DESC:FIELD OF THE INVENTION:
The present invention relates to a rapid process for producing and purifying Haemophilus influenzae type b polyribosyl ribitol phosphate polysaccharides (Hib-PRP). More specifically, the present invention relates to a process for preparing Hib-PRP capable of being used as such, or of being derivatized or linked to other molecules to make vaccines for the prevention of invasive diseases caused by Haemophilus influenzae type b bacteria (Hib).

BACKGROUND OF THE INVENTION:
Polysaccharides, especially antigenic polysaccharides, are used in preparation of vaccines. Monovalent, divalent and poly (multi) valent vaccines containing one, two or more polysaccharides are available in the market for prevention of certain diseases or infections caused by various microorganisms. The multivalent polysaccharide vaccines have been used for many years and have proved valuable in preventing diseases such as Pneumococcal, Meningococcal or Haemophilus influenzae diseases. Surveillance data gathered in the years following the introduction of the vaccine Prevnar has clearly demonstrated the reduction of invasive pneumococcal disease in US infants. However, in spite of several studies carried out on these polysaccharides, there is a need for improving yields as well as quality (purity) of the polysaccharides as evidenced by the continuing research.

Haemophilus influenzae is gram-negative pleomorphic coccobacilli, which causes an array of diseases such as meningitis, epiglottitis, pneumonia, septic arthritis, and respiratory tract infections such as bronchitis and otitis media. It has six capsular serotypes with nomenclature from type (a) through type (f). The serotype (b) designated as Haemophilus influenzae type b (Hib) has been observed as the most invasive. The immune response against Hib has been achieved by vaccination.

Immune response may be differentiated as T-cell dependent (TD) immune response and T-cell independent (TI) immune response. Proteins and peptides are known to elicit TD antigens by stimulating the helper T lymphocytes and generating memory T and B lymphocytes. In contrast, polysaccharides are TI antigens which do not involve T-cell activation and do not form memory B cells which is a major drawback while dealing with infants as they have an immature immune system

The production of the first Hib polysaccharide vaccine was accomplished in the early 1970s as there was an urgent need to combat this fatal disease. Later it was observed that the vaccines were not very efficient in children below two years of age as they have poorly developed immune system. This observation lead to further research resulting in the production of Hib polysaccharide-protein conjugates vaccines in late 1980s.

Evidence collected through numerous research findings defines the immunogenic aspect of the polysaccharide conjugate vaccine. However, there is very little information to aid the production and purification of the capsular polysaccharide of Hib-PRP.

The production of purified Hib-PRP is the foremost requirement for an effective conjugation with the carrier protein and its development as a conjugate vaccine. The cost of the cultivation and the purification of Hib-PRP is generally high and involves long working hours since it involves a series of production and purification steps. Any improvement in one or more of the steps of PRP production without compromising on quality would bring a significant change in the overall conjugate vaccine production and consequently makes the process relatively cost effective.

However, despite several studies that have been carried out on these polysaccharides, there has always been a need for improving yields as well as quality/purity of the polysaccharides in order to produce vaccines of high quality.

Studies such as the disclosure in the article published in Applied and Environmental Microbiology, Feb.2001, p. 969-971, Vol.67, No.2, titled “Production of capsular polysaccharide of Streptococcus pneumoniae Type 14 and it’s purification by Affinity chromatography,” mention a method which involves serotype specific anion exchange chromatography. This is a time consuming, and tedious process with lower yields.

Also the PCT Application No. WO 2011148382 A1 describes the method of preparing pure capsular polysaccharide using aluminium phosphate with alcohol for the purification of capsular polysaccharides of Haemophilus influenzae b, Neisseria meningitidis such as serotypes A, C, Y, W- 135 and other similar related capsular polysaccharides produced form both gram negative and gram positive microorganisms using aluminium phosphate with alcohol. Another patent US 7,582,459 B2 describes a method for producing a capsular polysaccharide for the use in conjugate vaccines using Hexa Cetyl trimethylammonium bromide (CTAB), ethanol and sodium deoxycholate taking a total time of 56-72 hours.

Presently, the various methods used for the production and purification of Hib-PRP take relatively long cultivation and purification time of about 40 hours to 72 hours thereby increasing the cost of production and making the process commercially unfeasible since they cannot be scaled up in a cost-effective manner.

Further the above disclosed prior arts teach a method which is more efficient at low temperature thereby requiring a controlled environment leading to addition of costs in research and production. Moreover, some of the current methods discuss the use of chromatography steps which leads to production of a polysaccharide with low yields which may increase the cost of production during scale-up, thereby increasing the process time to a great extent.

