Integrity and functionality test for adsorptive depth filter layers with an inorganic layered double hydroxide

Abstract

The present invention relates to a method for determining the functionality and integrity of depth filter sheets and depth filter sheet systems comprising inorganic layered double hydroxide for contaminant removal in biotechnological processes.

Claims

1. A method for determining the integrity and functionality of depth filter sheets and depth filter sheet systems comprising inorganic layered double hydroxides, comprising the steps of loading the depth filter sheet comprising inorganic layered double hydroxide with an adsorbate, comprising inorganic anions, under conditions under which the adsorbate is completely retrained by the adsorptive depth filter sheet until the breakthrough volume is reached, detecting the broken-through adsorbate by means of a secondary reaction, the limit of detection of a detected species, D, is p[D]4, wherein p[D] is the negative common logarithm of the concentration of species [D], and comparing the breakthrough characteristics with those of an adsorptive depth filter sheet of known integrity, wherein the depth filter sheet comprises, in addition to cellulose fibers and the inorganic layered double hydroxide, no further pulverulent adsorbent.

2. The method as claimed in claim 1, wherein the inorganic anions encompass phosphate ions from the group of oxygen acids of phosphorus.

3. The method as claimed in claim 2, wherein the secondary reaction encompasses a complex formation with a color reaction with formation of a phosphomolybedenum blue complex.

4. The method as claimed in claim 1, wherein the loading with the adsorbate is reversible.

5. The method as claimed in claim 1, further comprising the step of rinsing the depth filter sheet comprising inorganic layered double hydroxide with a carbonate-containing rinse solution, resulting in the depth filter sheet regaining at least 90% of its original binding capacity for a subsequent intended use.

6. The method as claimed in claim 1, wherein the proportion by weight of the inorganic layered double hydroxide in the depth filter sheet is 20% or more.

7. The method as claimed in claim 1, wherein the inorganic layered double hydroxide comprises hydrotalcite.

8. The method as claimed in claim 1, wherein the breakthrough of the inorganic ions correlates with the retention capacity with respect to biomolecules.

9. The method as claimed in claim 8, wherein the biomolecules are selected from the group of serum albumins, nucleic acids and immunoglobulins or combinations thereof.

Description

DESCRIPTION OF THE DRAWINGS

(1) The present invention will now be more particularly elucidated by means of the following exemplary embodiments and FIGS. 1 to 7, where

(2) FIG. 1 shows a standard curve in relation to the photometric determination of the concentration of phosphate as phosphomolybdenum blue at 820 nm using a Tecan photometer,

(3) FIG. 2 shows the phosphate breakthrough curves on the adsorptive depth filter sheet system comprising inorganic layered double hydroxide No. 4 during the application of phosphate ions at a concentration of 1 mmol/l as per Example 2 in comparison with conventional, commercially available depth filter sheets comprising kieselguhr,

(4) FIG. 3 shows the phosphate breakthrough curves on depth filter sheet systems comprising various concentrations of inorganic layered double hydroxide (No. 1 to No. 3) during the application of phosphate ions as per Example 2 in comparison with conventional, commercially available depth filter sheets comprising kieselguhr,

(5) FIG. 4 shows the phosphate breakthrough curves on the adsorptive depth filter sheet system comprising inorganic layered double hydroxide No. 4 during the application of phosphate ions as per Example 2 for various phosphate concentrations in the inflow,

(6) FIG. 5 shows the phosphate breakthrough curves for adsorptive depth filter sheet systems comprising various compositions of inorganic layered double hydroxide (No. 3 to No. 7) during the application of phosphate ions at a concentration of 1 mmol/l as per Example 2,

(7) FIG. 6 shows the correlation between the phosphate loading number L.sub.Phosphate and the dynamic binding capacities for BSA, DNA and IVIG (DBT 50%) for various adsorptive depth filter sheet systems comprising inorganic layered double hydroxide,

(8) FIG. 7 shows the phosphate breakthrough curves on the adsorptive depth filter sheet system comprising inorganic layered double hydroxide No. 4 during the application of phosphate ions at a concentration of 1 mmol/l as per Example 2 following the introduction of various defects.

