Method for separation of protein and other impurities from microbial capsular polysaccharides

Abstract

The invention relates to a method for the removal of protein and other impurities from microbial capsular polysaccharides. More particularly, the present invention relates to isolation of microbial capsular polysaccharides in pure form after removal of protein and other impurities.

Claims

1. A method for preparing an immunogenic composition comprising a purified capsular polysaccharide conjugated to a carrier protein selected from the group consisting of diphtheria toxoid, tetanus toxoid, and CRM.sub.197, the method comprising: exposing or contacting a solution of lysed Streptococcus pneumoniae cells comprising the capsular polysaccharide, proteins, nucleic acids, cell wall components and other impurities with silicone dioxide (SiO.sub.2); separating the capsular polysaccharide from the SiO.sub.2 by filtration or by centrifugation and without using chromatography to isolate the capsular polysaccharide in substantially pure form; and conjugating the purified capsular polysaccharide to the carrier protein.

2. The method of claim 1, wherein the Streptococcus pneumoniae comprises one or more serotypes selected from the group comprising of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F, and 33F.

3. The method of claim 1, wherein the SiO.sub.2 comprises particles each having a size ranging from 0.01 μm to 200 μm.

4. The method of claim 1, wherein the solution of lysed cells is exposed or contacted with the SiO.sub.2 at a temperature ranging from 15° C. to 60° C. for a period of time ranging from 10 minutes to 16 hours.

5. The method of claim 1, wherein the SiO.sub.2 is in an amount ranging from 0.5% to 20% weight/volume (w/v).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Comparative protein impurity levels from different pneumococcal serotypes before and after SiO.sub.2 treatment.

(2) FIG. 2: SDS-PAGE results for pneumococcal polysaccharide serotype 18C; protein reduction before and after SiO.sub.2 treatment.

(3) FIG. 3: SDS-PAGE results for pneumococcal polysaccharide serotype 23F; protein reduction before and after SiO.sub.2 treatment.

DETAILED DESCRIPTION

(4) The present invention provides a method for the isolation of polysaccharide, wherein the source of polysaccharide is from bacteria, yeast, filamentous fungus, algae or plant cells and the like, which comprises, exposing a solution comprising polysaccharide with SiO.sub.2 and optionally with other agents. The resulting solution after exposure to SiO.sub.2 and separation, is enriched in polysaccharide and reduced in one or more impurities such as protein, nucleic acid, cell wall polysaccharide, and other cell derived materials.

(5) The polysaccharides obtained according to the present invention are in substantially pure form.

(6) In a preferred embodiment, the invention relates to methods for the reduction or removal of protein impurities from a complex cellular Streptococcus pneumoniae lysate or centrate comprising one or more serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F, and 33F polysaccharides.

(7) In an embodiment, the SiO.sub.2 used may be in different forms/particle size such as fine particles ranging from 0.01 μm to 200 μm, preferably in the range of 3 to 40 μm. The amount of SiO.sub.2 used may range from 0.5 to 20% (w/v). SiO.sub.2 used may optionally be prepared by heating above 60° C. and for at least 1 hr and cooling prior to use. The SiO.sub.2 used may be pyrogenated or depyrogenated.

(8) In yet another embodiment, other agents used for the purification process of polysaccharide may be selected from sodium chloride, ammonium sulphate and the like at a concentration of at least 0.1% (w/v) or organic solvents such as alcohol at a concentration of at least 2% (v/V). The other agent may be used to further reduce the impurities and enrich the solution with polysaccharide.

(9) In another embodiment, the pH of the solution may be maintained in the range from acidic region to alkaline region, and preferably from 3.0 to 9.0. The pH may be adjusted using acids such as acetic acid, phosphoric, formic acid, hydrochloric acid and the like and alkalis such as sodium, potassium or ammonium hydroxide and the like.

