Protein A / G particles for affinity chromatography and methods of use thereof

Abstract

The present disclosure relates to non-porous polymer particles having an average particle size of from 1 micron to 10 microns and functionalized with an immunoglobulin binding protein. The functionalized particles of the present disclosure can be used to purify a range of immunoglobulins having affinity for immunoglobulin binding proteins.

Description

[0001] Cross-references to related applications

[0002] This patent application claims the benefit of U.S. Provisional Application No. 63 / 464,384, filed May 5, 2023, and U.S. Provisional Patent Application No. 63 / 587,864, filed October 4, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This technology relates to affinity capture materials. More specifically, this technology relates to chromatographic columns with non-porous particles for affinity chromatography. Background Technology

[0004] Chromatography is a separation technique with wide applicability across various industries, including pharmaceuticals, biotechnology, and the chemical industry. Chromatography involves two phases: a stationary phase and a mobile phase. Typically, the stationary phase contains porous or non-porous particles loaded into a column. The mobile phase then carries the sample through the stationary phase column. One specific application of affinity chromatography utilizes functionalized particles in the stationary phase. The fundamental principle behind affinity chromatography is that compounds or analytes within a sample have differential affinities for functional groups on the particles. These differences allow for the separation, isolation, and concentration of specific analytes from complex, heterogeneous samples.

[0005] In one instance, the analyte of interest to be separated using affinity chromatography is an antibody. Antibodies are typically separated from culture media (e.g., hybridoma cultures), serum, or ascites fluid, each of which contains unpredictable amounts and compositions of the analyte. However, due to the complexity and / or volume of antibody-containing samples, antibodies can be at such low concentrations, or contain sufficient impurities, that they are undetectable using standard analytical methods. Furthermore, the practicality of antibodies in experimental and therapeutic applications often requires homogenized, pure, and concentrated samples. Therefore, it is highly desirable to be able to separate antibodies that are substantially pure and at sufficiently high concentrations to allow for downstream use in applicable assays.

[0006] Current affinity chromatography methods are hampered by low-throughput and high-cost columns, and often result in antibody loss due to nonspecific binding and / or poor antibody retention. Other types of immunoassays, such as ELISA, can be complex, time-consuming, and highly variable. Therefore, there is a need in the art for affinity chromatography columns that are efficient and robust in separating, collecting, and concentrating antibodies independent of their target antigens. Summary of the Invention

[0007] In general, the present technology relates to a plurality of particles used in affinity chromatography. More specifically, the present technology relates to a chromatography column containing a plurality of particles having an average particle size of less than 10 pm, wherein an outer hydrophilic surface of each of the particles is conjugated to an immunoglobulin binding protein, such as protein A, which acts as a binding site for an antibody. Thus, the chromatography column of the present technology can be used for affinity chromatography of an antibody.

[0008] In one aspect, the present technology relates to a chromatography column. The chromatography column includes a column body formed from a metal or metal alloy and housing a plurality of particles. Each particle of the plurality of particles includes a non-porous polymeric core, a hydrophilic surface on an outer layer of the non-porous polymeric core; and one or more molecules of an immunoglobulin binding domain conjugated to the hydrophilic surface, wherein the average particle size of the particle is between 1.5 pm and 8 pm.

[0009] Other embodiments of the present technology are as follows. In some embodiments, the immunoglobulin binding protein is protein A, protein G, protein A / G, or protein L or binding domains thereof. In some embodiments, the non-porous polymeric core has a gradient composition. In some embodiments, the non-porous polymeric core comprises divinylbenzene (80%). In some embodiments, the hydrophilic surface is (3-glycidyloxypropyl)trimethoxysilane, (3- glycidyloxypropyl)triethoxysilane, polyacrylate, glycidol, glycerol triglycidyl ether, butyl diglycidyl ether, or poly(methyl acrylate). In certain embodiments, the one or more molecules of the immunoglobulin binding protein are conjugated to the hydrophilic surface of the particle via an epoxy linker. In some embodiments, the average particle size is between 2 pm and 5 pm. In one embodiment, the average particle size is 3.5 pm. In some embodiments, the immunoglobulin binding protein has a surface coverage of between 3 pg / mg of particles and 9 pg / mg of particles. In some embodiments, the immunoglobulin binding protein is protein A and has a surface coverage of between 3 pg / mg of particles and 9 pg / mg of particles.

[0010] In some embodiments, the chromatography column has at least a portion of an inner surface of the column body coated with an alkylsilyl material. In some embodiments, the column body comprises frits, wherein the frits are coated with the alkylsilyl material. In some embodiments, the alkylsilyl material is a hydrophilic non-ionic layer of polyethylene glycol silane.

[0011] In one aspect, disclosed herein is a chromatography apparatus comprising a chromatography column, a column injector positioned upstream of the chromatography column, and a conduit fluidly connected to the chromatography column and positioned downstream of the chromatography column, wherein a portion of an inner surface of the column injector and a portion of an inner surface of the conduit are coated with an alkylsilyl material.

[0012] In one aspect, disclosed herein is a method for enriching immunoglobulin, the method comprising four steps: steps a) through d). Step a) comprises selecting a chromatography column, wherein the chromatography column comprises a column body formed from a metal or metal alloy, the column body housing a plurality of particles, wherein each particle of the plurality of particles comprises a nonporous polymer core, a hydrophilic surface on an outer layer of the nonporous polymer core, and one or more molecules of an immunoglobulin binding protein conjugated to the hydrophilic surface, wherein the average particle size of the particles is between 1.5 pm and 8 pm. Step b) comprises washing the chromatography column with a binding buffer. Step c) comprises applying a solution containing immunoglobulin to the chromatography column. Step d) comprises washing the chromatography column with an elution buffer, such that the immunoglobulin elutes from the chromatography column.

[0013] In some embodiments, the immunoglobulin binding protein is protein A, protein G, protein A / G, protein L, or binding domains thereof. In some embodiments, the chromatography column is connected to a high performance liquid chromatography (HPLC) system, an ultra-high performance liquid chromatography (UHPLC) system, or a fast protein liquid chromatography (FPLC) system. In some embodiments, the elution buffer is at least 3 orders of magnitude more acidic than the binding buffer. In some embodiments, the pH of the binding eluent is between 7.0 and 8.0. In some embodiments, the pH of the elution buffer is between 1.3 and 3.5.

[0014] In some embodiments, the method further comprises step e) detecting the immunoglobulin with a UV spectroscopic detector, a fluorescence spectroscopic detector, and / or a mass spectrometry detector. In some embodiments, the method further comprises step f) washing the chromatography column with a binding buffer. In some embodiments, the method further comprises repeating steps b) through e).

[0015] In other embodiments, the method further comprises step e) washing the chromatography column with a binding buffer. In some embodiments, the method further comprises repeating steps b) through d).

[0016] In some embodiments, the washing of the chromatography column with the binding buffer is less than or equal to 10 minutes. In some embodiments, the method can be performed without the need for an organic modifier to reduce non-specific binding.

[0017] In some embodiments, the applying of the solution containing immunoglobulin to the chromatography column (step c) is performed in about 1 minute to 3 minutes. In some embodiments, the applying of the solution containing immunoglobulin to the chromatography column (step c) is performed in about 1 minute. BRIEF DESCRIPTION OF DRAWINGS

[0018] The present technology will be more fully understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0019] Figure 1A is a cross-sectional illustration of a particle according to an embodiment of the present technology prior to attachment of an immunoglobulin binding protein.

[0020] Figure 1B is a cross-sectional illustration of a particle according to an embodiment of the present technology after attachment of an immunoglobulin binding protein. Figure 1A

[0021] Figure 2A is a perspective view of a chromatography column packed with a plurality of particles (i.e., Figure 1B particles) having a cutaway portion 220 to illustrate the interior portion of the chromatography column.

[0022] Figure 2B is a cross-sectional view of a chromatography column taken along line BB of Figure 2A .

[0023] Figure 3 is a schematic illustration of a method of affinity capture of an antibody with a chromatography column of Figure 2B .

[0024] Figure 4A shows peak area of IgG eluted at different concentrations between 0.25 pg and 20 pg for the column of Figure 2 (black solid line) compared to the reference column 1 column (dashed line).

[0025] Figure 4B shows peak area of IgG eluted at different concentrations between 0.25 pg and 300 pg for the column of Figure 2 (black solid line) compared to the reference column 1 column (dashed line).

[0026] Figure 4C depicts percentage of IgG remaining between affinity capture assays for the column of Figure 2 compared to reference column 1.

[0027] Figure 5A shows elution peaks of eluted IgG for the column of Figure 2 (black dashed line) compared to the reference column 3 column (dashed line) and the reference column 2 column (solid line).

[0028] Figure 5B shows percentage of IgG remaining between affinity capture assays for the column of Figure 2 compared to reference column 2 and reference column 3.

[0029] Figure 6A depicts peak area of eluted IgG for the column of Figure 2 using a 1 minute binding phase (black solid line) or a 3 minute binding phase (black dashed line).

[0030] Figure 6B shows quantitative peak area of eluted IgG for the column of Figure 2 using a 1 minute binding phase or a 3 minute binding phase at different IgG concentrations.​

[0031] Figure 6C Residual percentage of IgG between affinity capture assays for the columns of Figure 2 using 1 minute binding phase or 3 minute binding phase at different IgG concentrations is depicted.

[0032] Figures 7A to 7C Chromatograms of a monoclonal antibody eluted from a column with particle type B using a phosphoric acid elution method Figure 7A ) or a glycine elution method Figure 7B . Figure 7C Linear correlation between monoclonal antibody concentration and peak area is shown.

[0033] Figures 8A to 8C Chromatograms of a monoclonal antibody eluted from a column with particle type D using a phosphoric acid elution method Figure 8A ) or a glycine elution method Figure 8B . Figure 8C Linear correlation between monoclonal antibody concentration and peak area is shown.

[0034] Figures 9A to 9C Chromatograms of a monoclonal antibody eluted from a column with particle type H using a phosphoric acid elution method Figure 9A ) or a glycine elution method Figure 9B . Figure 9C Linear correlation between monoclonal antibody concentration and peak area is shown.

[0035] Figures 10A to 10C Chromatograms of a monoclonal antibody eluted from a column with particle type E using a phosphoric acid elution method Figure 10A ) or a glycine elution method Figure 10B . Figure 10C Linear correlation between monoclonal antibody concentration and peak area is shown.

[0036] Figures 11A to 11C Chromatograms of a monoclonal antibody eluted from a column with particle type C using a phosphoric acid elution method Figure 11A ) or a glycine elution method Figure 11B . Figure 11C Linear correlation between monoclonal antibody concentration and peak area is shown.

[0037] Figures 12A to 12C Chromatograms of a monoclonal antibody eluted from a column with particle type F using a phosphoric acid elution method Figure 12A ) or a glycine elution method Figure 12B . Figure 12C Linear correlation between monoclonal antibody concentration and peak area is shown.

[0038] Figures 13A to 13C Chromatograms of monoclonal antibodies eluted from columns with particle type A using a phosphoric acid elution method Figure 13A ) or a glycine elution method Figure 13B Chromatograms comparing peak shape for particle types A, B, C, D, E, F, and H using a phosphoric acid elution method Figure 13C Linear correlation between monoclonal antibody concentration and peak area is shown.

[0039] Figures 14A to 14B Chromatograms comparing peak shape for particle types A, B, C, D, E, F, and H using a phosphoric acid elution method Figure 14A ) or a glycine elution method Figure 14B Chromatograms comparing peak shape for particle types A, B, C, D, E, F, and H using a phosphoric acid elution method

[0040] Figures 15A to 15B Peak height for particle types A, B, C, D, E, F, and G Figure 15A ) and peak volume Figure 15B ) for both elution methods are compared. DETAILED DESCRIPTION

[0041] To more readily understand the present technology, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter is set forth herein, the middles of the range and values listed are intended to be part of the disclosure, unless otherwise indicated. The term "about" means ±5% if not otherwise defined. It should also be noted that, as used herein and in the claims, the singular form "a", "an", and "the" include plural references unless the context clearly dictates otherwise.

[0042] Definitions

[0043] The term "non-porous" or "non-porous core" as used herein refers to a material or region of material (e.g., a core) having a pore volume of less than 0.1 cc / g. Preferably, in some embodiments, the non-porous polymeric core has a pore volume of less than 0.10 cc / g (e.g., 0.05 cc / g), and preferably less than 0.02 cc / g. Pore volume is determined using methods known in the art based on multipoint nitrogen adsorption experiments (Micromeritics ASAP 2400; Micromeritics Instruments Inc., Norcross, GA).

[0044] The term "rigid particle" as used herein refers to the strength of a particle to withstand an applied pressure under flow conditions. After exposure to a pressure of 3,500 psi, the rigid particles appear visually undamaged (i.e., maintain the same form factor without breaking, crushing, or changing) in a scanning electron microscope image, with less than 10% of the observed particles visually damaged. In addition, particles that break or deform in a packed bed result in a decrease in flow and an increase in pressure, as one would predict using the Kozeny-Carmen equation. Broken or deformed particles in a packed bed can cause the pressure to rise above levels suitable for HPLC or UHPLC.

