HK1178174A - Single unit antibody purification - Google Patents

Description

single unit antibody purification

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

Purification of monoclonal antibodies produced by cell culture for pharmaceutical applications is a process that involves a large number of steps. Such antibodies are necessarily free of all potentially harmful contaminants such as proteins and DNA derived from the cells producing them, media components such as insulin, PEG ethers and antifoams and any possible infectious agents such as viruses and prions (prions).

A typical procedure for purifying Antibodies from cultures of cells producing these proteins is described in BioPharmInternational Jun 1, 2005, Downstream Processing of Monoclonal Antibodies: from High Dilution to High Purity is described.

C 6. since the antibody is derived from cells such as hybridoma cells or transformed host cells (e.g., Chinese Hamster Ovary (CHO) cells, mouse myeloma-derived NS0 cells, juvenile hamster kidney (BHK) cells, human retina-derived perCells), the particulate cell material must be removed from the cell slurry, preferably early in the purification process. This part of the process is referred to herein as "clarification". Subsequently or as part of a clarification step, the antibody is roughly purified to at least about 80%, typically by a "bind and elute" chromatography step (typically using immobilized protein a in the case of IgG). This step, referred to herein as "capture", not only results in an initial large purification of the antibody, but can also result in a large reduction in volume and thus concentration of the product. Other alternative methods for capture are for example Expanded Bed Adsorption (EBA), 2-phase liquid separation (using for example polyethylene glycol) or fractional precipitation with lyotropic salts (such as ammonium sulphate).

After clarification and trapping, the antibody was further purified. Generally, at least two chromatographic steps after capture are necessary in order to adequately remove residual impurities. The chromatography step after trapping is usually referred to as an intermediate purification step, while the final chromatography step is usually referred to as a purification (refining) step. Each of these steps is typically performed as a single unit operation in batch mode, and at least one of these steps is performed in "bind and elute" mode. Furthermore, each chromatography step requires specific loading conditions, such as pH, conductivity, etc. Therefore, in order to adjust the load to the required conditions, additional treatments have to be performed before each chromatography step. All of the above makes the process time and labor consuming. The impurities that are typically substantially removed in these steps are all process-derived contaminants, such as host cell proteins, host cell nucleic acids, media components (if present), protein a (if present), endotoxins (if present), and microorganisms (if present).

Many such methods for purifying antibodies have been described in the prior art.

WO2007/076032 describes a method for purifying antibodies (CTLA4-Ig and variants thereof), in which method the cell culture supernatant or a fraction thereof obtained after affinity chromatography is subjected to anion exchange chromatography to obtain an eluted protein product, and the eluted protein product is subjected to hydrophobic interaction chromatography to obtain an enriched protein product. In this method, the eluted protein product is obtained by a method in which the antibody is first captured by an anion exchange chromatography material, followed by washing the exchange chromatography material with a wash buffer, after which the antibody is eluted therefrom by changing the process conditions (e.g. elution with an elution buffer).

US2008/016450 relates to a method for purifying Fc-containing proteins (such as antibodies) by: the protein was bound to a protein a column and eluted using a pH gradient elution system. This document describes the desirability of using hydrophobic interaction chromatography and anion exchange chromatography in flow-through mode (paragraphs 0058 to 0064).

WO2008/025747 relates to the purification of Fc-fusion proteins in a process comprising protein a or G chromatography, cation exchange chromatography, anion exchange chromatography and hydroxyapatite chromatography, in particular in this order. In this process, anion exchange chromatography and hydroxyapatite chromatography are applied in flow-through mode.

US2007/0167612 concerns the purification of proteins (such as antibodies) which are first captured to an affinity column, such as a protein a column. The eluate from the affinity column is then contacted with an anion exchange material, whereby the antibody binds thereto and is subsequently eluted. For further purification, additional chromatography columns and purification steps may be used, including additional cation-exchange chromatography, anion-exchange chromatography, size exclusion chromatography, affinity chromatography, hydroxyapatite chromatography and hydrophobic interaction chromatography.

WO2001/072769 describes the purification of highly anionized proteins, such as sulfated proteins. For this purpose, anion exchange chromatography and hydrophobic interaction chromatography in "bind-elute" mode were subsequently used.

