Claims:

We claim:
1. A method of increasing the production of Capsular polysaccharide from Haemophilus influenza without the use of external virulence enhancing agents.

2. The method according to claim 1, wherein the Haemophilus influenza is of type b.

3. The method according to claim 1, wherein the virulence of Haemophilus influenza type b is enhanced without the use of external virulence enhancement agents.

4. The method according to claim 3, wherein more specifically Haemoglobin and Mucin are not used as external virulence enhancement agents to enhanced the virulence of Haemophilus influenza type b.

5. The method according to claim 1, produced by the method comprising the steps of:
Culturing Haemophilus influenza type b in peritoneum of healthy Balb C mice;
growing the virulent Haemophilus influenza type b in the  fermentaion medium;
fermentation  of cultures which have undergone the in-vivo passage in Balb-C mice peritoneum ;
Culture which has undergone number of passages and then undergone in fermentation, yield of polysaccharide increase by at least  200% as in the  Table 2 and Fig 1;
fermentation culture was harvested using continuous centrifuge, the cell biomass discarded and supernatant used for recovering the polysaccharide and

drying under vacuum at low temperatures for improved shelf life of Polysaccharide.

6. The method according to claim 6, wherein the ribose content of polyribosyl ribitol phosphate (PRP) of the virulent  Haemophilus influenza type b increase at different stages of fermentation as shown in table 2  for Hib protein–polysaccharide conjugates and  further purification.
, Description:

FORM 2
THE PATENTS ACT 1970
(Act 39 of 1970)

COMPLETE SPECIFICATION
(SECTION 10; Rule 13)

Increase production of capsular polysaccharide from a virulent Haemophilus influenza type b without the use of external virulence enhancement agents

Techinvention Lifecare Pvt. Ltd.
#1004, The Summit Business Park,
Off WEH Metro Station, Andheri Kurla Road,
Andheri East, Mumbai 400093. INDIA

THE FOLLOWING SPECIFICATION DESCRIBES THE NATURE OF THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED

Field of the invention
The present invention relates to the field of the vaccine, the present invention relates to the increased production of Capsular polysaccharide from a virulent Haemophilus influenza type b without the use of an external virulence enhancement agents, its purification and conjugation with carrier protein.

Background of the invention
Background description includes information that may be useful in understanding the present 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 describe the state of the art to which this invention pertains.
Haemophilus influenza type b (Hib) is transmitted through the respiratory tract from infected 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 resource-poor countries, prior to the introduction of the vaccine into their national immunization programs.
Haemophilus influenza type b (Hib) is responsible for severe pneumonia, meningitis 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 programs. (Source WHO)

Usually the efficacy of a vaccine developed is evaluated in a laboratory animal model which is susceptible to the infection. Numerous publications have indicated lack of an appropriate and suitable animal model for studying the exact mechanism of virulence of different Hi strains. External virulence enhancement agents were proposed which could enhance the susceptibility of the animals to Hi infection and reduce the infective dose. External Virulence-enhancement agents studied includes ferric ammonium citrate, capsicum, Hematin, hemoglobin, iron dextran, iron sorbitol, lysed red blood cells, mineral oil, Mucin, Transferin, trypsin and Haemoglobin.
N Mojgani et al , reports that BALB/c mice appeared suitable for evaluating the virulence of Hib strains, and 2% hemoglobin with 4% Mucin an appropriate concentration for inducing infection in this animal model hence this animal could be considered suitable for testing the virulence and infectivity of Hib vaccines in future.
The lethality of the Hib isolates in study were significantly enhanced in the presence of hemoglobin and mucin in combination rather than hemoglobin alone.
In the absence of enhancement agents, all the Hib isolates appeared non-infective and were not able to kill the challenged mice even at high doses of 10 8 cfu/ml. However, a transient bacteremia was observed in few of the animals injected with high doses of Hib NM3 without hemoglobin and or mucin as reported in the studies of N Mojgani et al; 2013, Volume : 31 ;Issue : 2;Page : 148--153 ; Indian Journal of Medical Microbiology
Again, Howe J et al in Med Chem. 2008 Nov;4(6):520-5 discloses Hemoglobin enhances the biological activity of synthetic and natural bacterial (endotoxic) virulence factors Are included as prior art of the invention.
The focus of most of the research work was to increase the virulence of the organism by using Hemoglobin and Mucin as the Virulence-enhancement agents.
Most commonly used Hemoglobin is of bovine origin, while Mucin is of porcine origin. The main motive of using Hib is to produce Polysaccharide and subsequently use it for vaccine production. In the course of manufacturing of human vaccines, it is always recommended to avoid addition of any external components, unless they are absolutely essential for growth, expression or survival of the host organism.
Hemoglogin and Mucin is not essential for the growth, revival and survival of Hib culture.
Moreover since the origin of Mucin is porcine, use of such an agent will create subsequent challenges to remove during the purification process. Again, acceptability in certain user populations is also a matter of concern.
In view of the foregoing, it would be a significant advancement in the art of medicine to provide bacterial vaccines which are relatively free from external Virulence enhancement agents or other non-essential agents which are substantially avoided to be use in the virulent bacterial vaccines. It would be a further significant advancement in the art to provide virulent bacterial vaccines which increase the production of Capsular polysaccharide from a virulent Haemophilus influenza type b without the use of Virulence-enhancement agents

