TY - JOUR

T1 - A magnetic adsorbent-based process for semi-continuous PEGylation of proteins

A1 - Ottow,Kim Ekelund

A1 - Maury,Trine Lütken

A1 - Hobley,Timothy John

A1 - Lund-Olesen,Torsten

A1 - Hansen,Mikkel Fougt

AU - Ottow,Kim Ekelund

AU - Maury,Trine Lütken

AU - Hobley,Timothy John

AU - Lund-Olesen,Torsten

AU - Hansen,Mikkel Fougt

PB - Wiley - V C H Verlag GmbH & Co. KGaA

PY - 2011

Y1 - 2011

N2 - A semi-continuous magnetic particle-based process for the controlled attachment of PEG (PEGylation) to proteins is described for the first time. Trypsin and 2 kDa mono-activated PEG were used to systematically develop the steps in the process. Proof of concept was shown in a microfluidics system to minimize reagent consumption. Two streams containing (i) 1.2 g/L trypsin and (ii) 4 g/L magnetic adsorbents derivatized with the reversible affinity ligand benzamidine were pumped into a pipe reactor. At the exit, a third solution of activated PEG (0-40 g/L) was introduced and the solutions immediately fed into a second reactor. Upon exiting, the mixture was combined in a third reactor with a fourth stream of free amine groups to stop the reaction (50 mM lysine). The mixture continued into a high-gradient magnetic separator where magnetic supports, with PEGylated trypsin still attached, were captured and washing and elution steps were subsequently carried out. Analysis of the conjugates (with SDS-PAGE & LC-MS) showed that the extent of PEGylation could be controlled by varying the reaction time or PEG concentration. Furthermore, the PEG-conjugates had higher enzyme activity compared to PEGylation of non-immobilized trypsin.

AB - A semi-continuous magnetic particle-based process for the controlled attachment of PEG (PEGylation) to proteins is described for the first time. Trypsin and 2 kDa mono-activated PEG were used to systematically develop the steps in the process. Proof of concept was shown in a microfluidics system to minimize reagent consumption. Two streams containing (i) 1.2 g/L trypsin and (ii) 4 g/L magnetic adsorbents derivatized with the reversible affinity ligand benzamidine were pumped into a pipe reactor. At the exit, a third solution of activated PEG (0-40 g/L) was introduced and the solutions immediately fed into a second reactor. Upon exiting, the mixture was combined in a third reactor with a fourth stream of free amine groups to stop the reaction (50 mM lysine). The mixture continued into a high-gradient magnetic separator where magnetic supports, with PEGylated trypsin still attached, were captured and washing and elution steps were subsequently carried out. Analysis of the conjugates (with SDS-PAGE & LC-MS) showed that the extent of PEGylation could be controlled by varying the reaction time or PEG concentration. Furthermore, the PEG-conjugates had higher enzyme activity compared to PEGylation of non-immobilized trypsin.

KW - Trypsin

KW - Continuous process

KW - Protein modification

KW - High gradient magnetic fishing

U2 - 10.1002/biot.201000360

DO - 10.1002/biot.201000360

JO - Biotechnology Journal

JF - Biotechnology Journal

SN - 1860-6768

IS - 4

VL - 6

SP - 396

EP - 409

ER -