Glycan purification

Abstract

The present invention relates to a method for separating and purifying glycoforms using an ion exchange separation material having amino acid-based end groups.

Description

[0001] The present invention relates to a method for separating and purifying glycoforms using an ion exchange separation material having amino acid-based end groups.

[0002] Glycans are an essential part of glycoprotein molecules, ensuring their structure and function.

[0003] One of the most common glycoproteins is an immunoglobulin that contains two N-linked oligosaccharides at the conserved asparagine 297 in the CH2 domain of the Fc portion. The structure of the glycan consists of two N-acetylglucosamines (GlcNAc), three mannoses, and two GlcNAc residues. Other monosaccharides may also be present, such as fucose (Fuc), galactose (Gal), sialic acids including N-acetylneuraminic acid (NANA) or N-glycolylneuraminic acid (NGNA) residues. ( Figure 1 . Glycan structure). The glycan structure plays an important role in the affinity binding of glycoproteins and receptors. Alterations in glycan composition can cause conformational conversions of glycoproteins, affecting their specific receptor binding and leading to changes in effector functions. In addition, some glycan compositions initiate protective biological responses, such as terminal mannose binding to effectors carrying the mannose-binding receptor (ManR).

[0004] In addition, glycoproteins containing high levels of terminal mannose glycans, which are commonly 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 very important to control the level of high terminal mannose glycans in biopharmaceutical glycoproteins to avoid potential immunogenicity.

[0005] In addition, terminal mannose and mixed glycan structures reduce the conformational stability of the CH2 domain of monoclonal antibodies (mAbs). This may result in higher levels of enzymatic degradation or short storage times for such molecules (Fang, J. Richardson J, DuZ, 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).

[0006] Hybrid glycosylation variants are typically formed in the Golgi apparatus. Hybrid glycosylation variants show reduced or altered glycosylation, in other words, they do not have the desired glycosylation pattern. Some examples of the 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 aforementioned hybrid variants have terminal mannose, thus increasing the likelihood of a shorter lifespan in the bloodstream (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).

[0007] In addition, lower levels of galactose reduce complement-dependent cytotoxicity (CDC) activity, affecting glycoprotein activity. Some studies have shown that the activity can be reduced twofold between mAbs containing the G2-glycoform and mAbs containing the G0-glycoform (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 efficacy of glycoproteins.

[0008] One of the most obvious effects of the changes in glycan structure is 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 their binding to the FcγRIIIa receptor, thereby reducing antibody-dependent cell-mediated cytotoxicity (ADCC) activity. The specific binding to the FcγRIIIa receptor can be reduced by 50-fold, thus having a great impact on the efficacy 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).

[0009] It is also well known that the lack of glycosylation significantly reduces the binding affinity between glycoproteins and receptors. For example, the lack of mAb glycosylation significantly reduces the binding to the FcγRI receptor and abolishes the 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).

[0010] The Food and Drug Administration (FDA) assesses the glycosylation similarity of biosimilar drugs as one of the most critical requirements (FDA, 2012. Guidance for industry quality considerations in demonstrating biosimilarity to a reference protein product). In addition, glycosylation enhancement is one of the main trends of biobetters.

[0011] For all these reasons, there is a growing demand in the industry to enhance the efficiency of glycosylated biopharmaceutical molecules by controlling, enriching or separating various glycan species and making them have the best pharmacokinetics, efficacy, half-life and tolerance.

[0012] The current state of the art for glycoprotein separation based on its glycan variants can be carried out in preparative chromatography mode using ion exchange chromatography to enrich high-mannose glycoforms (WO 2014 / 100117). Unfortunately, in the given example, the enrichment of glycoforms with high mannose content overlaps with aggregate enrichment, making it difficult to identify if the separation of glycoproteins containing aggregates cannot be achieved. In addition, there is no indication that this technique can be used for the separation or removal of other glycan variants.

[0013] An alternative technique for high-mannose glycoforms is affinity chromatography using lectins (US 20020164328). Although this technique is more specific than ion exchange chromatography, it is limited to glycans containing high mannose and requires specific binding conditions. Additionally, the leaching, regeneration, and lifespan of the lectin in this resin hinder its application in the economical separation of high-mannose glycan variants.

[0014] Other preparative glycan variant separation methods include a combination of anion exchange and reverse-phase chromatography techniques (US20100151584) or using only anion exchange chromatography (IN 01066 ch2012). Unfortunately, none of the prior arts achieve the separation of more than one glycoform, which corresponds to a clear need for an effective and efficient technique for separating the glycoforms of glycoproteins.

[0015] It has been found that ion exchange materials carrying covalently linked leucine residues or similar residues can be used for the separation and enrichment of glycoforms, enabling effective separation of glycan substances at a capacity of >10 mg glycoprotein / mL of material. In a more preferred embodiment, the capacity is 10 - 80 mg / ml. Additionally, this innovative ion exchange material can be used at a high conductivity of >10 mS / cm, where in a more preferred embodiment, the conductivity is 10 - 60 mS / cm. Surprisingly, using a solvent pH gradient, glycan variants can be separated and enriched, including high-mannose-containing variants, terminal mannose-containing variants, fucose-containing variants, and non-glycosylated variants. Furthermore, this discovery enables us to explore a wide operating window where ionic and hydrophobic interactions contribute to the selectivity of glycan variant separation. Additionally, compared to the affinity chromatography mode, the application of this innovative ion exchange material shows significant economic advantages and, compared to the anion exchange mode, enables a wider selectivity and enhanced performance.

[0016] Accordingly, the present invention relates to the chromatographic purification and / or separation of protein glycoforms by contacting a sample containing protein glycoforms with a separation material comprising a hydroxy-containing base matrix, to which polymer chains are grafted by covalent bonding, characterized in that

[0017] a) the base matrix contains hydroxy,

[0018] b) The polymer chains are covalently bonded to the base matrix through hydroxyl groups,

[0019] c) The polymer chains contain terminal groups -N(Y)-R3, where

[0020] Y is independently H or CH for each, 3 , preferably H, and

[0021] R3 is -CHCOOMR4

[0022] where R4 is a C1 - C4 alkyl group, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, preferably isopropyl and isobutyl, very preferably isobutyl, or a C1 - C4 perfluoroalkyl group

[0023] and M is independently H, Na, K or NH 4 + .

[0024] In a preferred embodiment, the method of the present invention comprises the following steps

[0025] a) Contacting a sample containing protein glycoforms with a separation material, the separation material comprising a base matrix containing hydroxyl groups, and polymer chains are grafted onto the surface of the base matrix by covalent bonding, whereby one or more protein glycoforms bind to the separation material

[0026] b) Optionally washing the separation material

[0027] c) Contacting the separation material with an elution buffer under conditions where the bound protein glycoforms are eluted from the separation material

[0028] In a preferred embodiment, in step c), the elution buffer has a higher pH than the loading buffer. The elution buffer can be applied in a step - wise manner or in a gradient manner.

[0029] In a preferred embodiment, in step a), the contact between the sample and the separation material is carried out under conditions of increasing conductivity such that the sample loaded onto the separation material has a conductivity of 5 - 60 mS / cm, preferably 15 - 35 mS / cm. This is usually achieved by adding a salt such as sodium chloride to the sample solution.

[0030] In another preferred embodiment, the method comprises recovering the protein glycoforms that flow through the separation material.

[0031] In a preferred embodiment, the sample contains mannose - rich protein glycoforms.

[0032] In another preferred embodiment, the sample contains terminal mannose protein glycoforms.

[0033] In another preferred embodiment, the sample comprises fucosylated and non-fucosylated protein glycoforms.

[0034] In another preferred embodiment, the sample comprises glycosylated and non-glycosylated proteins.

[0035] In another preferred embodiment, the sample comprises an antibody and / or an Fc fusion protein and / or a viral protein glycoform that is isolated or on a virus or viral capsid.

[0036] Preferably, the monomer units of the polymer are linked in a linear manner and each monomer unit comprises a terminal group -N(Y)-R3.

[0037] Preferably, the ionic density of the separation material is between 10 - 1200 μeq / g.

[0038] In a preferred embodiment, Y is H, and R4 is isopropyl and / or isobutyl.

[0039] In a preferred embodiment, the hydroxy-containing base matrix comprises aliphatic hydroxyl groups.

[0040] In a preferred embodiment, the base matrix is a copolymer formed by copolymerization of b) and at least one compound from group a), where

[0041] a) at least one hydrophilic substituted alkyl vinyl ether of formula I

[0042] I

[0043] wherein R1, R2, R3 can each independently be H or a C1 to C6 alkyl group, preferably H or CH 3 ,

[0044] and R4 is a group bearing at least one hydroxyl group

[0045] and

[0046] b)

[0047] at least one crosslinking agent conforming to formula II and / or III and / or IV, where

[0048] Ⅱ

[0049] wherein X is a divalent alkyl group having 2 - 5 C atoms, preferably 2 or 3 C atoms, where one or more methylene groups that are not adjacent and not in direct proximity to the N can be replaced by O, C = O, S, S = O, SO 2 , NH, NOH or N, and one or more H atoms of the methylene groups can independently of one another be hydroxyl, C1 - C6 - alkyl, halogen, NH 2, C5-C10-aryl, NH-(C1-C8)-alkyl, N(C1-C8)-alkyl 2 , substituted by C1-C6-alkoxy or C1-C6-alkyl-OH, and

[0050] III

[0051] IV

[0052] wherein Y1 and Y2 in Formulas III and IV are independently of each other

[0053] C1-C10 alkyl or cycloalkyl, wherein one or more non-adjacent methylene groups or methylene groups not in direct proximity to N may be replaced by O, C=O, S, S=O, SO 2 , NH, NOH or N, and one or more H of the methylene groups may independently of each other be replaced by hydroxy, C1-C6-alkyl, halogen, NH 2 , C5-C10-aryl, NH(C1-C8)alkyl, N(C1-C8)alkyl 2 , substituted by C1-C6-alkoxy or C1-C6-alkyl-OH,

[0054] or C6-C18 aryl, wherein one or more H in the aryl system may independently of each other be replaced by hydroxy, C1-C6-alkyl, halogen, NH 2 , NH(C1-C8)alkyl, N(C1-C8)alkyl 2 , substituted by C1-C6-alkoxy or C1-C6-alkyl-OH, and

[0055] A is a divalent alkyl having 2-5 C atoms, preferably 2 or 3 C atoms, wherein one or more non-adjacent methylene groups or methylene groups not in direct proximity to N may be replaced by O, C=O, S, S=O, SO 2 , NH, NOH or N, and one or more H of the methylene groups may independently of each other be replaced by hydroxy, C1-C6-alkyl, halogen, NH 2 , C5-C10-aryl, NH(C1-C8)alkyl, N(C1-C8)alkyl 2 , substituted by C1-C6-alkoxy or C1-C6-alkyl-OH.

