Method for purifying or removing target components from whole plasma - Patents.com

Abstract

The present invention relates to a method for purifying a target component from a whole plasma sample.The present invention further relates to a method for removing one or more target components from a whole plasma sample.

Description

[Technical field]

[0001] The present invention relates to a method for purifying a target component from a full plasma sample.The present invention further relates to a method for removing one or more target components from a full plasma sample. [Background technology]

[0002] Until now, it has been extremely difficult to load a pool of thawed undiluted fresh frozen plasma or plasma of one patient directly into a chromatography unit or other purification device. The main challenges in processing and / or purifying fully undiluted human plasma are its viscosity, fragility, and precipitation of plasma components caused by the activation of the coagulation cascade and proteolytic enzymes. Monolithic supports, including also membrane-based materials, have recently been used to process cryopreserved plasma and / or other high-value biopolymers secreted by cultured cells [2, 3]. Usually, dilution of the sample is required before plasma processing. The reason for dilution is frequent clogging of the chromatography unit or low yield of the target molecule. Clogging occurs when bulk chromatography supports, as well as monolithic and / or membrane-based supports, are used for plasma processing. Moreover, plasma processing is routinely performed at low temperatures to prevent degradation of sensitive molecules or nanoparticles of interest [4, 5]. Almost all current plasma fractionation techniques still rely on a two-backbone process based on cryoprecipitation [6, 7] followed by low-temperature ethanol precipitation in conjunction with pH, ​​temperature, and osmolarity shifts that result in the selective precipitation of proteins. In the early days before the introduction of cryoglobulin precipitation, the most abundant plasma proteins albumin and intravenous immunoglobulin (IVIG) were produced [8].

[0003] During the past three decades, chromatography-based separation processes have been combined with precipitation, which has enabled the isolation of low-abundance proteins from cryoprecipitate, cryo-poor plasma, and different Cohn fractions [6, 9].

[0004] For example, reference

[10] discloses a monolith-based pseudo-affinity purification method for the separation of human plasma-derived and / or recombinant coagulation factor VIII (FVIII). A hydrophilic polymethacrylate-based monolith is used for the fast chromatographic separation of this plasma protein without any significant loss of separation efficiency. However, a notable difference of this process compared to the present invention is that the starting material is either cell culture supernatant and / or diluted human plasma. In both cases, extensive sample preparation by dilution and pH adjustment is necessary. This preparation requires additional steps, increasing both process time and costs [2].

[0005] The direct application of whole undiluted human plasma to the chromatography unit reduces both process costs and time. It also allows further sequential processing by a combination of different chromatographic methods to separate other biologically active proteins. Monoliths and membranes can also be stacked to separate different target components in one chromatographic run [2, 12]. This novel strategy allows for faster processes and reduces not only the costs but also the risk of degradation, modification, and / or activation of the plasma components of interest. This ultimately results in higher yields and better quality and safety of the product.

[0006] Direct (online) treatment of a patient's blood and / or plasma for the removal of small molecular toxic components that accumulate in body fluids in the event of insufficient renal function is a routine treatment known as hemodialysis [12, 13]. However, the removal or separation of macromolecules and nanoparticles directly from a patient's blood is a more challenging process, and the presented chromatographic method is a major step in this direction.

[0007] Plasmapheresis is the removal of blood or its components from the circulation, treatment, and return or exchange to the circulation

[14] . Autologous plasma exchange is defined as the temporary removal of plasma followed by the extracorporeal treatment of this fluid and its return to the same person as therapy. In plasma exchange, the patient's plasma is removed and replaced with donated plasma or other donated blood products. Autologous and exchange forms of plasmapheresis are used to treat a variety of disorders, including immune system and / or neurological disorders [14-17].

[0008] Using the methods described herein, patient plasma separated by plasmapheresis can be forced through a large pore chromatography unit having immobilized affinity ligands capable of binding to target macromolecules such as various pathological antibodies, and the purified plasma can be returned to the patient.

