Recombinant Fibrinogen

Abstract

The present invention relates to nucleotide sequences encoding a fibrinogen alpha, beta or gamma chain. The sequences are optimized for expression in a eukaryotic cell culture system. Such optimized nucleotide sequences allow for the efficient expression of recombinant fibrinogen and variants thereof in intact form in a eukaryotic cell culture system.

1 . A nucleotide sequence encoding a fibrinogen alpha, beta or gamma chain which is optimized for expression in a mammalian cell culture system. 
     
     
         2 . A nucleotide sequence according to  claim 1  which is optimized for expression in a COS cell, a BHK cell, a NSO cell, a CHO cell, a SP2/0 or a human cell culture system. 
     
     
         3 . A nucleotide sequence according to  claim 1  which is optimized for expression in a PER.C6 cell or a HEK293 cell culture system. 
     
     
         4 . A nucleotide sequence according to  claim 1  which is optimized by codon usage adaptation to CHO cells. 
     
     
         5 . A nucleotide sequence according to  claim 1  which has a GC content of at least 55%. 
     
     
         6 . A nucleotide sequence according to  claim 1  which shows at least 70% identity to its respective non-optimized counterpart. 
     
     
         7 . A nucleotide sequence according to  claim 1  wherein the codon optimized fibrinogen chains contain no cis-acting sites. 
     
     
         8 . A nucleotide sequence according to  claim 1  having a nucleotide sequence according to SEQ ID No. 4 or 7, or part thereof, or a nucleotide sequence which has a sequence which is at least 85% identical to SEQ ID NO. 4 or 7 and which encodes a fibrinogen alpha chain. 
     
     
         9 . A nucleotide sequence according to  claim 1  having a nucleotide according to SEQ ID No. 5 or part thereof, or a nucleotide sequence which has a sequence which is at least 85% identical to SEQ ID No. 5 and which encodes a fibrinogen beta chain. 
     
     
         10 . A nucleotide sequence according to  claim 1  having an nucleotide according to SEQ ID No. 6 or 12, or part thereof, or a nucleotide sequence which has a sequence which is at least 85% identical to SEQ ID No. 6 or 12 and which encodes a fibrinogen gamma chain. 
     
     
         11 . A nucleotide construct which comprises a nucleotide sequence according to  claim 1 . 
     
     
         12 . A cell comprising a nucleotide sequence according to  claim 1  or a nucleotide construct according to  claim 11 . 
     
     
         13 . A cell or cell line which produces intact recombinant fibrinogen at levels of at least 3 picogram per cell per day. 
     
     
         14 . A method for the production of fibrinogen in a eukaryotic cell culture system which method comprises culturing a cell according to  claim 12  under conditions wherein fibrinogen is produced, and optionally, recovering the fibrinogen produced. 
     
     
         15 . A fibrinogen preparation prepared by the method of  claim 14 . 
     
     
         16 . A fibrinogen preparation in which more than 10% of the alpha, beta or gamma chains are a variant type. 
     
     
         17 . A fibrinogen preparation according to  claim 16  wherein the variant type is a gamma prime chain or an alpha extended chain. 
     
     
         18 . A fibrinogen preparation according to  claim 14  for use as a medicament, as a tissue sealant or to facilitate tissue adherence. 
     
     
         19 . Method of using a fibrinogen preparation according to  claim 14  for the preparation of a medicament for the treatment or prevention of fibrinogen deficiency. 
     
     
         20 . A fibrinogen preparation produced by recombinant technology in which more than 85% of the fibrinogen is in the intact form. 
     
     
         21 . A method for the selection of a cell or cell line which produces intact recombinant fibrinogen, which method comprises the use of two antibodies, wherein one antibody selectively binds to the intact N-terminus of the alpha chain and the other antibody selectively binds to the intact C-terminus of the alpha chain. 
     
     
         22 . A nucleotide sequence according to  claim 4  with a codon adaptation index of at least 0.95. 
     
     
         23 . A method for the production of fibrinogen in a eukaryotic cell culture system which method comprises culturing a cell or cell line according to  claim 13  under conditions wherein fibrinogen is produced, and optionally, recovering the fibrinogen produced.