WO2009/058769 relates to a method for removing impurities from antibody preparations. In particular, the patent application relates to a method for purifying antibodies (containing hydrophobic variants). For this purpose, the sample is loaded on a protein a column; eluting from the protein A column with a suitable eluent, loading on a cation and/or anion exchange column; eluting from the ion exchange column, and loading on a Hydrophobic Interaction Chromatography (HIC) column, wherein the HIC column is in flow-through mode; the purified material was thereafter collected. Note that only the HIC column was applied in flow-through mode.

EP1614694 concerns the purification and isolation of immunoglobulins. In particular, it is of interest to purify antibodies from cell cultures in the order of a protein a column, an anion exchange column and a cation exchange column step and optionally a hydrophobic interaction column step. Of these steps, the anion exchange chromatography step was operated in flow-through mode, while all other steps were operated in "bind-elute" mode.

WO2008/051448 relates to the reduction of protein a in antibody preparations purified by protein a affinity chromatography. It has been suggested that this protein contamination can be removed using charge-modified depth filters. This removal step may be performed before or after the purification steps conventionally used for antibody preparations.

EP0530447 describes the purification of antibodies by a combination of anionic, cationic and hydrophobic interaction chromatography and a specific bactericidal step. The order of the chromatographic steps may vary. Each chromatography step was operated in "bind-elute" mode.

Kuczewski, M. et al (2009) [ Biotechn.Bioengn.105, 296-305] describes the use of a hydrophobic interaction membrane absorber for the fine purification of antibodies.

Chen, J. et al (2008) [ J.Chrom.A 1177, 272-281] compared conventional and new generation hydrophobic interaction chromatography resins (e.g.mixed mode) in antibody purification.

Zhou, j.x. et al (2006) [ j.chrom.a 113466-73] describes the use of a hydrophobic interaction membrane absorber as an alternative to hydrophobic interaction column chromatography.

Gottschalk, U. (2008) [ Biotechnol.Prog.24, 496. 503] discusses the disadvantages of column chromatography compared to the use of membrane absorbers in antibody purification.

Wang, c. et al (2007) [ j.chrom.a 1155, 74-84] use nucleated anion exchange chromatography in a flow-through process for the removal of trace contaminants from antibody materials (fine purification). Compared to non-nucleated anionic materials.

Azevedo, a. et al (2008) [ j. chrom. a.1213, 154-.

-Boi, c. (2007) [ j.chrom.b.848, 19-27] this review considers the use of membrane absorbers as an alternative technique to the capture and fine purification steps for the purification of monoclonal antibodies.

The disadvantages of the above method are: long operating times, variable costs (e.g. large amounts of expensive resin required due to the high column capacity necessarily required for the bind-elute step itself) and high immobilization costs (due to labor costs).

According to the present invention, a very efficient removal of residual impurities from antibodies produced from cell cultures can be achieved by operating both anion exchange chromatography (AEX) and Hydrophobic Interaction Chromatography (HIC) in series in a flow-through mode, preferably in one single unit operation. The lyotropic salts can be mixed in-line after AEX and before HIC to adjust the correct conditions for hydrophobic interaction chromatography.

The advantages of this approach using separate, series-connected, sequentially-connected AEX and HIC devices (both used in flow-through mode) are: the operating time and the labor and operating costs are considerably reduced. Furthermore, smaller (and thus less costly) chromatography units are required because all units operate in flow-through mode, requiring only sufficient capacity to bind impurities rather than product.

Thus, the present invention can be defined as a method for purifying an antibody from a cell slurry (cellbroth) produced in a bioreactor, the method comprising at least an intermediate purification step and a polishing purification step, wherein the novel purification step comprises a series of, sequentially connected, anion exchange chromatography (AEX) treatment that produces a separation mixture in the form of a flow fraction and Hydrophobic Interaction Chromatography (HIC) treatment that produces a purified antibody preparation in the form of a flow fraction, and wherein the purified antibody preparation is subjected to at least one further purification step.

In the context of the present invention, "separation mixture" refers to the solution resulting from the first ion exchange step of the present invention and "purified antibody preparation" refers to the solution resulting from the second ion exchange step of the present invention. This term is used throughout this application.

By "serially connected AEX and HIC" is meant that AEX and HIC are connected in series in such a way that the effluent of the AEX unit enters the HIC unit directly without intermediate storage.

