Method for Manufacturing a Fibrinogen Preparation

Abstract

A method for manufacturing a fibrinogen preparation from a fibrinogen containing source derived from blood plasma includes providing a liquid phase containing plasmatic fibrinogen; contacting the liquid phase with a cation exchange chromatography material under conditions resulting in binding of fibrinogen, wherein the liquid phase has a pH in the range of pH 5.6 to pH 7.0 which is near or above the pl of fibrinogen; optionally washing unbound compounds from the cation exchange chromatography material; and eluting the fibrinogen from the cation exchange material. The method is also suitable for reduction of von-Willebrand-factor.

Claims

1. Method for manufacturing a fibrinogen preparation from a fibrinogen containing source derived from blood plasma comprising: Providing a liquid phase containing plasmatic fibrinogen; Contacting the liquid phase with a cation exchange chromatography material under conditions resulting in binding of fibrinogen, wherein the liquid phase has a pH in the range of 5.6 to 7.0; Optionally washing unbound compounds from the cation exchange chromatography material; and Eluting the fibrinogen from the cation exchange material using an elution buffer.

2. Method according to claim 1, comprising reducing an amount of von-Willebrand-factor, when the source contains von-Willebrand-factor.

3. Method according to claim 1, comprising reducing an amount of prions.

4. Method according to claim 1, wherein the liquid phase of said contacting has a pH in the range of 6.3 to 6.9.

5. Method according to claim 1, wherein the liquid phase of said contacting has an ionic strength of 5 to 15 mS/cm.

6. Method according to claim 1, wherein the cation exchange chromatography material is a strong cation exchange chromatography material.

7. Method according to claim 1, wherein the cation exchange chromatography material is a macroporous material.

8. Method according to claim 1, wherein the cation exchange chromatography material is a material comprising sulfonate functional groups.

9. Method according to claim 8, wherein the cation exchange chromatography material comprises a resin backbone consisting of crosslinked polystyrenedivinylbenzene, wherein the sulfonate functional groups are linked as sulfopropyl via a polyhydroxyl surface.

10. Method according to claim 1, wherein the washing is performed using a wash buffer with a pH in the range of 5.6 to 7.0 and an ionic strength of 5 to 15 mS/cm.

11. Method according to claim 1, wherein the elution is performed using an elution buffer with a pH of at least 0.2 units above the conditions resulting in binding of fibrinogen.

12. Method according to claim 11, wherein the elution is performed using an elution buffer with an ionic strength at least 2 mS/cm higher than the conditions resulting in binding of fibrinogen.

13. Method according to claim 1, wherein the elution buffer comprises one or more drug formulation compounds.

14. Method according to claim 13, wherein the drug formulation compound is at least one amino acid.

15. Method according to claim 14, wherein the method further comprises formulating the fibrinogen into a pharmaceutical composition.

16. Method according to claim 1, wherein the manufacturing method further comprises at least one of: Using cryoprecipitate of human plasma as starting material; Al(OH)3 adsorption; S/D treatment; anion exchange chromatography and using the flow-through; glycine precipitation; UV-C treatment; ultrafiltration; lyophilisation; or heat treatment.

17. Method according to claim 16, wherein the cation exchange chromatography is performed between UV-C treatment and ultrafiltration.

18. Fibrinogen preparation obtained by the method of claim 1.

19. Fibrinogen preparation according to claim 18, wherein the fibrinogen preparation has a FXIII concentration of 0.5-2.0 FXIII:Ag (% of norm) and/or a FXIII activity of less than 16 FXIII:Ac (% of norm).

20. Fibrinogen preparation according to claim 18, wherein the fibrinogen preparation shows no detectable content of D-dimer.

21. Pharmaceutical composition obtainable from the fibrinogen preparation according to claim 18.

22. Pharmaceutical composition according to claim 21, which is a lyophilisate.

23. (canceled)

24. A method for the treatment of a haemostatic disorder or bleeding comprising administering the pharmaceutical composition according to claim 21.