[0009] The methods described herein utilize continuous chromatographic materials, such as synthetic monoliths or membrane-based supports, to process whole, undiluted human plasma. By using such chromatographic materials, the inventors have surprisingly found that the methods described herein are suitable for processing / purifying whole, undiluted human plasma without clogging the chromatographic materials and / or activating plasma components that result in activation of signaling pathways, including the coagulation cascade and the complement system. Summary of the Invention [Problem to be solved by the invention]

[0010] Based on the above, it is an object of the present invention to provide a method for purifying a target component from a whole plasma sample without the need to subject the plasma to any preparatory steps such as dilution and / or cryoprecipitation. [Means for solving the problem]

[0011] According to the present invention, the above object is to provide a method for purifying a target component from a whole plasma sample, comprising the steps of: applying whole plasma to a continuous chromatographic material, the continuous chromatographic material comprising channels having a diameter between 4 μm and 15 μm, the chromatographic material comprising chromatographic selection elements for target components; carrying out chromatography at a temperature between 30° C. and 39° C.; recovering said target component from the continuous chromatographic material; This is achieved by providing a method comprising:

[0012] According to this method, undiluted plasma is applied to a continuous chromatographic material. The components to be purified are bound to the chromatographic material and are recovered in a next step by elution. This method can also be used to recover several different components from undiluted plasma by using different chromatographic techniques such as ion exchange chromatography, affinity chromatography (e.g. protein A), hydrophobic interaction chromatography, reversed phase chromatography and / or different elution methods. Thus, this method is suitable for purifying and separating target molecules from undiluted plasma that can be used for therapeutic applications.

[0013] It is a further object of the present invention to provide a method for removing one or more target components from a whole plasma sample without subjecting the plasma to any preparatory steps such as dilution and / or cryoprecipitation.

[0014] According to the present invention, the above object is to provide a method for removing one or more target components from a whole plasma sample, comprising the steps of: applying whole plasma to a continuous chromatographic material comprising channels having a diameter between 4 μm and 15 μm, the chromatographic material comprising chromatographic selection elements for one or more target components; carrying out chromatography at a temperature between 30° C. and 39° C.; Recovering the plasma following removal of one or more target components; This is achieved by providing a method comprising:

[0015] This method can be used in therapeutic applications similar to apheresis, where removal of harmful components (e.g., autoimmune antibodies, allergens, etc.) from plasma is a critical step before the plasma is recirculated back to the patient.

[0016] The present invention, including preferred embodiments, will now be described in more detail in conjunction with the accompanying drawings. [Brief description of the drawings]

[0017] [Figure 1] Figure 2 shows the chromatogram of Example 1. Loading of 150 ml of human plasma through a 1 ml CIMmultus OH column with 6 μm pore size (solid line - 280 nm (mAU), dashed line - conductivity (mS / cm)). [Diagram 2] FIG. 1 shows SDS-PAGE analysis of the starting sample (undiluted human plasma), the flow-through fractions after 25 ml and 50 ml of plasma had passed through the column (FT25 and FT50), and the eluates E1 and E2 of Example 1. [Diagram 3] Figure 2 shows the chromatogram of Example 2. Loading of 150 ml of human plasma through a 1 ml CIMmultus QA column with 6 μm pore size (solid line - 280 nm (mAU), dashed line - conductivity (mS / cm)). [Figure 4] Figure 1 shows SDS-PAGE analysis of plasma and chromatography fractions from the QA column. Flow-through fractions after 25 ml, 75 ml and 100 ml of plasma have been passed through the column (FT25, FT75 and FT100) and eluates E1, E2 and E3 of Example 2 are analyzed. [Diagram 5] Example 3: Chromatograms of Protein G column chromatography. Separation of IgG from undiluted human plasma (solid line - 280 nm (mAU), dashed line - conductivity (mS / cm)). [Figure 6] Figure 1 shows SDS-PAGE analysis of human plasma, flow-through fraction FT and eluate. A heavy chain of approximately 50 kDa and a light chain of approximately 25 kDa are seen in the Protein G eluate lane (Example 3). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The present invention relates to the chromatographic separation and / or purification of whole plasma. Suitable starting materials are fresh frozen plasma and patient plasma after temporary removal of cells by autologous plasmapheresis. In a preferred embodiment, the plasma is human plasma.

