WO2025248017A1

WO2025248017A1 - Method of purifying recombinant proteins

Info

Publication number: WO2025248017A1
Application number: PCT/EP2025/064866
Authority: WO
Inventors: Curtis William PHIPPEN; Neil Alan WATSON
Original assignee: UCB Biopharma SRL
Current assignee: UCB Biopharma SRL
Priority date: 2024-05-31
Filing date: 2025-05-28
Publication date: 2025-12-04
Anticipated expiration: 2026-11-30

Abstract

The invention relates to the field of manufacturing recombinant proteins, including recombinant antibody molecules. In particular, methods are provided herein for purifying such recombinant proteins using expanded bed adsorption (EBA) chromatography where the cell culture fluid is loaded under low shear conditions.

Description

METHOD OF PURIFYING RECOMBINANT PROTEINS

FIELD OF THE INVENTION

The current invention relates to the field of manufacturing recombinant protein, including recombinant antibody molecules and, in particular, to a method of purifying such recombinant proteins using chromatography in expanded bed adsorption mode.

BACKGROUND OF THE INVENTION

In the field of therapeutics the use of biological entities such as proteins, including antibodies and antibody-derived molecules in particular, has been constantly gaining presence and importance, and, with it, the need for controlled large-scale manufacturing processes has developed in parallel.

Broadly speaking, these recombinant protein manufacturing processes can be divided into 2 stages, upstream processing which includes cell growth and production of the protein, and downstream processing that refers to protein purification. Traditionally in processes based on mammalian cell culture there is an intermediate harvesting step in which the supernatant containing the recombinant protein is separated from the cells and debris and then subjected to purification via chromatography. When using microbial cells to produce recombinant proteins it has been feasible to use a technique known as expanded bed adsorption (EBA) chromatography, which allows the combining of the primary recovery step with the first chromatography step, i.e. purification of biomolecules directly from unclarified mixtures, thus allowing to avoid steps such as filtration, centrifugation, associated holding times etc.

Contrary to more traditional packed-bed chromatography methods in which the resin is confined between the bottom of the column and the flow adapter, in EBA the columns are fed from below and the adapter is held away from the packed-resin level, giving the resin room to expand and thus creating spaces between the beads. EBA uses a series of pumps and valves connected through the adapter and the bottom of the column that control the flow rate and direction of the buffer and sample loading. As buffer is injected from below the resin becomes fluidized and the beads form a stable density gradient when their sedimentation velocity equals the upward liquid flow velocity.

The additional space generated using this technique allows particulate matter and debris to flow around the resin beads and avoid column blocking that takes place in the traditional packed-bed methods. This technique therefore allows for the processing of viscous and particulate liquids. However, although EBA was used when purifying recombinant proteins produced in microbial cells, with the use of mammalian host cells that allow higher productivity, there is also an increase in cell debris leading to column fouling when loading in upflow and pressure issues when attempting to elute the bound product in downflow. The cell debris blocks the bottom of the column and in addition lipid and DNA released by the lysed cells sticks to the resin causing pressure issues. As a consequence, EBA chromatography has been discarded in favour of harvesting techniques that separate the cells and other debris from the supernatant, such that a clarified cell culture fluid is applied to the first chromatography step.

There remains therefore a need in the art for improved processes of recombinant antibody production that enable the use of expanded bed adsorption chromatography when the cell culture fluid contains high levels of particulates.

SUMMARY OF THE INVENTION

The current invention refers to a method of purifying a recombinant protein comprising an expanded bed adsorption chromatography step characterized in that the loading of the cell culture fluid is performed under low shear conditions. The method of the invention further refers to a method of purifying a recombinant antibody comprising an expanded bed adsorption protein A chromatography step wherein the loading of the cell culture fluid is performed under low shear conditions, optionally through the use of a diaphragm pump.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a loading schematic according to the method of the invention, where a Quattroflow pump is used for loading onto the EBA resin.

Figure 2 shows an elution profile obtained for an exemplary IgG using the method of the invention with a loading schematic as shown in figure 1. 92.3% of IgG recovery was obtained with a cellular density of 18.3x10⁶ cells/mL.

Figure 3 shows an elution profile obtained for a TrYbe antibody using the method of the invention with a loading schematic as shown in figure 1. 90.2% of TrYbe recovery was obtained with a cellular density of 28.0x10⁶ cells/mL.

Figures 4 and 5 show a loading schematic according to the method of the invention, where the cell culture fluid is loaded via a flowpath that is independent from the pump. Figure 6 shows an elution profile obtained for an exemplary IgG using the method of the invention with a loading schematic as described in Example 3. 87.6% of IgG recovery was obtained with a cellular density of 14.1x10⁶ cells/mL

DETAILED DESCRIPTION OF THE INVENTION

The current invention solves the above-outlined problem by providing a novel method for purifying a recombinant protein comprising an expanded bed adsorption (EBA) chromatography step wherein the cell culture fluid is loaded into the column under low shear conditions, and optionally the load and equilibration buffer have an osmolality of 270-330 mOsm/kg. In the context of the present invention the EBA load is the cell culture fluid as recovered from the bioreactor. Thus, in the present invention, the cell culture fluid loaded on the EBA chromatography step is an unclarified cell culture fluid, i.e. the cells and debris have not been removed from the cell culture fluid through the use of techniques such as centrifugation or filtration.

