Method for purifying target products using affinity purification techniques

Abstract

The present invention relates to purification techniques using affinity purification techniques. In particular, the present invention relates to methods of purifying a target product using amphiphilic and polypeptides wherein the use of a solid matrix or column is avoided.

Description

Technical Field

[0001] The present invention relates to a general affinity purification technique using bivalent affinity polypeptides. In particular, the present invention relates to a rapid and efficient method for removing bivalent affinity proteins from target molecules in solution. Background Art

[0002] The most common method for affinity purification of biomolecules such as proteins at present is affinity column chromatography, in which the target biomolecule binds to an affinity ligand immobilized on a column resin or a solid matrix, is washed, and then recovered from the column resin by elution.

[0003] The affinity solid support, resin beads, matrix or solid phase is functionalized with a specific target-binding molecule or ligand and is usually packed in a column.

[0004] The resin beads used in affinity purification are prepared from polymers using chemical cross-linking reactions. Generally, these beads have a size of about 90 - 100 μm and contain multiple pores of about 30 nm. Both the outer surface and the inner surface of the beads can be used for attaching the affinity ligand.

[0005] The outer surface area of these beads is about 1 / 100 of the inner surface area. This means that only a small part of the bead surface is available for ligand attachment on the outside, while most of the ligands are immobilized inside the resin beads.

[0006] Therefore, the product molecules can only interact with the attached affinity ligand by undergoing diffusion-based mass transfer to reach the inner surface of the beads. This diffusion process takes time and is characteristic of all resin chromatography processes, whether on a laboratory scale, a pilot scale or an industrial scale. The present invention aims to eliminate or at least reduce these technical limitations.

[0007] Traditional affinity purification involves the following general steps:

[0008] A. Binding the target by incubating a crude sample (e.g., cell lysate or plasma) with an affinity solid support to allow the target molecule in the sample to bind to the immobilized ligand;

[0009] B. Washing away impurities (unbound sample components) from the solid support using an appropriate buffer that maintains the binding interaction between the target and the affinity ligand;

[0010] C. Eluting and recovering the target molecule from the immobilized ligand by changing the buffer conditions so that the binding interaction between the target and the ligand no longer occurs; and then

[0011] D. If the affinity ligand solid support is to be used again, regeneration or in-situ cleaning (CIP) is carried out.

[0012] EP 2220107 B1 discloses a method for purifying a target biomolecule, comprising the steps of: (a) contacting (i) the target biomolecule, (ii) a bifunctional polypeptide, and (iii) a solid matrix comprising a capture ligand; and (b) recovering the target biomolecule by elution, wherein the target biomolecule and the bifunctional polypeptide are contacted in solution before the mixture contacts the solid matrix.

[0013] EP 2427482 B1 discloses a method for purifying a target molecule, comprising the steps of: (a) contacting the target molecule and a population of target-binding polypeptides (TBPs) in solution for a sufficient time to allow complex formation; and (b) separating the target from the complex from (a) by subsequent purification steps, wherein (i) the target-binding polypeptide has at least two binding functionalities; a first binding functionality for the target and a second binding functionality for a capture ligand comprised in a solid matrix; and (ii) the first binding functionality comprises at least two binding sites for the target, and the target comprises at least two binding sites for the TBP.

[0014] Prior art methods utilize resin column or solid matrix purification processes, where the resin is optimized in terms of particle size to control surface area, binding capacity, and backpressure within the column. This is also done to optimize overall purification efficiency and minimize operating costs.

[0015] Due to resin properties, the most commonly used packed bed resin columns are prone to increased backpressure and non-uniform flow patterns upon scale-up, which can lead to resin collapse. Productivity in terms of the amount of target product purified per hour, in-situ cleaning time, and total operating cost is closely related to column size, format, and resin properties. Therefore, it is difficult to increase purification capacity or reduce the size and footprint of column-based systems when scaling up the purification process. Additionally, it is difficult to reduce process water consumption because in-situ cleaning (CIP) is required when reusing expensive affinity column resins.

[0016] Purifying a target by affinity chromatography using a packed column is well known. This is described, for example, in WO2023012321A1.

[0017] Various variants of traditional techniques attempt to address the aforementioned drawbacks.

[0018] EP 2220107 B1 and EP 2427482 B1 describe the purification of a target on a solid matrix using a protein A-streptavidin fusion protein, followed by elution and recovery of the target. These techniques use a solid resin matrix to capture the fusion protein.

[0019] Purification procedures using Protein A attached to a soluble polymer are described in US2009232737 A1 and WO2016049761 A1. The chromatographic material is a soluble polymer having a chemically attached binding moiety (e.g., an antigen) for the target to be purified. The polymer can be precipitated by changing the temperature or pH. The target product is purified by first binding the polymer in solution, precipitating the polymer, a washing step, and eluting the target protein from the polymer.

[0020] Other affinity purification procedures for target proteins are described in US2008108053 A1, WO2020037100A1, WO2012055854 A1, WO2018178991 A1, WO2021168270 A1, WO2006110292A2, WO2009078018A2, WO2008067591A1, which all describe various affinity purification systems using portions containing Protein A anchored fusion proteins, which can precipitate the entire target-Protein A fusion protein complex. The precipitation is accomplished by, for example, adding metal ions, changing the temperature or pH. The precipitated complex is collected and washed, and the target is eluted from the complex and collected. All of the above techniques, like conventional affinity purification procedures, focus on capturing and purifying the target protein by first binding to the target, a washing step, then an elution step and collecting the target protein.

[0021] Other potentially relevant disclosures include US2012238039 A1, WO0031128 A1, WO2017167960A1, US2013337528A1, WO2022179970A1, US2021024616A1, WO2021007484 A1 and WO2013177115A2.

[0022] Accordingly, there remains a need for a procedure for purifying target molecules where the reaction time and total cost can be significantly reduced and productivity can be increased.

[0023] The inventors have recognized that it is preferable to apply a soluble matrix with covalently bound ligands to address the time-consuming mass transfer problem by diffusion and establish a rapid and scalable purification procedure.

[0024] In a method for purifying a target biomolecule using a dual-affinity polypeptide (DAP), the inventors were surprised to find that the binding reaction in solution (DAP binding to a biotinylated linker-matrix) was much faster than when DAP reacted with a biotin ligand immobilized on the inner surface of resin beads. The affinity binding reaction of the soluble ligand-matrix was on the order of seconds (90 - 150 seconds) and was much faster than that of the ligand immobilized on resin beads. In the latter case, the reaction time (retention time in the column) was much longer and was in the order of minutes (50 - 70 minutes).

[0025] The extended reaction time is mainly due to the mass transfer of molecules reaching the inner surface of the beads by diffusion. This delay is exacerbated by the fact that chemical reactions in solution are known to occur approximately 1000 times faster than heterogeneous reactions on surfaces. (See Nygren, H. and Stenberg, M. (1989) Immunochemistry at Interfaces. Immunology, 66, 321 - 327).

[0026] In the present invention, a rapid affinity protocol is exemplified by removing DAP from a solution of the target biomolecule, which provides an example and background for new process modalities such as queuing processing and continuous process design for industrial applications.

[0027] Therefore, there is still a need for a protocol for purifying target molecules in which the reaction time and total cost can be significantly reduced and productivity can be increased.

[0028] Object of the Invention

[0029] An object of embodiments of the present invention is to provide a method for purifying a target molecule that is scalable, results in reduced processing time and thus increased productivity and target product stability, and results in reduced process footprint and wastewater consumption. Summary of the Invention

[0030] The inventors have found that by avoiding the use of solid matrices and resin columns and instead using a soluble capture matrix, an effective and industrially applicable scale-up method is provided that results in shorter overall processing times and reduced retention times for the target biomolecule, for example at low pH, which may reduce the risk of denaturation or degradation of the target biomolecule.

[0031] The inventors have recognized that it is preferable to apply a soluble matrix with covalently bound ligands to address the time-consuming mass transfer problem by diffusion and establish a rapid and scalable purification protocol.

[0032] In the method of purifying a target biomolecule using a dual-affinity polypeptide (DAP), to the inventors' surprise, the binding reaction in solution (DAP binding to the biotin linker-matrix) is much faster than when DAP reacts with a biotin ligand immobilized on the inner surface of resin beads. The affinity binding reaction of the soluble ligand-matrix is on the order of seconds (90 - 150 seconds) and is much faster than that of the ligand immobilized on resin beads. In the latter case, the reaction time (retention time in the column) is much longer and is in minutes (50 - 70 minutes).

[0033] In the present invention, a rapid affinity protocol is exemplified by removing DAP from a solution of the target biomolecule, which provides an example and background art for new process modes such as queuing processing and continuous process design for industrial applications.

