Biomedical subjects

N K Slater

Publications and source records attributed to N K Slater.

7 recordsLinked to original sources

PubMed2000

pH-responsive pseudo-peptides for cell membrane disruption.

We describe pseudo-peptides obtained by the copolymerisation of L-lysine and L-lysine ethyl-ester with various hydrophobic dicarboxylic acid moieties. In aqueous solution, when the carboxylic acid groups are charged, the polymers dissolve. When they are fully neutralised the hydrophobic moieties cause the polymer to precipitate. The pH range over which reversible precipitation occurs can be adjusted by changing the intramolecular hydrophilic/hydrophobic balance, by using a carboxylic acid moiety with a different pK(a) value or by changing the apparent pK(a) value of the polymer through chemical modifications of the backbone. These bio-degradable materials are well tolerated by a range of mammalian cell lines at physiological pH but display an ability to associate with the outer membranes of these cells, which they rupture to varying degrees at pH 5.5. Relative to the degree of lysis displayed by poly(L-lysine iso-phthalamide), lysis was reduced by partial esterification and increased by replacing the aromatic iso-phthaloyl moiety with a long chain aliphatic dodecyl moiety. Similar behaviour was observed for the pH-dependent rupture of human erythrocytes, where poly(L-lysine dodecanamide) displayed enhanced cell lysis at pH values <7.0 relative to poly(L-lysine iso-phthalamide).

Animals↗

PubMed1999

The affinity adsorptive recovery of an infectious herpes simplex virus vaccine.

The chromatographic purification of a recombinant Herpes Simplex Virus (type 2) from salt- and heparin-released harvests of infected complementing Vero (CR2) cells is addressed. Functionalized matrices and process operating conditions are identified that provide adequate virus titres in eluates that are significantly reduced in CR2 cell protein and DNA and possess a low level of HSV-2 protein. Virus from diluted salt-released harvests (0.14 M NaCl) was not appreciably adsorbed onto either heparin-Sepharose or Cellufine-heparin matrices but was virtually completely adsorbed onto Cellufine-sulfate and heparin-HP matrices. Virus was recovered by either a linear salt gradient elution (0.14-2 M NaCl) or by a single-step elution with 1.5 M NaCl in phosphate buffer. Recoveries of infectious virus with step elution were 21% and 89%, respectively, for these matrices. Virus from undiluted salt-released harvest (0.8 M NaCl) was substantially adsorbed onto Cellufine-sulfate gel (44% adsorption) and completely adsorbed onto heparin-HP matrices. This virus was recovered with high yield by either gradient or step elution with phosphate-buffered saline. Finally, heparin-harvested virus was fed directly to these matrices and quantitatively adsorbed. The virus could be completely recovered from the heparin-HP matrix with 1.5 M NaCl buffer to provide a purified preparation containing only 0.05 pg protein/pfu and 1.2 x 10(-4) pg DNA/pfu.

Adsorption↗

PubMed1999

Characterization of a generic monoclonal antibody harvesting system for adsorption of DNA by depth filters and various membranes.

The physical parameters governing adsorption of DNA by various positively charged depth filters and membranes have been assessed. Buffers that reduced or neutralised the depth filter or membrane charge, and those that impeded hydrophobic interactions were shown to affect their operational capacity, demonstrating that DNA was adsorbed by a combination of electrostatic and hydrophobic interactions. The adsorption profile of DNA by a Sartobind Q anion exchange membrane showed immediate breakthrough, irrespective of challenge DNA concentration or flow rate, and in this case adsorption was by electrostatic interactions only. The production-scale removal of DNA from harvest broths containing therapeutic protein by partitioning of cells and debris from protein in sequential centrifugation and filtration steps, and the concentration of DNA in process supernatant were assessed. Centrifugation reduced the quantity of DNA in the process material from 79.8 micrograms ml-1 to 9.3 micrograms ml-1 whereas the concentration of DNA in the supernatant of pre- and post-filtration samples had only marginally reduced DNA content: from 6.3 to 6.0 micrograms ml-1 respectively. DNA was concentrated to 27.3 micrograms ml-1 along with monoclonal antibody in the ultrafiltration step. Similar effects were observed in the harvest step for a second antibody.

Adsorption↗

PubMed1998

The primary production of an infectious recombinant Herpes Simplex Virus vaccine.

