INTEGRATED SEPARATION METHODS

Abstract: The present invention provides the use of membrane-based electrophoresis in combination with one or more conventional separation techniques to obtain a desired compound from a mixture of compounds. the use of one or more membrane-based electrophoresis steps results in faster and more efficient yields of the desired compound.

Description: INTEGRATED SEPARATION METHODS Technical Field The present invention relates to separation of compounds, particularly biomolecules, using a combination of separation techniques. Background Art Conventional plasma fractionation was until recently based upon the Cohn- Oncley ethanol precipitation methods first described in the late 1940's. Cohn fractionation was almost exclusively used in plasma fractionation until the early 1970's when column chromatography was introduced to improve the efficiency of the process and quality of the product. The use of chromatography for large- scale plasma processing has increased to such an extent that some companies have almost entirely removed conventional precipitation from their production schemes. There are, however, several major inherent problems using these methods including the cost of chromatography resin maintenance, pre-filtration required for chromatography columns and the limited product recoveries available using columns. Membrane-based electrophoresis is a new technology originally developed for the separation of macromolecules such as proteins, nucieotides and complex sugars. This unique preparative electrophoresis technology originally developed for macromolecule separation utilises tangential flow across a polyacrylamide membranes when an electric field or potential is applied across the membranes. The general design of the system facilitates the purification of proteins and other macromolecules under near native conditions. This results in higher yields and excellent recovery. The process provides a high purity, scalable separation that is faster, cheaper and higher yielding than current methods of macromolecule separation. Furthermore, the technology offers the potential to concurrently purify and detoxify macromolecule solutions. the present inventors have found that membrane-based electrophoresis technology can be integrated into conventional purification processes, such as those used for major blood fractionation, with out the need to replace the total purification process. This integration allows unexpected improvement in the overall speed of separation and purity of the end products. Disclosure of Invention In a general aspect, the present invention provides the use of membrane- based electrophoresis in combination with one,or more conventional separation techniques to obtain a desired compound from a mixture of compounds. The use of one more membrane-based electrophoresis steps results in faster and more efficient yields of the desired compound. In a first aspect, the present invention provides a process for large scale removal of at least one desired compound from a sample having a mixture of compounds, the process comprising: (a) treating the sample by one or more separation methods so as to obtain a sample fraction containing at least some of the desired compound; (b) placing at least some of the sample fraction containing the desired compound in a first interstitial volume of an electrophoresis apparatus comprising a cathode in a cathode zone; an anode in an anode zone, the anode disposed relative to the cathode so as to be adapted to generate an electric field in an electric field area therebetween upon application of an electric potential between the cathode and the anode; a separation membrane disposed in the electric field area; a first restriction membrane disposed between a first electrode zone and the separation membrane so as to define a first interstitial volume therebetween; a second restriction membrane disposed between a second electrode zone and the separation membrane so as to define a second interstitial volume therebetween; (c) providing a solvent to the first interstitial volume, wherein the solvent has a selected pH; (d) applying an electric potential between the first and second interstitial volumes wherein the application of the electric potential causes migration of a selected one of the selected compound and other components in the first interstitial volume through the separation membrane into the second interstitial volume while at a portion of the other of the selected compound and other components in the first interstitial volume are prevented from entering the second interstitial volume; and (e) maintaining step (d) until one of the interstitial volumes contains the desired amount of the selected compound to form a separation sample. Preferably, the process according to the first aspect of the present invention further comprises: (f) recovering at least a portion of the separation sample and subjecting the separation sample containing the desired compound to one more further separation methods so as to obtain a purified sample containing the desired compound. Preferably in steps (a) and (f), the one or more separation methods are selected from affinity chromatography, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, pseudo- affinity chromatography, membrane based ion exchange systems, preparative isoelectric focusing (IEF), buffer exchange / dialysis processing, precipitation, filtration, pasteurisation, salt/detergent treatment, centrifugation, ultrafiltration and combinations thereof. Preferably, step (c) results in one or more compounds in the sample fraction having a net charge or being substantially neutral. In a second aspect, the present invention provides a process for large scale removal of at least one desired compound from a sample having a mixture of compounds, the process comprising: (a) placing the sample containing the desired compound in a first interstitial volume of an electrophoresis apparatus comprising a cathode in a cathode zone; an anode in an anode zone, the anode disposed relative to the cathode so as to be adapted to generate an electric field