Correctly folded etanercept in high purity and excellent yield

Abstract

A mixed mode chromatography method for separating correctly folded from incorrectly folded conformations of a given protein is provided. The method is highly effective in separating correctly folded etanercept from incorrectly folded etanercept and aggregates in commercially attractive yields capable of affording etanercept preparations having very high purity in terms of correctly folded etanercept versus incorrectly folded etanercept. The invention is further directed to protein preparations and formulations comprising correctly folded proteins obtained using the present methods, and methods of treatment using the high purity preparations obtained from the mixed mode method.

What is claimed is: 
     
         1 . A mixed mode chromatography method for separating a correctly folded protein from an incorrectly folded protein, comprising the steps of:
 (a) binding a first protein mixture comprising both correctly folded and incorrectly folded conformations of a given protein to a mixed mode chromatography resin having both ion exchange moieties and hydrophobic moieties;   (b) eluting the correctly folded protein from the mixed mode resin to obtain a second protein mixture comprising a higher proportion of correctly folded protein than the first protein mixture.   
     
     
         2 . The method of  claim 1  wherein the mixed mode chromatography resin is a Capto™ MMC mixed mode chromatography resin. 
     
     
         3 . The method of  claim 1  wherein the mixed mode chromatography resin is a Capto™ Adhere mixed mode chromatography resin. 
     
     
         4 . The method of  claim 1  wherein the correctly and incorrectly folded protein conformations comprise correctly folded and incorrectly folded etanercept. 
     
     
         5 . The method of  claim 4  wherein the incorrectly folded etanercept constitutes less than about 10 wt. %, and preferably less than about 5 wt. % of the eluate obtained in step (b); the correctly folded etanercept constitutes more than about 90 wt % and preferably more than about 95 wt % of the eluate obtained in step (b); and a combined amount of correctly folded and incorrectly folded etanercept constitutes at least about 95 wt. percent and preferably at least about 98 wt. % of the eluate obtained in step (b). 
     
     
         6 . The method of  claim 5  wherein the mixed mode resin is Capto™ MMC and steps (a) and (b) of the method are conducted at a pH of between about 4.5 to about 7.5; and the elution step (b) is carried out by contacting the mixed mode resin with a salt solution. 
     
     
         7 . The method of  claim 5  wherein the mixed mode resin is Capto™ Adhere and steps (a) and (b) are conducted at a pH of about 4.5 to about 8.5; and the elution step (b) is carried out by contacting the mixed mode resin with a salt solution, said solution optionally further comprising arginine. 
     
     
         8 . The method of  claim 6  wherein the salt solution is applied in step (b) through a gradient whereby the salt concentration is gradually increased. 
     
     
         9 . The method of  claim 8  wherein the salt concentration gradient of step (b) causes an increase in the salt concentration of from about 0 to about 1 M. 
     
     
         10 . The method of  claim 6  wherein the salt is selected from sodium chloride and sodium sulfate. 
     
     
         11 . The method of  claim 1  wherein, during step (b), the pH of the solution contacting the resin in step (b) is gradually changed. 
     
     
         12 . The method of  claim 1  wherein the amount of correctly folded protein obtained in the eluate of step (b) is at least about 60 wt % of the amount of protein present in the protein mixture introduced to the resin in step (a). 
     
     
         13 . The method of  claim 12  wherein the amount of correctly folded protein is at least about 70 wt. % of the amount of protein present in the protein mixture introduced to the resin in step (a). 
     
     
         14 . The method of  claim 1  wherein a protein mixture comprising at least 90 wt % correctly folded etanercept and preferably less than about 5 wt. % incorrectly folded etanercept is obtained without performing, or without need to perform any chromatographic separation or purification steps to separate correctly folded from incorrectly folded etanercept, other than the following:
 (1) one or more purification steps, preferably comprising a protein A chromatographic purification step, where such step(s) are employed to purify a harvest cell culture fluid containing etanercept-based proteins, and where such purification step does not result in any appreciable separation of correctly from incorrectly folded etanercept. 
 (2) the mixed mode chromatographic steps (a) and (b) recited in  claim 1 ; and 
 (3) SEC, HIC or other analytical chromatographic steps performed solely for purposes of analysis. 
 