[0019] The target components to be purified or removed can be selected from the group consisting of proteins, such as antibodies and allergens, nucleic acids, viruses, macromolecular toxins, and nanoparticles. Nanoparticles are, for example, exosomes and other microvesicles. Suitable proteins for fast separation and therapeutic applications are clotting factors and / or clotting inhibitors and antibodies. One particular target component is clotting factor VIII.

[0020] According to the invention, a continuous chromatographic material is used. The continuous chromatographic material has channels within the material. Suitable materials are monolithic materials and / or membrane adsorber with continuous channels with a diameter of 4 μm to 15 μm. In a preferred embodiment, the continuous channels have a diameter in the range of 4 μm to 10 μm, more preferably 5 μm to 7 μm. The channel diameter represents the average diameter determined by mercury porosimety.

[0021] Monoliths are generally considered as an alternative to particle-based stationary phases in chromatography. Monoliths are manufactured in a single molded structure that features a large inlet surface area. Due to the absence of diffusion pores, mass transfer occurs solely by convective forces.

[0022] One of the main advantages of continuous chromatography materials, especially CIM monoliths and membranes, is their consistency. There is a notable difference between continuous and bulk chromatography materials. In monolithic and membrane-based materials, the interaction between ligands and ligates (components of the sample that are of interest, that should be separated, and that bind to the ligands) occurs mainly in channels on the surface of the continuous material. In "conventional" particle-based bulk materials, this interaction occurs within the pores inside the material and is limited by diffusion. This also means that the surface inside the pores of the bulk particle-based material is important for the separation performance of the bulk material. On the other hand, the increase in surface area also increases the possibility of non-specific interactions. These interactions are not only responsible for non-specific binding (which may be irreversible), but also for activation and aggregation of plasma components.

[0023] Furthermore, physical interactions occur within the pores of the bulk material. The driving force here is diffusion, and thus the physical interactions are limited by pore size, surface area, and flow rate. In monoliths and membranes, the interactions occur in the channels of the material rather than within the pores, and the driving force here is convection rather than diffusion (CIM - "Convective Interaction Media").

[0024] The pores have random orientation and may contain dead ends, increasing the surface area without benefiting the throughput of the column. In contrast, the channels of a continuous chromatographic material run from the beginning to the end of the column. In general, the larger the diameter of the channel, the smaller the surface area of ​​the chromatographic material. This means that the surface of a continuous chromatographic material is several orders of magnitude lower, which means that the level and risk of non-specific interactions and activation is likewise orders of magnitude lower

[18] .

[0025] In a preferred embodiment, the surface of the channel is hydrophilic and can contain specific ligands immobilized on the surface. Due to the tailored dimensions of the channels of the continuous chromatographic material, the hydrophobicity of the channel is reduced compared to bulk or micro / mesopore materials. Furthermore, the limiting factor of pore diffusion is eliminated, which is mainly responsible for the slow kinetics and indirectly responsible for non-specific binding and the build-up of aggregates. Due to the suitable tailoring of the channel, the destruction of fragile macromolecules and nanoparticles is significantly reduced.

[0026] In a further preferred embodiment, the continuous chromatographic material is essentially inert, thereby preventing direct interaction and activation of plasma components. An exemplary chromatographic material with inert and / or hydrophilic properties is prepared by radical copolymerization of glycidyl methacrylate and ethylene dimethacrylate in the presence of a pore-generating solvent according to the method of Svec et al. [19, 20].

[0027] According to the invention, the continuous chromatographic material includes a chromatographic selection element that is capable of selectively binding to a target component to be purified or removed from plasma.

