AU719422B2 - Adsorption method and separation medium - Google Patents

Description

WO 97/17132 PCT/SE96/01431 1 ADSORPTION METHOD AND SEPARATION MEDIUM TECHNICAL FIELD AND EARLIER KNOWN TECHNIQUES The invention relates to adsorption processes, particularly chromatography, on expanded/fluidized beds. The most advantageous bed is generated by expanding/fluidizing a bed consisting of sedimented beads by directing a flow of fluid against the force causing sedimentation. When the bed has been expanded or fluidized to the volume desired, a sample is introduced into the fluidizing flow. The sample-carried substance or substances to be separated out are therewith adsorbed on the beads so as to be delayed in relation to the sample in its passage through the bed. Adsorption is achieved by virtue of the ability of the matrix/beads to bind to the substance or substances to be separated out. A less effective bed can be generated by agitating suspendible beads with the aid of a turbulent flow or by mechanical agitation. This latter type of bed does not, of course, refer to chromatography, but to a batch-wise adsorption process.

In the context of the present invention the term "bead" refers to the population of particles making up the the bed.

Beads may be more or less spherical and encompasses thus also irregular forms, such as granules, crush-like forms etc, although in connection with the invention spherical or otherwise rounded forms are preferred.

By selecting a bead population which includes beads of mutually different sizes and/or densities and using this population in an expanded bed, it is possible to obtain a socalled classified bed in which the larger and heavier beads lie furthest down in the bed and the smaller and lighter particles are located further up in the bed. The beads will adopt a state of equilibrium when fluid distribution in the bottom of the fluidizing vessel is uniform. Each bead will move within a highly limited volume of the total bed, which means that back-mixing of beads will be very limited. This is the characteristic of a stable expanded bed (sometimes called a stable fluidized bed) and is in contrast to full backmixing which is more normal in the case of fluidized beds. It is important that back-mixing is kept to a minimum, in order WO 97/17132 PCT/SE96/01431 2 to achieve an effective chromatographic process. Reference is made to multi-stage adsorption and the occurrence of concentration gradients of adsorbed substance in the longitudinal direction of the bed. Back-mixing in a bed is often measured as axial dispersion, often expressed by "vessel dispersion number" (see Levenspiel, "Chemical Reaction Engineering" 2nd Edition, John Wiley Sons (1972)).

For stable expanded beds, the vessel dispersion number will preferably be 75x10 3 more preferably S 20x10 3 Backmixing in the bed can be illustrated with the vessels-inseries model, where each vessel, or tank, represents a theoretical plate (see Levenspiel), "Chemical Reaction Engineering" 2nd Edition, John Wiley Sons (1972)). The larger the number of plates, the more stable and more effective the bed. The number of plates of a bed is related directly to the vessel dispersion number. The number of plates for an expanded bed intended for chromatography is preferably 5, more preferably 30. In a fluidized bed with total back-mixing, the number of plates is 1i, i.e. complete agitation.

Expansion/fluidization of the bed is normally effected in a column having provided at its ends a net structure which covers the cross-sectional area of the column, or with some other perforated device which will not generate turbulence in the flow. See, for instance, WO-A-9218237 (Pharmacia Biotech AB, Uppsala, Sweden) in this regard.

Subsequent to adsorption, elution can be effected directly from the expanded bed. Alternatively, the bed may be allowed to settle and adsorbed material eluated from the bed with the aid of a fluid flow normally delivered in a direction opposite to that in which the bed is expanded.

Although aqueous liquids are normally used (for instance buffers dissolved in water), this does not exclude the use of other liquids.

In conjunction with expanded beds, mention is often made of the degree of expansion H/H 0 where H is the height of the expanded bed and Ho is the height of the sedimented bed. For practical reasons, the lowest possible degree of expansion is chosen, although without permitting the beads to pack WO 97/17132 PCT/SE96/01431 3 together or form agglomerates. The degree of expansion will normally lie in the range of 2-10, preferably 2.4-3.2, with a priority that the bed will, at the same time, be stable and achieve the number of bottoms necessary in context.

There are at present at least two commercial suppliers of systems and/or matrices for chromatography on expanded stable beds. Pharmacia Biotech AB (Uppsala, Sweden) market Streamline® which utilizes beads of cross-linked polysaccharide (agarose) with quartz particles (similar to glass crush with sharp edges) as filler. The beads have a density of about 1.2 with sizes lying in the range of 125-315 Pm. Streamline®V has been used primarily for ion exchange chromatography. See also WQ-A-9218237 (Pharmacia Biotech AB) in which there is given a description of suitable column constructions. The other main supplier is Bioprocessing Ltd. (Durham, England) whose porous glass beads (Prosep®D) can be used among other things for chromatography on expanded beds (Beyzavi, et al, Genetic Engineering News, March 1, 1994 17)) Still another supplier in the field is Sepracor.

Also described in the patent literature are similar systems for chromatography on unstable fluidized beds, i.e. beds with a smaller requirement on low back-mixing and low axial dispersion. See, for instance, U.S. 4,976,865 (Sanchez, et al, CNRS) which teaches the use of segmented columns and allows turbulence of the beads within each segment to a more or a less pronounced extent. in this variant, a concentration gradient is generated by virtue of allowing a series of batch adsorptions to be carried out with more or less heavily pronounced back-mixing in each segment.

WQ-A-9200799 (KEM-EN-TEK; Upfront Chromatography) teaches the use of a f luidized bed in which it is endeavoured to generate homogenous intermixing and distribution of f low by agitating the inlet f low to create a mixing zone that is -followed by a non-turbulent zone. The non-turbulent zone has later been claimed to behave as a stable expanded bed. The direction of flow may be either upward or downward utilizing beads that are heavier or lighter, respectively, than the f luid utilized. This latter publication discloses a large number of fillers and polymeric materials that can be WO 97/17132 PCT/SE96/01431 4 combined to produce beads usable for adsorption in fluidized beds.

