Development of a disposable micro-capillary film grafted with peptide ligands for immunoadsorption.

Traditional chromatographic techniques used in downstream processes of biomolecule manufacturing are often time-consuming and expensive. In this study, a cost-effective microporous micro-capillary film (MMCF) composed of ethylenevinyl alcohol (EVOH) was evaluated for its potential application in immunoadsorption with high process efficiency. A peptide ligand Ac-Phe-Tyr-His-Glu (Ac-FYHE) was immobilized on the inner surface of MMCF for selective binding of human immunoglobulin (hIgG). The porous structure and chemical properties of the prepared MMCF were confirmed by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). hIgG (2 mg/ml) adsorption studies demonstrated that the binding process followed a Langmuir isotherm with equilibrium adsorption capacities of 9.31 and 3.47 mg/ml adsorbent under static and dynamic conditions, respectively. Moreover, the membrane showed good flowrate tolerance when studied under flowrates of 0.5 ml/min to 10 ml/min. hIgG purity was 88.2% when obtained from an hIgG (2 mg/ml) and HSA (8 mg/ml) mixture and the purity remained over 80.0% when hIgG concentrations increased in the mixtures. Moreover, purity of 82.3% was achieved when removing hIgG directly from human serum. The MMCF-Ac-FYHE affinity column is expected to selectively remove hIgG from blood for the treatment of autoimmune diseases with high efficiency and cost effectiveness.

The intracellular fate of an amphipathic pH-responsive polymer: Key characteristics towards drug delivery.

Biopolymers have become important drug delivery systems for therapeutic molecules by enhancing their accessibility and efficacy intracellularly. However, the transport of these drugs across the cell membrane and their release into the cytosol remain a challenge. The trafficking of poly (l-lysine iso-phthalamide) grafted with phenylalanine (PP-50) was investigated using an osteosarcoma cell line (SAOS-2). Colocalisation of this amphipathic biopolymer with endocytosis tracers, such as transferrin and lactosylceramide, suggested that PP-50 is partially internalised by both clathrin and caveolin-mediated endocytosis. Macropinocytosis was also investigated, but a smaller correlation was found between this mechanism and PP-50 transport. A significant decrease in polymer-mediated calcein uptake was found when cells were pre-incubated with endocytosis inhibitors, suggesting also the use of a combination of mechanisms for cell internalisation. In addition, PP-50 colocalisation with endosome and lysosome pathway markers showed that the polymer was able to escape the endolysosomal compartment before maturation. This is a critical characteristic of a biopolymer towards use as drug delivery systems and biomedical applications.

The development of a weak anion micro-capillary film for protein chromatography.

In this study, the surface of a microporous walled micro-capillary film (MMCF) was modified into a weak anion exchanger by coupling cyanuric chloride and 2-diethylaminoethylamine (DEAE) to the ethylene-vinyl alcohol (EVOH) matrix. Fourier transform infrared spectroscopy (FTIR) measurements of modified and unmodified MMCFs confirmed the addition of a triazine ring and DEAE onto the membrane. Binding of bovine serum albumin (BSA) at pH 7.2 was found to follow a Langmuir isotherm with a maximum equilibrium binding of 12.4mg BSA/mL adsorbent and 8.2mg BSA/mL adsorbent under static and flow conditions, respectively. The ion exchange capacity, determined by Mohr's titration of chlorine atoms displaced from the functionalised surface, was found to be 195±21µmol Cl-/mL of adsorber, comparable to commercial ion exchangers. BSA adsorption onto the ion exchanger was strongly pH-dependant, with an observed reduction in binding above pH 8.2. Frontal experiments of a BSA (5mg/mL) and lysozyme (5mg/mL) mixture demonstrated successful separation of BSA from lysozyme at more than 97% purity as verified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Separation between similarly charged anionic molecules was also achieved using BSA (5mg/mL) and herring sperm DNA (0.25mg/mL). BSA was extracted at 100% purity, demonstrating the ability of MMCF-DEAE to remove significant DNA contamination from a protein solution. These experiments highlight the potential for MMCFs to be used for fast protein purification in preparative chromatography application.

Increased cryosurvival of osteosarcoma cells using an amphipathic pH-responsive polymer for trehalose uptake.

