Claims:1. A Process for production and purification of Capsular polysaccharide from Hae-mophilus influenza type b and method for producing a conjugate vaccine composi-tion using said polysaccharide comprising:
i)preparing modified inoculum seed in suitable media,
ii)fermenting Haemophilus influenza type b in medium,
ii) inactivating of culture
iii) harvesting and recovering the polysaccharide after the fermentation,
iv) purifying the polysaccharide
v) conjugating purified polysaccharide with antigenic protein for preparation of con-jugate vaccine.

2. A process as claimed in claim1, wherein a polysaccharide is Polyribosylribitol Phos-phate.

3. A process as claimed in claim 1 wherein, the polysaccharide is purified by using meth-ods selected from clarification, filtration and chromatography.
4. A process as claimed in claim 1 wherein the purified polysaccharide is conjugated with a carrier protein such as Tetanus Toxoid, Diphtheria toxoid and CRM protein for prep-aration of conjugate vaccine.
5. A composition of conjugate vaccine comprising

a. Polysaccharide such as Polyribosylribitol Phosphate obtained from Haemophi-lus influenza type b,
b. carrier protein such as Tetanus Toxoid, Diphtheria toxoid and CRM protein
, Description:Process for production and purification of Capsular polysaccharide from
Haemophilus Influenza type b

Field of the invention,
The present invention relates to the field of the vaccine, in the present invention relates to the production and purification of Capsular polysaccharide from Haemophilus influenza type b

Background of the invention
Background description includes information that may be useful in understanding the pre-sent invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced in this application is prior art. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully de-scribe the state of the art to which this invention pertains.
.
Haemophilus influenza type b bacteria is transmitted through the respiratory tract from in-fected to susceptible individuals. Hib also causes potentially severe inflammatory infections of the face, mouth, blood, epiglottis, joints, heart, bones, peritoneum, and trachea. Although this problem occurs worldwide the burden of Hib disease was considerably higher in re-source-poor countries, prior to the introduction of the vaccine into their national immuniza-tion programmes
Haemophilus influenza type b (Hib) is a bacteria responsible for severe pneumonia, meningi-tis and other invasive diseases almost exclusively in children aged less than 5 years
Vaccines are the only public health tool capable of preventing the majority of serious Hib disease. Hib vaccines are safe and efficacious even when administered in early infancy. In view of their demonstrated safety and efficacy, World Health Organization recommends that Hib conjugate vaccines to be included in all routine infant immunization programmes. (Source WHO)

The production and purification processes used in the present scenario are relatively ex-pensive, and include a long cultivation step of about 16-18 hours, see e.g. U.S. Pat. No. 4,644,059 and the period for culturing is typically based on arbitrary parameters, such as time or optical density, see e.g. U.S. Pat. No. 4,220,717. In this way, it is not possible to compensate for changes in culture conditions and suboptimal yields of polysaccharide are the inevitable result.
Again the current processes to produce polysaccharides such as Polyribosylribitol Phosphate (PRP) from Haemophilus influenza type b results in low yields. Process steps are cumber-some and lengthy as well as cost ineffective.
Further the conjugation of this polysaccharide with a carrier protein involves reaction be-tween PRP and TT and requires toxic substances/ reactants/chemicals in many instances see e.g. U.S. Pat. No. 4,695,624 and EP 0 528 635.The removal of these substances require many purification steps which result in the loss of conjugate product as well as poses health hazards to workers and environment. This also involves the in process and final testing of such reactants which establish the absence of such reactants.

Therefore present invention is aimed to resolve such inherent problem by making the poly-saccharide production process simpler, improving the recovery at final stages thus increasing the yield and making the process cost effective.

The conjugation process of present invention used no hazardous chemicals, applied simpler conjugation reaction and few but cost-effective purification steps. This helped us to gain re-markably good yield of PRP-TT conjugate

Objects of the invention
The objective of the present invention to provide a rapid, inexpensive, and efficient method for the purification of conjugates.

Yet another object of the present invention is to provide a method for conjugation of Polyri-bosylribitol Phosphate (PRP) with carrier protein such as Tetanus Toxoid, Diphtheria toxoid, CRM protein etc for preparation of conjugate vaccine.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, become better understood with reference to the following description, appended claims, and accompanying drawings in which like characters represent like parts throughout the drawings

Figure 1 represents the integrated process flow diagram for production of polysaccharide from Haemophilus influenza.

Figure 2 illustrates an exemplary method for conjugation of Polyribosyl Ribitol Phosphate (PRP) with Tetanus toxoid for preparation of HIBTT conjugate vaccine.

