Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/416—Systems
  • G01N27/447—Systems using electrophoresis
  • G01N27/44756—Apparatus specially adapted therefor
  • G01N27/44769—Continuous electrophoresis, i.e. the sample being continuously introduced, e.g. free flow electrophoresis [FFE]
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D57/00—Separation, other than separation of solids, not fully covered by a single other group or subclass, e.g. B03C
  • B01D57/02—Separation, other than separation of solids, not fully covered by a single other group or subclass, e.g. B03C by electrophoresis
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
  • B01D61/42—Electrodialysis; Electro-osmosis ; Electro-ultrafiltration; Membrane capacitive deionization
  • B01D61/425—Electro-ultrafiltration

Definitions

• the present invention relates to separation of compounds, particularly biomolecules, using a combination of separation techniques.
• 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.
• the technology offers the potential to concurrently purify and detoxify macromolecule solutions.
• 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.
• 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.
• 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:
• 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;
• step (e) maintaining step (d) until one of the interstitial volumes contains the desired amount of the selected compound to form a separation sample.
• the process according to the first aspect of the present invention further comprises:
• 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.
• affinity chromatography size exclusion chromatography
• ion exchange chromatography hydrophobic interaction chromatography
• pseudo- affinity chromatography hydrophobic interaction chromatography
• membrane based ion exchange systems membrane based ion exchange systems
• IEF isoelectric focusing
• step (c) results in one or more compounds in the sample fraction having a net charge or being substantially neutral.
• 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:
• 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;
• step (d) maintaining step (c) until one of the interstitial volumes contains the desired amount of the selected compound to form a sample fraction
• 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.
• step (b) results in one or more compounds in the sample fraction having a net charge or being substantially neutral.
• 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.
• 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.
• the sample is a recombinant product obtainable from any suitable source such as cells, culture supernatant, milk, or plant material.
• the sample contains monoclonal antibodies.
• membrane-based electrophoresis technology 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.
• steps (a) to (f) are carried out after one or more treatments of the original sample are carried out.
• steps (a) to (e) are further carried out after step (f) to obtain one or more compounds in substantially pure form.
• 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.
• restriction membranes can have the same molecular mass cut-off or different cut-offs therefore forming an asymmetrical arrangement.
• the present invention provides a macromolecule separated by the process according to the first or second aspects of the present invention.
• 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.
• FIG. 9 shows SDS PAGE analysis of strong anion exchange purification.
• Figure 10 shows Western Blot for plasminogen and the corresponding SDS PAGE separation.
• FIG. 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
• Electrophoresis System t A membrane-based electrophoresis apparatus suitable for use as in the present invention comprises:
• (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.
• the apparatus further comprises:-
• (h) means adapted for removing heat generated in the apparatus.
• 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.
• the zones and the interstitial volumes are configured to allow flow of the respective fluid/buffer and sample solutions forming streams.
• 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.
• peristaltic pumps are used as the pumping means for moving the sample, buffers or fluids.
• 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. .
• 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.
• 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.
• 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.
• the separation membrane is preferably an electrophoresis separation membrane comprised of polyacrylamide and having a defined molecular mass cut-off.
• 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.
• 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.
• 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.
• a distance of about 6 mm has been found to be suitable for a laboratory scale apparatus.
• 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.
• 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.
• 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.
• the first interstitial volume or stream is called stream 1 and the second interstitial volume or stream is called stream 2.
• 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 GradiflowTM technology.
• GradiflowTM is a trade mark of Gradipore Limited.
• 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.
• 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.
• VWF Factor Factor
• fibrinogen Factor VIII
• VWF fibrinogen
• 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.
• 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
• Multiple column steps could be removed and as a result efficiency would be improved and QA requirements reduced.
• membrane-based electrophoresis technology adds considerable advantage in the production of high quality albumin and IgG.
• 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.
• 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.
• Tris/Borate buffer approximately 1.8 L of it being loaded into the buffer stream.
• the three cartridge configurations used were:
• 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.
• the aim of these experiments was to use ion-exchange chromatography to partially purify IgG from the High MW fraction.
• 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:
• 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.
• Buffer Tris/Boric Acid @ pH 9.0 with 0.1 % Tween 20
• membrane-based electrophoresis 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
• Membrane Stack 5kd-200kD-5kD (upper restriction - 'separation membrane' - lower restriction membrane) x 5
• Running Conditions 590V, 25A, 10C, 5 hrs with 0.5 hr harvest
• 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.
• the nephelometry of the two IgG fractions showed that -80% of the IgG present in the start material was eluted in the fractions.
• the blot shows that the chromatography step purified the IgG with no visible plasminogen present in the sample.
• This system aims to bind the majority of the contaminant proteins and allow IgG to flow through during the binding step.
• the method used was based on an application template provided by APBiotech with the column.
• the following system was .devised for the experiments:
• 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)
• 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.
• the nephelometry of the IgG fractions showed greater than 70% of the IgG present in the start material was detected.
• 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.
• Protein G can also used for immunoglobulins that do not bind protein A.
• 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.
• Cohn typically results in loss of approximately 50% of available MG. As discussed above this equates to considerable monetary cost .

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