METHODS AND APPARATUS FOR CAPTURING TARGET SUBSTANCES FROM LIQUID MEDIA

Abstract: The application describes methods and apparatus for separating target substances from flowable media. Particular embodiments are directed to capturing biological molecules from unclarified cell culture broth. The target molecules are captured in the body of a microporous wall whose internal pore surfaces are adapted, e.g. by the provision of affinity binding sites, to capture target molecules as a filtrate permeates through. The filtrate may be recirculated. A preferred arrangement provides th emicroporous wall as tubes through which the flowable process medium is passed.

Description: METHODS AND APPARATUS FOR CAPTURING TARGET SUBSTANCES FROM LIQUID MEDIA FIELD OF THE INVENTION This invention relates to the capture, isolation and recovery of target substances from liquid media containing them. Particular embodiments relate to the capture of biologically-active substances from biological culture broths, e.g. proteins made by cell culture processes . BACKGROUND The isolation of chemical product species from reaction mixtures conventionally exploits the characteristic chemical behaviour of the substance to be captured, in a process such as chromatography. Chromatography has good selectivity, but requires careful preparation of the reaction mixture. In the context of biological production processes, for example, the "reaction mixture" is in effect a culture broth of the organisms which have been cultured to make the product. Depending on the cells and on the type of process, the mixture presented for purification may contain whole cells or cell debris resulting from lysis. Purification of such a mixture using chromatography requires preliminary stages of clarification, i.e. separating the solid particles from the liquid by methods such as centrifugation and filtering. Only then can the liquid be applied to a solid chromatography medium. More recently, proposals have been made for selective chemical capture of target species from unclarified broths. One such process is expanded bed adsorption, in which a bed of absorbent particles treated with selective binding functional groups is mixed with the broth in a through-flow process. Target substances are chemically captured on the absorbent particles as the broth, including the gross solids, passes through the loose bed. Subsequently the absorbent particles are retrieved and the bound target substance is desorbed by appropriately changing the prevailing conditions e.g. changing pH by means of a buffer. THE INVENTION We now propose novel methods and apparatus for capturing target substances from liquid media by chemical means . Broadly speaking, what we now propose is a procedure for capturing molecules of a target substance from a fluid process medium which incorporates the target substance in solution in a liquid component of the process medium, by applying the process medium to a microporous wall which has through-pores sized to accommodate the molecules of target substance, whereby the solution of target substance molecules permeates through the microporous wall. The pores of the microporous wall have binding surfaces adapted to bind the target substance molecules and which thereby capture such molecules in the body of the microporous wall as the solution thereof permeates through. The target substance is then recoverable from the microporous wall as appropriate, by subjecting it to conditions (e.g. washing at selected temperature and/or pH) which release the target substance from the binding surfaces in the pores. Aspects of the invention are the capture method itself, e.g. as applied to various kinds of process medium explained hereinafter, and apparatus adapted for carrying it out including the microporous wall. A particular virtue of the technique is that, because the target substance accumulates inside the porous wall, what happens at the surface of the porous wall becomes less critical and it is possible to use the method with process media containing gross particulates, e.g. unclarified broths from cell culture processes. A particular aspect of the invention is a cell culture process in which cell culture produces the target substance in a cell culture broth, and the target substance is recovered from solution in the cell culture broth by means of a technique as described herein, preferably without clarifying the broth. In the present technique only liquid dissolving the target substance need permeate through the microporous wall. Other matter contained in the process medium may be unable to pass through the microporous wall. Therefore it is preferable for the process to include one or both of - clearing accumulated non-permeating components of the original process medium from the microporous wall's upstream surface, and - recirculating the permeated filtrate for another passage through the wall. Having these desired features in mind, a particularly preferred embodiment of the invention provides a conduit having the microporous wall as a sidewall. The process medium is caused to flow along the conduit, the mentioned liquid component permeating out sideways via