WO2014009971A2 - Non-alcoholic vaccine compositions free from animal- origin and process for preparation thereof - Google Patents

Non-alcoholic vaccine compositions free from animal- origin and process for preparation thereof Download PDF

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WO2014009971A2
WO2014009971A2 PCT/IN2013/000418 IN2013000418W WO2014009971A2 WO 2014009971 A2 WO2014009971 A2 WO 2014009971A2 IN 2013000418 W IN2013000418 W IN 2013000418W WO 2014009971 A2 WO2014009971 A2 WO 2014009971A2
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polysaccharide
purification
purified
prp
polysaccharides
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PCT/IN2013/000418
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French (fr)
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WO2014009971A3 (en
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Krishna Murthy Ella
Venkatesan RAMASAMY
Mandalapu Gangadhara NAIDU
Annamraju Dattatreya SARMA
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Bharat Biotech International Limited
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Priority to IN2754/CHE/2012 priority Critical
Priority to IN2754CH2012 priority
Priority to IN2317CH2012 priority
Priority to IN2317/CHE/2012 priority
Application filed by Bharat Biotech International Limited filed Critical Bharat Biotech International Limited
Publication of WO2014009971A2 publication Critical patent/WO2014009971A2/en
Publication of WO2014009971A3 publication Critical patent/WO2014009971A3/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/02Bacterial antigens
    • A61K39/025Enterobacteriales, e.g. Enterobacter
    • A61K39/0275Salmonella
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/02Bacterial antigens
    • A61K39/09Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092Streptococcus
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/02Bacterial antigens
    • A61K39/095Neisseria
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/02Bacterial antigens
    • A61K39/102Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505Inorganic adjuvants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/60Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031Proteins
    • A61K2039/6037Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
    • Y02A50/46Medical treatment of waterborne diseases characterized by the agent
    • Y02A50/468The waterborne disease being caused by a bacteria
    • Y02A50/481The waterborne disease being caused by a bacteria of the genus Salmonella, i.e. Salmonellosis
    • Y02A50/482The waterborne disease being caused by a bacteria of the genus Salmonella, i.e. Salmonellosis the medicinal preparation containing antigens or antibodies, e.g. vaccines, antisera
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
    • Y02A50/46Medical treatment of waterborne diseases characterized by the agent
    • Y02A50/468The waterborne disease being caused by a bacteria
    • Y02A50/481The waterborne disease being caused by a bacteria of the genus Salmonella, i.e. Salmonellosis
    • Y02A50/483The waterborne disease being caused by a bacteria of the genus Salmonella, i.e. Salmonellosis the bacteria being Salmonella typhi, i.e. Typhoid fever
    • Y02A50/484The waterborne disease being caused by a bacteria of the genus Salmonella, i.e. Salmonellosis the bacteria being Salmonella typhi, i.e. Typhoid fever the medicinal preparation containing antigens or antibodies, e.g. vaccines, antisera

Abstract

Vaccine compositions and processes are disclosed for culturing the pathogenic bacteria containing virulent capsular polysaccharides in animal free culture medium, isolation, purification of polysaccharides and polysaccharide-protein conjugate. The purification of capsular polysaccharides may or may not employ 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. Also disclosed is a method for isolation, purification and conjugation of bacterial capsular polysaccharide of Haemophilus Influenza. The novel processes for purification, removal of endotoxin and formation of immuno-conjugates have also been used to generate novel compositions responsible to invoke immunogenicity against infections against Hib and prevention and treatment thereof.

Description

NON-ALCOHOLIC VACCINE COMPOSITIONS FREE FROM ANIMAL ORIGIN AND

PROCESS FOR PREPARATION THEREOF

This application claims priority from Indian Provisional Patent Application No. 2754/CHE/2012 filed on July 07, 2012 and Indian Provisional Patent Application No. 23 17/CHE/2012 filed on July 1 1 , 2012.

FIELD OF THE INVENTION The invention relates to non -alcoholic vaccines free from animal-origin. The invention also relates to a process for preparation of non -alcoholic vaccines free from animal-origin. In general the invention relates to the field of bacterial polysaccharide vaccines and culturing of organisms in novel media having improved characteristics and process of manufacture of bacterial polysaccharide and conjugate vaccines thereof. The invention also relates to downstream processing pertaining to purification of bacterial polysaccharides and their conjugate counterparts in the field of conjugate polysaccharide vaccines. Particularly, the present invention relates to a novel process 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 or using very low quantities of alcohol for limited periods, thereby negating all chances of presence of alcohol in the final vaccine preparations . The immunogenic formulations obtained from the process of the invention do not contain any sources of animal-origin and/or any other alcohol excipients since, in the purification steps, either alcohol is not used absolutely or is used in very limited quantities for limited periods of time.

BACKGROUND OF THE INVENTION

Bacterial pathogens can cause diseases that can be both endemic and epidemic. Most of the disease and mortality caused by these pathogens are high amongst children, especially in developing countries. Haemophilus influenzae type b (Hib) is a causative organism of pneumonia and meningitis in young children. Roughly 2 million cases of disease and 450,000 deaths occur in vast majority of developing countries; therefore the vaccine requirement for this disease is of great urgency at both national and international childhood immunization programs [Global programme for vaccine and immunization: The WHO position paper on Haemophilus influenzae type b conjugates vaccine (www.who.int/vaccine-documents/pp-wer/wer73 10.Pdf)]. Pathogenic bacteria like Neisseria meningitidis, Haemophilus influenzae type b (Hib), Salmonella typhi. Streptococcus pneumoniae contain virulent polysaccharides which causes meningitis, typhoid fever, pneumonia, bacteremia, cellulitis, osteomyelitis, epiglottitis, otitis media, sinusitis in infants, children and adults. Many gram negative bacteria like Streptococcus (pneumoniae, aureus). Neisseria meningitides, Salmonella entericus typhi, Haemophilus influenzae type b etc. possess cell surface capsular polysaccharide (CPS) or lipopolysaccharide (LPS) or both which help the pathogen to establish an infection and evasion of immune system. It has been observed that antibodies to most of these capsular polysaccharides have the ability to protect against the infection from encapsulated organism. 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 bacterial polysaccharides when purified are known to be protective antigens and hence capable to be used in bacterial vaccine preparations. However, most of the immune response elicited against the CPS is T-cell independent therefore induction of immunological memory, avidity maturation and isotype switching do not occur resulting in failure of immune response in infants and children. This problem is overcome by introduction of new generation of vaccine called conjugate vaccines, whereby the polysaccharide is conj ugated to a carrier group/moiety such as a protein, thereby introducing T-cell epitopes onto the polysaccharide and converting it to T-cell dependent antigen [Vaccine based on the cell surface carbohydrates of pathogenic bacteria (2005) Annals Brazilian Acd. Sci. 77, 293-324.].