(10) In another embodiment, contact or exposure of the solution comprising polysaccharide and other impurities to SiO.sub.2 is carried out at a temperature ranging from 15° C. to 60° C. for a period of 10 min to 16 hrs.

(11) In another embodiment, the present invention involves treatment of polysaccharide solution with activated charcoal for removing color and other impurities. This treatment is carried out before exposure to SiO.sub.2 or after exposure to SiO.sub.2.

(12) The polysaccharides purified using the method described in this invention may be used for different applications like cosmetics, food, pharma and biopharma industries.

(13) As used herein, the term “substantially pure form” refers to a polysaccharide lysate or centrate from which at least 30% of protein has been removed compared to the concentration of protein in the lysate or centrate prior to SiO.sub.2 exposure. Methods for the quantification of protein concentration in a cellular lysate or centrate are well known in the art and include, for example, biochemical methods such as Bradford assay, BCA assay, Lowry assay, analysis methods such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, chromatography, and electrophoresis (See, e.g., Deutscher, M. P. (ed.), Guide to Protein Purification, San Diego: Academic Press, Inc. (1990)).

(14) The invention also provides a process for purifying capsular saccharide from bacteria, wherein (a) the yield of the process is at least 10% and (b) the relative purity of the saccharide is at least 30%.

(15) In another embodiment, the present invention provides a method for the isolation of polysaccharide in a pure form which comprises, i). exposing a solution comprising polysaccharide, protein, nucleic acids cell wall components and other impurities with SiO.sub.2, ii). isolating the polysaccharide solution in a pure form and iii). separating the silica particles from polysaccharide by filtration or by centrifugation.

(16) The polysaccharide concentration obtained in the process of the present invention may be from 0.1 to more than 10 mg/ml.

(17) In another embodiment, the present invention provides a method for the isolation of polysaccharide in a pure form which comprises, i). preparing polysaccharide solution comprising polysaccharide, protein, nucleic acids, cell wall components and other impurities, ii). treating the polysaccharide solution with detergent to remove nucleic acid and other impurities iii). preparing a suspension of SiO.sub.2 in water or a buffer, iv). adding the suspension of SiO.sub.2 to the polysaccharide solution of step (i) and iv). isolating the polysaccharide solution in a pure form.

(18) The buffers used in the present invention for the isolation of polysaccharide includes sodium phosphate buffer, potassium phosphate buffer, tris buffer etc.,

(19) Proteins are having hydrophilic surfaces and hydrophobic pockets. When polysaccharide preparations incubated with Silicon dioxide, protein impurities get bound with silicon dioxide and separated from the polysaccharide, hydrophilic or hydrophobic or simple adsorption mechanism.

(20) The detergents used in the present invention includes CTAB (Cetyl trimethylammonium bromide), Cetrimonium chloride, Benzethonium chloride etc,

(21) The terms exposing or contacting means incubation of polysaccharide preparation with other components to treat the sample for the removal of impurities, to make pure polysaccharide.

(22) In a preferred embodiment, the present invention provides a method for the isolation of polysaccharide in a pure form which comprises, i) preparing polysaccharide solution comprising pneumococcal capsular polysaccharide, protein, nucleic acids cell wall components and other impurities, wherein the pH of the solution is maintained in the range of from 3.0 to 9.0. ii) optionally adding other reagent, iii) optionally treating the solution with activated charcoal, iv) preparing a suspension of SiO.sub.2 having particles ranging from 0.01 μm to 200 μm in water or a buffer, v) adding the suspension of SiO.sub.2 to the polysaccharide solution of step (i) at a temperature in the range of 15° C. to 60° C. for a period of 10 min to 20 hrs vi) optionally treating the solution with activated charcoal, vii) optionally adding other reagent and viii) isolating the polysaccharide solution in a pure form.

(23) The other reagents may be selected from sodium chloride, ammonium sulphate, alcohol and the like or mixture thereof.

(24) The purified capsular polysaccharide of the invention can be used as an immunogen with or without further modification for use in immunization. For immunization purposes it is preferred to conjugate the saccharide to a carrier molecule, such as a protein.

