|Title of Invention||
PROCESS OF ISOLATING AND PURIFYING POLYRIBOSYL RIBITAOL (PRP), THE CAPSULAR POLYSACCHARIDE OF HAEMOPHILUS INFLUENZAE TYPE B
|Abstract||A novel invention of process for the isolation and purification of immunologically active polyribosyl ribitol phosphate (PRP), the capsular polysaccharide of Haemophillus influenzae type b. The PRP has been purified with ethanol, cetavlon (hexadecyltrimethyl ammonium bromide) and a phosphate containing adsorbant, hydroxylapatite. The contaminants (nucleic acid, proteins and endotoxins) are removed to the minimum level by a treatment with hydroxylapatite. The sera with anti - PRP antibody exhibits a strong bacterial activity.|
THE PATENTS ACT, 1970
(39 of 1970)
The Patents Rules, 2003
__ (SECTION 10)
1. Title of the invention.- PROCESS OF ISOLATING AND PURIFYING POLYRIBOSYL
RIBITOL PHOSPHATE (PRP), THE CAPSULAR POLYSACCHARIDE OF Haemophilus influenzae type b
(a) NAME : CADILA HEALTH CARE LTD.
(b) NATIONALITY : INDIAN
ADDRESS : SARKHEJ-BAVLA N.H. No. 8a, MORAIYA, Tal.
SANAND, Dist.: AHMEDABAD, GUJARAT-382 210
3. PREAMBLE TO THE DESCRIPTION:
The following specification describes the invention
Field of the Invention:
The present invention relates to the field of the vaccine, in particular process for producing a purified polyribosyl ribitol phosphate (PRP,), the capsular polysaccharide of Haemophilus influenzae which is immunologically active principle for preparing Haemophilus influenzae type b (Hib) vaccine used for immunization against Haemophilus influenzae type b infection.
More specifically, this invention relates to:
1. Process of preparation of immunologically active polysaccharide polyribosyl ribitol phosphate. (PRP) from Haemophilus influenzae type b Rab strain ATCC ® 31512.
2. A novel method of PRP production using fermentation and processing step which uses semi synthetic liquid medium for fermentation devoid of raw blood.
3. A novel method of PRP purification yielding highly purified immunogenic PRP (contaminant removed to the minimum level) which can be easily combined / conjugate with carrier protein.
Background of Invention/ Prior Art:
Influenzae is a clinical syndrome associated with bacterial meningitis. Haemophilus influenza type b is a leading cause of bacterial meningitis and other invasive bacterial disease among 95% children less than 5 years of age. H. Influenzae Eire divided into two groups, those strain that [possess a polysaccharide capsule and those that do not. The encapsulated strains are typed by serological reaction of a capsule with reference antisera types a - f have been known.//. Influenzae type b is the most known virulent strain of H. influenza Influenzae caused by Haemophilus influenza type b, remains a common and serious disease in children in many parts of the world. Despite therapy with antibiotics, the mortality of haemophilus meningitis remains approximately 5%, and serious neurologic sequalae occur in as many as 10 to 25%of survivors. Since 1974, after the discovery of multiple antibiotic resistance strain of Haemophilus influenza type b. In a randomized, double blind clinical trail carried out in Finland, a type b polysaccharide vaccine was found to be 90% effective in children aged group of 2-6years of age. (Peltola, et al., n.Engl.J.med,
310:1561-1566(1984). The type b polysaccharide elicits a thymic-independent immune response (Anderson, P. et al., J.Infect.Dis.136, S57; 1977)
The concerned have intensified for the development of HIB vaccine, which is economically viable without compromising quality at all to serve the mankind.
Data indicates that type b capsule of Haemophilus influenzae, a heteropolymer of approximately equal amount of ribitol ribose and phosphate (PRP). Polysaccharide, polyribosylribitol phosphate, is a linear copolymer composed of repeated units of 3-b-D-ribofuranosyl- (l®l)-ribitol-5-phosphate [(C10H19O12P) n]. (British Pharmacopoeia).
The prior art method well known to people skilled in the art, for the purification of PRP with ethanol followed Cetavlon (hexadecyltrimethyl ammonium bromide). The contaminants, endotoxin and pyrogenic substances, were removed by use of cold phenol or t-butanol and chloroform which may result in loss antigenic nature of PRP.
New methods for the production of immunologically active PRP from H. influenzae type b describe herein after. The fermentation process having advantage in which effect of culture variable is minimized resulting in yielding high quality of immunologically active high quality PRP over the prior art procedures.
More importantly the process have distinct advantage in that Cetavlon in cold condition is used to precipitate the polysaccharide from culture broth prior to ethanol. Detergent, added to a molar ration 1:1 is sufficient to precipitate virtually all the polysaccharide. The Cetavlon react with polysaccharide to form cetyltrimethylammonium salt of polysaccharide which fairly stabilize the immunological properties PRP, which facilitate further purification steps and better yield.