[0056] R4 in Formula I is generally alkyl, alicyclic group or aryl having at least one hydroxy group.

[0057] In a highly preferred embodiment, the base matrix is formed by copolymerizing a hydrophilic substituted alkyl vinyl ether selected from 1,4 - butanediol mono vinyl ether, 1,5 - pentanediol mono vinyl ether, diethylene glycol mono vinyl ether or cyclohexanedimethanol mono vinyl ether with divinyl ethylene urea (1,3 - divinylimidazolin - 2 - one) as a crosslinking agent.

[0058] In a preferred embodiment, the separation material can be prepared by subjecting a hydroxy - containing base matrix to cerium(IV) - catalyzed graft polymerization of a monomer of formula V

[0059] V

[0060] wherein R 1 、R 2 and Y are each independently H or CH 3 , preferably H,

[0061] R 3 is - CHCOOMR4

[0062] wherein R 4 is a C1 - C4 alkyl group such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, preferably isopropyl and isobutyl, very preferably isobutyl, or a C1 - C4 perfluoroalkyl group

[0063] and M is H, Na, K or NH 4 + .

[0064] This graft polymerization is preferably carried out according to Example 8 on page 9 of US 5453186.

[0065] In a preferred embodiment, the protein glycoform binds to the separation material at pH 2 - 7 and is optionally washed and eluted by raising the pH value to an alkaline pH, preferably a value higher than 9, such as 9 - 11, preferably raised to about 10.

[0066] In a preferred embodiment, the sample is applied to the separation material at an ionic density of 10 - 1200 μeq / g.

[0067] In a preferred embodiment, 10 mg - 100 mg of glycoprotein binds per ml of the separation material.

[0068] Figure 1 Schematic diagram showing the glycan structure, where squares represent N - acetylglucosamine (GlcNAc), full - ring - mannose, triangles - fucose (Fuc) and open - ring - galactose (Gal).

[0069] Figure 2Shows a schematic diagram of the separation material according to the present invention, wherein the base material (dots) is functionalized with a linear polymer constructed by polymerization of acryloyl leucine monomers.

[0070] Figure 3 Shows a schematic diagram of the separation material according to the present invention, the separation material having a base material (dots) functionalized with a linear polymer constructed by polymerization of acryloyl valine monomers.

[0071] Figure 4 Shows the elution peak of glycoprotein bound to the separation matrix at 20 mg / ml CV loading and 250 mM NaCl. The UV absorption signal trace is shown as a solid line, and the pH signal trace is shown as a dashed line.

[0072] Figure 5 Shows the analysis results of characterizing the sample fractions using the LC-MS analysis method, shown as the quantitative area of the analyzed elution fractions (1A10 to 1E3), including the loaded glycoprotein (loaded) at 20 mg glycoprotein / ml CV loading and 250 mM NaCl.

[0073] Figure 6 Shows the elution peak of glycoprotein bound to the separation material at 20 mg / ml CV loading and 250 mM NaCl. The UV absorption signal trace is shown as a solid line, and the pH signal trace is shown as a dashed line.

[0074] Figure 7 Shows the analysis results of characterizing the sample fractions, using the LC-MS analysis method, showing the quantitative area of the analyzed elution fractions (1A10 to 1E3), including the loaded glycoprotein (loaded) at 20 mg glycoprotein / ml CV loading and 250 mM NaCl.

[0075] Figure 8 Shows the elution peak of glycoprotein (such as mAb05) bound to the separation material at 30 mg / ml CV loading and 250 mM NaCl. The UV absorption signal trace is a blue line.

[0076] Figure 9 Shows the analysis results of characterizing the sample fractions using the LC-MS analysis method, shown as the quantitative area of the analyzed elution fractions (1A4 to 4B2).

[0077] Figure 10 Shows the elution peak of glycoprotein (such as mAb05) bound to the separation material at 30 mg / ml CV loading and 200 mM NaCl. The UV absorption signal trace is shown as a solid line, and pH is shown as a dashed line.

[0078] Figure 11Shows the elution peak of glycoprotein (e.g., Rituximab®) bound to the separation material at 1 mg / ml CV loading and 150 mM NaCl. The trace of the UV absorption signal of partially deglycosylated Rituximab® is represented by a solid line, the UV absorption signal of native Rituximab® is represented by a dotted line, and the pH is represented by a dashed line.

[0079] Figure 12 Shows the breakthrough and elution peaks of glycoprotein (e.g., mAb05) bound to the separation material at 10 mg / ml CV loading and 450 mM NaCl. The trace of the UV absorption signal is represented by a solid line, and the pH is represented by a dashed line.

[0080] Figure 13 Shows the elution peak of glycoprotein (e.g., the spike S1 protein of SARS-CoV-2) bound to the separation material at 1 mg / ml CV loading and 150 mM NaCl. The trace of the UV absorption signal of the glycoprotein is represented by a solid line, and the pH is represented by a dashed line.

[0081] Figure 14 Shows the analysis results of sample fraction characterization using the HIC analysis method, shown as a UV signal trace, including a solid line for the loaded glycoprotein (loading) at 1 mg glycoprotein / ml CV and 250 mM NaCl and a dashed line for the elution fraction 1C6 of the glycoprotein (e.g., the spike S1 protein of SARS-CoV-2).

[0082] Before describing the present invention in detail, it should be understood that the present invention is not limited to specific compositions or method steps as they can vary. It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a ligand" includes a plurality of ligands, reference to "an antibody" includes a plurality of antibodies, and the like.

[0083] Unless otherwise defined, 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. For the purposes of the present invention as described herein, the following terms are defined.

[0084] As used herein, the term "target molecule" refers to any molecule, substance or compound that is to be separated, isolated or purified from one or more other components in a sample, such as impurities. Examples of target molecules are glycoproteins, also known as protein glycoforms or glycan species of glycoproteins. The target molecule can also be a non-glycosylated protein that is to be separated from the protein glycoforms that are also present in the sample. During production and / or purification, 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. In addition to the target molecule, the liquid can contain one or more impurities. The liquid can also be referred to as a sample. The composition of the liquid can change during production and / or purification depending on the process steps carried out. After a chromatographic step, the liquid typically contains additional solvents other than those present before, due to the eluent used in the chromatographic step. Typically, the target molecule can only be dried to prepare the final dosage form after the final purification step.

[0085] A glycoprotein or protein glycoform is a glycosylated protein. Glycosylation can be in the form of, for example, mono-, di- or oligosaccharide chains, containing one or more fucose, mannose, galactose, N-acetylglucosamine, sialic acid and / or neuraminic acid. For example, antibodies typically have complex N-linked oligosaccharides. Further information can be found in the above introduction. Examples of common glycan structures are N-glycosidically linked sugar chains, such as can be found in antibodies.

[0086] "N-glycosidically linked sugar chain" or "N-glycosidically linked glycan" is typically linked to asparagine 297 (according to Kabat numbering), although complex N-glycosidically linked sugar chains can also be linked to other asparagine residues. Complex N-glycosidically linked sugar chains typically have biantennary complex glycans, mainly having the following structure:

[0087]

[0088] where + / - indicates that a sugar molecule can be present or absent, and the numbers indicate the positions of connection between the sugar molecules. In the above structure, the end of the sugar chain that binds to asparagine is called the reducing end (right side), while the opposite side is called the non-reducing end. Fucose is typically bound to N-acetylglucosamine ("GlcNAc") at the reducing end, usually via an α1,6 bond (the 6-position of GlcNAc is linked to the 1-position of fucose). "Gal" refers to galactose, and "Man" refers to mannose.

[0089] Examples of protein glycoforms are proteins with different levels of fucosylation, such as different levels of core fucosylation, different levels of sialylation, different levels of galactosylation or different levels of mannoseylation. Preferably, the method of the present invention is used for separating or purifying high-mannose protein glycoforms.

[0090] High-mannose glycoprotein glycoforms are glycoproteins having glycan residues with five or more, typically five to nine mannose units. Examples of high-mannose glycoproteins are antibodies having N-linked oligosaccharides containing 5 to 9 mannose units. Another example of a mannose-rich protein is a viral glycoprotein, such as the mannose-rich envelope glycoprotein of HIV 1 or the spike (S1) protein of 2019-nCoV (SARS-CoV-2).

[0091] Endo-mannose glycoprotein glycoforms are glycoproteins having glycan residues with 3 mannose units in the core structure, where one or two mannose units in the branches are not bound to N-acetylglucosamine (“GlcNAc”). In some embodiments, this may refer to G0-N glycoproteins. In some embodiments, this may refer to G1-N glycoproteins. In some embodiments, this may refer to a mixed glycoform.

[0092] The glycoprotein or protein glycoform according to the invention can be an isolated glycoprotein linked to other moieties such as a drug, another protein, a virus or a viral capsid. In some embodiments, the protein glycoform is produced in mammalian cells, fungal cells, insect cells or plant cells.

[0093] The term “antibody” refers to a protein having the ability to specifically bind an antigen. “Antibody” or “IgG” also refers to a polypeptide substantially encoded by an immunoglobulin gene or a fragment thereof that specifically binds and recognizes an analyte (antigen). Immunoglobulin genes that are recognized include the κ, λ, α, γ, δ, ε, and μ constant region genes, as well as numerous immunoglobulin variable region genes. Light chains are divided into κ or λ. Heavy chains are divided into γ, μ, α, δ, or ε, which in turn define the IgG, IgM, IgA, IgD, and IgE classes of immunoglobulins, respectively.

[0094] An exemplary immunoglobulin (antibody) structural unit consists of two pairs of polypeptide chains, each pair having one “light” chain (about 25 kD) and one “heavy” chain (about 50 - 70 kD), which are stabilized, for example, by interchain disulfide bonds. The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids, which is mainly responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively.

