Characterisation of the post-translational modifications of a novel, human cell line-derived recombinant human factor VIII

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• 作者：Christoph Kannicht^a ; ^{christoph.kannicht@octapharma.com} ; Margareta Ramstr&#246 ; m^b ; Guido Kohla^a ; Maya Tiemeyer^c ; Elisabeth Casademunt^c ; Olaf Walter^d ; Helena Sandberg^b
• 关键词：¦Á-Gal ; Gal¦Á1-3Gal¦Â1-GlcNAc-R ; Asn/N ; asparagine ; BHK ; baby hamster kidney ; CHO ; Chinese hamster ovary ; EIC ; extracted ion chromatogram ; FVIII ; factor VIII ; HA ; Haemophilia A ; HEK ; human embryonic kidney ; HexNAcHexNAc ; hexosamine-hexosamine ; HPAEC-PAD
• 刊名：Thrombosis Research
• 出版年：2013
• 出版时间：January, 2013
• 年：2013
• 卷：131
• 期：1
• 页码：78-88
• 全文大小：1389 K

文摘

Introduction

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 Gal¦Á1-3Gal¦Â1-GlcNAc-R (¦Á-Gal) and N-glycolylneuraminic acid (Neu5Gc), which constitute epitopes antigenic to humans.

Materials and methods

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

Results and 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 ¦Á-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.