DETAILED DESCRIPTION OF THE INVENTION

The following is a detailed description and explanation of the present invention with some examples thereof. In order to better appreciate the invention, it is described with reference to the figures which illustrate various embodiments of the invention.

The present invention relates to the production and purification of Capsular polysaccharide from Haemophilus influenza type b and more particularly to the method for producing a conjugate vaccine composition using said polysaccharide.

Figure 1 shows the method for production of polysaccharide (10) comprising the step of preparing preinoculum (12) of an organism capable of producing polysaccharide, preinocu-lum is used for the preparation of inoculum (14) for fermentation and production of desired end product such as polysaccharide. Fermentation (16) takes place from about 12 to 24 hours after fermentation inactivation of culture takes place (18) followed by downstream processing such as harvest and centrifugation (20), clarification (22), concentration / Diafil-tration (24), precipitation (26), and centrifugation (28) and after centrifugation, precipitate (30) is subjected to homogenization (32) and pH is adjusted with stirring (34) after homoge-nization the homogenized material precipitate is subjected to centrifugation (36) followed by depth filtration (38) and precipitation (40). Precipitation is collected (42) and vacuum dried at low temperature (44) to obtain purified polyribosyl ribitol phosphate (PRP) (46) which is further kept for storage (48).

Figure 2 shows the preparation of conjugation of Polyribosylribitol Phosphate (PRP) with carrier protein (50) for preparation of conjugate vaccine such as Tetanus Toxoid, Diphtheria toxoid, CRM protein. As shown PRP is activated (52) and tetanus toxoid is modified (54) the modified tetanus toxoid is added to PRP (56) the mixture is concentrated and subjected to diafiltration (58) filtered conjugate is purified using chromatography method (60) and finally filtered in sterile environment (62).

The preparation of seed bank is performed keeping in view of high productivity. A well es-tablished fact about microorganism is that they are highly potent at youth stage of their life cycle i.e. Log phase.
Culture of Haemophilus influenzae type b such as ATCC 10211 (any other reference Hib strain such as Eagan strain, ATCC 31512, ATCC 51654 or any suitable clinical isolate strain of Hib may be used) was inoculated on plate with culture medium and incubated for about 12 about 24 hrs at about 30 to about 37ºC in incubator with slight CO2 tension. The growth of the plate was transferred to Erlenmeyer flask having culture medium and was incubated in incubator shaker at about 30 to about 37 ºC for about 6 to about 14 hrs. Once the culture attains the log phase, the incubation of the flask is stopped and the growth is used to inocu-late next set of flask with culture media. This step is repeated for about 6 to about 10 times followed by inoculation of the fermentor from the culture of the last set of flask. Fermenta-tion is carried out at about 30 to about 37 ºC for about 4 to about 12 hrs. Once the culture reaches to log phase, fermentation is terminated and culture is harvested aseptically. This culture is used for preparation of seed bank (liquid /freeze-dried).

The production of the PRP is performed under the principle of cell bank system wherein a vial of the Cell Bank of Haemophilus influenzae type b ATCC 10211 is taken for inocula-tion on Petri plates with Chocolate Agar culture medium are seeded and incubated for about 16 to about 24 hours at about 35±2ºC in incubator having the pressure of about 5% CO2. Erlenmeyer flasks of 2.0 L capacity each containing 330 ml of culture medium are inoculat-ed with individual colonies picked from Petri plates and incubated in a shaker at a tempera-ture range of about 33 to about 37 ºC at about 125 to about 225 rpm for about 6 to about10 hours. After the incubation, the Erlenmeyer flask is selected on the basis of Optical Density (0.5 -2.5) at 550 nm as well as by checking microbial purity. The content of one Erlenmeyer flask is aseptically transferred to an inoculation siphon which is used for the inoculation of inoculum fermentor.

Pre Inoculum is transferred in to the inoculum fermentor containing culture media and Fer-mentation Parameters are kept at Airflow at about 0.5 to about 1.0 vvm, temperature at about 33 to about 37º C, pH at about 6.5 to about 7.5, stirring at about 125 to about 225 rpm, PO2: =10. Fermentation is carried out till optical density reaches about 0.5 to about 2.5 at 550 nm (fermentation process in the inoculum fermentor takes about 4 to about 8 hours for comple-tion).

