title: Rapid quantification of protein-polyethylene glycol conjugates by multivariate evaluation of chromatographic data.
abstract: Size exclusion chromatography (SEC) is often applied for characterization of protein-polyethylene glycol (PEG) conjugates regarding the number of attached PEG chains (PEGamers). SEC analysis is advantageous as it is precise, robust, and straightforward to establish. However, most SEC based assays have a maximal throughput of a few samples per hour. We present a strategy to increase analytical throughput based on combining a short column with a fast flow rate, and finally multivariate calibration in order to compensate for the resolution lost in the trade off for speed. Different multivariate approaches were compared and multilinear regression was shown to result in the most precise calibrations. Further, a dynamic calibration approach was developed in order to account for changes in column performance over time. In this way, it was possible to establish a highly precise assay for protein PEGamer quantification with a throughput of 30 samples per hour.
authors.affiliation: Institute of Engineering in Life Sciences, Section IV Biomolecular Separation Engineering, Karlsruhe Institute of Technology-KIT, 76131 Karlsruhe, Germany.
authors.forename: Sigrid K
authors.initials: SK
authors.lastname: Hansen
authors.forename: Benjamin
authors.initials: B
authors.lastname: Maiser
authors.forename: Jürgen
authors.initials: J
authors.lastname: Hubbuch
mesh_terms: D002138:Calibration; D002850:Chromatography, Gel / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D015999:Multivariate Analysis; D011092:Polyethylene Glycols / Q000032:analysis* / Q000737:chemistry; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D012044:Regression Analysis; D012680:Sensitivity and Specificity
chemical_list: D011506:Proteins; D011092:Polyethylene Glycols