Characterisation of the post-translational modifications of a novel, human cell line-derived recombinant human factor VIII.
BACKGROUND
Host cell lines used for recombinant protein expression differ in their ability to perform post-translational modifications (PTMs). The currently available recombinant human FVIII (rhFVIII) products are produced in mammalian, non-human cell lines. For rhFVIII, glycosylation and sulfation are vital for functionality and von Willebrand factor (VWF)-binding affinity. Here we present the characterisation of the PTMs of a novel, human cell line-derived recombinant human FVIII (human-cl rhFVIII). rhFVIII expression in a human cell line avoids expression of undesirable mammalian glycoforms like Galα1-3Galβ1-GlcNAc-R (α-Gal) and N-glycolylneuraminic acid (Neu5Gc), which constitute epitopes antigenic to humans.


METHODS
We describe sulfation analysis, glycan profiling and characterisation using liquid chromatography-mass spectrometry and high performance anion exchange chromatography with pulsed amperometric detection.


CONCLUSIONS
Human-cl rhFVIII is confirmed to be sulfated and glycosylated comparable to human plasma-derived FVIII. Most importantly, human-cl rhFVIII is devoid of the antigenic Neu5Gc or α-Gal epitopes observed in Chinese Hamster Ovary- and Baby Hamster Kidney-derived rFVIII products. Both the avoidance of non-human glycan structures and the achievement of complete sulfation are proposed to lower the intrinsic immunogenicity of human-cl rhFVIII compared with current rFVIII products.
Octapharma, Berlin, Molecular Biochemistry, Germany. christoph.kannicht@octapharma.com
Christoph
C
Kannicht
Margareta
M
Ramström
Guido
G
Kohla
Maya
M
Tiemeyer
Elisabeth
E
Casademunt
Olaf
O
Walter
Helena
H
Sandberg
D000818:Animals; D000941:Antigens; D016466:CHO Cells; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D006224:Cricetinae; D003412:Cricetulus; D005169:Factor VIII / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D013431:Sulfates / Q000378:metabolism; D014162:Transfection; D014443:Tyrosine
D000941:Antigens; D011994:Recombinant Proteins; D013431:Sulfates; D014443:Tyrosine; C078147:F8 protein, human; D005169:Factor VIII