Method for stabilization of biological molecules

A method for lyophilization of a mixture containing at least one polysaccharide protein conjugate, sucrose at a concentration of approximately 3% w/v and sodium citrate at a concentration of approximately 0.5% w/v, the method comprising i) Freezing up to −45° C. with freezing rate of 5 to 10° C./minute;

• ii) Final freezing temperature between −40 to 50° C.;
• iii) Frozen mass held between −40 to −50° C. for up to 6 hrs;
• iv) Primary drying achieved by increasing the temperature from −45 to −30° C. in 30 min to 2 hr., holding for 8 to 12 hrs, at pressure 75 to 125 μbar;
• primary drying further achieved by increasing the temperature from −27 to −23° C. in 15 min to 1 hr., holding for 4 to 8 hrs, at pressure 50 to 100 μbar;
• primary drying further achieved by increasing the temperature from −5 to 5° C. in 50 min to 1.5 hr., holding for 3 to 4 hrs, at pressure 50 to 100 μbar;
• v) Heating ramp for secondary drying involving change in temperature from −5 to 35° C., achieved in 1 to 2 hrs, at pressure 30 to 70 μbar;
• vi) Secondary drying achieved at a temperature above 25° C., for 5 to 7 hrs, at pressure 30 to 70 μbar;
• vii)) Secondary drying further achieved at a temperature above 40° C., for 1 to 2 hrs, at pressure 30 to 70 μbar;
• and viii) Backfilling of vials with inert gases at pressure 500 to 100 μbar, wherein said mixture contains less than 11% free polysaccharide by weight over a six month period when stored at 40° C.

A method for lyophilization of a mixture containing at least one polysaccharide protein conjugate, sucrose at a concentration of approximately 3% w/v and sodium citrate at a concentration of approximately 0.5% w/v, the method comprising:

• i) Freezing up to −45° C. with freezing rate of 5 to 10° C./minute;
• ii) Final freezing temperature of −45° C.;
• iii) Frozen mass held between −40 to −50° C. for 5 hrs;
• iv) Primary drying achieved by increasing the temperature from −45 to −30° C. in 1 hr., holding for 10 hrs, at pressure of 100 μbar;
• primary drying further achieved by increasing the temperature from −27 to −23° C. in 30 min, holding for 6 hrs, at pressure of 70 μbar;
• primary drying further achieved by increasing the temperature from −5 to 5° C. in 100 min, holding for 3.5 hrs, at pressure of 70 μbar;
• v) Heating ramp for secondary drying involving change in temperature from −5 to 35° C., achieved in 1.5 hrs, at pressure of 50 μbar;
• vi) Secondary drying achieved at a temperature between 20 to 30° C., for 5 to 7 hrs, at pressure of 50 μbar;
• vii) Secondary drying further achieved at a temperature between 40 to 50° C., for 1 to 2 hrs, at pressure 30 to 70 μbar, preferably a 50 μbar;
• and viii) Backfilling of vials with nitrogen at pressure of 700 μbar, wherein said mixture contains less than 11% free polysaccharide by weight over a six month period when stored at 40° C.