OBJECT OF THE INVENTION:
The main object of the invention is to provide a rapid process for production and purification of Hib-PRP polysaccharide while eliminating the impurities in a very short time by simple, efficient, improved and commercially scalable method.

Yet another object of the invention is to provide the purification process which can be carried out at room temperature in contrast to the methods of production used in the existing state of art where the purification processes are mostly conducted at very low temperatures.

Yet another object of the present invention is to produce high quality polysaccharide with higher yield that meet the WHO specifications.

SUMMARY OF THE INVENTION:
Accordingly the present invention relates to the process of the production and purification of Hib-PRP. The invention discloses a rapid, simple yet cost effective and scalable process, wherein Hib-PRP is purified in significantly reduced time. Another important feature of the present invention is that the purification process is completed at room temperature.

The process of the instant invention commences with the selection of bacterial strain which has the potential to give consistent or reproducible results. For the propagation of strain, in this case, the glycerol stock culture is prepared using known processes and the bacterial strain is inoculated in the propagation media. The inoculated bacterial strain is then incubated at predefined temperature for an optimal time period. The selected bacterial strain is incubated and then cultured on cultivation media in the fermenter and then harvested. The cultivation media is optimized to accomplish higher growth of the bacterial strain. The harvest or the ‘fermented harvest’ is clarified to obtain the fermented broth or the supernatant. This fermented supernatant is subjected to ultrafiltration using molecular weight cut-off membranes to obtain concentrated supernatant.

The ultra-filtered concentrated supernatant is then precipitated with one of the selected cationic detergent which leads to the formation of precipitated polysaccharide pellet. These pellets are then collected and dissolved in saline solution. The polysaccharide in this saline solution is precipitated and treated with organic solvent preferably ethanol. The precipitated polysaccharide so obtained is then treated with one of the selected anionic detergents along with sodium acetate and ethanol. The supernatant thus obtained is subjected to filtration and ethanol precipitation/treatment. The precipitates obtained after ethanol treatment are dissolved in MilliQ water (MQW) and diafiltered to obtain purified polysaccharides in significantly reduced time.

The process exhibits a number of advantages over prior art, such as providing a robust and rapid method of preparing Hib polysaccharide having phosphorous backbone. The process is also cost effective as it reduces the total number of steps, does not require chromatographic screening and can be carried out at room temperature. An additional advantage is that this process is entirely scalable.

BRIEF DESCRIPTION OF DRAWINGS
Figure 1- depicts the process flow for production of PRP. The flowchart depicts the steps involved from the selection of bacterial strain till the collection of fermentation broth.
Figure 2- depicts the growth curve of H. influenzae type b.
Figure 3- depicts the purification scheme of PRP.
Figure 4- depicts the HPLC profile of purified PRP.
Figure 5- depicts the NMR of purified PRP.

DETAILED DESCRIPTION OF THE INVENTION WITH REFERENCE TO DRAWINGS AND EXAMPLES
Accordingly, the invention relates to the process of producing and purifying HiB-PRP from a number of distinctly different varieties of bacterial capsular polysaccharides and subjecting said HiB-PRP to an optimized process of purification. The invention need not to be limited to only these bacterial capsular polysaccharides.

The invention discloses a series of steps that have been optimized in a novel and inventive manner to enable the preparation and purification of Hib-PRP in considerably reduced time of less than 25 hours where the processes are carried out at room temperature and with a yield comparable to or more than in the existing state of the art processes. Also, the purified PRP so produced and purified meets the WHO specifications.

The process of production as shown in figure 1 commences with the step of selection of an appropriate bacterial strain. The bacterial strain is usually selected on the basis of immunogenicity with a preference for immunologically active strain and its ability to give consistent or reproducible results. The next step in the process involves the preparation of the media in which the selected bacterial strains are to be propagated. The said media has a numbers of nutritional and growth factors as well as pH stabilizing agents that can optimally sustain the selected bacterial culture and accelerate its propagation. Some of these factors and stabilizing agents include potassium chloride (KCl), sodium chloride (NaCl), yeast extracts, etc.

Once the medium for bacterial propagation is prepared, the selected bacterial strains are inoculated in flasks containing the necessary amount of the above mentioned propagation media. The inoculation is performed at temperatures of approximately 360C at a pH of 7.2. This seed culture is periodically tested for its optical density. As soon as the optical density is within the range of 0.8 to 1.0, the seed culture of the bacterial strain with maximum optical density is selected and transferred to the fermenter for the fermentation step.

