title: A high-throughput 2D-analytical technique to obtain single protein parameters from complex cell lysates for in silico process development of ion exchange chromatography.
abstract: The accelerating growth of the market for biopharmaceutical proteins, the market entry of biosimilars and the growing interest in new, more complex molecules constantly pose new challenges for bioseparation process development. In the presented work we demonstrate the application of a multidimensional, analytical separation approach to obtain the relevant physicochemical parameters of single proteins in a complex mixture for in silico chromatographic process development. A complete cell lysate containing a low titre target protein was first fractionated by multiple linear salt gradient anion exchange chromatography (AEC) with varying gradient length. The collected fractions were subsequently analysed by high-throughput capillary gel electrophoresis (HT-CGE) after being desalted and concentrated. From the obtained data of the 2D-separation the retention-volumes and the concentration of the single proteins were determined. The retention-volumes of the single proteins were used to calculate the related steric-mass action model parameters. In a final evaluation experiment the received parameters were successfully applied to predict the retention behaviour of the single proteins in salt gradient AEC.
authors.affiliation: Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
authors.forename: Frieder
authors.initials: F
authors.lastname: Kröner
authors.forename: Dennis
authors.initials: D
authors.lastname: Elsäßer
authors.forename: Jürgen
authors.initials: J
authors.lastname: Hubbuch
mesh_terms: D000818:Animals; D002852:Chromatography, Ion Exchange / Q000295:instrumentation / Q000379:methods*; D003198:Computer Simulation; D019476:Insect Proteins / Q000737:chemistry / Q000302:isolation & purification*; D018411:Spodoptera
chemical_list: D019476:Insect Proteins
keywords: High-throughput; In silico; Ion exchange chromatography; Multi-dimensional analysis; Protein purification; Steric mass-action