in an electric field area therebetween upon application of an electric potential between the cathode and the anode; a separation membrane disposed in the electric field area; a first restriction membrane disposed between a first electrode zone and the separation membrane so as to define a first interstitial volume therebetween; a second restriction membrane disposed between a second electrode zone and the separation membrane so as to define a second interstitial volume therebetween; (b) providing a solvent to the first interstitial volume, wherein the solvent has a selected pH; (c) applying an electric potential between the first and second interstitial volumes wherein the application of the electric potential causes migration of a selected one of the selected compound and other components in the first interstitial volume through the separation membrane into the second interstitial volume while at a portion of the other of the selected compound and other components in the first interstitial volume are prevented from entering the second interstitial volume; (d) maintaining step (c) until one of the interstitial volumes contains the desired amount of the selected compound to form a sample fraction; and (e) recovering the sample fraction and treating at least a portion of the sample fraction by one or more other separation methods so as to obtain a required amount of the desired compound in a separated sample. Preferably in step (e), the one or more separation methods are selected from affinity chromatography, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, pseudo-affinity chromatography, membrane based ion exchange systems, preparative isoelectric focusing (IEF), buffer exchange / dialysis processing, precipitation, filtration, pasteurisation salt/detergent treatment, centrifugation and ultrafiltration and combinations thereof. Preferably, step (b) results in one or more compounds in the sample fraction having a net charge or being substantially neutral. Preferably, the process according to the first or second aspects of the present invention results in at least 60 %, more preferably at least 80%, even more preferably at least 90% purity of the desired compound in the separated sample. In one preferred from, the sample is blood derived sample, particularly plasma and the compounds obtained are selected from Factor VIII, Factor IX, Factor II, Factor X, Protein C, albumin, immunoglobulin, fibrinogen, alpha 1 antitrypsin (AAT), antithrombin III (ATIII). More preferably, the compound is immunoglobulin G (IgG) obtained from Cohn fractions of plasma. In another preferred form, the sample is a recombinant product obtainable from any suitable source such as cells, culture supernatant, milk, or plant material. In yet another preferred from, the sample contains monoclonal antibodies. It will be appreciated, however, that other large scale processes for obtaining compounds including macromolecules from samples containing complex mixtures can be altered to include one or more steps utilising membrane-based electrophoresis technology. One advantage of the membrane-based electrophoresis technology is that it is possible to also remove pathogens or infectious agents from samples while separating one or more compounds. For example, viruses, bacteria, fungi, yeasts and prions can be removed efficiently and safely without the need for conditions or treatments that maybe detrimental to the function or integrity of the one or more biomolecules to be separated. In another preferred form, steps (a) to (f) are carried out after one or more treatments of the original sample are carried out. In another preferred form, steps (a) to (e) are further carried out after step (f) to obtain one or more compounds in substantially pure form. As a result of including one or more membrane-based electrophoresis technology steps in a process for the production of one or macromolecules, the overall efficiency of the process is improved. Preferably, the membranes are electrophoresis separation membranes or restriction membranes. The electrophoresis separation membrane is preferably made from polyacrylamide and has a molecular mass cut-off of at least about 3 kDa. The molecular mass cut-off of the separation membrane will depend on the sample being processed, the other compounds in the samples, and the type of separation carried out. The restriction membrane is also preferably formed from polyacrylamide. The molecular mass cut-off of the restriction membrane will depend on the sample being processed, the other compounds in the sample mixture, and the type of separation carried out. The membranes in the form of electrophoresis membranes may be formed as a multi-layer or sandwich arrangement. The thickness of the membranes can have an effect on the separation or movement of compounds. It has been found that the thinner the membrane, faster and more efficient movement occurs. The restriction membranes can have the same molecular mass cut-off or different cut-offs therefore forming an asymmetrical arrangement. In a third aspect, the present invention provides a macromolecule separated by the process according to the first or second aspects of the present invention. Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application. In order that the present invention may be more clearly understood, preferred forms will be described with reference to the following drawings and examples. Brief Description of the Drawings Figure 1 shows the chromatographic process for the commercial production of plasma proteins. Figure 2 shows SDS PAGE analysis of separation of IgG from other plasma components using membrane-based chromatography. Figure 3 shows non-reduced SDS PAGE analysis of separation of IgG from other plasma components using membrane-based chromatography and ion- exchange chromatography. Figure 4 shows SDS PAGE analysis of separation of IgG from Cohn II, III paste using Scheme 1. Figure 5 shows SDS PAGE analysis of separation of IgG from Cohn II, III paste using Scheme 2. Figure 6 shows SDS PAGE analysis of separation of IgG from Cohn II, III paste using Scheme 3. Figure 7 shows