Methods for producing streptococcus pneumoniae capsular polysaccharide carrier protein conjugates

a technology of capsular polysaccharide and pneumococcal polysaccharide, which is applied in the direction of pharmaceutical delivery mechanism, inorganic non-active ingredients, antibody medical ingredients, etc., can solve the problems of lyophilization preclude the ability to optimize individual formulations and cycle parameters, and the complication of pneumococcal diseases can be significant, etc., to achieve the ability to optimize individual formulations and cycle parameters, and facilitate handling

Inactive Publication Date: 2021-08-19
MERCK SHARP & DOHME LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0007]The present invention provides a process for producing multivalent pneumococcal polysaccharide protein conjugates for use as pneumococcal vaccines in which discrete lyophilization of pneumococcal polysaccharides and carrier protein using various sublimative processes are prepared and then used in a conjugation process to produce the multivalent pneumococcal polysaccharide protein conjugates for use in pneumococcal vaccines. The process of discrete drying provides various advantages over co-lyophilization of polysaccharides and carrier protein, including but not limited to, ability to optimize individual formulations and cycle parameters, convenient handling, and ability to produce individual polysaccharide and carrier protein preparations with a desired solution size.

Problems solved by technology

Furthermore, the complications of these diseases can be significant with some studies reporting up to 8% mortality and 25% neurologic sequelae with pneumococcal meningitis.
However, infants and young children respond poorly to unconjugated pneumococcal polysaccharides.
However, co-lyophilization precludes the ability to optimize individual formulations and cycle parameters with less convenient handling and ability to produce carrier protein and polysaccharide serotype supplies with desired solution sizes.

Method used

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  • Methods for producing streptococcus pneumoniae capsular polysaccharide carrier protein conjugates
  • Methods for producing streptococcus pneumoniae capsular polysaccharide carrier protein conjugates
  • Methods for producing streptococcus pneumoniae capsular polysaccharide carrier protein conjugates

Examples

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example 1

Preparation of S. Pneumoniae Capsular Polysaccharides 6A, 6B, 7F, 18C, 19A, 19F, and 23F

[0188]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, e.g., European Patent No. EP0497524. Isolates of pneumococcal subtypes are available from the American Type Culture Collection (Manassas, Va.). The bacteria are identified as encapsulated, non-motile, Gram-positive, lancet-shaped diplococci that are alpha-hemolytic on blood-agar. Subtypes can be differentiated on the basis of Quelling reaction using specific antisera. See, e.g., U.S. Pat. No. 5,847,112.

[0189]Cell banks representing each of the S. pneumococcus serotypes present are obtained from the Merck Culture Collection (Rahway, N.J.) in a frozen vial. A thawed seed culture is transferred to the seed fermentor containing a pre-sterilized growth media appropriate f...

example 2

[0191]Polysaccharide size reduction and activation is performed as follows.

[0192]About 6 g of purified pneumococcal capsular polysaccharide (Ps) powder is dissolved in water for injection (WFI) to a target concentration of approximately 4 g / L at room temperature. The solution is then 0.45-micron filtered to reduce bioburden. The Ps concentration of the filtered Ps solution is determined by HPSEC UV-MALS-RI.

[0193]For all serotypes except serotypes 18C and 19A, the solution is diluted to approximately 2.5 g / L and then homogenized to reduce the molecular mass of the Ps using a GEA-Niro Soavi Panda 2K homogenizer. All serotypes, except serotype 19A, were homogenized to reduce the molecular mass of the polysaccharide. Serotype 19A was not size reduced due to its relatively low starting size. Homogenization pressure and number of passes through the homogenizer were controlled to serotype-specific targets (150-1000 bar; 4-7 passes) to achieve a serotype-specific molecular mass. Size-reduce...

example 3

[0200]Preparation of CRM197 Carrier Protein may be performed as follows.

[0201]Frozen, purified CRM197, obtained through expression in Pseudomonas fluor escens as previously described (See International Patent Application Publication No. WO 2012 / 173876), is diafiltered against 10 diavolumes of 2 mM phosphate, pH 7.2 buffer using a 5 kDa NMWCO tangential flow ultrafiltration membrane and 0.22-micron filtered. In particular embodiments, 5 mM potassium phosphate, pH 6.4 (5 mM sodium phosphate, pH 7.0 is used for serotype 18C. The diafiltered solution is diluted in water with sucrose for a final concentration of between 1.0 and 5.3% (w / v) and lyophilized to produce dried CRM197 having a final moisture content of 6% or less. Table 2 below provides representative sucrose concentrations for CRM197 for conjugation against particular serotype polysaccharides.

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Abstract

A method is described for producing a pneumococcal capsular polysaccharide protein conjugate in which one or more activated pneumococcal polysaccharides of particular pneumococcal serotypes and carrier protein are separately lyophilized, the separately lyophilized polysaccharides and carrier protein are separately reconstituted in an organic solvent, and the reconstituted polysaccharide and carrier protein are then combined together by Tee-mixing and conjugated together to produce polysaccharide carrier protein conjugates. A plurality of conjugates, each comprising polysaccharides of a particular serotype, may be used to produce multivalent pneumococcal immunogenic compositions having a combination of conjugates for use in vaccines.

Description

BACKGROUND OF THE INVENTION(1) Field of the Invention[0001]The present invention relates to a method is described for producing a pneumococcal capsular polysaccharide protein conjugate in which one or more activated pneumococcal polysaccharides of particular pneumococcal serotypes and carrier protein are separately lyophilized, wherein the separately lyophilized polysaccharides and carrier protein are separately reconstituted in an organic solvent, and the reconstituted polysaccharide and carrier protein are then combined together by Tee-mixing and conjugated together to produce polysaccharide carrier protein conjugates. A plurality of conjugates, each comprising polysaccharides of a particular serotype, may be used to produce multivalent pneumococcal immunogenic compositions having a combination of conjugates for use in vaccines.(2) Description of Related Art[0002]Streptococcus pneumoniae, one example of an encapsulated bacterium, is a significant cause of serious disease world-wid...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/09A61K47/62A61K47/61
CPCA61K39/092A61K2039/70A61K47/61A61K47/62A61K2039/6037A61K2039/55505A61K9/19A61K9/0019A61K47/26A61K47/22A61K47/02A61K47/12A61K2039/64
Inventor AHL, PATRICK LEONARDBHAMBHANI, AKHILESHFARRELL, CHRISTOPHER JONMCHUGH, PATRICKJONES, MORRISA C.ROTH, DANIEL D.SINACOLA, JESSICA R.STANBRO, JUSTINWATSON, MATTHEW P.WEN, EMILYWINTERS, MICHAEL A.
Owner MERCK SHARP & DOHME LLC
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