THE PATENTS ACT, 1970
PROVISIONAL SPECIFICATION
Section 10
"Process for Preparation of Polysaccharide-carrier Protein Conjugate Vaccine"
Serum Institute of India Ltd. a Corporation organized and existing under the laws of India, of 212/2, Off Soli Poonawalla Road, Hadapsar, Pune 411 028 Maharashtra India.

The following specification particularly describes the nature of this invention:



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Provisional Patent Application
Title: Process for preparation of Polysaccharide-carrier protein conjugate vaccine
Objective:
To provide stable polysaccharide-carrier protein conjugate lyophilized vaccine substantially free of un-conjugated polysaccharide by lyophilizing the conjugate in the presence of one or more carbohydrates.
Brief Description:
Polysaccharide-carrier protein conjugates are known to release free polysaccharide after conjugation while further processing, lyophilization or storage in liquid as well as solid formulations. The instant invention provides a novel composition as well as a short and efficient method of lyophilization to afford a stable, readily soluble and aesthetically pleasing vaccine formulation.
The instant invention can be employed to produce vaccine against Heamophilus influenza, Meningococcal or Pneumococcal infections.
Composition I containing Mannitol and Sucrose
HiB PRP-TT Conjugate 20 to 240 (mg/ml (1 to 10 dose) , Sucrose 17.1 mg/ml, Mannitol 68.5 mg/ml, 5 to 25 mM Tris component, pH adjusted to 6-7, 0.5 ml fill volume.
The type-I tubular glass vials were used having 13 mm neck, 16.5 mm OD, 50 mm Height and 13 mm gray slotted rubber stoppers.
Composition II containing Mannitol and Trehalose
HiB PRP-TT Conjugate 20 to 240 mg/ ml (1 to 10 dose), Trehalose 17.1 mg/ml, Mannitol 68.5 mg/ml, 5 to 25 mM Tris component, pH adjusted to 6-7, 0.5 ml fill volume. The type-I tubular glass vials were used having 13 mm neck, 16.5 mm OD, 50 mm Height and 13 mm gray slotted rubber stoppers.
Composition III containing Trehalose

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PRP-TT Conjugate 20 to 240 mg/ ml (1 to 10 dose), Trehalose 80 to 100 mg/ml, 5-25 mM Tris component, pH adjusted to 6-7, 0.5 ml fill volume. The type-I tubular glass vials were used having 13 mm neck, 16.5 mm OD, 50 mm Ht and 13 mm gray slotted rubber stoppers.
Lyophilization cycle specifications for Formulation I and II are as outlined -
Freezing rate: 0-4°C/minute, preferred l-2°C/minute
Final freezing temperature: -30 to -60°C, preferred -50° C
Frozen mass was hold between -40 to -50°C for upto 0 to 5
hrs, preferred 3 hrs.
Condenser temperature: below -50°C, preferred -70 to -90 °C
Primary drying: below -20°C, preferred -45 to -20°C, 5-10 hrs, preferred 6-7 hrs, pressure 10 to 400 mbar, preferred 100 mbar.
Heating ramp for secondary drying: -20 to 40°C, 1 to 5 hrs preferred 2 hrs, Pressure 10 to 400 mbar, preferred 100 mbar.
Secondary Drying: above 35°C, preferred 35-45°C, 10-12 hrs, Pressure 10 to 200 mbar preferred 50 mbar.
Total cycle duration of less than 40 hrs, preferred less than 30, most preferred about 25.
Lyophilization cycle specifications for Composition III are as outlined -
Freezing rate: 0-4°C/minute, preferred l-2°C/minute
Final freezing temperature: below -20°C preferred -30 to -
40°C
Frozen mass was hold between less than -25°C preferred -25
to -40°C for 0 to 5 hrs, preferred 3 hrs.
Condenser temperature: below -50°C, -70 to -90°C
Primary drying: below -20°C, preferred -40 to -20°C over 0 to 20 hrs preferred 8-12 hrs, Pressure 10 to 400 mbar, preferred Pressure 100 mbar.

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Heating ramp for secondary drying: above -20°C preferred -20 to 30°C, 1 to 5 hrs preferred 2.5 hrs, Pressure 10 to 400 mbar, preferred Pressure 100 mbar.
Secondary Drying: above 15°C, preferred 15 to 40°C, most preferred 30 to 35°C, 2 to 15 hours preferred 6-8 hrs, Pressure 0 to 400 mbar preferred 25- 90 mbar.
Total cycle duration of less than 40 hrs, preferred less than 30, most preferred about 25.
Advantages of the instant invention
1. Short duration lyophilization cycle (less than 40 hrs)
2. Efficient lyophilization (residual moisture less than
2.5%)
3. Uniform lyophilized product
4. Release/movable pellet type of cake structure.
5. High solubility and aesthetically pleasant appeal
6. Intact conjugate containing free polysaccharide less
than 20% w/w of the polysaccharide used for conjugation

Dated this 07th day of August 2007


Of Anand And Anand Advocates
Attorney for the Applicant