Description

EXAMPLES

Example 1

[0121] A cryoprecipitate of human plasma is used as source for the fibrinogen. The cryoprecipitate is obtained by thawing frozen plasma at 0-4° C. and separation of the precipitate.

[0122] Per kg of cryoprecipitate a mixture of 2.91 kg of water (WFI), 114 g ethanol 25% (v/v) and 9,000 IU heparin is prepared. The cryoprecipitate is added to the WFI/ethanol/heparin solution under stirring. The pH value is adjusted to 7.0.

[0123] 108 g of a 2% aluminium hydroxide suspension are added per kg of cryoprecipitate used, and the mixture is stirred at 22.5° C. The pH value is adjusted to 6.55 and subsequently centrifuged by continuously operating centrifuges.

[0124] 1% Polysorbate 80 and 0.3% Tri-n-butyl phosphate are added while stirring. The protein solution is stirred at 25.0° C. over a period of at least 8 hours.

[0125] The anion-exchange gel Toyopearl TSK DEAE-650 (hydroxylated methacrylic polymer beads as matrix material with diethylaminoethyl groups) is used for further purification by column chromatography. The protein loading is about 50±10 mg of protein/ml anion exchange gel.

[0126] The chloride content of the protein solution is adjusted to 120 mmol/l by addition of NaCl solution. The protein solution is applied to the column and the flow through fraction is collected.

[0127] The resulting fibrinogen solution containing 10 mM tri-sodium-citrate, 120 mM NaCl, 120 mM Glycine, 1 mM CaCl.sub.2, 0.1% Polysorbat 80 and 0.3% TnBP, pH 7.0-7.1 is subjected to glycine precipitation. To precipitate fibrinogen, glycine is added to a final concentration of 1.2 M. NaCl is added to a final concentration of 2 M. The fibrinogen containing precipitate is then separated by centrifugation. The fibrinogen paste might be stored at a temperature ≤−70° C.

[0128] The precipitate is resuspended in a buffer (15 mM tri-sodium citrate dihydrate, pH value: 6.9+/−0.1, conductivity: 3.3+/−0.5 mS/cm). The composition beside the other proteins (e.g. 0.7-0.9 U/mg vWF) comprises TnBP, Polysorbate 80, glycine and NaCl. The composition is filtered and subjected to an UV-C treatment for virus inactivation using devices such as the UVivatec device (Sartorius Stedim Biotech). The irradiation is preferably performed at 254 nm±1 nm using 125-200 J/m.sup.2.

[0129] For the following cation exchange chromatography step the column (POROS™ 50 HS) is equilibrated with equilibration buffer (15 mM tri-sodium citrate dehydrate, 65 mM sodium chloride, pH value: 6.5+/−0.1, conductivity: 9.0+/−1.5 mS/cm, 2-5 column volumes).

[0130] The liquid phase containing fibrinogen resulting from the UV irradiation step is prepared by adjusting the composition to 15 mM tri-sodium citrate, pH value: 6.5+/−0.1 and conductivity: 9.0+/−1.5 mS/cm. The column is loaded with 10-20 g/l protein per liter gel volume.

[0131] The column is rinsed with wash buffer (15 mM tri-sodium citrate dehydrate, 65 mM sodium chloride, pH value: 6.5+/−0.1, conductivity: 9.0+/−1.0 mS/cm, 2-5 column volumes).

[0132] Then the fibrinogen is eluted using elution buffer (7.5 mM tri-sodium citrate dehydrate, 150 mM sodium chloride, 75 mM L-arginine monohydrochloride, pH value: 7.0+/−0.1, conductivity: 19.5+/−1.5 mS/cm). In this step the fibrinogen is eluted from the column. Most of the vWF still binds on the column.

[0133] The column is then rinsed with a buffer with higher salt concentration (15 mM tri-sodium citrate dehydrate; 1.5 M sodium chloride, pH value: 6.5+/−0.1, conductivity: 113.5+/−5.0 mS/cm). vWF is eluted from the column. The column is then cleaned using 1 M sodium hydroxide.