Anti bacterial vaccines are therefore regarded as the best preventive measure that can be used to control the spread of such diseases. Among the vaccine types, sub-unit vaccines for example protein antigens or polysaccharide vaccines are the most effective candidates, since these are not only safe and efficacious in infants and children but also are preventive against large number of pathogenic serotypes of the same organisms. A variety of Hib vaccines are widely used in the developed countries but relatively few of these vaccines are used routinely in under developed countries. This under utilization is mainly due to high cost and non-affordability of these vaccines. The prime reason for the high cost of these vaccines is due to sub optimal recovery of polysaccharide free of contaminants and sub-optimized conjugation methods. The present invention therefore proposes towards improvement of these two stages thereby building a production process that is, simple, gentle, efficient and cost effective in order to meet the high requirements of Hib vaccine at the same time without involving any animal derived product during the culture process. Purified CPS are used in the production of vaccines against these bacterial infections. Preparation of these bacterial vaccines which contain capsular polysaccharides as antigen are commonly prepared in media which contain animal sources preferefably of porcine origin, and involve alcohol in course of downstream processing. Hence, it is most likely that the finished product contain traces of porcine material and alcohol. Like hemine is added to the culture media of Hib, Meat media used for the culturing of clostredium tetani, and animal peptones used for cultivation of S. pneumoniae and n. meningitis. Purification procedures of cultivated polysachharides and conjugate proteins often include long term ethanol fractionation, enjyme digestion, and phenol extraction techniques. 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 about usage 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 containing alcohol.

Particularly, consumption of alcohol, intake of meat specifically derived from 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. Even pharmaceutical formulations which sometimes contain negligible quantities of alcohol or traces of porcine source are avoided by them. Therefore, it is obvious that, a certain portion of the population in those countries following strict Islamic faith remains to be treated, since they do not prefer to vaccinate themselves, against the above mentioned diseases due to possible chances of porcine and alcoholic content in the finished vaccine product. 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 high amounts of 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, meaning lawful or permissible under the Islamic law.

For example, 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 Islamic followers 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/ 1 1 98/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 absolutely negates the chances of occurrences of residual animal and alcoholic components in the final vaccine composition to nullity as there is no involvement of animal components and either no alcoholic components are involved during purification or may involve alcohol in very limited amounts 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 Clostridium et ni 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 foi l, 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 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 hydrolysate 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.

Furthermore, 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 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 precipitation 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 micron 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.

Many gram negative bacteria including Haemophilus influenzae type b produce extra cellular capsular polysaccharide that has been used as human vaccine. Often purification and downstream processing during large scale production of CPS is hampered by contaminants such as nucleic acids, cellular proteins and large amounts of highly variable (in terms of size, charge, hydrophobicity) amounts of lipopolysacchandes, referred as endotoxins during their purification steps. The methods employed according to the present state of the art for purification and removal of these contaminants are complicated, time consuming and expensive and thus in many ways compromise the yield of the precious CPS component. Some of the methods known to the art include: a) Deproteinization and extraction by phenol followed by ethanol precipitation resulting in undesirable phenolic toxic waste. Additionally use of chemical like phenol which can also lead to unwanted structural changes in the polysaccharide or protein carrier [U.S. 5045456 by Rienstra et al. and U.S. patent 4644059 by Gordon]. b) Ultrafiltration of culture broth and precipitation with cationic detergent followed by ethanol precipitation of the CPS. The CPS is dissolved in suitable buffer or solution and precipitated with an anionic detergent followed by second round of ethanol precipitation. These alternate detergent treatments of the CPS and ethanol precipitations suffer from unwarranted reproducibility, time consuming and costly processes [US6891 037B 1 and US7582459B2]. c) Enzymatic treatment fol lowed by ultrafiltration in the presence of chelating agents and detergents. These are again less cost effective and variable [Takagi M. et al. Purification of capsular polysaccharide produced by Haemophilus influenzae type b through a simple, efficient and suitable method for scale-up. J. Ind. Microbial . Biotechnology. (2008), 35, 1217- 1222.]. d) Use of column chromatography for purification. This drastically compromises the recovery of the CPS. [US422071 7 and US5 192540] .

Therefore, keeping in view of the drawbacks mentioned above, the present invention proposes for gentle, efficient, less time consuming processes which are disclosed. Moreover, use of high amounts of alcohol for extended periods of time 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 or reduction of involvement of alcohol to substantial levels 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 with or without employing alcohol for preparing immunogenic formulations.

The second critical and limiting step in the making of conjugate vaccines especially Haemophilus influenzae type B- polyribosylribitol phosphate (Hib-PRP) conjugate vaccine is the construction of the immuno-conjugate. This step is not only challenging in terms of restoring the immunological properties of the desired polysaccharide but also to ensure that the Hib-PRP satisfies reproducibility during large scale conjugation process, and stability of the conjugate.

Usually conjugation is initiated by chemical activation of the functional groups of both polysaccharide and carrier protein by derivatizing them with small chemical linkers or spacer reagents. This is followed by the conjugation between the two macromolecules using water soluble cross linking reagents. Choice of conjugation methods used in the art are frequently harsh and may destroy the native structure of polysaccharide or the carrier protein effecting either the immunogenicity or the yield. One particular method frequently used in the art of making polyribosylribitol phosphate (PRP) conjugate vaccine is the cynogen bromide (CNBr) activation method, wherein the polysaccharide is activated by using CNBr and immediately derivatized with bi-functional linker like adipic dihydrazide (ADH) and coupled to tetanus toxoid using a water soluble carbodi imide.

Typically, CNBr activation is carried out at high pH ( 10- 1 1 ), resulting in formation of cynate ester on hydroxyl groups of polysaccharide; these in turn reacts with bi-functional reagents like diamines or dihyrazides. These derivatized moieties are then cross-linked to carrier protein molecules. However, this process suffers several drawbacks which are as follows:

1 ) Alkaline hydrolysis results in several non-productive side reactions such as formation of carbamates, linear or cyclic imidocarbamates. 2) The reactive cynate ester formed on hydroxyl groups which ultimately reacts with linker reagents is highly unstable at high alkaline pH.

