FORM 2
THE PATENTS ACT, 1970
PROVISIONAL SPECIFICATION
Section 10
"Protein-Polysaccharide Conjugation"

Serum Institute of India Ltd., an Indian company, of 212/2, Off Soli Poonawalla Road, Hadapsar, Pune 411028 Maharashtra India.
The following specification particularly describes the nature of this invention:

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Title: Protein-Polysaccharide conjugation Objective:
To provide immunogenic conjugate/complex of protein polysaccharide by dry heating and removal of water.
Components of the invention: Protein, Polysaccharide, Maillard reaction, and Protein-polysaccharide conjugates.
Brief Description:
The conjugation reactions disclosed in the prior art involve use of harsh chemicals, cross linking agents, spacers as well as catalysts. Also the existing methods of conjugation have exhibited variations with respect to conjugate charachteristies and the yield respectively. These reactions need purification to remove the unreacted chemicals.
The instant invention overcomes the demerits of prior art by employing Maillard reaction, which is an initial condensation reaction accelerated by heat that involves interaction between carbonyl group of reducing sugar and an available amino group of the amino acid, producing N-substituted glycosylamine and water.
The first embodiment of the instant invention provides an alternative method of conjugation of polysaccharide and carrier protein, wherein the protein-polysaccharide conjugate/complex is produced by Maillard reaction due to dry heating, resulting in removal of water from the reaction mixture.
The second embodiment of the instant invention is a method of Maillard type conjugation that provides an immunogenic protein polysaccharide conjugate with yields more than 5%, approaching 100%, preferably 40%.
Another embodiment of the instant invention is a method of Maillard type conjugation that provides an immunogenic protein polysaccharide conjugate, wherein the method does not employ heating under vacuum but at different relative humidity conditions.
The carrier proteins of the instant inventions are, but not limited to, diphtheria toxoid, CDC peptide, tetanus toxoid.

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CRM197 or their derivatives e.g. mutants or other carrier proteins.
The Polysaccharide of the instant invention are, but not limited to, polysaccharide or their fragments selected from groups comprising cellular antigens such as carbohydrates obtained from a group of microorganisms comprising Streptococcus pneumoniae , Haemophilus influenzae type b, Neisseria meningitidis or E. coli and Combinations thereof.
Typically, the process of instant invention comprises:
a) Addition of native or activated polysaccharide or its fragment of molecular weight 5kDa to lOOOkDa to a native or activated carrier protein in ratio of 5:1 to 1:5 respectively; and preferably 1;1.
b) The pH of the above reaction mixture is the range of about 4 to 11.
c) Subjecting the reaction mixture to freezing followed by lyophillination resulting in moisture level of less than 1%.
d) The vial containing frozen dry mass is subjected to heating at a temperature below 100 °C, preferably in the range 60 to 85 °C, more preferably at 60 °C ; for a period ranging from 1hr to 15 days ;with 4 0 to 80% relative humidity;to obtain the final product.
Advantages of the invention:
1. The instant invention of conjugation is non-catalytic as well as non-chemical conjugation/complexing resulting in a stable product.
2. The process requires less or no purification steps after conjugation/complexing.
3. The method provides immunogenic protein-polysaccharide conjugate with reproducible yield.
Dated this 08th day of October 2008
Of Anand And Anand Advocates Agents for the Applicant