[0028] The hydrophilic and inert properties of continuous chromatographic materials may be further improved by applying specific surface chemistries. Suitable chemistries include polyethylene glycol (for neutral and inert surfaces) and the incorporation of various ion-exchange groups (ion exchangers), hydrophobic molecules, and small and / or large molecules such as enzymes, inhibitors, antibodies, allergens, polynucleotides and nucleic acids, as well as nanoparticles for affinity and pseudo-affinity chromatography

[21] .

[0029] In a preferred embodiment, the ligands can be immobilized on the surface of the chromatographic material and can function as chromatographic selection elements. Depending on the type of target component to be bound by the column, suitable chromatographic ligands include anion exchange ligands (strong or weak), cation exchange ligands (strong or weak), hydrophobic interaction ligands, small and / or polymeric affinity ligands (such as Protein A), oligonucleotides, aptamers, immobilized nanoparticles, and combinations thereof.

[0030] After applying the whole plasma to the chromatography material, the chromatography is carried out at a temperature between 30°C and 39°C. This prevents denaturation, aggregation, and precipitation of macromolecular plasma components and nanoparticles, and also reduces the viscosity of the plasma. The chromatography is preferably carried out at a temperature between 35°C and 37°C. The use of a physiological temperature range during plasma processing is one important difference compared to commonly known techniques that are carried out at lower temperatures, such as 4°C. At such low temperatures, undiluted plasma becomes highly viscous and processing without prior dilution is impossible.

[0031] In a preferred embodiment, the chromatography is carried out at a flow rate between 0.2 column volumes per minute (CV / min) and 2 column volumes per minute; i.e., for an 8 ml column, the flow rate is between 1.6 mL / min and 16 mL / min. Such a flow rate represents a good balance between the time taken for the chromatography and the column pressure obtained for the consistency of the plasma. Moreover, such a flow rate makes it possible to ensure the stability of plasma components such as macromolecules and nanoparticles (see references [1-3] and [18, 20, 21]).

[0032] In the final step of the method according to the invention, the plasma after removal of the target components may be collected and the column containing the target components may be discarded. Alternatively, the plasma may be discarded or used for further component separation and the target components are eluted from the column and collected.

[0033] A typical recovery method is to reduce the binding strength of the target component to the chromatographic material, which can be achieved by using high salt concentrations, pH changes in the elution buffer, and by components that have a higher affinity for the chromatographic support.

[0034] In summary, the method of the present invention can be used to purify target components from and remove one or more target components from whole plasma samples without the need for pre-treatment of the plasma, such as dilution and / or cryoprecipitation, and without the risk of clogging of the continuous chromatographic materials due to plasma viscosity and / or activation of the coagulation cascade.

[0035] The present invention is further illustrated in the following examples, without however being limited thereto. EXAMPLES

[0036] Materials used in Examples 1 to 3 Fresh frozen human plasma (FFP), 260mL CIMmultus OH, 1 mL column (6 μm pore diameter) (Sartorius BIA Separations doo, Ajdovščina, Slovenia) CIMmultus QA, 8 mL strong anion exchange column (6 μm pore diameter) (Sartorius BIA Separations doo, Ajdovščina, Slovenia) CIMmultus r-Protein G, 1 mL columns (1.3 μm and 6 μm pore diameter) (Sartorius BIA Separations doo, Ajdovščina, Slovenia) Coagulation factor VIII-deficient plasma (Siemens Healthcare GmbH, Erlangen, Germany), Lot 547664A Dade Actin FS activated PTT reagent (Siemens Healthcare GmbH, Erlangen, Germany), Lot 538564

[0037] Example 1: Flow of human plasma through a hydroxyl-modified column Fresh frozen plasma (FFP) was thawed and centrifuged at 10000g for 20 min at 36°C to remove the lipid layer. 150 ml of human plasma was pumped onto a 1 ml CIMmultus OH column with 6 μm pore size (available from Sartorius BIA Separations doo, Slovenia) at a flow rate of 1.0 mL / min to 5.0 mL / min (0.2 CV / min to 1.0 CV / min, see reference

[20] ). The pressure was 0.10 mPa to 0.20 mPa and the temperature was set to 36°C and maintained at this value during the entire process. An Aekta Explorer chromatography system (Cytiva, Freiburg / Breisgau, Germany) was used.

