Purification of Glycoform

Abstract

The present invention relates to a method for the separation and purification of glycoforms using ion exchange separation materials having amino acid-based terminal groups.

Description

Technical Field

[0001] The present invention relates to a method for the separation and purification of glycoforms using an ion exchange separation material having an amino acid-based end group.

Background Art

[0002] Glycans are an essential part of glycoprotein molecules and ensure their structure and function. One of the most common glycoproteins is immunoglobulin containing two N-linked oligosaccharides at conserved asparagine 297 in the CH2 domain of the Fc portion. The glycan structure is composed of two N-acetylglucosamines (GlcNAc), three mannoses and two GlcNAc residues. Additional monosaccharides such as fucose (Fuc), galactose (Gal), sialic acid containing N-acetylneuraminic acid (NANA) or N-glycolylneuraminic acid (NGNA) residues may also be present. (Figure 1. Glycan structure.) The glycan structure plays an important role in the affinity binding of glycoproteins to receptors. Changes in glycan composition can cause conformational changes in glycoproteins, affect their specific receptor binding, and result in changes in effector functions. Moreover, some glycan compositions initiate defensive biological reactions, for example, there is a terminal mannose that binds to effectors bearing the mannose-binding receptor (ManR).

[0003] On the other hand, high-mannose glycan-containing glycoproteins, which are generally found in glycoproteins derived from yeast, insect cells, and plants, may be highly immunogenic in humans (Durocher Y, Butler M. 2009. Expression systems for therapeutic glycoprotein production. Curr Opin Biotechnol 20: 700-707). Therefore, it is extremely important to control the level of high-mannose glycans in biopharmaceutical glycoproteins in order to avoid potential immunogenicity.

[0004] Furthermore, terminal mannose and hybrid glycan structures reduce the conformational stability of the CH2 domain of monoclonal antibodies (mAbs). This can lead to a higher level of enzymatic degradation or shorter storage times for such molecules (Fang, J. Richardson J, Du Z, Zhang Z. Effect of Fc-Glycan Structure on the Conformational Stability of IgG Revealed by Hydrogen / Deuterium Exchange and Limited Proteolysis, Biochemistry 2016, 55, 860-868).

[0005] Hybrid glycosylation variants are often formed in the Golgi apparatus. Hybrid glycosylation variants exhibit reduced or altered glycosylation, i.e., they do not have the desired glycosylation pattern. Some examples of hybrid forms include variants lacking N-acetylglucosamine in the G0 variant (e.g., G0-N) or lacking galactose in the G1 variant (e.g., G1-N) (Costa AR, Rodrigues ME, Henriques M, Oliveira R, Azeredo J. Glycosylation: impact, control and improvement during therapeutic protein production, Crit Rev Biotechnol. 2013, 1-19). Both of the hybrid variants mentioned have terminal mannose, thereby increasing the probability of a shorter lifespan in the blood (Goetze AM, Liu YD, Zhang Z, Shah B, Lee E, Bondarenko PV, Flynn GC. 2011. High mannose glycans on the Fc region of therapeutic IgG antibodies increase serum clearance in humans. Glycobiology 21: 949-959).

[0006] Moreover, lower levels of galactose reduce complement-dependent cytotoxicity (CDC) activity and affect the activity of glycoproteins. Multiple studies have shown that the activity can be reduced by half between mAbs containing G2-glycoforms and mAbs containing G0-glycoforms (Raju TS. 2008. Terminal sugars of Fc glycans influence antibody effector functions of IgGs. Curr Opin Immunol 20: 471-478). Therefore, the ability to control or reduce the amount of hybrid glycosylation variants, especially those with terminal mannose or lacking galactose, will increase the lifespan and effectiveness of glycoproteins.

[0007] One of the most obvious effects of glycan structural changes is seen by the presence or absence of fucose. Fucose is added to the glycan structure in the Golgi apparatus. The presence of fucose in the core glycan structure of mAbs is known to inhibit its binding to the FcγRIIIa receptor, thereby reducing antibody-dependent cell-mediated cytotoxicity (ADCC) activity. Specific binding to the FcγRIIIa receptor can be reduced by up to 50-fold, thereby having a great impact on the effectiveness of glycoproteins (Peipp et al, Antibody fucosylation differentially impacts cytotoxicity mediated by NK and PMN effector cells, BLOOD, September 15, 2008, Vol. 112, No. 6, 2390-2399).

[0008] It is also well known that the absence of glycosylation dramatically reduces the binding affinity between glycoproteins and receptors. For example, the lack of glycosylation on mAbs dramatically reduces binding to the FcγRI receptor and abolishes binding to the FcγRII and FcγRIII receptors (Liu L, Antibody Glycosylation and Its Impact on the Pharmacokinetics and Pharmacodynamics of Monoclonal Antibodies and FC-Fusion Proteins, Journal of Pharmaceutical Sciences 104: 1866-1884, 2015).

[0009] The US Food and Drug Administration (FDA) assesses the similarity of glycosylation as one of the most critical requirements for biosimilar drugs (FDA, 2012. Guidance for industry quality considerations in demonstrating biosimilarity to a reference protein product). Moreover, the improvement of glycosylation is one of the main trends of biobetters.

[0010] For all these reasons, there is a growing need in the industry to improve the efficiency of glycosylated biopharmaceutical molecules by controlling, enriching, or separating various glycan species to achieve optimal pharmacokinetics, efficacy, half-life, and tolerance.

[0011] Current state-of-the-art for glycoprotein separation based on such glycan variants can be performed in preparative chromatography mode using ion-exchange chromatography to concentrate high-mannose glycoforms (WO2014 / 100117). Unfortunately, the enrichment of high-mannose-containing glycoforms in the given examples overlaps with the enrichment of aggregates, which makes it difficult to recognize, even if the separation of glycoproteins without aggregates is also achieved. Moreover, there is no indication that this technique can be used for the separation or removal of other glycan variants.

[0012] An alternative technique for high-mannose-containing glycoforms is affinity chromatography using lectins (US20020164328). While this technique is more specific than ion-exchange chromatography, it is limited to high-mannose-containing glycans and requires specific binding conditions. In addition, the leaching of lectins, the regeneration and lifespan of this resin prevent its application to economically viable high-mannose-containing glycan variants.

[0013] Other preparative glycan variant separation methods include a combination of anion-exchange and reverse-phase chromatography techniques (US20100151584) or using anion-exchange chromatography only (IN 01066ch2012). Unfortunately, the prior art does not have a technique that achieves the separation of more than one glycoform corresponding to the obvious need for an efficient and effective technique for separating the glycoforms of glycoproteins. SUMMARY OF THE INVENTION

[0014] Ion exchange materials bearing covalently attached leucine residues or similar residues have been found to be useful for the separation and enrichment of glycoforms that enable efficient glycan species separation at a capacity of >10 mg glycoprotein / ml material. In a more preferred embodiment, the capacity is between 10 and 80 mg / ml. Moreover, this innovative ion exchange material can be used at high conductivity >10 mS / cm, and in a more preferred embodiment, the conductivity is between 10 and 60 mS / cm.

[0015] Surprisingly, it was possible to separate and enrich glycan variants including high mannose-containing variants, terminal mannose-containing variants, fucose-containing variants, and non-glycosylated variants using a solvent pH gradient. Moreover, this discovery enabled us to explore a wide window of operation where ionic and hydrophobic interactions contributed to the selectivity of glycan variant separation. In addition, the application of this innovative ion exchange material showed significant economic advantages compared to affinity chromatography mode and enabled improved performance compared to anion exchange mode with a broader selectivity.

[0016] The present invention is thus directed to the chromatographic purification and / or separation of protein glycoforms by contacting a separation material comprising a hydroxyl group-containing base matrix having polymer chains covalently grafted onto its surface with a sample containing protein glycoforms, a) the base matrix contains hydroxyl groups, b) the polymer chains are covalently bonded to the base matrix via the hydroxyl groups, c) the polymer chains contain a terminal group -N(Y)-R3, provided that in the formula, Y is independently H or CH3, preferably H, and R3 is -CHCOOMR4, However, in the formula, R4 is C1-C4 alkyl such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, preferably isopropyl and isobutyl, and most preferably isobutyl, or C1-C4 perfluoroalkyl, and M is independently of each other H, Na, K, or NH4+ characterized by this.

[0017] In a preferred embodiment, the method of the present invention a) contacting a separation material comprising a hydroxyl group-containing base matrix to which polymer chains are covalently bonded on its surface with a sample containing a protein glycoform, whereby one or more protein glycoforms bind to the separation material, b) optionally, washing the separation material, c) contacting the separation material with an elution buffer under conditions under which the bound protein glycoform elutes from the separation material including the steps of In a preferred embodiment, in step c), the elution buffer has a higher pH than the loading buffer. The elution buffer may be applied as a step or as a gradient.

[0018] In a preferred embodiment, in step a), the contact of the sample with the separation material is carried out under conditions of increased conductivity such that the sample loaded on the separation material has a conductivity between 5 and 60 mS / cm, preferably between 15 and 35 mS / cm. This is typically achieved by adding a salt such as sodium chloride to the sample solution. In another preferred embodiment, the method includes recovering the flow through protein glycoform through the separation material.

[0019] In a preferred embodiment, the sample contains a mannose rich protein glycoform. In another preferred embodiment, the sample contains terminal mannose protein glycoforms. In another preferred embodiment, the sample contains fucosylated and non-fucosylated protein glycoforms.

[0020] In another preferred embodiment, the sample contains glycosylated and non-glycosylated proteins. In another preferred embodiment, the sample is an antibody and / or an Fc fusion protein and / or a viral protein glycoform, which is either isolated or on a virus or a viral capsid.

[0021] Preferably, the monomer units of the polymer are linearly linked, and each monomer unit contains a terminal group -N(Y)-R3. Preferably, the ion density of the separation material is between 10 and 1200 μeq / g. In a preferred embodiment, Y is H, and R4 is isopropyl and / or isobutyl. In a preferred embodiment, the hydroxyl group-containing base matrix contains aliphatic hydroxyl groups.

