METHOD FOR PURIFICATION OF PLASMA PROTEINS

Abstract

The present invention relates to a method for purification of plasma proteins. More closely, the invention relates to a method using magnetic beads for separation of different plasma proteins from a plasma fraction, such as a cryoprecipitate or cryosupernatant of plasma, or alternatively directly from cell culture of recombinant plasma proteins.

Claims

1. A method for purification of plasma protein(s) from a crude sample, comprising binding of desired plasma protein(s) to ligands on magnetic beads and eluting said plasma protein(s), wherein the binding and eluting is performed in batch mode.

2. The method according to claim 1, wherein the ligands are anion exchange ligands, are preferably selected from diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) or quaternary ammonium (Q), most preferably the anion exchange ligands are Q-ligands.

3. The method according to claim 1, wherein the ligands bind to Factor VIII (FVIII).

4. The method according to claim 3, wherein the ligands have affinity for the light chain of FVIII.

5. The method according to claim 1, wherein the crude sample is a cryoprecipitate and the desired plasma protein(s) are Factor VIII (FVIII) and/or von Willebrand Factor (vWF).

6. The method according to claim 1, wherein the crude sample is a cryosupernatant and the desired plasma protein(s) are albumin, IgG or Factor IX (FIX), preferably FIX.

7. The method according to claim 1, wherein the crude sample is taken directly, without further purification besides settlement of the cells, from cell culture of recombinant plasma proteins.

8. The method according to claim 1, comprising a) adding a plasma protein fraction, such as a cryosupernatant or dissolved cryoprecipitate comprising at least one plasma protein, to a container or bag; b) adding magnetic beads provided with anion ligands by pouring or pumping said beads into said container or bag; c) incubating preferably at least 30 minutes with mixing; d) binding plasma protein(s) to ligands on the magnetic beads; e) retaining the magnetic beads with a magnetic field and washing away undesired material from the magnetic beads, optionally repeated; and f) elution of plasma protein(s) from the ligands on the magnetic beads in a yield of 92-100% active FVIII from cryoprecipitate or in yield of at least 85% F IX and a FIXa/FIX ratio of less than 1 ‰ from cryosupernatant.

9. The method according to claim 1, wherein the magnetic beads are porous agarose beads provided with embedded magnetic particles.

10. The method according to claim 8, wherein the anion ligands are Q ligands.

11. The method according to claim 1, wherein the magnetic beads are 8-300 um in diameter and the method is performed in large scale.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0018] The invention provides a magnetic bead capable of binding plasma proteins with a high binding strength and to a high binding capacity. This is achieved with a plasma protein-binding magnetic bead, comprising a porous matrix and one or more magnetic particles embedded in the matrix, where the matrix comprises a porous polymer, preferably agarose, and plasma protein-binding ligands coupled to the porous polymer.

[0019] One advantage is that the beads allow the selective binding of large amounts of plasma proteins directly from crude material. A further advantage is that the beads have a favourable adsorption isotherm for plasma proteins, giving a high yield of recovered plasma protein.

[0020] The size of the bead may suitably be such that a plurality of beads, to be used in the methods disclosed below, have a volume-weighted median diameter (d50,v) of 8-300 micrometers, such as 20-200, 20-100 micrometers or 20-80 micrometers. Beads of these sizes are easy to retain with a magnetic field, in particular compared to magnetic nanoparticles or micron-sized particles. The mass transport rates are however fast enough to give a rapid uptake of plasma protein by the bead. This applies in particular to beads with median diameter in the 20-100 micrometer and 20-80 micrometer intervals. The bead(s) may be spherical or essentially spherical, e.g. with a sphericity (the surface area of a sphere with the same volume as the bead divided by the surface area of the bead) of at least 0.9.

[0021] The invention will now be described in more detail in association with the Experimental part below.

[0022] Assays were performed for activity of Factor VIII, Factor IX, Factor IXa (FVIII, FIX, FIXa) or concentration of von Willebrand Factor (vWF).

[0023] Comparisons were made between MagSepharose prototypes and non-magnetic resins packed in columns in Experiments 1 and 3 below.

