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Protein Stabilization and Immobilization in Three Dimensions

  • Author(s): Mancini, Rock Joseph
  • Advisor(s): Maynard, Heather D
  • et al.

From bee venom to recombinantly produced replacement proteins, biologics have played a key role in the development of civilization. Advancement in two areas of this field is reported: three-dimensional (3-D) encapsulated hydrogel microstructures fabricated by electron-beam lithography and the stabilization of protein therapeutics to environmental insults by trehalose glycopolymer excipients.

A procedure for the fabrication of protein reactive hydrogels of orthogonal reactivity in an encapsulated configuration by electron beam (e-beam) lithography is described. Amine terminated 8-arm poly(ethylene glycol) (PEG) has been modified to produce polymers containing biotin (Biotin-PEG), and alkyne (Alkyne-PEG) end-groups. Hydroxyl terminated PEG has been used to synthesize a polymer with aminooxy functionality (AO-PEG). These polymers have in turn been employed to fabricate micro-gels by e-beam lithography, and it was determined via confocal microscopy that a nominal size exclusion effect exists for hydrogels of feature sizes ranging from 5 to 40 μ. Micro-realignment has been used to pattern Alkyne-PEG encapsulated as a core inside an outer shell of AO-PEG. Hydrogel micro-patterns with encapsulated architectures have been labeled with fluorescent dyes of complimentary reactivity and 3-D spatial orientation of the reactive end-groups has been confirmed via confocal microscopy. The proteins glucose oxidase (GOX) and horseradish peroxidase (HRP) have been modified to contain azide and carbonyl moieties along with green and blue fluorophores respectively. Upon incubation with encapsulated hydrogels it was determined that the modified GOX and HRP proteins reacted selectively with hydrogel end-groups of complimentary reactivity. Retention of enzyme activity was confirmed in this encapsulated configuration via an enzyme cascade reaction with glucose and Amplex Red substrates.

The synthesis of trehalose side chain polymers with stabilization of protein conjugates to environmental stressors is also reported. The glycomonomer 2-methacryl-4,6,4',6'-O-dibenzylidne-α,α-trehalose was synthesized over two steps in 14% yield through dibenzylidene protection and subsequent esterification with methacryloyl chloride. A thiol reactive glycopolymer was prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization using a thiol reactive chain transfer agent (CTA). Furthermore, the glycomonomer 4,6-O-(4-vinylbenzyl)-α,α-trehalose was synthesized in 40% yield over two steps without the use of protecting group chemistry. Polymers containing this glycomonomer were prepared via RAFT polymerization using three different protein reactive CTAs. A range of molecular weights from 4.2 to 49.5 kDa was obtained with well-defined polydispersities for all but the highest molecular weights attempted. The polymers were conjugated to thiolated hen egg white lysozyme and purified. The glycopolymers, when covalently attached to protein or added to the formulation without attachment, significantly increased stability towards lyophilization and heat relative to wild-type protein. Up to 100% retention of activity was observed when lysozyme was stressed ten times with lyophilization and 81% activity when the protein was heated at 90 �C for 1 hour; this is in contrast to 16% and 18% retention of activity, respectively, for the wild-type protein alone. The glycopolymers were compared to equivalent concentrations of trehalose relative to glycopolymer side-chain, and the glycopolymers were found to be superior at stabilizing the protein to lyophilization and heat. When compared to poly(ethylene glycol) (PEG) the glycopolymers were superior at heat stabilization and exhibited comparable lyoprotectant effects. In addition, the protein-glycopolymer conjugates exhibited significant increases in stability when compared to adding the same concentration of un-conjugated polymer to the protein.

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