3) CNBr reagent itself is highly unstable and spontaneously undergoes hydrolysis at high alkaline pH.

Thus, these limitations may results in variable yields and loss of integrity of the conjugate. Therefore, the present invention discloses a novel method of Hib-PRP conjugation technique to carrier protein Tetanus Toxoid, devoid of any alkaline environment, and without any use of Sodium Hydroxide. OBJECT OF THE INVENTION

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

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

Another object of the invention is to provide a novel method for isolation and purification of immunogenic polysaccharides and proteins for preparation of immunogenic formulations without employing alcohol in the purification steps or using very minimal quantities of alcohol for very limited periods of time.

Another object of the invention is to enhance the recovery of purified polysaccharide with or without the use of alcohol during downstream processing and to increase the cost to benefit ratio for the CPS polysaccharide conjugate vaccines.

One more objective of the invention is to provide a conjugation technique between Haemophilus influenzae type B capsular polysaccharide polyribosylribitol phosphate and carrier protein tetanus toxoid in absence of alkaline environment so that undesired formation of carbonate and carbamate derivatives is prevented. A further object of the invention is to provide an immunogenic formulation free from alcohol and animal-origin excipients that are termed as HALAL vaccines which can be used by all communities across all countries of the world without any religious or geographic limitations. 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 polysaccharides 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 or cultivation of the capsular polysaccharides.

A preferred embodiment of the invention provides a vaccine composition wherein the vaccine composition is made through a novel process without involving any phenolic components and optional use of alcoholic components at specified limits of time and quantity during purification of capsular polysaccharides.

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

Another preferred embodiment of the invention provides for fermentation and cultivation of N. Meningitidis A, C, Y, W l 35, X without any animal source. One another preferred embodiment of the invention provides for fermentation cultivation of Haemophilus influenzae type 'b' without any animal source.

Yet another preferred embodiment of the invention provides for fermentation cultivation of Salmonella typhi Type 2 without any animal source.

According to one of the other preferred embodiments of the invention, provides for fermentation and cultivation of Streptococcal pneumonia without any animal source.

According to another preferred embodiments of the invention, provides for fermentation and 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. Yet one other aspect of the invention provides purification and precipitation of polysaccharides in the presence of limited quantities of alcoholic components for very limited period of time.

According to one preferred embodiments of the invention, the invention provides novel method of conjugation of Hib-PRP-TT in absence of Sodium Hydroxide or alkaline pH.

The bacterial strains of Neisseria meningitidis serogroups A, C, Y, W 1 35, Haemophilus influenzae type b, Salmonella typhi Ty2, Streptococcus pneumonea serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F are isolated and cultivated under controlled conditions using a culture medium without any animal source. The harvested bacterial culture is processed by separation using a centrifuge and the supernatant was collected. From the supernatant, the polysaccharides were precipitated using a cationic compound hexadecyltrimethyl ammonium bromide (Cetrimide) by hydrophobic interactions. The isolation and purification of polysaccharides was achieved without employing harsh chemicals like chloroform, phenol, etc. In the present method, the endotoxins were removed using Triton-X 100/ 1 14 in a two layer separation method where endotoxins used to settle at the middle layer and the polysaccharides at the top layer. Further the collection of polysaccharide fraction is using a sterile tubing but not centrifugation. The purified polysaccharides were then formulated or conjugated with the carrier protein(s) derived preferably from the group consisting of tetanus toxoid, Diphtheria protein CRM 197 or any other recombinant proteins. The protein used for conjugation is also derived from a method where the organism was grown in animal origin free medium and then purified with or without the usage of alcohol.

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 against the targeted disease across all religious communities and countries.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 is schematic representation of general flow chart of the present invention for purification of bacterial polysaccharides: Once the target OD was obtained, the culture was centrifuged and the supernatant was concentrated to ¼lh of the original size. Hexadecyltrimethyl ammonium bromide (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 polysaccharide elute was further diluted with WFI to get 0.2 M NaCl concentration. The sample was centrifuged, the pellet was discarded and the supernatant was collected and further diafiltered against 1 0 mM Phosphate Buffer Saline (PBS). The sample was subjected for endotoxin removal in a phase separation using Triton-X 1 14. The Poly Ribosylribitol Phosphate layer (PRP layer) was collected and loaded onto hydrophobic interaction column to remove the residual Triton-Xl 14. The polysaccharide elute from the column was further diafiltered using 20 mM PBS buffer. The final polysaccharide bulk is sterile filtered using a 0.22μ capsule filter.

Figure2 : Purified polysaccharide of N. meningitidis sero group A when injected into SEC HPLC eluted at an average retention time of 14.05 minutes in the RID which indicates 500 KDa molecular size when compared with the standards graph and the single peak indicates the high purity of N. meningitidis serogroup A polysaccharide indicating presence of no other undesired proteins and nucleic acids.

Figure 3 : The purified polysaccharide of N. meningitidis sero group C when injected onto SEC HPLC RID, has eluted at an average retention time of 14.67 minutes which indicates approximetely 350 KDa molecular size when compared with the standards graph and the single peak profile indicates the high purity of polysaccharide where there is no presence of other proteins and nucleic acids. Figure 4: The purified polysaccharide of N. meningitidis sero group Y when injected onto SEC HPLC RID, has eluted at an average retention time of 13.72 minutes which indicates approximately 650 KDa molecular size when compared with the standard and the peak profile indicates the high purity of polysaccharide. Figure 5: The purified polysaccharide of N. meningitidis sero group W 1 35 when injected onto SEC HPLC RID, has eluted at an average retention time of 14.69 minutes which indicates approximately 320 KDa molecular size based on standard polysaccharide graph and the peak profile indicates the high purity of polysaccharide where there is no presence of proteins and nucleic acids.

Figure 6: The purified Vi-polysaccharide of Salmonella typhi Ty2 when injected · onto SEC HPLC RID, has eluted at an average retention time of 14.82 minutes which indicates approximately 280 KDa molecular size based on standard polysaccharide graph and the peak profile indicates the high purity of polysaccharide.

