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METHODOLOGY : The PEGylation processes used to date for protein conjugation can be broadly classified into two types: Solution phase Batch process On-column fed-batch process.

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SOLUTION PHASE BATCH PROCESS: The simple and commonly adopted batch process involves the mixing of reagents together in a suitable buffer solution, preferably at a temperature between a 4 and 6˚ C & under the presence of Nitrogen. followed by the separation and purification of the desired product.

Solution phase protein conjugation using PEG-NHS ester :

Solution phase protein conjugation using PEG-NHS ester To the clear solution of mono carboxy poly ethylene glycol N-hydroxy succinamide is added in the presence of nitrogen. and cool the mass at 0°C under stirring. and this results in the formation of PEG-NHS ester The above formed ester is then conjugated through N terminal amino group of the protein


Example: Cytochrome C conjugation with PEG-NHS ester: Stock solutions of 5ml each PEG-NHS (20 mg/ml) ester and cytochrome C (20 mg/ml) were mixed together at pH 7.4 in 25ml conical flask and stirred for 1 hr at 20°C. Reaction was terminated by adding a drop of 0.1 M Hcl

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ON-COLUMN FED-BATCH PROCESS : On-column PEGylation may be achieved by immobilising the protein to the solid phase during PEGylation, achieving at least partial separation by washing the column free of reactants and by-products in the mobile phase, and then eluting the product. T he native protein to an ion exchanger and then passed activated PEG through the column. Because PEGylation generally weakens (IEX) interactions, they were able to elute the PEGylated protein from the column at salt concentrations that did not elute the native form. They then replaced the equivalent amount of protein that was eluted and repeated the process.

Novel Approaches::

Novel Approaches: 1. Others have attempted to use on-column PEGylation to orientate the protein so that the active site of the protein is held towards the solid-phase interface, thus hindering conjugation at sites that might interfere with activity. This may result in a higher activity of the PEGylated form over forms that are PEGylated at random in free solution.


2 A latest approach to column PEGylation size exclusion reaction chromatography (SERC), was used by Fee. In this approach the protein and the activated PEG are injected into the column sequentially, lowest molecular size first. The larger reactant has a higher linear velocity so catches up to the smaller one such that a moving reaction zone is formed as they migrate through the column . The PEGylated protein formed is larger than either reactant, it moves ahead of the reaction zone. With a sufficient length of column, both reaction and separation of all species can be achieved in a single unit operation.

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Ideally, the activated PEG should be larger than the native protein so that a reaction front forms as the PEG catches up with the protein in the column . This way, the faster moving PEGylated form will move ahead of the reaction front through the (inert) protein band and so escape further PEGylation . In this mode, the native protein is converted from the trailing edge of the band toward the leading edge, leaving low molecular weight by-products behind.


Applications: Improved drug solubility Reduced dosage frequency, without diminished efficacy with potentially reduced toxicity Extended circulating life Increased drug stability Enhanced protection from proteolytic degradation.


CONCLUSION PEGylating small molecules is a feasible strategy for enhancing and optimizing current compounds. The technology can be effectively transferred from macromolecules to small molecules to provide a similar range of clinically important benefits such as greater efficacy, reduced side effects, a reduced dosing schedule, and increased patient compliance The universe of compounds that could be PEGylated is large, but extensive knowledge of structure-activity relationships and PEGylation properties is crucial to tapping this resource.


REFERENCES: ^ Abuchowski, A.; McCoy, J. R.; Palczuk, N. C.; van Es, T. ; Davis, F. F. Journal of Biological Chemistry. ^ Veronese, F. M. ; Pasut, G. "PEGylation, successful approach to drug delivery", Drug Discovery Today . ^ Veronese, F. M.; Harris, J. M. "Introduction and overview of peptide and protein pegylation", Advanced Drug Delivery Reviews. ^ Fee, C. J. ; Van Alstine, J. M. "PEG-proteins: Reaction engineering and separation issues", Chemical Engineering Sciences.



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