[0134] In this method Albumin and IgG do not bind to the CEX material. More than 50% of the vWF present in the liquid phase containing fibrinogen can be removed by using this method.

[0135] For the production of a drug substance the eluted fractions are concentrated using ultrafiltration and the protein concentration is adjusted to 33 g fibrinogen per liter by using citrate buffer. Further ingredients may be added to form the final drug substance.

[0136] The drug substance is filtrated (0.2 μm) into different vials and lyophilized. Then a final heat treatment in a steam autoclave (100° C., 30 min) was performed as a further virus inactivating step. The product is surprisingly stable.

[0137] The final product shows a good clot stability measured as maximum clot firmness (MCF) (Fib-tem assay at 2.5 g/L fibrinogen, 25 mm) compared to a standard human plasma control (27 mm) and a plasma pool (22 mm). This is strong evidence towards a good physiological activity.

[0138] Furthermore, the fibrinogen preparation has a FXIII concentration of 1.5 FXIII:Ag (% of norm) and a FXIII activity of less than 16 FXIII:Ac (% of norm). Moreover, the fibrinogen preparation shows no detectable content of D-Dimer. Both parameters are an indication of very good purity features of a fibrinogen product according to the invention. The data is shown in table 1 (Sample 1, 2, 3: preparations according to the method of the invention according to example 1—solubilised final product, 20 mg protein/ml).

TABLE-US-00001 TABLE 1 vWF: Ag vWF: Ag vWF: RiCo D-Dimer FXIII: Ag FXIII: Ac Sample [U/ml] [U/mg]* [U/ml] [mg/l] [% of norm] [% of norm] Sample 1 5.0 0.25 <0.1 <0.17 0.9 <15.1 Sample 2 6.5 0.33 0.38 <0.17 1.5 <15.1 Sample 3 5.1 0.26 0.15 <0.17 1.1 <15.1 *for 20 mg/ml fibrinogen

[0139] Commercially available fibrinogen product samples contain a much higher FXIII activity and higher D-Dimer content compared to the inventive product (data not shown).

Example 2

[0140] 25 g of a glycine precipitate prepared as described in Example 1 was treated with UV-C and purified using the cation exchange chromatography (POROS™ 50 HS) as described in Example 1. Table 2 shows the composition of the loading solution, flow-through and eluate.

TABLE-US-00002 TABLE 2 Protein Volume concentration vWF:Ag vWF:Ag Fib:Ag Sample [ml] [g/l] [U/ml] [U/mg] [g/l] Loading 38.4 7.98 3.8 0.45 9.26 solution Flow- 45.8 0.0484 0.4 8.26 0.0104 through Eluate 55.3 5.29 1.0 0.19 6.45

[0141] Given that total protein essentially corresponds to fibrinogen, based on the protein concentration measured with OD 280 the yield of protein in the eluate is 95.5%. 37.9% of the total vWF is present in the fibrinogen eluate. Measured as Fib:Ag 100.3% of the fibrinogen is in the eluate.

[0142] The depletion factor of vWF in this example is 2.4 (0.45 to 0.19 U/mg), because the vWF content in the load of this concrete example is relatively low. It has been demonstrated by the inventors, that in other examples the depletion factor of vWF is in the range of 3 to 6 when the vWF content in the load material is in the range of 0.6 to 0.9 U/mg total protein.

Example 3

[0143] A similar process as described in Example 1 with respect to the cation exchange chromatography step was performed except that Macro-Prep® High S was used as a strong cation exchange column material instead of POROS™ 50 HS. For an improved binding of the fibrinogen to the column during loading and washing the conductivity was set to 4.5 mS/cm and the pH was set to 6.0, which is above the pl of fibrinogen. Under elution conditions of pH 7 and conductivity of 19.5 mS/cm 94.8% of the fibrinogen is eluted from the column. The content of vWF in the eluted fractions is below 0.4 U/mg protein. Only around 18.3% of the vWF in the protein load of the CEX column could be found in the eluate. No polysorbate 80 or TnBP could be detected in the eluted fractions.