It would be yet another significant advancement in the art to provide relatively simple and inexpensive methods for preparing such bacterial vaccines which are both efficacious and affordable. Such bacterial vaccines, such methods for preparing virulent bacterial vaccines, and methods of Increase Production and Purification of Capsular polysaccharide from a virulent Haemophilus influenza type b without the use of Virulence-enhancement agents are disclosed and claimed herein.

Objects of the invention
The objective of the present invention is to provide increased production of Capsular polysaccharide from a virulent Haemophilus influenza type b without the use of an external virulence enhancement agents, its purification and conjugation with carrier protein.

Yet another object of the present invention is to provides a method for the production of virulent Haemophilus influenza type b without the use of hemoglobin and Mucin.
Yet another object of the present invention is to provide a method for the production and purification of Capsular polysaccharide from a more virulent Haemophilus influenza type b, its conjugation with carrier protein.

Summary of the invention
In a general aspect the present invention provides increased production of Capsular polysaccharide from a virulent Haemophilus influenza type b without the use of an external virulence enhancement agents, its purification and conjugation with carrier protein.

In an embodiment, the present invention provides a method for the production of virulent Haemophilus influenza type b without the use of Hemoglobin and Mucin.

In an embodiment, the present invention provides a method for the production and purification of Capsular polysaccharide from a more virulent Haemophilus influenza type b, its conjugation with carrier protein.

Detailed description of the invention
The following is a detailed description of some of the embodiments and explanation of the present invention with some examples thereof. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”
Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise.
In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.
The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
All processes described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
The term “Virulence’’is the ability of an organism to cause disease. Virulence of an organism frequently is tested by inoculation of different strains of a pathogen into groups of a rodent species and evaluation of lethality or invasiveness.
The term ‘carrier protein’ as used herein refers to the protein component to which the capsular polysaccharide of Hib is conjugated.
The term ‘stable’ as used herein means that each of the antigens of the vaccine composition has a potency/immunogenicity more than that set as the normal acceptance limit
Reference will now be made in detail to the exemplary embodiments of the present disclosure, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers are used in the drawings and the description to refer to the same or like parts.
The present invention relates to the increased production of Capsular polysaccharide from a virulent Haemophilus influenza type b without the use of an external virulence enhancement agents, its purification and conjugation with carrier protein 
In an embodiment, the present invention provides a method for the production of virulent Haemophilus influenza type b without the use of Hemoglobin and Mucin.

In an embodiment, the present invention provides a method for the production and purification of Capsular polysaccharide from a virulent Haemophilus influenza type b, its conjugation with carrier protein
Haemophilus type b conjugate
Haemophilus influenzae is a gram-negative Cocco bacilli which causes invasive blood borne infections and meningitis. The Hib antigen, which is derived from the capsular polysaccharide, may be conjugated or coupled to carrier proteins. The carrier proteins used for the conjugation of the Hib antigen is Tetanus Toxoid (TT). The conjugate of Hib antigen with Tetanus Toxoid is denoted as ‘Hib-PRP-T’.

While the foregoing description discloses various embodiments of the disclosure, other and further embodiments of the invention may be devised without departing from the basic scope of the disclosure. The invention is not limited to the described embodiments, versions or examples, the invention is described hereinafter, with reference to the following examples, which are to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art, such examples are illustrative only and should not be construed to the limit of the scope of present invention.

