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Biomedical subjects

P O Larsson

Publications and source records attributed to P O Larsson.

At least 19 recordsLinked to original sources
PubMed2001

Continuous superporous agarose beds in radial flow columns.

Continuous superporous agarose beds constitute a new support material for chromatography, biocatalysis and electrophoresis. The bed consists of a single piece of agarose gel, homogeneously transected by flow-carrying pores, which easily can be varied in the range of 10-100 microm. In this work, large diameter beds (60 mm) were prepared and used in specially designed radial flow columns. The basic chromatographic properties of the beds were investigated by size-exclusion chromatography experiments. In an affinity chromatography application one bed was derivatized with Cibacron Blue 3GA and used for the purification of lactate dehydrogenase from a crude bovine heart extract. In a biotransformation application one bed was provided with immobilized beta-galactosidase and used in the production of lactose-free milk.

Animals↗
PubMed2001

Theories of chromatographic efficiency applied to expanded beds.

Various quantities such as plate height (HETP), number of plates (N), axial dispersion coefficient (Dax) and Bodenstein number (Bo) are used to describe the efficiency of, and dispersion in chromatographic columns. Different quantities highlight different aspects of the performance. Due to the expansion of expanded-bed columns, the information contained in some of these quantities is not the same for expanded beds as for packed beds. In this article the mentioned quantities are described and discussed both theoretically and related to experimental data. It is concluded that they are often used in a confusing way. Quantities modified to be more informative when comparing beds of different expansions are developed (N(EB) = N/expansion2 and HETP(EB) = HETP x bed expansion) and recommendations of which quantity to use in what situation are given.

Chromatography↗
PubMed2000

Pellicular expanded bed matrix suitable for high flow rates.

A new type of expanded bed matrix with a heavy core of stainless steel covered with an agarose layer was prepared. Two bead size fractions, the smaller one (32-75 microm diameter) having a single particle core and the larger (75-180 microm diameter) with an agglomerate of stainless steel particles constituting the core, were chosen for further characterisation. The dispersion behaviour was determined both in packed bed and expanded bed modes by the retention time distribution method (RTD) and compared with the Streamline matrix (Amersham Pharmacia Biotech). The comparison turned out in favour of the new matrix. Flow rates as high as 3000 cm/h were used with the larger fraction, giving stable expanded beds with good mass transfer properties. The matrices were mechanically stable without any tendency to crack or peal, even after prolonged use.

Chromatography, Liquid↗
PubMed2000

Superporous agarose monoliths as mini-reactors in flow injection systems. On-line monitoring of metabolites and intracellular enzymes in microbial cultivation processes.

A new type of agarose material, superporous agarose, was used as a support material in an analytical system designed for monitoring of bioprocesses with respect to metabolites and intracellular enzymes. The superporous agarose was used in the form of miniaturised gel plug columns (15 x 5.0 mM I.D. monolithic gel bed). The gel plugs were designed to have one set of very large pores (about 50 microm in diameter) through which cells, cell debris and other particulate contaminants from the bioreactor could easily pass. The material also had normal diffusion pores (300 A) characteristic of all agarose materials, providing ample surface for covalent attachment of antibodies and enzymes used in the analytical sequence. The superporous agarose gel plug columns were characterised with respect to flow properties and handling of heavy cell loads as well as dispersion of injected samples (a Bodenstein number of about 40 was observed with acetone tracer at a flow rate of 1 ml min(-1)). To evaluate the practical performance of the superporous gel plug columns, two applications were studied: (1) on-line determination of glucose in cultivation broth (gel plug with immobilized glucose oxidase) and (2) immunochemical quantification of intracellular beta-galactosidase in E. coli (gel plug with lysozyme to achieve cell lysis and gel plug with antibodies against beta-galactosidase).

Bioreactors↗
PubMed1999

Faster isolation of recombinant factor VIII SQ, with a superporous agarose matrix.

A superporous agarose matrix was compared with a corresponding homogenous matrix in the isolation of recombinant factor VIII SQ (r-VIII SQ) by immunoaffinity chromatography. As a reference, the commercially available Sepharose FastFlow, used for a similar purification in the industry, was also evaluated. Breakthrough curves are described for flows between 50 and 400 cm/h with pre-purified r-VIII SQ and with cell culture broth. The superporous gel gave the best performance and a 1000-fold purification was obtained in a one-step procedure. The superporous matrix made it possible to increase the throughput about four-fold, presumably due to its better mass transfer properties. The importance of the ligand distribution profile is discussed based upon immunofluorescence microscopy data.

Chromatography, Affinity↗
PubMed1999

Continuous superporous agarose beds for chromatography and electrophoresis.