DESCRIPTION OF THE INVENTION

(9) Filter Materials

(10) The adsorptive depth filter sheets comprising inorganic layered double hydroxide that were used were produced according to DE 10 2008 037 678 A1. They differ in the composition of the recipe, i.e., in the type of hydrotalcite, cellulose and/or kieselguhr, the hydrotalcite content, the cellulose content, and the kieselguhr content. Table 1 shows an overview of the composition of the adsorptive depth filter sheets comprising inorganic layered double hydroxide that were used. Hydrotalcite type A has a magnesium oxide/aluminum oxide ratio of 1.65 and is not calcined. Hydrotalcite type B has a magnesium oxide/aluminum oxide ratio of 1.72 and is calcined. Hydrotalcite type C is chemically identical to type A and differs from type A in the particle size. Hydrotalcite type A has a d(1.0) value of 25 m and type C has a d(1.0) value of 280 m. The d(1.0) value is the value in m in which 100% of the particles of a sample are smaller than the specified d(1.0) value. The d(1.0) value therefore indicates the maximum size ranges for the largest particles of a sample.

(11) The comparative examples used were the conventional, commercially available depth filter sheets S9P from Sartorius Stedim Biotech GmbH and Beco Steril S100 from Begerow, which contain kieselguhr as pulverulent adsorbents and differ, as shown in Table 1, in the type and concentration of cellulose and kieselguhr.

(12) TABLE-US-00001 TABLE 1 Overview of components and compositions of the adsorptive depth filter sheets used. Hydro- Recipe Recipe Recipe talcite HT Cellulose Kieselguhr Filter (HT) [%] [%] [%] No. 1 comprising Type A 39 39 22 39% HT type A No. 2 comprising Type A 46 34 20 46% HT type A No. 3 comprising Type A 64 36 0 64% HT type A No. 4 comprising Type C 64 36 0 64% HT type C No. 5 comprising Type B 58 42 0 58% HT type B No. 6 comprising Type B 50.9 49.1 0 50.9% HT type B No. 7 comprising Type B 46.2 53.8 0 46.2% HT type B Sartorius S9P 0 52.5 47.5 Begerow Beco 0 42 58 Steril S100

Example 1 Determination of the Standard Curve for the Determination of Phosphate as Phosphomolybdenum Blue

(13) The following reagents were prepared:

(14) 1.1 TBS Buffer

(15) 10 mmol TBS buffer (TRIS-buffered saline solution), pH 7.4, composed of 1.21 g of tris(hydroxymethyl)aminomethane (TRIS), 8.8 g of NaCl with reverse osmosis water (RO water) made up to 1 liter, adjusted to pH 7.4 using HCl.
1.2 Phosphate Solution 1 mmol PO.sub.4.sup.3 solution in 10 mmol TBS composed of 0.178 g of Na.sub.2HPO.sub.4.Math.2H.sub.2O in 1000 ml of TBS buffer (from 1.1).
1.3 Detection Reagent Reagent A: 5 g of ascorbic acid is dissolved in 50 ml of water. Reagent B: 6 N sulfuric acid (12 ml of a 98% strength sulfuric acid are added to 60 ml of water). Reagent C: 1.25 g of ammonium heptamolybdate are dissolved in 50 ml of water.

(16) 50 ml of each of reagents A, B and C are thoroughly mixed with 100 ml of RO water. This working solution is freshly prepared prior to each determination series.

(17) To obtain a concentration series, the phosphate solution according to 1.2 was diluted with TBS buffer from 1.1 in accordance with Table 2 to give a standard solution of various phosphate concentrations. Thereafter, 1 ml of working solution was added to 1 ml in each case of said standard solution and thoroughly mixed. The preparations were placed in a 70 C. water bath for 10 min. The preparations were then measured in a suitable glass cuvette in a spectrophotometer at 820 nm against a reagent blank (1 ml of water+1 ml of working solution).

(18) A typical standard curve is shown in Table 2 and FIG. 1.

