title: Capillary electrophoresis-based assessment of nanobody affinity and purity.
abstract: Drug purity and affinity are essential attributes during development and production of therapeutic proteins. In this work, capillary electrophoresis (CE) was used to determine both the affinity and composition of the biotechnologically produced "nanobody" EGa1, the binding fragment of a heavy-chain-only antibody. EGa1 is an antagonist of the epidermal growth factor receptor (EGFR), which is overexpressed on the surface of tumor cells. Using a background electrolyte (BGE) of 50mM sodium phosphate (pH 8.0) in combination with a polybrene-poly(vinylsulfonic acid) capillary coating, CE analysis of EGa1 showed the presence of at least three components. Affinity of the EGa1 components towards the extracellular domain of EGFR was assessed by adding different concentrations (0-12 nM) of the receptor to the BGE while measuring the effective electrophoretic mobility of the respective EGa1 components. Binding curves obtained by plotting electrophoretic mobility shifts as a function of receptor concentration, yielded dissociation constants (Kd) of 1.65, 1.67, and 1.75 nM for the three components, respectively; these values were comparable to the Kd of 2.1 nM obtained for the bulk EGa1 product using a cellular assay. CE with mass spectrometry (MS) detection using a BGE of 25 mM ammonium acetate (pH 8.0) revealed that the EGa1 sample comprised of significant amounts of deamidated, bisdeamidated and N-terminal pyroglutamic acid products. CE-MS using a BGE of 100mM acetic acid (pH 2.8) in combination with a polybrene-dextran sulfate-polybrene capillary coating demonstrated the additional presence of minor products related to incomplete removal of the signal peptide from the produced nanobody. Combining the results obtained from affinity CE and CE-MS, it is concluded that the EGa1 nanobody product is heterogeneous, comprising highly-related proteins that exhibit very similar affinity towards EGFR.
authors.affiliation: Biomolecular Analysis, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands; Division of BioAnalytical Chemistry, AIMMS research group BioMolecular Analysis, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. Electronic address: r.haselberg@vu.nl.
authors.forename: Rob
authors.initials: R
authors.lastname: Haselberg
authors.affiliation: Division of Cell Biology, Department of Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Oliveira
authors.affiliation: Department of Pharmaceutics, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands.
authors.forename: Roy
authors.initials: R
authors.lastname: van der Meel
authors.affiliation: Biomolecular Analysis, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands; Division of BioAnalytical Chemistry, AIMMS research group BioMolecular Analysis, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
authors.forename: Govert W
authors.initials: GW
authors.lastname: Somsen
authors.affiliation: Biomolecular Analysis, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands.
authors.forename: Gerhardus J
authors.initials: GJ
authors.lastname: de Jong
mesh_terms: D000085:Acetates / Q000737:chemistry; D019342:Acetic Acid / Q000737:chemistry; D000595:Amino Acid Sequence; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D016264:Dextran Sulfate / Q000737:chemistry; D019075:Electrophoresis, Capillary; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000276:immunology; D006583:Hexadimethrine Bromide / Q000737:chemistry; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010710:Phosphates / Q000737:chemistry; D011145:Polyvinyls / Q000737:chemistry; D061905:Single-Domain Antibodies / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D013451:Sulfonic Acids / Q000737:chemistry
chemical_list: D000085:Acetates; D010710:Phosphates; D011145:Polyvinyls; D061905:Single-Domain Antibodies; D013451:Sulfonic Acids; C008959:lyapolate; D006583:Hexadimethrine Bromide; D016264:Dextran Sulfate; D066246:ErbB Receptors; D019342:Acetic Acid; C018824:ammonium acetate; C018279:sodium phosphate
keywords: Affinity capillary electrophoresis; Capillary coatings; Capillary electrophoresis–mass spectrometry; Epidermal growth factor receptor; Llama antibody