JPS62501196A - Affinity Chromatography Using Dry Calcium Alginate-Magnetite Separation Media in a Magnetically Stabilized Fluidized Bed - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Dry calcium alginate-magnetite in a magnetically stabilized fluidized bed. Affinity chromatography using standard separation media All of the beans In recent years, awareness of separation costs as part of the total cost in chemical processes has increased. is gradually increasing.

Sherwood et al. (Sherwood, Pigford, and Wilke, MassTransfer'', McGraw Old II, New York, 19 75) is, for example, the inverse of the value of a pure substance and its concentration in the crude mixture from which it was obtained. It shows the linear relationship in log-log coordinates between the numbers. This empirical pronto is It is suggested that separation cost is often the dominant part of the total cost of the target product. Heaven emphasizes the unusually low concentrations of natural biological substances and the great difficulties of purifying them; Subsequent research has tested several different bioproducts, including substances such as interferon. We showed that the correlation can be extended by several orders of magnitude using data from

Biochemical separation methods currently used for enzyme and protein purification pose additional difficulties. Ru. Currently, they are invariably highly labor intensive, slow and relatively non-selective. It is true. Typical minutes i! +il is gel filtration, ion exchange, or chromatography Including selective adsorption in ficolumns. The brittle column used in such columns Beads support pressure drop limits of less than 1 psi (approximately 0.07 kg/crA) Imposition with a low flow rate. Another common, but difficult process is protein This involves centrifugation and intermediate dialysis after fractional precipitation. Electrophoresis and isoelectric focusing Separation based on electrical charge, such as aerophoresis, is a relatively convenient technique at the laboratory level. However, there are scale-up problems. this The problem is that the heat generated by the passage of electric current is the square of the dimension (i.e., as a cross-sectional area). therefore increases, whereas the surface area for heat removal only increases linearly. It arises from Convection, band expansion, etc. also increase in proportion as the scale increases. increase Usually the process is stopped to see how far the separation has progressed and the protein is Must be stained for quality. Previously, bio-separation equipment was continuously processed. central location eliminates some of the manual labor normally associated with such operations. Attempts are being made to However, some of these devices are batch-oriented in nature. The elaborate ingenuity demonstrated by these attempts will lead to new refining techniques. It only serves to reinforce the recognition that it is necessary.

Probably the most efficient of the many techniques used in biochemical separations today. The specific and selective method is called affinity chromatography (AC). It is something that exists. It has a typical purification factor Pf (=product purity/raw material purity) of 2 to 10. Unlike other separation techniques mentioned above, affinity separation is preferred in cases where a single A Pf value of 10,000 is achieved in the process.

Unlike all of the technologies mentioned above and many others not mentioned, AC is does not rely on general molecular properties such as size, electrical charge, or density to effect separation. stomach. Instead, it is one of which is chemically bonded to a fixed support phase and the other A highly specific interaction between two biomolecules dissolved in a solution (usually aqueous) It includes action. Such interactions are an almost universal feature of biomolecules. Ru. Specific examples include antibodies and antigens, hormones and receptors, enzymes and substrates, coenzymes, and light pollution. DNA and its complement (representative of double-stranded DNA) Ser or catabolite gene activator protein or one! , *DNA complement ) and the binding between Mensenger DNA and liposomes.

The beauty of such biochemical combinations is that they involves many simultaneous interactions, which can be highly specific. Ru. Biomolecules typically share their functions in the presence of thousands of different types of molecules. and that this peculiarity is a necessary and natural part of their characteristics. It shows. Affinity chromatography is one molecule passed through one column. from weak interactions that simply retard the passage of It is a broad term that includes anything up to and including bonding. The latter is more appropriately bio - What is called a specific adsorption-desorption cycle. In such cases, pH, ionic strength, or temperature can be used to detach molecules from their complements on the solid phase. A drastic change in the concentration or the addition of counter-soluble molecules is required.

This strongly bonded system can be operated in a batch vessel in an adsorption-desorption mode, but only However, in most cases a column is used whether or not it is needed. Ru. Other molecules are usually unaffected by passing through the affinity column. Therefore, it is theoretically possible to use several columns in series to Several target molecules can be recovered.

Despite these huge advantages over other bio-based methods, affine Tichromatography still has some serious drawbacks. (1) Operate as a column Even when manufactured, it is the introduction of a “pulse” of the substance and the “pulse” of the product. It is a discontinuous chromatography or adsorption-desorption method characterized by recovery. Ru. The disadvantage of this type of operation is that the sample size is extremely limited. . Most of the time the column is in operation, product is not collected, leading to an inefficient system. (2) In such a system, any viscous debris containing broken cells from the fermentation that you wish to use may be suspension cannot be used. When exposed to such a mixture, the column will It gets blocked immediately. Debris and DNA (its extremely high molecular weight has a large effect on viscosity) Removal of carbonaceous substances (which have a large impact) is a serious problem in industrial-scale processes. . (3) Since peak appearance from the column is related to time, process control and Automation is more difficult than for steady state operation.

