Utilization of newly developed immobilized enzyme reactors for preparation and study of immunoglobulin G fragments.
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.
Korecká
Lucie
L
Department of Analytical Chemistry, University of Pardubice, nám. Cs. legií 565, 53210 Pardubice, Czech Republic. lucie.korecka@upce.cz
Bílková
Zuzana
Z
Holèapek
Michal
M
Královský
Josef
J
Benes
Milan
M
Lenfeld
Jiøí
J
Minc
Nicolas
N
Cecal
Roxana
R
Viovy
Jean-Louis
JL
Przybylski
Michael
M
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
D004798:Enzymes; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G