Formulation processes for pharmaceutical compositions of recombinant .beta. -
AssigneeSchering Corporation
InventorsZe'Ev Shaked, Tracy Stewart, Susan Hershenson, James W. Thomson, Terrance Taforo, Jody Thomson
Life of this patent
Estimating the term…
Abstract
Stable pharmaceutical compositions suitable for parenteral administration to animals or humans are prepared comprising a therapeutically effective amount of a recombinant IFN-.beta. protein dissolved in an inert carrier medium comprising as solubilizer/stabilizer(s) one or more biocompatible non-ionic polymeric detergents or a combination of one or more biocompatible non-ionic polymeric detergents with an additional solubilizing and/or stabilizing agent, such as sodium dodecyl sulfate or glycerol. The compositions are in liquid form or lyophilized.
Claims7
A method of screening for one or more biocompatible non-ionic polymeric detergents or for combinations of one or more of said detergents with another solubilizing or stabilizing agent capable of solubilizing and stabilizing pharmaceutical compositions of recombinant interferon-.beta. (IFN-.beta.), comprising the steps of:
- (a) passing extracted, purified IFN-.beta. in sodium dodecyl sulfate (SDS) on a desalting column equilibrated in sodium laurate in an elution buffer at pH 9.0-10.0 to form an eluate;
- (b) lowering the pH of the eluate to about pH 2-3.3;
- (c) centrifuging and filtering the eluate to remove the precipitated sodium laurate;
- (d) adding to the filtrate an effective amount of one or more of said detergents or said combination of one or more of said detergents with another solubilizing or stabilizing agent;
- (e) adjusting the pH of the filtrate to 3.5 to 9.5;
- and
- (f) allowing said filtrate to stand for about 24 hours at pH 3.5 to 9.5.
A method of preparing stable, pharmaceutical compositions of recombinant interferon-.beta. (IFN-.beta.) protein comprising the steps of:
- (a) extracting the IFN-.beta. from the disruptate of a host organism transformed to produce the protein;
- (b) purifying the IFN-.beta. using as the last purification step a desalting step at a pH range of about 8.5 to 10 employing an elution buffer containing a fatty acid salt having a carbon chain containing from about 10 to about 12 carbons to form a desalted pool;
- (c) lowering the pH of the desalted pool to a pH of about 2 to 4 thereby precipitating the fatty acid salt;
- (d) removing the precipitated salt from the pool by centrifugation and filtration;
- (e) adding to the desalted pool an effective amount of one or more non-ionic biocompatible polymeric detergents, or a combination of one or more biocompatible non-ionic polymeric detergents and another solubilizing or stabilizing agent, in effective amounts to
- stabilize and solubilize the IFN-.beta.;
- (f) adjusting the pH of the pool to a range of between 4.0 and 8.0;
- (g) adding an effective amount of a bulking/stabilizing agent to the pool;
- and
- (h) lyophilizing the formulation.
A method of preparing stable, pharmaceutical compositions of recombinant interferon-.beta. (IFN-.beta.) protein comprising the steps of:
- (a) isolating refractile bodies containing the IFN-.beta. from a host organism transformed to produce said IFN-.beta.;
- (b) solubilizing said refractile bodies by employing sodium laurate;
- (c) extracting and purifying said IFN-.beta. from the solubilized refractile material employing sodium laurate as the primary solubilizing agent;
- (d) lowering the pH of the purified IFN-.beta. to a pH from about 2 to about 4;
- (e) centrifuging and filtering the purified IFN-.beta. solution to remove the sodium laurate precipitate to create an IFN-.beta. pool;
- (f) desalting the IFN-.beta. pool at a pH from about 2 to 4;
- (g) adding to the IFN-.beta. protein solution an effective amount of one or more non-ionic biocompatible polymeric detergents, or of a combination of a non-ionic biocompatible polymeric detergent and another solubilizing or stabilizing agent to stabilize and solubilize the IFN-.beta.;
- (h) adjusting the pH of the IFN-.beta. solution to a pH range of 3.5 to 9.5;
- (i) adding to the solution an effective amount of a bulking/stabilizing agent;
- and
- (j) lyophilizing the formulation.
A method of extracting recombinant interferon-.beta. (IFN-.beta.) from a bacterial host transformed to produce it and then purifying and formulating said IFN-.beta. comprising the steps of:
- (a) growing the transformed bacterial hosts in an appropriate fermentation medium;
- (b) concentrating the bacterial host in the fermentation medium;
- (c) disrupting the cell wall and cell membrane of the bacterial host;
- (d) removing greater than 99% by weight of the salts from said disruptate by diafiltration or centrifugation;
- (e) redisrupting the desalted disruptate;
- (f) adding a material to the disruptate to increase the density or viscosity of, or to create a density or viscosity gradient in, the liquid within the disruptate;
- (g) separating the refractile material from the cellular debris by high-speed centrifugation;
- (h) solubilizing the refractile material in an aqueous buffer containing a reducing agent;
- (i) extracting the solubilized refractile material with 2-butanol or 2-methyl-2-butanol;
- (j) isolating said refractile material from the extractant;
- (k) solubilizing the resulting refractile material, which contains IFN-.beta., with an aqueous solution of sodium dodecyl sulfate at an IFN-.beta. to sodium dodecyl sulfate ratio of about 1:3 to form a solution;
- (l) adjusting the pH of the solution to about 9.5 and reducing the solubilized IFN-.beta. with dithiothreitol;
- (m) purifying the reduced IFN-.beta. by chromatography;
- (n) oxidizing the IFN-.beta. from step (m);
- (o) further purifying the oxidized IFN-.beta. by gel chromatography and collecting the eluate containing the purified IFN-.beta.;
- (p) desalting the purified IFN-.beta. eluate in a desalting column equilibrated and run in sodium laurate at pH 9.0 to 9.8;
- (q) lowering the pH of the eluate to pH 3.0;
- (r) centrifuging and filtering the solution to remove the precipitate;
- (s) adding to the filtrate an effective amount of one or more non-ionic biocompatible polymeric detergents or a combination of one or more of said detergents and another solubilizing or stabilizing agent, to solubilize and stabilize the IFN-.beta.;
- (t) adjusting the pH of the solution to 4.0 to 9.5;
- (u) adding an appropriate bulking/stabilizing agent in a concentration of from about 0.25% to about 10%;
- and
- (v) lyophilizing the IFN-.beta. solution.
Dependencies are inferred from the claim text.