[0045] The term "conjugated" as used herein refers to the linkage of two molecules formed by the chemical bonding of a reactive functional group of one molecule, such as protein A, to a suitably reactive functional group of another molecule, such as an epoxide.

[0046] The term "conjugate" as used herein refers to a compound formed by the chemical bonding of a reactive functional group of one molecule, such as protein A, to a suitably reactive functional group of another molecule, such as an epoxide. An example of a suitable reactive functional group pair is a nucleophile / electrophile pair. For example, the nucleophile can be an amine or thiol group of an amino acid from protein A, and the electrophile is an epoxide.

[0047] The terms "immunoglobulin" and "antibody" are used interchangeably as used herein, unless otherwise defined.

[0048] The term "antibody" as used herein refers to an immunoglobulin molecule that specifically binds to or is immunologically reactive with a particular antigen. This includes polyclonal, monoclonal, genetically engineered, and other modified configuration of antibodies, including but not limited to, chimeric, camelid, unibody, humanized, and heteroconjugate antibodies (e.g., bispecific, trispecific, and tetraspecific antibodies, diabodies). The term "monoclonal antibody" is intended to include intact molecules, as well as antibody fragments that are capable of specifically binding to a target protein, unless otherwise indicated.

[0049] 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 disclosure belongs.

[0050] Materials

[0051] The present technology relates to materials, columns, and apparatus for affinity chromatography. In particular, the present technology relates to materials for use in a high performance liquid chromatography (HPLC) system or an ultra-high performance liquid chromatography (UHPLC) system, and are modulated to allow affinity capture under high pressure and flow conditions associated with HPLC and / or UHPLC.

[0052] In the present technology, a plurality of particles are contained within a chromatography column. It is these particles, among other components, that have been designed to provide on-column affinity capture under high pressure and flow rates associated with HPLC and UHPLC systems.

[0053] Particles

[0054] To provide stability and surface area for affinity capture, the particles of the present technology are non-porous. Non-porous particles provide an appropriate surface area for attachment or coverage with one or more affinity binding groups. In some embodiments, each particle within the plurality of particles to be packed into a column can be highly spherical and have a smooth surface. In some embodiments, each particle within the plurality of particles to be packed into a column can be highly spherical and have a bumpy convex surface. Such materials have a surface area (measured in m 2 / g) that is close to their theoretical value. The theoretical surface area for a non-porous smooth sphere is equal to 6 / {particle diameter x particle density}. For example, a 1 micron polymer particle with a density of approximately 1 g / mL has a theoretical surface area of 6 m 2 / g, a 3.5 micron polymer particle with the same density has a theoretical surface area of 1.7 m 2 / g, and a 7 micron polymer particle with the same density has a theoretical surface area of 0.9 m 2 / g.

[0055] Without wishing to be bound by theory, it is believed that the use of non-porous spheres is advantageous because it improves the binding and elution kinetics of affinity binding groups attached to the surface of the spheres (with smooth or bumpy convex surfaces). It is believed that the shape factor of non-porous spheres prevents diffusion kinetics into the pores of the particles.

[0056] The particles are non-porous. While some pores or voids can be incorporated into the particles as discontinuities or as microporosities, non-porous particles are those particles of the material forming the particles that have a pore volume of less than 0.1 cc / g. Preferably, in some embodiments, the non-porous particles have a pore volume of less than 0.10 cc / g (e.g., 0.05 cc / g), and preferably less than 0.02 cc / g. Pore volume is determined using methods known in the art based on multipoint nitrogen adsorption experiments (Micromeritics ASAP 2400; Micromeritics Instruments Inc., Norcross, GA).

[0057] The average particle size of the particles of the present technology is less than 10 microns. For example, in one embodiment of the present technology, the average particle size of the plurality of particles packed within the column can be any value between 8 microns and 1.5 microns. In one embodiment, the average particle size of the plurality of particles is 7 microns. In another embodiment, the average particle size is 3.5 microns. In yet another embodiment, the average particle size is 1.7 microns.

[0058] The size (i.e., less than 10 microns), shape (i.e., spherical), and surface area (i.e., non-porous, smooth, or contoured convex outer surface) result in a shape factor that can be used for affinity capture from a flowing sample. To achieve high throughput and minimize assay development, the particles of the present technology are used in conjunction with LC systems, such as HPLC and UHPLC systems. These systems operate at high pressures (e.g., typically greater than 3,000 psi, such as, for example, 5,000 psi, 10,000 psi, 12,000 psi, 15,000 psi, etc.). Accordingly, the particles of the present technology need to be rigid particles such that the particles maintain their shape factor under HPLC and UHPLC operating conditions.

[0059] Generally, the particles of the present technology are rigid particles that maintain their shape factor (e.g., are not subject to breakage, crushing, extrusion, or change) under HPLC or UHPLC operating conditions (e.g., pressure and flow rate). For example, the rigid particles according to the present technology are not visibly altered in form (e.g., are not broken, crushed, or changed from spherical), as can be confirmed using a scanning electron microscope before (i.e., control) and after application of HPLC or UHPLC conditions.

[0060] The particular material used to form the core (e.g., center or base) of the particles of the present technology that satisfy shape factor considerations is a polymer, and in particular, an organic polymer. In one embodiment, the non-porous particles of the present technology comprise a non-porous polymer core. In one embodiment, the non-porous polymer core of the particle is divinylbenzene (DVB), such as 80% divinylbenzene. In some embodiments, the non-porous polymer core is formed to comprise two or more polymers. For example, in some embodiments, the non-porous polymer core comprises both divinylbenzene and polystyrene. In certain embodiments, the non-porous polymer core can be manufactured to include a gradient of polymer composition. For example, an inner portion of the core can be formed from 100% of a first polymer (i.e., Polymer A), and an outer portion of the core can be formed from 100% or some percentage greater than 0% of a second polymer (i.e., Polymer B). Radially from the inner portion to the outer portion of the core, the percentage of Polymer A and Polymer B can vary to form a gradient of polymer composition. Other embodiments of non-porous polymer cores and particles suitable for use in the present technology are described in U.S. Patent Publication No. 2019 / 0322783.

[0061] While examples and embodiments of the present technology illustrate the use of non-porous polymer cores for the particles, it should be noted that other non-porous materials can be utilized so long as the shape factor of the particles can be maintained under the operating conditions of HPLC or UPHLC. That is, other materials, such as silica, metal oxides, inorganic-organic hybrid materials, or combinations thereof can be used to produce non-porous spherical particles having an average particle size of less than 10 microns and having rigidity or strength to maintain their shape factor under high operating pressures.

[0062] To form the particles that can be used for affinity capture, the outer surface of the non-porous core of the particle is bonded or linked to an affinity attachment group. To this end, in one embodiment, the outer surface of the non-porous polymer core comprises a hydrophilic material. That is, a hydrophilic surface is created on this outer region of the non-porous polymer core. To the hydrophilic surface, one or more molecules of an immunoglobulin binding protein are conjugated. The one or more molecules of the immunoglobulin binding protein provide accessible binding sites for immunoglobulins. In some embodiments, the immunoglobulin binding protein is Protein A, Protein G, Protein A / G, or Protein L. In one aspect, any immunoglobulin binding protein that binds to a conserved portion of an antibody is suitable for use in the present technology.

[0063] The hydrophilic surface can also be referred to as a hydrophilic layer. The hydrophilic surface is on the outer surface of the non-porous polymer core and can be formed from a polymer, molecule, or siloxane with a high density of hydrophilic groups (e.g., hydroxyl, PEG, sugar, or carbohydrate). Immobilization of these hydrophilic groups can be performed by condensation (ester, amide, silanol, silyl ether), polymerization (methacrylates, acrylates, styryl), epoxy activation (epoxy chloropropane), or ether formation (direct attachment of PEG or carbohydrate groups via ether formation).

[0064] In one embodiment, the hydrophilic surface comprises a material selected from the group consisting of (3-glycidyloxypropyl)trimethoxysilane, (3- glycidyloxypropyl)triethoxysilane, polyacrylate, poly(methyl acrylate), and combinations thereof. In another embodiment, the hydrophilic surface comprises a material selected from the group consisting of glycidol, glycerol triglycidyl ether, and combinations thereof.

[0065] In embodiments where the immunoglobulin binding protein is used to provide a binding site for an antibody, a linker is typically used to immobilize or conjugate the immunoglobulin binding protein to the hydrophilic surface. Such linkers include, but are not limited to, epoxy linkers, hydroxyl linkers, and any other linker known in the art (see Hermanson G, “Bioconjugate Techniques” 3rd Edition, July 2013).

[0066] Figure 1A Embodiments of particles having a non-porous polymer core according to the present technology are illustrated. That is, Figure 1A The illustrated particles have a form factor (e.g., spherical, non-porous, and rigid) that withstands the operating conditions of HPLC and UHPLC. Figure 1A The illustrated particle 100 is a cross-sectional view prior to the addition of an immunoglobulin binding protein, such as, for example, Protein A. The particle 100 comprises a non-porous polymer core 112 having an inner core region 105 and a radially extending region 110 surrounding the inner core region 105. The inner core region 105 is typically formed from a polymer or homogenized blend of polymers, while the radially extending region 110 is typically formed from two or more polymers to form a gradient within the region. For example, the core region 105 can be formed from polystyrene, while the radially extending region 110 contains a gradient that transitions from 100% polystyrene to 80% to 100% DVB, with any remainder being polystyrene.

[0067] In one embodiment, the following three steps are used to form the non-porous polymer core 112. In step one: 561.1 g of reagent alcohol (90% ethanol, about 5% methanol and about 5% isopropanol), 16.9 g of polyvinylpyrrolidone (PVP-40, average molecular weight 40,000), 1.6 g of 2,2′-azobis(2-methylpropionitrile) (AIBN), and 6.7 g of Triton... ™ N-57, 80.1 g styrene, and 2.4 g poly(propylene glycol) dimethacrylate (average molecular weight 560) were loaded into the reactor. After purging with nitrogen, the reaction mixture was heated to 70°C with stirring and maintained at 70°C until all reaction steps were complete. In step two: After maintaining the reaction mixture at 70°C for 3 hours in step one, a solution containing 52.0 g DVB 80, 24.0 g styrene, 51.0 g PVP-40, 1080.4 g reagent alcohol (90% ethanol, approximately 5% methanol, and approximately 5% isopropanol), and 54.1 g p-xylene was added to the reaction mixture at a constant flow rate over two hours. Step 3: After completing the solution loading in Step 2, a primer coating solution containing 31.2 g glycidyl methacrylate (GMA), 6.2 g ethylene glycol dimethacrylate (EDMA), 12.9 g PVP-40, and 381.9 g reagent alcohols (90% ethanol, approximately 5% methanol, and approximately 5% isopropanol) was added to the reaction mixture at a constant flow rate over 1.5 hours. After maintaining the reaction mixture at 70°C for a total of 20 hours, the particles were separated from the reaction slurry by filtration. The particles were then washed with methanol, followed by tetrahydrofuran (THF), and then with acetone. The final product was dried overnight in a vacuum oven at 45°C. 91.8 g of monodisperse 2.3 μm polymer particles were obtained.

[0068] Although Figure 1A The illustrated embodiment exemplifies a nonporous polymer core 112 having two regions (core region 105 and radially extending region 110), but this is not always the case. Other embodiments may feature a nonporous polymer core with a single region, i.e., the nonporous polymer core extends from the center of the particle to the outer surface of the nonporous polymer core 112.

[0069] like Figure 1AA hydrophilic surface or layer 115 is formed on the outer surface of the non-porous polymer core (i.e., opposite the central region 105), as illustrated. In one embodiment, the hydrophilic surface 115 is formed by applying a hydrophilic primer coating solution containing 36.2 g of glycidyl methacrylate (GMA), 7.44 g of ethylene glycol dimethacrylate (EDMA), 8.21 g of PVP 360 (PVP 360, average molecular weight 360,000), and 489.4 g of reagent alcohol (90% ethanol, about 5% methanol, and about 5% isopropanol). The solution is added to the mixture containing the non-porous polymer core at a constant flow rate over about 1.5 hours to form the hydrophilic surface 115.

[0070] The above examples are provided for illustrative purposes only. Other types of hydrophilic surfaces can be applied. For example, the hydrophilic layer can also be formed from (3-glycidyloxypropyl)trimethoxysilane, (3-glycidyloxypropyl)triethoxysilane, polyacrylate and / or poly(methyl acrylate), glycidol, glycerol triglycidyl ether, butyl diglycidyl ether, or any other type of hydrophilic material.

[0071] To attach an immunoglobulin-binding protein, such as protein A, a linker is used to conjugate it to the hydrophilic surface 115. Referring to Figure 1B , a conjugated particle 150 is shown. That is, the particle 150 is the result of conjugating the immunoglobulin-binding protein 120 to the hydrophilic surface 115 using a linker. As described above, any type of linker can be used. Those of ordinary skill in the art will appreciate that there are a variety of ways to attach an immunoglobulin-binding protein to a hydrophilic layer. Many methods for conjugation are suitable for use in the present technology. For example, an epoxy linker can be used to link the immunoglobulin-binding protein to the hydrophilic group 115.

