Patent ID: 12186472

EXAMPLES

The invention is further described by the following examples. These are intended to present support for the workability of a number of preferred non-limiting embodiments or aspects of the invention without limiting the scope of the invention described herein.

Example 1: Immobilization of AP to Hemofilter Membranes

Membrane Preparation:

Alkaline Phosphatase (AP) was immobilized on various membranes in a proof of principle approach of the present invention. The table depicts the different components of the used membranes in percentage of the solution from which membranes are casted (in brackets the weight for a total solution weight of 50 g is given).

TABLEMembrane componentsPES/PVP/MaleicPES/PVP/chitosanPES/PVP membranesAnhydride membranesmembranes*80% (40.0 g) NMP78% (39.0 g) NMP74.57% (37.28 g) NMP12% (6.0 g) PES12% (6.0 g) PES13.24% (6.62 g) PES3% (1.5 g) PVP-3% (1.5 g) PVP-K852.84% (1.42 g) PVP-K85K855% (2.5 g) H2O5% (2.5 g) H2O3.78% (1.89 g) H2O2% (1.0 g) Poly(maleic5.48% (2.74 g)anhydride-alt-1-octadecene)Citric acid0.09% (0.05 g)Chitosan*Citric acid was first dissolved in H2O and chitosan was dissolved overnight, before addition of the rest of the mixture.

Components were dissolved overnight at 60° C. under continuous stirring. After completely dissolving all components, the solution was removed from the oil-bath and allowed to cool for 1 h. Flat-sheet membranes were casted using a 100 μm knife on an electronic casting machine. The knife was placed on a clean glass plate and approx. 10 mL of the polymer solution was applied. The motor was then engaged at 25 mm/s until the polymer film covered the entire glass plate. The motor was returned to its starting position, the knife removed, and the glass plated placed into a 500 mL bath of demineralized water. After 5 minutes, the water was replaced by fresh water, this process repeated after 2 hour and the membrane was left in the water bath for another 4 h, before being placed under vacuum at 60° C. overnight, to completely remove all NMP.

Alkaline Phosphatase Coupling to Membranes:

Membranes were cut into small pieces (approx. 1×1 cm) and their exact weight was determined (usually around 3-7 mg). A total of 5 mg of alkaline phosphatase (Santa Cruz Biotechnology) with an activity of 4000 U/mg and a protein content of 24.5 mg/mL was aliquoted into 5 μL aliquots, each containing 100 U/μL. For each immobilization experiment, a 5 μL aliquot was dissolved in 1 mL of 0.5M TRIS pH 8.5 containing 1 mM CaCl2, to obtain a 500 U/mL solution. Membranes were placed in an Eppendorf containing 900 μL Millipore H2O, 100 μL of 500 U/mL AP and 2.5 mg/mL of EDC. The membranes were then kept on a tumbler shaker at RT for 3-4 hours and placed at 4° C. overnight. After 18 h, the membranes were placed into a 50 mL 0.5M NaCl solution and left on a tumbler shaker in order to remove unbound AP. After 4 h, the wash solution was replaced with 1×PBS and left on the tumbler shaker for 2 more hours. Alternatively, membranes are placed in an Eppendorf containing 1 mL of MES buffer containing 2.5 mg/mL EDC for 30 minutes, removed and washed, and then placed into 1 mL of 50 U/mL AP overnight.

Activity Determination of Immobilized Alkaline Phosphatase:

After the final washing step, membranes were placed into 1 mL of p-Nitrophenyl phosphate (pNPP) Liquid substrate system (Sigma Aldrich). To determine the activity, 75 μL of the pNPP solution was removed and the absorbance was determined on t=10 min, 20 min, 30 min and 60 min. At each of these time points, the absorbance of the previous timepoint was determined again, and the change in absorbance over time was used to evaluate the release of Alkaline phosphatase from the membranes (since release results in continued color development and subsequent readings at later timepoints would show an increase in absorbance). The absorbance was then compared to the absorbance of standard concentrations of dephosporylated pNPP solution and the activity of the alkaline phosphatase on the membrane was determined in μmoles of pNPP converted per minute.

