Reactive PEGs for protein conjugation

Bonora, Gian Maria; Drioli, Sara

doi:10.1007/978-3-7643-8679-5_3

Cited by 7 publications

(2 citation statements)

References 37 publications

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“…Yielding amide or urethane bonds, this reaction type reduces the protein charge by the number of attached PEG chains. Alkylating reactions such as PEG‐aldehyde exclusively reacts with the ϵ‐amino side chains of lysine or the N‐terminal α‐amino group, leading to secondary amines, preserving the protein charge (Bonora and Drioli, ; Kinstler et al, ; Roberts et al, ). A major advantage of random reactions comprises the simple chemistry and the availability of binding sites to provide PEG attachment, without the need of an additional modification of the target molecule.…”

Section: Introductionmentioning

confidence: 99%

Optimization of random PEGylation reactions by means of high throughput screening

Maiser

Dismer

Hubbuch

2013

Biotech & Bioengineering

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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.

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Section: Introductionmentioning

confidence: 99%

Optimization of random PEGylation reactions by means of high throughput screening

Maiser

Dismer

Hubbuch

2013

Biotech & Bioengineering

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“… 11 , 15 The condition is more complicated with variation in PEG sizes and shapes that could employed for PEGylation. 16 , 17 Therefore, the examination of all possible sites is not practically achievable in a lab as it requires considerable time and cost. On one hand, the analysis of engineered protein is a problem.…”

Section: Discussionmentioning

confidence: 99%

Computational and nonglycosylated systems: a simpler approach for development of nanosized PEGylated proteins

Mirzaei

Kazemi

Bandehpour

et al. 2016

DDDT

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Cysteine PEGylation includes several steps, and is difficult to manage in practice. In the current investigation, the cysteine PEGylation of erythropoietin analogs was examined using computational and nonglycosylated systems to define a simpler approach for specific PEGylation. Two model analogs (E31C and E89C) were selected for PEGylation based on lowest structural deviation from the native form, accessibility, and nucleophilicity of the free thiol group. The selected analogs were cloned and the expression was assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot using Coomassie blue staining and anti-His monoclonal antibody, respectively. PEGylation with 20 kDa mPEG-maleimide resulted in 79% and 82% conjugation yield for E31C and E89C nonglycosylated erythropoietin (ngEPO) analogs, respectively. The size distribution and charge analysis showed an increase in size and negative charge of the PEGylated forms compared with nonconjugated ones. Biological assay revealed that E31C and E89C mutations and subsequent PEGylation of ngEPO analogs have no deleterious effects on in vitro biological activity when compared to CHO-derived recombinant human erythropoietin. In addition, PEG-conjugated ngEPOs showed a significant increase in plasma half-lives after injection into rats when compared to nonconjugated ones. The development of the cysteine-PEGylated proteins using nonglycosylated expression system and in silico technique can be considered an efficient approach in terms of optimization of PEGylation parameters, time, and cost.

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