[0034] Accordingly, in a first aspect, the present invention relates to a method for purifying a target biomolecule, which comprises the following steps: a. in solution, contacting i) a starting composition comprising the target biomolecule and ii) a dual-affinity polypeptide (DAP) to allow the DAP to form a 3D complex with the target biomolecule, the DAP having a first binding functionality for the target biomolecule and a second binding functionality for a capture ligand; b. washing the formed target biomolecule-DAP complex to remove any impurities and form a pre-purified target biomolecule-DAP complex; c. treating the pre-purified target biomolecule-DAP complex at a pH below 5.0 to separate the pure target product biomolecule from the DAP by dissolving the 3D complex, thereby providing a mixture of the target product biomolecule and DAP; d. contacting the mixture of step c. with a soluble matrix covalently bound to the capture ligand to form, in solution, a separate pure target biomolecule and a separate DAP-capture ligand-matrix product; and e. recovering the pure target biomolecule. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Shows the formation of a 3D complex in solution between the target biomolecule and DAP, i.e., the first step (step a.) of the claimed method.

[0036] Figure 2 Shows washing the formed target biomolecule-DAP 3D-complex to remove any impurities and capturing the complex to obtain a pre-purified target biomolecule-DAP 3D-complex, i.e., the second step (step b.) of the claimed method.

[0037] Figure 3Shows the treatment of the pre-purified biomolecule-DAP complex by dissolving it in a buffer composition with a pH below 5.0 to separate the target product biomolecule and DAP, which is the third step (step c.) of the claimed method. The resulting mixture is then subjected to a soluble matrix immobilized with a capture ligand to remove DAP from the target biomolecule, which is the fourth step (step d.) of the claimed method.

[0038] Figure 4a and 4b Shows the recovery of the pure target biomolecule (step e.), which uses, for example, a single semipermeable membrane (4a) or a crossflow filtration system with, for example, several semipermeable membranes to collect the target product biomolecule in continuous mode and remove the DAP-capture ligand-matrix complex (4b).

[0039] Figure 5 : Illustrates DAP binding (%) / subtraction (%) as a function of biotin-linker dextran concentration. The DAP binding ability is measured by SEC HPLC by determining the decrease in the DAP peak (AUC) as a function of the increase in the amount of biotin dextran. The curve rises to the point of 100% DAP binding (equivalence point) and then remains constant (horizontal part).

[0040] Figure 6 : Illustrates the filtrate content in Example 11. The SDS-PAGE gel shows the complete removal of DAP by filtration through the biotin-linker soluble matrix PBA 0268 in a C65 depth filter. Lane 1: (“M”): MW marker (10 μl Protein Ladder SeeBlueTM Plus2), Lane 2 (IgG + DAP): 5 μg Privigen (IgG) + DAP (starting material solution); Lane 3 (7a): 5 μg of protein loaded from the first filtrate fraction; and Lane 4 (7b): 5 μg of protein loaded from the filtrate 2. filtrate fraction. No DAP was detected in the first filtrate fraction.

[0041] Figure 7 : Illustrates the filtrate content in Example 13. The SDS-PAGE gel shows the partial removal of DAP by the biotin-linker soluble matrix PBA 0268. Lane 1: (“M”): MW marker (10 μl Protein Ladder SeeBlueTM Plus2), Lane 2 (IgG + DAP): 5 μg Privigen (IgG) and DAP (starting material solution); Lane 3 (7a): 5 μg of protein loaded from the first filtrate fraction, and Lane 4 (7b): 5 μg of protein loaded from the filtrate 2. filtrate fraction, etc. Detailed Description

[0042] The present invention completely changes the prior art affinity purification technology, which captures the target on a solid support packed in a column, washes the column and recovers the target by elution.

[0043] It should be clear that the present invention has different purification workflows: (1) First, react the target biomolecule in the crude solution with the added dual-affinity polypeptide (DAP molecule) in solution to form a 3D complex of the target biomolecule and DAP; (2) Capture the target biomolecule-DAP 3D complex by filtration such as depth filtration and wash away other impurities; then (3) Dissolve the target biomolecule-DAP 3D complex in a buffer solution to separate the target product biomolecule from DAP; and (4) Bind DAP to a soluble matrix, remove and discard it by filtration, and collect the purified target biomolecule in solution.

[0044] In this method, the added DAP (purifying agent) does not come from the raw material or crude material containing the target biomolecule. The added DAP is a reagent for assisting the purification process.

[0045] After dissolving the DAP-target biomolecule 3D complex, the mixture only contains the target biomolecule and DAP. The inventors have recognized that DAP can be effectively removed from the target biomolecule and discarded by binding DAP to a soluble matrix, which is easily removed by, for example, filtration or other unit operations suitable for large-scale operation.

[0046] Binding the DAP molecule to a soluble matrix converts it into a larger molecule. This conversion increases the difference in size between it and the target biomolecule, making the two easier to separate.

[0047] It should be understood that the DAP molecule is not the intended target to be purified in this affinity purification process. Instead, the intended target is the target biomolecule.

[0048] After the target biomolecule-DAP 3D complex is dissolved from, for example, a depth filter (step 3), the filter material can be discarded. In addition, after the second filtration step, the captured DAP bound to the soluble matrix can be discarded. Therefore, there is no reuse of materials, and no in-situ cleaning protocol is required at any step of the method.

[0049] The purification protocol allows for a shorter retention time of the target biomolecule and a faster binding reaction in solution. This is crucial for large-scale manufacturing, reducing manufacturing costs, and optimizing the processing of the effective target product.

[0050] The core of the present invention is the effective, specific, and rapid removal of the added purifying agent (DAP molecule) by capturing it on a soluble matrix, leaving the pure target biomolecule undisturbed in solution.

[0051] In bead-based protocols, in situ cleaning (CIP) is crucial for preventing cross-contamination when repeatedly reusing expensive affinity chromatography beads. Reusing the beads is necessary to make bead-based protocols economically viable.

[0052] The present invention addresses the significant drawbacks of affinity chromatography by introducing a fundamental shift away from bead-based affinity purification. By rapid elution, efficient DAP removal using a soluble matrix, and effective filtration, followed by pH adjustment, the present invention significantly reduces the overall processing time, especially under low pH conditions. This method ensures that the quality of the target biomolecule is maintained at an appropriate pH level.

[0053] This speed is achieved through a homogeneous binding step and efficient filtration, and it eliminates the need for expensive beads, column packing protocols for obtaining a uniform flow, and the mandatory CIP protocols and associated quality assurance checks upon reuse.

[0054] The definitions “purifying,” “purification,” or other grammatically different forms of these expressions are intended to refer to the complete or partial removal of a target biomolecule from at least one non-target product present in an initial composition containing the target biomolecule.

[0055] The “target product biomolecule” or “target biomolecule” can in principle be any compound for which its specific binding part is known and which is soluble, preferably soluble in water or an aqueous solution. Preferably, the target biomolecule is a peptide, polypeptide, antibody, virus particle, exosome (extracellular vesicle), cell, or cell component, more preferably an antibody.

[0056] The “initial composition containing the target biomolecule” can in principle be any such composition regardless of its origin. As preferred examples of the initial composition containing the target biomolecule, mention may be made of the cell-free culture broth of a cell culture producing the target product biomolecule or any partially purified fraction thereof, or for example human plasma.

[0057] In the case where the initial composition containing the target biomolecule is a culture broth, it is preferably pretreated before applying the method of the present invention to provide an initial composition containing the target biomolecule product and free of any particulate material. Such pretreatment methods are well known in the art and can be, for example, various conventional filtration techniques or, for example, centrifugation of a cell suspension in the case where the target biomolecule is extracellular, or, for example, cell homogenization followed by filtration or centrifugation in the case where the target biomolecule is intracellular.

[0058] In the case where the starting material is human blood, a filtration or centrifugation step may be included before practicing the present invention to remove blood cells and isolate the plasma fraction in which, for example, the target biomolecule is dissolved.

[0059] According to the present invention, "dual-affinity polypeptide" (DAP) is intended to mean a polypeptide having two or more different binding sites that are specific for two or more target molecule binding sites or ligands. Descriptions of DAP and DAP technology can be found in EP22201017 B1 and EP 2427482 B1. The teachings of these two documents are incorporated herein by reference.

[0060] According to the present invention, the term "first binding functionality" is intended to mean a binding functionality having an affinity for a target biomolecule or a group or portion of a target biomolecule bound by the first binding site of a DAP. The affinity of the first binding functionality of the DAP should be high enough to allow specific binding of the target biomolecule to the DAP. The equilibrium dissociation constant K of the DAP with the target biomolecule D,t is preferably in the range of 10 -8 -10 -4 more preferably in the range of 10 -7 -10 -5 and most preferably about 10 -6 .

[0061] In the context of the present invention, the equilibrium dissociation constant is measured according to the following reaction:

[0062]

[0063] A and B represent binding partners: the target biomolecule and the dual-affinity polypeptide or the dual-affinity polypeptide and a capture ligand covalently coupled to a soluble matrix.

[0064] The rate constants of the above reaction represent the rates of association and dissociation of the two molecules A and B:

[0065] Dissociation rate

[0066] Association rate:

[0067] When the rates are equal at equilibrium ka[A][B]=kd[AB], we get:

[0068]

[0069] In the context of the present invention, the term "second binding functionality" refers to the binding functionality of DAP that has a high binding affinity for a separate second ligand such as a capture ligand. The DAP according to the present invention has a first binding functionality that has an affinity for a target product biomolecule and another second binding functionality (i.e., another binding site) that has an affinity for a capture ligand covalently coupled to a matrix.