The fermentation is carried out in the fermenter which is equipped with automatic control of temperature, pH, dissolved oxygen concentration (DO), peristaltic pump for addition of feed, alkali for pH control, antifoam as well as addition of inoculum. The selected seed culture of strain from the inoculation stage is allowed to cultivate on the media components which provide the nutritional requirements to the strain. The optimized media components results in the significant growth of Hib bacteria.

The production of the capsular PRP is also dependent on the physical consideration such as pH and temperature and shows an exponential growth phase with the parameters within a specific range. The optimized fermentation media including the components like L-glutamic acid, L-cysteine, Hemin etc and the process parameter protocol results in the desired product.

After the completion of the growth phase, the fermentation comes to its termination stage which is indicated by the decrease in optical density of the strain. The termination is brought by treatment with formaldehyde (HCHO) for predefine time preferably 15 mins. The temperature of fermenter is then maintained at 350C for 15 minutes and the fermentation broth is collected in bottles. The fermented broth is subjected to centrifugation and the supernatant resulting from the centrifugation is collected.

The supernatant so collected is then subjected to diafiltration for the removal of the unwanted impurities and the recovered material so obtained is then processed for further purification.

Purification Process:
The purification scheme shown in Figure 3 is largely dependent on the physio-chemical properties of PRP, the quantum of impurities to be removed and the time taken to complete the whole process.

The culture broth having the crude PRP is processed with cationic detergent to remove impurities. The cationic detergent is preferably, but not limited to, CTAB (cetyltrimethyl-ammonium bromide). This culture broth precipitates down the polysaccharides as a pellet while removing most of the contaminants which forms the supernatant. The precipitated pellet is then treated at room temperature each with three treatments/cuts of organic solvent preferably ethanol, starting with 74% ethanol, 34% ethanol and 66% ethanol respectively.

These ethanol treatments/cuts enhance the purification characteristics of the polysaccharide as it facilitates the removal of Lipo-polysacchrides (LPS). The purified polysaccharide pellet is then subjected to zeta filtration which is achieved by activated carbon on supernatant facilitating the decolourization and elimination of nucleic acids.

The 1st and the 3rd ethanol treatments/ precipitation cuts yield pellets referred to as first Hib polysaccharide pellet and second Hib polysaccharide pellet respectively, while the 2nd ethanol treatment followed by centrifugation yields the supernatant. Further concentration and diafiltration of the second Hib polysaccharide pellet is performed to facilitate the removal of proteins and other impurities. The PRP sample so obtained from the second Hib polysaccharide pellet is then washed with MQW. Consequently sterile filtration is performed to obtain the purified Hib-PRP in a controlled pH environment of 6 and 7. Both production and purification processes are conducted at room temperature.

The Hib-PRP so produced and purified is of a quality that meets the WHO specification. The corroboration of the above employed procedures can be conveniently understood from Table 1 which clearly shows the purified PRP specifications meet the WHO standard.

Table 1: Purified PRP reference tests vis a vis WHO specifications for Hib-PRP.
TESTS Purified PRP Results WHO Specifications
PRP Content 8.99mg/ml Actual Value
Ribose content 34.7% Not less than 32%
Phosphorus 6.9% 6.8%-9.0%
Endotoxin 0.93 Eu/µg Less than 10 Eu/µg
Protein Impurity 0.1% Not more than 1%
NA Impurity 0.1% Not more than 1%
Average relative Mol wt. 481 kd Actual value from HPLC
Identity Positive Positive agglutination reaction

All above mentioned procedural steps performed results in the formation of purified pellet of polysaccharides which can further be employed for the production of both polysaccharide vaccines and conjugate vaccines.

Of particular interest is the observation that the time required to purify the Hib-PRP is significantly lower than those disclosed in the existing state of the art literature and practice. Further, the purification process protocol of the present invention is scalable with the yield being in compliance with the WHO set standard.

Analytical Procedures:
PRP obtained at different steps are constantly monitored and analyzed for their purity and yield. Different analytical procedures are reported but the preferred ones are as summarized below:

PRP concentration is measured by the modified Bial method, using ribose as standard. In Bial reaction, the ribose, as a pentose, reacts with concentrated HCl-Ferric Chloride solution to produce green colour with absorbance at 670nm.

Phosphorus concentration is determined by the colorimetric assay. In this assay, samples are initially acidified, then oxidized with perchloric acid or magnesium nitrate to activate and hydrolyze the polysaccharide prior to washing of the material at high temperature.