By "flow-through mode" is meant herein that the antibody to be purified is passed through a chromatographic apparatus. This is in contrast to the "capture mode" typically used in antibody purification, where the antibody is first bound to the chromatographic material and eluted in a subsequent step (i.e., released by changing the medium conditions or composition).

In a specific embodiment, the process of the present invention comprises the treatment of AEX and HIC as a single unit operation.

By "single unit operation" is meant herein that two serially connected chromatography apparatuses (AEX and HIC) are used in a single operation step.

Prior to the first ion exchange chromatography step, the cell slurry produced in the bioreactor is typically clarified (i.e. all cellular material, such as whole cells and cell debris, is removed).

Furthermore, prior to the first ion exchange chromatography step, a conditioning solution (added to the cell slurry or to the antibody-containing solution that has been separated from the cell material) may be added to ensure optimal pH and conductivity conditions for this first ion exchange step.

By "flow-through fraction" is meant herein at least a portion of the loaded antibody-containing fraction that leaves the chromatography column substantially unbound and/or leaves the chromatography column at substantially the same velocity as the elution fluid. Preferably, this fraction is not substantially retained on the column during elution. Thus, the conditions are selected such that impurities, but not antibodies, are bound to the anion exchange material and to the hydrophobic interaction material.

WO2006/020622 has disclosed the use of anion exchange chromatography and hydrophobic interaction chromatography to sequentially treat a mixture of proteins to separate proteins. However, in this patent application, both (AEX and HIC) columns are used in "bind-elute" mode. Furthermore, this treatment was described as a pre-purification prior to analysis of the protein mixture by 2D electrophoresis. It is therefore a (very) small scale separation.

We have found that for large scale production purposes the method of the invention (using flow-through mode) provides a much faster separation than the presently disclosed methods using binding and eluting the desired antibody.

Advantageously, the separation mixture containing the antibodies is supplemented with an appropriate amount of lyotropic/lyophilic (kosmotropic) salt prior to HIC treatment. The anion of the salt may preferably be selected from the group consisting of phosphate, sulfate, acetate, chloride, bromide, nitrate, chlorate, iodide and thiocyanate. The cation of the salt may preferably be selected from the group consisting of ammonium ion, rubidium ion, potassium ion, sodium ion, lithium ion, magnesium ion, calcium ion, and barium ion. Preferred salts are ammonium sulfate, sodium sulfate, potassium sulfate, ammonium phosphate, sodium phosphate, potassium chloride and sodium chloride.

Preferably, replenishing the separation mixture with an appropriate amount of lyotropic salt is part of a single unit operation, e.g. mixing the salt in-line in the process stream (e.g. in a mixing chamber) prior to the HIC step.

By "an appropriate amount of lyotropic salt" is meant herein a lyotropic salt that is sufficient to allow adsorption of a substantial portion of the relevant impurities to the hydrophobic interaction material, but is low enough not to cause binding or precipitation of the product. For each purification process, the optimum amount and preferred type of salt needs to be determined. In the case of ammonium sulphate, the concentration after in-line mixing is most likely between 0.1 and 1.0M.

AEX treatment according to the invention may be carried out in an AEX unit, which may be achieved by a classical packed bed column containing a resin, a column containing monolith material, a radial column containing suitable chromatographic media, an adsorption membrane unit, or any other chromatographic device known in the art with suitable media and ligands to function as an anion exchanger. In an AEX column, the chromatographic material may be in the form of a particulate support material having either strong or weak cationic ligands attached thereto. The anion exchangers in the form of membranes consist of a support material in the form of one or more sheets to which either strong or weak cation ligands are attached. The support material may comprise an organic material, or an inorganic material, or a mixture of organic and inorganic materials. Suitable organic materials are agarose based media and methacrylates. Suitable inorganic materials are silica, ceramics and metals. The membrane-form anion exchanger may be composed of hydrophilic polyethersulfone containing cationic ligands. Suitable strong cationic ligands are based, for example, on quaternary amine groups. Suitable weakly cationic ligands are for example based on primary, secondary or tertiary amine groups or any other suitable ligand known in the art.

