FIELD OF THE INVENTION

The present invention relates to vaccine compositions and processes for culturing the pathogenic bacteria containing virulent polysaccharides in animal free culture medium  isolation  purification of polysaccharides and polysaccharide-protein conjugate without employing alcohol for preparing immunogenic formulations. The immunogens obtained from the process of the invention were formulated and do not contain any sources of animal-origin and alcohol excipients.

BACKGROUND OF THE INVENTION

The Pathogenic bacteria like Haemophilus influenzae type ""b""  Salmonella typhi  Paratyphi A  B  Salmonella typhimurium  Neisseria meningitidis A  C  Y  W135  X and serotypes of streptococcal pnuemococcai contain virulent polysaccharides which can cause specific disease in infants  children and adults. These capsular polysaccharides defend the immune system by escaping the bacterium from the complement mediated lysis. Neisseria meningitidis and Streptococcus pneumoniae are human pathogens and the main cause of their virulence are the capsular polysaccharides (CPS). However  these purified bacterial polysaccharides are known to be protective antigens  which can be used in bacterial vaccine preparations. Purified CPS are used in the production of vaccines against these bacteria.

Consumption of alcohol and non-vegetarian food or food material which originates from any animal source is prohibited in many religions. In countries or states where consumption of animal products and alcohol is not prohibited by the Government or religious bodies  it is left to individual’s wish to take his own decision regarding consumption of these materials  however in those countries where there is no restriction on the usage of alcohol and animal products  citizens with deep religious faiths or firm followers of religious thoughts  restrain themselves from consumption of any food materials originated from animals or that contains alcohol. Even pharmaceutical formulations which sometimes contain negligible quantities of alcohol or animal source products are avoided by them.

Particularly  consumption of alcohol  intoxicants and intake of meat specifically swine or porcine is strictly prohibited in countries of Islamic region like Iran  Iraq  Egypt  Saudi Arabia  Malaysia  several other middle east and gulf countries. They term these things as HARAM which means unlawful or prohibited as a part of their religious belief.

Various vaccines being prepared and used in the prior art are prepared from the culture medium grown on sources of animal-origin  isolated and purified using alcohol therefore  contains excipients of alcohol and animal sources which limits their usage as widely accepted vaccine among Islamic races and religiously conservative communities. Therefore  an immunogenic formulation which is free from animal sources and alcohol excipients can be widely used in these regions without any religious or geographic limitations which may also be termed as HALAL vaccines  means which is lawful or permissible under the Islamic law. Additionally  the Health Authority of Abu Dhabi (HAAD) proposes to compulsorily produce a vaccination certificate for all Hajj pilgrims to Mecca to perform Al Hajj. Since millions of Muslims travel to Mecca for this auspicious reason  there is every chance of occurrence of the diseases in absence of any preventive methods. The vaccinations mandatory under HAAD Hajj responsibilities are Meningococcal vaccine  seasonal influenza vaccine  and pneumococcal vaccine to protect travellers from meningitis  flu and pneumococcal diseases. (http://www.haad.ae/HAAD/tabid/1198/Default.aspx) In such a scenario  a HALAL vaccine will always prove to be an additional benefit to the pilgrims and a potential need of the hour since HALAL vaccines reduce the chance of occurrences of residual animal and alcoholic components in the final vaccine composition to nullity as there is no involvement of animal and alcoholic components in the production and purification of HALAL vaccine components. Conventional meningitis vaccines have been certified by some of the countries as Halal vaccines since they accept usage of animal products during their preparation of the vaccine but without using any pork product. However  such Halal certification is being given only on the basis of non-usage of pork product but they may include sources from other animals. The present vaccination which is termed HALAL serves the literal meaning of the term in all aspects. It does not contain any kind of animal source as well as alcohol. Therefore  the inventors prefer to coin the term “HALAL VACCINE” in its true sense applicable to a wide range of communities across the globe without any cultural and religious barriers.