Acknowledging these shortcomings, various i! ! Continuing to devise chromatography techniques Attempts have been made to overcome these problems. The aim is to connect samples Eliminates inefficiencies in batch operations by continuously injecting and removing product It was to do. These are the movements (sometimes simulated) in each case. movement) is perpendicular to the solvent flow and simultaneously refines many different compounds. or countercurrent to the flow, in which case two It uses a mobile chromatographic bed that can only obtain the purest components. Ta. The advantage of each variant is relatively high throughput compared to repeated batch operations. Ru. The drawbacks of some of these technologies, e.g. simulated moving beds, are allowing them to operate elaborate and expensive mechanical moving seals or automatic pulps; It is necessary to. In addition to this added expenditure, if the system contains biomaterials, and there is a high risk of contamination if it is operated for a long period of time. or , the problem of clogging by debris is not eliminated by any of these continuous systems.

Eliminate clogging issues while retaining the other benefits of continuous chromatography or A recent development that can be advantageously used to substantially reduce This is a fluidized bed.

Conventional fluidized beds almost always use catalyst particles that are prone to contamination or poisoning, or use thermal effects. It has been used for many years in industrial operations when results are important. given fluid Once the velocity is exceeded, small particles of solids are suspended in the high velocity flow and the solid suspension becomes extremely fluid. The reactor acts in a similar manner to allow fluid to exit the reactor for regeneration or replacement. If fluid velocity If the degree of flow is increased above the critical flow value, bubbling and slugging etc. An undesirable phenomenon occurs. These create a bypass through the bed of reactants and This may result in particulate contamination in the water. These problems are caused by fluidized beds with liquids rather than gases. Although this is less confusing, fluidized particles still cause strong back mixing, resulting in The fruit bed behaves very similar to a continuous flow tank reactor. This turbulence is caused by heat exchange, etc. Although desirable for some types of chroma processes, it 1. It is also extremely harmful to graphical separation.

As early as 1961, Iershler discovered that liquid metal Experiments are carried out using a magnetic field applied to a magnetic field and a fluidized magnetically sensitive solid. Ru. He has published patent documents (U.S. Pat. No. 3,219,318 and U.S. Pat. No. 3,439,89). 9), such a liquid metal is stirred using a magnetic field formed using countercurrent. agitation to fluidize the bed even in the absence of supporting gas or liquid flow and ( Using some FM field in the column) to reduce bubbling and flow the material It has been reported that injection from the top of the floor can be prevented. these The mechanism of action of It is clear that they are not uniform at all.

Research on magnetic fields associated with fluidized beds is given by Tuthill (U.S. Pat. No. 3,440,731), however, it Rosensweig has developed magnetically stabilized materials in this field. After the late 1970s, when a careful and systematic study of fluidized beds began ``Magnetic stabilization of a state of uniform fluidization'' (Magnetic 5t) ability of the 5tate of Llniform Fluidization', Ind, Eng, Chem, Fund, +18: 260); “Fluidization: Hydrodynamic stabilization using magnetic fields” (“Flui-diz ation: Ilydrodynamic 5 tabilization Wi th A Magnetic Field”, 5science, 204:57: and "Magnetically Stabilized Fluidized Beds J" co-authored with Bilke-Nosi, Hatsuchi and Meyer (Lucchesi, l1atch.

and Mayer, “Magnetically 5 tabilized F "luidized Beds", A, I.

Ch, E, Sump, 5eries 77, +1205.8)]. Rosens Among the important discoveries of Baig and his collaborators are: firstly magnetic Fluidization of solids that are sensitive to It can be stabilized in a magnetic field.

Although the field gradient is not critical, axially oriented fields are preferred. Second, stabilization observed over a wide range of magnetic field strengths and fluidization velocities, demonstrating the applicability of important variables. The enclosure is currently being drawn by Rosenspeig. For most fluid velocities , whereas if the bed is stabilized a decrease in magnetic field strength will result in normal fluidization. This increase results in agglomeration of solid particles. The effect of this magnetic field is roughly parallel to the magnetic field lines. observed as creating a magnetic pole in each particle that causes it to become "sticky" in the direction can do. This creates what becomes a chain of beads parallel to the axis of the bed. do.

As in conventional fluidized beds, particles in magnetically stabilized fluidized beds have a wide range of It behaves as a fluid under the following conditions.

Their apparent density is not greater than the fluid phase, but more than the actual solid density. small. However, unlike a normal fluidized bed, particle dispersion and back-mixing are zero. It is valid to have one.

Magnetically stabilized fluidized beds are therefore very interesting new developments in their own right. This is a new phenomenon and deserves considerable further basic research. However In addition, the properties of magnetically stabilized fluidized beds make them useful for continuous chromatography systems. It's ideal to have. In this application, the fluid-like behavior of the solid is Allows flowing solid/solvent contact. Clogging caused by debris will cause the floor contents to filter out. Since it is continuously removed and replaced along with the debris, control is possible. should . All of these factors place a great need for improved processing operations, especially for biomaterials. Chromatography in magnetically stabilized fluidized beds in bioseparations for It is clear that Raffi is a very efficient separation method and that such a sufficiently complex method most easily justified for products with a high dollar value per pound. Suggests. However, prior to the present invention, the use of such techniques was limited to these Separation was not applied.

However, currently available for magnetically stabilized fluidized bed separation of biomaterials Prior to the present invention, testing of support media for bioseparation was not successful. Requirements, especially high density, accurate sphericity and uniform size, low porosity and standard fixation Chemistry that can be used to attach affinity ligands by conjugation reactions No magnetic particles were available that met the requirements of high concentrations of groups. gold such as nickel The genus lacks this last feature, and commercially available composites have too low a density and too little It was also porous. The various demands mentioned above were met through a series of theoretical predictions and actual experiments. This was achieved by Simply stated, they are dense and reasonably large. The size is necessary due to the use of a relatively dense and viscous fluid phase, such as water. It can be summarized by saying that it was essential. big big Low porosity can lead to undesirable chromatographic band broadening due to interparticle diffusion delays. controlled to prevent it from happening. Finally, non-porous particles have sufficient adsorption capacity. Requires a high concentration of surface binding sites.