Inoculum is transferred to production Fermentor containing culture media and Fermentation Parameters are kept at Airflow about 0.15 at about 0.70 vvm, Temperature at about 33 to about 37º C, pH at about 6.5 to about 7.5, stirring at about 80 to about 120 rpm, Pressure at about 350 to about 425 mbar and PO2 : = 10%,. Optical density at about 550nm of fermen-tation culture is monitored during fermentation. When the Optical density reaches at about 0.5 about 2.5, feed supplement (Glucose 20% and Yeast Extract 10%) is added to the fer-mentor. The feed is continued till O.D550 value becomes constant. The fermentation process in the production fermentor takes about 16 to about 24 hours for completion.

After the completion of fermentation, the fermented broth culture (volume ~400L) is sub-jected to inactivation at about 55 to about 65oC for about 15 minutes. The inactivated culture broth is then harvested and sampled for microbiological viability and ribose estimation.

The inactivated fermented broth is subjected to centrifugation (Continuous centrifugation) in disc stack centrifuge at Speed at about 7000 to about 10000 rpm, Temperature at about 3 to about 12 ºC and supernatant is collected for further processing.

Clarification of supernatant is carried out by depth filtration using Zeta plus LP series (LP50 and LP90) or equivalent filters. Clarification of supernatant is achieved by filtration, first through LP 50 and then through LP 90 filter. The process is carried out at a temperature of about 3 to about 12 ºC and takes about 4 to about10 hrs for completion

Concentration of clarified supernatant is performed using TFF (tangential flow filtration). 30 Kd size MWCO Cassette is used to carry out this process at temperature at about 3 to about 12 ºC. At this stage the retentate is concentrated to about 1/8th to about 1/12th of its starting volume. Diafiltration of the retentate is carried out against equal amount of purified water. This step is repeated 3-5 times to diafilter the retentate material. Concentration and dia-filtration process takes about 6 to about 12 hrs for completion.

Further about 7 to about 12% CTAB solution is added to the concentrated supernatant in about 4 to about 12 % v/v ratio and the mixture is agitated for at least for about 45 to about 75 min, Precipitates are allowed to settle for about 12 to abour16 hours at about 3 to about 12 ºC.

Further precipitates are centrifuged at about 5000 to about 7000 RCF for about 30 to about 45 minutes at about 3 to about 12 ºC temperature. The pellet is collected. These precipitates (~ 1.5 ± 3.2 kg) are then subjected to further purification by using ethyl alcohol.

Further ethanol (96 %) is added in the ratio of about 10 to about 14 % v/v to CTAB precipi-tates obtained from 30 KD concentrated and diafiltered broth. The pre-chilled precipitates are homogenized in about 3 to about 6 lots at low temperature of about 4 to about 18 °C for about 10 to abour15 minutes (for each lot) at about 6000 to about 10000 rpm. The approx-imate volume after this step would be about 5 to about 8 Litres.

Ethanol (96%) is added at about 30 to about 35 % v/v to the homogenized CTAB precipi-tates and pH is adjusted at about 6.5 to about 7.0 with 8M Acetic acid.

This extraction mixture kept on stirring (at about 300 to about 450 rpm) for about 12 to about 16 hrs at room temperature. The extraction mixture is then centrifuged in lots. Each lot is spinned for about 15 minutes at about 3 to about 12 ºC at about 5000 to about 7000 g and sampled for estimation of Ribose, Nucleic acid and Protein content.

The extraction mixture is filtered through the 10SP zeta plus cellulose filters (pre-treated with 96% ethanol). Post filtration, two washes are given to the filter, first with cold 96% ethanol (preferably chilled at about 5 to about 15 ºC) and second with warm 96% ethanol (prefera-bly warmed at about 30 to about 40°C). Both washes are then pooled, mixed with main fil-trate and sampled for the estimation of Ribose, Nucleic acid and Protein content.

The 10SP Zeta plus filtrate is passed through R53 SLP Zeta Carbon filters (pretreated with 96% ethanol). Post filtration, two washes are given to the filter, first with cold 96% ethanol (preferably chilled at about 5 to about 15 ºC) and second with warm 96% ethanol (prefera-bly warmed at about 30 to about 40°C). Both washes and filtrate are then pooled and mixed and sampled for Ribose, Nucleic acid and Protein content. Final recovered filtrate obtained after R53 SLP Zeta Carbon has the OD = 0.15 at 275nm.
The approximate volume at this stage would be about 19 to about 23 Litre.

4 M NaCl solution (the final concentration of 1% v/v) is added to R53 SLP Zeta Carbon fil-trate to precipitate the PRP. A white color lump or rubbery precipitate will appear which is allowed to settle at about 3 to about 10 ºC for about 12 to about14 hours.