WO2001/05997 discloses method for production of tetanus toxin using a media which is free from animal derived products. The invention talks about providing a system for the growth of C.tetani and production of tetanus toxin for use in formulation of tetanus toxoid preparations. The media used in the process as disclosed in this particular patent comprise hydrolyzed soy as a source of amino acids as a substitute to animal derived products. Additionally  it teaches use of iron powder  iron wire  iron foil  ferrous ammonium sulphate as various sources of iron. The fermentation media also contain sodium phosphate  magnesium sulphate  potassium phosphate and sodium chloride along with glucose including presence of nitrogen gas or a 90:10 mixture of nitrogen and hydrogen. Another WIPO publication WO2006/042542 also talks about production of tetanus  diphtheria  and pertusis toxins and toxoids using fermentation media containing non-animal derived and non-soy based components. The culture and fermentation media claims to use proteinaceous hydrolysates which are derived from wheat  or a mixture of rice and wheat  from potato  or from yeast. The teachings of the above mentioned patent or patent applications talks about processes those are applicable to only bacterial or viral proteins. Although prior art includes culture medium of non-animal derived products for only bacterial or viral proteins  there is a lacking in the present art in the area of bacterial polysaccharides which are devoid of any animal based products during their upstream and downstream operations.

Transmissible Spongiform Encephalopathy (TSE) is becoming an increased public health concern which is caused due to consumption or intake of meat products of cattle infected with Bovine Spongiform Encephalopathy (BSE). It is generally caused due to presence of a pathological protein known as a prion protein in many peripheral tissues of the cattle which infects the humans. Evidence of experimental transmission in humans from blood of rodents and sheep infected with BSE have also been recorded worldwide. It has also been reported that vaccines and other pharmaceutical products could spread TSE worldwide including even those countries where BSE is not yet reported. Bovine derived materials involved in the production of vaccines have been a potential cause of spread of the disease worldwide and poses a serious threat. In February 2003  WHO held consultations on medicinal products in relation to Human TSE with different forums  and revised the guidelines in this regard which was prepared in 1997.A new set of guidelines was also released in the year 2006 to prevent the human form of TSE. (http://www.who.int/bloodproducts/TSEPUBLISHEDREPORT.pdf) Although a few patent applications have been identified herein which talks about production of bacterial proteins useful as vaccine candidates without any animal source  yet there is no art available of such a development in the domain of bacterial polysaccharides which are required for the development of conjugate polysaccharide vaccines. Thence the current state of the art in the area of production and purification of conjugate polysaccharide vaccines is in serious requirement of developing a technology which is free from any kind of animal source.

There are several studies about the immunogenic characteristics of Conjugate Polysaccharides (CPS) vaccines. The available know-how of large scale production and purification is based on several selective precipitations steps with solvents like ethanol and phenol  and cationic detergents. After the bacteriae are cultivated in industrial bioreactors with appropriated controls  the CPS are required to be purified up to achieve the required purity levels  while maximizing the recovery and minimizing the production cost. Separations of solid from liquid are based on continuous centrifugation in explosion proof installations. A method disclosed to purify purification of capsular polysaccharide from Streptococcus pneumoniae consisted of using culture broth obtained by tangential microfiltration through a 0.22 microm membrane  broth microfiltrate concentration by tangential ultrafiltration in a 30 kDa spiral membrane  fractional ethanol precipitation (28-60%)  nuclease and proteinase treatment  and concentration/diafiltration in a 30 kDa cassette membrane. The final polysaccharide recovery was 89%. The final protein and nucleotide contamination was 1.5% (w/w) and 0.3% (w/w) respectively. (Goncalvese et al. Pubmed 2003 Jun; 37 (Pt 3); 283-7.) Phenols and acetal solutions are also usually involved in protein contamination of isolated capsular polysaccharides.

Moreover  use of alcohol in purification of bacterial polysaccharides to be used as particular vaccine candidates is commonly known and used widely. There exists a need for producing potential bacterial polysaccharides vaccine candidates which do not contain any animal source as well as free of alcohol during downstream processing of such bacterial polysaccharides. The present invention overcomes the problems with conventional vaccines  as specified in above paragraphs and discloses a novel method for culturing the pathogenic bacteria containing virulent polysaccharide in animal free culture medium  isolation  purification of polysaccharides and polysaccharide-protein conjugate without employing alcohol for preparing immunogenic formulations.

OBJECTS OF THE INVENTION

Primary object of the invention is to isolate and cultivate the target bacteria on a culture medium without animal source.

Another object of the invention is to provide a novel method for isolation and purification of polysaccharides and protein for preparation of immunogenic formulations without employing alcohol in the purification steps.
Another object of the invention is to provide an immunogenic formulation free from alcohol and animal-origin.

