HK1163737B - Method for controlling streptococcus pneumoniae polysaccharide molecular weight using carbon - Google Patents

Description

Method for controlling molecular weight of streptococcus pneumoniae polysaccharide by using carbon dioxide

Technical Field

The present invention relates to a method of increasing the molecular weight of an isolated streptococcus pneumoniae capsular polysaccharide having phosphodiester linkages between saccharide repeat units.

Background

In the preparation of a polyvalent conjugated pneumonia vaccine for the prevention of invasive diseases caused by Streptococcus pneumoniae (also known as pneumococcus) organisms, selected Streptococcus pneumoniae serotypes are grown to provide the polysaccharides required to produce the vaccine. Cells are grown in a fermentor and lysis is induced at the end of fermentation by addition of sodium deoxycholate or another lytic agent. The lysate broth is then harvested for downstream purification and recovery of the capsular polysaccharide surrounding the bacterial cells. After conjugation to the carrier protein, the polysaccharide is included in the final vaccine product and confers immunity to the selected streptococcus pneumoniae serotype to the vaccine target population.

Polysaccharide size is a quality attribute that is measured in each batch of product and must be properly controlled. The traditional treatment method uses NaOH (sodium hydroxide) as the alkaline titrant during fermentation. The advantage of using NaOH is that the pH of the deoxycholic acid lysate can be lowered without foaming, in order to remove proteins and improve filtration. This material was centrifuged and then filtered to remove most of the solids down to the nominal size of 0.45 μm (nominal size). However, this conventional treatment produces polysaccharides with lower molecular weights (< 450kDa) for serotypes with phosphodiester linkages between saccharide repeat units (e.g., 6A, 6B, 19A and 19F).

Accordingly, there is a need for improved methods for recovering high molecular weight capsular polysaccharides from cellular streptococcus pneumoniae lysates, particularly lysates containing streptococcus pneumoniae serotype 6A, 6B, 19A or 19F polysaccharides.

Brief description of the invention

The present invention provides an improved process for the recovery of high molecular weight capsular polysaccharides from streptococcus pneumoniae cell lysates containing serotypes with phosphodiester linkages between saccharide repeat units. In one method, a fermentation culture of a Streptococcus pneumoniae serotype containing phosphodiester linkages between saccharide repeat units is supplied with CO₂. Thus, in one embodiment of the invention, the method comprises the steps of: 1) preparing a fermentation culture of streptococcus pneumoniae bacterial cells that produce capsular polysaccharides comprising phosphodiester linkages between repeat units; 2) providing CO to the fermentation culture₂(ii) a 3) Lysing the bacterial cells in the fermentation culture; and 4) isolating the Streptococcus pneumoniae capsular polysaccharide from the fermentation culture, wherein a solution is produced comprising high molecular weight isolated Streptococcus pneumoniae capsular polysaccharides comprising phosphodiester linkages between repeat units.

In a specific embodiment, a fermentation culture of streptococcus pneumoniae bacterial cells that produce polysaccharide serotypes 19A, 6A, 19F, 6B, and combinations thereof, is prepared. In another specific embodiment, CO is provided to the fermentation culture₂Comprises adding bicarbonate ion (HCO) to the fermentation culture₃ ^-) For example, NaHCO is added to the fermentation culture₃(sodium bicarbonate). In a further embodiment, CO is provided to the fermentation culture₂Comprises adding carbonate ions (CO) to the fermentation culture₃ ^2-) For example, adding Na to the fermentation culture₂CO₃(sodium carbonate). In another embodiment, CO is provided to the fermentation culture₂Comprises first adding NaHCO₃Then adding Na₂CO₃. In another embodiment, CO is provided to the fermentation culture₂Comprising using CO₂The fermentation culture was overlaid. In another embodiment, the isolated streptococcus pneumoniae capsular polysaccharide has a molecular weight of at least 700 kDa. In another embodiment, there is provided a solution comprising high molecular weight isolated streptococcus pneumoniae capsular polysaccharides wherein the polysaccharides comprise phosphodiester linkages between repeat units, wherein the solution is produced by the process described above.