PROBLEMS RELATED TO EARLIER FLUIDISED BED SYSTEM In the case of adsorption processes on expanded and/or fluidized beds, there is an expressed desire to use beads that have an improved total capacity and/or breakthrough capacity. The desire for improved breakthrough capacity becomes particularly manifest when wishing to use rates of flow above those that can be achieved with current techniques, for instance rates of flow of 1,000 cm/h and preferably still higher, such as flow rates up to 1,500 cm/h. The requirement of stable expanded beds and low degrees of expansion described above also requires access to beads which are able to form sediments more readily. Beads of higher density are also desirable in batch adsorptions in fluidized beds having a high degree of back-mixing (unstable), since beds of this nature will sediment more rapidly at the end of the adsorption process. There is thus a need for bead populations whose beads have a higher density or are larger than is the case in earlier known techniques. At the same time, an increase in flow rate will in most cases result in a decrease in the breakthrough capacity, which in turn counteracts high productivity in the processes concerned. Parallel herewith is the need for beads that have an improved breakthrough capacity.

In Streamline®, there has been used hitherto a filler matrix comprising quartz particles in an agarose matrix. A total capacity of 24 mg/ml gel for IgG can be obtained when coupling Protein A to the beads. The breakthrough capacity for the same gel at a linear flow rate of 300 cm/h (with c/c 0 0.01, where c is the IgG concentration in eluate and co is the IgG concentration in applied liquid) is only 1 mg IgG per ml of gel. In the case of Prosep® (Bioprocessing Ltd.), the concentrations are 30 mg and 24 mg IgG per ml of gel, respectively. This figures refer to study on packed beds but the relation between beads with and without filler is transferrable to expanded beds. This illustrates that filler decreases breakthrough capacity. Although porous glass beads P:\OPER\PDB\75931-96.SPE 6/3/00 are able to give a high breakthrough capacity in relation to total capacity, the density cannot be increased to above that which glass itself affords since filler particles cannot be inserted in glass.

DISCLOSURE OF THE INVENTION We have now discovered that elevated flow rates can be applied and satisfactory breakthrough capacities obtained by using socalled filler matrices and by optimizing the filler with regard to form, diameter, density and quantity. Filler material in the form irregular particles with sharp edges lowers the breakthrough capacity more than spherical granules and other eoeee more or less rounded shapes. This does not exclude the fact that the filler matrices should also be optimized with respect to other variables generally accepted for chromatography, for instance the bead pore-sizes, pore volume and diameter. For instance, it is known that increasing pore sizes facilitate diffusion and permeation of substances to be adsorbed on the matrix. It is also known that decreasing bead diameters •g ~increase the number of pore openings per unit of gel volume, o* which in turn enhances the possibility of substances permating Sthe beads (enhances the breakthrough capacity) It is also known that optimal values with regard to pore diameter and bead diameter are also related to the substance to be bound/adsorbed to/on the beads.

Accordingly in a first aspect, the invention provides a method for adsorbing one or more substances of a sample solution to a fluidised bed comprising exposing said sample to a population of beads fluidised by a liquid flow and having affinity for said one ore more substances in order to separate out said one or more substances from the sample solution, wherein said beads having a density 1.2g/cm 3 comprise a polymer base matrix in which filler particles having a density 3 g/cm 3 are incorporated; and are porous with a Kav in the range 0.40-0.95 for the P:\OPER\PDB\75931-96.SPE 6/3/00 substance or substances.

In another aspect, the invention provides a population of beads having a density 1 g/cm 3 and constructed of a polymer base matrix in which filler particles are incorporated, and wherein the filler particles have a density of ;3 g/cm 3 In still another aspect, the invention provides a method for absorbing one or more substances of a sample solution to beads being suspended in a liquid and having affinity for said one or substances in order to separate out said one or more substances, wherein said beads having a density 1.2g/cm 3 comprise a polymer base matrix in which filler 15 particles having a density 3 g/cm 3 are incorporated; and are porous with a Kav in the range 0.40-0.95 for the substance or substances.

Advantageously, the invention may provide more rapid methods for 20 adsorption/desorption processes that involve fluidized beds. Of Sparticular importance in this regard is chromatography on expanded beds.

Additionally, one or more embodiments of the invention may provide filler matrices that have improved breakthrough capacity and are particularly adapted for chromatography on a stable expanded bed.

The invention may also make possible high yields in chromatography on stable expanded beds.

WO 97/17132 PCT/SE96/01431 6 filler in the form of granules or particles having a density 2 3 g/cm 3 preferably 4 g/cm 3 and thereabove. Those fillers that have been studied hitherto have all had a density g/cm 3 This does not exclude the possibility of utilizing other potential fillers with still higher densities, e.g. up to 25 g/cm 3 It is important that the filler particles are inert and undissolvable in those conditions applied to the use of the beads.

The material in suitable filler particles is often a heavy metal, either in the form of an alloy such as steel (e.g.

Anval®; Anval, Torsh&lla, Sweden) or an oxide zirconium oxide) or some other metal salt tungsten carbide). The filler may also comprise metal spheres tantalum).

The filler particles may vary in size and the size of said particles will always be much smaller than the size of the beads used. Typical sizes are 1-70 pm, with a preference to a range of 15-50 m.

The geometric shape of the filler is highly significant when wishing to retain a high breakthrough capacity in relation to corresponding beads which lack filler. Thus, preferred filler shapes are spheres, ellipsoids, droplets, noodle shapes, bean shapes and other rounded shapes including aggregates/agglomerates and irregular shapes thereof. A particular preference is given to rounded shapes which are continuously rounded.

The filler content of the beads is determined by the density to be achieved, i.e. the rates of flow that are conceivable for use.

