WO2009016651A1

WO2009016651A1 - Simplified means for obtaining prn from bordetella pertussis

Info

Publication number: WO2009016651A1
Application number: PCT/IN2008/000343
Authority: WO
Inventors: M.L. Mago; Rajesh Jain
Original assignee: Panacea Biotec Limited
Priority date: 2007-07-31
Filing date: 2008-05-30
Publication date: 2009-02-05
Also published as: CL2008002003A1; AR067471A1

Abstract

A component vaccine against pertussis obtaining Pertactin (PRN) antigen from Bordetella Pertussis. A simplified process for extraction and purification of Pertactin (PRN). Pertactin obtained by this method has a purity level of more than 90% and the process used in the present invention prevents the loss of PRN by avoiding complex processing steps. The outer membrane protein (OMP) purified by the method of present invention utilizes a simplified process and is useful in the preparation of Acellular Pertussis Vaccine either as stand alone or as peart of the combination Vaccines.

Description

SIMPLIFIED MEANS FOR OBTAINING PRN FROM BORDETELLA

PERTUSSIS

FIELD OF INVENTION The present invention relates to a component vaccine against pertussis obtaining

Pertactin (PRN) antigen from Bordetella Pertussis. The present invention particularly relates to a simplified process for extraction and purification of Pertactin (PRN). Pertactin obtained by this method has a purity level of more than 90% and the process used in the present invention prevents the loss of PRN by avoiding complex processing steps. The outer membrane protein (OMP) purified by the method of present invention utilizes a simplified process and is useful in the preparation of Acellular Pertussis Vaccine either as stand alone or as part of the combination Vaccines.

BACKGROUND OF THE INVENTION Whooping cough (Pertussis) is highly contagious respiratory infection that is caused by the bacteria Bordetella pertussis. Recent estimates from WHO suggest that, in 2003, about 17.6 million cases of pertussis occurred worldwide, more than 90% of which were n developing countries, and that about 279,000 patients died from this disease. It is further estimated that, in 2003, global vaccination against pertussis averted about 38.3 million cases and 607,000 deaths. Whooping cough commonly affects infants and young children, but can be prevented by immunization with the aforesaid Vaccine.

In the past, vaccines which were used to immunize infants and children were composed of whole cells of Bordetella pertussis inactivated by chemical agents or heat. The whole cell pertussis vaccine is prepared by growing known strain(s) of the Bordetella pertussis organism in a defined medium for several hours in a Fermentor, until the organism reaches certain previously defined parameters. At this point of time, the organism is harvested and treated with a chemical agent, such as formaldehyde, which kills the organism and detoxifies proteins present in the supernatant and in the organism itself. After allowing the harvest to stand for a specified time to ensure that this detoxification procedure is complete, the cells are separated from the supernatant by passing the mixture through a continuous centrifuge, to provide a packed mass of cells and the supernatant, which is discarded. The cells arc then resuspended in a solution of sodium chloride, that, when diluted to a known concentration usually determined by the opacity of the suspension, is used as an immunizing agent. This suspension then undergoes extensive Quality Control testing procedures and if found as per specifications is injected into animals in which it elicits antibodies that are protective against the disease.

The use of these vaccines, however, had a number of drawbacks in administration and in the adverse side effects that resulted from their administration. This "whole cell" vaccine is known for giving minor local reactions at the injection site with occasionally more severe overall reactions, such as elevated temperature and other complications. There has been speculation that the whole cell vaccine is also responsible for some neurological reactions in infants. Several investigations had lead to the findings, that these side effects were attributed to the presence of 'other substances' in addition to the antigenic components required for eliciting protective immunity.

This has lead to the need for a more defined vaccine and thus considerable effort has been directed by several manufacturers and researchers towards the development of an efficacious pertussis vaccine with minimal undesirable side effects. The pathogenic mechanisms of Bordetella pertussis have been studied to determine which antigenic components contribute to protective immunity. The specific antigens so identified and extracted were then incorporated in an 'Acellular Pertussis Vaccine' which would confer the desired immunity to disease without introducing irrelevant and possibly undesirable substances in the vaccine, hence free of possible side effects associated with them.