In another embodiment of the invention, a method is provided for producing a solution containing high molecular weight isolated streptococcus pneumoniae serotype 19A capsular polysaccharides. The method comprises the following steps: 1) preparing a fermentation culture of streptococcus pneumoniae bacterial cells that produces serotype 19A capsular polysaccharide; 2) providing CO to the fermentation culture₂(ii) a 3) Lysing the bacterial cells in the fermentation culture; and 4) isolating Streptococcus pneumoniae serotype 19A capsular polysaccharide from the fermentation culture; thereby producing a solution containing high molecular weight isolated streptococcus pneumoniae serotype 19A capsular polysaccharides. In another embodiment, there is provided a solution comprising high molecular weight isolated streptococcus pneumoniae serotype 19A capsular polysaccharides, wherein the solution is produced by the above-described process.

In another embodiment of the invention, a method is provided for producing a solution containing high molecular weight isolated streptococcus pneumoniae serotype 19F capsular polysaccharides. The method comprises the following steps: 1) preparing a fermentation culture of streptococcus pneumoniae bacterial cells that produces serotype 19F capsular polysaccharide; 2) providing CO to the fermentation culture₂(ii) a 3) Lysing the bacterial cells in the fermentation culture; and 4) isolating Streptococcus pneumoniae serotype 19F capsular polysaccharide from the fermentation culture; thereby producing a solution containing high molecular weight isolated streptococcus pneumoniae serotype 19F capsular polysaccharide. In another embodiment, there is provided a solution comprising a high molecular weight isolated streptococcus pneumoniae serotype 19F capsular polysaccharide, wherein the solution is produced by the above process.

In a further embodiment of the present invention,methods of producing a solution containing high molecular weight isolated streptococcus pneumoniae serotype 6A capsular polysaccharides are provided. The method comprises the following steps: 1) preparing a fermentation culture of streptococcus pneumoniae bacterial cells that produces serotype 6A capsular polysaccharide; 2) providing CO to the fermentation culture₂(ii) a 3) Lysing the bacterial cells in the fermentation culture; and 4) isolating Streptococcus pneumoniae serotype 6A capsular polysaccharide from the fermentation culture; thereby producing a solution containing high molecular weight isolated streptococcus pneumoniae serotype 6A capsular polysaccharides. In another embodiment, there is provided a solution comprising high molecular weight isolated streptococcus pneumoniae serotype 6A capsular polysaccharides wherein the solution is produced by the above process.

In another embodiment of the invention, a method is provided for producing a solution containing high molecular weight isolated streptococcus pneumoniae serotype 6B capsular polysaccharides. The method comprises the following steps: 1) preparing a fermentation culture of streptococcus pneumoniae bacterial cells that produces serotype 6B capsular polysaccharide; 2) providing CO to the fermentation culture₂(ii) a 3) Lysing the bacterial cells in the fermentation culture; and 4) isolating Streptococcus pneumoniae serotype 6B capsular polysaccharide from the fermentation culture; thereby producing a solution containing high molecular weight isolated streptococcus pneumoniae serotype 6B capsular polysaccharide. In another embodiment, there is provided a solution comprising high molecular weight isolated streptococcus pneumoniae serotype 6B capsular polysaccharides, wherein the solution is produced by the above-described process.

Brief Description of Drawings

FIG. 1 shows Optical Density (OD), alkali and glucose levels during the fermentation period from a 3L-scale laboratory study, where Na is used₂CO₃As titration base. For purposes of the drawing, the base in grams is divided by 10.

Figure 2 shows Optical Density (OD), base and glucose levels during the fermentation period from a 3L scale laboratory study, using NaOH as the titration base. For purposes of the drawing, the base in grams is divided by 10.