[0072] The immunoglobulin-binding protein provides accessible binding sites for immunoglobulins (i.e., antibodies), provided that the antibodies contain a conserved region that binds to the immunoglobulin-binding protein. In one embodiment, the immunoglobulin-binding protein is protein A. In another embodiment, the immunoglobulin-binding protein is protein G. In other embodiments, the immunoglobulin-binding protein is protein A / G or protein L.

[0073] Most immunoglobulins (Ig) are composed of four polypeptide chains: two identical heavy chains and two identical light chains connected by disulfide bonds. Within a given heavy or light chain, there are both variable and constant regions. The constant region, which contains 2 to 4 constant domains (depending on the isotype), is highly conserved within a given isotype. Thus, an immunoglobulin-binding protein that binds to a portion of the constant region is suitable for the affinity capture of antibodies independent of the target antigen of the antibody.

[0074] Generally, immunoglobulin binding proteins bind to the fragment crystallizable (Fc) region of antibodies, which comprises a portion of the heavy chain constant domains of antibodies. In at least one example, immunoglobulin binding proteins have been shown to bind to the constant domains of light chains as well.

[0075] Immunoglobulin binding proteins suitable for use in the present technology can exhibit strong binding affinity to the Fc portion of antibodies. The strength of this binding affinity varies by isotype and species. For example, Protein A exhibits strong binding affinity to IgG isotypes, but variable or even no binding affinity to IgA, IgD, IgE, and IgM isotypes. Even within the IgG isotypes, different subtypes can exhibit different binding affinities. Protein A has high binding affinity to human IgGl, IgG2, and IgG4, but very weak binding affinity to IgG3. In contrast, Protein A binds to murine IgG3, but not IgGl. Other examples of immunoglobulin binding proteins, such as Protein G, have high binding affinity to all four subtypes of IgG. Methods for characterizing protein-protein interactions, including binding affinity under a range of environmental conditions, are well known in the art.

[0076] The present technology enables any immunoglobulin binding protein to be conjugated to the hydrophilic surface of a non-porous polymeric particle. Thus, within the scope of the present technology, immunoglobulin binding proteins can bind to IgA, IgD, IgE, IgG, and / or IgM antibodies with varying levels of affinity. In some embodiments, the immunoglobulin binding protein is selected from the group consisting of Protein A, Protein G, Protein A / G, and Protein L. In some embodiments, the immunoglobulin binding protein can be a variant of Protein A, Protein G, Protein A / G, or Protein L. In some embodiments, the immunoglobulin binding protein can have 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% sequence identity to Protein A, Protein G, Protein A / G, or Protein L.

[0077] In some embodiments, the immunoglobulin binding protein has a surface coverage between 3 pg / mg of particles to 9 pg / mg of particles. In some embodiments, the immunoglobulin binding protein has a surface coverage of 3 pg / mg of particles to 3.5 pg / mg of particles, 3.5 pg / mg of particles to 4.0 pg / mg of particles, 4.0 pg / mg of particles to 4.5 pg / mg of particles, 4.5 pg / mg of particles to 5 pg / mg of particles, 5 pg / mg of particles to 5.5 pg / mg of particles, 5.5 pg / mg of particles to 6 pg / mg of particles, 6 pg / mg of particles to 6.5 pg / mg of particles, 6.5 pg / mg of particles to 7 pg / mg of particles, 7 pg / mg of particles to 7.5 pg / mg of particles, 7.5 pg / mg of particles to 8 pg / mg of particles, 8 pg / mg of particles to 8.5 pg / mg of particles, or 8.5 pg / mg of particles to 9 pg / mg of particles. In some embodiments, the immunoglobulin binding protein is Protein A and has a surface coverage between 3 pg / mg of particles to 9 pg / mg of particles. In some embodiments, the immunoglobulin binding protein is Protein G and has a surface coverage between 3 pg / mg of particles to 9 pg / mg of particles. In some embodiments, the immunoglobulin binding protein is Protein A / G and has a surface coverage between 3 pg / mg of particles to 9 pg / mg of particles. In some embodiments, the immunoglobulin binding protein is Protein L and has a surface coverage between 3 pg / mg of particles to 9 pg / mg of particles.

[0078] Example 1 and Example 2 provide exemplary methods of synthesizing non-porous polymer particles that can be functionalized with an immunoglobulin binding protein, such as, for example, Protein A, Protein G, Protein A / G, or Protein L.

[0079] Without being bound by any particular theory, it is understood that the binding affinity of an antibody to an immunoglobulin binding protein depends at least in part on the pH of the environment. Thus, by changing the pH of the in-column buffer of the present technology, the binding of an antibody to an immunoglobulin binding protein is reversible.

[0080] Column and apparatus

[0081] In the present technology, the above-described materials are typically packed into a chromatography device, such as, for example, a chromatography column. The chromatography device includes a column body formed from a metal or metal alloy (e.g., titanium, stainless steel). The column body houses a plurality of particles, such as, for example, Figure 1B The plurality of particles is shown.

[0082] Referring to Figure 2A A chromatography column 205 is shown. The chromatography column 205 has a stainless steel column body 210. In Figure 2AA portion of the column body 220 is removed to illustrate the location of the plurality of particles 225. Figure 2B A cross-sectional view of the chromatographic column 205 taken along line B-B in Figure 2A A cross-sectional view of the chromatographic column 205 taken along line B-B in

[0083] Figure 2B A cross-sectional view of the chromatographic column 205 taken along line B-B in In some embodiments, an alkylsilyl coating or other high performance surface is provided to limit or reduce non-specific binding of the sample to the walls or inner surface 230 of the column body 210. Without wishing to be bound by theory, it is believed that the alkylsilyl coating covering the metal surface prevents or minimizes contact between the fluid passing through the column body 210 and the inner surface 230. The alkylsilyl coating can be applied to the inner surface 230 of the metal column body 210, thereby defining the so-called wetted path of the column. The metal wetted path includes all surfaces formed of metal that are exposed to the fluid during operation of the chromatographic column. The metal wetted path includes not only the column body walls, but also metal frits disposed within the column. In embodiments, the alkylsilyl coating is applied not only to the walls of the column body 210, but also to the frits.

[0084] Generally, the alkylsilyl coating is applied by a vapor deposition technique. Vaporized precursors are loaded into a reactor in which the component to be coated is located. These vaporized precursors react on the surface of the component to be coated to form a first layer of deposition material. Vapor deposition can be applied in a step function to apply multiple layers of deposition material to the surface to increase the thickness of the coating and / or to apply layers of different materials (e.g., alternating between a first material and a second material) to form the coating.

[0085] In some embodiments, the alkylsilyl coating is applied to other parts of the liquid chromatography system. For example, the alkylsilyl coating can be applied to metal components located upstream and downstream of the column. In particular, the alkylsilyl coating can be applied to the injector of the liquid chromatography system, and to the post-column tubing and connectors (e.g., tubing and connectors leading from the column to downstream components, such as detectors). Furthermore, the affinity chromatographic columns of the present technology do not require the addition of additional organic modifiers to reduce non-specific binding. Typically, for the use of adsorbents in affinity chromatography, it can be necessary to add an organic modifier (e.g., acetonitrile) to reduce non-specific binding. Since the non-specific binding of the columns of the present technology is already low, no organic modifier is required.

[0086] In one embodiment, the alkylsilyl coating comprises a hydrophilic non-ionic layer of polyethylene glycol silane. In another embodiment, the alkylsilyl coating is formed from one or more of the following precursor materials: bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane. Other embodiments of alkylsilyl coatings suitable for use with the present technology are described in U.S. Patent Publication No. 2019 / 0086371 and U.S. Application Publication No. 2022 / 0118443.

[0087] The dimensions of the chromatography apparatus of the present technology can be suitably set for use in an HPLC system, a UHPLC system, or a FPLC (fast protein liquid chromatography) system. For example, in an embodiment in which the average particle size of a plurality of particles packed into a column is 3.5 microns or 1.7 microns, the column body of the present technology can have an inner diameter between 1 mm and 4.6 mm and a column length between 5 cm and 50 cm. In certain embodiments, the column body has an inner diameter between 1 mm and 2.1 mm and a length between 15 cm and 50 cm.

[0088] Method using affinity chromatography column

[0089] The resulting affinity chromatography column of the present technology can be used to perform affinity capture of antibodies that bind to a specific immunoglobulin binding protein conjugated to the particles of Figure 1B The affinity chromatography column is suitable for use in conjunction with any liquid chromatography system as described above. In some embodiments, the liquid chromatography system is an HPLC, UHPLC, or FPLC system.

[0090] Figure 3 An overview of a method (300) of performing affinity capture of antibodies using a chromatography column (305) having a plurality of particles functionalized with an immunoglobulin binding protein (225) is provided. The column (305) is connected to a liquid chromatography system (310), and a solution containing antibodies (315) is flowed onto the column. Due to the binding affinity of the antibodies to the immunoglobulin binding protein conjugated to the particles (320), the antibodies are immobilized on the column. This can be reversed by changing the environment such that the binding affinity is reduced, for example by making the pH acidic. In doing so, the antibodies are no longer immobilized and will elute from the column.

[0091] The examples described below use three reference columns, referred to herein and throughout as Reference Column 1, Reference Column 2, and Reference Column 3.

[0092] Reference Column 1 is a polymer-based Protein A monolith column in a 5.6 mm x 5 mm stainless steel column.

[0093] Reference Column 2 comprises Protein A conjugated to 12 micron non-porous polystyrene / divinylbenzene (PS / DVB) particles in a 4 mm x 35 mm polyether ether ketone column.

[0094] Reference Column 3 comprises Protein A conjugated to 20 micron fully porous (500 Angstrom to 1000 Angstrom pore) polystyrene / divinylbenzene (PS / DVB) particles in a 2.1 mm x 30 mm polyether ether ketone column.

[0095] Example 3 describes a method of performing an affinity capture assay using an affinity chromatography column prepared with Protein A-functionalized particles. The column is used to bind and elute a range of concentrations of purified IgG. This is then compared to Reference Column 1 using similar conditions and samples.

[0096] Figure 4A A comparison of peak volumes for the experiments described in Example 3 for IgG between 0.25 pg and 20 pg is shown. Figure 4B A comparison of peak volumes for the experiments described in Example 3 for IgG between 0.25 pg and 300 pg is shown. The affinity chromatography column of the present technology provides better peak volumes and enables more robust titration calculations compared to Reference Column 1.

[0097] Example 4 describes a method of performing an affinity capture assay using an affinity chromatography column prepared with Protein A-functionalized particles. The column is used to bind and elute a range of concentrations of purified IgG. This is then compared to Reference Column 2 and Reference Column 3 using similar samples and supplier recommended protocols. Figure 5A It is shown that the column of the present technology provides significantly improved peak shape and width relative to the reference columns. Figure 5B It is shown that the column of the present technology results in significantly reduced carryover between affinity capture cycles. The affinity chromatography column of the present technology provides better peak volumes and enables more robust titration calculations relative to the reference columns. The column of the present technology results in a >5-fold increase in sensitivity, reaching detector saturation at an IgG injection amount of 25 pg due to the enhanced sensitivity of the column.

[0098] Furthermore, as described in Example 5, the column of the present technology allows for faster affinity capture of IgG than columns available in the art without sacrificing sensitivity or performance. Figure 6A It is shown that the column of the present technology allows for affinity capture of IgG using a 1 minute binding phase, which results in comparable peak area ( Figure 6B ) and carryover ( Figure 6C ) to a 3 minute binding phase. Thus, the column of the present technology allows for rapid determination of IgG titers using a binding phase time of about 1 minute.

[0099] Buffer solutions suitable for liquid chromatography are well known in the art. It will be appreciated that a range of binding buffers are suitable for use with the disclosed technology, and that one of ordinary skill in the art can determine, without undue experimentation, an appropriate binding buffer suitable for a given antibody or sample. In one aspect, the pH of the binding buffer is between 7.0 and 8.0. In some embodiments, the pH of the binding buffer is between 7.0 to 7.1, 7.1 to 7.2, 7.2 to 7.3, 7.3 to 7.4, 7.4 to 7.5, 7.5 to 7.6, 7.6 to 7.7, 7.7 to 7.8, 7.8 to 7.9, or 7.9 to 8.0. In one aspect, the pH of the elution buffer is between 1.0 and 3.5. In some embodiments, the pH of the elution buffer is between 1.0 to 1.2, 1.2 to 1.4, 1.4 to 1.6, 1.6 to 1.8, 1.8 to 2.0, 2.0 to 2.2, 2.2 to 2.4, 2.4 to 2.6, 2.6 to 2.8, 3.0 to 3.2, 3.2 to 3.4, or 3.4 to 3.5. Importantly, the pH of the elution buffer is at least 3 orders of magnitude more acidic than the binding buffer.