In second part of the assay, ascorbic acid acts to reduce a phosphomolybdate complex to a blue colored compound. The absorbance is taken at 820 nm with D-ribose -5-phosphate disodium dehydrate as a standard reagent against which the actual concentration of the phosphorus present in the PRP is analysed. Phosphorus comprises 8.3-8.5% of PRP, and the assay can detect 5-10 nmoles phosphorus, or PRP monomeric unit. Lipopolysaccharide (LPS) is determined using compact and simple Endosafe ®-PTS ™. It is a handheld spectrophotometer that utilizes FDA-licenced disposable cartridges for accurate, convenient endotoxin testing. The quantitative endotoxin results are available in 15 minutes. Protein impurity is determined by Lowry’s method using bovine serum albumin (BSA) as a standard and the absorbance is taken at 750nm. The assay can detect protein upto 6.25µg and looking at the sensitivity of the assay, the sample is tested at high/neat concentration. Nucleic acids (NA) are estimated at 260 nm and the amount is calculated assuming an absorbance of 1.0A=50µg/mL.

The Relative Average Molecular size (Mw) of PRP is determined using HPLC as shown in Table 2. The columns used are from Tosoh Bioscience, PWXL-3000 and PWXL-4000 in series, using a range of 5 kD to 800 kD Pullulans as standard. The buffer used is 0.1 m Sodium Nitrate with a run time of 30 min at a flow rate of 1ml/min. The identity of a polysaccharide are carried out using rapid latex agglutination test (Remel Wellcogen Bacterial Antigen Kit). As the WHO specification to determine the purity and to characterize the polysaccharide is based on dry weight basis, the polysaccharides are first lyophilized and then tested. The moisture content is thus subtracted to extract the dry weight. Moisture content of lyophilized cake is determined by thermo Gravimetric Analyser (TGA) from Perkin Elmer.

The said purified Hib polysaccharide is dried and can be used for the preparation of polysaccharide-protein conjugate vaccines.

Various aspects of the invention described in details above are now illustrated with non-limiting examples:

Example 1:
Selection of Bacterial Strain:
Haemophilus influenza type b (Hib) strain #10211, is obtained from ATCC (American Type Cell Collection) USA. The culture of Hib is maintained in 10% glycerol and stored at below -70ºC. The culturing media used for maintaining the culture consists of Mueller Hinton Broth as 21.0 g/L (Becton Dickinson), Hemin as 0.015 g/L (Sigma Aldrich), Yeast Extract as 5.0 g/L (Fluka), Iso Vitale X as 10 ml (Becton Dickinson) , and milliQ water (MQW) as 900ml.

Example 2:
Preparation of Propagation Media:
The media is prepared for the bacterial strain propagation, which is maintained with base or acid whenever so required at constant value of pH equivalent to 7.2 ± 0.1.

The medium for bacterial propagation consists of disodium hydrogen phosphate as 2.5 g/L, KCl as 0.5 g/L, NaCl as 3.3 g/L, NH4Cl as 0.70g/L, Yeast Extract as 2.0g/L, MgSO4 as 0.60g/L, L-cysteine as 0.015 g/L, L-Glutamic acid as 1.2 g/L, D-glucose as 5.0 g/L, Hemin as 0.020 g/L, NAD as 0.010 g/L.

Example 3:
Preparation of Propagation Media:
The media is prepared for the bacterial strain propagation which is maintained with base or acid whenever so required at constant value of pH equivalent to 7.2 ± 0.1.

The medium for bacterial propagation consists of Di sodium hydrogen phosphate as 2.5 g/L, KCl as 0.5 g/L, NaCl as 3.3 g/L , NH4Cl as 0.7g/L, Bacteriological Peptone as 15.0 g/L , Yeast Extract as 2.0g/L, L-cysteine as 0.015 g/L, D-glucose as 5.0 g/L, L-Glutamic acid as 1.2 g/L, NAD as 0.010 g/L ,

Example 4:
Preparation of Propagation Media:
The media is prepared for the bacterial strain propagation, which is maintained with base or acid whenever so required at constant value of pH equivalent to 7.2 ± 0.1.

The medium for bacterial propagation consists of Di sodium hydrogen phosphate as 2.5 g/L, KCl as 0.5 g/L, NaCl as 3.3 g/L, NH4Cl as 0.7 g/L , Bacteriological Peptone as 15.0 g/L , Yeast Extract as 2.0 g/L , L-Glutamic acid as 1.2 g/L , L-cysteine as 0.015 g/L, D-glucose as 5.0 g/L, Hemin as 0.020 g/L, NAD as 0.010 g/L.