[0038] The continuous chromatography unit used has hydrophilic inert OH groups immobilized on the inner surface of a monolith with large channels, the average diameter of the monolith channels being 6 μm. This chromatography material has an inert surface, allowing the flow-through of undiluted human plasma without activating the coagulation cascade. To demonstrate this embodiment, human plasma was pumped through the chromatography unit without binding, clogging, aggregation, activation of plasma components and / or precipitation of proteins and other macromolecules and nanoparticles contained in this biological fluid, i.e. dissolved and / or emulsified.

[0039] Prior to application, the CIMmultus column was washed and equilibrated with 50 mM phosphate buffer, pH 7.2, containing 155 mM NaCl at a conductivity of 15.9 mS / cm (buffer A). After adjusting the temperature to 36° C. and the flow rate to 3 mL / min, 150 ml of human plasma was passed through the column without clogging (no increase in back pressure was recorded). Samples were taken after thawing (FFP-1), before centrifugation (FFP-2), after centrifugation (FFP-3) and after pumping the following volumes of FFP through the column: 25 ml, 50 ml, 100 ml, and 150 ml. Non-specifically bound proteins were eluted in two steps with an elution buffer containing 0.25 M NaCl in 50 mM phosphate buffer, pH 6.95 and conductivity 25.8 mS / cm (buffer B) and an elution buffer containing 0.5 M NaCl in 50 mM phosphate buffer, pH 7.2 and conductivity 25.8 mS / cm (buffer C).

[0040] Figure 1 shows the chromatogram obtained and Table 1 shows the values ​​of the plasma coagulation status and possible complement activation measured before and after sample preparation and further processing of the human plasma. The tests for the determination of clotting factor VIII, partial thromboplastin time (PTT) and activated partial thromboplastin time (APTT) were carried out on a BCS XP, Siemens (Siemens Healthineers, Munich, Germany) at the Transfusion Department of the Pula General Hospital.

[0041] Principle of the coagulation assay: Factor VIII-deficient plasma, an in-vitro diagnostic reagent for the determination of the activity of coagulation factor VIII, was used to determine the clotting activity of the spiked samples according to the manufacturer's protocol. For details, see reference

[21] . For the determination of PTT and APTT, the Dade Actin FS activated PTT reagent (Siemens) was used. Factors of the intrinsic coagulation system are activated by incubating plasma with optimal amounts of phospholipids and surface activators. The coagulation process is triggered by the addition of calcium ions and the clotting time is measured. For details, see references [22, 23].

[0042] Tests for complement C1 (esterase inhibitor), C3 and C4 were performed on a Cobas C503 analytical unit (Roche Diagnostics, Rotkreuz, Switzerland) at the Department of Clinical Chemistry, Pula General Hospital. The Berichrom C1-Inhibitor Kit (Sigma Aldrich, Merck,KGaA, Darmstadt, Germany) is a human C1 esterase-based assay that determines the presence of C1 inhibitor in patient samples

[24] . This chromogenic activity reagent is used for the diagnosis of angioneurotic edema and for monitoring replacement or steroid therapy in angioneurotic edema. Complement C3 determination was performed using an immunoturbidimetric assay (Abcam, Cambridge, UK) in which human C3c forms a precipitate with a specific antiserum determined turbidimetrically. Complement C4 activity was determined using an immunoturbidimetric procedure that measures the increase in turbidity of the sample caused by the formation of insoluble immune complexes when an antibody against C4 (Abcam) is added to the sample

[24] .

[0043] [Table 1]

[0044] As shown in Table 1, pumping through the OH column did not affect the coagulation state of the plasma (FVIII activity, PT, and APTT) nor did it cause activation of complement components C1, C3, and C4.