SDS PAGE analysis of separation of IgG from Cohn II, I II paste using Scheme 4 having two separation phases. Figure 8 shows SDS PAGE analysis of albumin depleted plasma. Figure 9 shows SDS PAGE analysis of strong anion exchange purification. Figure 10 shows Western Blot for plasminogen and the corresponding SDS PAGE separation. Figure 11 shows SDS PAGE analysis of weak anion exchange purification. Figure 12 shows SDS PAGE analysis of Protein A chromatography purification of IgG. Mode(s) for Carrying Out the Invention APPARATUS Electrophoresis System t A membrane-based electrophoresis apparatus suitable for use as in the present invention comprises: (a) a cathode in a cathode zone; (b) an anode in an anode zone, the anode disposed relative to the cathode so as to be adapted to generate an electric field in an electric field area therebetween upon application of an electric potential between the cathode and the anode; (c) a separation membrane disposed in the electric field area; (d) a first restriction membrane disposed between a first electrode zone and the separation membrane so as to define a first interstitial volume (stream 1) therebetween; (e) a second restriction membrane disposed between a second electrode zone and the separation membrane so as to define a second interstitial volume (stream 2) therebetween; (f) means adapted to provide solvent to the cathode zone, the anode zone and at least one of the first and second interstitial volumes (stream 1 and stream 2); (g) means adapted to provide a sample constituent in a selected one of the first interstitial and second interstitial volumes wherein upon application of the electric potential, a selected separation product is removed from the sample constituent through at least one membrane and provided to the other of the first and second interstitial volumes or the cathode or anode zones. In one preferred form, the apparatus further comprises:- (h) means adapted for removing heat generated in the apparatus. Preferably, samples and fluids are passed through heat exchangers to remove heat produced by the apparatus during electrophoresis. The cathode zone and the anode zone are supplied with suitable solvent or buffer solutions by any suitable pumping means. A sample to be processed is supplied directly to the first or second interstitial volumes by any suitable pumping means. Preferably, the zones and the interstitial volumes are configured to allow flow of the respective fluid/buffer and sample solutions forming streams. In this form, large volumes can be processed quickly and efficiently. The solutions are typically moved or recirculated through the zones and volumes from respective reservoirs by suitable pumping means. In a preferred embodiment, peristaltic pumps are used as the pumping means for moving the sample, buffers or fluids. In one embodiment, electrode buffer, other buffers and sample solutions are cooled by any suitable means to ensure no inactivation of the micromolecules, compounds or macromolecules occurs during the separation process and to maintain a desired temperature of the apparatus while in use. . Preferably, in order to collect and/or concentrate separated constituents, solution in at least one of the volumes or streams containing any separated components or molecules is collected and replaced with suitable solvent to ensure that electrophoresis can continue in an efficient manner. In use, a sample is placed in the first interstitial volume (stream 1), buffer or solvent is provided to the electrode zones and the second interstitial volume (stream 2), an electric potential is applied to the electric field area causing at least one constituent in the sample to move to buffer/solvent in the cathode zone or buffer/solvent in the second interstitial volume. It will be appreciated that the order of interstitial volumes can be reversed where a sample is placed in the second interstitial volume, buffer or solvent is provided to the electrode zones and the first interstitial volume, an electric potential is applied to the electric field area causing at least one constituent in the sample to move to buffer in the anode zone or buffer in the first interstitial volume. It is also feasible to place sample in one (or both) of the electrophoresis zones and movement into one or more of the interstitial volumes achieved during the application of the voltage potential. The separation membrane is preferably an electrophoresis separation membrane comprised of polyacrylamide and having a defined molecular mass cut-off. Preferably, the electrophoresis separation membrane has a molecular mass cut-off from about 1 kDa to about 2000 kDa. The selection of the molecular mass cut-off of the separation membranes will depend on the sample being processed and the other molecules in the mixture. It will be appreciated, however, that other membrane chemistries or constituents can be used for the present invention. The first and second restriction membranes are preferably formed from polyacrylamide and usually having a molecular mass cut-off less than the separation membrane, preferably from about 1 kDa to about 1000 kDa. The selection of the molecular mass cut-off of the restriction membranes will depend on the sample being processed and the size of the macromolecules to be removed. The restriction membranes can have the same molecular mass cut-off or different cut-offs forming an asymmetrical arrangement. In one preferred form, the membranes forming the first and second interstitial volumes are provided as a cartridge or cassette positioned between the electrode zones of the apparatus. The configuration of the cartridge is preferably a housing with the separation membrane positioned between the first and second restriction membranes thus forming the required interstitial volumes. Preferably, the cartridge or cassette is removable from an electrophoresis apparatus adapted to contain or receive the cartridge. The distance between the electrodes has an effect on the separation or movement of sample constituents through the membranes. The shorter the distance between the electrodes, the faster