[0022] In a preferred embodiment, the base matrix is a copolymer formed by copolymerization of at least one compound from groups a) and b), where a) at least one hydrophilic substituted alkyl vinyl ether represented by formula I

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0023] R4 in formula I is typically an alkyl radical, cycloaliphatic radical or aryl radical bearing at least 1 hydroxyl group. In a highly preferred embodiment, the base matrix is formed by copolymerization of a hydrophilic substituted alkyl vinyl ether selected from the group of 1,4-butanediol monovinyl ether, 1,5-pentanediol monovinyl ether, diethylene glycol monovinyl ether or cyclohexanedimethanol monovinyl ether with divinylethyleneurea (1,3-divinylimidazolin-2-one) as crosslinking agent.

[0024] In a preferred embodiment, the separation material is a hydroxyl group-containing base matrix of formula V

Chemical formula

[0025] This graft polymerization is preferably carried out according to Example 8 on page 9 of US5453186.

[0026] In a preferred embodiment, the protein glycoform binds to the separation material at a pH between 2 and 7 and is optionally washed and eluted by increasing the pH value to an alkaline pH, preferably a value greater than 9, for example, between 9 and 11, preferably around 10. In a preferred embodiment, the sample is applied to the separation material at an ionic density between 10 and 1200 μeq / g. In a preferred embodiment, between 10 mg and 100 mg of glycoprotein binds per ml of the separation material.

Brief Description of the Drawings

[0027]

Figure 1

Figure 2

Figure 3

[0028]

Figure 4

Figure 5

Figure 6

[0029]

Figure 7

Figure 8

Figure 9

[0030]

Figure 10

Figure 11

Figure 12

[0031]

Figure 13

Figure 14

Mode for Carrying Out the Invention

[0032] Before describing the present invention in detail, it should be understood that the invention is not limited to a particular composition or process step, and such may vary. It should be noted that the singular forms "a", "an", and "the" as used in this specification and the appended claims include the plural forms of the indicated objects unless the context clearly dictates otherwise. Thus, for example, reference to "ligand" includes a plurality of ligands, and reference to "antibody" includes a plurality of antibodies, and so on.

[0033] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The following terms are defined for the purposes of the invention as described herein.

[0034] As used herein, the term "target molecule" refers to any molecule, substance, or compound that is to be isolated, separated, or purified from one or more other components, such as impurities, in a sample. Examples of target molecules are glycoproteins, also referred to as protein glycoforms, or glycan species of glycoproteins. The target molecule can also be a non-glycosylated protein to be separated from a protein glycoform that is also present in the sample. In a production and / or purification process, the target molecule is typically present in a liquid. The liquid can be water, a buffer, a non-aqueous solvent such as ethanol, or any mixture thereof. Alongside the target molecule, the liquid can contain one or more impurities.

[0035] The liquid is also sometimes referred to as the sample. The composition of the liquid can vary depending on the process steps performed during production and / or purification. After a chromatography step, the liquid typically contains a different solvent than before due to the eluent used in the chromatography step. Typically, the target molecule can only be dried to prepare the final dosage form only after the very last purification step.

[0036] A glycoprotein or protein glycoform is a glycosylated protein. Glycosylation can be in the form of monosaccharides, disaccharides, or oligosaccharide chains, for example, comprising one or more fucose, mannose, galactose, N-acetylglucosamine, sialic acid, and / or neuraminic acid. For example, antibodies typically have complex-type N-linked oligosaccharides. Further information can be found in the above introduction. An example of a common glycan structure is the N-glycosidically linked sugar chain as found in antibodies.

[0037] Although complex-type N-glycosidically linked sugar chains can also link to other asparagine residues, "N-glycosidically linked sugar chains" or "N-glycosidically linked glycans" typically bind to asparagine 297 (according to Kabat numbering). Complex-type N-glycosidically linked sugar chains typically have a biantennary complex sugar chain and mainly have the following structure:

Chemical formula

[0038] Examples of protein glycoforms are proteins with different levels of fucosylation, for example, different levels of core fucosylation, different levels of sialylation, different levels of galactosylation, or different levels of mannosylation. Preferably, the method of the present invention is used to isolate or purify high-mannose protein glycoforms.

[0039] High-mannose protein glycoforms are glycoproteins having glycan residues with 5 or more, typically 5 - 9, mannose units. An example of a high-mannose protein is an antibody having an N-linked oligosaccharide containing 5 - 9 mannose units. Another example of a mannose-rich protein is a viral glycoprotein such as the mannose-rich envelope glycoprotein of HIV1 or the spike (S1) protein of 2019-nCoV (SARS-CoV-2).

[0040] Terminal mannose protein glycoforms are glycoproteins having glycan residues with 3 mannose units in the core structure, wherein one or both of the mannose units at the branch are not linked to N-acetylglucosamine (“GIcNAc”). In some embodiments, this can refer to a G0-N glycoprotein. In some embodiments, this can refer to a G1-N glycoprotein. In some embodiments, this can refer to a hybrid glycoform.

[0041] The glycoproteins or protein glycoforms according to the present invention can be isolated glycoproteins or glycoproteins linked to other moieties such as drugs, other proteins, viruses or virus capsids. In some embodiments, the protein glycoforms are produced in mammalian cells, in fungal cells, in insect cells or in plant cells.

[0042] The term "antibody" refers to a protein having the ability to specifically bind to an antigen. "Antibody" or "IgG" further refers to a polypeptide substantially encoded by an immunoglobulin gene(s) that specifically binds to and recognizes an analyte (antigen), or a fragment thereof. The immunoglobulin genes recognized include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. The light chain is classified as either kappa or lambda. The heavy chain is classified as gamma, mu, alpha, delta, or epsilon, and thus defines immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively.

[0043] An exemplary immunoglobulin (antibody) structural unit consists of two pairs of polypeptide chains, each pair having 1 "light" (about 25 kD) and 1 "heavy" chain (about 50 - 70 kD), and the chains are stabilized, for example, by interchain disulfide bonds. The N-terminus of each chain defines a variable region of about 100 - 110 or more amino acids that is mainly involved in antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively.

[0044] Antibodies may be monoclonal or polyclonal and may exist in monomeric or polymeric forms, for example, IgM antibodies that exist in pentameric form and / or IgA antibodies that exist in monomeric, dimeric, or multimeric forms. Antibodies may also include multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, provided that they retain or are modified to contain a ligand-specific binding domain.

[0045] The term "fragment" refers to a part or portion of an antibody or antibody chain that contains fewer amino acid residues than an intact or full antibody or antibody chain. Fragments can be obtained via chemical or enzymatic treatment of an intact or full antibody or antibody chain. Fragments can also be obtained by recombinant means. When produced recombinantly, fragments may be expressed alone or as part of a larger protein called a fusion protein. Exemplary fragments include Fab, Fab’, F(ab’)2, Fc and / or Fv fragments. Exemplary fusion proteins include Fc fusion proteins. According to the present invention, fusion proteins are also encompassed by the term "antibody".

[0046] In some embodiments, the antibody is an Fc region-containing protein, such as an immunoglobulin. In some embodiments, the Fc region-containing protein is a recombinant protein that includes the Fc region of an immunoglobulin fused to another polypeptide or a fragment thereof.

[0047] Exemplary polypeptides include, by way of example, renin; growth hormones including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; α-1-antitrypsin; insulin α-chain; insulin β-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factors such as factor VIIIC, factor IX, tissue factor, and von Willebrand factor; anticoagulation factors such as protein C; atrial natriuretic factor; lung surfactant; plasminogen activators such as urokinase or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factors; tumor necrosis factor-α and -β; enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted); human macrophage inflammatory protein (MIP-1-α); serum albumins such as human serum albumin; Müllerian inhibiting substance; relaxin α-chain; relaxin β-chain; prorelaxin; mouse gonadotropin-related peptide; microbial proteins such as, for example, β-lactamase; DNase; IgE; cytotoxic T lymphocyte-associated antigen (CTLA) (e.g., CTLA-4); inhibin; activin; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; protein A or D; rheumatoid factors; neurotrophic factors such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or nerve growth factor such as NGF-β; platelet-derived growth factor (PDGF); fibroblast growth factors such as αFGF and βFGF; epidermal growth factor (EGF); transforming growth factors (TGF) such as TGF-α, and TGF-β including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growth factors-I and -II (IGF-I and IGF-II); des(l-3)-IGF-I (brain IGF-I), insulin-like growth factor binding protein (IGFBP); CD proteins such as CD3, CD4, CD8, CD19, CD20, CD34, and CD40; erythropoietin; bone inductive factors; immunotoxins;Bone morphogenetic protein (BMP); interferons such as interferon-α, -β, and -γ; colony-stimulating factors (CSF), such as M-CSF, GM-CSF, and G-CSF; interleukins (IL), such as IL-1 to IL-10; superoxide dismutase; T cell receptor; surface membrane protein; decay-accelerating factor; viral antigens such as portions of the AIDS envelope; transport protein; homing receptor; addressin; regulatory protein; integrins such as CD11a, CD11b, CD11c, CD18, ICAM, VLA-4 and VCAM; tumor-associated antigens such as HER2, HER3 or HER4 receptor; and fragments and / or variants of any of the polypeptides listed above.

[0048] In addition, the antibodies according to the present invention are any protein or polypeptide, fragment or variant thereof that specifically binds to any of the polypeptides listed above.

[0049] As used herein and unless otherwise stated, the term "sample" refers to any composition or mixture containing a target molecule. Samples may be of biological or other origin. Biological sources include eukaryotic sources such as animals or humans. Preferred samples are blood or plasma samples derived from mammals. Samples may also include diluents, buffers, detergents, and contaminating species, etc. found mixed with the target molecule. Samples may be "partially purified" (i.e., subjected to one or more purification steps such as filtration or centrifugation steps) or obtained directly from the biological entity producing the target molecule. A plasma sample is any sample containing plasma or a portion of plasma.

