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A. Khalili, A. Hamid, S. Balan, J. Fletcher, P. T. Khaw, S. Brocchini; Disulfide Bridging PEGylation of Avastin. Invest. Ophthalmol. Vis. Sci. 2008;49(13):778. doi: https://doi.org/.
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To site-specifically PEGylate Avastin at the accessible disulfides to determine if subconjunctival residence time can be increased compared to the native antibody. Disulfide bridging PEGylation (Balan et al 2007) has been shown to site-specifically PEGylate proteins. The method relies on the reduction of an accessible protein disulfide followed by the formation of a 3 carbon bridge that spans the 2 cysteines from the original disulfide with PEG attached to the bridge.
The partial reduction of Avastin was conducted using dithiothreitol (DTT) at pH 7.8 for 30 minutes at ambient temperature. The DTT was removed by passing the reaction solution over a PD-10 column. Protein fractions were collected and then a 10 kDa PEGylation reagent was added (1-5 eq.). The solution was incubated at 4°C or ambient temperature for 12 hours, and then a solution of reduced and oxidised glutathione (1:1) was added. The pH was reduced to 6.2 while being run over a PD-10 column to remove reagents. Reduction and PEGylation were followed by SDS-PAGE using colloidal blue to stain for protein and barium iodide to stain for poly(ethylene glycol).
Disulfide bridging PEGylation requires that an intact disulfide be reduced to liberate the two cysteine sulphur atoms so they can be conjugated to a PEG molecule. In a protein such as Avastin with multiple disulfides, reducing conditions must be found that do not cause protein denaturation while reducing the accessible disulfides. Partial reduction of the accessible heavy chain and Fab disulfides in Avastin was possible with DTT. These disulfides are distal from the binding region of the antibody. Subsequent incubation at ambient temperature with 10 kDa PEG reagent (2-5 eq.) resulted in near quantitative PEGylation of Avastin as observed by SDS-PAGE. In control reactions, no PEGylation was observed unless a disulfide was first reduced.
These results indicate that Avastin can be PEGylated at high conversion at an accessible disulfide. Reduction conditions are critical to ensure that only the accessible disulfides are reduced.
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