Methods for producing streptococcus pneumoniae capsular polysaccharide carrier protein conjugates from lyospheres

12144,855

17/049208

April 25, 2019

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 in the form of lyospheres of carrier protein and lyospheres of the one or more polysaccharides. A predetermined amount of carrier protein lyospheres and activated polysaccharide lyospheres are mixed together and the mixture reconstituted in an organic solvent 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.

Merck Sharp & Dohme LLC (Rahway, NJ, US)

Merck Sharp & Dohme LLC (Rahway, NJ, US)

1. A method for making a composition comprising one or more Streptococcus pneumoniae polysaccharides covalently linked to a carrier protein, the method comprising: (a) providing a first lyosphere composition comprising activated Streptococcus pneumoniae polysaccharides from one or more Streptococcus pneumoniae serotypes and a second lyosphere composition comprising carrier protein, wherein the first and second lyosphere compositions are prepared by a sublimative drying process selected from lyophilization and radiant energy vacuum (REV) dehydration comprising: (i) providing a first aqueous solution comprising activated Streptococcus pneumoniae polysaccharides from one or more Streptococcus pneumoniae serotypes and a second aqueous solution comprising a carrier protein and a buffer, wherein the first aqueous solution comprises about 4% to 6% sucrose and the second aqueous solution comprises about 4% to 8% sucrose, and (ii) subjecting the first and second aqueous solutions to the sublimative drying process to produce the first and second lyosphere compositions: (b) combining an amount of the first lyosphere composition and an amount of the second lyosphere composition together to provide a lyosphere mixture having a predetermined ratio of the one or more activated polysaccharides to the carrier protein; (c) reconstituting the lyosphere mixture in an organic solvent and mixing to provide a reconstituted mixture; and (d) adding a reducing agent to the reconstituted mixture to produce a conjugate solution comprising carrier protein conjugated to the polysaccharides of the one or more Streptococcus pneumoniae serotypes, wherein the conjugated carrier protein has a lysine loss value of greater than five (mole/mole).

2. The method of claim 1 , wherein the buffer is a Histidine, Succinate, MES, MOPS, HEPES, or Acetate buffer in a pH range of 5.0-7.0.

3. The method of claim 1 , wherein the buffer is a Phosphate or a Citrate buffer in a pH range of 5.0-7.0.

4. The method of claim 1 , wherein the first and second lyosphere compositions each have a moisture content of less than 6%.

5. The method of claim 1 , wherein the organic solvent is dimethyl sulfoxide (DMSO).

6. The method of claim 1 , wherein the reconstituting is performed in eight minutes or less and the mixing is performed in 120 minutes or less.

7. The method of claim 1 , wherein the conjugate solution comprises polysaccharide conjugated to the carrier protein at a ratio from about 0.6 to about 1.5 polysaccharide to carrier protein on a weight to weight basis.

8. The method of claim 1 , wherein the conjugate solution comprises a free polysaccharide amount that is less than about 15% of the total polysaccharide in the solution.

9. The method of claim 1 , wherein the one or more Streptococcus pneumoniae serotypes are selected from the group consisting of Streptococcus pneumoniae serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6G, 6H, 7F, 7A, 7B, 7C, 8, 9A, 9L, 9N, 9V, 10F, 10A, 10B, 10C, 11F, 11A, 11B, 11C, 11D, 11E, 12F, 12A, 12B, 13, 14, 15F, 15A, 15B, 15C, 16F, 16A, 17F, 17A, 18F, 18A, 18B, 18C, 19F, 19A, 19B, 19C, 20A, 20B, 21, 22F, 22A, 23F, 23A, 23B, 24F, 24A, 24B, 25F, 25A, 27, 28F, 28A, 29, 31, 32F, 32A, 33F, 33A, 33B, 33C, 33D, 33E, 34, 35F, 35A, 35B, 35C, 36, 37, 38, 39, 40, 41F, 41A, 42, 43, 44, 45, 46, 47F, 47A, 48, CWPS1, CWPS2, and CWPS3.

