Glycan Modulation of Pharmaceutical Glycoproteins by In Vitro Enzymatic Approaches
Abstract
Many therapeutic proteins are glycoproteins which are proteins that have chains of sugars (i.e. glycans) attached to them at specific sites in a process known as glycosylation. The structure and distribution of glycans on pharmaceutical glycoproteins have a dramatic effect on their efficacy, safety, immunogenicity, and stability. In this project, therapeutic glycoproteins that are known to act as protective bioscavengers against chemical and biological toxins are produced recombinantly in plant-based production systems. Plant cells, as higher eukaryotes, can glycosylate proteins and the initial steps are identical between humans and plants. In addition, they do not harbor or propagate mammalian pathogens. Furthermore, plants can be cultured in mass scales. They are ideal expression systems for the low-cost and large-scale production of glycoproteins that are important for counteracting weapons of mass destruction, particularly chemical and biological agents. The goals of this project are to develop novel methods for the production of two bioscavenger glycoproteins, human butyrylcholinesterase (BuChE), a bioscavenger for organophosphorus (OP) nerve agents, and a chimeric anthrax antitoxin (CGM2-Fc), with improved efficacy against their binding targets by altering the glycan structures that are attached to the proteins. BuChE is produced in transgenic rice cell culture bioreactors and CGM2-Fc is produced transiently in tobacco plants in an indoor facility. Following production and purification of the bioscavengers, advanced analytical tools (liquid chromatography and mass spectrometry) are used to determine precise glycan structures at each site. In vitro enzymatic processes are then used to modify the glycan structures, while computer modeling using atomistic molecular dynamic simulations and coarse grain modeling approaches predicts the effects of glycan structures on glycoprotein structure and target binding energy and is used to direct the glycan modification studies. Finally, experimental studies are used to test the efficacy of the various therapeutic bioscavenger glycoforms (including target binding rate constants, dissociation rates, thermodynamic parameters of interaction, pharmacokinetics and challenge studies). Improved fundamental understanding of how the glycan structures impact binding efficiency for therapeutic bioscavengers will allow us to design protective bioscavengers with improved efficacy. This project will provide fundamental understanding of the structure-function relationship of glycans on glycoprotein therapeutics, and supply technologies for large-scale production of glycoproteins in plants and in vitro enzymatic modification of glycans on glycoproteins. Improved therapeutics may be discovered and the approach is generalizable to other bioscavenger glycoproteins.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- May 26, 2016
- Source ID
- HDTRA11510054
Entities
People
- Karenann Mcdonald
Organizations
- Defense Threat Reduction Agency
- University of California, Davis