title: In-situ protein determination to monitor contamination in a centrifugal partition chromatograph.
abstract: Centrifugal partition chromatography (CPC) works with biphasic liquid systems including aqueous two-phase systems. Metallic rotors are able to retain an aqueous stationary phase able to purify proteins. But the adhesion of proteins to solid surface may pose a cross-contamination risk during downstream processes. So it is of utmost importance to ensure the cleanliness of the equipment and detect possible protein contamination in a timely manner. Thereby, a direct method that allows the determination of the effective presence of proteins and the extent of contamination in the metallic CPC rotors was developed. This in-situ method is derived from the Amino Density Estimation by Colorimetric Assay (ADECA) which is based on the affinity of a dye, Coomassie Brillant Blue (CBB), with protonated N+groups of the proteins. In this paper, the ADECA method was developed dynamically, on a 25 mL stainless-steel rotor with various extents of protein contaminations using bovine serum albumin (BSA) as a fouling model. The eluted CBB dye was quantified and found to respond linearly to BSA contamination up to 70 mg injected. Limits of detection and quantification were recorded as 0.9 mg and 3.1 mg, respectively. While the non-specific interactions between the dye and the rotor cannot currently be neglected, this method allows for in situ determination of proteins contamination and should contribute to the development of CPC as a separation tool in protein purification processes.
authors.affiliation: Univ Lyon, CNRS, Université Claude Bernard Lyon 1, Ens Lyon, Institut des Sciences Analytiques, UMR 5280, 5 rue de la Doua, F-69100 Villeurbanne, France; Kromaton Sarl, Groupe Rousselet-Robatel, 45 Avenue Rhin et Danube, 07100 Annonay, France.
authors.forename: Feriel
authors.initials: F
authors.lastname: Bouiche
authors.affiliation: Univ Lyon, CNRS, Université Claude Bernard Lyon 1, Ens Lyon, Institut des Sciences Analytiques, UMR 5280, 5 rue de la Doua, F-69100 Villeurbanne, France. Electronic address: karine.faure@isa-lyon.fr.
authors.forename: Karine
authors.initials: K
authors.lastname: Faure
mesh_terms: D000818:Animals; D002417:Cattle; D002498:Centrifugation / Q000379:methods*; D003377:Countercurrent Distribution; D004866:Equipment Contamination / Q000517:prevention & control*; D007202:Indicators and Reagents / Q000378:metabolism; D012394:Rosaniline Dyes / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000378:metabolism*
chemical_list: D007202:Indicators and Reagents; D012394:Rosaniline Dyes; D012710:Serum Albumin, Bovine; C004692:coomassie Brilliant Blue
keywords: Bovine serum albumin; Centrifugal partition chromatography; Coomassie brillant blue; Countercurrent chromatography; Proteins contamination