Calcium alginate gel has traditionally been used for many different immobilized enzyme and cell preparation systems. It has been used as a support for biomaterials. This support is biochemically inert. It is easy to handle and can be applied to affinity chromatographs like other gels. can be filled. Immobilization (a technique that has been widely reported in the past) is usually done by capture. will be achieved. That is, the desired enzyme or cell population is mixed with an alginate solution, and It polymerizes and becomes "trapped" in the gel matrix. The gel itself is largely resistant to matrix diffusion. Does not give sex.

However, for many reactions and separation systems, the introduction of reactants into the interior of the support is difficult. Diffusion is undesirable or impossible. Enzymes that react with large substrate molecules are If the quality is immobilized in areas of the gel that cannot be penetrated, it will be wasted. . Affinity cell separation systems that are dispersed in supports or contain Gantts It is also inefficient since the cells only contact the surface of the gel. these The system will be more efficient if the active species is linked only to the surface of the beads.

Some types of separated wave 4JJs currently in use also require magnetic support for efficient operation. I need a body. For example, high gradient magnetic filtration can be used to filter and explore lysed cell fractions. One such technique allows for both purification of enzymes. This technology The affinity matrix attached support is then added to the disrupted cell mixture. added. The solution and support are then held by a magnetic support, however, It is passed through a high gradient magnetic filter through which insoluble proteins and debris continue to pass. magnetic field is then removed and the purified enzyme is obtained after being desorbed from the support.

Supports used in the past for such separations have been metals or various gels, and magnetic particles have adsorbed on their surfaces or dispersed in the gel matrix. Ta.

The support described in the present invention is used in the field of biotechnology to use alginic acid. This provides a new application for salt.

Although similar in some respects to others currently available, these beads are unique. It has special and highly desirable characteristics. is the polymeric material from which beads are made Alginate β-D-mannuronate (M) and α-L-guluronate (G) residues It is a block copolymer extracted from kelp residue.

When LWn is applied to calcium ions in solution, the acid residues of alginate molecules are cross-linked into a gel. to produce a sufficiently stable support. Magnetite (magnetic oxide of iron) u When particles of esoa) are mixed with a gelling part alginate solution (herein Generalization of the Disclosed Method) Beads are transferred from a cloudy white support to an opaque black support. Change. When the beads are dried, the support becomes highly spherical and highly magnetically sensitive. It irreversibly shrinks from the hydrogel state to a synthetic solid while remaining receptive. drying The density of the support is on the order of glass, but the reactivity is considerably greater.

The porosity of the support is limited, but the filtration surface is microscopically very small. It is rough and provides many sites for protein or cell attachment.

Therefore, it is an object of the present invention to provide a new magnetic chromatographic separation support material. It is.

A further object of the present invention is to develop affinity chromatography for bioproducts using novel magnetic support materials. It is to disclose the use in Raffi.

Furthermore, it is an object of the present invention to provide an aftertreatment method for bioproducts carried out in a magnetically stabilized fluidized bed. The purpose of the present invention is to disclose identity chromatography.

The following drawings and description of specific examples will provide a better understanding of the subject matter and experimental procedures of the invention. It is shown for the purpose of understanding. The drawings and examples illustrate embodiments of the invention. However, it is not intended to limit the scope of the present invention.

Concave dish ■■ place FIG. 1 shows a magnetically stabilized fluidized bed chromatograph according to one embodiment of the invention. It is a schematic diagram of the Raffi system.

More specifically, Figure 1 shows the magnetic field used to obtain the results in the following experiments. 1 is a schematic representation of a fluidized bed chromatography system stabilized in FIG. However, this systems in which, for example, solids are recycled or solids and feed streams are passed upwardly through a bed. It may also be modified to accommodate other arrangements, such as flowing systems.

Many of these alternatives are known from the prior art, such as U.S. Pat. Same No. 4,261,109, Same No. 4,272,893, Same No. 4.261.10 No. 9, No. 4,247,987, No. 4,115,927 and references thereto. found in the published literature. Magnetically stabilized flow as shown in Figure 1 Bed 1 is contained in chromatographic column 11. This column sets the bed temperature. A water jacket 7 is provided to provide a normal temperature source 14 to maintain within the specified parameters. will be attached. An upper circular magnetic coil 5 is arranged at the top of the column, and a lower circular magnetic coil 5 is arranged at the top of the column. The column 5' is located at the bottom of the column. Controls the current flowing through these coils. control, adjust the distance between these coils, and adjust the flow of solvent, solids and raw materials. This change maintains the integrity of the floor.

In the specific example below, a uniform magnetic field covers an approximately 5-inch inner diameter and an approximately two-inch outer diameter. This pair of Helmholtz coils is formed to have a diameter. these The coils are approximately inches thick and spaced 2 to 3 inches apart. coil The dimensions of and the number of turns in each are calculated to give a uniform magnetic field. Kara The system 11 has a diameter of about a hand inch and includes a bed 1 about 4 inches long. 0-5 volts Using a stable, C, power supply that can give 1 ampere that can vary in the range of Activate these coils to the required low field strength. These are shown in Figure 1. design parameters of the system, but clearly they depend on the final application of the invention. and such modifications are within the scope of the present invention. .

