title: Quantification of PEGylated proteases with varying degree of conjugation in mixtures: An analytical protocol combining protein precipitation and capillary gel electrophoresis.
abstract: PEGylation, i.e. the covalent attachment of chemically activated polyethylene glycol (PEG) to proteins, is a technique commonly used in biopharmaceutical industry to improve protein stability, pharmacokinetics and resistance to proteolytic degradation. Therefore, PEGylation represents a valuable strategy to reduce autocatalysis of biopharmaceutical relevant proteases during production, purification and storage. In case of non-specific random conjugation the existence of more than one accessible binding site results in conjugates which vary in position and number of attached PEG molecules. These conjugates may differ considerably in their physicochemical properties. Optimizing the reaction conditions with respect to the degree of PEGylation (number of linked PEG molecules) using high-throughput screening (HTS) technologies requires a fast and reliable analytical method which allows stopping the reaction at defined times. In this study an analytical protocol for PEGylated proteases is proposed combining preservation of sample composition by trichloroacetic acid (TCA) precipitation with high-throughput capillary gel electrophoresis (HT-CGE). The well-studied protein hen egg-white lysozyme served as a model system for validating the newly developed analytical protocol for 10kDa mPEG-aldehyde conjugates. PEGamer species were purified by chromatographic separation for calibrating the HT-CGE system. In a case study, the serine protease Savinase(®) which is highly sensitive to autocatalysis was randomly modified with 5kDa and 10kDa mPEG-aldehyde and analyzed. Using the presented TCA protocol baseline separation between PEGamer species was achieved allowing for the analysis of heterogeneous PEGamer mixtures while preventing protease autocatalysis.
authors: Institute of Process Engineering in Life Sciences, Section IV: Molecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany. Electronic address: Josefine.Morgenstern@kit.edu.
authors: Josefine
authors: J
authors: Morgenstern
authors: Institute of Process Engineering in Life Sciences, Section IV: Molecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
authors: Markus
authors: M
authors: Busch
authors: Institute of Process Engineering in Life Sciences, Section IV: Molecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany. Electronic address: Pascal.Baumann@kit.edu.
authors: Pascal
authors: P
authors: Baumann
authors: Institute of Process Engineering in Life Sciences, Section IV: Molecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany. Electronic address: juergen.hubbuch@kit.edu.
authors: Jürgen
authors: J
authors: Hubbuch
mesh_terms: D000818:Animals; D055162:Biocatalysis; D011232:Chemical Precipitation; D002645:Chickens; D019075:Electrophoresis, Capillary / Q000379:methods*; D005260:Female; D009113:Muramidase / Q000032:analysis* / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D014233:Tricarboxylic Acids
chemical_list: D011506:Proteins; D014233:Tricarboxylic Acids; D011092:Polyethylene Glycols; C028210:monomethoxypolyethylene glycol; C421864:hen egg lysozyme; D009113:Muramidase; D012697:Serine Endopeptidases; C032923:microbial serine proteinases
keywords: Autocatalysis; High-throughput capillary gel electrophoresis; PEGamer quantification; Protease PEGylation; TCA precipitation