Cells are typically cultured in a relatively low shear stress environment and high levels of shear stress may lead to considerable attrition and release of intracellular components that can then contribute to blocking of the chromatography column. It is further hypothesized that cell lysis is limited during the loading phase through the use of the conditions of the current invention and consequently fouling due to bound cell debris, lipid and DNA is reduced.

Shear stress occurs when a force is applied to a material tangentially to the area of said material, in contrast to normal stress when the force is applied perpendicularly. Any fluids, such as a cell culture fluid, moving along a solid boundary, such as boundaries present in tubing, pumps or chromatography columns, will incur a shear stress at that boundary.

In the context of the current invention, loading a cell culture fluid under “low shear conditions" means loading the cell culture fluid under conditions that provide low shear stress, i.e. conditions that minimize frictional forces exerted on the cells by the flow of the cell culture fluid during loading. Typically, the chromatography bed is fluidized by adding equilibration buffer through the use of one or more pumps, and once this has been achieved the cell culture fluid is loaded onto the chromatography column. Generally speaking in EBA chromatography methods the cell culture fluid is added through the bottom of the column.

Low shear conditions during loading of the expanded bed adsorption chromatography may be obtained through different means. One possibility is to introduce the cell culture fluid into the flow path to the chromatography resin after the pump. Alternatively, it is possible to use a low shear pump, such as for example a diaphragm pump. In the context of the present invention a diaphragm pump, also known as a membrane pump, is a type of positive displacement pump. It uses a combination of the reciprocating action of a flexible diaphragm, typically made of rubber, thermoplastic, or Teflon, and suitable valves on either side of the diaphragm. The pump operates by flexing the diaphragm, which causes the volume of the pump chamber to increase and decrease. This action draws fluid into the chamber when the volume increases (the diaphragm moving up) and forces the fluid out when the chamber pressure increases from decreased volume (the diaphragm moving down). This cycle repeats, allowing the pump to transfer, compress, and evacuate the medium.

A low shear pump suitable in the context of the present invention is for example a Quattroflow® pump.

Therefore, in a first aspect the present invention refers to a method for the purification of a recombinant protein wherein the method comprises an expanded bed adsorption chromatography step characterized by loading the cell culture fluid onto the expanded bed adsorption chromatography column under low shear conditions.

High levels of shearing during loading of the cell culture fluid can result in cell lysis and decreased cell viability. Cell lysis and/or a decrease in cell viability may be avoided by loading the cell culture fluid under low shear conditions. In a particular embodiment of the invention, the cell density of the cell culture fluid is decreased by less than 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, or 5% after loading. In a particular embodiment, the cell density of the cell culture fluid is decreased by less than 5% after loading. In a particular embodiment of the invention, the cell viability of the cell culture fluid is decreased by less than 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% after loading. In a particular embodiment, the cell viability is decreased by less than 1% after loading.

In a particular embodiment of the invention the cell culture fluid is loaded onto the column via a flowpath that is independent from the pump (i.e. , without going through a pump).

In a particular embodiment of the invention the cell culture fluid is loaded onto the column directly from the bioreactor (i.e., without going through a pump).

In an alternative embodiment the pump maintaining the expanded bed is a diaphragm pump. In a further particular embodiment the diaphragm pump is a Quattroflow® pump. In a particular embodiment of the method of the invention, the expanded bed adsorption chromatography column is equilibrated using an equilibration buffer having an osmolality of 270-330 mOsm/kg. In a particular embodiment of the invention, the chromatography step comprises a first wash step to remove impurities. In a further particular embodiment, said first wash step is performed with equilibration buffer.

Once the majority of the cells have been washed off the column, as is readily determined by visual inspection, a further wash step may be implemented. Hence, in a further particular embodiment the method of the invention additionally comprises a second wash step wherein the wash buffer comprises a detergent.

Detergents are often used in bioprocessing due to their ability to break lipid-lipid and lipid-protein interactions without disrupting protein-protein interactions. This makes them ideal for processes where maintaining protein structure is important. During affinity chromatography detergents are frequently used to prevent non-specific binding during purification. Typically detergents can be divided in ionic vs non-ionic detergents. Some examples of non-ionic detergents suitable for use in the method of the invention include:

Polyoxyethylene-based detergents: These detergents contain a neutral, polar head group and the tails are composed of hydrophobic oxyethylene chains. They are effective at isolating active membrane proteins.

Glycosidic detergents: These detergents use a sugar as the head group, such as glucose or maltose, and contain an alkyl polymer tail. They are also effective at breaking lipid-lipid and lipid- protein interactions.

NP-40: This is a non-denaturing, mild lysis agent that works well when performing protein analysis.

Tween 20 and Tween 80: These are mild lysis agents that are good for cell lysis and protein isolation.

Examples of ionic detergents include:

Anionic detergents such as Alkyl sulfates that include sodium dodecyl sulphate and sodium octyl sulphate. Alkyl Sulphonates sodium heptane sulphonate.

Bile acids and salts deoxycholate acid and sodium cholate hydrate.

Cationic detergents such as Benzalkonium chloride, tetradecyltrimethylammonium bromide or methylbenzethonium.

