Selective Measurement of Human Recombinant Von Willebrand Factor (rVWF) in the Presence of Plasma-Derived Von Willebrand Factor (pdVWF)

Abstract

rVWF expressed in Chinese hamster ovary (CHO) cells can be hardly distinguished from pdVWF. This holds true for the primary amino acid sequence and for posttranslational modifications including N- and O-linked glycans. However, because CHO cells lack a a2,6-sialyltransferase, the enzyme attaching N-acetylneuraminic acid (Neu5Ac) in a a2,6- linkage to the galactose residues of complex N-glycans, Neu5Ac will be found in CHO-cell derived glycoproteins only in a2,3-linkage. We used this minute difference in glycosylation to develop a lectin-based immunoassay that allows the selective measurement of human rVWF in human plasma based on the lack of a2,6-bound Neu5Ac on rVWF. At equal VWF:Ag concentrations, mixtures of rVWF and pdVWF will show lower ratios of a2,6-bound Neu5Ac to VWF:Ag than those in pdVWF alone. The difference in ratio is directly related to the concentration of rVWF in the mixture.We used the Sambucs nigra agglutinin (SNA) to measure VWF-bound a2,6-linked Neu5Ac. This lectin binds to Neu5Ac on N-glycans only when Neu5Ac is a2,6-linked to galactose. rVWF/pdVWF was captured from the sample by a plate-immobilized polyclonal rabbit anti-VWF antibody. a2,6-bound Neu5Ac was then specifically detected using biotinylated SNA and streptavidin peroxidase. The assay setup resulted in a linear dose-response curve ranging from 0.2 to 2.9 mU pdVWF:Ag/mL in human plasma. In contrast, rVWF showed no binding, even when tested at concentrations about 100-times higher. Subjecting plate-adsorbed pdVWF to enzymatic desialylation with neuraminidase completely abrogated the binding of SNA. The linkage specificity of the SNA binding was shown in an inhibition study using the trisaccharides 3′-sialyllactose and 6′-sialyllactose as hapten sugars. As expected only 6′-sialyllactose proved able to inhibit the binding of SNA to plate-immobilized pdVWF. This result confirmed the linkage specificity of the SNA binding essential for the discrimination of plasma-derived and rVWF. To demonstrate the feasibility of this approach we spiked a sample of a normal human plasma pool with rVWF in several concentrations ranging from 0 to 1.5 IU/ml. In these samples the VWF:Ag concentrations and the levels of VWF-bound a2,6-Neu5Ac with the SNA binding assay were measured. Then, we calculated the ratio of SNA binding to VWF:Ag on the assumption that all VWF was pdVWF, i.e. contained only a2,6-linked Neu5Ac. The difference between the expected and the actually found ratios of SNA binding to VWF:Ag correlated (R2=0.99) with the amount of rVWF present in the mixtures. Thus, we obtained a calibration curve that allowed the specific measurement of rVWF in a range of 0.2 to 1.5 IU/ml in the presence of 1 IU/ml pdVWF. Both assays, the VWF:Ag ELISA and the SNA-based immunoassay for the measurement of VWF-bound a2,6-bound Neu5Ac, showed acceptable precision.The data show that rVWF in a human plasma sample can readily be differentiated from endogenous pdVWF based on minute, linkage-specific differences of the N-glycan structures. This type of lectin-based immunoassay could be useful in the clinical setting to determine the circulating concentration of therapeutic CHO-cell-derived rVWF and to specifically measure its concentration in the presence of the endogenous glycoprotein.