TECHNICAL DOMAIN

This disclosure concerns a method for protein purification.

BACKGROUND

With the increasing number of protein therapeutic candidates, especially monoclonal antibodies (mAbs) entering various stages of development, biopharmaceutical companies are increasingly looking at innovative solutions to deliver this pipeline. For antibody manufacturing process development, maintaining desired quality attributes while reducing time to market, maintaining cost effectiveness, and providing manufacturing flexibility are key issues in today's competitive market. Since antibody therapies may require large doses over a long period of time, the drug substance must be produced in large quantities with cost and time efficiencies to meet clinical requirements and pave the way toward commercialization. This is also the case for other recombinant therapeutic proteins including but not limited to fusion proteins, therapeutic enzymes and antibody fragments.

Generally, proteins are produced by cell culture, using animal cell lines, bacterial cell lines or viruses engineered to produce the protein of interest. The cell lines are in the presence of a complex growth medium comprising sugars, amino acids, and growth factors. For use as a human therapeutic, the target molecules or target protein expressed by the cultured cells must be treated to reach a high level of purity. Known processes for protein purification currently include a large number of steps starting with initial purifications, viral inactivation steps and final purification steps, which are often referred to as polishing. Although required for achieving a high level of purity, polishing may lead to significant protein yield loss, which is to be avoided from a commercial point of view.

It is the aim of the present disclosure to provide a protein production process that provides a high yield of the desired protein while reducing the operation expenses (OPEX). The present disclosure further aims to allow the whole process of protein purification to be performed in a safe and limited amount of space. SUMMARY

Disclosed herein are methods for purifying a molecule of interest. In some embodiments, the molecule of interest is a protein of interest. In some embodiments, the protein is present in a feed. In some embodiments, the method comprises the step of (a) performing a clarification step on a cell culture harvest comprising a protein of interest by an anion exchange step, thereby obtaining a feed comprising the protein of interest. In some embodiments, the method comprises (b) performing a protein capture step on the feed of step (a) or a feed derived thereof by chromatography, thereby obtaining an eluate comprising the protein of interest. In some embodiments, the method comprises (c) performing a polishing step on the eluate of step (b) or an eluate derived thereof. In some embodiments, the polishing comprises an ion exchange step. In some embodiments, the method comprises obtaining the purified protein of interest. In some embodiments, in step (a) the anion exchange step is a liquid anion exchange step. In some embodiments, in step (a), electropositive compounds are added to the cell culture harvest. In some embodiments, the electropositive compound is polydiallyl dimethylammonium chloride (pDADMAC or polyDDA). In some embodiments, in step (a) one or more compounds selected of the group of fatty acids having 7 to 10 carbon atoms and derivatives thereof or ureides are further added to the cell culture harvest. In some embodiments, the cell culture harvest is filtered through a filter. In some embodiments, the filter comprises a layer of diatomaceous earth (DE). In some embodiments, the chromatography step of step (b) is an affinity chromatography step or an ion exchange chromatography step. In some embodiments, the chromatography step is a protein A chromatography step or a cation exchange chromatography step. In some embodiments, the chromatography step is combined with a simultaneous viral inactivation step. In some embodiments, the chromatography step is performed in batch or in continuous mode. In some embodiments, during the polishing step the feed is contacted with a multimodal ion exchanger. In some embodiments, the multimodal ion exchanger comprises a ligand with a positively or negatively charged moiety. In some embodiments, the multimodal ion exchanger step is the sole polishing step of the method. In some embodiments, the multimodal ion exchanger comprises a ligand with a hydrophobic moiety. In some embodiments, the multimodal ion exchanger comprises a ligand with a non-hydrophobic moiety. In some embodiments, the eluate is loaded on the multimodal ion exchanger under high conductivity conditions. In some embodiments, the multimodal ion exchanger is a multimodal anion exchanger. In some embodiments, the protein is a monoclonal antibody. In some embodiments, a concentration step occurs prior to the protein capture step. In some embodiments, viruses present in the feed are inactivated during step (b). In some embodiments, the protein of interest is a monoclonal anti-TNFa antibody or a fragment thereof.

In some embodiments, disclosed herein are methods comprising the step of: (a) performing a clarification step on a cell culture harvest comprising a protein of interest by the addition of one or more compounds selected of the group of fatty acids having 7 to 10 carbon atoms and derivatives thereof, ureides and/or electropositive compounds to the cell culture harvest and subsequently filtering of the harvest through a diatomaceous earth layer, thereby obtaining a feed comprising the protein of interest. In some embodiments, the method comprises (b) performing a protein capture step by chromatography on the feed of step (a) or a feed derived thereof, thereby obtaining an eluate comprising the protein of interest. In some embodiments, the method comprises (c) performing a polishing step on the eluate of step (b) or an eluate derived thereof on a multimodal chromatography resin. In some embodiments, the resin comprises a ligand with a positively or negatively charged moiety or both, thereby allowing binding of the protein of interest to the resin. In some embodiments, the method comprises (d) obtaining the purified protein of interest by eluting the protein from the resin of step c. In some embodiments, viruses present in the feed are inactivated during step b. In some embodiments, the protein of interest is a monoclonal anti-TNFa antibody or a fragment thereof.

Disclosed herein are methods for purifying a protein of interest. In some embodiments, the protein is present in a feed. In some embodiments, the method comprises performing a clarification step on a cell culture harvest comprising a protein of interest by the addition of one or more compounds selected of the group of fatty acids having 7 to 10 carbon atoms and derivatives thereof, ureides and/or electropositive compounds to the cell culture harvest and subsequently filtering of the harvest through a diatomaceous earth layer, thereby obtaining a feed comprising the protein of interest. In some embodiments, the method comprises performing a protein capture step by chromatography on the feed of step (a) or a feed derived thereof, thereby obtaining an eluate comprising the protein of interest. In some embodiments, the method comprises performing a polishing step on the eluate of step (b) or an eluate derived thereof on a multimodal chromatography resin. In some embodiments, the resin comprises a ligand with a positively charged moiety, thereby allowing binding of the protein of interest to the resin. In some embodiments, the method comprises obtaining the purified protein of interest by eluting the protein from the resin of step. In some embodiments, viruses present in the feed are inactivated. In some embodiments, the protein of interest is a monoclonal anti-TNFa antibody or a fragment thereof.

Disclosed herein are purification platforms. In some embodiments, the purification platform comprises at least a clarification unit, a protein capture unit and/or a polishing unit. In some embodiments, the polishing unit comprises a device capable of performing multimodal ion exchange chromatography. In some embodiments, the polishing unit comprises a multimodal anion exchange chromatography. In some embodiments, the device capable of performing multimodal ion exchange chromatography is the sole polishing device. In some embodiments, the protein capture unit and polishing unit are serially connected, wherein an outlet of the protein capture unit is connected to an inlet of the polishing unit. In some embodiments, an outlet of the clarification unit is connected to an inlet of the protein capture unit. In some embodiments, the chromatography compartment comprises a protein A affinity chromatography resin or a cation exchange chromatography resin.

FIGURES

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

Figure 1A is a flow chart depicting a protein purification process according to the prior art and an embodiment of the current disclosure.

Figure IB is a flow chart depicting a protein purification process according to an embodiment of the current disclosure.

Figure 2 depicts a protein purification platform suitable for performing a process according to an embodiment of the current disclosure.

Figure 3 shows a comparison between a conventional purification process and a process as described herein, quantifying the impurities present in the feed during the substeps of both processes. Figure 4 shows an embodiment of a protein platform.

Figure 5 shows the reduction of impurities due to the use of an advanced clarification according to an embodiment of the disclosure.

Figure 6 shows the results of a twin column continuous capture according to an embodiment of the current disclosure.

Figure 7 shows the effect of a SP-TFF step prior to the capture step on capture productivity.

Figure 8 shows the results of polishing by multimodal anion exchange (AEX). Figure 9 shows the potential capacity of a platform according to an embodiment of the current disclosure.

DETAILED DESCRIPTION

Methods and a platform are disclosed for production and purification of a biomolecule, in some embodiments, protein production and purification - such as the production of monoclonal antibodies - from a cell culture harvest produced in a bioreactor, using specific cell culture clarification, protein purification and protein polishing steps. In some embodiments, the methods allow high quality protein purification with a minimal amount of steps, thereby lowering the operational costs and allowing the process of protein purification to occur safely in a limited amount of space and time.

Unless otherwise defined, all terms used herein, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present disclosure.

As used herein, the following terms have the following meanings:

"A", "an", and "the" refers to both the singular and plural unless the context clearly dictates otherwise. By way of example, "a compartment" refers to one or more than one compartment.

"About" as referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/-20% or less, preferably +/-10% or less, more preferably +/-5% or less, even more preferably +/-1% or less, and still more preferably +/-0.1% or less of and from the specified value, in so far such variations are appropriate to perform in the disclosed invention. However, it is to be understood that the value to which the modifier "about" refers is itself also specifically disclosed.

"Comprise," "comprising," and "comprises" and "comprised of" are synonymous with "include", "including", "includes" or "contain", "containing", "contains" and are inclusive or open-ended terms that specifies the presence of what follows e.g. component and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein. The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints.

The expression "% by weight" (weight percent), here and throughout the description unless otherwise defined, refers to the relative weight of the respective component based on the overall weight of the formulation. The expression "1% w/w" refers to what can be understood as lg of respective component per 100 g of the formulation, the expression "1% w/v" refers to what could be understood as lg of respective component per 100 ml. of the formulation, the expression "1% v/v" refers to what can be understood as 1 ml. of respective component per 100 ml. of formulation.

"Cell culture harvest", "culture harvest" and "harvest" are used as synonyms and refer to the unclarified cell culture obtained from culturing cells in a bioreactor. The cultured cells or the grown cells also are referred to as host cells.

"Bioreactor" refers to any device or system that supports a biologically active environment, for example for cultivation of cells or organisms for production of a biological product. This would include cell stacks, roller bottles, shakes, flasks, stirred tank suspension bioreactors, high cell density fixed bed perfusion bioreactors, etc.