[0050] As used herein, the terms "impurity" or "contaminant" refer to any foreign or undesirable molecule, which may be present in a sample containing a target molecule separated from one or more foreign or undesirable molecules, including biological macromolecules such as DNA, RNA, one or more host cell proteins, nucleic acids, endotoxins, lipids, impurities of synthetic origin, and one or more additives. The terms "impurity" or "contaminant" also apply to certain immunoglobulins, such as immunoglobulin A and immunoglobulin M, which need to be separated from the target molecule and cause allergic reactions in patients. In addition, such impurities may include any reagents used in the steps of the production and / or purification process.

[0051] As used herein interchangeably, the terms "purify", "separate" or "isolate" refer to increasing the purity of a target molecule by separating it from a composition or sample containing the target molecule and one or more other components, such as impurities for example. Typically, the purity of the target molecule is increased by removing (completely or partially) at least one impurity from the composition.

[0052] The term "chromatography" refers to any type of technique for separating an analyte of interest (such as a target molecule) from other molecules present in a mixture. Generally, the target molecule is separated from other molecules as a result of differences in the rates at which the individual molecules of the mixture move through a stationary medium or separation material under the influence of a mobile phase or in a binding - elution process. Examples of chromatography separation processes are reverse - phase chromatography, ion - exchange chromatography, size - exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, and mixed - mode chromatography.

[0053] "Buffer" is a solution that resists changes in pH due to the action of its acid-base conjugate components. For example, various buffers that can be used depending on the desired pH of the buffer are described in Buffers. A Guide for the Preparation and Use of Buffers in Biological Systems, Gueffroy, D., ed. Calbiochem Corporation (1975). Non-limiting examples of buffers include MES, MOPS, MOPSO, Tris, HEPES, phosphates, acetates, citrates, succinates, glycine and ammonium buffers, and combinations thereof.

[0054] The terms "separation material" or "chromatography matrix" are used interchangeably herein and refer to any type of granular adsorbent, resin, matrix or solid phase that separates a target molecule (e.g., an Fc region-containing protein such as an immunoglobulin) from other molecules present in a mixture in a separation process. Generally, the target molecule is separated from other molecules as a result of differences in the rates at which the individual molecules of the mixture move through and interact with the separation material under the influence of the mobile phase or in a binding-elution process. Separation materials, for example, composed of resin particles, membranes or monolithic adsorbents can be placed in a column or cartridge. Typically, the separation material comprises a base matrix as a substrate and one or more types of ligands attached to the base matrix.

[0055] A "ligand" is or contains a functional group that attaches to a chromatography matrix and determines or affects the binding properties of the matrix. Preferably, the ligand is a polymer chain having one or more, preferably a plurality of functional groups. Most preferred are ligands made of polymer chains having at least one functional group per monomer unit that constructs them.

[0056] Examples of "ligands" include, but are not limited to, ion exchange groups, hydrophobic interaction groups, hydrophilic interaction groups, thiophilic interaction groups, metal affinity groups, affinity groups, bioaffinity groups, and mixed mode groups (combinations as described above). Preferred ligands that can be used herein include, but are not limited to, weak ion exchange groups such as carboxylic acids.

[0057] The terms "ion exchange" and "ion exchange chromatography" refer to a chromatographic process in which a target molecule in a mixture (e.g., an Fc region-containing target protein) interacts with a charged compound linked to an ion exchange matrix (such as by covalent attachment), whereby the target molecule non-specifically interacts with a compound that is more or less charged than the solute impurities or contaminants in the mixture. The impurities in the mixture elute from the column of the ion exchange material either faster or slower than the target molecule, or relative to the target molecule, bind to the resin or are removed from the resin.

[0058] Specifically, "ion exchange chromatography" includes cation exchange, anion exchange, and mixed mode ion exchange chromatography. Ion exchange chromatography can either bind to a target molecule (e.g., an Fc region-containing target protein) followed by elution, or can mainly bind to impurities while the target molecule "flows through" the column. Preferably, the steps of ion exchange chromatography are performed in a bind - elute mode.

[0059] The phrase "ion exchange matrix" refers to a chromatographic medium or separation material that is negatively charged (i.e., a cation exchange resin) or positively charged (i.e., an anion exchange resin). The charge may be provided by attaching one or more charged ligands to the matrix, such as by covalent linkage. Alternatively, or in addition, the charge may be an inherent property of the matrix. The term "cation exchange matrix" is used herein to refer to a separation material that has, as an example of a negatively charged moiety, one or more negatively charged ligands such as carboxylic acid groups attached thereto.

[0060] When "loading" a separation column in the bind - elute mode, the buffer is used to load a sample or composition containing a target molecule (e.g., an Fc - region - containing target protein) and one or more impurities onto a chromatography column (e.g., an ion - exchange column). The buffer has a conductivity and / or pH such that the target molecule binds to the separation material while ideally all of the impurities do not bind to the column and flow through the column.

[0061] When "loading" a separation column to "flow - through" the target molecule, the buffer is used to load a sample or composition containing a target molecule (e.g., an Fc - region - containing target protein) and one or more impurities onto a chromatography column (e.g., an ion - exchange column). The buffer has a conductivity and / or pH such that the target molecule does not bind to the separation material and flows through the column while ideally all of the impurities bind to the column.

[0062] When "loading" a separation column to "bind and elute" the target molecule, the buffer is used to load a sample or composition containing a target molecule (e.g., an Fc - region - containing target protein) and one or more impurities onto a chromatography column (e.g., an ion - exchange column). The buffer has a conductivity and / or pH such that the target molecule binds to the separation material. Separation from one or more impurities occurs following a conductivity and / or pH such that the target molecule is washed or eluted before or after one or more impurities. Typically, the buffer in which the sample is loaded onto the separation material is called the loading buffer or sample buffer.

[0063] The term "equilibration" refers to the use of a buffer to equilibrate the separation material prior to loading the target molecule. Typically, a loading buffer is used for equilibration. "Washing" or "washing" the separation material means passing a suitable liquid, such as a buffer, through or over the separation material. Typically, washing is used after loading to remove weakly bound contaminants from the separation material prior to eluting the target molecule and / or to remove unbound or weakly bound target molecules.

[0064] In this case, typically, the washing buffer is the same as the loading buffer. When using a virus inactivation buffer, it is used to inactivate any existing viruses prior to eluting the target molecule. In this case, typically, the virus inactivation buffer is different from the loading buffer as it may contain a detergent(s) or may have different properties (pH / conductivity / salt and their amounts).

[0065] Washing can also be used to remove contaminants from the separation material after elution of the target molecule. This is done by passing a suitable liquid, such as a buffer, through or over the separation material after elution of the target molecule. In this case, typically, the washing buffer is different from the loading buffer. It may contain a detergent(s) or may have various properties (pH / conductivity / salt and their amounts). The washing buffer is, for example, an acidic buffer.

[0066] "Eluting" a molecule (e.g., a polypeptide or impurity of interest such as immunoglobulin G) from a separation material means removing the molecule therefrom. Elution can be performed directly in flow-through mode when the target molecule is eluted with a solvent on the front side of the loading buffer, or by changing the solution conditions such that a buffer different from the loading buffer competes with the molecule of interest for the ligand site on the separation material. A non-limiting example is eluting a molecule from an ion exchange material by changing the ionic strength of the buffer surrounding the ion exchange material such that the buffer competes with the molecule for the charged site of the ion exchange material.

[0067] The term "average particle size" or d50 means the average particle size distribution value at 50% of the cumulative particle size distribution. The particle size is determined by laser diffraction, preferably using a Malvern 'Master Sizer'. The term "average pore size" means the average pore size distribution value at 50% of the cumulative pore size distribution. The terms "flow-through process", "flow-through mode" and "flow-through operation", when used interchangeably herein, refer to a separation technique in which at least one target molecule (e.g., an Fc region-containing protein or antibody) contained in a sample together with one or more impurities is intended to flow through a separation material that binds generally one or more of the impurities and elutes (i.e., flows through) the separation material together with the loading buffer without generally binding the target molecule.

[0068] The terms "bind and elute mode" and "bind and elute process", when used herein, refer to a separation technique in which at least one target molecule (e.g., an Fc region-containing protein) contained in a sample binds to a suitable separation material (e.g., an ion exchange chromatography medium) and is then eluted with a buffer different from the loading buffer.

[0069] As used herein, the term "ion density" refers to the number of ions per unit volume or mass of a given separation material, more specifically, the number of ions of a given type (e.g., cations or anions) per unit volume or mass of the separation material. Generally, the number of ions is estimated by titrating a given separation material. Moreover, the amount of ions is given in equivalents (eq) per mass or per unit volume of the separation material.

[0070] As used herein, the term "conductivity" refers to an inherent property of most materials that quantifies how strongly a material resists or conducts an electric current. In an aqueous solution such as a buffer, an electric current is carried by charged ions. Conductivity is determined by the number of charged ions, the amount of charge they carry, and how fast they move. Thus, for most aqueous solutions, the higher the concentration of dissolved salts, the higher the conductivity. Increasing the temperature enables the ions to move faster and thus increases the conductivity. Typically, unless otherwise indicated, conductivity is defined at room temperature. The fundamental unit of conductivity is the Siemens (S). This is defined as the reciprocal of the resistance, expressed in ohms, measured between the opposing faces of a 1 cm cube of the liquid. Thus, values are estimated in S / cm.

[0071] The present invention provides a method for separating or concentrating protein glycoforms. This means that one or more protein glycoforms can be separated from other protein glycoforms and / or from other impurities in a sample. Preferably, at least one protein glycoform is separated from at least one other glycoform, for example, one high-mannose glycoform is separated from other protein glycoforms. This is done by chromatographic separation on a predetermined separation material, also called a resin, which comprises a base matrix to which polymer chains are attached in a double-bonded manner. The polymer chains are made of monomers that contain amino acids and polymerizable double bonds.

[0072] By the method of the present invention, glycoforms can be separated, concentrated and / or purified, enabling efficient separation of glycan species. In certain aspects of the present invention, it is useful for separating glycan variants containing high mannose or terminal mannose molecules that are more hydrophobic and demonstrate faster clearance. Moreover, such glycan variants can contribute to higher toxicity and lower glycoprotein efficacy. In another aspect of the present invention, it is useful for separating glycoproteins that retain fucose, which ensures better control of antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In other aspects of the present invention, it is useful for removing proteins that are not glycosylated but are otherwise identical to the target glycoprotein.

