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Novel formulations which mitigate agitation-induced aggregation of immunogenic compositions

A preparation and conjugation technology, applied in animal repellents, drug combinations, plant growth regulators, etc., can solve the problems of decreased total vaccine concentration, unusable protein products, vaccine protein aggregation and precipitation, etc.

Inactive Publication Date: 2013-11-13
MERCK & CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Protein-based vaccines, including polysaccharide-protein conjugates, are prone to protein aggregation and precipitation, and the precipitated protein product cannot be utilized, resulting in a significant decrease in the total concentration of the vaccine

Method used

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  • Novel formulations which mitigate agitation-induced aggregation of immunogenic compositions
  • Novel formulations which mitigate agitation-induced aggregation of immunogenic compositions
  • Novel formulations which mitigate agitation-induced aggregation of immunogenic compositions

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Methods of culturing pneumococci are well known in the art. See, e.g., 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. EPO497524. Isolates of pneumococcal subtypes are available from the ATCC.

[0100] The bacterium was identified as a capsulated, inactive, Gram-positive, lancet-shaped diplococcus that was alpha-hemolytic on blood agar. Subtyping was performed based on the Quelling reaction using specific antisera. See, eg, US Patent No. 5,847,112.

[0101] cell bank

[0102] Cell banks of each S. pneumoniae serotype present in PCV-15 were obtained from the Merck Culture Collection (Rahway, NJ) in cryovials.

[0103] inoculation

[0104] Thawed seed cultures were transferred to seed fermenters containing appropriate pre-sterilized growth medium.

[0105] Seed Fermentation

[0106] Cultures were grown in temperature and pH co...

Embodiment 2

[0114] activation operation

[0115] Individually conjugate saccharides of different serotypes to purified CRM using conventional protocols 197 on the carrier protein. In this operation, sugars are dissolved and sized to target molecular weights, chemically activated and buffer exchanged by ultrafiltration. The purified CRM is then 197 Conjugated with an activated sugar, the obtained conjugate was purified by ultrafiltration and finally filtered through a 0.2 μm membrane filter. As described in this example, various operating parameters within each step, such as pH, temperature, concentration and time, are serotype specific.

[0116] Step 1: Dissolve

[0117] The purified polysaccharide was dissolved in water at a concentration of 2-3 mg / mL. The dissolved polysaccharides are passed through a mechanical homogenizer with a pre-set pressure of 0-1000 bar. After size reduction, sugars were concentrated on a 10 kDa MWCO ultrafilter and diafiltered with sterile water. The...

Embodiment 3

[0133] The volume required for the volumetric concentrate is calculated based on the batch volume and the volumetric sugar concentration. The combined 15 conjugates were further diluted to the target absorption concentration by adding excipients (such as poloxamer), the excipients include buffer solution containing sodium chloride and L-histidine, pH 5.8. After thorough mixing, the mixture was sterile filtered through a 0.2 μm filter. The aseptically prepared volume was mixed gently while and after mixing with the volume of aluminum phosphate. The prepared vaccine is stored at 2-8°C.

[0134] Example 4: Effect of Excipients on Agitation-Induced Aggregation

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PUM

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Abstract

The present invention provides novel formulations which mitigate agitation-induced aggregation of immunogenic compositions particularly those having polysaccharide-protein conjugates. Specifically, the novel formulations comprise a poloxamer within a molecular weight range of 1100 to 17,400 which provides significant advantages over previously used surfactants including polysorbate 80. In one embodiment, the present invention provides a multivalent immunogenic composition having 15 distinct polysaccharide-protein conjugates and a poloxamer. Each conjugate consists of a capsular polysaccharide prepared from a different serotype of Streptococcus pneumoniae (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F or 33F) conjugated to a carrier protein, preferably CRM197.

Description

technical field [0001] The present invention provides novel formulations that alleviate agitation-induced aggregation of immunogenic compositions containing polysaccharide-protein conjugates. In particular, the new formulation contains the surfactant poloxamer in the molecular weight range of 1100 to 17400, which has significant advantages over previously used surfactants including polysorbate 80. technical background [0002] Vaccine formulations often must be stable and maintain a uniform consistency to meet the need for long shelf life and the use of multi-dose containers. Protein-based vaccines, including polysaccharide-protein conjugates, are prone to protein aggregation and precipitation, and the precipitated protein products cannot be utilized, resulting in a significant decrease in the total concentration of the vaccine. Polysaccharide-protein conjugate vaccines, in particular, appear to have a stronger tendency to aggregate than carrier proteins alone. See Berti e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N25/00
CPCA61K33/06A61K47/4833A61K47/10A61K47/48261A61K39/385A61K47/6415A61K47/646A61P31/04A61P37/04A61P43/00A61K2300/00A61K47/50A01N25/00A61K39/116A61K47/34
Inventor J.T.布卢J.肯农W.J.史密斯E.J.格林-特克斯勒B.西格弗里德
Owner MERCK & CO INC
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