TW201302777A - Single unit chromatography antibody purification - Google Patents

Abstract

The present invention relates to a method for the purification of antibodies from a protein mixture produced in a bioreactor, at least comprising the steps of intermediate purification and polishing, wherein the intermediate and polishing step comprises in-line anion exchange chromatography (AEX) treatment and mixed mode chromatography (MiMo) treatment in flow through mode. The present invention further relates to a single operational unit comprising both an anion exchange chromatography part and a mixed mode chromatography part, which are serially connected, wherein the unit comprises an inlet at the upstream end of the anion exchange chromatography part and an outlet at the downstream end of the mixed mode chromatography part and wherein the unit also comprises an inlet between the anion exchange chromatography part and the mixed mode chromatography part.

Description

Single unit chromatography antibody purification technology

The present invention relates to a method for purifying antibodies in a single unit and a device that can be used in this method.

Purification of monoclonal antibodies (produced by cell culture) for pharmaceutical applications is a method involving many steps. Such antibodies are essentially from all potentially harmful contaminants {such as proteins and DNA derived from cells producing such antibodies, medium components (such as insulin), PEG ethers and antifoaming agents, and any potentially existing The vector (such as viruses and pathogenic protein prions) is released.

A typical method for purifying antibodies from a culture of cells producing these proteins is described in BioPharm International June 1, 2005, "Downstream Processing of Monoclonal Antibodies: from High Dilution to High Purity".

Since the antibody is made up of cells {such as hybridoma cells or transformed host cells [like Chinese Hamster Ovary (CHO) cells, mouse myeloma-derived NSO cells (mouse myeloma-derived) Produced by NSO cells, baby hamster kidney cells, and human retina-derived PER.C6 ^® cells], the particulate cell material will have to be removed from the cell culture fluid (preferably in the early stages of the purification process). This portion of the method is here indicated as "clarification". Subsequently or as part of the purification step, the antibodies are typically roughly purified to at least about 80% in a combined plus elution chromatography step (in the case of IgG, typically using immobilized protein A). . This step (indicated herein as "capture") not only results in an initial substantial purification of the antibody, but can also result in a substantial reduction in volume, thus concentrating the product. Alternative methods for capture are, for example, Expanded Bed Adsorption (EBA), 2-phase liquid separation (using, for example, polyethylene glycol), or liquid-based salts (lyotropic). Fractionated precipitation of a salt such as ammonium sulphate.

These antibodies were further purified after purification and capture. In general, at least 2 chromatography steps are required after capture to adequately remove residual impurities. The chromatography step after capture is commonly referred to as the intermediate purification step, and the final chromatography step is generally referred to as the polishing step. Each of these steps is generally performed in a batch mode as a single unit operation, and at least one of these steps is generally performed in a combined plus elution mode. In addition, each chromatography step requires specific loading conditions [with respect to, for example, pH, conductivity, etc.]. Therefore, in order to adjust the loading to the required conditions, additional processing must be performed prior to each chromatography step. All of the mentioned mentioned things make the method complicated and time consuming. The impurities that are generally substantially removed during these steps are method-derived contaminants such as: host cell proteins, host cell nucleic acids, medium components (if present), protein A (if present), endotoxin ( Endotoxin) (if present) and microorganisms (if present). Several methods for this purified antibody have been described in the current patent publication: WO 2010/062244 relates to an aqueous two phase extraction augmented precipitation process for isolating and purifying proteins (like monoclonal antibodies). For subsequent purification of the antibody, two options are described: (1) cation exchange chromatography in the binding and elution mode, followed by anion exchange in the overflow mode, or (2) in the overflow The first multi-mode (or mixed-mode) chromatography of the channel mode, followed by anion exchange in the overflow channel mode. The two chromatographic units selected (2) were not operated as a single unit operation and none were used for purification purposes.

WO 2005/044856 relates to the selective combination of hydroxyapatite resin to remove high molecular weight aggregates from an antibody preparation by anion exchange chromatography (anion exchange chromatography) ( Aggregates)). Of these, the two chromatographic processes are described as overflow path methods, however they are described as being carried out as if they were operated separately.

WO 2011/017514 is directed to the purification of antibodies and other Fc-containing proteins by subsequent in-line cation and anion exchange chromatography steps. Although the second step can be operated as a spillway method, the two chromatographic treatments are generally carried out as if they were combined-and-washed.