[0100] In some embodiments, the binding buffer comprises 20 mM to 100 mM sodium phosphate with 125 mM NaCl (pH 7.4). In some embodiments, the binding buffer comprises 100 mM sodium phosphate buffer (pH 7.4). In some embodiments, the binding buffer comprises 20 mM to 200 mM ammonium acetate (pH 7.0 to 8.0). In some embodiments, the binding buffer comprises 20 mM to 200 mM ammonium formate (pH 7.0 to 8.0). In some embodiments, the binding buffer is compatible with mass spectrometry.

[0101] In some embodiments, the elution buffer comprises 20 mM to 50 mM sodium phosphate (pH 2.3). In some embodiments, the elution buffer comprises 20 mM to 50 mM sodium phosphate with 100 mM to 500 mM NaCl (pH 2.3). In some embodiments, the elution buffer comprises 6 mM hydrochloric acid (pH 2.5). In some embodiments, the elution buffer comprises 24 mM phosphoric acid (pH 1.93 to 2.05). In some embodiments, the elution buffer comprises 100 mM glycine (pH 2.5). In some embodiments, the elution buffer is an aqueous solution of 0.1% to 2% acetic acid (pH 1.0 to 3.0). In some embodiments, the elution buffer is an aqueous solution of 0.1% formic acid (pH 1.0 to 3.0). In some embodiments, the elution buffer is compatible with mass spectrometry.

[0102] In some embodiments, the target analyte is eluted from the column using step elution, in which the mobile phase is switched from the binding buffer to the elution buffer. In some embodiments, the target analyte is eluted from the column using gradient elution, in which the binding buffer is transitioned to the elution buffer in a gradient.

[0103] In one aspect, the affinity chromatography columns of the present technology provide elution of immunoglobulins with small peak volumes. In some embodiments, the peak volume is less than 50 µL. In some embodiments, the peak volume is less than 40 µL, less than 30 µL, or less than 20 µL. In some embodiments, the peak volume is less than 10 µL.

[0104] In one aspect, the affinity chromatography columns of the present technology are capable of being reused for affinity capture assays. In this regard, after elution of the antibody from the column, the column is then re-equilibrated. Re-equilibration involves flowing a buffer through the column such that any residual material from the previous assay is removed. In some embodiments, re-equilibration is performed with the binding buffer. In some embodiments, re-equilibration is performed with the equilibration buffer. The pH of the equilibration buffer can be equal to or lower than the binding buffer. Due to the relationship between pH and protein conformational stability and folding, it can be desirable to immediately neutralize the eluted sample with a base.

[0105] In one aspect, the present technology allows for rapid re-equilibration of the affinity chromatography column. In some embodiments, the affinity chromatography column is re-equilibrated for 1 minute to 10 minutes, 1 minute to 2 minutes, 2 minutes to 3 minutes, 3 minutes to 4 minutes, 4 minutes to 5 minutes, 5 minutes to 6 minutes, 6 minutes to 7 minutes, 7 minutes to 8 minutes, 8 minutes to 9 minutes, or 9 minutes to 10 minutes. In some embodiments, it can be desirable to re-equilibrate the chromatography column for a longer period of time.

[0106] After re-equilibration, the affinity chromatography column is ready to be reused for affinity capture assays. The affinity chromatography columns of the present technology can be reused 10 times to 20 times, 20 times to 30 times, 30 times to 40 times, 40 times to 50 times, 50 times to 60 times, 60 times to 70, 70 times to 80 times, 80 times to 90 times, 90 times to 100 times, 100 times to 110 times, 110 times to 120 times, 120 times to 130 times, 130 times to 140 times, 140 times to 150 times, 150 times to 160 times, 160 times to 170 times, 170 times to 180 times, 180 times to 190 times, 190 times to 200 times, or 200 times or more without decay or significant loss of specificity. In one aspect, the affinity chromatography columns of the present technology can be stored intermittently between uses. Buffers suitable for storing affinity chromatography columns are well known in the art.

[0107] In one aspect, the affinity chromatography columns of the present technology result in a significant reduction in carryover of samples applied after the re-equilibration process.

[0108] Figure 4C and Figure 5B The affinity chromatography columns of the present technology were shown to reduce carryover between uses to 1 / 5 to 1 / 2 compared to reference columns 1, 2, and 3.

[0109] Method and examples

[0110] Example 1 : Addition of an epoxy linker to hydrophilic particles

[0111] Non-porous epoxy-modified hydrophilic particles for use in the disclosed methods were prepared as follows. As a first step, 1500 g of reagent alcohol (90% ethanol, about 5% methanol, and about 5% isopropanol), 45.1 g of polyvinylpyrrolidone (PVP-40), 4.8 g of 2,2'-azobis(2-methylpropionitrile), 5.9 g of Triton N-57, and 81.7 g of styrene were charged to a reactor. After purging the reactor with nitrogen, the reaction mixture was heated to 70 °C and maintained at that temperature for 3 hours with stirring. ™ N-57, 81.7 g of styrene were charged to a reactor. After purging the reactor with nitrogen, the reaction mixture was heated to 70 °C and maintained at that temperature for 3 hours with stirring.

[0112] After three hours, a solution containing 110.4 g of divinylbenzene 80 (DVB), 39.7 g of PVP-40, 510 g of reagent alcohol, and 100.2 g of p-xylene was added to the reaction mixture at a constant flow rate over two hours. After this step, a primer coating solution containing 26.0 g of glycidyl methacrylate (GMA), 26.0 g of ethylene glycol dimethacrylate (EDMA), 36.4 g of PVP-40, and 560 g of reagent alcohol was added to the reaction mixture at a constant flow rate over 1.5 hours.

[0113] The reaction mixture was maintained at 70 °C for a total of 20 hours, after which the particles were isolated from the reaction slurry by filtration. The particles were then washed sequentially with methanol, tetrahydrofuran (THF), and acetone. The final product was dried in a vacuum oven at 45 °C to yield monodisperse 3.5 μιη polymer particles. These particles contained a gradient polystyrene / DVB core with a poly(GMA / EDMA) primer. While the above reaction conditions produced 3.5 μιη polymer particles, it will be understood that particles ranging in size from 1.5 μιη to 8 μιη are within the scope of the present disclosure. By varying the concentrations of PVP-40, 2,2'-azobis(2-methylbutyronitrile), and Triton N-57, one of ordinary skill in the art can produce a range of particle sizes.

[0114] The 3.5 μιη resulting polystyrene / DVB particles with poly(GMA / EDMA) primer were then coated with a hydrophilic layer. 70 g of particles were hydrolyzed in 0.5 M H2SO4 at 60 °C for 1 to 20 hours. The hydrolyzed particles were washed sequentially with MilliQ water and methanol, and then dried under vacuum at 45 °C overnight. The dried particles were added to a 1 L three-necked round bottom flask with an overhead stirring motor, stirring shaft and stirring blade, water-cooled condenser, nitrogen inlet, and probe-controlled heating mantle. 700 mL of anhydrous diglyme was added, the flask was sealed, and purged with nitrogen for 15 minutes with moderate stirring. 2.0 g of potassium tert-butoxide was added, and the reaction was warmed to 70 °C. To create the hydrophilic layer, a mixture of 10.5 g of glycidol, 2.6 g of glycerol triglycidyl ether, and 14.9 g of anhydrous diglyme was prepared separately and added to the particle mixture in four equal aliquots at 30 minute intervals. The reaction was held at 70 °C for 20 hours, cooled to room temperature, and filtered. The resulting particles were washed sequentially with water six times, with methanol three times, and then dried under vacuum at 45 °C overnight. The following procedure created a hydrophilic layer that was 2% to 4% (by weight) of the overall particle.

[0115] At room temperature, 20 g of 3.5 μιη resulting particles with the hydrophilic coating were added to a mixture of 100 g of ethylene glycol diglycidyl ether (EGDGE) and 100 g of MeOH. 1 mL of a 50% aqueous solution of sodium hydroxide was added, and the reaction was continuously stirred for 20 hours. The particles were isolated by filtration, washed with 400 mL of MeOH ten times, and partially dried under a stream of nitrogen. The particles were stored in a methanol wet bed at 4 °C for later use. The resulting particles had sufficient epoxide content to enable functionalization of the particle surface.

[0116] Example 2: Protein A functionalised particles

[0117] Particles were prepared and functionalized with Protein A as described in Example 1. 1.5 g of particles were mixed in 8.3 mL of 50 mM sodium phosphate buffer (pH 8). To this was added 0.3 mL of a 50 mg / mL solution of Protein A (15 mg). Next, 21.4 mL of a sodium phosphate / ammonium sulfate solution was added dropwise. The reaction was then stirred for 20 hours between 24 °C and 37 °C. The final concentration of ammonium sulfate and the reaction temperature can be adjusted to modulate the extent of Protein A coverage on a given particle.

[0118] After 20 hours of incubation, 1 g of ethanolamine in 5 mL of sodium phosphate buffer can be added and the reaction stirred for 3 hours at room temperature. The particles can then be isolated by filtration and sequentially washed three times with water (pH 4, adjusted with HC1), twice with water, and twice with storage buffer (20 mM PBS, pH 7.3, 0.02% sodium azide). The particles can be stored as a slurry in storage buffer (about 10 mL buffer / g particles) in a sealed container at 4°C. The protein A coverage of the particles can be determined using a standard bicinchoninic acid assay (BCA). The maximum surface coverage of protein A on the particles is determined to be 4.2 pg to 5.0 pg protein A / mg particles. An exemplary protein A particle has a surface coverage of about 4.8 pg protein A / mg particles (particle type A).

[0119] Alternatively, particles prepared as described in Example 1 can be functionalized with protein G. 1.5 g of particles can be mixed in 8.3 mL of 50 mM sodium phosphate buffer (pH 8). To this can be added 0.3 mL of a 50 mg / mL solution of protein G (15 mg). Next, 21.4 mL of a sodium phosphate / ammonium sulfate solution can be added dropwise and the reaction stirred for 20 hours between 24°C and 37°C. The final concentration of ammonium sulfate and the reaction temperature can be adjusted to modulate the degree of protein G coverage on a given particle. After 20 hours of incubation, 1 g of ethanolamine in 5 mL of sodium phosphate buffer can be added and the reaction stirred for 3 hours at room temperature. The particles can be isolated by filtration and sequentially washed three times with water (pH 4, adjusted with HC1), twice with water, and twice with storage buffer (such as 20 mM PBS, pH 7.3, 0.02% sodium azide). The protein G coverage of the particles can be determined using methods known in the art, such as a standard BCA assay.

[0120] Alternatively, the particles prepared as described in Example 1 can be functionalized with protein A / G. 1.5 g of particles are mixed in 8.3 mL of 50 mM sodium phosphate buffer (pH 8). To this, 0.3 mL of a 50 mg / mL solution of protein A / G (15 mg) is added. Next, 21.4 mL of a sodium phosphate / ammonium sulfate solution can be added dropwise, and the reaction is stirred for 20 hours between 24°C and 37°C. The final concentration of ammonium sulfate and the reaction temperature can be adjusted to modulate the extent of protein A / G coverage on a given particle. After 20 hours of incubation, 1 g of ethanolamine in 5 mL of sodium phosphate buffer can be added, and the reaction is stirred for 3 hours at room temperature. The particles can be isolated by filtration and sequentially washed three times with water (pH 4, adjusted with HC1), twice with water, and twice with a storage buffer such as 20 mM PBS, pH 7.3, 0.02% sodium azide. The protein A / G coverage of the particles can be determined using methods known in the art, such as a standard BCA assay.

[0121] Alternatively, the particles prepared as described in Example 1 can be functionalized with protein L. 1.5 g of particles are mixed in 8.3 mL of 50 mM sodium phosphate buffer (pH 8). To this, 0.3 mL of a 50 mg / mL solution of protein L (15 mg) is added. Next, 21.4 mL of a sodium phosphate / ammonium sulfate solution can be added dropwise, and the reaction is stirred for 20 hours between 24°C and 37°C. The final concentration of ammonium sulfate and the reaction temperature can be adjusted to modulate the extent of protein L coverage on a given particle. After 20 hours of incubation, 1 g of ethanolamine in 5 mL of sodium phosphate buffer can be added, and the reaction is stirred for 3 hours at room temperature. The particles can be isolated by filtration and sequentially washed three times with water (pH 4, adjusted with HC1), twice with water, and twice with a storage buffer such as 20 mM PBS, pH 7.3, 0.02% sodium azide. The protein L coverage of the particles can be determined using methods known in the art, such as a standard BCA assay.

[0122] Example 3: Binding of IgG to a protein A affinity chromatography column

[0123] Affinity capture of IgG is performed using the particles prepared in Example 2. The protein A-functionalized particles are packed in a 2.1 mm x 20 mm hardware. This is connected to a liquid chromatograph and equilibrated with 20 mM sodium phosphate (pH 7.4) and 125 mM sodium chloride at a flow rate of 1 mL / minute for 5 minutes.