(25) Preferred carrier proteins are bacterial toxins or toxoids, such as diphtheria toxoid or tetanus toxoid or CRM197 mutant of diphtheria toxin etc.

(26) In yet another embodiment, the present invention provides an immunogenic composition comprising capsular polysaccharide prepared according to the present invention conjugated to carrier protein selected from diphtheria toxoid or tetanus toxoid or CRM197.

(27) In yet another preferred embodiment, the present invention provides an immunogenic composition comprising capsular polysaccharides from one or more serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F, and 33F conjugated to CRM197 carrier protein.

(28) Some common brand names of SiO.sub.2 (silicon dioxide) available in the market are Aerosil®, Aeroperl® may be used in the present invention.

(29) Polysaccharide solution comprising polysaccharide, protein, nucleic acids cell wall components and other impurities can be prepared by any of the methods known the art.

(30) The isolation of the capsular polysaccharide in pure form after exposing or contacting with SiO.sub.2 is carried out by conventional methods.

(31) The present invention is more specifically illustrated with reference to the examples given below. However, it should be understood that the present invention is not limited by an example in any manner.

Example 1

(32) Streptococcus pneumoniae fermentation broth, cell lysis was carried out by adding Deoxycholate (0.005% to 2%). Post Deoxycholate incubation broth was centrifuged at 10000 to 15000 g and supernatant was collected. Supernatant pH was adjusted with acids like orthophosphoric acid, hydrochloric acid etc. to pH 4-6 and incubated for 3 hrs to overnight. Few serotypes pH was again adjusted to neutral and heated up to 60° C. for 10 to 150 min. Centrifuged the polysaccharide at 10000 to 15000 g, pellet was discarded. Further clarified the supernatant by passing through depth filter or 0.22 or 0.45 μm filter. Concentrated the filtrate 4 to 15 folds on ultrafiltration membrane 30 to 300 kDa. Concentrate was buffer exchanged up to 4 to 12 dia volumes on Phosphate buffer. To the Concentrated and buffer exchanged polysaccharide, CTAB was added, i.e. 0.2% to 5%, incubated for 2 hr to overnight at 4° C. to 40° C. Sodium chloride was added to few polysaccharides before CTAB addition in the range of 0.05M to 2M.

(33) After CTAB treatment pellet was separated by centrifugation at 10000 to 15000 g. Supernatant was passed through charcoal column/filters. Activated silicon dioxide added to charcoal filtered polysaccharide in the range of 3 to 10% (W/V) and added NaCl from 0.5M to 3M. Polysaccharide preparation was exposed to silicon dioxide for 2 hrs to 26 hrs at temperature 5° C. to 40° C. Silicon dioxide was separated from polysaccharide solution by centrifugation/cloth filtration/bag filtration. Filtrate was passed through depth filter, carbon filter and 0.22 to 5μ.Math.η filter. Filtered Polysaccharide was concentrated and diafiltered on 10 kDa to 500 kDa membrane. Polysaccharide was buffed exchanged into phosphate buffer or WFI. Purified polysaccharide preparation was passed through 0.22 μm filter and collected into LDPE bag under LAFU. The purified polysaccharide was stored at >−20° C.

(34) TABLE-US-00001 TABLE 1 Protein removal from different Pneumo polysaccharides Serotype Pneumococcal Pneumococcal Pneumococcal Pneumococcal Serotype 1 Serotype 6A Serotype 7F Serotype 19A Pre Post Pre Post Pre Post Pre Post Treatment Treatment Treatment Treatment Treatment Treatment Treatment Treatment Polysaccharide 1.9 1.77 2.82 2.62 3.6 2.72 2.67 2.5 (mg/mL) Protein 0.34 BDL 0.26 BDL 0.26 0.05 0.59 BDL (mg/mL) Protein % 17.89 BDL 9.22 BDL 7.22 1.84 22.10 BDL (per mg of PS) BDL: Below detection limit

(35) This embodiment describes the influence of depyrogenation on impurity removal from the polysaccharide preparation. As depicted in Table 2 protein impurity was removed by both depyrogenated and pyrogenated SiO.sub.2. Hence depyrogenated as well as pyrogenated SiO.sub.2 can be used for the removal of impurities.