In a particular embodiment of the current invention the detergent is a non-ionic detergent, in a further particular embodiment the non-ionic detergent is Triton or tergitol. The appropriate amount of detergent may be determined empirically by the skilled person depending on the chosen detergent and the specific cell culture fluid to be applied to the EBA chromatography. In a particular embodiment of the method of the invention the wash buffer comprises between about 0.05% and about 3% tergitol, between about 0.06% and about 2.9% tergitol, between about 0.065% and about 2.8% tergitol, between about 0.07% and about 2.7% tergitol, between about 0.075% and about 2.6% tergitol, between about 0.08% and about 2.5% tergitol, between about 0.085% and 2.4% about tergitol, or between about 0.09% and about 2.3% tergitol. Alternatively the wash buffer comprises between about 0.05% and about 2% tergitol, between about 0.06% and about 1.9% tergitol, between about 0.065% and about 1.8% tergitol, between about 0.07% and about 1.7% tergitol, between about 0.075% and about 1.6% tergitol, between about 0.08% and about 1.5% tergitol, between about 0.085% and about 1.4% tergitol, or between about 0.09% and about 1.3% tergitol, between about 0.09% and about 1.2% tergitol, or between about 0.09% and about 1.1% tergitol. In a particular embodiment of the method of the invention the wash buffer comprises 0.1 % tergitol.

In a further embodiment of the method of the invention, the first and second wash steps of the EBA column are performed in upflow.

In the context of the current invention performing a step in “downflow” means applying the fluid through the top of the column such that the fluid will flow downwards, i.e. in the case of a wash step performed in downflow the corresponding wash buffer will be added through the top of the chromatography column. Similarly, performing a step in “upflow” means applying the fluid through the bottom of the column such that the fluid will flow upwards.

As with any chromatography there is typically a loading step, a wash step and an elution step designed to recover the bound protein.

In the case of EBA one would typically allow the bed to settle after the last wash step and before starting the elution step.

In a particular embodiment the method of the invention additionally comprises an elution step performed in downflow.

In a further particular embodiment the method of the invention comprises a last wash step that is performed in downflow prior to elution.

The inventors have surprisingly found that the EBA chromatography method of the invention provides a higher binding capacity for the resin than what has previously been described. Therefore, in a particular method of the invention the binding capacity of the resin is at least 20 grams of protein per liter of resin, e.g. 20 g/L, at least 25 g/L, at least 30 g/L, at least 35 g/L, at least 40 g/L, at least 45 g/L, or at least 50 g/L.

The protein binding principles in EBA are the same as in classical column chromatography, and common ion-exchange, hydrophobic interaction, and affinity chromatography ligands can be used. As a skilled artisan would know the binding of proteins to any of these resins is primarily dependent on the pH of the buffers used during the chromatography steps and the pl of the protein to be purified, it is therefore within the skilled artisan’s reach to choose a suitable buffer and then achieve the desired osmolality range of the current invention through the addition of salt.

Many recombinant protein therapeutics are based on antibodies or antibody derived molecules which are typically purified via a first affinity capture step based on protein A.

Protein A is a cell wall component produced by several strains of Staphylococcus aureus that binds to the Fc region of antibody molecules. Affinity chromatography using immobilized protein A as a ligand has been extensively used for antibody purification and remains to date the core purification step in most antibody purification processes, allowing a high elimination of impurities from the starting material. Furthermore, protein A is also known to bind VH3 regions present on antibody molecules, giving rise to certain purification strategies for alternative antibody formats that lack an Fc region based on an affinity interaction between the VH3 region and protein A.

Therefore, in a particular embodiment of the method of the invention the expanded bed adsorption chromatography step comprises a protein A resin.

There are multiple providers of protein A chromatography materials available to the skilled person. In a further particular embodiment of the method of the invention the resin is protein A Endure from Biotoolomics.

Protein A has high affinity for Fc domains at neutral pH. As a consequence, starting material containing the antibody to be purified is typically loaded on the protein A resin at neutral pH. A typical process will be followed by one or more steps of washing the chromatography material with a buffer that is also at neutral pH to ensure removal of as many impurities as possible. And finally, an elution step is necessary to recover the bound antibody from the protein A. This elution step involves the use of an elution buffer with an acidic pH (typically from about 2.5 to about 4.0) that will disrupt the interaction between the antibody and protein A.

In a particular embodiment of the invention, the protein A EBA chromatography step the equilibration buffer is selected from PBS, sodium acetate, phosphate, HEPES, Histidine, Tris, or any buffer that the pKa is within the range pH 5.0 to pH 9.0. As stated previously it is within the skilled person’s reach to adjust the buffering solution to the desired osmolality. In a particular embodiment of the current invention the equilibration buffer is sodium acetate with 0.1M NaCI at pH 7.0. In a further particular embodiment, the equilibration buffer is PBS.

In a specific embodiment, the current invention refers to a method for the purification of a recombinant antibody wherein the method comprises a protein A expanded bed adsorption chromatography step comprising:

Loading the cell culture fluid under low shear conditions and

Using equilibration buffers having an osmolality of 270-330 mOsm/kg

Wherein a first wash is performed using equilibration buffer,

- A second wash is performed using a buffer containing a non-ionic detergent such as tergitol, and

- An elution step from which the recombinant antibody is obtained.

In a further particular embodiment of the method for the purification of an antibody according to current invention, the loading and first and second wash steps are performed in upflow.

In a further particular embodiment, the method for the purification of an antibody according to current invention optionally comprises a third wash step performed in downflow.

In a further particular embodiment of the method for the purification of an antibody according to current invention, a diaphragm pump, such as a Quattroflow® pump, is used to fluidize the chromatography bed. Optionally the cell culture fluid is loaded onto the chromatography column through the diaphragm pump. Alternatively, the cell culture fluid is loaded onto the chromatography column via a flowpath that is independent from the path.