[0124] Once equilibrated, a 10 pg / mL IgG was injected in a 10 pL injection volume onto the column and allowed to bind for 0.4 minutes at a flow rate of 2 mL / min. The column effluent was monitored using a UV detector (280 nm). Following the 0.4 binding phase, elution buffer (20 mM sodium phosphate (pH 2.7)) was flowed onto the column at a flow rate of 2 mL / min for 0.4 minutes. Following this, the column was re-equilibrated with 20 mM sodium phosphate (pH 7.4) and 125 mM sodium chloride at a flow rate of 2 mL / min for 0.4 minutes.

[0125] Following re-equilibration of the column, a 10 pg / mL IgG was injected in a 5 pL injection volume onto the column and the binding, elution, and equilibration processes were repeated as described above. This was repeated using volumes that resulted in total concentrations of 0.5 pg, 1 pg, 3 pg, 5 pg, 10 pg, 20 pg, 50 pg, 75 pg, 100 pg, 300 pg, and 500 pg of IgG. As a comparison, this was performed using Reference Column 1 under similar conditions.

[0126] The Protein A column of the present technology (black solid line) resulted in improved peak area relative to Reference Column 1 (dashed line) Figures 4A to 4B and further allowed robust antibody titer calculation. The Protein A column of the present technology resulted in significantly reduced carryover of target antibody after the elution step and equilibration step (reduced to 1 / 3 to 1 / 2) compared to Reference Column 1 Figure 4C .

[0127] Example 4: Binding of IgG to a protein A affinity chromatography column

[0128] Affinity capture of IgG was performed using the particles prepared in Example 2. The Protein A-functionalized particles were packed in 2.1 mm x 20 mm hardware. This was connected to a liquid chromatograph and equilibrated with binding buffer (100 mM sodium phosphate (pH 7.4)) at a flow rate of 1 mL / min for 10 minutes. The binding buffer can be sodium phosphate (pH 7.4) in the range of 20 mM to 100 mM with 125 mM sodium chloride or 100 mM sodium phosphate. As a reference, Reference Column 3 (also referred to herein as particle type G) and Reference Column 2 were used. The Reference Column 3 column was equilibrated with 20 mM to 100 mM sodium phosphate (pH 7.4) with 120 mM to 150 mM NaCl. The Reference Column 2 column was equilibrated with 50 mM sodium phosphate (pH 7.4), 150 mM NaCl, and 5% acetonitrile.

[0129] Once equilibrated, a 10 μL injection of 10 μg / mL IgG was injected onto the column and flowed for 1 minute at a flow rate of 1 mL / minute to allow for binding. The column effluent was monitored using a UV detector (280 nm). Following the 1 minute binding phase, elution buffer (24 mM phosphoric acid (pH 1.93)) was flowed onto the column at a flow rate of 1 mL / minute for 1 minute. Following this, the column was re-equilibrated with 100 mM sodium phosphate (pH 7.4) at a flow rate of 1 mL / minute for 10 minutes.

[0130] Following re-equilibration of the column, different concentrations of IgG were injected onto the column in 10 μL injections and the binding, elution and equilibration processes repeated as described above. This was performed using different concentrations of IgG resulting in total IgG concentrations of 0.5 μg, 1 μg, 2.5 μg, 5 μg, 10 μg and 25 μg of IgG. As the sensitivity of the Protein A column increased, concentrations of IgG above 25 μg resulted in saturation of the UV detector.

[0131] As a comparison, this process was performed using reference column 2 and reference column 3 with similar samples. For the reference column 3 column, 10 μL of 10 μg / mL IgG was injected onto the column and flowed for 0.6 minutes at a flow rate of 1 mL / minute. The column effluent was monitored using a UV detector (280 nm). Following the binding phase, elution buffer (50 mM sodium phosphate, pH 2.3) was flowed onto the column at 1 mL / minute for 1.2 minutes. The column was re-equilibrated with binding buffer for 2.4 minutes. For the reference column 2 column, 10 μL of 10 μg / mL IgG was injected onto the column and flowed for 0.6 minutes at a flow rate of 1 mL / minute. The column effluent was monitored using a UV detector (280 nm). Following the binding phase, elution buffer (50 mM sodium phosphate, pH 2.3, 150 mM NaCl, 5% acetonitrile) was flowed onto the column at 1 mL / minute for 1.2 minutes. The column was re-equilibrated with binding buffer for 2.4 minutes. This process was repeated using injection volumes resulting in total IgG amounts of 0.5 μg, 1 μg, 2.5 μg, 5 μg, 10 μg, 25 μg, 50 μg, 75 μg and 100 μg of IgG. Unlike the column of the present technology, no saturation of the UV detector was observed on reference column 2 and reference column 3 even at the highest concentrations tested.

[0132] The Protein A column of the present technology (black dashed line) produced an improved peak shape and width (i.e. peak area) relative to reference column 3 (dashed line) and reference column 2 (black solid line) Figure 5A and further allowed robust antibody titer calculations. The Protein A column of the present technology resulted in a significant reduction (one order of magnitude) in the residual target antibody after the elution and equilibration steps relative to the reference columns Figure 5BCompared to the reference column, the protein A column of this technique further results in a significant increase in sensitivity (>5-fold), and the peak width is reduced by an order of magnitude after the elution and equilibration steps compared to reference column 3.

[0133] Example 5: Protein A column provides rapid affinity capture of IgG

[0134] IgG affinity capture was performed using the particles prepared in Example 2. Protein A-functionalized particles were packed into a 2.1 mm × 20 mm hardware. The column was connected to a liquid chromatograph and equilibrated for 10 min at a flow rate of 0.5 mL / min with binding buffer (100 mM sodium phosphate (pH 7.4)). Once equilibrated, 10 μL of 10 μg / µL IgG was injected onto the column and flowed at 1 mL / min for 1 min to allow binding. The column effluent was monitored using a UV detector (280 nm). The captured IgG was eluted with elution buffer (100 mM glycine (pH 2.5)) at a flow rate of 1 mL / min for 0.5 min. After this, the column was reequilibrated with binding buffer at a flow rate of 1 mL / min for 0.5 min. After reequilibration, 10 μL of different concentrations of IgG were injected onto the column, and the binding, elution, and equilibration processes were repeated as described above. This process was performed using concentrations that produced total IgG amounts of 0.5 μg, 1 μg, 2.5 μg, 5 μg, 10 μg, and 25 μg. For comparison, these processes were also repeated using a binding phase in which IgG was injected onto the column and flowed at a flow rate of 1 mL / min for 3 minutes to allow binding.

[0135] like Figure 6A As shown, the 1-minute (solid black line) and 3-minute (dashed black line) combined phases both produce similar peak widths and sensitivities. Figures 6B to 6C As shown, peak area ( Figure 6B ) and residues ( Figure 6C The results were similar across the different binding phase times tested. Therefore, the protein A column of this technique can effectively capture IgG using a 1-minute binding phase.

[0136] Example 6: Additional protein A functionalised materials

[0137] Non-porous silica particles with hybrid coating, diol coating and hydrophilic coating - particle type B

[0138] Nonporous epoxy-modified hydrophilic particles based on silica were prepared. Nonporous 3.6 micron silica particles were heat treated (900 °C) in air for 10 hours. The surface of the particles was rehydroxylated using 10% v / v nitric acid at 100 °C for 16 hours. The reaction was then allowed to cool to below 40 °C and the particles were isolated via filtration. The product was washed with water until the pH of the filtrate was greater than 5, after which the product was washed with acetone 3 times. The isolated particles were dried in a vacuum oven at 80 °C overnight.

[0139] The hydroxylated silica particles were coated with 1,2-bis(triethoxysilyl)ethane (BTEE) and tetraethyl orthosilicate (TEOS). The silane reagent was first prepared from incomplete (about 68%) hydrolysis condensation of BTEE with tetraethyl orthosilicate (TEOS). Ethanol (3.1 mol ethanol / mol silane), TEOS (molar ratio to BTEE of 1:4), and 0.1 M HC1 (19.7 g / mol silane reagent) were added to BTEE. The solution was heated at 70 °C under inert atmosphere for 18 hours. The reaction was warmed to 90 °C to remove ethanol. The reaction was further warmed to 100 °C under inert atmosphere for 1 hour. The mixture was cooled to room temperature (RT) to obtain the condensation product.

[0140] The silica particles were fully dispersed in toluene (21 mL / g particles). Residual water was removed from the material by azeotropic distillation (110 °C for 1 hour). The reaction was maintained at 40 °C while the silane reagent (0.821 g / g particles) was added and stirred for 10 minutes. Catalytic aqueous NH4OH (0.05 g / g particles) was added, stirred for an additional 10 minutes at 40 °C, and an additional 2 hours at 60 °C. The reaction was allowed to cool to room temperature and the particles were isolated via filtration. The particles were washed twice with ethanol (10 mL / g) and then dispersed in 70:30 water:ethanol (v / v) at 10 mL / g particles. Ammonium hydroxide solution (1 g / g particles) was added and the mixture was stirred at 50 °C for 2 hours. The reaction was allowed to cool to below 40 °C and the particles were isolated via filtration. The isolated particles were washed twice with 1:1 methanol:water (v / v) at 10 mL / g particles and twice with methanol. The surface-modified particles were vacuum dried at 70 °C for 16 hours.

[0141] To ensure uniformity of the hybrid coating, the modified particles were exposed to elevated temperature (100 °C to 140 °C) and elevated pH (8 to 9.8) after the hydrothermal treatment process according to the methods described in U.S. Patent Nos. 6,686,035, 7,223,473, and 7,919,177, and International Patent Publication No. WO 2008 / 103423, each of which is incorporated herein by reference.

[0142] The modified particles were dispersed in 1 M HC1 (8.4 mL / g of particles) and the mixture was stirred at 100 °C for 20 hours. The reaction was allowed to cool to below 40 °C and the particles were isolated via filtration. The isolated particles were washed with water until the pH of the filtrate was above 5, after which the particles were washed with acetone 3 times. The isolated surface modified particles were vacuum dried at 70 °C for 16 hours.

[0143] The resulting 3.6 micron particles were then coated with glycidyloxypropyltrimethoxysilane (GPTMS). 20 mM sodium acetate buffer (pH 5.5) (about 5 mL / g of particles) was added to a round bottom flask equipped with a thermometer, condenser, and mechanical stirring apparatus. The solution was heated to 70 °C, after which GPTMS was added to the flask at a concentration of 10.22 μmol / m 2 of particles and mixed at this temperature. After 1 hour, the particles were added to the flask (1 g of particles / 5 mL of buffer solution). The mixture was stirred at 70 °C for 20 hours, after which the reaction was cooled to below 40 °C and filtered. The product was washed with water 3 times and transferred to a new flask equipped with a thermometer, condenser, and mechanical stirring apparatus. Acetic acid (0.1 M) was added to the flask (5 mL / g of particles) and the slurry was heated to 70 °C for 20 hours. The flask was cooled to below 40 °C and filtered. The product was washed with water until the pH of the supernatant increased to above 5 and then washed with methanol 3 times. The isolated particles were placed in a vacuum oven at 70 °C to dry for 16 hours.

[0144] Ten grams of the resulting particles were added to a 1 L three necked round bottom flask with an overhead stirring motor, stirring shaft, stirring blade, water cooled condenser, nitrogen inlet, and probe control heating mantle. 100 mL of anhydrous diglycol dimethyl ether (diglyme) was added. The flask was sealed, purged with nitrogen for 15 minutes with moderate stirring. 0.45 g of potassium t-butoxide was added and the reaction was warmed to 70 °C. To create the hydrophilic layer, a mixture of 1.5 g of glycidol, 0.4 g of glycerol triglycidyl ether, and 2.2 g of anhydrous diglycol dimethyl ether was prepared separately and added to the particle mixture in 4 equal aliquots using 30 minute intervals. The reaction was held at 70 °C for 20 hours, cooled to room temperature, and filtered. The resulting particles were washed with water 6 times in succession, washed with methanol 3 times, and dried under vacuum at 45 °C overnight. This process created a hydrophilic layer that was 2% to 4% by weight of the entire particle.

[0145] At room temperature, 5 g of the resulting 3.6 micron particles with a hydrophilic coating were added to a mixture of 25 g ethylene glycol diglycidyl ether (EGDGE) and 25 g methanol. 0.25 mL of a 50% aqueous sodium hydroxide solution was added and the reaction was continuously stirred for 20 hours. The particles were isolated by filtration, washed with methanol 10 times, and partially dried under a stream of nitrogen. The particles were stored in a methanol wet bed at 4 °C. The resulting particles had sufficient epoxide content to enable functionalization of the particle surface.

[0146] The particles were functionalized with Protein A as described in Example 2, resulting in approximately 8.21 pg Protein A / mg of particles.

