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Title: Site-specific, efficient and stable PEGylation
Author: Khayrzad, Farzad Ahmad
ISNI:       0000 0004 7660 1603
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2019
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Protein-based medicines have shown clinical benefit in almost all fields of medicine. However, a key limitation for many therapeutic proteins lies in their short circulation half-life after administration. Several strategies have been investigated to address this issue, which include use of (i) external pumps, (ii) parenterally administered colloidal and microparticulate associated depots and (iii) conjugation of the proteins/peptides to a water-soluble polymer. Of all the strategies available for extending half-life of proteins, PEGylation has shown to be the most versatile in the clinic. PEGylation involves the covalent conjugation of polyethylene glycol (PEG) to the protein of interest. It is recognised that key needs in protein and peptide PEGylation reactions are to achieve site-specificity, high efficiency and conjugate stability to avoid de-conjugation of the PEG from the protein. Development of mono and bis-alkylation PEGylation reagents that undergo thiol specific conjugation on proteins and peptides has been accomplished. PEG reagents based on a mono-thiol alkylation linker (M1-PEG) were developed to (i) liberate the reactive functionality in a controlled way to match protein/peptide reactivity, (ii) to give stable PEG-polypeptide conjugates that address the well-known instability issues that are associated with maleimide reagents and (iii) to be useful for practical radiolabelling experiments during preclinical studies. PEG reagents based on bis-thiol alkylation linker (B1-PEG) were developed to undergo reaction with the two thiols derived from the cysteines from the disulfide bond. B1-PEG reagents result in a 3-carbon bridge spanning between the two thiols from the original disulfide. The 3-carbon bridge helps to stabilise the replaced natural disulfide linkages with PEG conjugated to the middle carbon of the bridge. M1-PEG and B1-PEG reagents undergo conjugation by a Michael addition-elimination pathway. An aryl-ketone acts as an electron withdrawing group to allow the Michael addition. Reagents derived from different PEG molecular weights, ranging from 2-40 kDa were prepared in good purity and yield. These thiol-selective PEG reagents were prepared with mono-functional, homo-bifunctional and tetra homo-functional linkers that were characterised by NMR, RP-HPLC, MALDI TOF MS and SDS-PAGE. The aryl ketone was evaluated to develop a locking strategy for PEGylated proteins based on mild borohydride reduction of the ketone to a secondary alcohol. The locking strategy can play a pivotal role to give stable conjugates that do not undergo competitive retro-Michael reaction with other nucleophiles, which can allow a modified protein to better exert its biopharmaceutical effect(s). The M1-PEG and B1-PEG reagents were examined with a peptide and a modified M-1 linker was used to prepare an antibody-drug conjugate (ADC). Monomeric and multimeric versions of an antimicrobial peptide (AMP) were prepared using M1-PEG and B1-PEG reagents. AMP PEGylation reactions and keto-reduction stabilisation reactions were performed in scales ranging from microgram to milligram. Mono-PEGylated AMP was further characterised before and after performing the locking strategy by means of RP-HPLC and LC/MS to produce a stable PEG-AMP conjugate that was shown to retain activity. In addition, a multimeric PEGylated AMP conjugate also showed that site specific, efficient and stable conjugates could be prepared in good yield and retain microbiological activity. A mono-alkylation M-1 linker was prepared and then used to prepare an ADC derived from trastuzumab. The M1-ADC was prepared with an average drug to antibody ratio of 4 (DAR-4). Post locking step, the M1-ADC demonstrated retained binding affinity and cytotoxicity in in-vitro assays. A maleimide based DAR-4 ADC (Mal-ADC) was prepared for a comparative stability study with M1-ADC DAR-4 in simulated serum and it was shown that locked M1-ADC was more stable than its maleimide comparator.
Supervisor: Brocchini, S. ; Godwin, A. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available