A further object of the invention is to provide HALAL vaccines free from alcohol and sources of animal-origin which can be used by all communities across all countries of the world without any religious or geographic limitations.

The overall object of the invention is to provide a method for Isolation and purification of polysaccharides and polysaccharide-protein conjugates free from alcohol  animal-origin for immunogenic formulations thereof.

SUMMARY OF THE INVENTION

According to one embodiment of the invention  it provides for a vaccine composition wherein the components of the vaccine composition are devoid of any animal or alcoholic component.

According to one other embodiment of the invention  the invention provides for a vaccine composition  wherein the antigenic components are capsular polyschharides and conjugated proteins thereto.

According to one other embodiment of the invention  it provides for a vaccine composition wherein the vaccine composition is made through a novel process without incorporation of any animal derived products during culture  fermentation and production of the capsular polysachharides.

A preferred embodiment of the invention provides a vaccine composition wherein the vaccine composition is is made through a novel process without involving any phenolic and/or alcoholic components during purification of capsular polysaccharides.

Another preferred embodiment of the invention relates to conjugate polysaccharide vaccine compositions wherein the conjugate protein is also produced and purified through a process in which no animal components are involved in the culture media.

Another preferred embodiment of the invention relates to optimization of the culture media for fermentation/Cultivation of N. Meningitidis A  C  Y  W135  X without any animal source.

One another preferred embodiment of the invention relates to optimization of the culture media for fermentation/Cultivation of Haemophilus influenzae type ""b""without any animal source.

Yet another preferred embodiment of the invention relates to optimization of the culture media for fermentation/Cultivation of Salmonella typhi  Salmonella Paratyphi A  B  and Salmonella typhimurium without any animal source.

According to one of the other preferred embodiments of the invention  provides optimization of the culture media for fermentation/Cultivation of Streptococcal pneumonia without any animal source.

According to another preferred embodiments of the invention  provides optimization of the culture media for fermentation/Cultivation of Clostredium tetani without any animal source.

Yet one other aspect of the invention provides purification and Precipitation of polysaccharides in the absolute absence of any alcoholic components.

BRIEF DESCIPTION OF THE DRAWINGS

Fig.1: schematic representation of general flow chart of culturing the bacteria in animal free culture medium. Once the target OD was obtained  the culture was centrifuged and the supernatant was concentrated to ¼ th of the original size. Cetrimide was added to the concentrated supernatant and kept for incubation at room temperature under stirring condition and loaded onto depth filters. The collected cetrimide precipitate was washed with sufficient WFI at room temperature. The precipitate was further dissolved and eluted using 0.5 M NaCl. The elute was further diluted with WFI to get the 0.2 M NaCl concentration. The sample was centrifuged  the pellet was discarded and the supernatant was collected and further diafiltered against 10 mm PBS buffer. The sample was subjected for endotoxin removal in a phase separation using triton- X 100/114. The PRP layer was collected and loaded onto XAD column to remove the residual triton-X 100/114. The elute was further diafiltered using 20 mm PBS buffer. The sample was subjected for dissolving and in the polysaccharide precipitate was dissolved and further purified purification method of the invention wherein  process of polysaccharide purification comprises. The obtained crude polysaccharide fraction is treated by hexadecyltrimethylammonium bromide (Cetavlon) followed by Triton-X100/114 treatment. The triton-X100/114 treated polysaccharide is passed through an XAD column to remove all chemical residues and endotoxin residues. The final polysaccharide bulk is sterile filtered using a 0.22µ capsule filter.

Figure 2: illustrates the elution profile of Meningococcal polysaccharide (HPLC  NMR profile).

Figure 3: illustrates the elution profile of Hib polysaccharide (HPLC  NMR profile).