FIG. 3 shows for alternating Na₂CO₃Or total protein and polysaccharide results with NaOH feed at different pH adjustments.

Detailed Description

Streptococcus pneumoniae (Streptococcus pneumoniae) is a gram-positive, lanceolated (lantshaped) coccus that is usually paired (diplococcus), but also short-chained or unicellular. It grows easily on blood agar plates, has flashing colonies (glistening colonies), and shows alpha hemolysis except under anaerobic growth conditions, and in anaerobic growth it shows beta hemolysis. The cells of most pneumococcal serotypes have a capsule, which is a polysaccharide envelope (coating) surrounding each cell. This capsule is a determinant of virulence in humans, as it interferes with phagocytosis by preventing antibodies from attaching to bacterial cells. There are currently more than 90 known pneumococcal capsular serotypes identified, with the 23 most common serotypes accounting for approximately 90% of invasive diseases worldwide.

As a vaccine, the pneumococcal polysaccharide envelope may confer moderate immunity to streptococcus pneumoniae in individuals with a mature or undamaged immune system, but proteins conjugated to polysaccharides generate an immune response in infants and the elderly who are also at greatest risk for pneumococcal infection. Pneumococcal cells were grown in fermenters and lysis was induced at the end of fermentation. The lysate broth is then harvested for downstream purification and the capsular polysaccharide is recovered.

Polysaccharide size is a quality attribute measured in each preparation batch and must be properly controlled. The molecular weight of serotypes with phosphodiester linkages between saccharide repeat units (e.g., 6A, 6B, 19A, and 19F) is influenced by the parameters of the fermentation process. The process of the invention allows for the recovery of high molecular weight capsular polysaccharides from cellular streptococcus pneumoniae lysates containing serotypes with phosphodiester linkages between saccharide repeat units, such as serotype 6A, serotype 6B, serotype 19A, serotype 19F, and combinations thereof.

In the development of the method of the invention, the concentration of HySoy and the choice of alkaline titrant were modified in an attempt to modify the final polysaccharide yield and molecular weight. Four fermentation protocols were tested. The first protocol used a baseline NaOH method with 28g/L HySoy. The second protocol used 20% sodium carbonate as the base titrant and 20g/L HySoy. The third option combines the advantages of the first two methods, the introduction of carbonate by the addition of sodium bicarbonate in portions and the use of a mixed NaOH/carbonate base titrant. The fourth protocol used carbonate as a base titrant and 10mM bicarbonate was added to support growth.

The advantage of using NaOH as the alkaline titrant during fermentation is that it enables deoxycholate lysate to be lowered to pH5.0 without foam formation to remove proteins and improve filtration, but produces lower molecular weight polysaccharides (< 450 kDa). Na (Na)₂CO₃Higher molecular weights are provided but there is a problem with foam formation if the pH of the deoxycholate lysate is lowered. At a higher pH, maintaining pH6.6, Na is used₂CO₃Fermentation is carried out to form a gel-like material, followed by filtration problems. By using NaOH with Na₂CO₃The mixture of (A) is used as a pH titrant to react Na₂CO₃Is minimized, Na is provided₂CO₃And due to the large amount of CO₂The sudden release eliminates the foam formation and filtration problems. 20% Na was used₂CO₃(w/v) as base titrant and 10mM NaHCO was added₃Supporting growth (fourth method), produces a sustained high yield of high molecular weight polysaccharides.