[0073] In one aspect of the present invention, the purified glycoprotein does not contain high mannose glycan variants. In another aspect of the present invention, the purified glycoprotein contains low levels of hybrid glycan variants with terminal mannose. In another aspect of the present invention, the purified glycoprotein contains low levels of non-fucosylated glycan variants. In another aspect of the present invention, the purified glycoprotein contains low levels of non-glycosylated proteins.

[0074] The separation material of the present invention comprises a substrate to which tentacle-like structures are attached, preferably grafted.

[0075] The substrate, also referred to as the base matrix, contains reactive groups available for graft polymerization reactions, especially OH groups, preferably aliphatic OH groups. The substrate can thus also be prepared, for example, from organic polymers.

[0076] This type of organic polymer can be a polysaccharide such as agarose, dextran, starch, cellulose, etc., or a synthetic polymer such as poly(acrylamide), poly(methacrylamide), poly(acrylate), poly(methacrylate), hydrophilically substituted poly(alkyl allyl ether), hydrophilically substituted poly(alkyl vinyl ether), poly(vinyl alcohol), poly(styrene), and copolymers of the corresponding monomers. These organic polymers can also preferably be employed in the form of a crosslinked hydrophilic network. This includes polymers made from styrene and divinylbenzene, which can preferably be employed in a hydrophilized form, like other hydrophobic polymers.

[0077] Alternatively, inorganic materials such as silica, zirconium oxide, titanium dioxide, aluminum oxide, etc., can be employed as the substrate. It is also equally possible to employ composite materials, i.e., particles that can be magnetized by themselves, for example, by copolymerization of magnetizable particles or a magnetizable core. It is also possible to use core - shell materials where the shell, i.e., at least the surface or coating, has OH groups.

[0078] However, since the materials according to the present invention should preferably withstand alkaline cleaning or regeneration, for example, at a basic pH, over an extended period of use, the use of a hydrophilic substrate that is stable to hydrolysis or cannot be hydrolyzed without difficulty is preferred.

[0079] The base matrix can consist of irregularly shaped or spherical particles, and the particle size can be between 2 and 1000 μm. An average particle size between 3 and 300 μm is preferred, and in the most preferred embodiment, the average particle size is between 20 and 63 μm. The base matrix can in particular be in the form of non-porous or preferably porous particles. The average pore diameter can be between 2 and 300 nm. A pore diameter between 5 and 200 nm is preferred, and the most preferred average pore diameter is between 40 and 110 nm.

[0080] The base matrix can equally well be in the form of a membrane, fiber, hollow fiber, coating or monolithic molding. The monolithic molding is preferably in the form of a porous three-dimensional body, for example in the form of a cylinder.

[0081] In a preferred embodiment, the base matrix is a copolymer formed by copolymerization of at least one compound from groups a) and b), where a) at least one hydrophilic substituted alkyl vinyl ether of formula I

Chemical formula

Chemical formula

[0082] R4 in formula I is typically an alkyl radical, cycloaliphatic radical or aryl radical bearing at least one hydroxyl group.

[0083] In a highly preferred embodiment, the base matrix is formed by copolymerization of a hydrophilic substituted alkyl vinyl ether selected from the group consisting of 1,4 - butanediol monovinyl ether, 1,5 - pentanediol monovinyl ether, diethylene glycol monovinyl ether or cyclohexanedimethanol monovinyl ether with divinylethyleneurea (1,3 - divinylimidazolin - 2 - one) as a crosslinking agent. Examples of suitable commercially available vinyl ether - based substrates are Eshmuno®, Merck KGaA, Germany.

[0084] wherein linear polymer chains are covalently bonded to the surface of the substrate such that a separation material is produced, a) the substrate preferably contains aliphatic hydroxyl groups, b) the polymer is covalently bonded to the support, c) the polymer contains amino acid residues, d) the monomer units of the polymer are linearly linked.

[0085] The actual separation material comprising the substrate and the covalently attached linear polymer chains can be prepared in various ways. In the case of the "grafting onto" method, the polymer chains first have to be formed from monomers and, in a second step, attached to the surface. In the case of the "grafting from" method, the polymerization reaction is initiated on the surface and the graft polymer is constructed directly from the individual monomers. Other polymerization methods enabling the attachment to the surface of the substrate can also be employed.

[0086] The "grafting from" method is preferred, and in particular preferred are modified methods in which only minor by-products such as non-covalently bound polymers that must be removed by separation are formed. Processes involving controlled free radical polymerization, such as the atom transfer radical polymerization (ATRP) method, for example, are seen as particularly interesting. Here, the initiating groups are covalently bound to the support surface at the desired density in the first step. The initiating groups are, for example, halides bound via an ester functional group as in 2-bromo-2-methylpropionic acid ester. The graft polymerization is carried out in the presence of a copper(I) salt in the second step.

[0087] A highly preferred one-step graft polymerization reaction suitable for the production of the separation materials used in the present invention can be initiated by cerium(IV) on a hydroxyl-containing substrate without the need to activate the substrate.

[0088] This cerium(IV)-initiated grafting is preferably carried out according to EP0337144 or US5,453,186. The chains produced are linked to the substrate via monomer units. For this purpose, the substrates according to the invention are suspended in a solution of the monomer, preferably in an aqueous solution. The grafting-on of the polymer material can be done in the course of a conventional redox polymerization involving the exclusion of oxygen. The polymerization catalyst employed is cerium(IV) ions, since this catalyst forms free radical sites on the surface of the substrate from which the polymerization of the monomer starts. This reaction is usually carried out in dilute mineral acid. For carrying out this graft polymerization, the acid is generally employed in an aqueous solution with a concentration in the range of 1 to 0.00001 mol / l, preferably 0.1 to 0.001 mol / l. The use of dilute nitric acid is particularly highly preferred and is employed at a concentration of 0.1 to 0.001 mol / l.

[0089] For the preparation of the separation material according to the present invention, the monomers are usually added in an excess amount relative to the substrate. Typically, 0.05 to 100 mol of total monomers per liter of the deposited polymer material are employed, preferably 0.05 to 25 mol / l are employed.

[0090] The polymerization is terminated by a termination reaction involving a cerium salt. For this reason, the (average) chain length can be affected by the concentration ratios of the substrate, initiator and monomers. Furthermore, either a uniform monomer or a mixture of different monomers can also be employed; in the latter case, a graft copolymer is formed.

[0091] The monomers advantageously used for the preparation of the separation material used in the present invention have the formula V

Chemical formula

Chemical formula

[0092] Perfluoroalkyl means that all H atoms of the alkyl residue are replaced by F atoms. Exemplary preferred structures of formula V are Va

Chemical formula

[0093] Such monomers can also be described as acryloyl valine (Formula Va), acryloyl leucine (Formula Vb), acryloyl alanine, acryloyl norleucine, methacryloyl valine, methacryloyl leucine, methacryloyl alanine, methacryloyl norleucine, dimethacryloyl valine, dimethacryloyl leucine, dimethacryloyl alanine, dimethacryloyl norleucine. Among them, acryloyl valine and acryloyl leucine are preferred, and acryloyl leucine is particularly preferred.

[0094] The separation material used in the method of the present invention preferably contains only a tentacle-like linear polymer structure grafted on a substrate constructed from monomers according to Formula V. Preferably, they contain a linear polymer constructed from only one type of monomer according to Formula V.

[0095] However, it is also possible that a linear polymer is constructed by copolymerization of two or more different monomers according to Formula V. A linear polymer can also be constructed by copolymerization of one or more different monomers according to Formula V and one or more other polymerizable monomers (such as other acrylamides, methacrylates, acrylates, methacrylates functionalized (for example, with ionic groups, hydrophilic groups or hydrophobic groups), etc.).

[0096] Exemplary structures of the separation materials according to the present invention are shown in FIGS. 2 and 3. FIG. 2 shows a substrate (dots) functionalized with a linear polymer constructed by polymerization of a croloyl leucine monomer. In addition, for clarity, the carboxyalkyl end groups of each polymer unit are shown. FIG. 3 shows a substrate (dots) functionalized with a linear polymer constructed by polymerization of an acryloyl valine monomer. In addition, for clarity, the carboxyalkyl end groups of each polymer unit are shown.

[0097] In a preferred embodiment, the separation material used in the method of the present invention is an ion exchange material with various amounts of ion density bands ranging from 10 to 1200 μeq / g, where in a more preferred embodiment, the ion density band is between 400 and 900 μeq / g. In a preferred embodiment, the ion exchange material can contain an ion exchange functional group and a hydrophobic functional group, where in a more preferred embodiment, both functional groups are on a single surface functional unit resulting from a monomeric unit constructed in the polymer chain, and where in the most preferred embodiment, the functional group is part of an amino acid residue.

[0098] Typically, a chromatography column containing the separation material described above is used in the method according to the invention. Chromatography columns are known to those skilled in the art. They typically comprise a cylindrical tube or cartridge filled with the separation material, as well as filters and / or means for fixing the separation material in the tube or cartridge, and connections for solvent delivery to and from the inside and outside of the tube. The size of the chromatography column varies depending on the application, for example, analytical or preparative. The chromatography column can also be a membrane containing cartridge.

[0099] The materials used in the method of the present invention can also be described as substrates provided with separation effectors. They are aimed at separating the sample solution out for the selective, partially selective or non-selective binding or adsorption of one or more target components, or for the selective, partially selective or non-selective binding or adsorption of one or more secondary components, for the separation of secondary components out of the matrix, the isolation, concentration and / or depletion of biopolymers from natural sources, the isolation, concentration and / or depletion of biopolymers from recombinant sources, the isolation, concentration and / or depletion of proteins or polypeptides, the isolation, concentration and / or depletion of monoclonal and polyclonal antibodies, the isolation, concentration and / or depletion of viruses, the isolation, concentration and / or depletion of host cell proteins, or the isolation, concentration and / or depletion of glycoproteins, and can be used. Preferred target molecules are glycoproteins.