Figure 7 describes NMR spectra of purified Vi-polysaccharide of Salmonella typhi Ty2. The spectrum of the Vi polysaccharide showed the expected peaks. The N-acetyl and acetate anion resonances were resolved and integration suggested that the degree of O-acetylation of the original sample was close to 1 00%. The O-acetyl was arised at 2 ppm. The residual water as arised at 4.7 ppm. The presence of O-acetyl group confirms Vi-polysaccharide of S. Typhi Type 2. Figure 8: The purified polysaccharide of Haemophilus influenzae type b when injected onto SEC HPLC RID, has eluted at an average retention time of 12.50 minutes which indicates approximately 1600 KDa molecular size and the peak profile indicates the high purity of polysaccharide.

Figure 9 describes NMR profile of purified PRP polysaccharide of Haemophilus influenzae type b. The spectrum of Hib polysaccharide showed the expected peaks. The ribosyl and ribitol phosphate groups resonances were resolved and integration suggested that the degree of ribosylation of the original sample was close to 100%. The ribosyl and ribitol phosphate groups were arised at 4.7 ppm to 5. 1 ppm,. The presence of ribosyl and ribitol phosphate groups (HI , H2, H3) confirms capsular polysaccharide of Haemophilus influenzae type 'b'.

Figure 10: The purified protein of Clostridium tetani when injected onto SEC HPLC UV, has eluted at an average retention time of 1 8.02 minutes which indicates approximately 150 KDa molecular size and the peak profile indicates the high purity of protein .

Figure 1 1 : The purified protein of diphtheria protein CRM 197 when injected onto SEC HPLC UV, has eluted at an average retention time of 1 8.88 minutes which indicates approximately 60 KDa molecular size and the peak profile indicates the high purity of CRM 1 97 protein.

Figure 12: SDS page TT: The purified Tetanus toxoid in the present invention run through the non-reducing PAGE and have shown 1 50 kD when compared with the standard markers. The tetanus toxoid up to l Ofold also has shown prominent band. Figure 13: SDS CRM 197: The purified CRM 1 97 in the present invention run through the non- reducing PAGE and have shown equivalent to 66 kD when compared with the standard markers. The concentrated CRM 1 97 was also loaded to check the purity of the band. 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 Neisseria meningitidis serogroups A, C, Y, W 135, Haemophilus influenzae type b, Salmonella typhi Ty2, Streptococcus pneumonea serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F 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 animal free source 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. The bacterial cultures are harvested by inactivating with formaldehyde or by heat kill at early stationary phase before the secondary metabolites are produced. The novel features of the invention comprise the culture medium used for growth of the bacterium without any animal sources and isolation, purification of polysaccharides without employing alcohol in purification steps.

In the process of polysaccharide extraction, the bacterial culture is centrifuged and supernatant is either concentrated or directly treated with hexadecyltrimethyl ammonium bromide. The obtained crude polysaccharide is further purified for the removal of host cell impurities like nucleic acids, proteins and lipo-polysaccharides.

Definition of 'HALAL VACCINE' : 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 any risks involved with TSE- Transmissible Spongiform Encephalopathies (TSE's), a fatal neurodegenerative disease affecting human beings and Bovine Spongiform Encephalopathy (BSE), popularly known as "Mad Cow Disease" or "Prion Disease".

The invention is further described in the following examples. Person skilled in the art would appreciate that minor modifications of the following examples are to be construed as non- limiting to the scope of the invention.

EXAMPLES EXAMPLE 1 : Cultivation of bacterial organisms.

According to the present invention specific media was obtained 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 those cultures were used for obtaining the specific derivative polysaccharide through inoculation into the fermentation media (seed lots). The growth of the cultures were determined by measuring the optical density (OD) at specific wavelength (nanometer) using a spectrophotometer. The bacterial cultures were harvested based on OD at the pH that was established for each organism in the fermentor. Each organism was grown in respective medium in a seed lot system. The seed was revived either on a solid media plate or in a liquid medium.

EXAMPLE 1.1 : Cultivation of N. meningitidis sero groups A, C, Y, and W135.

N. meningitidis sero group A (Strain No: CCUG 42379), sero group C (Strain No:CCUG32912), sero group Y, (Strain No:CCUG38303) sero group W 135 (Strain No:41483) procured from University of Goteberg, Sweden are based on seed lot system and initially grown at 35°C for about 1 8-20 hours on Agar plate which contains plant peptones - 1 7.5 g/L, Starch soluble- 1 .5 g/L, Agar- 17.0 g/L, Sodi um Chloride -2.0 g/L. The culture from the plate was transferred to production medium that contains plant peptones- 1 7.5 g/L, Starch soluble- 1 .5 g/L, Sodium chloride-2.0 g/L). The production culture was harvested at 12(±2) hours of fermentation and at an OD of 10±2 measured at 600 nm. EXAMPLE 1.2: Cultivation of S. typhi Type 2.

Cultivation of Salmonella typhi Type 2 procured from NIH (USA) are based on seed lot system and the seed development was done in two stages grown on medium that contains plant peptones - 17.0 g/L, papic digest of soyabean meal-3.0 g/L, Dextrose-2.5 g/L, Sodium Chloride-5.0 g/L, 2P04-2.5 g/L), Disodium hydrogen orthophosphate dihydrate (Na2HP04.2H20) -3.5 g/L, pH 7.3±0.2 at 37 °C. The final seed was transferred to the same production 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 reached to 100± 1 0 OD measured at 600 nm. EXAMPLE 1.3: Cultivation of Haemophilus- influenzae type b.

The cultivation of H. influenzae type b CS 68 strain collected from CMC Hospital, Vellore, India are based on seed lot system and grown in L-Glutamic acid- 1 .5g/L, Ammonium sulphate (NH3S04) - 1 .25 g/L, Disodium hydrogen orthophosphate dihydrate (Na2HP04. 2H20)- 1 1 .0 g/L, Disodium dihydrogen orthophosphate dihydrate (NaH2P04.2H20) -3.3 g/L, Yeast extract - 5 g/L, Potassium chloride (KCL)- 100.0 mg/L, Sodium chloride ( aCl)- 6 g/L, Plant extract peptone- 10 g/L, Nicotinamide Adenocine Dinucleotide (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 measured at 550 m and harvested at 12±2 hours of fermentation when the OD reaches to 5± 1 .

EXAMPLE 1.4 : Cultivation of Streptococcus pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F.