When a stable expanded bed is used, the beads should preferably vary in size and/or in density so that they are able to more easily position themselves stably, with lighter and/or smaller beads above heavier and/or larger beads. No compartimentalization in the form of column segmentation is necessary. Neither is it necessary to use magnetic filler in combination with external magnetic fields. Thus, there can be used a bead fraction having sizes within a given range where the proportion of beads in the lower part of the range will be larger than the beads in the upper part thereof.

WO 97/17132 PCT/SE96/01431 7 Typical particle size distributions for a bead population used to create a stable expanded bed are normally such that of the beads fall within a range whose width is 0.1 to times the mean bead diameter, preferrably 0.3 to 3 times the mean bead diameter. The exact particle size distribution to be selected will depend on factors, such as flow rate, mean bead diameter, density of beads, density of fluid etc. A too wide particle size distribution will result in elutriation and/or sedimenting of large proportions of beads. A too narrow particle size distribution will counteract stabilization of the expanded bed. This implies that the population of beads can only be monodisperse in case the individual beads of a population have density distribution within a given density interval.

The ratio between total surface area the beads (outer pus inner surface) and the total bead volume is highly significant to breakthrough capacity. Larger relative contact surface areas (small beads) lead to a higher breakthrough capacity. The total capacity, on the other hand, is only marginally affected. The mean particle size of the beads should generally lie in the range of 10-1,000 pm, with preference to a range of 50-700 pm. The lower limit is determined with a view to the fact that the beads shall not be able to escape from the column in which the expanded bed has been created. Other factors which influence the choice of range limits for bead sizes and distribution within said range include the desired capacity and the substance or substances to be separated from the sample. Although less preferred, another alternative is to vary the density of the beads used in one and the same bed. Bead fractions in which both the size and the density of the individual beads vary can also be used.

The density (mean density) of the beads will always be 1 g/cm 3 for instance 1.1 g/cm 3 such as 2 1.2 g/cm 3 and upwards (measured in the buffer used to maintain the bed in a fluidized state). Beads that are used in one and the same bed will preferably have generally the same density. The amount of filler required can be readily determined from a given polymer base matrix and from the density desired.

WO 97/17132 PCT/SE96/01431 8 It is preferred that the beads are porous with open pores.

Optimal porosity can be determined on the basis of the substance or substances to be adsorbed among other things, and can be calculated conventionally. In order to achieve effective adsorption and/or separation, Kav should lie in the range of 0.40-0.95 for the substance or substances to be adsorbed. For a definition of Kay see L. Hagel in "Protein Purification, Principles, High Resolution, and Applications", J-C Janson and L Ryden (Eds), VCH Publishers Inc. New York, 1989, p. 99.

The beads are normally comprised of a polymer base matrix in which the filler is enclosed. The polymer in the base matrix may be hydrophobic, for instance a bead which is comprised of styrene-divinyl benzene copolymer and which is hydrophilized on the surface by coating it with an appropriate hydrophilic polymer (preferably a polymer having hydroxy or amino groups), for instance. Alternatively, the base matrix may be comprised of an insoluble or soluble hydrophilic polymer, for instance agarose, cellulose, dextran starch, etc., which has been cross-linked to the degree of porosity and stability desired in a known manner, when necessary. At the time of filing the instant application, agarose was the polymer preferred, preferably in a cross-linked form.

The beads often have some form of affinity to the substance or substances to be separated out. This normally means that the base matrix is substituted with one or more groups that have affinity to the substance/substances concerned. Typical groups are: 1.Positively charged groups (primary, secondary, tertiary or quartenary amine groups).

2.Negatively charged groups (for instance carboxy, phosphonic acid, sulfonic acid, etc.).

3.Amphoteric groups.

4.Groups having a specific affinity (for instance bioaffinity groups), such as between IgG-binding protein (Protein A, G, L, etc.) and IgG, lectin and carbohydrates, antigen/hapten and antibody, (strep) avidin and biotin.

nucleic acids/oligonucleotide).

WO 97/17132 PCT/SE96/01431 9 6.Groups which exhibit pi-electron systems.

7.Chelate groups.

8.Hydrophobic groups, etc.

The matrix itself may also have an affinity to the substance or substances to be adsorbed, this affinity being expressed by virtue of the substance concerned being delayed when it shall pass a bed of non-substituted beads. With the aid of these groups, the inventive method can be performed as affinity chromatography, such as ion-exchange chromatography, biospecific affinity chromatography, hydrophobic chromatography, "Reverse Phase Chromatography", chelate chromatography, covalent chromatography, etc. At the filing date of this application, the use of beads according to the above substituted with IgG-binding protein, such as Protein A, G, H or L, preferably produced by recombinant techniques and optionally containing cysteine (for instance rProtein A-cys) was particularly preferred to affinity-purify IgG, particularly monoclonal antibodies. Alternatively, adsorption can be effected batch-wise in a vessel in which the particles are fluidized by subjecting them to a flow of liquid or by agitation.

The samples to be purified may be of the same type as those earlier used in chromatographic processes on packed or expanded beds, or in batch adsorption processes on fluidized beds. The invention can be applied to great benefit in the direct treatmen of supernatants/culture media from fermentators and other cell culture vessels, particularly in chromatographic processes on expanded beds.

The invention will function for the separation of compounds of various molecular weights and types. Examples are polysaccharides, proteins/polypeptides and nucleic acids and synthetic water-soluble polymers, e.g. with molecular weights 5,000 dalton. Typically preferred molecular weights with regard to substances to be adsorbed on the beads in accordance with the invention are 50,000, preferably 100,000. There is normally no upper limit, even though the process is normally limited to the removal of compounds that have a molecular weight below 350,000.