Example 4

[0144] For testing the ability of the cation exchange chromatography step in reducing the content of vWF in the eluted fibrinogen fraction the cation exchange chromatography with loading material enriched in vWF (spiked) was performed using POROS™ 50 HS as described in Example 1. The vWF content of the load was 1.09 U/mg total protein (non-spiked material) up to 4.49 U/mg total protein in several spiking samples (see table 3). After selective elution of fibrinogen (150 mM NaCl, pH 7.0) vWF was eluted with the high-salt fraction (1.5 M NaCl, pH 6.5). As can be taken from table 3 the content of vWF in the eluted fibrinogen fraction (Fibrinogen Eluate) remains essentially constant with increasing vWF content of the load varying between 0.21 U/mg total protein (non-spiked material) and 0.35 U/mg total protein (spiked with 4.49 U/mg total protein in the load). The depletion factor for vWF increased with spiking of the load material starting from 5 for the non-spiked load material up to 12 obtained for the spiked load material with 4.49 U/mg total protein.

[0145] Table 4 shows depletion factors compared to the dissolved cryoprecipitate for a typical sample on larger scale. Over the total process a depletion factor of 24.4 is achieved compared to the dissolved cryoprecipitate.

TABLE-US-00003 TABLE 3 Load Fibrinogen Eluate vWF vWF per vWF vWF per measured protein Protein measured protein Protein Depletion Exp. (U/ml) (U/mg) (mg/ml) (U/ml) (U/mg) (mg/ml) Factor vWF 1 6.5 1.09 5.94 1.2 0.21 5.71 5 2 9.9 1.70 5.84 1.7 0.30 5.68 5 3 12.6 2.14 5.89 1.8 0.32 5.62 6 4 14.6 2.49 5.87 1.8 0.32 5.58 7 5 17.5 2.99 5.85 1.4 0.25 5.56 11 6 25.8 4.49 5.75 1.9 0.35 5.41 12

TABLE-US-00004 TABLE 4 vWF depletion factor compared dissolved Step cryoprecipitate Cryoprecipitate — Anion exchange 1.3 chromatography Dissolved glycine 6.4 precipitate Drug product 24.4

Example 5

[0146] In order to demonstrate the robustness of the cation exchange chromatography step in view of the pH value and the conductivity when loading the column (POROS™ 50 HS) a range of pH 6.3 up to 6.7 was tested when loading the column according to Example 1. The conductivity when loading the column was between 7 and 11 mS/cm. The protein load was between 20 g and 22 g protein per liter column material. In all tested conditions vWF was depleted below 0.5 U/mg total protein as can be taken from FIG. 1 (FIG. 1 shows the relation of amount of vWF for different values of conductivity and pH of the loading solution for different protein loads (upper row for pH 6.4-6.6 and 8-10 mS/cm; lower row pH 6.3-6.7 and 7-11 mS/cm). The results show, that a range of 6.4 to 6.6 is especially advantageous for reduction of vWF in the fibrinogen eluate, wherein vWF is depleted below 0.4 U/mg. Moreover, the results show, that a range of conductivity of 8 to 10 mS/cm is especially advantageous for reduction of vWF in the fibrinogen eluate, resulting in a depletion of vWF below 0.4 U/mg. Moreover, it is advantageous in view of an effective depletion of vWF, that the total protein load is not more than 21 g/l, especially not more than 20 g/l column material.