The present invention therefore provides an improved process for recovering high molecular weight capsular polysaccharides from cellular streptococcus pneumoniae lysates containing serotypes with phosphodiester linkages between saccharide repeat units. In one method, a fermentation culture of a Streptococcus pneumoniae serotype containing phosphodiester linkages between saccharide repeat units is provided with CO₂. Exemplary having repeat units of sugar betweenPhosphodiester-linked streptococcus pneumoniae serotypes include serotypes 6A, 6B, 19A, and 19F. Accordingly, in one embodiment of the invention, there is provided a process for producing a solution containing high molecular weight isolated streptococcus pneumoniae capsular polysaccharides comprising phosphodiester linkages between repeat units, the process comprising the steps of: 1) preparing a fermentation culture of streptococcus pneumoniae bacterial cells that produce capsular polysaccharides comprising phosphodiester linkages between repeat units; 2) providing CO to the fermentation culture₂(ii) a 3) Lysing the bacterial cells in the fermentation culture; and 4) isolating Streptococcus pneumoniae capsular polysaccharide from the fermentation culture; thereby producing a solution containing high molecular weight isolated streptococcus pneumoniae capsular polysaccharides having phosphodiester linkages between repeat units. In another embodiment, the invention relates to a solution comprising high molecular weight isolated streptococcus pneumoniae capsular polysaccharides having phosphodiester linkages between repeat units, wherein the solution is produced by the above process.

The methods of the invention produce high molecular weight streptococcus pneumoniae capsular polysaccharides that include phosphodiester linkages between repeat units (e.g., serotypes 6A, 6B, 19A, and 19F). As used herein, "high molecular weight" refers to a molecular weight of at least about 480kDa, about 490kDa, about 500kDa, about 510kDa, about 520kDa, about 525kDa, about 550kDa, about 575kDa, about 600kDa, about 625kDa, about 650kDa, about 675kDa, about 700kDa, about 725kDa, about 750kDa, about 775kDa, about 800kDa, about 825kDa, about 850kDa, about 875kDa, about 900kDa, about 925kDa, about 950kDa, about 975kDa, or about 1000 kDa.

In certain methods, CO is provided to a fermentation culture₂Comprising adding bicarbonate ions (HCO) to the fermentation culture₃ ^-) For example, NaHCO is added to the fermentation culture₃. 5-50mM NaHCO may be added₃E.g., 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, or 50 mM. In other methods, CO is provided to the fermentation culture₂Comprising culturing in said fermentationAdding Carbonate (CO)₃ ^2-) Ions, e.g. addition of Na to the fermentation culture₂CO₃. 0.1M-2.0M of Na may be added₂CO₃Such as 0.1M, 0.2M, 0.4M, 0.6M, 0.7M, 0.9M, 1.0M, 1.5M, 1.8M or 2.0M. Weight/volume (w/v) equivalents may also be used, such as 5% (w/v) Na₂CO₃、10％(w/v)Na₂CO₃Or 20% (w/v) Na₂CO₃. In other methods, CO is provided to the fermentation culture₂Comprising first adding NaHCO to the fermentation culture₃And then adding Na₂CO₃. In a further method, CO is supplied to the fermentation culture₂Comprising using CO₂Covering the fermentation culture. 5% -100% CO can be used₂Coverage, e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

In the methods of the invention, the bacterial cells may be lysed by using any lysing agent. A "lytic agent" is any agent that facilitates cell wall disruption and release of autolysin that results in cell lysis, including, for example, detergents. As used herein, the term "detergent" refers to any anionic or cationic detergent capable of inducing lysis of bacterial cells. Representative examples of such detergents for use in the methods of the invention include sodium Deoxycholate (DOC), N-dodecylsarcosine (NLS), sodium chenodeoxycholate, and saponins.

In one embodiment of the invention, the lytic agent used to lyse the bacterial cells is DOC. DOC is the sodium salt of the bile acid deoxycholic acid, which is generally derived from biological sources such as cows or bulls. DOC activates the LytA protein, an autolysin involved in cell wall growth and division in streptococcus pneumoniae. LytA has a choline binding domain in its C-terminal part, and mutation of the LytA gene is known to produce a LytA mutant, which is resistant to DOC cleavage.

