阅读说明：本技术 纯化血浆蛋白的方法 (Method for purifying plasma proteins ) 是由 M·哈尔 M·安德尔 M·贝格 S·克里斯蒂安松 于 2020-05-11 设计创作，主要内容包括：本发明涉及一种纯化血浆蛋白的方法。更准确地说,本发明涉及使用磁珠从血浆部分,例如血浆的冷沉淀物或冷上清液,或者直接从重组血浆蛋白的细胞培养物中分离不同血浆蛋白的方法。(The present invention relates to a method for purifying plasma proteins. More precisely, the invention relates to a method for separating different plasma proteins from a plasma fraction, such as a cryoprecipitate or a cold supernatant of plasma, or directly from a cell culture of recombinant plasma proteins, using magnetic beads.)

1. A method of purifying one or more plasma proteins from a crude sample comprising binding one or more desired plasma proteins to ligands on magnetic beads and eluting the one or more plasma proteins, wherein the binding and eluting are performed in a batch mode.

2. The method according to claim 1, wherein said ligand is an anion exchange ligand, preferably selected from Diethylaminoethyl (DEAE), Quaternary Aminoethyl (QAE) or quaternary ammonium (Q), most preferably said anion exchange ligand is a Q-ligand.

3. The method according to claim 1 or 2, wherein the ligand binds to factor viii (fviii).

4. A method according to claim 3, wherein the ligand has affinity for the light chain of FVIII.

5. The method according to one or more of the preceding claims, wherein the crude sample is a cryoprecipitate and the one or more desired plasma proteins are factor viii (fviii) and/or von willebrand factor (vWF).

6. The method according to one or more of the preceding claims 1-4, wherein the crude sample is a cold supernatant and the one or more desired plasma proteins are albumin, IgG or Factor IX (FIX), preferably FIX.

7. The method according to one or more of the preceding claims 1-4, wherein the crude sample is taken directly from the cell culture of the recombinant plasma protein without further purification, except for cell sedimentation.

8. The method according to one or more of the preceding claims, comprising a) adding a plasma protein fraction, such as a cold supernatant or a dissolved cryoprecipitate comprising at least one plasma protein, to a container or bag; b) adding the magnetic beads provided with anionic ligands by pouring or pumping the magnetic beads into the container or bag; c) incubating preferably for at least 30 minutes with mixing; d) binding one or more plasma proteins to a ligand on a magnetic bead; e) retaining the magnetic beads with a magnetic field and washing unwanted material from the magnetic beads, optionally repeatedly; and F) eluting one or more plasma proteins from the ligand on the magnetic beads in a yield of 92-100% active FVIII from the cryoprecipitate or in a yield of at least 85% FIX and less than 1% FIXa/FIX ratio from the cold supernatant.

9. A method according to one or more of the preceding claims, wherein the magnetic beads are porous agarose beads provided with embedded magnetic particles.

10. A method according to claim 8 or 9, wherein the anionic ligand is a Q ligand.

11. The method according to one or more of the preceding claims, wherein the diameter of the magnetic beads is between 8 and 300 μm and the method is performed on a large scale.

Technical Field

The present invention relates to a method for purifying plasma proteins. More precisely, the invention relates to a method for separating different plasma proteins from a plasma fraction, such as a cryoprecipitate or a cold supernatant of plasma, or directly from a cell culture of recombinant plasma proteins, using magnetic beads.

Background

Blood contains different types of cells and molecules essential for the functioning of a living body and is therefore collected for therapeutic purposes, for example for blood transfusions. However, different fractions, such as red blood cells or cell-free plasma, can be isolated and prepared from the blood, which enables a more direct therapeutic treatment of medical conditions. Several proteins in plasma can also be further isolated and used for specific therapeutic treatments, such as albumin for restoring blood volume, immunoglobulins for immunodeficiency, coagulation factors for coagulopathy.