The precipitate obtained from previous step is teased off to small pieces and subjected to drying for about16 to about 24 hrs under vacuum and at low temperature (about -70 to about -80°C). A simple lab scale freeze drier can be used for the purpose. The dried material ob-tained is called purified PRP and sampled for estimation of Ribose, Nucleic acid, Protein content, Phosphorus content, Moisture, Endotoxin, identity, and molecular size distribution. Purified PRP is stored at temperature of about -20°C or below. The dried weight of obtained PRP is about 90 to about 120 g.

Dried PRP quantity is taken as per batch size on the basis of PRP content. An ideal reaction can carried out by dissolving the dried purified PRP (having 10 g PRP content) in WFI (~ 500 ml) at about 3 to about 10 ºC (Preferably at about 4 to about 8 ºC) to get the final concentration of 20±2 mg/ml on the basis of PRP content. Allow to dissolve the PRP at about 250 to about 300 rpm at prescribed temp.

The activation of 10 g of PRP (in terms of PRP content) is carried out with 150 ml CDAP solution (50 mg/ml prepared in Acetonitrile) by gentle stirring for about 5 to about 8 min at about 3 to about 10 ºC (Preferably at about 4 to about 8 ºC) followed by addition of equal volume i.e. 150 ml of 0.2 M Triethylamine and mixing by gentle stirring for about 5 to about 8 min at about 3 about 10 ºC (Preferably at about 4 to about 8 ºC).

The modification of tetanus toxoid (For 1g of PRP content add 2.2 ±0.2 g of tetanus toxoid (in terms of total protein) is carried with EDAC solution on the basis of total protein available as TT. For 1 g of total protein, add 1g of EDAC (10 mg/ml) to the tetanus toxoid with constant stirring at about 3 to about 10 ºC (Preferably at about 4 to about 8 ºC) for about 60±10 minutes. For 10 g PRP reaction 22±2 g TT (n terms of total protein) is used with equal amount of EDAC.

Modified tetanus toxoid (TT-EDAC mix) is added to the PRP-CDAP complex, slowly with constant gentle stirring at about 3 to about 10 ºC (Preferably at about 4 to about 8 ºC). The reaction mixture is incubated for about 16 to about18 hours at about 3 to about 10 ºC (Preferably at about 4 about 8 ºC) with continuous gentle stirring. Total reaction mixture volume at this stage would be approx. 5-6 L (mainly depending upon the total protein concentration of TT). After completion of the reaction (for about 16 to about18 hours) take sample of the conjugate for PRP and free PRP.

Concentration and Diafiltration of purified PRP-TT has 2 steps; Concentration and Diafiltration of purified PRP-TT followed by Filtration of the concentrated-diafiltered PRP-TT conjugate.

PRP-TT conjugate is concentrated up to about 60 to about 80 % of the initial conjugated volume through 100 kD PES cassette (Min 0.5 m2 used in this case which may go up as per the scalability of the process) followed by diafiltration (of about 12 to about15 cycle) with physiological saline and is filtered through 0.45 micron filter.

The filtered concentrate is sampled and tested for microbiological control, PRP content, Free PRP and protein and stored at 5 ? 3°C till further purification step.

PRP-TT concentrate is applied to Sephadex G 25 matrix. Up to 25 % of sample (v/v of matrix volume) can be loaded on the matrix @ 20 L/hr on BPG glass column 200/500. Multiple runs can be carried out on the matrix as per the PRP-TT concentrate and pack bed volume of matrix. As soon as UV starts increasing, fraction is collected and it is collected until base line is reached. Collected elute is sampled for PRP content and molecular size distribution.

The last stage of the production process of the Haemophilic influenzae type b conjugate is the sterile filtration which is carried out using PES capsule or cartridge filter, pore size 0.22-?m. The final filtered material is sampled to test/analyze various parameters and stored in cold room at 3 – 10 ºC (Preferably 4-6 ºC).

Preferably, the purified polysaccharide fraction contains at least 70% (w/w) polysaccharide, more preferably at least 75, 80 or 85% (w/w) polysaccharide, based on the dry weight. The Endotoxin content is preferably less than 25 IU/microgram, more preferably less than 15, less than 10, less than 5 IU/microgram; most preferably, it is less than 2 IU/microgram poly-saccharide. The nucleic acid content is preferably less than 1% (w/w)

Advantages of the method according to the present invention are that the method is repro-ducible and cost-effective and gives optimal yields, even after a change in culture condi-tions. Furthermore, the bacteria are cultivated using a simple medium which does not contain components from animal origin, except for hemin. This yields a clean medium which is a big advantage, because the trend nowadays is to minimise transfer of animal disease, such as BSE, by using as much as possible media free from animal components.

