Chromatography medium

Abstract

The present invention provides a chromatography medium comprising one or more electrospun polymer nanofibres which in use form a stationary phase comprising a plurality of pores through which a mobile phase can permeate and use of the same.

Claims

1. A chromatography medium comprising a non-woven fabric comprising one or more polymer nanofibres wherein the polymer nanofibers are fused together at points where the nanofibers intersect one another, and which form a stationary phase comprising a plurality of pores through which a mobile phase can permeate, and wherein the chromatography medium has affinity chromatography protein ligand surface functionality interactive with target molecules.

2. The medium according to claim 1, wherein the stationary phase is in the form of a membrane.

3. The medium according to claim 1, wherein the affinity chromatography protein ligand surface functionality comprises a Protein A ligand, Protein G ligand, Protein A/G ligand, or Protein L ligand.

4. The medium according to claim 3, wherein the affinity chromatography protein ligand surface functionality comprises a Protein A ligand.

5. The medium according to claim 2, wherein the membrane has a thickness of 100 μm to 1 mm.

6. The medium according to claim 1, wherein the polymer is selected from: nylon, poly(acrylic acid), polyacrylonitrile, polystyrene and polyethylene oxide.

7. The medium according to claim 1, wherein the polymer is cellulose.

8. The medium according to claim 1, wherein the polymer nanofibres are covalently cross-linked.

9. The medium according to claim 1, wherein the nanofibres have a diameter of 10 nm to 1000 nm.

10. The medium according to claim 1, wherein the nanofibres have a diameter of 200 nm to 800 nm.

11. The medium according to claim 1, wherein the nanofibres have a diameter of 300 nm to 400 nm.

12. The medium according to claim 1, wherein the mean length of nanofibres is greater than 10 cm.

13. The medium according to claim 1, wherein the pores have a diameter of 10 nm to 10 μm.

14. A chromatography method using the medium according to claim 1.

15. The method according to claim 14, wherein the chromatography is affinity capture chromatography.

16. The method according to claim 14, wherein the medium is used to isolate biological molecules from the mobile phase.

17. The method according to claim 16, wherein the biological molecules have a molecular weight of 1 kDa to 200 kDa.

18. The method according to claim 14, wherein the biological molecules are selected from the group comprising: recombinant proteins, monoclonal antibodies, viral vaccines and plasmid DNA.

19. The method according to claim 14, wherein a simulated moving bed system is employed.

20. A cartridge for use in chromatography comprising: two or more membranes including the medium according to claim 2 arranged in series; and a holding member to fix the membranes in place relative to one another.

Description

DESCRIPTION

(1) The invention will now described by reference to the following figures:

(2) FIG. 1 shows a schematic image of the cartridge of the invention;

(3) FIG. 2 shows an alternative embodiment of the cartridge of the invention; and

(4) FIG. 3 shows an exploded image of the embodiment of FIG. 2.

(5) FIG. 4a shows binding/elute profiles of a nanofibre cartridge of the invention at varying flow rates in which, starting from light grey full line to dark grey dotted line: 72 cm/h (2,400 CV/h), 96 cm/h (3,200 CV/h), 120 cm/h (4,000 CV/h), 240 cm/h (8,000 CV/h) and 360 cm/h (12,000 CV/h). FIG. 4b shows a chromatogram showing 10 repeat bind/elute profiles of a nanofibre cartridge of the invention at a fixed loading flow rate of 240 cm/h (8,000 CV/h).

(6) FIG. 5 shows SEM images of a nanofibre cartridge of the invention and Sartobind IEXD membrane before and after a feed stream of centrifuged and 0.45 μm filtered yeast homogenate. Top SEM images show clean membranes at two magnifications, while the bottom images show membranes after 50 cycles of clarified homogenate loading. The scale bar indicates 10 μm.

(7) FIG. 6a shows the results of a filtered load investigation showing pressure flow relationships for equivalent volumes of: 0 Sartobind IEXD, 0 Sartobind Epoxy-DEAE, and ⋄ DEAE nanofibre of the invention. Error bars are ±1 standard deviation of the sample population. FIG. 6b shows two bind/elute profiles before and two after clarified homogenate loading and CIP of the DEAE nanofibre cartridge of the invention at a fixed loading flow rate of 240 cm/h to demonstrate reproducibility after clarified yeast homogenate loading (2 mL) and CIP (20 mL 1M NaOH).