Amphipathic pH-responsive polymers have shown to increase the permeability of cell membranes to trehalose hence improving the cryopreservation of mammalian cells. However, the trafficking of both the polymer and trehalose across the cell membrane has not yet been thoroughly analysed. The objective of this study was to investigate the effect on cryopreservation of the trafficking of the disaccharide trehalose along PP-50, an amphipathic polymer, through an osteosarcoma cell line (SAOS-2). Confocal microscopy analysis confirmed the presence of intracellular labelled trehalose only when incubated in the presence of PP-50. Further analysis confirmed that both trehalose and PP-50 localised in the cytoplasm, accumulated mainly in the perinuclear area. Quantitative analysis of the colocalisation between trehalose and PP-50 showed Pearson and Manders coefficients of 0.862 ± 0.008 and 0.766 ± 0.033, respectively, suggesting a high degree of intracellular colocalisation between these molecules. Cryopreserved cells pre-incubated with trehalose and PP-50 showed increased cryosurvival when compared with cells pre-incubated in the absence of the polymer. PP-50 showed to be directly involved in the uptake of trehalose, a critical characteristic towards use in cryopreservation and biomedical applications.

Effect of bore fluid composition on microstructure and performance of a microporous hollow fibre membrane as a cation-exchange substrate.

Micro-capillary film (MCF) membranes are effective platforms for bioseparations and viable alternatives to established packed bed and membrane substrates at the analytical and preparative chromatography scales. Single hollow fibre (HF) MCF membranes with varied microstructures were produced in order to evaluate the effect of the bore fluid composition used during hollow fibre extrusion on their structure and performance as cation-exchange adsorbers. Hollow fibres were fabricated from ethylene-vinyl alcohol (EVOH) copolymer through solution extrusion followed by nonsolvent induced phase separation (NIPS) using bore fluids of differing composition (100wt.% N-methyl-2-pyrrolidone (NMP), 100wt.% glycerol, 100wt.% water). All HFs displayed highly microporous and mesoporous microstructures, with distinct regions of pore size <1µm, 5-15µm and up to 50µm in diameter, depending upon proximity to the bore fluid. Scanning electron microscopy (SEM) revealed skins of pore size <1µm at the inner surface of HFs produced with water and glycerol, while NMP bore fluid resulted in a skinless inner HF surface. The HFs were modified for chromatography by functionalising the polymer surface hydroxyl groups with sulphonic acid (SP) groups to produce cation-exchange adsorbers. The maximum binding capacities of the HFs were determined by frontal analysis using lysozyme solutions (0.05-100mgml(-1)) for a flow rate of 1.0mlmin(-1). The NMP-HF-SP module displayed the largest maximum lysozyme binding capacity of all the fibres produced (40.3mg lysozyme/ml adsorbent volume), a nearly 2-fold increase over the glycerol and 10-fold increase over the water variants at the same sample flow rate. The importance of NMP as a bore fluid to hollow fibre membrane performance as a result of inner surface porosity was established with a view to applying this parameter for the optimisation of multi-capillary MCF performance in future studies.

Optimised concentration and purification of retroviruses using membrane chromatography.

The ability of an anion exchange membrane to purify a γ-retrovirus was assessed and optimised with respect to different loading and wash buffers. Recoveries of infectious virus greater than 50% were consistently obtained, while specific titre was increased up to one thousand fold when compared to the material loaded. Specific proteins removed and retained by this optimised process were identified by mass spectrometry. It was possible to successfully bind and elute the equivalent of 1.27 × 10(8) Ifu/ml of ion exchange membrane. This could then be highly concentrated, with infectious virus concentrated to a maximum of 420-fold compared to the load.

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.

Dual affinity method for plasmid DNA purification in aqueous two-phase systems.

The DNA binding fusion protein, LacI-His6-GFP, together with the conjugate PEG-IDA-Cu(II) (10 kDa) was evaluated as a dual affinity system for the pUC19 plasmid extraction from an alkaline bacterial cell lysate in poly(ethylene glycol) (PEG)/dextran (DEX) aqueous two-phase systems (ATPS). In a PEG 600-DEX 40 ATPS containing 0.273 nmol of LacI fusion protein and 0.14% (w/w) of the functionalised PEG-IDA-Cu(II), more than 72% of the plasmid DNA partitioned to the PEG phase, without RNA or genomic DNA contamination as evaluated by agarose gel electrophoresis. In a second extraction stage, the elution of pDNA from the LacI binding complex proved difficult using either dextran or phosphate buffer as second phase, though more than 75% of the overall protein was removed in both systems. A maximum recovery of approximately 27% of the pCU19 plasmid was achieved using the PEG-dextran system as a second extraction system, with 80-90% of pDNA partitioning to the bottom phase. This represents about 7.4 microg of pDNA extracted per 1 mL of pUC19 desalted lysate.