Example-1
Increasing productivity of Polysaccharide production using in-vivo techniques without the use of external virulence enhancement agents
Haemophilus influenza type b is grown in peritoneum of healthy Balb C mice preferably female by infecting appropriately diluted culture. Mice colony is maintained with proper access to feed and water in good laboratory conditions. The strain of H. influenza is grown in suitable rich medium (Chocolate Agar, Brain Heart Infusion Broth preferably supplemented with 3 and 1.5 mg/ml of Hemin and nicotinamide adenine dinucleotide) respectively at 37°C in the presence of 5% CO2 till culture reaches logarithmic phase. After incubation the culture broth is harvested by centrifugation at 4°C and pellet washed twice with Phosphate buffer saline (PBS)
Groups of mice, aged 4-5 weeks and weighing 17-22 g were housed under standard conditions of temperature and relative humidity with a 12 h lighting schedule. The culture is diluted appropriately in PBS to achieve different concentrations in colony forming unit cfu/ml. The diluted culture is injected into the peritoneum of female Balb C mice and observed. Percentage survival was recorded in each group of mice by observing the survival and death. After incubation of 5 days, the peritoneum fluid of mice is harvested by tapping method-using hypodermic syringe.
The peritoneal fluid harvested is cultured on selective media (Chocolate Agar, Brain Heart Infusion Broth supplemented with 3 and 1.5 mg/ml of Hemin and nicotinamide adenine dinucleotide) to isolate the pure Haemophilus influenza type b Strain by picking an isolated single colony. The media is incubated at 37°C in the presence of 5% CO2.
The isolated strain is further passaged on the selective media (Chocolate Agar, Brain Heart Infusion Broth supplemented with 3 and 1.5 mg/ml of Hemin and nicotinamide adenine dinucleotide) and incubated at 37°C in the presence of 5% CO2 .

There is significant difference observed in the yields of polysaccharide at the end of fermentation derived from cultures which have undergone the in-vivo passage in Balb-C mice peritoneum shown in Table 1 and 2.
As the number of passage increase the yield of polysaccharide increase upto 200% at 6th passage in Table: 2. Thereafter it appears to reach a plateau between 5 and 6 the passage in Fig: 1

Table 1: Polysaccharide production without in-vivo passage

Table 2: Polysaccharide production at various stages of in-vivo experiments

Fig 1: Graphical representation of the yield of polysaccharide with the number of in-vivo passage in Balb C mice

Example -2
Use of modified media composition for optimized growth of modified organism
The isolated strains of Haemophilus influenza type b at each passage are cultivated using fermentor with a control system and the harvest is subjected to purification. The fermentor is filled with the basal medium (consisting of carbon, nitrogen, salts, and proteins) before being sterilized of media. The media is sterilized in situ at 121ºC. Just before inoculation the proper amount of sterilized stock solutions (consisting of L-Glutamic acid, L-Cysteine, Nicotinamide adenine dinucleotide (NAD), D+ Glucose monohydrate, Hemin chloride) were added to the medium. The fermentor was inoculated using the strain isolated.
The fermentation parameters: pH, temperature, dissolved oxygen (DO), airflow and rpm are controlled and monitored during the entire fermentation process. The modified seed culture is grown in suitable culture medium at a suitable pH and temperature adjusting the pH of the culture medium to a constant value with base or acid and feed is added to provide the culture required nutrient for growth for fermentation and fermentation is continued until culture reaches late logarithmic phase. The fermentation culture is heat inactivated for inactivation of the live culture in the medium. Thereafter the fermentation culture was harvested using a continuous centrifuge, the cell biomass discarded and supernatant used for recovering the polysaccharide.
The polyribosyl ribitol phosphate (PRP) capsule of Hib is a linear copolymer composed of repeated units of?3)-ß-D-Ribf-(1?1)-D-ribitol-(5-OPO3?[(C10H19O12P)n] with a defined molecular size. The fermentation medium is composed of complex components and does not contain any ribose. Estimation of ribose content in the different stages of fermentation and purification provides a reasonably accurate estimation of the polysaccharide content in the solutions. Hence ribose was selected as a marker molecule for PRP content.
Five fermentation batches (1-5) were taken and samples were withdrawn at different time intervals of fermentation for Ribose content (µg/ml) estimation. Samples withdrawn at zero hour and 6th hour of fermentation did not showed the presence of ribose. The sample withdrawn at 8th hour of fermentation and after that at every two hour interval till end of fermentation showed the presence of ribose. The results are shown in Table: 3
Table 3: Ribose content (µg/ml) at various stages of Fermentation