In a further embodiment the method of the invention will comprise one or more additional chromatography steps to remove remaining impurities. Generally, such steps will employ a nonaffinity chromatography step using a solid phase with appropriate functionality for use in gel filtration chromatography, cation chromatography, anion chromatography, mixed-mode chromatography, hydrophobic chromatography, and hydrophobic charge induction chromatography. These may be operated in bind and elute mode or in flow-through mode. In flow- through mode, the impurities bind to or have reduced mobility in the solid phase whereas the target protein is recovered in the flow-through fraction. Appropriate solid phases for use in chromatography such as beaded resins or membranes with the appropriate functionality are readily available to the skilled artisan. In a particular embodiment according to the method of the invention, the method additionally comprises a step of anion exchange chromatography operated in the flow-through mode. In a further particular embodiment the method of the invention comprises a protein A chromatography step followed by a first chromatography step that is an anion exchange chromatography producing a flow-through containing the protein and a second chromatography step that is a cation exchange chromatography from where an eluate containing the protein is recovered.

In another embodiment, the method of the invention comprises a protein A chromatography followed by a first chromatography step that is a cation exchange chromatography from where an eluate containing the protein is recovered, and a second chromatography step that is an anion exchange chromatography to produce a flow-through containing the protein.

In another embodiment, the method of the invention comprises a protein A chromatography followed by a first chromatography step that is a hydrophobic interaction chromatography from where a flow-through fraction containing the protein is recovered, and a second chromatography step that is a cation exchange chromatography to produce an eluate containing the protein.

In a further embodiment, the method of the invention comprises a protein A chromatography followed by a first chromatography step that is an anion exchange chromatography from where an eluate containing the protein is recovered, and a second chromatography step that is a hydrophobic interaction chromatography to produce a flow-through containing the protein.

In a further embodiment, the method of the invention comprises a protein A chromatography followed by a first chromatography step that is an anion exchange chromatography from where an eluate containing the protein is recovered, and a second chromatography step that is a hydrophobic interaction chromatography to produce an eluate containing the protein.

In another embodiment, the method of the invention comprises a protein A chromatography followed by a first chromatography step that is an anion exchange chromatography from where a flow-through fraction is collected containing the protein, and a second chromatography step that is a hydrophobic interaction chromatography to produce a flow-through containing the protein.

In another embodiment, the method of the invention comprises a protein A chromatography followed by a first chromatography step that is an anion exchange chromatography from where a flow-through fraction is collected containing the protein, and a second chromatography step that is a hydrophobic interaction chromatography to produce an eluate containing the protein.

Antibodies

The terms "antibody" or "antibodies" as used herein include monoclonal and polyclonal antibodies. Furthermore, the terms "antibody" or "antibodies" as used herein include, but are not limited to, recombinant antibodies that are generated by recombinant technologies as known in the art. "Antibody" or "antibodies" include antibodies' of any species, in particular of mammalian species; such as human antibodies of any isotype, including IgD, IgGi, lgG2a, lgG2b, IgGs, lgG4 IgE and antibodies that are produced as dimers of this basic structure including IgGAi, lgGA₂, or pentamers such as IgM and modified variants thereof, non-human primate antibodies, e.g. from chimpanzee, baboon, rhesus or cynomolgus monkey; rodent antibodies, e.g. from mouse, or rat; rabbit, goat or horse antibodies; and camelid antibodies (e.g. from camels or llamas such as Nanobodies™) and derivatives thereof; or of bird species such as chicken antibodies or of fish species such as shark antibodies. The term "antibody" or "antibodies" also refers to "chimeric" antibodies in which a first portion of at least one heavy and/or light chain antibody sequence is from a first species and a second portion of the heavy and/or light chain antibody sequence is from a second species. Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g. Old World Monkey, such as baboon, rhesus or cynomolgus monkey) and human constant region sequences. "Humanized" antibodies are chimeric antibodies that contain a sequence derived from non-human antibodies. For the most part, humanized antibodies are human antibodies (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region [or complementarity determining region (CDR)] of a non-human species (donor antibody) such as mouse, rat, rabbit, chicken or non-human primate, having the desired specificity, affinity, and activity. In most instances residues of the human (recipient) antibody outside of the CDR; i.e. in the framework region (FR), are additionally replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody properties. Humanization reduces the immunogenicity of non-human antibodies in humans, thus facilitating the application of antibodies to the treatment of human disease. Humanized antibodies and several different technologies to generate them are well known in the art. The terms "antibody" or "antibodies" also refer to human antibodies, which can be generated as an alternative to humanization. For example, it is possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of production of endogenous murine antibodies. Other methods for obtaining human antibodies/antibody fragments in vitro are based on display technologies such as phage display or ribosome display technology, wherein recombinant DNA libraries are used that are either generated at least in part artificially or from immunoglobulin variable (V) domain gene repertoires of donors. Phage and ribosome display technologies for generating human antibodies are well known in the art. Human antibodies may also be generated from isolated human B cells that are ex vivo immunized with an antigen of interest and subsequently fused to generate hybridomas which can then be screened for the optimal human antibody. The term “antibody” or “antibodies” as used herein, also refers to an aglycosylated antibody.