Example 5:
Procedure for Inoculation:
The Hib strain so obtained is subjected for inoculation. The Hib inoculum is prepared in three 250 ml conical flask, each flask having 100ml of said bacterial propagation media in liquid state. The media is inoculated with 0.5 ml of glycogen stock culture and incubated at 350C, for 4 to 6 hours for 150 revolutions per minute (rpm). These Hib-inoculum flasks are constantly checked for their optical density at 550nm using UV-Spectrophotometer (Optical Density (OD) 550nm). As soon as the optical density of the inoculum flasks reached to 0.8 to 1.0, the seed culture with maximum OD is picked and used for the inoculation in fermenter.

Example 6:
Cultivation Procedure/Fermentation Process:
The fermentation of the above prepared shake flask is carried out in 5 liters Biostat B Sartorius glass fermenter equipped with automatic control of temperature, pH, dissolved oxygen concentration (DO), four peristaltic pump for addition of feed, alkali for pH control, antifoam and for inoculum. The media components used for cultivation are following in their preferred concentration:

The medium for bacterial cultivation consists of disodium hydrogen phosphate as 2.5 g/L, KCl as 0.5 g/L, NaCl as 3.3 g/L, NH4Cl as 0.7 g/L, Bacteriological Peptone as 15.0 g/L, Yeast Extract as 2.0 g/L, L-Glutamic acid as 1.2 g/L , L-cysteine as 0.015 g/L, Hemin as 0.020 g/L, NAD as 0.010 g/L , D-glucose as 5.0 g/L. All the listed cultivation media components are sterilized and cooled to 350C.

The various process parameters are controlled during the automated fermentation run. For instance, the pH of the medium is controlled at 7.2 + 0.1 by the addition of 2 M NaOH, temperature is maintained at 350C, air flow rate is 2L/M, DO is maintained at 30% by cascade mode of the fermenter and agitation speed is between 100 to 150 rpm. The seed culture with maximum optical density (OD) is inoculated in fermenter. OD of the culture is measured every hour at 550 nm using UV-Spectrophotometer 1800 (Shimadzu). As soon as the OD of culture reached to a level of 0.8 to 1.0, the DO level is maintained by pure oxygen at a flow rate of 0.2 to 0.5 L/min, glucose and yeast extract pulse are then initiated and the level in the culture are maintained between 0.2g/L to 0.5g/L. The fermentation process which goes maximum for 10 hrs with an achieved OD of 8.10 during growth phase is brought to its termination by treatment with formalin by simple addition during the decline phase.

After the treatment with formalin, 0.2% formaldehyde (HCHO) is added. The temperature of fermenter is maintained at 350C for 15 minutes and the fermentation broth is collected in pre-sterilized 1L centrifuge bottles. Centrifugation of fermented broth is performed in pre-sterilized centrifuge bottles at 8000g for 30 minutes using RC 6+ Thermo Scientific centrifuge.

After centrifugation, the supernatant is collected in sterile glass bottle and pellet is collected in biowaste bags and decontaminated by autoclave which is maintained preferably at 1340C for a time period of about 10 mins before disposing off.

Example 7:
Concentration of Harvest by Tangential Flow Filtration:
After centrifugation the collected supernatant with a mass ranging from 5.5L to 6.0 L is concentrated up to 1:10 volumes by using 100Kda cut off pellicon 0.11m2 (Millipore) Poly Ether Sulfone (PES) slice membrane.

Concentrated supernatant is diafiltered 4 to 5 times with equal volume of MQW or with 0.01M PBS at a pH 7.2 ± 0.1 to remove low molecular weight contaminants. Transmembrane pressure (TMP) of system is maintained preferably at 0.2 bar throughout the process. After the diafiltration, the material is recovered and further processed for the purification of capsular polysaccharide.

Example 8:
PRP purification and recovery:
The clarified fermentation broth (FB) of Haemophilus influenzae type b (Hib) culture is concentrated and diafiltered with MilliQ water (MQW) using 100 KDa 0.1m2 slice cassettes. The cationic detergent CTAB preferably in final concentration of 0.5-1.5% (w/v) is added to the fermentation broth and left overnight for precipitation at temperature range from 2?C to 8?C. The solution is subject to centrifugation at 5000rpm/4690 xg for 30 mins at the end of CTAB precipitation to obtain the pellet.