(19) TABLE-US-00002 TABLE 2 Values of the standard curve for the determination of phosphate as phosphomolybdenum blue by means of UV absorbance Phosphate in mmol/l Absorbance at 820 nm 0.00781 0.0818 0.01563 0.1592 0.03125 0.3148 0.06250 0.6257 0.12500 1.2403 0.25000 2.4496

(20) It is apparent that there is a linear dependency of the UV absorbance in relation to the amount of phosphate used. The coefficient of determination for the curve is R.sup.2=1.000. The phosphate ions are captured here as a blue phosphomolybdenum blue complex as per T. G. Cooper, The Tools of Biochemistry, John Wiley & Sons, 1977, pages 55-56, and C. H. Fiske and Y. P. Subbarow, J. Biol. Chem. 66, (1925), 375-400.

Example 2 Breakthrough Curve on a Depth Filter Sheet Comprising Inorganic Layered Double Hydroxide During the Application of Phosphate Ions

(21) The depth filter sheet comprising inorganic layered double hydroxide No. 4, produced according to DE 10 2008 037 678 A1, having the composition shown in Table 1 was clamped in a suitable device and used as follows:

(22) 1. Rinsing of the adsorptive depth filter sheet system comprising inorganic layered double hydroxide (13.2 cm.sup.2) with 100 ml of a TRIS buffer solution (10 mmol, pH 7.4) at a flow rate of 5 ml/min. 2. Application of phosphate solution (5 mmol KH.sub.2PO.sub.4 solution in 10 mmol TRIS, pH 7.4) to the adsorptive depth filter sheet system comprising inorganic layered double hydroxide at a flow rate of 4 ml/min. 3. Collection of the filtrate in 1 ml fractions. 4. Determination of the phosphate concentrations in the individual fractions as per Example 1. The concentration c.sub.i of fraction i is calculated from the absorbance E.sub.i and the gradient m of the standard curve according to
c.sub.i=E.sub.i/m.(1) 5. Plotting of the phosphate concentration in the filtrate against the filtrate volume on a graph in the form of a breakthrough curve. 6. Determination of the breakthrough value V.sub.D. V.sub.D is the filtrate volume at which a phosphate concentration having an absorbance A.sub.820 nm=0.03 can be detected for the first time (corresponds to 1% of the absorbance that is maximally detectable here). It is determined from the two fractions, the absorbance of which is just below or just above the value of 0.03. Between these two pairs of values x.sub.1/y.sub.1 and x.sub.2/y.sub.2, linear interpolation is carried out and the x value for y=0.03 is determined. The following apply:

(23) $\begin{array}{cc}{V}_D=x_1+\left(0.03-y_1\right).Math.\left(\frac{x_2-x_1}{y_2-y_1}\right)\mathrm{or}{V}_D=x_2+\left(0.03-y_2\right).Math.\left(\frac{x_2-x_1}{y_2-y_1}\right)& \left(2\right)\end{array}$ 7. Determination of the loading number L.sub.phosphate for phosphate. The loading number L.sub.Phosphate in mg/cm.sup.3 is calculated according to

(24) $\begin{array}{cc}{L}_{\mathrm{Phosphate}}\left[\mathrm{mg}\text{/}{\mathrm{cm}}^3\right]=\frac{c_{\mathrm{Phosphate}}\left[\mathrm{mol}\text{/}l\right].Math.V_D\left[\mathrm{ml}\right].Math.M_{\mathrm{Phosphate}}\left[g\text{/}\mathrm{mol}\right]}{t\left[\mathrm{cm}\right].Math..Math.{r\left[\mathrm{cm}\right]}^2}& \left(3\right)\end{array}$ where the following apply: radius of the punch-out r=2.35 cm average molar mass of phosphate at pH=7.4 M.sub.phosphate=96.37 g/mol concentration of phosphate in the test solution C.sub.Phosphate=0.001 mol/l filter thickness t in cm breakthrough V.sub.D in ml determined according to 6.

(25) FIG. 2 shows the profile of the phosphate concentration in the outflow during the application of KH.sub.2PO.sub.4 solution in the inflow to the adsorptive depth filter sheet system containing inorganic layered double hydroxide. The concentration of the phosphate ions in the outflow during the application to the adsorptive depth filter sheet system comprising inorganic layered double hydroxide is initially zero before then sharply rising when saturation of the adsorptive depth filter sheet system comprising inorganic layered double hydroxide is reached. The position of the phosphate breakthrough on the x-axis can be detected very accurately on the basis of the emerging phosphate ions in the outflow. By comparison, FIG. 2 shows the profiles of the phosphate concentration in the outflow during the application of KH.sub.2PO.sub.4 solution in the inflow to conventional, commercial adsorptive depth filter sheets S9P from Sartorius Stedim Biotech GmbH and Beco Steril S100 from Begerow, which both contain kieselguhr. The breakthrough of the phosphate ions takes place immediately for the non-hydrotalcite-containing depth filter sheets of the comparative examples.