EXPERIMENTAL PART

[0024] Synthesis of Magnetic Bead Prototypes:

[0025] MagSepharose Q Prototype, LS-000672

[0026] MagSepharose base matrix was washed with 2 M NaOH. Glycidyl trimethylammonium chloride (GMAC) was added and the coupling reaction proceeded at room temperature over night. The GMAC reaction was repeated two more times. The resin was washed with distilled water. The reaction scheme is shown below.

##STR00001##

[0027] VIII MagSepharose Prototype, LS-034256

[0028] NHS-activated MagSepharose was washed with cold 1 mM HCI. VIII ligand was added and the coupling reaction proceeded at pH 8.2 at 24° C. for 2 h 15 min. The resin was washed with 0.1 M acetic acid/0.15 M NaCI pH 4.5, 50 mM Tris/0.15 M NaCI pH 8.5 and distilled water. The reaction scheme is shown below.

##STR00002##

[0029] Sample Preparation: Plastic bags with recovered plasma were thawed slowly in ice water (temp approx 0-4° C.), to obtain liquid plasma with cryoprecipitate. The thawed plasma was centrifuged, to collect the cryoprecipitate in the pellet. The supernatant was poured off (cryosupernatant) and the pellets were dissolved in Equilibration buffer (dissolved cryoprecipitate), 1/5 of the original plasma volume.

[0030] Analyses: FVIII activity, vWF ELISA (concentration), FIX and FIXa activity: The presence of FVIII, vWF, FIX and FIXa (activated FIX) were analysed using commercial kits according to the manufacturer's instructions. The activities/concentrations are listed as mU/mL (mUnits/mL) in tables below. FVIII activity was determined using the Coamatic FVIII kit from Chromogenix. vWF concentration was determined using the Technozym vWF:Ag ELISA kit from Technoclone. FIX and FIXa activities were determined using commercial kits from Rossix: Rox Factor IX, Rox FIX-A, Factor IXa Calibrator, Factor IXa Control.

[0031] Experiment 1: Purification of Factor VIII and von Willebrand Factor From Dissolved Cryoprecipitate Using Q-ligand

[0032] A. Packed Chromatography Columns

[0033] Chromatography conditions for tests with packed columns:

[0034] Columns: HiTrap Q HP 5 mL, HiTrap Capto Q ImpRes 5 mL.

[0035] Column volume (CV) 5 mL.

TABLE-US-00001 Method Volume step Sample/Buffer (CV or mL) Equilibration 20 mM Na-citrate, 0.15M NaCl, 2.6 Column mM CaCl2, 0.1% Tween 80, pH 7.0 pre-equilibrated Sample Cryoprecipitate dissolved in 10 mL Equilibration buffer Wash1 See Equilibration above 2 CV Wash2 20 mM Na-citrate, 0.20M NaCl, 2.6 7 CV mM CaCl2, 0.1% Tween 80, pH 7.0 Elution 0.1M Lysine, 1M NaCl, 10 mM CaCl2, 5 CV 0.1% Tween 80, 12% glycerol, pH 6.0 CIP 0.5M NaOH 3 CV Equilibration See Equilibration above 10 CV

[0036] B. Batch Adsorption With Magnetic Beads

[0037] Magnetic Beads: MagSepharose Q prototype resin LS-000672, 5 mL resin/tube in tests MagSepharose Q tests were made with 5 mL resin in a 50 mL plastic tube with screw cap. The incubation and mixing of resin and buffer/sample was performed manually by shaking the tube, or in an end-over-end rotating mixer. The tube was then placed in the Sepmag A 200 mL (Sepmag), where the magnetic beads were magnetically attracted to the side of the tube. The clear liquid was removed by plastic Pasteur pipette.

