Chromatographic methods for purification of proteins from plasma

Abstract

The present invention relates to the field of chromatography. More closely, the invention relates to a chromatographic method for purification of plasmaproteins, such as Factor VIII, von Willebrand factor and Factor IX. The chromatographic method is performed on a matrix comprising an inner porous core and outer porous lid surrounding said core.

Claims

1. A chromatographic method comprising the following steps: loading plasma comprising plasma proteins on a chromatography column packed with a resin comprising porous lid beads having an inner porous core and an outer porous lid, wherein the inner core is provided with anion exchange ligands, and wherein the porosity of the lid and core does not allow entering of molecules larger than 500 kD; adsorbing Factor IX (FIX) on the anion exchange ligands in the core; collecting separated plasma proteins in the flow through; and eluting FIX from the ligands in the core.

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

3. The method of claim 2, wherein the anion exchange ligands are Q-ligands.

4. The method of claim 1, wherein other plasma proteins besides FIX are collected in the flow through separated from each other by the sieving effect of the core and shell and comprise Factor VIII (FVIII) and von Willebrand factor (vWF), IgG, human serum albumin (HSA) and Complement C3 (C3).

5. The method of claim 1, wherein the loading of plasma is repeated 1-20 times, followed by running buffer to obtain a corresponding number of flow throughs (FT's), before the FIX is eluted from the ligands in the core.

6. The method of claim 5, wherein other plasma proteins besides FIX are collected in the flow through separated from each other by the sieving effect of the core and shell and comprise Factor VIII (FVIII) and von Willebrand factor (vWF), IgG, human serum albumin (HSA) and Complement C3 (C3), and wherein specific fractions of respective FT are pooled to obtain FVIII/vWF, IgG/HSA and C3 respectively.

7. The method of claim 1, wherein the total lid bead thickness is 40-100 μm in diameter, and the lid thickness is 2-10 μm.

8. The method of claim 1, wherein the ligand concentration in the core is 50-200 μmol/ml.

9. The method of claim 1, wherein the lid is provided with affinity ligands, hydrophobic interaction ligands, IMAC ligands, cation exchange ligands or multimodal ligands; and at least one plasma protein other than FIX are adsorbed on the ligands in the lid in the same step as FIX is adsorbed on the ligands in the core; and wherein plasma proteins are sequentially eluted from the ligands in the lid and FIX is eluted from ligands in the core.

10. The method of claim 9, wherein the ligands in the lid are ligands having immunoglobulin affinity.

11. The method of claim 1, wherein the lid and core are made of agarose of the same porosity.

12. The method of claim 1, wherein the porosity of the lid is larger than of the core.

13. The method of claim 1, wherein the porosity of the lid is smaller than of the core.

14. The method of claim 5, wherein the loading of plasma is repeated 5-10 times.

15. The method of claim 9, wherein the at least one plasma protein other than FIX comprises FVIII/vWF, IgG, HSA, or C3.

16. The method of claim 1, wherein the lid is provided with ligands comprising at least one of Protein A or G or variants thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A: Chromatogram of plasma applied to prototype 99 column. Numbers 1-6=flow through (FT) peaks from the 6 sample applications; E=elution peak; CIP=cleaning-in-place.

(2) Absorbance 280 nm—solid line; conductivity—dotted line; pH—dashed line.

(3) FIG. 1B: Partial enlargement of chromatogram of plasma applied to prototype 99 column according to FIG. 1A, showing final (6th) sample application and elution. 6=flow through peaks from the 6.sup.th sample application; A=large molecules, eg FVIII/vWF; B=smaller molecules, eg albumin, IgG; C=partially retained material; E=eluted molecules, eg FIX.

(4) Absorbance 280 nm—solid line; conductivity—dotted line; pH—dashed line.

DETAILED DESCRIPTION OF THE INVENTION

(5) The invention will now be described more closely in connection with some non-limiting examples and the accompanying figures.

Example 1: Synthesis of Prototypes

(6) General

(7) Volumes of matrix refer to settled bed volume and weights of matrix given in gram refer to suction dry weight. For large scale reaction stirring is referring to a suspended, motor-driven stirrer since the use of magnet bar stirrer is prompt to damage the beads. Conventional methods were used for the analysis of the functionality and the determination of the degree of allylation, or the degree of ligand content on the beads.

