Lentivirus capture directly from cell culture with Q-functionalised microcapillary film chromatography.

A new disposable adsorbent material for fast anion-exchange capture of nano-complexes without prefiltering, clarification or pre-processing of samples was developed based on plastic microcapillary films (MCFs). An MCF containing 19 parallel microcapillaries, each with a mean internal diameter of 142 ± 10 µm, was prepared using a melt extrusion process from an ethylene-vinyl alcohol copolymer (EVOH). The MCF internal surfaces were functionalised using branched chain chemistries to attach quaternary amine groups producing an anion-exchange adsorbent. The purification of nano-complexes using this newly fabricated MCF-EVOH-Q was successfully demonstrated with the capture of lentivirus from pre-filtered culture harvest. This 5m chromatographic substrate was found to bind and elute ∼40% of bound lentivirus or 2.5 × 10(6)infectious units (ifu). The unique properties of this chromatographic substrate that allow the passage of large particulates was further demonstrated with the capture of lentiviral particles from unfiltered un-processed culture media containing cells and cell debris. Using this approach, 56% or 1 × 10(7)ifu of captured lentivirus was eluted. A device based on this new material might be used at an early stage in clinical lentiviral production to harvest lentiviral particles, directly from bioreactors.

Fast cation-exchange separation of proteins in a plastic microcapillary disc.

A novel disposable adsorbent material for fast cation-exchange separation of proteins was developed based on plastic microcapillary films (MCFs). A MCF containing 19 parallel microcapillaries, each with a mean internal diameter of 142 µm, was prepared using a melt extrusion process from an ethylene-vinyl alcohol copolymer (EVOH). The MCF was surface functionalized to produce a cation-exchange adsorbent (herein referred as MCF-EVOH-SP). The dynamic binding capacity of the new MCF-EVOH-SP material was experimentally determined by frontal analysis using pure protein solutions in a standard liquid chromatography instrument for a range of superficial flow velocities, u(LS)=5.5-27.7 cm s⁻¹. The mean dynamic binding capacity for hen-egg lysozyme was found to be approximately 100 µg for a 5 m length film, giving a ligand binding density of 413 ng cm⁻². The dynamic binding capacity did not vary significantly over the range of u(LS) tested. The application of this novel material to subtractive chromatography was demonstrated for anionic BSA and cationic lysozyme at pH 7.2. The chromatographic separation of two cationic proteins, lysozyme and cytochrome-c, was also performed with a view to applying this technology to the analysis or purification of proteins. Future applications might include separation based on anion exchange and other modes of adsorption.

Detection of endogenous magnetic nanoparticles with a tunnelling magneto resistance sensor.

The magnetotactic bacterium Magnetospirillum sp. has been cultured and the properties of its endogenous magnetic nanoparticles characterized. Electron-microscopic analyses indicate that the endogenous magnetite nanoparticles in Magnetospirillum sp. are coated with a 3-4 nm thick transparent shell, forming a magnetosome. These magnetite nanoparticles had diameters of 50.9+/-13.3 nm, in good agreement with the diameter of 40.6+/-1.2 nm extracted from magnetometry. Each Magnetospirillum sp. bacterium contained chains of 5-25 magnetosomes. Superconducting quantum interference device magnetometry results indicate that the extrinsic superparamagnetic response of the bacterial solution at room temperature can be attributed to the reversal of the magnetization by physical rotation of the nanoparticles. The intrinsic blocking temperature of a sample of freeze-dried bacteria was estimated to be 282+/-13 K. A tunnelling magneto resistance sensor was used to detect the stray fields of endogenous magnetic nanoparticles in static and quasi-dynamic modes. Based on the tunnelling magneto resistance sensor results, the magnetic moment per bacterium was estimated to be approximately 2.6 x 10(-13) emu. The feasibility of this detection method either as a mass-coverage device or as part of an integrated microfluidic circuit for detection and sorting of magnetosome-containing cells was demonstrated.