USE OF A CITRATE SOLUTION FOR AFFINITY CHROMATOGRAPHIC PURIFICATION OF CRP USING PHOSPHOCHOLINE AND DERIVATIVES THEREOF

Abstract

The invention relates to the use of a citrate solution for affinity-chromatographic removal of C-reactive protein (CRP) from biological fluids, wherein the CRP is affinity-chromatographically removed using (Ca.sup.2+-dependent) binding of CRP to a column material functionalized with ω-phosphonooxyalkyl ammonium groups and/or with ω-ammoniumalkoxy-hydroxy-phosphoryloxy groups.

Claims

1. A method for affinity-chromatographic removal of CRP from a biological fluids comprising running the biological fluid through a column material functionalized with a ω-phosphonooxyalkyl ammonium groups and/or with ω-ammoniumalkoxy-hydroxy-phosphoryloxy groups, wherein the citrate solution serves as a binding buffer.

2. The method according to claim 1, wherein the ω-phosphonooxyalkyl ammonium groups have the following general formula (I): ##STR00029## wherein n is selected from 2 and 3; R.sup.1 and R.sup.2 are independently selected from the group consisting of: —H, —CH.sub.3, —C.sub.2H.sub.5, —C.sub.3H.sub.7, —C.sub.4H.sub.9, —C.sub.5H.sub.11, and —C.sub.6H.sub.13, or R.sup.1 and R.sup.2 together with the nitrogen atom to which they are attached can forms a heterocycle selected from the group consisting of: ##STR00030## and wherein one or more hydrogen atom(s) is optionally replaced by (a) fluorine atom(s).

3. The method according to claim 1, wherein the ω-ammoniumalkoxy-hydroxy-phosphoryloxy groups have the following general formula (II): ##STR00031## wherein n is selected from 2 and 3; R.sup.1, R.sup.2 and R.sup.3 are independently selected from the group consisting of: —H, —CH.sub.3, —C.sub.2H.sub.5, —C.sub.3H.sub.7, —C.sub.4H.sub.9, —C.sub.5H.sub.11, and —C.sub.6H.sub.13, or R.sup.1 and R.sup.2 together with the nitrogen atom to which they are attached forms a heterocycle selected from the group consisting of: ##STR00032## R.sup.3 is selected from the group consisting of: —H, —CH.sub.3, —C.sub.2H.sub.5, —C.sub.3H.sub.7, —C.sub.4H.sub.9, —C.sub.5H.sub.11, and —C.sub.6H.sub.13; and wherein one or more hydrogen atom(s) is optionally replaced by (a) fluorine atom(s).

4. The method according to claim 1, wherein the citrate solution comprises at least one citrate compounds selected from the group consisting of citric acid, sodium dihydrogen citrate, disodium hydrogen citrate, trisodium citrate, trisodium citrate dihydrate, potassium dihydrogen citrate, dipotassium hydrogen citrate, tripotassium citrate, lithium dihydrogen citrate, dilithium hydrogen citrate, trilithium citrate, ammonium dihydrogen citrate, diammonium hydrogen citrate, triammonium citrate, tricalcium dicitrate (calcium citrate), trimagnesium dicitrate (magnesium citrate), and partial citrate esters.

5. The method according to claim 4, wherein the at least one citrate compound is citric acid or citrate salts with monovalent metal cations.

6. The method according to claim 4, wherein the citrate solution contains no additional Ca.sup.2+ chelators apart from the citrate compound(s).

7. The method according to claim 1, wherein the citrate solution comprises citric acid, trisodium citrate, D-glucose and water.

8. The method according to claim 1, wherein the citrate solution comprises at least one citrate compound with a total concentration in a range from 50 mM to 200 mM.

9. The method according to claim 1, wherein the citrate solution has a pH-value in a range from pH 4.0 to pH 7.0.

10. The method according to claim 1, wherein the ratio of the citrate solution and the biological fluid is in a range from 1:50 to 1:5.