WO2005/082483 is directed to the purification of antibodies by two subsequent mixed mode chromatography steps, wherein the chromatographic material of the first step is a cation exchange group and an aromatic group that binds to the bindable antibodies. The mixed mode cation exchange resin of the two groups, and the chromatographic material of the second step is a mixed mode anion exchange resin. This second chromatography step can be performed in the overflow channel mode. The two chromatographic steps are described Such as separate operations.

Disadvantages of the methods described above are long operating times, high variable costs, and high fixed costs (due to labor costs).

According to a specific embodiment of the present invention, very efficient removal of residual impurities from antibodies produced by cell culture can be achieved by using in-line anion exchange chromatography (AEX) and mixing in an overflow channel mode. - Mode (MiMo) chromatography was achieved. Prior to the MiMo chromatography step, the overflow channel is in-line adjusted from the AEX step (e.g., by mixing a suitable buffer) to adjust the overflow to pH and conductivity for MiMo chromatography. The correct condition.

The advantage of this novel method is that it considerably reduces operating time and labor, and therefore reduces operating costs. Furthermore, since all cells operate in an overflow mode that only requires sufficient binding capacity for impurities rather than products, smaller (and therefore less expensive) chromatography units are needed.

Accordingly, the present invention can be defined as a method for purifying an antibody from a cell culture fluid produced in a bioreactor, which comprises at least a step of intermediate purification and polishing. Wherein the novel purification step comprises a combined in-line AEX and MiMo chromatography. This can be performed by applying an AEX chromatography step to produce a separation mixture such as a flow-through fraction, a tandem in-line followed by a MiMo chromatography step to produce a purified antibody preparation. There is an overflow channel separation portion, and wherein the purified antibody preparation is subjected to at least one further purification step.

Thus, in the context of the present invention, a "separation mixture" is a solution produced from a first chromatography step according to the invention, and the "purified antibody preparation" is from the invention according to the invention The solution produced by the second chromatography step. What is desired is to support this term throughout this application.

Prior to the first chromatography step, the cell culture fluid produced in the bioreactor will generally be purified [i.e., released from all cellular material (such as whole cells and cell debris)].

Also, prior to the first chromatography step, in order to ensure optimal conditions regarding the pH and conductivity of this first step, a conditioning solution may be added to the cell culture solution or the antibody-containing solution. Solution.

In a particular embodiment, the method according to the invention involves a combined chromatography with AEX and MiMo being performed as a single unit operation.

In this context, the invention is intended to mean any protein that has the ability to specifically bind an antigen with "antibody" and plural "antibodies". An antibody [or immunoglobulin] in its natural form is secreted into a blood or lymphoid reaction-antigen stimulus (such as a bacterium, virus, parasite or transplanted organ) and is specifically bound to it by its specificity. While neutralizing the antigen, the Y-shaped protein on the surface of the B cell. The term antibody as used herein also encompasses an antigen binding portion of a native or artificial antibody. The term antibody also encompasses a non-natural (and thus artificial) protein that has the ability to specifically bind to an antibody according to a similar interaction mechanism as a natural antibody, and thus also includes, for example, An antigen-binding portion derived from a species (eg, a mouse) and a chimeric antibody derived from a non-antigen-binding portion of a species (eg, a human).

With regard to "mixed-mode chromatography (MiMo)", we mean the type of chromatography that utilizes more than one interaction to occur for materials that adsorb and/or desorb proteins. These interactions can be of the following types: anionic, cationic, hydrophobic, affinity, π-π, thiophilic, size exclusion. Examples of well known materials are mixed mode hydroxyapatite (Hydroxyapatite) (metal affinity, anion and cation interactions), Capto ^TM adhere (anionic and hydrophobic interaction) and the MEP HyperCel ^TM (cationic and hydrophobic interaction ).

With regard to "in-line AEX and MiMo", we mean that AEX and MiMo are connected in series with the effluent of the AEX device being fed into the MiMo device without intermediate storage.

By "flood separation portion" is herein meant: at least a portion of a packed, antibody-containing separation portion that exits the chromatography column at substantially the same rate as the elution fluid. This separated portion is not substantially retained on the column during elution. These conditions are therefore chosen such that instead of the antibodies, the impurities are bound to the individual chromatography material.

It has been found that for the purpose of mass production, the method according to the invention (with overflow mode) provides a faster separation than previously disclosed methods with binding and elution of the desired antibody.

According to the invention, the separation mixture containing the antibody is in-line Section. For this purpose, the separation mixture is supplemented with a sufficient amount of a suitable conditioning solution in order to modify its composition and/or properties (such as pH and/or conductivity) and/or special ionic components. The presence or amount is preferably used for optimal execution in the second chromatography step according to the invention.

