Downstream process for purifying polysaccharides

Abstract

The present invention relates to a novel process for purifying bacterial polysaccharide. It is an efficient and scalable process for removing impurities from Neisseria meningitidis serogroup C (Men-C) polysaccharide which is capable of being used as such in a derivatized form or linked to other molecules, for the preparation of vaccines, more particularly conjugate vaccines for N. meningitidis infection.

Claims

1. A process for purifying Men C-Polysaccharide, wherein said process comprises the steps of: (a) centrifugation of the fermented harvest to clarify the fermented broth; (b) concentrating the fermented supernatant by ultrafiltration; (c) deacetylation and incubating said concentrated supernatant of step (b) at a temperature ranging from 70 C. to 80 C.; (d) collecting the supernatant of step (c) and subjecting to diafiltration and concentration; (e) purification of diafiltered supernatant of step (d) by chromatography; (f) diafiltration and concentration to obtain purified polysaccharides; and (g) sterile filtration of said purified polysaccharides, wherein the said purification process is completed within 61 hours and wherein the purification process is completed without the use of a detergent.

2. The process as claimed in claim 1 wherein said step of centrifugation of the fermented harvest is carried at 4500g to 5000g.

3. The process as claimed in claim 1 wherein said step of deacetylation is carried with NaOH solution at a concentration which ranges from 1 to 1.5 M.

4. The process as claimed in claim 1 wherein the incubation in the deacetylation step is performed with a total incubation time ranging from 1 hour 30 mins to 2 hours 30 min.

5. The process as claimed in claim 1 wherein the diafiltration in the collecting step after the deacetylation step is carried out 20 to 25 times with Milli-Q water followed by 8 to 10 times with 25 mM Tris HCl buffer.

6. The process as claimed in claim 1 wherein said chromatography used in the purification step is hydrophobic interaction chromatography (HIC) Phenyl sepharose 6 fast flow.

7. The process as claimed in claim 1 wherein the diafiltration and concentration to obtain purified polysaccharides step is carried with 100 KDa PES membrane (0.1 m.sup.2), with 6 to 8 volumes of Milli-Q water.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 Depicts the process flow for the MenC-PS purification and recovery.

(2) FIG. 2 Depicts the HPLC Chromatogram of MenC-PS

(3) FIG. 3 Depicts the NMR spectrum of MenC-PS.

DETAILED DESCRIPTION OF THE INVENTION

(4) The invention discloses two steps that have been optimized to enable the purification of MenC polysaccharide (MenC-PS) in lesser time as shown in FIG. 1.

(5) The strain of bacterial polysaccharide is inoculated in the fermenter containing appropriate media components required for bacterial growth. After achieving the maximum optical density, in the range of 8 to 10 depicting substantial bacterial growth, it is subjected to termination by adding prerequisite concentration of formaldehyde and the resultant FB is obtained. FB is then centrifuged at high speed to clarify the FB of cell debris followed by diafiltration and concentration using molecular weight cut-off membranes.

(6) The diafiltered and concentrated FB having the crude polysaccharide is treated with NaOH at high temperature e.g up to 80 C. for deacetylation. Concentration and diafiltration of the crude polysaccharide is performed with polyether sulfone (PES) membrane, with milliQ water (MQW), followed by Tris HCl buffer pH 7.40.1. After diafiltration crude polysaccharide is concentrated and filtered with 0.22 m filter.

(7) Deacetylated crude polysaccharide is further purified by Hydrophobic interaction chromatographic (HIC) technique.

(8) The separation of two or more components of a mixture based on differences in polarity is well known to those skilled in the art. For example, using hydrophobic-interaction chromatography, compounds of relatively greater hydrophobicity are retained longer on the column relative to those compounds that are more hydrophilic. Conversely, using hydrophilic-interaction chromatography, hydrophilic compounds are retained longer on the column relative to those compounds that are more hydrophobic. Using both methods consecutively allows for the removal of impurities that are both less polar and more polar relative to the compound of interest

(9) The extracted CPS present in the base extraction reagent can be separated from impurities resulting from the cellular components by chromatography. Non-limiting examples of the chromatographic separation methods are ion-exchange (cationic or anionic), hydrophilic-interaction, hydrophobic-interaction or gel-permeation chromatography. More preferred is hydrophobic-interaction chromatography (HIC) on phenyl sepharose which will remove most of the high molecular weight, uv-active contaminants from the base extract. Capsular polysaccharide will elute in the beginning, while, the more hydrophobic protein and nucleic acids will be retained. The preferred method in this invention is hydrophobic-interaction chromatography.

(10) Further concentration and diafiltration of the polysaccharide is performed to facilitate the removal of proteins and other impurities, and the polysaccharide sample is then washed with MQW.

(11) Consequently sterile filtration is done to obtain the purified Men C polysaccharide depicted by FIGS. 2 and 3.

(12) The corroboration of the above employed procedures can be conveniently understood from Table 1 which clearly shows that the purified polysaccharide specifications meet the WHO standard.

