Process For the Preparation of Glycosylated Interferon Beta

Abstract

The present invention relates to a process for the production of interferon beta, and to an interferon beta composition having a unique glycosylation pattern.

1 - 32 . (canceled) 
     
     
         33 . A process for the manufacturing of glycosylated recombinant human interferon beta, comprising a step of culturing a human interferon beta producing cell in a serum-free medium, the serum-free medium comprising:
 a) about 10 to about 30 mM HEPES;   b) about 0.5 to about 3 mM Proline; and   c) about 5500 to about 7000 mg/L sodium chloride.   
     
     
         34 . The process according to  claim 33 , the serum-free medium further comprising about 10 to about 20 mg/L Phenol Red. 
     
     
         35 . The process according to  claim 33 , said method comprising a growth phase I, a growth phase II and a production phase, wherein growth phase I is carried out at about 37° C., growth phase II is carried out at about 35° C., and the production phase is carried out at about 33° C. 
     
     
         36 . The process according to  claim 35 , wherein the process is a perfusion process with a dilution rate ranging from about 1 to about 10. 
     
     
         37 . The process according to  claim 36 , wherein the dilution rate is increased within the first two to three weeks of cell culture from an initial value of about 1 to 2 per day to a value of about 7 to 10 per day. 
     
     
         38 . The process according to  claim 33 , further comprising:
 a) subjecting the medium containing human interferon beta to affinity chromatography and eluting said human interferon beta;   b) subjecting the human interferon beta containing eluate to cation exchange chromatography and eluting said human interferon beta; and   c) subjecting the eluate of the cation exchange chromatography to hydrophobic chromatography by RP-HPLC and eluting said human interferon beta.   
     
     
         39 . The process according to  claim 38 , comprising, before step (a), clarifying of the medium by filtration. 
     
     
         40 . The process according to  claim 39 , further comprising the steps of:
 d) performing ultrafiltration and dialysis;   e) subjecting the dialysate to size exclusion chromatography; and   f) subjecting the eluate of the size exclusion chromatography to microfiltration.   
     
     
         41 . The process according to  claim 38 , wherein step (a) is carried out on Blue Sepharose and step (b) is carried out on Carboxymethyl Sepharose. 
     
     
         42 . The process according to  claim 33 , wherein said human interferon beta producing cell comprises:
 a) a human interferon beta coding sequence functionally linked to a SV40 T Ag early polyadenylation region, wherein the nucleic acid does not comprise the human interferon beta polyadenylation signal;   b) a human interferon beta coding sequence functionally linked to a SV40 T Ag early polyadenylation region, wherein the nucleic acid does not comprise the human interferon beta polyadenylation signal and wherein said nucleic acid does not comprise the human interferon beta 3′ UTR;   c) a nucleic acid comprising a SV40 promoter/enhancer functionally linked to a human interferon beta coding sequence, wherein the human interferon beta coding sequence is functionally linked to the SV40 T Ag early polyadenylation region and the nucleic acid does not comprise the human interferon beta polyadenylation signal or the human interferon beta 3′ UTR;   d) a nucleic acid according to a), b) or c), wherein said nucleic acid further comprises a mouse DHFR gene;   e) a nucleic acid according to d), wherein said mouse DHFR gene is functionally linked to a SV40 T Ag polyA-containing early polyadenylation region; or   f) a nucleic acid according to e), further comprising a SV40 promoter/enhancer functionally linked to the mouse DHFR gene.   
     
     
         43 . A process for the purification of recombinant human interferon beta from a fluid, comprising the steps of:
 a) subjecting the fluid to affinity chromatography;   b) subjecting the eluate of the affinity chromatography to cation exchange chromatography; and   c) subjecting the eluate of the cation exchange chromatography to hydrophobic chromatography by RP-HPLC.   
     
     
         44 . The process according to  claim 43 , comprising, before step (a), clarifying the fluid by filtration. 
     
     
         45 . The process according to  claim 44 , further comprising the steps of:
 d) performing ultrafiltration and dialysis to form a dialysate;   e) subjecting the dialysate to size exclusion chromatography; and   f) subjecting the eluate of the size exclusion chromatography to microfiltration.   
     
     
         46 . The process according to  claim 43 , wherein step (a) is carried out on Blue Sepharose and step (b) is carried out on Carboxymethyl Sepharose. 
     
     
         47 . The process according to  claim 43 , further comprising the step of formulating the purified interferon beta into a pharmaceutical composition, optionally together with a pharmaceutically acceptable carrier. 
     
     
         48 . A glycosylated recombinant interferon beta composition, obtainable by a process according to  claim 33 . 
     
     
         49 . The interferon beta composition according to  claim 48 , comprising an oligosaccharide structure comprising two or three fucose saccharides. 
     
     
         50 . The interferon beta composition according to  claim 48 , wherein said oligosaccharide structure comprises a disialyl biantennary trifucosylated glycan (Neu 2 Ac.Hex5.HexNAc 4 .Fuc 3 ). 
     