     
     
         15 . The method of  claim 1  wherein the amount of protein present in the eluate of step (b) is determined by UV absorbance at A 280; the amount of correctly folded etanercept in the eluate of step (b) is determined by hydrophobic interaction chromatography; and the combined amount of correctly and incorrectly folded etanercept present in the eluate of step (b) is determined by size exclusion chromatography. 
     
     
         16 . The method of  claim 1  wherein the method is practiced two or more times in the following manner:
 performing a first mixed mode separation (separation #1) by carrying out steps (a) and (b); followed by 
 performing a second mixed mode separation (separation #2): by carrying out steps (a) and (b) again; 
 
       wherein the eluate obtained in step (b) of separation #1 is used as the solution containing a protein mixture in step (a) of separation #2. 
     
     
         17 . The method of  claim 16  wherein the mixed mode resin used in separation #1 is the same as, or different from, the mixed mode resin used in separation #2. 
     
     
         18 . The method of  claim 16  wherein separation #1 and separation #2 are carried out in a manner selected from the following combinations:
 Separation #1 uses CAPTO MMC as the mixed mode chromatography resin and Separation #2 uses CAPTO ADHERE as the mixed mode chromatography resin; 
 - - - 
 Separation #1 uses CAPTO ADHERE as the mixed mode chromatography resin and Separation #2 uses CAPTO MMC as the mixed mode chromatography resin; 
 - - - 
 Separation #1 uses CAPTO MMC as the mixed mode chromatography resin and Separation #2 uses CAPTO MMC as the mixed mode chromatography resin; or 
 - - - 
 Separation #1 uses CAPTO ADHERE as the mixed mode chromatography resin Separation #2 uses CAPTO ADHERE as the mixed mode chromatography resin. 
 
     
     
         19 . The method of  claim 18  wherein separation #1 and separation #2 are carried out in the following manner: Separation #1 uses CAPTO MMC as the mixed mode chromatography resin; and Separation #2 uses CAPTO ADHERE as the mixed mode chromatography resin. 
     
     
         20 . An etanercept-containing protein mixture, or a pharmaceutically acceptable formulation comprising said mixture, obtained by the method of  claim 1  and wherein said protein mixture comprises correctly folded etanercept in amount constituting greater than about 90 wt. % of the protein mixture; and comprising incorrectly folded etanercept in an amount constituting less than about 5 wt % of the protein mixture; and wherein the protein mixture has a combined amount of correctly folded and incorrectly folded etanercept constituting at least about 95 and preferably at least about 98 wt. % of the etanercept-containing protein mixture. 
     
     
         21 . A pharmaceutically acceptable formulation containing highly pure etanercept suitable for administration to a subject requiring treatment for a TNF alpha mediated condition, said formulation containing a protein mixture comprising a major amount of correctly folded etanercept and a minor amount of incorrectly folded etanercept, wherein:
 (i) the incorrectly folded etanercept constitutes less than about 10 wt. %, preferably less than about 8 wt. % and most preferably less than about 5 wt. % of the protein mixture;   (ii) the correctly folded etanercept constitutes more than 90 wt. % and preferably more than about 92 wt % and preferably more than about 95 wt % of the protein mixture; and   (iii) the total amount of correctly folded etanercept and incorrectly folded etanercept (but not including aggregates thereof) constitutes at least 95 and preferably at least 98% by weight of the protein mixture;   
       wherein the formulation further comprises pharmaceutically acceptable inactive ingredients, excipients or carriers rendering the formulation suitable for administration to the subject. 
     
     
         22 . The formulation of  claim 21  wherein the etanercept preparation constitutes about 25 to about 75 mg/ml of the formulation, and the formulation further comprises sucrose, sodium chloride, L-arginine hydrochloride, and sodium phosphate. 
     