"Purification" refers to the substantial reduction of the concentration of one or more target impurities or contaminants relative to the concentration of a biomolecule of interest, particularly a protein.

"A single operation unit" means serially connected devices used in a single operation step.

"Protein" refers to any of a class of nitrogenous organic compounds which have large molecules composed of one or more chains of amino acids. "Protein" may be any sort of protein such as (monoclonal) antibodies, antibody fragments, fusion proteins, enzymes, recombinant proteins, peptides, polypeptides or other biomolecules expressed by cells.

"Antibody" refers to any immunoglobulin molecule, antigen-binding immunoglobulin fragment or immunoglobulin fusion protein, monoclonal or polyclonal, derived from human or other animal cell lines, including natural or genetically modified forms such as humanized, human, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies. Commonly known natural immunoglobulin antibodies include IgA (dimeric), IgG, IgE, IgG and IgM (pentameric).

An "antibody charge variant" as used herein is an antibody or fragment thereof wherein the antibody or fragment thereof has been modified from its native state such that the charge of the antibody or fragment thereof is altered. Antibody charge variants are often referred to as acidic, neutral and/or basic antibody species.

"IgG" refers to any immunoglobulin G molecule, monoclonal or polyclonal, derived from human or other mammalian cell lines, including natural or genetically modified forms such as humanized, human, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies.

"Protein aggregate" or "aggregate of a protein" refers to an association of at least two protein molecules. The association may be either covalent or non-covalent without respect to the mechanism by which the protein molecules are associated. The association may be direct between the protein molecules or indirect through other molecules that link the protein molecules together. Examples of the latter include but are not limited to disulfide linkages with other proteins, hydrophobic associations with lipids, charge associations with DNA, affinity associations with leached protein A, or mixed mode associations with multiple components.

"Feed" refers to a clarified cell culture harvest or a clarified protein sample comprising a biomolecule of interest such as a protein or raw protein harvest.

"Viral inactivate" means that a feed comprising a protein of interest further comprises inactivated viruses and/or viral particles previously existing in their active form in a bioreactor.

"Particulates" refers to solid particles suspended in a liquid.

"Flocculation" refers to the aggregation, precipitation and/or agglomeration of soluble (e.g., chromatin components) and/or insoluble (e.g., cells) particles caused by the addition of a suitable flocculating agent to a suspension. By increasing the particle size of the insoluble components present in the suspension, the efficiency of solid/liquid separations, such as by filtration or centrifugation, is improved. Flocculation of a cell culture leads to the formation of "floccules" which comprise host cell impurities such as cell material including cells and cell debris or host cell proteins, DNA or other components present therein.

"Filtration" or "separation" refers to the removal of an aqueous phase, comprising soluble biomolecules of interest, from insoluble particles, for example following flocculation of a cell culture harvest.

"Fatty acid" refers to an unmodified or a modified fatty acid wherein the fatty acids may be saturated or unsaturated. "Fatty acid" also includes fatty acid derivatives which comprise a salt of the fatty acid or a fatty acid whose carboxylic acid group is reversibly converted into another group to form amides, esters, glycerides, sugars. The term "fatty acid" also refers to fatty acids that are provided with side-chains, such as, without limitation, one or more alkyl groups.

"Ureide" refers to any class of organic compound derived from urea by replacing one or more of its hydrogen atoms by organic groups including compounds derived from the acylation of urea, and diureide compounds which contain two molecules of urea, or radicals thereof. Ureides can have a cyclic or acylclic structure and include but are not restricted to allantoin and allantoic acid.

"Electropositive compound" is to be understood as molecules having a positive net charge and able to bind to or form complexes with negatively charged molecules.

As used herein, "polishing" refers to a step, preferably a chromatographic step, used to remove residual host cell impurities, product-related impurities (product fragments and/or aggregated species and/or charge variants) and/or virus contaminants, thereby (further) purifying the biomolecule (such as a protein) of interest. Polishing is often preceded by a protein capture step.

"Mixed-mode" and "multimodal" chromatography are used interchangeably herein and refer to a chromatographic method wherein separation of components in a mobile phase is based on more than one interaction type between a stationary phase and components of the mobile phase. Mixed-mode chromatography is, for example, based on simultaneous hydrophobic and/or non-hydrophobic interactions and electrostatic interactions between the mobile phase and the stationary phase. A "multimodal or mixed-mode anion exchanger" and "multimodal or mixed-mode anion exchange ligand" refers to a component of the solid phase which is suited for use in multimodal chromatography and which has a positively charged moiety.

A "multimodal or mixed-mode cation exchanger" and "multimodal or mixed-mode cation exchange ligand" refers to a component of the solid phase which is suited for use in multimodal chromatography and which has a negatively charged moiety.

"High Conductive Conditions" are to be understood as those conditions or measures that exist during loading of a feed or buffer of such feed and binding of a feed or buffer of such feed to a resin whereby the conductivity of the feed or buffer of said feed is above 75mS/cm, more preferably above 85mS/cm. Often, high conductivity is obtained by having an increased salt concentration of the feed buffer, e.g. above 1 M NaCI and more. The skilled person is aware that a different salt will have a different effect on conductivity.

The term "solid phase" is used to mean any non-aqueous matrix to which one or more ligands can adhere or alternatively, in the case of size exclusion chromatography, it can refer to the gel structure of a resin. The solid phase can be any matrix capable of adhering ligands in this manner, e.g., a purification column, a discontinuous phase of discrete particles, a membrane, filter, gel, etc. Examples of materials that can be used to form the solid phase include polysaccharides (such as agarose and cellulose) and other mechanically stable matrices such as silica (e.g. controlled pore glass), poly(styrenedivinyl)benzene, polyacrylamide, ceramic particles and derivatives of any of these.

"Buffering compound" refers to a chemical compound employed for the purpose of stabilizing the pH of an aqueous solution within a specified range. Phosphate is one example of a buffering compound. Other common examples include but are not limited to compounds such as acetate, citrate, borate, MES, Tris, and HEPES, among many others.

"Buffer" refers to an aqueous formulation comprising a buffering compound and other components required to establish a specified set of conditions to mediate control of a chromatography method. The term "equilibration buffer" or "conditioning buffer" refers to a buffer formulated to create the initial operating conditions. "Wash buffer" refers to a buffer formulated to displace unbound contaminants from a chromatography support. "Elution buffer" refers to a buffer formulated to displace the one or more components from the chromatography matrix.

The terms "equilibration" and "conditioning" are used herein interchangeably and refer to adjusting the conditions to create the initial operating conditions.

An "isolator" or "biosafety cabinet" are used herein as synonyms and refer to a containment system, which prevents the escape of a biological agent into the immediate environment. This ventilated workspace for safely working with materials contaminated with (or potentially contaminated with) pathogens requiring a defined biosafety level is usually equipped with high efficiency particulate air (HEPA) filters and may or may not be open-fronted.

An "isolator" or "biosafety cabinet" are used herein as synonyms and refer to a containment system, which prevents the escape of a biological agent into an immediate environment. An isolator or biosafety cabinet may be a ventilated and may provide a workspace for safely working with materials contaminated with (or potentially contaminated with) pathogens that require a defined biosafety level and which are usually equipped with high efficiency particulate air (HEPA) filters and may or may not be open-fronted.

A "containment enclosure" refers to a system of containment, usually involving specialized air handling, airlocks, and secure operating procedures, which prevents the escape of a biological agent into the external environment or into other working areas. Said containment enclosure may serve as a cleanroom. In certain embodiments, containment enclosure may be used as a synonym for an isolator or biosafety cabinet.

In a first aspect of the present disclosure, a method for purifying a protein is disclosed, wherein said protein is present in a feed, and wherein said method comprises the steps of:

(a) Performing a clarification step on a cell culture harvest comprising a protein of interest by an anion exchange step, thereby obtaining a feed;

(b) Performing a protein capture step on the feed of step (a) or a feed derived thereof by chromatography, thereby obtaining an eluate; (c) Performing a polishing step on the eluate of step b or an eluate derived thereof, wherein said polishing comprises an ion exchange step; thereby obtaining said purified protein of interest.

In some embodiments, the cell culture harvest of the disclosure comprises at least one biological target such as a protein of interest such as antibodies, antibody fragments, fusion proteins, enzymes, recombinant proteins, or other proteins expressed by the cells. In one embodiment, the protein of interest is a monoclonal antibody. In a further embodiment, the protein of interest is a monoclonal anti-TNFa antibody or a fragment thereof.

Cell culture harvest treatment and clarification - step (al

In some embodiments, the clarification step performed on the cell culture harvest (step a) ensures removal of cell debris and other contaminants from the crude cell culture harvest in a filtration step based on the use of diatomaceous earth (DE) as a filtration aid. In some embodiments, as a result of clarification, a feed comprising the protein of interest is obtained and is suited for downstream processing steps. Preferably, the current method uses a clarification step according to PCT/EP2018/058366 and US62/670,220 which content is incorporated herein by reference in its entirety.

In some embodiments, the clarification step disclosed herein is based on the formation of floccules in the cell culture harvest, followed by efficient removal of the floccules, thereby resulting in a feed that comprises the protein of interest.

In some embodiments, the clarification step of the current method includes an anion exchange step. In one embodiment, the anion exchange step is a liquid anion exchange step. This liquid anion exchange step is performed, in an embodiment, by addition of electropositive compounds to the cell culture harvest. Electropositive compounds are thought to bind negatively charged components derived from host cells such as, but not limited to, nucleic acids including host cell DNA and RNA, and host-cell viruses. Accordingly, an advantage of including an anion exchange step during cell clarification is the potential contribution of the electropositive compounds to the reduction of viral components in the cell culture harvest. In some embodiments, electropositive compounds are provided during the clarification either in a solid phase such as bound to beads or to a depth filter, or as soluble compounds thereby performing a liquid anion exchange step. Suitable electropositive compounds are any electropositive charged compounds such as electropositive polysaccharide, electropositive polymer, chitosan, chitosan derivatives such as deacetylated chitosan, synthetic polymers such as polydiallyl dimethylammonium chloride (pDADMAC or polyDDA), benzylated poly(allylamine) and polyethylenimine, commercially available particles like TREN (BioWorks, WorkBeads TREN, high) or cationic surfactants like hexadecyltrimethylammonium bromide (also known as CTAB) or any combination thereof. Without wishing to be bound by theory, electropositive polymers are thought to act as flocculation agents because they can simultaneously bind several negatively charged contaminants such as host cell DNA and RNA causing formation of a floe. In some embodiments, the abovementioned electropositive compounds perform extremely well (floe formation and floe size) when combined with filtration using DE according to the method of the disclosure.