The term "antibody" or "antibodies" as used herein not only refers to full-length antibodies of any species, including from human (e.g. IgG) and other mammalian species, but also refers to an antibody fragment. A fragment of an antibody comprises at least one heavy or light chain immunoglobulin domain as known in the art and binds to one or more antigen(s). Examples of antibody fragments according to the invention include Fab, Fab', F(ab')2, and Fv and scFv fragments; as well as diabodies, triabodies, tetrabodies, minibodies, domain antibodies (dAbs), such as sdAbs, V_HH and V_NAR fragments, single-chain antibodies, bispecific, trispecific, tetraspecific or multispecific antibodies formed from antibody fragments or antibodies, including, but not limited to, Fab-Fv or Fab-Fv-Fv constructs. Antibody fragments as defined above are known in the art.

In one embodiment, the antibody that is purified using the method of the invention does not comprise any of the following motifs: a polyhistidine motif, an HQ motif, an HN motif or a HAT motif, wherein a polyhistidine motif is a sequence of five or more consecutive histidine residues, a HQ motif is a sequence comprising at least three alternations of histidine and glutamine (HQHQHQ (SEQ ID NO:7)), a HN motif is a sequence comprising at least three alternations of histidine and asparagine (HNHNHN (SEQ ID NO:8)) and a HAT motif is the sequence KDHLIHNVHKEEHAHAHNK (SEQ ID NO:9).

In one embodiment of the method of the invention, the antibody to be purified is an antibody comprising an Fc region, or at least comprising a CH2 and a CH3 domain.

In another embodiment, the antibody to be purified is an antibody that contains a VH3 region and binds the affinity chromatography resin via the VH3 region.

In another embodiment, the antibody is selected from: IgG, Fab’, F(ab’)2, scFv, Fab-Fv, Fab-scFv, Fab-(scFv)2, Fab-(Fv)2, diabodies, triabodies, and tetrabodies.

In one embodiment of the method of the invention the antibody is a FabFv or disulfide stabilized form thereof as disclosed in PCT/EP2014/074409, incorporated herein by reference.

In one embodiment, the antibody comprises a binding domain specific to human serum albumin, in particular with CDRs or variable regions as disclosed in WQ2013/068563, incorporated herein by reference.

In one embodiment the antibody, such as a Fab-dsFv format is one disclosed in PCT/EP2014/074409 or WQ2014/019727, incorporated herein by reference. In another embodiment the antibody is a Fab-scFv fusion protein format disclosed in WO20 13/068571 , incorporated herein by reference.

In another embodiment the antibody is a multi-specific antibody molecule comprising or consisting of: a) a polypeptide chain of formula (I):

VH-CH1-X-V1 ; and b) a polypeptide chain of formula (II):

VL-CL-Y-V2; wherein:

VH represents a heavy chain variable domain;

CH1 represents a domain of a heavy chain constant region, for example domain 1 thereof;

X represents a bond or linker;

Y represents a bond or linker;

V1 represents a dsFv, a sdAb, a scFv or a dsscFv;

VL represents a light chain variable domain;

CL represents a domain from a light chain constant region, such as Ckappa;

V2 represents dsFv, a sdAb, a scFv or a dsscFv; wherein at least one of V1 or V2 is a dsFv or dsscFv, described in WO2015/197772 incorporated herein by reference.

"Single chain variable fragment" or "scFv" as employed herein refers to a single chain variable fragment comprising or consisting of a heavy chain variable domain (VH) and a light chain variable domain (VL) which is stabilised by a peptide linker between the VH and VL variable domains. The VH and VL variable domains may be in any suitable orientation, for example the C- terminus of VH may be linked to the N-terminus of VL or the C-terminus of VL may be linked to the N-terminus of VH.

"Disulphide-stabilised single chain variable fragment" or "dsscFv" refers to a single chain variable fragment which is stabilised by a peptide linker between the VH and VL variable domain and also includes an inter-domain disulphide bond between VH and VL. "Disulphide-stabilised variable fragment" or "dsFv" refers to a variable fragment which does not include a peptide linker between the VH and VL variable domains and is instead stabilised by an interdomain disulphide bond between VH and VL.

In one particular embodiment, the antibody is the multispecific antibody of the format Fab-2x dsscFv described in WO2015/197772, incorporated herein by reference.

In a further particular embodiment, the multispecific antibody of the format Fab-2x dsscFv is a trivalent antibody, i.e. each Fv binds to a different epitope.

In a further particular embodiment the multispecific antibody has a Fab-dsscFv-dsFv format as described in WO2015/197772, incorporated herein by reference.

In one embodiment, the antibody to be purified comprises a sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

SEQ ID NO:1 : (a) Heavy chain variable domain of anti-albumin antibody (no ds)

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTFYATWAK GRFTISRDNSKNTVYLQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTLVTVSS

SEQ ID NO:2: (b) Heavy chain variable domain of anti-albumin antibody (ds)

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKCLEWIGIIWASGTTFYATWAK GRFTISRDNSKNTVYLQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTLVTVSS

SEQ ID NO:3: (c) Light chain variable domain of anti-albumin antibody (no ds)

DIQMTQSPSSVSASVGDRVTITCQSSPSVWSNFLSWYQQKPGKAPKLLIYEASKLTSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCGGGYSSISDTTFGGGTKVEIKRT

SEQ ID NO:4: (d) Light chain variable domain of anti-albumin antibody (ds)

DIQMTQSPSSVSASVGDRVTITCQSSPSVWSNFLSWYQQKPGKAPKLLIYEASKLTSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCGGGYSSISDTTFGCGTKVEIKRT