WO 97/17132 PCT/SE96/01431 Manufacture of the beads, cross-linking, introduction of binding groups, addition of filler, etc., are effected in a known manner, while ensuring that the beads will be suited for adsorption purposes in accordance with the aforegoing.

The beads are sieved when necessary, to obtain a suitable size fraction.

The affinity group concerned may be introduced on a bead by activating an appropriate hydrophilic group, such as carboxy, amino, hydroxy, etc., with a suitable bifunctional reagent, such as CNBr, bisepoxide or corresponding epihalohydrin, etc., which in is turn reacted with a compound that exhibits the affinity group concerned.

A second aspect of the invention, is a bead population (bead fraction) according to the above, which is suitable for use as a matrix in adsorption processes, particularly chromatographic processes, effected on expanded/fluidized beds in accordance with the aforegoing. This aspect of the invention also includes the population in the form of a stable expanded bed according to the above, placed in a chromatographic column. The aspect also includes bead populations which lack affinity groups, including preactivated forms. In this latter case, the customer/user can himself introduce a desired group.

A particularly important embodiment of this second aspect of the invention includes beads which are substituted with IgG-binding protein, particularly an IgG-binding protein containing the amino acid cystein for mediating binding to the beads. Proteins of this type have earlier been produced by recombinant techniques, e.g. cysteine-variants of Protein A (Ljungquist, et al, (Eur. J. Biochem. 186 (1989) 557-561) and Profy (EP 284,368), and cysteine-variants of Protein G (Fahnestock, et al 4,977,247)). It is known to bind cysteine-containing proteins covalently to solid phases via thiol groups. It is also known that certain coupling methods will give high selectivity for bonding via thiol, for instance via reactive disulphides (for instance pyridyl disulfide) or maleimide groups. Other methods, such as via epoxy or epihalo hydrin, are less selective and also give coupling via primary amine (lysyl).

WO 97/17132 PCT/SE96/01431 11 With the aid of IgG-binding protein, particularly recombinant Protein A-cys, we have been successful in producing filler matrices whose total capacity lies in the range above 30 mg IgG/ml gel, for instance between 40-60 mg IgG/ml gel, and whose breakthrough capacity has been above mg IgG/ml gel, for instance in the range of 20-30 mg IgG/ml gel (with c/co 0.01, where c is the IgG concentration in eluate and c o is IgG concentration in applied fluid). The base matrix comprised cross-linked agarose. In these instances, the filler had the aforesaid particle form. See also the experimental part of this document.

The following experimental part discloses the manufacture of the bead population most preferred at the priority filing of this application. When derivatized further or used in different studies, this bead population is referred to as standard gel.

EXPERIMENTAL PART SYNTHESIS METHODS Fillers used: o Anval 600® (Anval, Torshalla, Sweden) is a chromiumnickel alloy having the chemical composition (weight-%) C 0.02%, Si 0.41%, Mn 0.31%, P 0.007%, S 0.001%, Cr 15.5%, Ni 75.0%, Cu 0.02% and Fe 8.68%. The alloy has a density of 8.4 g/cm 3 The particles have a spherical shape and preparations with two diameters were used (16-44 pm 16 pm).

o Tantalum (Ta) (Novakemi AB, Enskede, Sweden), having a density of 16.5 g/cm 3 and consisting of sintered spherical particles. Diameter 5-44 pm.

o Tungsten carbide (WC) (AB Sandvik Coromant, Stockholm, Sweden) having a density of. 15.6 g/cm 3 and consisting of small (in relation to tantalum) sintered spherical particles. Two preparations were used (diameter 10-50 pi and 15 pm).

o Zirconium oxide (ZrO,) (MEL Chemicals, Manchester, England) having a density of 5.6 g/cm 3 and consisting of small (in relation to tantalum) sintered spherical WO 97/17132 PCT/SE96/01431 12 particles. There was used a preparation having a diameter of 5-30 pm.

1. Emulsion Step 1.1. Dissolution of ethyl cellulose in the emulsifying reactor.

Equipment: Cylindrical glass reactor 2.5 1 with flat bottom (emulsifying vessel), water bath, armature agitator and thermometer.

Method: 1050 ml of toluene here charged to the reactor, whereafter the agitator was activated and a 49.5 g of ethyl cellulose were added in a "fine jet". The mixture was heated to 60 0 C until all ethyl cellulose had dissolved (about two hours).

Step 1.2. Preparing a solution containing agarose and filler.

Ecuipment: Mantled glass reactor with bottom valve (the reactor was positioned so that its contents could be emptied into the emulsifying vessel), agitator, circulating water and thermometer.

Method: 900 ml of distilled water were charged to the reactor. The agitator was then activated and 36 g of agarose were delivered. The mixture was heated to until the agarose had dissolved (about one hour). 378 g Anval® were then added to the mixture and the mixture was agitated for a further fifteen minutes, after which the temperature was lowered to Step 1.3. Transferring the solution containing agarose and filler to the emulsifying vessel.

Method: The speed of the agitator in the emulsifying vessel was adjusted to 130 r.p.m. and the solution (70 0

C)

was transferred to the emulsifying vessel.

Step 1.4. Adjusting particle size.

This was effected conventionally, by varying the agitator speed.

WO 97/17132 PCT/SE96/01431 13 Target values: When desiring a gel in which the beads of the main bead fraction have a diameter of 80-160 pm, the emulsifying process is interrupted when 95% (volume) of the beads have a diameter 200 pm (standard gel). When desiring bead diameters of 80-200 pm in the main bead fraction, the emulsifying process is interrupted when of the beads have a diameter 250 pm.

Step 1.5. Cooling.

Cooling: Heating of the water bath was stopped. With the equipment used (laboratory scale), the temperature of the bath was lowered from 60 to 30°C in about seven hours.

Step 1.6. Working up.