[0095] Antibodies can be monoclonal or polyclonal and can exist as monomers or multimers, such as IgM antibodies, which exist as pentamers, and / or IgA antibodies, which exist as monomers, dimers or multimers. Antibodies can also include multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, provided that they retain or are modified to include a ligand-specific binding domain. The term "fragment" refers to a portion or part of an antibody or antibody chain that contains fewer amino acid residues than the complete or full antibody or antibody chain. Fragments can be obtained by chemical or enzymatic treatment of the complete or full antibody or antibody chain. Fragments can also be obtained by recombinant methods. When produced recombinantly, fragments can 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 included within the term "antibody".

[0096] In some embodiments, the antibody is a protein containing an Fc region, such as an immunoglobulin. In some embodiments, the protein containing an Fc region is a recombinant protein that includes the Fc region of an immunoglobulin fused to another polypeptide or a fragment thereof. Exemplary polypeptides include, for example, renin; growth hormone, 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; blood 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, normal T cell expressed and secreted); human macrophage inflammatory protein (MIP-1-α); serum albumin, such as human serum albumin; Mullerian-inhibiting substance; relaxin α-chain; relaxin β-chain; prorelaxin; murine gonadotropin-associated peptide; microbial proteins, such as 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 factor; 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 factor (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 proteins (IGFBPs); CD proteins such as CD3, CD4, CD8, CD19, CD20, CD34 and CD40; erythropoietin; bone inductive factors; immunotoxins; bone morphogenetic proteins (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 receptors; surface membrane proteins; decay accelerating factor; viral antigens such as part of the AIDS envelope; transport proteins; homing receptors; addressins; regulatory proteins; integrins such as CD11a, CD11b, CD11c, CD18, ICAM, VLA-4 and VCAM; tumor associated antigens such as HER2, HER3 or HER4 receptors; and fragments and / or variants of any of the above polypeptides. In addition, an antibody according to the invention is any protein or polypeptide, fragment or variant thereof, which specifically binds any of the above polypeptides.;

[0097] As used herein, unless otherwise specified, the term "sample" refers to any composition or mixture containing a target molecule. Samples can be from biological or other sources. Biological sources include eukaryotic sources such as animals or humans. Preferred samples are blood or plasma samples from mammals. Samples can also include diluents, buffers, detergents and contaminants found mixed with the target molecule. Samples can be "partially purified" (i.e., one or more purification steps such as filtration or centrifugation steps have been performed) or can be obtained directly from the organism producing the target molecule. A plasma sample is any sample containing plasma or a plasma fraction.

[0098] As used herein, the terms "impurity" or "contaminant" refer to any foreign or unwanted molecule, 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, which may be present in a sample containing a target molecule separated from one or more foreign or unwanted molecules. The terms "impurity" or "contaminant" as used herein can also apply to certain immunoglobulins that need to be separated from the target molecule, such as immunoglobulin A and immunoglobulin M that cause allergic reactions in patients. In addition, such impurities can include any reagent used in the production and / or purification process steps.

[0099] The terms “purify,” “isolate,” or “separate,” as used interchangeably herein, refer to increasing the purity of a target molecule by separating the target molecule from a composition or sample that contains the target molecule and one or more other components, such as impurities. Typically, the purity of the target molecule is increased by removing at least one impurity (fully or partially) from the composition.

[0100] The term “chromatography” refers to any kind of technique for separating an analyte of interest (e.g., a target molecule) from other molecules present in a mixture. Typically, the target molecule is separated from other molecules due to differences in the rate at which the individual molecules of the mixture migrate through a stationary medium or separation material under the influence of a mobile phase, or during binding and elution processes. Examples of chromatographic separation methods are reverse-phase chromatography, ion-exchange chromatography, size-exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, and mixed-mode chromatography.

[0101] “Buffer” is a solution that resists changes in pH through the action of its acid-base conjugate components. Various buffers that can be used depending on, for example, 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, phosphate, acetate, citrate, succinate, glycine, and ammonium buffers, and combinations thereof.

[0102] The terms “separation material” or “chromatography matrix” are used interchangeably herein and refer to any kind of particulate 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 during a separation process. Typically, the target molecule is separated from other molecules due to differences in the rate at which the individual molecules of the mixture migrate through the separation material and interact with the separation material under the influence of a mobile phase, or during binding and elution processes. Separation materials composed of, for example, resin particles, membranes, or monolithic adsorbents can be placed in columns or cartridges. Typically, the separation material comprises a base matrix as the base material and one or more types of ligands attached to the base matrix.

[0103] A "ligand" is or comprises a functional group and is attached to a chromatographic matrix and determines or affects the binding properties of the matrix. Preferably, the ligand is a polymeric chain carrying one or more, preferably a plurality of functional groups. Most preferably, the ligand is constituted by a polymeric chain in which each monomer unit carries at least one functional group, wherein the polymeric chain is constituted by the monomer units. Examples of "ligands" include, but are not limited to, ion exchange groups, hydrophobic interaction groups, hydrophilic interaction groups, thiophilic interaction groups, metal affinity groups, bioaffinity groups, and mixed mode groups (combinations of the above groups). Preferred ligands useful herein include, but are not limited to, weak ion exchange groups such as carboxylic acid.

[0104] The terms "ion exchange" and "ion exchange chromatography" refer to such chromatographic methods in which a target molecule in a mixture (e.g., an Fc region containing a target protein) interacts with a charged compound attached (e.g., by covalent attachment) to an ion exchange matrix such that the non-specific interaction of the target molecule with the charged compound is more or less than that of the solute impurities or contaminants in the mixture. The impurities in the mixture are eluted from the column of the ion exchange material faster or slower than the target molecule, or bind to or are excluded from the resin relative to the target molecule. "Ion exchange chromatography" specifically includes cation exchange, anion exchange, and mixed mode ion exchange chromatography. Ion exchange chromatography can bind a target molecule (e.g., an Fc region containing a target protein) and then elute it, or can primarily bind impurities when the target molecule "flows through" the column. Preferably, the ion exchange chromatography step is carried out in a bind and elute mode.

[0105] 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 can be provided by attaching one or more charged ligands to the matrix, e.g., by covalent attachment. Alternatively or additionally, the charge can be an inherent property of the matrix.

[0106] The term "cation exchange matrix" is used herein to refer to a negatively charged separation material, such as one having one or more negatively charged ligands, such as carboxylic acid groups, attached thereto.

[0107] When "loading" a separation column in a bind and elute mode, a sample or composition containing a target molecule (e.g., an Fc region containing a target protein) and one or more impurities is loaded onto a chromatographic column (e.g., an ion exchange column) using a buffer. The buffer has a conductivity and / or pH such that the target molecule binds to the separation material while ideally all impurities do not bind and flow through the column.

[0108] When "loading" a separation column to "flow through" a target molecule, a sample or composition containing the target molecule (e.g., the Fc region of a target protein) and one or more impurities is loaded onto a chromatography column (e.g., an ion exchange column) using a buffer. 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 impurities bind to the column.

[0109] When "loading" a separation column to "bind and elute" a target molecule, a sample or composition containing the target molecule (e.g., the Fc region of a target protein) and one or more impurities is loaded onto a chromatography column (e.g., an ion exchange column) using a buffer. The buffer has a conductivity and / or pH such that the target molecule binds to the separation material. The separation from one or more impurities is accompanied by a change in conductivity and / or pH such that the target molecule is washed or eluted before or after one or more impurities.

[0110] Generally, the buffer in which the sample is loaded onto the separation material is referred to as the loading buffer or sample buffer.

[0111] The term "equilibrate" refers to using a buffer to equilibrate the separation material before loading the target molecule. Generally, the loading buffer is used for equilibration.

[0112] "Washing" or "washing" the separation material means passing or flowing a suitable liquid, such as a buffer, through or over the separation material. Generally, washing is used to remove weakly bound contaminants from the separation material before eluting the target molecule and / or to remove unbound or weakly bound target molecules after loading.

[0113] In this case, generally, the wash buffer and the loading buffer are the same. If a virus inactivation buffer is used, it is used to inactivate certain present viruses before eluting the target molecule. In this case, generally, the virus inactivation buffer is different from the loading buffer as it can contain detergents or have different properties (pH / conductivity / salts and their amounts).

[0114] Washing can also be used to remove contaminants from the separation material after eluting the target molecule. This is done by passing or flowing a suitable liquid, such as a buffer, through or over the separation material after eluting the target molecule. In this case, generally, the wash buffer is different from the loading buffer. It can contain one or more detergents or have different properties (pH / conductivity / salts and their amounts). The wash buffer is, for example, an acidic buffer.

[0115] "Eluting" molecules (e.g., a target polypeptide such as immunoglobulin G or an impurity) from a separation material is intended to remove the molecules therefrom. When eluting the target molecule with the solvent front of the loading buffer, elution can be carried out directly in a flow-through mode. Alternatively, by changing the solution conditions, a buffer different from the loading buffer is made to compete with the molecule of interest for the ligand sites on the separation material. A non-limiting example is by changing the ionic strength of the buffer around an ion exchange material such that the buffer competes with the molecule for the charged sites on the ion exchange material, thereby eluting the molecule from the ion exchange material.

[0116] The term "average particle size" or D50 refers to 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'.

[0117] The term "average pore size" refers to the average pore size distribution value at 50% of the cumulative pore size distribution.

[0118] The terms "flow-through method", "flow-through mode" and "flow-through operation", as 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 is intended to flow through a separation material together with one or more impurities, the separation material typically binding one or more impurities, where the target molecule typically does not bind (i.e., flows through) and is eluted from the separation material with the loading buffer.

[0119] As used herein, the terms "binding and elution mode" and "binding and elution method" refer to a separation technique in which at least one target molecule (e.g., containing the Fc region of a protein) contained in a sample binds to a suitable separation material (e.g., an ion exchange chromatography medium) and is subsequently eluted with a buffer different from the loading buffer.

[0120] The term "ionic density" as used herein 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., positive or negative ions) per unit volume or mass of the separation material. Typically, the number of ions is estimated by titrating the given separation material. In addition, the amount of ions is given in equivalents (eq) per unit mass or volume of the separation material.