The production and extracellular release of a recombinant Herpes Simplex Virus (type 2) from monolayers of infected complementing Vero cells (CR2) are addressed. Growth and virus production conditions are identified that provide adequate virus titers with cell seeding densities and viral multiplicities of infection that could be reasonably handled in manufacturing. Harvesting by sonication of cell monolayers is shown to give the highest recovery of infectious virus (to 2.5 x 10(6) pfu/mL) but leads to process stream contamination by cellular proteins through the rupturing of cells (to 28 pg protein/pfu). By comparison, freeze-thaw cycles and osmotic rupture by hypotonic saline or glycerol shock procedures yield only low virus recovery (typically <10% of that by sonication), and are accompanied by yet higher levels of protein contamination (up to 30-fold higher pg protein/pfu). Addition of the polyanionic polymers, heparin or dextran sulphate to a harvest using either hypotonic saline, glycerol shock or isotonic phosphate buffered saline increased the yield of infectious virus in the supernatant. By contrast, addition of polycationic poly-L-lysine resulted in negligible increase in the supernatant virus titer. The highest virus titers (4.7 x 10(7) pfu/mL) were achieved following treatment of roller bottle cultured cells displaying a high cytopathic effect with heparin at 50 microg/mL for at least 3 h post harvest. This procedure also gave the lowest levels of protein contamination (<2 pg protein/pfu). The fivefold lower yield of infectious virus from cultures displaying a low cytopathic effect (<70% CPE) indicates the importance of cell physiological state at harvest.

Animals↗

PubMed1996

Strategies for the isolation and purification of retroviral vectors for gene therapy.

Viral gene therapy vectors promise new opportunities for treatment of hitherto debilitating and life threatening illnesses. To enable early and rapid clinical evaluation of the therapeutic potential of the technology, the initial objectives of process development have so far largely concerned vector assembly, product quality and safety, and manufacturing consistency appropriate to modest scales. The first of such vectors are under test in clinical trials approved through the regulatory CTX/IND route and thus conform to the standards specified for purity and contaminant removal. Process optimisation, scale-up and operability have been of secondary concern and the establishment of a scientific basis for the mechanistic development of future vector manufacturing processes has yet to be seriously addressed. This review considers the manufacturing demands of retroviral vectors and the candidate separation technologies which could facilitate preparation of clinical grade materials. Note is made that the practising community appears to place implicit confidence in the capability of conventional membranes and chromatographic supports developed for protein purification to perform adequately for large-scale purification of viruses. In particular, these are expected to deliver virus preparations to product standards currently required of therapeutic proteins. It is argued that the basis for this confidence may be ill-placed, since the physical and chemical characteristics of viral particles differ significantly from macromolecular proteins. The specific requirements for separation systems and materials for processing of retroviral vectors are considered, and specific routes to more efficient manufacturing processes are proposed.

Animals↗

PubMedDate not supplied

Polyvinyl alcohol-coated perfluorocarbon supports for metal chelating affinity separation of a monoclonal antibody.

The preparation, characterisation and testing of stable non-porous coated perfluorocarbon supports functionalised with the metal chelate, iminodiacetic acid (IDA) is described. Polyvinyl alcohol (PVA), a neutral hydrophilic polymer was esterified with perfluorooctanoyl chloride and anchored to the surface of solid perfluorocarbon particles through multiple fluorophilic interactions. The PVA-coated particles were then activated by epoxidation and coupled with IDA. The presence of surface-attached chelates was clearly demonstrated by the binding and selective desorption of Zn2+ ions. Three particulate perfluorocarbons were selected as potential starting materials and the conditions for preparation of metal chelating adsorbents optimised with respect to ease of manufacture, ligand density and binding capacity towards a monoclonal antibody known to bind to commercial Zn(2+)-IDA supports. The choice of base particle strongly influenced the ligand densities and specific binding capacities towards the monoclonal antibody that could be achieved under optimal preparative conditions. Possible ways in which these metal chelating adsorbents may be employed to recover the monoclonal antibody directly from culture vessels are discussed.

Animals↗

PubMedDate not supplied

Mixing in process vessels used in biopharmaceutical manufacturing.

The use of nonbaffled vessels for mixing applications is becoming common in the biopharmaceutical industry but is not sufficiently well studied. Orientation of the impellers off-centered and/or at an angle is necessary to enhance mixing and eliminate swirling that would result without a baffle in a standard tank. This study focuses on characterizing mixing in vessels with the hydrofoil axial flow impellers mounted off-center at 10 degrees to the vertical. Geometrically similar vessels ranging from 100 to 5000 L working volume were used in this study. Mixing performance was successfully correlated to vessel geometric factors.

Biopharmaceutics↗