[0070] According to the present invention, "capture ligand" is intended to refer to a ligand that is different from the target product biomolecule and is bound by the "second binding functionality" of DAP. According to the present invention, the capture ligand is covalently linked to a soluble matrix. The capture ligand according to the present invention may also be covalently linked to the soluble matrix through a linker. The capture ligand can in principle be any moiety or group having the ability to specifically bind to / with DAP. The affinity of the capture ligand for DAP should also be high enough to allow efficient binding of DAP to the soluble matrix. The equilibrium dissociation constant K of DAP and the capture ligand D，s is preferably in the range of 10 -16 -10 -10 , more preferably in the range of 10 -15 -10 -11 , and most preferably in the range of about 10 -14 -10 -12 .

[0071] The ratio [K D,t / K D,s of the binding coefficient of the first ligand to DAP to the binding coefficient of the second ligand to DAP is preferably at least 10, such as greater than 10, preferably greater than 100, more preferably greater than 1000, more preferably greater than 5000, even more preferably greater than 10000, and most preferably greater than 20000.

[0072] The term "3D complex with the target biomolecule" is intended to refer to a three-dimensional complex formed by a binding reaction between the target biomolecule and DAP.

[0073] The term "pre-purified biomolecule" is intended to refer to a mixture of target biomolecule-DAP 3D-complexes present in solution without any other impurities.

[0074] The term "dissolve" or "dissolution" of the target biomolecule-DAP complex refers to the step of treating the target biomolecule-DAP complex in a buffer solution to separate the target product biomolecule from DAP to obtain a mixture of the target biomolecule and DAP.

[0075] The term "mixture of target biomolecule and DAP" is intended to refer to a two-component mixture of a target biomolecule in solution in the presence of DAP. The target biomolecule at this stage is no longer bound or linked to DAP molecules, and thus DAP can be regarded as an impurity in the mixture, which will subsequently be removed by the subsequent steps of the method of the present invention to produce a pure target product biomolecule.

[0076] A final filtration step is performed on a solution containing only buffer components, a target biomolecule, and DAP bound to a soluble matrix. The primary purpose of the final filtration step is to be rapid, gentle, and non-interactive with the target biomolecule. Ideally, the target biomolecule should remain largely unaffected during the removal of the DAP-soluble matrix complex.

[0077] Key characteristics of the soluble matrix include water solubility or solubility in a solution with a water content of more than 50%, availability in industrial-scale quantities, ease of modification with binding ligands, and non-toxicity. To increase the reaction rate of binding, the soluble matrix should be in a non-solid state, rather than in the form of amorphous particles, and should exist as molecules in a nearly perfect solution. This property allows for efficient mixing, convection, and thus rapid binding of DAP to the matrix in solution.

[0078] In one embodiment of the present invention, the soluble matrix is a polymer.

[0079] Non-limiting examples of suitable polymers include dextran, xanthan gum, pectin, chitin and chitosan, carrageenan, guar gum, cellulose ethers, hyaluronic acid, albumin, hydroxyethyl starch and other starch derivatives, polyacrylic acid, polyacrylamide, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, divinyl ether-maleic anhydride, polyoxazoline, polyphosphates, polyphosphazenes, their copolymers, and their mixtures. Preferably, the matrix is a polymer selected from the group consisting of dextran, carrageenan, pectin, cellulose ethers, polyacrylic acid, polyacrylamide, polyvinyl alcohol, their copolymers, and their mixtures, and even more preferably, the matrix is a dextran polymer.

[0080] The soluble matrix in polymer form can be partially crosslinked to increase the total molecular weight and change the molecular size, flexibility, and density, up to the extent that it remains soluble.

[0081] Filtration in this context is a separation process that separates and removes the matrix bound to DAP from the target biomolecule.

[0082] Filters have a wide range of applications in water treatment, food processing (including dairy, brewing, and juice production), chemical processes, and the life sciences, particularly in the downstream processing of recombinant proteins, polishing steps, and virus removal.

[0083] A variety of filtration processes are generally understood within the field of chemical and biological unit operations. Filtration is widely used in industrial applications and encompasses a variety of methods, such as coarse particle filters, asymmetric depth filters, depth filters using filter aids, or dead-end filters, as well as nanofiltration, ultrafiltration, and microfiltration using specialized membranes. It is noted that the terms "filter" and "membrane" and "filtration" and "membrane filtration" may be used interchangeably herein.

[0084] The membrane can be in the form of, for example, flat sheets, hollow fibers, and arranged in, for example, plate-and-frame modules or spirals in a housing having a single or multiple chambers.

[0085] Whether in batch or continuous operation, the filtration system offers a variety of options. It can combine controlled flow rates and pressures, use cross-flow or counter-current arrangements, conventional or single-pass tangential flow filtration, with or without recycle, and optimize factors such as the target biomolecule collection rate, retention time, fouling, energy consumption, and operating costs.

[0086] Other advanced filtration systems are well-known to industry experts, such as large-scale setups with automatic scraper rough filters, overflow and recycle configurations, stack filters, variable filter sizes, and automatic monitoring and control of flow rates and pressures. These combinations are readily available in industrial applications.

[0087] Considering that the target biomolecule is in the eluate while the DAP-matrix is retained, continuous filtration operation is preferred. This option helps to reduce the downstream processing volume, reduce the retention time, and minimize the environmental and physical footprint of the operation.

[0088] Industrial filtration employs a wide range of materials with various shapes and properties, from basic choices such as raw paper, cloth, polymer, and glass fiber matrices to advanced polymer membranes with precise pore structures and permeation properties, such as ceramics, polysulfone, regenerated cellulose, cellulose acetate, nitrocellulose, cellulose esters, polysulfone, polyethersulfone, polyacrylonitrile, polyamide, polyimide, polyethylene, polypropylene, polytetrafluoroethylene, polyvinylidene fluoride, polyvinyl chloride, and their blends.

[0089] Filter aids are used, for example, in depth filtration operations. Filter aids are typically low-density, fibrous, or fine-particle materials used to prevent fouling and blockage of the filter, increase the flow rate, and improve the operating performance and filtration quality.

[0090] Filter aids are used to fill depth filters, which improve the permeability of the filter cake and sometimes the porosity, improve the transparency of the filtrate, and help prevent clogging of the filter medium or the filter. Filter aids include relatively porous particles such as diatomaceous earth, perlite, celite, kieselguhr, or activated carbon, and are filtered onto the medium as a precoat or, alternatively, mixed with the solution or suspension as a body feed during a pretreatment stage.

[0091] In most filtration processes, the separation mechanism depends on a combination of size and interaction with the filter membrane, filter aid, or system. This means that it is often challenging to clearly distinguish between size-based separation and other factors such as ionic or hydrophobic interactions when molecules pass through the filter or membrane.

[0092] The inventors have recognized the importance of both the size and size distribution of the soluble matrix. A greater size difference between the target biomolecule and the DAP-matrix simplifies the filtration process.

[0093] Polymers or polymer populations typically exhibit a size distribution and an average molecular weight. In this context, the average molecular weight is less critical than, for example, the average size of the smallest 10% of the distribution. A wider distribution means a higher proportion of smaller molecules in the population.

[0094] Therefore, it is preferred to apply a matrix such as a DAP polymer that has a narrow molecular weight distribution and an average size large enough to optimize the effectiveness of the filtration separation. Even more preferably, a polymer population from which the smallest molecules have been removed is used. Such polymers can be characterized by both the average molecular weight and the molecular weight interval of the lowest 10% of the population.

[0095] To eliminate the smallest molecules, techniques such as crossflow filtration with an appropriate membrane cutoff can be employed. This results in a polymer matrix population with a size not lower than the molecular cutoff value. It is important to note that the amount of DAP bound to the matrix increases the average molecular size and broadens the size distribution of the DAP-matrix population.

[0096] The ultimate goal is to establish a substantial size difference between the DAP-matrix and the target biomolecule, ensuring that all DAP-matrix molecules within the population are significantly larger than the target biomolecule. This facilitates rapid and efficient separation by filtration.

[0097] The core of the present invention is to add a soluble matrix to a solution containing a target biomolecule and a DAP purifying agent. The DAP binds to the matrix, converting it into a much larger molecule with significantly different properties compared to the target biomolecule. The matrix-bound DAP can be considered a very large polymeric DAP molecule, containing specific properties of DAP, including hydrophobicity, ionic nature, or other physical or chemical properties.

[0098] The choice of an effective filtration method is well-known to those skilled in the art. The solution of the target biomolecule and the DAP bound to the soluble matrix is pumped into a filtration system, and the target biomolecule and the DAP-bound matrix are separated and measured in the retentate and filtrate, respectively. The performance can be fine-tuned by varying, for example, the filter type and arrangement, the type and volume of the filter aid, or the membrane type or area, pressure, circulation, and flow rate. The aim is to find the conditions for recovering the target biomolecule and removing the DAP-matrix as much as possible. Preferably, the DAP content in the filtrate is less than 1%, more preferably less than 100 ppm, and even more preferably less than 5 ppm.