Further advantage of present invention is that has a revolutionary step in ethanol purification. Which is completed in following steps: ethanol extractions, gentle on PRP at the same time efficiently remove contaminants. The advantage of this is that it preserves the immunological properties of PRP. Followed by ethanol precipitation. The partially purified PRP is subjected to hydroxylapatite treatment to obtained PRP with desired purity.
Yet another advantage of this invention is by dissolving crude PRP in 1M NaCl. Facilitate further purification and conjugation procedure instead of conventionally used Calcium salt. The use of calcium salt interferes with the use of carbonate buffer in conjugation procedure.
Thus, in accordance with one aspect, the present invention gives a method of purifying PRP, the capsular polysaccharide of H. influenzae type b , obtained by fermenting, purifying the PRP a novel method.
Objectives of the Invention:
The objective of present invention to provide a bacterial sub unit vaccine (HIB) for use against Haemophilus influenzae infections.
The present invention further provides a process for the isolating and purification of PRP, capsular polysaccharide from Haemophilus influenzae type b.
Detailed Description of the invention
In the present invention a novel technique of fermentation, purification and extraction has been developed that makes it possible to obtain an immunologically active sub-unit, PRP.
Following schemes summarizes the process of the present invention:
PROCESS FOR PRODUCTION AND PURIFICATION OF POLYRIBOSYL RIBITOL PHOSPHATE, THE CAPSULAR POLYSACCHARIDE OF Haemophilus influenzae type b :
In the first step of the production process of the present invention, the working seed is prepared from master seed lot. The preparation of master seed and working seed is summarized in Scheme 1. The master seed lot is obtained by inoculating, a vial of seed culture obtained from ATCC (ATCC No: 31512;Rab strain) suspended in 40 ml of enrich B.H.I, medium (Diffco)+ 0.4 ml of haemophilus Growth supplement (Himedia) and incubating it for 22-24 hrs at 370C+i°C.with moderate shaking. To the above seed culture sterile (70 -80 %) glycerol and (20-25%) lactose solution was added, 10 ml of each for preservation. The above preparation was distributed into cryovials each containing one ml. Master seed lot undergoes Purity, biochemical and serological tests.
Working seed lot is prepared by inoculating 1ml of the master seed lot in 40 ml of B.H.I + 0.4 ml of haemophilus Growth supplement to BHI broth at 37 ±1°C for 22-24h. After obtaining of suitable growth 10ml of Lactose (20-25%) and 10 ml of Glycerol (70-80 %) added to broth and then dispense 1 ml in Cryovials. The working seed lot undergoes further QC analysis as per master seed before it is approved and is ready for use.
Following this the Production of PRP is carried out
• The fermentation is carried out using the seed lot principle. In this, each production run is initiated from a vial of Haemophilus influenza type b working seed lot.
• The working seed is produced by the method as explained hereinbefore.
• Fermentation is preformed in a fermentor using a semi-synthetic broth medium having composition that is known to the people skilled in the art.
• Fermentation is preformed in a fermentor using a semi-synthetic broth medium having following composition:
Sr.No Name of component Quantity (in gram / L)
1 Yeast Extract 5.0
2 Casamino acid 22.5
3 DiSodium Hydrogen Orthphosphate (Anhydrous) 14.4
4 Amonium Choloride 0.046
• Production Media Sterilized in fermenter at 12l'C for 20 min:
• Nutritive Media for production added after filter sterilization.
• The process of production involves a transfer of matured seed lot to the production fermentor,
The time of transfer depends upon the OD of the growth of seed culture.
• The fermentation is carried out at appropriate temperature in the range of 37 ±1 C with aeration.
• The pH during the fermentation is maintained between7.0 ± 0.5 by using sterile 3M NaOHsolution.
• The aeration and agitation is controlled to achieve the dissolved oxygen levels up to 50% of saturation.
• A backpressure of 0.2 is maintained throughout fermentation to facilitate the oxygen transfer and to control the foaming.
• Samples are drawn at regular intervals throughout the exponential growth phase, for checking identity, pH, OD, reducing sugar and microbial purity.
• The total duration of the fermentation is 8-10 hrs and final OD is around 5.0 at 600nm.
• The fermentation batch is terminated by immediate inactivation using formaldehyde at a concentration of 1% (final) for 16-18 hours at 2°C to 8°C and checked for inactivation.
• The overall summary of Purification of PRP-polysaccharide process is given in scheme: 3
Purification: Cell Separation:
• Formaline inactivated broth is centrifuged in refrigerated centrifuge Sorvoll RC3C plus at
4500 rpm with 4°C. Separate the liquid and bacterial residue, discarded residue.