In another embodiment, one or more compounds selected from the group of fatty acids having 7 to 10 carbon atoms and derivatives thereof or ureides are further added to the cell culture harvest during step (a). In some embodiments, the different compounds of step (a) might be mixed prior to their simultaneous addition to the cell culture. This is advantageous as it reduces the number of steps necessary for obtaining a clarified cell culture. In some embodiments, the compounds of step (a) might be added separately, sequentially and/or alternatingly to the cell culture.

Ureides and fatty acids as described above are especially suited to (further) induce precipitation and flocculation of host cell culture associated impurities in step (a) of the method according to the disclosure. Fatty acids having 7 to 10 carbon atoms, on the one hand, are thought to exert hydrophobic interactions with hydrophobic host cell derived impurities, causing their agglomeration. Suitable fatty acids may be enanthic acid (heptanoic acid), caprylic acid (octanoic acid), pelargonic acid (nonanoic acid), capric acid (decanoic acid) or any combination thereof. The fatty acid may be added in the form of a fatty acid derivative for example a fatty acid salt, such as a sodium salt, for example sodium caprylate. Ureides, on the other hand, are thought to function as binding agents by interacting with impurities in solution, for example through hydrogen bonding. Ureides are organic compounds derived from urea and can have a cyclic or acylclic structure. Ureides include, but are not restricted to, allantoin and allantoic acid.

In some embodiments, the compounds which are added to the cell culture will stimulate the precipitation of impurities and/or the aggregation or agglomeration of impurities, precipitates or particulates present in the harvest. Specifically, precipitated fractions contain host cell impurities, e.g. host cells, host cell proteins and host cell DNA. In some embodiments, flocculation of host cell impurities is achieved due to the ability of the added compounds to exert hydrophobic interactions, ionic interactions and/or hydrogen bonding with host cells related impurities present in the cell culture harvest or other mechanisms of interaction. Precipitated fractions can further include viral components present in the host cells. In some embodiments, the cell clarification in step (a) contributes to the viral clearance of the feed in multiple ways. First, medium chain fatty acids are known to have antiviral activity and have been previously shown to cause precipitation of viral particles. Furthermore, in some embodiments, electropositive compounds used in step (a) are able to bind viral particles based on electrostatic interactions with the latter. Accordingly, step (a) significantly contributes to viral clearance of the feed.

In some embodiments, simultaneous to the addition of the compounds described above or in a subsequent step, DE is added to the cell culture harvest and allowed to settle as a DE layer or cake on a surface, preferably a support filter having a filter surface (impermeable to DE) wherein the filter surface may not need to be a horizontal, flat or disc-shaped surface but can for example be candle-shaped. An embodiment of a specific filter is disclosed in US62/670,220 which is in its entirety incorporated by reference herein. In short, such vessel may comprise a flexible liner wherein said filtration vessel includes at least one filter having a surface on which the dynamic filter media accumulates into a cake, said cake and at least one filter adapted, during filtration operation, to permit a filtrate including target molecules to pass therethrough and said cake, during filtration operation, adapted to prevent unwanted solid materials from passing therethrough; and a backflush source including a backflush fluid and fluidly connected to the filtration vessel via the at least one filter, said backflush source, during backflush operation, adapted to supply backflush fluid back through the at least one filter for removing the cake formed on the filter. In an embodiment, said filtration vessel includes at least one candle filter having a surface on which the dynamic filter media accumulates into a cake, said cake and at least one filter are adapted, during normal operation, to permit a filtrate including target molecules to pass therethrough and said cake, during normal operation, adapted to prevent unwanted solid materials from passing therethrough, the filtration vessel including a flexible liner for receiving the cell culture harvest solution and in fluid communication with the at least one candle filter. In an embodiment, the filtration vessel comprises a rigid or semi-rigid outer container for receiving the flexible liner. In an embodiment, an actuator is present for collapsing the flexible liner. Said flexible liner may include a drain or an agitator. In an embodiment, at least one candle filter is suspended within the flexible liner.

Such DE cake will contain a structure comprising a plurality of channels or paths. Upon filtration of the cell culture harvest comprising the floccules through the DE layer or cake, the large matter such as cells, cell debris, and other large non-target compounds of the solution obtained) are retained by the DE cake structure, whereas the target proteins, having a smaller size, flow through the channels of the DE cake structure. In some embodiments, to facilitate such flow, a pump or other pressure dispense aid or other fluid driving mechanism is used as further described in embodiments below.

In some embodiments, the use of DE to facilitate filtration overcomes the limitations imposed by the physical changes of the cell culture after flocculation which tend to render routine clarification complex. For instance, in case of depth filtration, a very significant surface of depth filters is needed to clarify the first solution comprising the formed precipitates. Use of DE allows achieving significant operational advantages including shorter processing time, less process steps, less process materials, less equipment and solutions, without compromising the quality of the cell clarification step. Furthermore, filtration of the flocculated cell culture harvest results in a feed comprising the protein of interest.

In an embodiment, the feed obtained in step (a) can be optionally subjected to a single-pass tangential-flow filtration step in order to concentrate the proteins in the feed prior to performing protein capture.

In some embodiments, step (a) of the method for purifying a protein according to the present disclosure assures fast and easy removal of impurities including host cell derived impurities such as the cells themselves, cell debris, and other unwanted materials allowing efficient further processing of the cell culture which would otherwise be threatened by the host cell derived impurities. Use of this robust and high-throughput cell culture clarification step allows to reduce the number of post- clarification steps needed to obtain the molecule of interest, therefore reducing complexity of and time of protein production when compared to the use of cell culture clarification steps of the prior art. In addition, elimination of additional post- clarification steps results in a higher yield and the reduction of operation expenses (OPEX). Protein capture - step ( b )

In some embodiments, the method for purifying a protein according to the present disclosure comprises a protein capture step (b) which is performed on the feed or clarified harvest obtained in step (a) or a feed derived thereof by chromatography, thereby obtaining an eluate. The removal of key impurities in step (a) of the method allows for an improved performance of step (b) of the method. More in particular, preceding step (b) by the particular clarification step of (a) as described above, allows for an improved performance of the chromatography process used for capturing the protein in step (b). The protein capture step is based on a chromatographic process wherein the feed obtained in step (a) or a feed derived thereof forms the mobile phase and the solid phase comprises any non-aqueous matrix or support to which one or more ligands can adhere, for example but not limited to, a porous particle, non-porous particle, membrane or resin.

In an embodiment, the chromatography step of step (b) comprises a cation exchange chromatography step or an affinity chromatography step wherein such chromatography step comprises a column, resin, particles, media and/or a membrane.

In some embodiments, the chromatography step of step (b) may be an affinity chromatography step such as a protein A or a protein G step, preferably protein A. When making use of a clarification step as described above, the performance of protein A as capture step is considerably improved. The capacity of the resin increases due to a reduction in impurities in the feed, and as a consequence, the quality and purity of the eluate is increased. In an embodiment, the resin is washed prior to elution. In another embodiment, the resin is washed prior to elution, preferably with a buffer comprising Tris and NaCI, i.e. 50mM TRIS, 2M NaCI with pH 8. In another or a further embodiment, washes comprising a potassium salt of polyvinyl sulfate (PVS-K) and/or polyethylene imine (PEI) prior to elution from Protein A may be used to further remove DNA and histone-associated complexes and viral particles. In another embodiment, said chromatography step is an ion exchange chromatography step, preferably a cation exchange chromatography step. Use of cation exchange chromatography results in a significant reduction of the operational costs as these resins are less costly than affinity chromatography resins, have a longer lifetime and a higher productivity.

In a further embodiment, said chromatography step is combined with a simultaneous viral inactivation step. An example of such method has been extensively described in PCT/EP2018/058365 which is incorporated by reference herein. In some embodiments, the method provides for simultaneous purification and viral inactivation of a feed comprising a protein of interest in one step. This is advantageous as it reduces the amount of processing steps required to obtain a purified protein of interest.

An embodiment of such method for simultaneous viral inactivation and chromatography comprises the steps of:

(a) contacting the feed with negatively charged (separation) particles or media, such as a cation exchange chromatography step; thereby binding said protein of interest to said particles or media

(b) conditioning the particles or media such that their pH is acidic for a sufficient time after contact to inactivate one or more viruses present in said feed, and

(c) eluting said protein of interest from said particles or media.

As a result, a feed with the protein of interest and inactivated viruses is obtained.

In a further embodiment, said method for simultaneous viral inactivation and chromatography comprises the steps of:

conditioning the feed such that its conductivity is at most 15 mS/cm and its pH is at least 3,

contacting the feed with negatively charged particles or media, thereby binding the protein of interest to the particles or media (cation exchange chromatography step),

conditioning the separation particles or media such that its pH is acidic [at most 4.5] and maintaining said pH for at least 1 min, or at least 5 min, thereby inactivating viruses,

conditioning the separation particles or media such that its conductivity is at least 5 mS/cm and its pH is of from 5 to 8 thereby eluting the protein of interest.

As a result, a feed with the protein of interest and inactivated viruses is obtained. The non-limiting conditions as described in the embodiment above allow good binding of the protein of interest to the separation particles or media on the one hand and for the simultaneous inactivation of the virus on the other hand.

The method of simultaneous viral inactivation and chromatography is particularly advantageous when enveloped viruses are being used, as these viruses are easily inactivated by a low pH. In some embodiments, non-enveloped viruses may only partially be impacted by such low pH and may require further downstream handling such as viral filtration and/or anion exchange.

In an embodiment, the capture step (b) precedes the polishing step (c).

