Complete flow-through process for purification of recombinant proteins

Abstract

The present invention relates to a method for purifying proteins, which method comprises in a continuous manner: a filtration step involving the use of at least one chelating agent; an exchange step involving the use of at least one diafiltration membrane; and a fine purification step involving the use of a membrane adsorber combination, wherein the two membrane adsorbers of the combination are orthogonal in terms of the mechanism of action.

Description

Technical Field

[0001] The present invention relates to a complete flow-through purification process for small-scale and large-scale purification of proteins, particularly monoclonal antibodies. Background Art

[0002] Antibody purification can be one of the most expensive aspects of bioproduction. Monoclonal antibodies (mAbs) are generally purified using a three-step three-resin chromatography process, each of which uses at least one specific buffer system. This conventional purification process includes a capture step, followed by a moderate purification step, and ends with a fine purification step, and typically takes 3 to 5 working days (including storage and open stages). In such conventional processes, these three steps are performed in a series of different unit operations, which cannot be operated in a continuous manner due to the need to adjust pH, molar concentration, and protein concentration between each step. Therefore, conventional purification processes generally require many different buffers and many storage units between each discontinuous step and several systems. Therefore, these conventional purification processes are susceptible to contamination, technical failures, and human errors. In addition, due to the need to interrupt between each step to concentrate the eluent, adjust pH and conductivity, and store the eluent before the next step, and because one step cannot be started before the previous step is completed, such conventional purification processes are particularly lengthy and costly.

[0003] The high cost of conventional processes is also due to the fact that protein A matrix is ​​generally used as the first step of purification. In fact, historically, the most selective resin (which is the case with protein A) is generally placed early in the process to remove as many impurities as possible. However, even though protein A is the most selective medium for purifying monoclonal antibodies, contaminants can still remain in the process. This step is also the most expensive in the entire process, also because it is used as the first step in contact with a large number of contaminants. In addition, it is difficult to remove the remaining contaminants to meet pharmaceutical specifications.

[0004] With the increase in cell culture titers and the increase in cell culture volumes for production, downstream processing is seen as an industry bottleneck. This is particularly relevant to the production of monoclonal antibodies, where the focus has shifted from batch volume to downstream processing capabilities. In addition, early preclinical and clinical stage studies require large quantities of antibodies that can be produced more quickly. Therefore, there is a need in the industry for a lower cost process that can be performed in a continuous manner for protein purification, particularly for antibody purification, and for reducing the time it takes to obtain batches, reducing the risk of contamination, technical failures and human errors, and reducing the need for process scale-up. Summary of the invention

[0005] The inventors have discovered a novel method for purifying proteins, in particular antibodies, which comprises only three steps in a continuous complete flow-through manner, none of which involve Protein A, and advantageously uses only one buffer, while still allowing a high yield of purified antibodies with good purity. Therefore, the purified protein is suitable for medical applications. Therefore, the method can be used to purify proteins for clinical trials and / or for the manufacture of pharmaceutical compositions comprising the protein. In addition, the method does not require any adjustments between the steps, and therefore the process from harvesting the protein to be purified to the final product can be carried out in a closed system.

[0006] In brief, the process consists of only three consecutive steps, with a lower cost technology aimed at removing the bulk of the contaminants being used as the first step, and the technologies used for the second and third steps becoming increasingly selective to remove the remaining small amounts of contaminants and therefore being used in smaller quantities than in conventional processes.

[0007] Therefore, the method of the present invention comprises in a continuous manner: a filtration step involving the use of at least one chelating agent; an exchange step involving the use of at least one diafiltration membrane; and a fine purification step involving the use of a membrane adsorber combination, wherein the two membrane adsorbers of the membrane adsorber combination are orthogonal in terms of the mechanism of action. Figure 1 The method of the present invention is shown. These three purification steps are advantageously implemented in this particular order. In addition, it has been found that only one buffer, i.e. the buffer for the exchange step, needs to be used in the whole process. In other words, the balance buffer optionally used in the fine purification step is advantageously identical to the buffer for the exchange step. This buffer advantageously comprises Bis Tris, Tris, Tris-HCl, phosphate and / or citric acid, for example, in combination with NaCl, acetic acid and water. More particularly, only one buffer can be used in the whole process, thereby ensuring compatibility between all steps, and enabling the supply chain to be saved in manufacturing and quality control and reducing storage requirements.