SEQ ID NO:5: 645 gH5gL4 specific to albumin

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIGIIWASGTTFYATWAK GRFTISRDNSKNTVYLQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTLVTVSSGGGGS GGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCQSSPSVWSNFLSWYQQKPGKAP KLLIYEASKLTSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGGGYSSISDTTFGGGTKVEIKR T

SEQ ID NO:6: 645 gH5gL4ds specific to albumin EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKCLEWIGIIWASGTTFYATWAK GRFTISRDNSKNTVYLQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTLVTVSSGGGGS GGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCQSSPSVWSNFLSWYQQKPGKAP KLLIYEASKLTSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGGGYSSISDTTFGCGTKVEIKR T

A recombinant antibody or antibody derivative, such as an antibody fragment, manufactured for large-scale commercial purposes can be produced by culturing eukaryotic host cells transfected with one or more expression vectors encoding the recombinant antibody. The eukaryotic host cells are preferably mammalian cells, more preferably Chinese Hamster Ovary (CHO) cells.

Mammalian cells may be cultured in any medium that will support their growth and expression of the antibody, preferably the medium is a chemically defined medium that is free of animal-derived products such as animal serum and peptone. There are different cell culture mediums available to the person skilled in the art comprising different combinations of vitamins, amino acids, hormones, growth factors, ions, buffers, nucleosides, glucose or an equivalent energy source, present at appropriate concentrations to enable cell growth and protein production. Additional cell culture media components may be included in the cell culture medium at appropriate concentrations at different times during a cell culture cycle that would be known to those skilled in the art.

The osmolarity of mammalian cell culture medium is a critical parameter for maintaining cell health and function. Osmolarity is the number of osmoles of solute per liter of solution, which is different than osmolality, which is the osmoles of solute per kilogram of solution. Osmolality refers to the concentration of solute particles (such as salts, sugars and other molecules) in a solution. In cell culture, osmolality directly affects the cell volume and overall cellular homeostasis; the typical osmolality range for mammalian cell culture media is approximately 280-300 mOsm/kg.

Mammalian cell culture can take place in any suitable container such as a shake flask or a bioreactor, which may or may not be operated in a fed-batch mode depending on the scale of production required. These bioreactors may e.g. be stirred-tank or air-lift reactors. Various large scale bioreactors are available with a capacity of more than 1 ,000 L to 50,000 L, preferably between 5,000 L and 20,000 L, or between 2,000L and 10,000 L. Alternatively, bioreactors of a smaller scale such as between 2 L and 200L, such as 80L or 100 L may also be used to manufacture an antibody to be purified according to the method of the invention.

Mammalian cells are typically cultured until a cell density of at least 0.1x10⁷ is reached. In a particular embodiment, the method of the invention comprises loading a cell culture fluid with a cell density of at least 0.1x10⁷ cells/mL, 0.5x10⁷ cells/mL, 1x10⁷ cells/mL, 2x10⁷ cells/mL, 3x10⁷ cells/mL, 4x10⁷ cells/mL or 5x10⁷ cells/mL, preferably the cell density is at least 0.1 x10⁷ to 0.3x10⁷ cells/mL. Different methods and equipment, which are well known to the skilled person are available for the determination of the cell density, such as the Cell Viability Analyzer or the Vi- CELL BLU or Vi-CELL XR from Beckman Coulter. The latter provides an automatic means to determine the cell density with the Trypan Blue Dye Exclusion method.

An antibody or antigen-binding fragment thereof that can be manufactured in accordance with the methods of the present invention is typically found in the supernatant of a mammalian host cell culture, typically a CHO cell culture.

Large-scale antibody production methods typically involve the use of cells which express high amounts of antibody. Mammalian cells, such as CHO cells, used for industrial scale antibody production typically have an expression titer of at least 1 g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7 g/L, 8g/L, 9g/L or 10g/L. In a particular embodiment of the current invention, the cell culture fluid comprises antibody in a concentration of at least 1 g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8 g/L, 9g/L or 10g/L. In one embodiment of the invention, the method comprises culturing CHO cells that express the antibody of interest, recovering the cell culture fluid, and purifying said antibody from the mixture wherein said purification comprises at least one affinity chromatography step performed according to the method of the invention.

The method of the present invention is particularly suitable for industrial scale (also referred to herein as large-scale) production of antibodies. Accordingly, in a particular embodiment, the method of the invention is an industrial-scale method.

Industrial scale methods are typically carried out on a very large scale. Commercial bioreactors for producing antibodies, typically contain hundreds, thousands or even tens of thousands liters of culture medium. The large volume of cell culture fluid, which comprises large amounts of antibody, subsequently needs to be purified on industrial scale, which requires a properly sized chromatography column.

In a particular embodiment of the method of the invention, the bioreactor has a capacity of at least 2,000L, preferably between 2,000L and 50,000L, preferably between 5,000L and 20,000L, or 15,000L. In a particular embodiment, 2,000, 5,000L, 10,000L, 15,000L or 20,000L of cell culture fluid is loaded onto an expanded bed adsorption chromatography column under low shear conditions.

Columns used for industrial scale purification methods typically have a packed resin bed height of between 15 to 35 cm. In a particular embodiment, the chromatography column used in the method of the invention has a packed resin bed height of at least 15 cm, 18 cm, 20 cm, 25 cm, 30 cm or 35 cm. The packed resin bed height is the height of the resin bed in packed mode, i.e. when the resin is not fluidized due to the upward liquid flow.