Although there is no indication that the polysaccharide is pure in Streptococcus pneumoniaeThe use of DOC during chemotherapy constitutes a health risk, but the use of such biologically derived preparations can create potential regulatory issues (regulatory issues). Thus, in one embodiment of the invention, the lytic agent used to lyse the bacterial cells is a non-animal derived lytic agent. Non-animal derived lytic agents useful in the methods of the invention include preparations from non-animal sources that act in a pattern similar to DOC (i.e., affect LytA function and cause lysis of streptococcus pneumoniae cells). Such non-animal derived lytic agents include, but are not limited to, analogs of DOC, surfactants, detergents, and structural analogs of choline, and can be determined using methods as described in the experimental section below. In one embodiment, the non-animal derived lytic agent is selected from the group consisting of: decane sulfonic acid (decanoesulfonic acid), tert-octylphenoxy poly (oxyethylene) alcohols (e.g. Igepal)CA-630, CAS #: 9002-93-1 from Sigma Aldrich, St.Louis, Mo.), an octylphenol ethylene oxide condensate (e.g., TritonX-100 from Sigma Aldrich, St.Louis, MO), N-dodecyl sarcosine sodium (NLS), sodium N-dodecyl-iminodipropionate, sodium dodecyl sulfate, chenodeoxycholate, hyodeoxycholate, glycodeoxycholate, taurodeoxycholate, and cholate. In another embodiment, the non-animal derived lytic agent is NLS.

In the methods of the invention, the streptococcus pneumoniae capsular polysaccharide is isolated using standard techniques known to those skilled in the art. For example, after fermentation of bacterial cells that produce the capsular polysaccharide of streptococcus pneumoniae, the bacterial cells are lysed to produce a cell lysate. The capsular polysaccharides are then isolated from the cell lysate using purification techniques known in the art, including the use of centrifugation, precipitation, ultrafiltration, and column chromatography techniques (see, e.g., U.S. patent app. pub. nos.20060228380, 20060228381, 20070184071, 20070184072, 20070231340, and 20080102498).

Variations in the above process allow recovery of high molecular weight capsular polysaccharides such as serotype 6A, serotype 6B, serotype 19A, serotype 19F and combinations thereof from cellular streptococcus pneumoniae lysates containing serotypes having phosphodiester linkages between saccharide repeat units. This strongly improves the fermentation/recovery process and can greatly enhance the production of pneumococcal polysaccharides.

The following examples are intended to illustrate the invention, but not to limit the scope of the invention.

Examples

Selected streptococcus pneumoniae serotypes are grown to provide the polysaccharides required to produce a vaccine to actively immunize invasive disease caused by streptococcus pneumoniae with capsular serotypes contained in the vaccine. Cells were grown in a fermentor and lysis was induced at the end of fermentation. The lysate broth is then harvested for downstream purification and the capsular polysaccharide is recovered. Since polysaccharide size is a quality attribute determined in each batch of preparation, it is necessary to control the polysaccharide size appropriately. It was found that the molecular weight of serotypes having phosphodiester linkages between saccharide repeat units (e.g., 6A, 6B, 19A, and 19F) is influenced by the parameters of the fermentation process. The following examples describe the provision of CO during fermentation of Streptococcus pneumoniae serotypes having phosphodiester linkages between repeat units₂To improve the molecular weight of the polysaccharide.

Example 1: providing the effect of carbon dioxide on the molecular weight of polysaccharides

Fermentation of

Laboratory runs were performed in a 3L Braun Biostat B fermenter (B. The fermenter was filled with 1.8L HYS medium (20g/L HySoy, 2.5g/L NaCl, 0.5g/L KH)₂PO₄，0.013g/L CaCl₂·2H₂O, 0.15 g/L-cysteine HCl hydrochloride). Then will beThe fermenter was autoclaved at 121 ℃ for 60 minutes. After cooling, 40 or 60mL/L of a 50% glucose + 1% magnesium sulfate (w/v) (DMS) solution was added to each device. Sodium bicarbonate is added prior to inoculation if necessary.