(8) FIG. 7 shows a design drawing of a simulated moving bed (SMB) system showing different phases of operation.

(9) FIG. 8 shows the average separation results for DEAE nanofibre adsorbent of the invention (Black) and Sartobind IEXD (Dark Grey). ±1 standard deviation of the sample population is shown by the shaded grey area around each curve.

(10) FIG. 9 shows an SDS Page Gel showing denatured components of 12 protein samples.

(11) FIG. 10 shows chromatogram data from the SMB operation of 2-component separation; UV absorbance traces were a snapshot for a 25 second time period taken from the three different adsorbent columns during one SMB operation.

(12) FIG. 11 shows photographs from a coomassie blue dye flow distribution study utilising different pore size frits.

(13) As shown in FIG. 1, cartridge (1) comprises a holding member (2) within which multiple membranes (3) are arranged in series. In this embodiment, one or more frits (6) are also present, in a 1:1 ratio with the membranes of the invention. The cartridge also has an inflow port (4) through which a mobile phase can be passed and a corresponding outflow port (5).

(14) FIGS. 2 and 3 show an alternative embodiment of the cartridge of the invention. In this embodiment holding member (2) comprises a support column (7) onto which the membranes (3) and, where present, frits (7), may be placed.

(15) Unless otherwise stated each of the integers described in the invention may be used in combination with any other integer as would be understood by the person skilled in the art. Further, where numerical ranges are provided, it is intended that these represent a specific disclosure not only of the end points of the range, but of each value, in particular integers, within the range. In addition, although all aspects of the invention preferably “comprise” the features described in relation to that aspect, it is specifically envisaged that they may “consist” or “consist essentially” of those features outlined in the claims.

EXAMPLES

(16) A solution of cellulose acetate (CA) (Mr=29,000; 40% acetyl groups; 0.20 g/mL) in acetone/dimethylformamide/ethanol (2:2:1) was electrospun to obtain CA nanofibre non-woven membranes.

(17) Sartorious Stedim Sartobind membranes (Sartorius Stedim UK Ltd. Epsom, UK) were cut to size and used as a comparison.

(18) Electrospinning

(19) The process was carried out in a ClimateZone climate control cabinet (a1-safetech Luton, UK) which allows the process to be performed under controlled atmospheric conditions. The temperature and RH were selected and kept constant throughout the fabrication at 25° C. and 60% RH.

(20) A 50 mL polymer solution was loaded into a sterile syringe and attach to a Harvard PHD 4400 syringe pump (Harvard Apparatus Ltd. Kent, UK), with a programmable flow rate range from 0.0001 up to 13.25 L/h, to deliver the polymer solution to a 0.5-mm ID stainless steel micro needle. The pump is set at a flowrate of 800 μL/h. The tip of the needle was placed 30 cm above the grounded collector. The collector used was an earthed aluminium rotating drum (15 cm diameter×25 cm width) covered with low surface friction polymer rotating at a speed of 100 rpm. The process was run for 60 h. These conditions were selected based on preliminary experiments and are known to yield solid dry nanofibres with diameters from 300-400 μm.

(21) Post Electrospinning Modification

(22) Once electrospun the nanofibres are removed from the collection drum and placed into a drying oven at 213° C. for ten minutes. This is below the glass transition temperature of cellulose acetate but it is hot enough to begin fusing joints where nanofibres intersect, thereby increasing the structural stability of the fibre matt giving improved manual manipulation characteristics.

(23) After this process the fibre matt is cut into multiple 25 mm diameter discs using a wad punch. These discs are then ready for packing into a PALL Easy Pressure Syringe Filter Holder (Pall Life Sciences, Portsmouth, United Kingdom).