The CTAB pellet is liquefied in minimal volume of NaCl solution, preferably in final concentration of 4% to 7% w/v. Thereafter ethanol (EtOH) is added in three distinct steps to precipitate and purify the polysaccharide. These ethanol additions or cuts are referred as 1st EtOH precipitation cut, 2nd EtOH treatment and 3rd EtOH precipitation cut. In the 1st EtOH precipitation cut, the ethanol added to the liquefied solution preferably in the concentration range of 60% to 90% v/v. The pH of the solution is adjusted with 8M acetic acid in the range of 6.7 to 6.9. The solution is then subjected to incubation for overnight at temperatures of 2?C to 8?C. Subsequently centrifugation is performed at 5000rpm/4690 xg for 30 mins to obtain the first Hib-polysaccharide pellet.

The first Hib polysaccharide precipitated pellet obtained from 1st EtOH precipitation cut is dissolved in MQW. The suitable anionic detergent used is sodium deoxycholate (DOC) to remove the proteins impurities and is used preferably at a concentration range of 0.5 to 1.5% w/v with 5% to 20% w/v sodium acetate. After the removal of the proteins, ethanol is further added in a concentration range of 10% to 50% v/v to the solution which is referred to as 2nd EtOH treatment. The pH of the 2nd EtOH treatment is adjusted with 8M acetic acid in the range of 6.3 to 6.5. The solution is incubated overnight at temperature of 2 ?C to 8?C. The incubated solution is then subjected to centrifugation at 5000rpm/4690 xg for 30 mins to obtain the supernatant.

Supernatant from the 2nd EtOH treatment is subjected to (zeta) carbon filtration. The zeta carbon filtration is followed by filtration at 0.22µm. The optical density (OD) of the resultant solution is analysed at 260nm at constant time and maintained below 0.2. After ascertaining the required OD, the solution is subjected to ethanol preferably at the concentration range of 40 %-80 % v/v to the solution which is referred to as the 3rd EtOH precipitation cut. The pH of the 3rd EtOH precipitation cut is adjusted with 8M acetic acid and maintained at 6.1 to 6.3. The solution is incubated for overnight at temperature of 2?C to 8?C and thereafter the solution is subjected to centrifugation at 5000rpm/4690 xg for 30mins to obtain the second Hib polysaccharide pellet.

The second Hib polysaccharide pellet so collected after treatment with the ethanol is mixed with MQW to obtain a non-viscous solution. Finally the solution is subjected to 100 kDa diafiltration and washed with 10 to 20 volumes of MQW. The volume approximately to 1 lit is prepared with MQW for above diafiltered solution and again subject to filtration through 0.22µm filter to obtain the purified Hib polysaccharide.

Example 9:
PRP purification and recovery:
The clarified FB of Hib culture is concentrated and diafiltered with MQW using 100 KDa 0.1m2 slice cassettes. The cationic detergent CTAB preferably in final concentration of 0.8 to 1.2% (w/v) is added in the FB. The solution is incubated for 8 to 10 hours for precipitation at 4?C temperature. The solution is subject to centrifugation at 5000rpm/4690 xg for 30 mins to obtain the pellet.

The pellet is then liquefied in minimal volume of NaCl solution, preferably in final concentration of 5% to 6% w/v. The liquefied solution is subjected to treatment with ethanol for the 1st EtOH precipitation cut preferably in a concentration range of 65% to 75% v/v to obtain the first Hib-polysaccharide pellet. The pH of the solution is adjusted with 8M acetic acid in the range of 6.7 to 6.9. The solution is then incubated for 8 to 10 hours for precipitation at 4?C temperatures. Subsequently centrifugation is performed at 5000rpm/4690 xg for 30 mins to obtain the first Hib-polysaccharide pellet.

The first Hib-polysaccharide precipitated pellet so obtained from 1st EtOH precipitated cut is dissolved in pyrogen free distill water or MQW. The desirable anionic detergent having the property to remove the undesirable proteins is used preferably DOC. The anionic detergent so used is in the concentration range of 0.9% to 1.2% w/v along with 10 to 15% w/v of sod acetate. The solution obtained after the treatment with anionic detergent is subject to 2nd EtOH treatment and treated with ethanol preferably in the concentration range of 20% to 40% v/v. The solution is then incubated for 8 to 10 hours at 4?C and the supernatant is collected after centrifuging the solution for 5000rpm/4690 xg for 30 mins.

