EP0203163A1 - Chromatographie par affinite par utilisation d'agents de separation entre la magnetite et l'alginate de calcium seche dans un lit fluidise et stabilise magnetiquement - Google Patents

Chromatographie par affinite par utilisation d'agents de separation entre la magnetite et l'alginate de calcium seche dans un lit fluidise et stabilise magnetiquement

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Publication number
EP0203163A1
EP0203163A1 EP85906143A EP85906143A EP0203163A1 EP 0203163 A1 EP0203163 A1 EP 0203163A1 EP 85906143 A EP85906143 A EP 85906143A EP 85906143 A EP85906143 A EP 85906143A EP 0203163 A1 EP0203163 A1 EP 0203163A1
Authority
EP
European Patent Office
Prior art keywords
beads
magnetic
support
magnetite
dried
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP85906143A
Other languages
German (de)
English (en)
Other versions
EP0203163A4 (fr
Inventor
David J. Graves
Mark A. Burns
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University Patents Inc
Original Assignee
University Patents Inc
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Filing date
Publication date
Application filed by University Patents Inc filed Critical University Patents Inc
Publication of EP0203163A1 publication Critical patent/EP0203163A1/fr
Publication of EP0203163A4 publication Critical patent/EP0203163A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3804—Affinity chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
    • B01D15/1807—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using counter-currents, e.g. fluidised beds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
    • B01J20/28009—Magnetic properties
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B01J20/3255—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising a cyclic structure containing at least one of the heteroatoms nitrogen, oxygen or sulfur, e.g. heterocyclic or heteroaromatic structures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B01J20/3274—Proteins, nucleic acids, polysaccharides, antibodies or antigens
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/328—Polymers on the carrier being further modified
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3291—Characterised by the shape of the carrier, the coating or the obtained coated product
    • B01J20/3293—Coatings on a core, the core being particle or fiber shaped, e.g. encapsulated particles, coated fibers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated

Definitions

  • AC does not rely on general molecular properties such as "size, electrical charge or density to carry out a separation. Instead, it involves a very specific interaction between two biomolecules, one of which is chemically attached to a solid support phase and the other of which is dissolved in solution (usually aqueous) . Such interactions are almost a universal feature of biomolecules. Specific examples would include binding between antibodies and antigens, hormones and receptors, enzymes and either sub ⁇ strates, coenzymes, inhibitors or activators, DNA and its complement (a repressor or catabolite gene activator protein for double-stranded DNA or the complement of a single strand of DNA) and messenger RNA and ribosomes.
  • biochemical pairing since it involves a number of simultaneous interac ⁇ tions between amino acid or nucleotide residues, it can be highly specific. Biomolecules typically perform their functions in the presence of thousands of different types of molecules, indicating that this specificity is "both a necessary and a natural part of their character. Affinity chromatography is a broad term which involves everything from a weak interac ⁇ tion which simply retards one molecule's passage through a column to a strong, almost nonreversible binding to the column packing. The latter would more properly be termed a bio-specific adsorption-desorp ⁇ tion cycle.
  • Drastic changes in pH, ionic strength, or temperature, or the addition of a competing soluble molecule are needed in such a case to release the molecule from its complement on the solid phase.
  • This strong binding system could be operated in a batch vessel in an adsorption-desorption mode, but in most cases a column is used whether it is needed or not. Since other molecules are not usually affected -by passage through the affinity column, in theory, several columns in series could be used to recover several molecules of interest from a given fermenta ⁇ tion broth.
  • affinity chromatography still has several serious disadvantages: (1) Even when operated as a column, it is a discontinuous chromato- graphic or adsorption-desorption process charac ⁇ terized by the introduction of a "pulse" of material and the recovery of a "pulse” of product. The disad ⁇ vantage of this type of operation is that the size of the sample is severely limited. Most of the time the column is in operation no product is being collected, leading to an inefficient system. (2) One cannot, in such a column, use the viscous, debris-laden suspen ⁇ sion of broken cells from a fermentation that one might hope to. A column would almost immediately plug if subjected to such a mixture.
  • a recent development which might be used to advantage to eliminate or substantially reduce the problem of clogging while retaining the other advan ⁇ tages of continuous chromatography is the magnetical ⁇ ly stabilized fluidized bed.
  • the ordinary fluidized bed has been used in industrial processing for many years, mostly with catalytic particles which tend to foul or become poisoned or where thermal effects are important. Above a certain critical fluid velocity, small particles of a solid become suspended in a high velocity stream and the solids suspension acts much like a fluid, permitting it to flow out of the reactor for regeneration or replacement. If the fluid velocity is increased above the critical fluidization value, undesirable effects such as bubbling and slugging occur. These cause bypassing of reactants through the bed and can result in particle entrainment in the gas.
  • the particles in a magnetically stabilized fluidized bed behave as a fluid over a wide range of conditions. Their apparent density is greater than the fluid phase but less than the actual solid density. Unlike the ordinary fluidized bed, however, the dispersion and back-mixing of particulates is effectively zero.
  • the magnetically stabilized fluidized bed is there ⁇ fore an extremely interesting new phenomenon in its own right and is worthy of considerable further basic study.
  • the properties of a magnetically stabilized fluidized bed are ideal for use in a continuous chromatography system. In this application, the fluid-like behavior of the solids would allow countercurrent solids/solvent contacting. Clogging by debris should be controllable, because the bed contents, along with debris that they filter
  • Calcium alginate gels have been previously used • as a biomaterial support for many different immo ⁇ bilized enzyme and cell preparation systems.
  • the support is biochemically inert, easy to handle, and can be packed, like any other gel, into affinity chromatography columns.
  • Immobilization (the tech ⁇ niques for which have been reported extensively) is usually accomplished by entrapment; the desired enzyme or cell population is mixed with the alginate solution and, upon polymerization, is "trapped" in the gel matrix.
  • the gel itself offers little resis ⁇ tance to substrate diffusion.
  • High gradient magnetic filtration is one such technique which allows both filtering of lysed cell parts and purification of the enzyme being sought.
  • the support with an affinity matrix attached is added to the disrupted cell mixture.
  • the solution and support are then passed through a high gradient magnetic filter where the magnetic support is retained but the insoluble proteins and debris continue through.
  • the field is -then removed and the purified enzyme is obtained after desorption from the support.
  • the supports used in the past for such separations have been metals or various gels with magnetic particles either adsorbed on their surface or dispersed throughout the gel matrix.
  • Alginate the polymeric material from which the beads are made, is a block copolymer extracted from kelp consisting of / 3-D-mannuronate (M) and -L-guluronate (G) residues. Exposure to calcium ions in solution crosslinks the acid residues of the alginate molecules into a gel, producing a fairly stable support.
  • the beads change from a cloudy white support to an opaque black magnetic support.