Example 6

[0147] For testing the ability of the cation exchange chromatography step to reduce the content of Polysorbat 80 and TnBP in the eluted fibrinogen fraction the cation exchange chromatography with loading material enriched in both Polysorbat 80 and TnBP (spiked) was performed using POROS™ 50 HS as described in Example 1, with the exception that the UV-C irradiation step was omitted. The Polysorbat 80 and TnBP content of the load was 1.11 mg/mL and approx. 20 μg/mL, respectively (non-spiked material) and was raised up to 10.74 mg/mL and 2590 μg/mL, respectively, in several spiking samples (see table 5). After selective elution of fibrinogen (150 mM NaCl, pH 7.0) vWF was eluted with the high-salt fraction (1.5 M NaCl, pH 6.5). The yield of fibrinogen in the column eluate remained constant over all experiments, as was deduced from chromatograms (data not shown). As can be taken from table 5 the content of Polysorbat 80 and TnBP in the eluted fibrinogen fraction (Fibrinogen Eluate) is below the limit of detection of the analytical methods for all applied spikes of Polysorbat 80/TnBP, with the exception of the highest spike of TnBP where approx. 30 μg/mL TnBP were found in the eluate. Hence Polysorbat 80 and TnBP could be depleted by up to factor 1000 using POROS™ 50 HS-chromatography on fibrinogen-containing samples. At the same time, the level of vWF found in the fibrinogen eluate remained mostly unaffected, with only a slight increase observed for the highest spikes of Polysorbat 80/TnBP.

TABLE-US-00005 TABLE 5 Load Fibrinogen Eluate Polysorbat Polysorbat 80 TnBP 80 TnBP Experi- measured measured measured measured vWF ment (mg/mL) (μg/mL) (mg/mL) (μg/mL) (U/mL) 1 1.11 approx. 20  <0.01 <0.8 0.9 2 1.78 approx. 620 <0.01 <0.8 0.8 3 6.26 1230 <0.01 <0.8 0.9 4 10.74 2590 <0.01 approx. 30 1.1

Example 7

[0148] The inventors were able to demonstrate that prions are effectively removed to below the limit of detection by the cation exchange chromatography step. To show this effect hamster prions (strain 263K) as a well-established test model for variant Creutzfeldt-Jacob disease were tested. The removal capacity was analyzed by prion-spiked test material. The prion titers were determined by Western blot. In the eluate of the cation exchange chromatography step prions are removed from the fibrinogen by ≥3.27 log.sub.10 to below the limit of detection demonstrating the reliable removal of prions by the cation exchange chromatography step. The prions were stripped from the column by 1.5 M NaCl (high salt).

[0149] Table 6 summarizes the results of the experiment. The test material was spiked with prions (Hamster adapted scrapie isolate, strain 263K, supplier ViruSure GmbH, Austria) and samples were taken from the prion stock and spiked test material to determine the prion titers by Western blot. A volume of approximately 94 ml of spiked test material was loaded on the column and the column was washed. The flow through and wash was collected and the volume determined. The fibrinogen was eluted from the column in a volume of 50 ml and the column was washed with 1.5 M NaCl. Of each fraction, a sample was taken for prion titer analysis. The results of this study are listed in the following table 6, wherein prions are removed from the fibrinogen by 3.27 log.sub.10 to below the limit of detection.

TABLE-US-00006 TABLE 6 Endpoint Titer (log.sub.10) Actual Determined Titer for Total by Western Undiluted Volume Titer Reduction Sample Treatment Blot Sample (mL) (log.sub.10).sup.a (log.sub.10).sup.a  11.00a Prion 4.0 4.0 1.8 4.3 spike 11.0a spiked, 2.0 2.0 93.9 4.0 untreated 11.11 Flow 0.0 0.0 163.9 ≤2.2 ≥1.8 through and wash 11.21 Eluate 0.0.sup.b <−1.0 50.0 ≤0.7 ≥3.3 11.31 Strip 0.5.sup.c 1.0 70.0 2.8 1.1 .sup.arounded after calculation to one decimal place .sup.b10-fold concentrated sample (−1.0log) was non-reactive .sup.cSample was diluted

[0150] Analytical Methods

[0151] Protein Determination

[0152] Protein determination is performed by the UV absorption method (Spektralphotometer Genesys™ 6, Spektralphotometer Genesys™ 10). Proteins in solution adsorb UV light at a wavelength of 280 nm due to the presence of aromatic amino acids, mainly tyrosine and tryptophan. This property is the basis of the protein determination at 280 nm. The accuracy of the UV spectroscopic determination of protein can be decreased by the scattering of light by the test specimen. For the compensation of this effect the absorption at 360 nm is subtracted from the absorption at 280 nm.

