Intensification of Bioprocess
Removal of endotoxin with aptamer functionalized superparamagnetic nanoparticles
Endotoxins or lipopolysaccharides (LPS) are a major constituent of the outer membrane of Gram-negative bacteria widely used in biotechnology for production of recombinant therapeutic peptides/proteins and plasmid DNA (pDNA) vaccines. The removal of endotoxins from these products is critical because of their potent biological activities as pyrogens. Various procedures for endotoxin removal, such as ion-exchange, ultrafiltration, extraction, and sucrose gradient centrifugation, have been developed. These, however, are unsatisfactory with respect to selectivity, adsorption capacity, and target product recovery. Aptamers are oligonucleic acid or peptide molecules capable of binding to target molecules (e.g. small molecules, peptides and protein) with high affinity and selectivity. We identified aptamers with specific binding affinity to endotoxin by in vitro selection procedure called capillary electrophoresis (CE) based non-SELEX (systematic evolution of ligands by exponential enrichment). In our previous study, we developed iminodiacetic (IDA) functionalized superparamagnetic iron oxide nanoparticles (NPs) which shows particle size-dependent varying affinity and adsorption capacity for various biomolecules. Superparamagnetic NPs with appropriate surface chemistry have been widely used for numerous applications including MRI, tissue repair, immunoassay, detoxification of biological fluids, hyperthermia, and drug delivery. Besides, magnetic NPs provides many advantages for selective capture or removal of specific compounds from complex biological liquor primarily due to its decoupling of adsorption and recovery steps, fouling resistant non-porous surface, and facilitated separation of target-loaded NPs. In this project, we focus on functionalizing in-house superparamagnetic NPs with endotoxin cognitive aptamers and using the aptamer functionalized NPs for removal of endotoxins to intensify various biological downstream processes and/or dialysis of endotoxin contaminated blood where selective endotoxin capture is critical. Besides, we also work on harnessing endotoxin cognitive aptamers for development of aptamer functionalized conducting polymer based biosensor capable of directly and selectively detecting endotoxin molecules in a complex mixture.
Purification of plasmid DNA using immobilized metal affinity chromatography
Plasmid DNA (pDNA) vaccine comprises gene of an antigen inserted into a bacterial plasmid. Some potential advantages of pDNA vaccines over conventional vaccines include higher safety and ability to induce long-lived immune response against multiple diseases in a single inoculation. With gene therapies and pDNA vaccines moving toward the stage of market approval, a simpler and intensified purification process for pDNA from impurities such as RNA and endotoxins in large-scale is required. Various types of chromatography harnessing differences in physicochemical properties (e.g. size, charge, hydrophobicity and affinity) of separands in a mixture have been employed to purify pDNA. However, similar properties of pDNA and major contaminants such as RNA and endotoxins render selective purification of pDNA to a desired extent challenging. In our previous study, differential interactions of pDNA, RNA and endotoxins with various immobilized and free metal ions were investigated and it was found that Cu2+-charged IDA (iminodiacetic acid) exhibited the best preferential capture for endotoxins, RNA and pDNA in the decreasing order. This hierarchical binding of pDNA and other impurities was harnessed for developing a simple pDNA purification process comprising 1) CuCl2-mediated precipitation of pDNA, RNA and endotoxins; 2) selective resolubilization of pDNA and RNA; 3) selective recovery of pDNA with the use of Cu2+-charged IDA beads. On the other hand, the advent of new chromatographic matrices (e.g. monolith or membrane adsorber) with higher porosity and throughput than those of conventional packed-bed supports has the potential to provide a new and efficient pDNA purification platform. This is primarily because convective flow driven separation and purification on the monolithic support is conducive to flow-independent resolution and dynamic binding capacity, providing competitive advantages for processing of large molecules such as pDNA, RNA and endotoxins. In the present study, we explore dynamic binding capacity of pDNA, RNA and endotoxins to Cu2+-charged IDA monolithic column. The dynamic binding capacity of RNA and endotoxins (determined from the frontal analysis of breakthrough curves) increases with the increase in RNA or endotoxin concentration, while that for pDNA is negligible. In addition, the breakthrough curves of RNA and endotoxins are found to be independent of the flow rate. Taken together, it is expected that an efficient combination of metal affinity chemistry and monolith platform will radically intensify the conventional pDNA purification process, giving rise to a faster and economically more viable process alternative ideal for large-scale processing at high concentration and flow rate.