Protein polishing - step (cl

In some embodiments, the method for purifying a protein according to the present disclosure comprises a protein polishing step (c) which is performed on the eluate obtained in step (b) or an eluate derived thereof. In some embodiments, polishing comprises an ion exchange chromatography step, either an anion or cation exchange step, thereby obtaining the protein of interest.

In an embodiment, the ion exchange chromatography step comprises an ion exchange chromatography step wherein such chromatography step comprises a column, resin or membrane.

In an embodiment the eluate obtained in step (b) or an eluate derived thereof is contacted, during the protein polishing step (c), with a mixed-mode or multimodal chromatographic resin, membrane, particle or media comprising a ligand with a positively (anion) or negatively (cation) charged moiety or a combination thereof. In an embodiment, the mixed-mode chromatographic step is a mixed-mode cation resin. In another, preferred embodiment, the mixed-mode chromatographic resin is a mixed-mode anion resin. Multimodal or mixed-mode chromatography allows separation of components in a mixture (i.e. the eluate of step (b) or an eluate derived thereof) based on different types of interactions. The separation properties of the ligand are modulated by adjusting the conditions wherein interaction takes place between components of said eluate and said resin. In the currently disclosed method, the driving forces for binding and elution of the protein of interest to the resin are specifically adapted by adjusting the chromatography conditions in order to improve the chromatographic separation such that it can be performed more accurately and more selectivity towards a specific protein of interest (e.g. the monomeric form of an antibody) and even towards specific forms of the protein of interest. Simultaneously, all major attributes of ion exchange chromatography such as reduction of host cell proteins and DNA, as well as reduction of the number of viral particles are preserved as well. Hence, the method allows obtaining a highly pure protein sample, of a high quality.

In some embodiments, in a large scale process, a feed or cell culture harvest comprising a protein of interest and viruses will be subjected to a virus inactivation step or process. After virus inactivation, the virus or viral particles are still present in the feed and need to be removed in an efficient manner to ensure high protein yield and purity. Viral inactivation may be achieved in a previous step by use of a low pH, thereby inactivating the viruses in the feed, accordingly the eluate of step (b) or an eluate derived thereof may comprise inactivated viruses or viral particles. In some embodiments, by means of the current polishing step after viral inactivation, inactivated viruses and/or viral particles are further removed, thereby resulting in a very pure protein with a high yield.

In an embodiment, to achieve optimal viral clearance, the polishing step (c) comprises a mixed-mode anion chromatography step.

In some embodiments, the positively charged moiety of the ligand of the multimodal resin is suitable to perform anion exchange chromatography. Anion exchange chromatography is a powerful tool to remove impurities such as host cell proteins, DNA, endotoxin and leached Protein A. Additionally, separation using an anion exchanger is selective, and the resins used are relatively inexpensive. Accordingly, In some embodiments, during the protein polishing step an additional chromatography step is performed on the eluate obtained in step (c), thereby further enhancing the final purity of the purified protein of interest.

In an embodiment, the method comprises a single polishing step using the above described mixed-mode or multimodal resin. The use of such multimodal ion exchange chromatography (IEX) step, preferably anion exchange chromatography, provides significant polishing and removal of contaminants, thereby enhancing the quality of the purified product while the reduction of the polishing steps to a single step (compared to the prior art) assures a minimal amount of protein loss, thus enhancing the yield of the protein production process. Multimodal resins can combine different types of interactions between components of the mobile and the solid phase such as ionic interactions, hydrogen bonding, hydrophobic interaction, interactions based on size and/or molecular weight and/or interactions based on affinity interaction between components of the mobile phase and the solid phase. Accordingly, in another or further embodiment, the ligand of the multimodal resin further comprises hydrophobic or non-hydrophobic moieties such as a hydrogen moiety, which may allow further interaction with the protein of interest, depending on the binding conditions used. Hydrophobic interaction chromatography (HIC) is a useful tool for separating proteins based on their hydrophobicity, and is complementary to other techniques that separate proteins based on charge, size or affinity. In another or further embodiment, the ligand of the multimodal resin further comprises hydrophobic and non-hydrophobic moieties in addition to a charged (positively, negatively or both) moiety. The ion exchange moiety (anion and/or cation) and the hydrophobic and/or hydrogen-bonding moiety can be located on the same ligand or located on two or more different ligands comprised on the chromatography resin.

Various multimodal chromatography ligands are available commercially. Ligands well known in the art and which are compatible with the methodology as currently described are Capto-Adhere™ or Capto-Adhere™, Capto MMC HiScreen, ImpRes, Nuvia™ aPrime™ or Toyopearl NH2-750F. The choice of resin will define the loading conditions used. Resins which are able to function in both hydrophobic interaction chromatography (HIC) and ion exchange (IEX) mode allow loading in both high and low salt conditions, whereas resins which do not have a hydrophobic moiety such as NH2-750F are mainly loaded under low salt conditions, and use high salt conditions for elution from the resin. In an embodiment, loading of said eluate on the multimodal ion exchanger occurs under high conductivity conditions.

Conditions of high conductivity, according to the present disclosure refer to conditions where the conductivity is above 75 mS/cm, more preferably above 85 mS/cm. High conductivity is often obtained by having an increased salt concentration, e.g. above 1 M NaCI and more. Preferred salts are ammonium sulfate, sodium sulfate, potassium sulfate, ammonium phosphate, sodium phosphate, potassium phosphate, potassium chloride, sodium chloride or a mixture thereof. The skilled person will be aware that a different salt will affect the conductivity to a different degree. In some embodiments, the salt is selected from the group comprising sodium chloride, ammonium sulfate and potassium phosphate. These salts are especially well suited for the currently disclosed method as they generate ions which strongly promote hydrophobic interactions. Most chromatography devices or units are adapted with equipment to monitor the conductivity of the mobile phase or buffers used during chromatography, as well as the salt concentration, and/or the temperature thereof. In a further embodiment step c comprises monitoring the mobile phase.

A multimodal ion exchange ligand can be provided on a solid phase chromatography support which can be a porous particle, nonporous particle, membrane, or monolith. The solid phase support comprising the multimodal ion exchange ligand may be practiced in a packed bed column, a fluidized/expanded bed column, and/or a batch operation wherein the multimodal ion exchange resin is mixed with the protein feed prior to separation. In some embodiments, the solid phase support comprising the multimodal ligand is packed in a column of at least 5 mm internal diameter and a height of at least 25 mm. Such embodiments are useful, e.g., for evaluating the effects of various conditions on a particular protein (e.g. antibody). Another embodiment employs the solid phase support comprising the multimodal ligand, packed in a column of any dimension required to support applications and further scaled-up operations. In some embodiments, column diameter may range from less than 5 mm cm to more than 1 meter, and column height may range from less than 1 cm to more than 30 cm depending on the requirements of a particular application. Commercial scale applications will typically have a column diameter (ID) of 20 cm or more and a height of at least 20 cm. Appropriate column dimensions can be determined by the skilled artisan. In an embodiment, tow or more columns may be linked, operating in series.

In one embodiment a multimodal anion exchange resin is used. In some embodiments, the multimodal anion exchange comprises a positively charged moiety (preferably an amine or a quaternary ammonium ion) and an aromatic ring structure. These functionalities can be accommodated either in separate substituents/ligands as a more or less stochastic mixture of ligands or with both functionalities present in the same substituent/ligand.

Particularly useful are ligands defined by the formula

RI-R ₂ -N(R ₃ )-R ₄ -R5

wherein

Ri is a substituted or non-substituted phenyl group;

R ₂ is a hydrocarbon chain comprising 0-4 carbon atoms; R. ₃ is a hydrocarbon chain comprising 1-3 carbon atoms

R. ₄ is a hydrocarbon chain comprising 1-5 carbon atoms and

R. ₅ is OH or H.

Examples of such ligands include but are not limited to N-Benzyl-N-methyl ethanol amine or N,N-dimethyl benzylamine.

In another embodiment, the multimodal exchanger comprises both a charged moiety (positively, negatively, or both) and a hydrophobic moiety. In a further embodiment, the eluate of step (b) or an eluate derived thereof is loaded on the multimodal ion exchanger under high conductivity or high salt concentration. Accordingly, binding of the protein of interest to the resin occurs under high conductivity conditions or high salt concentration. Often, multimodal chromatography uses the electropositive or electronegative properties of the resin as major mechanisms of interaction for separating the components of the mixture, the mixture in this case is the eluate of step (b) or an eluate derived thereof. By loading and binding said eluate onto a multimodal ion exchange resin under high conductivity or high salt concentration, a shift from ion exchange properties towards the hydrophobic properties of the multimodal resin is forced, whereby interaction of said protein with the multimodal ion exchange resin is based on the hydrophobic properties of the protein and the hydrophobic moiety of the multimodal ion exchange ligand.

The above mentioned shift in chromatography mode allows for successful modulation of the separation properties of the multimode chromatography resin, e.g. selectivity towards the monomeric form of a protein, e.g. antibody. In addition, the chromatography conditions can be adapted in order to enable selective separation of protein/antibody charge variants from one another. These antibody charge variants are often referred to as the acidic, the neutral or the basic antibody species. Simultaneously and advantageously, all major attributes of anion exchange chromatography such as reduction of host cell proteins and DNA, as well as reduction in viral particles are preserved. Hence, a highly pure protein sample of a high quality is obtained using the method of the disclosure.

In an embodiment, the loading and binding of the eluate of step (b) or an eluate derived thereof occurs at a pH of about 6 to 9 and a salt concentration, preferably sodium chloride or potassium phosphate, of between 0.5 to 3 M or a salt concentration corresponding to a conductivity of at least 0.5 mS/cm, preferably at least 1 mS/cm. In a further embodiment, the salt concentration will be between 1M and 2M NaCI or 0.5M and 1M potassium phosphate, respectively, or a salt concentration corresponding to a conductivity of above 75mS/cm, more preferably above 85mS/cm. In some embodiments, binding below 1M NaCI may be too low to switch the interaction mode from anion exchange chromatography (AEX) to hydrophobic interaction chromatograph (HIC), whereas, above 2M NaCI, a salting out effect can be observed and buffers will become less cost effective. In some embodiments, a pH lower than 6 may result in limited binding to the resin, whereas a pH above 9 results in protein deamidation which is an undesired modification of the protein of interest. A typical condition could be an eluate of step (b) or an eluate derived thereof at a 1M to 2M NaCI concentration in a (20 to 100 mM) HEPES or Tris buffer of pH 7 to 8. The conditions of the eluate of step (b) or an eluate derived thereof may be adjusted or conditioned towards the conditions as described above or may already comply to the conditions as described due to upstream processing steps, in order to be compatible with the conditions for loading and binding of the polishing step (c). Preferably, the conditions used in step (b) are selected in order to be compatible with step (c), thereby making a substep comprising conditioning of the eluate before performing polishing unnecessary thus saving time and resources.