Yet another advantage is that the method is scaled-up without substantial problems and has potential for further scale up especially because harvesting is based on growth cycle of the organism. Furthermore the method results in a very stable bulk polysaccharide that can be purified using a relatively simple process. The purification process is based on the concen-trated supernatant; the amount of auxiliary materials is therefore minimal. The purification results in a purified polysaccharide that is stable for a long time and that passes all the WHO requirements.

A polysaccharide which is produced using the method of the invention may be used to in-crease the ability of the human or animal immune system to fight infections. In particular, it may be used for the preparation of a pharmaceutical composition for administration to a human or animal subject. The polysaccharide or a conjugate thereof is preferably adminis-tered parenterally, e.g. by injection or infusion by intravenous, intraperitoneal, intramuscu-lar, intra arterial or intralesional route. The polysaccharide or a conjugate thereof may be combined with a pharmaceutically acceptable medium or delivery vehicle by conventional techniques known in the art. Methods for preparing parenterally administrable compositions are well known in the art and described in more detail in various sources, including, for ex-ample, Remington's Pharmaceutical Sciences, Ed. AR Gennaro, 20th edition, 2000, Williams & Wilkins, PA, USA. The polysaccharide is preferably administered in a therapeutically ef-fective dose, i.e. one that will increase the ability of the human or animal immune system to fight infections.

Preferably, it is used for the production of a vaccine, for example a polysaccharide conju-gate vaccine. Methods for producing conjugate vaccines are known in the art and described in e.g. Ada et al (2003) Clin. Microbiol. Infect. 9(2): 79-85,Dick et al (1986) Contributions to Microbiology and Immunology, vol. 10: Conjugate Vaccines: 48-114, and Jennings et al (1994) Neoglycoconjugates: Preparation and Applications: 325-371. Although there are slight variations in the methods used for producing conjugate vaccines, production methods typically comprise: activation of the polysaccharide and/or the protein carrier conjugation of the (activated) polysaccharide to the (activated) protein carrier optionally, purification of the polysaccharide-protein conjugate optionally, formulation of the polysaccharide-protein con-jugate.

The polysaccharide, which is preferably a polyribosyl ribitol phosphate (PRP), may be cou-pled to any protein carrier. Suitable protein carriers increase its immunogenicity and include immunogenic membrane proteins, viral protein subunits, synthetic polypeptides and other immunogenic proteins. Most preferably, the protein carrier is a toxoid. Well-known toxoids used in conjugate vaccines are tetanus toxoid and diphtheria toxoid.

It is common to size down the polysaccharide to a consistent molecular mass before conju-gation, by using controlled depolymerization methods known in the art. Suitable depolymer-ization methods comprise oxidation of vicinal diols, ultra-sonication, and acid or alkaline hydrolysis. These depolymerization reactions can be conducted at room temperature, but preferably in the cold, such as 5±3°C, to minimize unwanted side reactions, and preferably under semi vigorous agitation.

But the method described in the art, slightly acidic hydrolysis of the polysaccharide is pref-erably allowed between pH 5.5 and 6.5 and at low temperature preferably 5±3°C.
The polysaccharide may be activated before conjugation or before sizing down by activation methods known in the art, such as for example by using a cyanylating reagent (Kohn et al (1986) Appl. Biochem. Biotechnol. 9: 285-305). Suitable cyanylating agents include cyano-gen bromide (CNBr), 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate (CDAP), N-cyano-N,N,N-triethylammonium tetrafluoroborate (CTEA), and p-nitrophenylcyanate (pNPC). Alternatively, terminal aldehyde groups may be formed on the polysaccharide via oxidative cleavage of vicinal diols and conjugation may then be effected by reductive ami-nation with a suitable reducing reagent, such as sodium cyanoborohydride.

The polysaccharide may be conjugated to the protein carrier directly or after (further) activa-tion via spacer or linker molecules, introduced either on the (activated) polysaccharide and/or the (activated) protein carrier. Alternatively, these spacers can be introduced onto the protein carrier by an amidation reaction.

Removal of excess spacers can be effected by purification methods known in the art, such as gel permeation chromatography, differential precipitation, and diafiltration. A suitable diafiltration system makes use of the tangential flow filtration principle on microporous membranes. Physiological saline has been used as a preferred choice of solution to facilitate this purification process. After cyanylating the polysaccharide, conjugation to the protein carrier can be carried out by the mediation of a carbodiimide amidation reagent. A suitable amidation reagent is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimidehydrochloride (EDAC), which can be supplemented by N-hydroxysuccinimide (NHS) to facilitate the reac-tion.

