Immunogenic glycoprotein conjugates

Abstract

The present invention relates generally to glycoconjugates comprising a saccharide covalently conjugated to a carrier protein through a spacer containing ((2-oxoethyl)thio)). In an aspect the invention provides oxo-eT linked glycoconjugates comprising a saccharide covalently conjugated to a carrier protein through a ((2-oxoethyl)thio) spacer having the formula (I): wherein: A is a group (C═X) m wherein X is S or O and m is 0 or 1; B is a bond, O, or CH 2 ; and when m is 0, B can also be (C═O); R is a C 2 -C 16 alkylene, C 2 -C 16 heteroakylene, NH—C(═O)—C 2 -C 16 alkylene, or NH—C(═O)—C 2 -C 16 heteroakylene, wherein said alkylene and heteroalkylene are optionally substituted by 1, 2 or 3 groups independently selected from COOR′ where R′ is selected from H, methyl, ethyl or propyl. The invention further relates to immunogenic compositions comprising such glycoconjugates, and to methods for the preparation and use of such glycoconjugates and immunogenic compositions.

1 - 28 . (canceled) 
     
     
         29 . A method of making a glycoconjugate comprising a carboxyl containing saccharide conjugated to a carrier protein through an (((2-oxoethyl)thio)alkyl)amide (oxo-eTAAD) spacer, comprising the steps of:
 (a) reacting the carboxyl group containing saccharide to generate an activated saccharide with a carbodiimide or a derivative thereof;   (b) reacting the activated saccharide with a heterobifunctional linker containing amine and thiol functionalities (in protected or free form) from the amino end, to produce a thiolated saccharide;   (c) reacting the thiolated saccharide with a deprotecting or reducing agent (if protected) to produce an activated thiolated saccharide comprising one or more free sulfhydryl residues;   (d) reacting the activated thiolated saccharide with an activated carrier protein comprising one or more α-haloacetamide groups, to produce a thiolated saccharide-carrier protein conjugate; and   (e) reacting the thiolated saccharide-carrier protein conjugate with (i) a first capping reagent capable of capping unconjugated α-haloacetamide groups of the activated carrier protein; and/or (ii) a second capping reagent capable of capping unconjugated free sulfhydryl residues of the activated thiolated saccharide;   whereby an oxo-eTAAD linked glycoconjugate is produced.   
     
     
         30 - 32 . (canceled) 
     
     
         33 . The method of  claim 29 , wherein the carbodiimide derivative of step (a) is selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N,N′-dicyclohexylcarbodiimide (DCC), and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide. 
     
     
         34 . The method of  claim 33 , wherein the carbodiimide derivative is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). 
     
     
         35 . The method of  claim 29 , wherein step (a) is conducted in a polar aprotic solvent selected from dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), N-methyl-2-pyrrolidone (NMP), acetonitrile, 1,3-Dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU), and hexamethylphosphoramide (HMPA), or a mixture thereof. 
     
     
         36 . The method of  claim 35 , wherein the polar aprotic solvent is dimethylsulfoxide (DMSO). 
     
     
         37 . The method of  claim 29 , wherein step (a) is accomplished by carbodiimide and thiazolidinone thione. 
     
     
         38 . The method of  claim 29 , wherein step (a) is accomplished by N-ethyl-3-phenylisoxazolium-3′-sulfonate (Woodward's reagent K). 
     
     
         39 . The method of  claim 29 , wherein step (e) comprises capping with N-acetyl-L-cysteine as the first capping reagent and iodoacetamide (IAA) as the second capping reagent. 
     
     
         40 . The method of  claim 29 , further comprising purification of the thiolated polysaccharide produced in step (c) via diafiltration. 
     
     
         41 . The method of  claim 29 , wherein the method further comprises purification of the glycoconjugate via diafiltration. 
     
     
         42 . The method of  claim 29 , wherein the saccharide is a capsular polysaccharide derived from  S. pneumoniae.    
     
     
         43 . The method of  claim 42 , wherein the capsular polysaccharide is selected from pneumococcal (Pn) serotype 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 12, 15B, 18C, 19A, 19F, 22F, 23F and 33F capsular polysaccharides. 
     
     
         44 . The method of  claim 43 , wherein the capsular polysaccharide is a Pn-serotype 10A capsular polysaccharide. 
     
     
         45 . The method of  claim 43 , wherein the capsular polysaccharide is a Pn-serotype 11A capsular polysaccharide. 
     
     
         46 . The method of  claim 43 , wherein the capsular polysaccharide is a Pn-serotype 22F capsular polysaccharide. 
     
     
         47 . The method of  claim 43 , wherein the capsular polysaccharide is a Pn-serotype 33F capsular polysaccharide. 
     
     
         48 . The method of  claim 29 , wherein the carrier protein is selected from the group consisting of TT, DT, DT mutants,  H. influenzae  protein D, PhtX, PhtD, PhtE fusions, detoxified pneumolysin, PorB, N19 protein, PspA, OMPC, toxin A or B of  C. difficile , and PsaA. 
     
     
         49 . The method of  claim 29 , wherein the carrier protein is CRM 197 . 
     
     
         50 . A glycoconjugate produced by the method of  claim 29 . 
     
     
         51 . An immunogenic composition comprising the glycoconjugate of  claim 50  and a pharmaceutically acceptable excipient, carrier or diluent.