(26) Therefore, the depth filter sheets of the comparative examples cannot have their integrity tested using the method according to the invention, whereas depth filter sheets comprising inorganic layered double hydroxide can have their integrity tested in a reproducible and reliable manner using the method according to the invention.

Example 3 Breakthrough Curves on Adsorptive Depth Filter Sheet Systems Comprising Various Concentrations of Inorganic Layered Double Hydroxide During the Application of Phosphate Ions

(27) Adsorptive depth filter sheet systems comprising various concentrations of inorganic layered double hydroxide were used. The procedure was as per Example 2. KH.sub.2PO.sub.4 solution was applied as per the procedure in Example 2. FIG. 3 shows the breakthrough curves for adsorptive depth filter sheet systems comprising various concentrations of inorganic layered double hydroxide. It is clearly evident that the position of the phosphate breakthrough shifts toward greater filtrate volumes as the concentration of hydrotalcite increases as a result of their differing adsorption capacity. It is further evident that a content of, for example, 39% hydrotalcite allows the testability of the depth filter, compared to the comparative-example filters which contain only kieselguhr and for which integrity is not testable.

Example 4 Breakthrough Curve on an Adsorptive Depth Filter Sheet System Comprising Inorganic Layered Double Hydroxide During the Application of Phosphate Ions of Different Concentrations in the Inflow

(28) The depth filter sheet comprising inorganic layered double hydroxide No. 4, produced according to DE 10 2008 037 678 A1, having the composition shown in Table 1 was used.

(29) The procedure was as per Example 2. KH.sub.2PO.sub.4 solution of various concentrations was applied, and the outflow was collected in a fractionated manner in 1 ml volume units. The phosphate concentration was determined as in Example 1 and plotted against the filtrate volume. The breakthrough curves are shown in FIG. 4.

Example 5 Breakthrough Curves on Various Compositions of Adsorptive Depth Filter Sheet Systems Comprising Inorganic Layered Double Hydroxide During the Application of Phosphate Ions

(30) Various variants of adsorptive depth filter sheet systems comprising inorganic layered double hydroxide were used, differing in the components and their composition as per Table 1.

(31) The procedure was as per Example 2. KH.sub.2PO.sub.4 solution was applied as per the procedure in Example 2. FIG. 5 shows the breakthrough curves for various compositions of adsorptive depth filter sheet systems comprising inorganic layered double hydroxide. The depth filter sheet systems differ in the type and composition of the hydrotalcite and of the cellulose and are characterized by the position of their phosphate breakthrough as a result of their differing adsorption capacity.

Example 6 Dynamic Protein Binding

(32) To determine the dynamic binding capacities of the depth filter sheets, punch-outs having the diameter of 47 mm and an effective filter area of 13.2 cm.sup.2 are wetted with 10 ml of RO water, inserted into a stainless steel filtration housing (from Sartorius Stedim Biotech GmbH) and prerinsed with 100 ml of TBS (as per Example 1) at 4 ml/min and then rinsed through with a protein or DNA test solution. The test solutions used are a) a freshly prepared solution of BSA (bovine serum albumin from Roth) of a concentration of 1 g/l in TBS buffer (as per Example 1) b) a solution of salmon sperm DNA (Na salt, size distribution 500-1000 base pairs, product number 54653 from Biomol) of a concentration of 0.5 mg/ml and c) a solution of intravenous immunoglobulin IVIG of a concentration of 1 g/l (Cytoglobin from Bayer Vital, Leverkusen).

(33) The absorbances of the filtrate is recorded in fractions in a computer-controlled photometer at a wavelength of a) 280 nm for BSA, b) 260 nm for DNA and c) 280 nm for IVIG, and the concentration of the particular test substance is determined using standard series. Evaluation is carried out by dividing the absorbance of the filtrate by the absorbance of the test solution used and plotting this value against the filtration volume. The breakthrough curves are evaluated by determining the 50% dynamic breakthrough (DBT) and the cumulative binding over the entire course of filtration. The results are shown in Table 3 and in FIG. 6.

