title: High-performance liquid chromatography as a technique to determine protein adsorption onto hydrophilic/hydrophobic surfaces.
abstract: The purpose of this study is to evaluate the potential of simple high performance liquid chromatography (HPLC) setup for quantification of adsorbed proteins on various type of plane substrates with limited area (<3 cm(2)). Protein quantification was investigated with a liquid chromatography chain equipped with a size exclusion column or a reversed-phase column. By evaluating the validation of the method according to guidelines of the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), all the results obtained by HPLC were reliable. By simple adsorption test at the contact of hydrophilic (glass) and hydrophobic (polydimethylsiloxane: PDMS) surfaces, kinetics of adsorption were determined and amounts of adsorbed bovine serum albumin, myoglobin and lysozyme were obtained: as expected for each protein, the amount adsorbed at the plateau on glass (between 0.15 μg/cm(2) and 0.4 μg/cm(2)) is lower than for hydrophobic PDMS surfaces (between 0.45 μg/cm(2) and 0.8 μg/cm(2)). These results were consistent with bicinchoninic acid protein determination. According to ICH guidelines, both Reversed Phase and Size Exclusion HPLC can be validated for quantification of adsorbed protein. However, we consider the size exclusion approach more interesting in this field because additional informations can be obtained for aggregative proteins. Indeed, monomer, dimer and oligomer of bovine serum albumin (BSA) were observed in the chromatogram. On increasing the temperature, we found a decrease of peak intensity of bovine serum albumin as well as the fraction of dimer and oligomer after contact with PDMS and glass surface. As the surface can act as a denaturation parameter, these informations can have a huge impact on the elucidation of the interfacial behavior of protein and in particular for aggregation processes in pharmaceutical applications.
authors.affiliation: Institut de Science des Matériaux de Mulhouse (IS2M)-UMR 7361 CNRS-UHA, 15 rue de Jean Starcky B.P. 2488, 68057 Mulhouse Cedex, France; Aptar Pharma, Route Des Falaises, 27100 Le Vaudreuil, France.
authors.forename: Tongtong
authors.initials: T
authors.lastname: Huang
authors.affiliation: Institut de Science des Matériaux de Mulhouse (IS2M)-UMR 7361 CNRS-UHA, 15 rue de Jean Starcky B.P. 2488, 68057 Mulhouse Cedex, France.
authors.forename: Karine
authors.initials: K
authors.lastname: Anselme
authors.affiliation: Aptar Pharma, Route Des Falaises, 27100 Le Vaudreuil, France; Aptar Stelmi, 22 Avenue des Nations, BP 59415 Villepinte, 95944 Roissy CDG Cedex, France.
authors.forename: Segolene
authors.initials: S
authors.lastname: Sarrailh
authors.affiliation: Institut de Science des Matériaux de Mulhouse (IS2M)-UMR 7361 CNRS-UHA, 15 rue de Jean Starcky B.P. 2488, 68057 Mulhouse Cedex, France. Electronic address: arnaud.ponche@uha.fr.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Ponche
mesh_terms: D000327:Adsorption; D000818:Animals; D002417:Cattle; D002645:Chickens; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase; D000068598:Data Accuracy; D004129:Dimethylpolysiloxanes / Q000737:chemistry*; D005898:Glass / Q000737:chemistry*; D006736:Horses; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D057230:Limit of Detection; D009113:Muramidase / Q000737:chemistry*; D009211:Myoglobin / Q000737:chemistry*; D020836:Protein Structure, Quaternary; D011804:Quinolines / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D013499:Surface Properties
chemical_list: D004129:Dimethylpolysiloxanes; D009211:Myoglobin; D011804:Quinolines; D012710:Serum Albumin, Bovine; C013830:baysilon; C047117:bicinchoninic acid; D009113:Muramidase
keywords: Aggregation; Denaturation; HPLC; Kinetics; Protein adsorption