Figure 4: illustrates the elution profile of Salmonella typhi polysaccharide (HPLC  NMR profile).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the isolation and purification of bacterial polysaccharides in preparation of immunogenic formulations for human use. The capsular polysaccharide of many pathogenic bacteria like Haemophilus influenzae type ""b""  Salmonella typhi  Paratyphi A  B  Salmonella typhimurium  N. Meningitidis A  C  Y  W135  X and serotypes of Streptococcal pneumoniae are grown on a suitable medium and the actively grown cells  usually called as inoculum is developed. The inoculum is transferred to fermenter containing pre-sterilized medium. In the fermenter  the bacterial cells are grown under controlled conditions of the fermentation parameters like pH  dissolved oxygen concentration  agitation and temperature. The fermentation process is carried out till the required optical density (OD) is obtained. Mostly all the bacterial cultures are harvested by inactivating with formaldehyde or by heat kill at early stationary phase before the secondary metabolites are produced. The invention provides two alternative purification methods for polysaccharides.

The novel features of the invention comprise optimization of the culture medium used for growth of the bacterium without any animal sources and isolation  purification of polysaccharides without employing alcohol in intermediate purification steps.

In the process of polysaccharide extraction  the bacterial culture is centrifuged and supernatant is treated with hexadecyltrimethylammonium bromide (Cetavlon). The obtained crude polysaccharide is further purified for the removal of host cell impurities like nucleic acids  proteins and lipo-polysaccharides.

The immunogenic formulations prepared from the polysaccharides  polysaccharide protein conjugate isolated and purified by the present method of the invention do not contain any sources of animal-origin and alcohol excipients. Therefore  these immunogenic formulations can be used and commercialized in the all communities without any religious or geographic limitations. Due to absence of animal sources and alcohol  these immunogenic formulations can also be termed as HALAL vaccines or vegetarian vaccines  which are free from TSE- Transmissible Spongiform Encephalopathies (TSE""s)  a fatal neurodegenerative diseases affecting human beings and Bovine Spongiform Encephalopathy (BSE)  popularly known as "Mad Cow Disease" or "Prion Disease".

The targeted bacterial strains are isolated and cultivated individually under controlled conditions using a culture medium without any animal source. The harvested bacterial culture is processed without alcohol  which includes simplified precipitation steps i.e.  Usage of cationic compounds  Hydrophobic interactions etc. The purified polysaccharides and polysaccharide-protein obtained by the method of the invention are used for preparation of HALAL immunogenic formulations free from alcohol and animal-origin. Such immunogenic formulations can be used for inducing immunogenicity against the targeted disease across all religious communities and countries.

EXAMPLES

According to the present invention media optimization was done where no animal derived raw material was used for the selective bacterial strains. The bacterial strains were obtained and further subjected to serial passages in the selective medium. The cultures that have shown adoption and viability were selected and prepared for a cell bank. Only these seed lots were used for obtaining the specific derivative polysaccharide.

EXAMPLE-1A

Cultivation of N. Meningitidis A  C  Y  W135 and X
N.Meningitidis serotypes A  C  Y  W135  X are based on seed lot system and initially grown at 35°C for about 18-20 hours in Mueller Hinton HiVeg Agar plate which contains HiVeg acid hydrolysate-17.5 g/L  HiVeg infusion-2.0 g/L  Starch soluble-1.5 g/L  Agar-17.0 g/L. The culture from the plate was transferred to production medium (Mueller Hinton HiVeg broth-contains HiVeg acid hydrolysate-17.5 g/L  HiVeg infusion-2.0 g/L  Starch soluble-1.5 g/L). The production culture was harvested at 10-14 hours of fermentation.

EXAMPLE-1B

Cultivation of S. typhi  S. Paratyphi A & B  S. typhimurium.
Cultivation of Salmonella typhi  Salmonella Paratyphi A& B  Salmonella typhimurium are based on seed lot system and the seed developemt was in two stages grown on Soyabean HiVeg medium which contains-HiVeg hydrolysate-17.0 g/L  Papic digest of soyabean meal-3.0 g/L  Dextrose-2.5 g/L  Sodium Chloride-5.0 g/L  Dipotassium phosphate-2.5 g/L)  pH 7.3±0.2 at 37 °C. The final seed was transferred to the production medium (Soyabean HiVeg medium)  pH 7.3±0.2. The fermentation was carried out at 37 °C. The fermentor culture was harvested at 24 hours when the cell density reaches to 100 OD.