(8) Support Particles

(9) The support particles used were highly crosslinked agarose beads, prepared according to the methods described in U.S. Pat. No. 6,602,990, which is hereby incorporated by reference in its entirety. The beads had a volume-weighted average diameter (D50, v) of 88 micrometers and a pore size distribution such that 69% of the pore volume was available to dextran molecules of Mw 110 kDa. This can also be expressed such that Kd for dextran 110 kDa on the beads was 0.69, when measured according to the methods described in “Handbook of Process Chromatography, A Guide to Optimization, Scale-Up and validation” (1997) Academic Press, San Diego. Gail Sofer & Lars Hagel eds. ISBN 0-12-654266-X, p. 368.

(10) Allylation (Prototype 76)

(11) 250 mL (g) of of support particles were washed 6× gel volumes (GV) with distilled water, and then 3×GV with 50% NaOH. The gel was then sucked dry and transferred to a 2 L round bottom flask. 485 mL of 50% NaOH was added, mechanical propeller stirring was applied and the flask was immersed into a water bath at 50° C. After 30 minutes 80 mL of allyl glycidyl ether (AGE) was added. The reaction progressed for 18.5 h. The gel was washed 1×GV with distilled water, 3×GV with ethanol and then 8×GV with distilled water.

(12) The allyl content, 276 μmol/mL, was measured by titration.

(13) Prototype 99

(14) Partial Bromination and Shell Inactivation

(15) 171.8 g of allylated gel slurry (prototype 76) was transferred to a glass filter (por. 2) and sucked dry. The dry gel is transferred to a 1000 mL round bottom flask fitted with a mechanical stirrer. 571 g of distilled water is added and the suspension is stirred at 300 rpm. 83.7 g of the 1.6% bromine solution is added during 1.5 min. After the addition the suspension is still white. The reaction proceeded for 15 min at rt. The round bottom flask was immersed in a bath and when the temperature had reached 50° C., 52 g of 50% NaOH was added. The reaction was let to stand for 17 hours. The reaction is transferred to a glass filter (por. 2) and washed with distilled water 10×1 GV. The remaining allyl content, 216 μmol/mL, was measured by titration. This corresponds to a theoretical shell thickness of 3.5 μm.

(16) Core Bromination and Q Coupling

(17) 41.5 mL (g) of partial allylated base matrix from above was transferred drained to a 250 mL round bottom flask fitted with a mechanical stirrer. 10.37 g of distilled water and 1.66 g of sodium acetate was added. After stirring for a couple of minutes, 0.66 mL bromine is added with a pipette and the reaction is stirred at 300 rpm for additionally 20 min. Excess bromine is consumed by adding 4.15 mL of 40% sodium formate solution. The reaction is colourless. After 15 min, 8.30 mL of trimethyl ammonium chloride (TMAC) is added and the pH is adjusted to 11-11.5 by adding 50% NaOH. The reaction is stirred at 250 rpm at 30° C. for 18 h. The reaction is neutralized by adding 60% acetic acid to pH 5-7 before transferring to a glass filter (por. 3). The gel was washed with distilled water 10×1 GV, followed by 20% EtOH 2×1 GV. Titration of the ion exchange groups gave a Q ligand density of 125 μmol/ml.

(18) Table 1 shows the lid thickness, ligand type and concentration in Prototype 99.

(19) TABLE-US-00001 TABLE 1 Lid thickness Lid ligand Core ligand Resin (μm) (type, conc.) (type, conc.) Prototype 99 3.5 No ligand Q, 125 μmol/ml

Example 2: Chromatography of Plasma on Prototype 99

(20) Sample

(21) The sample was human plasma. Frozen human plasma was thawed and filtered through cotton, and applied to the column.

(22) Buffers and Running Conditions

(23) Column: Tricorn 10/300 with prototype 99, bed height 28.6 cm, column volume (CV) 22.5 mL.

(24) Chromatography: Sample volume 6×5.2 mL (6×0.23 CV). Flow rate 50 cm/h (0.65 mL/min).