[0147] The particles were tested for their ability to bind and elute a monoclonal antibody (NIST mAb standard, purchased from Sigma-Aldrich). The particles were packed in a 2.1 mm x 200 mm column body and connected to an ultra-high performance liquid chromatography system (Acquity Premier, purchased from Waters Technologies Corporation, Milford MA). The resulting column was flushed at 0.5 mL / min with water (pH 4) for 10 minutes to remove unbound Protein A. The column was then equilibrated in 0.1 M sodium phosphate (pH 7.5) at 0.5 mL / min for 10 minutes. Different concentrations (ranging from 0.001 pg / pL to 1 pg / pL) of the monoclonal antibody standard were injected onto the column using 0.1 M sodium phosphate (pH 7.4) buffer at a flow rate of 1 mL / min in 10 pL injection volumes. Between samples, a blank injection of binding buffer was used to calculate carryover. Elution was performed with 24 mM phosphoric acid (pH 2) or 100 mM glycine (pH 2.4) for 1 minute. The column was equilibrated with 0.1 M sodium phosphate buffer (pH 7.4) for 1 minute.

[0148] As Figure 7A (24 mM phosphoric acid (pH 2)) and Figure 7B (100 mM glycine (pH 2.4)), poor peak shape, fronting, and shoulder peaks were observed due to Protein A leaching from the column. Significant peak tailing was present for both conditions due to non-specific binding of the monoclonal antibody sample. Thus, even with a hybrid material, a diol, and a hydrophilic layer coating the silica surface, high non-specific binding by the silica particles was still observed. Low detection limits of 0.025 pg and 0.05 pg were observed for the phosphoric acid and glycine elution buffers, respectively. At low concentrations, >40% carryover was observed due to Protein A leaching. At higher concentrations, >6% carryover was observed. Figure 7C Linear correlations between monoclonal antibody concentration and peak area are shown for both elution methods.

[0149] Non-porous silica particles with hybrid coating and diol coating (particle type D)

[0150] Non-porous silica-based epoxy-modified hydrophilic particles were prepared. Non-porous 3.6 micron silica particles were heat treated (900 °C) in air for 10 hours. The surface of the particles was re-hydroxylated using 10% v / v nitric acid at 100 °C for 16 hours. The reaction was then allowed to cool to below 40 °C and the particles were isolated via filtration. The product was washed with water until the pH of the filtrate was greater than 5, after which the product was washed with acetone three times. The isolated particles were dried in a vacuum oven at 80 °C overnight.

[0151] The hydroxylated silica particles were coated with 1,2-bis(triethoxysilyl)ethane (BTEE) and tetraethyl orthosilicate (TEOS). The silane reagent was first prepared from the incomplete (about 68%) hydrolysis condensation of BTEE with tetraethyl orthosilicate (TEOS). Ethanol (3.1 mol ethanol / mol silane), TEOS (molar ratio to BTEE of 1:4), and 0.1 M HC1 (19.7 g / mol silane reagent) were added to BTEE. The solution was heated at 70 °C under inert atmosphere for 18 hours. The reaction was warmed to 90 °C to remove ethanol. The reaction was further warmed to 100 °C for 1 hour under inert atmosphere. The mixture was cooled to room temperature (RT) to obtain the condensation product.

[0152] The silica particles were fully dispersed in toluene (21 mL / g particles). Residual water was removed from the material by azeotropic distillation (110 °C for 1 hour). The reaction was maintained at 40 °C while the silane reagent (0.821 g / g particles) was added and stirred for 10 minutes. Catalytic aqueous NH4OH (0.05 g / g particles) was added, stirred for an additional 10 minutes at 40 °C, and an additional 2 hours at 60 °C. The reaction was allowed to cool to room temperature and the particles were isolated via filtration. The particles were washed twice with ethanol (10 mL / g) and then dispersed in 70:30 water:ethanol (v / v) at 10 mL / g particles. Ammonium hydroxide solution (1 g / g particles) was added and the mixture was stirred at 50 °C for 2 hours. The reaction was allowed to cool to below 40 °C and the particles were isolated via filtration. The isolated particles were washed twice with 1:1 methanol:water (v / v) at 10 mL / g particles and twice with methanol. The surface-modified particles were vacuum dried at 70 °C for 16 hours.

[0153] To ensure uniformity of the hybrid coating, the modified particles were exposed to elevated temperature (100°C to 140°C) and elevated pH (8 to 9.8) after the hydrothermal treatment process according to the methods described in U.S. Patent Nos. 6,686,035, 7,223,473, and 7,919,177, and International Patent Publication No. WO 2008 / 103423, each of which is incorporated herein by reference.

[0154] The modified particles were dispersed in 1 M HC1 (8.4 mL / g of particles) and the mixture was stirred at 100°C for 20 hours. The reaction was allowed to cool to below 40°C and the particles were isolated via filtration. The isolated particles were washed with water until the pH of the filtrate was above 5, after which the particles were washed 3 times with acetone. The isolated surface modified particles were vacuum dried at 70°C for 16 hours.

[0155] The resulting 3.6 micron particles were then coated with glycidyloxypropyltrimethoxysilane (GPTMS). 20 mM sodium acetate buffer (pH 5.5) (about 5 mL / gram of particles) was added to a round bottom flask equipped with a thermometer, condenser, and mechanical stirring apparatus. The solution was heated to 70°C, after which GPTMS was added to the flask at a concentration of 10.22 μmol / m 2 After 1 hour, the particles were added to the flask (1 g of particles / 5 mL of buffer solution). The mixture was stirred at 70°C for 20 hours, after which the reaction was cooled to below 40°C and filtered. The product was washed 3 times with water and transferred to a new flask equipped with a thermometer, condenser, and mechanical stirring apparatus. Acetic acid (0.1 M) was added to the flask (5 mL / gram of particles) and the slurry was heated to 70°C for 20 hours. The flask was cooled to below 40°C and filtered. The product was washed with water until the pH of the supernatant increased to above 5, and then washed 3 times with methanol. The isolated particles were placed in a vacuum oven at 70°C to dry for 16 hours.

[0156] At room temperature, 5 g of the resulting 3.6 micron particles with a hydrophilic coating were added to a mixture of 25 g of ethylene glycol diglycidyl ether (EGDGE) and 25 g of methanol. 0.25 mL of 50% aqueous sodium hydroxide solution was added and the reaction was continuously stirred for 20 hours. The particles were isolated by filtration, washed 10 times with methanol, and partially dried under a stream of nitrogen. The particles were stored in a methanol wet bed at 4°C. The resulting particles had sufficient epoxide content to enable functionalization of the particle surface.

[0157] The particles were functionalized with Protein A as described in Example 2, resulting in approximately 1.84 μg of Protein A / mg of particles.

[0158] The ability of particles to bind and elute monoclonal antibodies (NISTmAb standards, purchased from Sigma-Aldrich) was tested. Particles were packed into a 2.1 mm × 200 mm column body and connected to an ultra-high performance liquid chromatography system (Acquity Premier, purchased from Waters Technologies Corporation, Milford, MA). The column was rapidly washed with water (pH 4) at 0.5 mL / min for 10 min to remove unbound protein A. The column was then equilibrated in 0.1 M sodium phosphate (pH 7.5) at 0.5 mL / min for 10 min. 10 μL of different concentrations (ranging from 0.001 μg / μL to 1 μg / μL) of monoclonal antibody standards were injected onto the column at a flow rate of 1 mL / min using 0.1 M sodium phosphate (pH 7.4) buffer. Residue was calculated using blank injections of binding buffer between samples. Elution was performed for 1 min with either 24 mM phosphate (pH 2) or 100 mM glycine (pH 2.4). Equilibrate the column for 1 minute using 0.1M sodium phosphate buffer (pH 7.4).

[0159] The detection limit is 0.01 μg for the phosphate elution method and 0.25 μg for the glycine elution method. (See phosphate elution method for details). Figure 8A In contrast, the retention time in the glycine elution method shifts (see...). Figure 8B The maximum pressure that can be applied is 2950 psi. Figure 8C A linear correlation between monoclonal antibody concentration and average peak area was shown for both elution methods. Residue from glycine elution was less than 3%, while residue from phosphate elution was <22%. Tailing was caused by the lack of a hydrophilic layer due to the non-specific binding of monoclonal antibodies to the particle surface.

[0160] Non-porous silica particles with diol coating (particle type H)

[0161] Non-porous epoxy-modified hydrophilic particles based on silica were prepared. Non-porous 3.6 μm silica particles were heat-treated in air (900 °C) for 10 h. The surface of the particles was then re-hydroxylated at 100 °C for 16 h using 10% v / v nitric acid. The reaction was then cooled to below 40 °C, and the particles were separated by filtration. The product was washed with water until the pH of the filtrate was greater than 5, and then washed three times with acetone. The separated particles were dried overnight in a vacuum oven at 80 °C.

[0162] The resulting 3.6 micron particles were then coated with glycidyloxypropyltrimethoxysilane (GPTMS). 20 mM sodium acetate buffer (pH 5.5) (about 5 mL / gram of particles) was added to a round bottom flask equipped with a thermometer, condenser, and mechanical stirring apparatus. The solution was heated to 70 °C, after which GPTMS was added to the flask at a concentration of 10.22 μmol / m 2 After 1 hour, the particles were added to the flask (1 g of particles / 5 mL of buffer solution). The mixture was stirred at 70 °C for 20 hours, after which the reaction was cooled to below 40 °C and filtered. The product was washed with water 3 times and transferred to a new flask equipped with a thermometer, condenser, and mechanical stirring apparatus. Acetic acid (0.1 M) was added to the flask (5 mL / gram of particles), and the slurry was heated to 70 °C for 20 hours. The flask was cooled to below 40 °C and filtered. The product was washed with water until the pH of the supernatant increased to above 5, and then washed with methanol 3 times. The isolated particles were placed in a vacuum oven at 70 °C to dry for 16 hours.

[0163] At room temperature, 5 g of the resulting 3.6 micron particles with a hydrophilic coating were added to a mixture of 25 g of ethylene glycol diglycidyl ether (EGDGE) and 25 g of methanol. 0.25 mL of 50% aqueous sodium hydroxide solution was added, and the reaction was continuously stirred for 20 hours. The particles were isolated by filtration, washed with methanol 10 times, and partially dried under a stream of nitrogen. The particles were stored in a methanol wet bed at 4 °C. The resulting particles had sufficient epoxide content to enable functionalization of the particle surface.

[0164] The particles were functionalized with Protein A as described in Example 2, resulting in approximately 2.7 μg of Protein A / mg of particles.

[0165] Test the ability of the particles to bind and elute a monoclonal antibody (NIST mAb standard, purchased from Sigma-Aldrich). The particles were packed in a 2.1 mm x 200 mm column body and connected to an ultra-high performance liquid chromatography system (Acquity Premier, purchased from Waters Technologies Corporation, Milford, MA). The column was flushed at 0.5 mL / min with water (pH 4) for 10 minutes to remove unbound protein A. The column was then equilibrated in 0.1 M sodium phosphate (pH 7.5) at 0.5 mL / min for 10 minutes. Different concentrations (ranging from 0.001 pg / pL to 1 pg / pL) of the monoclonal antibody standard were injected onto the column using 0.1 M sodium phosphate (pH 7.4) buffer at a flow rate of 1 mL / min in 10 pL injection volumes. A blank injection of the binding buffer was used between samples to calculate the carryover. Elution was performed with 24 mM phosphoric acid (pH 2) or 100 mM glycine (pH 2.4) for 1 minute. The column was equilibrated with 0.1 M sodium phosphate buffer (pH 7.4) for 1 minute.

[0166] As Figure 9A (24 mM phosphoric acid (pH 2)) and Figure 9B (100 mM glycine (pH 2.4)), significant peak tailing was observed for both elution methods. For phosphoric acid elution, peak splitting was observed from 0.25 pg / pL, while for glycine elution, peak splitting was observed from 1 pg / pL. The maximum pressure that can be applied is 2500 psi. Figure 9C Linear correlation between the monoclonal antibody concentration and the average peak area is shown for the glycine elution method. The carryover was >3% for both elution conditions. The lack of a hydrophilic layer due to non-specific binding of the monoclonal antibody on the particle surface resulted in peak distortion and significant tailing.

[0167] Non-porous silica particles with diol coating and hydrophilic coating (particle type E)

[0168] Prepare non-porous, silica-based, epoxy-modified hydrophilic particles. Non-porous 3.6 micron silica particles are heat treated (900 °C) in air for 10 hours. The surface of the particles is re-hydroxylated using 10% v / v nitric acid at 100 °C for 16 hours. The reaction is then allowed to cool to below 40 °C and the particles are isolated via filtration. The product is washed with water until the pH of the filtrate is greater than 5, after which the product is washed with acetone 3 times. The isolated particles are placed in a vacuum oven at 80 °C to dry overnight.