Results:

Results from two different membranes show the measured activity between 0.06 and 0.10 μmole min−1. To clarify results and make up for different sample membrane weights, the activity was extrapolated to the activity for a whole dialyzer (containing 20 gram of membrane), indicating activities around 560 μmole min−1. Release of alkaline phosphatase was in all cases minimal and, with one exception, did not exceed standard deviations of average activity. Results of these experiments are shown inFIG.2A.

Example 2a: Immobilization of AP to an Epoxy-Functionalized Adsorber Resin

First, the resin is equilibrated. The resin is washed with immobilization buffer and filtered. A resin/buffer ratio of 1/1 (w/v) is preferable. The immobilization buffer is chosen to be compatible with AP. The process is repeated 2-4 times. The AP solution is prepared by dissolving the protein in immobilization buffer. For example, 100-200 mg AP can be loaded per gram of wet resin. Protein concentration can be determined by using standard protein content assays. The AP is dissolved in a sufficient amount of buffer to obtain a ratio resin/buffer of 1:4 (w/v). This ratio can be optimized depending on the protein used (range can vary from 1:1-1:4). Immobilization begins with the transfer of the immobilization buffer containing the AP protein into the immobilization vessel. The epoxy-functionalized resin, for example the Purolite® Lifetech™ resin described herein, is then added. The slurry is gently mixed at 70-80 rpm for 18 h and afterwards left without mixing for another 20 h. Magnetic stirring during protein immobilization should be avoided as this can damage the beads. Immobilization can be performed at temperatures of 20° C.-30° C., depending on the protein stability. Immobilizations should not be performed at high temperatures as this can cause degradation of the epoxy rings (hydrolysis) and facilitate microbial growth. Finally, the liquid phase is filtered off and collected. The protein content in the liquid is determined and the immobilization yield calculated. The resin is then washed with washing buffer. The process is repeated 2-4 times under gentle stirring or in column wash. An additional washing step using a 0.5 M NaCl containing buffer for complete desorption of non-covalently bound proteins can be performed. Excess water is removed by filtration. The immobilized AP protein can then be characterized in terms of moisture content and specific binding activity.

Example 2b: Immobilization of AP to an Amino-Functionalized Adsorber Resin

First, the resin is equilibrated. The resin is washed with immobilization buffer and filtered. A resin/buffer ratio of 1:1 (w/v) is preferable. The immobilization buffer is chosen to be compatible with the AP protein. In a second step 2% glutaraldehyde buffer is prepared starting from a solution of 25% (w/v) glutaraldehyde. A 2% glutaraldehyde (v/v) solution is prepared using the immobilization buffer. In a third step, the amino resin is activated by adding the 2% glutaraldehyde buffer prepared in step 2 to the resin. The optimal volume of 2% glutaraldehyde buffer should be in the range of resin/buffer ratio of 1:4 (w/v). The slurry is left to mix for 60 min at 20° C.-25° C. The beads are then filtered and washed with immobilization buffer using a resin/buffer ratio of 1:4 (w/v). It should be avoided to store pre-activated resin for a period longer than 48 h. Beads are then ready for the immobilization step. In a fourth step the AP protein solution is prepared. To that end, the protein is dissolved in immobilization buffer. For example, between 1 mg and 100 mg AP protein can be loaded per gram of wet resin. The protein concentration can be determined by using standard protein content assays.

The protein is dissolved in buffer to obtain a ratio resin/buffer of 1:4 (w/v). Optimization of this ratio can be pursued in further trials (range can vary from 1:1-1:4). In a fifth step, the protein is immobilized. The immobilization buffer is transferred into the immobilization vessel and the pre-activated amino resin (e.g. from Purolite®, Lifetech™) as prepared in step 3 is added. The slurry is gently mixed for 18 h at 70-80 rpm. Magnetic stirring should be avoided during immobilization as this can damage the beads. The immobilization can be performed at 20° C.-30° C. accordingly to AP protein stability. The immobilization should not be performed at high temperatures since this might cause side reactions of the aldehyde groups on the resin formed during step 3. Finally, the liquid phase is filtered off and collected. The protein content in the liquid is determined and the immobilization yield calculated. The resin is then washed with washing buffer. The process is repeated 2-4 times under gentle stirring or in column wash. An additional washing step using a 0.5 M NaCl containing buffer for complete desorption of non-covalently bound proteins can be performed. Excess water is removed by filtration. The immobilized AP protein can then be characterized in terms of moisture content and specific enzymatic activity.