the microporous sidewall to emerge as a filtrate. A particular virtue of this arrangement is that the bulk of the process medium flow need not be detained at the microporous wall. It continually passes away along the conduit. This helps to keep the inner surface of the microporous sidewall clear, and enable continuous processing. The retentate, i.e. that part of the process medium which has gone along the conduit without passing through the wall, can be recirculated to pass through it again, as often as is necessary. Since recirculation is a technically simple matter it is not necessary that a large proportion of the target substance be captured at each pass. Provided the binding surface has sufficient absorptive capacity, the process medium can be passed and re-passed through the conduit until a maximum, preferably essentially all, of the target substance is taken up or until the absorptive capacity of the microporous wall for the target substance has been taken up. By using the porous interior of the wall layer a significant increase in effective surface area for capture can be achieved, compared with size filters which capture at the surface. The filtrate can be returned for a further pass through the microporous wall, e.g. by addition to the process medium, as a further opportunity for take-up of any target substance which may have passed through the wall and/or for re-diluting the process liquid. Re- dilution is often important since the process liquid will frequently contain solids which will not pass through the microporous wall and hence form an increasing proportion of the process liquid as filtration proceeds unless re- diluted. The process medium is applied to the wall/passed along the conduit under physical and chemical conditions in which the target substance will bind to the binding surface. Typically this will involve control of temperature, and/or pH in the process medium. After the capture process has proceeded to the desired extent, preferably corresponding to capture of substantially all of the target substance from the process liquid, the microporous wall can be separated from the process medium environment, preliminarily washed if desired, and subjected to chemical conditions appropriate for desorption of the target substance from the binding surface. Again, typically this may involve adjustment of pH and/or temperature. A suitable conduit preferably is or includes one or more tubes consisting of the microporous wall material. For ease of handling, the conduit is preferably a stiff or self-supporting body. The microporous wall needs to be selected/prepared in line with the type of process medium and the type of target substance involved. Where insoluble particulates are contained in the process medium, the size of openings in the microporous wall is preferably adjusted so that these cannot pass through, and sufficiently smaller than the particle size to avoid blockage by such particles. Conversely the pores should be sufficiently large for the target substance molecules to pass readily into the pores. Opening sizes on the micron scale are typically appropriate, for example an average pore size of at least O.lμm, preferably at least 0.2μm. Preferably the average pore size is not more than 5μm, more preferably not more than 3μm. (Average pore sizes may be as determined from a size distribution measured by a liquid displacement porometer, which monitors expulsion of a wetting liquid e.g. isopropyl alcohol from the wetted material under progressively increasing differential air pressure) . However pore sizes excluding passage of particles even down to viral cell size, e.g. about 0.1 μm may be useful. Since molecular sizes may be of the order of e.g. 2- lOnm, in general any pore size which admits these may be suitable . The wall thickness (i.e. transverse to the process liquid flow direction in the conduit embodiment) of the microporous wall is not particularly limited provided that adequate specific surface area is available to collect the target and flow can pass. Typical wall thicknesses are in the range 0.1 to 2mm, or up to 3mm. As to the material of the microporous wall, its essential features are that openings/pores of the requisite dimensions can be provided and that the material can provide the desired selective binding surface. We prefer to use a material having the nature of a porous body rather than a fibrous material. Synthetic polymeric materials are preferred because of the variety of structural and chemical properties readily available, and because technologies for providing selective binding surfaces on polymer surfaces for chemical substance capture are already established. A particularly preferred microporous construction is one created by forming an interpenetrating network (IPN) by shearing a blend of a polymer with an immiscible fluid substance, preferably an immiscible polymer, and leaching the substance away to leave an open porous network of the polymer. Such techniques are described in EP-A-246752 ; IPN-type porous bodies have been proposed as filter media and also (in EP-A-276560) as substrates for enzymes for causing chemical reactions, but not for methods