the electrophoretic movement of constituents. A distance of about 6 mm has been found to be suitable for a laboratory scale apparatus. For scale up versions, the distance will depend on the number and type of separation membranes, the size and volume of the chambers for samples, buffers and separated products. Preferred distances would be in the order of about 6 mm to about 10 cm. The distance will also relate to the voltage applied to the apparatus. The effect of the electric field is based on the equation: e = V/d (e = electric field, V = voltage, d = distance) Therefore, the smaller the distance between the electrodes the faster the separation. Preferably, the distance between the electrodes should decrease in order to increase electric field strength, thereby further improving transfer rates through the membranes. Flow rate of sample/buffer/fluid has an influence on the separation of constituents. Rates of millilitres per minute up to litres per minute are used depending on the configuration of the apparatus and the nature and volume of , the sample to be separated. Currently in a laboratory scale instrument, the preferred flow rate is about 20 + 5 mL/min. However, flow rates from about 0 mL/min to about 50,000 mL/min are used across the various separation regimes. The maximum flow rate is even higher, depending on the pumping means and size of the apparatus. The selection of the flow rate is dependent on the product to be transferred, efficiency of transfer, pre- and post- positioning with other applications. Selection or application of the voltage and/or current applied varies depending on the separation. Typically up to several thousand volts are used but choice and variation of voltage will depend on the configuration of the apparatus, buffers and the sample to be separated. In a laboratory scale instrument, the preferred voltage is about 250 V. However, depending on transfer, efficiency, scale-up and particular method from about 0 V to about 5000 V are used. Higher voltages are also considered, depending on the apparatus and sample to be treated. Optionally, the electric potential may be periodically stopped and/or reversed to cause movement of a constituent having entered a membrane to move back into the volume or stream from which it came, while substantially not causing any constituents that have passed completely through a membrane to pass back through the membrane. Reversal of the electric potential is an option but another alternative is a resting period. Resting (a period without an electric potential being applied) is an optional step that can replace or be included before or after an optional electrical potential reversal. This resting technique can often be practised for specific separation applications as an alternative or adjunct to reversing the potential. For convenience, the first interstitial volume or stream is called stream 1 and the second interstitial volume or stream is called stream 2. Typically, sample was placed in stream 1 and constituents caused to move through the separation membrane into stream 2. The above system is produced by Gradipore Limited, Australia and is referred to as Gradiflow™ technology. Gradiflow™ is a trade mark of Gradipore Limited. APPLICATIONS One application of the present invention is in the commercial processing of plasma to produce a number of blood products used in the health industry. Other applications include but not limited to separation of recombinant proteins from milk sources and separation of monoclonal antibodies. An initial survey of column chromatography scheme as used in the production of plasma proteins has identified several points of application for membrane-based electrophoresis technology. These areas are highlighted Figure ! Point of integration 1 Cryo-precipitation is the initial fractionation step and is the only point in the purification scheme that still utilises precipitation. As a result, it is the only stage at which centrifugation and/or filtration is required, thereby acting as a bottleneck in the entire scheme. It is, however the step where Factor VIII is separated from von Willibrand Factor (VWF) and fibrinogen. Factor VIII is one of the traditional cost drivers of plasma fractionation market and much of the other downstream processing is based upon Factor VIII demand and supply. Replacement of cryo-precipitation with membrane-based electrophoresis technology will ensure a cheap and concurrently more efficient method of protein prefractionation. Point of Integration 2 The intermediate ion exchange steps in the production of the haemostatic factors II, IX, X and protein C is a possible point of use for membrane-based electrophoresis technology. The need for two or more column steps in the process may be replaced by a single membrane-based electrophoresis process, thereby, removing the need for extra Quality Assurance (QA), minimising the risk of pathogen contamination as the membrane-based electrophoresis can act as a concurrent decontamination step. This point of integration will also shorten processing time and improve the efficiency of the process and the nativity of the product. Point of Integration 3 The final intermediate steps in the production of albumin and IgG are the most likely candidates for replacement with membrane-based electrophoresis technology. The ion exchange and gel filtration steps are relatively non-specific (unlike affinity chromatography) and hence may be easily substituted with a new technique. The risk of deactivating other valuable plasma components (ie haemostatic factors) is removed as they are purified earlier in the scheme using conventional techniques. Multiple column steps could be removed and as a result efficiency would be improved and QA requirements reduced. In addition to this, the present inventors know that membrane-based electrophoresis technology adds considerable advantage in the production of high quality albumin and IgG. Although Cohn fractionation is the major process used to fractionate plasma, other processes use ammonium sulfate and PEG precipitation. Accordingly, membrane-based electrophoresis