[0121] As used herein, the term "conductivity" refers to an inherent property of most materials that quantifies the strength with which they tolerate or conduct an electric current. In an aqueous solution such as a buffer, the 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 salt, the higher the conductivity. Raising the temperature enables the ions to move faster and thus increases the conductivity. Unless otherwise indicated, conductivity is generally defined at room temperature. The fundamental unit of conductance is the siemens (S). It is defined as the reciprocal of the ohmic resistance measured between the opposing faces of a 1 cm cube of liquid. Thus, these values are estimated in S / cm.

[0122] The present invention provides methods for separating or purifying protein glycoforms. This means that one or more protein glycoforms in a sample can be separated from one or more other protein glycoforms and / or from other impurities. Preferably, at least one protein glycoform is separated from at least one other glycoform, for example, a high-mannose glycoform is separated from another protein glycoform. This is accomplished by chromatographic separation on certain separation materials (also referred to as resins) that comprise a base matrix to which polymer chains are covalently attached. The polymer chains are made from monomers that contain amino acids and polymerizable double bonds.

[0123] By the methods of the present invention, glycoforms can be separated, enriched, and / or purified, enabling effective separation of polysaccharides. In a specific aspect of the present invention, it is beneficial to separate glycan variants containing high-mannose or terminal mannose molecules, which are more hydrophobic and exhibit faster clearance. Additionally, such glycan variants can contribute to higher toxicity and lower glycoprotein potency. In other aspects of the present invention, it is beneficial to separate fucosylated glycoproteins to ensure better control of antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In other aspects of the present invention, it is beneficial to remove proteins that are identical to the target glycoprotein but not glycosylated. In one embodiment of the present invention, the purified glycoprotein does not contain high-mannose glycan variants. In another embodiment of the present invention, the purified glycoprotein contains low levels of mixed glycan variants with terminal mannose. In another embodiment of the present invention, the purified glycoprotein contains low levels of non-fucosylated glycan variants. In another embodiment of the present invention, the purified glycoprotein contains low levels of non-glycosylated proteins.

[0124] The isolation material of the present invention comprises a base material to which whisker-like structures are attached, preferably grafted.

[0125] The base material, also known as the base matrix, contains reactive groups capable of undergoing graft polymerization reactions, in particular OH groups, preferably aliphatic OH groups. Thus, the base material can also be prepared from, for example, organic polymers. Organic polymers of this type can be polysaccharides, such as agarose, dextran, starch, cellulose, etc., or synthetic polymers, 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 used in the form of a crosslinked hydrophilic network. This also includes polymers made from styrene and divinylbenzene, which can preferably be used in a hydrophilized form, like other hydrophobic polymers.

[0126] Alternatively, inorganic materials, such as silica, zirconia, titanium dioxide, alumina, etc., can be used as the base material. Composite materials can also be used, i.e., particles that can be magnetized, for example, by copolymerization with magnetizable particles or a magnetizable core. Core-shell materials can also be used, where the shell, i.e., at least the surface or coating, has OH groups.

[0127] However, it is preferred to use hydrophilic base materials that are stable to hydrolysis or can only be hydrolyzed with difficulty, since the materials of the present invention should preferably withstand alkaline cleaning or regeneration at, for example, alkaline pH during extended use.

[0128] The base matrix can consist of irregularly shaped or spherical particles, and its particle size can be between 2 and 1000 μm. The average particle size is preferably 3 - 300 μm, and in the most preferred embodiment, the average particle size is 20 - 63 μm.

[0129] The base matrix can particularly be in the form of non-porous or preferably porous particles. The average pore diameter can be between 2 and 300 nm. The pore diameter is preferably 5 - 200 nm, and most preferably the average pore diameter is 40 - 110 nm.

[0130] The base matrix can also be in the form of a membrane, fiber, hollow fiber, coating, or monolith. The monolith is preferably a porous three-dimensional body, such as cylindrical.

[0131] In a preferred embodiment, the base matrix is a copolymer formed by copolymerization of b) and at least one compound from group a), where

[0132] a) at least one hydrophilically substituted alkyl vinyl ether of formula I

[0133] I

[0134] where R1, R2, R3 can each independently be H or C1 to C6 alkyl, preferably H or CH3 ,

[0135] and R4 are groups with at least one hydroxyl group

[0136] and

[0137] b)

[0138] at least one crosslinking agent conforming to formula II and / or III and / or IV, wherein

[0139] Ⅱ

[0140] wherein X is a divalent alkyl group having 2 - 5 C atoms, preferably 2 or 3 C atoms, and one or more methylene groups that are not adjacent and not in the immediate vicinity of N can be replaced by O, C=O, S, S=O, SO 2 , NH, NOH or N, and one or more H atoms of the methylene group can be independently replaced by hydroxyl, C1 - C6 - alkyl, halogen, NH 2 , C5 - C10 - aryl, NH-(C1 - C8)-alkyl, N(C1 - C8)-alkyl 2 , C1 - C6 - alkoxy or C1 - C6 - alkyl - OH, and

[0141] III

[0142] IV

[0143] wherein Y1 and Y2 in formulas III and IV are independent of each other,

[0144] C1 - C10 alkyl or cycloalkyl, and one or more non - adjacent methylene groups or methylene groups not in the immediate vicinity of N can be replaced by O, C=O, S, S=O, SO 2 , NH, NOH or N, and one or more H of the methylene group can be independently replaced by hydroxyl, C1 - C6 - alkyl, halogen, NH 2 , C5 - C10 - aryl, NH(C1 - C8)alkyl, N(C1 - C8)alkyl 2 , C1 - C6 - alkoxy or C1 - C6 - alkyl - OH,

[0145] or C6 - C18 aryl, and one or more H in the aryl system can be independently replaced by hydroxyl, C1 - C6 - alkyl, halogen, NH 2 , NH(C1 - C8)alkyl, N(C1 - C8)alkyl 2 , C1 - C6 - alkoxy or C1 - C6 - alkyl - OH, and

[0146] A is a divalent alkyl group having 2 to 5 carbon atoms, preferably 2 or 3 carbon atoms, wherein one or more non-adjacent methylene groups or methylene groups not in direct proximity to N may be replaced by O, C=O, S, S=O, SO 2 , NH, NOH or N, and one or more H of the methylene group may independently of one another be replaced by hydroxyl, C1-C6-alkyl, halogen, NH 2 , C5-C10-aryl, NH(C1-C8)alkyl, N(C1-C8)alkyl 2 , C1-C6-alkoxy or C1-C6-alkyl-OH.

[0147] R4 in formula I is generally an alkyl group, an alicyclic group or an aryl group bearing at least one hydroxyl group.

[0148] In a highly preferred embodiment, the base matrix is formed by copolymerization of a hydrophilically substituted alkyl vinyl ether with divinylethyleneurea (1,3-divinylimidazolin-2-one) as crosslinking agent, said hydrophilically substituted alkyl vinyl ether being selected from 1,4-butanediol mono vinyl ether, 1,5-pentanediol mono vinyl ether, diethylene glycol mono vinyl ether or cyclohexanedimethanol mono vinyl ether.

[0149] An example of a suitable commercially available vinyl ether-based base material is Eshmuno® from Merck KGaA, Germany.

[0150] Linear polymer chains are covalently bonded to the surface of the base material so as to produce a separation material, whereby

[0151] a) the base material preferably contains aliphatic hydroxyl groups,

[0152] b) the polymer is covalently bonded to the support,

[0153] c) the polymer contains amino acid residues,

[0154] d) the monomer units of the polymer are linked in a linear manner.

[0155] The actual separation material comprising the base material and the covalently linked linear polymer chains can be prepared in various ways. In the case of "grafting onto", the polymer chains must first be formed from monomers and in a second step be attached to the surface. In the case of "grafting from", the polymerization reaction is initiated on the surface and the graft polymer is directly composed of individual monomers. Other polymerization methods allowing attachment to the surface of the base material can also be used.

[0156] The "grafting from" method is preferred, and a variant in which only a small amount of by-products are formed, such as non-covalently bonded polymers, which must be separated, is particularly preferred. Methods with controlled radical polymerization, such as atom transfer radical polymerization (ATRP) methods, appear to be particularly interesting. Here, in the first step, initiator groups are covalently bonded to the carrier surface at the desired density. The initiator groups can be, for example, halides bonded through an ester functional group, such as in 2-bromo-2-methyl propionate. The graft polymerization is carried out in the second step in the presence of a copper(I) salt.

[0157] A very preferred one-step graft polymerization reaction suitable for preparing the separation material used in the present invention can be initiated by cerium(IV) on a hydroxyl-containing base material without activating the base material.

[0158] This cerium(IV)-initiated grafting is preferably carried out according to EP0337144 or US 5,453,186. The resulting chains are connected to the base material through monomer units. For this purpose, the base material of the present invention is suspended in a monomer solution, preferably in an aqueous solution. In a conventional redox polymerization process, in the absence of oxygen, the grafting of the polymer material is achieved. The polymerization catalyst used is cerium(IV) ions because the catalyst forms free radical sites on the surface of the base material, thereby initiating the graft polymerization of the monomer. This reaction is usually carried out in a dilute inorganic acid. For carrying out this graft polymerization, the acid is usually used as an aqueous solution with a concentration of 1 - 0.00001 mol / l, preferably 0.1 - 0.001 mol / l. Very particularly preferably, dilute nitric acid with a concentration of 0.1 - 0.001 mol / l is used.

[0159] To prepare the separation material of the present invention, an excess of monomer is usually added to the base material. Generally, 0.05 - 100 mol of total monomer is used per liter of the settled polymer material, preferably 0.05 - 25 mol / l.

[0160] The polymerization is terminated by a termination reaction involving a cerium salt. Therefore, the (average) chain length may be affected by the concentration ratio of the base material, initiator, and monomer. In addition, a homogeneous monomer or a mixture of different monomers can be used; in the latter case, a graft copolymer is formed.

[0161] Monomers advantageously used for preparing the separation material used in the present invention are those of formula V

[0162] V

[0163] where R 1 、R 2 and Y are each independently H or CH3, preferably H.

[0164] R 3-CHCOOMR4

[0165]

[0166] wherein R 4 is a C1-C4 alkyl group such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, preferably isopropyl and isobutyl, very preferably isobutyl, or a C1-C4 perfluoroalkyl group

[0167] and M is H, Na, K or NH 4 + .

[0168] A perfluoroalkyl group means that all H atoms of the alkyl residue are replaced by F atoms.

