Polysaccharide precipitation process

Details Polysaccharide precipitation process Polysaccharide precipitation process

1998-06-01

2002-06-25

Geist, Gary (Department: 1623)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Conjugate or complex

C424S184100, C424S197110, C424S256100

Reexamination Certificate

active

06410025

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a process of preparing bacterial polysaccharides for a vaccine. Specifically, it related to a process of precipitating bacterial polysaccharides in solution.
BACKGROUND OF THE INVENTION
Bacterial polysaccharides are known in the art to be a component of various vaccines, including PNEUMOVAX® (a pneumococcal conjugate vaccine) and PedVax HIP® (an
H. influenzae
conjugate vaccine), both sold by Merck & Co., Inc.
Purified bacterial capsular polysaccharides, including those of
Haemophilis influenzae
type b and
Streptococcus pneumoniae
, are generally not sufficiently immunogenic to use as a vaccine. To solve this problem, the capsular polysaccharides are conjugated to a protein carrier, such as immunogenic membrane proteins, viral protein subunits, synthetic polypeptides, bacterial toxoids, or other suitable immunogenic proteins. Examples of processes for making suitable immunogenic conjugates are well known in the art and include those detailed in U.S. Pat. Nos. 4,695,624, and 4,882,317.
In general, to make an immunogenic conjugate, a bacterial polysaccharide is first isolated and purified from the source bacteria. Such polysaccharides are also available commercially. The polysaccharide may then be subjected to a round of further purification, and/or size reduction steps. Next it will undergo a series of chemical reactions to add functional groups so that they can be joined to the immunogenic protein carrier. After the coupling occurs, the conjugate is subsequently purified and an adjuvant such as aluminum hydroxide may be added to produce the final vaccine.
One of the problems which has been encountered during synthesis of the conjugate is that the polysaccharides themselves are water soluble, but the later derivatization steps are best performed in an organic solvent. In the past, this required two steps in a manufacturing process, both of which were rather difficult: 1) conversion of the polysaccharide (in its calcium salt form) to its tetrabutylammonium salt (tBuAM) form using calcium oxalate metathesis or by column ion exchange; 2) and subsequent removal of water by either vacuum distillation and flushing with dimethyl formamide (DMF) or by lyophilization. However, not all of the calcium salt was converted into the tBuAM form, resulting in an oil emulsion. Some of the polysaccharide types are not sufficiently soluble in DMF, so the distillation step is not feasible, and another solvent such as DMSO is not practical due to the significantly higher boiling point of DMSO. Further, lyophilization was not feasible at a manufacturing scale.
It would be desirable to develop a process that can accommodate the transition from an aqueous process to an organic solvent without sacrificing yield, purity, and ease of manufacture.
DETAILED DESCRIPTION OF THE INVENTION
This invention is directed to a process for precipitating bacterial capsular polysaccharides from an aqueous solution comprising adding a precipitating amount of a long chain detergent to the solution. In an optional step, the invention also comprises separating the precipitated polysaccharide paste from the aqueous solution and extracting the water from the paste to form a dry polysaccharide powder. The powder may be then be further processed to produce vaccines, such as pneumoconjugate vaccines.
This invention is suitable for use with polysaccharides from any bacterial source. Preferred polysaccharides are from Pneumococcus and
Haemophilis influenzae
, particularly the negatively charged polysaccharides. Especially preferred are anionic Pneumococcus polysaccharides. Such polysaccharides are well known in the art and include those designated as 3, 4, 5, 6b, 9V, 18C, 19f, and 23f. Some 84 serotypes of Pneumococcus are known, and the anionic polysaccharides from any of the serotypes may be used in this invention. These polysaccharides are soluble in water, but are not soluble in organic solutions. In general, the polysaccharides are dissolved in pyrogen free water (PFW) during the vaccine manufacturing process, and while concentrations may vary during processing, are generally present in amounts of from about 0.2% to 1% by weight in the PFW.
The long chain detergent may be virtually any long chain detergent. Particularly preferred ones include cetylpyridinium chloride (CPC) and cetyltrimethylammonium bromide (CETAB). Generally, any amount of long chain detergent may be added to precipitate the polysaccharide from the aqueous solution, but in most applications, a 1-10% solution of the detergent, added to a molar ratio of about 1:1 is sufficient to precipitate virtually all the polysaccharide. The detergents react with the negatively-charged polysaccharide to form a polysaccharide-salt which is insoluble in water and which precipitates out of solution. The insoluble salt form of the polysaccharides, particularly the cetylpyridinium salt and the cetyltrimethylammonium salt of the polysaccharides form yet another aspect of this invention. In particular, this invention also includes the cetylpyridinium salt form of Pneumococcus polysaccharides 3, 4, 5, 6b, 9V, 19f, and 23f as well as the cetyltrimethylammonium salt form of 4, 6b, 9V, 19f, and 23f.
The precipitate is paste-like in consistency. It may be removed from the aqueous solution by any convenient means, such as by centrifugation or the like. Next, it is dried by any convenient means, such as by mixing with acetone or ether to extract any water which may be present (trituration). Thus, a preferred process of this invention comprises precipitation of an aqueous solution of bacterial polysaccharides by adding an effective amount of a long chain detergent, and subsequently drying the precipitate using acetone trituation to obtain a slurry.
The slurry may also be filtered and vacuum dried to afford the cetylpyridinium salt or cetyltrimethylammonium salts as a dry powder. The dry powder is soluble in the solvents which are commonly used in derivatization chemistry, such as dimethylsulfoxide (DMSO) and dimethylformamide (DMF).
A further advantage of this invention is that the method affords a further level of purification of the polysaccharides during the aqueous precipitation step, especially with respect to impurities which bear a net positive charge, such as some non-capsular membrane proteins and C-Polysaccharide.
Yet another advantage is that by providing a dry powder form of the polysaccharide salt, an accurate amount of polysaccharide can be measured and used in the initial derivatization reactions, where precision is most desirable.
The precipitation process and the polysaccharide salts of this invention are essentially intermediate processes and products in a lengthy method to make vaccines which comprises the steps of 1) preparation of the starting polysaccharides; 2) the precipitation process of this invention; 3)derivatization of the polysaccharides; 4) conjugation of the derivatized polysaccharides to a protein; and 5) alum formulation. Each of the process steps 1, 3, 4, and 5 which do not form a part of this invention are summarized below.
Preparation of the Starting Polysaccharides. The starting point in the entire process of making the vaccine is the isolation of bacterial capsular polysaccharides from a fermentation broth. Alternatively, these polysaccharides are also commercially available and may be used as starting material. Individual native polysaccharides (in a powder form) are dissolved in water, and incubated with sodium chloride to dissociate residual impurities which are then removed by membrane diafiltration. Each PsPn solution is then diafiltered against water to establish well-defined conditions for size reduction and the precipitation steps. The polysaccharides are preferably size-reduced by passage through a high pressure orifice using a mechanical homogenizer. This step facilitates downstream processing as well as provides for a product of uniform size. Solubility in organic solvents such as DMSO is also increased by reducing the polysaccharide's molecular weight of the polysaccharide. The

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