A carbodiimide-mediated conjugation reaction can take place at slightly acidic pH, typically pH 5.5 to 6.8, thereby insuring preferential amidation of activated cyanylated polysaccha-ride groups over amino groups found on the protein carrier.
Removal of unreacted polysaccharide and protein can be effected by purification methods known in the art, such as gel permeation or gel filtration chromatography followed by con-centration and diafiltration. A suitable gel permeation chromatography system makes use of Sephadex G-25M, Sephacryl S-500 HR (GE Healthcare), or equivalent gel media, with a neutral physiological saline solution as eluent.

The polysaccharide produced using the method of the invention may be used to produce a monovalent as well as multivalent vaccine. A suitable example of a monovalent vaccine is a polysaccharide or a conjugate vaccine only against Haemophilus influenzae type b (Hib). Alternatively, the polysaccharide of the invention may be used to produce a multivalent vaccine. It may for example be used to produce a tetravalent vaccine, such as diphtheria-tetanus-polio-Hib or diphtheria-pertussis-tetanus-Hib, or a pentavalent vaccine, such as diph-theria-pertussis-tetanus-polio-Hib, or diphtheria-pertussis-tetanus-hepatitis B-Hib.

The invention is described hereinafter, with reference to the following examples, which are illustrative only and should not be construed to the limit of the scope of present invention.

Example 1

Growth test of Haemophilus influenzae type B

A Haemophilus influenzae type b strain (ATCC 10211) was cultivated using a 22 L bioreac-tor (working volume of 15 L) with a control system. This strain was identified as being a Haemophilus influenzae type b using commonly used tests, such as immune- and serotyp-ing, and morphology testing. The bioreactor was first filled with the basal medium (com-pound 1 to 6 in Table 1) before being sterilized in situ for 30 minutes at 121.degree. C. Just before inoculation the proper amount of stock solutions (Compound 7 to 11 in table 1) were added to the medium. The bioreactor was inoculated using pre-culture, cultivated on a 300 ml scale using the same medium and a frozen -70.degree. C. seed lot of the Hib strain.

The pH was kept constant at 7.2 using 5 mol NaOH. The temperature was kept constant at 35.degree. C. The dissolved oxygen (DO) was kept constant at =10% using Airflow: 0.5 – 1.0 vvm. The stirrer speed was kept 150 rpm.

Samples were collected at an interval of 1 hr. As the growing culture reached the OD550 1, feeding was started using a peristaltic pump. The feed rate was monitored and manually con-trolled to maintain glucose level 0.2- 0.7 gm/l. The culture was grown to an OD550 when three consecutive reading showed =0.5 units and allow to 2-4 hrs after stationary phase.

Table 1 : Composition of media
S.No. Reagents Concentration
1. Na2 HPO4.2H2O 2.5g/L
2. KCl 0.5g/L
3. NaCl 3.3g/L
4. NH4Cl 0.7g/L
5. Peptone Bacteriological(Granular) 15.0g/L
6. Yeast Extract 2.0g/L
7. L-Glutamic acid 1.2g/L
8. L-Cysteine 0.015g/L
9. Nicotinamide adenine dinucleotide (NAD) 0.020g/L
10. D+ Glucose monohydrate 5.0g/L
11. Hemin chloride 0.040g/L
Notes: compounds 1 to 6 can be dissolved in water, autoclaved after adjusting the pH to 7.5 and stored (basal medium). Compound 7 to 11 needs separate solution preparation and steri-lization.

Example 2

Production of Polyribosyl Ribitol Phosphate (PRP)

PRP was produced using 400 L bioreactor. The inoculum for 400 L bioreactor prepared un-der the conditions of Example 1 (batch fermentation). The bioreactor was first filled with the basal medium (compound 1 to 6 in Table 1) before being sterilized in situ for 30 minutes at 121.degree. C. Just before inoculation the proper amount of stock solutions (Compound 7 to 11 in table 1) were added to the medium. The bioreactor was inoculated using inoculum, cul-tivated on a 15 L bioreactor scale culture.

The pH was kept constant at 7.2 using 5 mol/l NaOH. The temperature was kept constant at 35.degree. C. The dissolved oxygen (DO) was kept constant at =10% using Airflow: 0.25 – 0.5 vvm. The stirrer speed was kept 100 rpm.

Samples were collected at an interval of 1 hr. As the growing culture reached the OD550 1, feeding was started using a peristaltic pump. The feed rate was monitored and manually con-trolled to maintain glucose level 0.2- 0.7 gm/l. The culture was grown to an OD550 when three consecutive reading showed =0.5 units and allow to 2-4 hrs after stationary phase.