Method: The beads were washed by agitation and thereafter decantered (3x) with 3 1 of 99.5% ethanol. Washing of the beads was continued on nutsch with 4 x 2 1 of ethanol with self-draining. The beads were finally transferred to distilled water, via agitation and decantering.

2. Cross-linking.

EquiDment: Glass reactor, water bath, agitator, glass cooler and thermometer.

Method: 100 ml of 75% gel slurry and 34 g of sodium sulphate were charged to the reactor and stirred for two hours, whereafter the temperature was raised to 50 0 C. 1 ml of 45% NaOH solution and 0.1 g of sodium boron hydride were then added. 7 ml (10.5 g) of 45% NaOH solution and ml of epichlorohydrin were added at the same time with the aid of a pump and over a period of six to eight hours. The reaction was allowed to continue overnight while stirring the system (for about sixteen hours) at OC. Upon completion of the reaction, the gel was slurried (washed) with 7 x 150 ml water, whereafter the gel was acidified with 60% acetic acid to pH 5-6 and wet-sieved (80-160 pm or 80-200 pm).

WO 97/17132 PCT/TISE96/01431 14 3. Coupling Protein A to the finished gel.

Native Protein A was from Pharmacia Biotech AB, Sweden.

Recombinant Protein A-cys (rProtein A-cys) contained the four IgG-binding domains D, A, B and C) of the native form, followed by the first eight amino acids of the X-domain, followed by a non-Protein A sequence of five amino acids and cysteine in the C-terminal (the Protein A sequence corresponded to alanine in position 18 up to and including proline in position 316 in accordance with EP 284,368. The process was analogous with the process earlier described by Profy T (EP 284,268) and Lungquist, et al (Eur. J. Biochem.

186 (1989) 557-561). Solutions of rProtein A-cys were stored in a reduction buffer.

A. Native Protein A Couplina with N-hvdrolxvsuccinimide (NHS): 30 ml of standard gel were washed with distilled water, mixed with 3.67 g NaOH dissolved in 18 ml distilled water while stirring, and the temperature was adjusted to 24 0 C. After some minutes, 7.2 ml of epichlorohydrin were added while vigorously stirring the mixture. After two hours, the gel was washed on a glass filter with 300 ml of distilled water. The washed gel was then mixed with 6-aminocapronic acid (6-ACS; 30 ml solution 1 M 6-ACS, 1 M NaC1 pH 11.5) and the mixture stirred for 17-24 hours and then finally washed with 200 ml 0.5 M NaCl. The gel was then again washed, now with 2 x 30 ml acetone, whereafter the gel was mixed with 15 ml acetone and activated with 559 mg NHS and 1007 mg of dicyclohexylcarbodiimide while stirring the system. After 4-17 hours at 31°C, the gel was washed with 2 x 30 ml acetone 450 ml isopropanol and cooled with 210 ml of ice-cold 1 mM HC1. The resultant activated gel was mixed with 30 ml solution containing native Protein A (Protein A dissolved in 0.2 M NaHCO 3 1 M NaCl pH and stirred at room temperature for two hours, and then washed with Tris buffer pH 8, and acetate buffer pH 3, and finally with distilled water.

WO 97/17132 PCT/SE96/01431 B. CouDlina of rProtein A-cvs Activation with the aid of 1.4-bis (2.3-epoxvropoxy) butane (BPR butane). One litre of drained Sepharose® FF (agarose in bead form cross-linked with epichlorohydrin, Pharmacia Biotech AB, Uppsala, Sweden) was washed on nutsch with distilled water and mixed in a thermostated reaction vessel with 55 g NaOH dissolved in 300 ml distilled water, 35°C, while stirring the system. 390 ml BPR butane were added. The system was stirred for two hours at 35°C, followed by washing with 15 1 water.

Coupling of rProtein A-cvs. The activated gel was washed on nutsch with 3 x 1 1 nitrogen-gas saturated 0.1 M Na phosphate, 1 mM EDTA, pH 8.5 and allowed to drain. The gel was then mixed with 5.5 g rProtein A-cys dissolved in nitrogen-gas saturated aqueous solution of 0.1 M Na phosphate, 1 mM EDTA, pH 8.5. The system was stirred at 37°C while blowing-in nitrogen gas. Sodium sulphate (370 g) was added. After stirring the system for two hours at 37C, the gel was washed with 3 1 of distilled water and sucked dry until cracks formed.

Deactivation: The dry-sucked gel was mixed with 100 ml glycerol dissolved in 900 ml 0.2 M sodium bicarbonate, M NaC1, 1 mM EDTA, pH 10, while stirring the system.

The system was stirred overnight at 37C, whereafter the gel was pH-washed on nutsch with 0.1 M Tris, 0.15 M NaC1, pH 8, and 0.05 M acetic acid in three cycles with a 3 x 1 gel volume in each cycle. The gel was finally washed with water.

The aforesaid synthesis conditions were varied as disclosed in the following Tables.

Analysis methods Function tests on finished gel.

Degree of expansion Equipment: Pump P 6000 and column XK 26/60 (Pharmacia Biotech AB, Uppsala, Sweden).

Method: About 70 ml of finished gel were delivered to the column. An upper adapter (without net) was fitted, such that WO 97/17132 PCTISE96/01431 16 the distance to the lower adapter was about 52 cm. Elutration was effected over a period of one hour at a rate of flow of about 100 cm/h higher than that at which it is desired to determine the degree of expansion. At the end of the elutration process, the rate of flow was lowered to the flow rate at which the degree of expansion shall be determined. The gel height was read-off after a further thirty minutes and the pump shut-off. When the gel had settled to form a sediment, fifteen minutes, the gel height (H 0 was again read-off. The degree of gel expansion is determined from the

H/H

0 relationship.

Breakthrough capacity Q, (binding of IgG) at 300 cm/h (c/co=0.01). Packed bed. Native Protein A and rProtein A-cys.