In some embodiments, in preparation for contacting the mobile phase, the eluate from step (b) or an eluate derived thereof, with the chromatographic resin, the multimodal ion exchange resin, it is common practice in the art to condition the mobile phase and to equilibrate the resin, in order to attain compatible conditions for loading and binding.

It is to be understood that conditioning of the eluate of step (b) or an eluate derived thereof might be achieved by any method known to the person skilled in the art including dilution, buffer exchange, titration or any combination thereof. A conditioning aimed at decreasing the conductivity of an eluate of step (b) or an eluate derived thereof could be achieved by diluting the feed with ultrapure water or with low-salt buffering solution. A conditioning that aims at increasing the conductivity of the eluate of step (b) or an eluate derived thereof might be achieved by titration of the feed with a salt solution such as a 5 M sodium chloride solution until reaching the desired conductivity. The pH of a solution might be lowered using a (0.2M to 1.0M) hydrochloric acid solution or (1M) acetic acid. The pH of a solution might be increased using a (0.2M to 1.0M) sodium hydroxide or with 2M TRIS base pH 9.50. Equilibration of the chromatography resin may be accomplished by flowing an equilibration buffer through the resin to establish the appropriate pH, conductivity, concentration of salts etc. The equilibration buffer may be chosen depending on the binding requirements of a particular protein. The equilibration buffer will normally include a buffering compound to confer adequate pH control. Buffering compounds may include but are not limited to MES, HEPES, BICINE, imidazole, Tris, phosphate, citrate, or acetate, or some mixture of the foregoing or other buffers. The concentration of a buffering compound in an equilibration buffer commonly ranges from 20 to 100 mM depending of the protein of interest. The pH of the equilibration buffer may range from about pH 4.0 to pH 9.5, more preferably 6 to 9. When hydrophobic interaction is involved, a pH of 6.0 to 9 is preferable. For interactions based on ion exchange, a pH of 7.5 to 9 is preferred. As mentioned above, in some embodiments, a pH lower than 6 will generally compromise binding while a pH above 9 will result in unwanted modifications of the protein. The equilibration buffer may also comprise a salt to adjust ionic strength or conductivity of the solution as needed. Examples of suitable salts include ammonium sulfate, sodium sulfate, potassium sulfate, ammonium phosphate, sodium phosphate, potassium phosphate, potassium chloride, sodium chloride or combinations thereof.

Elution of the protein of interest from the chromatography resin or column can be performed with the aid of an elution buffer. Either gradient elution or isocratic elution is an elution option. During isocratic elution, the mobile phase composition remains constant throughout the procedure. In contrast, during gradient elution the composition of the mobile phase is altered.

In some embodiments, elution may either be done by gradually decreasing pH in the buffer or gradually decreasing the salt concentration (such as NaCI) or both. This is particularly useful when the binding of the protein is based on a hydrophobic interaction. In a further embodiment, elution may be attained by both decreasing the pH and decreasing the salt concentration of the elution buffer.

Alternatively, elution may be done by gradually decreasing the pH in the elution buffer, while maintaining the salt concentration constant or by decreasing the pH in the elution buffer and increasing the salt concentration. This is particularly useful when the interaction is based on the AEX mechanism of the multimodal resin. Yet another option for elution when operating the multimodal resin in AEX mode is to increase the salt concentration in the elution buffer and maintaining a constant pH. In an embodiment, elution occurs by gradually and stepwise decreasing the pH of the elution buffer below 7. In a further embodiment, the pH of the elution buffer is stepwise decreased from a pH of about 9, more preferably 8 to at least a pH of about 5.5 or lower such as pH 2. In another or further embodiment, elution occurs by gradually and stepwise decreasing the salt concentration of the elution buffer, to a salt concentration of below 0.5 M. In a further embodiment, the salt concentration will go from the used loading conditions such as 2 M or 1 M to below 0.5 M, up to even 0 M. In some embodiments, a NaCI solution is used. In an embodiment, said elution buffer will have a salt concentration of between 250 and 500 mM, more preferably 300 and 450 mM. In some embodiments, NaCI is used. In another embodiment, pH of a the elution buffer will be between about 5.5 and 6.5, such as 6. An example is a MES buffer of pH 6. In another embodiment, both a combination of pH and salt concentration as described above is used for eluting the protein. An example is a 50 mM MES and 350 mM NaCI buffer, of about pH 6.

The amount of steps needed during gradient elution or isocratic elution and the pH gradient as well as the salt gradient will depend on the nature of the protein to be purified as well as the ion exchange ligand used.

Elution typically occurs over about 3 to about 20 column volumes. After use, the multimodal resin may optionally be cleaned, stripped, sanitized, and stored in an appropriate agent, and optionally, re-used.

In between loading/binding of the protein and eluting, one or more wash steps may be performed. Washing may be advantageous to re-equilibrate the column and to remove weakly bound impurities prior to elution. In some embodiments, the wash buffer is either the same as the buffer used to equilibrate the chromatography resin or is conditioned to have a pH and conductivity that will result in desorption of weakly bound impurities without desorption of the target compound from the chromatography media. The wash buffer may contain for example Tris, HEPES, phosphate, BICINE, triethanolamine, sodium chloride, ammonium phosphate, sodium sulfate and/or potassium phosphate. In some embodiments, wash conditions can be experimentally selected for each resin/protein of interest combination. Once established, such conditions may be built into an automated protocol for said protein, e.g. a software package.

When the buffering agent and the salt are the same chemical, the additional advantage is attained that altering of conditions such as the conductivity or salt concentration during elution can be performed without the need to re-condition the sample. Therefore, in an embodiment, the buffering chemical is the same as the salt used for driving elution from the resin. For example, when phosphate is used as buffering agent, potassium phosphates are preferably used as salts to engage elution from the resin. This results in a faster more efficient purification process by reducing the number of steps as well as the number of chemicals/buffers required during the purification procedure, eventually also reducing the process costs.

In one embodiment, chromatography steps forming part of the disclosed methods comprise a solid phase and a mobile phase wherein the solid phase can be for example, but is not limited to, a purification column, a discontinuous phase of discrete particles, a membrane, filter, gel, etc. In other embodiments, the chromatography step comprises a classical packed bed column containing a resin, a column containing monolith material, a radial column containing suitable chromatographic medium, an adsorption membrane unit, or any other chromatography device known in the art with the appropriate medium and ligands as disclosed. In the chromatographic column the chromatographic material may be present as particulate support material to which the ligands according to the disclosure are attached. The current disclosure is provided with respect to chromatography steps comprising a column containing a resin, however, it is clear to the skilled artisan that the teachings of the disclosure can equally be applied with respect to chromatography steps comprising a column containing an adsorption membrane.

In some embodiments, the solid phase particles or media are packed in a column of any dimension required to support preparative applications. Column diameter may range from less than 1 cm to more than 1 meter, and column height may range from less than 1 cm to more than 50 cm depending on the requirements of a particular application. Appropriate column dimensions can be determined by the skilled artisan.

The membrane-type chromatography columns consist of a support material in the form of one or more sheets to which a suitable ligand is attached. The support material may be composed of organic material or inorganic material or a mixture of organic and inorganic material. In some embodiments, suitable organic materials include agarose based media and methacrylate. In some embodiments, suitable inorganic materials include silica, ceramics and metals. In an embodiment of the current method, the method comprises at least the steps of:

a. Performing a clarification step on a cell culture harvest comprising a protein of interest by the addition of one or more compounds selected from the group of fatty acids having 7 to 10 carbon atoms and derivatives thereof, ureides and/or electropositive compounds to said cell culture harvest and subsequently filtering of the harvest through a DE layer, thereby obtaining a feed comprising the protein of interest;

b. Performing a protein capture step on the feed of step (a) or a feed derived thereof by chromatography, thereby obtaining an eluate comprising the protein of interest;

c. Further purifying the eluate of step (b) or an eluate derived thereof by performing a polishing step by means of a mixed-mode or multimodal chromatography resin comprising a ligand with a charged moiety, either positively or negatively, thereby allowing binding of the protein to said resin, and eluting said protein from said resin thereby obtaining the purified protein of interest. In some embodiments, the chromatography of step (b) may be an affinity chromatography step such as Protein A, or may be an ion exchange step, such as cation exchange. The chromatography step may be combined with simultaneous viral inactivation of the feed obtained from the clarification step in (a), or alternatively, an additional viral inactivation or filtration step (e.g. microfiltration) may be present between step (b) and step (c).

By combining three method steps (a), (b) and (c) as described above, a more efficient process for protein purification is described, with a high protein yield (over 80% or more). Removal of key impurities by step (a) allows to efficiently use cation exchange chromatography as capture step (b). This ion exchange resin is cheaper than the Protein A affinity resin, therefore reducing the operation expenses (OPEX) without compromising the quality of the purified protein of interest. In addition, due to the advanced clarification according to step (a), the need to condition the feed prior to the subsequent chromatography is eliminated, thus again reducing the number of steps during the purification process resulting in an improved process efficiency. Method steps (a) to (c), (a) to (b) or (b) to (c) or portions thereof may be performed serially, inline and/or as a single operation unit, wherein the sub steps result in fractions which are readily useable in the subsequent step or with minimal conditioning. Advantages of this novel method are considerable reduction of the operation time, size and labor and hence lower operational costs.

