, including fermentation stage of growing strain of Corynebacterium diphtheriae in the medium in the fermenter under conditions of agitation sufficient to maintain a homogeneous culture, and limited aeration, so that the partial pressure of oxygen (pO2in culture drops to less than 4% throughout the greater part of the fermentation stage, and the allocation of diphtheria toxin or a mutant or fragment from the culture.

2. The fermentation method according to claim 1, where Rho2drops to levels close to zero, during the greater part of the fermentation stage.

3. The method according to claim 1 or 2, where ro2less than 4% support from the time when the culture of Corynebacterium diphtheriae grown to a density sufficient to Rho2fell to less than 4% because of the rapid consumption of oxygen, until the end of fermentation stage.

4. The method according to claim 1, where the fermentation stage is carried out at constant values of volumetric mass transfer coefficient of oxygen (KLa).

5. The method according to claim 1, where the fermentation stage is carried out at a constant speed of agitation speed and aeration.

6. The method according to claim 1, where the fermentation stage of conducting the ri conditions varying KLa.

7. The method according to claim 1, where the fermentation stage is carried out at KLa 10-50 h-1.

8. The method according to claim 1, where the fermentation stage is carried out in a 10 to 30-liter fermenter and KLa 10-30 h-1.

9. The method according to claim 1, where the fermentation stage is carried out in a 100-250-liter fermenter at KLa 30-60 h-1.

10. The method according to claim 1, where the fermentation stage is carried out in 250-800-liter fermenter at KLa 60-150 h-1.

11. The method of claim 8, where the fermentation stage is carried out using compressed air stream 1-5 l/min and a stirring speed of 200-400 rpm

12. The method according to claim 9, where the fermentation stage is carried out using compressed air stream 15-25 l/min and a stirring speed of 150-250 rpm

13. The method according to claim 1, where the environment represents CY, SOC or similar environment, and pH in the fermenter support when the value of 7.0 to 7.8 intensity aeration without the need of adding acid or base.

14. The method according to claim 1, where the strain Corynebacterium diphtheriae causes diphtheria toxin or a mutant, in particular CRM 197.

15. The method according to claim 1, where the strain Corynebacterium diphtheriae is ATSS.

16. The method according to claim 1, where the culture is quite resistant to the presence of iron salts in the environment, so the application does not require processing environment for removal of iron.

17. The method according to claim 1, where cremasteric 10 through 4000 billion -1iron.

18. The method according to claim 1, comprising a stage of adding one or more than one additional antigen(s) to diphtheria toxin or fragment or mutant.

19. The method according to p, including additional phase conjugation diphtheria toxin or fragment or mutant with one or more than one additional antigen(s).

20. A method of manufacturing a pharmaceutical composition for treating or preventing diseases caused .diphtheriae, comprising a step for diphtheria toxin or mutant using fermentation method according to any one of claims 1 to 19 and mixing it with a pharmaceutically acceptable carrier.



 

Same patents:

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and can be used in immunodiagnosis of Marburg haemorrhagic fever. A strain of hybrid animal cells Mus musculus L. 3F9 is formed, which is deposited in the collection of cell cultures of The State Research Center of Virology and Biotechnology VECTOR. The hybridoma strain produces monoclonal antibodies which are specific to the VP35 protein of the Marburg virus (Popp strain) (hereinafter MCA). MCA 3F9 produced by hybrid animal cells Mus musculus L. 3F9 relate to a subclass of immunoglobulins IgGI, having a heavy 55 kDa and a light 25 kDa chain and having a unique feature of detecting the VP35 protein of the Marburg virus (Popp strain) in a "sandwich" immunoenzymometric system format owing to antigen "capture" properties and simultaneously be an indicator, labeled biotin. The antigen epitope for MCA 3F9 produced by the 3F9 hybridoma is localised between 252 and 278 aminoacid residues.