Equipment Instruments: FPLC with two P 6000 pumps (Pharmacia Biotech AB), UV-2 and printer.

Column: XK 16/20 (Pharmacia Biotech AB).

Buffer A: 20 mM sodium phosphate, pH Buffer B: 0.1 M glycine, pH Protein: hIgG (Pharmacia Biotech AB).

Flow: 10 ml/min. 300 cm/h.

Procedure: 25 ml of drained-off Protein A gel (or rProtein A-cys gel produced from standard gel in accordance with the above) .were allowed to settle and form a sediment in the column. The adapter was adjusted to the gel surface and a flow containing buffer A was applied stepwise to 10 ml/min, whereafter the adapter was further adjusted. A flow of 0.5 g IgG per ml in buffer A was then applied through the column.

The column was initially bypassed and the flow delivered directly to the UV-monitor, wherein an absorbance value (280 nm) for non-absorbed solution was measured whereafter the flow together with sample was allowed to pass through the column. When the absorbance value for the flow through the column (280 nm) was 1% of the absorbance c o the test was interrupted, the gel was washed and IgG bound to the gel was eluated with buffer B and the gel washed with 0.1 M glycine, pH 2.5, 30% isopropanol and 20% ethanol. The eluate was collected and its IgG content determined, which in turn gave WO 97/17132 PCT/SE96/01431 17 the amount of absorbed IgG per ml of gel (breakthrough capacity for c/c 0 The breakthrough capacity Q, (IgG) for an expanded bed.

Native Protein A.

Equipment: Instrument: Pump P 6000, UV-2; 280 nm and printer (Pharmacia Biotech AB).

Column: XK 26/60 (Pharmacia Biotech AB).

Buffer A: 20 mM sodium phosphate, pH Buffer B: 0.1 M glycine, pH Protein: 0.5 mg/ml hIgG (Pharmacia Biotech AB) in buffer

A.

Flow: 300 cm/h.

Procedure: 12.8 cm of Protein A gel sediment (produced from the standard gel in accordance with the above) was elutriated at 400 cm/h and expanded stepwise in XK 26/60 (Pharmacia Biotech AB). The column was initially bypassed and the flow delivered direct to the UV monitor, in which an absorbance value (280 nm) of non-absorbed solution was measured (Co), whereafter the flow together with sample was allowed to pass through the column. When the absorbance of the flow through the column (280 nm) was 1% of the absorbance c o the test was interrupted, the gel was washed and the IgG bound to the gel eluated with buffer B and washed with 0.1 M glycine, pH isopropanol and 20% ethanol. The eluate was collected and its IgG content determined, which in turn gave the amount of absorbed IgG per ml of gel (the breakthrough capacity for c/co=0.01).

Total capacity (IgG). Packed bed. Native Protein A and rProtein A-cys.

Equipment: Instrument: FPLC system with superloop (Pharmacia Biotech AB).

Column: 1 ml Hitrap (Pharmacia Biotech AB).

Buffer A: 10 mM sodium dihydrophosphate, 0.15 M sodium chloride and 10 mm EDTA, pH Buffer B: 0.5 M acetic acid (gives a pH of about 2.7).

WO 97/17132 PCT/SE96/01431 18 IgG solution: 150 mg hIgG in 10 ml of buffer A.

Procedure: 1.0 ml of Protein A gel (or Protein A-cys gel produced from standard gel in accordance with the aforegoing) was packed in the column and equilibrated with buffer A, whereafter IgG solution was delivered through the superloop at a flow rate of 0.15 ml/min. After washing with buffer A at the same rate of flow, adsorbed IgG was eluated with 9 ml buffer B at a flow rate of 0.30 ml/min. The eluate was collected and its volume determined. A 280 was read-off after diluting to 1:10 and the total IgG capacity (mg (IgG/ml gel) was determined as the eluate volume (ml) x A 280 (diluted eluate) x 7.244.

Table 1. Presentation of synthesis conditions and results from examining the relationships between filler (type and diameter) and the IgG capacity of Protein A (native) gel.

Agarose sol. Filler Emulg. Sieving Capacity Type Diam Amount QB Total ml Pu g g1) Pm mg IgG/ml gel 400 4 None 7.5 80-160 14.80 24.0 300 4 Quartz <25 17 6.7 80-160 3.00 20.50 400 4 W 2 <15 40 10 80-160 5.40 23.40 400 .4 2) 10-50 40 7.5 80-160 10.60 22.50 400 4 Tantalum 5-45 40 10 80-160 14.60 26.70 300 4 Anval <16 40 5.8 80-160 11.60 19.80 300 4 Anval 16-20 40 7.5 80-160 14.90 23.30 400 4 Anval 16-44 40 10 60-160 15.30 23.20 300 4 Anval 44-105 40 7.5 3) 3) 300 4 Z 4 <15 40 7.3 80-160 9 17.00 1) Amount per 100 ml agarose solution; 2) Tungsten carbide; 3) Low emulsifying yield and hence cross-linking and coupling to Protein A were not carried out; 4) Zirconium oxide.

Table 2. Presentation of representative synthesis conditions and results from tests concerning the relationships between WO 97/17132 PCTSE96/01431 19 added amounts of Anval to agarose solution and the degree of expansion of the cross-linked gel at 500 cm/h and 600 cm/h.

Filler" Bead fraction Degree of expansion (H/H 0 amount g 2 pm Flow 500 cm/h Flow 600 cm/h 60 80-200 3.3 3.7 125-250 3.3 3.6 125-250 2.0 2.2 80-250 3.0 3.2 125-250 2.1 1 Anval diameter 16-44 pm; 2)Amounts in Table calculated on 100 ml of agarose solution The density varies between 1.6-1.8.

Table 3. Total capacity and breakthrough capacity Q. (mg hIgG/ml sedimented gel, c/c 0 for Protein A gel (produced from standard gel in accordance with the experimental part).