[0099] Surprisingly, it was found that both membrane systems and traditional depth filters with filter aids proved highly effective in separating the DAP bound to the matrix from the target biomolecule. The target biomolecule was successfully recovered in the filtrate, while the DAP bound to the matrix remained in the retentate or within the depth filter matrix and could subsequently be discarded.

[0100] Specific embodiments of the present invention

[0101] The method according to the present invention provides reduced reaction and incubation times, which results in increased productivity. This process is easy to scale up. In addition, the described method reduces production costs and allows implementation on an industrial scale.

[0102] Step a. of the method -- Figure 1

[0103] In the first step of the method of the present invention, an initial composition containing a target biomolecule is contacted with a DAP having an affinity for the target biomolecule in solution, and the mixture of the initial composition containing the target biomolecule and the DAP is maintained in solution for a time suitable for forming a 3D complex of the DAP and the target biomolecule. The target biomolecule can be, for example, a protein, or preferably an antibody, a virus particle, an exosome, or a cell or cell component. Since the binding reaction is rapid in solution, this contact can in principle be carried out using any protocol and equipment capable of efficiently mixing the two liquids, including, for example, both traditional mixers and static mixers. Such operations are well-known, and those skilled in the art will understand to select appropriate equipment and conditions for this step.

[0104] The 3D complex is formed immediately after mixing the components and thus does not require a long incubation time for 3D complex formation.

[0105] Step b. of the method -- Figure 2

[0106] The 3D complex formed in step a of the method is washed to remove any impurities and form a pre-purified target biomolecule-DAP 3D complex. Preferably, the washing is carried out by filtration, such as via depth filtration. Filter aids can be used to establish a depth filter to improve the filtration capacity and efficiency.

[0107] Since the 3D complex formed in step a. is partially insoluble, forming a turbid suspension, filtration is possible.

[0108] Buffers can be used to wash the captured biomolecule-DAP 3D-complex and wash away impurities.

[0109] Step c. of the method -- Figure 3

[0110] In the third step, the target biomolecule is dissolved from the DAP, i.e., the target biomolecule-DAP bond is broken using a method that depends on the specific nature of the binding site of the target biomolecule and the DAP. For example, the pre-purified target biomolecule-DAP 3D-complex can be treated with a buffer. The 3D complex thus dissolves in the buffer, which dissociates the target product from the DAP. This buffer can be the same buffer used in the second step, or this buffer can be a buffer different from the buffer used in the second step. The solution at this time contains only the target biomolecule and the DAP.

[0111] Preferably, the target biomolecule-DAP-3D-complex can be dissolved into the target biomolecule and the DAP by changing the pH in the elution buffer and more preferably by lowering the pH below 5.0. Preferably, the pH in step c. is in the range of 2.8 - 4.7, preferably in the pH range of 3.1 - 4.5, more preferably in the pH range of about 3.4 - 4.3.

[0112] Step d. of the method described above—— Figure 3

[0113] In the next step, the mixture containing the target biomolecule and the DAP in solution is contacted with a soluble matrix covalently bound with a capture ligand to form a target biomolecule and DAP-capture ligand-matrix product. In this step, only the DAP will bind to the soluble matrix through the second binding functionality, and the target biomolecule will remain unaffected in solution.

[0114] The soluble matrix can be a water-soluble matrix or soluble in an aqueous solution. However, other solvents can also be used, such as organic solvents, as long as the solvent is compatible with the target biomolecule and the matrix. The matrix can be linear, non-linear, branched or cross-linked.

[0115] The soluble matrix can be a polymer selected from the group consisting of dextran, polyacrylic acid, polyacrylamide, polyvinyl alcohol, polyethylene glycol, copolymers thereof and mixtures thereof. Preferably, the matrix is a dextran polymer.

[0116] The polymer can have an average molecular weight (Mw) of 200 - 5,000 kDa. Preferably, the polymer has an average molecular weight (Mw) of 200 - 5,000 kDa, where the smallest 10% fraction in the distribution is greater than 200 kDa. Even more preferably, the polymer has an average molecular weight (Mw) of 500 - 4,000 kDa, where the smallest 10% fraction in the distribution is greater than 500 kDa.

[0117] The dextran polymer can have an average molecular weight (Mw) of 200 - 5,000 kDa. Preferably, the dextran polymer has an average molecular weight (Mw) of 200 - 5,000 kDa, where the smallest 10% fraction in the distribution is greater than 200 kDa. Even more preferably, the dextran polymer has an average molecular weight (Mw) of 500 - 4,000 kDa, where the smallest 10% fraction in the distribution is greater than 500 kDa.

[0118] Optionally, the size difference between the matrix and the target biomolecule, such as the difference in average molecular weight (Mw), is at least 3-fold, such as at least 5-fold, such as at least 7-fold, such as at least 10-fold. The size and size distribution can be estimated by a variety of methods, including, for example, size exclusion chromatography, dynamic light scattering, hydrodynamic chromatography and field flow fractionation.

[0119] To provide efficient recovery of the pure target biomolecule, the size difference between the DAP soluble matrix and the target biomolecule, such as the difference in average molecular weight (Mw), is at least 3-fold, such as at least 5-fold, such as at least 7-fold, such as at least 10-fold. The molecular size can be described by the Stokes radius.

[0120] The matrix can be linked to the capture ligand via a linker. However, conjugation chemistry can be used to directly link the capture ligand to the matrix without a linker.

[0121] In the case of using a linker molecule to create space between the soluble matrix and the capture ligand, conjugation chemistry is used to covalently link the linker to the soluble matrix. A preferred method for covalently linking the linker to the soluble matrix can be, for example, coupling an amino-functionalized capture linker to carboxyl-modified dextran using carbodiimide or an active ester.

[0122] When the matrix is connected to the capture ligand via a linker molecule, the linker preferably has a length of 10 - 25 bonds, such as a length of 10 - 24 bonds, such as a length of 10 - 23 bonds, such as a length of 10 - 22 bonds, such as a length of 10 - 21 bonds, such as a length of 10 - 20 bonds, such as a length of 10 - 19 bonds, such as a length of 10 - 18 bonds, such as a length of 10 - 17 bonds, such as a length of 10 - 16 bonds, such as a length of 10 - 15 bonds, such as a length of 10 - 14 bonds, such as a length of 10 - 13 bonds, such as a length of 10 - 12 bonds, such as a length of 10 - 11 bonds, such as a length of 11 - 25 bonds, such as a length of 12 - 25 bonds, such as a length of 13 - 25 bonds, such as a length of 14 - 25 bonds, such as a length of 15 - 25 bonds, such as a length of 16 - 25 bonds, such as a length of 17 - 25 bonds, such as a length of 18 - 25 bonds, such as a length of 19 - 25 bonds, such as a length of 20 - 25 bonds, such as a length of 21 - 25 bonds, such as a length of 22 - 25 bonds, such as a length of 23 - 25 bonds, such as a length of 24 - 25 bonds.

[0123] The linker can also be a peptide.

[0124] The linker can be selected from the group consisting of: 2 - Vinyl - 4,4 - dimethyl - 5 - oxazolone (VDMA), vinyl azlactone derivatives, acrylic derivatives, hexandiisocyanate (HDI) derivatives, diisocyanate derivatives, or mixtures thereof.

[0125] The capture ligand can be selected from the group consisting of biotin and biotin analogs. Preferably, the linker is biotin - C17 - NH2.

[0126]

[0127] The structure of biotin - C10 - NH2.

[0128]

[0129] The structure of biotin - C13 - NH2.

[0130]

[0131] The structure of biotin - C17 - NH2.

[0132]

[0133] Structure of biotin-C24-NH2.

[0134] Step e. of the method—— Figure 4a and 4b 。

[0135] In the final step, the DAP-ligand matrix is removed from the target biomolecule in solution.

[0136] Preferably, the separation of the target biomolecule and the DAP-capture ligand-matrix product is by filtration, such as by membrane filtration or depth filtration. Preferably, the filtration allows the pure target biomolecule to pass through and retains the DAP-capture ligand-matrix product. This type of filtration is a procedure well known to those skilled in the art, and examples are, for instance, filtration using a semipermeable membrane or depth filtration established using a filter aid.

[0137] Membrane filtration techniques using a membrane that is permeable to the pure target biomolecule but impermeable to the complex consisting of the individual DAP-capture ligand-matrix are particularly useful in the method of the present invention. Preferably, the permeable filter is a semipermeable membrane. Preferably, recovery is carried out using filtration such as diafiltration, crossflow diafiltration, hollow fiber filtration, microfiltration, or ultrafiltration.

