Optimization of random PEGylation reactions by means of high throughput screening.
Since the first FDA approval of a PEGylated product in 1990, so called random PEGylation reactions are still used to increase the efficacy of biopharmaceuticals and represent the major technology of all approved PEG-modified drugs. However, the great influence of process parameters on PEGylation degree and the PEG-binding site results in a lack of reaction specificity which can have severe impact on the product profile. Consequently, reproducible and well characterized processes are essential to meet increasing regulative requirements resulting from the quality-by-design (QbD) initiative, especially for this kind of modification type. In this study we present a general approach which combines the simple chemistry of random PEGylation reactions with high throughput experimentation (HTE) to achieve a well-defined process. Robotic based batch experiments have been established in a 96-well plate format and were analyzed to investigate the influence of different PEGylation conditions for lysozyme as model protein. With common SEC analytics highly reproducible reaction kinetics were measured and a significant influence of PEG-excess, buffer pH, and reaction time could be investigated. Additional mono-PEG-lysozyme analytics showed the impact of varying buffer pH on the isoform distribution, which allowed us to identify optimal process parameters to get a maximum concentration of each isoform. Employing Micrococcus lysodeikticus based activity assays, PEG-lysozyme33 was identified to be the isoform with the highest residual activity, followed by PEG-lysozyme1 . Based on these results, a control space for a PEGylation reaction was defined with respect to an optimal overall volumetric activity of mono-PEG-lysozyme isoform mixtures.
Institute of Engineering in Life Sciences, Section IV: Biomolecular Separation Science, Karlsruhe Institute of Technology (KIT), 76131, Karlsruhe, Germany.
Benjamin
B
Maiser
Florian
F
Dismer
Jürgen
J
Hubbuch
D002850:Chromatography, Gel; D057166:High-Throughput Screening Assays / Q000379:methods*; D007700:Kinetics; D008956:Models, Chemical; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D020033:Protein Isoforms; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D009113:Muramidase
PEGylation; high throughput experimentation; high throughput process development; lysozyme; quality-by-design