Activity Determination of Immobilized Alkaline Phosphatase:

Both epoxy and amino-functionalized resins treated as described above to immobilize AP were subsequently placed into 1 mL of p-Nitrophenyl phosphate (pNPP) Liquid substrate system (Sigma Aldrich).

As described above, to determine the activity, 75 μL of the pNPP solution was removed and the absorbance was determined over time. The absorbance was then corrected for any background signal obtained when unmodified resins had not been treated with AP.

Results

Results from both epoxy and amino-functionalized resins show that AP is effectively immobilized and active based on the commercial pNPP solution used in the assay. Results of these experiments are shown inFIG.2B.

Example 3: Immobilization of AP to Membranes Under Varying pH Conditions

Polyacrylonitrile AN69 membranes were employed to determine optimized pH conditions for AP binding to a membrane, verified by subsequent AP activity tests. The tests on pH optimization were carried out with “M-Filter” membranes (MF150), which are prepared as “minimodules”, namely small filters commonly used for testing membrane characteristics. The tests described were carried out on AN69 minimodules each comprising a bundle of fibers, whereby in each bundle approx. 40 hollow fibers are present.

AN69 is an established polymer used in dialysis applications and is a copolymer of acrylonitrile and sodium methyl allyl sulfonate. The sulfonate groups result in a hydrophilic membrane with negative charges, which forms a hydrogel structure through water retention. This composition of the membranes allows for medium-sized proteins to be adsorbed. A scanning electron microscope (SEM) image of an AN69 hollow fiber is shown inFIG.7together with a schematic setup of how such bundles are employed in dialysis. The AN69 minimodules have an inner surface of approximately 1 cm2per fiber. This corresponds to an inner surface of 40 cm2per minimodule.

The minimodules were attached to a pumping station using common hosing (Promedt), through which liquid could be pumped (pump from Ismatec) at controlled rates through the minimodules.

In order to prepare the immobilized AP on the AN69 minimodules, the minimodules were coated with polyether imide (PEI) prior to attaching glutaraldehyde (GA) and AP. Due to the negative charges present on the AN69 membrane, a positively charged polymer can be attached through ionic interactions. In the present case PEI was employed, which carries positively charged amino groups. The PEI attachment is typically carried out in an acidic medium, as a result of which the amino groups are protonated, and the charge density is increased. By using citric acid in this process, a larger amount of PEI can be bound on the membrane than in alkaline medium. Optimization of the PEI treatment is discussed in more detail below.

After PEI treatment the AN69 membrane was treated with glutaraldehyde (GA) prior to immobilizing AP. Glutaraldehyde serves as a bifunctional spacer, generating a pre-activated polymer surface to bind the enzyme. Following the pre-activation of the surface, the free aldehyde groups of the glutaraldehyde are available in order to be able to bind another component via the same reaction. In the present case, AP is subsequently covalently bound in a second modification step via the free amino groups.

The AN69 minimodules were therefore:Flushed to remove glycerin (with reverse osmosis water, 5 mL per minimodule at 1.5 mL/min, followed by 5 to 20 mL at 3 mL/min). Volume visually adjusted in order to remove air,Treated with PEI (PEI at 200 mg/kg in citric acid solution at 200 mg/kg, using 20 mL per minimodule at 3 mL/min),Flushed (in PBS, 20 mL per minimodule at 3 mL/min)Treated with glutaraldehyde (2.5% GA solution, 40 mL per minimodule at 1.5 mL/min),Flushed (in PBS, 50 mL per minimodule at 1.5 mL/min),Treated with AP (1000 U AP per minimodule in PBS, 20 mL recirculated at 1.5 mL/min for 16 h), andFinally rinsed (in PBS, 50 mL per minimodule at 1.5 mL/min).