(36) TABLE-US-00002 TABLE 2 Influence of aeroeprl ® depyrogenation on protein removal from different pneumococcal polysaccharides. Pneumococcal Pre Post Serotype 6B Description treatment treatment Pyrogenated Protein (mg/mL) 0.21 BDL SiO.sub.2 Protein % (per mg of PS) 9.86 BDL Polysaccharide (mg/mL) 2.13 1.27 Depyrogenated Protein (mg/mL) 0.23 BDL SiO.sub.2 Protein % (per mg of PS) 8.07 BDL Polysaccharide (mg/mL) 2.85 1.5  BDL: Below detection limit
Conditions: Aeroperl® 5% (w/v), NaCl 1M, Incubated at room temperature for Ih.

(37) Aerosil® can be used in a range from 0.1% to 10% or higher concentration. Protein was completely removed by treatment with SiO.sub.2 in an hour to more than 17 hrs. Impurity removal can be improved by the addition of NaCl to the SiO.sub.2 suspension.

(38) TABLE-US-00003 TABLE 3 Influence of Aerosil ® concentration on Protein removal from Pneumococcal polysaccharide serotype 6B Pre- Post 2% Post 3% Post 4% Post 5% Aeroperl Aeroperl Aeroperl Aeroperl Aeroperl S. No. Parameter treatment treatment treatment treatment treatment 1 Protein 0.23 BDL BDL BDL BDL (mg/ml) 2 Protein % 8.07 BDL BDL BDL BDL (per mg of PS.) 3 PS (mg/ml) 2.85 1.68 1.62 1.52 1.49 BDL: Below detection limit Conditions: NaCl 1M; Incubated at room temperature for lh.

(39) This embodiment also supports that depyrogenated Aeroeprl® can remove the protein effectively in presence of NaCl ranging from 0.1M to 2.5M or above. SiO.sub.2 particles can be used in the range or size from 0.1 μm to 100 s of microns.

(40) TABLE-US-00004 TABLE 4 Influence of aeroperl concentration on protein removal from pneumococcal polysaccharide, serotype 6B Pre- Post 2% Post 3% Post 4% Post 5% Aeroperl Aeroperl Aeroperl Aeroperl Aeroperl S. No. Parameter treatment treatment treatment treatment treatment 1 Protein 0.23 BDL BDL BDL BDL (mg/ml) 2 Protein % 8.07 BDL BDL BDL BDL (per mg of PS.) 3 PS (mg/ml) 2.85 1.68 1.62 1.52 1.49 BDL: Below detection limit Condition: NaCI 1M, Incubated at room temperature for lh

(41) Fermentation broth contains number of contaminants. These contaminants can be removed in series of steps like centrifugation, precipitation, chromatography etc.

(42) Current invention was carried at small scale (50-100 ml) and pilot (15 L) level. At both volumes of polysaccharide, contaminants were efficiently removed by the different forms of SiO.sub.2 (Table 5 and FIG. 1). SiO.sub.2 treatment can be introduced at different stages of the′ polysaccharide purification. Further SiO.sub.2 particles can be separated by simple centrifugation or filtration or by any other method such as physical settling, pressure settling etc.