In the prior art documents cited above, there is no in-line adjustment between the two chromatography steps being applied or suggested, and it has surprisingly been found that very good separation results can be entered into the second according to the invention. This is achieved by in-line adjustment of the fluid (separation mixture) prior to the chromatography step.

Accordingly, the present invention is directed to a method for purifying an antibody from a protein mixture produced in a bioreactor comprising at least a step of moderate purification and purification, wherein the intermediate purification and purification are performed. The step comprises in-line anion exchange chromatography (AEX) to produce a separation mixture such as an overflow separation section, followed by mixed-mode chromatography (MiMo) to produce a purified antibody preparation such as an overflow. a separation step, and wherein the purified antibody preparation is subjected to at least one further purification step, wherein the mixed-mode chromatography is used for adjusting the pH and/or conductivity and/or the concentration or type of the particular ionic component The impurities are removed from the antibodies in a method step which is supplemented with a sufficient amount of a suitable adjustment solution prior to mixing mode chromatography.

The terms "adjustment solution" and "adjustment solution" are used interchangeably and mean here a solution which is added to the separation mixture before feeding the separation mixture to the second (MiMo) chromatography step according to the invention.

A "sufficient amount of a suitable adjustment solution" is hereby referred to as Any acidic, neutral or alkaline solution which optionally contains one or more salts or any other additives, when mixed with the separation mixture, will cause adsorption of most of the relevant impurities to the MiMo material, but it will not promote The substantial combination of products. With regard to the various purification methods, the optimum pH, the preferred type of salt system, and the optimum amount of the conditioning solution must be established.

Preferably, the pH of the solution mentioned will be the same as that of the separation mixture containing the antibody, and the optimum conductivity value will be the addition of a sufficient amount of one or more salts or dilution present in the The result of separating the salts of the mixture(s). The anion of the salt may preferably be selected from the group consisting of phosphates, sulfates, acetates, chlorides, bromides, nitrates, chlorates, iodides and thiocyanides. Ion (thiocyanate ions). The cation of the salt may preferably be selected from the group consisting of ammonium, rubidium, potassium, sodium, lithium, magnesium, calcium and barium ions. Preferred salts are ammonium sulfate, sodium sulfate, potassium sulfate, ammonium phosphate, sodium phosphate, potassium phosphate, potassium chloride and sodium chloride. Other additives that can be used are ethanol, ethylene glycol, propylene glycol, polyethylene glycol or any other compound known in the art to optimize the MiMo chromatography step. .

The acidic component for an acid-adjusting solution may be selected from a compound such as citric acid (or its mono- or dibasic sodium or potassium salt), phosphoric acid (or its mono- or di-basic sodium or Potassium salt), acetic acid, hydrochloric acid, sulfuric acid].

The basic component for an alkaline adjustment solution may be selected from the group consisting of a compound {such as sodium hydroxide or potassium hydroxide (or its mono- or dibasic sodium or potassium salt), tris(hydroxymethyl)aminomethane. [tris(hydroxymethyl)aminomethane]}, However, any other alkaline component known in the art can be used for this purpose.

Preferably, the desired conditioning solution will be replenished in a small amount to have a minimum dilution of the product.

Preferably, supplementing the separation mixture with a sufficient amount of a suitable conditioning solution in this example is part of the single unit operation, such as the adjustments mentioned by in-line mixing prior to the MiMo chromatography step. The solution is in a process stream (e.g., in a mixing chamber).

The AEX chromatography according to the present invention can be carried out in an AEX unit which can be composed of a standard packed bed column containing a resin, a column containing a monolith material, and the like. A radiographic column containing a suitable chromatographic medium, an adsorption membrane unit, or any medium known in the art to have an appropriate medium and ligand to act as an anion exchanger Other anion exchange chromatography devices were embodied. In the AEX column, the chromatography material may be present as a particulate support material to which a strong or weak cationic ligand is attached. The membrane type anion exchanger is comprised of a support material in the form of one or more sheets to which a strong or weak cationic ligand is attached. The support material may be composed of an organic material or an inorganic material or a mixture of organic and inorganic materials. Suitable organic materials are agarose based media and methacrylate. Suitable inorganic materials are silica, ceramics and metals. A membrane form anion exchanger can be composed of a hydrophilic polyethersulfone containing an AEX ligand to make. Suitable strong AEX ligands are based, for example, on quaternary amine groups. Suitable weak AEX ligands are based on, for example, primary, secondary or tertiary amine groups or any other suitable ligand known to those skilled in the art.