EFFECT: invention enables to obtain MCA with specificity to VP35 protein of the Marburg virus (Popp strain), suitable for immunodiagnosis of Marburg haemorrhagic fever.

2 cl, 3 dwg, 1 tbl, 5 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and can be used in immunodiagnosis of human cytomegalovirus. The strain of hybrid animal cells Mus musculus L.5F10 is obtained by merging mouse myeloma cells p3-X63/Ag8.653 (NS/1) with mouse spleen cells BALBc, immunised by an affinity purified recombinant protein pp65. The hybridoma strain is deposited in the collection of cell cultures of The State Research Center of Virology and Biotechnology VECTOR and is used as a producer of monoclonal antibodies for detecting the pp65 protein of human cytomegalovirus.

EFFECT: invention enables to widening of range of strains of hybrid cells Mus musculus L - producers of MCA for detecting the pp65 protein of human cytomegalovirus and production of domestic diagnostic test-systems for detecting cytomegaly.

2 dwg, 1 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: strain of Propionibacterium freudenreichii subsp.shermanii Ac-103 is deposited into the Russian national Collection of microorganisms used in veterinary medicine and animal husbandry numbered VGNKI - 08/02/1957 DEP. This strain is a producer of feed protein.

EFFECT: invention enables to eliminate pollution at production of protein feed, to increase relative protein yield, to reduce energy costs at preparation of protein feed, to simplify medical equipment, to dispose wastes of manufactures using natural raw materials.

1 tbl, 9 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention pertains to bioengineering. In particular, the invention relates to method of obtaining recombinant mutant horse cytochrome c. This method is realised by introduction of K27E/E69K/K72E/K86E/K87E/E90K or K8E/E62K/E69K/K72E/K86E/K87E or K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K mutations through site-directed mutagenesis into the horse cytochrome c gene which is contained in pBPCYCS/3 plasmid DNA. Further, the Escherichia coli JM-109 strain of the obtained recombinant plasmid DNA is transformed and the target protein is expressed and introduced through cation-exchange and adsorption chromatography.

EFFECT: invention enables use of recombinant mutant horse cytochrome c as a test system for measuring the rate of generation of superoxide in membrane preparations.

3 dwg, 5 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and can be used in agriculture when making feed. The Lactobacillus plantarum 578/26 strain is deposited in the collection of Russian State Centre for Quality and Standardisation of animal medicines and feed under number VGNKI 08.02.54.-DEP. This strain is a feed protein producer.

EFFECT: invention prevents environmental pollution when making protein feed, increases protein specific output, reduces power consumption during protein feed production, simplifies and speeds up the manufacturing process, simplifies the equipment, enables recycling production wastes used natural material.

2 tbl, 9 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention relates to a process and apparatus for isolating and purifying protein of interest in a stream of a tissue culture liquid obtained from a continuous perfusion fermentation process. The proposed apparatus, in which sterile conditions are maintained, includes a continuous perfusion fermentation system, a continuous particle removal system integrated with the perfusion fermentation system and adapted for continuous reception of tissue culture liquid therefrom and continuous production of clarified tissue culture liquid, and a continuous purification system integrated with the particle removal system and adapted for continuous reception clarified tissue culture liquid therefrom and constant production of the extracted product which contain the protein of interest, where the continuous purification system is an ultrafiltration system. The process involves obtaining heterogeneous tissue culture liquid mixture containing the protein of interest during a continuous perfusion fermentation process, continuous removal of large particle impurities from the liquid mixture to obtain clarified tissue culture liquid containing protein of interest, and purification of the protein from the clarified culture liquid through ultrafiltration. Specific flow rate of the tissue culture liquid mixture during continuous perfusion fermentation, continuous removal of impurities and continuous purification is kept constant.

EFFECT: design of an efficient method of isolating and purifying protein.

9 cl, 17 dwg, 2 tbl

FIELD: biotechnologies.