• The liquid portion is precipitated with a detergent (0.2%w/v), the solute is used in a weak concentration and a pH is adjusted between 7.0 to 7.5 with a strong acid. The process is performed at lowered temperature 0°C to 4°C, to avoid any enzymatic or microbial development.
• Precipitates of the detergent precipitate are collected by centrifugation at 4500 rpm at + 4 °C, which is subsequently dissolved lM NaCl.
Alcohol Extraction and Precipitation:
• The dissolved material is subjected to 1st ethanol extraction (final concentration of ethanol about 50%) for 16-18 hours at 2°C to 4°C.
• Centrifugation at 4500 rpm to separate, the supernatant liquid collected further treated with the ethanol (final concentration of ethanol 80%) for 16-18 hours at 2°C to 4°C
• Centrifuge to collect precipitates from above step and dissolved in Phosphate Buffer Saline (20 mmol pH 6.9)
• To the above Dissolved material hydroxylapatite is added to give final concentration 1 % of
hydroxylapatite, vortex four an hour at 2°C to 4°C, centrifuge to collect soup. Repeat this
step atleast three times.
• The supernatant obtained is subjected to dialyze against pyrogen free water for injection using 10 KD Millipore membrane.
• Later quality control testing is performed and it has been ascertained in in-house testing using standard, generally accepted methods that the batches manufactured according to the present scheme pass the criteria according to WHO guidelines Ref: WHO/TRS/814. & British pharmacopoeia.
Assessment of Quality of Polysaccharide:
Sample is withdrawn from the bulk PRP and tested for following criteria
Criteria for acceptance include:
(a) Analysis of ribose (Orcinol method): greater than 30 percent,
(b) Analysis of protein (Lowry method): less than 1 percent,
(c) Analysis of nucleic acids (U.V. adsorbance): less than 1 percent, and
(d) precipitation with specific immune sera by the (counterimmunoelectrophoresis method).
In addition, the molecular size is determined by suitable gel premeation chromatography. The polysaccharide is monitored for endotoxin by Limulus Lysate testing and by rabbit pyrogenicity testing.
1. Anderson, P. and Smith, D. H. Feb. 1977.Isolation of the capsular polysaccharide from Culture supernatant of Haemophilus influenzae type b. J. Infection and immunity. 15 No.2: 472-477.
2. Liesbeth, V., Birgitta, D., Forien, G., Paul, E., Henk J., Jacob D and Loek van Alpphen. Mar. 1996. Immune selection for Antigen drift of major Outer membrane protein P2 of Haemophilus influenzae during persistence in subcutaneous tissue cages in rabbit. J. Infection and immunity.64 No.3: 980-986.
3. George, R.S., Donna, M.A., James, M., Thomas J.E., Gerald, S., Stephen, S.S. and George, F. G. July 1984. Preparation of Human Hyperimmune Globulin to Haemophilus influenzae b, Streptococcus pneumoniae, and Neisseria meningitidis. J. Infection and Immunity. 64 No. 1:248-254.
4. P. Anderson, G. Peter, R., B. Johnston, Jr., Liesle H. Wetterlow, and David J.H. Immunization of Humans with, Polyribophosphate, the Capsular Antigen of Hemophilus influenzae, type b The Journal of Clinical Investigation Vol. 57. 1972.
5. R. M. Herriott, E. M. Meyer, AND M. Vogt. Feb. 1970. Defined Non growth Media for Stage II Development of Competence in Haemophilus influenzae. J of Bacteriology. Vol. 101, No. 2. p 517-524
6. R. M. Herriott, E.Y.Meyer, M.Yogt, and M. MOD AN. Feb. 1970. Defined Medium for Growth of Haemophilus influenzae. Journal of Bactreriology. Vol. 101, No. 2. p. 513-516
7. Eugene Rosenberg and Stephen Zamenhof. November 1961. Further Studies on Polyribophosphate. The Journal of Biochemistry. Vol. 236, No. 11.
8. G. Toennies, B. Bakay, AND G. D. Shockman. Dec 1959. Bacterial Composition and Growth Phase. The J. Biochemistry. Vol: 234 No. 12.
Dated this 15th day of September 2006.
|Indian Patent Application Number||1557/MUM/2006|
|PG Journal Number||39/2013|
|Date of Filing||26-Sep-2006|
|Name of Patentee||CADILA HEALTHCARE LIMITED|
|Applicant Address||SARKHEJ-BAVLA N.H. NO. 8A, MORAIYA, TAL. SANAND, DIST-AHMEDABAD|
|PCT International Classification Number||C12N1/20|
|PCT International Application Number||N/A|
|PCT International Filing date|