MiMo chromatography according to the present invention can occur in a MiMo unit which can be comprised of a standard column containing a resin, a column of monolithic material, a column of radioactivity containing a suitable chromatographic medium, Adsorption membrane units, or any other mixed mode chromatography apparatus known in the art to have suitable ligand interactions, such as a mixed mode material, are embodied. In the MiMo column, the chromatography material may be present as a particulate support material to which the MiMo ligand is attached. The chromatograph of the membrane is constructed of a support material in the form of one or more sheets to which the MiMo ligand is attached. The support material may be composed of an organic material or an inorganic material or a mixture of organic and inorganic materials. Suitable organic support materials are, for example, hydrophilic carbohydrates [such as crosslinked agarose, cellulose or dextran] or synthetic copolymer materials {such as poly(alkylaspartamide) [poly(alkylaspartamide) )], a copolymer of 2-hydroxyethyl methacrylate and ethylene dimethacrylate or an acylated polyamine. Suitable inorganic support materials are, for example, alumina, ceramics and metals. A membrane form of MiMo can be composed of a hydrophilic polyether oxime containing a MiMo ligand. Suitable examples of MiMo ligands are hydroxyapatite, fluorapatite, 4-mercapto ethyl pyridine, hexylamino, phenylpropylamino, 2 -2-mercapto-5-benzamidazole sulfonic acid, N-benzyl-N- N-benzyl-N-methyl ethanolamine or any other ligand known to the art having multiple functions.

The antibody which can be purified according to the method of the present invention is an antibody having an isoelectric pH of 6.0 or higher (preferably 7.0 or higher, more preferably 7.5 or higher). These antibodies may be immunoglobulins of the G, A or M class. The antibodies may be human, or non-human [such as rodent] or chimeric (eg, "humanized") antibodies, or may be the immunoglobulins mentioned above. Subunits, or may be hybrid proteins composed of an immunoglobulin moiety and a moiety derived from or identical to another (non-immunoglobulin) protein.

Surprisingly, the antibody material produced by the combined AEX and MiMo chromatography will generally have a value of at least 98%, preferably at least 99%, more preferably at least 99.9%, even more preferably at least 99.99. Very high purity of % (reference protein content).

The AEX chromatography step according to the invention is preferably carried out in a neutral or slightly alkaline pH. It will remove negatively charged impurities {like DNA, host proteins, protein A (if present), viruses (if present), proteinacous medium components (such as insulin and insulin like growth factor (insulin like Growth factor)] (if present)}.

During this MiMo chromatography step, the major remaining macromolecular impurities (mainly product aggregates) will be removed using the pH and conductivity characteristics of the correct conditions, and they will bind to the product when it overflows. Chromatography unit.

Subsequently, in order to remove all residual low molecular weight impurities, this (height) The purified antibody preparation will generally have to be treated by ultrafiltration and diafiltration to replace the buffer with the final formulation buffer and adjust the desired final product concentration.

Again, the purified antibody preparation will generally also have to be treated to ensure complete removal of potentially existing infectious agents (such as viruses and/or infectious proteins).

The invention also relates to a single operating unit comprising both an anion exchange chromatography component (AEX) and a mixed mode chromatography component (MiMo) connected in series. The single unit of operation further includes an inlet at an upstream end of the first ion exchange chromatography component and an outlet at a downstream end of the second ion exchange chromatography component. The single unit of operation also includes a connection between the first ion exchange chromatography component and the second ion exchange chromatography component further comprising an inlet for supplementing a conditioning solution to the separation mixture.

During the process according to the invention, the liquid stream can be passed through any commercially available dual pump chromatographic system (e.g., a ÅKTA detector (GE), a BIOPROCESS (GE), any pair A pump HPLC system or any custom device that conforms to the diagram of Figure 1 is established. Most of these chromatography devices are designed to operate a single chromatography unit (i.e., a column or membrane). With a simple modification, an additional connection can be made to place the first ion exchange unit after pump A and before the mixing chamber.

Figure 1 shows the basic configuration. A tandem connection of two chromatography devices plus a selective pre-filter as shown in Figure 1 The location can occasionally lead to undesired pressure increases. Therefore, additional technical modifications (e.g., an additional pump after the AEX unit and a pressure reducing device prior to the AEX unit) may have to be included in this figure in some cases.