  • the support shrinks irreversibly from the hydrogel state to a rigid solid while remaining quite spheri ⁇ cal and highly magnetically susceptable.
  • the density of the dried support is on the order of glass, but the reactivity is considerably greater.
  • the porosity of the support is limited, but the exposed surface is microscopically very rough, providing many sites for protein or cell attachment.
  • Fig. 1 is a schematic diagram of a magnetically stabilized fluidized bed chromatography system according to one embodiment of the present invention.
  • Fig. 1 depicts the schematic representation of the magnetically stabilized fluid ⁇ ized bed chromatography system used to obtain the results in the experiments which follow.
  • This system may be modified to conform to other configurations, as for example, a system wherein the solids are recirculated, or wherein the solids and feed flow upwardly through the bed.
  • a magnetically stabilized fluidized bed 1 is contained in a chromatographic column 11.
  • the column may be water-jacketed 7 by providing a constant temperature source 14 in order to maintain the temperature of the -bed within set parameters.
  • an upper circular magnetic coil 5 At the upper portion of the column is located an upper circular magnetic coil 5, and a lower circular magnetic coil 5' is located at the lower portion of the column.
  • solid chromatographic support materials are introduced into bed 1 by entraining the solids in a small amount of solvent stream 3.
  • Both the solvent stream 3 and the feed stream 2 containing the crude bioproduct enter the column through indi ⁇ vidual adjustable ports 6 which may be raised or -lowered to vary the relative lengths of the "enrich ⁇ ing" and "stripping" sections of the column.
  • a solvent stream 9 enters at the lower portion of the column through a porous fluid distribution plate 8, passes through the bed, and exits through line 12.
  • a solids removal means 10 which, when activated, as for example by vertical movement as shown in the figure, raises or lowers a seal means 16 allowing the solids to be removed from the column.
  • a seal means 16 allowing the solids to be removed from the column.
  • alternative means are also available for removing the solids from the column.
  • An offline 13 is also included in the downflow stream between the seal 16 and the collection vessel 15 which allows the excess fluids coming off with the solids to be drained for either analysis or disposal.
  • a total of 12.8 g FeCl 2 * 4H-0 and 34.56 g FeCl- * 6 H-0 was mixed in 1600 ml distilled water. This solution was then heated to 70 C and 32 g NAOH dissolved in 320 ml of distilled water was added. A black precipitate immediately formed and settled out after standing at room temperature for 1 hr. After part of the supernatant was aspirated, the remaining magnetite suspension was rinsed with several volumes of water (without drying) and transferred to a 500 ml volumetric flask. The amount of magnetite formed was calculated from the density of the magnetite/water mixture and the volume of the mixture was adjusted to form a 4.4% magnetite suspension. The magnetite could be resuspended at any time with vigorous shaking.
  • the 'resulting beads were placed in two 200 mm Petri dishes and rinsed several times with distilled water. The solution was then slowly aspirated using a Pasteur pipet and the support was air-dried in a hood 12 hrs. The beads were removed with gentle scraping, resulting in 3 ml of dried support.
  • Nonmagnetic beads may also be prepared by substituting 50 ml of 1% sodium alginate solution for the alginate-magnetite solution. Without the magne ⁇ tite, the total volume of the support following the drying procedure described previously was 1 ml) .
  • the magnetic beads may also be prepared accord ⁇ ing to a number of different protocols. A number of these appear in Example III.
  • Spraying - a 50/50 mixture of 4.4% magnetite and 2% alginic acid was prepared and placed in a 50 ml syringe.
  • a 25 gauge needle was cut to a length of 2 mm and affixed to the end of the syringe.
  • a high pressure (15-50 PSIG) was used to force the liquid out in a steady stream of 12 ml/min. This stream was directed at a solution of 0.2M CaCl 2 at an angle of 15° below horizontal and the resulting gel dried as previously described.
  • Bead size and shape were analyzed by transfer ⁇ ring 1-2 ml of dried beads to an 80 mm Petri dish and then placing the dish on top of a fluorescent light box.
  • the macro-viewer assembly of a Cambridge Instruments Quantimet image analyzer was positioned above the dish and focussed to give a 15 x 15 mm field.
  • the image obtained was edited and the pro ⁇ jected area per bead calculated along with the
  • Bead density was measured by the following. A volumetric flask (10 or 25 ml) was filled to the calibration mark with distilled water and weighed. A number of beads was then added and the flask re- weighed. Finally, water was aspirated until the original level was obtained and a third weight taken. The difference between the second and first readings was the weight of beads while the difference between the second and third readings, upon division by the density of water, was the volume.
  • Magnetic affects were studied. 1.13 g of dried 0.9 mm diameter magnetic beads were placed in a vial 0.6 cm x 3 cm and inserted into an 60 Hz oscillating magnetic field. M vs H measurements were made at 77 K (liquid nitrogen temperature) and 300 K.
  • Pore Structure was determined. Steady state porosity measurements were made using a solution (approx. 0.3 g/ml, absorbance of 0.79) of crys ⁇ tallized egg albumin as the penetrant. Known volumes of beads and albumin solution were mixed and allowed to equilibrate overnight. An absorbance reading of the supernatant solution was taken using an ISCO UA-4 absorbance monitor. The bead volume accessible to molecules of size similar to that of albumin (0.01 in dia.) was then calculated.