technology can also be used in such processes to obtain useful products. EXPERIMENTAL I. Replacement of Cohn-Oncley Cold Ethanol Precipitation Background & Aims Cohn-Oncley ethanol fractionation is still used in a modified form in the early stages of large-scale plasma fractionation. A number of commercial producers use this old method in their current immunoglobulin production scheme along with anion exchange chromatography, pasteurisation and chemical treatment. There is the potential to replace the Cohn fractionation component of the process with membrane-associated electrophoresis-based technology. Using IgG as a model, this project aimed to investigate the potential use of electrophoresis and compare the resulting product with that from conventional schemes and the established IgG purification by membrane-associated electrophoresis. Size Cuts The aim of these experiments was to use membrane-associated electrophoresis to process plasma into high and low molecular weight fractions. This segmentation of the proteins from plasma would simplify their further purification, mimicking the Cohn/Oncley ethanol precipitation process. Method Three experiments were carried out to determine which cartridge configuration would produce the desired separation. Fifteen mL of 1 :3 dilute plasma was used as the starting material in the stream 1 with 10 L of buffer in the stream 2. The experiments were carried out at pH 9.0 using 65 mM Tris/Borate buffer, approximately 1.8 L of it being loaded into the buffer stream. The three cartridge configurations used were: 5 -'100'- 5 (upper restriction - 'separation membrane' - lower restriction membrane) 5-'200'-5 (upper restriction - 'separation membrane' - lower restriction membrane) 5-'500'-5 (upper restriction - 'separation membrane' - lower restriction membrane) All separations were ran for ~500 min at 250V, 150 mA under forward polarity. The stream 2 was harvested hourly being replaced with 10 mL of buffer. Analysis was carried out using Absorbance at OD 280 nm, SDS-PAGE, and Nephelometry. Results All separations resulted in the retention of the majority of the IgG in the start sample to the stream 1 during the course of the run. The separation which used the 5-'200'-5 cartridge was chosen as having the best result as it retained >80% of the IgG to the Stream 1 while allowing almost complete transfer of albumin to the Stream 2. This is advantageous, as albumin is the most abundant protein in human plasma. Figure 2 shows two gels analysing the Stream 1 and Stream 2 samples for the 5-'200'-5 run. Conclusions The separation carried out with the 5-'200'-5 cartridge resulted in two fractions. One fraction was the high MW sample containing the majority of the protein of a size greater than -150 kDa. The other was the low MW sample containing the majority of the protein of a size lower than ~120 kDa. These fractions could be used as a start material for further purification. Partial Purification of IgG from High MW Fraction using Ion-Exchange The aim of these experiments was to use ion-exchange chromatography to partially purify IgG from the High MW fraction. Method The chromatography system used was the AKTA Prime unit from Amersham Pharmacia. The column was a 5 mL HiTrap-Q Sepharose HP ion- exchange column from Pharmacia. The binding buffer was 20 mM Tris/HCI pH 8.0, the elution buffer was 20 mM Tris/HCI pH 8.0 + 1 M NaCI. The following protocol was used: 100% elution buffer 100% binding buffer Inject sample with binding buffer 40-90% elution gradient, 25 mL @ 3 mLΛnin 3 mL fraction 100% elution buffer The fractions were analysed using non-reduced SDS-PAGE, native PAGE, and Western Blot for IgG on non-reduced SDS-PAGE. Results A single peak was seen spanning fractions 3-6 (Figure 3). The non- reduced SDS-PAGE shown in Figure 4 indicated the majority of the protein was in fractions 4 and 5. A Western Blot of this gel resulted in a positive signal for most of the protein in fractions 4 and 5. Conclusions Pure IgG was achieved from the starting material by integration into the chromatography system with only precautionary syringe filtration carried out prior to the run. This is in contrast to most Cohn/Oncley preparations which require centrifugation before being passed onto to further chromatographic processing. II. Purification of IgG from Cohn II, III paste using the membrane-based electrophoresis Aim Use an electrophoresis apparatus (Gradipore BF200) to purify IgG from Cohn II, III paste to a high degree of purity replacing the position of chromatography in a conventional purification scheme. Method The development of the IgG purification schemes was carried out on Cohn II, III paste sourced from Sigma. Several strategies were developed, each providing a slightly different combination of results. Several schemes included membranes made specifically for the application. Below is a summary of the final protocols selected. Results Scheme 1 Cartridge Configuration: 5 -'250'- 5 (upper restriction - 'separation membrane' - lower restriction membrane) - separation membrane allows passage of IgG Buffer: GABA/Acetic Acid @ pH 5.0 with 0.1% Tween 20 Run Time: 160 min Yield: >90% in Stream 2 Scheme 2 Cartridge Configuration: 5 -'100'- 5 (upper restriction - 'separation membrane' lower restriction membrane) separation membrane prevents passage of IgG Buffer: Tris/Boric Acid @ pH 9.0 with 0.1 % Tween 20 Run Time: 180 min Yield: >90% in Stream 1 Scheme 3 Cartridge Configuration: 5 -'100'- 5 (upper restriction - 'separation membrane' lower restriction membrane) - separation membrane prevents passage of IgG Buffer: GABA/Acetic Acid @ pH 5.0 + 0.1% Tween 20 Run Time: 180 min Yield: >90% in Stream 2 Scheme 4 - Two Phase 1 st Phase Cartridge Configuration: 5 -'100'- 5 (upper restriction - 'separation membrane' - lower restriction. membrane) - separation membrane prevents passage of IgG 1 st Phase Buffer: Tris/Boric Acid @ pH 9 .0 with 0.1% Tween 20 1 st Phase Run Time: 180 min 1 st Phase Yield: 1 st Phase >90% 2 nd Phase Cartridge Configuration: 5 -'100'- 5 (upper restriction - 'separation membrane' - lower restriction membrane) separation