The HIC treatment according to the invention may be carried out in a HIC unit, which may be achieved by a classical column containing resin, a column based on monolith material, a radial column containing suitable chromatographic media, an adsorption membrane unit, or any other chromatographic device known in the art with a suitable ligand acting as a hydrophobic interaction material. In a HIC column, the chromatographic material may be in the form of a particulate support material having hydrophobic ligands attached thereto. The membrane-like chromatographic device consists of a support material in the form of one or more sheets to which hydrophobic ligands are attached. The support material may comprise an organic material, or an inorganic material, or a mixture of organic and inorganic materials. Suitable organic materials include, for example, hydrophilic carbohydrates (e.g., crosslinked agarose, cellulose, or dextran) or synthetic copolymer materials (such as poly (alkyl asparagine), copolymers of 2-hydroxyethyl methacrylate and ethylene dimethacrylate, or acylated polyamines). Suitable inorganic support materials are, for example, silica, ceramics, and metals. The membrane-form HIC may be composed of hydrophilic polyethersulfone containing hydrophobic ligands. Suitable ions of hydrophobic ligands are straight or branched alkanes (such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl or octyl), aromatic groups (such as phenyl), ethers or polyethers such as polypropylene glycol.

Antibodies that can be purified according to the method of the invention are antibodies 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 antibody may be a human or non-human (such as rodent) or chimeric (e.g. humanized) antibody, or may be a subunit of an immunoglobulin as described above, or may be a hybrid protein consisting of an immunoglobulin part and a part derived from or identical to another protein (non-immunoglobulin).

Surprisingly, the antibody material resulting from the combined AEX and HIC treatment typically has a very high purity (referring to protein content) of at least 98%, preferably at least 99%, more preferably at least 99.9%, even more preferably at least 99.99%.

The anion exchange chromatography step according to the invention is preferably carried out at neutral or slightly alkaline pH. It will remove negatively charged impurities such as DNA, host cell proteins, protein a (if present), viruses (if present), proteinaceous media components such as insulin and insulin-like growth factors (if present).

In the subsequent hydrophobic interaction chromatography step, the majority of the remaining macromolecular impurities (the majority of product aggregates) will be removed, which takes advantage of the more hydrophobic nature of macromolecular impurities than monomeric products and sets the conditions so that they bind to the chromatography apparatus while the product flows through.

Subsequently, the highly purified material typically has to be processed by ultrafiltration and diafiltration (diafiltrations) to remove all residual low molecular weight impurities, replace the buffer with the final formulation buffer, and adjust the desired final product concentration. This step also ensures the removal of the added lyotropic salt.

In addition, the highly purified material typically has to be treated to ensure complete removal of infectious agents (such as viruses and/or prions) that may be present.

The invention also relates to a single operating unit containing both an anion exchange chromatography fraction (AEX) and a hydrophobic interaction chromatography fraction (HIC), the two fractions being connected in series. This single operation unit also comprises an inlet at the upstream end of the anion exchange chromatography section and an outlet at the downstream end of the hydrophobic interaction chromatography section. This single operation unit also comprises a connection between the anion exchange chromatography part and the hydrophobic interaction chromatography part, which connection further comprises an inlet for supplying a lyotropic salt solution to the latter part and thus to the separation mixture.

In the process according to the invention, the liquid stream may be passed through any commercial dual pump chromatography system, for exampleexplorer (ge), bioprocess (ge), any dual pump HPLC system, or any custom device consistent with fig. 1 (consistent with fig. 1). Most of these chromatography devices are designed to operate a single chromatography unit (i.e. column or membrane). With a simple adaptation, an additional connection can be made to place the anion exchanger after the pump a, before the mixing chamber.

Fig. 1 shows a basic structure. The series sequential connection of two chromatographic devices plus an optional pre-filter in the positions indicated in fig. 1 may lead to an undesirable pressure build-up. Therefore, under some conditions, additional technical adaptations (e.g., an additional pump after the AEX unit and a pressure relief device before the AEX unit) may have to be included in the figure.

Drawings

FIG. 1: a single operating unit comprising both an anion exchange chromatography part and a hydrophobic interaction chromatography part. Buffer a is a conditioning and washing buffer suitable for optimal operation of the AEX step. Buffer B contains lyotropic salts and is mixed with load/buffer a in the ratio necessary to obtain the optimal conditions for the operation of the HIC step. The mixing ratio may be achieved with a fixed volumetric mixing flow or may be automatically controlled by a material feedback loop based on, for example, conductivity output results. MC is an optional mixing chamber that may contain any type of static mixer.