EXAMPLE-1C

Cultivation of Haemophilus influenzae type ""b"" 
The cultivation of H. Influenzae type b are based on seed lot system and grown in HiVeg mixed with synthetic medium which contains-L-Glutamic acid-1.5g/L  NH3Cl-1.25 g/L  K2HPO4-2.5 g/L  Na2HPO4-11.0 g/L  NaH2PO4-3.3 g/L  Yeast extract HiVeg- 5 g/L  KCL-100.0 mg/L  NaCl- 6 g/L  Hiveg special peptone-10 g/L  NAD-3.0 mg/L  Synthetic Hemin-5.0 mg/L Glucose-5.0g/L  L-Cystine-100 mg/L) at 37°C. The pH was maintained at 7.0. The cultures are harvested at 12(2) hours of fermentation.

EXAMPLE- 1D

Cultivation of different serotypes of Streptococcal pneumonia
S.Pneumonia serotypes A  C  Y  W135  X are based on seed lot system and grown in Mueller Hinton HiVeg Agar plate contains Mueller Hinton HiVeg Agar plate which contains HiVeg acid hydrolysate-17.5 g/L  HiVeg infusion-2.0 g/L  Starch soluble-1.5 g/L  Agar-17.0 g/L. The production medium is a Mueller Hinton HiVeg broth which contains HiVeg acid hydrolysate-17.5 g/L  HiVeg infusion-2.0 g/L  Starch soluble-1.5 g/L at 35°C.

EXAMPLE 1E

Cultivation of Clostredium tetani
Clostredium tetani are grown on HySoy veg medium which containing no animal source at 35°C for 7 days in both static mode fermentor.

EXAMPLE- 2

Purification of polysaccharides: According the present invention the precipitation of polysaccharides was done in the absence of any traces of alcohol. The raw materials (buffers  sanitization solutions  dissolving agents  washing agents etc. ) used herein all the purification steps also doesn’t contain any traces of alcohol materials.

EXAMPLE - 2.1

Precipitation of polysaccharides
The bacterial culture was centrifuged above 8000 rpm at 4°C using a continuous centrifuge and the supernatant was collected. In cooling conditions the supernatant was concentrated to 40 times of the original supernatant volume using a 100 KD cutoff membrane. To the concentrated supernatant  a solution of 10% hexadecyltrimethylammonium bromide (Cetrimide) was added under stirring at 15% (v/v) and kept for 3 hours incubation at room temperature continuing the stirring condition. The crude polysaccharide precipitate obtained was loaded onto 5.0 µdepth filters at a flow rate of 3L/min.

EXAMPLE- 2.2

Removal of protein and nucleic acid impurities from crude polysaccharide precipitate
The collected crude polysaccharide precipitate was washed with sufficient cool WFI until the conductivity of the filtrate was zero. The precipitate was further made dissolved using cool 0.5 M NaCl and eluted out. The elute was further diluted with WFI to achieve a 0.2 M NaCl concentration. The sample was centrifuged at 4000 rpm at 4°C for 30 minutes  the resulting pellet was discarded and the supernatant was collected and further diafiltered against 6.0-10.0 diavolumes of 10 mM phosphate buffered saline  pH7.0  and hence the partially purified polysaccharide was obtained.

EXAMPLE- 2.3

Removal of endotoxin impurities from partially purified polysaccharide
The partially purified polysaccharide sample was subjected for endotoxin removal in a phase separation using triton- X 100/114. After the addition of the triton X 100/114 the reaction sample was kept in for incubation at above room temperatures in a static condition. The endotoxin removed polysaccharide layer was collected and loaded onto XAD matrix ion exchange column to remove the residual triton-X 100/114. The polysaccharide elute was collected and further diafiltered against 6.0-10.0 diavolumes of 20 mM PBS buffer  pH7.0. The final purified polysaccharide bulk is sterile filtered using a 0.22µ capsule filter and stored.

EXAMPLE- 3

Conjugation of purified polysaccharides to proteins: According the present invention the polysaccharides were selected from N. Meningitidis A  C  Y  W135  X  Haemophilus influenzae type b  Salmonella typhi  Streptococcal pnuemococcai  Salmonella Paratyphi A  B  Salmonella typhimurium.
was done in the absence of any traces of alcohol. The raw materials (buffers  sanitization solutions  dissolving agents  washing agents etc. ) used herein all the purification steps also doesn’t contain any traces of alcohol materials.

Dated this 7th day of July 2012

Afzal Hasan
of HASAN AND SINGH
IN/PA-1328