Continuous agarose beds (monoliths) were prepared by casting agarose emulsions designed to generate superporous agarose. The gel structures obtained were transected by superpores (diameters could be varied in the range 20-200 microns) through which liquids could be pumped. The pore structure and the basic properties of the continuous gel were investigated by microscopy and size exclusion chromatography. The chromatographic behaviour was approximately the same as for beds packed with homogeneous agarose beads with a particle diameter equivalent to the distance between the superpores. In one application, the superporous continuous agarose bed was derivatized with a NAD+ analogue and used in the affinity purification of bovine lactate dehydrogenase from a crude extract. In another application, a new superporous composite gel material was prepared by adding hydroxyapatite particles to the agarose phase. The composite bed was used to separate a protein mixture by hydroxyapatite chromatography. In a third application, the continuous superporous agarose material was used as an electrophoresis gel. Here, a water-immiscible organic liquid was pumped through the superpores to dissipate the joule heat evolved, thus allowing high current densities.

Animals↗
PubMed1999

Superporous agarose beads as a hydrophobic interaction chromatography support.

Superporous agarose beads were used as a support for hydrophobic interaction chromatography. These beads have large connecting flow pores in addition to their normal diffusion pores. The flow pores, which are approximately one fifth of the overall diameter of the superporous agarose beads, were earlier shown to give the beads improved mass transfer properties relative to homogeneous agarose beads (Gustavsson and Larsson, J. Chromatogr. A, 734 (1996) 231-240). Superporous agarose beads and homogeneous agarose beads of the same particle size range (106-180 microns) were derivatized with phenyl groups. The properties of the superporous beads were then compared with the homogeneous beads in the separation of a mixture of three model proteins (ribonuclease A, lysozyme and bovine serum albumin) at various superficial flow velocities from 30 to 600 cm/h. The superporous beads gave satisfactory separation at flow velocities five times higher than was possible for homogeneous beads. The performance of the two types of beads was also compared in the purification of lactate dehydrogenase from a beef heart extract at a superficial flow velocity of 150 cm/h. The superporous beads performed considerably better, leading to twice the purification factor and twice the concentration of the desired product. The results were interpreted using the theoretical treatment given by Carta and Rodrigues (Carta and Rodrigues, Chem. Eng. Sci., 48 (1993) 3927).

Adsorption↗
PubMed1998

Direct measurements of convective fluid velocities in superporous agarose beads.

Superporous agarose beads contain two sets of pores, diffusion pores and so-called superpores or flow pores, in which the chromatographic flow can transport substances to the interior of each individual bead [Gustavsson and Larsson, J. Chromatogr. A 734 (1996) 231]. The existence of pore flow may be proven indirectly by the chromatographic performance of beads but it has never been directly demonstrated in a chromatographic bed. In this report, pore flow was directly measured by following the movement of micro-particles (dyed yeast cells) in a packed bed. The passage of the micro-particles through the superpores and through the interstitial pores was followed by a microscope/video camera focused on beads which were situated four layers from the glass wall. The video recordings were subsequently used to determine the convective fluid velocities in both the superpores and the interstitial pores. Experiments were carried out with three different bead size ranges, all of which contained superporous beads having an average superpore diameter of 30 microns. The superpore fluid velocity as % of interstitial fluid velocity was determined to be 2-5% for columns packed with 300-500-micron beads (3% average value), 6-12% for columns packed with 180-300-micron beads (7% average value) and 11-24% for columns packed with 106-180-micron beads (17% average value). These data were compared to and found to agree with theoretically calculated values based on the Kozeny-Carman equation. In order to observe and accurately measure fluid velocities within a chromatographic bed, special techniques were adopted. Also, precautions were made to ensure that the experimental conditions used were representative of normal chromatography runs.

Chromatography, Liquid↗
PubMed1998

Direct measurement of intraparticle fluid velocity in superporous agarose beads.

Superporous agarose beads contain both normal diffusion pores and special, very wide superpores through which part of the chromatographic flow is transported, a situation that may greatly improve the chromatographic performance. For the first time such pore flow was measured directly by following the movement of microparticles (dyed yeast cells) through superporous beads packed in a chromatographic bed. The passage of the microparticles through the superpores and through the interstitial pores was recorded by a microscope/video camera. The video recordings were subsequently used to determine flow paths as well as the convective fluid velocities in both the superpores and the interstitial pores. The superpore fluid velocity was found to be proportional to the ratio between the squares of the respective pore diameters, which is in agreement with the Kozeny-Carman equation. Values for two-dimensional and three-dimensional tortuosity of the flow paths were measured and calculated respectively.

Chromatography, Agarose↗
PubMed1991

New methods for separation and recovery of biomolecules.

New methods and applications in the separation of biomolecules are reviewed, with an emphasis on the large-scale recovery of proteins. Highlights include the advent of flow-through particles in perfusion chromatography, which allows for very high flow rates, while retaining a high chromatographic efficiency.