5". pneumoniae serotypes 4, 6B, 9V, 14, 1 8C, 19F, and 23 F are collected and developed by our in-house R&D are based on seed lot system and grown in Agar plate which contains plant peptones - 17.5 g/L, Starch soluble- 1 .5 g/L, Agar- 17.0 g/L, NaCl-2.0 g/L. The production medium containing plant peptones - 1 7.5 g/L, Starch soluble- 1.5 g/L, Sodium Chloride (NaCl) - 2.0 g/L at 35°C. Periodical fermentation samples are checked for microbial purity and optical density. The samples were subjected for quelling test by using specific anti pneumococcal capsule serum to determine specific serotype of streptococcus pneumoniae. The production cultures were grown up to 10±2 OD and then harvested. EXAMPLE 1.5: Cultivation of Clostridium tetani:

Clostredium tetani are collected from Pasteur Institute, Coonoor, India, grown on medium which contains Soy peptones- l OOg/L, NaCl- l .Og/L, D-Glucose-2.0g/L, Disodium hydrogen orthophosphate dihydrate (Na2HP04 2H2Or 1 .Og/L, Potassim phosphate dihydrate ( P04.2H20) -0.15 g/L, Magnesium sulphate (Mg2SO4)-0.15 g/L, L-Cystine ( 10%)-2.5mL/L, Uracil (25mg %) 10 mL/L, Calcium D pantothenate ( 100 mgm%) 1 .0 mL, Thiamin (25 mgm%)- 1 .0 mL, Pyridoxin(25 mgm%)- l mL, Riboflavin(25 mgm%)- 1 .0 mL, Niacin(25 mgm%)- 1 .0 mL, Folic acid-(25 mgm%)- 1 .0 mL, Cyanocobalamin-(25 mgm%)- 1 .0 mL, Biotin( 1 .25 mgm%)-0.2 mL, Fe2S04- 40 mg/L, pH 8.0±0. at 35°C for 7 days in a fermentor and also in static mode and the OD is at 5±2 measured at 600 nm.

EXAMPLE 1.6: Cultivation of Escherichia Coli expressing CRM197 protein

Clones of recombinant E. coli and yeast that expresses CRM 197 were developed and grown in Minimal salt media that contain plant peptones - 4.0 g/L, Disodium hydrogen orthophosphate dihydrate (Na2HP04.2H20)- 6.8 g/L, Potassium orthophosphate (KHPO4.2H2O)-3.0 g/L, Sodium Chloride (NaCl) - 0.5 g/L, Ammonium Chloride (NH3C12)- 1 .0 g/L, Dextrose-4.0 g/L, Magnesium sulphate (Mg2SO4)-0.24 g/L. or the medium which contains plant peptone - 10.0 g/L, Yeast extract-5.0 g/L, Sodium chloride ( aCl)-5.0 g/L, pH7.0 ±0.2. The culture was grown in the fermentor at 37°C for about 24 hours. The culture is tested for purity on streaking onto agar plates. The identity was checked by gram staining, agglutination test using specific antibodies. The culture was harvested when the OD reaches to 80± 1 0 measured at 600 nm.

EXAMPLE 2: Purification of polysaccharides without any use of alcohol

The harvested bacterial culture of the above mentioned bacterial capsular polysachharides are processed by separation using a centrifuge followed by collection of the supernatant. From the supernatant, the polysaccharides were precipitated using a cationic compound hexadecyltrimethyl ammonium bromide (Cetrimide) by hydrophobic interactions. The isolation and purification of polysaccharides was achieved without employing harsh chemicals like chloroform, phenol, etc. The raw materials (buffer solutions, sanitization solutions, dissolving agents, washing agents etc..) used herein all the purification steps also doesn't contain any traces of alcoholic components. In the present method, the endotoxins were removed using Triton- X I 14 in a two layer separation method where endotoxins settle at the middle layer and the polysaccharides at the top layer. Further the col lection of polysaccharide fraction is achieved using a sterile tubing but not centrifugation. EXAMPLE 2.1 : Precipitation and collection of polysaccharides

The harvested bacterial cultures were centrifuged above 8000 rpm at 4°C using either continuous centrifuge or by a batch mode centrifuge and the supernatant was collected. Further they were purified using two precipitation and collection methods. The first precipitation and collection method comprises of that the supernatant was concentrated to 40 times of the original supernatant volume using a 100 KD cutoff membrane in cooling conditions. To the concentrated supernatant, a solution of 10% hexadecyltrimethylammonium bromide was added under stirring at and kept for 3 hours incubation at room temperature continuing the stirring condition. The crude polysaccharide precipitate was loaded and collected onto 5.0 μ depth filters at a flow rate of 3 L/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 Water for Injection (WFI) until the conductivity of the filtrate was zero. The precipitate was further dissolved using cool 0.5 M NaCl and collected out. The polysaccharide sample 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 10 niM phosphate buffered saline, at pH 7.0 to obtain the partially purified polysaccharide.

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 by adding triton-X l 14. After the addition of the triton X 1 14 the reaction sample was kept for incubation at 30±3°C in a static condition. The upper layer was collected which contains the polysaccharide free from endotoxins and loaded onto ion exchange column (XAD matrix) to remove the residuals of triton X I 14. . The polysaccharide elute from the colum was collected and further diafiltered against 20 mM PBS buffer, pH 7.0. The final purified polysaccharide bulk is sterile filtered using a 0.22μ capsule filter and further stored at 2-8°C. EXAMPLE 3: Purification of polysaccharides with limited use of alcohol

As described above the one of the objective of this invention is to improve the large scale purification of PRP and subsequent conjugation method so as to enhance the cost benefit ratio for vaccine production. After the separation of cells from the fermentation broth, the culture supernatant containing PRP is mixed with known amounts of cetrimide and the resultant precipitated complex of PRP-cetrimide is allowed to pass through specified depth filters. Nearly 90-95% of this complex remains trapped in the pores of the filters. As the PRP-Cetrimide complex is insoluble in water, this property is exploited by washing the filters extensively by hot (37°C) water. This step not only removes a considerable amount of proteins but also washes away uncomplexed cetrimide and other unwanted media components. Once the conductivity of the feed reached to that of water the PRP-cetrimide complex was eluted in NaCl solution. To get rid of cetrimide from PRP two volumes of absolute ethanol is slowly mixed with PRP-cetrimide solution. As the concentration of ethanol reaches the cloud point the PRP salts out of the solution and forms heavy deposit of precipitate. After allowing the mixture to settle for 3-4 Hr, the supernatant containing cetrimide in ethanol is slowly decanted and the precipitated PRP is solubilized in known volumes of appropriate buffer. Though at this stage the protein and nucleic acid content is nearly reduced to the recommended pharmacopeial levels but the levels of endotoxins still remain far higher than expected to be present in the final PRP bulk substance. As described above several methods in the prior art has been described for removal of endotoxins, however, these methods are both lengthy and less cost effective or with unwarranted reproducibility, harsh and produce undesirable waste. In order to tackle this challenge we introduced a novel method of endotoxin removal by employing an aqueous two phase micellar system using the surfactant Triton x 1 14. Use of Tritonx l H in removal of endotoxin from recombinant proteins and nucleic acid preparations is an extensively worked process but it has not been used in the case of capsular polysaccharide till date. Only known example of this technique being used in endotoxin removal from polysaccharide is for detoxification of an unknown Klebsiella Sp. However, the large scale operation and use of this technique in vaccine making is neither reported nor documented.