membrane prevents passage of IgG 2 nd Phase Buffer: GABA/Acetic Acid @ pH 5.0 + 0.1% Tween 20 2 nd Phase Run Time: 240 min 2 nd Phase Yield: -70% The yield results were obtained using Nephelometry and the purity established by using SDS-PAGE analysis. Runs resulted in a greater than 90% recovery in IgG except for the two-phase protocol. The degree of purity achieved was similar compared to a therapeutic MG product Gamimune (Bayer). The separation membrane used in schemes 1 , 3, and 4 was also shown in an experiment in which porcine parvovirus was added to restrict porcine parvovirus to the Stream 1 while allowing the transfer of IgG to the Stream 2. Results of separation from Schemes 1 , 2, 3 and 4 are shown in Figures 4, 5, 6 and 7 respectively. Conclusions The above protocols can be used to purify IgG from Cohn II, III with a high degree of purity and high yield. The majority of contaminants have been removed to a significant level. This was a good result given the low grade of the start material. III. Purification of IgG from Albumin-depleted plasma using various chromatography methods Background & Aim One commercial application of membrane-based electrophoresis is the implementation into a conventional blood fractionation scheme. To improve separation, methods were devised to cover the implementation of one or more electrophoresis steps at different points within the process. In this experiment a sample obtained by membrane-based electrophoresis was further purified using chromatography methods (similar to those used routinely in commercial production). The aim of this project was to purify IgG from an albumin depleted plasma sample derived using the membrane-based electrophoresis technology. Start Material A large scale (100x) membrane-based electrophoresis run was carried out with the following configuration: Membrane Stack: 5kd-200kD-5kD (upper restriction - 'separation membrane' - lower restriction membrane) x 5 Buffer: Tris Borate pH 8 with 0.1% Tween 20 Start Sample: 2 L 1 :3 Clinisys Plasma (diluted 1 :3 in buffer) Running Conditions: 590V, 25A, 10C, 5 hrs with 0.5 hr harvest The aim of this run was to transfer the majority of the albumin into stream 2. The sample remaining in the Stream 1 is albumin depleted plasma. Figure 8 is a SDS-PAGE analysis of this sample. Purification of IgG from albumin depleted human plasma (prepared using membrane-based electrophoresis technology) using strong anion exchange chromatography Aim To purify IgG from albumin depleted human plasma using strong anion exchange chromatography under commercially documented conditions. Method The following system was devised for the experiments: Unit: AktaPrime APBiotech Chromatography System Column: 5 mL HiTrapQ HP Strong Anion Exchange from APBiotech Binding Buffer: 20 mM Bis-Tris Propane pH 6.5 (with HCI). Elution Buffer: 20 mM Bis-Tris Propane pH 6.5 (with HCI) + 1 M NaCI Start Material: 5 mL albumin depleted plasma diluted 1 :5 with binding buffer This system aims to bind the majority of the contaminant proteins and allow IgG to flow through during the binding step. Results A shouldered peak eluting into fractions 2 and 3 was achieved. The SDS PAGE of the fractions can be seen in lanes 3 and 4 in Figure 9. The nephelometry of the two IgG fractions showed that -80% of the IgG present in the start material was eluted in the fractions. Analysis of plasminogen removal was also carried out using the western blot technique. The IgG-rich fraction was compared to plasma and the albumin depleted plasma start material. The result of the blot and the corresponding SDS-PAGE is shown below in Figure 10. The blot shows that the chromatography step purified the IgG with no visible plasminogen present in the sample. Conclusion Strong anion exchange was used to successfully purify IgG from an albumin-depleted plasma sample obtained by membrane-based electrophoresis. The purity of the IgG was excellent with single band purity on SDS PAGE stained with a coomassie based stain. The yield was greater than 80% of the IgG in the start material recovered. The purification was also shown to reduce the level of plasminogen to below detectable limits. This illustrates the advantage of polishing the membrane-based electrophoresis product with chromatography. Purification of IgG from albumin-depleted human plasma using weak anion exchange chromatography Aim To purify IgG from albumin-depleted human plasma using weak anion exchange chromatography under commercially documented conditions. Method The following system was devised for the experiments: Unit: AktaPrime APBiotech Chromatography System Column: 20 mL HiPrep 16/10 DEAE FF Weak Anion Exchange from APBiotech Binding Buffer: 20 mM L-Histidine pH 5.20(with HCI) Elution Buffer: 20 mM L-Histidine pH 5.20 (with HCI) + 1 M NaCI Start Material: 5 mL albumin-depleted plasma diluted 1 :5 with binding buffer This system aims to bind the majority of the contaminant proteins and allow IgG to flow through during the binding step. Results A broad peak eluting into fractions 3 to 5 was produced. The SDS PAGE of the fractions can be seen in lanes 4 to 6 in Figure 11. The nephelometry of the IgG fractions showed that only -30% of the IgG present in the start material was detected. Conclusion Weak anion exchange was used to successfully purify IgG to a high degree of purity from an albumin-depleted plasma sample obtained by membrane-based electrophoresis. The low recovery can be typical of ion exchange chromatography procedures.. Purification of IgG from albumin depleted human plasma using Protein A affinity column chromatography Aim To purify IgG from albumin depleted human plasma using Protein A affinity column chromatography and a developed application method. Method The method used was based on an application template provided by APBiotech with the column. The following system was .devised for the experiments: Unit: AktaPrime APBiotech Chromatography System Column: 1 mL HiTrap Protein A from APBiotech Binding Buffer: 100 mM Sodium Phosphate + 100 mM Sodium Citrate pH 7.0 (with NaOH) Elution Buffer: 100 mM Sodium Phosphate + 100 mM