Commonly purified and isolated antigenic components include pertussis toxin (PT), also known as lymphocytosis promoting factor (LPF), filamentous haemagglutinin (FHA), Fimbriae (FIM) and 69 kD outer membrane proteins also known as Pertactin (PRN) and are preferred for inclusion in an Acellular Pertussis Vaccine.

A large amount of work done on Acellular Pertussis Vaccines has concentrated on a PT-based vaccine. However, results indicated that a vaccine consisting exclusively of PT-toxoid only partially protected children from the infection. A PT/FHA combination showed slightly higher efficacy but this was still lower than that obtained for the whole- cell vaccine.

Another antigenic component which is studied extensively for incorporation in the Acellular Pertussis Vaccine is an outer membrane protein, with a molecular weight of around 69,000 daltons known as Pertactin (PRN) which is found in almost all virulent strains of Bordetella pertussis. Many scientific papers reveal the purification process of this membrane protein from fermentation broth as well as from cell biomass. US patent no. 6,444,211, describes method for extraction with 4 molar urea and purification of Pertactin using hydroxyapatite column. This process at our hands, leads to lower yields and low purity profile of the antigen.

U.S. patent no. 5,101,014 describes extraction and purification involving heat treatment and centrifugation, followed by DEAE-Sepharose ion-exchange chromatography and protein-specific, dye-ligand gel chromatography. This method however was regarded as complex and resulted in lower yields and was economically non- viable. After heat treatment it was identified that the 69 kD protein is released into solution as a very small percentage of the total protein.

European Patent EP-A-O 437 687 sets out to improve the efficiency of release of 69 kD protein from the culture broth by using a repetitive heat extraction process involving a plurality of extraction steps in series.

It is a known fact that established procedures for purification of Pertactin do not lead to higher yields as compared to the other antigens such as Pertussis toxin and Filamentous haemagglutinin.

Thus, there still existed a need for the development of an efficient method for the significant quantity of extraction and purification of Pertactin. Also there is a need of the method so developed, to be well suited and be applicable to commercial scale production.

Through a series of experimentations and expenditure of intellectual efforts, it has been possible to develop a procedure by which purification of PRN can be achieved using simple, easy and reliable steps. The present invention provides a simplified process of extraction and purification of Pertactin (PRN) from Bordetella pertussis. The process used is simple and utilizes purification steps which provide at the same time significant yield of PRN of good purity level. The protein (PRN) purified by the method of present invention is useful in the preparation of acellular Bordetella pertussis vaccine comprising of PRN as a key component. Moreover, the present invention is an attempt to overcome the shortcomings of Pertactin purification production by way of providing a simple process for the manufacture of an outer membrane protein which is more than 90% pure and has a yield better than produced by other methods heretofore known in the art.

SUMMARY OF INVENTION

It is an objective of the present invention to provide a simplified process of obtaining more than 90% pure PRN wherein the process of obtaining PRN comprises of the following steps a) centrifuging the harvested culture of Bordetella pertussis Tohama I strain to segregate the cell biomass from the supernatant, b) preparing a suspension of cell biomass which is stirred with urea to get an extract comprising mixture of PRN and other membrane proteins, c) subjecting directly the extract to a centrifugation cycle, and providing supernatant containing 69kD membrane protein, d) diafϊltering the supernatant by using single membrane filter (30 kD cassette), e) precipitating PRN (by ammonium sulphate) and subjecting it to centrifugation at high speed, f) preparing the aqueous solution of PRN and passing it through a series of Hydroxyapatite and ion exchange matrix systems, under conditions sufficient to retain high molecular weight proteins contaminants, g) load the effluent mixture comprising PRN into affinity column, wherein it is bound to the matrix h) recovering the bound PRN with an elutant.

It is also an objective of the present invention to provide a simplified process of obtaining more than 90% pure PRN wherein the cell biomass of B. pertussis is suspended in a concentration of about 20% w/v, in the phosphate buffer saline comprising urea.

It is also an objective of the present invention to provide a simplified process of obtaining more than 90% pure PRN wherein the extract is directly subjected to a centrifugation cycle, thereby avoiding ultrasonification step and providing a least contaminated supernatant containing 69kD membrane protein.