SUBSTANCE: in modified molecule IL-4RA, which inhibits mediated IL-4 and IL-13 activity, amino-acid remains 37, 38 or 104 represent cysteine. Polynucleotide, which codes specified antagonist, in composition of expression vector, is used to transform host cell and produce IL-4RA. Produced molecule IL-4RA is PEGylated and used to eliminate abnormalities that are related to high activity of IL-4 and IL-13.

EFFECT: invention makes it possible to produce antagonist with longer period of half-decay compared to non-modified IL-4RA.

17 cl, 1 dwg, 7 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: invention relates to humanised anti-TGF-beta-antibody which is linked to TGF-beta. The humanised antibody has a variable domain VH which contains residues of the hypervariable region (non-human), which are contained in the human domain VH which includes a modified framework region (FR) (amino acid and nucleotide sequences are given in the list of sequences). The humanised antibody can contain residues of the complementarity determining region (CDR) of the variable domain of the light strand VL. The invention also relates to a composition for treating TGF-beta mediated disorders, e.g. malignant tumours, nucleic acid, coding monoclonal antibody, and a method of obtaining the latter using host cells. The invention provides a method of treating and detecting TGF-beta in a sample from the body using the disclosed antibody, as well as to a product which contains the humanised antibody and directions for use for treating TGF-beta mediated disorders.

EFFECT: invention enables control of TGF-beta molecules, which can prevent possible changes in antibodies, enables preparation of high-affinity humanised antibodies which act as TGF-beta antagonists.

57 cl, 45 dwg, 4 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention aims at preparation of new strain of hybrid cells Mus. Musculus 6F3 - a producer of monoclonal antibody (MCA) to hemagglutinin protein of high-pathogen avian influenza virus A/duck/Novosibirsk/56/05. Strain 6F3 is prepared by fusing murine myeloma cells Sp2/0 with murine spleen cells BALB/c, immunised with a purified and inactivated preparation of avian influenza virus A/H5N1 (strain A/duck/Novosibirsk/56/05). Hybridoma produced MCA belong to IgA class. Strain 6F3 is deposited in the Collection of cell culture of Ivanovsky State Research Institution of Virology of the Russian Academy of Medical Sciences, No. 8/2/3. Using hybridoma allows producing specific monoclonal antibodies to hemagglutinin protein of avian influenza virus A/H5N1.

EFFECT: possibility to use antibodies to studying the antigenic structure of hemagglutinin for differential diagnostics of avian influenza virus A/H5 serotype.

1 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: invention represents a recovered nucleic acid with a nucleotide sequence coding human lysosomal glucocerebrosidase protein continuously bound with C-terminal vacuolar target signal and N-terminal signal peptide of endoplasmic reticulum. The invention concerns host-cells, vectors and methods for expression and production of high-yield biologically active glucocerebrosidase (GCD) with high mannose content. The invention also concerns pharmaceutical compositions and methods of treating Gaucher disease.

EFFECT: invention allows high-efficient treatment of Gaucher disease.

54 cl, 22 dwg, 3 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to pyrido[1,2-a]benzimidazole derivatives of general formula I, where R=alkyl; R1=alkyl, aryl; R2=alkyl. The invention also relates to a method of producing formula I compounds.

EFFECT: novel to pyrido[1,2-a]benzimidazole derivatives with antibacterial activity are obtained.

2 cl, 2 tbl

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to treatment of infectious diseases, and can be used in determination of non-specific anti-infectious action of immunomodelling immunobiological preparation (IIP). Essence of method consists in the following: IIP in therapeutically efficient for immunocorrection day dose is perorally introduced to a group of 5-7 monkeys with diarrhea syndrome, in combination with efficient in case of diarrhea syndrome antibiotic, in day dose, which constitutes 1/2 of the dose efficient in case of diarrhea syndrome. After that in case if carried out treatment is inefficient during 2 days, day dose of antibiotic is increased to 3/4 of the dose efficient in case of diarrhea syndrome. Determined is either absence of non-specific anti-infectious IIP action in case when combined application of IIP and antibiotic proves to be inefficient during 7 days, or presence of non-specific anti-infectious IIP action in case when combined application of IIP and antibiotic proves to be efficient.