In operation, solid chloride provided in a fixed storage or other supply means according to the invention The matoographic support material is introduced into the bed 1 by mixing the solids into a small volume of the solvent stream 3. entered. Both solvent stream 3 and feed stream 2 containing the crude bioproduct are raised or lowered to change the relative lengths of the into the column through individual adjustable inlets.

Solvent stream 9 enters the bottom of the column through porous fluid distribution plate 8 and passes through the bed to the line Exit through 12.

A solids removal means 10 is arranged in the lower part of the column, which is illustrated in FIG. For example, the sealing means 16 can be raised or lowered when moved by an up-and-down movement. Allow solids to be removed from the column. Of course, to remove solids from the column Other means are also available. Once the solids are removed, they are placed in collection vessel 1 5, further processed to elute bound iosomes and recycled to solid storage 4. It is circulated. Off-line 13 between seal 16 and collecting vessel 15 in the downward flow Also included is the removal of excess fluid along with the solids for analysis or disposal. make it possible.

As mentioned above, prior to the development of the magnetically stabilized fluidized chromatographic system described above, There are no magnetic particles that meet the requirements for ion separation using this technology. Ta. The following specific examples are necessary for magnetically stabilized fluidized bed separation of biomaterials. 1 illustrates a support medium according to the present invention that meets the established standards.

Husband blood spot -± Ekushie Fukudara Alliance 12.8 g of FeCRz'411zO and 34.56 g (D FeC j! s' 611zO was mixed in distilled water at 1600 mA. This solution is 32 g of Na0II dissolved in 320 mf of distilled water was added. added. A black precipitate formed immediately and settled after standing at room temperature for 1 hour. Kamisumi no Ichi After aspirating the remaining magnetite solution, rinse the remaining magnetite solution with several times the volume of water (and let it dry. ), 500m! ! volumetric flask. Amount of magnetite formed is calculated from the density of the magnetite/water mixture and adjusted for the volume of the mixture to 4.4 % magnetite suspension was formed. This magnetite is produced by vigorous stirring from time to time. It was possible to resuspend.

Pointed plate - plate Made in Shori, Danni Ward 50mj! 50rrl of 2% alginic acid mixed with 4.4% magnetite solution of Sodium was added to a 50ml syringe equipped with a hypodermic needle. This syringe is outside Slightly pressurized (less than 5 PSIG) by an air source, the solution was 　M　CaCj! + Add dropwise to the solution to approximately 50 mm below the tip of the needle. This operation Repeated with another 50 mA' sodium alginate solution and obtained 1 A mixture of 00 mA alginate beads and 100 ml Ca (1° solution) was reduced. Both were stored for 2 hours to allow complete reaction with Ca'' ions.

Place the obtained beads in a 200ml veterinary dish and add several times distilled water. TAif, :.

This solution is then slowly aspirated using a bathstool (bathroom) and the support is placed under a hood. It was air-dried for 12 hours.

The beads were removed by gentle scraping to obtain a 3 mA dry support.

(Non-magnetic beads can also be used instead of alginate-magnetite solution in 50 ml of 1% acetate. Prepared by using sodium ruginate solution. Where there is no magnetite In this case, the total volume of the support obtained after said drying operation was 1 mA. ) magnetic vinyl The seeds can also be prepared according to many different recipes. is seen in Example ■.

Leave plate - see 1 out of each Koseifi Spray method - Prepare 50150 mixture of 4,4% magnetite and 2% alginic acid and put it into a 50ml syringe. Cut a 25 gauge needle into a length of 21 and attach it to the end of the syringe. I attached it. Using high pressure (15-50 PSTG), this liquid was pumped at a constant flow of 12 m//min. It was forcibly released as a result. This flow is 0.2 MCa at an angle of 15° below the horizontal plane. C1, and the resulting gel was dried as before.

Stabilization method - 1m! ! DuPont Tyzor TER() Reethanol minced titanium chelate) solution with 1 mff of 4.4% magnetite suspension and 2 mA of 2% alginic acid. Quickly transfer this liquid to the syringe device. (TER hydrolyzes rapidly in aqueous media), apply an additional 2 mA through a 21G needle. Added dropwise into 20ml of CaClz containing ``Tyzor TE''. After adding 1 MCl (sufficient to reach pl+7) and reacting for 2 hours, the beads were The mixture was boiled for 1 hour (additional water was added if necessary) and dried by a conventional method.

Blow drying method - Dry approximately 10 ml of wet beads with blotting paper to dry the droplets. into a syringe used for

Air is connected to the needle end of the syringe while a porous cloth is connected to the other end in place of the plunger. Attached to the end. Constant air flow in the center of the tube using an air pressure of 10-15 PSIG kept shouting. The beads were dry within 90 minutes.

The beads were examined under various conditions to characterize their behavior and structure.

The size and shape of the pieces are determined by transferring 1 to 2 rrl of dried beads to an 80 mm Petri dish. The dish was then analyzed on top of a fluorescent box. Cambridge Instruments Quantimet image analyzer macro Place the viewer on the plate and focus it to give a field of view of 15 x 15 mf. Ta. Edit the obtained image and calculate the projected area per bead by the roundness factor (perimeter). It was calculated together with 2/4 [ou] (projected area). These calculations apply to each set of beads. repeated for at least four fields of view, totaling 15 to 133 fields for each set. beads were analyzed. Calculate the mean square radius, mean roundness factor and their standard deviation. I calculated it.