[0008] The method of the present invention further allows the elimination of the open phase (i.e., the step of opening the purification system to perform manual operations such as preparing a chromatographic column for a new buffer, diluting the sample, or adjusting its pH), thereby reducing the risk of contamination and providing the possibility of working in a less classified environment. In addition, since the method of the present invention advantageously does not involve the use of any column and mainly uses a disposable and ready-to-use membrane adsorber, there is no need for storage or reuse validation, no need to prepare or fill columns or related controls, no need for cleaning validation and limited hardware. Therefore, the process cycle time is shortened, the need for process expansion is minimized, and operating and storage costs can be reduced. Therefore, the method of the present invention allows both fast and low-cost batch production and can reduce the occupancy time of the purification system. Therefore, the method is suitable for expansion from laboratory scale to industrial scale and purification of recombinant proteins.

[0009] A specific scheme has been developed and implemented for two different antibodies. In this scheme, the filtered protein solution obtained at the end of the first filtering step is passed directly through the at least one diafiltration membrane, i.e., without any treatment such as pH adjustment, buffer exchange or dilution, and the retentate obtained at the end of the exchange step is also passed directly through the combination of at least two membrane adsorbers, i.e., without any treatment such as pH adjustment, buffer exchange or dilution. The advantage of this scheme is that it is extremely fast (several hours), resulting in high yields (more than 70%), purity meets pharmaceutical industry standards, and can reduce both the buffer and storage facilities used. In addition, this process has the advantage of being very flexible and cost-saving because it does not involve the use of protein A matrix, which is generally the most expensive element in conventional processes. In addition, the process can be fully automated, run in a continuous manner, and does not include any open phase. In addition, the process has been successfully performed for two different antibodies.

[0010] Therefore, the present invention provides a method for purifying a protein from a solution, the method comprising:

[0011] (a) a filtering step, comprising:

[0012] - passing said solution through at least one chelating agent matrix in said flow-through manner,

[0013] - recovering a filtered protein solution from the flow-through of the at least one chelator matrix;

[0014] (b) an exchanging step, comprising:

[0015] - passing said filtered protein solution obtained at the end of step (a) through at least one diafiltration membrane using only one buffer for said exchange,

[0016] - recovering the retentate of the at least one diafiltration membrane containing the partially purified protein;

[0017] (c) a fine purification step, which comprises:

[0018] - passing the retentate obtained at the end of step (b) in said flow-through manner through a membrane adsorber assembly, wherein the two membrane adsorbers of said membrane adsorber assembly are orthogonal in terms of the mechanism of action and said membrane adsorber assembly has been previously equilibrated with the same equilibration buffer as said one buffer used for the exchange in step (b),

[0019] - recovering the purified protein from the flow-through of the membrane adsorber assembly;

[0020] wherein the purification method does not include a Protein A chromatography step.

[0021] The present invention also provides a method for purifying protein from a solution, the method comprising:

[0022] (a) a filtering step, comprising:

[0023] - passing said solution through at least one chelating agent matrix in said flow-through manner,

[0024] - recovering a filtered protein solution from the flow-through of the at least one chelator matrix;

[0025] (b) an exchanging step, comprising:

[0026] - passing said filtered protein solution obtained at the end of step (a) through at least one diafiltration membrane using only one buffer for said exchange, said only one buffer being the same as the equilibration buffer used in said polishing step (c);

[0027] - recovering the retentate of the at least one diafiltration membrane containing the partially purified protein;

[0028] (c) a fine purification step, which comprises:

[0029] - passing the retentate obtained at the end of step (b) in the flow-through manner through a membrane adsorber assembly, wherein the two membrane adsorbers of the membrane adsorber assembly are orthogonal in terms of the mechanism of action and the membrane adsorber assembly has been previously equilibrated with the equilibration buffer,

[0030] - recovering the purified protein from the flow-through of the membrane adsorber assembly;

[0031] wherein the purification method does not include a Protein A chromatography step.

[0032] The present invention particularly provides a method for purifying a protein from a solution, the method comprising:

[0033] (a) a filtering step, comprising:

[0034] (i) passing said solution through said at least one chelating agent matrix in said flow-through manner,

[0035] (ii) recovering a filtered protein solution from the flow-through of the at least one chelator matrix,

[0036] (b) an exchanging step, comprising:

[0037] (i) passing said filtered protein solution obtained at the end of step (a) through said at least one diafiltration membrane using only one buffer for said exchange,

[0038] (ii) recovering the retentate containing the partially purified protein from the at least one diafiltration membrane;

[0039] as well as

[0040] (c) a fine purification step, which comprises:

[0041] (i) passing an equilibration buffer through the membrane adsorber assembly, wherein the equilibration buffer is the same as the one buffer used for exchange in step (b),

[0042] (ii) passing the retentate obtained from step (b) through the membrane adsorber assembly in said flow-through mode,

[0043] (iii) recovering purified protein from the flow-through of the membrane adsorber assembly;

[0044] wherein the purification method does not include a Protein A chromatography step.