[0100] The target molecule is separated from one or more other substances in the sample, where the sample containing the target molecule is dissolved in a liquid, which is brought into contact with the material according to the invention. The contact time is generally in the range of 30 seconds to 24 hours. It is advantageous for it to act according to the principle of liquid chromatography by passing the liquid through a chromatography column containing the separation material according to the invention. The liquid can pass through the column simply by its gravity or by being pumped through it.

[0101] An alternative method is batch chromatography, where the liquid is mixed by stirring or shaking for the length of time necessary for the target molecule or biopolymer to be able to bind to the separation material. Similarly, it is possible for the liquid to be separated to act according to the principle of chromatographic fluidized bed, for example by introducing the liquid to be separated into a suspension containing the separation material, where the separation material is selected to be suitable for the desired separation due to its high density and / or magnetic core.

[0102] When the chromatography process is operated in the binding - elution mode, the target molecule binds to the separation material. The separation material can then be washed with a washing buffer, which preferably has the same ionic strength and the same pH as the liquid in which the target molecule is brought into contact with the separation material. The washing buffer removes all substances that do not bind to the separation material. Further washing steps with other suitable buffers may follow without desorbing the target molecule. The desorption of the bound target molecule is carried out by changing the ionic strength in the eluent and / or by changing the pH in the eluent and / or by changing the solvent. The target molecule is thus obtained in a purified and concentrated form in the eluent. The target molecule generally has a purity of 70% - 99%, preferably 85% - 99%, particularly preferably 90% - 99% after desorption.

[0103] It is also possible to use the binding - elution process described above to separate or purify more than one target molecule, in which case a group of target molecules binds to the separation material and is separated from one or more impurities as described above.

[0104] However, if the chromatographic process is operated in flow - through mode, the target molecule or group of different types of target molecules remains in the liquid while other accompanying substances bind to the separation material. The target molecule is then obtained directly by collecting the column eluate in the through - flow. It is known to those skilled in the art how to adapt the conditions, particularly pH and conductivity, in order to bind a specific biopolymer to the separation material, or whether it is advantageous for the purification task of not binding the target molecule.

[0105] The present invention is preferably directed to the use of the separation material described above for the separation and purification of glycoproteins, and to a method for the separation and / or purification of glycoproteins by liquid chromatography, which comprises contacting the separation material with a sample containing one or more glycoproteins, preferably under acidic conditions.

[0106] Unexpectedly, we have found that the ion - exchange material according to the present invention can be used for the separation or enrichment of glycoforms that enable efficient glycan species separation with a capacity of > 10 mg glycoprotein / ml material, and in a more preferred embodiment, the capacity is between 10 and 80 mg / ml. Moreover, this innovative ion - exchange material can be used at high conductivity > 5 mS / cm, and in a more preferred embodiment, the conductivity is between 5 and 60 mS / cm.

[0107] Surprisingly, it was possible to separate and concentrate glycan variants including high mannose-containing variants, terminal mannose-containing variants, fucose-containing variants and non-glycosylated variants, preferably using a pH change from 4 - 7 to above 9, preferably up to a pH between 9 - 11, most preferably up to pH 10, in a gradient or step mode. In addition, the application of this separation material showed significant economic advantages compared to affinity chromatography mode, and enabled a broader selectivity and improved performance compared to anion exchange mode.

[0108] Furthermore, the application is not limited to the separation of different glycan variant species and can also be used to remove non-glycosylated variants from glycosylated ones.

[0109] In addition, the application of this material is not limited to binding - elution applications and can also be used in flow-through mode, resulting in the adsorption of higher mannose-containing species or higher terminal mannose-containing glycan variants onto an ion exchange material using a buffer having a pH < 6 and / or a high conductivity (> 20 mS / cm). Furthermore, surprisingly, only this ion exchange material, which consists essentially of amino acids covalently attached to the material, has the required selectivity for glycan species, and in a more preferred embodiment, these amino acids are valine or leucine (including their combinations and derivatives), and most preferably leucine.

[0110] Furthermore, the present invention provides a chromatography-based glycoprotein purification step that can be regenerated and is applicable in a broad operation window, for example, pH 3 - 10; conductivity 5 - 60 mS / cm. In a preferred embodiment, the ion exchange material used in the present invention is incorporated into a chromatography column and used for a glycoprotein purification process in a binding - elution mode for separating glycan species.

[0111] In a preferred embodiment, the ion exchange material used in the present invention is incorporated into a chromatography column and used for a glycoprotein purification process in a binding - elution mode for separating glycan species, where the span of the operation window is between pH 2 and 10, and in a more preferred embodiment the pH is between 4 and 7. In a preferred embodiment, the ion exchange material used in the present invention is incorporated into a chromatography column and used for a glycoprotein purification process in a binding - elution mode for separating glycan species, where solvent pH elution is used to recover the bound components.

[0112] In a preferred embodiment, the ion exchange material used in the present invention is incorporated into a chromatography column and used for a glycoprotein purification process in a binding - elution mode for separating glycan species, where the span of the binding window is between 10 mg glycoprotein / ml material and 100 mg glycoprotein / ml material, and in a more preferred embodiment the span of the binding window is between 20 mg glycoprotein / ml material and 80 mg glycoprotein / ml material.

[0113] In a preferred embodiment, the ion exchange material used in the present invention is incorporated into a chromatography column and used for a glycoprotein purification process in a binding - elution mode for separating glycan species, where the span of the conductivity band is between 5 and 60 mS / cm, and in a more preferred embodiment the conductivity range is between 15 and 35 mS / cm.

[0114] In another preferred embodiment, the ion exchange material used in the present invention is incorporated into a chromatography column and used for a glycoprotein purification process in a flow-through mode for separating glycan species, where low-mannose-containing species are in the flow-through and higher-mannose-containing species bind to the separation material.

[0115] In another preferred embodiment, the ion exchange material used in the present invention is incorporated into a chromatography column and used for a glycoprotein purification process in a flow-through mode for separating glycan species, where non-hybrid species are in the flow-through and hybrid (e.g., terminally mannose-containing) species bind to the separation material.

[0116] In another preferred embodiment, the ion exchange material used in the present invention is incorporated into a chromatography column and used for a glycoprotein purification process in a flow-through mode for separating glycan species, where low-fucosylated species are in the flow-through and highly fucosylated-containing species bind to the separation material.

[0117] In another preferred embodiment, the method of the present invention is a) applying a sample containing a mixture of protein glycoforms, at least one of which is a high-mannose glycoform, to a chromatography column containing the ion exchange material used in the present invention; b) separating and eluting at least one high-mannose glycoform in the flow-through; c) eluting the glycoforms bound from the separation material, whereby the high-mannose glycoforms are reduced in the eluted glycoforms.

[0118] In another preferred embodiment, the method of the present invention is a) Applying a sample containing a mixture of protein glycoforms to a chromatography column containing an ion exchange material used in the present invention, wherein at least one of the glycoforms is a high-mannose glycoform, and wherein at least one high-mannose glycoform, and typically also other protein glycoforms, bind to the separation material. b) Contacting an elution buffer with the separation material, whereby one or more glycoforms different from the high-mannose glycoform elute, while at least one high-mannose glycoform remains bound to the separation material. c) Optionally, contacting the separation material with a second elution buffer, typically different from the first elution buffer, and thereby eluting at least one high-mannose glycoform.

[0119] In this embodiment, the eluate obtained in step b) has a reduced high-mannose glycoform compared to the sample applied in step a), and is concentrated in the eluate obtained in step c). The mannose glycoforms separated in the flow-through or bound elution as described above are preferably antibodies having different degrees of mannosylation, or viruses and / or virus capsids carrying proteins with different degrees of mannosylation.

[0120] In another preferred embodiment, the ion exchange material used in the present invention is incorporated into a chromatography column for use in a flow-through or bound elution mode in a glycoprotein purification process for separating viruses and / or virus particles containing glycoprotein forms from viruses and / or virus particles containing other types of glycoproteins or no glycoproteins. Preferably, the separation occurs based on the different amounts of mannose present in the glycan structure.

[0121] In another preferred embodiment, the method of the present invention a) Applying a sample containing a mixture of protein glycoforms, at least one of which is a terminal mannose glycoform, to a chromatography column containing an ion exchange material used in the present invention, wherein at least one terminal mannose glycoform, and typically also other protein glycoforms, bind to the separation material. b) Contacting an elution buffer with the separation material, whereby one or more glycoforms different from the terminal mannose glycoform elute, while at least one terminal mannose glycoform remains bound to the separation material. c) Optionally, contacting the separation material with a second elution buffer, typically different from the first elution buffer, and thereby eluting at least one terminal mannose glycoform.

[0122] In this embodiment, in the eluate obtained in step b), the terminal mannose glycoform is reduced compared to the sample applied in step a), and in the eluate obtained in step c) it is concentrated. The terminal mannose glycoforms separated in the flow-through or binding elution as described above are preferably antibodies having different degrees of mannosylation, or viruses and / or virus capsids carrying proteins with different degrees of mannosylation.

[0123] In another preferred embodiment, the ion exchange material used in the present invention is incorporated into a chromatography column for use in a flow-through or binding elution mode in a glycoprotein purification process for separating viruses and / or virus particles containing glycoprotein forms from viruses and / or virus particles containing other types of glycoproteins or no glycoproteins. Preferably, the separation occurs based on the different amounts of terminal mannose present in the glycan structure.

[0124] In another preferred embodiment, the method of the present invention a) Applying a sample containing a mixture of protein glycoforms, wherein at least one of the glycoforms is a glycoform having fucose, to a chromatography column containing a separation material used in the present invention, wherein at least one glycoform having fucose, and typically also other protein glycoforms, bind to the separation material. b) Contacting an elution buffer with the separation material, whereby one or more glycoforms different from the fucose-free glycoforms elute, while at least one glycoform having fucose remains bound to the separation material. c) Optionally, contacting the separation material with a second elution buffer, typically different from the first elution buffer, and thereby eluting at least one fucose-free glycoform.