(34) TABLE-US-00003 TABLE 3 Results of the dynamic binding capacities of BSA, DNA and IVIG and of the phosphate-binding capacity for various adsorptive depth filter sheets comprising inorganic layered double hydroxide DBT DBT DBT BSA DNA IVIG Thickness V.sub.D L.sub.Phosphate 0.5* 0.5* 0.5* Filter [cm] [ml] [mg/cm.sup.3] [mg/cm.sup.2] [mg/cm.sup.2] [mg/cm.sup.2] No. 1 (39% 0.37 69.09 1.028 8.79 6.74 8.17 hydrotalcite type A) No. 2 (46% 0.39 105.93 1.513 10.72 8.80 9.67 hydrotalcite type A) No. 3 (64% 0.33 140.02 2.096 15.33 10.96 12.77 hydrotalcite type A) No. 4 (64% 0.29 76.79 1.385 10.50 6.92 9.42 hydrotalcite type C) No. 5 (58% 0.35 51.23 0.818 5.23 5.19 3.89 hydrotalcite type B) No. 6 (50.9% 0.41 26.10 0.351 4.45 4.16 3.22 hydrotalcite type B) No. 7 (46.2% 0.38 16.72 0.244 2.86 2.68 2.37 hydrotalcite type B) *Corresponds to the loading at 50% dynamic breakthrough of the starting concentration

(35) It is apparent that there is a linear correlation between the phosphate loading number L.sub.Phosphate (as calculated in Example 2) and the dynamic binding capacities of BSA, DNA and IVIG, which allows a precise and reliable prediction of the binding capacities for said biomolecules depending on the experimentally determined phosphate loading number.

Example 7 Restoration of Binding Capacity after Prior Phosphate Loading for the Purpose of Checking Adsorption Capacity

(36) To restore (reload) binding capacity after prior application of phosphate during the integrity test (as per Example 2), a filter punch-out having a diameter of 47 mm and an effective filter area of 13.2 cm.sup.2 is inserted into a stainless steel filtration housing (from Sartorius Stedim Biotech GmbH) while still wetted or left in said housing directly after the prior phosphate test. Binding capacity is restored by rinsing with 50 ml of a 500 mmol/l potassium carbonate solution and subsequent rinsing with 100 ml of TBS (as per Example 1) at a flow rate of 5 ml/min. After this treatment, the BSA binding capacity of the depth filter treated with the phosphate test is 91% of the BSA binding capacity of an untreated depth filter punch-out (see Table 4).

(37) TABLE-US-00004 TABLE 4 Restoration of the binding capacity of adsorptive depth filter sheet systems comprising inorganic layered double hydroxide. Influence of concentration and rinse volume of the carbonate-containing rinse solution on the restoration of binding capacity (reload). DBT BSA DBT BSA DBT 0.5 BSA Concentration/ 0.5* 0.03** % loading after rinse volume [mg/cm.sup.3] [mg/cm.sup.3] regeneration Reference 0 ml 11.257 7.500 1.00 TBS rinse 50 ml 6.698 4.387 0.60 Carbonate/TBS rinse 7.309 4.816 0.65 50 ml 50 mmol/l Carbonate rinse 8.978 6.385 0.80 50 ml 50 mmol/l Carbonate rinse 10.231 7.266 0.91 50 ml 500 mmol/l Carbonate rinse 2 10.761 7.185 0.96 25 ml 500 mmol/l *DBT 0.5: Loading of the filter at 50% dynamic breakthrough of the starting concentration **DBT 0.03: Loading of the filter at 3% dynamic breakthrough of the starting concentration

(38) Table 4 shows that rinsing with a carbonate-free buffer solution leads to a 60% regeneration of BSA binding capacity.

(39) Rinsing with carbonate-containing buffer solution and subsequent rinsing with carbonate-free buffer solution improves the regeneration of BSA binding capacity to 65%. In the case of use of nonbuffered carbonate solution, the regeneration of BSA binding capacity rises from 80% at 50 mmol/l to 91% at 500 mmol/l. According to the last row in Table 4, an improvement in the regeneration of BSA binding capacity to a level of 96% is reached by applying two portions comprising in each case 25 ml of nonbuffered carbonate solution (500 mmol/l) to the depth filter, with an exposure time of 30 minutes between the two portions. The method according to the invention thus allows a simple, cost-effective and almost quantitative regeneration of the depth filter for the subsequent intended use to adsorb biomolecules.