[0045] In one embodiment of the invention, the entire purification process uses only one buffer. In a specific embodiment, the buffer comprises Tris, Tris-HCl, Bis Tris, phosphate and / or citric acid. In another embodiment, the buffer comprises or consists of the following: (i) Bis Tris, Tris or Tris-HCl, (ii) acetic acid, (iii) water and (iv) optional salt.

[0046] In one embodiment, the at least one chelating agent matrix of the filtering step is selected from activated carbon, diatomaceous earth, free cation exchange resin, free anion exchange resin and free mixed mode resin. In a specific embodiment, the at least one chelating agent matrix is ​​a combination of two different chelating agent matrices. In a more specific embodiment, the combination of two different chelating agent matrices is a combination of activated carbon and free anion exchange resin. In another specific embodiment, the at least one chelating agent matrix is ​​a combination of more than two (i.e., three, four, five or more than five) different chelating agent matrices.

[0047] In one embodiment, the at least one diafiltration membrane of the exchanging step is a single pass tangential flow filtration (SPTFF) module or a tangential flow filtration (TFF) module. In a specific embodiment, the at least one diafiltration membrane of the exchanging step is in the form of a cassette, a hollow fiber or a spiral wound. In a specific embodiment, the filtered protein solution is concentrated in the exchanging step.

[0048] In one embodiment, the combination of membrane adsorbers of the polishing step is a combination of two membrane adsorbers or a combination of at least two membrane adsorbers (eg, three, four, or at least four membrane adsorbers).

[0049] In one embodiment, the membrane adsorber of the membrane adsorber combination is selected from a cation exchange membrane adsorber, an anion exchange membrane adsorber, a multimodal membrane adsorber, a hydrophobic interaction membrane adsorber and a combination thereof. In a specific embodiment, the membrane adsorber combination of the fine purification step is a combination of a cation exchange membrane adsorber and an anion exchange membrane adsorber.

[0050] In one embodiment, the membrane adsorber combination of the polishing step is a combination of at least two membrane adsorbers selected from the group consisting of a cation exchange membrane adsorber, an anion exchange membrane adsorber, a multimodal membrane adsorber and a hydrophobic interaction membrane adsorber.

[0051] In one embodiment of the invention, the method further comprises a nanofiltration step after step (c) and optionally a final ultrafiltration and / or diafiltration step after the nanofiltration step. In another embodiment of the invention, the method further comprises a low pH inactivation step after step (c), after the nanofiltration step and / or after the final ultrafiltration and / or diafiltration step. In one embodiment of the invention, the method comprises a step of carrying out cell culture in a liquid culture medium, preferably in a bioreactor, to provide a liquid culture medium containing the protein before step (a). The cultured cells may be mammalian, bacterial or yeast cells. In a preferred embodiment, the cultured cells may be mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, HEK293 cells, etc., including different subtypes of these cell lines) and primary or established mammalian cell cultures (e.g., produced by lymphoblasts, fibroblasts, embryonic cells, epithelial cells, neural cells, adipocytes, etc.).

[0052] In one embodiment of the invention, the protein being purified is an antibody. In another embodiment, the antibody is a monoclonal antibody.

[0053] Thus, the present invention also provides an integrated process for producing purified proteins from liquid culture medium.

[0054] In certain embodiments of the invention, the one buffer comprises 5 mM to 40 mM (particularly 20 mM) BisTris, 15 mM to 150 mM (particularly 75 mM) NaCl, the pH adjusted to 6-9 (particularly 7.5) with acetic acid.

[0055] In certain embodiments of the invention, the one buffer comprises 5 mM to 40 mM (particularly 20 mM) Tris or Tris-HCl, 15 mM to 150 mM (particularly 75 mM) NaCl, the pH adjusted to 6-9 (particularly 7.5) with acetic acid.

[0056] In certain embodiments of the invention, the one buffer comprises 5 mM to 40 mM (particularly 20 mM) Tris or Tris-HCl, 15 mM to 150 mM (particularly 75 mM) NaCl, the pH adjusted to 6-9 (particularly 7.5) with citric acid.

[0057] In certain embodiments of the present invention, the one buffer comprises 15mM to 150mM (particularly 75mM) NaCl, and the pH is adjusted to 6-9 (particularly 7.5) with 2HPO4 / NaH2PO4 (preferably 10mM / 90mM Na2HPO4 / NaH2PO4 to 90mM / 10mM Na2HPO4 / NaH2PO4).

