Dac hyp compositions and methods

Abstract

The present disclosure relates to compositions of daclizumab suitable for subcutaneous administration and methods of manufacturing thereof.

What is claimed is: 
     
         1 . A modified NS0 cell that has been adapted to grow in serum- and cholesterol-free media and that is engineered to express a recombinant protein, said cell being capable of achieving a volumetric productivity exceeding 100 mg/L/day recombinant protein in a culture of 100 L in a 10-day fed-batch process when grown in serum- and cholesterol-free media. 
     
     
         2 . The modified NS0 cell of  claim 1  which is capable of achieving a volumetric productivity exceeding:
 (a) 100 mg/L/day recombinant protein in a culture of 1,000 L in a 10-day fed-batch process when grown in media free of cholesterol and animal-derived components; 
 (b) 100 mg/L/day recombinant protein in a culture of 16,000 L in a 10-day fed-batch process when grown in media free of cholesterol and animal-derived components; 
 (c) 200 mg/L/day recombinant protein in a culture of at least 100 L in a 13-day fed-batch process; 
 (d) 200 mg/L/day recombinant protein in a culture of 1,000 L in a 13-day fed-batch process when grown in cholesterol-free media; or 
 (e) 100 mg/L/day recombinant protein in a culture of 16,000 L in a 10-day fed-batch process when grown in serum- and cholesterol-free media. 
 
     
     
         3 . The modified NS0 cell of  claim 1 , wherein said fed-batch process comprises adding a feed medium is added according to the following schedule, where the volume added represents the percentage of the initial cell culture volume: 
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                   Day 
                   Volume added 
                 
                     
                     
                 
                     
                 
                 
                 
                 
               
                     
                   0 
                   0 
                 
                     
                   1 
                   0 
                 
                     
                   2 
                   4 
                 
                     
                   3 
                   7.8 
                 
                     
                   4 
                   7.8 
                 
                     
                   5 
                   7.8 
                 
                     
                   6 
                   11 
                 
                     
                   7 
                   13 
                 
                     
                   8 
                   15 
                 
                     
                   9 
                   15 
                 
                     
                   10 
                   0 
                 
                     
                     
                 
             
                
                
                
               
               
                
               
            
             
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         4 . The modified NS0 cell of  claim 1  which is stably transfected with a nucleic acid useful for expressing an anti-CD25 monoclonal antibody, optionally in which the anti-CD25 monoclonal antibody comprises a V L  chain corresponding in sequence to positions 21-233 of SEQ ID NO:2 and a V H  chain corresponding in sequence to positions 20 to 465 of SEQ ID NO:4. 
     
     
         5 . A method of producing a recombinant protein, comprising culturing the modified NS0 cell of  claim 1 :
 (a) under conditions that result in the production of at least 100 mg/L/day recombinant protein in a 100 L, 1,000 L or 16,000 L culture in a 10-day fed-batch process, or at least 200 mg/L/day recombinant protein in a 100 L, 1,000 L or 16,000 L culture in a 13-day fed-batch process;   (b) in the absence of serum and cholesterol, and optionally in the absence of tropolone and hydrocortisone;   (c) in a basal and/or feed medium containing 10-35 g/L glucose; or   (d) in a basal medium containing 15 g/L glucose and/or a feed medium containing 28 g/L glucose, optionally wherein the basal medium is composed of the components of PFBM2±10% or PFFM3±10% and wherein the cell is cultured in basal medium for 1-3 days, and then in feed medium for 10-13 days.   
     
     
         6 . A vector useful for recombinantly expressing a protein of interest, comprising a weak promoter driving expression of a selectable marker operable in mammalian cells and a strong promoter driving expression of a protein of interest, optionally wherein the vector is pAbX.gpt or pHAT.IgG1.rg.dE and/or the protein of interest is (a) a therapeutic antibody; (b) an anti-CD25 antibody; (c) an anti-CD25 antibody which comprises the CDRs of daclizumab; or (d) daclizumab. 
     