Example 8 Restoration of Binding Capacity after Prior Phosphate Loading for the Purpose of Checking Adsorption Capacity

(40) To restore (reload) binding capacity after prior application of phosphate ions during the integrity test (as per Example 2), a filter punch-out having a diameter of 47 mm and an effective filter area of 13.2 cm.sup.2 is inserted into a stainless steel filtration housing (from Sartorius Stedim Biotech GmbH) while still wetted or left in said housing directly after the prior phosphate test. Reloading is achieved by rinsing with 225 ml of a 500 mmol/l potassium carbonate solution at a flow rate of 5 ml/min, with the flow rate being set to 0 ml/min for the period of 30 min between the first 25 ml rinse volume and the second 25 ml rinse volume and rinsing subsequently being carried out with 100 ml of TBS (as per Example 1) at a flow rate of 5 ml/min. After this treatment, the BSA binding capacity of the depth filter treated with the phosphate test is 96% of the BSA binding capacity of an untreated depth filter punch-out (see Table 4).

Example 9 Phosphate Breakthrough Curves on Adsorptive Depth Filter Sheet Systems Comprising Inorganic Layered Double Hydroxide During the Application of Phosphate Ions Following Introduction of Artificial Defects

(41) In the dry state, a punch-out of an adsorptive depth filter sheet system comprising inorganic layered double hydroxide having the diameter of 47 mm and an effective filter area of 13.2 cm.sup.2 is provided with a single hole in the center of the filter punch-out by means of a stainless steel needle. This involved using needles having a diameter of 200 m or 400 m. The rest of the procedure to determine the phosphate breakthrough is as described in Example 2. The breakthrough curve is shown in FIG. 7.

Example 10 Phosphate Breakthrough Curves on Adsorptive Depth Filter Sheet Systems Comprising Inorganic Layered Double Hydroxide During the Application of Phosphate Ions Following Introduction of Artificial Defects

(42) A punch-out of an adsorptive depth filter sheet system comprising inorganic layered double hydroxide having the diameter of 47 mm and an effective filter area of 13.2 cm.sup.2 is wetted with 10 ml of RO water and provided with a single hole in the center of the filter punch-out by means of a stainless steel needle having a diameter of 400 m. The rest of the procedure to determine the phosphate breakthrough is as described in Example 2. The breakthrough curve is shown in FIG. 7.

Example 11 Phosphate Breakthrough Curves on Adsorptive Depth Filter Sheet Systems Comprising Inorganic Layered Double Hydroxide During the Application of Phosphate Ions Following Introduction of Artificial Defects

(43) In the dry state, a punch-out of an adsorptive depth filter sheet system comprising inorganic layered double hydroxide having the diameter of 47 mm and an effective filter area of 13.2 cm.sup.2 is provided with a crease, breaking the surfaces of the depth filter sheet. The rest of the procedure to determine the phosphate breakthrough is as described in Example 2. The breakthrough curve is shown in FIG. 7.

Example 12 Phosphate Breakthrough Curves on Adsorptive Depth Filter Sheet Systems Comprising Inorganic Layered Double Hydroxide During the Application of Phosphate Ions Following Introduction of Artificial Defects

(44) In the dry state, the surface of a punch-out of an adsorptive depth filter sheet system comprising inorganic layered double hydroxide having the diameter of 47 mm, a thickness of 0.37 mm and an effective filter area of 13.2 cm.sup.2 is destroyed on the upstream side. To this end, tweezers were used to remove from the depth filter material in the dry state approximately the top half of the material across the entire inflow area. This avoided a complete physical breakthrough through the entire thickness of the depth filter sheet. The rest of the procedure to determine the phosphate breakthrough is as described in Example 2. The breakthrough curve is shown in FIG. 7.

(45) FIG. 7 illustrates that the high sensitivity of the method according to the invention makes it possible to detect very easily even small defects in the filter, because the position of the breakthrough point responds very sensitively to the presence of defects.