Immobilized metal affinity chromatography of histidine-tagged lentiviral vectors using monolithic adsorbents.

Histidine-tagged lentiviral vectors were separated from crude cell culture supernatant using labscale monolithic adsorbents by immobilized metal affinity chromatography. The capture capacity, concentration factor, purification factor, and elution efficiency of a supermacroporous cryogel monolith were evaluated against the BIA Separations convective interaction media (CIM) disc, which is a commercial macroporous monolith. The morphology of the polymeric cryogel material was characterised by scanning electron microscopy. Iminodiacetic acid was used as the metal chelating ligand in both monoliths and the chelating capacity for metal ions was found to be comparable. The CIM-IDA-Ni(2+) adsorbent had the greatest capture capacity (6.7 x 10(8) IU/ml of adsorbent), concentration factor (1.3-fold), and elution efficiency (69%). Advantages of the cryogel monoliths included rapid, low pressure processing as well low levels of protein and DNA in the final purified vector preparations.

Affinity partitioning of plasmid DNA with a zinc finger protein.

The affinity isolation of pre-purified plasmid DNA (pDNA) from model buffer solutions using native and poly(ethylene glycol) (PEG) derivatized zinc finger-GST (Glutathione-S-Transferase) fusion protein was examined in PEG-dextran (DEX) aqueous two-phase systems (ATPSs). In the absence of pDNA, partitioning of unbound PEGylated fusion protein into the PEG-rich phase was confirmed with 97.5% of the PEGylated fusion protein being detected in the PEG phase of a PEG 600-DEX 40 ATPS. This represents a 1322-fold increase in the protein partition coefficient in comparison to the non-PEGylated protein (Kc = 0.013). In the presence of pDNA containing a specific oligonucleotide recognition sequence, the zinc finger moiety of the PEGylated fusion protein bound to the plasmid and steered the complex to the PEG-rich phase. An increase in the proportion of pDNA that partitioned to the PEG-rich phase was observed as the concentration of PEGylated fusion protein was increased. Partitioning of the bound complex occurred to such an extent that no DNA was detected by the picogreen assay in the dextran phase. It was also possible to partition pDNA using a non-PEGylated (native) zinc finger-GST fusion protein in a PEG 1000-DEX 500 ATPS. In this case the native ligand accumulated mainly in the PEG phase. These results indicate good prospects for the design of new plasmid DNA purification methods using fusion proteins as affinity ligands.

On the mechanical properties of bovine serum albumin (BSA) adhesives.

Biological adhesives, natural and synthetic, are of current active interest. These adhesives offer significant advantages over traditional sealant techniques, in particular, they are easier to use, and can play an integral part in the healing mechanism of tissue. Thus, biological adhesives can play a major role in medical applications if they possess adequate mechanical behavior and stability over time. In this work, we report on the method of preparation of bovine serum albumin (BSA) into a biological adhesive. We present quantitative measurements that show the effect of BSA concentration and cross-linker content on the bonding strength of BSA adhesive to wood. A comparison is then made with synthetic poly(glycidyl methacrylate) (PGMA) adhesive, and a commercial cyanoacrylate glue, which was used as a control adhesive. In addition, BSA samples were prepared and characterized for their water content, tensile strength, and elasticity. We show that on dry surface, BSA adhesive exhibits a high bonding strength that is comparable with non-biological commercial cyanoacrylate glues, and synthetic PGMA adhesive. Tensile testing on wet wood showed a slight increase in the bonding strength of BSA adhesive, a considerable decrease in the bonding strength of cyanoacrylate glue, and negligible adhesion of PGMA. Tests performed on BSA samples demonstrate that initial BSA concentration and final water content have a significant effect on the stress-strain behavior of the samples.