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Pegylation of proteins: investigation of coupling techniques
Pegylation, i.e. covalent attachment of polyethylene glycol (PEG) to proteins is commonly performed to improve the protein properties. Increased circulating half-life, increased resistance to proteolytic degradation, and decreased immunogenicity in vivo as well as increased stability in vitro may be achieved by pegylation. Problems associated with pegylation are the difficulties achieving selective coupling to a protein, which often leads to conjugates with great heterogeneity and reduced biological activity.
This work primarily aims at evaluating known coupling chemistries for pegylating proteins. The focus will be to learn about different coupling techniques, to find parameters that affect coupling selectivity and to develop methods to direct the coupling to certain sites on the protein.
The practical work involves three parts:
Coupling reactions will be performed using common PEG-reagents and two model proteins. The effect of PEG size and PEG structure (linear or branched) on the selectivity will be investigated. Selective reagents to N-terminus will be evaluated, parameters that affect coupling will be determined, and the reactions will be optimised.
Suggestions on parameters to be investigated are:
How is the degree of pegylation affected by the excess of PEG?
Changes of pH and ionic strength, which may have effect on which lysins, that are exposed for pegylation.
Purification of conjugates
Purification methods (chromatographic methods i.e. gel permeation chromatography and ion exchange chromatography) will be set up for separation of pegylated and non-pegylated variants.
Characterising the conjugates
For evaluation of coupling selectivity the conjugates will be characterised with regards to degree of modification and sites of attachment. The effect of pegylation on stability will also be investigated. This will involve development or modification of available characterisation methods.
Techniques involved for characterization may be:
Protein structure methods such as SDS-PAGE, IEF, RP-HPLC and Peptide Map.
Biophysical methods for analysis of stability and aggregation
For this project we are looking for a student with education in the biotech area with a large interest in protein chemistry. The assignment will include experiment planning, laboratory work in purification and analysis laboratories, evaluation and interpretation of obtained data. The assignment is based in the Purification and Characterization department, Biopharmaceutical development unit at Biovitrum in Stockholm.
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