Bead density was measured as follows. Volumetric flask (10 or 25mA ) was filled up to the calibration mark with distilled water and weighed. Then add many beads, The flask was reweighed. Finally, aspirate the water until the original level is obtained, and the third The weight was measured. Whereas the difference between the second and first reading is the weight of the beads , the difference between the second and third readings was the density of water divided by volume.

Thermal properties were investigated as follows. Wet and dry beads at 80°C to 265°C. Placed in a Petri dish in a Fisher isothermal oven at constant temperature. Beads then Check for any size or color change, place in pHIO carbonate buffer solution, Check for solubility or structural changes.

The effects of pH and salt were investigated. Approximately 0.3g of beads at 2.5mf! Various types of 0.1 Placed in several tens of ml test tubes together with M buffer and left for 30 minutes to 25 hours. . For medium to high pH solutions, two different buffers are used, one with calcium One contained ion removal ions, while the other did not. Also some NaC4 in separate test tubes at a concentration of . In each case, check the size and stiffness of the support. Beta.

Mechanical effects were considered. Place each bead tested on an Ainsworth electronic balance. The beads were then compressed under hand pressure using the flat end of a 2 cm diameter glass rod. . The force was read when the bead height was reduced to approximately half of its original value. This scale is Since a magnetic field servo system is used for weighing, there is almost no movement of the scale during this operation. won.

Magnetic effects were considered. 1.13 g of dry 0.9 ml diameter magnetic beads (15 It was placed in a cm x 3 cm vial and inserted into an oscillating magnetic field of 60 tlz. M H measurements were carried out at 77K (liquid nitrogen temperature) and 300K.

The surface structure was investigated. Magnetic beads made with a 21G needle and dried on a Pel-IJ dish were 1. mm diameter support and vacuum shaded with gold for electron microscopy scanning. did. Initialize and calibrate the device once, then view the photos at 40x, 1250x and ], 0.0 The images were taken at 00x magnification.

At 1250x magnification, two photographs, one 14° from vertical and one vertical to obtain a stereoscopic appearance.

The pore structure was determined. Steady-state porosity measurements using crystallized egg albumen as an interstitial agent It was carried out using a solution of Min (approximately 0.3 g/mj!, absorbance 0.79). known The volumes of beads and albumin solution were mixed and allowed to equilibrate overnight. of supernatant solution Absorbance readings were taken using a 15CODA-4 absorbance monitor. Albumi bead volume accessible to molecules of a size similar to that of the 100 mm (0.01 mm diameter) was then calculated.

The results in Table 1 represent density and mechanical strength studies.

Obviously the strength of dry magnetic beads is much better than that of wet gel. child Their strength depends on whether they have been stored dry or submerged in water for 24 hours. (Displayed as “rehydrated” in the table) The condition of the beads varies depending on the condition, and there may be slight swelling. Some strength loss was observed due to

Judgment-m-1 Non-magnetic, wet 1.067 Magnetic, wet 1.10 20 Non-magnetic, dry 1.7' -- Magnetic, dry 2.2 1000 Topsoil - Density and machine of calcium alginate beads extruded in droplet form from 21G needle mechanical strength The mechanical properties of this non-magnetic wet gel are similar to that of dextran or The strength of the wet magnetic gel is slightly higher, whereas the strength of the wet magnetic gel is similar to that of polyacrylamide. It's bigger than that.

The wet Tyzor-stabilized gel is less elastic than the other two alginate gels. , collapses when compressed instead of just flattening.

The magnetic properties of this bead were found to be attractive. M (magnetization) vs. H (magnetic field strength) The plot shows that while the beads have relatively high magnetism, they have a characteristic characteristic of small magnetic particles. It retains some kind of super-paramagnetic properties. Super-paramagnetism refers to the magnetic mode of magnetite particles. This implies that the ment is sufficiently small that it fluctuates rapidly due to thermal vibrations. this Beads exhibit little remanence when the magnetic field is removed, which is important for most applications. This is a decisive advantage. This hypothesis suggests that the loop at liquid nitrogen temperature is This is confirmed by observing a small hysteresis loop of M vs. H at temperature.

Previously, residual magnetism in polyacrylamide/magnetite beads has been shown to be undesirable. It was reported that this caused the agglomeration of the particles. A small number of beads according to the invention can be used in a magnetic field (3<em>M Although the particles remained slightly magnetized when taken out from It was.

The surface structure of the support, visible at 40x magnification, was slightly altered by drying on a Petri dish. It has a very spherical appearance except for a few small flat spots. 1250 times Detailed surface +14 structures and surface irregularities can be seen in the area. 10.000 times At magnification, the valleys on the surface are approximately 2-5 microns wide and at least 2 microns deep. It appears to be. The representation is extremely rough in this way, and many enzymes, biomolecules, and fine particles are present. Provides a large filtration surface area for cell binding.

The porous nature of the beads was investigated using steady-state albumin permeation. This egg The infiltration of white matter into the beads showed the porosity values listed in Table H. Ordinary alginate While the lucium beads were relatively porous, the wet magnetite was clearly The porosity is slightly smaller due to the internal volume of the beads occupied by the netite particles. It was. Dry spheroids were virtually impermeable by albumin porosity measurements . However, porosity below 5% would not have been apparent due to measurement errors. . Also, since the beads are soft and stretch very slightly after rehydration, some pores of small diameter may occur. There is a possibility that it exists.