  • the mechanical properties of the non-magnetic wet gel are similar to those of dextran or polyacryl- amide of comparable percent solids, while the strength of the wet magnetic gel was slightly great ⁇ er.
  • the wet Tyzor-stabilized gel was less elastic then the other two alginate gels, disintegrating when compressed instead of merely flattening.
  • the surface structure of the support as revealed at 40x magnification appears quite spherical with only minor flat spots due to drying on the Petri dishes.
  • At 1250x magnification detailed surface structure and surface irregularities are seen.
  • the valleys in the surface seem to be about 2-5 microns wide and at least 2 microns deep. The surface is thus quite rough. providing a large exposed surface area for attachment of many enzymes, biomolecules, and cells.
  • the porous nature of the beads was investigated using steady state albumin permeation.
  • the pene ⁇ tration of this protein into the beads revealed the porosity values listed in Table II. While normal calcium alginate beads were found to be relatively porous, undried magnetite beads were slightly less porous, undoubtedly due to the internal volume of the bead occupied by magnetite particles.
  • the dried spheres, by albumin porosity measurements, were essentially impermeable. However, a porosity of 5% or less would have been obscured by measurement error. Also, since the beads are soft and do expand very slightly after rehydrating, it is possible that some pores of smaller size are present.
  • the disadvantage of this technique is that the homogeneous size of the support achieved by dropwise production is lost.
  • the diameter of the beads formed in a dropwise manner has a standard deviation of only about 3%, but that of the spray-formed beads is almost 20%.
  • the correct calcium concentration is essential for optimum crosslinking of the polymer chains.
  • Calcium is another factor that governs the shape of both the wet and dried beads. A low calcium concentration will cause the beads to develop tails as they pass through the surface of the polymerizing solution, but these tails are eliminated when the Ca concentration is raised.
  • Other ions used in the receiving solution such as Fe , Mg and Mn formed spherical beads but their rigidity was not as good as that of the calcium alginate spheres. Even a highly acidic solution made with HCl was able to form a gel.
  • the method of drying probably has the most noticeable effect on the shape of the beads. If the magnetic spheres were left in a beaker to dry, the resulting small beads agglomerated, making separation difficult. However, if just enough wet beads to form a monolayer were placed in a Petri dish, the dried beads obtained were isolated shperes which were easily removed. The major drawback of this drying technique was that small "flat spots" formed where the beads touched the glass surface. The size of this flat spot on magnetic beads varied. When non-magnetic alginate beads were used, however, the flat spot was so pronounced that flat discs instead of round spheres formed.
  • Table VI shows effects of various buffer solutions at several different pH values.
  • the nonmagnetic dried beads' properties were not as attractive as those of the magnetic spheres. In almost all solutions, the beads increased at least 20% in size and became quite soft. However, in some solutions (such as pH 7: tris buffer solution with CaCl- added) the beads did not lose their rigid shape or expand more than about 5%. It appears the addi ⁇ tion of magnetite to the alginate solution strength ⁇ ens the bead's overall structure.
  • Protease from Actinomyces fradiae was immo ⁇ bilized on dried calcium alginate/magnetite beads using known techniques with both glutaraldehyde and TiCl, and assayed for activity.
  • ⁇ -amylases bound to a number of supports ranged from
  • the temperature optimum of immobilized x-amylase was the same as that for the soluble enzyme (55°C) and the pH optimum and pH stability of both x-amylase and protease were not changed.
  • Ten-fold repetition of the specific reaction on 5% substrates did not change the activities of o-amylase and proteases immobilized to the support. It was concluded from these results that the immobilization of these enzymes to calcium alginate/magnetite beads did not affect the enzymes properties appreciably.
  • Cibacron Blue F3GA Dye attachment was achieved by a modified form of Bohme's procedure. Magnetic dried beads (1.4 g) were placed in 48 ml H_0 and heated to 60°C. Cibacron Blue F3GA (0.27 g) in 8.2 ml distilled water was added dropwise and the solu ⁇ tion stirred for 30 min. Calcium chloride (7.9 g) was added and the mixture stirred for an additional hour. At this point, the temperature was increased to 80°C and 0.2 g NaOH in 2 ml H_0 added. After two (2) hours of stirring, the support was extensively washed with a 6M urea and 1M CaDl- solution until no blue color was observed leaching from the beads.
  • a plexiglas column h" in inside diameter and 4" long was used for the separation.
  • the column was situated in the center of a pair of coils, each of which had an inside diameter of 5h" an outside diameter of 7V and a width of 3/4".
  • the coils were placed 2%" apart.
  • Each coil had approximately 300 turns, and a current of 1.3 amps produced a field strength of about 40 Oersteds.
  • the support withstood not only temperatures of up to 120°C, but also most pH values and common solvents. While some solutions, such as phosphate buffers, dissolved the spheres, stabilization eliminated this problem.
  • the physical properties of the beads include a glasslike density of 2.2 g/ml, excellent sphericity, low porosity, and a narrow size distribution.
  • the magnetite present in the support allows the beads to be used for magnetic separations such as high gradi- ent magnetic filtration. Their high degree of micro-roughness provides a large exposed surface area for enzyme and ligand binding.
  • the magnetic bead supports of the present invention should have a wide range of applications in bioseparation and immo ⁇ bilized biochemical technology.

