Chemical Engineering News

Dr. David W. Wood

Ohio State University

Chemical & Biomolecular Engineering

Friday-March 22, 2013

10:00 a.m.

 MUB-Alumni Lounge

New Technologies from Engineered Self-Modifying Proteins

Professor Wood’s work seeks to apply biological concepts of protein function, cell metabolism, genetics and evolution to the molecular-scale development of new technologies.  These goals are achieved through the development of designer fusion proteins that combine domains and functions from unrelated proteins and enzymes.  We typically combine rational protein engineering with genetic selection to create and fine-tune the desired activities.  In oseparations, we have combined a previously developed pH-sensitive self-cleaving protein with a variety of purification tags to produce simple and economical methods for purifying recombinant proteins.  Our most recent work involves rational and evolutionary approaches to optimizing our self-cleaving tags for use in a wider variety of expression hosts.  In biosensing, we have developed allosteric proteins that incorporate human hormone receptors, and have used these proteins to generate Escherichia coli strains that are growth-dependent on hormones and hormone-like compounds.  Remarkably, this genetically simple bacterial sensor can differentiate agonist from antagonist activities and has been effective in detecting a wide variety of strong and weak estrogenic compounds.  More recently, we have applied this system to the discovery of thyroid active compounds, as well as the evaluation of environmental endocrine disruptors in humans and animals, and even the discovery of possible autism-associated environmental factors.  Applications of our designed proteins are far-reaching, and include drug discovery, biosensing, drug activation, reversible knockouts for metabolic research, new genetic selection systems, and advanced cellular control strategies.

Dr. Todd M. Przybycien

 Carnegie Mellon University

Departments of Chemical Engineering and Biomedical Engineering 

Friday-February 22, 2013

10:00am

MUB- Alumni Lounge

Unconventional Applications of Poly(ethylene glycol)-modified Proteins in BioProcessing and Drug Delivery

The covalent attachment of poly(ethylene glycol) (PEG) polymer chains, or “PEGylation,” improves the efficacy of protein drugs by extending their half-lives in the circulation without adversely affecting biological binding activity: the PEG chains are thought to hinder recognition by proteases, inhibitors and antibodies through steric interactions and to retard renal clearance through increased molecular size.  We used a more complete understanding of the solution and interfacial adsorption behavior of PEG-protein conjugates to explore new applications of protein PEGylation in bioprocessing and drug delivery. 

We have developed new, high selectivity protein affinity chromatography media by PEGylating immobilized protein affinity ligands outside of the target binding site.  This discourages the non-specific binding of contaminant species without decreasing target binding.  We find selectivity enhancements for IgG-class antibodies of 2x to 3x for Protein A affinity chromatography media modified with 5 kDa and 20 kDa PEG chains relative to the un-modified media, without loss of antibody binding affinity.  Increased contaminant rejection by Protein A media has important implications for simplifying downstream processing operations for monoclonal antibody production and for extending the operating lifetime of this expensive class of bioseparations media.

We have exploited PEGylation to reduce denaturing adsorptive interactions between proteins and interfaces that limit the successful delivery of protein drugs from poly(lactide-co-glycolide) (PLG) microsphere delivery systems. Oil/water interfaces are present during the generation of protein-loaded PLG microspheres by the double emulsion technique and solid/water interfaces are present as the microspheres erode during delivery.  The depressed adsorption isotherms of conjugates reduce the extent of adsorption at denaturing interfaces and the attached PEG random coils serve as steric diluents at interfaces.  While PEGylation with 20 kDa PEG has little effect on protein behavior at ethyl acetate/water interfaces, at PLG/water interfaces we find decreased extents of adsorption, increased reversibility of adsorption and decreased tendency to aggregate.  These results have translated to ~50% and ~100% improvements in active protein release for monoPEGylated and diPEGylated ribonuclease A, respectively.