10. The method of claim 1 , wherein the carrier protein is an inactivated bacterial toxoid selected from the group consisting of tetanus toxoid, diphtheria toxoid, pertussis toxoid, bacterial cytolysins, and pneumolysin.

11. The method of claim 10 , wherein the diphtheria toxoid is CRM 197 .

12. The method of claim 1 , wherein the conjugate solution is sterile filtered.

13. The method claim 1 , wherein the Streptococcus pneumoniae polysaccharides are activated by reacting with an oxidizing agent.

6146902 November 2000 McMaster
6517526 February 2003 Tamari
7709001 May 2010 Hausdorff et al.
8808707 August 2014 Siber
9669084 June 2017 Siber
10406220 September 2019 Siber et al.
20070042031 February 2007 MacLachlan et al.
20070231340 October 2007 Hausdorff et al.
20090010966 January 2009 Davis et al.
20110195086 August 2011 Caulfield et al.
20110201791 August 2011 Prasad
20120045479 February 2012 Sievers et al.
20150190521 July 2015 Biemans et al.
20150202309 July 2015 Emini et al.
20160009768 January 2016 Davis et al.
20160252300 September 2016 Bhambhani et al.
20160324948 November 2016 Gu et al.
20160324950 November 2016 Anderson et al.
20170220463 August 2017 Malina et al.
20180099039 April 2018 Emini et al.
102068690 May 2011
3096786 July 2021
200038711 July 2000
2006110352 October 2006
2006110381 October 2006
2007127665 November 2007
2008045852 April 2008
2008118752 October 2008
2009009629 January 2009
2010080484 July 2010
2010080486 July 2010
2011041003 April 2011
2011100151 August 2011
2011151760 December 2011
2012078482 June 2012
2014097099 June 2014
2015110940 July 2015
2015110941 July 2015
2015110942 July 2015
2016113644 July 2016
2016178123 November 2016
2017013548 January 2017
2017085586 May 2017
2017085602 May 2017
2018134693 July 2018
2018144438 August 2018
2018144439 August 2018
2018156465 August 2018
2018156467 August 2018
2018156468 August 2018
2018156491 August 2018
2018212846 November 2018
2019036313 February 2019
2019050813 March 2019
2019050814 March 2019
2019050815 March 2019
2019050816 March 2019
2019050818 March 2019
2019083865 May 2019
2019139692 July 2019
2019212842 November 2019
2019212844 November 2019
2019212846 November 2019
2019217183 November 2019
2019236435 December 2019
2020121159 June 2020
2020131763 June 2020
2020208502 October 2020
2020247299 December 2020
2020247301 December 2020

Arditi et al., Three-Year Multicenter Surveillance of Pneumococcal Meningitis in Children: Clinical Characteristics, and Outcome Related to Penicillin Susceptibility and Dexamethasone Use, Pediatrics, 1998, 1087-1097, 102(5). cited by applicant
Lei et al., Quantification of free polysaccharide in meningococcal polysaccharide-diphtheria toxoid conjugate vaccines, Dev. Biol., 2000, Abstract—1/1, 103. cited by applicant
MMWR, Prevention of Pneumococcal Disease: Recommendations of the Advisory Committee on Immunization Practices (ACIP), Centers for Disease Control and Prevention, 1997, 1-25, 46(RR-8). cited by applicant
Shivu, Bhavana, Distinct /J-Sheet Structure in Protein Aggregates Determined by ATR-FTIR Spectroscopy, Biochemistry, 2013, 5176-5183, 52. cited by applicant
Lei, Q.P. et al., Quantification of Free Polysaccharide in Meningococcal Polysaccharide-Diphtheria Toxoid Conjugate Vaccines, Dev. Biol., 2000, 259-264, 103. cited by applicant
Nagaki, Aiichiro et al., Fast Micromixing and Flow Synthesis, Nagare: Journal of Japan Society of Fluid Mechanics, 2015, 3-9, 34. cited by applicant
Steven P. Schwendeman et al., Stabilization of tetanus and diphtheria toxoids against moisture-induced aggregation, Proc. Natl. Acad. Sci. USA, 1995, 11234-11238, 92. cited by applicant

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