Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.647295
Title: New strategies for protein-to-protein conjugation
Author: Farys, M.
ISNI:       0000 0004 5366 1988
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2015
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Abstract:
Chimeric proteins (e.g. fusion proteins, bispecific antibodies) often have more than one therapeutic function. All of the clinically validated examples (e.g. Enbrel®) are produced using recombinant strategies. Considering the recombinant fusion of two different proteins, there is a need to consider the design of the construct, specifically the polypeptide linker. Challenges remain to make small families of chimeric proteins for preclinical studies as well as to produce these proteins at manufacturing scale. A possible alternative strategy is a recombinant-chemical conjugation approach using separate proteins and heterobifunctional reagents. The concept of conjugating two proteins together has been known for several decades, but there have been limitations because many protein conjugation reagents were not site-specific. To explore strategies to prepare chimeric proteins more efficiently, site-specific heterobifunctional reagents were developed. These heterobifunctional reagents were derived from PEG and were designed to undergo site-specific protein conjugation at one PEG terminus by either mono- or bis-alkylation Michael reaction. The other reagent terminus comprised a functional moiety that was capable of undergoing a reaction with a reactive group not found on a protein (e.g. aldehyde and hydrazine). These bio-orthogonal moieties would then be matched to undergo reaction to give Protein1-(PEG)2×5k-Protein2 conjugates with PEG being used as a spacer. To prepare Protein1-(PEG)2×5k-Protein2 it was first necessary to generate protein-PEG5k-X(Y) intermediates. This was accomplished using only 1-2 molar equivalents of either mono- or bis-alkylating-PEG5k-X/Y reagents with a conversion achieved up to 75%. The X and Y functional groups were hydrazine and aldehyde. Two heterodimers (IFN-(PEG)2×5k-HSA and amylase-(PEG)2×5k-lipase) were then prepared by coupling two of the protein intermediates together. Purified amylase-(PEG)2×5k-lipase heterodimer was active when tested for both lipase and amylase activity. IFN-(PEG)2×5k-HSA heterodimer was shown to have comparable specific activity to Pegasys®.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.647295  DOI: Not available
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