In an embodiment, a concentration and/or filtration step is added prior to capture step b. Such concentration/filtration could be any type of concentration/filtration suitable in the art. The system's concentrator/filter can be a chosen from a number of devices known to the skilled person which are suited for reducing the volume of the liquid in which the target biomolecule resides. In some embodiments, the concentrator/filter comprises one type of concentration/filter device (e.g., tangential flow filter). In some embodiments, the concentrator comprises more than one type of concentration device (e.g., tangential flow filter and dead-end filter). Most of these devices are based on filtration and/or size exclusion chromatography. In one embodiment the concentrator is a filtration device, more preferably a micro-filtration device, or an ultra-filtration device or a combination of both micro- and ultra- filtration device. When the system is provided with an ultra-filtration device for reducing the volume of the liquid in which the target biomolecule resides, the membrane of the device is adapted as to allow flow through of water and low molecular weight solutes, which are in general referred to as the permeate, while macromolecules such as biomolecules are retained on the membrane in the retentate. In a further embodiment, the system is provided of a tangential flow filtration device (TFF). In an embodiment, said TFF is equipped with at least one hollow fiber having pores with a porosity sufficient to retain practically all of the target biomolecules, while permitting smaller contaminants such as growth medium and solutes to pass through the pores of the membrane. In contrast to dead-end filtration, in which the liquid is passed through a membrane or bed, and where the solids are trapped on the filter, tangential flow across the surface of the filter is allowed in the TFF device, rather than directly through the filter. Accordingly, formation of a filter cake in the TFF is avoided. In another embodiment, said TFF may be equipped with a cassette allowing tangential flow filtration. In yet another embodiment, said TFF is a single pass tangential flow filtration (SP-TFF). This device is especially advantageous when purifying proteins such as antibodies.

Chromatography steps according to the disclosed method can be performed in batch mode. Alternatively, said chromatography steps are performed in continuous mode. In some embodiments, when performed in batch mode, the chromatography step is repeated over one column (single-column batch) or multiple columns (parallel batch) until all the feed (in case of step b) or eluate (in case of step c) has been loaded and subsequently eluted. The eluates or pooled eluates, respectively, are optionally pooled before proceeding to the next purification step. Alternatively, the chromatography steps can be performed as a continuous process where each step of the purification method is performed simultaneously and the column (single- column continuous) or columns (parallel column continuous) are loaded and eluted continuously. While the classical batch-operation sequence does not require specific adaptations of the equipment and often results in a protein of high purity, the current method is well suited to be performed in a parallel column continuous mode process. A continuous mode offers the additional advantages that higher productivity can be achieved as the efficiency of the protein production process is increased. In addition, the continuous mode helps reduce the production costs by reducing the amount of consumables needed for a larger production scale. Continuous mode chromatography, for example, allows for a reduction in the size of the chromatography column without sacrificing the process productivity and reduces the size of the system.

While the purified protein of interest obtained after step (c) will be virtually free of contaminants, further downstream steps may be implemented if desired. Such further downstream steps may include viral filtration such as nanofiltration, ultrafiltration, diafiltration, formulation, packaging etc. to complete the protein production process for use in a pharmaceutical context.

In a second aspect, the current disclosure relates to a protein purification or a protein production and purification platform comprising at least one unit selected from the group comprising a cell culture harvest clarification unit, a protein capture unit and a polishing unit, wherein the polishing unit comprises a device capable of performing multimodal anion exchange chromatography. In some embodiments, the protein production and/or purification platform is able to execute one or more of the methods as described above. The platform further allows for purification of a protein from a cell culture harvest in a safe, efficient and cost-effective manner. In some embodiments, the protein purification platform of the present disclosure allows down-scaling of the infrastructure required for protein production on an industrial level. Accordingly, the protein production and/or purification platform or one or more of the units of the current disclosure may be portable and/or can be provided in an embodiment as a portable platform e.g. in a container or trailer. Therefore, the current platform for protein production and purification can be a mobile platform. In another or further embodiment, the units of the platform can also be mobilized, for example, by placing each unit on a mobile skid.

The protein purification platform of the disclosure allows rapid purification of (recombinant) proteins using significantly smaller equipment compared to the prior art platforms. In addition, high yield of the purified protein is obtained using the protein purification platform thereby reducing the costs of the final product. This eventually results in a lower investment and production cost, which is a considerable advantage.

By combining down-scaling of the infrastructure required for protein production on an industrial level, with rapid purification of recombinant proteins using significantly smaller equipment compared to the prior art platforms. The protein purification platform of the disclosure can be provided as a portable platform. In addition, high yield of the purified protein is obtained using the protein purification platform thereby reducing the costs of the final product. This eventually results in a lower investment and production cost, and higher efficiency, which is a considerable advantage.

In another or further embodiment, the polishing unit comprises a device capable of performing multimodal ion exchange chromatography, such as an anion or cation exchange. In an embodiment, multiple columns may be provided. In an embodiment, the AEX column (whether single or in multiple mode) is the sole polishing device present in the disclosed protein purification platform. Due to the enhanced efficiency of the polishing step according to the method described herein, in some embodiments, one single device to carry out this complex task suffices in the currently disclosed platform. Accordingly, the cost of building or manufacturing the disclosed protein purification platform decreases as well as the space required to locate all the necessary equipment. Therefore, in some embodiments, there is a reduction in the number of required pieces of equipment due to a reduction of the number of steps performed during the protein purification method and due to the enhanced compatibility of conditions used in the different steps (a) to (c) which further results in a reduction of the production cost of a protein purification platform as presently disclosed.

In an embodiment, said protein capture unit and polishing unit are connected, wherein an outlet of the protein capture unit is connected to an inlet of the polishing unit. In another or further embodiment, an outlet of the clarification unit is connected to an inlet of the protein capture unit. In an embodiment, a concentrator may be positioned between the polishing and clarification unit. In an embodiment, such concentrator may be part of said polishing, capture or clarification unit. In an embodiment, the concentrator may be a TFF.

The design of the one or more units allows for efficient protein purification at a high yield. In some embodiments, the connections between the different units of the platform further contribute to the rapid protein purification when using this platform.

In an embodiment, the protein capture unit comprises a protein A affinity chromatography column, resin or membrane or a cation exchange chromatography column, resin or membrane. In some embodiments, when a protein A affinity chromatography column is used in the protein capture unit, it may be desirable to include an additional viral inactivation unit in the protein purification platform. In an embodiment, one or more interconnected (also known as twin) capture columns, resins or membranes are provided allowing twin capturing. In this set-up, a first capture column, resin or membrane is loaded beyond its capacity observed in batch and product breakthrough is captured by the second interconnected column, resin or membrane. In this set-up, the feed is continuously loaded, wherein a first column, resin or membrane undergoes recovery and regeneration while the second or further column, resin or membrane is loaded.

In an embodiment, one or more units are fluidly connected to each other. To this purpose, the production and purification platform is provided with conduits, pumps, manifolds, valves, pipings etc that allow transport of the liquid feed from one unit to another and which interconnects the units in the platform. In an embodiment the conduits of the system are fitted with one or more pumps to provide directional liquid flow and to allow control or induce differential pressure between different parts of the platform. In a further embodiment, the pumps can operate both forward and backwards. In an embodiment, the conduits of the system are preferably fitted with one or more pumps to provide cross-flow of the liquid through the concentrator.

The conduits of the platform here disclosed, may be provided with sensors for measuring parameters important for cell growth and for the purification process including but not limited to liquid flow rate, temperature, pH, oxygen saturation and pressure. In addition conduits of the system may be provided with valves to control flow distribution. The valves further allow engaging or disengaging a specific system segment or conduit. In some embodiments, the valves are metered valves or discrete valves (e.g., on or off valves). In an example, the valves are discrete valves. In some embodiments the valves allow sampling of the liquid feed from the respective conduit, for example for quality control.

In an embodiment, an outlet of the protein capture unit is connected to an inlet of this viral inactivation unit, and an outlet of the latter is connected to an inlet of the polishing unit.

Advantageously, the platform may be combined with or may comprise a bioreactor unit. Additional downstream units such as one or more microfiltration and/or viral filtration and/or packaging and/or system and/or waste decontamination units may equally be provided in the platform. Any bioreactor suitable in the art may be used. In an embodiment, a shaken-type bioreactor is used, such as an orbital shaken- type bioreactor. Said bioreactor may be provided with a bag structure allowing production of cells in said structure. In an embodiment, said bag structure is single use. Said bag structure may be provided with sensors for monitoring parameters such as DO and pH. The orbital or shaker motion ensures efficient liquid mixing and facilitates high oxygen transfer rates with low shear forces. Microstructures or carriers may be provided to ensure optimal cell growth. In an embodiment, said bioreactor may have a volume of between 10 and 200 L.

Due to the optimization of each process in the platform, the reduction in space required for each unit allows all units belonging to the protein purification platform, including any additional units such as the bioreactor, to be incorporated in a single safety cabinet or containment enclosure. This not only contributes to reduction of space required but also to the enhanced safety when using this platform. In addition, the connections between the units allow the purification steps to be performed without the (intermediate) product and consumables exiting the containment enclosure thus ensuring minimal safety risks.

In another or further embodiment, the one or more units are placed in one or more isolators which may or may not be present within a containment enclosure. Each unit may be present in a separate isolator, or one or more units may be combined in one isolator. In an embodiment, the units or isolators are configured to be clean rooms which may or may not allow entrance of users. In some embodiments, to allow sample taking for quality control procedures, the isolated units or isolators are, in an embodiment, provided with flexible sleeves through which a user is allowed limited indirect access to the units, whose contents remain isolated from a user. In another or further embodiment, one or more isolators used in the system are provided with one or more liquid transport ports and/or rapid transfer port / rapid transfer container (RTP/RTC) systems to allow safe transport of liquids and/or solids into and out of the isolators. In some embodiments, liquids that may be transported from outside the isolator to inside the isolators include for example but are not limited to growth media, infection media, cells, buffers, products such as formaldehyde. In some embodiments, liquids which may be transported from inside the isolator to outside the isolator include, but are not limited to, liquid waste after decontamination thereof has been achieved and possible samples taken throughout the production and purification steps. In some embodiments, liquid containing the target biomolecule (also referred to as the product flow) is transported from one isolator to another during the production and/or purification process through conduits.