     
         23 . A method for producing an etanercept-containing protein mixture having high purity with respect to the amount of correctly folded versus incorrectly folded etanercept present therein, said method comprising the steps of:
 (1) expressing etanercept in a mammalian expression system to obtain a harvest cell culture fluid containing an etanercept-containing protein mixture comprising both correctly folded and incorrectly folded etanercept;   (2) subjecting the harvest cell culture fluid obtained in step 1 to a purification process whereby an etanercept-containing protein mixture is obtained with a reduced amount of, or substantially free of, undesired impurities present in the harvest cell culture fluid produced in step (1);   (3) contacting the etanercept-containing protein mixture obtained in step (2) one or more times with a mixed mode chromatographic resin having both ion exchange moieties and hydrophobic interaction moieties in order to affix proteins contained in the mixture to the resin; and   (4) contacting the resin having protein bound thereon from step 3 with a solution to elute correctly folded etanercept from the mixed mode resin to obtain an eluate comprising an etanercept-containing protein mixture having a higher proportion of correctly folded etanercept versus incorrectly folded etanercept than the etanercept-containing mixture introduced to the resin in step 3;   wherein:
 (i) the amount of protein present in the etanercept-containing protein mixture obtained from purification of step 2 is at least about 80 wt % of the amount of the etanercept-based protein mixture present in the harvest cell culture fluid obtained in step 1. 
 (ii) the combined amount of correctly and incorrectly folded etanercept protein present in the protein mixture eluted in step 4 is at least about 60 wt. % of the amount thereof present in the protein mixture obtained from step 2; 
 (iii) the amount of correctly folded etanercept present in the eluate of step 4 is at least about 30 wt. %, and preferably at least about 35 wt % of the amount of etanercept-containing protein mixture present in the harvest cell culture fluid obtained in step 1; and 
 (iv) said correctly folded etanercept constitutes at least about 90 wt % and preferably at least about 95 wt. % of the eluate obtained in step 4. 
   
     
     
         24 . The method of  claim 23  wherein the mixed mode resin is selected from the group consisting of CAPTO MMC and CAPTO ADHERE. 
     
     
         25 . The method of  claim 24  comprising the following additional steps:
 Step (5): contacting the protein mixture obtained in the eluate of step 4 with a mixed mode chromatographic resin having both ion exchange moieties and hydrophobic interaction moieties in order to affix the proteins contained in the mixture to the resin, and then; 
 step (6) contacting the resin with a solution to elute correctly folded etanercept therefrom to obtain an eluate comprising a protein mixture having a higher proportion of correctly folded versus incorrectly etanercept; 
 
       wherein the mixed mode resin used in said additional steps 5 and 6 is the same as, or different from the mixed mode resin used in steps 3 and 4. 
     
     
         26 . The method of  claim 25  wherein the mixed mode resin used in step (3) and (4) is CAPTO MMC and the resin used in steps (5) and (6) is CAPTO ADHERE. 
     
     
         27 . The method of  claim 1  which excludes use of single mode hydrophobic interaction chromatography as a means of separating correctly folded etanercept from incorrectly folded etanercept, except when performed solely for purposes of analysis. 
     
     
         28 . A method for treating a subject suffering from a TNF mediated disease, comprising the steps of administering to such individual a pharmaceutical formulation containing a protein mixture comprising correctly folded etanercept and incorrectly folded etanercept wherein the amount of incorrectly folded etanercept in the protein mixture is less than about 5 wt % of said mixture. 
     
     
         29 . The method of  claim 28  wherein the amount of incorrectly folded etanercept in the protein mixture is less than about 3 wt % of said mixture and the amount of correctly folded etanercept in the mixture is greater than about 95 wt % of the mixture. 
     
     
         30 . A method for separating correctly folded etanercept from incorrectly folded etanercept, wherein chromatographic means are used to achieve such separation, and wherein the chromatographic means consist solely or essentially of mixed mode chromatography in which a mixture comprising correctly folded and incorrectly folded etanercept is contacted with a mixed mode chromatographic resin having ion exchange and hydrophobic moieties, and then eluted therefrom, to obtain an eluate comprising at least about 85 and preferably at least about 90, and most preferably at least about 95 wt % correctly folded etanercept. 
     
     
         31 . The method of  claim 30  wherein the mixed mode resin is selected from CAPTO MMC and CAPTO ADHERE; the elution is conducted with a salt solution, optionally applied using a gradient of increasing salt concentration; the pH of the salt solution is in the range of about 4 to about 8.5, applied optionally in a gradient in which the pH is gradually increased or decreased; and wherein the eluate is obtained from the resin over a period of time, and eluate collected early in said period is free or essentially free of incorrectly folded etanercept.