Simple illustration

Figure 1: A single unit of operation comprising both an anion exchange chromatography component and a cation exchange chromatography component. Buffer A is a conditioning and wash buffer suitable for optimal operation of the AEX step. Buffer B contains an acidic solution and is mixed in a ratio to the desired loading/buffer A to obtain the optimum conditions for operating the MiMo step. The mixing ratio can be implemented using a fixed volume mixing stream or can be automatically controlled by a feedback loop (according to, for example, pH output). MC is an optional mixing chamber that can contain any type of electrostatic mixer.

Example Materials and Method:

All experiments were carried out using an IgG produced by a CHO cell strain. Cultured in chemically defined medium used in the XD ^® mode is performed (see Genetic Engineering & Biotechnology news, Apr 1 2010, No.7).

Rough purification of XD ^® harvested and used to capture Rhobust ^® EBA technology and protein A is carried out in a single step like (see, Innovations in Pharmaceutical Technology, March 2011) . The product was eluted with 35 mM NaCl, 0.1 M acetate; pH 3.0 elution buffer. The eluate contained 5 g/L IgG and was stored at 2-8 °C.

Using the materials thus obtained, 6 experiments were each carried out: 1. Establishment of conditions for preferential binding of aggregates in a MiMo chromatography using a hydroxyapatite resin (Experiment 1). 2. Run a MiMo chromatography using a hydroxyapatite resin in an overflow mode with in-line mixing (Experiment 2). 3. AEX and MiMo chromatography using a combination of monohydroxyapatite resin as a single unit operation (Example 1). 4. Establish the optimum conditions for MiMo chromatography using an anion-HIC resin in the overflow mode (Experiment 3). 5. Run a MiMo chromatography using an anion-HIC resin in an overflow mode with in-line mixing (Experiment 4). 6. AEX and MiMo chromatography using an anion-HIC resin in combination as a single unit operation (Example 2).

The optimum conditions for AEX chromatography in the overflow channel mode have previously been determined and applied in the experiments of Example 1 and Example 2.

Protein (product) concentration of UV / Vis spectroscopy (Spectroscopy) is measured by measuring the absorption of ²⁸⁰ nm (A 280) and extinction coefficient (extinction coefficient) of a 1.63.

The IgG and aggregate concentrations of the monomers were determined by size exclusion chromatography (HP-SEC) according to standard procedures.

HCP was measured by CHO HCP ELISA Assay, 3G (Cygnus Technologies).

Experiment 1 Establish conditions for preferential binding of aggregates in MiMo chromatography using a hydroxyapatite resin

For this experiment, pre-purified IgG was diluted to one by demineralized water. Conductivity of 5 mS/cm and adjusted to pH 6.5 using a 2 M Tris pH 9.0. MiMo chromatography was carried out in a binding-elution mode. Is filled with a hydroxyapatite chromatography HA Ultrogel ^® adsorbent bed length of 4 cm method ^{(HA Ultrogel ® Hydroxyapatite Chromatography Sorbent)} (Pall, Life Sciences) a VL11 (Millipore) column was used in a In the KTA detector. The column was equilibrated and washed with a 10 mM sodium phosphate, pH 7.0 at a flow rate of 3 mL/min. The product was loaded at a flow rate of 2 mL/min. The initial loading contained 2.6 g/L IgG and a 2.2% initial amount of aggregate. After filling, the product was eluted with a linear gradient from 0 to 100% with 10 mM sodium phosphate, pH 7.0 (buffer A) and 10 mM sodium phosphate, 1 M NaCl, pH 7.0 (buffer B).

During the elution step, the separated fraction is collected and analyzed as a function of the presence of aggregate and protein (product) content as a function of conductivity.

The results of the analysis of the samples (shown in Table 1) clearly indicate that a conductivity value of 26.9 mS/cm is reached, and the eluent does not contain or contains an insignificant amount of aggregates.

Experiment 2 Aggregate removal using a hydroxyapatite resin in MiMo chromatography in a spillway mode with in-line mixing

For this experiment, pre-purified IgG was diluted to a conductivity of 2.4 mS/cm with demineralized water and adjusted to pH 7.4 using a 2 M Tris pH 9.0 buffer. A 4 cm is filled with the adsorbent bed length HA Ultrogel ^® hydroxyapatite chromatography (Pall, Life Sciences) a VL11 (Millipore) column was used in a In the KTA detector. The column was equilibrated with demineralized water and 10 mM sodium phosphate, 0.8 M NaCl, pH 7.4 (buffer B). Demineralized water and Buffer B were in-line mixed at a fixed volume ratio of 30% Buffer B at a flow rate of 5 mL/min. After equilibration, the product is filled. In order to adjust the conductivity to a value of 25 mS/cm, the product stream was in-line mixed with buffer B during the filling. The product stream and Buffer B were mixed at a fixed volume ratio of 30% Buffer B at a flow rate of 1 mL/min. The initial loading contained 0.78 g/L of IgG and an initial amount of aggregate of 2.97%.