Two 2L inoculum flasks containing 1L of HYS medium were inoculated with Type19A or Type 6A cryopreserved (frozen seed stock) and incubated at 36 ℃ for approximately 6-8 hours without shaking. By using slave OD₆₀₀The fermentor was inoculated in 100mL (. about.5.2% v/v) volumes in one flask between 0.3-0.9 and pH between 4.75-5.60. The fermentation temperature and pH are controlled at desired selected points. Standard conditions of 36 deg.C, 1L/min air blanket, pH control to 7 and stirring at 75rpm were used. Two impellers were placed below and in the middle of the agitator shaft. Turn on (Hooked up) the solution containing the appropriate base titrant (3NNaOH, 3N NaOH mixed with NaHCO at different concentrations₃3N NaOH mixed with Na of different concentrations₂CO₃And NaHCO₃And 20% Na₂CO₃) The culture flask of (2) to automatically control pH. For external pH, OD at different time points₆₀₀Glucose, polysaccharide and protein were sampled from the fermentor. The run was terminated when the glucose concentration approached depletion or when it was noted that the OD no longer increased over time.

Optical Density (OD)₆₀₀) Measuring

The cell density of the fermentation broth was determined by reading the absorbance of the sample at 600nm using Shimadzu (Columbia, MD) UV-1601(2nm bandwidth) or Spectronics (Westbury, NY) Genesys 5 spectrophotometer (5nm bandwidth). The blank for the device was HYS medium diluted with Deionized (DI) water to match the dilution required for the sample. The sample was diluted to keep the absorbance read below 0.4, which is well within the linear range of the spectrophotometer.

Glucose concentration

Glucose levels were determined by centrifugation of the cells and using either the direct supernatant or the supernatant diluted 3 x with DI water. Samples were analyzed on Nova biomedicalal (Waltham, MA) BioProfile 400.

Polysaccharide analysis

Samples were taken at the final fermentation reading, treated with 12% sodium Deoxycholate (DOC) to a concentration of 0.13% (w/v) and gently stirred. It was maintained at 5 ℃ for 8-24 hours, and then the pH was adjusted to 5.0 with 50% acetic acid to precipitate most of DOC and protein. After another 12-24 hours at 5 ℃, the samples were centrifuged (14000rpm, Sorvall (Thermo Fisher Scientific, Waltham, MA) SS34 rotor, 10 minutes at 15 ℃). The pH of the supernatant was adjusted to 6.0. The supernatant was then filtered through a 0.45 μm Pall (East Hills, NY) HT Tuffryn Membrane needle filter, which is low protein binding. The filtered product is analyzed by high performance size exclusion chromatography (HPLC-SEC) using standard Methods well known in the art (see, e.g., Aquilar, M. "HPLC of Peptides and proteins: Methods and Protocols" Totowa, NJ: Humana Press (2004)).

Protein analysis

Protein levels are analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) methods well known in The art (see, e.g., Walker, J.M. "The Protein Protocols Handbook" Totowa, NJ: Humana Press (2002)). An aliquot of the filtered cell lysate (supernatant) prepared as described above was placed in a microfuge tube at 65 μ L/tube. Reducing agent (10. mu.L Dithiothreitol (DTT)) and NuPAGE were added to each sample(Invitrogen, Carlsbad, Calif.) 4 × Lithium Dodecyl Sulfate (LDS) sample buffer (25 μ L). Samples were vortexed and heated for 10 min before loading at 10. mu.L/lane on NuPAGE4-12% Bis-Tris 12-well gel. Gel on NuPAGEThe run was run at 150V in MES-SDS buffer for about 60 minutesBell, then stained using the Zoion staining protocol (Zoion Biotech, Worcester, MA). UVP Imager (UVP Inc., Ugland, CA) and LabWorks were used^TM(UVP Inc.) v.3 software sample analysis was performed to obtain the appropriate concentration of the particular protein band of interest. Bovine Serum Albumin (BSA) fraction V was used to generate a protein standard curve to calculate the appropriate protein value (in the lysed cell broth) for the sample.

