Concentrated protein lyophilates, methods, and uses

Abstract

The invention provides, among other things, lyophilized compositions of high surface area that comprise a protein and that reconstitute quickly and efficiently to solution of high protein concentration with minimal formation, if any, of foam, effervescence, bubbles, turbidity, or particulates that might be deleterious. The invention also provides, among other things, methods for making the lyophilized compositions. The invention in additional aspects also provides Raman Imaging Spectrographic methods for real time analyses of polymorphs in a sample using PLS algorithms. By way of particular example, the use of the method for the analysis of mannitol polymorphs is described, and the use of the analysis to determine optimum compositions and lyophilization methods for producing lyophilates of pharmaceutical proteins having a predefined distribution of mannitol polymorphs and having the aforementioned reconstitution properties is also described.

1 . A method for producing a lyophilized protein, comprising:
 (a) annealing a composition comprising a protein under reduced pressure at a first annealing temperature;   (b) drying the annealed composition under further reduced pressure at a first drying temperature for a time effective to reduce the moisture content to 75% or less; and   (c) further drying the composition under the further reduced temperature at a second drying temperature for a time effective to reduce the moisture content of the composition to less than 3.5%, thereby producing the lyophilized protein composition.   
     
     
         2 . A method according to  claim 1 , comprising:
 (a) reducing the temperature of a composition comprising a protein and a bulking agent to a first temperature, of −40° C. or less;   (b) raising the temperature of the composition to a second temperature, between −25° C. and 0° C., and maintaining the compositions at that temperature for a period of time;   (c) reducing the temperature of the composition to a third temperature, of −40 or less;   (d) raising the temperature of the composition to a fourth temperature, of or above −10° C. and holding the composition at that temperature for a period of time;   (e) raising the temperature of the composition to a fifth temperature higher than the fourth temperature and holding the compositions at that temperature until the moisture content of the composition is less than 3.5%, thereby producing the lyophilate.   
     
     
         3 . A method according to  claim 1 , comprising:
 (a) applying a vacuum to a composition comprising water, a protein, and a lyoprotectant or bulking agent;   (b) cooling the composition under partial vacuum to or below a first reduced temperature;   (c) maintaining the composition under partial vacuum at or below the first reduced temperature until it is equilibrated thereto and frozen;   (d) raising the temperature of the composition under partial vacuum to an annealing temperature;   (e) maintaining the composition at the annealing temperature under partial vacuum for a time effective for programmed crystallization of more than 60% of the lyoprotectant or bulking agent in the composition;   (f) cooling the composition under partial vacuum to a second reduced temperature, keeping the composition below the glass transition temperature throughout the cooling process;   (g) maintaining the composition at the second reduced temperature at least until it is equilibrated thereto;   (h) raising the temperature of the composition under vacuum to a first drying temperature less than the glass transition temperature, keeping the composition below the glass transition temperature throughout the heating process;   (i) maintaining the composition under vacuum at the first drying temperature and below the glass transition temperature until the water content of the composition is 10% or less;   (j) raising the temperature of the composition under vacuum to a second drying temperature, keeping the composition below the glass transition temperature throughout the heating process;   (k) maintaining the composition at the second drying temperature until the water content of the composition is 3% or less, maintaining the composition below the glass transition temperature throughout the process, thereby producing the lyophilate.   
     
     
         4 . A method according to  claim 1 , comprising:
 (a) reducing the temperature of a composition comprising a protein and a bulking agent to a first minimum temperature of −40° C. or less;   (b) raising the temperature of the composition to an annealing temperature between −25° C. and 0° C.;   (c) holding the composition at the annealing temperature for one hour or more;   (d) reducing the temperature of the composition to a second minimum temperature of −40° C. or less;   (e) raising the temperature of the composition to a first drying temperature of or above −10° C.;   (f) holding the composition at the first drying temperature for five hours or more;   (g) raising the temperature of the composition to a second drying temperature;   (h) holding the composition at the second drying temperature for two hours or more until the composition is dry, thereby producing the protein lyophilate.   
     
