title: Utilization of newly developed immobilized enzyme reactors for preparation and study of immunoglobulin G fragments.
abstract: The newly developed immobilized enzyme reactors (IMERs) with proteolytic enzymes chymotrypsin, trypsin or papain were used for specific fragmentation of high molecular-mass and heterogeneous glycoproteins immunoglobulin G (IgG) and crystallizable fragment of IgG (Fc). The efficiency of splitting or digestion were controlled by RP-HPLC. The specificity of digestion by trypsin reactor was controlled by MS. IMERs (trypsin immobilized on magnetic microparticles focused in a channel of magnetically active microfluidic device) was used for digestion of the whole IgG molecule. The sufficient conditions for IgG digestion in microfluidic device (flow rate, ratio S:E, pH, temperature) were optimized. It was confirmed that the combination of IMERs with microfluidic device enables efficient digestion of highly heterogeneous glycoproteins such as IgG in extremely short time and minimal reaction volume.
authors.affiliation: Department of Analytical Chemistry, University of Pardubice, nám. Cs. legií 565, 53210 Pardubice, Czech Republic. lucie.korecka@upce.cz
authors.forename: Lucie
authors.initials: L
authors.lastname: Korecká
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Bílková
authors.forename: Michal
authors.initials: M
authors.lastname: Holèapek
authors.forename: Josef
authors.initials: J
authors.lastname: Královský
authors.forename: Milan
authors.initials: M
authors.lastname: Benes
authors.forename: Jiøí
authors.initials: J
authors.lastname: Lenfeld
authors.forename: Nicolas
authors.initials: N
authors.lastname: Minc
authors.forename: Roxana
authors.initials: R
authors.lastname: Cecal
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Viovy
authors.forename: Michael
authors.initials: M
authors.lastname: Przybylski
mesh_terms: D000595:Amino Acid Sequence; D019149:Bioreactors; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004798:Enzymes / Q000378:metabolism*; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000378:metabolism*; D007074:Immunoglobulin G / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D004798:Enzymes; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G