L-load

PA ═ pump a

PB is pump B

AEX ═ anion exchange unit

HIC ═ hydrophobic interaction chromatography unit

pH sensor

Sigma-conductivity sensor

PF (optional Pre-Filter)

Examples

Materials and methods

All experiments were performed with IgG1 produced by clone P419 of the human cell line per.

The culture was performed in fed-batch format using chemically defined medium, after which the cells were removed by three-step depth filtration with the filter sequences Zetaplus 10M02P, Zetaplus 60ZA05 and SterAssure PSA020, all from Cuno (3M).

The thus clarified harvest contained 7.5g/L IgG and was stored at 2-8 ℃.

First, primary purification by standard protein a chromatography was performed using mabselect (ge) using standard procedures (loading of clarified harvest, first wash with 20mM Tris +150mM NaCl, second wash with ph5.5 buffer, and elution with buffer ph 3.0). In order to find optimal buffer conditions for subsequent purification, the second wash and elution was performed with 100mM acetate buffer or with 100mM citrate buffer.

After MabSelect elution, the eluted peak was collected and kept at pH 3.5 for 1 hour. Thereafter, the sample was neutralized to pH7.4 with 2M Tris pH 9.0 and diluted with demineralised water to set the conductivity to 5.0mS, and then the sample was filtered through 0.22 μ M.

The material thus obtainable was pre-purified IgG in acetate Tris buffer or in citrate Tris buffer. With this material, 3 series of experiments were performed: 1. determining the optimal conditions for AEX chromatography in flow-through mode (experiment 1); 2. determination of the optimal conditions for the flow-through mode of HI-chromatography (experiment 2); 3. the optimal AEX and HIC conditions were combined in one single unit operation experiment (experiment 1).

HCP was measured by ELIZA using polyclonal anti-per.c6hcp.

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

Experiment 1.

Determination of the optimal conditions for anion exchange chromatography in flow-through mode

AEX chromatographic separation in flow-through mode was performed using the above mentioned pre-purified IgG in acetate Tris buffer or in citrate Tris buffer. The following AEX media were tested: mustang Qcoins (0.35ml) (Pall), Sartobind Q capsule (1ml), ChromaSorb capsule (0.08ml) (Millipore) (all membrane adsorbers), and packed bed columns (applied Biosystems) (1ml packed bed) using Poros 50HQ resin were used.

All AEX medium utilizationThe explorer was operated with a 40 bed volume/hr flow through. The conditioning and washing buffers were either 100mM acetate Tris pH7.4 (for product runs in acetate buffer) or 100mM citrate Tris pH7.4 (for product runs in citrate buffer). The amount of product loaded in each AEX medium was 1.5g/ml membrane or bed volume.

HCP was measured before and after the chromatographic separation step. HCP removal is considered to be the most important for AEX chromatographic separation performance. For the aforementioned anion exchangers (all single experiments), the log reductions for HCP were 1.9, 1.7, 1.8 and 2.1, respectively. All AEX media performed quite poorly with the citric acid media, HCP log reductions of Mustang Q, Chromasorb, and Poros 50HQ were 1.2, 0.2, and 1.3, respectively. These results indicate that all AEX chromatographic media tested were suitable for gross removal of HCP using acetate buffer, and that HCP log reduction was almost comparable under these conditions.

Experiment 2

Determination of optimal conditions for hydrophobic interaction chromatography in flow-through mode

For the HIC step, 4 resins were tested: phenyl Sepharose FF lowclub (GE), Toyopearl PPG 600(Tosoh), Toyopearl Phenyl 600(Tosoh), Toyopearl butyl600 (Tosoh).

For these experiments, the prepurified IgG was in 100mM Tris acetate buffer at pH7.4 with a conductivity of 5.0 mS. In addition, to increase the amount of aggregates to about 20%, the MabSelect prepurified IgG-containing material was incubated at pH4 and 50 ℃ for 40 min.