(13) TABLE-US-00001 TABLE 1 Purified Men-C polysaccharide specifications in accordance with WHO specification are shown below Tests MenC-PS WHO Specifications Polysaccharide content 4 mg/ml Actual value Sialic acid content 100% The sialic acid for MenC content not less than 80% of the dry weight Endotoxin <0.06 EU/g Less than 100 EU/g Protein % 0.14% Not more than 1% Nucleic Acid % 0.42% Not more than 1% Average relative 351 kd Actual value from HPLC Molecular Weight Identity Positive Positive agglutination reaction with specific sera; Defined .sup.1H- NMR spectrum

(14) Of particular interest is the observation that the time required to purify the Men C polysaccharides is significantly less from those disclosed in the prior arts whereas the hallmark of the present invention is the novel, rapid and scalable purification process protocol which can be completed within 61 hours and the PS so produced is in compliance with the WHO set standard.

(15) Analytical Procedures:

(16) Polysaccharide 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:

(17) Total sialic acid of MenC-PS is determined by a colorimetric method. The assay is based on the reaction between sialic acid and resorcinol at 100 C. in the presence of hydrochloric acid and copper (II) ions for 30 minutes; this leads to the formation of a blue-violet complex which exhibits a strong absorbance at 564 nm. The total sialic acid concentration is determined from a calibration curve obtained using a series of sialic acid standards [(Svennerholm, 1957)]. Lipopolysaccharide (LPS) is determined using compact and simple Endosafe-PTS apparatus. Protein impurity is determined by Lowry's method [(Lowry et al., 1951)] using bovine serum albumin as a standard and the absorbance is taken at 750 nm. Nucleic acids (NA) is estimated at 260 nm and the amount is calculated assuming an absorbance of 1.0 A=50 g/mL [(Frasch, 1990)].

(18) Relative Average molecular size (Mw) of HibPRP is determined using High-performance liquid chromatography (HPLC) (Alliance, Waters). The columns used are PWXL-4000 and PWXL-5000 in series (Tosoh Bioscience). Furthermore, a range of 5 kD to 800 kD Pullulans (Shodex) are used as standards for MenC-PS. The HPLC is performed using 0.1 M sodium nitrate with a run time of 30 min at a flow rate of 1 ml/min. The identity of MenC-PS is verified by 1H-NMR spectroscopy. NMR yields a spectrum of magnetic sensitive nuclei (e. g. 1H).

(19) The MenC-PS is identified serologically by combining with the reference antisera against each polysaccharide. As the WHO specifications [WHO, 2004] to determine the purity and to characterize the polysaccharide is based on dry weight basis, the MenC-PS is first lyophilized and then tested. The moisture content is thus subtracted to get the exact dry weight. Moisture content of lyophilized cake is determined by Thermo gravimetric Analyzer (TGA) from Perkin Elmer. The analytical results for MenC-PS are given in Table-1 and are in accordance and as specified by WHO.

(20) The said Men C polysaccharide can be used for the preparation of polysaccharide-protein conjugate vaccines.

(21) Various aspects of the invention described in details above is now illustrated with non-limiting examples.

EXAMPLE-1

(22) Polysaccharide Purification Using Anionic Detergent with Tris Buffer and Hydrophobic Interaction Chromatography (HIC):

(23) The diafiltered and concentrated FB having the crude polysaccharide is processed with anionic detergent, such as, sodium deoxycholate at a concentration 0.3% to 0.6% (w/v) and incubated at 50 C.-60 C. for 1 hour. Subsequently it is cooled to below 40 C. followed by concentration and diafiltration of the crude polysaccharide with 0.1 m.sup.2 poly ether sulfone (PES) membrane, with 6-8 volumes of milliQ water (MQW).

(24) Deacetylation is then performed on the crude polysaccharide with 0.5M sodium hydroxide (NaOH) at 50 C. for 10 hours. Subsequently it is cooled to below 40 C. followed by diafiltration and concentration of the crude polysaccharide simultaneously with 6-8 volumes of MQW and 20 mM Tris HCL buffer using 0.1 m.sup.2 PES membrane. Afterwards the polysaccharide is further purified by Hydrophobic Infraction Chromatography (HIC), Such as, Phenyl sepharose 6 Fast Flow. Finally, concentration and diafiltration of the purified polysaccharide was performed with 0.1 m.sup.2 PES membrane, with 6 to 8 volumes of MQW. Accordingly, sterile filtration is done with 0.22 m filter and the final purified polysaccharide is stored at 20 C. The total time taken to purify the polysaccharide including HIC chromatography using the above process was 16-18 hours.

EXAMPLE-2

(25) Polysaccharide Purification Using Anionic Detergent with HEPES Buffer (4-(2-Hydroxyethyl)-1-Piperazineethanesulfonic Acid) and Hydrophobic Interaction Chromatography (HIC):

(26) The diafiltered and concentrated FB having the crude polysaccharide is processed with anionic detergent, for example, sodium deoxycholate at a concentration 0.3% to 0.6% (w/v) and incubated at 50 C.-60 C. for 1 hour. Subsequently it is cooled to below 40 C. followed by concentration and diafiltration of the crude polysaccharide with 0.1 m.sup.2 poly ether sulfone (PES) membrane, with 6-8 volumes of MQW.