It is also an objective of the present invention to provide a simplified process of obtaining more than 90% pure PRN wherein the diafiltration and concentration is done by using 30 kD membrane filter. Use of a single diafiltration step avoids the loss of PRN that occurs during multiple or repetitive diafiltration steps.

It is also an objective of the present invention to provide a simplified process of obtaining more than 90% pure PRN wherein aqueous solution of PRN passed through a series of hydroxyapatite and Q-Sepharose column at a conductivity of between 1 to 50 mS/cm preferably between 3 to 5 mS/cm and the pH ranges from between 7 to 10, preferably between 7.5 to 8.5. The use of specific conductivity and pH are important to minimize the loss of PRN during extraction and the process gives unbound PRN in the flow through.

It is also an objective of the present invention to provide a simplified process of obtaining more than 90% pure PRN wherein the flow through containing PRN along with other proteins is loaded on to the Cibracron blue F3GA dye affinity column. The PRN is obtained as a bound fraction, which is finally eluted with suitable elutant.

It is also an objective of the present invention to provide a method of preparation of Acellular Pertussis Vaccine against whooping cough comprising PRN as one of the antigenic component obtained by the simplified process of the present invention.

It is yet another objective of the present invention to provide a method of preparation of Acellular Pertussis Vaccine against whooping cough comprising PRN obtained by the simplified process of the present invention in combination with the other components selected amongst PT, FHA, FIM and thereof.

A further objective of the present invention is to provide a multivalent vaccine composition comprising PRN and other Acellular Pertussis components obtained by the simplified process of the present invention in combination with at least one of the immunogens selected amongst the group of diphtheria toxoid, tetanus toxoid, capsular polysaccharide of Haemophilus influenzae, outer membrane protein of Haemophilus influenzae, hepatitis B surface antigen, polio, mumps, measles and/or rubella. The multivalent vaccine compositions as provided herein may further comprise an adjuvant selected amongst aluminum phosphate, aluminum hydroxide, Quil A, QS21, calcium phosphate, calcium hydroxide, zinc hydroxide, a glycolipid analog, an octodecyl ester of an amino acid or a lipoprotein.

It is yet another objective of the present invention to provide a method of preventing whooping cough in the human, by administering vaccine composition that comprises of PRN as one of the antigenic component obtained by the simplified process of the present invention.

The details of one or more embodiments are set forth in the description below. Other features, objects and advantages will be apparent from the description and claims.

DESCRIPTION OF THE INVENTION

The present invention is not limited to the particular process steps and materials disclosed herein, but are extended to equivalents thereof as would be recognized by those skilled in the relevant arts. It should be understood that the terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting.

It has been observed through series of experimentation that PRN though can be obtained from the conventional processes described in many prior publications, however, the reported procedure so far are not only complex and expensive but also time consuming. It was also noticed that due to heat or chemical inactivation procedures used in the past and the multiple steps of Ultrafiltration and Diafiltration used in many of the current processes would ultimately results in low yield of PRN. Thus, we have continuously worked on these shortcomings and now came out with a procedure which is simple, reliable, less time consuming and economic. The present invention relates to the simplified process for the purification of PRN requiring less complex processing steps such as using minimal Diafiltration steps, avoiding heat or chemical treatment. To further improve the yield of PRN, single membrane cassettes have been used for the diafiltration process instead of the repetitive and multiple membrane cassettes used in previously described methods. Additionally, we have combined ion exchange chromatography with AFFIGEL BLUE affinity chromatography under regulated conditions to obtain pure PRN. With the present invention good amount of PRN, which was more than 90% pure was obtained.

The genus Bordetella includes seven species. The most studied species are B. pertussis, B. parapertussis, and B. bronchiseptica. Whooping cough is caused by Gram negative bacteria Bordetella pertussis. In particular, Bordetella pertussis Tohama phase I strain

(Infection and Immunity, Vol. 6, p. 89, (1972)] is maintained in the stock culture collection of National Institute of Health, Ministry of Social Welfare, Tokyo, Japan

(NIHJ 1052), deposited under accession number IFO 14073 and also the strain is available in the stock culture collection of ATCC (American Type Culture Collection),

USA as Strain No. BAA-589 which has been employed in the present invention.

