US2014273092A1PendingUtilityA1

Manufacturing methods to control c-terminal lysine, galactose and sialic acid content in recombinant proteins

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Assignee: JANSSEN BIOLOG B VPriority: Mar 15, 2013Filed: Mar 13, 2014Published: Sep 18, 2014
Est. expiryMar 15, 2033(~6.6 yrs left)· nominal 20-yr term from priority
Inventors:Marcel FlikweertCharles GoocheeFrancis C. MaslankaFranciscus Johannes Ignatius NagelJames R. RylandEugene J. Schaefer
C07K 2317/92C07K 2317/21C07K 16/241C07K 16/00C07K 2317/52C07K 2317/76C07K 2317/41C07K 2317/14C07K 2317/40C07K 2317/54C07K 2317/24C07K 2317/55
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Claims

Abstract

Provided herein is a method for producing an antibody, such as an anti-TNFα antibody (e.g., infliximab) having a C-terminal lysine content of about 20% to about 70%, and a sialic acid content of about 1% to about 20%, comprising culturing a zinc-responsive host cell transfected with DNA encoding the antibody in a culture medium comprising at least 0.5 μM zinc; and controlling the concentration of zinc in the culture medium, thereby producing the antibody.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
         1 . A method for producing an antibody having a C-terminal lysine content of about 20% to about 70%, and a sialic acid content of about 1% to about 20%, the method comprising:
 culturing a zinc-responsive host cell transfected with DNA encoding the antibody in a culture medium comprising at least 0.5 μM zinc; and   controlling the concentration of zinc in the culture medium, thereby producing the antibody.   
     
     
         2 . The method of  claim 1 , wherein the C-terminal lysine content of the antibody is about 40% to about 70%. 
     
     
         3 . The method of  claim 2 , wherein the C-terminal lysine content of the antibody is about 55% to about 65%. 
     
     
         4 . The method of  claim 3 , wherein the C-terminal lysine content of the antibody is about 60%. 
     
     
         5 . The method of  claim 1 , wherein the sialic acid content of the antibody is about 3% to about 14%. 
     
     
         6 . The method of  claim 1 , wherein the antibody is an anti-TNFα antibody, or antigen-binding fragment thereof, wherein said anti-TNFα antibody or antigen-binding fragment thereof (i) competitively inhibits binding of A2 (ATCC Accession No. PTA-7045) to human TNFα; and (ii) binds to a neutralizing epitope of human TNFα with an affinity of at least 1×10 8  liter/mole, measured as an association constant (Ka). 
     
     
         7 . The method of  claim 6 , wherein the anti-TNFα antibody or antigen-binding fragment thereof is a human antibody. 
     
     
         8 . The method of  claim 6 , wherein the anti-TNFα antibody is a humanized or chimeric antibody. 
     
     
         9 . The method of  claim 6 , wherein the anti-TNFα antibody or antigen-binding fragment thereof is of immunoglobulin class IgG1, IgG2, IgG3, IgG4 or IgM. 
     
     
         10 . The method of  claim 9 , wherein the anti-TNFα antibody or antigen-binding fragment thereof comprises an IgG1 constant region. 
     
     
         11 . The method of  claim 6 , wherein the anti-TNFα antibody or antigen-binding fragment thereof is selected from the group consisting of Fab, Fab′, F(ab′) 2  and Fv. 
     
     
         12 . The method of  claim 6 , wherein
 a) the light chain comprises all antigen-binding regions of the light chain of A2 (ATCC Accession No. PTA-7045);   b) the heavy chain comprises all antigen-binding regions of the heavy chain of A2 (ATCC Accession No. PTA-7045); or   c) the light chain comprises all antigen-binding regions of the light chain of A2 (ATCC Accession No. PTA-7045) and the heavy chain comprises all antigen-binding regions of the heavy chain of A2 (ATCC Accession No. PTA-7045).   
     
     
         13 . The method of  claim 12 , wherein the anti-TNFα antibody or antigen-binding fragment thereof comprises a non-human variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 5. 
     
     
         14 . The method of  claim 13 , wherein the non-human variable region comprises a polypeptide encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4. 
     
     
         15 . The method of  claim 6 , wherein the anti-TNFα antibody or antigen-binding fragment thereof has epitopic specificity identical to monoclonal antibody cA2. 
     
     
         16 . The method of  claim 15 , wherein the anti-TNFα antibody or antigen-binding fragment thereof is monoclonal antibody cA2. 
     
     
         17 . The method of  claim 1 , wherein the concentration of zinc in the culture medium is in the range of about 0.6 μM to about 6.5 μM. 
     