The separated portion of the overflow channel is collected and analyzed for the presence of aggregate and protein (product) content.

The results of the analysis of these samples (shown in Table 2) clearly indicate that the product-containing loading was mixed in a spillway mode and in-line with a fixed volume ratio of 10 mM sodium phosphate at a rate of 30%, 0.8 Removal of aggregates using a hydroxyapatite resin under the product of M NaCl, pH 7.4 1%.

Example 1 Purification of IgG by a single unit operation using AEX and MiMo chromatography using monohydroxyapatite resin

Use one as depicted in the diagram of Figure 1 The KTA detector, an AEX unit and a MiMo unit are coupled in series. For the AEX, a Sartobind Q capsule (1 mL) was used, and for which the MiMo, the adsorbent is filled with a bed length of 4 cm HA Ultrogel ^® hydroxyapatite chromatography (Pall, Life Sciences) of VL11 (Millipore The column is used. Regarding the conditioning prior to product loading and prior to attachment of the AEX unit, the MiMo unit was demineralized water (pumped with pump A) and 10 mM sodium phosphate, 0.8 M NaCl, pH 7.4 (buffer B ). balance. Go mineral water and Buffer B in Buffer B in a fixed volume ratio of 30% are mixed in-line as a 5 mL / min flow rate. The AEX unit was rinsed and equilibrated with 100 mL of 0.05 M Tris, pH 7.4 buffer before connecting it to the system. An experiment can be made in which the balance of the individual units is not made separately.

For this experiment, the prepurified IgG was diluted to a conductivity of 2.4 mS/cm with demineralized water, adjusted to pH 7.4 with a 2 M Tris pH 9.0 buffer and filtered at 0.22 μm. The pre-purified IgG loading was initiated by pumping at a rate of 1 mL/min. Buffer B was pumped at a flow rate of 30% at the same flow rate. A quantity of 240 mL (containing 0.6 g/L of IgG) was filled. After the filling is completed, the AEX unit is removed to initiate the cleaning. An experiment can be made where the AEX unit does not need to be removed for cleaning. The MiMo unit was washed with a linear gradient from 0 to 30% of 10 mM sodium phosphate, pH 7.4 (buffer A ) and buffer B , and was buffered with a 0.5 M sodium phosphate, 1.5 NaCl, pH 6.8 buffer. Stripping. Filling, overflow channels and cleaning were analyzed regarding the presence of aggregates, HCP content and protein (product) content. The fill has an HCP concentration of 2179 ng/mg IgG. The overflow channel plus the wash separation portion has an HCP concentration of 447 ng/mg IgG. The amount of aggregates in the charge was 2.93% and 0.76% in the overflow plus cleaning. The stripping contained 54.97% of aggregates. The total product recovery in the overflow channel plus cleaning was 88.2% and the cleaning plus the stripping was 90%.

This experiment shows that when the separation mixture is in-line supplemented with a sufficient amount of a suitable conditioning solution, a final purity of 99.2% of the antibody material is obtained by using in-line anion exchange chromatography followed by in-line anion exchange chromatography. MiMo (hydroxyapatite) chromatography operations are achieved as a single unit operation. The initial purity of the charge was 97%.

Experiment 3 Optimum conditions in MiMo chromatography using an anion-HIC resin in the overflow channel mode

For this set of experiments, the pre-purified IgG was diluted with demineralized water to a conductivity of 2.29 mS/cm and the pH was adjusted to pH 7.4 using a 2 M Tris pH 9.0 buffer. Is filled with a length of 6.3 Capto ^TM adhere (GE Healthcare) a VL11 (Millipore) column was used in a KTA detector. The column was equilibrated and washed with 25 mM sodium phosphate, pH 7.4 (buffer A) and 100 mM sodium phosphate, pH 7.4 (buffer B). Buffer A and Buffer B were run in a mixture of 0, 5, 15 and 25% by volume at a flow rate of 5 mL/min as if they were separated. After equilibration, the product is filled. This product stream was mixed in-line with Buffer B during the filling. The product stream and Buffer B were run in in-line mixing at a flow rate of 3 mL/min at a volume ratio of 0, 5, 15 and 25%, such as a separation operation. Due to in-line mixing with Buffer B, the initial charge contained 1.09 g/L IgG and an initial amount of 3.13% polymer prior to dilution.