Sodium Citrate pH 3.0 (with HCI) Start Material: 5 mL albumin depleted plasma diluted 1:5 with binding buffer This system aims to bind IgG to the column and allow the majority of the contaminants to pass through the column. The IgG is then eluted and collected. Results A narrow peak eluting mainly into the 2 nd and 3 rd fractions was achieved. The SDS PAGE of the fractions can be seen in lanes 5 and 6 in Figure 12. The nephelometry of the IgG fractions showed greater than 70% of the IgG present in the start material was detected. Conclusion Protein A affinity chromatography was used to successfully purify IgG from an albumin depleted plasma sample obtained by membrane-based ,. electrophoresis. The purity of the IgG was good with only a slight protein smear below the single band of IgG (as seen on SDS PAGE stained with a coomassie based stain). The yield was greater than 70% of the IgG in the start material detected in the main IgG fractions. It will be appreciated that Protein G can also used for immunoglobulins that do not bind protein A. Advantages of Integration of Membrane-Based Electrophoresis into Existing Plasma Fractionation Schemes Cohn Fraction Replacement Advantages of the present invention in this mode include: -Higher Recoveries from Cohn fraction than obtainable using chromatography. Chromatography alone typically yields no greater than 70% of the starting product whilst membrane-based electrophoresis can produce 80% and greater recoveries. The difference is considerable seeing that every 1% of MG product equates with approximately $US 9 million in sales. -Less sample preparation for membrane-based electrophoresis than with columns ie pre-filtering. -High purity obtained on the basis of size and/or charge -Specific contaminant removal -In situ viral, bacterial and prion and pyrogen clearance -Linearly scalable Integration with conventional methods -Higher recoveries than Cohn fractionation. Cohn typically results in loss of approximately 50% of available MG. As discussed above this equates to considerable monetary cost . -Single phase as opposed to multiple precipitation steps. This allows infrastructure to freed up, results in less chance of contaminant introduction and minimises quality assurance costs -No centrifugation/filtration required to remove precipitous materials as target molecules are retained in solution throughout process. This is one of the major bottlenecks in the current processes -More refined product after membrane-based electrophoresis gross capture than obtained by Cohn. This potentially results in a higher quality product after several polishing steps than is currently available -In scheme viral and prion and pyrogen clearance -Specific contaminant removal using chromatography Overall advantage of integration of membrane-based electrophoresis with other purification methods -Minimal interruption to overall processing scheme -Less capital expenditure due to the existing Cohn and chromatography technologies -Less regulatory interference as existing technologies are already accepted -Safer product -Column longevity -Column efficiency improvement, -Freeing up infrastructure -No need to repack columns as replaceable/disposable cartridges are used -No column sterilisation process required Although the separation of blood products from plasma has been exemplified, the present invention is applicable for other large scale commercial purification processes including production of recombinant proteins from cells and cell lysates, milk and dairy products, antibiotics and other microbial products. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims: Claims: 1. A process for large scale removal of at least one desired compound from a sample having a mixture of compounds, the process comprising: (a) treating the sample by one or more separation methods selected from the group consisting of affinity chromatography, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, pseudo- affinity chromatography, membrane based ion exchange systems, preparative isoelectric focusing (lEF), buffer exchange / dialysis processing, precipitation, filtration, pasteurisation, salt/detergent treatment, centrifugation, ultrafiltration and combinations thereof so as to obtain a sample fraction containing at least some of the desired compound; (b) placing at least some of the sample fraction containing the desired compound in a first interstitial volume of an electrophoresis apparatus comprising a cathode in a cathode zone; an anode in an anode zone, the anode disposed relative to the cathode so as to be adapted to generate an electric field in an electric field area therebetween upon application of an electric potential between the cathode and the anode; a separation membrane disposed in the electric field area; a first restriction membrane disposed between a first electrode zone and the separation membrane so as to define a first interstitial volume therebetween; a second restriction membrane disposed between a second electrode zone and the separation membrane so as to define a second interstitial volume therebetween; (c) providing a solvent to the first interstitial volume, wherein the solvent has a selected pH; (d) applying an electric potential between the first and second interstitial volumes wherein the application of the electric potential causes migration of a selected one of the selected compound and other components in the first interstitial volume through the separation membrane into the second interstitial volume while at a portion of the other of the selected compound and other components in the first interstitial volume are prevented from entering the second interstitial volume; and (e) maintaining step (d) until one of the interstitial volumes contains the desired amount of the selected compound to form a separation sample. 