Plasma contains proteins of different functions, different sizes, different amounts, etc., and thus there are different methods of purifying different plasma proteins. Purification methods are usually designed to obtain several proteins of interest from a single starting pool of plasma. The process typically comprises a precipitation or chromatography step or a combination thereof. Chromatography is commonly used to improve the purity of target proteins and reduce the risk of harmful side effects. Many plasma proteins exhibit very strong activity and, if present as contaminants, they can cause adverse effects even at very low levels when administered to a patient.

The collected human plasma was stored frozen and the initial steps in the plasma protein purification process were thawing and pooling of the plasma. When thawed at low temperatures (typically 1-6 ℃), some of the plasma proteins precipitate and can be collected by, for example, centrifugation. The collected precipitate, called cryoprecipitate, may be used as a source of, for example, coagulation factor viii (fviii) and von willebrand factor (vWF). Most FVIII in plasma exists as a complex with large vWF multimers, and therefore both proteins are usually co-purified. The remaining liquid after removal of the cryoprecipitate is commonly referred to as cold depleted plasma or cold supernatant and this may be used as a source of, for example, albumin, immunoglobulin g (igg), coagulation factor ix (fix).

Purification of many plasma proteins can be challenging. This may depend on the presence of small amounts of contaminants with undesirable but strong activity, or the protein sometimes loses its activity or acquires an undesirable activity. For example, FVIII is prone to loss of activity and known methods for purification are unsatisfactory in many respects. Thus, there is a need for improved methods that can be operated under conditions where the protein retains its activity in order to obtain plasma products in good yield.

Disclosure of Invention

The present invention relates to magnetic beads for purifying plasma proteins by batch adsorption of proteins in a crude sample, which has been shown to be a mild technique that can preserve sensitive proteins. The beads are of the chromatography bead type provided with embedded magnetic particles and plasma protein binding ligands.

In a first aspect, the present invention relates to a method for purifying plasma proteins from a crude sample, comprising binding desired plasma proteins to ligands on magnetic beads and eluting said plasma proteins, wherein said binding and elution are performed in a batch mode. The process can be carried out on a large scale to provide large quantities of the desired plasma protein.

The ligand is preferably an anion exchange ligand and is preferably selected from Diethylaminoethyl (DEAE), Quaternary Aminoethyl (QAE) or quaternary ammonium (Q), most preferably the anion exchange ligand is a Q-ligand.

Alternatively, the ligand may have affinity for FVIII, for example for the light chain of FVIII.

Preferably, the crude sample is cryoprecipitate and the one or more desired plasma proteins are factor viii (fviii) and/or von willebrand factor (vWF).

The crude sample may also be a cold supernatant and the one or more desired plasma proteins are albumin, IgG or factor ix (fix). Alternatively, the crude sample may be whole plasma.

In a preferred embodiment, the present invention relates to a method according to one or more of the preceding claims, comprising a) adding a plasma fraction comprising at least one plasma protein, such as a cold supernatant or a dissolved cryoprecipitate, to a container, such as a bag or a tank; b) adding magnetic beads provided with anionic ligands, preferably Q ligands, by pouring or pumping the magnetic beads into the container; c) incubating for at least 30 minutes with mixing; d) binding plasma proteins to magnetic beads; e) holding the magnetic beads with a magnetic field and washing away unwanted material, optionally repeated; f) eluting plasma proteins from the magnetic beads at a yield of 92-100% active FVIII from cryoprecipitate or at a yield of at least 85% FIX and less than 1% FIXa/FIX ratio from cold supernatant.

The magnetic beads are preferably porous agarose beads provided with embedded magnetic particles. The present invention enables large scale purification by providing magnetic beads of suitable size and large containers with their attached equipment, such as Wave bags (Wave bags).

A magnetic chromatography resin prototype with anion exchange (Q) or affinity (VIII Select) ligands was used to purify factor VIII (fviii), von willebrand factor (vWF) or factor ix (fix) from human recovered plasma. Conventional packed bed chromatography was referenced as a test with Q ligand as shown in the experimental section below.