[0045] In summary, based on the above experiments and the obtained experimental data, it was concluded that after optimization of temperature [36°C] and flow rate [3mL / min], 150mL of human plasma passed through the column without clogging and, surprisingly, no changes in the activity of coagulation FVIII and PTT and APTT, nor activation of complement components occurred. Non-specific binding and loss of plasma components was virtually zero, as shown by the absence of any substantial bands in the elution fractions E1 and E2 in SDS-PAGE in Figure 2.

[0046] It was further concluded that the OH-derivatized CIMmultus column could therefore be used for the direct processing of undiluted human plasma without the activation of the coagulation cascade and complement. If necessary, the processed human plasma could be returned to the patient's blood. The described experiments therefore provide a proof of concept that monolithic materials with surfaces immobilized with inert hydrophilic ligands could be used for the processing and / or purification of fresh undiluted human plasma, providing a powerful tool in various therapeutic applications.

[0047] Example 2: Treatment of undiluted plasma with a strong ion exchanger Fresh frozen plasma was thawed, centrifuged and the lipid layer was removed. 150 ml of the sample was pumped at a flow rate of 3.0 ml / min onto an 8 ml CIMmultus QA column with a pore size of 6 μm (available from Sartorius BIA Separations doo, Slovenia). FVIII and other proteins with affinity for the column bound and could be eluted (see FIG. 3). Coagulation factor VIII was chosen as a model protein for this experiment. The equilibration and washing buffer for the CIMmultus QA column was 20 mM phosphate buffer, pH 7.0, containing 155 mM NaCl, pH 7.0, with a conductivity of 17.6 mS / cm. Step elution was performed with 280 mM NaCl in 20 mM phosphate buffer at pH 7.0 and 27.9 mS / cm (buffer 1), 360 mM NaCl in 20 mM phosphate buffer at pH 7.0 and 34.7 mS / cm (buffer 2) and 500 mM NaCl in 20 mM phosphate buffer at pH 7.0 and 47.0 mS / cm. The chromatograms are shown in Figure 3. Fractions were collected and analyzed for protein content and clotting factor VIII activity (see Table 2) and the protein pattern was analyzed by SDS-PAGE (see Figure 4).

[0048] [Table 2]

[0049] In Table 2, γ represents the protein concentration in the sample. Samples ending with -1 were taken at the beginning of the elution of the fractions, and samples ending with -2 were taken at the end of the elution.

[0050] As shown in Table 2, after loading of 25 mL of plasma, factor VIII bound to the column and was not detected in the flow-through fraction (see fraction QA25 in Table 2). Upon overloading, FVIII is displaced from the column by other proteins with higher affinity for this anion exchanger and can be detected in subsequent fractions. The bound proteins can be eluted. As can be seen in the SDS-PAGE in Figure 4, the plasma proteins are stable throughout the experiment and are not affected by passing through the column, as suggested by the unchanged profiles of the initial plasma and flow-through fractions.

[0051] Conclusion: From experiment 1 and experiment 2 it can be derived that binding and subsequent elution of plasma components is possible without any clogging or precipitation during the processing of this biological fluid. The described experiments therefore provide a demonstration that the method described herein is suitable for processing whole undiluted plasma for therapeutic applications and / or for purifying / removing (harmful) target substances from whole undiluted human plasma.

[0052] Example 3: Processing of undiluted human plasma with a protein G affinity column Fresh frozen plasma was thawed, centrifuged and the lipid layer was removed. 150 ml of the sample was pumped at a flow rate of 3 mL / min onto an 8 mL CIMmultus Protein G column with 1.3 μm and 6 μm pore sizes modified with recombinant Protein G. The equilibration buffer (Buffer A) was 155 mM NaCl in 100 mM Tris.HCL buffer at pH 7.2 with a conductivity of 21.0 mS / cm. After pumping of undiluted human plasma, the column was washed with 10 column volumes of a wash buffer containing 0.5 M NaCl in 100 mM Tris.HCL buffer at pH 7.2 with a conductivity of 49.0 mS / cm. Bound antibodies (human IgG) were eluted with 0.1 M glycine-HCl, pH 2.08 and 14.25 mS / cm. The corresponding chromatogram is shown in FIG. 5. To demonstrate the stability of the plasma during this chromatography, the clotting factor VIII activity and the activated partial thromboplastin time were measured. The results are shown in Table 3. To confirm the recovery of IgG from the Protein G column, SDS-PAGE analysis was performed (Figure 6).