(24) Once packed into a ˜100 mg cartridge (˜6 layers ˜0.4 mm bed height) the membrane is treated with 200 mL 0.1M NaOH in a solvent mixture of 2:1 De-ionised (DI) H.sub.2O:Ethanol for 24 hours fed continuously in a cyclical manner using a Watson Marlow 205U Peristaltic pump (Watson-Marlow Pumps Group Falmouth, UK) at a rate of 15 mL/min. After the saponification/deacetylation process to form a regenerated cellulose membrane 200 mL DI H.sub.2O is passed through the membrane at the same flowrate. Anion-exchange surface functionality is then obtained by recycling 20 mL warm (40° C.) 15% DEACH aqueous solution at 20 mL/min for 10 minutes. Cartridges are subsequently removed from the filter holder housing and left in 20 mL hot (80° C.) 0.5M NaOH on a hot plate stirrer with gentle agitation. Finally the membrane cartridges are rinsed in multiple volumes of DI H.sub.2O before being packed ready for use.

(25) Permeability

(26) The permeabilities of load and buffer solutions through packed nanofibre were compared with the permeabilities of a commercially available alternative membrane, Sartorious Sartobind membrane, using an AKTA Explorer (GE Healthcare Life Sciences, Buckinghamshire, UK) with online pressure measurement capabilities. The pressure drop of the system was first evaluated using the empty membrane holder evaluated at flow rates ranging from 1 mL/min-100 mL/min. The different membranes were then evaluated with the system pressure drop being subtracted to calculate the permeability of each membrane at the varying flow rates.

(27) Equilibrium Binding

(28) The previously prepared 25 mm diameter AEX membrane cartridges have a total film surface area of 4.91 cm.sup.2, an approximate mass of 100 mg, and an approximate wet bed height of 0.3 mm suggesting a bed volume ˜0.17 mL. Equilibrium binding studies were carried out to find the total capacity of the DEAE AEX membranes for of a model protein Bovine Serum Albumin (BSA). This was carried out in the sealed filter holder system using sterile disposable syringes (BD biosciences) and a Harvard PHD 4400 syringe pump (Harvard Apparatus Ltd. Kent, UK). The DEAE membrane was equilibrated with 10 mL wash buffer 10 mM Tris, pH 8.0 buffer a rate of 40 mL/min 20 mL of lmg/mL BSA (in wash buffer) was then loaded onto the membrane at a rate of 40 mL/min. This load sample was then pulled back through the membrane at the same rate with this process being repeated continuously for 1 hour in order to expose the membrane to the model protein for a sufficient length of time to reach maximum binding capacity. Collection of the load stage was followed by five wash stages with 1 mL wash buffer before desorption of the model protein was carried out by three elution stages each with 1 mL 1M NaCl 10 mM Tris, pH 8.0 elution buffer at a rate of 40 mL/min.

(29) All wash and elution stages followed the same dual flow pattern used with the loading for a period of two minutes each. At each stage in this process the eluate was collected and UV absorbance readings at 280 nm were taken using Jasco V-630 UV spectrophotometer (Jasco (UK), Essex, United Kingdom). Studies were repeated on three occasions in order to ensure replicates were reproducible. Mass balances were conducted to ensure the traceability of all model protein introduced into the system. The same protocol was run with Sartobind DEAE membranes and Sartobind epoxy membrane functionalised in house for comparison. Control samples were run under the same conditions to discount possible binding to the filter holder surface or non-specific binding of BSA to non-functionalised membrane, regenerated cellulose (RC) membrane.

(30) Dynamic Breakthrough

(31) A more useful determination of the binding capacity of these nanofibre adsorbents is their dynamic binding capacity employing operational flowrates observed as suitable in previous permeability studies. Experiments were completed using the AKTA Explorer (GE Healthcare Life Sciences, Buckinghamshire, UK) with online measurement of UV absorbance (280 nm), pH, and conductivity.

(32) DEAE nanofibre membrane cartridges were prepared in the same way as before in order to determine the dynamic capacity of the membranes for of a model protein Bovine Serum Albumin (BSA). The DEAE membrane was equilibrated with 10 mL wash buffer 10 mM Tris, pH 8.0 buffer a rate of 6000 CV/h. 1 mg/mL load sample of BSA were then loaded onto the membrane until 100% breakthrough at various flow rates from 24,000 CV/h-4000 CV/h. 10 mL wash buffer was then passed through the saturated membrane before 5 mL 100% 1M NaCl 10 mM Tris, pH 8.0 elution buffer was introduced to the membrane at a range of flow rates from 8000 CV/h-2000 CV/h.