For conditioning and washing, 100mM Tris acetate buffer (buffer A) at pH7.4, conductivity 5.0mS was used, mixed in-line with a volume percentage of buffer B. Buffer B contained 2M ammonium sulfate in 100mM acetate Tris buffer, pH 7.4. All resins were tested during product loading using in-line mixing (on a volume basis) with buffer B. For each resin, several percent ratios of load/buffer a and buffer B were tested. All columns were 1ml in volume, flow rate was 100ml/hr, amount of IgG in the load was 0.29g/l, load was 100 ml.

Both the load and the flow-through were sampled and analyzed.

At 0% B, both Toyopearl phenyl 600, Toyopearl butyl600 already bound IgG as well as the majority of aggregates. Therefore, it was concluded that: these resins are not suitable for aggregate removal using P419IgG in flow-through mode under the conditions applied.

Phenyl Sepharose FF lowpub (not shown), Toyopearl PPG 600 (see table 1) both obtained good aggregate clarification in a flow-through manner using in-line mixing of buffer B containing ammonium sulphate at a certain ratio.

TABLE 1 aggregate clarification with ammonium sulphate containing buffer B mixed in-line at different volume ratios using Toyopearl PPG 600.

Example 1

Purification of IgG in a single unit operation at optimal AEX and HIC conditions.

The AEX unit and HIC unit are coupled in series in the sequence shown schematically in FIG. 1. For AEX, Mustang Q coin was used, and for HIC, a column containing 3ml of Toyopearl PPG 600 resin was used.

For resin conditioning before product loading, 100mM Tris acetate buffer (buffer A) at pH7.4, conductivity 5.0mS was used. At the same time, buffer B was mixed in-line at 22% volume ratio. Buffer B contained 2M ammonium sulfate in 100mM acetate Tris buffer, pH 7.4.

The loading of pre-purified IgG was started by pumping IgG at a similar flow rate as buffer a, while the pumping of buffer a was stopped. 362ml of solution containing 4.37g IgG was loaded. After loading was complete, the pump was switched back to buffer a in order to recover all product from the system. Thereafter, the in-line mixing of buffer B was stopped, thus 100% buffer a (collected separately) was used to desorb (strip) HIC units. Throughout the run, the flow rate through the HIC was 185 ml/hr. The total time (including conditioning, washing and stripping) was 3.5 hours. The load and flow-through were analyzed for IgG aggregate ratio, DNA content, HCP content and protein (product) content (a)²⁸⁰). HCP reduction > log 2.3 (amount of HCP in flow through was below LoD). The amount of aggregate was 5.8% in the load and 1.2% in the flow-through. Based on A²⁸⁰The overall recovery of product was 86.7% without desorption and 90.1% with desorption included.

Abbreviations used:

Claims (6)

1. A method for purifying antibodies from a protein mixture produced in a bioreactor, comprising at least an intermediate purification step and a polishing step, wherein the intermediate purification step and polishing step comprise anion exchange chromatography (AEX) and Hydrophobic Interaction Chromatography (HIC), connected in series in sequence, the anion exchange chromatography producing a separation mixture in the form of a flow-through fraction, the hydrophobic interaction chromatography producing a purified antibody preparation in the form of a flow-through fraction, and wherein the purified antibody preparation is subjected to at least one further purification step.

2. The method of claim 1, wherein anion exchange chromatography and hydrophobic interaction chromatography are performed in two separation devices connected in series.

3. The method of claim 1, wherein the series of sequentially connected AEX and HIC are performed in a single unit operation.

4. A method according to any one of claims 1 to 3, wherein the separation mixture is supplemented with an appropriate amount of lyotropic salt prior to HIC.

5. A method according to any one of claims 1 to 4, wherein the separation mixture is supplemented with appropriate amounts of ammonium sulphate, sodium sulphate, potassium sulphate, ammonium phosphate, sodium phosphate, potassium chloride and sodium chloride prior to HIC.

6. A single operation unit for use in the method of any one of claims 1 to 5, comprising both an anion exchange chromatography part and a hydrophobic interaction chromatography part connected in series, wherein the outlet of the anion exchange chromatography part is connected to the inlet of the hydrophobic interaction chromatography part, 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 hydrophobic interaction chromatography part, and wherein the unit further comprises an inlet between the anion exchange chromatography part and the hydrophobic interaction chromatography part.