Performance of R‐N(R′)‐R′′ functionalised poly(glycidyl methacrylate‐co‐ethylene glycol dimethacrylate) monolithic sorbent for plasmid DNA adsorption

Abstract

High-throughput plasmid DNA (pDNA) manufacture is obstructed predominantly by the performance of conventional stationary phases. For this reason, the search for new materials for fast chromatographic separation of pDNA is ongoing. A poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) (GMA-EGDMA) monolithic material was synthesised via a thermal-free radical reaction, functionalised with different amino groups from urea, 2-chloro-N,N-diethylethylamine hydrochloride (DEAE-Cl) and ammonia in order to investigate their plasmid adsorption capacities. Physical characterisation of the monolithic polymer showed a macroporous polymer having a unimodal pore size distribution pivoted at 600 nm. Chromatographic characterisation of the functionalised polymers using pUC19 plasmid isolated from E. coli DH5alpha-pUC19 showed a maximum plasmid adsorption capacity of 18.73 mg pDNA/mL with a dissociation constant (KD) of 0.11 mg/mL for GMA-EGDMA/DEAE-Cl polymer. Studies on ligand leaching and degradation demonstrated the stability of GMA-EGDMA/DEAE-Cl after the functionalised polymers were contacted with 1.0 M NaOH, which is a model reagent for most 'cleaning in place' (CIP) systems. However, it is the economic advantage of an adsorbent material that makes it so attractive for commercial purification purposes. Economic evaluation of the performance of the functionalised polymers on the grounds of polymer cost (PC)/mg pDNA retained endorsed the suitability of GMA-EGDMA/DEAE-Cl polymer.

Chromatographic Removal of Endotoxins: A Bioprocess Engineer's Perspective

“…Both filtrations show relatively good endotoxin clearance [13]. The choice of a stationary phase material is based on the chemical nature, physical characteristics and chromatography performance indicators [29]. Other important factors to be considered are chromatographic affinity of endotoxin and protein, affinity of endotoxin for protein, temperature, pH, types of detergent and solvent [1,30].…”

“…Dispersion, emulsion and suspension polymerisations involving glycidyl methacrylate (GMA) and ethylene glycol dimethylacrylate (EDMA) make PGMA [31]. PGMA has large pores, which translate to rapid mass transfer, high adsorption kinetics and negligible lowflow resistance [29]. Past researchers have functionalised PGMA with urea, ammonia and DEAE-Cl as well as covalent coupling with imminodiacetic acid-metal (IDAmetal) chelator for the purification of plasmid DNA [29,31].…”

“…PGMA has large pores, which translate to rapid mass transfer, high adsorption kinetics and negligible lowflow resistance [29]. Past researchers have functionalised PGMA with urea, ammonia and DEAE-Cl as well as covalent coupling with imminodiacetic acid-metal (IDAmetal) chelator for the purification of plasmid DNA [29,31]. Adsorption isotherm was determined as Langmuir due to the strong plasmid molecules-ligands interactions.…”

“…Adsorption isotherm was determined as Langmuir due to the strong plasmid molecules-ligands interactions. During the functionalisation of the monolith, temperature reduction will increase the reaction time and reduce the ligand densities [29]. DEAE-Cl ligand has a lower energy cost when compared to ammonia and it displays higher efficiency and capability of plasmid DNA (pDNA) adsorption when compared to urea [29].…”

“…During the functionalisation of the monolith, temperature reduction will increase the reaction time and reduce the ligand densities [29]. DEAE-Cl ligand has a lower energy cost when compared to ammonia and it displays higher efficiency and capability of plasmid DNA (pDNA) adsorption when compared to urea [29]. PGMA functionalised with IDAmetal chelator has been used in Immobilised Metal Affinity Chromatography (IMAC) for protein adsorption and has shown favourable results.…”