Molecular weight analysis

1-2L of the fermentation sample was treated with 12% sodium DOC to a concentration of 0.13% (w/v) and stirred at 200 rpm. The samples were held at 5 ℃ or 20 ℃ for 8-24 hours. The sample was then adjusted to ph5.0 or ph6.6 with 50% acetic acid to precipitate most of the DOC and protein. After another 12-24 hours at 5 ℃, the samples were centrifuged (11000rpm, Sorvall (Thermo Fisher Scientific, Waltham, MA) SLA-3000rotor, 15 minutes at 10 ℃). The supernatant sample was adjusted to pH 6.0 with 3N NaOH and filtered through a 0.45 μm Millipore (Billerica, MA) MP60 filter. The sample was then subjected to a modified purification method comprising 100K Molecular Weight Cut Off (MWCO) diafiltration (5 x concentration followed by 7.5 x diafiltration with DI water), 0.1% HB precipitation and carbon filtration. The purified material was then subjected to Multi Angle Laser Light Scattering (MALLS) analysis.

Study of fermentation Process

Based on previous studies, by adding Na₂CO₃Conversion to NaOH as the base titrant optimized the fermentation process. The use of NaOH allowed the recovery pH to drop to 5.0, resulting in significant protein precipitation. Na (Na)₂CO₃Will release CO at low pH (< 6.6)₂Resulting in foam formation. The effect of alkaline titrant on Type19A polysaccharide and protein levels was examined. Two 3L fermentors were set up, one of which served as the original process control, using 20% Na₂CO₃The solution (w/v) was fed as base. Another fermentor used 3n naoh as base feed.

During the recovery phase, the cells were lysed in the fermentor with DOC (final concentration 0.13% (w/v)) and the fermentor was kept at 36 ℃ for 30 minutes. After this step, the lysate was left to stir overnight at room temperature (22 ℃). After lysate retention, a pH titration was performed from the unadjusted to 4.5 range and samples were taken at different pH selection points (pull). These samples were kept overnight at room temperature before processing and analysis for polysaccharide and protein concentrations. The OD, alkali and glucose levels during the fermentation phase are shown in fig. 1 and 2. The main difference is the higher final OD of the carbonate run.

The effect of pH adjustment of the lysate after DOC on total protein levels was also examined and the results are shown in figure 3. In NaOH operating and Na₂CO₃In operation, lower pH levels all reduced the protein loading in the cell-free broth. Lower pH (< 6.6) had no negative effect on polysaccharide yield. Fermentation analysis results showed that NaOH base supplementation was an acceptable alternative to Na during fermentation₂CO₃Alkali supplementation, but with Na₂CO₃The yield obtained with feeding gives a lower yield than with feeding.

Effect of alkaline titrants on 19A and 6A molecular weights

A series of fermentations were performed on a 3L scale to determine if the base titrant, HySoy concentration and pH maintenance steps affected serotype 19A molecular weight. After the modified purification method, molecular weight determination was performed using MALLS assay. The results are shown in table 1. For serotype 6A, only the base titrant was evaluated. The results are shown in table 2.

Table 1: effect of alkaline titrant on molecular weight of 19A (L29331-94)

Table 2: effect of alkaline titrant on 6A molecular weight

Action of bicarbonate with Mixed base pH titrant

In the first study (runs L29331-122 and L29331-139), different levels of initial sodium bicarbonate and sodium hydroxide in combination with a sodium carbonate base mixture at pH5.0 were used after the DOC maintenance step. The initial bicarbonate addition was in the range of 10-50mM, sodium carbonate was added with 3N sodium hydroxide as a base titrant in the range of 0.2-1.8M. One run contained 50mM initial bicarbonate and NaOH as the base titrant. The carbonate level at the end of these fermentations ranged from 14 to 111 mM. Serotype 19A molecular weights ranged from 520 and 713 kDa. The operating parameters and results are shown in table 3.