TABLE-US-00002 Method Volume step Sample/Buffer (mL) Equilibration 20 mM Na-citrate, 0.15M NaCl, 2.6 Resin pre- mM CaCl2, 0.1% Tween 80, pH 7.0 equilibrated Sample Cryoprecipitate dissolved in 10 mL Equilibration buffer Wash1 See Equilibration above 3 × 10 mL pooled Wash2 20 mM Na-citrate, 0.20M NaCl, 2.6 3 × 15 mL mM CaCl2, 0.1% Tween 80, pH 6.99 pooled (spec pH 7.0), cond 23.37 mS/cm (spec 21.0 mS/cm?) Elution 0.1M Lysine, 1M NaCl, 10 mM CaCl2, 3 × 10 mL 0.1% Tween 80, 12% glycerol, pH 6.0 pooled + 10 mL separately CIP 0.5M NaOH 5 × 20 mL Equilibration See Equilibration above 25 mL multiple times, until pH approx 7

TABLE-US-00003 TABLE 1 Results from Experiment 1 The low limit for quantification (LOQ) was 9 mU/mL for FVIII and 170 mU/mL for vWF. Values below limit of quantification (LOQ) are indicated by <LOQ. FVIII FVIII FVIII vWF vWF Volume act total Yield vWF total Yield Fraction (g = mL) (mU/mL) (mU) (%) (mU/mL) (mU) (%) Q Sepharose HP Cryoprecipitate 10.0 4276 42760 100 4622  46220 100 Flow through 12.2 <LOQ <LOQ Wash1 + 2 43.6 <LOQ 489 21320 46 Eluate 4.3 8497 36537 85 2717  11683 25 Capto Q ImpRes Cryoprecipitate 10.0 4276 42760 100 4622  46220 100 Flow through 12.6  10 126 0.3 <LOQ Wash1 + 2 43.1 <LOQ 520 22412 48 Eluate 6.1 6407 39083 91 515 3142 6.8 MagSepharose Q Cryoprecipitate 10.0 4276 42760 100 4622  46220 100 Flow through 10.0 <LOQ <LOQ Wash1 30.0 <LOQ <LOQ Wash2 45.0 <LOQ 320 14400 31 Eluate 1-3 30.0 1454 43620 102 833 24990 54 Eluate 4 10.0  114 1140 2.7 <LOQ

[0038] As shown in Table 1, there were high yields of FVIII in the eluate fractions, and highest yield with the MagSepharose Q prototype resin.

[0039] vWF is partially removed during the wash steps without any loss of FVIII.

[0040] The results from Experiment 1 surprisingly show that FVIII was obtained in 10-15% higher yields with magnetic beads with Q-ligands than conventional Q-resin.

[0041] Experiment 2: Purification of Factor VIII From Dissolved Cryoprecipitate Using VIII Select-ligand

[0042] A test was also made with an affinity ligand for FVIII coupled to MagSepharose beads. This magnetic prototype resin was called MagSepharose VIII Select prototype LS-034256. The test was made only with the MagSepharose VIII Select prototype, no comparison was made with a packed column with VIII Select resin. The conditions were comparable to the conditions in Experiment 1, but with different buffers.

TABLE-US-00004 TABLE 2 Results from Experiment 2 The low limit for quantification (LOQ) was 9 mU/mL for FVIII and 170 mU/mL for vWF. Values below limit of quantification (LOQ) are indicated by <LOQ. FVIII FVIII FVIII vWF vWF Volume act total Yield vWF total Yield Fraction (g = mL) (mU/mL) (mU) (%) (mU/mL) (mU) (%) MagSepharose VIII Select Cryoprecipitate 10 4122* 41220 100 4942 49420 100.0 Eluate 1-3 30 553 16590 40.2 <LOQ <LOQ <LOQ Eluate 4 10 435 4350 10.6 <LOQ <LOQ <LOQ *FVIII activity value from a cryoprecipitate dissolved in equilibration buffer from Experiment 1. Value used for yield estimation in the test with MagSepharose VIII Select prototype.

[0043] The FVIII yield was 51% in the eluate fractions (Eluate 1-3 and Eluate 4).

[0044] The vWF yield was below LOQ, and a low yield was expected as the affinity ligand binds to FVIII, and vWF which is not in complex with FVIII should not co-purify.

