title: Development of a lectin binding assay to differentiate between recombinant and endogenous proteins in pharmacokinetic studies of protein-biopharmaceuticals.
abstract: Human glycoproteins, expressed in hamster cell lines, show similar glycosylation patterns to naturally occurring human molecules except for a minute difference in the linkage of terminal sialic acid: both cell types lack α2,6-galactosyl-sialyltransferase, abundantly expressed in human hepatocytes and responsible for the α2,6-sialylation of circulating glycoproteins. This minute difference, which is currently not known to have any physiological relevance, was the basis for the selective measurement of recombinant glycoproteins in the presence of their endogenous counterparts. The assay is based on using the lectin Sambucus nigra agglutinin (SNA), selectively binding to α2,6-sialylated N-glycans. Using von Willebrand factor (VWF), factor IX (FIX), and factor VIIa (FVIIa), it was demonstrated that (i) the plasma-derived proteins, but not the corresponding recombinant proteins, specifically bind to SNA and (ii) this binding can be used to deplete the plasma-derived proteins. The feasibility of this approach was confirmed in spike-recovery studies for all three recombinant coagulation proteins in human plasma and for recombinant VWF (rVWF) in macaque plasma. Analysis of plasma samples from macaques after administration of recombinant and a plasma-derived VWF demonstrated the suitability and robustness of this approach. Data showed that rVWF could be selectively measured without changing the ELISAs and furthermore revealed the limitations of baseline adjustment using a single measurement of the predose concentration only. The SNA gel-based depletion procedure can easily be integrated in existing procedures as a specific sample pre-treatment step. While ELISA-based methods were used to measure the recombinant coagulation proteins in the supernatants obtained by depletion, this procedure is applicable for all biochemical analyses.
authors.affiliation: Baxter Innovations GmbH, Benatzkygasse 2-6, 1220 Vienna, Austria. Electronic address: alfred_weber@baxter.com.
authors.forename: Alfred
authors.initials: A
authors.lastname: Weber
authors.affiliation: Baxter Innovations GmbH, Benatzkygasse 2-6, 1220 Vienna, Austria. Electronic address: eva_minibeck@baxter.com.
authors.forename: Eva
authors.initials: E
authors.lastname: Minibeck
authors.affiliation: Baxter Innovations GmbH, DC Tower, Donau-City-Straße 7, 1220 Vienna, Austria. Electronic address: fritz_scheiflinger@baxter.com.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Scheiflinger
authors.affiliation: Baxter Innovations GmbH, DC Tower, Donau-City-Straße 7, 1220 Vienna, Austria. Electronic address: peter_turecek@baxter.com.
authors.forename: Peter L
authors.initials: PL
authors.lastname: Turecek
mesh_terms: D000818:Animals; D001779:Blood Coagulation Factors / Q000378:metabolism; D001798:Blood Proteins / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006023:Glycoproteins / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D008251:Macaca; D019158:N-Acetylneuraminic Acid / Q000737:chemistry; D037121:Plant Lectins / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism*; D054788:Ribosome Inactivating Proteins / Q000378:metabolism*
chemical_list: D001779:Blood Coagulation Factors; D001798:Blood Proteins; D006023:Glycoproteins; D037121:Plant Lectins; D011994:Recombinant Proteins; C050600:Sambucus nigra lectins; D054788:Ribosome Inactivating Proteins; D019158:N-Acetylneuraminic Acid
keywords: Factor IX; Hamster cell lines; Lectin; Sambucus nigra agglutinin (SNA); von Willebrand factor