Columns for use in industrial scale purification methods typically have a diameter ranging from 10 to 125 cm. In a particular embodiment, the chromatography column used in the method of the present invention has a diameter of at least 10 cm, 20 cm, 30 cm, 40 cm, 50 cm, 75 cm, 100 cm, 125 cm.

In a specific embodiment of the invention, the EBA chromatography resin of the invention has a binding capacity of at least 20 grams of protein per liter of resin, e.g. 20 g/L, at least 25 g/L, at least 30 g/L, at least 35 g/L, at least 40 g/L, at least 45 g/L, or at least 50 g/L.

Industrial scale purification methods are typically performed using several EBA columns simultaneously. In a further particular embodiment of the invention, a single pump is used to load cell culture fluid on two or more EBA columns simultaneously. In a particular embodiment the pump is a diaphragm pump, such as Quattroflow® pump.

Alternatively, host cells are preferably prokaryotic cells, preferably Gram-negative bacteria. More preferably, the host cells are E. coli cells. Prokaryotic host cells for protein expression are well known in the art (Terpe, K. (2006). Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 72, 211-222.). The host cells are recombinant cells which have been genetically engineered to produce the protein of interest such as an antibody fragment. The recombinant E. coli host cells may be derived from any suitable E. coli strain including from MC4100, TG1. TG2, DHB4, DH5a, DH1 , BL21 , K12, XLI BIue and JM109. One example is E. coli strain W3110 (ATCC 27,325) a commonly used host strain for recombinant protein fermentations. Antibody fragments can also be produced by culturing modified E. coli strains, for example metabolic mutants or protease deficient E. coli strains.

An antibody fragment that can be purified in accordance with the methods of the present invention is typically found in either the periplasm of the E. coli host cell or in the host cell culture supernatant, depending on the nature of the protein, the scale of production and the E. coli strain used. The methods for targeting proteins to these compartments are well known in the art (Makrides.S.C. (1996). Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev 60, 512-538.). Examples of suitable signal sequences to direct proteins to the periplasm of E. coli include the E. coli PhoA, OmpA, OmpT, LamB and OmpF signal sequences. Proteins may be targeted to the supernatant by relying on the natural secretory pathways or by the induction of limited leakage of the outer membrane to cause protein secretion examples of which are the use of the pelB leader, the protein A leader, the co-expression of bacteriocin release protein, the mitomycin-induced bacteriocin release protein along with the addition of glycine to the culture medium and the co-expression of the kil gene for membrane permeabilization.

Expression of the antibody in the E. coli host cells may also be under the control of an inducible system, whereby the expression of the recombinant antibody in E. coli is under the control of an inducible promoter. Many inducible promoters suitable for use in E. coli are well known in the art and depending on the promoter expression of the recombinant protein can be induced by varying factors such as temperature or the concentration of a particular substance in the growth medium. Examples of inducible promoters include the E.coli lac, tac, and trc promoters which are inducible with lactose or the non-hydrolyzable lactose analog, isopropyl-b-D-1 -thiogalactopyranoside (IPTG) and the phoA, trp and araBAD promoters which are induced by phosphate, tryptophan and L-arabinose respectively. Expression may be induced by, for example, the addition of an inducer or a change in temperature where induction is temperature dependent. Where induction of recombinant protein expression is achieved by the addition of an inducer to the culture the inducer may be added by any suitable method depending on the fermentation system and the inducer.

E. coli host cell cultures (fermentations) may be cultured in any medium that will support the growth of E. coli and expression of the recombinant protein. The medium may be any chemically defined medium such as e.g. described in Durany O,C.G.d.M.C.L.-S.J. (2004). Studies on the expression of recombinant fuculose-1-phosphate aldolase in Escherichia coli. Process Biochem 39, 1677-1684.

Culturing of the E. coli host cells can take place in any suitable container such as a shake flask or a fermenter depending on the scale of production required. Various large scale fermenters are available with a capacity of more than 1 ,000 liters up to about 100,000 liters. Preferably, fermenters of 1 ,000 to 50,000 liters are used, more preferably 1 ,000 to 25,000, 20,000, 15,000, 12,000 or 10,000 liters. Smaller scale fermenters may also be used with a capacity of between 0.5 and 1 ,000 liters.

Fermentation of E. coli may be performed in any suitable system, for example continuous, batch or fed-batch mode depending on the protein and the yields required. Batch mode may be used with shot additions of nutrients or inducers where required. Alternatively, a fed-batch culture may be used and the cultures grown in batch mode pre-induction at the maximum specific growth rate that can be sustained using the nutrients initially present in the fermenter and one or more nutrient feed regimes used to control the growth rate until fermentation is complete. Fed-batch mode may also be used pre-induction to control the metabolism of the E. coli host cells and to allow higher cell densities to be reached. If desired, the host cells may be subject to collection from the fermentation medium, e.g. host cells may be collected from the sample by centrifugation, filtration or by concentration.

In one embodiment the process according to the present invention comprises a step of centrifugation and cell recovery prior to extracting the antibody.

For E. coli fermentation processes wherein the protein of interest such as an antibody fragment is found in the periplasmic space of the host cell it is required to release the protein from the host cell. The release may be achieved by any suitable method such as cell lysis by mechanical or pressure treatment, freeze-thaw treatment, osmotic shock, extraction agents or heat treatment. Such extraction methods for protein release are well known in the art.

Definitions

In order that the present invention may be more readily understood, certain terms are defined below. In the absence of a definition, terms may be construed as they would be by the skilled person working in the present technical field.