[0169] The resulting 3.6 micron particles were then coated with glycidyloxypropyltrimethoxysilane (GPTMS). 20 mM sodium acetate buffer (pH 5.5) (about 5 mL / gram of particles) was added to a round bottom flask equipped with a thermometer, condenser, and mechanical stirring apparatus. The solution was heated to 70 °C, after which GPTMS was added to the flask at a concentration of 10.22 μmol / m 2 After 1 hour, the particles were added to the flask (1 g of particles / 5 mL of buffer solution). The mixture was stirred at 70 °C for 20 hours, after which the reaction was cooled to below 40 °C and filtered. The product was washed with water 3 times and transferred to a new flask equipped with a thermometer, condenser, and mechanical stirring apparatus. Acetic acid (0.1 M) was added to the flask (5 mL / gram of particles), and the slurry was heated to 70 °C for 20 hours. The flask was cooled to below 40 °C and filtered. The product was washed with water until the pH of the supernatant increased to above 5, and then washed with methanol 3 times. The isolated particles were placed in a vacuum oven at 70 °C to dry for 16 hours.

[0170] Ten grams of the resulting particles were added to a 1 L three-necked round bottom flask with an overhead stirring motor, stirring shaft, stirring blade, water-cooled condenser, nitrogen inlet, and probe control heating mantle. 100 mL of anhydrous diglyme was added. The flask was sealed, purged with nitrogen for 15 minutes with moderate stirring. 0.45 g of potassium t-butoxide was added, and the reaction was warmed to 70 °C. To create the hydrophilic layer, a mixture of 1.5 g of glycidol, 0.4 g of glycerol triglycidyl ether, and 2.2 g of anhydrous diglyme was prepared separately and added to the particle mixture in 4 equal aliquots using 30 minute intervals. The reaction was held at 70 °C for 20 hours, cooled to room temperature, and filtered. The resulting particles were washed with water 6 times in succession, washed with methanol 3 times, and vacuum dried at 45 °C overnight. This process created a hydrophilic layer that was 2% to 4% of the entire particle by weight.

[0171] Five grams of the resulting 3.6 micron particles with the hydrophilic coating were added to a mixture of 25 g of ethylene glycol diglycidyl ether (EGDGE) and 25 g of methanol at room temperature. 0.25 mL of 50% sodium hydroxide aqueous solution was added, and the reaction was continuously stirred for 20 hours. The particles were isolated by filtration, washed with methanol 10 times, and partially dried under a stream of nitrogen. The particles were stored in a methanol wet bed at 4 °C. The resulting particles had sufficient epoxide content to enable functionalization of the particle surface.

[0172] The particles were functionalized with Protein A as described in Example 2, resulting in approximately 1.47 μg of Protein A / mg of particles.

[0173] Test the ability of the particles to bind and elute a monoclonal antibody (NIST mAb standard, purchased from Sigma-Aldrich). Fill the particles in a 2.1 mm x 200 mm column body and connect to an ultra-high performance liquid chromatography system (Acquity Premier, purchased from Waters Technologies Corporation, Milford, MA). Flush the column with water (pH 4) at 0.5 mL / min for 10 minutes to remove unbound protein A. Then equilibrate the column in 0.1 M sodium phosphate (pH 7.5) at 0.5 mL / min for 10 minutes. Use 0.1 M sodium phosphate (pH 7.4) buffer to inject 10 pL injection volume of different concentrations (ranging from 0.001 pg / pL to 1 pg / pL) of the mAb standard onto the column at a flow rate of 1 mL / min. Use a blank injection of the binding buffer between samples to calculate the carryover. Elute with 24 mM phosphoric acid (pH 2) or 100 mM glycine (pH 2.4) for 1 minute. Equilibrate the column with 0.1 M sodium phosphate buffer (pH 7.4) for 1 minute.

[0174] The limit of detection was 0.01 pg for the phosphoric acid elution method and 0.25 pg for the glycine elution method. There was a shift in the retention time in the glycine elution method compared to the phosphoric acid elution method (see Figure 10A ). The maximum pressure that can be applied was 3100 psi. Figure 10B The linear correlation between the monoclonal antibody concentration and the average peak area is shown for both elution methods. The carryover was less than 7% for both elution methods. The amount of protein A immobilized on the surface was very low, indicating that less epoxide was available for protein A conjugation. The high carryover and significant tailing indicate that the hydrophilic layer was not optimal. Figure 10C

[0175] In general, silica-based materials are not ideal for affinity chromatography due to the tendency of silica to have high levels of non-specific binding. The particle types described above (B, D, H, and E) indicate that masking the silica surface with a hybrid material layer, a glycol layer, and / or a hydrophilic coating is not sufficient to eliminate non-specific binding. Without wishing to be bound by any particular theory, silica-based particle types can also have lower protein A conjugation efficiency, which can result in high leaching.

[0176] Non-porous polymer particles with diol coating and hydrophilic coating (particle type C)

[0177] ​Non-porous polymer-based particles were prepared. 65.1 grams of polyvinylpyrrolidone (PVP-40), 8.9 grams of Triton N-57, 2452.6 grams of reagent alcohol (90% ethanol, 5% methanol, 5% isopropanol), and 153.3 grams of p-xylene were charged into a 4 L round bottle reactor equipped with mechanical stirring, condenser, and thermocouple. The mixed mixture was stirred at 200 rpm and the dissolved oxygen in the solution was removed by sub-surface purging with nitrogen. After the dissolved oxygen was below 1 ppm, 2.1 grams of azobisisobutyronitrile and 105 grams of divinylbenzene were charged into the reaction. The reaction was warmed to 70 °C and held for 20 hours.

[0178] After 1.8 grams of azobisisobutyronitrile was added to the reaction, a solution of 67.6 grams of divinylbenzene (DVB) and 20.3 grams of PVP-40 in 202.7 grams of reagent alcohol was metered in at a constant flow rate over 120 minutes via a pump. The reaction was continued at 70 °C for at least 12 hours. The particles were isolated from the reaction slurry by filtration and washed sequentially with methanol, tetrahydrofuran (THF), and acetone. The final product was dried in a vacuum oven at 45 °C overnight.

[0179] Polymer cores were dispersed in 231 grams of ethanol by ultrasonication for 4 minutes using an ultrasonic horn and transferred to a round bottom flask equipped with mechanical level stirring, condenser, and thermocouple. 49.1 grams of ammonium hydroxide and 462 grams of toluene were added to the reaction and mixed at room temperature for 60 minutes. The reaction was warmed to 35 °C and a mixture of 26.06 grams of TEOS and 66.34 grams of BTEE diluted with 148.9 grams of ethanol and 148.9 grams of toluene was charged via peristaltic pump at a constant flow rate of 0.95 mL / minute. After the reaction, the particles were isolated by filtration, washed 6 times with methanol, and dried in a vacuum oven at 45 °C overnight.

[0180] The resulting polymer core particles were dispersed in a 0.3 M aqueous solution of tris(hydroxymethyl)aminomethane at a slurry concentration of 5 mL / g. The pH was adjusted to 9.8 using acetic acid. The slurry was then sealed in a stainless steel autoclave and heated to 155 °C for 20 hours. After cooling to room temperature, the product was isolated on a 0.5 micron filter paper and washed with water and methanol. The particles were vacuum dried at 80 °C for 16 hours.

[0181] After the hydrothermal treatment, 50 g of the particles were refluxed in 1 M hydrochloric acid at a slurry concentration of 10 mL / g for 10 hours. The reaction was allowed to cool to room temperature and the particles were isolated via filtration. The particles were washed with water until the pH of the filtrate was greater than 5. The semi-neutralized particles were washed 3 times with acetone (10 mL / g). The resulting particles were fractionated to remove any agglomerates, resulting in a final particle size of 2.3 μm.

[0182] The resulting 2.3 micron particles were then coated with glycidyloxypropyltrimethoxysilane (GPTMS). 20 mM sodium acetate buffer (pH 5.5) (about 5 mL / gram of particles) was added to a round bottom flask equipped with a thermometer, condenser, and mechanical stirring apparatus. The solution was heated to 70 °C, after which GPTMS was added to the flask at a concentration of 10.22 μmol / m 2 After 1 hour, the particles were added to the flask (1 g of particles / 5 mL of buffer solution). The mixture was stirred at 70 °C for 20 hours, after which the reaction was cooled to below 40 °C and filtered. The product was washed with water 3 times and transferred to a new flask equipped with a thermometer, condenser, and mechanical stirring apparatus. Acetic acid (0.1 M) was added to the flask (5 mL / gram of particles), and the slurry was heated to 70 °C for 20 hours. The flask was cooled to below 40 °C and filtered. The product was washed with water until the pH of the supernatant increased to above 5, and then washed with methanol 3 times. The isolated particles were placed in a vacuum oven at 70 °C to dry for 16 hours.

[0183] Five grams of the resulting particles were added to a 1 L three-necked round bottom flask with an overhead stirring motor, stirring shaft, stirring blade, water-cooled condenser, nitrogen inlet, and probe control heating mantle. 50 mL of anhydrous diglycol dimethyl ether (diglyme) was added. The flask was sealed, purged with nitrogen for 15 minutes with moderate stirring. 0.224 g of potassium t-butoxide was added, and the reaction was warmed to 70 °C. To create the hydrophilic layer, a mixture of 0.75 g of glycidol, 0.19 g of glycerol triglycidyl ether, and 1.1 g of anhydrous diglycol dimethyl ether was prepared separately and added to the particle mixture in 4 equal aliquots using 30 minute intervals. The reaction was held at 70 °C for 20 hours, cooled to room temperature, and filtered. The resulting particles were washed with water 6 times in succession, washed with methanol 3 times, and vacuum dried at 45 °C overnight. This process created a hydrophilic layer that was 2% to 4% by weight of the entire particle.

[0184] Five grams of the resulting particles with the hydrophilic coating were added to a mixture of 25 g of ethylene glycol diglycidyl ether (EGDGE) and 25 g of methanol at room temperature. 0.25 mL of 50% sodium hydroxide aqueous solution was added, and the reaction was continuously stirred for 20 hours. The particles were isolated by filtration, washed with methanol 10 times, and partially dried under a stream of nitrogen. The particles were stored in a methanol wet bed at 4 °C. The resulting particles had sufficient epoxide content to enable functionalization of the particle surface.

[0185] The particles were functionalized with Protein A as described in Example 2, resulting in approximately 2.5 μg of Protein A / mg of particles.

[0186] Test the ability of the particles to bind and elute a monoclonal antibody (NIST mAb standard, purchased from Sigma-Aldrich). The particles are packed in a 2.1 mm x 200 mm column body and connected to an ultra-high performance liquid chromatography system (Acquity Premier, purchased from Waters Technologies Corporation of Milford, MA). The column is flushed at 0.5 mL / min with water (pH 4) for 10 minutes to remove unbound protein A. The column is then equilibrated in 0.1 M sodium phosphate (pH 7.5) at 0.5 mL / min for 10 minutes. A 10 pL injection of different concentrations (ranging from 0.001 pg / pL to 1 pg / pL) of the monoclonal antibody standard is injected onto the column using 0.1 M sodium phosphate (pH 7.4) buffer at a flow rate of 1 mL / min. A blank injection of the binding buffer is used between samples to calculate carryover. Elution is performed with 24 mM phosphoric acid (pH 2) or 100 mM glycine (pH 2.4) for 1 minute. The column is equilibrated with 0.1 M sodium phosphate buffer (pH 7.4) for 1 minute.

[0187] The limit of detection is 0.01 pg for the phosphoric acid elution method and 0.25 pg for the glycine elution method. The maximum pressure that can be applied is 3100 psi. Figure 11A The elution profile is shown for 24 mM phosphoric acid (pH 2), and Figure 11B The elution profile is shown for 100 mM glycine (pH 2.4). Figure 11C The linear correlation between the monoclonal antibody concentration and the average peak area is shown for both elution methods. The carryover is less than 1% for glycine and 2-10% for phosphoric acid. The hydrophilic layer causes some tailing, which indicates non-specific binding of the monoclonal antibody on the surface of the particles.

[0188] Non-porous polymer particles with diol coating (particle type F)

[0189] A polymer-based non-porous particle is prepared. 65.1 grams of polyvinylpyrrolidone (PVP-40), 8.9 grams of Triton N-57, 2452.6 milliliters of reagent alcohol (90% ethanol, 5% methanol, 5% isopropanol), and 153.3 grams of p-xylene are charged into a 4 L round bottle reactor equipped with mechanical stirring, condenser, and thermocouple. The mixed mixture is stirred at 200 rpm and the dissolved oxygen in the solution is removed by subsurface purging with nitrogen. After the dissolved oxygen is below 1 ppm, 2.1 grams of azobisisobutyronitrile and 105 grams of divinylbenzene are charged into the reaction. The reaction is warmed to 70 °C and held for 20 hours.

[0190] To the reaction was added 1.8 g of azobisisobutyronitrile after which a solution of 67.6 g of divinylbenzene (DVB) and 20.3 g of PVP-40 in 202.7 g of reagent alcohol was metered in at a constant flow rate over 120 minutes via a pump. The reaction was continued at 70 °C for at least 12 hours. The particles were isolated from the reaction slurry by filtration and successively washed with methanol, tetrahydrofuran (THF) and acetone. The final product was dried in a vacuum oven at 45 °C overnight.