Example-3
Modified process for drying under vacuum at low temperatures for improved shelf life of Polysaccharide
After salt precipitation, the Haemophilus influenza type b polysaccharide, which appears as white to off white rubbery substance like chewing gum/cheesy mass, settles in the bottom of the bottle.
This mass is then tweaked or broken into small pieces with SS scissors or SS knives and filled into pre chilled round bottom flasks.
These pre chilled round bottom flasks with small pieces of polysaccharide are then rotated in liquid nitrogen in polystyrene box containing 5-10 L liquid Nitrogen preferably 7-8 L for 5 to 15 minutes by preferably 10-12 min quick deep freezing.
The individual round bottom flask is then fixed to manifold of deep freezer already set at -80deg. C. The drying is carried out at low temperature for 24 – 30 hrs. , preferably 26-27 hrs. at 60-120 m torr preferably 80-100 m torr.
After completion of vacuum drying, dried lumps are collected in pre weighed glass bottles and stored at or below -20 degree.C. The residual moisture is measured using Karl Fisher apparatus.
So the advantage of vacuum drying of polysaccharide is improved shelf life of the dried polysaccharide. There is no need to store in liquid/dissolved condition which requires larger bottles. Reduced volume by drying requires smaller storage area as stored in small glass bottles. Ease of operation increases as larger volumes of dissolved Polysaccharide is difficult to handle whereas small pieces/lumps of PS can be weighed and used for conjugation reaction and easy to transport to different facilities or rooms. Another significant advantage of dried Polysaccharide with low moisture content is increased thermal stability of the PRP. Reduced moisture contents in turn increases the shelf life of dried polysaccharide. PRP stored in liquid form or frozen form has been shown to have a shelf life of lesser than of 18 month. Thereafter there is degradation in the polysaccharide and free ribose content increases.

Accelerated stability studies on PRP which was exposed to long term storage at 37. Degree C +/- 1 degree C. The ribose content was estimated at weekly intervals. The real time shelf life was predicted based on Arrhenius equation.
Moisture content was evaluated for several samples subjected to the drying and storage process as above. Results are provided in Table 4 and shown in Fig: 2.
Table 4: Moisture content of samples after modified drying process

Fig 2: Graphical representation of moisture content is various test samples stored after modified drying method