EFFECT: method allows to determine non-specific anti-infectious IIP action due to determination of minimal efficient in case of diarrhea syndrome day dose of antibiotic in its combination with IIP

3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine, particularly to pharmaceuticals applied for skin protection against chemical and biological damage factors, bacterial or fungal infections, for prevention of occupational skin diseases, for prevention of contact dermatitis, including allergic dermatitis, and also for first aid and following treatment of wound defects of skin, soft tissues or mucous membranes and can be used in the industry, building, agriculture, medicine and everyday life. Substance of the invention is a therapeutic dermatological composition for local administration which contains an active principle in the form of complex rare-earth salt compounds and a pharmaceutically acceptable carrier.

EFFECT: when applying the pharmaceutical composition based on complex rare-earth salt compounds with polyoxy-compounds in the form of a liquid or soft dosage form, an evident protective and preventive, dermatoprotective and medical effect, no adverse reactions, good tolerance and fast skin repair are observed.

6 cl, 10 tbl, 22 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, and can be used for integrated treatment of purulent cholangitis by external percutaneous transhepatic drainage and laser and antibiotic therapy. That is ensured by ultrasound-aided percutaneous transhepatic drainage. Then Cefoperazone 2 g in 100 ml of 0.9% saline solution in a plastic package is exposed to low-intensity infra-red pulse laser light at wave length 0.89 mcm, power 200 mWt and frequency 1500 Hz for 6 minutes. Thereafter, the exposed antibiotic substance is introduced intravenously drop-by-drop for 30 minutes. Simultaneously, blood is exposed intravenously to continuous laser light at wave length 0.63 mcm, power 2 mWt for 30 minutes. The therapeutic course of laser and antibiotic therapy is 5-10 days.

EFFECT: method allows preventing purulent-septic complications represented by hepatic abscesses and hepatic failure.

3 cl, 2 ex

FIELD: medicine.

SUBSTANCE: invention belongs to vaccine manufacture for infectious diseases prophylaxis. Salmonella vaccine manufacture includes detoxication of exo- and endotoxins in suspention of autoclaved salmonells consecuently with two detoxicants - 0.2% formalin solution at 40±1 C° during 6-7 days and 0.5±0.1% aethonium solution at 41±1 C° during 7-8 days, followed by sterilisation at 1.0 atm during 20 minutes. Derived anatoxins are sorbed on aluminium hydroxide and sterilised at 1 atm during 20 minutes.

EFFECT: anatoxin-vaccine with enhanced effectivity and safety is used for specific prevention of salmonellosis in piglets, calves, carnivorous and poultry.

3 ex

FIELD: medicine.

SUBSTANCE: invention belongs to medicine, notably to pharmaceutical composition for treatment and prevention of bacterial infections induced by gram-positive bacteria. Composition includes effective doses of cholanic acid or its salt, phosphatidylcholine and neutral lipids. Neutral lipids and phospholipids are associated in lipoprotein-like particles, which does not include proteins or peptides.

EFFECT: composition effectivity is caused by its ability to bind lipoteichoic acid of gram-positive bacteria, neutralising or preventing their pathogenic action.

7 cl, 2 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention relates to pharmaceutical industry, in particular to composition for prevention and treatment of bacterial infections of oral cavity. Composition for prevention and treatment of bacterial infections of oral cavity, containing anthocyanosides, extracted from Vaccinium myrtillus or Vaccinium myrtillus or procyanides extracted from Vitis vinifera, sanguinarines and heleretrines extracted from Sanguinaria canadensis, Macleaya cordata or Macleaya microcarpa and lypophylic extract of Echinacea angustifolia. Application of said components for production of composition for prevention and treatment of bacterial infections of oral cavity. Application of said components for production of composition for oral cavity hygiene. Application of said composition for oral cavity hygiene.