[0191] Polymer cores were dispersed in 231 g of ethanol by ultrasonication for 4 minutes using an ultrasonic horn and transferred to a round bottom flask equipped with mechanical level stirring, condenser and thermocouple. To the reaction was added 49.1 g of ammonium hydroxide and 462 g of toluene and mixed at room temperature for 60 minutes. The reaction was warmed to 35 °C and charged with a mixture of 26.06 g of TEOS and 66.34 g of BTEE diluted with 148.9 g of ethanol and 148.9 g of toluene at a constant flow rate of 0.95 mL / minute via a peristaltic pump. After the reaction, the particles were isolated by filtration, washed with methanol 6 times and dried in a vacuum oven at 45 °C overnight.

[0192] The resulting polymer core particles were dispersed in a 0.3 M aqueous solution of tris(hydroxymethyl)aminomethane at a slurry concentration of 5 mL / g. The pH was adjusted to 9.8 using acetic acid. The slurry was then sealed in a stainless steel autoclave and heated to 155 °C for 20 hours. After cooling to room temperature, the product was isolated on a 0.5 micron filter paper and washed with water and methanol. The particles were vacuum dried at 80 °C for 16 hours.

[0193] After the hydrothermal treatment, 50 g of the particles were refluxed in 1 M hydrochloric acid at a slurry concentration of 10 mL / g for 10 hours. The reaction was allowed to cool to room temperature and the particles were isolated via filtration. The particles were washed with water until the pH of the filtrate was greater than 5. The semi-neutralized particles were washed with acetone (10 mL / g) 3 times. The resulting particles were classified to remove any agglomerates to give a final particle size of 2.3 μm.

[0194] The resulting 2.3 micron particles were then coated with glycidyloxypropyltrimethoxysilane (GPTMS). 20 mM sodium acetate buffer (pH 5.5) (about 5 mL / gram of particles) was added to a round bottom flask equipped with a thermometer, condenser and mechanical stirring apparatus. The solution was heated to 70 °C after which GPTMS was added at 10.22 μmol / m 2The concentration of the reagent was added to the flask and mixed at the temperature. After 1 hour, the particles were added to the flask (1 g particles / 5 mL buffer solution). The mixture was stirred at 70 °C for 20 hours, after which the reaction was cooled to below 40 °C and filtered. The product was washed with water 3 times and transferred to a new flask equipped with a thermometer, condenser, and mechanical stirring apparatus. Acetic acid (0.1 M) was added to the flask (5 mL per gram of particles) and the slurry was heated to 70 °C for 20 hours. The flask was cooled to below 40 °C and filtered. The product was washed with water until the pH of the supernatant increased to above 5 and then washed with methanol 3 times. The isolated particles were placed in a vacuum oven at 70 °C to dry for 16 hours.

[0195] At room temperature, 5 g of the resulting particles with a hydrophilic coating were added to a mixture of 25 g ethylene glycol diglycidyl ether (EGDGE) and 25 g methanol. 0.25 mL of a 50% aqueous sodium hydroxide solution was added and the reaction was continuously stirred for 20 hours. The particles were isolated by filtration, washed with methanol 10 times, and partially dried under a stream of nitrogen. The particles were stored in a methanol wet bed at 4 °C. The resulting particles had sufficient epoxide content to enable functionalization of the particle surface.

[0196] The particles were functionalized with Protein A as described in Example 2, resulting in approximately 4.66 pg Protein A / mg particles.

[0197] The particles were tested for their ability to bind and elute a monoclonal antibody (NIST mAb standard, purchased from Sigma-Aldrich). The particles were packed in a 2.1 mm x 200 mm column body and connected to an ultra-high performance liquid chromatography system (Acquity Premier, purchased from Waters Technologies Corporation of Milford, MA). The column was flushed at 0.5 mL / min with water (pH 4) for 10 minutes to remove unbound Protein A. The column was then equilibrated in 0.1 M sodium phosphate (pH 7.5) at 0.5 mL / min for 10 minutes. Different concentrations (ranging from 0.001 pg / pL to 1 pg / pL) of the monoclonal antibody standard were injected onto the column using 0.1 M sodium phosphate (pH 7.4) buffer at a flow rate of 1 mL / min in 10 pL injection volumes. Between samples, a blank injection of binding buffer was used to calculate carryover. Elution was performed with 24 mM phosphoric acid (pH 2) or 100 mM glycine (pH 2.4) for 1 minute. The column was equilibrated with 0.1 M sodium phosphate buffer (pH 7.4) for 1 minute.

[0198] The limit of detection was 0.01 pg for the phosphoric acid elution method and 0.25 pg for the glycine elution method. For the glycine elution, the retention time shifted at lower concentrations (see Figure 6). The limit of detection was calculated by determining the lowest concentration at which the peak area was 3 times the baseline noise. Figure 12B), which was not observed in the phosphoric acid elution method (see Figure 12A The maximum pressure that can be applied is 4900 psi. Figure 12C Linear correlation between monoclonal antibody concentration and average peak area is shown for both elution methods. Residual glycine was less than 1.5% and residual phosphoric acid was less than 6%. Low residual was observed, but significant tailing was caused by non-specific binding of the monoclonal antibody on the surface of the particles.

[0199] Example 1 particles functionalised with protein A (particle type A)

[0200] As a comparison, the particles of Example 1 to Example 2 were tested using the same binding and elution methods. The particles were packed in a 2.1 mm x 200 mm column body and connected to an ultra-high performance liquid chromatography system (Acquity Premier, available from Waters Technologies Corporation of Milford, MA). The column was flushed at 0.5 mL / min with water (pH 4) for 10 minutes to remove unbound protein A. The column was then equilibrated in 0.1 M sodium phosphate (pH 7.5) at 0.5 mL / min for 10 minutes. Different concentrations (ranging from 0.001 pg / pL to 1 pg / pL) of monoclonal antibody standards were injected onto the column using 0.1 M sodium phosphate (pH 7.4) buffer at a flow rate of 1 mL / min for a 10 pL injection volume. A blank injection of binding buffer was used between samples to calculate residual. Elution was performed with 24 mM phosphoric acid (pH 2) or 100 mM glycine (pH 2.4) for 1 minute. The column was equilibrated with 0.1 M sodium phosphate buffer (pH 7.4) for 1 minute.

[0201] The limit of detection was 0.01 pg for the phosphoric acid elution method and 0.1 pg for the glycine elution method. The maximum pressure that can be applied was 5000 psi. Residual glycine was less than 2% and residual phosphoric acid was less than 6%. As shown in Figure 13A (24 mM phosphoric acid (pH 2)) and Figure 13B (100 mM glycine (pH 2.4)), high sensitivity and detector saturation (10 pg) were observed at low amounts of monoclonal antibody. Figure 13C Linear correlation between monoclonal antibody concentration and average peak area is shown.

[0202] Comparison between particle types A to H

[0203] Figures 14A to 14B A comparison of peak shape at the highest concentration of the tested sample (5 pg of monoclonal antibody) is provided using the 24 mM phosphoric acid elution method ( Figure 14A ) and the 100 mM glycine elution method ( Figure 14B ). As shown inFigure 14A As shown, using phosphoric acid elution, particle types B, C, D, and E exhibited retention time shifts and peak tailing, while particle type H caused peak splitting. Similarly, as Figure 14B As shown, particle types B and C, D, E, and F exhibit shifts in retention time. Particle types B, H, C, D, and E also have broadened peaks with peak tails. Notably, particle type A results in a narrower peak with higher intensity compared to the other materials tested.

[0204] Figure 15A Peak heights are shown for particle types A, B, C, D, E, F, and G (porous particles as described in Example 4). Figure 15B The peak volume for the particle is shown. For example... Figure 15A As shown, particle type A has a peak height >1.4 times higher than all other particle types tested for both elution methods. Particle types A, C, D, E, and F have similarly small peak volumes. Without being theoretically constrained, the low peak volumes may be partly due to the increased kinetics of small, non-porous particles. In contrast, particle type G has a significantly higher peak volume—almost four times that of particle type A.

Claims

1. A chromatographic column, the chromatographic column comprising: A column body formed of metal or metal alloy, the column body containing a plurality of particles, each of the plurality of particles comprising: Non-porous polymer core; The hydrophilic surface on the outer layer of the non-porous polymer core; and One or more molecules of immunoglobulin-binding proteins conjugated to the hydrophilic surface. The average particle size of the particles is between 1.5 μm and 8 μm.

2. The particle according to claim 1, wherein the immunoglobulin-binding protein is selected from the group consisting of: protein A, protein G, protein A / G, protein L, or their binding domains.

3. The particles according to claim 1 or 2, wherein the nonporous polymer core has a gradient composition.

4. The particles according to any one of claims 1 to 3, wherein the nonporous polymer core comprises divinylbenzene (80%).

5. The particles according to any one of claims 1 to 4, wherein the hydrophilic surface is selected from the group consisting of: (3-glycidoxypropyl)trimethoxysilane, (3-glycidoxypropyl)triethoxysilane, polyacrylate, glycidyl, glyceryl triglycidyl ether, butyl diglycidyl ether, and poly(methyl acrylate).

6. The particle according to any one of claims 1 to 5, wherein one or more molecules of the immunoglobulin-binding protein are conjugated to the hydrophilic surface of the particle via an epoxy linker.

7. The particles according to any one of claims 1 to 6, wherein the average particle size is between 2 μm and 5 μm.

8. The particles according to claim 7, wherein the average particle size is 3.5 μm.

9. The particles according to any one of claims 2 to 8, wherein the immunoglobulin-binding protein has a surface coverage concentration between 3 μg immunoglobulin-binding protein / mg particle and 9 μg immunoglobulin-binding protein / mg particle.

10. The particle according to claim 9, wherein the immunoglobulin-binding protein is protein A.

11. The chromatographic column according to any one of claims 1 to 10, wherein at least a portion of the inner surface of the column body is coated with an alkylsilyl material.

12. The chromatographic column of claim 11, further comprising a sieve plate within the column body, wherein the sieve plate is coated with the alkylsilyl material.

13. The chromatographic column according to claim 11 or 12, wherein the alkylsilane material is a hydrophilic nonionic layer of polyethylene glycol silane.

14. A chromatographic apparatus, the chromatographic apparatus comprising: chromatographic column according to any one of claims 1 to 13; A column injector and tubing, wherein the column injector is positioned upstream of the chromatographic column, and the tubing is fluidly connected to the chromatographic column and located downstream of the column. A portion of the inner surface of the column injector and a portion of the inner surface of the conduit are coated with an alkylsilyl material.

15. A method for enriching immunoglobulins, the method comprising: a) Selecting a chromatographic column, wherein the chromatographic column comprises a column body formed of metal or metal alloy, the column body containing a plurality of particles, each of the plurality of particles comprising: Non-porous polymer core; The hydrophilic surface on the outer layer of the non-porous polymer core; and One or more molecules of immunoglobulin-binding proteins conjugated to the hydrophilic surface. The average particle size of the particles is between 1.5 μm and 8 μm; b) Wash the column with binding buffer; c) Apply a solution containing immunoglobulins to the chromatographic column; as well as d) Wash the column with elution buffer to elute the immunoglobulin from the column.

16. The method of claim 15, wherein the immunoglobulin-binding protein is selected from the group consisting of: protein A, protein G, protein A / G and protein L or their binding domains.

17. The method according to claim 15 or 16, wherein the immunoglobulin-binding protein has a surface coverage concentration between 3 μg immunoglobulin-binding protein / mg particle and 9 μg immunoglobulin-binding protein / mg particle.

18. The method of claim 17, wherein the immunoglobulin-binding protein is protein A.

19. The method according to any one of claims 15 to 18, wherein the chromatographic column is connected to a high-performance liquid chromatography (HPLC) system, an ultra-high-performance liquid chromatography (UHPLC) system, or a rapid protein liquid chromatography (FPLC) system.

20. The method according to any one of claims 15 to 19, wherein the elution buffer is at least three orders of magnitude more acidic than the binding buffer.

21. The method according to any one of claims 15 to 20, wherein the pH of the binding buffer is between 7.0 and 8.

0.

22. The method according to any one of claims 15 to 21, wherein the pH of the elution buffer is between 1.3 and 3.

5.

23. The method of claim 15, further comprising step e) detecting the immunoglobulin using an ultraviolet spectroscopy detector, a fluorescence spectroscopy detector, and / or a mass spectrometry detector.

24. The method of claim 23, further comprising step f) washing the chromatographic column with the binding buffer.

25. The method of claim 24, further comprising repeating steps b) through e).

26. The method of claim 15, further comprising step e) washing the chromatographic column with the binding buffer.

27. The method of claim 26, further comprising repeating steps b) through d).

28. The method of claim 24 or 26, wherein the chromatographic column is washed with the binding buffer for less than or equal to 10 minutes.

29. The method according to any one of claims 15 to 28, wherein the application of the solution containing immunoglobulin to the chromatographic column (step c) is carried out within about 1 minute to 3 minutes.

30. The method of claim 29, wherein the solution containing immunoglobulin is applied to the chromatographic column within about 1 minute.

31. The method according to claim 29 or 30, wherein the immunoglobulin is eluted from the column in a peak volume of less than 50 μL.

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