Immunogenic glycoprotein conjugates
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. A method of making a glycoconjugate comprising a saccharide conjugated to a carrier protein through a (((2-oxoethyl)thio)alkyl)carbamate (oxo-eTAC) spacer, comprising the steps of:
(a) reacting a saccharide with a carbonic acid derivative or cyanogen derivative, to produce an activated saccharide;
(b) reacting the activated saccharide with L-cystine, mercaptopropionylhydrazide, 2-(2-aminoethoxy)ethane-1-thiol, or 4-Amino-1-butanethiol, or a salt thereof, to produce a thiolated saccharide;
(c) reacting the thiolated saccharide with a deprotecting or reducing agent 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-eTAC linked glycoconjugate is produced.
2. The method of claim 1 , wherein the carbonic acid derivative of step (a) is selected from the group consisting of 1,1′-carbonyl-di-(1,2,4-triazole) (CDT), 1,1′-carbonyldiimidazole (CDI), disuccinimidyl carbonate (DSC), and N-hydroxysuccinimidyl chloroformate.
3. The method of claim 1 , wherein step (e) comprises capping with N-acetyl-L-cysteine as the first capping reagent and iodoacetamide (IAA) as the second capping reagent.
4. The method of claim 1 , further comprising purification of the thiolated polysaccharide produced in step (c) via diafiltration.
5. The method of claim 1 , wherein the method further comprises purification of the glycoconjugate via diafiltration.
6. The method of claim 1 , wherein step (a) is conducted in a polar aprotic solvent selected from the group consisting of 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.
7. The method of claim 1 , wherein the saccharide is a capsular polysaccharide derived from S. pneumoniae.
8. The method of claim 7 , wherein the capsular polysaccharide is selected from the group consisting of 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.
9. The method of claim 7 , wherein the capsular polysaccharide is a Pn-serotype 10A capsular polysaccharide.
10. The method of claim 7 , wherein the capsular polysaccharide is a Pn-serotype 11A capsular polysaccharide.
11. The method of claim 7 , wherein the capsular polysaccharide is a Pn-serotype 22F capsular polysaccharide.
12. The method of claim 7 , wherein the capsular polysaccharide is a Pn-serotype 33F capsular polysaccharide.
13. The method of claim 1 , wherein the carrier protein is CRM 197 .
14. A method of making a glycoconjugate comprising a Pn-serotype 33F capsular polysaccharide conjugated to a carrier protein through a (((2-oxoethyl)thio)alkyl)carbamate (oxo-eTAC) spacer, comprising the steps of:
(a) reacting a Pn-serotype 33F capsular saccharide with a carbonic acid derivative or cyanogen derivative, to produce an activated saccharide;
(b) reacting the activated saccharide with L-cystine, mercaptopropionylhydrazide, 2-(2-aminoethoxy)ethane-1-thiol, or 4-Amino-1-butanethiol, or a salt thereof, to produce a thiolated saccharide;
(c) reacting the thiolated saccharide with a deprotecting or reducing agent 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-eTAC linked glycoconjugate is produced.
15. The method of claim 14 , further comprising purification of the thiolated polysaccharide produced in step (c) via diafiltration.
16. The method of claim 14 , wherein the method further comprises purification of the glycoconjugate via diafiltration.
17. The method of claim 14 , wherein step (a) is conducted in a polar aprotic solvent selected from the group consisting of 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.
18. The method of claim 14 , wherein the carrier protein is CRM 197 .