2. The process according to claim 1 further comprising: (f) recovering at least a portion of the separation sample and subjecting the separation sample containing the desired compound to one more further separation methods selected from the group consisting of affinity chromatography, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, pseudo-affinity chromatography, membrane based ion exchange systems, preparative isoelectric focusing (lEF), buffer exchange / dialysis processing, precipitation, filtration, pasteurisation, salt/detergent treatment, centrifugation, ultrafiltration and combinations thereof so as to obtain a purified sample containing the desired compound. 3. The process according to claim 1 or 2 wherein step (c) results in one or more compounds in the sample fraction having a net charge or being substantially neutral. 4. The process according to any one of claims 1 to 3 resulting in at least 70% purity of the desired compound in the separation sample. 5. The process according to claim 4 resulting in at least 80% purity of the desired compound in the separation sample. 6. The process according to claim 5 resulting in at least 90% purity of the desired compound in the separation sample. 7. The process according to any one of claims 1 to 6 wherein the sample is a blood-derived sample. 8. The process according to claim 7 wherein the blood-derived sample is plasma. 9. The process according to claim 8 wherein the compound obtained is selected from the group consisting of Factor VIII, Factor IX, Factor II, Factor X, Protein C, albumin, immunoglobulin, fibrinogen, alpha 1 antitrypsin (AAT) and antithrombin III (ATI 11). 10. The process according to claim 9 wherein the compound is immunoglobulin G (IgG) obtained from Cohn fractions of plasma. 11. The process according to any one of claims 1 to 10 wherein step (a) is cryo-precipitation. 12. The according to any one of claims 1 to 10 wherein step (a) is ammonium sulphate precipitation and polyethylene glycol precipitation. 13. The process according to any one of claims 1 to 10 wherein the sample is milk containing a recombinant protein. 14. The process according to any one of claims 1 to 10 wherein the sample . contains a monoclonal antibody. 15. A process for large scale removal of at least one desired compound from a sample having a mixture of compounds, the process comprising: (a) placing the sample containing the desired compound in a first interstitial volume of an electrophoresis apparatus comprising a cathode in a cathode zone; an anode in an anode zone, the anode disposed relative to the cathode so as to be adapted to generate an electric field in an electric field area therebetween upon application of an electric potential between the cathode and the anode; a separation membrane disposed in the electric field area; a first restriction membrane disposed between a first electrode zone and the separation membrane so as to define a first interstitial volume therebetween; a second restriction membrane disposed between a second electrode zone and the separation membrane so as to define a second interstitial volume therebetween; (b) providing a solvent to the first interstitial volume, wherein the solvent has a selected pH; (c) applying an electric potential between the first and second interstitial volumes wherein the application of the electric potential causes migration of a selected one of the selected compound and other components in the first interstitial volume through the separation membrane into the second interstitial volume while at a portion of the other of the selected compound and other components in the first interstitial volume are prevented from entering the second interstitial volume; (d) maintaining step (c) until one of the interstitial volumes contains the desired amount of the selected compound to form a sample fraction; and (e) recovering the sample fraction and treating at least a portion of the sample fraction by one or more separation methods selected from the group consisting of affinity chromatography, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, pseudo- affinity chromatography, membrane based ion exchange systems, preparative isoelectric focusing (lEF), buffer exchange / dialysis processing, precipitation, filtration, pasteurisation, salt/detergent treatment, centrifugation, ultrafiltration and combinations thereof so as to obtain a required amount of the desired compound in a separated sample. 16. The process according to claim 15 wherein step (b) results in one or more compounds in the sample fraction having a net charge or being substantially neutral. 17. The process according to claim 15 or 16 resulting in at least 70% purity of the desired compound in the separation sample. 18. The process according to claim 17 resulting in at least 80% purity of the desired compound in the separation sample. 19. The process according to claim 18 resulting in at least 90% purity of the desired compound in the separation sample. 20. The process according to any one of claims 15 to 19 wherein the sample is a blood-derived sample. 21. The process according to claim 20 wherein the blood-derived sample is plasma. 22. The process according to claim 21 wherein the compound obtained is selected from the group consisting of Factor VIII, Factor IX, Factor II, Factor X, Protein C, albumin, immunoglobulin, fibrinogen, alpha 1 antitrypsin (AAT) and antithrombin III (ATIII). 23. The process according to claim 22 wherein the compound is immunoglobulin G (IgG) obtained from Cohn fractions of plasma. 24. The process according to claim 22 wherein the compound is immunoglobulin G (IgG) obtained from ammonium precipitation and polyethylene glycol precipitation. 25. The process according to any one of claims 15 to 24 wherein step (e) is ion-exchange chromatography. 26. The process according to claim 25 wherein the ion-exchange chromatography is anion exchange chromatography. 27. The process according to any one of claims 15 to 24 wherein step (e) is affinity chromatography. 28. The process according to claim 27 wherein the affinity chromatography is Protein A affinity chromatography or Protein G affinity chromatography.

Inventors: GILBERT ANDREW MARK, NAIR CHEMICHERI HARIHARAN, GILBERT, ANDREW, MARK, NAIR, CHEMICHERI, HARIHARAN
Applicants: GRADIPORE LTD, GRADIPORE LIMITED
Application year: 2001
Main classification: B01D57
IPC codes: B01D57, B01D61, B03C5, G01N27, G01N30