     
         7 . A method for obtaining a mammalian host cell that has a high volumetric productivity of a protein of interest, comprising transfecting the cell with the vector of  claim 6 , and selecting a cell that is capable of producing at least 100 mg/L/day protein of interest in a 100 L, 1,000 L or 16,000 L culture in a 10-day fed-batch process or at least 200 mg/L/day recombinant protein in a 100 L, 1,000 L or 16,000 L culture in a 13-day fed-batch process. 
     
     
         8 . A composition comprising daclizumab, wherein the daclizumab is characterized by the presence of a pE/Q heavy chain N-linked isoform and/or a Q/VHS heavy chain N-terminal isoform, optionally wherein the pE/Q heavy chain N-terminal isoform constitutes approximately 3-17%, approximately 3-15%, approximately 6-15%, approximately 5-15%, approximately 5-12%, or approximately 7-12% of the daclizumab, and, optionally wherein the Q/VHS heavy chain N-terminal isoform constitutes approximately 1-15% or approximately 3-12% of the daclizumab. 
     
     
         9 . The composition of  claim 8  in which the heavy chain of daclizumab exists in the following N-terminal isoforms: 
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                   Isoform 
                   Prevalence 
                 
                     
                     
                 
                     
                 
                 
               
                   (a) 
                 
                 
                 
                 
               
                     
                   pE/pE 
                   25%-50%  
                 
                     
                   pE/Q 
                   3%-15% 
                 
                     
                   pE/VHS 
                   25%-48%  
                 
                     
                   Q/VHS 
                   1%-15% 
                 
                     
                   VHS/VHS 
                   0.5%-25%     
                 
                 
               
                   or (b) 
                 
                 
                 
                 
               
                     
                   pE/pE 
                    31-46% 
                 
                     
                   pE/Q 
                   5%-12% 
                 
                     
                   pE/VHS 
                   31%-42%  
                 
                     
                   Q/VHS 
                   3%-12% 
                 
                     
                   VHS/VHS 
                   1%-17% 
                 
                     
                     
                 
             
                
                
                
               
               
                
               
            
             
                
               
            
             
                
                
                
                
                
               
            
             
                
               
            
             
                
                
                
                
                
                
               
            
           
         
       
     
     
         10 . The composition of  claim 8 , wherein the daclizumab is characterized by a cation exchange chromatography isoform profile substantially similar to that of  FIG. 18 , optionally wherein the daclizumab is DAC HYP. 
     
     
         11 . A composition comprising daclizumab, wherein the daclizumab is characterized by an N-linked glycosylation HPLC profile containing two main peaks, one corresponding to oligosaccharide G0-GlcNAc and one corresponding to oligosaccharide G0, wherein the combined AUC of these two peaks constitutes about 75-100%, about 80-100%, about 85-100%, or about 88-99.5% of the total AUC of all peaks, optionally in which
 (a) the AUC of the G0-GlcNAc peak constitutes about 5-20%, about 5-18%, about 7-15%, or about 6-16% of the total AUC of all peaks and the AUC of the G0 peak constitutes about 70-99.2%, about 75-92%, about 75-90%, about 78-90%, or about 81-88% of the total AUC of all peaks, and optionally, in which the N-linked glycosylation profile has less than about 3% of Man5 or less than about 0.5% G2, Man6 and/or Man7;   (b) the N-linked glycosylation profile contains a third peak corresponding to sialylated oligosaccharides, and the AUC of the sialylated oligosaccharide peak constitutes 1% or less of the total AUC of all peaks, or in which the N-linked glycosylation profile contains a third peak corresponding to oligosaccharide G1, and the AUC of the G1 peak constitutes less than about 10%, less than about 5%, less than about 4%, less than about 3%, or about 1-5% or about 1-4% or about 1-3% of the total AUC of all peaks;   (c) the N-linked glycosylation profile contains peaks corresponding to Man5, Man6, and Man7 glycoforms which together are less than about 6% of the total AUC; or   (d) the daclizumab has an N-linked glycosylation HPLC profile substantially similar to that of  FIG. 19  or to that of the lower panel of  FIG. 21  and/or is DAC HYP.   
     
