Conjugation process of bacterial polysaccharides to carrier proteins

• US 9,265,839 B2
• Filed: 03/10/2014
• Issued: 02/23/2016
• Est. Priority Date: 03/09/2010
• Status: Active Grant

• Current Assignee: Glaxosmithkline Biologicals SA (GlaxoSmithKline PLC)
• Original Assignee: Glaxosmithkline Biologicals SA (GlaxoSmithKline PLC)
• Inventors: Biemans, Ralph Leon, Duvivier, Pierre, Gavard, Ollivier Francis Nicolas
• Primary Examiner(s): Baskar, Padma V
• Application Number:
  US14/202,119
• Publication Number:
  US 20140186389A1
• Time in Patent Office: 715 Days
• US Class Current: 1/1
• CPC Class Codes: A61K 2039/6037 Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
• CPC Class Codes: A61K 2039/62 characterised by the link between antigen and carrier
• CPC Class Codes: A61K 39/092 Streptococcus
• CPC Class Codes: A61K 47/61 the organic macromolecular compound being a polysaccharide or a derivative thereof
• CPC Class Codes: A61K 47/6415 Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
• CPC Class Codes: A61K 47/646 the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/16 for influenza or rhinoviruses

Abstract

Process for conjugation of bacterial saccharides including Streptococcus pneumoniae and Haemophilus influenzae saccharides by reductive amination are provided herein.

Claims

1. A process for conjugating a bacterial saccharide and reducing the sizing effect on bacterial saccharide comprising the steps of

a) reacting the bacterial saccharide with 0.001-0.7 molar equivalents of periodate to form an activated bacterial saccharide;

b) mixing the activated bacterial saccharide with a carrier protein;

c) reacting the activated bacterial saccharide and the carrier protein with a reducing agent to form a conjugate;

wherein step a) occurs in a buffer which does not contain an amine group, and the buffer has a concentration between 1-100 mM and wherein the bacterial saccharide is S. pneumoniae capsular saccharide 6B.

2. The process of claim 1 wherein the buffer is selected from the group consisting of phosphate buffer, borate buffer, acetate buffer, carbonate buffer and citrate buffer.

3. The process of claim 1 wherein the pH in step a) is pH 3.5-8.0.

4. The process of claim 1 wherein the average molecular weight of the bacterial saccharide is between 1-1100 kDa after step a).

5. The process of claim 1 wherein the carrier protein is selected from the group consisting of tetanus toxoid, fragment C of tetanus toxoid, diphtheria toxoid, CRM197, Pneumolysin, protein D, PhtD, PhtDE and N19.

6. The process of claim 1 wherein the reducing agent comprises sodium cyanoborohydride or sodium triacetoxyborohydride.

7. The process of claim 1 comprising a further step e) of purifying the conjugate.

8. The process of claim 1 containing a further step of mixing the conjugate with further antigens.

9. The process of claim 8 wherein the further antigens comprise one or more S. pneumoniae proteins selected from the group consisting of the Poly Histidine Triad family (PhtX), Choline Binding Protein family (CbpX), CbpX truncates, LytX family, LytX truncates, CbpX truncate-LytX truncate chimeric proteins (or fusions), pneumolysin (Ply), PspA, PsaA, Sp128, Sp101, Sp130, Sp125 and Sp133.

10. The process of claim 1 wherein the conjugate is mixed with an adjuvant or a pharmaceutically acceptable excipient.