[0138] A particularly preferred separation method is continuous crossflow filtration, especially crossflow diafiltration, in which the product stream containing the separated DAP-capture ligand-matrix and the target biomolecule complex flows over a membrane that is impermeable to the complex but permeable to the target biomolecule. Washing filtration can be carried out until the target biomolecule is collected in the filtrate and the retentate contains practically only the compounds that cannot pass through the membrane, i.e., the individual DAP-capture ligand-matrix.

[0139] A variety of membranes can be selected according to the size of the target biomolecule. Preferably, the membrane is selected to allow the target molecule to penetrate the membrane while retaining the DAP-capture ligand-matrix product.

[0140] Suitable membranes typically have a cut-off value of >50 kDa, preferably >100 kDa, more preferably >150 kDa, even more preferably >300 kDa or >500 kDa, depending on the DAP-capture ligand-matrix and the target biomolecule under discussion.

[0141] When the target biomolecule is, for example, IgG, suitable membranes typically have a typical cut-off value of >150 kDa, suitably >300 kDa, more suitably >500 kDa. When the target biomolecule is, for example, albumin, suitable membranes typically have a typical cut-off value of >50 kDa, suitably >100 kDa.

[0142] Another preferred method for performing the separation and / or recovery step is by using hollow fiber filtration, where the product stream is introduced into the hollow fibers made of a semipermeable material that is impermeable to the DAP-capture ligand-matrix but permeable to the target biomolecule. Figure 4a and 4b shows an exemplary recovery process.

[0143] The membranes can be arranged as a dead-end or depth filter with a filter aid as described above, or as a hybrid system combining filtration types such as the 3M Emphaze Hybrid Purifier filter, or as having a continuous flow through the membrane to avoid fouling and more easily control the pressure. Examples include diafiltration, cross-flow filtration, hollow fiber systems, etc. The membranes can be stacked together, arranged in a spiral, plate and frame, or hollow fiber, or cascaded arrangement (see continuous arrangement, Figure 4B). Pressure can be applied during filtration, or it can be not applied. Typically, the filtrate passing through the membrane contains the target biomolecule, and the DAP-capture ligand-matrix remains in the retentate. The retentate can be recycled into the feed stream to increase the separation yield. The retentate can also be discarded without being recycled.

[0144] The flux through the membrane and thus the rate of membrane filtration depends to a large extent on factors such as the volume being processed, the membrane area, the pore size and distribution, the pressure, and the likelihood of fouling. For example, using a larger filtration area can significantly reduce the filtration time.

[0145] Typically, the retention time in the membrane system is short, for example at most 60 minutes, at most 50 minutes, at most 40 minutes, at most 30 minutes, at most 20 minutes, at most 15 minutes, at most 10 minutes, at most 5 minutes, however the membrane system can be further optimized to suit a particular protocol.

[0146] It should be understood that without departing from the scope of the present invention, other suitable membrane filtration systems can be used, i.e., different types of membranes with a suitable size cut-off can be used, arranged in series and having a recycle stream to optimize the flow rate, retention time, and separation efficiency.

[0147] After recovering the target biomolecule, a suitable buffer can be added to the target biomolecule solution to adjust the pH and salt conditions to stabilize the pure target biomolecule. Preferably, the pH is raised to near neutral pH. Even more preferably, for IgG purified from plasma, the pH is adjusted to 4-5. However, the pH can be raised to other suitable pHs that are most suitable for maintaining the stability and quality of the specific target molecule.

[0148] After this step, the pure target product can optionally be further polished and filtered to remove any residual impurities, such as virus particles, etc.

[0149] After the purification method of the present invention, the target biomolecule can be formulated using techniques well known in the art as needed. The DAP-capture ligand matrix can be discarded.

[0150] Due to the mass transfer process optimized in solution (where mass transfer occurs by convection rather than by diffusion, as in conventional affinity purification using solid resin-based systems), the purification method according to the present invention is rapid. DAP can be fermented in large-scale production facilities to be a low-cost item. Exemplary DAP production methods are disclosed in EP 2220107 B1. The method of the present invention improves the overall production efficiency both economically, environmentally, and in terms of time.

[0151] Example 1

[0152] General procedure for synthesizing carboxymethyl dextran 500 (CM-dextran 500). The example is substitution every 15 - 30 glucose units.

[0153] Prepare dextran (T500, Pharmacosmos A / S), KOH (Fluka), and chloroacetic acid solution.

[0154] Also freshly prepare the chloroacetic acid solution: Add chloroacetic acid (Aldrich, 9.45 g; MW 94.49 g / mol) to type 1 water (75 ml) in an ice bath, and add Na2CO3 (4.77 g; 0.9 mol equivalent).

[0155] Slowly add 25 ml of 4N KOH solution to 50 ml of 10% dextran 500. Then add 25 ml of 1M chloroacetic acid solution with gentle stirring. Keep the reaction mixture (total 100 ml, 5% dextran, 0.25 mol chloroacetic acid, and 1 mol KOH) in a 65 °C water bath for 1 h. Stir the solution gently from time to time.

[0156] Terminate the reaction by gently adding 1M HCl to adjust the solution to pH 3.0. Keep the solution cold (below room temperature), and precipitate the CM dextran by very slowly adding the cold solution to 500 ml of methanol. Collect the CM dextran 500 by suction through a Buchner funnel and wash it twice with cold methanol (20 ml). Vacuum dry the collected CM-dextran overnight and weigh the collected CM-dextran. Repeat the precipitation procedure by reprecipitation of CM-dextran 500, by redissolving the CM-dextran in a few milliliters of 5 mM KOH and reprecipitating by slowly adding the KOH solution of dextran 500 to cold methanol (5x CM-dextran 500 in 5 mM KOH).

[0157] Characterization was carried out by simple pH titration using a pH meter (Metler). Dextran was modified by carboxymethyl groups approximately every 15 - 30 glucose units.

[0158] Example 2

[0159] Biotin-C13-linker-NH2 was coupled to CM-dextran 500 by pre-activation with N-hydroxysuccinimide ester (NHS). The following example is given for 1 / 25 equivalent linker modification. Biotin-C13-linker-NH2 (1 / 25 mol equivalent per glucose unit) was added to NHS and pre-activated CM-dextran 500 in 0.5 M MES at pH 6.0 to produce a biotin-linked soluble dextran matrix.

[0160] First, EDAC (Aldrich, 59.4 mg, 1 / 10 mol equivalent per glucose unit) and N-hydroxysuccinimide (NHS) (14.27 mg, 1 / 25 mol equivalent) were added to CM-dextran 500 (500 mg, 3.1 mmol glucose units) to synthesize NHS-CM-dextran "in situ". The reaction mixture was kept under mixing at room temperature for 2 hours.

[0161] Second, after 2 hours, the biotin-C13-NH2 linker (1 - 25 mol equivalent, 485 g / mol, 60.26 mg) in 0.5 ml of 0.5 M MES buffer at pH 6.0 was added, and the mixture was mixed overnight at room temperature in a "blood sample mixer". Biotin-C13-NHCO-CH2-dextran 500 (1 - 25 mol equivalent) was purified by precipitation in methanol (ratio 5:1), filtration, and washing with cold MeOH (5×5 ml) and cold acetone (5×5 ml). The precipitated biotin-C13-linker NHCO-dextran 500 was then collected by vacuum filtration on a Büchner funnel, washed with 5x5 ml cold methanol and 5x cold acetone, and then vacuum dried in a desiccator. The powder was then "vacuum" dried in the desiccator overnight.

[0162] Dextrans were produced with 1 / 25, 1 / 50, and 1 / 100 equivalents of biotin linker per CM-dextran 500.

[0163] Example 3

[0164] The equivalence point of DAP molecules relative to the biotin-linked soluble dextran matrix in solution was determined as determined by SEC-HPLC

[0165] A fixed amount of DAP molecules (2.13 mg / mL DAP stock solution) was diluted to 0.80 mg / mL and titrated against a known dilution series of soluble capture matrix in microtiter plate wells (Dex 1 - 13). The recombinant dual - affinity polypeptide (DAP) molecule consists of multiple binding domains from protein A fused to streptavidin. DAP was expressed by an E. coli cell line and the purified protein was obtained from CHRETO Aps, Denmark.

[0166] Samples were analyzed by SEC - HPLC (TSKgel 300 column, Tosoh Bioscience) and the areas under the curves (AUC) of free DAP, matrix, and DAP - matrix were recorded.

[0167] The various retention times and AUC are summarized in Table 1 below.

[0168]

[0169]

[0170] Table 1. Absorbance measurement data: Soluble biotin - linker dextran matrix for DAP titration. AUC is the area under the curve.

[0171] Example 4

[0172] The basic method for purifying the target molecule is shown below for purifying IgG from plasma using DAP and a soluble capture matrix (biotin - linker dextran matrix).

[0173] Recombinant DAP was prepared as described, for example, in EP2427482. The recombinant DAP molecule consists of a binding domain from protein A fused to streptavidin. Plasma samples were donated by the local municipal blood bank.