The supernatant so obtained from the 2nd EtOH treatment is subjected to (zeta) carbon filtration. The zeta carbon filtration is followed by filtration at 0.22µm. The optical density (OD) of the resultant solution is analysed at 260nm at constant time and maintained below 0.2. The OD of the solution is constantly measured at 260nm and maintained below 0.2. The solution is then subject to 3rd EtOH precipitation cut i.e. treated with ethanol preferably in the concentration range of 50% to 70 %. The pH is adjusted with 8M acetic acid in the range of 6.1 to 6.3. The solution is incubated for 8 to 10 hours at 4?C temperature and subjected to centrifugation at 5000rpm/4690 xg for 30 mins to obtain the second Hib-polysaccharide pellet.

The second Hib-polysaccharide pellet obtained from 3rd EtOH precipitation cut is collected and mixed with MQW to form a non-viscous solution. Finally the solution is subject to 100 kDa diafiltration and washed with 10 to 20 volumes of MQW. The volume approximately to 1 lit is prepared with MQW for above diafiltered solution and again subject to filtration through 0.22µm filter to obtain the purified Hib polysaccharide.

Example 10:
PRP purification and recovery:
The clarified FB of Hib culture is concentrated and diafiltered with MQW using 100 KDa 0.1m2 slice cassettes. The cationic detergent CTAB preferably in final concentration of 0.8 to 1.2% (w/v) is added in the fermentation broth. The solution is then incubated for 4 to 6 hours for precipitation at room temperature (RT). The solution is subject to centrifugation is performed at 5000rpm/4690 x g for 30 mins to obtain the pellet.

The pellet is then liquefied in minimal volume of NaCl solution, preferably in final concentration of 5% to 6% w/v. The liquefied solution is subject to ethanol preferably in the concentration range of 65% to 75% referred as 1st EtOH precipitation cut. The pH of the solution is adjusted with 8M acetic acid in the range of 6.7 to 6.9. The solution is incubated for 4 to 6 hrs for precipitation at RT and centrifuge subsequently at 5000rpm/4690 xg for 30 mins to obtain the first Hib polysaccharide pellet.

The first Hib-polysaccharide pellet so obtained from 1st EtOH precipitation cut is dissolved in pyrogen free distilled water or MQW. The desirable anionic detergent used is DOC (w/v) to remove the proteins impurities and is used preferably in the concentration range of 0.9% to 1.2% with 10% to 15% w/v of sod acetate. The solution obtained after the treatment with anionic detergent is subject to 2nd EtOH treatment and treated with ethanol preferably in the concentration range of 20% to 40% v/v. The pH of the solution is adjusted with 8M in the range of 6.3 to 6.5. The solution is incubated for 4 to 5 hours at room temperature and centrifuge at 5000rpm/4690 xg for 30 mins to obtain the supernatant.

The supernatant of 2nd EtOH treatment is obtained and subjected to zeta filtration on activated carbon for decoloration and removal of nucleic acids and thereafter subjected to 0.22µm filtration. The OD of the solution is constantly measured at 260nm and maintained below 0.2. The solution is then subject to 3rd EtOH precipitation cut i.e. treated with ethanol preferably in the concentration range of 50 % to 70 % v/v. The pH is adjusted to 6.1 to 6.3 with 8M acetic acid. The solution is incubated for 4 to 6 hours at RT and then subjected to centrifugation at 5000rpm/4690 x g for 30mins to obtain the second Hib-polysaccharide pellet.

The second Hib-polysaccharide pellet obtained from 3rd EtOH precipitation cut is collected and mixed with MQW to form a non-viscous solution. Finally the solution is subject to 100 kDa diafiltration and washed with 10 to 20 volumes of MQW. The volume approximately to 1 lit is prepared with MQW for above diafiltered solution and again subject to filtration through 0.22µm filter to obtain the purified Hib polysaccharide.

Example 11:
PRP purification and recovery:
The clarified FB of Hib is concentrated and diafiltered with MQW using 100 KDa 0.1m2 slice cassettes. Further, 1.1% CTAB (w/v) is added in the FB and left for precipitation for 2 hrs at RT. The centrifugation is performed at 5000rpm/4690 x g for 30mins in the end of CTAB precipitation to obtain the pellet.

The CTAB pellet is liquefied in minimal volume of NaCl having concentration 5.85% w/v and the liquefied solution is subjected to 1st EtOH precipitation cut i.e. ethanol of 74%v/v concentration is added to the liquefied solution. This is referred to as first ethanol precipitation cut. The pH of the solution is adjusted with 8M acetic acid in the range of 6.7 to 6.9. Thereafter the solution is incubated at room temperature for 3 hrs. The incubated solution is subject to centrifugation is at 5000 rpm / 4690 xg for 30 mins to obtain the first Hib-polysccharide pellet.