     
         12 . A composition comprising daclizumab which exhibits (a) less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% ADCC average cytotoxicity or (b) 5-30%, 10-30%, 10-25%, or 15-25% ADCC average cytotoxicity as measured in an in vitro cellular assay using effector cells from at least 3 healthy donors and Kit 225 K6 cells as target cells, at a daclizumab concentration of 1 μg/mL and an effector to target cell ratio of about 25:1, optionally wherein said assay uses effector cells from at least 6 or at least 10 healthy donors. 
     
     
         13 . The composition of  claim 12 , in which the daclizumab is DAC HYP. 
     
     
         14 . A composition useful for making a daclizumab drug formulation, comprising about 150-190 mg/mL daclizumab and quantities of excipients such that dilution of the composition with a dilution buffer yields a diluted composition that contains about 85-165 mg/mL, or 85-115 mg/mL, or 150±15 mg/mL daclizumab and has an osmolality in the range of about 267-327 mOsm/kg and a pH in the range of about pH 5.8-6.2 at 25° C., and in which at least about 95% of the daclizumab is in monomer form, as measured by size exclusion chromatography. 
     
     
         15 . A composition comprising:
 (a) about 4 to 15 mg/mL daclizumab, wherein 0.1% or less of the daclizumab is in aggregate form, optionally wherein the composition is obtained by purifying a daclizumab composition comprising about 4 to 15 mg/mL daclizimab, wherein up to 2.5% of the daclizumab is in aggregate form, via column chromatography on a weak cation exchange resin; or   (b) about 85-165 mg/mL or about 85-115 mg/mL or about 135-165 mg/mL daclizumab; and about 0.02-0.04% (w/v) polysorbate 80, wherein the composition has an osmolality in the range of about 267-327 mOsm/kg and a pH in the range of about pH 5.8-6.2 at 25° C., and at least about 95% or at least about 99% of the daclizumab is in monomer form, as measured by size exclusion chromatography, wherein the composition is suitable for administration to humans, optionally wherein the composition consists essentially of about 100 mg/mL or about 150 mg/mL daclizumab, about 40 mM sodium succinate, about 100 mM sodium chloride, and about 0.03% (w/v) polysorbate 80, and has a pH of about 6.0 at 25° C.   
     
     
         16 . A pharmaceutical composition suitable for subcutaneous administration comprising about 85-165 mg/mL, about 85-115 mg/mL, or about 135-165 mg/mL daclizumab, wherein the percentage of daclizumab in aggregate form does not exceed about 2% or about 3% following storage for a period of about 12 months at a temperature in the range of about 2-8° C., or about 3% following storage for a period of about 18 months at a temperature in the range of about 2-8° C. 
     
     
         17 . A process for harvesting a recombinant protein from a cell culture, comprising the steps of:
 (i) adjusting the pH of a cell culture that expresses and secretes a recombinant protein to a pH in the range of about pH 4.5-5.5;   (ii) incubating the pH-adjusted cell culture for about 30-90 minutes at a temperature in the range of about 4 to 15° C.; and   (iii) centrifuging the incubated pH-adjusted cell culture to remove cell debris.   
     
     
         18 . A process for producing a purified daclizumab composition, comprising the steps of:
 (i) adsorbing daclizumab from a crude daclizumab preparation onto affinity chromatography resin;   (ii) washing the affinity chromatography resin with a wash buffer to remove contaminants;   (iii) eluting the adsorbed daclizumab with an elution buffer;   (iv) inactivating viruses in the eluate by adjusting the pH to a pH in the range of about pH 3-4 and incubating the pH-adjusted eluate at specified temperature for a period of time sufficient to inactivate viruses;   (v) neutralizing the virus-inactivated eluate to a pH in the range of about pH 7.7-7.9 (measured at 25° C.) or a pH in the range of about pH 7.7-8.5 (measured at 25° C.);   (vi) flowing the neutralized eluate across a strong anion exchange chromatography resin;   (vii) adsorbing the daclizumab of the eluate of step (vi) onto a weak cation exchange chromatography resin; and   (viii) eluting the adsorbed daclizumab from the weak cation exchange chromatography resin; optionally comprising the steps of   (ix) filtering the eluted daclizumab composition of step (viii) to remove viruses; and   (x) concentrating the filtered solution via ultrafiltration to yield a purified, daclizumab composition comprising about 85-180 mg/mL daclizumab.   
     