of chemical capture/isolation of target substances. Incidentally the present techniques should not be confused with membrane affinity ultrafiltration, in which the affinity membrane is not permeable to the target species which are therefore trapped only its upstream surface. The polymeric material is not particularly limited but may be for example a nylon such as nylon 6, 6 which contains amino groups useful for provision of binding sites . The benefit of a porous body, such as one obtained from an IPN, is that it provides a large specific surface area and therefore enables capture of a large quantity of target substance in a small, easily handled volume of material. The nature of the selective binding surface will naturally be chosen in accordance with the target substance to be captured. The skilled person can exploit a variety of existing technologies in this respect. We particularly envisage the provision of affinity binding sites in accordance with known principles for purifying or capturing biologically-active molecules by selective adsorption. Thus, the binding surface may have affinity binding sites which can specifically and reversibly adsorb the target substance, e.g. created by covalently attaching an appropriate ligand molecule to the pore surface. This can be done by exploiting the chemical characteristics of a polymer surface in the microporous wall. Immobilisation techniques for this purpose, which may involve physical as well as chemical factors, constitute known technology. With nylon, for example, specific affinity groups (ligands) can be bonded to the nylon surface by means of known linking agents such as glutaraldehyde and difunctional diimidates such as dimethyl pimelimidate or carbodiimide . Particular ligands of interest which can be immobilised in this way include those which exploit Fc- specific binding such as Protein A, Protein G and variant forms of these, which will bind specifically (that is, relative to the process liquid) to several different immunoglobulins . A further possibility which may enable high specificity/selectivity is to exploit an immunospecific agent immobilised on the pore surface. This might be for example an agent consisting of or containing an antigen to an antibody protein which is to be captured. While this can give high selectivity and secure binding, care needs to be taken that one can practically desorb the target protein. A further possibility is to provide an ion exchange surface in the pores. Ion exchange is well-established as a chromatographic technique and the person skilled in the art of preparation of chromatographic media can provide useful anion exchange and cation exchange activity on polymer surfaces useable in microporous walls as proposed herein. A further possibility is to exploit a characteristic hydrophobicity of the target substance relative to other substances in the process liquid, and again persons skilled in the preparation of chromatography media are familiar with providing domains or surfaces of controlled hydrophobicity on polymer substrates for this purpose. Preferred apparatus for carrying out the process generally comprises the conduit having the microporous wall - which in itself is a separate aspect of the present disclosure - in combination with a flow motive arrangement for causing a flow of the process liquid through the conduit, and a filtrate collection arrangement for collecting filtrate passing out through the microporous wall. Preferably the flow arrangement includes a re-circulation conduit for passing the retentate from an outlet of the conduit for re- circulation to the inlet thereof, optionally via a reservoir of the process liquid. A return conduit may also be provided to return filtrate for re-circulation. The arrangement may also include means for establishing a pressure differential across the microporous wall to urge the permeation of filtrate. This may be provided simply by the flow motive pump for the process liquid, but a pump downstream to draw the filtrate is also possible. The skilled person will appreciate that the volume flow of filtrate permeating through the microporous wall will usually be generally slow in relation to the volume flow of process liquid along the microporous wall. For an efficient capture process it is therefore desired to have a large exposure of the process liquid to microporous wall for binding. As previously mentioned, the conduit is desirably a tube consisting of microporous wall. It is further preferred that plural conduits are provided in parallel, the flow from a single process liquid feed being sub-divided to pass through these conduits simultaneously with a respective outflow of filtrate from each conduit. Most conveniently a set of conduits e.g. tubes of the microporous material is provided as a bundle in a surrounding container or manifold having a common inlet and a common outlet. This manifold containing a set of binding-active conduits is a convenient processing unit which desirably can be quickly connected and disconnected into and from the flow arrangement for replacement with a further process unit and for post-processing to desorb the target substance. An