(25) Running buffer: 20 mM Na-citrate, 0.15 M NaCl, 2.6 mM CaCl.sub.2, pH 7.0.

(26) Elution buffer: 20 mM Na-citrate, 0.5 M NaCl, 2.6 mM CaCl.sub.2, pH 7.0.

(27) Cleaning-in-place (CIP): 0.5 M NaOH.

(28) The column was equilibrated with running buffer prior to the first sample application. 0.23 CV of plasma was applied to the column, followed by 1.8 CV of running buffer. This procedure, application of 0.23 CV of plasma followed by 1.8 CV of running buffer, was repeated 5 times, resulting in a total of 6 plasma sample applications. After the final 1.8 CV of running buffer, the column was eluted with 1.5 CV of high salt elution buffer. The column was then subjected to CIP by applying 1.5 CV of 0.5 M NaOH. Finally, the column was re-equilibrated by 4 CV of running buffer.

(29) Analysis

(30) Selected fractions were analyzed for FVIII activity (Chromogenix Coamatic Factor VIII kit), vWF (Technozym vWF:Ag ELISA kit), FIX (ROX Factor IX kit), and by SDS PAGE, and liquid chromatography-mass spectrometry (LC-MS).

(31) The prototype 99 was packed in a Tricorn 10/300 column. The bed height was 28.6 cm (CV 22.5 mL) and the flow rate was 50 cm/h, to enable size-dependent group separation. The run consisted of 6 plasma sample applications (6×0.23 CV) resulting in 6 group separations, and one final high salt elution from the Q ligand in the core. The fractions from the 6 group separations were pooled so that one pooled fraction A with very large molecules, one pooled fraction B with smaller proteins, and one pooled fraction C with slightly retained smaller proteins, were obtained. The high salt elution resulted in one eluted fraction E. All fractions were analysed for FVIII, vWF and FIX. The chromatogram is shown in FIG. 1A and a partial enlargement is shown in FIG. 1B.

(32) See Table 2 below for analytical results and yield calculations. The yields of FVIII and vWF in the fraction A pool was 37% and 40%, respectively. Fraction B and C pools had FVIII and vWF activities below level of quantification. The eluted fraction E contained 22% of the FVIII activity, and 13% of the vWF activity. This indicated that most of the large FVIII/vWF complexes do not enter the beads, and pass in the flow through, separated from the smaller molecules such as albumin and IgG in fraction B, which enter the beads but do not bind to the Q ligand in the core under these conditions. SDS PAGE and LC-MS showed that albumin and IgG were the main proteins in fraction B. LS-MC indicated that the major components in Fraction C were C3 and albumin. However, some FVIII/vWF complexes enter the pores and bind to the Q ligand, this could depend on the varying size of the FVIII/vWF complexes and the low flow rate. Fraction E consisted of molecules eluted with high salt, and it was the only fraction with FIX activity, 91% yield. This shows that the smaller FIX molecules enter the chromatography beads and bind to the Q ligand in the core. This demonstrates that by using a chromatography media with an inactive lid and ligand-containing core, it is possible to separate large (FVIII/vWF) and small (FIX) plasma proteins which both bind to the core ligand, as the large proteins are collected in the flow through, and the smaller molecules can be collected by elution.

(33) TABLE-US-00002 TABLE 2 Plasma on prototype 99. FVIII, vWF and FIX activity. For fractions, see FIG. 1 A and B. X = not measured. LOQ = level of quantification (FVIII: 8 mU/mL, vWF: 0.10 U/mL, FIX: 30 mU/mL). FVIII Vol (mU/ FVIII FVIII vWF vWF vWF FIX FIX FIX Sample (mL) mL) (mU) (%) (U/mL) (U) (%) (mU/mL) (mU) (%) Plasma 31.0 875 27125 100 1.00 31.0 100 1360 42160 100 Fraction A 35.7 280 9996 37 0.35 12.5 40 <LOQ Fraction B 66.6 <LOQ <LOQ <LOQ Fraction C 47.3 <LOQ <LOQ <LOQ Fraction E 8.2 743 6093 22 0.49 4.0 13 4673 38319 91