The fermentation culture was heat inactivated at 550C for 15 minutes, There after the fer-mentation culture was harvested using a continuous under centrifuge. The supernatant was collected and clarified with cellulose filters using depth filtration method. Concentration and diafiltration of the filtrate was carried out using 30 kD MWCO PES membrane. The cation precipitation preferably using CATB is carried out to obtain precipates of polysaccharide. Centrifugation is carried out
preferably at 5000-7000 rpm

Example 3

Purification of Polyribosyl Ribitol Phosphate (PRP)

Purification mainly involved the operations of extraction by ethyl alcohol, centrifugation, and filtration through SP 10 filters and R53 SLP Zeta Carbon filters, precipitation with 4M sodium Chloride and drying at low temperature under vacuum to get PRP.

CTAB precipitates were further processed to extract PRP. Ethanol (96 %) was added in the ratio of 12.5 % v/v i.e. ~ 5 L to CTAB precipitates obtained from 30 KD concentrated and diafiltered broth. The prechilled precipitates were homogenized in 3-4 lots at low tempera-ture i.e. Temp between 4 and 15 °C for ~ 10-15 minutes (for each lot) at 6000 ± 500 rpm. The final volume after this step was approximate ~ 6- 8L.

Ethanol (96%) was added 32.5% v/v i.e. ~13 L to the homogenized CTAB precipitates and pH was adjusted to 6.8±0.3 with 8M Acetic acid. IKA homogenizer/Ultraturrax of working capacity of 30 L and which can attain up to 10000 rpm was used to homogenize the materi-al.

This extraction mixture kept on stirring (300 – 450 rpm) for 12-14 hrs at room temperature. The extraction mixture was then centrifuged in lots. Each lot was spinned for ~ 15 minutes at 5±3°C at 5000-6000 g and sampled for estimation of Ribose, Nucleic acid and Protein con-tent.

The extraction mixture was filtered through the 10SP zeta plus filters (cellulose filters pre-treated with 96% ethanol). Post filtration two washes were given to the filter, first with 96% ethanol (cold wash 12-20 °C) and second with 96% ethanol (warm wash 35-40 °C). Both washes and filtrate were then pooled, mixed with main filtrate and sampled for the estima-tion of Ribose, Nucleic acid and Protein content.

The 10SP Zeta plus filtrate was passed through R53 SLP Zeta Carbon filters (pretreated with 96% ethanol). Two washes were given to the filter, first with 96% ethanol (cold wash 12-20 °C) and second with 96% ethanol (warm wash 35-40 °C).. Both washes and filtrate were then pooled and mixed and sampled for Ribose, Nucleic acid and Protein content Final re-covered filtrate obtained after R53 SLP Zeta Carbon had the OD = 0.15 at 275nm. The ap-proximate volume this stage was 19-22 L. 4 M NaCl solutions (the final concentration of 1% v/v) was added to R53 SLP Zeta Carbon filtrate to precipitate the PRP. A white off white or ivory color lump or rubbery precipitate will appear which was allowed to settle at 5± 3°C for 12-14 hours. The white to off white or ivory color precipitate obtained from previous step was teased off to small pieces and subjected to drying for 16-18 hrs under vacuum and at low temperature (-70 to -80°C). A simple lab scale freeze drier was used for the purpose. The dried material obtained was identified as purified PRP and sampled for estimation of Ribose, Nucleic acid, Protein content, Phosphorus content, Moisture, Endotoxin, identity, and molecular size distribution (WHO TRS 814 Annex 1 1991 and TRS 897 Annex 1, 2000).

Purified PRP is stored at temperature of -20°C or below. The dried weight of obtained PRP ranges between 100 and 120 g.
Table – 3
Parameter Acceptance Criteria
Moisture Content < 3 %
Ribose Content ?32% w/w (Dry weight basis)
Phosphorus Content 6.8 -9.0% w/w (Dry weight basis)
Protein Content < 1% w/w (Dry weight basis)
Nucleic acid Content < 1% w/w (Dry weight basis)
KD ( Molecular size distribution) PRP ? 50 % ; Kd ? 0.3
Endotoxin content ? 25 IU / µg PRP
Identity Positive

Example 4
Conjugation of polyribosylribitol phosphate (PRP) with Tetanus Toxiod:
(TT) Conjugation process was carried out in 4 steps; PRP dissolution, TT modification, acti-vation of PRP and conjugation of PRP with TT as described below:

Dissolution of PRP:

Dried PRP quantity was taken as per batch size preferably 10 g on the basis of PRP content result specified for the particular PRP lot or batch used.
Dried purified PRP (having 10 g PRP content) was dissolved in WFI (~ 500 ml) at low temperature preferably at 5±3oC to get the final concentration of 20±2 mg/ml on the basis of PRP content. (PRP takes longer time to get dissolved at prescribed temperature. Allow to dissolve the PRP at 250-300 rpm at prescribed temp)