The separated portion of the overflow channel is collected under different ratios of buffer B And analysis about the presence of aggregates and protein (product) content.

The results of the analysis of these samples (shown in Table 3) clearly indicate that aggregates were removed in an anion-HIC MiMo resin when the product-containing loading was in-line mixed with a phosphate-adjusted buffer. Up to <1%.

Experiment 4 AgMO chromatography for removal of aggregates using an anion-HIC resin in overflow mode and in-line mixing

For this experiment, the purified IgG was diluted with demineralized water to a conductivity of 2.4 mS/cm and adjusted to pH 7.4 using a 2 M Tris pH 9.0 buffer. Is filled with a length of 6.3 Capto ^TM adhere (GE Healthcare) a VL11 (Millipore) column was used in a On the KTA detector. The column was equilibrated and washed with 25 mM sodium phosphate, pH 7.4 (buffer A) and 100 mM sodium phosphate, pH 7.4 (buffer B). Buffer A and Buffer B were in-line mixed at a fixed volume ratio of 15% of Buffer B at a flow rate of 5 mL/min. After equilibration, the product is filled. The product was mixed in-line with buffer B during the filling. The product stream and Buffer B were in-line mixed at a flow rate of 3 mL/min at a fixed volume ratio of Buffer B of 15%. The initial charge contained an initial amount of 0.93 g/L IgG and a 3.15% polymer. The column was stripped with a 100 mM sodium phosphate, pH 3.0 buffer.

The results of the analysis of these samples (shown in Table 4) clearly indicate: mixing in a spillway mode with in-line mixing at a fixed volume ratio of 100 mM sodium phosphate at 30%, pH 7 Removal of aggregates in the overflow channel in an anion-HIC MiMo resin 1%. The percentage of aggregates was 0.18% of the overflow volume.

Example 2 Purification of IgG as a single unit operation using AEX and MiMo chromatography using an anion-HIC resin

Use one as depicted in the diagram of Figure 1 The KTA detector, an AEX unit and a MiMo unit are coupled in series. For the AEX, a Sartobind Q capsule (1 mL) was used, and for which the MiMo, are used to be filled with a 6.3 cm bed length Capto ^TM adhere (GE Healthcare) a VL11 (Millipore) column. Regarding the adjustment prior to product loading and prior to attachment of the AEX unit, the MiMo unit was equilibrated with 25 mM sodium phosphate, pH 7.4 (buffer A) and with 100 mM sodium phosphate, pH 7.4 (buffer B). Buffer A and Buffer B in Buffer B in a fixed volume ratio of 15% is mixed in-line as a 5 mL / min flow rate. The AEX unit was rinsed and equilibrated with 100 mL of 0.05 M Tris, pH 7.4 buffer before connecting it to the system. An experiment can be made in which the balance of the individual units is not made separately.

For this experiment, the prepurified IgG was diluted to a conductivity of 2.29 mS/cm with demineralized water, adjusted to pH 7.4 with a 2 M Tris pH 9.0 buffer and filtered at 0.22 μm. The pre-purified IgG loading was initiated by pumping at a rate of 3 mL/min. Buffer B was pumped at a flow rate of 15% at the same flow rate. A quantity of 479 mL (containing 0.91 g/L of IgG) was filled. To initiate the cleaning, after the filling is completed, the AEX unit is removed and the stream is converted back to buffer A and the line is primed. An experiment can be made where the AEX unit does not need to be removed for cleaning. After washing, the MiMo unit was stripped by adding a 100 mM sodium phosphate, pH 3.0 buffer via pump A and pump B was stopped. Filling, overflow channels and cleaning were analyzed regarding the presence of aggregates, HCP content and protein (product) content. The fill has an HCP concentration of 1711 ng/mg IgG. The overflow channel plus the wash separation portion has an HCP concentration of 206 ng/mg IgG. The amount of aggregates in the charge was 3.13% and was 0.18% in the overflow plus cleaning. The stripping contained 14.23% of aggregates. The total product recovery at the overflow plus cleaning was 82.9% and the cleaning plus stripping was 99.9%.