Table 3: na (Na)₂CO₃For mixed alkali as pH titrant

The second study (L29331-159 and L29331-185) used an initial addition of 15-30mM bicarbonate and a 0.4-1.0M Na₂CO₃A mixture of bases. The carbonate level at the end of the fermentation was in the range 24-62 mM. Serotype 19A molecular weights ranged from 502-763 kDa. The operating parameters and results are shown in table 4.

Table 4: NaHCO 2₃Mixed with alkali as pH titrant

Comparison of Mixed and pure carbonate titration alkali fermentation Processes

Experiments were performed to compare the base mixing method (0.7M Na)₂CO₃3N NaOH) with carbonate titrant method (20% Na)₂CO₃Solution, w/v). Results (Table 5)The molecular weight obtained from the carbonate titrant method was confirmed to be higher and more consistent (778, 781kDa) than the molecular weight obtained from the alkali mixing method (561-671 kDa). Using Na₂CO₃The method has high yield of polysaccharide.

Table 5: run L29399-1 Na₂CO₃For mixed alkali

Pilot scale run

Several serotype 19A pilot scale (100L) runs were performed using various base titrants. Molecular weight determination was performed after the complete purification process using MALLS assay and reports were obtained from the final purification batch. The results are shown in table 6.

Table 6: effect of base titrant on 19A molecular weight at Pilot Scale

Effect of alkaline titrant and covering on 19A molecular weight

A series of fermentations were performed on a 3L scale to determine if base titrant and atmospheric coverage affected molecular weight. Molecular weight determination was performed using MALLS assay after the modified purification process. The results are shown in table 7.

Table 7: effect of alkaline titrant and covering on 19A molecular weight

The articles "a" and "an" herein refer to one or more than one (i.e., to at least one) of the objects in accordance with the syntax. For example, "an element" means one or more elements.

All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

Claims (9)

1. A process for producing a solution containing high molecular weight isolated Streptococcus pneumoniae (Streptococcus pneumoniae) capsular polysaccharides wherein the polysaccharides comprise phosphodiester linkages between repeat units, the process comprising:

a) preparing a fermentation culture of streptococcus pneumoniae bacterial cells that produce capsular polysaccharides comprising phosphodiester linkages between repeat units;

b) providing CO to the fermentation culture₂；

c) Lysing the bacterial cells in the fermentation culture; and

d) isolating streptococcus pneumoniae capsular polysaccharide from the fermentation culture;

wherein the streptococcus pneumoniae capsular polysaccharide is serotype 19A, serotype 6A, serotype 19F, or serotype 6B; and

wherein CO is provided to the fermentation culture₂Comprises first adding NaHCO₃And adding Na again₂CO₃(ii) a And

wherein the isolated Streptococcus pneumoniae capsular polysaccharide has a molecular weight of at least 480 kDa.

2. The method of claim 1, wherein the streptococcus pneumoniae capsular polysaccharide is serotype 19A.

3. The method of claim 1, wherein the streptococcus pneumoniae capsular polysaccharide is serotype 6A.

4. The method of claim 1, wherein the streptococcus pneumoniae capsular polysaccharide is serotype 19F.

5. The method of claim 1, wherein the streptococcus pneumoniae capsular polysaccharide is serotype 6B.

6. The method of any one of claims 1-5, further wherein the pH of the fermentation culture is between 6.0-6.6.

7. The method of claim 1, wherein the fermentation culture is provided with CO₂Comprising using CO₂Covering the fermentation culture.

8. The method of claim 1, wherein lysing Streptococcus pneumoniae in the fermentation culture comprises adding sodium deoxycholate to the fermentation culture.

9. A solution comprising a high molecular weight isolated streptococcus pneumoniae capsular polysaccharide, wherein the polysaccharide comprises phosphodiester linkages between repeat units, wherein the solution is produced by the process of any one of claims 1-8.