As a read-out, the immobilized AP activity was assessed. A test solution comprising pNPP substrate (para-nitrophenylphosphate disodium salt hexahydrate) with a matrix of magnesium chloride and zinc chloride in PBS buffer was used. To quantify the AP activity on the membrane, a single fiber was cut out of the minimodules. The modules were opened, the fibers were washed with PBS and then each fiber was placed in a plate well. The fibers were cut into appropriately sized pieces and pNPP test solution was applied. Readings were taken at 60 min and 120 min. As usual, the samples were measured against standard solutions of the free enzyme and therefore the results expressed as an activity of free AP under ideal standard (alkaline) conditions.

In order to assess optimal pH conditions for AP immobilization, two processes were modified, namely (1) coating of the membrane with glutaraldehyde (designated below as “GA-pH”) and (2) subsequent immobilization of AP (designated below as “AP-pH”). For each of these treatments, multiple pH values of the relevant solutions were tested.

In the table below, the results from several protocols (I-IV) and the results for AP activity in U/cm2after 60 and 120 minutes are shown. In order to simplify the discussion, the corresponding average values are calculated and also displayed.

Activity/U/cm2ExperimentModuleGA-AP-t =t =##pHpH60 Min120 MinAvg.I737.47.40.090.100.10740.040.040.04750.150.180.16760.130.130.13II779.90.410.410.41780.430.380.40790.440.400.42800.390.360.37III819.97.40.100.140.12820.150.200.18830.130.170.15840.730.920.82IV859.90.140.160.15860.160.150.16870.680.730.70880.800.820.81

The experiments under II gave reproducibly high AP activities in the range of from 0.38 to 0.45 U/cm2, which is about ten-fold higher than the lowest limit which was empirically estimated as a therapeutically relevant activity (0.05 U/cm2). Alternative pH conditions also appear effective, although less reproducible than in experiment II.

Example 4: Immobilization of AP to Beads Under Varying pH Conditions

Experiments similar to Example 3 above were conducted using amino-functionalised beads (Purolite Lifetech ECR 8408F) as a matrix for immobilization of the AP. Beads were pre-treated using GA at varying pH values and subsequently treated with AP, also at different pH values.

Experiments were conducted in 96-well plates with GA/AP-treated beads in 900 μL of pNPP solution under constant stirring. 75 μL of reaction was removed and analyzed in a separate 96-well plate at 10, 30, 60 and 120 minutes, using the photometric activity measurement described above.

The table below outlines the AP activity determined after 10 minutes using the beads as a solid matrix.

ExperimentExtinctionActivity in U/mLReference-Beads (washed)0.278750.05Beads + GA pH 7.4 + AP pH 7.40.35050.62Beads + GA pH 7.4 + AP pH 9.90.83564.51Beads + GA pH 9.9 + AP pH 9.90.89384.98Beads + GA pH 9.9 + AP pH 7.40.417651.16Beads + AP pH 7.40.286550.11Beads + AP pH 9.90.26645−0.05

As can be observed from the above results, AP activity was maintained after coupling to the beads, the most effective immobilization conditions being GA pH 9.9+AP pH 9.9 or GA pH 9.9+AP pH 7.4, similar to the AN69 minimodule experiments above. The GA step is important when using these beads (homobifunctional spacer glutadialdehyde), as no enzyme binding respectively activity can be observed by bringing the beads directly together with the AP.

Example 5: Adjustment of Cofactors for AP Activity

The AP used in the above examples was commercially available and shipped with cofactor concentrations of 1 mM MgCl2and 0.1 mM ZnCl2. In the activity test for AP described above, cofactor concentrations are 0.5 mM MgCl2and 0.1 mM ZnCl2. In the PBS simulated use tests, cofactors Mg2+and Zn2+are also added in a concentration of 0.5 mmol/L and 0.1 mmol/L, respectively (see below for simulated use stability tests).

