Methods of producing long acting ctp-modified polypeptides

Abstract

Disclosed herein is a method for manufacturing a recombinant polypeptide of interest modified by a CTP extension in a mammalian cells culture system.

1 . A method of manufacturing a human CTP-modified polypeptide of interest, the method comprising the steps of:
 (a) stably transfecting a predetermined number of cells with an expression vector comprising a coding portion encoding said CTP-modified polypeptide of interest;
 (i) wherein said transfected cells express and secrete said CTP-modified polypeptide of interest; 
   (b) obtaining cell clones that overexpress said CTP-modified polypeptide of interest;   (c) expanding said clones in solution to a predetermined scale;   (d) harvesting said solution containing said clones;   (e) filtering said solution containing said clones to obtain a clarified harvest solution; and,   (f) purifying said clarified harvest solution to obtain a purified protein solution having a desired concentration of a CTP-modified polypeptide of interest;   thereby manufacturing the CTP-modified polypeptide of interest.   
     
     
         2 . The method of  claim 1 , wherein the purity of the CTP-modified polypeptide of interest is at least 70%. 
     
     
         3 . The method of  claim 1 , wherein said CTP-modified polypeptide of interest is highly glycosylated. 
     
     
         4 . The method of  claim 3 , wherein the glycosylation pattern of each CTP comprises at least glycosylation at 4 O-linked glycosylation sites. 
     
     
         5 . The method of  claim 1 , wherein said CTP-modified polypeptide of interest is highly sialylated. 
     
     
         6 . The method of  claim 1 , wherein when said polypeptide of interest is an erythropoietin (EPO), an interferon (IFN), or a glucagon-like peptide 1 (GLP-1), said CTP-modified polypeptide of interest polypeptide consists of two CTPs attached to the carboxy terminus of said polypeptide of interest, and one CTP attached to the amino terminus of said polypeptide of interest. 
     
     
         7 . The method of  claim 1 , wherein when said polypeptide of interest is a coagulation factor, a coagulation Factor VII (FVII), an activated coagulation Factor VII (FVIIa) or a coagulation Factor IX (FIX), said CTP-modified polypeptide of interest polypeptide consists of one to five CTPs attached to the carboxy terminus of said polypeptide of interest. 
     
     
         8 . The method of  claim 7 , wherein said CTP-modified polypeptide of interest polypeptide consists of three CTPs attached to the carboxy terminus of said polypeptide of interest. 
     
     
         9 . The method of  claim 1 , wherein when said polypeptide of interest is a dual GLP-1/Glucagon receptor agonist or OXM (OXM), said CTP-modified polypeptide of interest polypeptide consists of one to five chorionic CTPs attached to the carboxy terminus of said polypeptide of interest and/or one to five CTPs attached to the amino terminus of said polypeptide of interest. 
     
     
         10 . The method of  claim 1 , wherein in said step (a)(i) the secreted CTP-modified polypeptide of interest lack a signal peptide. 
     
     
         11 . The method of  claim 1 , wherein said step (c) comprises expanding clones from a working cell bank (WCB) that optimally expresses and secretes said CTP-modified polypeptide of interest. 
     
     
         12 . The method of  claim 1 , wherein said step (c) comprises expanding clones obtained from a master cell bank (MCB) that optimally expresses and secretes said CTP-modified polypeptide of interest. 
     
     
         13 . The method of  claim 1 , wherein said method of manufacturing is an animal-free process. 
     
     
         14 . The method of  claim 1 , wherein at step (c) said CTP-modified polypeptide of interest is expressed at a level of at least 30 mg/ml. 
     
     
         15 . The method of  claim 1 , wherein at step (c) expanding said clones in solution comprises expanding said clones in solution through a series of sub-cultivating steps up to production bioreactor level. 
     
     
         16 . The method of  claim 15 , wherein said bioreactor comprises a disposable bioreactor, a stainless steel bioreactor, a fed-batch mode bioreactor, a batch mode bioreactor, a repeated batch mode bioreactor, or a perfusion mode bioreactor, or any combination thereof. 
     
     
         17 . The method of  claim 1 , wherein at step (f) at least 60% of the purified CTP-modified polypeptides of interest in said clarified harvest comprises a high glycosylation form of said CTP-modified polypeptide. 
     
     
         18 . The method of  claim 1 , wherein said purification (step f) comprises sequentially performing the following steps comprising:
 (g) concentrating, diafiltering and purifying said clarified harvest solution;
 i. wherein said concentration, diafiltration and purifying is accomplished by sequentially passing said clarified harvest solution through an anion exchange column and a hydrophobic interaction column; 
   (h) obtaining said clarified harvest obtained following step (g) and inactivating viruses present in said clarified harvest by incubating in a solution toxic to said viruses;   (i) obtaining said clarified harvest solution from step (h) and concentrating, diafiltering, and purifying said clarified harvest solution,
 i. wherein said concentration, diafiltration and purifying is followed by sequentially passing said clarified harvest solution through a multimodal or mixed-mode protein chromatography column and a cation exchange column; 
   (j) obtaining said clarified harvest solution following step (i) and physically removing said clarified harvest solution from viruses by nanofiltration; and   (k) obtaining said clarified harvest solution following step (j) and concentrating and diafiltering said clarified harvest solution to arrive at a maximally purified clarified harvest containing said highly glycosylated form of CTP-modified polypeptide of interest.   
     
     
         19 . The method of  claim 1 , wherein said method achieves at least a 20% recovery rate of highly glycosylated CTP-modified polypeptide of interest. 
     
     
         20 . The method of  claim 18 , wherein said viral clearance shows a viral log reduction factor (LRF) of more than 12. 
     
     
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