Although experiments have shown purification of plasma-derived proteins, the present invention also contemplates purification of recombinant plasma proteins directly from cell cultures, i.e., without further purification prior to binding of the plasma proteins to the selected ligand on the magnetic beads.

Detailed Description

The present invention provides a magnetic bead capable of binding plasma proteins with high binding strength and capable of having high binding ability. This is achieved with a plasma protein-binding magnetic bead comprising a porous matrix and one or more magnetic particles embedded in the matrix, wherein the matrix comprises a porous polymer, preferably agarose, and a plasma protein-binding ligand coupled to the porous polymer.

One advantage is that the beads allow selective binding of large amounts of plasma proteins directly from the crude material. Another advantage is that the beads have a favorable adsorption isotherm for plasma proteins, resulting in high yields of recovered plasma proteins.

The size of the beads may suitably be such that a plurality of beads to be used in the methods disclosed hereinafter have a volume weighted median diameter (d50, V) of 8-300 microns, for example 20-200, 20-100 microns or 20-80 microns. Beads of these sizes are easily retained with a magnetic field, especially compared to magnetic nanoparticles or micron-sized particles. However, the mass transfer rate is fast enough for the beads to rapidly absorb plasma proteins. This applies in particular to beads having median diameters in the interval 20 to 100 microns and 20 to 80 microns. The one or more beads may be spherical or substantially spherical, for example having a sphericity (surface area of a sphere having the same volume as the bead divided by the surface area of the bead) of at least 0.9.

The present invention will now be described in more detail in connection with the experimental section below.

Assays for factor VIII, factor IX, factor IXa (FVIII, FIX, FIXa) activity or von willebrand factor (vWF) concentration were performed.

In experiments 1 and 3 below, a comparison was made between a MagSepharose prototype packed in a column and a non-magnetic resin.

Experimental part

Synthesis of magnetic bead prototype:

MagSepharose Q prototype, LS-000672

The MagSepharose base matrix was washed with 2M NaOH. Glycidyltrimethylammonium chloride (GMAC) was added and the coupling reaction was carried out overnight at room temperature. The GMAC reaction was repeated two more times. The resin was washed with distilled water. The reaction scheme is shown below.

VIII MagSepharose prototype, LS-034256

NHS activated MagSepharose was washed with cold 1 mM HCl. VIII ligand was added and the coupling reaction was carried out at 24 ℃ for 2 hours 15 minutes at pH 8.2. The resin was washed with 0.1M acetic acid/0.15M NaCl, pH 4.5, 50 mM Tris/0.15M NaCl, pH 8.5 and distilled water. The reaction scheme is shown below.

Sample preparation: the plastic bag with recovered plasma was slowly thawed in ice water (temperature about 0-4 ℃) to obtain liquid plasma with cryoprecipitate. The thawed plasma was centrifuged and the cryoprecipitate in the pellet was collected. The supernatant (cold supernatant) was decanted and the pellet was dissolved in equilibration buffer (dissolved cryoprecipitate) for an initial plasma volume 1/5.

And (3) analysis: FVIII activity, vWF ELISA (concentration), FIX and FIXa activity: the presence of FVIII, vWF, FIX and FIXa (activated FIX) was analyzed using a commercial kit according to the manufacturer's instructions. The activities/concentrations are listed in the table below in mU/mL (m units/mL). FVIII activity was determined using the Coamatic FVIII kit from Chromogenix. The concentration of vWF was determined using a Technozym vWF Ag ELISA kit from Technoclone. FIX and FIXa activities were determined using a commercial kit from Rossix: rox factor IX, Rox FIX-a, factor IXa calibrator, factor IXa control.

Experiment 1: purification of factor VIII and von Willebrand factor from solubilized cryoprecipitate using Q-ligand

A. Packed chromatography column

Chromatographic conditions tested with packed column:

column: HiTrap Q HP 5 mL, HiTrap Capto Q ImpRes 5 mL.