[0125] In this embodiment, in the eluate obtained in step b), the fucose-free glycoforms are reduced compared to the sample applied in step a), and are concentrated in the eluate obtained in step c). The glycoforms having fucose, separated in the flow-through or bound elution as described above, are preferably antibodies having different degrees of fucosylation, or viruses and / or virus capsids carrying proteins with different degrees of fucosylation.

[0126] In another preferred embodiment, the ion exchange material used in the present invention is incorporated into a chromatography column for use in a flow-through or bound elution mode in a glycoprotein purification process for separating viruses and / or virus particles containing glycoprotein forms from viruses and / or virus particles containing other types of glycoproteins or no glycoproteins. Preferably, the separation occurs based on different amounts of fucose present in the glycan structure.

[0127] In another preferred embodiment, the method of the present invention a) applying a sample comprising a mixture of glycoprotein-bearing and non-glycoprotein proteins, wherein at least one of the glycoprotein-bearing proteins, a protein glycoform, binds to a chromatography column comprising the separation material used in the present invention, b) contacting an elution buffer with the separation material, whereby at least one glycoprotein-bearing protein elutes, while at least one non-glycoprotein remains bound to the separation material, c) optionally, contacting the separation material with a second elution buffer, typically different from the first elution buffer, and thereby eluting at least one non-glycoprotein.

[0128] In this embodiment, in the eluate obtained in step b), the glycoprotein-bearing proteins are enriched compared to the sample applied in step a), and are reduced in the eluate obtained in step c). The glycoforms separated in the flow-through or binding elution as described above are preferably antibodies having different degrees of glycosylation, or viruses and / or virus capsids carrying proteins with different degrees of glycosylation.

[0129] In another preferred embodiment, the ion exchange material used in the present invention is incorporated into a chromatography column for use in a flow-through or binding elution mode in a glycoprotein purification process for separating viruses and / or virus particles containing glycoprotein forms from other viruses and / or virus particles containing other types of glycoproteins or no glycoproteins. Preferably, the separation occurs based on different amounts of glycosylation present in the glycan structure.

[0130] In another preferred embodiment, the ion exchange material used in the present invention is incorporated into a chromatography column and used for a glycoprotein purification process in a flow-through mode for separating glycan species, where proteins containing glycosylation are in the flow-through and proteins not containing glycosylation bind to the separation material.

[0131] In a preferred embodiment, the ion exchange material can be of various particle sizes ranging from 1 to 200 μm, and in a more preferred embodiment, the average particle size is between 20 and 63 μm. In a preferred embodiment, the ion exchange material according to the present invention can have various pore sizes ranging from 4 to 1500 nm, and in a more preferred embodiment, the average pore size is between 10 and 120 nm, and in a most preferred embodiment, the average pore size is between 40 and 110 nm.

[0132] The method of the present invention is very flexible. A suitable chromatography mode (binding - elution or flow-through) can be applied, and the conditions can vary within the wide range described above. In addition, the target molecule can also be of various types. As discussed above, in any case, the sample contains at least one protein glycoform. However, the target molecule can be any of a protein glycoform, a group of different protein glycoforms, or a non-glycosylated protein.

[0133] Typically, in the method of the present invention, at least one protein glycoform binds to the separation material, but this protein glycoform does not necessarily have to be the target molecule. It may be the target molecule, but the target molecule can also be another protein glycoform or a non-glycosylated protein that also binds to the separation material or is in the flow-through. Since the process of the present invention enables the separation of different protein glycoforms, as well as glycosylated and non-glycosylated proteins, the target molecule can be defined as necessary.

[0134] The present invention is further illustrated by the following figures and examples, but is not limited thereto. The entire disclosures of all applications, patents, and publications cited in the above and below, as well as in the corresponding EP patent application No. 19184130.3 filed on July 7, 2019, are incorporated herein by reference.

Examples

[0135] Example The following examples represent practical applications of the present invention.

[0136] 1. Synthesis of Separation Material For the preparation of monomers used for grafting from the process, an amino acid, for example, valine, leucine or alanine, is dissolved in VE water, and the pH is adjusted to a pH above 13 by adding NaOH (32%). An acrylic compound such as acrylate chloride or acrylic acid is added at a temperature between 0 and 5 °C, and the mixture is stirred for 1 hour. The reaction schemes with acrylate chloride for valine and leucine are shown in Figures 2 and 3.

[0137] Then, the pH is adjusted to about 2.2 by adding nitric acid. Thereafter, an OH-containing substrate, for example, Eshmuno® particles, is added. The polymerization starts by adding cerium(IV) nitrate. The reaction is carried out at 30 - 50 °C for 4 hours. After the polymerization reaction, the unreacted components and initiator are removed by extensive washing with acidic, basic, and solvent mixtures at room temperature or elevated temperature.

[0138] 2. Separation of Rituximab® from high - mannose glycan forms An ion - exchange material (for example, having an average particle size between 20 - 63 μm, an average pore size between 40 - 110 nm, and an ion density between 400 - 900 μeq / g) prepared as per Example 1 was evaluated for its ability to separate the high - mannose - containing species of the commercial drug Rituximab®. The ion - exchange material was packed into a 5 × 100 mm - sized chromatographic column with an asymmetry between 0.8 - 1.2 and > 3000 plates / m. After packing the ion - exchange material, the resulting chromatographic column was washed with 1M NaOH solution for 30 minutes and pre - equilibrated with a loading buffer solution having a pH of 4.75 and 250 mM NaCl. The buffer solution contained a combination of salts such as sodium dihydrogen phosphate, TRIS, and glycine to obtain a pH of 4.5. Using the same solution, the Rituximab® sample was diluted to a concentration of 4.8 mg / ml.

[0139] This solution was loaded onto the prepared chromatographic column until a loading of 20 mg Rituximab® per 1 ml of resin was reached. These steps and the subsequent steps were carried out at a buffer velocity of 150 cm / h. After loading 20 mg / ml Rituximab®, the chromatographic column was washed with a buffer solution at pH 4.75 and then eluted using gradient elution with a buffer having a pH of 8.5 and 250 mM NaCl. This elution buffer was prepared using different salts such as sodium dihydrogen phosphate, TRIS, and glycine. The conductivity and pH values were traced during the experimental setup, which indicates that elution of Rituximab® from the column was achieved due to the pH change during gradient elution. The sample eluate was fractionated and the resulting fractions were evaluated for each line using LC-MS method for glycan species identification (Figure 4).

[0140] Analytical evaluation using LC-MS analysis of the collected fractions is shown in Figure 5, which indicates that the main glycoprotein elution peaks, further characterized by representative 1B9 and 1C7 fractions, contained no high-mannose variants. Most of the high-mannose glycan variants eluted at a higher pH, which is further characterized by representative 1D10 and 1E3 fractions (Figure 5).

[0141] As shown in Figure 4, separation / concentration of high-mannose-containing glycan species is possible while using linear pH gradient elution. No high-mannose variants were detected in the main glycoprotein-containing fractions (e.g., 1B9 and 1C7). All high-mannose-containing variants eluted in the separated fractions (e.g., 1D10).

[0142] 3. Separation of high-mannose glycan forms of Erbitux® in a wide application window within the range of 200 mM to 600 mM sodium sulfate An ion exchange material prepared as per Example 1 (for example, having an average particle size between 20 - 63 μm, an average pore size between 40 - 110 nm, and an ion density between 400 - 900 μeq / g) was evaluated for its ability to separate the high mannose-containing species of the commercially available drug Erbitux®. The ion exchange material was packed into a 5 × 100 mm sized chromatographic column with an asymmetry between 0.8 - 1.2 and >3000 plates / m. After packing the ion exchange material, the resulting chromatographic column was washed with 1M NaOH solution for 30 minutes and pre-equilibrated with a loading buffer solution having a pH of 4.5 and 250 mM NaCl. The buffer solution contained a combination of salts such as sodium dihydrogen phosphate, TRIS, and glycine to obtain a pH of 4.5.

[0143] Onto the prepared chromatographic column, a pre-purified Erbitux® sample was loaded until a loading of 5 mg of pre-purified Erbitux® per 1 ml of resin was reached. These steps and subsequent steps were carried out at a buffer velocity of 150 cm / h. After loading 5 mg / ml of pre-purified Erbitux®, the chromatographic column was washed with a buffer solution of pH 4.5 and various amounts of Na2SO4. The amount of Na2SO4 was varied between 200 - 600 mM. The column was then eluted using a gradient elution with a buffer having a pH of 8.5 and the corresponding amount of Na2SO4. This elution buffer was prepared using different salts such as sodium dihydrogen phosphate, TRIS, and glycine. The conductivity and pH values were traced during the experimental setup, which indicates that elution of Erbitux® from the column was achieved due to the pH change during gradient elution.

[0144] In Table 1, the main peak and the maximum elution value of the high mannose-containing peak at the corresponding pH are shown.

Table 1

[0145] Table 1 shows the main peak and the maximum elution value of the high-mannose-containing peak at the corresponding pH.

[0146] As shown in Table 1, separation / concentration of high-mannose-containing glycan species is possible over a wide range of conductivities while using linear pH gradient elution. Fractions with high-mannose-containing variants eluted at higher pH values compared to glycan species without high mannose over the entire range investigated.

[0147] 4. Separation of Rituximab® and Hybrid Glycan Forms An ion-exchange material (e.g., having an average particle size between 20 - 63 μm, an average pore size between 40 - 110 nm, and an ion density between 400 - 900 μeq / g) prepared as per Example 1 was evaluated for its ability to separate the hybrid glycan species of the commercially available drug Rituximab®. The ion-exchange material was packed into a 5 × 100 mm size chromatographic column with an asymmetry between 0.8 - 1.2 and >3000 plates / m. After packing the ion-exchange material, the resulting chromatographic column was washed with 1M NaOH solution for 30 minutes and pre-equilibrated with a loading buffer solution having a pH of 4.75 and 250 mM NaCl. The buffer solution contained a combination of salts such as sodium dihydrogen phosphate, TRIS, and glycine to obtain a pH of 4.75. Using the same solution, the Rituximab® sample was diluted to a concentration of 4.8 mg / ml.