Further experiments were conducted using the AN69 membrane minimodules from Example 3, in order to titrate Mg2+concentration in dilution series from 5 mM to 0.005 mM. Although some slowing down of AP reaction was observed, clear differences between the kinetic profiles of different magnesium concentrations could not be observed, indicating that the immobilized AP is likely to exhibit suitable activity at the cofactor concentration found in human blood samples.

Blood plasma concentration in healthy individuals is similar to serum, ranging from 0.7 to 1.0 mM. Accordingly, when the device is in clinical use, blood would have high enough levels of magnesium to support AP activity. The same is true for zinc. Serum zinc concentrations are 12.4±1.4 μmol/L (9-22 μmol/L in women; 12-26 μmol/L in men).

This example also demonstrates that a device for extracorporeal therapy containing immobilized AP, may preferably be filled with or stored in a buffer or other solution, wherein the matrix comprising the immobilized AP is immersed in said buffer or solution, with the presence of Mg2+in a concentration of 0.1 to 2, preferably 0.1 to 1 mmol/L, and a Zn2+in a concentration of 5 to 150, preferably 10 to 100 μmol/L.

Example 6: Optimizing PEI Density on Membranes

Optimal PEI coating and density was tested using the “TNBS”-Test for APA-CRRT. The TNBS test is known to a skilled person (based on 2,4,6-Trinitrobenzene sulfonic acid) and can be used for quantitative analysis of amino functions e.g. on the membrane surface. TNBS reacts with reactive amines molecules to from a highly chromogenic (orange) product, whose absorbance at 335 to 345 nm can be measured with a plate reader or spectrophotometer. The amino function is derived from the PEI in the present examples.

A 50 g/L TNBS solution was used for the test. The absorbance of the solution was measured at 340 nm on a photometer blanked against reverse osmosis water. In 15 mL of the 0.1 g/L TNBS solution, all 40 fibers from one minimodules are removed and added to the solution in approximately 1 cm long pieces. After shaking for 30 minutes at room temperature in the dark, the supernatant of the respective solution was removed, and the absorbance measured. The decrease in absorbance correlates directly with the number of reactive amines. An optimal PEI density on the membrane of 1-3 μmol/g of hollow fiber membrane was achieved.

It was found that membranes which are coated with the process as described in Example 3 can be AP-functionalized very efficiently. The PEI coating process based on citric acid (see Example 3; preferably PEI at 200 mg/kg in citric acid solution at 200 mg/kg) is effective. Further experiments were also conducted using functionalization with PEI/GA/AP based on a pre-coated (PEI-coated) ST-Filter, which is the same AN69 membrane, but pre-coated with PEI in commercial production. AP immobilization to pre-treated PEI membranes was also successful. However, those hollow fibers with AP showed significantly lower enzymatic activity as the ones mentioned above.

Example 7: Simulated Use and Stability of AP Activity in PBS and Human Plasma

In order to assess whether immobilized AP is stable over time, the following experiments were conducted. Minimodules were prepared as described above under Experiment II of Example 3, and then attached to a pumping station, as in Example 3.

In all experiments, the minimodules were closed on the dialysate side and perfused recirculating on the lumen side with the appropriate medium. The flows and volumes were set accordingly in order to simulate a 72-hour dialysis, which corresponds to a maximum permitted use time of a standard dialysis filter for acute treatment.

With standard dialysis, flows of about 200 mL/min-500 mL/min are used with a blood volume of about 5 L. The inner surface of a dialyzer used here is, for example, 1.5 m2. For a minimodule with an area of 40 cm2, this results in a flow of 0.5 mL/min-1.3 mL/min and a pool volume of 13 mL. Since the calculated values are too low for the laboratory test, the flow was set to 1.5 mL/min and the pool volume to 20 mL.

A PBS buffer with 0.5 mmol/L MgCl2and 0.1 mmol/L ZnCl2was used as the medium for the perfusion. The modules were then perfused under a warming hood at 37° C. for the specified period. At certain times, samples were taken from the pool and measured for activity in order to investigate possible leaching or washout processes. The fibers from the corresponding modules were then subjected to the activity test described above without further treatment.