Suichi n Non-magnetic, wet 55 Magnetic, wet 25 Magnetic, dry less than 5 ■-Various types of algi extruded in droplet form from a 21G needle calculated from the degree of albumin penetration Porosity of calcium phosphate beads The shape of dried magnetic beads is extremely spherical, and their size is reproducible; There are numerous factors that influence these parameters. One factor is the hypodermic needle This is the method used to extrude alginate solutions from. extruded in drops In this case, the size of the generated beads is determined by the needle size (Table ■) and the flow rate of the solution (Table ■) It can be predicted from

20 0.549 Sat 0.044 1.06±0.03 0.84±0.0321 0.608±0.033 1.04±0.01 0.88±0.0222 0 ．． 53G±0.0/14 1.06±0.03 0.83±0.0323 0. 455±0.0714 1.05±0.03 0.76±0.0425 0.3 5B±0.027 1.06±0.03 0.6B±0.02 Table■-Various gauges Dry calcium alginate/magnetite resin extruded in droplets from a hypodermic needle. size and shape of the , Table--1- 20,567±0.039 1. ．． 04±0.02 0.85±0.033 0 , G24±0.040 1.04±0.02 0.139±0.034 0.6 56±0.046 1.05±0.03 0.91±0.035 0.643± 0.051 1.04±0.03 0.90±0.046 0.71.0±0. 043 1.04±0.02 0.95±0.03B 0.693±0.052 1.05±0.03 0.94±0.0310 0.720±0.054 1 ．． 04 ± 0.02 0.96 ± 0.03 7- from 21G hypodermic needle at different flow rates Size and size of dried calcium alginate/magnetite beads extruded in droplet form shape This flow rate increases so that it deviates from this particular pattern and a spray of small droplets is formed. As a result, beads of considerably small size were made (Table ■).

Dry alginate calcium extruded in spray and drop form from IL-25G hypodermic needle. Size and shape of lucium/magnetite beads A disadvantage of this technique is that the uniform size of the support achieved by dropwise manufacturing is lost. That is. The diameter of the beads formed by the dropwise method has a standard deviation of only about 3%. However, that of spray-formed beads is almost 20%.

Correct calcium depth is essential for optimal crosslinking of the polymer ti'i. However However, calcium is another factor that governs the shape of both wet and dry beads. It is. Low calcium concentrations generate tails as the beads pass over the surface of the polymerization solution However, these tails are removed when the Ca4 + tM degree is increased.

Other species such as Fe3+, M, , 2- and Mn”+ used in the receiving solution on formed spherical beads, but their rigidity was lower than that of calcium alginate. It wasn't as good as the sphere.

Even highly acidic solutions made with 11Cβ were able to form gels.

The drying method is probably the one that gives the most noticeable effect to the shape of the beads. be exposed. Small beads obtained if magnetic spheres are left to dry in a beaker will aggregate and make separation difficult. However, just enough wet solution to form a monolayer If the beads are placed in a vetri dish, the resulting beads are isolated beads that are easily removed. It was the body. The main drawback of this drying technique is that the beads are in contact with the glass surface. It was a small "flat spot" formed by This flat strip on the magnetic beads The pots were of various sizes. However, non-magnetic alginate beads can be used If the flat spot is too prominent, a flat circle is formed instead of a round sphere. A board was formed.

Drying at 80°C and 120°C in a Petri dish removes this flat spot. However, it did not reduce the time required for drying (required for air drying). 60 minutes and 15 minutes respectively for approximately 10 hours). However, the temperature of the beads above 160°C When dried at room temperature or heated and then dissolved as described above, the supernatant solution turns yellow. Color - orange, indicating that the calcium alginate has been affected by excessive heat. are doing.

In some applications, such as high pressure liquid chromatography (IIPLC), the optimum Precise spherical shape and monodisperse diameter are required for splitting. We therefore dry We have developed a technology to remove the irregularities caused by this. Continuous air explained in Example ■ Using a magnetic flux, we created magnetic beads that appeared to be perfectly spherical, with no flat spots. was manufactured. When non-magnetic particles are dried in a similar manner, irregular, coarse spherical beads are formed. Obtained.

The stability of dried beads in aqueous solutions and other solvents is an important consideration. It was. If the beads simply swell to their original size when added to the solvent , the unique characteristics described here would be irrelevant. In order to investigate these characteristics, Samples of beads were then placed in distilled water and the solution was stirred for one week. at least every day The water was removed, replaced with fresh distilled water, and tested for rupture due to calcium diffusion. . At the end of this period the beads were still rigid and could not be crushed with the fingers. Bee When it was first placed in water, it swelled by about 1%, but no further change was observed after that. won. Similar results can be obtained by soaking the beads in solvents such as methanol and acetone. was gotten.

However, other substances can alter the structure of the beads.

Table ■ shows the effect of various buffer solutions at several different pH values.