In one embodiment, an isolator or containment enclosure is adapted for producing a biomolecule of interest such as a protein, wherein said isolator or containment enclosure comprises an upstream process cabinet comprising one or more units chosen from a bioreactor, a harvest treatment or clarification unit; a downstream process cabinet comprising one or more units chosen from a viral inactivation unit, polishing unit or viral filtration unit; and a virus free cabinet allowing formulation of the biomolecule.

In an embodiment, the protein production and purification platform according to the current disclosure comprises an upstream (USP) cabinet, a downstream (DSP) cabinet and virus-free cabinet. In an embodiment, the USP cabinet allows for harvest treatment and clarification. As a consequence, the USP cabinet may comprise a harvest treatment unit and a clarification unit. In an embodiment, said DSP cabinet allows for capture, viral inactivation, polishing and viral filtration and thus comprises the corresponding units and devices. In an embodiment, the virus free cabinet allows for the formulation of the produced protein, such as an antibody.

In a particular embodiment of the current invention, the platform comprises a clarification unit equipped for anion exchange and cake filtration (diatomaceous earth), such as a filtration device; a capture unit equipped with a chromatography device (affinity or ion exchange, such as protein A or cation exchange, in single or twin mode) and a polishing unit equipped with one or more multimodal ion exchanger devices (in single or twin mode). In some embodiments, the platform may further be equipped to perform harvest treatment, viral inactivation and filtration, as well as sample preparation.

In an embodiment the process flow in the platform is controlled by a process controller or process control device. The controller controls and operates bioreactor parameters as well as process flow parameters and monitors and records data from one or more sensors described above (pH, temperature and/or DO). To that purpose, said controller is provided with software allowing monitoring, controlling and recording the process flow and parameters of the system. The controller is able to manage liquid flow through the subsequent units of the platform thereby controlling the production and purification of the target biomolecule, being the protein. Preferably, liquid flow is managed by the controller in the system by controlling the functioning of the pumps and or valves present therein. In an embodiment the process control device provides automated control of the system's process flow.

Access to the controller can be provided to the user via a computer which can be connected to the controller. The controller allows export of data through one or more data transfer devices which can be wireless such as a Wifi or Bluetooth connection or wired such us a USB connection present on said controller. Data connections on the controller can in another or further embodiment allow access to an IT network. In a further embodiment, a screen is connected to the controller which allows the system's user or operator to follow the process flow and measured parameters as well as to manually operate the system, e.g. by starting or stopping certain sub- processes.

It is supposed that the present disclosure is not restricted to any form of realization described previously and that some modifications can be added without reappraisal of the appended claims.

DETAILED DESCRIPTION OF FIGURES

Figure 1A is a flow chart depicting a protein purification process according to the prior art and the corresponding steps of a method according to an embodiment of the current disclosure. Prior art processes and platforms are typically characterized by their large footprint, including their need to use of large equipment, such as massive centrifuges, large filters and columns and a high number if unit operations.

The depicted embodiment of the disclosed method for the purification of a protein of interest employs three steps. In a first step (a) 1, a clarification step is performed on a cell culture harvest comprising the protein of interest, thereby obtaining a feed. The obtained feed or a feed derived thereof, which comprises the protein of interest (e.g. a monoclonal antibody), is subsequently loaded on a chromatography column to perform the second step (b) of the purification method, the protein capture step 2. The cell culture harvest clarification method according to the current embodiment ensures a high degree of cell culture harvest clarification which allow for the performance of an intensified protein capture step which, according to the current embodiment, is based on a protein A affinity chromatography step 2. Alternatively, the protein capture step may also make use of ion-exchange chromatography such as cation exchange chromatography. A microfiltration of the eluate inactivates viruses present in the eluate.

Optionally, the method includes an efficient viral inactivation step performed by a low pH hold 3. This step simplifies further processing of the purified protein since only an additional step comprising microfiltration of the eluate microfiltration step is performed to obtain the desired level of viral inactivation.

In step (c) the eluate of the intensified protein capture step 2 or an eluate derived thereof is submitted to an intensified polishing step 4. Whereas the methods of the prior art combine two polishing steps and an intermediate ultrafiltration or depth filtration step to achieve a high degree of purity of the purified protein of interest, the current method employs a single polishing step which makes use of a multimodal ion exchange chromatography step 4. Figure 1 depicts anion exchange but it will be obvious to the one skilled in the art that other methods such as multimodal cation exchange equally belongs to the options. Accordingly, the three steps of the prior art methods are reduced to a single step in the method according to the disclosure, thereby reducing purification process time, space and consumables without compromising the degree of protein purity at the end of the purification process. As a result of the intensified protein polishing of the current method, the purified protein of interest is obtained. Optionally, the purification process can include an additional step of viral filtration by performing nanofiltration 5 and a formulation step 6. Referring now to Figure IB is an alternative to the process of Figure 1A. Cells are grown in a bioreactor and the harvest is incubated with the addition of allantoin, caprylic acid and pDADMAC in a specific concentration (harvest treatment 16). Optionally, the pH may be adjusted. Secondly, the harvest is clarified by an advanced clarification process using DE cake filtration 1 which could be performed in multiple cycles or in one cycle depending on the application. The output of the clarification is subsequently put on a concentration device 17, which in the current case is a TFF, more preferably a SP-TFF. Subsequently, the output will be brought over a capture unit, which in the embodiment of figure IB is a twin-column affinity chromatography step. After capturing, viral inactivation will occur in alternative mixing tanks. In a subsequent step, an intensified polishing step takes place by means of a mixed-mode chromatography 4 as extensively explained above. Finally, a nanofiltration device 5 will allow viral filtration. In a viral free cabinet, the obtained protein will further be formulated 6 and prepared 18 according to the required specifications.

Figure 2 shows an embodiment of a purification platform 7. The purification platform 7 according to this embodiment comprises at least a clarification unit 8, a protein capture unit 9 and a polishing unit 10 which are serially connected to each other.

A cell culture harvest is obtained from a bioreactor 11 and is clarified in the clarification unit 8. In this embodiment, a clarification vessel 12 is provided where a diatomaceous earth filtration cake is formed during clarification.

Subsequently, the obtained feed comprising the protein of interest or a derivative thereof is transferred to the protein capture unit 9 where it is subject to a chromatography step. In an embodiment, a vessel 23 may be present to store the clarified harvest. The proteins comprised in the feed obtained from the clarification unit 8 can be optionally concentrated using a device for single-pass tangential flow filtration 13, thereby obtaining a derived feed prior to transfer to the protein capture unit 9. Either affinity chromatography or ion-exchange may be used as the chromatography step in the protein capture unit 9 and as such, this unit 9 will be equipped with adequate tools to enable such chromatography. The tools of the protein capture unit 9 of the current embodiment include multiple chromatography columns 14 which are operated in a continuous mode. Alternatively, the protein capture unit 9 can consist of a single chromatography column 14 or multiple columns 14 operated in batch mode.

The eluate obtained in the protein capture unit 9 or a derivative thereof can subsequently be transferred to the polishing unit 10 comprising a single polishing device capable of performing multimodal ion exchange chromatography, preferably multimodal anion exchange. According to the currently depicted embodiment of the platform 7, this device comprises a number of multimodal anion exchange chromatography columns 15 which are operated in batch mode.

Optionally, the platform may be provided with a viral inactivation unit between the protein capture unit 9 and the polishing unit 10. Preferably, this unit 10 is additionally equipped with systems that allow pH adjustment of the eluate resulting from the protein capture unit 9. Due to the enhanced efficiency of the method to which the platform 7 has been adapted, the platform enables high quality protein purification on an industrial scale using only a restricted amount of space. Finally, the platform may further be provided with additional units such as units for viral filtration 24, and finally formulation of the end product 25.

Figure 3 shows a comparison between a conventional protein purification process and a protein purification process as described herein, quantifying the impurities present in the fractions comprising the protein of interest during the substeps of both processes.

As can be seen from the graph, the process according to the disclosure shows in each step a significant lower level of impurities compared to the conventional process. In addition, the single polishing step (polishing 1) of the disclosed method leads to a purified protein fraction that has a lower level of impurities than the purified protein fraction after two consecutive polishing steps (polishing 1 and polishing 2) are performed in the conventional protein purification process. The significant reduction in the level of impurities present in the final purified protein fraction does not compromise the yield of the protein purification process. The protein purification method and process according to the disclosure result in 90% yield, 99.9% purity and a protein production of 30 g/L/h.

Reference is made to Figure 4 which shows a possible embodiment of one or more cabinet(s) or isolators wherein the above mentioned platform is implemented and wherein a USP cabinet 19, a DSP cabinet 20 and a virus-free cabinet 21 is present. In this particular embodiment, the USP cabinet 19 comprises a flush vessel 22, a bioreactor 11 and a clarification vessel 12 which allows executing the harvest treatment and clarification process as described above. In an embodiment (not shown on the figures) the clarification vessel includes at least one candle filter having a surface on which the DE accumulates into a cake. The clarification vessel may be a rigid or semi-rigid container. The clarified harvest is subsequently transferred to the DSP cabinet 20. To that purpose, the USP cabinet 19 is in fluid connection with the DSP cabinet 20. In the embodiment of Figure 4, the clarification vessel 12 will be connected to the clarified harvest vessel 23. It will be apparent that such clarified harvest vessel could also be part of the DSP cabinet 19. From the clarified harvest vessel 23, the clarified harvest will be transferred to the capture unit 9 which in the current example is equipped with multiple (in batch) chromatography columns 14. From the capturing unit 9 the feed is transferred to a viral inactivation unit 26. This is an optional feature as in certain embodiments, viral inactivation may occur during the capture step. Subsequently, the obtained feed is polished in the polishing unit 10, comprising multimodal anion exchange chromatography columns 15 in batch mode. After polishing, the polished feed is transferred to a viral filtration unit 24. Finally, formulation of the end product is performed in the virus-free cabinet 21, which comprises one or more units 25 for further processing of the purified product. Such units may for instance comprise ultrafiltration, nanofiltration, diafiltration, packaging or formulation units. As shown in figure 4, the various units in the platform are connected with manifolds, conduits, tubings, pumps etc. for allowing transport of the feed from one unit to another, and from one cabinet to a second cabinet.