     
         18 . The method of  claim 17 , wherein the concentration of zinc in the culture medium is in the range of about 0.6 μM to about 1.1 μM. 
     
     
         19 . The method of  claim 1 , wherein the culture medium further comprises total EDTA in a concentration range of about 2.5 μM to about 30 μM, and the method further comprises controlling the concentration of iron-free EDTA in the culture medium. 
     
     
         20 . The method of  claim 19 , wherein the culture medium further comprises free EDTA in a concentration range of about 5 μM to about 16 μM. 
     
     
         21 . The method of  claim 1 , further comprising recovering the antibody. 
     
     
         22 . The method of  claim 21 , wherein the antibody is recovered when the zinc-responsive host cells in the culture medium reach a cell density of about 1.5 million cells per mL to about 11 million cells per mL. 
     
     
         23 . The method of  claim 22 , wherein the antibody is recovered when the zinc-responsive host cells in the culture medium reach a cell density of about 3 million cells per mL to about 11 million cells per mL. 
     
     
         24 . The method of  claim 21 , wherein the concentration of zinc is controlled until the antibody is recovered. 
     
     
         25 . The method of  claim 1 , wherein the concentration of zinc is controlled during an exponential growth phase of the zinc-responsive host cells. 
     
     
         26 . The method of  claim 1 , wherein controlling the concentration of zinc comprises monitoring the concentration of zinc in the culture medium, and regulating the concentration of zinc in the culture medium, such that the concentration of zinc in the culture medium is at least 0.5 μM. 
     
     
         27 . The method of  claim 26 , wherein the concentration of zinc in the culture medium is in the range of about 0.6 μM to about 6.5 μM. 
     
     
         28 . The method of  claim 1 , wherein the antibody has a galactose content of about 50% to about 90%. 
     
     
         29 . The method of  claim 28 , wherein the antibody has a galactose content of about 45% to about 85%. 
     
     
         30 . The method of  claim 1 , wherein the antibody has a ratio of sialic acid to galactose of about 0.05 to about 0.20. 
     
     
         31 . The method of  claim 1 , wherein the zinc-responsive host cell is an SP2/0 cell. 
     
     
         32 . A method for controlling C-terminal lysine content of an antibody having a C-terminal lysine content of about 20% to about 70%, in a process for biosynthesizing the antibody in a culture medium, the method comprising:
 monitoring a level of zinc in the culture medium during biosynthesis of the antibody; and   regulating the level of zinc in the culture medium during biosynthesis of the antibody.   
     
     
         33 . A method for controlling sialic acid content of an antibody having a sialic acid content of about 1% to about 20% in a process for biosynthesizing the antibody in a culture medium, the method comprising:
 monitoring a level of zinc in the culture medium during biosynthesis of the antibody; and   regulating the level of zinc in the culture medium during biosynthesis of the antibody.   
     
     
         34 . A method for controlling galactose content of an antibody having a galactose content of about 50% to about 90% in a process for biosynthesizing the antibody in a culture medium, the method comprising:
 monitoring a level of zinc in the culture medium during biosynthesis of the antibody; and   regulating the level of zinc in the culture medium during biosynthesis of the antibody.   
     
     
         35 . A method for controlling the ratio of sialic acid to galactose in an antibody having a ratio of sialic acid to galactose of about 0.05 to about 0.20 in a process for biosynthesizing the antibody in a culture medium, the method comprising:
 monitoring a level of zinc in the culture medium during biosynthesis of the antibody; and   regulating the level of zinc in the culture medium during biosynthesis of the antibody.   
     
     
         36 . The method as in one of  claims 33 - 35 , wherein the antibody is an anti-TNFα antibody or antigen-binding fragment thereof. 
     
     
         37 . The method of  claim 36 , wherein the antibody is an anti-TNFα antibody, or antigen-binding fragment thereof, wherein said anti-TNFα antibody or antigen-binding fragment thereof (i) competitively inhibits binding of A2 (ATCC Accession No. PTA-7045) to human TNFα; and (ii) binds to a neutralizing epitope of human TNFα with an affinity of at least 1×10 8  liter/mole, measured as an association constant (Ka). 
     
     
         38 . The method of  claim 37 , wherein the antibody is an anti-TNFα antibody or antigen-binding fragment thereof having epitopic specificity identical to monoclonal antibody cA2. 
     
     
         39 . The method of  claim 38 , wherein the antibody is monoclonal antibody cA2. 
     
     
         40 . The method of  claim 39 , wherein the antibody is biosynthesized by an SP2/0 cell line.

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