Multivalent pneumococcal polysaccharide-protein conjugate composition
Provided are mixed carrier, multivalent pneumococcal conjugate compositions comprising 21 different pneumococcal capsular polysaccharide-protein conjugates, wherein each of the conjugates includes a capsular polysaccharide from a different serotype of Streptococcus pneumoniae conjugated to either tetanus toxoid (TT) or CRM 197 , wherein the Streptococcus pneumoniae serotypes are selected from 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11 A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, and 33F, where the capsular polysaccharides of two of serotypes 1, 3, and 5 and one or both of serotypes 15B and 22F are conjugated to TT and the remaining capsular polysaccharides are conjugated to CRM 197 . Also provided are methods of producing the mixed carrier, multivalent pneumococcal conjugate compositions and methods of using the same for prophylaxis against Streptococcus pneumoniae infection or disease in a subject.
1. A method for preparing an immunogenic serotype 9N conjugate, the method comprising:
(a) lysing a bacterial cell producing Streptococcus pneumoniae serotype 9N capsular polysaccharide by fermentation;
(b) purifying Streptococcus pneumoniae serotype 9N capsular polysaccharide from the lysed bacterial cell;
(c) activating the Streptococcus pneumoniae serotype 9N capsular polysaccharide by reacting with an oxidizing agent to achieve a degree of oxidation of 2-19, wherein the activated Streptococcus pneumoniae serotype 9N capsular polysaccharide has a molecular weight of 200-700 kDa; and
(d) mixing the activated Streptococcus pneumoniae serotype 9N capsular polysaccharide with a CRM 197 carrier protein to conjugate the CRM 197 carrier protein to the activated Streptococcus pneumoniae serotype 9N capsular polysaccharide and thereby form the immunogenic serotype 9N conjugate.
2. The method of claim 1 , wherein the CRM 197 mixed in the step (d) is reacted with a reducing agent to form the conjugate with the activated Streptococcus pneumoniae serotype 9N capsular polysaccharide.
3. The method of claim 1 , wherein in step (c), 0.02-0.19 μg of periodate is reacted with 1 μg of the Streptococcus pneumoniae serotype 9N capsular polysaccharide at 20-25° C. for 15-20 hours.
4. The method of claim 1 , wherein the Streptococcus pneumoniae serotype 9N capsular polysaccharide reacted with the oxidizing agent in step (c) has a molecular weight of 400-900 kDa.
5. The method of claim 1 , wherein the immunogenic serotype 9N conjugate formed in step (d) has a molecular weight of 500-4,000 kDa.
6. The method of claim 1 , wherein an initial input ratio of the CRM 197 to the activated serotype 9N capsular polysaccharide is 0.5-2.5:1.
7. The method of claim 1 , wherein at least 15-60% of the immunogenic serotype 9N conjugate has a K d of 0.3 or below as measured in a CL-4B column.
8. The method of claim 1 , wherein in step (c), 0.02-0.19 μg of periodate is reacted with 1 μg of the Streptococcus pneumoniae serotype 9N capsular polysaccharide, and wherein the immunogenic serotype 9N conjugate formed in step (d) has a molecular weight of 500-4,000 kDa, a molecular weight distribution of 15-60% with a K d of 0.3 or below as measured in a CL-4B column, and a CRM 197 /polysaccharide ratio of 0.5-2.5.
9. The method of claim 1 , wherein the degree of oxidation is 5-10.