[0153] Measurement of Fibrinogen (Fib:Ag)

[0154] The Fib:Ag concentration is determined by nephelometry at the BN Prospec (Siemens) Nephelometer. Fibrinogen forms a complex with a specific antibody. This complex causes a dispersion of irradiated light. The increased dispersion is correlated to a fibrinogen concentration.

[0155] Measurement of Activity of Fibrinogen by Clottable Protein

[0156] For the assay of fibrinogen activity (=clottable protein), the sample preparation is mixed with a suitable buffer solution containing sufficient thrombin and incubated at 37° C. The residual protein is determined in the supernatant by UV spectrometry at 280/360 nm and the result is subsequently subtracted from the total protein content (see above) to calculate the clottable protein.

[0157] Determination of Specific Activity of Fibrinogen

[0158] The specific activity of fibrinogen is determined by clottable protein activity related to total protein as measured by UV absorption (280 nm).

[0159] Measurement of Clot Stability

[0160] The clot stability is measured using the Fib-tem assay with the ROTEM whole blood analyzer (Tem Innovations GmbH, Munich). This is an established viscoelastic method for hemostasis testing in whole blood.

[0161] The test is performed according to the manufacturer's instruction. The validity of the method is tested with control preparations (Rotrol N and P).

[0162] For the measurement of Fib-tem Fibrinogen samples are dissolved according to the manufacturer's instructions and further dilutions are performed in Fibrinogen deficient plasma to obtain Fibrinogen concentrations of 1.5 g/l, 2.0 g/l and 2.5 g/l.

[0163] Measurement of the vWF Activity (vWF:Aq)

[0164] The testkit “vWF Ag” contains reagents for the immunoturbidimetric determination of von Willebrand factor antigen (vWF:Ag) in human plasma or plasma products measured with the Behring coagulation system (BCS XP).

[0165] The test is performed according to the manufacturer's instruction including the reagents provided as well as the predefined test definition and measurement instruction of the coagulation system BCS XP. The results of the samples are evaluated with a standard/reference curve.

[0166] For the preparation of the standard/reference curve Standard Human Plasma (Siemens) is used in different dilution steps in duplicate determination. The coagulation system (BCS XP) automatically dilutes the calibrator in the range of 10-200% of the norm. The validity of the reference curve is tested with the control preparation (Control Plasma N).

[0167] For the measurement a dilution series of fibrinogen concentrate samples at dilutions of 11:1, 1:10 in Owren's Veronal Buffer is prepared. All other dilutions, incubations, usage of different reagents provided in the reagent kit are automatically prepared by the test system (BCS XP).

[0168] Measurement of the Activity of FXIII (FXIII:Ac)

[0169] The activity of FXIII is determined with the photometric test (Berichrom FXIII, Siemens Healthcare Diagnostics GmbH) measured with the Behring coagulation system (BCS XP, Siemens Healthcare).

[0170] The test is performed according to the manufacturer's instruction including the reagents provided as well as the predefined test definition and measurement instruction of the coagulation system BCS XP. The samples are evaluated with a standard/reference curve.

[0171] For preparation of the calibration/reference curve, Standard Human Plasma (Siemens) is used in different dilution steps in duplicate determination. The coagulation system (BCS XP) automatically dilutes the calibrator in the range of 15-130% of the norm. The validity of the reference curve is tested with the control preparation (Control Plasma N). As used herein 100% of the norm (Siemens Standard Human Plasma (CoA)) correspond to 1 IU/ml according to WHO Standard.

[0172] For the measurement a dilution series of fibrinogen concentrate samples at dilutions of 1:1, 1:3 and 1:5 in NaCl solution (0.9%, w/v) is prepared. All other dilutions, incubations, usage of different reagents provided in the test kit are automatically prepared by the coagulation system (BCS XP).

[0173] Measurement of the Concentration of FXIII:Aq

[0174] The measurement is based on a Sandwich Elisa assay using a Matched-Pair Antibody set and VisuLize Buffer Pak (both Affinity Biologicals). The test is performed according to the manufacture's instruction including the reagents provided as well as the predefined test definition and measurement instruction. For calibration purposes a Standard Human Plasma (Siemens), 1:100 over seven steps in a 1:2 dilution, is used.