(27) Deacetylation is then performed on the crude polysaccharide with 0.5M sodium hydroxide (NaOH) at 50 C. for 10 hours. Subsequently it is cooled to below 40 C. followed by diafiltration and concentration of the crude polysaccharide simultaneously with 6-8 volumes of MQW and 20 mM HEPES containing 3M NaCl buffer using 0.1 m.sup.2 PES membrane. Afterwards the polysaccharide is further purified by Hydrophobic Interaction Chromatography (HIC), Such as, Phenyl sepharose 6 Fast Flow. Concentration and diafiltration of the purified polysaccharide was performed with 0.1 m.sup.2 PES membrane, with 6-8 volumes of MQW. Consequently, sterile filtration was done with 0.22 m filter and the final purified polysaccharide is stored at 20 C. The total time taken to purify the polysaccharide including HIC chromatography using the above process was 16-18 hours.

EXAMPLE-3

(28) Polysaccharide Purification Using Sodium Hydroxide with HEPES Buffer ((4-(2-Hydroxyethyl)-1-Piperazineethanesulfonic Acid) and Hydrophobic Interaction Chromatography (HIC):

(29) The diafiltered and concentrated FB having the crude polysaccharide is deacetylated with 0.8M NaOH for 6 hours at 80 C. Subsequently it is cooled to below 40 C. followed by diafiltration and concentration of the crude polysaccharide simultaneously with 6-8 volumes of MQW and 20 mM HEPES containing 3M NaCl buffer using 0.1 m.sup.2 PES membrane. Afterwards the polysaccharide is further purified by Hydrophobic Interaction Chromatography (HIC), Such as, Phenyl sepharose 6 Fast Flow. Concentration and diafiltration of the purified polysaccharide was performed with 0.1 m.sup.2 PES membrane, with 6-8 volumes of MQW. Consequently, sterile filtration was done with 0.22 m filter and the final purified polysaccharide is stored at 20 C. The total time taken to purify the polysaccharide including HIC chromatography using the above process was 10-12 hrs. Moreover the polysaccharide is partially purified with the above mentioned process. The process may require few more steps for complete purification of the polysaccharide and may not be a cost effective process.

EXAMPLE-4

(30) Polysaccharide Purification Using Sodium Hydroxide with Tris Buffer and Hydrophobic Interaction Chromatography (HIC):

(31) The diafiltered and concentrated FB having the crude polysaccharide is deacetylated with 0.8M NaOH for 6 hours at 80 C. Subsequently it is cooled to below 40 C. followed by diafiltration and concentration of the crude polysaccharide is done simultaneously with 6-8 vols of MQW and 20 mM Tris HCL buffer using 0.1 m.sup.2 PES membrane. Afterwards the polysaccharide is further purified by Hydrophobic Infraction Chromatography (HIC), Such as, Phenyl sepharose 6 Fast Flow. Concentration and diafiltration of the purified polysaccharide was performed with 0.1 m.sup.2 PES membrane, with 6 to 8 volumes of MQW. Accordingly, sterile filtration is done with 0.22 m filter and the final purified polysaccharide is stored at 20 C. The total time taken to purify the polysaccharide including HIC chromatography using the above process is 10-12 hours. Moreover the polysaccharide is partially purified with the above mentioned process. The process may require few more steps for complete purification of the polysaccharide and may not be a cost effective process.

EXAMPLE-5

(32) Polysaccharide Purification Using Sodium Hydroxide with Tris Buffer and Hydrophobic Interaction Chromatography (HIC):

(33) The diafiltered and concentrated FB having the crude polysaccharide was deacetylated with 1M NaOH for 2 hrs at 755 C. The deacetylated polysaccharide was then cooled to a temperature below 40 C. After cooling, the concentration and diafiltration of the crude deacetylated polysaccharide was performed through 100 KDa PES membrane (0.1 m.sup.2) with 20 to 25 volumes of MQW, followed by 8-10 volumes of 20 mM Tris HCl buffer (pH 7.40.1). Consequently sterile filtration was done with 0.22 m PES membrane (0.1 m.sup.2).

(34) Daifiltered deacetylated polysaccharide was further purified by hydrophobic interaction chromatrography (HIC) through XK-16 column packed with phenyl sepharose 6 fast Flow (FF) using chromatography system. The column was equilibrated with 20 mM Tris HCl buffer pH 7.40.1 containing 20% Ammonium Sulphate. The material was loaded at 60 cm/hr. and the flow through containing the purified polysaccharide was collected. Finally column was regenerated with 20 mM Tris HCl buffer pH 7.40 and stored in 20% ethanol for further use. Concentration and diafiltration of the purified polysaccharide was performed with 100 KDa PES membrane (0.1 m.sup.2), with 6-8 volumes of MQW and sterile filtration is done with 0.22 m filter and the final purified polysaccharide is stored at 20 C. The total time taken to purify the polysaccharide is 61 hours and the PS qualifies to the WHO specifications.