The removal of endotoxins using Tritonx l 14 relies on the fact that at low concentration this non- ionic detergent is miscible in aqueous buffered solution at low temperatures (below 20°C) but as the solution containing tritonx l 14 is warmed above 20°C (cloud point) it aggregates into micellar structures forming a separate phase that sequesters the hydrophobic molecules like endotoxins. The endotoxin rich phase can then be separated mechanically which leaves the hydrophilic polysaccharide (endotoxin free) in the aqueous phase. In all the methods so far known in the art had used several separate steps of dissociation and dissolution for endotoxin removal, however, the method described in this invention can achieve both these processes (dissociation and dissolution of endotoxin) simultaneously resulting in reduced process cycle as well as increased recoveries. Furthermore, the use of ethyl alcohol is also brought down by several hundred liters, thus making this invention more environmental friendly process. Use of ethanol precipitation in known methods is compared with the process of the present invention, wherein we see that, the step of ethanol precipitation is repeated twice for a total of atleast 48 hrs in the case of currently known processes.

According to the process of PRP purification currently followed in the art, post cell separation by centrifugation, concentration and diafiltration is carried for 10 hours, followed by cetrimide precipitation for 20 hours, then employing ethanol precipitation for 20 hours, which is followed by DOC precipitation for 20 hours, and again employing ethanol precipitation for the second time for 24 hours. Thereafter, concentration, diafiltration and sterile filtration is dons for 7 hours. Subsequently, Low Vacuum Drying takes place for a period of 8 days to get the PRP suitable for preparation of vaccines. Hence, we see that the presently followed process known in the art requires a total of approximately 12 days or 288 hours to complete the whole process. It is obviously very much lengthy process and also associated with high costs involved in the process.

Whereas, according to the present invention, post cell separation by centrifugation, cetrimide precipitation and passing through depth filters takes 7 hours, followed by only 4 hours of ethanol precipitation. Thereafter dissolution of precipitate in PBS and aqueous two phase separation using TritonX 1 14 takes 1 6 hours, and then collection of aqueous phase and passing through XAD column needs only 3 hours, followed by sterile filtration for 2 hours. Hence, the total process of purification and recovery of PRP is achieved within only 32 hours (approximately 2 days) as opposed to 288 hours i.e. 12 days required in the known process. Additionally, since, the process involves only 4 hours of ethanol precipitation, therefore, the process is more environment friendly, cost effective, and minimizes the chances of alcohol residues to nullity in the final product, as compared to repeated ethanol precipitation processes involved for almost 2- 3 days in the currently followed process. In both the above methods relating to purification of the capsular polysaccharide PRP of Hib disclosed above (without alcohol and with limited amounts of alcohol), it was observed that, in a production batch size of 700 litres, 70-80 gm of crude PRP was obtained, which after undergoing the purification process, yielded about 40-50 gm of PRP purified bulk suitable for further stages of vaccine formulation was recovered. This resulted in 50% to 60% recovery of purified PRP polysaccharide. Therefore, the disclosed methods of purification give considerable yield as well even without using alcohol or using drastically reduced quantities of alcohol, thereby making the process, less time consuming, environment friendly and economically cheaper process to purify and obtain pure capsular polysaccharides for vaccine formulations. Finally, when tested for quality attributes for purified PRP it was found to be in complete agreement with the recommendations by WHO for Hib PRP bulk substance which has been presented in the following examples.

EXAMPLE 3: Purification of carrier proteins

According to the present invention the purification of carrier proteins were done in the absence of alcohol. The raw materials (buffer solutions, sanitization solutions, dissolving agents, washing agents etc.,) used herein all the purification steps also doesn't contain any traces of alcoholic materials. The final purified tetanus toxoid and CRM 197 was analyzed using SEC HPLC UV detector at 280 nm. TT (Tetanus Toxoid): Following harvest, the toxin is kept for 4 weeks incubation at 37°C and kept at room temperature for 2 weeks. Impurities were removed by treating with ammonium sulphate precipitation ( 1 5%W/V). The supernatant was collected and the precipitate was discarded. The tetanus protein was precipitated by adding 37% ammonium sulphate (w/v) and dissolved in 0.5 M NaCl solution. The solution was finally diafiltered with physiological saline. The antigenic purity and formaldehyde contents were measured. The sample was subjected for SEC HPLC test for purity and further tested for absence of toxicity reversion. The purity was also checked on SDS PAGE and has shown a single major band corresponding to the 150 KD standards (Figure 12).

CRM197: After harvest, wash cells very gently with iso-osmotic 0.5M sucrose buffered with 0.03 M Tris-HCl, pH 7.8±0.2. Centrifuge to remove traces of buffered sucrose solution completely. The cells are re-suspended rapidly in cool water for osmotic shock, and vortex or agitate the whole content. The protein comes entirely in the periplasmic fraction due to this treatment. The protein is a -61 KD protein and it carries a His tag at the C-terminus and was purified to homogeneity. The protein is purified by chromatography technique. The sample was tested for DNA and endotoxin contents, SEC HPLC test for purity and further tested for absence of toxicity reversion. The purity was also checked on SDS PAGE (Figure 13) and has shown a single major band corresponding to the 67 KD standards. The purified proteins are stored in 2- 8°C according to the formulation requirement.

EXAMPLE 4: Qualitative and Quantitative Analysis of the purified bacterial CPS.

The final purified polysaccharides derived from Neisseria meningitidis, Haemophilus influenzae type b, Salmonella typhi, Streptococcus pneumonia were analyzed using Size Exclusion high Performace Liquid Chromatography (SEC HPLC) for establishing purity of the products. The results are given in the accompanying figures 2 to 1 1 which shows purity of polysaccharide and the protein used for the conjugation. All polysaccharides were measured using the RID detector of Size Exclusion Chromatography system (SEC HPLC). Existence of a sharp and single peak in the HPLC profiles confirms the presence of only the desired polysaccharide in each case, and negating all possible chances of existences of any other impurities in the samples. The purified polysaccharides are either stored in 2-8°C or lyophilized and stored in -20°C according to the formulation requirement.