B. Pertussis causes immunogenic responses in body due to various factors, such as pertussis toxin (PT), also known as lymphocytosis promoting factor (LPF), filamentous hemagglutinin (FHA), Fimbriae (FIM), adenylate cyclase and 69 kD outer membrane proteins also known as Pertactin (PRN) wherein preferred embodiment suggests inclusion of PRN (Pertactin) in an Acellular Pertussis Vaccine..

The term "PRN" as used herein is Pertactin. It is the protein present on the outer membrane of the Gram negative bacterium Bordetella pertussis and acts as an antigen in the mammalian body and has a molecular weight of 69,000. It is an important component of the vaccine described by the preferred embodiment of the present invention.

The term "pure PRN" as used herein, refers to the Pertactin which is substantially or at least 90% pure and is essentially free of other proteins such as PT, FHA and other contaminating proteins. The pure PRN obtained after the purification and detoxification can be used as one of the antigenic component for the preparation of Acellular Pertussis Vaccine.

In the present invention the term "Culture" refers to the growing of Bordetella pertussis cells under artificially controlled conditions in the Fermentor i.e. in vitro which also includes addition of carbon, oxygen and energy sources for the growth of said cells.

In the present invention, after the centrifugation of cultured cells, the Fermentor harvest gets separated into a clear supernatant from which PT and FHA are purified and the Cell Biomass from which the Pertactin is purified.

Bordetella pertussis can be cultured by various methods. Various culture media known in the art can be employed, such as Cohen- Wheeler, Stainer-Scholte, Verwey Medium, B2 Medium or other similar liquid media, with the preference given to modified Stainer-Scholte medium. Conventional methods and temperature conditions for harvesting the culture of virulent B. pertussis were used.

The term "elutant" used herein refers to range of solvent systems used in the preferred embodiment of the present invention for the recovery of PRN from the various chromatographic columns used in the present invention.

In one of the embodiment of the present invention, Bordetella pertussis culture is subjected to centrifugation and filtration and is separated into cell Biomass and culture supernatant. However, prior to centrifugation cell culture is not subjected to any inactivation procedure by any means. The supernatant contains FHA and PT along with other impurities and the cell biomass contains PRN along with other impurities. In the preferred embodiment of the present invention, cell biomass is used as a preferred source for obtaining PRN. However, their centrifuged supernatant is rendered bacteria- free by passing it through membranes which can retain bacteria.

The cell paste obtained from the cell culture is used for the preparation of a 20% w/v cell suspension in 0.01 M phosphate buffer saline at pH 7.5 containing around 4M urea. After the subsequent stirring for 2 hours the suspension is subjected to centrifugation to obtain the supernatant. The use of 20% w/v cell suspension is an advantage over the previous methods as it leads to the optimized and highest reported extraction of PRN from the cell biomass. Further this single step extraction using urea for membrane disruption is advantageous in making process simpler and less time consuming. The repetitive and plurality of steps used for extraction of PRN does not act as advantageous is terms of yields.

The supernatant hence obtained is diafiltered and concentrated through single 30 kD.

Cassette membrane against phosphate buffer saline of low molarity is at pH from about 7 to 10. The use of single 30 kD cassette membrane minimizes the loss of PRN which occurred if multiple diafiltration membranes are used. In a preferred embodiment of the present invention supernatant is filtered only once with 30 IcD membrane, which is an advantage over existing prior art, where supernatant is subjected to at least two different Diafiltration steps, which leads to drastic decrease in the yield of PRN. This loss of PRN is usually attributed to the ability of proteins to bind with the membrane filters to some extent from where it is not recovered. This particular exclusion of multiple diafilters not only saves time but is an economic means for the Diafiltration and concentration of PRN.