EFFECT: said composition is efficient for prevention and treatment of bacterial infections of oral cavity.

5 cl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention concerns veterinary medicine. A vaccine contains cell suspensions of pure cultures of activators of pseudomonose Pseudomonas aeruginosa and enterococcal infection Enterococcus faecalis prepared by sampling involved organs from dead nutrias of a local epizootic centre, preparation of a suspension, inoculation for differential diagnostic mediums, recovery of pure cultures of activators and their separate cultivation in a plain broth with glucose to concentration of microbial cells 4-5 billion per 1 cm3. The vaccine also contains formalin and aluminium hydroxide in the following ratio, wt %: cell suspensions of pure cultures of the activator of pseudomonose Pseudomonas aeruginosa recovered from involved organs from dead nutrias from the local epizootic centre, in a nutrient medium with titre 4-5 billion of microbial cells per 1 cm3 - 38.0-41.5, cell suspensions of pure cultures of the activator of enterococcal infection Enterococcus faecalis recovered from involved organs from dead nutrias from the local epizootic centre in the nutrient medium with titre of 4-5 billion of microbial cells per 1 cm3 - 38.0-41.5, glucose - 2.0-1.0, formalin - 2.0-1.5, aluminium hydroxide - the rest.

EFFECT: prepared vaccine is safe, specific and immunogenic.

1 tbl, 5 ex

Composition // 2389481

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention concerns pharmacy and medicine and represents an antimicrobial composition containing at least 3 various diols wherein specified diols have general formula (CH2)nH2O2 where n is a number of groups CH2 within 3 to 6, in total approximately 0.1 to approximately 50% vol/vol.

EFFECT: invention provides production of the composition which exhibits improved antimicrobial properties, is highly effective, non-toxic, anallergic, and ecologically safe.

16 cl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to the novel tri-indolylmethane derivatives of general formulae I and II. The compounds can be used during bacterial or fungal infection and for protecting different products from harmful effect of bacteria or fungi, particulary as antiseptics or for disinfection. In general formulae

I

or II , where R1; R7; R13 independently represent hydrogen, alkyl, substituted alkyl, R2; R8; R14 independently represent hydrogen, alkyl, substituted alkyl, -OH, -OR, C1-C4acyl, where R is alkyl or substituted alkyl, R3-R6; R9-R12; R15-R18 independently represent hydrogen, alkyl, substituted alkyl, -OH, -OR, C1-C4acyl, where R represents alkyl or substituted alkyl, Y is an anion of a pharmacologically acceptable organic or inorganic acid; R19 is hydrogen, alkyl, substituted alkyl acyl, metal ion. The invention also relates to methods of obtaining compounds of formulae I and II, a pharmaceutical composition and use. The invention relates to a method for synthesis of tri-indolylmethane of formula III mono-substituted in the methane group which is an intermediate compound.

EFFECT: obtaining tri-indolylmethanes of general formulae I or II having antibacterial and antifungal activity.

17 cl, 4 dwg, 4 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: group of inventions concerns medicine, namely immunoprophylaxis and can be used for Neisseria meningitidis immunisation. The method under the invention provides introduction to the patient pre-immunised with diphtheritic anatoxin or its derivative, of conjugate meningococcal capsular saccharides with diphtheritic anatoxin or its derivative. Herewith the patient is pre-immunised with diphtheritic anatoxin (or its derivative). Application under the invention concerns manufacturing of a medical product for prevention of the disease caused by Neisseria meningitidis.

EFFECT: application of the inventions allows crating the immune response against Neisseria meningitidis in the diphtheria-immunised patients without immunological interference between meningococcal conjugates and diphtheria immunity.

42 cl, 5 tbl

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