Table 1. Results of N. meningitidis serogroups A, C, Y, W135 polysaccharides.

Figure imgf000025_0001

The Table 1 (Figures 2, 3, 4 and 5)describes the results of the purified N. meningitidis A, C, Y, W 135 polysaccharides. The nucleic acids and protein contents of all the serogroup A, C, Y, and W 135 are less than 1 %. The phosphorus content of the serogroup was 87 mg/g of polysaccharide whereas the remaining serogroups doesn't contain the phosphorus. The sialic acid content of serogroup C was found to be 850 mg/g of polysaccharide, 610 g/g of polysaccharide and 620 g/g of purified polysaccharide. The O-acetyl content of serogroup A was found as 2.2 mmole/g of purified polysaccharide, 1 .9 mmole/g of polysaccharide for serogroup C, 0.45 mmole/g of purified polysaccharide, 0.62 mmole/g of purified polysaccharide.

Table 2. Results of S. typhi Ty2 polysaccharides.

Figure imgf000025_0002
The Table 2 (Figure 6 and Figure 7) describes the results of the purified S. typhi Ty2 polysaccharides. The nucleic acid content of the purified 5'. typhi Ty2 shows that 8.02 mg/g, 6.32 mg/g of purified polysaccharide. The moisture content has shown 2.14% and the O-acetyl content has shown 2. 1 8 mmol/g of purified polysaccharide. The endotoxins were less than 150 IU/ of purified polysaccharide which is the allowed limit.

Table 3. Results of H. influenzae type b polysaccha

Figure imgf000026_0001

The Table 3 (figure 8 and figure 9) describes the results of the purified H. influenzae type b polysaccharides The nucleic acid content is less than 1 % and the protein content is also less than 1 %. The phosphorus content is less than 7.52%. The ribose content which is the specific test shows to be 33.32%. The endotoxin content is less than 25 EU/ng of PRP.

Table 4. Results of Streptococcus pneumoniae serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F Polysaccharides.

Figure imgf000026_0002
S. pneumonia type < 1 .0% < 1 .0% 3.75 3.9

19F

5". pneumonia type < 1 .0% < 1 .0% 3.21 0.67

23F

The nucleic acid and the protein contents in the final purified polysaccharide of all the serogroups (4, 6B, 9V, 14, 18C, 19F, 23F) are less than 1 %. The phosphorus contents are in the range of 0.38 to 3.75 mg/g of purified polysaccharides. The total nitrogen content is in the range of 0.63 to 5.9 mg/g of purified polysaccharide. The sialic acid and O-acetyl were found only in the serotype 9V and serogroup l 4. The sialic acid was found as 12% in the serotype 9V and 14% in type 14. The O-acetyl content was found as 1 .5 mmole/g, 1 .62 mmole/g of purified polysaccharide. Table 5: Results of purified Clostridium tetani toxoid

Figure imgf000027_0001

The antigenic purity of purified tetanus toxoid achieved was 1 800 Lf/mg of protein nitrogen and the free formaldehyde was found to be 0.05 g/L. (Table 5). Table 6. Results of purified CRM197 protein

Figure imgf000027_0002
The purity of CRM 197 protein on SDS PAGE has shown > 95% and the nucleic acid content is less than 1%. The endotoxin content is less than 100 EU/mg of protein. (Table 6).

EXAMPLE 5: Conjugation of polysaccharides to carrier proteins.

According the present invention the polysaccharides were selected from N. meningitidis serogroups A, C, Y, W135, Haemophilus influenzae type b, Salmonella typhi Ty2, Streptococcus pneumonea serotypes 4, 6B, 9V, 14, 1 8C, 19F and 23F. The conjugation of polysaccharides was done using the carrier proteins (Tetanus toxoid or CRM 197 which are also derived according the present invention) which is performed in controlled conditions and in the entire process of conjugation no alcohol was used.

Once it is ascertained that the PRP bulk substance fulfills all the quality requirements it is imperative to have a reliable method to produce immuno-conjugate that again is reproducible, mild, maintains the epitopic integrity of the polysaccharide and carrier protein. Towards reaching these goals the second aspect of this invention is related to the advantageous process for making a successful immuno-conjugate. The novel conjugation technique as disclosed and practiced according to the present invention relies on the finding that Cyanogen Bromide (CNBr) can very effectively cyanalate hydroxyl groups of polysaccharides in the presence of triethylamine (TEA) at neutral pH. At this pH not only the cyanate esters are very stable but also the formation of imidocarbamates and carbamates were almost negligible. This approach of cyano-transfer thereby avoids all base dependent, undesirable side reactions making this a promising method towards making an immuno-conjugate that is gentle, easy to perforin and reproducible.

According to the process generally followed in the current status of the art, Cyanogen bromide (CNBr) is used for the conjugation of Hib-PRP to Tetanus Toxoid at basic pH conditions (pH 10- 1 1 ) in presence of Sodium Hydroxide, which reacts with the hydroxyl groups to form cyanate esters. Cyanate esters formed during the conjugation process requires to be highly stable, which could further get hydrolysed and result in the formation of unwanted imidocarbonates, cyclic imido imidocarbamates or N-substituted imidocarbamates or N-substituted carbamates in presence of alkaline conditions (pH 10- 1 1 ) which is highly undesirable. Because cyanate esters are more reactive than are cyclic imidocarbamates, the amine will react mostly with the ester, yielding isourea derivatives, and partially with the less reactive imidocarbamates, yielding substituted imidocarbamates.

In TEA mediated activation, TEA acts on CNBr to form the highly reactive triethylammoniumitrile complex to yield active cynate groups and since the pH of the reaction medium remains neutral the hydrolysis of active cynate esters is prevented. Each PRP repeat units has five hydroxyl groups that are potentially amenable to activation by CNBr and eventually modification by the ADH linker.