11. The method according to claim 1, wherein the biological fluids is blood or blood plasma.

Description

DESCRIPTION OF THE FIGURES

[0200] FIG. 1: The results of several chromatography runs for the purification of CRP are shown, the CRP depletion in % is plotted against the matrix volume in a semi-logarithmic representation. A citrate solution was added to human blood plasma with a CRP concentration of 50 mg/L, 1:5, 1:10, 1:20 or 1:50, and applied to a chromatography column containing 0.5 g of APPC-coupled epoxy-Ultraflow-4 Agarose (Sterogen) as a matrix. A sample without citrate addition served as a control. The column run was collected at a flow rate of 5, 10, 25, 50, and 75 matrix volumes in 1 ml fractions. The CRP concentration in the fractions was then determined and the CRP depletion in % was determined on the basis of the initial concentration of CRP (before the application). The data points represent mean values from at least two independent experiments.

[0201] FIG. 2: The bar graph shows the depleted amount of CRP in blood plasma purified by affinity chromatography with and without the addition of citrate by means of a column material functionalized with ω-phosphonooxyalkyl ammonium groups and/or with ω-ammoniumalkoxy-hydroxy-phosphoryloxy groups.

[0202] FIG. 3: shows an SDS gel applied to the blood plasma that was purified on three different days with and without the addition of citrate by means of a column material functionalized with ω-phosphonooxyalkyl ammonium groups and/or with ω-ammoniumalkoxy-hydroxy-phosphoryloxy groups, by affinity chromatography. The arrow marks the C1q band. This band contains less protein in the eluate from the blood plasma, which was mixed with ACD-A.

EXAMPLES

[0203] The term “matrix volume” (also abbreviated as MV) as used herein refers to the volume of column material used in the respective affinity chromatography.

Example 1

Comparative Study on the Effect of Addition of Citrate Solution on the Affinity-Chromatographic Purification of CRP from Blood Plasma

[0204] As a starting material, human blood plasma with a CRP concentration of approximately 50 mg/L was filtered using a 35 μm-filter, and optionally citrate solution was added (see below).

General Procedure of Affinity-Chromatography

[0205] For chromatography, a low-pressure chromatography system (BioLogic™ LP System, Bio-Rad, Munich Germany, catalogue number 731-8300), together with a column (Mobicol “Classic”, MoBiTec GmbH, Göttingen, Germany, product number M1002) with a 35 μm-filter (MoBiTec GmbH, product number M523515) was used, which was equipped with 0.5 g of APPC-coupled agarose (epoxy-Ultraflow-4 agarose, Sterogene) as column material (1.6 mg APPC/ml), i.e. a column material with a sepharose-matrix to which the phosphocholine derivative p-aminophenylphosphocholine (APPC) was coupled. The column was equilibrated with 3 matrix volumes (MV) wash buffer (0.1 M Tris, 0.2 M NaCl, 2 mM CaCl.sub.2, pH 8.0) and the flow was discarded.

[0206] The sample was then applied to the column. The flow of the column was divided into fractions of 1 ml each by means of a fraction collector (BioFrac™ Fraction Collector, Bio-Rad, catalogue number 741-0002) connected downstream of the column. The 1 ml fractions (also referred to as flow fractions) at a flow rate of 5 MV, 10 MV, 25 MV, 50 MV, 75 MV and 100 MV were collected while the remaining fractions were combined to form the so-called “flow pool”.

[0207] The column was then washed with 25 MV wash buffer after the sample application. This was followed by elution of the CRP bound to the column by application of 7.5 MV elution buffer 1 (0.1 M Tris 0.2 M NaCl, 2 mM EDTA, pH 8.0). Here the eluate was fractionated and collected only when the UV absorption was above 0.01.

[0208] Subsequently, 5 MV elution buffer 2 (glycine buffer pH 2.8) was used to regenerate the column. If elution by the elution buffer 1 is not complete, proteins and in particular CRP may also be present in this fraction. Neutralization buffer (3.5 M Tris, pH 9.0) is added to this eluate, so that the CRP does not denature at the acid pH of the elution buffer 2 of 2.8. Finally, the column is washed again with 20 MV of the wash buffer.

[0209] The CRP content of the filtered starting material, of the flow pool and of the collected fractions was determined by means of a competitive and human-specific immunoassay (see, for example, Example 2).

[0210] Starting from the mean CRP concentration (also referred to as mean [CRP]) in the starting material, the percent CRP depletion was then determined for each fraction according to the following equation.