[0174] A mixture of plasma solution (46 mL, 6.5 g IgG / L), DAP solution (42.7 mg DAP, 6.1 mL), and 40 mL of reaction buffer (0.1 M sodium phosphate buffer, 0.15 M NaCl, 0.1% Tween - 20, pH 7.2) was mixed on a static mixer and incubated for 5 minutes using the static mixer.

[0175] In a first alternative, the suspension of the formed DAP-IgG complex was added to 6 g of filter aid (Celpure C65 filter aid from Advanced Minerals purchased by Filtrox AG, Sct. Gallen, Switzerland) and collected on a depth filter and washed three times (50 mL) with different washing buffers: Washing buffer 1: 0.1 M sodium phosphate buffer, 0.15 M NaCl, 0.1% Tween-20, pH 7.2.

[0176] Washing buffer 2: 0.1 M Tris-HCl + 2 M NaCl, pH 7.4,

[0177] Washing buffer 3: 50 mM Tris-HCl, pH 7.4. Then 50 mL of type 1 water.

[0178] In a second alternative, the suspension of the formed DAP-IgG 3D complex was mixed with diatomaceous earth high-purity filter aid (HPFA) (6 g, Celpure C65, Advanced Minerals / Imerys) and established as a depth filter and washed repeatedly with different washing buffers (50 mL) until other plasma proteins were completely washed out of the homogeneous filter cake, according to SDS-PAGE analysis: standard Tris buffer (pH 7.4), phosphate / NaCl (with or without 0.1% Tween20, pH 7.2), and finally MilliQ water of type 1.

[0179] The IgG-DAP 3D complex was dissolved in an elution buffer (0.1 M citrate, 0.03% Tween-20, pH 3.4).

[0180] Then a mixture of IgG and DAP (0.80 mg / mL) was contacted with a soluble capture matrix (biotin-C13 (linker)-dextran 500 matrix) conjugated with a capture ligand (equivalent to approximately 0.0201 mg biotin / mL). After a 2-minute incubation, the free target IgG was separated from the DAP-capture ligand-matrix and collected as the permeate from a Merck Pelicon cassette XL50 with a 1000 kDa Millipore membrane.

[0181] Hereinafter, a soluble matrix material made of a larger dextran with a controlled molecular weight and molecular weight distribution was prepared, functionalized with chloroacetic acid, conjugated with a biotin linker, and tested for its ability to capture a dual-affinity polypeptide (DAP) and removed by filtration using a depth filter or a membrane filter.

[0182] Numerous soluble matrices and combinations were prepared to illustrate the utility and possible variations of the methods according to the present invention. Additionally, the following examples describe the systematic optimization of individual unit operations by varying only a few parameters of each step. It should be clear that those skilled in the art of conjugation and filtration unit operations are able to further vary the various parameters.

[0183] Example 5

[0184] Purification of high molecular weight dextran:

[0185] Various large dextrans with reduced amounts of low molecular weight chains or narrow molecular weight distributions were obtained from PK Chemicals A / S, Denmark or Pharmacosmos A / S, Denmark. The average size and distribution of the dextran were measured by size exclusion chromatography (phosphate buffer pH 7, flow rate 0.6 mL / min, 2.50 mg / ml, Waters Ultra hydrogel Linear 7.8 x 300 mm)) and a refractive index (Waters) detector for concentration and an online multi-angle light scattering (MALS) size detector (triple laser MiniDawn Treos, Wyatt Technology Corp) were used.

[0186] Examples of both the technical quality and high molecular weight dextran washed 4 times in a 100 kDa cut-off membrane system are listed in the following table, with the average molecular weight size and the average molecular weight of the lowest and highest 10% fractions.

[0187]

[0188] Table 2: Technical quality and filtered high molecular weight dextran.

[0189] The example illustrates the possibility of obtaining and preparing soluble polymers (in this case dextran) with very different sizes as the base material for soluble matrices, and having different molecular weight distributions and cut-off values. The exact selection of a particular size of soluble matrix depends on the subsequent DAP loading of each soluble matrix, the size of the specific target biomolecule, the filtration unit operation, and the required DAP removal criteria.

[0190] In the following examples, one of several methods for activating, purifying, and conjugating a capture ligand to a soluble dextran matrix is described.

[0191] Example 6

[0192] Preparation and analysis of CM dextran

[0193] As in Example 1, various carboxymethyl glucans (CM-glucans) were prepared using chloroacetic acid and KOH. After recovery by precipitation and drying in methanol, the acid content was measured by pH titration.

[0194] Briefly, CM-glucan was dissolved in Type 1 water (10% (w / w), 10 mL), and a few drops of HCl (1.0 M) were added to ensure that all carboxylic acids were protonated. Precipitation was carried out by adding CM-glucan dropwise to a magnetically stirred cold methanol solution (5x volume, 50 ml), collected by filtration in a Buchner funnel and washed with cold methanol until the methanol effluent was neutral (1:1 dilution in Type 1 water, Mettler-Toledo pH meter). The CM-glucan was dried to constant weight in a vacuum desiccator. CM-glucan was titrated with HCl. NaOH (0.010 N, 10 ml) was added to the CM-glucan solution (1.00% (w / w), 10 ml) and titrated with HCl solution (0.010 N) using a burette, while recording the pH as a function of the volume of HCl added to determine the equivalence point. Unmodified glucan was titrated for reference. The equivalence point was calculated from the CM-glucan pH curve compared to the glucan pH curve.

[0195] Using the exact stoichiometry and conditions as in Example 1, for all sizes and distributions of glucan, the CM loading per gram of CM-glucan was measured to be 310 - 320 μmol carboxylic acid / g. This corresponds to the modification of approximately 6% of the glucose subunits.

[0196] By varying the reaction time or temperature, the degree of substitution can be changed.

[0197] Example 7

[0198] Molecular weight refined CM-glucan

[0199] In the following example, high molecular weight carboxymethyl glucan (CM-glucan) was further refined to remove chemical reactants and lower molecular weight fractions. In this example, refined CM-glucan was prepared from glucan with an average Mw of 1.903,410 kDa (#5 in Example 5).

[0200] Briefly, a tangential flow filtration system (Minimate EVO TFF, Pall, equipped with three different MW cut-off membrane capsules with an effective filtration area of 50 cm 2 : Minimate TM Tangential Flow Filtration Capsules, Pall, 100K (OA100C12), 300K (OA300C12) and 500K (OA500C12).

[0201] In summary, for each of the three membranes, a 2% CM-dextran solution (in type 1 water, 50 ml) was loaded into the storage vessel of the system. The system was operated at 1 bar pressure to drive flow through the membrane.

[0202] During filtration, a 40 mL filtrate volume was collected, and the retentate (approx. 50 ml) was collected after filtration, lyophilized and the mass determined to estimate the mass balance.

[0203] Subsequently, the sizes of the two native and purified CM-dextrans from the retentate and filtrate were analyzed using the method outlined in Example 1.

[0204] Examples of CM-dextrans with different Mw cut-off values are listed in the table below:

[0205]

[0206] Table 3: Filtration of carboxymethyl dextran with three different membranes, and the resulting average molecular weights and average molecular weights of the lowest and highest 10% fractions.

[0207] The molecular weight of the purified CM-dextran cannot be directly compared to the unmodified dextran as the measured sizes are significantly larger. For these specific membranes, no large differences were observed between the 300 and 500 kDa membranes for the purified CM-dextran.

[0208] Example 8

[0209] Coupling of biotin-C17 linker to high molecular weight CM-dextran

[0210] In biotin-C17-NH2 linker (8 molar equivalents per mole of carboxyl group, 5-((3aS,4S,6aR)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazol-4-yl)-N-(2-(2-(3-(3-aminopropionamido)-2-methyl-propionamido)propionamido)-2-methylpropionamido)ethyl)pentanamide hydrochloride, 635.2 g / mol, C 26 H 46N8O6S·HCl, obtained from NCK A / S, Denmark, with HPLC area + 98% purity. In the presence of, carboxymethyl dextran (from 5, 5a, 5b, 5c of the above Example 7, 320 μmol COOH / g, 25 mg / ml) was activated and coupled with a biotin linker through a "one-pot" coupling reaction using carbodiimide (16 molar equivalents per mole of carboxyl group, EDAC, Merck) and N-hydroxysuccinimide (16 molar equivalents per mole of carboxyl group, Aldrich) in 0.5 M MES buffer (Sigma-Aldrich) at pH 6.0. The reaction mixture was mixed overnight at room temperature using an inclined laboratory mixer.

[0211] The solution was added dropwise to a magnetically stirred cold methanol solution (5× volume, 50 mL) for precipitation, and the biotin-dextran was collected by filtration in a Buchner funnel and washed with cold methanol. The washing was repeated 3 times. The biotin-dextran was collected and dried to a constant weight in a vacuum desiccator.

[0212] A sample of the biotin-dextran, the soluble matrix, was dissolved in type 1 water and measured by UV at 205 nm. The biotin-linker content in the prepared biotin-dextran was calculated by interpolation on a biotin-C17-NH2 linker standard curve.

[0213] Examples of the soluble matrix of biotin-dextran are summarized in Table 5 of Example 9 below.

