Method for the production of immunoglobulin single variable domains

Abstract

The present disclosure relates to an improved method for the manufacture of immunoglobulin single variable domains. More specifically, the present disclosure relates to a method of producing immunoglobulin single variable domains in which the proportion of carbamylated variants is strongly reduced or absent and to improved immunoglobulin single variable domains obtainable by methods of the present disclosure.

1 . Method for producing an immunoglobulin single variable domain in a host or a host cell comprising
 a) applying conditions that reduce carbamylation of one or more amino acid residues in the immunoglobulin single variable domain, or   b) removing the immunoglobulin single variable domain comprising one or more carbamylated amino acid residues, or   c) a combination of (a) and (b).   
     
     
         2 . Method according to  claim 1 , wherein
 a) carbamylation of one or more amino groups, preferably an N-terminal amino group and/or one or more amino groups in a side-chain of a lysine and/or an arginine residue, in the immunoglobulin single variable domain is reduced, or   b) the immunoglobulin single variable domain comprising one or more carbamylated amino groups, preferably an N-terminal carbamylamino group and/or one or more carbamylamino groups in a side-chain of a lysine and/or an arginine residue, are removed, or   c) a combination of (a) and (b).   
     
     
         3 . Method according to  claim 1 , wherein carbamylation is reduced by lowering the pH in one or more steps of the production process as compared to the standard pH for the host organism used in said one or more steps of the production process. 
     
     
         4 . Method according to  claim 3 , wherein the pH is lowered at least in the step of inducing the host to produce the immunoglobulin single variable domain. 
     
     
         5 . Method according to  claim 3 , wherein the pH is lowered to a pH of about 6.45 or less. 
     
     
         6 . Method according to  claim 1 , wherein carbamylation is reduced by reducing the culturing time, in particular the (glycerol fed-)batch time and/or the induction time, preferably the induction time, as compared to the standard culturing, (glycerol fed-)batch time or induction time for the host organism. 
     
     
         7 . Method according to  claim 6 , wherein the culturing time, in particular the (glycerol fed-)batch time and/or the induction time, is lowered by 30 to 80% as compared to the standard culturing, (glycerol fed-)batch time or induction time. 
     
     
         8 . Method according to  claim 6 , wherein the induction time is lowered from about 96 hours to a period between 24 to 96 hours. 
     
     
         9 . Method according to  claim 6 , wherein the glycerol fed-batch time is lowered from about 16 to 18 hours to a period between 2 to 4 hours. 
     
     
         10 . Method according to  claim 1 , wherein carbamylation is reduced by lowering the temperature in one or more steps of the production process as compared to the standard temperature for the host organism used in said one or more steps of the production process. 
     
     
         11 . Method according to  claim 10 , wherein the temperature is lowered at least in the step of inducing the host to produce the immunoglobulin single variable domain. 
     
     
         12 . Method according to  claim 10 , wherein the temperature is lowered by 1 to 15° C. 
     
     
         13 . Method according to  claim 1 , wherein carbamylation is reduced by decreasing the dissolved oxygen concentration in one or more steps of the production process as compared to the standard dissolved oxygen concentration for the host organism used in said one or more steps of the production process. 
     
     
         14 . Method according to  claim 1 , wherein carbamylation is reduced by decreasing the percentage complex substrate in the glycerol feed in one or more steps of the production process as compared to the standard percentage complex substrate in the glycerol feed for the host organism used in said one or more steps of the production process, and/or by decreasing the glycerol feed rate in the glycerol fed-batch phase as compared to the standard glycerol feed rate for the host organism used in said glycerol fed-batch phase. 
     
     
         15 . Method according to  claim 1 , wherein carbamylation is reduced by adapting the methanol feed rate and/or composition in the induction phase as compared to the standard methanol feed rate for the host organism used in said induction phase. 
     
     
         16 . Method according to  claim 1 , wherein carbamylation is reduced by optimizing the medium composition in one or more steps of the production process. 
     
     
         17 . Method according to  claim 1 , wherein carbamylation is reduced in the step of culturing the host. 
     
     
         18 . Method according to  claim 17 , wherein carbamylation is reduced in the step of inducing the host to produce the immunoglobulin single variable domain. 
     
     
         19 . Method according to  claim 1 , wherein carbamylation is reduced in the culture broth after fermentation. 
     
     
         20 . Method according to  claim 1 , wherein carbamylation is reduced in the supernatant comprising the immunoglobulin single variable domain after removal of the host. 
     
     
         21 . Method according to  claim 1 , wherein carbamylation is reduced in any step of purification of the immunoglobulin single variable domain. 
     
     
         22 . Method according to  claim 1 , wherein carbamylation is reduced at the stage of the purified immunoglobulin single variable domain. 
     
     
         23 . Method according to  claim 1 , wherein the immunoglobulin single variable domain comprising one or more carbamylated amino acid residues is removed by one or more chromatographic techniques. 
     
     
         24 . Method according to  claim 23 , wherein the chromatographic techniques are chromatographic techniques based on shifts in pi or hydrophobicity, most preferably ion-exchange chromatography. 
     
     
         25 . The method according to  claim 1 , wherein one or more of the conditions that reduce carbamylation of one or more amino acid residues in the immunoglobulin single variable domain are applied alone, or in combination with one or more techniques to remove the immunoglobulin single variable domain comprising one or more carbamylated amino acid residues. 
     
     
         26 . The method according to  claim 1 , wherein the host is selected from a prokaryotic or a eukaryotic host. 
     
     
         27 . The method according to  claim 26 , wherein the prokaryotic host is  E. coli.    
     
     
         28 . The method according to  claim 26 , wherein the eukaryotic host is selected from insect cells, mammalian cells, and lower eukaryotic hosts comprising yeasts such as  Pichia, Hansenula, Saccharomyces, Kluyveromyces, Candida, Torulopsis, Torulaspora, Schizosaccharomyces, Citeromyces, Pachysolen, Debaromyces, Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, Endomycopsis , preferably  Pichia pastoris.    
     
     
         29 . Method according to  claim 1 , wherein the immunoglobulin single variable domain is a light chain variable domain sequence or a heavy chain variable domain sequence. 
     
     
         30 . Method according to  claim 29 , wherein the immunoglobulin single variable domain is a heavy chain variable domain sequence that is derived from a conventional four-chain antibody or a heavy chain variable domain sequence that is derived from a heavy chain antibody. 
     
     
         31 . Method according to  claim 30 , wherein the immunoglobulin single variable domain (or an amino acid sequence that is suitable for use as a immunoglobulin single variable domain) is a Nanobody (including but not limited to a VHH sequence). 
     
     
         32 . The method according to  claim 1 , at least comprising the steps of culturing the host or the host cell to produce the immunoglobulin single variable domain comprising:
 i) cultivating the host or host cell under conditions that are such that the host or host cell will multiply;   ii) maintaining the host or host cell under conditions that are such that the host or host cell expresses and/or produces the immunoglobulin single variable domain;   iii) optionally followed by isolating and/or purifying the secreted immunoglobulin single variable domain from the medium.   
     
     
         33 . The method according to  claim 32 , wherein the conditions that reduce carbamylation of one or more amino acid residues in the immunoglobulin single variable domain are applied at one or more of step i), step ii), after step ii), or at or after step iii), preferably at step ii). 
     
     
         34 . The method according to  claim 33 , wherein the techniques to remove the immunoglobulin single variable domain comprising one or more carbamylated amino acid residues are applied after step ii). 
     
     
         35 . Immunoglobulin single variable domain obtainable by the method of  claim 1 .