     
         51 . The interferon beta composition according to  claim 48 , comprising one or more of the following oligosaccharide structures:
 a) a non sialylated biantennary structure (Hex 5 .HexNAc 4 .Fuc);   b) a disialylated triantennary structure or disialylated biantennary with N-acetyl lactosamine repeat structures (NeuAc2.Hex6.HexNAc5.Fuc);   c) a trisialylated triantennary structure (NeuAc 3 .Hex6.HexNAc 5 .Fuc);   d) a trisialylated triantennary structure with N-acetyl lactosamine repeat structures or trisialylated tetrantennary (NeuAc 3 .Hex 7 .HexNAc 6 .Fuc); or   e) a mono sialylated and disialylated biantennary structure with two fucose units (NeuAc.Hex 5 .HexNAc 4 .Fuc 2 ,NeuAc 2 .Hex 5 .HexNAc 4 .Fuc 2 ).   
     
     
         52 . The interferon beta composition according to  claim 48 , characterized by a sialylation profile comprising about 1 to about 5% of unsialylated N-glycans, about 5 to about 25% of mono-sialylated glycans, about 55 to about 75% of di-sialylated N-glycans, and about 10 to about 25% of tri-sialylated N-glycans. 
     
     
         53 . A method of treating a disease selected from multiple sclerosis, viral diseases or cancer comprising the administration of a composition comprising the interferon β according to  claim 48  to an individual with said disease. 
     
     
         54 . The method according to  claim 53 , wherein said multiple sclerosis is selected from the group consisting of relapsing, non-relapsing and early onset multiple sclerosis. 
     
     
         55 . An article of manufacture comprising packaging material and a therapeutically effective amount of an isolated, purified recombinant interferon beta according to  claim 48 , wherein said packaging material comprises a label or package insert indicating that said recombinant human interferon beta can be administered to a human for treatment thereof. 
     
     
         56 . A composition of matter comprising:
 a) a nucleic acid comprising:
 i) a human interferon beta coding sequence functionally linked to a SV40 T Ag early polyadenylation region, wherein the nucleic acid does not comprise the human interferon beta polyadenylation signal; 
 ii) a human interferon beta coding sequence functionally linked to a SV40 T Ag early polyadenylation region, wherein the nucleic acid does not comprise the human interferon beta polyadenylation signal and wherein said nucleic acid does not comprise the human interferon beta 3′ UTR; 
 iii) a nucleic acid comprising a SV40 promoter/enhancer functionally linked to a human interferon beta coding sequence, wherein the human interferon beta coding sequence is functionally linked to the SV40 T Ag early polyadenylation region and the nucleic acid does not comprise the human interferon beta polyadenylation signal or the interferon beta 3′ UTR; 
 iv) a nucleic acid according to i), ii) or iii), wherein said nucleic acid further comprises a mouse DHFR gene; 
 v) a nucleic acid according to iv), wherein said mouse DHFR gene is functionally linked to a SV40 T Ag polyA-containing early polyadenylation region; or 
 vi) a nucleic acid according to v), further comprising a SV40 promoter/enhancer functionally linked to the mouse DHFR gene; 
   b) an expression vector comprising:
 i) a human interferon beta coding sequence functionally linked to a SV40 T Ag early polyadenylation region, wherein the nucleic acid does not comprise the human interferon beta polyadenylation signal; 
 ii) a human interferon beta coding sequence functionally linked to a SV40 T Ag early polyadenylation region, wherein the nucleic acid does not comprise the human interferon beta polyadenylation signal and wherein said nucleic acid does not comprise the human interferon beta 3′ UTR; 
 iii) a nucleic acid comprising a SV40 promoter/enhancer functionally linked to a human interferon beta coding sequence, wherein the human interferon beta coding sequence is functionally linked to the SV40 T Ag early polyadenylation region and the nucleic acid does not comprise the human interferon beta polyadenylation signal or the human interferon beta 3′ UTR; 
 iv) a nucleic acid according to i), ii) or iii), wherein said nucleic acid further comprises a mouse DHFR gene; 
 v) a nucleic acid according to iv), wherein said mouse DHFR gene is functionally linked to a SV40 T Ag polyA-containing early polyadenylation region; or 
 vi) a nucleic acid according to v), further comprising a SV40 promoter/enhancer functionally linked to the mouse DHFR gene; or 
   c) an isolated host cell comprising an expression vector comprising:
 i) a human interferon beta coding sequence functionally linked to a SV40 T Ag early polyadenylation region, wherein the nucleic acid does not comprise the human interferon beta polyadenylation signal; 
 ii) a human interferon beta coding sequence functionally linked to a SV40 T Ag early polyadenylation region, wherein the nucleic acid does not comprise the human interferon beta polyadenylation signal and wherein said nucleic acid does not comprise the human interferon beta 3′ UTR; 
 iii) a nucleic acid comprising a SV40 promoter/enhancer functionally linked to a human interferon beta coding sequence, wherein the human interferon beta coding sequence is functionally linked to the SV40 T Ag early polyadenylation region and the nucleic acid does not comprise the human interferon beta polyadenylation signal or the human interferon beta 3′ UTR; 
 iv) a nucleic acid according to i), ii) or iii), wherein said nucleic acid further comprises a mouse DHFR gene; 
 v) a nucleic acid according to iv), wherein said mouse DHFR gene is functionally linked to a SV40 T Ag polyA-containing early polyadenylation region; or 
 vi) a nucleic acid according to v), further comprising a SV40 promoter/enhancer functionally linked to the mouse DHFR gene. 
   
     
     
         57 . The composition of matter according to  claim 56 , wherein said host cell is a Chinese Hamster Ovary (CHO) cell.