(33) Online UV absorbance readings at 280 nm were taken throughout the experiment.

(34) A simulated moving bed system was designed and built using a series of Burkert solenoid valves (Bürkert Fluid Control Systems, Stroud, UK) 1/16″ Peek tubing, Peek connectors, and a Perimax 12 peristaltic pump (Spetec GmbH, Erding, Germany). UV sensors were placed on the exit of each of the three filter holders which was connected to a National Instruments analogue input module (National Instruments Corporation (U.K.) Ltd, Newbury, UK) to record the UV absorbance at 280 nm. Three National Instrument digital output modules were used to control the valve positions with NI Labview 2010 software used to sequence the control and compute the analogue input signals.

(35) Chemical Surface Derivatisation (FT-IR)

(36) ATR-FTIR spectrum of the CA, RC and modified RC membrane was obtained on a Thermo Scientific Nicolet iS10 FT-IR Spectrometer fitted with an attenuated total reflectance (ATR) module (Loughborough, UK) The Attenuated total reflectance technology allows for direct analysis of solid, liquid or gas samples without further preparation. Spectra were recorded in the range 4000-500 cm.sup.−1 by an accumulation of 50 scans. A background was measured with 10 scans prior to each sampling. The manufacturer supplied software OMNIC was used to normalise and analyse the spectra.

(37) Scanning Electron Microscopy (SEM)

(38) A Hitachi TM-1000 Tabletop microscope (Hitachi High-Technologies Europe Gmbh) was used to monitor the physical properties of the nanofibres after electrospinning and during/post modification to ensure than the nanofibre form remained consistent. Samples were analysed from three SEM images each with 20 individual measurements of nanofibre diameters.

(39) Protein Concentration (UV)

(40) UV spectrophotometer Jasco V-630 (Jasco (UK), Essex, United Kingdom) was used to determine the concentration of BSA in solution. A full spectrum from 320 nm-240 nm was recorded with a scan speed of 200 nm/min and a step of 1 nm.

(41) AKTA Explorer (GE Healthcare Life Sciences, Buckinghamshire, UK) was used to measure online absorbance at 280 nm, pressure, feed rates and conductivity throughout the experiments allowing for full run profiles to be analysed.

(42) Reproducibility, Mass Transfer, and Life Cycle Performance

(43) Reproducibility in performance was shown based upon 10 bind/elute runs of the DEAE nanofiber cartridge at a fixed loading flow rate of 240 cm/h. The absorbance flow profiles shown in FIG. 4b clearly show that the adsorbent operates in a reproducible manner during laboratory scale experiments using standard liquid chromatography apparatus.

(44) Bind/elute profile of the DEAE nanofibre cartridge at varying flow rates was also investigated and is shown in FIG. 4a: starting from light grey full line to dark grey dotted line; 72 cm/h (2,400 CV/h), 96 cm/h (3,200 CV/h), 120 cm/h (4,000 CV/h), 240 cm/h (8,000 CV/h) and 360 cm/h (12,000 CV/h). The nanofibre adsorbents demonstrated high mass transfer characteristics that are desired for a high productivity separation with no observed peak broadening in the separation with increased flow rate when plotting the chromatogram against time indicating that mass transfer is a minimal component of the resistance in the column. The integrated areas of the elution peaks in the UV absorbance profiles remained constant demonstrating equivalent capture and elution over the range of flow rates tested. The adsorbents tested had a bed height of 0.3 mm and column volume of ˜0.15 mL.

(45) Fouling studies utilized clarified yeast homogenate to give an understanding of how the adsorbent would perform with complex load conditions. Initial adsorbent fouling studies conducted in a conventional process manner showed no change in trans-bed pressure over 50 cycles of loading, washing, eluting, cleaning-in-place (CIP), and equilibration. Scanning electron microscopy images showed no visible build-up of homogenate on the nanofibre adsorbents after 50 cycles. Conversely, the Sartobind IEXD membranes showed some fouling (FIG. 5) illustrating that with standard CIP conditions (1M NaOH) nanofibre adsorbents were more easily cleaned.