The graphs of Figure 5 show the reduction of impurities due to the use of the advanced clarification as described above, by anion exchange and DE cake filtration. The first graph shows a conventional filtration used in the art, the bottom graph the results of advanced clarification according to an embodiment of the current invention. Advanced clarification enables superior process performance, as shown in the table of Figure 5.

Figure 6 shows the results of a twin column continuous capture according to an embodiment of the current disclosure, wherein a first column is loaded beyond its binding capacity and protein breakthrough is captured by the second interconnected column (CaptureSMB). The feed is continuously loaded such that when the first column undergoes recovery and regeneration, the second column is loaded. It was found that this may lead to a 60% increase in productivity, 4 times improvement in resin utilization and only a fourth of the buffer demand compared to a conventional capture system.

Figure 7 shows the effect of a SP-TFF step prior to the capture step on capture productivity. SP-TFF may be used as a tool to increase the titer of the midstream product, thereby enhancing the capture productivity. The SP-TFF step will concentrate the feed stream volume and reduce capture loading time. In certain embodiments, productivity may be improved up to 50 g/L/h, for example going from a 100L volume of IgG concentration of 3g/L (low titer) to a 20 L volume with IgG concentration of 15g/L (high titer).

Figure 8 shows two cases of polishing by multimodal anion exchange (AEX) wherein in a first case the AEX is used in hydrophobic mode and the protein is eluted by a salt gradient. In a second case, the column is used in AEX mode and the protein is eluted by pH gradient.

Figure 9 shows an example of the potential capacity of a platform according to an embodiment of the current disclosure. This high productivity did not come at a quality compromise as purity and yield of the product is comparable to other systems known in the art. The small footprint of the system allows for a simple and low CAPEX facility. The platform is suited for scale-up and scale-out.

EXAMPLES

The following examples are meant to further clarify the disclosure but are not be seen as a limitation of the latter.

Example 1: Purification of a monoclonal anti-TNFa antibody from fresh CHO culture

1. Advanced clarification

To an unclarified CHO cell culture, the following was added :

between 0.5-1.5% w/v of Allantoin,

between 0.1-0.9 % v/v octanoic acid, and

between 0.05-0.1% v/v of pDADMAC, thereby the first solution was obtained. The pH of the first solution was adjusted to between 5.0-6.0 and conductivity was kept at physiological values of between 11-13 ±1 mS/cm. The first solution was agitated and between 15 - 17 g/L of diatomaceous earth (DE) was added (approximately 78% of WCW) and agitated to form a second solution that was connected to a single use filtration device comprising a depth filter membrane of cellulose and filtered to form a clarified harvest. Anti-TNFa antibody recovery was of 90%. The pH of the clarified harvest was adjusted to around 7.0 and then processed with a sterilizing grade filtration device.

2. Intensified Capture

The feed obtained as a clarified harvest comprising the anti-TNFa antibody was passed through a Vantage Lll column (1.1 cm x 30 cm) packed with mAbselect Prisma®, with a 19-cm bed height (26 ml) using an AKTA 150 chromatography system (GE Lifesciences). More than 93% of recovery, with a combined recovery for advance clarification and capture step of more than 85% was achieved. HCP and purity values were consistent around 75 ppm and 90%, respectively.

3. Viral inactivation

The eluate obtained from Protein A chromatography comprising the protein fraction was incubated under room temperature condition at low pH for viral inactivation, after which the protein solution was neutralized and filtered.

4. Intensified Polishing

After reconditioning, the protein solution containing monoclonal anti-TNFa antibody was passed through a multimodal anion exchange chromatography matrix, Capto Adhere Impres®, GE Lifesciences, for further purification in bind-elute mode using a AKTA 150 chromatography system. The column was equilibrated with a 50mM HEPES buffer, 2 M NaCI, pH 7.0. Following binding to the column matrix at 30g/l, the column was washed with a 50mM HEPES buffer, 1.5 M NaCI, pH 7.0. Target molecule was eluted from the column with a 50mM HEPES buffer, 0.35 M NaCI, pH 7.0. More than 98% purity of monoclonal anti-TNFa antibody is achieved after this column step, as assessed by SE-HPLC.

5. Viral Filtration

After the multimodal anion exchange step, the purified protein solution containing the desired monoclonal antibody underwent a nano-filtration step and purity of anti- TNFa antibody was observed to remain more than 98% with a recovery of over 96%. 6. Formulation

After nano-filtration, protein solution was concentrated and diafiltered for the preparation of bulk drug substance. The final purified monoclonal anti-TNFa antibody exhibits more than 98% purity, as assessed by SE-HPLC.

Example 2: Purification of an IgGl antibody

1. Advanced Clarification

To an unclarified CHO cell culture, the following was added :

between 0.5-1.5% w/v of Allantoin,

between 0.1-0.9 % v/v octanoic acid, and

between 0.05-0.1% v/v of pDADMAC, thereby the first solution was obtained.

The pH of the first solution was adjusted to between 5.0-6.0 and conductivity was kept at physiological values of between 11-13 ±1 mS/cm. The first solution was agitated and between 15 - 17 g/L of diatomaceous earth (DE) was added (approximately 78% of WCW) and agitated to form a second solution that was connected to a single use filtration device comprising a depth filter membrane of cellulose and filtered to form a clarified harvest. IgGl recovery was of 90%. The pH of the clarified harvest was adjusted to around 7.0 and then processed with a sterilizing grade filtration device 2. Intensified Capture

The target molecule was captured from the feed comprising the protein of interest obtained as the clarified harvest with a Vantage L16 column (Merck Millipore) packed with 26ml_ of MabSelect PrismA media (1.6 cm x 13.4 cm bed height, Merck Millipore) using AKTA 150 chromatography system (GE Lifesciences). IgG recovery was of 98%. HCP were found to be 18 ppm and monomer content higher than 97.6%.

3. Viral Inactivation

The eluate obtained from Protein A chromatography comprising the protein fraction was incubated under room temperature condition at low pH for viral inactivation, after which the protein solution was neutralized and filtered.

4. Intensified polishing Protein A eluate at 6mS/cm conductivity and pH 7.30, containing 15.6 g/L IgGl antibody was prepared as follows: pH was adjusted to 8.0 with 2M Tris-HCI pH 9.50 solution and sample was diluted 1.75x fold with UP H20 to a final conductivity of starting conductivity of binding buffer. Multimode anion exchange matrix NH2-750F (Tosoh) was used for polishing in bind-elute mode using a AKTA 150 chromatography system. The column was equilibrated with 0.1M Tris, 4.20 mS/cm, pH 8.0 solution and 33 g/L resin were loaded at 3 min residence time. Following sample application, the column matrix was washed with equilibration buffer. Target molecule was eluted in a salt linear gradient from 0M to 0.6M NaCI in 20 CVs and column was striped with 1M NaCI solution. More than 99.5% purity and recovery of over 95% was achieved for the monomer as assessed by SE-HPLC.

Example 3: Monoclonal antibody IgG4 subclass purification

1. Advanced Clarification

To an unclarified CHO cell culture, the following was added :

between 0.5-1.5% w/v of Allantoin,

between 0.1-0.9 % v/v octanoic acid, and

between 0.05-0.1% v/v of pDADMAC, thereby the first solution was obtained.

The pH of the first solution was adjusted to between 5.0-6.0 and conductivity was kept at physiological values of between 11-13 ±1 mS/cm. The first solution was agitated and between 15 - 17 g/L of diatomaceous earth (DE) was added (approximately 78% of WCW) and agitated to form a second solution that was connected to a single use filtration device comprising a depth filter membrane of cellulose and filtered to form a clarified harvest. IgG4 recovery was of 90%. The pH of the clarified harvest was adjusted to around 7.0 and then processed with a sterilizing grade filtration device.

2. Intensified Capture

The target molecule was captured from the feed obtained as clarified harvest with a Vantage L22 column (Merck Millipore) packed with 76mL of Eshmuno A media (2.2 cm x 20 cm bed height, Merck Millipore) using AKTA 150 chromatography system (GE Lifesciences). IgG4 recovery was higher than 95% leading to a combined recovery with advanced clarification of 85%. HCP were consistently below 85 ppm and monomer content higher than 98%. 3. Viral Inactivation

Protein A eluate fraction was incubated and turbidity of titrated protein A eluate was removed by microfiltration Viral inactivate filtrate turbidity was 1 NTU and total protein recovery was more than 95%.

4. Intensified polishing

After conditioning, the protein solution containing IgG4 was polished using multimode anion exchange matrix NH2-750F (Tosoh) in bind-elute mode using a AKTA 150 chromatography system. The column was equilibrated with 20mM Bis- Tris, 6 mS/cm, pH 6.50 solution and loaded at 50 g/L resin at 2 min residence time. Following sample application, the column matrix was washed with 20mM Bis-Tris, 20 ms/cm, pH 6.50 Target molecule was eluted with 20mM Bis-Tris, 60 mS/cm, pH 6.50 in 5 CVs. More than 99% monomer purity is achieved after this step, as assessed by SEC-HPLC. Monomer recovery was higher than 85%.

5. Viral Filtration

After the multimodal anion exchange step, the protein solution containing the desired monoclonal antibody underwent a nano-filtration step and monomer purity of IgG4 was observed to remain more than 99%.

Example 4: Monoclonal antibody IgG4 subclass purification

Example 4 is similar to example 3, with the exception that during the polishing step, a multimodal cation exchange step is used. After conditioning the protein solution was loaded onto a mixed mode cation exchanger Capto MMC HiScreen (0.8x10cm) column, with a bed volume of 4.7ml_ fitted to AKTA 150 chromatography system (GE Lifesciences), at 200cm/hr. Prior to loading, the column was equilibrated with a 1M KP04 buffer, pH 7.0. The loading was done at 14 mg/ml_ of matrix and 3 min residence time, the column was washed with the same buffer (first wash). Following the first wash step, the protein was eluted from the column with a lOmM KP04 buffer, pH 7.0.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.