How Full-Length FVIII Benefits from Its Heterogeneity \u2013 Insights into the Role of the B-Domain

Julia Anzengruber,Josenato Ilas,Norbert Haider,Philipp Bärnthaler,Friedrich Scheiflinger,Karima Benamara,Martin Feichtinger,Birgit M Reipert,Nina Pruckner,Mantas Malisauskas

doi:10.1007/s11095-019-2599-2

Julia Anzengruber, Josenato Ilas + Show 8 more

Open Access

https://doi.org/10.1007/s11095-019-2599-2

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Journal: Pharmaceutical research	Publication Date: Apr 1, 2019
Citations: 5	License type: open-access

Affiliation: Takeda (Austria)

Abstract

PurposeTo explore how the natural heterogeneity of human coagulation factor VIII (FVIII) and the processing of its B-domain specifically modulate protein aggregation.MethodsRecombinant FVIII (rFVIII) molecular species containing 70% or 20% B-domain, and B-domain-deleted rFVIII (BDD-rFVIII), were separated from full-length recombinant FVIII (FL-rFVIII). Purified human plasma-derived FVIII (pdFVIII) was used as a comparator. Heterogeneity and aggregation of the various rFVIII molecular species, FL-rFVIII and pdFVIII were analysed by SDS-PAGE, dynamic light scattering, high-performance size-exclusion chromatography and flow cytometry-based particle analysis.ResultsFL-rFVIII and pdFVIII were heterogeneous in nature and demonstrated similar resistance to aggregation under physical stress. Differences were observed between these and among rFVIII molecular species. FVIII molecular species exhibited diverging aggregation pathways dependent on B-domain content. The propensity to form aggregates increased with decreasing proportions of B-domain, whereas the opposite was observed for oligomer formation. Development of cross-β sheet-containing aggregates in BDD-rFVIII induced effective homologous seeding and faster aggregation. Naturally heterogeneous FL-rFVIII and pdFVIII displayed the lowest propensity to aggregate in all experiments.ConclusionsThese results demonstrate that pdFVIII and FL-rFVIII have similar levels of molecular heterogeneity, and suggest that heterogeneity and the B-domain are involved in stabilising FVIII by modulating its aggregation pathway.

Highlights

MATERIALS AND METHODSHuman factor VIII (FVIII) is an essential plasma glycoprotein in the blood coagulation cascade, serving as a co-factor for factor IXa in the conversion of factor X to factor Xa [1,2]
Heterogeneous FL-recombinant FVIII (rFVIII) and plasma-derived FVIII (pdFVIII) displayed the lowest propensity to aggregate in all experiments. These results demonstrate that pdFVIII and FL-rFVIII have similar levels of molecular heterogeneity, and suggest that heterogeneity and the B-domain are involved in stabilising FVIII by modulating its aggregation pathway
Page 5 of 12 77 within the B-domain. rFVIII molecular species containing 100% (B100-), 70% (B70-), 20% (B20-) or 0% B-domain were the main rFVIII species found in FL-rFVIII, with heavy chain (HC) migration levels at 180, 150, 110 and 90 kDa, respectively (Fig. 1b)

Summary

Introduction

MATERIALS AND METHODSHuman factor VIII (FVIII) is an essential plasma glycoprotein in the blood coagulation cascade, serving as a co-factor for factor IXa in the conversion of factor X to factor Xa [1,2]. A defect or deficiency in FVIII results in haemophilia A, one of the most common severe bleeding disorders [3]. Recombinant protein technology has generated recombinant FVIII (rFVIII) products to treat haemophilia A by protein replacement. Products differ mainly in glycosylation [4] and the presence or absence of the B-domain sequence in the FVIII cDNA, commonly referred to as fulllength (FL-) rFVIII and B-domain-deleted (BDD-) rFVIII [5,6,7,8,9]. All protein-based drugs, including FVIII, bear a certain risk to aggregate during manufacturing and shelf storage, and a susceptibility to mishandling during treatment [10,11]. The presence of aggregates in protein therapeutics has induced unwanted immune responses in some patients, which may affect the therapy’s efficacy [12,13,14,15,16,17,18,19,20]

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Discussion

Conclusion