absorption plate and flow apparatus for direct application of process medium, i.e. without cross-flow, is also comprehended herein. Specific embodiments of our proposals will now be described with reference to the accompanying drawings, in which: Fig. 1 is a fragmentary view, enlarged, of the end of a microporous polymeric tube; Fig. 2 is a schematic view of a process module having several microporous tubes in a housing; Fig. 3 is a section at III-III of Fig. 2; Fig. 4 is a cross-section at IV-IV of Fig. 2; Fig. 5 is a schematic flow diagram of the system for activating the microporous substrate in the process module with an affinity binding reagent, and Fig. 6 is a schematic flow diagram of a system for recovering a target substance from a process liquor. The invention is illustrated by an example in which Protein G is used to provide affinity binding sites for capturing IgG as a test target module. Figs 1 to 4 show the construction of a membrane binding module 2 consisting of a steel housing tube 21 in which a set of porous binding tubes 1 is installed. This embodiment uses nylon 6,6 porous tubes created by leaching a second polymer (polypropylene) from an IPN using the technique described in EP-A-246752. Each binding tube 1 has an ID of 2mm defining a flow conduit 12 and an OD of 2.5mm (wall thickness 0.25mm), is 200mm long and weighs 1.15g. Five of the porous tubes 1 are arranged side-by-side along inside the housing tube 21, which has a side port 23 half way along. Alternatively plural side ports may be provided distributed along the unit. The ends of the housing tube 21 are fitted with pipe coupling unions 25. Near to the ends of the tube 21 the bundle of porous tubes 1 is sealed in relation to the bore of the outer steel tube 21 {see Fig 3) so that liquid flow between the two end unions 25 of the tube 21 is constrained to be within the flow conduits 12 inside the porous tubes 1, subject to permeation of liquid though those tubes, of course. Around the porous tubes 1 the outer tube 21 defines a filtrate collection space 22 communicating to the side port 23. It will be appreciated that a unit of this kind may be adapted to contain different numbers of porous tubes at any appropriate length. The function of the binding module is to collect a target compound within the porous walls of the porous tubes 1, using a specific binding property of the pore surface . In the present embodiment the nylon surface is given a selectively-binding functionality by immobilising Protein G to the nylon surface. As shown schematically in Fig 5, the tubular binding module 2 was made up as described with untreated nylon porous tubes and installed in a tube preparation flow circuit with a pump for circulating liquid through the module 2 (shown here only schematically) at excess pressure relative to its side port 23, and with means for monitoring the chemical content of the filtrate from the side port by UV spectrometry . In a first stage, the nylon pore surface was glutaraldehyde-activated by pumping aqueous glutaraldehyde around the circuit with differential transmembrane pressure to ensure filtrate flow. In a second stage an aqueous solution containing ethanolamine and dithiothreitol @ pH 7.5 and containing 1 g/1 of Protein G is circulated to bind to the gluteraldehyde- activated sites. The extent of binding in the porous material can be determined by monitoring UV absorbance at 280mm, using a similar ethanolamine/dithiothreitol buffer without Protein G to establish a zero line. Unbound Protein G can be washed out using aqueous ethanolamine . Fig 6 shows the experimental setup for an affinity binding procedure. The Protein G-bearing tube unit 2 is installed in a pumped process liquor circuit. Filtrate from the filtrate outlet 23 can selectively be connected to a filtrate recycle line, an analysis line or a concurrent flow pump for creating a concurrent flow in the outer space 22. IgG is used as test target molecule, made up at 1 g/1 in 0.02M phosphate buffer at pH 7.4. After equilibrating the unit by circulating plain phosphate buffer, the IgG solution is applied and, as in the binding procedure, the transmembrane pressure controlled to achieve a flow of filtrate which is mostly recycled while a certain proportion passes to analysis to determine the rate of take-up of IgG. After capture, IgG can be liberated from absorption in the porous tubes by flushing with an acidic buffer, e.g. glycine/HCl. In practical applications of the procedure to capture target substances from cell broths, there is a combination of substance capture and medium filtration. This gives further benefits in filtering the broth and concentrating solids. Biotechnologically-produced molecules can be purified/captured without the need to clarify the broth initially. The volume of the circulating process liquor is limited only by the concentration of suspended solids, i.e. it must be at least flowable under pressure. Because of the ease of recirculation and continued "clearing" of the porous surface, the process could be used to capture target molecules from very large volumes of liquor containing very low target molecule concentrations.