[0053] [Table 3]

[0054] Conclusion: Example 3 shows that binding of antibodies (including pathological antibodies) from patient plasma and further analysis for diagnostic purposes is possible without any clogging or precipitation during the processing of this biological fluid. The data suggests that the method described herein is suitable for the purification of target molecules such as antibodies that can then be used as drug molecules in various therapeutic applications. Furthermore, the data provides a demonstration that the method described herein is suitable for removing harmful target substances such as autoimmune antibodies from human plasma that can then be recycled into the patient's body.

[0055] References 1. EP 2 925 777 B1 2. Sidney, AL, Continuous Downstream Processing for High Value Biological Products, Biotechnol. Bioeng. 2016; 113(3): 465-475. 3. Vogel, J., Nguyen, H., Giovannini, R., Ignowski, J., Garger, S., Salgotra, A. Tom, J., A New Large-scale Manufacturing Platform for Complex Biopharmaceuticals Biotechnol. Bioeng. 2015; 109(12): 3049-3058. 4. M. Srajer Gajdosik, J. Clifton, Dj. Josic, Sample Displacement Chromatography as a Method for Purification of Proteins and Peptides from Complex Mixtures, J. Chromatogr. A. 2012, 1239, 1-9. 5. Dj. Josic, L. Breen, J. Clifton, M. Srajer Gajdosik, D. Gaso Sokac, M. Rucevic, E. Mueller, Separation of Proteins from Human Plasma in Hydrophobic Interaction Mode, Electrophoresis 2012, 33, 1842-1849. 6. Burnouf, T. Modern Plasma Fractionation, Transf. Med. Reviews, 2007, 21(2) 101-117. 7. Pool, J., Hershgold, E.J., Pappenhagen, A.R., High-potency Antihaemophylic Factor Concentrate prepared from Cryoglobulin Precipitate, Nature 1964, 203, 312. 8. Cohn, E., Strong, L., Hughes, W. Preparation and Properties of Serum and Plasma Proteins.IV. A System for the Separation into Fractions of the Protein and Lipoprotein Components of Biological Tissues and Fluids, J. Am. Chem. Soc. 1946, 68, 459-475. 9. Josic, Dj. Hoffer, L. et al. Manufacturing of a prothrombin complex concentrate aiming at low thrombogenicity, Thromb. Res. 2000, 100(5), 433-441. 10. Janakiraman, V.N., Prasanna, R.P., Kamalanathan, A.S., Vijayalakshmi, M.A. Monolith-based Pseudo-bioaffinity Purification Methods for Factor VIIIand Applications Thereof, WO14083510. 11. Rajamanickam V., Hervig, C., Spaduit, O., Monoliths in Bioprocess Technology, Chromatography, 2015, 2(2), 195-212. 12. National Institute of Diabetes and Digestive and Kidney Diseases, Hemodialysis, https: / / www.niddk.nih.gov / health-information / kidney-disease / kidney-failure / hemodialysis 13. Ciceri, P., Cozzolino, Expandend Haemodialysis as a Current Strategy to Remove Uremic Toxins, Toxins (Basel) 2021, 13(6) 380. doi: 10.3390 / toxins13060380 14. Lehmann, H.C., Kieseier, B.C., Hetzel, G.R., Grabensee, B., Hartung, H.-P. Plasmapheresis Therapy in the Neurology, Arzneimitteltherapie 2004, 22, 92-90. 15. Assessment of plasmapheresis. Report of the Therapeutics and Technology Assessment Subcommittee of the American Academy of Neurology. Neurology 1996, 47, 840-843. 16. Franz E, Hoerl W. editors. Blutreinigungsverfahren. Stuttgart, New York: Georg Thieme Verlag, 1997. 17. Drew, M.J. "Plasmapheresis in the Dysproteinemias". Therapeutic Apheresis, 2002, 6(1): 45-52. 18. Josic, Dj. Strancar, A., Application of Monoliths and Compact Porous Units for the Separation of Biopolymers", Industrial and Engineering Chemistry, 1999, 38(2), 333-342. 19. Svec, F., Jelinkova, M., Votavova, E., Macroporous Membranes, I, Reactive Macroporous Membranes Based on Glycidyl Methacrylate-Ethylene Dimethacrylate Copolymer for High-Performance Membrane Chromatography, Angew. Macromol. Chem. 1991, 188, 167-176. 20. Strancar, A., Koselj, P., Schwinn, H., Josic, Dj. Application of Compact, Porous Disks for Fast Separations of Biopolymers and In-Process Control in Biotechnology, Anal. Chem. 1996, 68(19), 3483-3488. 21. Sartorius / BIASeparations, Calculating Linear Flow Rate for CIM monoliths, https: / / www.biaseparations.com / en / technology / calculating-linear-flow-rate-for-cimr-monoliths 22. Potgieter, J.J. Damgaard, M., Hillarp, A. One-stage vs. Chromogenic Assays in Haemophilia A, Eur. J. Haematol. 2015, 94(S77) 38-44. 23. 1-stage APTT-based Factor Assays https: / / practical-haemostasis.com / Factor%20Assays / 1_stage_aptt_factor_assay.html 24. Horstl, J. Measurement of Prothrombin Time in EDTA Plasma with Combined Thromboplastin Reagent, Cli. Chem. 2000, 46(11) 1844-1846. 25. https: / / www.testing.com / tests / complement /