(46) In the continuous homogenate loading experiments both the DEAE nanofibre cartridge and Sartobind IEXD membrane performed well with the clarified homogenate; running for over 9,000 column volumes with only a small increase in trans-bed pressure (FIG. 6a). The Sartobind Epoxy-DEAE derivatized membrane was affected more by this homogenate most likely due to the smaller pore sizes in this membrane. The chromatogram of BSA capture by nanofibre adsorbent before and after clarified homogenate loading and CIP shown in FIG. 6b demonstrates that the nanofibre cartridge performed reproducibly irrespective of the complex feed and harsh CIP conditions employed, confirming the observation shown in FIG. 5 that there was little build-up of homogenate on the nanofibre surface during these studies.

(47) Simulated Moving Bed Operation

(48) A simulated moving bed (SMB) system was designed and built using a series of Burkert solenoid valves (Bürkert Fluid Control Systems, Stroud, UK), 1/16″ Peek tubing, Peek connectors, and three Dionex P580 P HPLC pumps (Dionex Softron GmbH, Germany). UV sensors were placed on the exit of each of the three adsorbent holders which were connected to a National Instruments analogue input module (National Instruments Corporation (U.K.) Ltd, Newbury, UK) to record the UV absorbance at 280 nm. Three National Instrument digital output modules were used to control the valve positions with NI Labview 2010 software used to sequence the control and compute the analogue input signals. FIG. 7 shows the SMB design layout operating at 3 different phases of the process.

(49) Use of Nanofibre Adsorbents in the SMB System

(50) The mass transfer properties of these nanofibre adsorbents make them ideally suited to SMB operation where product can be rapidly loaded and removed in a multiple reuse fashion. FIG. 8 shows the flow through of cytochrome C+ unbound BSA as the first peak during loading of 2-component mixture and the elution of BSA as the second peak. The graphs show the reproducibility over 12 equivalent runs for each adsorbent type. This shows that for the conditions chosen, the nanofibre adsorbents performed favourably for an equivalent volume of adsorbent. The nanofibre adsorbent captured and eluted 99% of the BSA loaded, whereas the Sartobind IEXD membrane only captured 70%. Over the 12 runs for each adsorbent the standard deviation was calculated to show that the nanofibre adsorbent operated more consistently, though both types demonstrated high levels of consistency. The results shown in FIG. 8 demonstrate that for the conditions chosen the nanofibre adsorbent captured and eluted more of the target molecule (BSA) from the 2-component mixture. The tighter shaded region also indicates a more consistent performance in separation. In each case the adsorbent tested had a bed height of 0.3 mm and column volume of ˜0.15 mL

(51) Samples from the 2-component separation studies were separated by SDS Page for analysis and the gel shown in FIG. 9 confirms the results shown in FIG. 8. The samples run in the gel included samples from both ATKA and SMB studies, and from both nanofibre IEXD and sartobind IEXD adsorbents. The Sartobind IEXD membranes did not capture all of the BSA during the loading, which is expressed by the 66 kDa band that can be seen in the flow through sample (well 6) on the gel. In contrast the flow through sample from the nanofibre adsorbent (well 4) indicates no BSA present. The same is observed in wells 9 and 10 showing the flow through from 2 Nanofibre DEAE SMB runs. Wells 7 and 8 show the product streams from 2 Nanofibre DEAE SMB runs which indicated the same result as the product stream of the separation carried out on the AKTA Basic (well 3), where BSA was present in the eluate but Cytochrome C was not. Wells 11 and 12 show the components of the product and waste streams during an SMB run using Sartobind IEXD membranes. The product stream contained only BSA but the waste stream appeared to contain both Cytochrome C and BSA suggesting that not all the BSA was being captured as seen with the AKTA Basic separation in well 6. The gel indicates that the nanofibre adsorbents performed preferentially in both the modes of operation.

(52) Throughput and Productivity

(53) The SMB system relies on a series of valves to switch at given time points to direct the flow of the different mobile phases. In order to optimise the performance of the system, and therefore productivity, the 18 valves must direct the different phases to different locations at exactly the right time. Once optimisation of productivity for the 2-component system was complete 200 mg BSA was repeatedly purified from the two-component protein mixture in 7.5 minutes using three column volumes of 0.15 mL implying an overall system productivity of 1.72 g/hour. This relates to a system productivity of 3.92 g (product)/mL (adsorbent)/hour.