[0148] This solution was loaded onto the prepared chromatographic column until reaching a loading of 20 mg Rituximab® per 1 ml of resin. These steps and the subsequent steps were carried out at a buffer velocity of 150 cm / h. After loading 20 mg / ml Rituximab®, the chromatographic column was washed with a buffer solution at pH 4.75 and then eluted using gradient elution with a buffer having a pH of 8.5 and 250 mM NaCl. This elution buffer was prepared using different salts such as sodium dihydrogen phosphate, TRIS, and glycine. The conductivity and pH values were traced during the experimental setup, which indicates that the elution of Rituximab® from the column was achieved due to the pH change during gradient elution. The sample eluate was fractionated, and the obtained fractions were evaluated using an LC-MS method for glycan species identification (Figure 7).

[0149] The analytical evaluation using LC-MS analysis of the collected fractions is shown in Figure 7, and the main glycoprotein elution peaks, further characterized by representative 1B9 and 1C7 fractions, showed no hybrid glycan variants (e.g., those containing terminal mannose). Most of the hybrid glycan variants eluted at a higher pH, which is further characterized by representative 1D10 and 1E3 fractions (Figure 7).

[0150] As shown in Figures 6 and 7, separation / concentration of hybrid glycan species is possible while using linear pH gradient elution. No hybrid glycan variants were detected in the main glycoprotein-containing fractions (e.g., 1B9 and 1C7). All hybrid glycan variants eluted in the separated fractions (e.g., 1D10 and 1E3).

[0151] 5. mAb05 and Hybrid Glycan Form Separation An ion exchange material prepared as per Example 1 (for example, having an average particle size between 20 - 63 μm, an average pore size between 40 - 110 nm, and an ion density between 400 - 900 μeq / g) was evaluated for its ability to separate the hybrid glycan species of mAb05. The ion exchange material was packed into a 5 × 100 mm size chromatographic column with an asymmetry between 0.8 - 1.2 and >3000 plates / m. After packing the ion exchange material, the resulting chromatographic column was washed with 1M NaOH solution for 30 minutes and pre-equilibrated with a loading buffer solution having a pH of 4.75 and 250 mM NaCl. The buffer solution contained a combination of salts such as sodium dihydrogen phosphate, TRIS, and glycine to obtain a pH of 4.75. Using the same solution, the mAb05 sample was diluted until a concentration of 5 mg / ml was reached.

[0152] This solution was loaded onto the prepared chromatographic column until a mAb05 load of 30 mg per 1 ml of resin was reached. These steps and subsequent steps were performed at a buffer velocity of 150 cm / h. After loading 30 mg / ml of mAb05, the chromatographic column was washed with a buffer solution of pH 4.75 and then eluted using gradient elution with a buffer having a pH of 8.5 and 250 mM NaCl. This elution buffer was prepared using different salts such as sodium dihydrogen phosphate, TRIS, and glycine. The conductivity and pH values were traced during the experimental setup, which indicates that the elution of mAb05 from the column was achieved due to the pH change during gradient elution. The sample eluate was fractionated and the resulting fractions were evaluated for each line using an LC-MS method for glycan species identification (Figure 9).

[0153] The analytical evaluation using LC-MS analysis of the collected fractions is shown in Figure 9, indicating that the first elution fractions 1A4 to 2A4 contained no hybrid glycan variants at all (as examples, terminal mannose-containing G0F-N and G1F-N). Most of the hybrid glycan variants eluted at a higher pH, which was further characterized by the typical fractions 3B5 to 4B2 (Figure 9). In addition to the hybrid form, the mannose 5-containing glycan variants were also eluted at a similarly higher pH.

[0154] As shown in Figure 8, separation / concentration of hybrid glycan species is possible while using linear pH gradient elution. No hybrid glycan variants were detected in the main glycoprotein-containing fractions (as examples, 1A4 to 2A4). All hybrid glycan variants eluted at a higher pH value (as examples, 3B5 to 4B2).

[0155] Moreover, the obtained fractions were characterized using analytical size exclusion chromatography and the level of fragmentation or aggregation was monitored. The results showed the presence of less than 1% aggregates in the samples used or in the obtained fractions (Table 2).

Table 2

[0156] Table 2 shows the results of analytical size exclusion chromatography presented as area for the obtained fractions of the analyzed elution fractions (1B4 to 1F12) and as % for fragmented or aggregated species. All fractions had a purity > 99%.

[0157] 6. Separation of mAb05 into fucosylated and non-fucosylated forms An ion-exchange material prepared as per Example 1 (for example, having an average particle size between 20 - 63 μm, an average pore size between 40 - 110 nm, and an ion density between 400 - 900 μeq / g) was evaluated for its ability to separate fucosylated and non-fucosylated glycan species of mAb05. The ion-exchange material was packed into a 5 × 100 mm sized chromatographic column with an asymmetry between 0.8 - 1.2 and >3000 plates / m. After packing the ion-exchange material, the resulting chromatographic column was washed with 1M NaOH solution for 30 minutes and pre-equilibrated with a loading buffer solution having a pH of 4.75 and 200 mM NaCl. The buffer solution contained a combination of salts such as sodium dihydrogen phosphate, TRIS, and glycine to obtain a pH of 4.75. Using the same solution, the mAb05 sample was diluted until it reached a concentration of 5 mg / ml.

[0158] This solution was loaded onto the prepared chromatographic column until it reached a mAb05 load of 30 mg per 1 ml of resin. These steps and the subsequent steps were carried out at a buffer velocity of 150 cm / h. After loading 30 mg / ml mAb05, the chromatographic column was washed with a buffer solution of pH 4.75 and then eluted using gradient elution with a buffer having a pH of 8.5 and 200 mM NaCl. This elution buffer was prepared using different salts such as sodium dihydrogen phosphate, TRIS, and glycine. The conductivity and pH values were traced during the experimental setup, which indicates that the elution of mAb05 from the column was achieved due to the pH change during gradient elution (Figure 10). The flow-through and sample eluate were fractionated, and the resulting fractions were evaluated for each line using LC-MS method for glycan species identification (Table 3).

[0159] The analytical evaluation using LC-MS analysis of the collected fractions is shown in Table 3, indicating that the first elution fractions 1B4 to 1D5 contained fewer non-fucosylated forms (G0 and G1) compared to the loaded glycoproteins, as well as (G0F and G1F). Larger amounts of fucosylated glycan variants (G0F and G1F) eluted at higher pH values, which were further characterized by the representative 1E4 and 1F12 fractions (Table 3). In addition, mannose 5-containing glycan variants were also eluted at similarly higher pH values.

[0160]

Table 3

[0161] Table 3 shows the analysis results of the sample fraction characteristics evaluation using LC-MS analysis presented as the quantitative area of the analyzed elution fractions (1B4 to 1F12).

[0162] As shown in Figure 10 and Table 3, separation / concentration of non-fucosylated glycan species is possible while using linear pH gradient elution. Fewer fucosylated glycan variants were detected in the major glycoprotein-containing fractions (e.g., 1B4 to 1D5). More fucosylated variants eluted at higher pH values (e.g., 1E4 to 1F12).

[0163] 7. Separation of Native and Deglycosylated Rituximab® An ion exchange material prepared as per Example 1 (for example, having an average particle size between 20 - 63 μm, an average pore size between 40 - 110 nm, and an ion density between 400 - 900 μeq / g) was evaluated for its ability to separate glycosylated and non-glycosylated Rituximab®. The ion exchange material was packed into a 5×100 mm sized chromatographic column with an asymmetry between 0.8 - 1.2 and >3000 plates / m. After packing the ion exchange material, the resulting chromatographic column was washed with 1M NaOH solution for 30 minutes and pre-equilibrated with a loading buffer solution having a pH of 4.75 and 150 mM NaCl. The buffer solution contained a combination of salts such as sodium dihydrogen phosphate, TRIS, and glycine to obtain a pH of 4.75. Using the same solution, native and deglycosylated Rituximab® samples were diluted to a concentration of 4 mg / ml.

[0164] This solution was loaded onto the prepared chromatographic column until a Rituximab® load of 1 mg per 1 ml of resin was reached. These steps and subsequent steps were carried out at a buffer velocity of 150 cm / h. After loading 1 mg / ml Rituximab®, the chromatographic column was washed with a buffer solution of pH 4.75 and then eluted using gradient elution with a buffer having a pH of 8.5 and 150 mM NaCl. This elution buffer was prepared using different salts such as sodium dihydrogen phosphate, TRIS, and glycine. Conductivity and pH values were traced during the experimental setup, indicating that Rituximab® elution from the column was achieved due to the pH change during gradient elution.

[0165] To prepare the sample, Rituximab® was partially deglycosylated using an endoglycosydase digest. Partially deglycosylated and native Rituximab® were both loaded in two different applications and the retention time was compared (Figure 11).

[0166] The elution profile of native glycoproteins (e.g., Rituximab®) in a linear pH gradient shows a lower elution pH for native glycosylated proteins. The deglycosylated portion of the glycoprotein elutes at a higher pH of the gradient.

[0167] As shown in Figure 11, separation / concentration of deglycosylated and native glycoproteins is possible while using linear pH gradient elution.

[0168] 8. mAb05 and High-Mannose Glycan Form Separation in Flow-Through Mode An ion exchange material as prepared according to Example 1 (e.g., having an average particle size between 20 - 63 μm, an average pore size between 40 - 110 nm and an ion density between 400 - 900 μeq / g) was evaluated for its ability to separate high-mannose glycan species of mAb05 in flow-through mode. The ion exchange material was packed into a 5 x 100 mm dimension chromatographic column with an asymmetry between 0.8 - 1.2 and >3000 plates / m. After packing the ion exchange material, the resulting chromatographic column was washed with 1M NaOH solution for 30 minutes and pre-equilibrated with a loading buffer solution having a pH of 4.75 and 400 mM NaCl. The buffer solution contained a combination of salts such as sodium dihydrogen phosphate, TRIS, and glycine to obtain a pH of 4.75. Using the same solution, the mAb05 sample was diluted to a concentration of 4.8 mg / ml.