Claims

1. 1. A method for purifying a target component from a whole plasma sample, comprising: applying whole plasma to a continuous chromatographic material, said continuous chromatographic material comprising channels having a diameter of between 4 μm and 15 μm, said chromatographic material comprising chromatographic selective elements for said target components; carrying out the chromatography at a temperature of 30°C to 39°C; recovering the target component from the continuous chromatographic material; A method comprising:

2. 1. A method for removing one or more target components from a whole plasma sample, comprising: applying whole plasma to a continuous chromatographic material comprising channels having a diameter of between 4 μm and 15 μm, said chromatographic material comprising chromatographic selective elements for said one or more target components; carrying out the chromatography at a temperature of 30°C to 39°C; collecting the plasma after removal of the one or more target components; A method comprising:

3. 3. The method of claim 1 or 2, wherein the whole plasma is human plasma.

4. 3. The method of claim 1 or 2, wherein the whole plasma is selected from fresh frozen plasma or patient plasma.

5. 3. The method of claim 1 or 2, wherein the one or more target components are selected from the group consisting of proteins, allergens, nucleic acids, viruses, toxins and nanoparticles.

6. 6. The method of claim 5, wherein the nanoparticle is an exosome.

7. 6. The method of claim 5, wherein the protein is selected from the group consisting of clotting factors, clotting inhibitors, antibodies, and other biologically active proteins.

8. The method of claim 7, wherein the protein is clotting factor VIII.

9. 3. The method of claim 1 or 2, wherein the one or more target components comprise one or more harmful components selected from the group consisting of pathological antibodies, macromolecular toxins, nucleic acids, and nanoparticles.

10. 3. The method of claim 1 or 2, wherein the chromatographic selection element is selected from the group consisting of anion exchange ligands, cation exchange ligands, affinity ligands, HIC ligands, oligonucleotides, aptamers, and combinations thereof.

11. 3. The method of claim 1 or 2, wherein the continuous chromatographic material is a compact monolithic material or a membrane adsorber.

12. 3. The method of claim 1, wherein the continuous chromatographic material is a continuous methacrylate chromatographic material.

13. The method of claim 1 or 2, wherein the channel has a diameter in the range of 4 μm to 10 μm.

14. 3. The method of claim 1, wherein the chromatography is carried out at a temperature of 35°C to 37°C.

15. 3. The method of claim 2, wherein the plasma is returned to the patient after removal of the one or more target components.

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