[0175] An affinity-purified polyclonal antibody to FXIII A subunit is coated in a microtitre plate. The remaining binding sites are blocked with bovine serum albumin. After washing standard and samples are applied. The bound FXIII is detected with a peroxidase conjugated antibody to FXIII. The peroxidase activity is processed with OPD (o-Phenylenediamine) and stopped with H2504. The OD is measured at 490 nm. As used herein 100% of the norm correspond approximately to 1 IU/ml according to WHO Standard.

[0176] Measurement of D-Dimer

[0177] INNOVANCE D-Dimer is a particle-enhanced, immunoturbidimetric assay for the quantitative determination of cross-linked fibrin degradation products (D-dimers) in human plasma or plasma products measured with the Behring coagulation system (BCS XP).

[0178] The test is performed according to the manufacture's instruction including the reagents provided as well as the predefined test definition and measurement instruction of the coagulation system BCS XP. The samples are evaluated with a standard/reference curve.

[0179] For the preparation of the standard/reference curve, INNOVANCE D-dimer Calibrator is used in different dilution steps in duplicate determination. The coagulation system (BCS XP) automatically dilutes the calibrator in the range of 0.17-4.4 mg/l. The validity of the reference curve is tested with the control preparation (Control Plasma N).

[0180] For the measurement a dilution series of fibrinogen concentrate samples at dilutions of 1:1, 1:5 in diluent (provided in the test kit) is prepared. All other dilutions, incubations, usage of different reagents provided in the test kit are automatically prepared by the test system (BCS XP).

[0181] Measurement of TnBP Concentration

[0182] N-Hexan is used for the extraction of TNBP from the sample solutions. Reference solutions with amounts identical to the specification limits are prepared and subjected to the same extraction procedure as other samples. The hexanoic phase from reference and sample solutions are analyzed by gas chromatography.

[0183] The evaluation of the conformity to the specification is carried out with a comparison of the heights of TNBP peaks in the chromatograms from the samples and the corresponding reference solution. The reference solutions are prepared from a valid standard solution from the TNBP-Assay-Test be dilution.

[0184] Measurement of Polysorbat 80 Concentration

[0185] The determination of Polysorbate 80 is performed by photometry according to the Ph. Eur., current edition, 2.2.25.

[0186] Polysorbate 80 in protein solutions is determined by a photometric assay. Polyoxylated compounds like Polysorbate 80 are forming a blue colored complex with ammonium cobalt thiocyanate.

[0187] Interferences due to high protein content are avoided by a deproteination with ethanol. After precipitation of protein with ethanol, the supernatant is evaporated to near dryness. The complex is extracted by dichloromethane followed by a photometric measurement at 620 nm. The calibration function is prepared without the deprotonation step.

[0188] Ristocetin Cofactor Activity (RCoF, vWF:RiCo)

[0189] The cofactor activity of von Willebrand factor is measured with BC von Willebrand Reagent, which is an in-vitro test for the determination of Ristocetin cofactor activity of von Willebrand factor in human plasma or plasma products through platelet agglutination measured with the Behring coagulation system (BCS XP).

[0190] The test is in principle performed according to the manufacture's instruction including the reagents provided. The reference curve is modified from 20-150% to 5-100% of the norm. The test definition and measurement instruction of the coagulation system BCS XP is adapted accordingly. The samples are evaluated with a standard/reference curve.

[0191] For the preparation of the calibration/reference curve Standard Human Plasma is used in different dilution steps in duplicate determination. The coagulation system (BCS XP) automatically dilutes the calibrator in the range of 5-100% of the norm. The validity of the reference curve is tested with the control preparation (Control Plasma N).

[0192] For the measurement a dilution series of fibrinogen concentrate samples at dilutions of 1:1 and 1:5 in NaCl solution (0.9%, w/v) is prepared. All other dilutions, incubations, usage of different reagents provided in the test kit are automatically prepared by the test system (BCS XP).