θ~2 11cff Magnetite dissolution 4 Phthalate 1% swelling Marinate bead dissolution 7 Tris monkey 100% swelling plus 7 Tris” 10 CaCp2 1% swelling 8 Tris” 100% swelling plus 8 Tris”+CaC/2 1% swelling 9 Boric acid 1% swelling 10 Carbonate bead dissolution 10 Boric acid 1% swelling 11 Boric acid 1% swelling 12-14 Na011 1% swelling “　Tris-tris(hydroxymethyl)aminomethane ■-Dry alginate calcium Stability of sia/magnetite beads in aqueous solution. Beads are 21G subcutaneous It was extruded from the syringe needle in the form of drops.

Although some buffers caused bead lysis, at least one buffer solution Does not dissolve beads or affect their structure at pH found. For all cases tested, the addition of buffer solution CaCi, The drying solution also removes calcium from the beads once a lucium precipitate has formed, and and dissolved them. Calcium chelating agents such as EDTA also Dissolved. Other solutions, such as polyvalent metal ions or NaCβ at high temperatures, are Not only the alginate gel but also the structure of the dry spheres was weakened. Simply swell the beads or For the softened solution, a small amount of CaCj! , the addition of usually eliminates this problem Ta.

The properties of non-magnetic dry beads were not as attractive as those of magnetic spheres. Almost In all solutions, the beads increase in size by at least 20% and become extremely soft. It got dark. However, some solutions (e.g. pl+7, CaC#z added Tris) buffer solution), the beads do not elongate more than about 5% without losing their rigid shape. It was. The addition of magnetite to the alginate solution strengthens the overall structure of the beads. It seems like that.

Although these magnetic beads are stable in many different solutions, another material As techniques for introducing additional crosslinks using materials are essential for certain applications. (phosphate and other typical buffer ions destroy the support).

Glutaraldehyde and other reagents have been unsuccessful as cross-linking agents, but A product called "Tyzor TE" from DuPor+t was effective. Tyzor-treated beads prepared as shown in Table 1 are treated with p++ lO carbonate buffer or other calcium. Robust support that is resistant to sium chelators and can withstand a variety of conditions He gave his body. Of course, not all materials have been tested as crosslinkers, just Ty It is not the intention of the present invention to limit the Hydrin as a technology to stabilize non-magnetic wet calcium alginate beads This material has also been reported to have utility in stabilizing magnetic beads. It may be something to do.

In order to demonstrate the usefulness of the magnetic beads according to the present invention as biomaterial supports, a number of We conducted an experiment.

From Actinomyces fradiae The protease of glutaraldehyde and TiCj2. techniques known by both was immobilized on dry calcium alkyl phosphate/magnetite using analyzed.

Glutaraldehyde Cu. 61.0 Glutaraldehyde Zn, 4 3.0 Glutaraldehyde Co+. 35.0 Glutaraldehyde Cd, , 25.0 l-Actinomyces frasiaebu immobilized on a Ca-based support Rotease activity. The beads used were extruded dropwise from a 21G needle and dried. . The concentration of all metal ions used was 0.013M. Their activity was 2 ．． On other supports ranging from 4 to 102 units/g support with an average of 44 units/g support. This is a preferable contrast to the activity obtained. For each method used, It was possible to bind 20-25 mg of protease per g of support.

α-amylase can also be bound to a support according to the invention using other immobilization compounds. Ta.

Glutaraldehyde Ca’° 133TiCj! 3 78 TiC13Ca・◆167 Aspergillus nijini immobilized on a magnetic support illus niger) α-amylase activity. The beads used were 21G. ! The depth of the calcium ions used was 0. It was 013M.

This immobilization in the presence and absence of calcium It is interesting to consider α-amylase from It was something. If the enzyme loses calcium it also loses activity. calcium Immobilization in the absence of is usually not successful, however, in the support according to the invention The presence of calcium in the protein resulted in a binding protein with relatively good activity. fixed Addition of extra Ca” ions during oxidation increased this activity, but the binding The total amount of protein was not increased (residual activity was approximately 45%). numerous branches The activity of other α-amylases bound to carriers is 15-4500 U/H. There was an average of about 200 U/g. Average 20mg for each gram of support of α-amylase was bound.

The optimal temperature for immobilized χ-amylase is the same as that for the soluble enzyme (55°C). , the optimal pl+ and pH stability of both χ-amylase and protease remain unchanged. It was. 10 replicates of specific reaction on 5% substrate immobilized on support α-Amylase and protease activities were not changed. From these results , the immobilization of these enzymes on calcium alginate/magnetite beads is very difficult. It was concluded that this did not affect the enzyme properties.

In view of the results obtained in the immobilization of enzymes, the potential usefulness of magnetic beads according to the present invention has been demonstrated. Its usefulness has become clear.

However, before using these magnetic beads for affinity chromatographic separation, "Binding ligands" that act to bind bioproducts to beads are attached to beads. It is necessary to. Although a large number of these compounds are known [e.g. Dean and Watson, “Protein purification using immobilized triazine dyes J. and Watson, “Protein purification usi ngimmobilized triazine dies’, J, of c chromatography, 155:301). reference. The disclosure content of this document applies mutatis mutandis to this specification. ), special dye C1b acbrontlluc F3G is a type of protein with a very restricted range (" that only those with "dinucleotide cleavage" can be bound by this dye. Since it is well known, I did UtR. Therefore, the test protein, human serum albumin It would be possible to separate the molecule (IIsΔ) from a mixture of other substances. Of course , other binding substances such as antibodies (e.g. monoclonal antibodies) can be combined with this dye. May be used instead of.