Type of bed Flow rate QB Total capacity Packed bed 300 cm/h 15.8 mg/ml 25.1 mg/ml Expanded bed 300 cm/h 13.4 mg/ml Not determined Table 4. Packed bed. Total capacity and breakthrough capacity Q (mg hIgG/ml sedimented gel, c/c 0 for rProtein A-cys gel (produced from epoxy-activated standard gel according to the experimental part).

Epoxy Protein A-cys mg hIgG per ml gel Standard gel pmol/ml gel Charged mg/ml gel Total QB 80-200 p 6.0 6.5 52.7 30.0 80-200 I 6.1 6.5 48.2 27.1 100-200 i 5.9 6.5 49.2 21.1 80-200 7.7 6.5 51.4 26.6 WO 97/17132 PCT/SE96/01431 Characterizing the stability of an expanded bed A column (200 mm, 100 cm height) containing a distributor whose hole area was 0.3% of the total area, was filled with cm (4.7 1 rProtein A-cys gel) having a particle size distribution in the range of 80-160 pm. The gel was expanded to 38 cm at a linear flow rate of 300 cm/h (20 mM Na phosphate, pH The pressure drop across the distributor was 100 Pa. A positive step-response injection with 0.25% acetone solution was introduced into the column as a stimulus experiment. When 100% of the acetone solution could be detected at the column outlet, the flow was switched back to buffer solution for a negative step response. The plate number for the column and system was calculated on the basis of the negative step response in accordance with the same principle as that applied with pulse injection (Chemical Reaction Engineering, 2nd Edition, John Wiley Sons (1971)).

The number of plates for the column plus system was Compensation was then made for the number of plates for the system contribution, resulting in 34 plates for the column.

In turn, this corresponded to a vessel dispersion number of 17 x 10- 3

DISCUSSION

Degree of expansion The test results show that the inventive filler matrices are able to achieve degrees of expansion that lie within the ranges used in practice in expanded beds, even in the case of elevated flow rates 300 cm/h).

Filler Synthesis conditions and results are apparent from Table 1.

The fillers used were Anval®, tantalum, tungsten carbide, zirconium oxide and quartz.

There seems to be no differences for various fillers in the total capacity of the gel to bind IgG.

The breakthrough capacity is significantly influenced by the type of filler used and its diameter and shape. Studied fillers can be divided into groups: WO 97/17132 PCT/SE96/01431 o Group 1 Anval®: Anval®: Tantalum: o Group 2 Anval®: Tungsten carbide: Zirconium oxide: Approximate breakthrough capacity (IgG) for gel with and without filler.

Diameter 16-20 pm spherical particles.

Diameter 16-44 pm spherical particles.

Diameter 5-44 pm sintered spherical particles.

About 25% lower breakthrough capacity (IgG) than Group 1.

Diameter 16 pm spherical particles.

Diameter 10-50 pm sintered small" spherical particles.

Diameter 5-30 pm sintered small" spherical particles.

Roughly a 70% lower breakthrough capacity (IgG) than Group 1.

Diameter 15 pm sintered small" spherical particles.

Diameter 25 in "crush-like particles".

o Group 3 Tungsten carbide: Quartz: "In relation to tantalum.

Comparison between fillers of equivalent diameters.

o Anval® (16-44 pm) and tantalum (5-44 pm) give the same breakthrough capacity (IgG) as gel that lacks a filler.

o Tungsten carbide (10-50 pm) gives a breakthrough capacity (IgG) which is about 30% lower than Anval® (16-44 pn).

o Tungsten carbide 15 pm) gives a breakthrough capacity (IgG) which is about 50% lower than Anval® 16 pm).

o Quartz 25 pm) gives a breakthrough capacity (IgG) which is about 75% lower than Anval® 16 pm) and about lower than tungsten carbide 15 pm).

The usefulness of the various fillers with respect to breakthrough capacity (IgG) can be ranked on this basis: Anval® tantalum tungsten carbide quartz. If it is assumed that the breakthrough capacity is influenced solely by shape, a smooth, spherical surface (Anval®) is better than WO 97/17132 PCr/SE96/01431 22 the surface of small, spherical sintered particles (tungsten carbide). Worst of all are the irregular particles ("crush" (quartz)). It can be seen from the study that quartz included in existing matrices for Streamline® is least suited as a filler.

When comparing the same type of fillers with different diameters, it is found that: o Anval® Having a particle diameter 16 pm gives a breakthrough capacity (IgG) which is about 25% lower than particles having a diameter of 16-44 pm.

o Tungsten carbide Having a particle diameter 15 pm gives a breakthrough capacity (IgG) which is about lower than particles sizes of 10-50 pm.

Filler having a particle diameter 15 pm thus lowers the breakthrough capacity (IgG).

Very low yields were achieved when emulsifying together with Anval® having a particle size distribution in the interval of 45-105 mn, because the filler sedimented instead of being incorporated in the beads.

The study carried out on the matrices shows that the optimal diameter of the filler particles is 15-40 pm with respect to manufacturing yield.

In order to achieve high mass transfer and thereby high breakthrough capacity at high flow rate, a preferred mode employs, bead diameters 80 pm, when not taking the entire system solution into account. There is often used at the inlet and outlet of chromatographic expanded beds a net which functions to retain the bed while allowing fermentor solution to pass through (including particulate impurities). In the case of existing Streamline® this means that particulate impurities of up to 56 pmn in diameter are able to pass through. The upper bead diameter should preferably lie as close as possible to the lower bead diameter, if optimal capacity is to be obtained. In this study, we have obtained good yields and high breakthrough capacity with the upper limit of 160-200 pm. The end choice of an upper limit will depend on the particle size distribution obtained by scalingup relative the costs for the to base matrix and performance.