[0169] An exemplary preferred structure of formula V is Va

[0170] Va

[0171] wherein R1, R2 and Y are H

[0172] R 3 is -CHCOOMR 4

[0173] R 4 is isopropyl

[0174] M is H

[0175] and formula Vb

[0176] Vb

[0177] wherein R1, R2 and Y are H

[0178] R3 is -CHCOOMR4

[0179] R4 is isobutyl

[0180] M is H

[0181] These 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, and thus acryloyl valine and acryloyl leucine are preferred, and acryloyl leucine is particularly preferred.

[0182] The separation material for the method of the present invention preferably contains only whisker-like linear polymer structures grafted onto a base material, which are constructed from monomers according to formula V. Preferably, they contain linear polymers constructed from only one type of monomer according to formula V.

[0183] However, the linear polymer may also be formed by copolymerization of two or more different monomers according to formula V. The linear polymer can also be constructed by copolymerization of one or more different monomers of formula V with one or more other polymerizable monomers, such as other acrylamides, methacrylates, acrylates, methacrylates, etc., which are functionalized, for example, with ionic, hydrophilic or hydrophobic groups.

[0184] Exemplary structures of the separation materials of the present invention are shown in Figure 2 and 3 in, Figure 2 showing a base material (dots) functionalized with a linear polymer constructed by polymerization of acryloyl leucine monomers. Additionally, for clarity, the carboxy-alkyl end groups of each polymer unit are shown. Figure 3 showing a base material (dots) functionalized with a linear polymer constructed by polymerization of acryloyl valine monomers. Additionally, for clarity, the carboxy-alkyl end groups of each polymer unit are shown.

[0185] In a preferred embodiment, the separation material for the method of the present invention is an ion exchange material in which the amount of the ion density window varies in the range of 10 - 1200 μeq / g, and in a more preferred embodiment, the ion density window is between 400 - 900 μeq / g.

[0186] In a preferred embodiment, the ion exchange material may contain ion exchange functional groups and hydrophobic functional groups, and in a more preferred embodiment, both functional groups are on a single surface functional unit produced by one monomer unit incorporated into the polymer chain, and in a most preferred embodiment, the functional groups are part of an amino acid residue.

[0187] Generally, in the method according to the present invention, a chromatographic column containing the above separation material is used. Chromatographic columns are known to those skilled in the art. They generally include 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 connectors for delivering the solvent to and from the tube. The size of the chromatographic column varies according to the application, such as analysis or preparation. The chromatographic column can also be a membrane-containing column.

[0188] The materials used in the method of the present invention can also be described as base materials for separating effectors. They can be used for selectively, partially selectively or non-selectively binding or adsorbing one or more target components for the purpose of separating them from a sample liquid, or for selectively, partially selectively or non-selectively binding or adsorbing one or more second components for the purpose of separating the second components from a matrix, separating, enriching and / or depleting biopolymers from natural sources, separating, enriching and / or depleting biopolymers from recombinant sources, separating, enriching and / or depleting proteins and peptides, separating, enriching and / or depleting monoclonal and polyclonal antibodies, separating, enriching and / or depleting viruses, separating, enriching and / or depleting host cell proteins, or separating, enriching and / or depleting glycoproteins. Preferred target molecules are glycoproteins.

[0189] The target molecule is separated from at least one or more other substances from the sample, wherein the sample containing the target molecule is dissolved in a liquid and brought into contact with the material according to the invention. The contact time is typically from 30 seconds to 24 hours. It is advantageous to operate according to the principle of liquid chromatography by passing the liquid through a chromatographic column containing the separation material of the present invention. The liquid can flow through the column solely by its gravity or be pumped through by means of a pump. An alternative method is batch chromatography, where the separation material is mixed with the liquid by stirring or shaking as long as the target molecule or biopolymer needs to be able to bind to the separation material. It can also be operated according to the principle of a chromatographic fluidized bed by introducing the liquid to be separated into, for example, a suspension containing the separation material, where the separation material is selected to be suitable for the required separation due to its high density and / or magnetic core.

[0190] If the chromatographic process is run in a binding and 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 contacts the separation material. The washing buffer removes all substances that do not bind to the separation material. Further washing steps can then be carried out with other suitable buffers without desorbing the target molecule. 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. Thus, the target molecule can be obtained in a purified and concentrated form in the eluent. The purity of the target molecule after desorption is typically 70% - 99%, preferably 85% - 99%, particularly preferably 90% - 99%.

[0191] The above-described binding and elution method can also be used to separate or purify more than one type of target molecule, whereby the group of target molecules is then bound to the separation material and separated from one or more impurities, as described above.

[0192] However, if the chromatographic process is run in a flow-through mode, the target molecule or groups of different types of target molecules remain in the liquid, while other accompanying substances bind to the separation material. The target molecules are then obtained directly by collecting the column eluate in the flow-through. It is known to the person skilled in the art how he can adjust the conditions, in particular the pH and / or the conductivity, in order to bind specific biopolymers to the separation material, or whether it is advantageous for the purification task not to bind the target molecules.

[0193] The present invention preferably relates to the use of the above separation material in the separation and purification of glycoproteins, and to a method for purifying and / or separating glycoproteins by liquid chromatography, which method comprises contacting the separation material with a sample containing one or more glycoproteins, preferably under acidic conditions.

[0194] Surprisingly, we have found that the ion-exchange material according to the invention can be used for the purification, separation or enrichment of glycoforms, enabling effective separation of glycan substances at >10 mg glycoprotein / mL material capacity, where in a more preferred embodiment, this capacity is between 10 mg / mL and 80 mg / mL. Furthermore, this innovative ion-exchange material can be used at a high conductivity of >5 mS / cm, where in a more preferred embodiment, the conductivity is 5 - 60 mS / cm.

[0195] Surprisingly, a pH change from 4 - 7 to a pH above 9, preferably 9 to 11, most preferably to pH 10, is preferably used to separate and enrich glycan variants, including high-mannose-containing variants, terminal-mannose-containing variants, fucose-containing variants and non-glycosylated variants, in a gradient or stepwise mode. Additionally, compared to the affinity chromatography mode, the application of this separation material shows significant economic advantages, and compared to the anion-exchange mode, it enables a wider selectivity and enhanced performance.

[0196] Furthermore, the present application is not limited to separating different glycan variant species, but can also be used to remove non-glycosylated variants from glycosylated variants.

[0197] In addition, the application of this material is not limited to binding and elution applications, but can be used in a flow-through mode, resulting in the adsorption of higher-mannose-containing substances or glycan variants containing terminal mannose on the ion-exchange material using a buffer with pH <6 and / or high conductivity (>20 mS / cm). Additionally, surprisingly, this ion-exchange material consisting mainly of amino acids covalently linked to the material has the necessary selectivity for glycan substances, where in a more preferred embodiment, these amino acids are valine or leucine, including their combinations and derivatives, most preferably leucine.

[0198] In addition, the present invention provides a chromatographic-based glycoprotein purification step which can be regenerated and is applicable to a wide operating window, such as pH 3 - 10; conductivity of 5 - 60 mS / cm.

[0199] In a preferred embodiment, the ion exchange material to be used in the present invention is bound to a chromatography column and used in a binding and elution manner for the glycoprotein purification process to separate glycan species.

[0200] In a preferred embodiment, the ion exchange material to be used in the present invention is bound to a chromatography column and used for the glycoprotein purification process of separating glycan species in a binding and elution mode, wherein the operating window span is between pH 2 - 10, and in a more preferred embodiment, the pH is between 4 - 7.

[0201] In a preferred embodiment, the ion exchange material to be used in the present invention is bound to a chromatography column and used for the glycoprotein purification process of separating glycan species in a binding and elution manner, wherein solvent pH elution is used to recover the bound components.

[0202] In a preferred embodiment, the ion exchange material to be used in the present invention is bound to a chromatography column and used in a binding and elution mode for the glycoprotein purification process to separate glycan species, wherein the binding window span is between 10 mg glycoprotein / mL material to 100 mg glycoprotein / mL material, and in a more preferred embodiment, the binding window span is between 20 mg glycoprotein / mL material to 80 mg glycoprotein / mL material.

[0203] In a preferred embodiment, the ion exchange material to be used in the present invention is bound to a chromatography column and used in a binding and elution manner for the glycoprotein purification process to separate glycan species, wherein the conductivity window span is between 5 - 60 mS / cm, and in a more preferred embodiment, the conductivity range is between 15 - 35 mS / cm.

[0204] In another preferred embodiment, the ion exchange material for use in the present invention is bound to a chromatography column and used for the glycoprotein purification process of separating glycan substances in a flow-through mode, wherein the low-mannose-containing substances flow through, and the higher-mannose-containing substances bind to the separation material.

[0205] In another preferred embodiment, the ion exchange material to be used in the present invention is incorporated into a chromatography column and used in a glycoprotein purification process for separating glycan species in a flow-through mode, where the non-hybrid species are in the flow-through type and the hybrid (e.g., containing terminal mannose) species bind to the separation material.

[0206] In another preferred embodiment, the ion exchange material for use in the present invention is introduced into a chromatography column and used in a glycoprotein purification process for separating glycan substances in a flow-through mode, where the low-fucosylated substances are in the flow-through mode and the highly fucosylated substances bind to the separation material.

[0207] In another preferred embodiment, the method of the present invention comprises

[0208] a) applying a sample containing a mixture of protein glycoforms to a chromatography column containing the ion exchange material for use in the present invention, where at least one glycoform is a high-mannose glycoform

[0209] b) separating and eluting at least one high-mannose glycoform in the flow-through

[0210] c) eluting the bound glycoforms from the separation material, whereby the eluted glycoforms are depleted in high-mannose glycoforms.

[0211] In another preferred embodiment, the method of the present invention comprises

[0212] a) applying a sample containing a mixture of protein glycoforms to a chromatography column containing the ion exchange material for use in the present invention, where at least one glycoform is a high-mannose glycoform, where at least one high-mannose glycoform, and typically also other protein glycoforms, bind to the separation material

[0213] b) contacting the separation material with an elution buffer, whereby at least one glycoform different from the high-mannose glycoform is eluted while at least one high-mannose glycoform remains bound to the separation material

[0214] 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.