The first Hib-polysccharide pellet so obtained from the 1st EtOH precipitation cut is dissolved in MQW. Further 1% DOC (w/v), and 10% of sodium acetate (w/v) are added. The solution is subjected to 2nd Ethanol treatment wherein, 34% EtOH is added to the above liquefied solution in step wise manner. The pH of the solution is adjusted with 8M acetic acid in the range of 6.3 to 6.5. The solution is then incubated at RT for 3 hours and centrifuged at 5000rpm/4690 x g for 30 mins is performed to obtain the supernatant.

The resultant supernatant of 2nd EtOH treatment is obtained and allowed for (zeta filteration) carbon filtration for decolourization and nucleic acid removal and subject to 0.22µm filtration. The OD of the solution is constantly measured at 260nm and maintained below 0.2. The solution is then subject to 3rd EtOH precipitation cut i.e. treated with 66% ethanol. The pH of 3rd EtOH precipitation cut is adjusted with 8M acetic acid and maintained at 6.1 to 6.3. The solution is incubated at RT for 3 hours and centrifugation is performed at 5000rpm/4690 xg for 30 mins to obtain the second Hib-polysaccharide pellet.

The second Hib-polysaccharide pellet obtained from 3rd EtOH precipitation cut is collected and mixed with MQW to form a non-viscous solution. Finally the solution is subject to 100 kDa diafiltration and washed with 10 to 20 volumes of MQW. The volume approximately to 1 lit is prepared with MQW for above diafiltered solution and again subject to filtration through 0.22µm filter to obtain the purified Hib polysaccharide.
,CLAIMS:We Claim:

1. A rapid process for producing and purifying Hib-PRP -wherein said process comprises the following steps:
(a) producing and harvesting concentrated supernatant of Hib polysaccharide by known processes
(b) precipitating said concentrated supernatant of Hib polysaccharide of step (a) with at least one cationic detergent to obtain Hib polysaccharide pellet
(c) liquefying said Hib polysaccharide pellet of step (b) in the saline solution and subjecting it to an organic solvent for first precipitation cut to obtain first Hib polysaccharide pellet after incubation,
(d) treating said first Hib polysaccharide pellet obtained in step (c) with at least one anionic detergent in presence of sodium acetate and said organic solvent to obtain supernatant after incubation,
(e) subjecting said supernatant of step (d) to filtration and treatment with said organic solvent for third precipitation cut to obtain second Hib polysaccharide pellet after incubation,
(f) dissolving said second Hib polysaccharide precipitate in MilliQ water followed by diafiltration to obtain purified polysaccharide
such that the entire process of production and purification of Hib-PRP is completed rapidly within 25 hours at room temperature .

2. The rapid process as claimed in 1, wherein said cationic detergent is hexadecyl trimethyl ammonium bromide (CTAB) in concentration range of 0.8% to 1.2% (w/v) and more preferably in the concentration of 1.1%.

3. The process as claimed in 1, wherein the total time required for step (b) ranges from 1 hour 30 minutes to 2 hours at room temperature.

4. The process as claimed in 1, wherein said saline solution in step (c) is NaCl solution having a concentration in the range of 5.5 % to 6.5% (w/v) and more preferably in the concentration of 5.85%.

5. The process as claimed in 1, wherein said organic solvent is ethanol.

6. The process as claimed in 1 and 5, wherein said ethanol in step (c) has concentration in the range of 70% to 75% v/v, concentration of said ethanol being in step (d) being in the range of 30% to 40% v/v.

7. The process as claimed in 1 and 5, wherein said ethanol in step (e) has concentration in the range of 60% to 70% v/v.

8. The process as claimed in 1, wherein said anionic detergent in step (d) comprises sodium deoxycholate (DOC) in a concentration range of 0.8% to 1.2% (w/v) and more preferably with a concentration of 1%.

9. The process as claimed in 1 wherein the incubation time in step (c) is 2 hours to 3 hours at room temperature with pH being maintained at 6.5 to 6.9, incubation time in step (d) is 2 hours to 3 hours at room temperature with pH being maintained in a range of 6.3 to 6.5 and incubation time in step (e) is 2 hours to 3 hours at room temperature with pH being maintained in the range of 6.1-6.3.

10. The process as claimed in claim 1, wherein said process of purification is completed within 9 to 15 hours, preferably within 12 hours such that said entire process of production and purification is completed in a range of 19 to 25 hours, preferably within 22 hours to yield Hib-PRP of high purity.