[0214] Compared with extra-refined dextran, the biotin loading of PBA0263 is 30% lower, which may be due to chemical reaction impurities. The further refined CM-dextran has a slightly higher biotin-linker loading.

[0215] The amount of the linker can be changed by further changing the coupling reagent, the stoichiometry of the biotin-linker, or by changing the pH or concentration.

[0216] Example 9

[0217] Determination of binding capacity

[0218] The following examples illustrate the determination of the equivalence point of DAP molecules in solution relative to a biotin-linked soluble dextran matrix, as determined by SEC-HPLC.

[0219] Recombinant DAP was prepared as explained, for example, in EP2427482. The recombinant DAP molecule consists of the binding domain from protein A fused with streptavidin.

[0220] Dilute the stock solution of DAP molecule (purified recombinant protein obtained from CHRETO Aps, Denmark) to 0.80 mg / mL and titrate it against a known dilution series of biotin-linker soluble matrix in microtiter plate wells (Dx-3, Dx-5, Dx-7, Dx-9 and Dx-11) in citrate buffer (0.1 M citrate, 0.03% Tween-20, pH 3.4).

[0221] Analyze samples of the solution by SEC-HPLC (TSKgel 300 column, Tosoh Bioscience) and record the retention times and areas under the curve (AUC) at 280 nm for free DAP and DAP bound to the biotin-linker soluble matrix.

[0222] Examples of SEC-HPLC retention times and AUC (PBA0268) are summarized in the table below and plotted, ( Figure 5 ) showing DAP binding (%) / subtraction (%) as a function of biotin-dextran concentration.

[0223]

[0224] Table 4: Retention times and areas under the curve (AUC) recorded by SEC-HPLC analysis of the reaction between DAP (0.8 mg / mL) and biotin-linker-dextran at different concentrations.

[0225] The maximum binding capacity of the soluble matrix is determined by the minimum amount of biotin matrix sufficient to reduce the free DAP AUC to zero.

[0226] The maximum binding capacity can then be calculated as the maximum amount of DAP bound per biotin-dextran matrix. The biotin density and binding capacity to many soluble matrices are summarized below.

[0227]

[0228] Table 5: Summary of biotin loading, number of glucose subunits between biotin linkers and binding capacity of 4 different biotin-dextrans to DAP. DAP binding of PBA0267 was not detected.

[0229] The table shows the relationship between biotin density and binding capacity.

[0230] Example 10

[0231] Removal of DAP on a biotin resin column

[0232] For comparison purposes, commercially available prior art biotin affinity resin was used for the removal of DAP from solution. A DAP solution (43 mg in 50 mL of 0.1 M citrate buffer, pH 3.4) was provided and DAP molecules were removed from the solution by using a high-performance cross-linked biotin agarose resin (12.5 mL, product code number 4BCL-BI-100, Agarose Bead Technologies, Spain) packed in a column (XK16, Cytiva Sweden AB).

[0233] Briefly, the DAP solution was introduced into the biotin agarose bead column at different pump speeds to ensure efficient and complete DAP removal. The entire protocol was limited to a maximum of 60 minutes to minimize both processing time and subsequent exposure of the target IgG to the low pH buffer.

[0234] According to the manufacturer's recommendations, Novex TM Tris-glycine SDS sample and running buffers and NovexTMWedgeWellTM 12% Tris-glycine gel (1.0 mm × 12 wells and SeeBlueTM Protein Ladder (Plus2 prestained standards) and an XCell SureLock Mini-Cell electrophoresis system with an LKB Bromma 2301 Macrodrive 1 power supply (all from Invitrogen / ThermoFisher Scientific) were used for SDS-PAGE analysis. In a typical protocol, each well was loaded with sample (20 μl, 2.5 μg / μl) or protein standard (10 μl), and the gel was run at 225 V for 40 minutes. After Coomassie blue staining (Serva Blue R / acetic acid / ethanol) overnight and subsequent decolorization (glycerol / ethanol / acetic acid) and washing with type 1 water, the gel was scanned for recording.

[0235] The efficiency of DAP removal was evaluated by SDS-PAGE, showing that no DAP was detected at a flow rate of 1 mL / min. However, as confirmed by SDS-PAGE, at a higher flow rate (4 mL / min), trace amounts of DAP were observed in the flow-through. Attempts to increase DAP removal at lower pump speeds exceeded the 60-minute limit.

[0236] This example of the DAP removal protocol using the biotin bead system corresponds to an actual binding capacity of approximately 3.4 mg DAP / mL of biotin agarose resin, but also illustrates the slower binding reaction of the resin bead-based system.

[0237] Example 11

[0238] DAP Removal Using Soluble Matrices in Depth Filters.

[0239] In this experiment, the effects of dextrans with different molecular weight (MW) values were tested in a rapid depth filter application.

[0240] A depth filter device was used to capture DAP through three different soluble matrices. Soluble matrices (PBA0263, PBA0266, and PBA0268) derived from the same high Mw dextran had different Mw values (see Examples 7 and 8). Additionally, an antibody sample representing the target biomolecule was introduced to evaluate the efficiency of separating DAP and the target antibody using the soluble matrices in the depth filter under low pH conditions.

[0241] Samples were prepared by mixing DAP and the soluble matrix with or without antibody (Privigen Immune Globulin Intravenous (Human), 10% (w / v) IgG, CSL Behring AG).

[0242] Briefly, a biotin - linker - dextran solution (182 μL of 9.1 mg of PBA0263, 128 μL of 6.4 mg of PBA0266, or 128 μL of 6.4 mg of PBA0268) and a DAP solution (53 mL, 0.8 mg DAP / mL, 42.7 mg) were mixed under magnetic stirring for 120 seconds. The ratio between biotin - linker - dextran and DAP corresponded to the equivalence point.

[0243] A filter aid (6 g Filtrox C65, Advanced Minerals, in 50 mL of 0.1 M citrate buffer, pH 3.4) was added to the DAP - biotin - linker - dextran solution, and the suspension was pumped (80 mL / min) into the filter chamber to build a uniform filter cake (volume approximately 20 mL and thickness 1 cm).

[0244] The flow - through (filtrate) from the filter chamber was collected in 40 mL fractions, and the filter cake was washed with additional buffer (2 × 40 mL citrate buffer, pH 3.4, pump speed 50 mL / min) and the filtrate was collected.

[0245] The duration of DAP removal by soluble biotinylated dextran in the depth filter protocol was 140 seconds in total (2 minutes 20 seconds).

[0246] The free antibody and trace DAP in the filtrate were analyzed by UV (280 nm, Nanodrop One, Thermo Fisher) and SDS - PAGE (see previous protocol).

[0247] The results are summarized in the table below:

[0248]

[0249] Table 6: Summary of the removal of DAP using three different soluble matrices (biotin-dextran) in a depth filter in the presence and absence of the target biomolecule IgG.

[0250] Capture without added IgG yields a low UV signal, which may be due to trace amounts of DAP. Alternatively, this low UV signal may be filter aid debris.

[0251] According to SDS-PAGE, the capture of DAP using a depth filter with the soluble matrices PBA0266 or PBA0268 and the C65 filter aid is complete. No visible DAP residues are present, and the antibody can be recovered in high yield. Figure 6 SDS-PAGE analysis of the filtrate from an experiment using the soluble matrix PBA0268 is shown.

[0252] According to SDS-PAGE analysis, the less refined PBA0263 soluble matrix with a slightly lower biotin density results in almost complete capture of DAP since only trace amounts of DAP are found in the filtrate.

[0253] This example is used to demonstrate the effect of biotin-linker loading and Mw values of different soluble matrices when using a depth filter.

[0254] In addition, compared to the DAP capture using the biotin resin column protocol (Example 10), the soluble matrix and depth filter protocol is approximately 25 times faster. In addition, this protocol utilizes simple solution mixing and filtration, making it easy to scale up the process.

[0255] Example 12

[0256] Removal of DAP Using a Soluble Matrix in a Membrane Filter

[0257] In this experiment, the utility of capturing and removing DAP by a soluble matrix was tested using a membrane filtration system.

[0258] Using a tangential flow filtration system (Pall Minimate EVOTFF and flow filtration capsule, OA500C12, Pall) with a 500K membrane and a 50 cm 2 effective filtration area.

[0259] In summary, the biotin-linker-dextran solution (PBA0268, 128 μL, 6.82 mg) was mixed with the DAP solution (53 mL, 0.8 mg DAP / mL, 46.4 mg) in 0.1 M citrate buffer (pH 3.4) and incubated for 120 seconds with magnetic stirring.

[0260] The DAP and biotin-linker-dextran solutions were loaded into the storage container of the system and mixed with an additional 300 mL of 0.1 M citrate buffer (pH 3.4). The system was operated at a pressure of 0.5 - 1 bar to drive flow through the membrane.

[0261] During the filtration process, a filtrate volume of 40 mL of filtrate fraction was collected, and after the filtration process, approximately 50 mL of the retentate was collected.