[00001]$\mathrm{CRP}.Math.\text{-}.Math.\mathrm{Depletion}.Math..Math.\left(\mathrm{in}.Math..Math.\%\right)=100-\left(\frac{\mathrm{mean}.Math.\left[\mathrm{CRP}\right].Math..Math.\mathrm{in}.Math..Math.\mathrm{fraction}.Math..Math.X}{\mathrm{mean}.Math.\left[\mathrm{CRP}\right].Math..Math.\mathrm{in}.Math..Math.\mathrm{the}.Math..Math.\mathrm{starting}.Math..Math.\mathrm{material}}\times 100\right)$

[0211] Thus, conclusions were drawn about the retention of CRP through the column by means of the CRP concentration in a fraction, i.e. the flow from the column, taking into account the likewise determined CRP concentration in the starting material.

[0212] The total CRP quantity remaining on the column can be calculated by subtracting the total CRP amount of the flow-pool and the individual fractions from the total CRP amount in the starting material.

Influence of Citrate

[0213] In order to investigate the effect of citrate on the affinity-chromatographic purification of CRP from biological fluids e.g. blood plasma, by means of a column material functionalized with ω-phosphonooxyalkyl ammonium groups and/or with ω-ammoniumalkoxy-hydroxy-phosphoryloxy groups, the human blood plasma was mixed with citrate solution in different mixing ratios. The used citrate solution was as follows: 7.3 g of citric acid (anhydrous) per liter (i.e. 38 mM citric acid), 22.0 g of trisodium citrate dihydrate per liter (i.e., 74.8 mM trisodium citrate dihydrate), and 1 L with water. Experiments in which the citrate solution additionally contained 24.5 g of dextrose (monohydrate) per liter (i.e. 123.6 mM dextrose monohydrate) yielded identical results. The following mixing ratios were investigated:

[0214] The following mixing ratios were investigated:

[0215] 1:50 i.e. 1 Part citrate solution+49 parts blood plasma

[0216] 1:20 i.e. 1 Part of citrate solution+19 parts of blood plasma

[0217] 1:10 i.e. 1 Part citrate solution+9 parts blood plasma

[0218] 1:5 i.e. 1 Part citrate solution+4 parts blood plasma

[0219] Human blood plasma without citrate addition served as reference sample. The citrate solution was added to the human blood plasma before the column using a gradient mixer.

[0220] For the reference sample (no citrate addition) and for each citrate concentration, two independent affinity chromatography runs were performed. Results

[0221] It can be seen in FIG. 1 that an efficient CRP depletion from the blood has taken place during the reference measurement (i.e. without citrate). The percentage depletion at the beginning of the sample application to the column was over 90% (see FIG. 5 GV) and then declined to marginally below 50% in the further course of the sample application. Such a drop can be explained by the fact that the used column has bound more and more CRP with persistent sample application and is thus becoming more and more saturated.

[0222] Surprisingly, however, at a mixing ratio of 1:50 to 1:5 a CRP depletion corresponding to the depletion performance under reference conditions (see also FIG. 1) is found. Contrary to all expectations, an efficient CRP removal from blood plasma under the use according to the invention of a citrate solution for the affinity-chromatographic removal of CRP from biological fluids could be effected by means of a column material functionalized with ω-phosphonooxyalkyl ammonium groups and/or with ω-ammoniumalkoxy-hydroxy-phosphoryloxy groups.

Example 2

hu-CRP ELISA for the Determination of the CRP Concentration in Samples

[0223] In order to determine the effectiveness of an affinity-chromatographic purification of CRP from biological fluids e.g. blood plasma, the concentration of human CRP (hu-CRP) in the appropriate samples were determined by means of an immunoassay (ELISA, Enzyme Linked Immunosorbent Assay).

[0224] To this end, the wells of a microtiter plate were first coated with an antibody directed against human CRP by adding 100 μI of a solution of rabbit polyclonal anti-human CRP antibody (Dako, Hamburg, Germany, product number Q0329), diluted 1:500 in coating buffer (0.1 M NaHCO.sub.3), incubated for 1 h at room temperature. It was then washed four times with at least 250 μI of PBST (0.1% Tween 20 in PBS).

[0225] For the determination of the concentration, sample buffer (5 mM EDTA in PBST) as a zero calibration and a standard series with concentrations of 300, 200, 100, 50, 30, 15 and 8.3 ng CRP/ml (in sample buffer) were used. For the standard series, a commercially available CRP standard (Human Serum C Reactive Protein Calibrator, 162 mg CRP/L, Dako, product number X0923) was diluted accordingly with sample buffer.