[0215] In this embodiment, the eluate obtained in step b) is depleted in high-mannose glycoforms compared to the sample applied in step a), and the eluate obtained in step c) is enriched. The mannose glycoforms separated in the flow-through or bound eluate as described above are preferably antibodies with different degrees of mannose glycosylation or viruses and / or virus capsids carrying proteins with different degrees of mannose glycosylation.

[0216] In another preferred embodiment, the ion exchange material used in the method of the present invention is incorporated into a chromatography column and used in a flow-through or binding elution mode for the glycoprotein purification process to separate viruses and / or virus particles containing glycoproteins from viruses and / or virus particles containing other types of glycoproteins or no glycoproteins. Preferably, the separation is based on the different amounts of mannose present in the glycan structure.

[0217] In another preferred embodiment, the method of the present invention comprises

[0218] a) applying a sample containing a mixture of protein glycoforms to a chromatography column containing the ion exchange material for the present invention, wherein at least one glycoform is a terminal mannose glycoform, and wherein at least one terminal mannose glycoform, and typically also other protein glycoforms, bind to the separation material

[0219] b) contacting the separation material with an elution buffer, whereby at least one glycoform different from the terminal mannose glycoform is eluted while at least one terminal mannose glycoform remains bound to the separation material

[0220] 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.

[0221] In this embodiment, the eluate obtained in step b) has a reduced amount of terminal mannose glycoform compared to the sample applied in step a), and the eluate obtained in step c) is enriched. As described above, the terminal mannose glycoforms separated in the flow-through or binding eluate are preferably antibodies with different degrees of mannosylation or viruses and / or virus capsids carrying proteins with different degrees of mannosylation.

[0222] In another preferred embodiment, the ion exchange material used in the method of the present invention is incorporated into a chromatography column and used in a flow-through or binding elution mode for the glycoprotein purification process to separate viruses and / or virus particles containing glycoproteins from viruses and / or virus particles containing other types of glycoproteins or no glycoproteins. Preferably, the separation is based on the different amounts of terminal mannose present in the glycan structure.

[0223] In another preferred embodiment, the method of the present invention comprises

[0224] a) applying a sample containing a mixture of protein glycoforms to a chromatography column containing the separation material for the present invention, wherein at least one glycoform is a fucose-carrying glycoform, and wherein at least one fucose-carrying glycoform, and typically also other protein glycoforms, bind to the separation material

[0225] b) Contact the separation material with an elution buffer, thereby eluting at least one fucose-containing glycoform different from the fucose-free glycoform, while at least one fucose-free glycoform remains bound to the separation material.

[0226] c) Optionally, contact the separation material with a second elution buffer that is generally different from the first elution buffer, thereby eluting at least one fucose-free glycoform.

[0227] In this embodiment, the eluate obtained in step b) is free of fucose-bearing glycoforms compared to the sample applied in step a), and the eluate obtained in step c) is enriched. As described above, the fucose-bearing glycoforms separated in the flow-through or bound eluate are preferably antibodies with different degrees of fucosylation or viruses and / or virus capsids carrying proteins with different degrees of fucosylation.

[0228] In another preferred embodiment, the ion exchange material used in the method of the present invention is incorporated into a chromatography column and used in a flow-through or bound elution mode for the purification process of glycoproteins to separate viruses and / or virus particles containing glycoproteins 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.

[0229] In another preferred embodiment, the method of the present invention comprises

[0230] a) Applying a sample comprising a mixture of proteins with and without glycans, wherein at least one of the proteins with glycans, i.e., the protein glycoform, binds to a chromatography column comprising the separation material to be used in the present invention.

[0231] b) Contact the separation material with an elution buffer, thereby eluting at least one glycoprotein-containing protein while at least one glycoprotein-free protein remains bound to the separation material.

[0232] c) Optionally, contact the separation material with a second elution buffer that is generally different from the first elution buffer, thereby eluting at least one glycoprotein-free substance.

[0233] In this embodiment, the eluate obtained in step b) is enriched in protein-carrying glycoforms compared to the sample applied in step a), and the eluate obtained in step c) is reduced. As described above, the glycoforms separated in the flow-through or bound eluate are preferably antibodies with different degrees of glycosylation or viruses and / or virus capsids carrying proteins with different degrees of glycosylation.

[0234] In another preferred embodiment, the ion exchange material used in the method of the present invention is incorporated into a chromatography column and used in a flow-through or binding and elution mode for the glycoprotein purification process to separate viruses and / or virus particles containing glycoproteins from viruses and / or virus particles containing other types of glycoproteins or no glycoproteins. Preferably, the separation occurs based on the different amounts of glycosylation present in the glycan structure.

[0235] In another preferred embodiment, the ion exchange material for the present invention is introduced into a chromatography column and used in a glycoprotein purification process for separating glycan substances in a flow-through mode, where the glycosylated proteins are in the flow-through mode and the non-glycosylated proteins bind to the separation material.

[0236] In a preferred embodiment, the ion exchange material can have various particle sizes from 1 to 200 μm, where in a more preferred embodiment, the average particle size is 20 to 63 μm.

[0237] In a preferred embodiment, the ion exchange material of the present invention can have various pore sizes from 4 to 1500 nm, where in a more preferred embodiment, the average pore size is 10 to 120 nm, and in a most preferred embodiment, the average pore size is 40 to 110 nm.

[0238] The method of the present invention is extremely flexible. The chromatography mode (binding - elution or flow-through) can be appropriately applied, and the conditions can vary within the wide ranges described above. Additionally, the target molecule can also vary. As described above, in any case, the sample contains at least one protein glycoform. However, the target molecule can be a protein glycoform, a group of different protein glycoforms, or a non-glycosylated protein. Generally, in the method of the present invention, at least one protein glycoform binds to the separation material, but this protein glycoform does not have to be the target molecule. It can be the target molecule, but the target molecule can also be another protein glycoform or non-glycosylated protein that also binds to the separation material or is in the flow-through. Since the method of the present invention allows for the separation of different protein glycoforms as well as glycosylated and non-glycosylated proteins, the target molecule can be defined as needed.

[0239] The present invention is further illustrated by the following figures and examples. However, the present invention is not limited thereto.

[0240] All applications, patents, and publications cited above and below, as well as the entire disclosure of the corresponding EP patent application 19184130.3 filed on July 7, 2019, are incorporated herein by reference. Examples

[0241] The following examples represent the practical applications of the present invention.

[0242] 1. Synthesis of the separation material

[0243] To prepare the monomer for grafting from the method, an amino acid, such as valine, leucine or alanine, is dissolved in VE water and the pH is adjusted to a pH above 13 by adding NaOH (32%). At a temperature of 0-5 °C, an acrylic acid compound such as acryloyl chloride or acrylic acid is added and the mixture is stirred for one hour.

[0244] The reaction flow diagrams of valine and leucine with acryloyl chloride are shown in Figure 2 and Figure 3 .

[0245] Then the pH is adjusted to about pH 2.2 by adding nitric acid. Then a base material containing OH, such as Eshmuno® particles, is added.

[0246] The polymerization is started by adding cerium(IV) nitrate. The reaction is carried out at 30-50 °C for 4 hours.

[0247] After the polymerization reaction, the unreacted components and starting materials are removed by thorough washing with an acidic, basic and solvent mixture at room temperature or elevated temperature.

[0248] 2. Rituximab® isolated with high-mannose glycan

[0249] Evaluate the ability of the ion-exchange material prepared according to Example 1 (e.g., with an average particle size of 20 - 63 μm, an average pore size of 40 - 110 nm, and an ion density of 400 - 900 μeq / g) to separate the glycoforms of the commercially available drug Rituximab® containing high mannose. The ion-exchange material was packed in a chromatography column with a size of 5 × 100 mm, with an asymmetry between 0.8 - 1.2 and > 3000 plates / m. After packing the ion-exchange material, the obtained chromatography column was washed with 1 M NaOH solution for 30 minutes and pre-equilibrated with a loading buffer 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. The Rituximab® sample was diluted to a concentration of 4.8 mg / ml using the same solution. This solution was loaded onto the prepared chromatography column until 1 ml of resin reached a loading of 20 mg of Rituximab®. These steps and subsequent steps were carried out at a buffer velocity of 150 cm / h. After loading 20 mg / ml of Rituximab®, the chromatography column was washed with a buffer of pH 4.75 and then eluted with a buffer gradient of pH 8.5 and 250 mM NaCl. This elution buffer was prepared using different salts such as sodium dihydrogen phosphate, TRIS, and glycine. During the experimental setup, the conductivity and pH values were tracked, indicating the elution of Rituximab® from the column due to the change in pH during the gradient elution. The sample eluate was fractionated, and the obtained fractions were serially evaluated using the LC-MS method for glycoform identification ( Figure 4 ).

[0250] Figure 5 The analytical evaluation of the collected fractions using the LC-MS analysis method is shown, showing the main glycoprotein elution peaks, further characterized by the representative 1B9 and 1C7 fractions, and not containing any high mannose glycan variants. Most of the high mannose glycan variants eluted at a higher pH, further characterized by the representative 1D10 and 1E3 fractions ( Figure 5 ).

[0251] As Figure 4 shown, the separation / enrichment of high mannose-containing glycoforms is possible when eluting with a linear pH gradient. In the fractions mainly containing glycoproteins (e.g., 1B9 and 1C7), no high mannose variants were detected. All variants with high mannose content eluted in separate fractions (e.g., 1D10).

[0252] 3. Erbitux® separated with high mannose glycan types in a wide application window within 200 mM to 600 mM sodium sulfate

[0253] Evaluate the ability of the ion-exchange material prepared according to Example 1 (e.g., with an average particle size of 20 - 63 μm, an average pore size of 40 - 110 nm, and an ion density of 400 - 900 μeq / g) to separate the commercially available drug Erbitux® substance containing high mannose. The ion-exchange material is packed in a chromatography column with dimensions of 5 × 100 mm, with an asymmetry between 0.8 - 1.2 and > 3000 plates / m. After packing the ion-exchange material, the obtained chromatography column is washed with 1 M NaOH solution for 30 minutes and pre-equilibrated with a loading buffer having a pH of 4.5 and 250 mM NaCl. The buffer solution contains a combination of salts, such as sodium dihydrogen phosphate, TRIS, and glycine, to obtain a pH of 4.5. The pre-purified Erbitux® sample is loaded onto the prepared chromatography column until 5 mg of pre-purified Erbitux® is loaded per 1 ml of resin. These steps and subsequent steps are carried out at a buffer velocity of 150 cm / h. After adding 5 mg / ml of pre-purified Erbitux®, the chromatography column is washed with a pH 4.5 buffer and different amounts of Na2SO4. The amount of Na2SO4 varies between 200 - 600 mM. Then, the column is eluted with a buffer having a pH of 8.5 and a corresponding Na2SO4 amount gradient. This elution buffer is prepared using different salts such as sodium dihydrogen phosphate, TRIS, and glycine. During the experimental setup, the conductivity and pH values are tracked, showing that the elution of Erbitux® from the column is achieved due to the change in pH during the gradient elution. Table 1 shows the values of the elution maxima of the main peak and the high-mannose peak at the corresponding pH values.