(43) TABLE-US-00005 TABLE 5 Protein impurities diminution using aeroperl from Pneumococcal polysaccharides Serotype Pneumo Serotype 1 Pneumo Serotype 6B condition Pre Post Pre Post Aeroperl Aeroperl Aeroperl Aeroperl Parameter treatment treatment treatment treatment Protein (mg/mL) 0.21 BDL 0.23 BDL Protein % 9.86 BDL 8.07 BDL (per mg of PS) Polysaccharide 2.13 1.88 2.85 1.52 (mg/mL) BDL: Below detection limit. Conditions: Aeroperl: 5% (w/v), NaCl 1M, Incubated at room temperature for lh

(44) TABLE-US-00006 TABLE 6A Protein impurity removal from capsular polysaccharide of different pneumococcal serotypes Serotype Pneumococcal Pneumococcal Pneumococcal Pneumococcal Serotype 6A Serotype 7F Serotype 9V Serotype 14 Condition Parameter Pre Post Pre Post Pre Post Pre Post Protein 0.88 0.14 1.15 0.21 0.86 0.07 0.67 0.01 (mg/mL) Protein % 9.64 1.72 12.43 2.69 9.83 0.86 12.91 0.30 (per mg of PS) Polysaccharide 9.13 8.15 9.25 7.81 8.75 8.11 5.19 3.38 (mg/mL) Pre: Pre aeroperl treatment; Post: Post aeroperl treatment Condition: Aeroperl 5% (w/v), NaCl 1M, Incubated at room temperature for lh.

(45) TABLE-US-00007 TABLE 6B Protein impurity removal from capsular polysaccharide of different Pneumococcal polysaccharide serotypes Serotype Pneumococcal Pneumococcal Pneumococcal Pneumococcal Serotype 18C Serotype 19A Serotype 19F Serotype 23F condition Parameter Pre Post Pre Post Pre Post Pre Post Protein 0.89 BDL 0.48 0.1 0.67 0.11 0.1 BDL (mg/mL) Protein % 17.66 BDL 5.63 1.46 10.11 1.81 3.30 BDL (per mg of PS) Polysaccharide 5.04 4.48 8.52 6.85 6.63 6.09 3.03 2.51 (mg/mL) BDL: Below detection limit Pre: Pre aeroperl treatment; Post: Post aeroperl treatment ND: Not detected Condition: Aeroperl 5% (w/v), NaCl 1M, Incubated at room temperature for lh

(46) Limit of contaminants have been set for the purified polysaccharide of each serotype to reduce the risk of adverse events from the vaccine. Among contaminants CWPS is one. Current invention has been taken care of CWPS. CWPS was removed at room temperature by simple mixing and followed by separation of S1O2 from the polysaccharide sample. Polysaccharide sample with SiO.sub.2 contact time may vary from 10 min to more than 18 hrs (Table 7).

(47) TABLE-US-00008 TABLE 7 Removal of cell wall polysaccharide (CWPS) from different serotypes of pneumococcal polysaccharides Serotype Pneumo Serotype 19A Pneumo Serotype 19F Condition Pre Post Pre Post Aerosil Aerosil Aerosil Aerosil Parameter treatment treatment treatment treatment CWPS (mg/ml) 0.484 0.031 0.108 0.038 CWOP %(per mg of 12.94 1.40 3.45 1.65 PS) Polysaccharide 3.74 2.22 3.13 2.3 (mg/mL) Conditions: Aerosil 5% (w/v), NaCl 1M; Incubated at room temperature for lh

(48) Protein impurity removal can be visualised by SDS-PAGE. Pneumococcal polysaccharide serotype 18C and 23F protein impurity was reduced to limit of specification. As depicted in the FIGS. 2 and 3, clear removal of protein can be seen in lane 2 and 3. Results were represented in table 8.

(49) TABLE-US-00009 TABLE 8 Protein concentration before and after aeroperl ® treatment (FIG. 2 and 3) Pnumococcal Before aeroperl ® treatment After aeroperl ® treatment Polysaccharide PS Protein Protein %/ PS Protein Protein %/ Serotype mg/ml mg/ml mg of PS mg/ml mg/ml mg of PS 23F 3.03 0.1 3.30 2.4 BDL BDL 18C 4.49 0.33 7.35 4.79 0.02 0.42