     
         19 . The process of  claim 18  in which the crude daclizumab preparation is harvested from a cell culture, optionally using the method of  claim 17 . 
     
     
         20 . The process of  claim 18 , wherein steps (i) to (x) are carried out, which further comprises the step of diluting the purified daclizumab composition with a dilution buffer so as to obtain a composition comprising about 85-165 mg/mL daclizumab; and about 0.02-0.04% (w/v) polysorbate 80, wherein the composition has an osmolality in the range of about 267-327 mOsm/kg and a pH in the range of about pH 5.8-6.2 at 25° C., and at least about 95% of the daclizumab is in monomer form, as measured by size exclusion chromatography, optionally, wherein the composition obtained has less than 50 ppm of host cell proteins from a recombinant source of daclizumab, less than 10 ppm of protein A, and no more than 3% of the daclizumab in the composition is in aggregate form. 
     
     
         21 . A medium which is a basal medium PFBM2 or a feed medium PFFM3. 
     
     
         22 . A daclizumab composition, which is obtained by a process comprising the step of culturing a host cell according to  claim 1  under conditions in which daclizumab is secreted into the culture medium, optionally in which the process further comprises the step of isolating the secreted daclizumab from the cell culture medium. 
     
     
         23 . A buffer useful for sanitizing a protein A affinity chromatography resin, comprising about 100-500 mM sodium citrate, about 10-30 mM NaOH and about 0.5-3% (v/v) benzyl alcohol. 
     
     
         24 . A method of sanitizing a protein A affinity chromatography column, comprising washing the column with the sanitization buffer of  claim 23  at a flow rate and for a period of time sufficient to sanitize the column, optionally in which the column is washed with approximately 1.8 column volumes of sanitization buffer at a flow rate of about 150 cm/hr, the washed column incubated without flow for a period of about 30-45 min., and then equilibrated with an equilibration buffer. 
     
     
         25 . A method of treating a patient suffering from multiple sclerosis, comprising administering to the patient an amount of a DAC HYP composition sufficient to provide therapeutic benefit. 
     
     
         26 . The method of  claim 25  in which the DAC HYP composition is administered:
 (a) intravenously; 
 (b) in amount corresponding to about 0.8-0.9 mg/kg or about 1 mg/kg DAC HYP; 
 (c) once per week for a period of at least 6 weeks, at least 12 weeks, at least 24 weeks; 
 (d) as monotherapy; or 
 (e) according to any combination of (a)-(d). 
 
     
     
         27 . The method of  claim 26  in which DAC HYP is administered as monotherapy and the patient has either failed to respond to prior treatment with interferon-beta or has discontinued prior treatment with interferon-beta. 
     
     
         28 . The method of  claim 26  in which the DAC HYP is administered adjunctively to interferon-beta. 
     
     
         29 . The method of  claim 25  in which the DAC HYP composition is administered:
 (a) subcutaneously; 
 (b) in an amount corresponding to about 1 mg/kg DAC HYP, optionally once every two weeks or in an amount corresponding to about 2 mg/kg DAC HYP, optionally once every 4 weeks; 
 (c) for a total of about 24 weeks; or 
 (d) according to any combination of (a)-(c). 
 
     
     
         30 . The method of  claim 29 , in which the DAC HYP composition is administered in an amount corresponding to 75 mg to 300 mg DAC HYP, or 150 mg, or 300 mg. 
     
     
         31 . The method of  claim 30  wherein the DAC HYP composition is administered once every 4 weeks, optionally for a total of at least 48 weeks. 
     
     
         32 . The method of  claim 30  in which the DAC HYP is administered as monotherapy, optionally, in which the patient has either failed to respond to prior treatment with interferon-beta or has discontinued prior treatment with interferon-beta. 
     
     
         33 . The method of  claim 30  in which the DAC HYP is administered adjunctively to interferon-beta.