[0254]

[0255] Table 1 shows the elution maxima of the main peak and the high-mannose peak at the corresponding pH values.

[0256] As shown in Table 1, when eluting with a linear pH gradient, it is possible to separate / enrich the high-mannose-containing glycan species over a wide range of conductivities. Compared with the glycan species without high mannose, the fractions with high-mannose variants are eluted at higher pH values throughout the study range.

[0257] 4. Rituximab® Separated with a Mixed Glycan Type

[0258] Evaluate the ability of the ion-exchange material prepared according to Example 1 (e.g., with an average particle size of 20 - 63 μm, an average pore size of 40 - 110 nm, and an ion density of 400 - 900 μeq / g) to separate the mixed glycan substances of the commercially available drug Rituximab®. The ion-exchange material is packed in a chromatographic column with a size of 5 × 100 mm, with an asymmetry between 0.8 - 1.2 and > 3000 plates / m. After packing the ion-exchange material, the obtained chromatographic column is washed with 1 M NaOH solution for 30 minutes and pre-equilibrated with a loading buffer having a pH of 4.75 and 250 mM NaCl. The buffer solution contains a combination of salts such as sodium dihydrogen phosphate, TRIS, and glycine to obtain a pH of 4.5. The Rituximab® sample is diluted to a concentration of 4.8 mg / ml using the same solution. This solution is loaded onto the prepared chromatographic column until 20 mg of Rituximab® is loaded per 1 ml of resin. These steps and subsequent steps are carried out at a buffer velocity of 150 cm / h. After loading 20 mg / ml of Rituximab®, the chromatographic column is washed with a buffer of pH 4.75 and then eluted with a buffer gradient of pH 8.5 and 250 mM NaCl. This elution buffer is prepared using different salts such as sodium dihydrogen phosphate, TRIS, and glycine. During the experimental setup, the conductivity and pH values are tracked, indicating the elution of Rituximab® from the column due to the change in pH during the gradient elution. The sample eluate is fractionated, and the obtained fractions are serially evaluated using the LC-MS method for glycan species identification ( Figure 7 ).

[0259] The analytical evaluation of the collected fractions using the LC-MS analysis method is shown in Figure 7 , showing that the main glycoprotein elution peaks further characterized by the representative 1B9 and 1C7 fractions do not contain any mixed glycan variants (e.g., containing terminal mannose). Most of the mixed glycan variants elute at a higher pH and are further characterized by the representative 1D10 and 1E3 fractions ( Figure 7 ).

[0260] As Figure 6 and Figure 7 shown, when eluting with a linear pH gradient, mixed glycan species can be separated / enriched. In the main glycoprotein-containing fractions (e.g., 1B9 and 1C7), no mixed glycan variants are detected. All the mixed glycan variants elute in the separated fractions (e.g., 1D10 and 1E3).

[0261] 5. mAb05 separated by mixed glycan type

[0262] Evaluate the ability of the ion exchange material prepared according to Example 1 (e.g., average particle size of 20 - 63 μm, average pore size of 40 - 110 nm, ion density of 400 - 900 μeq / g) to separate the mAb05 mixed glycan substances. The ion exchange material is packed in a chromatographic column with a size of 5 × 100 mm, the asymmetry is between 0.8 - 1.2 and > 3000 plates / m. After packing the ion exchange material, the obtained chromatographic column is washed with 1M NaOH solution for 30 minutes and pre-equilibrated with a loading buffer having a pH of 4.75 and 250 mM NaCl. The buffer solution contains a combination of salts such as sodium dihydrogen phosphate, TRIS, and glycine to obtain a pH of 4.75. The mAb05 sample is diluted to a concentration of 5 mg / ml using the same solution. This solution is loaded onto the prepared chromatographic column until 30 mg of mAb05 is loaded per 1 ml of resin. These steps and subsequent steps are carried out at a buffer velocity of 150 cm / h. After loading 30 mg / ml of mAb05, the chromatographic column is washed with a buffer solution of pH 4.75 and then eluted with a buffer gradient of pH 8.5 and 250 mM NaCl. This elution buffer is prepared using different salts such as sodium dihydrogen phosphate, TRIS, and glycine. During the experimental setup, the conductivity and pH values are tracked, indicating that mAb05 is eluted from the column due to the change in pH during the gradient elution. The sample eluate is fractionated, and the obtained fractions are serially evaluated using LC-MS method for glycan species identification( Figure 9 ).

[0263] The analytical evaluation of the collected fractions using the LC-MS analysis method is shown in Figure 9 , showing that the first elution fraction 1A4 - 2A4 does not contain any mixed glycan variants (e.g., G0F-N and G1F-N containing terminal mannose). Most of the mixed glycan variants are eluted at higher pH, further characterized by the representative fractions 3B5 to 4B2( Figure 9 ). In addition to the mixed forms, the glycan variants containing mannose 5 are also eluted at higher pH.

[0264] As Figure 8 shown, when eluting with a linear pH gradient, the mixed glycan species can be separated / enriched. In the main glycoprotein-containing fractions (e.g., 1A4 to 2A4), no mixed glycan variants are detected. All the mixed glycan variants are eluted at higher pH values (e.g., 3B5 to 4B2).

[0265] In addition, the obtained fractions are characterized using analytical size exclusion chromatography and the level of fragmentation or aggregation is monitored. The results show that there are less than 1% aggregates in the samples used or the obtained fractions (Table 2).

[0266]

[0267] Table 2 shows the results of size exclusion chromatography analysis, showing the area of the fractions obtained and the % of fragmented or aggregated material. The purity of all fractions was >99%.

[0268] 6. mAb05 separated in fucosylated and non-fucosylated forms

[0269] Evaluate the ability of the ion exchange material prepared according to Example 1 (e.g., average particle size of 20 - 63 μm, average pore size of 40 - 110 nm, ion density of 400 - 900 μeq / g) to separate the fucosylated and non-fucosylated glycan species of mAb05. The ion exchange material was packed in a chromatography column with a size of 5 × 100 mm, with an asymmetry between 0.8 - 1.2 and >3000 plates / m. After packing the ion exchange material, the obtained chromatography column was washed with 1M NaOH solution for 30 minutes and pre-equilibrated with a loading buffer 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. The mAb05 sample was diluted to a concentration of 5 mg / ml using the same solution. This solution was loaded onto the prepared chromatography column until 30 mg of mAb05 was loaded per 1 ml of resin. These steps and subsequent steps were carried out at a buffer velocity of 150 cm / h. After loading 30 mg / ml of mAb05, the chromatography column was washed with a buffer solution at pH 4.75 and then eluted with a buffer gradient of pH 8.5 and 200 mM NaCl. This elution buffer was prepared using different salts such as sodium dihydrogen phosphate, TRIS, and glycine. During the experimental setup, the conductivity and pH values were tracked, indicating that the elution of mAb05 from the column was achieved due to the change in pH during the gradient elution ( Figure 10 ). The sample eluate was fractionated, and the obtained fractions were serially evaluated using LC-MS method for glycan species identification (Table 3).

[0270] The analytical evaluation of the LC-MS analysis method for the collected fractions is listed in Table 3, showing that the first elution fraction 1B4 - 1D5 indeed contains less non-fucosylated forms (G0 and G1) than the loaded glycoprotein (G0F and G1F). Higher amounts of fucosylated glycan variants (G0F and G1F) eluted at higher pH were further characterized with representative 1E4 and 1F12 fractions (Table 3). Additionally, glycan variants containing mannose 5 also eluted at higher pH.

[0271]

[0272] Table 3 shows the analytical results of the sample fraction characterization, using the LC-MS analytical method, presented as the quantitative area of the elution fractions (1B4 to 1F12) analyzed.

[0273] As Figure 10 shown in and Table 3, the separation / enrichment of non-fucosylated glycan species is possible when eluting with a linear pH gradient. Fewer fucosylated glycan variants were detected in the major glycoprotein-containing fractions (e.g., 1B4 to 1D5). More fucosylated glycan variants eluted at higher pH values (e.g., 1E4 to 1F12).

[0274] 7. Isolation of native and deglycosylated Rituximab®

[0275] Evaluate the ability of the ion exchange material prepared according to Example 1 (e.g., with an average particle size of 20 - 63 μm, an average pore size of 40 - 110 nm, and an ion density of 400 - 900 μeq / g) to separate glycosylated and non-glycosylated Rituximab®. The ion exchange material was packed in a chromatography column with a size of 5 × 100 mm, an asymmetry between 0.8 - 1.2, and > 3000 plates / m. After packing the ion exchange material, the obtained column was washed with 1 M 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. Native and deglycosylated Rituximab® samples were diluted to a concentration of 4 mg / ml using the same solution. This solution was loaded onto the prepared chromatography column until 1 mg of Rituximab® was loaded per 1 ml of resin. These steps and subsequent steps were carried out at a buffer velocity of 150 cm / h. After loading 1 mg / ml Rituximab®, the chromatography column was washed with a buffer solution of pH 4.75 and then eluted with a buffer of pH 8.5 and a 150 mM NaCl gradient. This elution buffer was prepared using different salts such as sodium dihydrogen phosphate, TRIS, and glycine. The conductivity and pH values were tracked during the experimental setup, indicating the elution of Rituximab® from the column due to the change in pH during the gradient elution.

[0276] To prepare the sample, Rituximab® was partially deglycosylated using Endoglycosydase digestion.

[0277] Both partially deglycosylated and native Rituximab® were loaded in two different applications, and the retention times ( Figure 11 ) were compared.

[0278] The elution curves of native glycoproteins (e.g., Rituximab®) in a linear pH gradient show a lower elution pH for native glycosylated proteins. The deglycosylated portion of the glycoprotein elutes at a higher pH gradient.