This experiment shows that when the separation mixture is in-line supplemented with a sufficient amount of a suitable adjustment solution, a final purity of 99.72% of the antibody material is obtained by using in-line anion exchange chromatography followed by MiMo The (anion-HIC) chromatography operation is achieved as a single unit operation. The initial purity of the charge was 96.8%.

Abbreviation used

(light) absorption of A ²⁸⁰ at 280 nm

AEX anion exchange

BHK cells, baby hamster kidney cells

CHO cell Chinese hamster ovary cell

EBA expanded bed adsorption

HCP host cell protein

HIC hydrophobic interaction chromatography

HPLC high pressure liquid chromatography

IgG immunoglobulin G

MiMo mixed mode

TFF tangential flow filtration

Tris tris(hydroxymethyl)methylamine

L ‧‧‧Loading

PA ‧‧‧Pump A

PB ‧‧‧Pump B

AEX ‧‧‧ anion exchange unit

MiMo ‧‧‧Cation exchange unit

pH ‧‧‧pH sensor

σ ‧‧‧ conductivity sensor

PF ‧‧‧Selective pre-filter

Figure 1: A single unit of operation comprising both an anion exchange chromatography component and a cation exchange chromatography component. Buffer A is a conditioning and wash buffer suitable for optimal operation of the AEX step. Buffer B contains an acidic solution and is mixed in a ratio to the desired loading/buffer A to obtain the optimum conditions for operating the MiMo step. The mixing ratio can be implemented using a fixed volume mixing stream or can be automatically controlled by a feedback loop (according to, for example, pH output). MC is an optional mixing chamber that can contain any type of electrostatic mixer.

L ‧‧‧Loading

PA ‧‧‧Pump A

PB ‧‧‧Pump B

AEX ‧‧‧ anion exchange unit

MiMo ‧‧‧Cation exchange unit

pH ‧‧‧pH sensor

σ ‧‧‧ conductivity sensor

PF ‧‧‧Selective pre-filter

Claims (10)

A method for purifying an antibody from a protein mixture produced in a bioreactor, comprising at least a step of moderately purified and purified, wherein the intermediate purification and purification steps comprise a series of inline anions Exchange chromatography (AEX) to produce a separation mixture as a separate portion of the overflow channel, followed by a mixed mode chromatography (MiMo) to produce a purified antibody preparation as a spill separation portion, and wherein the purified portion The antibody preparation is subjected to at least one further purification step, wherein to adjust the pH and/or conductivity and/or the concentration or type of the particular ionic component to remove impurities from the antibodies in the mixed-mode chromatography step, The separation mixture is supplemented with a suitable amount of a suitable conditioning solution prior to mixing mode chromatography. The method of claim 1, wherein the anion exchange chromatography and the mixed mode chromatography occur in two separate devices connected in series. The method of claim 1, wherein the in-line AEX and MiMo are performed as a single unit operation. The method of any one of claims 1 to 3 wherein the separation mixture is supplemented with a sufficient amount of a salt or a combination of salts prior to the MiMo. The method of claim 4, wherein the separation mixture is supplemented with a sufficient amount of ammonium sulfate, sodium sulfate, potassium sulfate, ammonium phosphate, sodium phosphate, potassium phosphate, potassium chloride, and sodium chloride before being added to MiMo. . The method of claim 1 to 3, wherein the separation mixture is supplemented with a sufficient amount of an acidic solution prior to the MiMo chromatography. The method of claim 6, wherein the separation mixture is supplemented with a sufficient amount of citric acid (or its monobasic or dibasic sodium or potassium salt), phosphoric acid (or It is a solution of monobasic or dibasic sodium or potassium salt), acetic acid, hydrochloric acid or sulfuric acid. The method of claim 1 to 3, wherein the separation mixture is supplemented with a sufficient amount of an alkaline solution prior to the mixed mode chromatography. The method of claim 8, wherein the separation mixture is supplemented with a sufficient amount of sodium hydroxide or potassium hydroxide (or a mono- or dibasic sodium or potassium salt thereof) or prior to MiMo chromatography. A solution of tris(hydroxymethyl)aminomethane. A single operating unit that can be used in the method of any one of claims 1 to 9 comprising an anion exchange chromatography component and a mixed mode chromatography component that are connected in series, Wherein the outlet of the anion exchange chromatography component is coupled to the inlet of the mixed mode chromatography component, wherein the unit comprises an inlet at the upstream end of the anion exchange chromatography component and a mixed mode chromatography An outlet of the downstream end of the component, and wherein the unit also includes an inlet between the anion exchange chromatography component and the mixed mode chromatography component.