Abstract

Agents de séparation entre la magnétite et l'alginate séché pour utilisation dans la chromatographie par affinité et l'immobilisation d'enzymes. Les agents de séparation sont aussi utiles dans des séparations chromotographiques exécutées dans un lit fluidisé et stabilisé magnétiquement.
EP19850906143 1984-11-28 1985-11-27 Chromatographie par affinite par utilisation d'agents de separation entre la magnetite et l'alginate de calcium seche dans un lit fluidise et stabilise magnetiquement. Withdrawn EP0203163A4 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US67550584A 1984-11-28 1984-11-28
US675505 1996-06-28

Publications (2)

Publication Number Publication Date
EP0203163A1 true EP0203163A1 (fr) 1986-12-03
EP0203163A4 EP0203163A4 (fr) 1987-09-10

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP19850906143 Withdrawn EP0203163A4 (fr) 1984-11-28 1985-11-27 Chromatographie par affinite par utilisation d'agents de separation entre la magnetite et l'alginate de calcium seche dans un lit fluidise et stabilise magnetiquement.

Country Status (3)

Country Link
EP (1) EP0203163A4 (fr)
JP (1) JPS62501196A (fr)
WO (1) WO1986003136A1 (fr)

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0611870B2 (ja) * 1986-06-27 1994-02-16 徳山曹達株式会社 無機化合物／染料複合体粒子
SE9101149D0 (sv) 1991-04-17 1991-04-17 Pharmacia Lkb Biotech Beads for down stream processing
US5213683A (en) * 1991-08-08 1993-05-25 Chromaflow, Inc. Apparatus for charging and discharging of chromatography column bed
GB9223334D0 (en) 1992-11-06 1992-12-23 Hybaid Ltd Magnetic solid phase supports
US6706188B2 (en) 1993-05-03 2004-03-16 Amersham Biociences Ab Process and means for down stream processing
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WO1986003136A1 (fr) 1986-06-05
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