Activity measurements showed an activity prior to 72 h perfusion of 0.4 U/cm2, whereas the activity after the test was 0.7-2.0 U/cm2. No activity was observed in the eluate pool, indicating that leaching or washing out of immobilized AP does not occur or is negligible.

When the minimodule with an AP-functionalized membrane is perfused with PBS in the presence of the AP cofactors, the immobilized activity is therefore reliably retained (or even reproducibly increased) over 72 h. It is assumed that the AP before perfusion is already somewhat depleted from cofactors, which the enzyme is subsequently provided through the buffer and therefore not only maintains but increases activity over time. In any case, the AP on the hollow fiber membrane is highly stable.

In human plasma, the effect is even more pronounced. The experiments were repeated using human plasma in place of PBS buffer. Human plasma, by nature, already contains the required cofactors of AP. Activity measurements showed an activity prior to 72 h perfusion in plasma of 0.4 U/cm2, whereas the activity after the test was 1.6-2.4 U/cm2. No activity was observed in the eluate pool, indicating that leaching or washing out of immobilized AP does not occur or is negligible.

Example 8: Stability of AP Activity After Sterilization

Sterilization techniques are required for clinical products to inactivate potential microorganismal pathogens. Due to the necessary AP activity, heat treatment is possible, but not preferred, as steam sterilization could damage the membrane and/or denature the enzyme.

Sterilization using gamma radiation was assessed as a promising approach. During gamma sterilization, gamma radiation is generated by a radioactive radiation source, which then reaches the product. Gamma rays have a high penetration depth and can therefore be used for the sterilization of products in the final packaging.

The gamma sterilization was carried out on minimodules as described above using a dose of approximately 30 kGy. With this sterilization method, the minimodules, still moistened by the modification processes (approx. 0.5 g moisture/minimodule), can be closed with stoppers and do not need to be filled with glycerin. After the coating (as in Example 3), the minimodules on the blood and dialysate side were filled with reverse osmosis water and closed with stoppers and sent for gamma sterilization.

After sterilization with a dose of 30.6 kGy, the water contained in the minimodules was collected and stored. The two minimodules 91 and 92 were pre-rinsed with the salt solution of MgCl2and ZnCl2. For this purpose, the minimodules were connected to hose sets and filled with a flow of 3 mL/min. The hoses were then disconnected and the minimodules left to stand for 30 minutes, before being pumped empty and subjected to the activity test. The results are shown inFIG.8.

The two minimodules 89 and 90 were directly subjected to the activity test, as described above. The two flushed minimodules 91 and 92 showed an activity of about 0.5 U/cm2. The non-flushed minimodule 89 showed a very low activity of 0.02 U/cm2. Minimodule 90 showed a higher activity of about 0.3 U/cm2. Minimodules modified with the same procedure and not sterilized showed an activity of about 0.4 U/cm2. Sterilization of water-filled minimodules therefore shows little influence on 3 of the 4 minimodules when assessed for AP activity.

Alternatively, sterilization with ethylene oxide (EtO) gas was also carried out. The minimodules were first filled with glycerin, to prevent the gel membrane from drying out, and subsequently treated using standard techniques. Ethylene Oxide (EtO) is a common gas used for low temperature sterilization. It is a colorless, poisonous gas that attacks the cellular proteins and nucleic acids of microorganisms. It is most commonly used to sterilize medical instruments with long lumens and materials that have to be sterilized but cannot withstand higher temperature.

EtO sterilization was carried out on minimodules and the activity of AP was subsequently determined, as described above. EtO sterilization led to a slight reduction of AP activity. After testing 5 minimodules (two without and three with EtO sterilization), the AP activity was lost in only one minimodule (likely due to an unknown technical error). In the two minimodules that retained AP activity, initial data revealed that after 120 minutes in the activity test, 40% of AP activity was maintained after EtO sterilization, compared to unsterilized controls. These test results are preliminary and being repeated.

In summary, both gamma and EtO sterilization appear feasible in order to maintain AP activity immobilized on membranes.