[0279] As Figure 11 shown, it is possible to separate / enrich deglycosylated and native glycoproteins when eluting with a linear pH gradient.

[0280] 8. mAb05 isolated with high-mannose glycan type in flow-through mode

[0281] Evaluate the ability of the ion exchange material prepared according to Example 1 (e.g., with an average particle size of 20 - 63 μm, an average pore size of 40 - 110 nm, and an ion density of 400 - 900 μeq / g) to separate high-mannose glycan substance mAb05 in flow-through mode. The ion exchange material is packed in a chromatography column with dimensions of 5 × 100 mm, with an asymmetry between 0.8 - 1.2 and > 3000 plates / m. After packing the ion exchange material, the obtained chromatography column is washed with 1M NaOH solution for 30 minutes and pre-equilibrated with a loading buffer having a pH of 4.75 and 400 mM NaCl. The buffer solution contains a combination of salts, such as sodium dihydrogen phosphate, TRIS, and glycine, to obtain a pH of 4.75. The mAb05 sample is diluted to a concentration of 4.8 mg / ml using the same solution. This solution is loaded onto the prepared chromatography column until 10 mg of mAb05 is loaded per 1 ml of resin. These steps and subsequent steps are carried out at a buffer velocity of 150 cm / h. After loading 10 mg / ml of mAb05, the chromatography column is washed with a buffer solution of pH 4.75 and then eluted with a buffer gradient of pH 8.5 and 400 mM NaCl. This elution buffer is prepared using different salts such as sodium dihydrogen phosphate, TRIS, and glycine. During the experimental setup, the conductivity and pH values are tracked, indicating that due to the change in pH during gradient elution, the high-mannose mAb05 glycoforms are eluted from the column. The glycoforms without mannose do not bind to the column and are in the flow-through. The flow-through and sample eluates are fractionated, and the obtained fractions are evaluated using LC-MS methods for glycan species identification (Table 4).

[0282]

[0283] Table 4 shows the analytical evaluation of the collected fractions using LC-MS analysis methods. It shows that the flow-through fraction 1F8 indeed contains < 1% of mannose-containing glycoforms. Most of the mannose-containing glycoforms eluted at a higher pH were further characterized with the representative fraction 1H9 (Table 4). As Figure 12As shown, it is possible to separate / enrich low-mannose-containing glycan species in the flow-through when using a linear pH gradient elution. High-mannose glycan variants were detected in the bound fractions (e.g., 1H9). The high-mannose glycan variants elute at higher pH values (e.g., 1H9).

[0284] 9. Separation of the spike S1 protein of SARS-CoV-2 in a bind-elute mode

[0285] Evaluate the ability of the ion exchange material prepared according to Example 1 (e.g., with an average particle size of 20 - 63 μM, an average pore size of 40 - 110 nm, and an ion density of 400 - 900 μeq / g) to bind the spike S1 protein of SARS-CoV-2. The ion exchange material was packed in a chromatography column with dimensions of 5 × 100 mm, with an asymmetry between 0.8 - 1.2 and > 3000 plates / m. After packing the ion exchange material, the obtained chromatography column was washed with 1 M NaOH solution for 30 minutes and pre-equilibrated with a loading buffer having a pH of 4.75 and 250 mM NaCl. The buffer solution contains a combination of salts, such as sodium dihydrogen phosphate, TRIS, and glycine, to obtain a pH of 4.75. The S1 protein sample was reconstituted with the same solution until a concentration of 0.6 mg / ml. This solution was loaded onto the prepared chromatography column until 1 mg of S1 protein was loaded per 1 ml of resin. 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 chromatography column was washed with a buffer at pH 4.75 and then eluted with a buffer gradient of pH 10.5 and 250 mM NaCl. This elution buffer was prepared using different salts such as sodium dihydrogen phosphate, TRIS, and glycine. During the experimental setup, the conductivity and pH values were tracked, showing that the elution of the S1 protein from the column was achieved due to the change in pH during the gradient elution. The sample eluate was fractionated, and the obtained fractions were serially evaluated using an analytical HIC method for protein identification ( Figure 13 ).

[0286] Figure 14 The analytical evaluation of the collected fractions using an analytical HIC method is shown, showing that the main elution peak (dashed line) further characterized with the representative 1C6 fraction indeed contains only the S1 protein (solid line). ( Figure 14 )

[0287] As Figure 13 shown, it is possible to bind / elute the spike S1 protein of SARS-CoV-2 despite using a linear pH gradient elution. The elution peak contains only the S1 protein.

[0288] 10. mAb03 separated with high-mannose glycan type in a flow-through mode

[0289] Evaluate the ability of the ion-exchange material prepared according to Example 1 (e.g., average particle size of 20 - 63 μm, average pore size of 40 - 110 nm, ion density of 400 - 900 μeq / g) to separate the high-mannose glycan substance mAb03 in the flow-through mode. The ion-exchange material was packed in a chromatography column with dimensions of 5 × 100 mm, with an asymmetry between 0.8 - 1.2 and > 3000 plates / m. After packing the ion-exchange material, the obtained chromatography column was washed with 1 M NaOH solution for 30 minutes and pre-equilibrated with a loading buffer 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. The mAb03 sample was diluted to a concentration of 5.0 mg / ml using the same solution. This solution was loaded onto the prepared chromatography column until 100 mg of mAb03 was loaded per 1 ml of resin. These steps and subsequent steps were carried out at a buffer velocity of 150 cm / h. After loading 100 mg / ml of mAb03, the chromatography column was washed with a buffer solution of pH 5.12 and then eluted with a buffer gradient of pH 8.5 and 250 mM NaCl. This elution buffer was prepared using different salts such as sodium dihydrogen phosphate, TRIS, and glycine. During the experimental setup, the conductivity and pH values were tracked, indicating that due to the change in pH during the gradient elution, the high-mannose mAb03 glycoforms were eluted from the column. The glycoforms without mannose did not bind to the column and were in the flow-through. The flow-through was fractionated, and the amount of high-mannose substance in the obtained fractions was evaluated (Table 5).

[0290]

[0291] Table 5 shows the analytical evaluation of the representative amount of high-mannose substance in the fractions collected after a certain mAb03 breakthrough. The starting concentration of the high-mannose substance was 7.68%. It was thus shown that 66.2 mg of the flow-through mAb03 sample indeed contained < 1% of high-mannose glycoforms.

[0292] 11. Gammanorm® Static Binding Capacity

[0293] Measure the static binding capacity of Gammanorm® using the material prepared from hexafluoro-valine for grafting. Dissolve hexafluoro-valine in VE water, add NaOH (32%), and adjust the pH to a pH above 13. At a temperature between 0 - 5 °C, add acrylic compounds such as acryloyl chloride or acrylic acid, and stir the mixture for one hour. Then adjust the pH to approximately pH 2.2 by adding nitric acid. Then add a OH-containing base material, such as Eshmuno® particles. Start the polymerization by adding cerium(IV) nitrate. The reaction was carried out at 30 - 50 °C for 4 hours.

[0294] An exact amount of this material was soaked in a solution containing 5 mg / ml Gammanorm, pH 5.0, and various amounts of NaCl. After incubating for 4 hours, the material was removed, and the amount of Gammanorm® remaining in the solution was measured to estimate the amount of bound Gammanorm®. The measured static binding capacity was as follows:

[0295] At 0 mM NaCl, 64.5 mg Gammanorm® / ml hexafluoro-valine grafted material was bound; at 30 mM NaCl, 50.9 mg Gammanorm® / ml hexafluoro-valine grafted material was bound; at 75 mM NaCl, 50 mg Gammanorm® / ml hexafluoro-valine grafted material was bound.

Claims

1. A method for chromatographically purifying and / or separating protein glycoforms by contacting a sample containing protein glycoforms with a separation material, the separation material comprising a base matrix to which polymer chains are covalently bonded on the surface, characterized in that the polymer chains are composed of monomer units, and the monomer units of the polymer chains are connected in a linear manner, and each monomer unit contains a terminal group -N(Y)-R3, where Y is independently H or CH 3 , and R3 is -CHCOOMR4 where R4 is a C3 to C4 alkyl group and M is H, Na, K or NH 4 .

2. The method of claim 1, comprising the steps of a) contacting a sample containing protein glycoforms with the separation material, whereby one or more protein glycoforms bind to the separation material c) contacting the separation material with an elution buffer under conditions in which the bound protein glycoforms are eluted from the separation material.

3. The method according to claim 2, further comprising step b) washing the separation material before step c) and after step a).

4. The method according to claim 2 or 3, characterized in that the method further comprises recovering the protein glycoforms that flow through the separation material in step a).

5. The method according to claim 2 or 3, characterized in that the elution buffer has a higher pH than the loading buffer used to contact the sample with the separation material in step a).

6. The method according to claim 2 or 3, characterized in that, in step a), the sample contacted with the separation material in step a) has a conductivity of 5 mS / cm to 60 mS / cm.

7. The method according to claim 1, 2 or 3, characterized in that, the sample comprises one or more of the following: - mannose-rich protein glycoforms - terminal mannose protein glycoforms - fucosylated and non-fucosylated protein glycoforms - glycosylated and non-glycosylated proteins.

8. The method according to claim 1, 2 or 3, characterized in that the sample contains glycosylated antibodies and / or viral protein glycoforms.

9. The method according to claim 1, 2 or 3, characterized in that Y is H and R4 is isopropyl and / or isobutyl.

10. The method according to claim 1, 2 or 3, characterized in that, the ionic density of the separation material is between 10 - 1200 μeq / g.

11. The method according to claim 1, 2 or 3, characterized in that the protein glycoforms bind to the separation material at a pH of 2 to 7.

12. The method according to claim 1, 2 or 3, characterized in that the protein glycoforms bind to the separation material at a pH of 2 to 7, and are washed and eluted by increasing the pH value to a value of 9 to 11.

13. The method according to claim 1, 2 or 3, characterized in that, the sample is applied to the separation material at an ionic density between 10 - 1200 μeq / g.

14. The method according to claim 1, 2 or 3, characterized in that 10 mg to 100 mg of protein glycoforms are bound per ml of separation material.

15. The method according to claim 1, 2 or 3, characterized in that the sample contains SARS-CoV-2 protein glycoforms.

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