Column Volume (CV) 5 mL.

B. Batch adsorption with magnetic beads

Magnetic beads: MagSepharose Q prototype resin LS-000672, 5 mL resin/tube in test

The MagSepharose Q test was performed with 5 mL of resin in 50 mL of plastic tubing with a screw cap. The incubation and mixing of the resin and buffer/sample is done manually by shaking the tube or in an end-to-end rotating mixer. The tube was then placed in Sepmag a 200 ml (Sepmag) where the magnetic beads were magnetically attracted to the side of the tube. Clear liquid was removed with a plastic pasteur pipette.

Table 1: results of experiment 1

The lower limit of quantitation (LOQ) is 9 mU/mL for FVIII and 170 mU/mL for vWF. Values below the limit of quantitation (LOQ) are denoted by < LOQ.

As shown in table 1, high yields of FVIII were present in the eluate fraction and the highest yield was obtained using the MagSepharose Q prototype resin.

vWF was partially removed in the washing step without any loss of FVIII.

The results of experiment 1 surprisingly show that FVIII yields are obtained with 10-15% higher using magnetic beads with Q-ligand than using conventional Q-resin.

Experiment 2: purification of factor VIII from solubilized cryoprecipitate Using VIII Select ligand

Tests were also performed with affinity ligands for FVIII coupled to MagSepharose beads. This magnetic prototype resin was designated as MagSepharose VIII Select prototype LS-034256. The test was performed with only the MagSepharose VIII Select prototype, and not compared to a packed column with VIII Select resin. The conditions were comparable to those in experiment 1, but different buffers were used.

Table 2: results of experiment 2

The lower limit of quantitation (LOQ) is 9 mU/mL for FVIII and 170 mU/mL for vWF. Values below the limit of quantitation (LOQ) are denoted by < LOQ.

FVII activity values of cryoprecipitate dissolved in equilibration buffer from experiment 1. Values for yield estimation in the test with the MagSepharose VIII Select prototype.

FVIII yield was 51% in the eluate fractions (eluates 1-3 and eluate 4).

The yield of vWF is lower than LOQ and low yields are expected when affinity ligand binds FVIII and vWF that is not complexed with FVIII should not be co-purified.

Experiment 3: purification of factor IX from Cold supernatant

A. Packed chromatography column

Chromatographic conditions tested with packed column:

column: HiTrap Q FF 5 mL, HiTrap Capto Q5 mL. Column Volume (CV) 5 mL.

A. Batch adsorption with magnetic beads

Magnetic beads: MagSepharose Q prototype resin LS-000672, 5 mL resin/tube in test

The MagSepharose Q test was performed with 5 mL of resin in 50 mL of plastic tubing with a screw cap. The incubation and mixing of the resin and buffer/sample is done manually by shaking the tube or in an end-to-end rotating mixer. The tube was then placed in a Sepmag a 200 mL (Sepmag) device with an adapter for a 50 mL tube, where the magnetic beads were magnetically attracted to the side of the tube. Clear liquid was removed with a plastic pasteur pipette.

Table 3: results of experiment 3

The lower limit of quantitation (LOQ) is 30 mU/mL for FIX and 0.2 mU/mL for FIXa. Values below the limit of quantitation (LOQ) are denoted by < LOQ.

As shown in the table, FIX was obtained in good yield and the FIXa/FIX ratio was low.

The yield of FIX activity in the eluate fraction was 80-85%. The lowest ratio of FIXa/FIX in the eluate fraction of the MagSepharose Q prototype resin indicates a low activation of FIX to FIXa.

Summary of the invention

Batch adsorption with magnetic beads is considered a mild technique, which is advantageous in the purification of sensitive plasma proteins. The inventors have shown excellent results for the purification of FVIII and vWF in solubilized cryoprecipitate and FIX yield and activity in cold supernatant using magnetic beads with suitable ligands. The use of large volumes of magnetic beads and instruments for separation enables large scale applications not previously possible.