| Summary: | Biologics are an increasing class of pharmaceuticals that possess many therapeutic benefits over typical, small‐molecule drugs, such as; high specificity, reduced frequency of off‐target effects, and the ability to mimic the body’s own physiological system. Despite these advantages, biologics still suffer from limitations due in part to their inherent protein composition, including; rapid circulatory degradation, renal clearance, and immunogenicity. Consequently, methods to modify biologics have been sought that can ameliorate these limitations yet still maintain their efficacy. PEGylation, defined as the conjugation of polyethylene glycol (PEG) to biologics, is one such modification. The coupling of PEG increases the overall molecular weight (MW) of the biologic, resulting in reduced renal clearance, whilst simultaneously acting as a shield to protect against proteolytic degradation. Both of these effects extend the half‐life of coupled biologics and improve their bioavailability. Furthermore, the extended residence times of PEGylated agents leads to an increase in efficacy in comparison to the non‐PEGylated counterpart; since the therapeutic moiety has more opportunities to elicit a response. The shielding effect of PEG is also reported to reduce the immunogenicity of biologics through preventing immune recognition of antigenic epitopes present on the biologic’s surface. The benefits of PEGylation have been well validated and there are a number of PEGylated agents clinically available, with more in various stages of development. Despite this, however, there is still little known concerning the disposition, metabolism and biological fate of PEGylated proteins. Furthermore, whilst PEGylation can reduce the immunogenicity of a protein, this has not precluded the onset of a new immunogenicity raised against the PEG moiety itself, nor indeed has PEGylation been shown to universally reduce the immunogenicity of coupled proteins. Anti‐drug antibodies (ADAs) and hypersensitivity reactions have been reported in both animal studies and patients receiving PEGylated agents. Furthermore, these adverse drug reactions (ADRs) occur against both the PEG and protein moieties of the conjugate. Consequently, there is a real need to understand the fundamental mechanics behind the metabolism, disposition and biological fate of PEGylated biologics, as well as defining the effect of PEGylation on the immune recognition, processing and presentation of the coupled protein. The bioanalysis of PEGylated proteins is hindered by inherent difficulties associated with the PEG moiety. PEG is transparent, non‐fluorescent, contains no ultra‐violet (UV) chromophore, is polydisperse, and is not easily ionised; making analysis by spectroscopy and mass spectrometry difficult. The utility of radiolabeling is also limited due to issues arising from placement of the radiolabel within the conjugate. Consequently, alternative analytical tools are required for the comprehensive bioanalysis of PEGylated proteins. In light of these issues the studies described in this thesis aimed to develop methodologies that can: 1) provide quantitative information concerning the biological fate and disposition of both the PEG and protein moieties of a PEGylated protein, and 2) define the effect of PEGylation on the processes involved in generating an immune response. The first investigation in this thesis involved developing and optimising gel‐based methodology and 1H nuclear magnetic resonance (NMR) spectroscopy to monitor the kinetics, disposition and biological fate of a model PEGylated protein, 40KPEG‐insulin, in a rodent disposition study. Male Wistar rats were intravenously administered a single dose of 40KPEG‐insulin and maintained over a period of 28 days. Plasma and urine samples were collected almost daily and liver and kidneys harvested on days 14 and 28. 1H NMR and gelbased analysis, incorporating western blotting for both PEG and insulin, and a barium iodide (BaI2) stain for PEG, revealed that PEG persists in both biological tissues and fluids across 28 day days. However, the anti‐insulin western blots revealed that the insulin moiety was either metabolically cleaved or sequentially degraded from PEG to the extent that no immunodetectable insulin was detected by day 7 in urine, or by day 14 in plasma, and could not be detected at all in liver and kidney tissue on days 14 and 28. However, an in vitro plasma stability study found the insulin moiety of 40KPEG‐insulin to be stable in plasma over 7 days, indicating that the loss of insulin signal observed in vivo must be occurring following cellular internalisation; potentially allowing for the liberated protein to be immunologically processed. The second investigation in this thesis concerned the effect of PEGylation on in vitro cellular internalisation. A range of different PEG MWs were incubated with dendritic cells (DCs) – a model antigen presenting cell (APC) – and internalisation was assessed by flow cytometry and fluorescence microscopy. These data revealed that DC internalise PEG regardless of MW, suggesting that PEGylation, in terms of MW, may have little effect on the internalisation of a PEGylated biologic by APC – over the clinically relevant PEG MW range used in this study. Thirdly, the effect of PEGylation was assessed on the lysosomal and proteasomal pathways of antigen processing and presentation, events en route to producing an immune response which occur after cellular internalisation. Insulin was conjugated to a range of different MW PEGs and incubated with either lysosomes or proteasomes. PEG was shown to be stable to lysosomal proteolysis over a period of 7 days; however insulin was completely degraded within 2 hours. When coupled to PEG, the insulin moiety was again degraded completely within 2 hours regardless of PEG MW. No PEG MW effect was observed when analysing the peptide repertoires generated between the PEGylated insulin conjugates. Indeed, it was shown that PEGylation provides only local protection against lysosomal degradation at the site of attachment. However, when degraded by proteasomes a PEG MW‐dependent effect was observed. Over 48 hours, insulin was nearly degraded to completion when conjugated to 20 kDa PEG. But when coupled to either 30 or 40 kDa PEG, only ~50% of the insulin moiety was degraded. When analysing the peptide repertoires, again PEG, regardless of MW, provided partial protection at the site of attachment. However, there was much more variation in the peptides generated between each PEGylated insulin. In conclusion, the methods developed in this thesis represent facile, inexpensive analytical tools to comprehensively analyse the disposition, kinetics and biological fate of both the PEG and protein moieties of PEGylated proteins. Furthermore, the investigation presented in this thesis was the first of its kind to demonstrate that cleavage and/or degradation of a PEGylated protein can occur in vivo. Consequently, the methods described in this thesis could be easily used to provide a measure of the pharmacokinetics and tissue retention of PEGylated biologics in man. When analysing the effect of PEGylation on the processing of PEGylated proteins, there was shown to be little difference when processed via the lysosomal pathway of antigen processing. However, based on the peptide repertoires generated PEG did provide local protection against degradation at the site of attachment – suggesting that site‐specific PEGylation, tailored for individual biologics, may provide a viable route to reduce the immunogenicity of the attached protein. Future investigation is warranted to further elucidate the potential effects of PEGylation on immunological events occurring downstream of lysosomal/proteasomal processing.
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