In a preferred embodiment of the present invention, ammonium sulphate is slowly added to the concentrate obtained after Diafiltration, at the room temperature for precipitation of the PRN. Polyethylene Glycol (PEG) may also be used as a different precipitating agent. Particularly, ammonium sulphate used in the present invention is in the concentration range from about 25% w/v to about 35% w/v is used and more preferably, 30 % w/v ammonium sulphate. The solution is then stirred continuously at 2-8⁰C for 2 hours. The precipitated PRN is subjected to centrifugation at higher speed of about 8,000 rpm to about 10,000 rpm and dissolving it in Tris Buffer at a pH from about 7 to 10 and more preferably from about 7.5 to 8.5, containing Ammonium sulphate to achieve final conductivity between 3 to 5 mS/cm and the supernatant containing the PRN is filtered through 0.2μ membrane. The molarity of Tris buffer may range from 0.01 M to 3.0 M, preferably from 0.01M to about 0.05M. The use of high speed centrifugation is important to completely extract the PRN which is substantially free of other protein contaminants.

In one of the preferred embodiments of the present invention, the precipitate comprising PRN is dissolved in Tris Buffer of molarity between 0.01M to 3.0M, pH 7.5 and conductivity 3 to 5 mS/cm and is subjected to further purification by the use of sequential chromatographic steps. The hydroxyapatite and Q-Sepharose columns both are packed and equilibrated with Tris Buffer containing 0.015 M ammonium sulphate such that final conductivity is between 3 mS/cm to 5 mS/cm, more preferably conductivity is 3.4 mS/cm. Now the solution prepared after dissolving the precipitate in Tris buffer is loaded on the hydroxyapatite column and flow through is collected which contains the PRN. The flow through collected from hydroxyapatite column is loaded onto Q-Sepharose column and collected thereafter. Buffer systems used in present system may be any of those commonly used for the extraction and purification of biological and well known in the art. Examples of buffers used include phosphate, borate, acetate, carbonate, tris, and ammonium buffers. More preferably, phosphate buffer and tris buffers are used, having molarity ranges from 0.01M to 3.0M, preferably 0.01M to about 0.05M. In an embodiment of the present invention, the pH may range from 7 to 10, more preferably the pH ranges from 7.5 to 8.5. The mild conditions of pH and ionic strength are important during the chromatographic extraction of PRN, since it is obtained as an unbound fraction in the flow through, whereas other high molecular weight pertinacious impurity gets bound on the columns.

In one of the preferred embodiments of the present invention, PRN thus obtained is further recovered by using suitable affinity column chromatography, containing PRN specific binding medium/ligand. The ligand is selected from but not limited to a group comprising Affi-Gel.Blue (Bio Rad), Blue-Sepharose CL-6B, Red Sepharose CL-6B or combination thereof. In a preferred embodiment, Affigel Blue is used, which has good adsorbent characteristics and the capability to retain 69 kD protein. The flow through obtained after the treatment with Q sepharose column is filtered through 0.2μ membrane filter. The affinity column is then equilibrated with suitable buffer preferably Tris Buffer of low ionic strength and mild pH, followed by loading of the PRN solution on to it, and the column is washed with the equilibration buffer to wash off the undesirable proteins.

The PRN now needs to be eluted from the matrix of Affigel Blue. The elutant is selected from but not limited to a group comprising various salt solutions such as Magnesium chloride, Sodium chloride, Potassium chloride or combination thereof. In a preferred embodiment, the elutant used is Magnesium chloride, at a concentration gradient from 0.1M to 0.5 M in 0.05 M Tris Buffer at a pH of 7.4. This differential elution of PRN using increased concentration of Mg++ ions is important for the high purity of PRN. The PRN so obtained is dialyzed against 0.01 M PBS having a pH of 7.4 to remove Magnesium chloride. The dialyzed residue is filtered through a 0.2 μ membrane and is further subjected to detoxification step known in the art. PRN obtained after purification process is detoxified by using one of the conventional method so that it can be used for further vaccine preparation. In a preferred embodiment of the present invention, PRN was taken at a concentration of around 100 μg/ml in 0.01M PBS buffer having pH of 7.2. To this solution 1% formaldehyde is added till its final concentration in the resultant solution reaches 0.025%. The solution is mixed well and kept stable for 7 days at a temperature of 37⁰C. Then solution is dialyzed against 0.01M PBS buffer at pH of 7.2 having 0.01% formaldehyde and 0.02% Tween-80 at a temperature of 2° to 8° C. The solution obtained is filtered through 0.2μ membrane filter. To the filtrate 2-phenoxyethanol is added to make the resultant solution having a concentration of 5 mg/ml of 2- Phenoxyethanol.