Biotechnology↗
PubMed1990

Magnetic aqueous two-phase separation in preparative applications.

Magnetic aqueous two-phase separation is a new technique to speed up the separation of aqueous two-phase systems (Anal. Biochem. 1987, 167, 331-339). It is based on the addition of magnetically susceptible material (e.g. 1-micron iron oxide particles) which induces rapid phase separation when a mixed system is placed in a magnetic field. The technique has been applied to a number of two-phase systems. The time for phase separation was decreased by a factor of 5-240,000, with the largest improvement for systems containing high concentrations of protein and for systems with viscous or nearly isopycnic phases. An apparatus for preparative multistage extraction with magnetic separation was constructed and tested on glycolytic enzymes present in a yeast extract using a dextran/Cibacron blue-polyethylene glycol system. The presence of iron oxide particles did not adversely affect the extracted enzymes. An electromagnet-based apparatus for continuous phase separation on a larger scale was also designed. A phase system containing crude dextran and unpurified cell homogenate was effectively processed. The apparatus also allowed effective separation when the phase containing iron oxide particles was only a small fraction (4%) of the total phase system.

Alcohol Dehydrogenase↗
PubMed1990

Desmopressin: a new principle for symptomatic treatment of urgency and incontinence in patients with multiple sclerosis.

Thirteen patients with advanced multiple sclerosis and urge urinary incontinence were treated with desmopressin--a synthetic analogue of antidiuretic hormone--in a double-blind cross-over study. The micturition frequency decreased significantly (p less than 0.05). Less leakage was considered valuable for daily life. Peroral medication was favourable in these patients with muscular dysfunction. Side-effects were few.

Administration, Oral↗
PubMed1989

Synthesis of 2-acetamido-2-deoxy-3-O-beta-D-galactopyranosyl-D-galacto se by the sequential use of beta-D-galactosidases from bovine testes and Escherichia coli.

beta-D-Galp-(1----3)-D-GalNAc (1) was synthesised from lactose and GalNAc on a mmolar scale by transgalactosylation using beta-D-galactosidase from bovine testes. The large proportions of unwanted oligosaccharides in the product mixture were removed by treatment with beta-D-galactosidase from E. coli, which left 1, monosaccharides, and a small proportion of trisaccharides. Carbon-Celite chromatography then gave 1 in a yield of 21% based on the GalNAc added.

Acetylgalactosamine↗
PubMed1989

Synthesis of Gal beta 1-3GlcNAc and Gal beta 1-3GlcNAc beta-SEt by an enzymatic method comprising the sequential use of beta-galactosidases from bovine testes and Escherichia coli.

Gal beta 1-3GlcNAc (1) and Gal beta 1-3GlcNAc beta-SEt (2) were synthesized on a 100 mg scale by the transgalactosylation reaction of bovine testes beta-galactosidase with lactose as donor and N-acetylglucosamine and GlcNAc beta-SEt as acceptors. In both cases the product mixtures contained unwanted isomers and were treated with beta-galactosidase from Escherichia coli which has a different specificity, under conditions favouring hydrolysis, yielding besides the desired products, monosaccharides and traces of trisaccharides. The products were purified to greater than 95% by gel filtration, with a final yield of 12% of 1 and 17% of 2, based on added acceptor. In a separate experiment Gal beta 1-6GlcNAc beta-SEt (3) was synthesized by the transglycosylation reaction using beta-galactosidase from Escherichia coli. No other isomers were detected. Compound 3 was purified by HPLC.

Animals↗
PubMed1988

High-performance liquid affinity chromatography: rapid immunoanalysis of transferrin in serum.

We describe a new method for quantitatively measuring substances of clinical interest by high-performance liquid affinity chromatography (HPLAC). As a model system we selected analysis for transferrin in human serum with immobilized antibodies in a high-performance liquid chromatographic system. SelectiSpher-10 Activated Tresyl columns (5 or 10 x 0.5 cm) were used for in situ coupling of polyclonal antibodies to transferrin. The amount of transferrin eluted was determined by integrating the eluted peak at 280 nm. The whole analytical procedure--including injection of sample, washing, elution, and analysis of data--takes only 7 min. We characterized the HPLAC system for analysis of transferrin in several ways: intra-assay CV approximately 3%; inter-assay CV 2-9%; linear response up to 1 mg/mL column volume; detection limit approximately 3 micrograms; analytical recovery 98% +/- 2%; purity of eluted sample greater than 95% (SDS-PAGE). The HPLAC method was compared with "rocket" immunoelectrophoresis, a commonly used method of analysis for transferrin, and there was excellent correlation between the two methods (r = 0.96, n = 60). Benefits of this HPLAC technique include high precision, rapid analysis, and simplified sample handling.

Chromatography, High Pressure Liquid↗