[0045] Experiment 3: Purification of Factor IX From Cryosupernatant

[0046] A. Packed Chromatography Columns

[0047] Chromatography conditions for tests with packed columns:

[0048] Columns: HiTrap Q FF 5 mL, HiTrap Capto Q 5 mL. Column volume (CV) 5 mL.

TABLE-US-00005 Method Volume step Sample/Buffer (CV or mL) Equilibration 20 mM Na-citrate, 70 mM NaCl, Column pH 7.0 pre-equilibrated Sample Cryosupernatant 40 mL Wash See Equilibration above 7 CV Elution 20 mM Na-citrate, 500 mM NaCl, 5 CV pH 7.0 CIP 0.5M NaOH 3 CV PreEquilibration 20 mM Na-citrate, pH 4.5 2 Equilibration See Equilibration above 5 CV

[0049] A. Batch Adsorption With Magnetic Beads

[0050] Magnetic beads: MagSepharose Q prototype resin LS-000672, 5 mL resin/tube in tests

[0051] MagSepharose Q tests were made with 5 mL resin in a 50 mL plastic tube with screw cap. The incubation and mixing of resin and buffer/sample was performed manually by shaking the tube, or in an end-over-end rotating mixer. The tube was then placed in a Sepmag A 200 mL (Sepmag) device with adapter for 50 mL tubes, where the magnetic beads were magnetically attracted to the side of the tube. The clear liquid was removed by plastic Pasteur pipette.

TABLE-US-00006 Method Volume step Sample/Buffer (mL) Equilibration 20 mM Na-citrate, 70 mM NaCl, pH 7.0 Resin pre- equilibrated Sample Cryosupernatant 40 mL Wash See Equilibration above 3 × 15 mL pooled Elution 20 mM Na-citrate, 500 mM NaCl, pH 7.0 3 × 10 mL pooled + 10 mL separately CIP 0.5M NaOH 5 × 20 mL Equilibration See Equilibration above 25 mL multiple times, until pH approx 7

TABLE-US-00007 TABLE 3 Results from Experiment 3 The lower limits for quantification (LOQ) were 30 mU/mL for FIX and 0.2 mU/mL for FIXa. Values below limit of quantification (LOQ) are indicated by <LOQ. FIX FIX FIX FIXa FIXa FIXa FIXa/ Vol act total Yield act total Yield FIX Fraction (g = mL) (mU/mL) (mU) (%) (mU/mL) (mU) (%) (‰) Q Sepharose FF Cryosupernatant 40.0 1365 54600 100 6.2 248 100 4.5 Flow through 44.1 <LOQ 0.4 18 7.1 13 Wash 30.7 <LOQ <LOQ Eluate 11.2 4148 46458 85 5.7 64 26 1.4 Capto Q Cryosupernatant 40.0 1365 54600 100 6.2 248 100 4.5 Flow through 43.2 <LOQ 1.4 61 24 47 Wash 32.2 <LOQ <LOQ Eluate 13.8 3176 43829 80 2.7 37 15 0.9 MagSeph Q tube 1 Cryosupernatant 40.0 1365 54600 100 6.2 248 100 4.5 Flow through 40.0 <LOQ 0.6 24 9.7 20 Wash 45.0 <LOQ <LOQ Eluate 1-3 30.0 1501 45030 82 0.8 24 9.7 0.5 Eluate 4 10.0  183 1830 3.3 <LOQ

[0052] As shown in the table, FIX was obtained in good yields and the FIXa/FIX ratio was low.

[0053] There was a 80-85% yield of FIX activity in the eluate fractions. The FIXa/FIX ratio was lowest in the eluate fraction from the MagSepharose Q prototype resin, indicating low activation of FIX to FIXa.

CONCLUSION

[0054] Batch adsorption with magnetic beads is considered to be a gentle technique, which is an advantage in the purification of sensitive plasma proteins. The present inventors have shown excellent results in yield and activity in the purification of FVIII and vWF in dissolved cryoprecipitate and FIX in cryosupernatant using magnetic beads with suitable ligands. Using large volumes of magnetic beads and instruments for separation enables large-scale applications not possible before.