Vaccine compositions comprising PRN obtained using the process of the present invention is within the scope of the present invention. Further, the vaccine compositions of the invention are not limited to inclusion of PRN and may also comprise a mixture of other antigenic factors of Bordetella species, in an amount which is sufficient to induce an immunogenic or protective response in vivo and a pharmaceutically acceptable carrier therefore and where appropriate, with an adjuvant selected amongst aluminum phosphate and aluminum hydroxide gel etc.

In one of the preferred embodiment, the whooping cough or pertussis vaccine as herein described may also be used in combination with other vaccines currently available in the market, for example, those of the vaccine against diphtheria, polio, tetanus, Hepatitis B or diseases caused by Haemophilus or, with any other immunogenic constituent, for example, a particular inactivated pathogenic agent or toxin.

The state of the art show that the Acellular Pertussis Vaccines tested, monovalent (PT), bivalent (PT, FHA), trivalent (PT, FHA, PRN), or pentavalent (PT, FHA, PRN, AGG2, AGG3) induce very few side effects, are all immunogenic and all have an efficacy against the disease (according to WHO definition) which is greater than or equal to 70%. The compositions of the preferred embodiment can be included in these vaccines and other Acellular Pertussis Vaccines. In one of the preferred embodiment of the present invention, these multivalent vaccine composition may comprise about 3 to about 15 μg protein nitrogen of PRN, about 5 to about 30 μg protein nitrogen of Pertussis Toxoid, about 5 to about 30 μg nitrogen of Filamentous Haemagglutinin, and optionally about 1 to about 10 μg protein nitrogen of agglutinogens .

The immunization schedule will depend upon several factors, such as the susceptibility of the host to infection and the age of the host. A single dose of the vaccine of the invention can be administered to the host or a primary course of immunization can be followed in which several doses at intervals of time are administered. Subsequent doses used as boosters can be administered as need following the primary course.

The PRN hence obtained is of high purity in the range of at least 90%, more preferably PRN obtained through the process as described in the present invention can have a purity profile of 90% or more. Thus, the products and processes of the present invention are characterized by the advantages of simplicity of the process steps and of the techniques involved, common availability of the materials and the apparatus used, rapid processing and easy scale up for industrial production while providing for good yields, protection against external contamination, and purity by specific selectivity for the antigens rather than unwanted substances from the fermentation broth.

The following examples are used to further illustrate the present invention and advantages thereof. The following specific examples are given with the understanding that these are intended to be illustration without serving as a limitation on the scope of present invention.

Example 1

This example illustrates the growth of B. Pertussis cells in defined culture medium A culture of B. Pertussis Tohama I strain is fermented in modified Stainer Scholte medium according to the methods and conditions described in literature (Stainer, D. W. and Scholte, M. J. 1971 J. Gen. Micro biol. 63: 211-220), which is incorporated herein by reference. 4% of grown culture of B. Pertussis is inoculated in the Fermentor containing medium (modified Stainer Scholte) for 44- 48 hours, the temperature of the fermentation is 35^°C and stirrer speed is about 100 to 200 revolution per minute depending upon the oxygen requirement which is kept around 50 %. The process may include supply of Oxygen from outside sources. During the process of fermentation many growth factors, energy and carbon sources are added in the medium to increase the antigenic yield. After the fermentation process, the harvested culture is centrifuged to obtain bacterial biomass and the supernatant. Cell biomass obtained from centrifugation is further processed for extraction and purification of PRN protein.

Example 2 This example illustrates the extraction of PRN from cell biomass.

Cell paste obtained from example 1 (20% w/v) is suspended in phosphate buffered saline (0.01M sodium phosphate, pH 7.5) containing 4M urea and the suspension stirred for 2 hrs. at room temperature. Cell debris are removed by centrifugation and Supernatant obtained from centrifugation diafiltered through 30 kDa NWML cassette membrane against phosphate buffered saline, preferably 0.0 IM sodium phosphate at pH 7.5.

Example 3

This example illustrates the precipitation and centrifugation of PRN using Ammonium sulfate.