Addition of this dye to the magnetic beads of the invention was accomplished as described in the Examples below. It was done.

Departure hearing-N C4bacron Blue F3GA dye binding is Bohme This was accomplished through a modified form of law. Magnetic dry beads (1.4 g) in 1 of 48 mff (C4 bacron in 8.2 ml of distilled water. Blue F3GA (0.27 g) was added dropwise and the solution was stirred for 30 minutes. chloride Calcium (7.9 g) was added and the mixture was stirred for a further hour. At this point 0.2 g NaOH in 2 ml IIzO was added. After 2 hours of stirring, the support was thoroughly treated with 6M urea and IMCaD7! Z melt The beads were thoroughly washed with solution until the blue color leached from the beads was no longer observed.

disaster relief Binding C1bacbron 131ueF3 to remove albumin from solution Separation using alginate-magnetite beads with G.

A plexiglass column with an internal diameter of 172 inches and a length of 4 inches was used for the separation. child columns each having an inner diameter of 5172 inches and an outer diameter of 7172 inches and a diameter of 37 It was located in the center of a pair of coils having a width of 4 inches. These parts Ile was spaced 2172 inches apart. Each coil has approximately 300 turns; 1. A current of 3 amps resulted in a magnetic field strength of about 40 Oersteds.

Calcium alginate-magnetite-Cibacb with a diameter of approximately 200 microns ron Blue beads are automatically added to the column to maintain a bed height of 50 cm. Ta. They range from 0.3 to 0 with an automatically pulsed solid valve at the bottom. ．． It was taken out at a rate of 6 g/min. Solvent (Tris/11Cρ 0.05M 0.05 M CaC1z in pH 8 plus 11□0) into the base of the column. ~15mI! /min. The feed solution is 1 mg/mp concentrated in the same solution as a solvent. The level of human serum albumin (IISA') was It is in the same position as the feed liquid. 0.2-1.0 mg rubumin/min was introduced into the column. The liquid is It was removed from the top of the column at a rate of 8-13 m#/min.

Case 1 (1 mg albumin/min, solids flow 0.3 g/min) will appear. Analysis of liquid and solid components showed that approximately 40% of albumin was adsorbed within a contact time of 30 seconds. It was shown that Albumin is transported at a lower rate of 0.2 mg/min and solidified. When the form is passed through the column at 0.6 g/min, all albumin is solid beads. adsorbed by.

In summary, the present invention is made from an alginate solution containing magnetite particles. dried spheres have excellent potential as supports for enzyme immobilization and chromatographic applications. demonstrated that they have the ability to do so. These beads are made of polyacrylamide and dex Much stronger than gels such as Tolan, allowing high flow rates and pressures in column separations. It shows that it can be used. This support is suitable for temperatures up to 120°C. It not only withstood water, but also withstand most pH values and 9 different solvents. a A solution of some kind, e.g. phosphate buffer, would dissolve this sphere, but stabilization would eliminate this problem. did. The physical properties of these beads are 2.2g/m7! glass-like density, excellent sphericity , low porosity and narrow diameter distribution. The magnetite present in this support This allows the beads to be used in magnetic separations such as high gradient magnetic filtration. that Their high degree of fine-roughness provides a large filtration surface area for enzyme and ligand binding. Ru. Mixed actinomycetes, two representative enzymes of the class that attack large substrates. ・Fraziche proteases and Aspergillus nizhnii α-amylase It was immobilized on the bead surface with high activity and stability.

Cyanuric dyes, which can be used in chromatographic applications, also support this support. was easily attached to the surface with good yield. In short, the magnetic beads of the present invention The support has a wide range of applications in bioseparation and immobilization biochemical techniques. be.

While the preferred embodiments of the invention have been illustrated and described in this manner, the invention may be modified. and modifications are possible, and there is no desire to be limited to the exact terminology shown. Such changes and modifications may be made to adapt the invention to various uses and conditions. We hope that we will make use of it ourselves. Therefore, such changes and modifications are It is appropriate that the claims should be included within the entire scope of the individual, and therefore the following claims: It is included in the scope of.

As described above, the present invention and the method for producing and using the same are related to the present invention, or the present invention is most applicable to the present invention. such that it can be made and used by a person skilled in the art of closely related art. Explained in complete, clear, concise and precise terms.

Brief description of the drawing Procedural amendment (formality) March 7th, 1985 Patent Office Official Black 1) Akio-dono 1.Display of the incident PCT/US 85102360 2. Name of the invention Dry calcium alginate-magnetite in a magnetically stabilized fluidized bed. Use separate separation media. Affinity chromatography/'3, person who makes corrections Relationship to the incident: Patent applicant Name: University Batents.

Incoholated/ 4. Agent Address: 8-10-6 Toranomon-chome, Minato-ku, Tokyo 105, subject to amendment Translation of the description and claims 7. Contents of correction Translation of the specification and claims (No change in content) 8. List of attached documents Translation of the description and claims - one copy each of the international search report

Claims (5)

[Claims] 1. Beads with a generally central inner core of magnetite and an exterior of alginate A solid support material for the binding of bioproduct materials consisting of 2. The support of claim 1 further comprising a binding ligand. 3. 3. The support according to claim 2, wherein the ligand is a triazine dye. 4. 3. The support according to claim 2, wherein the ligand is an antibody. 5. It is characterized by bringing the support according to claim 2 into contact with the biological material. A method for performing affinity chromatographic separation of biomaterials.