P:\OPER\PDB\75931-96.SPE 6/3/00 23 Stability of expanded beds comprising inventive beads.

The test results showed that expanded beds which fulfil conditions for stabilized beds can be obtained with good margins.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

9O ooo

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Claims (24)

1. A method for adsorbing one or more substances of a sample solution to a fluidised bed comprising exposing said sample to a population of beads fluidised by a liquid flow and having affinity for said one ore more substances in order to separate out said one or more substances from the sample solution, wherein said beads having a density 1.2g/cm 3 comprise a polymer base matrix in which filler particles having a density 3 g/cm 3 are incorporated; and are porous with a Kav in the range 0.40-0.95 for the substance or substances.

2. The method according to claim 1 wherein the filler particles of the beads used have a rounded shape or are S aggregate/agglomerate of particles of such shapes.

3. The method according to claim 1 wherein the filler particles of the beads used have spherical or ellipsoidal shape. S cooa *5So

4. The method according to any one of claims 1-3 wherein the filler particles of the beads used further comprising a heavy metal. The method according to any one of claims 1-4 wherein the filler particles of the beads used contain a heavy metal salt.

6. The method according to claim 4 wherein the filler particles of the beads used contain a heavy metal in the form of a carbide, an oxide, an alloy or a pure heavy metal.

7. The method according to any one of claims 1-6 wherein said liquid flow is directed upwards and is at a liquid flow rate in excess of 300 cm/hr.

8. The method according to claim 7 wherein the liquid flow P:\OPER\PDB\75931-96.SPE 6/3/00 rate is 1000 cm/hr or greater.

9. The method according to any one of claims 1-8 wherein the fluidised bed is a stabilized expanded bed. The method according to any one of claims 1-8 wherein the fluidised bed is an expanded bed having a vessel dispersion number of 75 x 10 3 or less.

11. The method according to any one of claims 1-8 wherein the fluidised bed is an expanded bed having a plate number 5 or greater.

12. The method according to any one of claims 1-8 wherein the fluidised bed is an expanded bed having degree of expansion which is in the range of 2-10.

13. The method according to any one of claims 1-12 wherein the beads further comprise functional groups that exhibit affinity to the substance or substances to be separated out. .14. The method according to any one of claims 1-13 wherein the individual beads of the population of beads have densities within a given density range and/or different sizes within a given size range. The method according to any one of claims 1-14 wherein the population of beads has a bead size distribution such that of the beads fall within a range of 0.1 to 10 times the mean bead diameter of the population.

16. The method according to claim 14 wherein the beads of the population of beads used have a mean size of between 10-1,000 gm. S 17. The method according ot claim 16 wherein the beads of the 1V population of beads used have a mean size between 50-700 /m. P:\OPER\PDB\75931.96.SPE -6/3100 26

18. The method according to any one of claims 1-17 wherein the filler particles of the beads used have a particle size within the range of 1-70 pm.

19. The method according to claim 18 herein the filler particle of the beads used have a particle size within the range 15-50 kum. The method according to any one of claims 1-19 wherein the polymer base matrix includes a plurality of hydroxy groups.

21. A method according to claim 9 wherein no external magnetic field is applied to the fluidized bed.

22. A method according to any one of claims 1-21 wherein said S: one or more substances has a molecular weight 5,000 dalton.

23. A population of beads having a density 1 g/cm 3 and constructed of a polymer base matrix in which filler particles are incorporated and wherein the filler particles have a density **of 3 g/cm-3. A

24. A population of beads according to claim 23, wherein the individual beads have different densities within a given density Srange and/or different sizes within a given size range. A population of beads according to claim 23 or claim 24 wherein the beads have a mean density of 2 1.2 g/cm 3

26. A population of beads according to any one of claims 23-25 wherein the beads have a mean size of between 10-1,000 gm.

27. A population of beads according to claim 26 wherein the beads have a mean size of between 50-700 Mm. A population of beads according to any one of claims 23-27 P:\OPER\PDB\75931-96.SPE 6/3/00 27 wherein the filler has a particle size in the range of 1-70 Im.

29. A population of beads according to claim 28 wherein the filler has a particle size in the range of 15-50 /m. A population of beads according to any one of claims 23-29 wherein the filler particles have a rounded shape or an aggregate/agglomerate of particles of such shapes.

31. A population of beads according to any one of claims 23-30 wherein the filler particles have a spherical or ellipsoidal shape.

32. A population of beads according to any one of claims 23-31 wherein the filler particles contain a heavy metal, a carbide or e an oxide, or an alloy or a pure metal. %e

933. A population of beads according to claim 32 wherein the heavy metal is in the form of a heavy metal salt. *o 34. A population of beads according to claim 32 wherein the heavy metal is in the form of a carbide or an oxide or an alloy or a pure metal. 35. A population of beads according to any one of claims 23-34 wherein the polymer base matrix includes a plurality of hydroxy groups. 36. A population of beads according to any one of claims 23-35 wherein the beads are porous. 37. A method for absorbing one or more substances of a sample solution to beads being suspended in a liquid and having affinity for said one or substances in order to separate out said one or more substances, wherein said beads having a density 1.2g/cm 3 comprise a polymer base matrix in which fillerRA comprise a polymer base matrix in which filler P:\OPER\PDB\75931-96.SPE 6/3/00 28 particles having a density 3 g/cm 3 are incorporated; and are porous with a Kav in the range 0.40-0.95 for the substance or substances. 38. A method according to claim 37 wherein the beads are as defined in any one of claims 1 to 6 or 13 to 39. A method according to claim 1 or claim 37 substantially as hereinbefore described. A population of beads according to claim 23 substantially as hereinbefore described. DATED this 6th day of March 2000 Pharmacia Biotech AB By its Patent Attorneys .*C "DAVIES COLLISON CAVE *eeo*