Where the term “comprising” is used in the present description and claims, it does not exclude other elements. For the purposes of the present disclosure, the term “consisting of” is considered to be a preferred embodiment of the term “comprising of”. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group which preferably consists only of these embodiments. Also provided are embodiments, consisting essentially of what is set out.

Where an indefinite or definite article is used when referring to a singular noun, e.g. “a”, “an” or “the”, this includes a plural of that noun unless something else is specifically stated. The terms “about” or “approximately” denote an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value of ±10%, and preferably of ±5%.

EXAMPLES

Example 1

The below example details how to load unclarified mammalian cell culture from a bioreactor onto a Protein A EBA column. The Protein A EBA columns used for lab experiments has a diameter of 2.5cm up to 10cm but can be scaled up where needed. The packed bed height of the column is <25cm with PrA EBA Endure ™ resin provided by Biotoolomics.

A typical process would see an IgG molecule loaded utilizing the solutions and phases shown in table 1 & 2 to capacities <50g/L resin. The setup would resemble that shown in figure 1 , where a Quattroflow® pump (diaphragm pump) is used to transfer the cell culture feed from the bioreactor into the EBA column. This method is expected to work for a range of different antibody molecules and formats. To date we have purified multiple IgGs according to this method, including lgG1s and lgG4s.

Table 1 - Typical Process Solutions

PBS is typically used for the equilibration, as its osmolality is ~290mOsm/kg. Note that these solutions can be optimized based on the product that is being purified.

Small adaptations may be required for different molecules. For instance, experiments for one lgG4 utilized 50mM Sodium phosphate, 0.1 M NaCI, pH 7.0, as a lower pH is required, this buffer was designed with an osmolality of ~300mOsm/kg. Table 2 - PrA EBA Phases

Product is collected during the elution phase based on optimized absorbances. This should result in eluate volumes ~2CV (column volumes). Figure 2 shows an elution profile obtained for an IgG using the low shearing pump.

The same experiment was performed with a standard pump classically used in the pharmaceutical industry (i.e. AKTA AVANT 150 which is a piston pump). With the standard pump, an increasing amount of cell debris caused the EBA column to clog during the loading phase. Cell viability and cell density of the cell culture fluid were measured and compared before and after loading with the diaphragm pump and the standard AKTA piston pump (see Table 3). To this effect, cell viability and cell density were measured before and after the low shearing pump (i.e. Quattroflow® Pump) and the standard pump (i.e. AKTA pump). Table 3: Effect of Diaphragm Pump and Standard Pump on cell viability and cell density

Table 3 shows that the Quatroflow pump has negligible impact on the cell density (3.3% decreased after going through the pump) and on cell viability (0.7% decreased after going through the pump), whereas the standard AKTA pump has a significant impact on the cell density (50% decreased after going through the pump) and on the cell viability (40% decreased after going through the pump).

Example 2

Where example 1 utilized an IgG format molecule, example 2 was performed with a Fab-2x dsscFv as described in WO 2015/197772, also referred to as a TrYbe molecule. The solutions and phases were the same as described in Tables 1 and 2 above, with the exceptions of load challenge being <25g/L, the equilibration buffer was optimized for this molecule to 0.2M Glycine, 50mM NaCI pH8.3, osmolality 310mOsm/kg. Figure 3 shows an elution profile for a TrYbe antibody using the above-described process.

Example 3

Cell Loading After Pump (CLAP) is performed with the same solutions as those used in example 1. The phases are also the same with the following exception during the loading, which was setup in such a way to ensure very low levels of shear stress on the cells.

Instead of the cell culture being pumped through a diaphragm pump and then onto the EBA, in CLAP the cell culture is loaded into an offline hold tank via gravity or pressure. Once the EBA has completed the upflow equilibration the flow path is adjusted to include the hold tank. The cell culture is then loaded onto the EBA, as the pump fills the hold tank with a chase buffer (equilibration buffer), which displaces the cell culture due to the linear flow. See figure 4 & 5. CLAP can be operated with the flow in either direction within the hold tank, as long as the EBA flow remains in upflow. Displacing the hold tank via upflow, as shown in the schematics, however, causes back mixing leading to an increase in chase buffer required to empty the tank. The volume of chase buffer required will therefore, be equal or greater than the hold tank to ensure complete loading.

Claims

1. A method for the purification of a recombinant protein wherein the method comprises an expanded bed adsorption chromatography step characterized by loading the cell culture fluid under low shear conditions.

2. A method according to claim 1 comprising using an equilibration buffer having an osmolality of 270-330 mOsm/kg.

3. A method according to claim 1 or 2 further comprising a wash step using equilibration buffer.

4. A method according to claim 1 or 2 further comprising a wash step wherein the wash buffer comprises a detergent.

5. A method according to 4 wherein the detergent is a non-ionic detergent such as tergitol.

6. A method according to any preceding claim, wherein a diaphragm pump is used to fluidize the chromatography bed, such as a Quattroflow pump.

7. A method according to any preceding claim wherein all steps are performed in upflow.

8. A method according to any preceding claim further comprising an elution step.

9. A method according to any preceding claim wherein the chromatography step is a protein A expanded bed adsorption chromatography.

10. A method according to any preceding claim wherein the recombinant protein is a recombinant antibody.

11. A method according to claim 10 wherein the antibody is selected from: IgG, Fab’, F(ab’)2, scFv, Fab-Fv, Fab-scFv, Fab-(scFv)2, Fab-(Fv)2, diabodies, triabodies, and tetrabodies