     
         5 . A method according to  claim 1 , comprising:
 (a) reducing the temperature of a composition comprising a protein and a bulking agent at between 0.05 and 1.0° C. per minute to a first minimum temperature of −40° C. or less;   (b) raising the temperature of the composition to an annealing temperature between −25° C. and 0° C.;   (c) holding the composition at the annealing temperature for more then 1 hour;   (d) reducing the temperature of the composition at a rate between 0.05 and 1.0° C. per minute to a second minimum temperature of 40° C. or less;   (e) raising the temperature of the composition at a rate between 0.05 and 1.0 per minute to a first drying temperature of or above −10° C.;   (f) holding the composition at the first drying temperature for 5 or more hours;   (g) raising the temperature of the composition at a rate between 0.05 and 1.0° C. per minute to a second drying temperature;   (h) holding the composition at the second drying temperature for at least two hours until the composition is dry, thereby producing the protein lyophilate.   
     
     
         6 . A method according to  claim 1 , wherein the composition for lyophilization is degassed prior to the initial freezing step. 
     
     
         7 . A method according to  claim 1 , wherein the composition for lyophilization is cooled to 5° C. and held at that temperature until it is equilibrated thereto prior to bringing the composition to the annealing temperature. 
     
     
         8 . A method for producing, a lyophilized composition of an Fc fusion protein, comprising:
 (a) formulating a composition comprising an Fc fusion protein, a buffering agent, a bulking agent, and a protectant in solution, degassing the composition, and then equilibrating the composition to a temperature approximately five degrees above the freezing point;   (b) cooling the composition under partial vacuum above 250 mTorr to a first freezing temperature at or between −60 and −40° C.;   (c) maintaining the composition under partial vacuum at or below the first reduced temperature until it is completely frozen;   (d) raising the temperature of the composition under partial vacuum to a temperature at or between −10 to 15° C.;   (e) maintaining the composition at the temperature in (d) under partial vacuum for a time effective for crystallization of more than 60% of the bulking agent in the composition;   (f) cooling the composition under partial vacuum to a second reduced temperature at or between −40 and −60° C.;   (g) maintaining the composition at the second reduced temperature under partial vacuum for a period of time;   (h) under vacuum less than 250 mTorr, raising the temperature of the composition to a first drying temperature, keeping the composition below the glass transition temperature throughout the heating process;   (i) maintaining the composition under vacuum at the first drying temperature always below the glass transition temperature until the water content of the composition is 10% or less;   (j) raising the temperature of the composition wider vacuum to a second drying temperature, keeping the composition below the glass transition temperature throughout the heating process;   (k) maintaining the composition at the second drying temperature until the water content of the composition is 3% or less, maintaining the composition below the glass transition temperature throughout the process; thereby producing the lyophilate.   
     
     
         9 . A method for producing a protein lyophilate, comprising:
 (a) equilibrating an approximately 100 mg/ml sample comprising the protein for lyophilization to approximately 4° C.;   (b) cooling the sample to approximately −50° C. at approximately—0.5° C./min and then holding the sample at approximately −50° C. for approximately 120 minutes;   (c) warming the sample to approximately −12° C. at approximately 1.3° C./min and then holding the sample at approximately −12° C. for approximately 360 minutes;   (d) cooling the sample to approximately −50° C. at approximately 0.6° C./min and then holding the sample at approximately −50° C. for approximately 120 minutes;   (e) adjusting the ambient pressure on the sample to approximately 100 mTorr;   (f) maintaining the pressure at approximately 100 mTorr, warming the sample to approximately −25° C. at approximately 0.2° C./min and then holding the sample at approximately −25° C. for approximately 1600 minutes;   (g) warming the sample to approximately −25° C. at approximately 0.03° C./min, reducing the pressure to approximately 50 mTorr and then holding the sample at approximately −25° C. and approximately 50 mTorr for approximately 800 minutes.   
     
     
         10 . A method according to  claim 8 , wherein the protein comprises an Fc region of an antibody or a variant, derivative, fragment, or mimetic thereof. 
     
     
         11 . A method according to  claim 8 , wherein the protein comprises an Fc region of an antibody. 
     
     
         12 . A method according to  claim 8 , wherein, the protein is Fc-IL-1ra.