[0262] The effectiveness of DAP removal was evaluated by analyzing the retentate and filtrate fractions by UV (Nanodrop One) at 280 nm.

[0263] No free DAP or DAP-biotin-linker-dextran was detected in the filtrate, while all DAP or DAP-biotin-linker-dextran remained in the retentate.

[0264] Example 13

[0265] DAP Removal Using a Soluble Matrix in a Membrane Filter

[0266] In this experiment, the utility of capturing and removing DAP by a soluble matrix was tested using a 1000K membrane filtration system. Additionally, an antibody sample representing the target biomolecule was introduced to evaluate the efficiency of separating DAP and the target antibody under low pH conditions. Samples were prepared by mixing DAP and the soluble matrix with the antibody (Privigen Immune Globulin Intravenous (Human), 10% (w / v) IgG, CSL Behring AG) in 0.1 M citrate buffer at pH 3.4.

[0267] A tangential flow filtration system (Minimate EVO TFF and Flow Filtration Capsule, OA990C12, Pall) with a 1000K membrane and a 50 cm 2 effective filtration area was used.

[0268] In summary, the biotin-linker-dextran solution (PBA0268, 128 μL, 6.4 mg) was mixed with the DAP solution (51 mL, 42.4 mg DAP) and 3.0 mL of Privigen, 300 mg IgG) in 0.1 M citrate buffer at pH 3.4 and incubated for 120 seconds with magnetic stirring.

[0269] Load DAP, IgG, and biotin-linker-dextran solution into the storage container of the system and mix with an additional 300 mL of buffer (0.1 M citrate buffer, pH 3.4). The system is operated at a pressure of 0.5 - 1 bar to drive flow through the membrane.

[0270] During filtration, collect a filtrate volume of 40 mL filtrate fractions, and after the filtration process, collect approximately 80 mL of the retentate.

[0271] Analyze the retentate and filtrate fractions by SDS-PAGE analysis to evaluate the effectiveness of DAP removal.

[0272] A small amount of DAP or DAP-biotin-linker-dextran was detected in the filtrate by SDS-PAGE. ( Figure 7 ). In this experiment, the cut-off of the 1000K membrane was too high to achieve complete removal. The recovery of IgG in the filtrate was evaluated by visual inspection to be approximately 80 - 90%, and the removal of DAP was better than 50%. The retentate showed a high content of DAP-biotin dextran and some IgG residues.

[0273] This example illustrates the importance of the combination of soluble matrix, DAP, target, and filtration membrane. For example, since DAP-biotin-dextran separates in the retentate, various cut-off values of the membrane can be applied to design a process that allows both high recovery of the target molecule in the flow-through and the desired level of DAP removal.

[0274] List of references

[0275] EP 2220107 B1 (CHRETO)

[0276] EP 2427482 B 1 (CHRETO)

[0277] WO 2020 / 127311 A1

[0278] US2008108053 A1 (AFFISINK BIOTECHNOLOGY LTD), 2008-05-08

[0279] WO2020037100 A1 (ISOLERE BIO INC.), 2020-02-20

[0280] WO2012055854 A1 (SPIBER TECHNOLOGIES AB etc.), 2012-05-03

[0281] US2009232737 A1 (MOYA WILSON et al.), September 17, 2009

[0282] WO2018178991 A1 (YISSUM RESDEV CO OF HEBREW UNIV JERUSALEM LTD), October 4, 2018

[0283] WO2021168270 A1 (ISOLERE BIO INC et al.), August 26, 2021

[0284] WO2006110292 A2 (UNIV CALIFORNIA et al.), October 19, 2006

[0285] WO2009078018 A2 (AFFISINK BIOTECHNOLOGY LTD et al.), June 25, 2009

[0286] WO2008067591 A1 (INNOVATION PURIFICATION TECHNO et al.), June 12, 2008

[0287] WO2023012321 A1 (ROCHE DIAGNOSTICS GMBH et al.), February 9, 2023

[0288] WO2016049761 A1 (BIOASTRA TECHNOLOGIES INC), April 7, 2016

[0289] Nygren, H. and Stenberg, M. (1989) Immunochemistry at Interfaces. Immunology, 66, 321 - 327).

Claims

1. A method for purifying a target biomolecule, comprising the following steps: a. In a solution, contacting i) a starting composition comprising the target biomolecule and ii) a dual-affinity polypeptide (DAP) to allow the DAP to form a complex with the target biomolecule, the DAP having a first binding functionality for the target biomolecule and a second binding functionality for a capture ligand, wherein the ratio of the equilibrium dissociation constants of the dual-affinity polypeptide D,t / K D,s is at least 10 under standard conditions 1 ; b. Washing the formed target biomolecule-DAP complex to remove any impurities and form a pre-purified target biomolecule-DAP complex; c. Treating the pre-purified target biomolecule-DAP complex at a pH below 5.0 to separate the target biomolecule from the DAP by dissolving the 3D complex, thereby providing a mixture of the pure target biomolecule and DAP; and d. Contacting the mixture of step c. with a soluble matrix bound to the capture ligand to form a separate pure target biomolecule and a separate DAP-capture ligand-matrix product; and e. Recovering the pure target biomolecule.

2. The method according to claim 1, wherein the ratio between the equilibrium dissociation constants of the two parental polypeptides [K D,t / K D,s is at least 10 1 under standard conditions.

3. The method according to any one of the preceding claims, wherein the washing in step b. is carried out by filtration, such as via depth filtration.

4. The method according to any one of the preceding claims, wherein the pH in step c. is in the range of 2.8 - 4.7, preferably in the pH range of 3.1 - 4.5, more preferably in the pH range of about 3.4 - 4.

3.

5. The method according to any one of the preceding claims, wherein the soluble matrix is a water-soluble matrix or soluble in an aqueous solution.

6. The method according to claim 5, wherein the soluble matrix is a polymer selected from the group consisting of dextran, xanthan gum, pectin, chitin and chitosan, carrageenan, guar gum, cellulose ether, hyaluronic acid, albumin, hydroxyethyl starch and other starch derivatives, polyacrylic acid, polyacrylamide, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, divinyl ether-maleic anhydride, polyoxazoline, polyphosphate, polyphosphazene, copolymers thereof and mixtures thereof, preferably selected from the group consisting of dextran, carrageenan, pectin, cellulose ether, polyacrylic acid, polyacrylamide, polyvinyl alcohol, copolymers thereof and mixtures thereof, more preferably, the soluble matrix is a dextran polymer.

7. The method according to any one of the preceding claims, wherein the size difference between the soluble matrix and the target biomolecule is at least 3-fold, such as at least 5-fold, such as at least 7-fold, such as at least 10-fold size difference.

8. The method according to any one of the preceding claims, wherein the size difference between the DAP-soluble matrix and the target biomolecule is at least 3-fold, such as at least 5-fold, such as at least 7-fold, such as at least 10-fold size difference.

9. The method according to any one of the preceding claims, wherein the soluble matrix is linked to the capture ligand via a linker having a linker length of 10 - 25 bonds, such as a length of 10 - 24 bonds, such as a length of 10 - 23 bonds, such as a length of 10 - 22 bonds, such as a length of 10 - 21 bonds, such as a length of 10 - 20 bonds, such as a length of 10 - 19 bonds, such as a length of 10 - 18 bonds, such as a length of 10 - 17 bonds, such as a length of 10 - 16 bonds, such as a length of 10 - 15 bonds, such as a length of 10 - 14 bonds, such as a length of 10 - 13 bonds, such as a length of 10 - 12 bonds, such as a length of 10 - 11 bonds, such as a length of 11 - 25 bonds, such as a length of 12 - 25 bonds, such as a length of 13 - 25 bonds, such as a length of 14 - 25 bonds, such as a length of 15 - 25 bonds, such as a length of 16 - 25 bonds, such as a length of 17 - 25 bonds, such as a length of 18 - 25 bonds, such as a length of 19 - 25 bonds, such as a length of 20 - 25 bonds, such as a length of 21 - 25 bonds, such as a length of 22 - 25 bonds, such as a length of 23 - 25 bonds, such as a length of 24 - 25 bonds.

10. The method according to claim 9, wherein the linker is selected from the group consisting of 2 - vinyl - 4,4 - dimethyl - 5 - oxazolone (VDMA), vinyl azlactone derivatives, acrylic derivatives, peptides, polyamides, hexamethylene diisocyanate (HDI) derivatives, diisocyanate derivatives, or mixtures thereof.

11. The method according to any one of the preceding claims, wherein the capture ligand is selected from the group consisting of biotin and biotin analogs.

12. The method according to any one of the preceding claims, wherein step e. is carried out using filtration such as membrane filtration, diafiltration, cross - flow diafiltration, hollow fiber filtration, depth filtration, microfiltration, or ultrafiltration.

13. The method according to any one of the preceding claims, wherein the target biomolecule is a protein, preferably an antibody, a virus particle or exosome, a cell, or a cell component.

14. The method according to any one of the preceding claims, wherein at least steps c. - e. of the method, and preferably the entire method, are performed as a continuous operation.

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