The filtrate from example 2 is precipitated by the slow addition 30% w/v of ammonium sulphate at room temperature and the mixture is allowed to stir at 2° to 8° C for 2 hrs. After that the precipitate is centrifuged at 9000 rpm and dissolved in 0.01 M Tris Buffer, at pH 7.5, and saturated ammonium sulphate solution added to achieve final conductivity corresponding to that of 0.01 M Tris Buffer at pH 7.4, containing 15 mM ammonium sulphate (3.5 mS/cm). After centrifugation at 9000 rpm, the supernatant containing the PRN is filtered through 0.2μ membrane. The PRN containing solution is then subjected to chromatographic purification through hydroxyapatite and Q- Sepharose column as described in Example 4.

Example 4

This example illustrates the purification of PRN by using hydroxyapatite and Q- Sepharose column chromatography.

The PRN obtained from example 3 is further mixed with aqueous solution containing 0.01 M Tris Buffer, pH 7.5, containing 15 mM ammonium sulphate (conductivity approximately 3.4 mS/cm). Hydroxyapatite is packed into a suitable size column, preferably 5 cm [D] x 10 cm [H] and equilibrated with 0.01 M Tris Buffer, pH 7.5, containing 15 mM ammonium sulphate (conductivity approximately 3.4 mS/cm). The aqueous solution is then loaded on to Hydroxyapatite column and flow through containing PRN is collected. This flow through containing PRN is further subjected to Q-Sepharose column, which is packed into a similar column and equilibrated with the same buffer as that of Hydroxyapatite column. Both the columns can be connected in series. The PRN containing solution (from Example 3) when subjected to chromatographic purification on the two columns in series did not bind to the matrices but is obtained as an unbound fraction in the flow through.

Example 5 This example illustrates the purification of PRN by using Affigel Blue chromatography.

The flow through of Q-Sepharose column as described in the example 4 is passed through 0.2μ membrane filter and is further subjected to another step of chromatographic purification through Affigel Blue column, wherein the Affigel Blue column is packed and equilibrated with 0.05M Tris Buffer at pH 7.4. The filtrate is loaded on the Affigel Blue column that results in adsorption of PRN antigen on to the matrix of Affigel blue thereafter column is washed with the equilibration buffer to ward off the undesired proteins. The PRN which has been adsorbed by the Affigel Blue matrix is eluted using MgCl₂ at a linear concentration gradient from 0. IM to 0.5 M in 0.05 M Tris Buffer ( pH -7.4). The PRN so obtained is dialyzed against 0.01 M PBS, pH of 7.4 to remove excessive Magnesium chloride. The dialyzed residue is filtered through a 0.2 μ membrane and is further subjected to detoxification step.

Example 6 This example illustrates the Detoxification of PRN.

Although the PRN obtained from the down streaming process is highly purified but it is not suitable to be used for the preparation of vaccines as it is still toxic. So, it is further subjected to the process of detoxification wherein PRN is taken at a concentration of about 100 μg/ml in 0.01M PBS buffer having pH of 7.2. To this solution 1% formaldehyde is added till the final concentration of the resultant solution becomes 0.025%.

The solution is mixed well and kept stable for 7 days at a temperature of 37° C. After 7 days the solution is dialyzed against 0.01M PBS buffer at pH of 7.2 having 0.01% formaldehyde and 0.02% Tween-80 at temperature of 2° to 8°C. The solution obtained is filtered through 0.2μ membrane ^v and to the filtrate 2-phenoxyethanol is added to make the resultant solution having a concentration of 5 mg/ml. The final solution of formalin treated PRN is stored at 2° to 8° C.

Particular steps and process for purification of PRN from Bordetella species have been described and also Pertactin as one of the essential component of Acellular Pertussis Vaccine or of multivalent vaccines. It is apparent that various modifications and combinations of the purification and extraction steps detailed in the text can be carried out without departing from the spirit and scope of the invention.