TT Modification:
The modification of tetanus toxoid (For 1g of PRP content add 2.2 ±0.2 g of tetanus toxoid (in terms of total protein) was carried out with EDAC solution on the basis of total protein available as TT. For 1 g of total protein, 1g of EDAC (10 mg/ml) was added to the tetanus toxoid with constant stirring at low temperature preferably at 5±3oC for 60±10 minutes. For 10 g PRP reaction 22±2 g TT (n terms of total protein) was used with equal amount of EDAC.

Activation of PRP:
The activation or cyanylation of 10 g of PRP (in terms of PRP content) was carried out with 150 ml CDAP solution (50 mg/ml prepared in Acetonitrile) by gentle stirring for ~ 5 min low temperature preferably at 5±3oC followed by addition of equal volume i.e. 150 ml of 0.2 M Triethylamine and mixing by gentle stirring for ~ 5 min at low temperature preferably at 5±3oC

Conjugation of PRP with TT:
Modified tetanus toxoid (TT-EDAC mix) was added to the PRP-CDAP complex, slowly with constant gentle stirring at low temperature preferably at 5±3oC. The reaction mixture was incubated for 16-18 hours at 5±3oC with continuous gentle stirring. Samples of the conjugate were analyzed for PRP and free PRP at this stage.

Purification of conjugate:
Concentration and Diafiltration of PRP-TT Conjugate:
Concentration and Diafiltration of purified PRP-TT was carried out in 2 steps; Concentration and Diafiltration of purified PRP-TT followed by Filtration of the concentrated-diafiltered PRP-TT conjugate. PRP-TT conjugate was concentrated up to 60-80 % of the initial conjugated volume through TFF system having 100 kD MWCO PES cassette/cartridge (Min 0.5 m2 to max 1.5 m2 can be used) followed by diafiltration (12-15 cycle) with physiological saline and was filtered through 0.45 micron filter. (Concentration-diafiltration can be carried out on simple cassette holder of relevant size with external peristaltic pump or an automated system from Pall/Sartorius/Millipore can be used).

The filtered concentrate was sampled and tested for microbiological control, PRP content, Free PRP and protein and stored at 5 ? 3°C till further purification step.

Gel Filtration Chromatography:
Chromatography step was carried out using AKTA process or equivalent system. PRP-TT concentrate was applied to Sephadex G 25 M matrix. Any other equivalent media can be used for chromatography. Maximum up to 25 % of sample (v/v of matrix volume) can be loaded on the matrix @ 20 L/hr on BPG glass column 200/500 (GE healthcare). Multiple runs can be carried out on the matrix as per the PRP-TT concentrate and pack bed volume of matrix. Collected eluate is sampled for PRP content and molecular size distribution. This was the last stage of the production process of the Drug Substance (DS) or Active Raw Material of Haemophilus influenzae type b conjugate vaccine. Sterile filtration is carried out using PES capsule or cartridge filter, pore size 0.22-?m and stored at 5 ± 3°C.

TABLE: 4 Composition of PRP-TT conjugate WHO specifications

Parameter Acceptance Criteria
PRP Content Not less than 0.5 mg/ml
Protein content Not less than 0.92 mg/ml
PRP to Protein ratio 0.30 - 0.55
Molecular size distribution > 85 % elution at KD = 0.5
Free polysaccharide < 20 %
Free Protein < 3 %
EDAC Content < 1.0 µg/ml
Identity Positive
CDAP Content <200 ng/dose (10 µg PRP)
pH 6.0-7.5
Endotoxin content < 25 EU/ µg of PRP

The present invention is used for expression and purification of polysaccharide that can be scaled-up without substantial problems and has potential for further scale up especially be-cause harvesting is based on growth cycle of the organism. The method results in a stable bulk polysaccharide that can be purified using a relatively simple process thus making a commercially viable option. The purification of PRP and conjugate vaccine involves simpler and cost effective steps without using any harsh or hazardous chemicals. The manufacturing process of PRP gives higher yield in terms of both dry weight as well as PRP content. The purification results in a purified polysaccharide which that is stable for 18 months at 5 +/- 3 deg.C and passes all the regulatory requirements such as WHO.

It is to be noted that the present invention is susceptible to modifications, equivalent chang-es, adaptations, by those skilled in the art. Such modifications, equivalent changes, adapta-tions are within the scope of the present invention which is further set forth under the claims as follows.