[0226] In addition, a reference sample with a high CRP concentration (Human Serum C Reactive Protein High Control, Dako, product number X0926) was diluted 1:600 and 1:1500 with sample buffer and a reference sample with a low CRP concentration (Human Serum C-Reactive Protein Low Control, Dako, product number X0925) diluted 1:250 and 1:1000 with sample buffer and used as additional reference samples for the ELISA. The actual samples to be measured (i.e. starting material, flow pool, flow fractions) were applied to the microtiter plate in three dilutions (in sample buffer).

[0227] For each well of the microtiter plate, 100 μl each were applied, incubated for 1.5 hours at room temperature and agitation on a shaker (600 revolutions per minute) and then washed four times with at least 250 μl of PBST.

[0228] For the detection, a peroxidase (POD)-labelled antibody (rabbit anti-huCRP-POD conjugate, 20 μg/ml stock solution, diluted 1:1000) and a competitive polyclonal anti-human CRP antibody from rabbits (Dako, Product number Q0329, diluted 1:2000) in blocking solution (1% casein, 0.9% NaCl, 0.001% thiomersal) were added subsequently. 100 μI per well were again applied, incubated for 1.5 h at room temperature and agitation on a shaker (600 revolutions per minute) and then washed again four times with at least 250 μI of PBST.

[0229] Subsequently, the substrate was added for the detection reaction. A solution of 3,3′,5,5′-tetramethylbenzidine (TMB, 25 mg/ml in DMSO and EtOH) was diluted in substrate buffer (0.01% H.sub.2O.sub.2 in 0.2 M citric acid, pH 3.95) and 100 μl thereof was pipette into each well. After an incubation for 10 to 15 minutes in the dark, the detection reaction was stopped by the addition of 50 μl of stop solution per well. The photometric detection was then carried out by means of a microtiter plate reader (BioRad 680 XR, BioRad, Munich, Germany) by determining the extinction at 450 nm (reference 655 nm). A double determination of each microtiter plate was carried out in each case in order to eliminate measuring inaccuracies caused by the device. In addition, each experiment was repeated three times to increase the reproducibility of the results.

[0230] In preliminary experiments, an influence of the citrate concentrations used on the reliability of the hu-CRP-ELISA was excluded.

[0231] It should be pointed out here that, of course, other methods for the determination of CRP can also be used to determine the CRP concentration in the samples to be examined.

Example 3

CRP Depletion Under Citrate

[0232] A blood plasma with a defined CRP concentration (38.8 mg/l) was divided into 12.5 ml each. One half was diluted with the addition of citrate (acid-citrate-dextrose-solution A (ACD-A)-solution 1:15) over the affinity chromatography column (0.5 ml matrix volume of agarose coupled with 4-aminophenylphosphocholine), the other half was passed without ACD-A over an identical column. The CRP content was determined before and after apheresis. From this, the distant amount of CRP was calculated. It was found that after addition of citrate, more CRP binds to the column and is removed. The results are shown in FIG. 2

[0233] The eluates were also applied to an SDS gel, the photo of which is shown in FIG. 3. It is shown that in the apheresis with citrate (ACD-A) less complement (C1q) is removed than in apheresis without citrate solution.

[0234] The capacity increase of the column due to the citrate is explained by the present inventors, without claim to correctness, as follows. Complement binds to CRP bound to ω-phosphonooxyalkyl ammonium groups or ω-ammoniumalkoxy-hydroxy-phosphoryloxy groups. It seems that in the presence of citrate, less complement is activated and less of the C1q/C1r/C1s complexes bind. If the C1-4/C1r/C1s complexes bind directly or indirectly to the column material, less steric hindrance of the CRP bond also occurs due to these rather large complexes.

[0235] Overall, the addition of a citrate solution to the biological fluid to be purified leads to an increase in the capacity of the column material functionalized by means of ω-phosphonooxyalkyl ammonium groups and/or ω-ammoniumalkoxy-hydroxy-phosphoryloxy groups. Furthermore, less complement is activated. In a comparison with peptidic ligands, this increase in capacity was not observed by citrate.