Example 7

Procedure for Extraction and Purification of PRN

Take B. Pertussis cell paste i

Prepare a 20 % w/v cell suspension in 0.01 M Phosphate buffer saline, pH 7.5 containing 4 M Urea

1

Keep on stirring for 2 hrs. Centrifuge and take the supernatant

I

Diafilter and concentrate the supernatant through 30 kD cassette membrane ;

Add 30% w/v Ammonium Sulphate and keep it on stirring for 2 hours.

Centrifuge at 9000 rpm and dissolve the prec Iipitate in a buffer filter through 0.2 μ membrane . I

Pack Hydroxyapatite column (Bio-Rad) and equilibrate with 0.01 M Tris Buffer, pH 7.5 containing 15 mM Ammonium sulphate such that final conductivity is 3.4 mS/cm i

Load the precipitate solution on Hydroxyapatite column and collect the flow through I

Pack Q-Sepharose column and equilibrate with 0.01 M Tris Buffer, pH 7.5 containing 15 mM Ammonium sulphate such that final conductivity is 3.4 mS/cm

I

Load the flow through of hydroxyapatite column on Q-Sepharose column and collect the flow through

I

Filter the flow-through through 0.2 μ. Membrane

I Pack Affigel Blue column and equilibrate with 0.05 M Tris Buffer, pH 7.4 and load the sample.

I

Wash the column with equilibration buffer

Elute PRN from Affigel blue colum In using a gradient OfMgCl₂

I

Dialyze the PRN elutes against 0.01 M PBS, pH 7.4

Filter through 0.2 μ

Purified Pertactin Toxin is rea Idy for detoxification.

Claims

1. A component vaccine against pertussis comprising Pertactin (PRN) from acellular Bordetella pertussis membrane protein.

2. A simplified process of obtaining more than 90% pure Pertactin (PRN) according to claim 1, which comprises of : a. centrifuging the harvested culture of Bordetella pertussis Tohama I strain to segregate the Cell Biomass from supernatant, b. preparing a suspension of Cell Biomass which is stirred to get an extract comprising mixture of PRN and other membrane proteins, c. subjecting directly the extract to a centrifugation cycle, and providing a less contaminated supernatant containing 69 IcD membrane protein, d. diafiltering and concentrating the supernatant by using a single membrane filter, e. precipitating PRN and subjecting it to centrifugation at high speed, f. preparing the aqueous solution of PRN and passing it through a series of hydroxyapatite and ion exchange matrix systems under conditions sufficient to retain high molecular weight proteins contaminants, g. loading the flow through mixture comprising PRN into affinity column, wherein PRN gets bound to the matrix, h. recovering the bound PRN with a suitable elutant.

3. The process according to claim 2, wherein the cell biomass of B. pertussis is suspended in a concentration of about 20% w/v, in the phosphate buffer saline comprising urea.

4. The process according to claim 2, wherein the single membrane filter is having a molecular weight cutoff of about 30 kD.

5. The process according to claim 2, wherein the PRN is precipitated by adding ammonium sulphate to the filtrate.

6. The process according to claim 5, wherein filtrate is further subjected to centrifugation at about 9000 rpm.

7. The process according to claim 5, wherein the precipitated PRN is dissolved in Tris Buffer solution at pH not less than 7.

8. The process according to claim 7, wherein the buffer solution has the conductivity of about 3 to 5 mS/cm.

9. The process according to claim 2, wherein the aqueous solution of PRN is passed through hydroxyapatite at conductivity of 3.4 mS/cm and pH of about 7.4.

10. The process according to claim 9, wherein flow through obtained from hydroxyapatite column is further subjected to ion exchange column at conductivity of 3.4 mS/cm and pH of about 7.4.

11. The process according to claim 2 or 10, wherein the ion exchange column is Q- Sepharose column.

12. The process according to claim 2, wherein affinity column is packed with cross- linked gel covalently attached to Cibacron blue F3GA dye.

13. The process according to claim 2, wherein the elutant is Tris-buffer containing ammonium sulfate.

14. The process according to claim 13, wherein the elutant comprises salt solution in Tris-buffer having pH of 7.4.

15. A method of using component vaccine according to claim 1, which comprises administering to a subject in need thereof an effective amount of the component vaccine.

16. The process for the preparation of obtaining more than 90% pure Pertactin (PRN) according to claim 1, substantially as herein described and illustrated by the examples.