It was observed in our experiments that CNBr surface activation using TEA as a transfer reagent instead of NaOH resulted in higher activated hydroxyls and thereby subsequent higher yields of PRP-TT immuno-conjugate almost equal to 30% to 40% immuno-conjugate. Preliminary testing of the PRP-TT conjugate produced by this new conjugation method showed good immune response in pre-clinical studies. In addition, it also meets wel l with the specification required under the prescribed guidelines of WHO. The novel process for purification, removal of endotoxin and formation of immuno-conj ugates have also been used to generate novel compositions responsible to invoke immunogenicity against infections against Hib and prevention and treatment thereof in combination with other bacterial and viral infections.

EXAMPLE 6: Formulation of polysaccharide protein conjugates:

According the present invention the vaccines were formulated both as a native polysaccharides and as well as polysaccharide conjugate vaccines. The polysaccharides were selected from N. meningitidis serogroups A, C, Y, W1 35, Haemophilus influenzae type b, Salmonella typhi Ty2, Streptococcus pneumonea serotypes 4, 6B, 9V, 14, 1 8C, 19F, and 23F. Hib-PRP-TT conjugate vaccines were formulated with appropriate stabilizers according to the present invention. The formulations were performed in controlled conditions and in the entire process no alcohol was used for any purpose. Also any raw material which derived from any animal source was not used as a buffer. Stabilizers like sucrose, trehalose maltose and lactose were used in the formulations. Both meningococcal A polysaccharide and meningococcal A polysaccharide conjugate was formulated as a liquid and then subjected for lyophilization. N. meningitidis serogroups C, Y, W 135 polysaccharides were formulated as a liquid form with or without adjuvant but not subjected for lyophillization. Pneumococcal serotypes are prepared as a liquid with or without an adjuvant (Aluminium phosphate or Aluminium Hydroxide) with no preservatives. The formulated polysaccharides and polysaccharide conjugate vaccines are stored in 2-8°C . and shown to have good stabi lity for six months at 25°C, and 1 month at 37°C and 2 years at 2-8°C.

Claims

We claim:
1 . Vaccine composition for prophylaxis against infections caused due to of N. Meningitidis, Haemophilus influenzae type 'b', Salmonella typhi, Streptococcal pneumonia comprising capsular polysachharides of N. Meningitidis, Haemophilus influenzae type 'b', Salmonella typhi, Streptococcal pneumonia conjugated to a carrier protein selected from diphtheria toxoid and tetanus toxin as antigens in presence or absence of a suitable adjuvant; and combination of sugars as stabilizers selected from sucrose, glucose, lactose, maltose and trehalose, wherein the said vaccine formulations do not contain any animal component, and alcohol.
2. Vaccine composition according to claim 1 , wherein the antigen is capsular polysaccharides of N. Meningitidis consists ofN. Meningitidis serotypes A, C, Y, W 135 cultivated in absence of any animal source in the culture media.
3. Vaccine composition according to claim 1 , wherein the antigen is capsular polysaccharides of of Haemophilus influenzae type 'b' consists of Haemophilus influenzae type 'b' polysibosylribitol phosphate (Hib-PRP) cultivated in absence of any animal source in the culture media.
4. Vaccine composition according to claim 1 , wherein the antigen is capsular polysaccharides of Salmonella typhi type 2 consists of Vi polysaccharide cultivated in absence of any animal source in the culture media.
5. Vaccine formulations according to claim 1 , wherein the antigen capsular polysaccharides of Streptococcal pneumonia consists of serotypes selected from Streptococcal pneumonia 4, Streptococcal pneumonia 6B, 9V, 14, 1 8C, 19F and 23F cultivated in absence of any animal source in the culture media
6. A method of purification, endotoxin removal and recovery of capsular polysaccharides from the crude harvest without involving alcohol comprising the following steps: a. centrifugation of culture at 8000 rotations per minute (RPM) using 100 Kd cut-off membrane in cooling conditions and collecting the supernatant;
b. concentrating the supernatnant through continuous mode centrifugation or batch mode centrifugation to one-fourth of its original size;
c. addition of 10% hexadecyltrimethylammonium bromide (cetrimide) to the concentrated supernatant of step b and incubating the same at room temperature for 3 hours at stirring conditions;
d. loading of the supernatant of step c onto depth fi lters at a flow rate of 3 L/minute till cetrimide precipitate was collected and washed with water for injection (WFI) at room temperature until conductivity of filtrate is 0;
e. dissolving the precipitate of step b and eluted using 0.5 M NaCl to give the polysaccharide elute;
f. adding the polysaccharide elute of step e with WFI diluted to get 0.2 M NaCl concentration to collect the supernatant followed by diafiltration of the supernatant against 1 0 mM Phosphate Buffered Saline (PBS) at pH 7 to obtain the partially purified capsular polysachharides;
g. endotoxin removal of the partially purified polysaccharide of step f using Triton X I 14 phase separation technique;
h. incubating the reaction sample at 30+3°C in static condition and collecting the upper layer and loaded onto ion-exchange XAD matrix to remove residual triton X I 14; i. diafiltration of the capsular polysaccharide elute of step h using 20 mM PBS buffer at pH 7;
j. sterile filtration of the polysaccharide of step i using a 0.22 μιτι capsule filter to obtain the purified capsular bulk polysaccharide.
7. A method of purification, endotoxin removal and recovery of capsular polysaccharide polyribosylribitol phosphate of Haemophilus influenzae type 'b' (Hib-PRP) comprising:
a. cetrimide precipitation and passing through depth filters;
b. ethanol precipitation for 4 hours;
c. dissolution of precipitate in PBS buffer followed by aqueous two phase separation using TritonX l 14; d. collection of aqueous phase and passing the partially purified polysaccharide through XAD4 column followed by sterile filtration to obtain the purified Hib-PRP polysaccharide.
8. The method according to claim 5, 6 and 7, wherein the recovery of purified Hib-PRP is 40%- 50% from the crude Hib-PRP.
9. The method according to claim 6 and 7, wherein the recovery of purified Hib-PRP is completed within 32 hours post cell harvest including ethanoi precipitation for maximum of 4 hours involved in the recovery process.
1 0. A method of purification of conj ugate proteins selected from Tetanus Toxoid and CRM 197, wherein the purification of conjugate proteins do not involve the use of alcohol.
1 1 . A method of conjugation of Hib-PRP polysaccharide to conjugate protein, the said conjugate protein selected from Tetanus Toxoid and CRM 1 97, through bifunctional linker ligand adipic dihydrazide (ADH), in the presence of Cyanogen Bromide at a neutral pH using Tri- Ethanol Amine so that the alkaline hydrolysis of cyanate esters to carbamide impurities are prevented.
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