[0169] This solution was loaded onto the prepared chromatographic column until a loading of 10 mg mAb05 per 1 ml of resin was reached. These steps and the subsequent steps were carried out at a buffer velocity of 150 cm / h. After loading 10 mg / ml mAb05, the chromatographic column was washed with a buffer solution at pH 4.75 and then eluted using a gradient elution with a buffer having a pH of 8.5 and 400 mM NaCl. This elution buffer was prepared using different salts such as sodium dihydrogen phosphate, TRIS, and glycine. The conductivity and pH values were traced during the experimental setup, which indicates that the elution of high-mannose mAb05 glycovariants from the column was achieved due to the pH change during the gradient elution. On the other hand, the glycovariants without mannose did not bind to the column and were in the flow-through. The flow-through and the sample eluate were fractionated, and the obtained fractions were evaluated using LC-MS method for glycan species identification (Table 4).

[0170]

Table 4

[0171] Table 4 shows the analytical evaluation using LC-MS analysis of the collected fractions. It indicates that the flow-through fraction 1F8 contained <1% mannose-containing glycovariants. Most of the mannose-containing glycovariants eluted at a higher pH, which was further characterized by the representative 1H9 fraction (Table 4).

[0172] As shown in Figure 12, it is possible to separate / concentrate low-mannose-containing glycan species in the flow-through while using linear pH gradient elution. In the bound fractions (e.g., 1H9), high-mannose glycan variants were detected. The high-mannose glycan variants eluted at a higher pH value (e.g., 1H9).

[0173] 9. Separation of the Spike S1 Protein of SARS-CoV-2 in the Binding-Elution Mode An ion-exchange material as prepared according to Example 1 (e.g., having an average particle size between 20 and 63 μm, an average pore size between 40 and 110 nm, and an ion density between 400 and 900 μeq / g) was evaluated for its ability to bind the Spike S1 protein of SARS-CoV-2. The ion-exchange material was packed into a 5×100 mm size chromatographic column with an asymmetry between 0.8 and 1.2 and >3000 plates / m. After packing the ion-exchange material, the resulting chromatographic column was washed with 1M NaOH solution for 30 minutes and pre-equilibrated with a loading buffer solution having a pH of 4.75 and 250 mM NaCl. The buffer solution contained a combination of salts such as sodium dihydrogen phosphate, TRIS, and glycine to obtain a pH of 4.75. Using the same solution, the S1 protein sample was reconstituted to a concentration of 0.6 mg / ml.

[0174] This solution was loaded onto the prepared chromatographic column until a loading of 1 mg of S1 protein per 1 ml of resin was reached. These steps and subsequent steps were carried out at a buffer velocity of 150 cm / h. After loading 1 mg / ml of S1 protein, the chromatographic column was washed with a buffer solution at pH 4.75 and then eluted using gradient elution with a buffer having a pH of 10.5 and 250 mM NaCl. This elution buffer was prepared using different salts such as sodium dihydrogen phosphate, TRIS, and glycine. The conductivity and pH values were traced during the experimental setup, indicating that the elution of the S1 protein from the column was achieved due to the pH change during gradient elution. The sample eluate was fractionated, and the resulting fractions were evaluated for each line using an analytical HIC method for protein identification (Figure 13).

[0175] The analytical evaluation using analytical HIC method of the collected fractions is shown in Figure 14, and the main elution peak (dashed line) further characterized by a representative 1C6 fraction indicates that it contained only the S1 protein (solid line). (Figure 14)

[0176] As shown in Figure 13, the separation / concentration of the SARS-CoV-2 spike S1 protein is possible while using linear pH gradient elution. The elution peak contains only the S1 protein.

[0177] 10. Separation of high-mannose glycan forms of mAb03 in flow-through mode The ion-exchange material as prepared according to Example 1 (for example, having an average particle size between 20 - 63 μm, an average pore size between 40 - 110 nm, and an ion density between 400 - 900 μeq / g) was evaluated for its ability to separate the high-mannose glycan species of mAb03 in flow-through mode. The ion-exchange material was packed into a 5 × 100 mm sized chromatographic column with an asymmetry between 0.8 - 1.2 and >3000 plates / m. After packing the ion-exchange material, the resulting chromatographic column was washed with 1M NaOH solution for 30 minutes and pre-equilibrated with a loading buffer solution having a pH of 5.12 and 250 mM NaCl. The buffer solution contained a combination of salts such as sodium dihydrogen phosphate, TRIS, and glycine to obtain a pH of 5.12. Using the same solution, the mAb03 sample was diluted until a concentration of 5.0 mg / ml was reached.

[0178] This solution was loaded onto the prepared chromatographic column until reaching an mAb03 loading of 1 ml resin. These steps and the subsequent steps were carried out at a buffer velocity of 150 cm / h. After loading 100 mg / ml mAb03, the chromatographic column was washed with a buffer solution at pH 5.12 and then eluted using a gradient elution with a buffer having a pH of 8.5 and 250 mM NaCl. This elution buffer was prepared using different salts such as sodium dihydrogen phosphate, TRIS, and glycine. The conductivity and pH values were traced during the experimental setup, which indicates that the elution of high-mannose mAb03 glycovariants from the column was achieved due to the pH change during gradient elution. On the other hand, the non-mannose-containing glycovariants did not bind to the column and were in the flow-through. The flow-through was fractionated and the resulting fractions were evaluated for the quantification of high-mannose species (Table 5).

[0179]

Table 5

[0180] Table 5 shows an analytical evaluation of the cumulative high-mannose species amount of the fractions collected after a given mAb03 breakthrough. The initial concentration of the high-mannose species was 7.68%. This indicated that 66.2 mg of the flow-through mAb03 sample contained <1% high-mannose-containing glycovariants.

[0181] 11. Gammanorm (registered trademark) static binding capacity Using the material prepared from hexafluorovaline used for grafting, the static binding capacity of Gammanorm® was measured. Hexafluorovaline was dissolved in VE water, and the pH was adjusted to above 13 by adding NaOH (32%). An acrylic compound such as acryloyl chloride or acrylic acid was added at a temperature between 0 and 5 °C, and the mixture was stirred for 1 hour. Then, the pH was adjusted to about 2.2 by adding nitric acid. Thereafter, an OH-containing substrate, for example, Eshmuno® particles were added. Polymerization was initiated by adding cerium(IV) nitrate. The reaction was carried out at 30 to 50 °C for 4 hours. An exact amount of such material was immersed in a solution containing 5 mg / ml Gammanorm and various amounts of NaCl at pH 5.0. After 4 hours of incubation, the material was removed, and the amount of remaining Gammanorm® in the solution was measured, whereby the amount of bound Gammanorm® was estimated.

[0182] The measured static binding capacity was as follows: At 0 mM NaCl - 64.5 mg Gammanorm® / ml hexafluorovaline graft material; at 30 mM NaCl - 50.9 mg Gammanorm® / ml hexafluorovaline graft material; at 75 mM NaCl - 50 mg Gammanorm® / ml hexafluorovaline graft material was bound.

Claims

**Claim 1** A method for chromatographic purification and / or separation of protein glycoforms by contacting a separation material comprising a base matrix to which polymer chains are covalently bonded with a sample containing protein glycoforms, comprising: - terminal mannose protein glycoforms, - fucosylated protein glycoforms, - non-fucosylated protein glycoforms, - glycosylated proteins, and - non-glycosylated proteins selected from one or more of the above, wherein the polymer chain comprises a terminal group -N(Y)-R3, where in the formula: R3 is -CHCOOMR4, Y is, independently of one another, H or CH ３ and where in the formula, R4 is C1-C4 alkyl or C1-C4 perfluoroalkyl, characterized in that the method is as described above. and M is H, Na, K, or NH ４ is **Claim 2** a) contacting the separation material with a sample containing protein glycoforms, whereby one or more protein glycoforms bind to the separation material, and b) contacting the separation material with an elution buffer under conditions such that the bound protein glycoforms elute from the separation material The method according to claim 1, comprising the steps of: **Claim 3** The method according to claim 2, further comprising the step of washing the separation material to which one or more protein glycoforms are bound. **Claim 4** The method according to any one of claims 1 to 3, characterized in that the method further comprises recovering the protein glycoforms that flow through the separation material in step a). **Claim 5** The method according to any one of claims 1 to 4, characterized in that the elution buffer has a higher pH than the loading buffer used to contact the separation material with the sample in step a). **Claim 6** The method according to any one of claims 1 to 5, characterized in that in step a), the sample contacted with the separation material in step a) has a conductivity of 5 to 60 mS / cm. **Claim 7** The sample is characterized by comprising one or more of the following: - protein glycoforms rich in mannose - terminal mannose protein glycoforms - fucosylated and non-fucosylated protein glycoforms - glycosylated and non-glycosylated protein glycoforms The method according to any one of claims 1 to 6. **Claim 8** ​ The method according to any one of claims 1 to 7, wherein the sample comprises a glycosylated antibody and / or a viral protein glycoform.

9. The method according to any one of claims 1 to 8, wherein the polymer chain is constructed of monomer units, the monomer units of the polymer chain are linearly linked, and each monomer unit comprises a terminal group -N(Y)-R3.

10. The method according to any one of claims 1 to 9, wherein Y is H and R4 is isopropyl and / or isobutyl.

11. The method according to any one of claims 1 to 10, wherein the ionic density of the separation material is between 10 and 1200 μeq / g.

12. The method according to any one of claims 1 to 11, wherein the protein glycoform binds to the separation material at a pH between 2 and 7.

13. The method according to any one of claims 1 to 11, wherein the protein glycoform binds to the separation material at a pH between 2 and 7 and is washed and eluted by increasing the pH value to a value between 9 and 11.

14. The method according to any one of claims 1 to 13, wherein the sample is applied to the separation material at an ionic density between 10 and 1200 μeq / g.

15. The method according to any one of claims 1 to 14, wherein 10 mg to 100 mg of the protein glycoform per ml of the separation material binds.

16. The method according to any one of claims 1 to 15, wherein the sample comprises a SARS-CoV-2 protein glycoform.

Images