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Processes for the formulation of pneumococcal polysaccharides for conjugation to a carrier protein

A polysaccharide technology for Streptococcus pneumoniae and Streptococcus, which is applied to medical preparations with non-active ingredients, medical preparations containing active ingredients, and pharmaceutical formulas, and can solve problems such as the increase in the prevalence of pneumococcus

Pending Publication Date: 2020-04-21
MERCK SHARP & DOHME BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the prevalence of pneumococci expressing serotypes not present in the vaccine is increasing

Method used

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  • Processes for the formulation of pneumococcal polysaccharides for conjugation to a carrier protein
  • Processes for the formulation of pneumococcal polysaccharides for conjugation to a carrier protein
  • Processes for the formulation of pneumococcal polysaccharides for conjugation to a carrier protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0140] Embodiment 1: Preparation of Streptococcus pneumoniae capsular polysaccharide

[0141] Methods of culturing pneumococci are well known in the art. See, eg, Chase, 1967, Methods of Immunology and Immunochemistry 1:52. Methods of preparing pneumococcal capsular polysaccharides are also well known in the art. See eg European Patent No. EP 0 497 524 B1. The method described below generally follows that described in European Patent No. EP 0497 524 B1 and is generally applicable to all pneumococcal serotypes unless specifically modified.

[0142] Isolates of pneumococcal serotypes 3, 8, 12F were obtained from the University of Pennsylvania (Dr. Robert Austrian). Isolates of pneumococcal serotypes 15A, 16F, 23A, 24F, 35B were obtained from Merck Culture Collection. Isolates of pneumococcal serotypes 23B and 31 were obtained from the Centers for Disease Control and Prevention (Atlanta, GA). Isolates of pneumococcal serotype 17F were obtained from the FDA Office of Biologic...

Embodiment 2

[0149] Example 2: General conjugation method

[0150] Polysaccharide size reduction and oxidation

[0151] The purified pneumococcal capsular polysaccharide powder was dissolved in water and subjected to 0.45 micron filtration. Polysaccharides were homogenized to reduce polysaccharide molecular weight unless otherwise stated. Homogenization pressure and number of passes through the homogenizer were controlled to serotype specific targets (150-1000 bar; 4-7 passes).

[0152] Size-reduced polysaccharides were subjected to 0.2 micron filtration, concentrated and diafiltered with distilled water using 5 kDa or 10 kDa NMWCO tangential flow ultrafiltration membranes.

[0153] The polysaccharide solution was then adjusted to 22 °C and pH 5 with sodium acetate buffer to minimize polysaccharide size reduction due to activation.

[0154] Purified polysaccharides were prepared for conjugation, ie, activation, using sodium metaperiodate oxidation (see Anderson et al., 1986, J. Immunol....

Embodiment 3

[0164] Example 3: Acid hydrolysis of polysaccharides from serotypes 12F, 23A, 24F and 31

[0165] Conjugation of pneumococcal polysaccharides to proteins by reductive amination in aprotic solvents such as DMSO has been previously described. Activated polysaccharides (Ps) and proteins (Pr) are typically lyophilized, resuspended in DMSO, then mixed and incubated with sodium cyanoborohydride and sodium borohydride to achieve conjugation. The polysaccharide can be mechanically reduced in size (eg, by homogenization) prior to oxidation to reduce the Ps molecular weight and provide a consistent Ps size for conjugation. For many pneumococcal serotypes, conjugation of mechanically reduced size and oxidized Ps produced conjugates meeting the targeted properties of size, lysine consumption, free polysaccharide and free protein. However, for some serotypes, it was found difficult to achieve the target conjugation properties using this process even after optimizing the process parameters...

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Abstract

The present invention provides a number of process improvements related to the conjugation of capsular polysaccharides from Streptococcus pneumoniae to a carrier protein. These processes are serotypespecific and include acid hydrolysis, addition of sodium chloride to the reductive amination reaction, and addition of sucrose to dissolve polysaccharides. Polysaccharide-protein conjugates prepared using the processes of the invention can be included in multivalent pneumococcal conjugate vaccines.

Description

technical field [0001] The present invention provides a number of process improvements related to the conjugation of capsular polysaccharides from S. pneumoniae to carrier proteins. Polysaccharide-protein conjugates prepared using the methods of the invention can be included in multivalent pneumococcal conjugate vaccines. Background technique [0002] Streptococcus pneumoniae is an encapsulated bacterium and is an important cause of severe disease worldwide. In 1997, the Centers for Disease Control and Prevention (CDC) estimated that there were 3,000 cases of pneumococcal meningitis, 50,000 cases of pneumococcal bacteremia, 7,000,000 cases of pneumococcal otitis media, and 500,000 cases of pneumococcal pneumonia in the United States each year. See Centers for Disease Control and Prevention, MMWR Morb Mortal Wkly Rep 1997, 46(RR-8): 1-13. In addition, complications of these diseases can be severe, with some studies reporting mortality rates as high as 8% for pneumococcal me...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/02A61K39/085A61K39/116
CPCA61K39/092A61K2039/6037A61K2039/62A61P11/00A61P31/04Y02A50/30A61K47/6415A61K47/646A61K2039/70A61K47/62A61K47/549C07H1/00
Inventor P·麦克休M·A·温特斯J·科涅特兹克
Owner MERCK SHARP & DOHME BV
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