Example 4: Recovery process
a) Using a cationic detergent to precipitate the polysaccharide or part of the contaminants from the supernatant;
b) Using alcohol to solubilize the polysaccharide in two steps. Step 1 comprises of using 12.5% of alcohol (96% ethanol) v/v followed by homogenization at 5500 – 6500 rpm at low temperature. Step 2 comprises the further addition of 32.5% alcohol (96% ethanol) v/v, pH adjustment by 8 M Acetic acid to get the range of 6.5 -7.1 and gentle stirring at room temperature for over night preferably 12-16 hrs.
c) Subjecting the solubilized polysaccharide to centrifugation at 5000-6000 rpm at low temperature preferably 5±3°C to recover the supernatant. Supernatant is then filtered through cellulose filters sheets and followed by filtration through carbon (activated charcoal) filters sheets to get the decolorized fraction. The degree of decolourization of the filtrate is determined by OD = 0.15 at 275nm against water. In the last step of precipitation i.e. salt precipitation, 4M Sodium Chloride is mixed at 1 % v/v to the solubilized polysaccharide. The pellet is preferably separated from the supernatant by decanting and not by centrifugation
Advantages of the method according to the present invention are that the method is reproducible and cost-effective and gives optimal yields, even after a change in culture conditions.
The method for production of polysaccharide comprising the step of preparing pre inoculum of Haemophilus influenza type b. Pre inoculum is used for the preparation of inoculum for fermentation and production of desired end product i.e. polysaccharide.
The cultivation of Haemophilus influenza type b is carried out in suitable fermentor by keeping parameters under control. The pH is kept constant at 7.0- 7.2 . The temperature is kept constant at 35 degree Celsius. The dissolved oxygen (DO) is kept constant at =10% using Airflow: 0.25 - 1.0 vvm. The stirrer speed is kept 100-150 rpm.
The feed rate is monitored and manually controlled to maintain glucose level 0.2- 0.7 gm/l.
Fermentation takes place from about 12 to 24 hours, after fermentation, inactivation of culture takes place followed by downstream processing such as harvest and centrifugation, clarification, concentration / Diafiltration, precipitation, and centrifugation.
After centrifugation, precipitate is subjected to homogenization and pH is adjusted. The homogenized material is subjected to centrifugation followed by depth filtration and salt precipitation. Precipitate is collected and vacuum dried at low temperature to obtain purified poly ribosyl ribitol phosphate (PRP) which is further kept for storage at or below minus 20 degree Celsius..
The Poly ribosyl ribitol Phosphate (PRP) is subjected to chemical reaction with carrier protein such as Tetanus Toxoid, Diphtheria Toxoid and CRM protein for preparation of conjugate vaccine.
The PRP is activated and tetanus toxoid is modified, the modified tetanus toxoid is added to PRP and the mixture is concentrated and subjected to diafiltration followed by filtration and purified using size exclusion chromatography method. The purified chromatographic material is then filter sterilized and stored at 2 to 8 degree Celsius.
Inactivation of fermentation culture is done preferably by heating the culture at suitable temperature and for suitable time, of about 50 degree C to about 60 degree C for about 10 to about 30 minutes. There after the culture broth is harvested by separating the cell mass by centrifugation at speed of 4000-8000 followed by clarification with cellulose filters using depth filtration method. This is followed by Concentration and diafiltration of the filtrate using desired MWCO PES membrane 30kD .
The cation precipitation preferably using CATB is carried out to obtain precipates of polysaccharide. Centrifugation is carried out preferably at 5000 – 7000 rpm at lower temperature.
The polysaccharide purification does not require any harsh chemicals such as phenol and simple steps of precipitation and filtration makes the purification process safe and economically viable.

Example 5: Method of producing conjugate vaccine
The method of producing conjugate vaccine comprise: activation of the polysaccharide and/or the protein carrier conjugation of the (activated) polysaccharide to the (activated)
protein carrier i.e. Tetanus Toxoid followed by purification of the polysaccharide-protein conjugate and formulation of the polysaccharide-protein conjugate.
The polysaccharide is sized down to a consistent molecular mass using high alkaline medium before conjugation at low temperature such as 5±3°C or room temperature and under gentle agitation.
1 g of dried PRP quantity is taken on the basis of PRP content. Dried purified PRP (having 1 g PRP content) is dissolved in WFI (~ 50 ml) at low temperature preferably at 5±3°C to get the final concentration of 20±2 mg/ml on the basis of PRP content at 250-300 rpm)
The polysaccharide is then activated by using a cyanylating reagent 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate (CDAP). The polysaccharide is then conjugated to the protein carrier which is also modified chemically to introduce space by amidation reaction which employ amidation reagent is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimidehydrochloride (EDAC).
The activation or cyanylation of 1 g of PRP (in terms of PRP content) is carried out with 15 ml CDAP solution (50 mg/ml prepared in Acetonitrile) by gentle stirring for ~ 5 min low temperature preferably at 5±3°C followed by addition of equal volume i.e. 15 ml of 0.2 M Triethylamine and mixing by gentle stirring for ~ 5 min at low temperature preferably at 5±3°C.
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 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) is added to the tetanus toxoid with constant stirring at low temperature preferably at 5±3 degree Celsius for 60±10 minutes.
Modified tetanus toxoid (TT-EDAC mix) is added to the PRP-CDAP complex, slowly with constant gentle stirring at low temperature preferably at 5±3°C. The reaction mixture is incubated for 16-18 hours at 5±3°C with continuous gentle stirring.

Unreacted moieties are removed by purification methods such as gel permeation chromatography, using Sephadex G-25M or Sephacryl S-500 HR or equivalent gel media. Physiological saline is used as eluent to facilitate this purification process.

Concentration and Diafiltration of purified PRP-TT is 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 is 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 is filtered through 0.45 micron filter.
Chromatography step is carried out using Sephadex G 25 M matrix or 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 at 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 is 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- µm and stored at 5 ± 3°C.
The polysaccharide produced using the method of the invention may be used to produce a monovalent as well as multivalent vaccine.