Sandoz Ag

Johann Holzmann

Michael Fuchs

Clemens Achmüller

Alfred Rupprechter

William Lamanna

Christoph Posch

Hansjorg Toll

Robert Mayer

Stefan Hutwimmer

Hansjörg Toll

The present invention relates in general to a nucleic acid encoding human granulocyte-colony stimulating factor (G-CSF), wherein the first leucine residue occurring on the N-terminal end of the encoded G-CSF is encoded by a codon other than the CTG/CUG codon, and wherein the nucleic acid does neither comprise the nucleic acid sequence according to SEQ ID NO: 1, nor according to SEQ ID NO: 2, nor according to SEQ ID NO: 3, nor according to SEQ ID NO: 4. The present invention also relates to a nucleic acid 100% complementary to the aforementioned nucleic acid, as well as to vectors and host cells comprising the aforementioned nucleic acids. Finally, the present invention relates to methods for producing human G-CSF using these nucleic acids, vectors and/or host cells and resulting G-CSF compositions.

DNA coding sequence for human G-CSF

Amino acid residue misincorporations are necessarily found in sequence variants at low concentrations in admixture with expressed polypeptides, resulting from one or more base mismatches within codons susceptible to amino acid residue misincorporation during transcription and/or translation. The invention provides a method of optimizing the coding sequences of a polynucleotide that encodes a polypeptide, wherein at least one codon is susceptible to amino acid residue misincorporation. The method of the invention can be used to reverse-engineer an unknown coding sequence, which encodes the same polypeptide, but differs in said at least one codon from the known coding sequence. The method can further be used to alter the immunogenic potential of an expressed polypeptide. Thus, the invention is useful in engineering optimized polynucleotides encoding polypeptides.

Sequence variants

The present invention is directed to methods for determining the relative amount of wrongly disulphide bridged TNFR2:Fc in a sample of TNFR2:Fc, a fusion protein which is used in a variety of therapeutic applications. In addition, the invention pertains to a method for purifying TNFR2:Fc using said method for determining the percentage of wrongly disulphide bridged TNFR2:Fc, and to TNFR2:Fc compositions obtained thereby.

Quantification of misfolded TNFR2:Fc

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Kundl, Austria

Johann Holzmann