Claims: CLAIMS 1. A method of capturing molecules of a target substance from a fluid process medium which incorporates the target substance in solution in a liquid component of the medium, comprising applying the process medium to a microporous wall which has through-pores able to accommodate the target substance molecules whereby solution of the target substance in said liquid component permeates through the microporous wall, the pores of the microporous wall having internal binding surfaces adapted to bind the target substance molecules and which thereby capture such molecules in the body of the microporous wall as the solution thereof permeates through. 2. A method according to claim 1 in which the microporous wall is provided as a side wall of a conduit, and the process medium is caused to flow along the conduit . 3. A process according to claim 1 or claim 2 in which the binding surfaces in the pores of the microporous wall have binding sites at which ligand molecules with affinity for the target molecule are covalently attached. . A process according to claim 3 in which the target substance is an immunoglobulin . 5. A process according to claim 1 or claim 2 in which the binding surfaces in the pores of the microporous wall have an immunospecific agent immobilised on the pore interior surface. 6. A process according to claim 1 or claim 2 in which the binding surfaces in the pores of the microporous wall have ion exchange surfaces or hydrophobic domains on a polymer surface of the microporous wall. 7. A process according to any one of the preceding claims comprising collecting a filtrate which has permeated through the microporous wall and returning it for a further passage therethrough. 8. A process according to any one of the preceding claims comprising applying a pressure differential across the microporous wall. 9. A method of preparing a target substance comprising culturing cells to produce the target substance in a culture broth, capturing the target substance from the broth by means of a process according to any one of the preceding claims, and recovering the target substance from the microporous wall. 10. Apparatus for capturing molecules of a target substance from a flowable medium which incorporates the target substance in solution in a liquid component thereof, the apparatus comprising a microporous wall having through-pores permeable to the molecules of the target substance and binding surfaces in the pores adapted to bind the target substance, in order to capture such molecules in the body of the microporous wall from a solution of the target substance permeating through it. 11. Apparatus according to claim 10 comprising a conduit having the microporous wall as a sidewall thereof. 12. Apparatus according to claim 10 or claim 11 in which the conduit is or comprises a tube consisting of the microporous wall. 13. Apparatus according to any one of claims 10 to 12 in which the microporous wall is of natural or synthetic polymeric material. 14. Apparatus according to claim 13 in which the microporous wall has an open porous polymeric network structure which is the residue of an interpenetrating network of a polymer with an immiscible fluid substance, which immiscible fluid substance has subsequently been removed. 15. Apparatus according to any one of claims 10 to 14 in which the binding surfaces in the pores of the microporous wall have affinity binding sites for covalent attachment of ligand molecules to the pore surface, immobilised immunospecific agent, ion exchange surface properties, or hydrophobic domains. 16. Apparatus according to any one of claims 11 to 15 comprising said conduit in combination with a flow motive arrangement for causing a flow of the flowable process medium through the conduit and a filtrate collection arrangement for collecting filtrate permeating out through the microporous wall. 17. Apparatus according to claim 16 including a recirculation conduit for passing a retentate from the outlet of the conduit for re-circulation to the inlet thereof. 18. Apparatus according to any one of claims 10 to 17 comprising means for establishing a pressure differential across the microporous wall to encourage the permeation of filtrate. 19. Apparatus according to any one of claims 11 to 18 in which plural conduits are provided in parallel, the flow from a process medium feed being divided among the plural conduits . 20. Apparatus according to claim 19 in which the plural conduits are provided as a bundle in a surrounding container or manifold providing a common filtrate collection volume for the bundle.

Inventors: HOFMANN MARTIN JOHN, HOFMANN, MARTIN, JOHN
Applicants: DOWNSTREAM MEDIA LTD, HOFMANN MARTIN JOHN, DOWNSTREAM MEDIA LIMITED, HOFMANN, MARTIN, JOHN
Application year: 2000
Main classification: B01D15
IPC codes: B01D15, B01D61, B01D63, B01D67, B01J20