(54) TABLE-US-00001 Dynamic Binding Operating Capacity Flowrate Productivity Adsorbent (mg/mL) (CV/h) (cm/h) (g/mL/h) Nano-DEAE 10 12000 360 3.92 Sarto-IEXD 6.5 6000 180 1.22 DEAE Sepharose 110 24 60 0.1

(55) Table 1 shows data comparing the average productivities of the DEAE nanofibre adsorbents and the Sanobind IEXD achieved during SMB operation. Operational flow rates were chosen to maintain a standard pressure drop of 0.125 bar across the different adsorbents. Productivity calculations (expressed as Grams of product per Millilitre of adsorbent per Hour) were based on the following column dimensions: Porous beaded system 0.7×2.5 cm (W×H). other adsorbents 2.5×0.204 cm (W×H).

(56) Data collected by the three SMB UV sensors is shown in the chromatogram of FIG. 10. The signal created by the sensors was fed back into the NI analogue input module and recorded by a Labview program that was specifically written for this purpose. The traces show how all three adsorbent columns operated similarly and how the different phases ran through each column at a particular time. The flow through was observed as the smaller peak containing Cytochrome C and the larger elution peak containing the target molecule, BSA. The high level of consistency observed highlights the suitability of these nanofibre adsorbents for operation in continuous processing applications.

(57) The productivity that was achieved was limited by the simple design of the SMB system which utilised only three adsorbent modules in sequence and could only be run at 360 cm/h due to the limitations of the SMB system. Productivity calculations were based on chromatography cycle times for the loading, washing, elution, and regeneration of the adsorbents for set flow rates. Using the productivity ratios that were established at the SMB system's limits productivities for higher flow rates were extrapolated based on the known pressure drop limitations of each type of adsorbent. In a previous study it was shown that the nanofiber adsorbents could operate at a flowrate of 2,400 cm/h with a pressure drop across the adsorbent of less than 0.5 bar [31]. For the DEAE Sepharose comparison phase lengths were taken from recommended values in literature [42].

(58) TABLE-US-00002 Dynamic Binding Operating Calculated Capacity Flowrate Productivity Adsorbent (mg/mL) (CV/h) (cm/h) (g/mL/h) Nano-DEAE 10 (±0.3)  80000 2,400 26 Sarto-IEXD 6.5 (±0.71) 40000 1,200 5 DEAE Sepharose 110 150 375 0.6

(59) Table 2 shows data comparing the potential productivity of the adsorbents operating at their maximum established flow rate for a pressure drop of 0.5 bar. Productivity calculations (expressed as Grams of product per Millilitre of adsorbent per Hour) were based on the following column dimensions: Porous beaded system 0.7×2.5 cm (W×H), other adsorbents 2.5×0.204 cm (W×H).

(60) Flow Distribution

(61) To facilitate suitable flow distribution of media through these adsorbent materials a bespoke adsorbent holder was machined. In addition to this suitable packing of the adsorbent into bespoke adsorbent holders is required to promote flow distribution. To evaluate flow distribution, a dye solution (Coomassie Brilliant Blue dissolved in 20 mM Bis-Tris buffer, pH 5.8) was loaded onto the adsorbent.

(62) After loading with a dye solution the adsorbent holder was disassembled to show the suitable coverage of the flow showing even flow distribution. FIG. 11 shows the photographs of the adsorbents after the dye flow distribution study which utilised 3 different stainless steel frits containing different pore sizes. The formation of air bubbles underneath the frit also presented an issue for flow distribution but these would be removed by flowing 20% ethanol through the packed bed prior to normal operation.

(63) The photographs from the flow distribution study shown in FIG. 11 demonstrate the requirement for creating distribution prior to loading onto these adsorbents. A 5 μm stainless steel frit offered the most even distribution that would be required to utilise the adsorbents to their maximum efficiency. The removal of air bubbles underneath the frit is also required by the flow through of 20% ethanol prior to normal operation.

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