Method for Cell Line Development

Abstract

The present invention relates to an improved method for cell line development (CLD) which is generally applicable to production of any therapeutic protein that can be produced using mammalian Cell lines and in particular Chinese Hamster Ovary (CHO) cells. The method combines site-directed integration (SDI), expression construct components improving the post-transcriptional processing of the gene of interest (GOI) and the introduction of a onetime pre-CLD host cell line selection workflow to generate a production competent cell line that can then be used in multiple CLD efforts using SDI from that point on.

1 . A method to obtain a cell suitable for expressing a protein interest comprising the following steps:
 a) integration of a nucleic acid molecule encoding a first protein of interest into a pre-defined site in the genome of a recipient cell;   b) identification of cells expressing the first protein of interest;   c) excision of the nucleic acid molecule encoding the first protein of interest from the genome of the identified cells and simultaneously introducing one or several functional sequence elements Q enabling targeted introduction of a nucleic acid molecule encoding a second protein of interest into said pre-defined site in the genome,   wherein the genome of the recipient cell does not comprise the one or several sequence elements Q prior to excision of the nucleic acid molecule encoding the first protein of interest; and   d) subsequent to step c), integration of the nucleic acid molecule encoding the second protein of interest into the pre-defined site in the genome of the resulting cell.   
     
     
         2 . The method according to  claim 1 , further comprising creating of a cell line suitable for production of said second protein of interest as a recombinant protein. 
     
     
         3 . The method according to  claim 2 , wherein the recombinant protein is an active pharmacological ingredient. 
     
     
         4 . The method according to  claim 2 , further comprising he recombinant protein as an ingredient in a diagnostic reagent. 
     
     
         5 . The method according to  claim 2 , further comprising providing the recombinant protein as a reagent in an industrial process. 
     
     
         6 . The method according to  claim 1 , wherein the first protein of interest is a fluorescent protein. 
     
     
         7 . The method according to  claim 1 , wherein the first protein of interest is an immunoglobulin or immunoglobulin-like protein. 
     
     
         8 . The method according to  claim 1 , further comprising selecting a cell with increased capacity to express the first protein of interest relative to other cells identified as expressing the first protein of interest prior to excision of the nucleic acid molecule encoding the first protein of interest. 
     
     
         9 . The method according to  claim 8 , wherein selecting the cell with increased capacity to express the first protein of interest comprises isolating clones from a culture of said identified cells expressing the first protein of interest. 
     
     
         10 . The method according to  claim 8 , wherein selecting the cell with increased capacity to express the first protein of interest comprises isolating pools or clones from said identified cells expressing the first protein of interest after (1) different culture time in a continuous culture format such as a perfusion culture or (2) different number of individual cultures starting with inoculation of a first culture using said identified cells expressing the first protein of interest and using a volume from a finished culture to inoculate a next culture or (3) a combination of (1) and (2). 
     
     
         11 . The method according to  claim 8 , wherein selecting the cell with increased capacity to express the first protein of interest comprises performing targeted engineering by applying gene editing methods to introduce, remove or modify genetic material in the genome of said identified cells expressing the first protein of interest. 
     
     
         12 . The method according to  claim 1 , wherein a selection marker is simultaneously introduced with the one or several functional sequence elements Q. 
     
     
         13 . The method according to  claim 1 , wherein the nucleic acid molecule encoding the first protein of interest, when integrated into the recipient cell, comprises flanking sequences X and Y flanking the nucleic acid molecule encoding the first protein of interest, wherein the flanked region comprises a sequence z being unique or rare in the genome, and step c) comprises forming a double strand break with a specific gene editing DNA nuclease at sequence z and performing a cassette exchange with a template DNA construct containing the one or several functional sequence elements Q. 
     
     
         14 . The method according to  claim 1 , wherein the one or several functional sequence elements Q comprises one or more recombinase recognition sequences. 
     
     
         15 . The method according to  claim 1 , wherein the first protein of interest and the second protein of interest are of the same protein class. 
     
     
         16 . The method according to  claim 1 , wherein only a single copy of the nucleic acid molecule encoding the first protein of interest and/or the nucleic acid molecule encoding the second protein of interest is integrated.