[0058] The present invention provides a kit comprising: at least one chelating agent matrix, at least one diafiltration membrane and a membrane adsorber combination, wherein the two membrane adsorbers of the membrane adsorber combination are orthogonal in terms of the mechanism of action; and a buffer comprising Tris, Tris-HCl, Bis Tris, phosphate and / or citric acid, in particular comprising or consisting of: (i) Bis Tris, Tris or Tris-HCl, (ii) acetic acid, (iii) water and (iv) optional NaCl. In some embodiments, the kit is used to purify proteins from a solution using the method of the present invention.

[0059] The present invention provides a kit comprising: at least one chelating agent matrix, at least one diafiltration membrane and a membrane adsorber combination, wherein the two membrane adsorbers of the membrane adsorber combination are orthogonal in terms of the mechanism of action; and instructions for preparing a buffer comprising Tris, Tris-HCl, Bis Tris, phosphate and / or citric acid, in particular comprising or consisting of: (i) Bis Tris, Tris or Tris-HCl, (ii) acetic acid, (iii) water and (iv) optional NaCl. In some embodiments, the kit is used to purify proteins from a solution using the method of the present invention.

[0060] Also provided herein are isolated proteins, pharmaceutical preparations, and pharmaceutical compositions obtained by any of the methods described herein.

[0061] These and other features and advantages of the disclosed purification method will be more fully understood from the following detailed description and in conjunction with the appended claims. It should be noted that the scope of the claims is defined by the description therein and not by the specific discussion of the features and advantages set forth in the specification.

[0062] In the context of the present invention, the terms "comprising", "having", "including" and "containing" are to be considered open-ended terms (i.e., meaning "including but not limited to") unless otherwise indicated. In addition, the term "comprising" encompasses "consisting of" (e.g., a composition "comprising" X may consist of X alone or may include other substances, such as X+Y). DETAILED DESCRIPTION

[0063] In order to simplify and reduce the cost of protein purification processes, the inventors have developed a new purification process that is continuous, complete flow-through and does not include a Protein A chromatography step.

[0064] The present invention relates to a method for purifying a protein from a solution, the method comprising:

[0065] (a) a filtering step, comprising:

[0066] - passing said solution through at least one chelating agent matrix in said flow-through manner,

[0067] - recovering a filtered protein solution from the flow-through of the at least one chelator matrix;

[0068] (b) an exchanging step, comprising:

[0069] - passing said filtered protein solution obtained at the end of step (a) through at least one diafiltration membrane using only one buffer for said exchange,

[0070] - recovering the retentate of the at least one diafiltration membrane containing the partially purified protein;

[0071] (c) a fine purification step, which comprises:

[0072] - passing the retentate obtained at the end of step (b) in said flow-through manner through a membrane adsorber assembly, wherein the two membrane adsorbers of said membrane adsorber assembly are orthogonal in terms of the mechanism of action and said membrane adsorber assembly has been previously equilibrated with the same equilibration buffer as said one buffer used for the exchange in step (b),

[0073] - recovering the purified protein from the flow-through of the membrane adsorber assembly;

[0074] wherein the purification method does not include a Protein A chromatography step.

[0075] In the exchange step (b), the expression "the filtered protein solution obtained at the end of step (a) is passed through at least one diafiltration membrane using only one buffer for the exchange" means that the filtered protein solution and the one buffer are passed through the at least one diafiltration membrane.

[0076] In certain embodiments, the methods of the present invention comprise:

[0077] (a) a filtering step, comprising:

[0078] (i) passing said solution through said at least one chelating agent matrix in said flow-through manner,

[0079] (ii) recovering a filtered protein solution from the flow-through of the at least one chelator matrix,

[0080] (b) an exchanging step, comprising:

[0081] (i) passing the filtered protein solution obtained from step (a) through the at least one diafiltration membrane using only one buffer,

[0082] (ii) recovering the retentate containing the partially purified protein from the at least one diafiltration membrane;

[0083] as well as

[0084] (c) a fine purification step, which comprises:

[0085] (i) passing an equilibration buffer through the membrane adsorber assembly, wherein the equilibration buffer is the same as the one buffer used for exchange in step (b),

[0086] (ii) passing the retentate obtained from step (b) through the membrane adsorber assembly in said flow-through mode,

[0087] (iii) recovering the purified protein from the flow-through of the membrane adsorber assembly,

[0088] wherein the purification method does not include a Protein A chromatography step.

[0089] As mentioned above, the above method of the present invention comprises only three steps, none of which is a Protein A chromatography step. Even though the method according to the present invention comprises only three steps and no Protein A chromatography step, it can still obtain a purified protein suitable for pharmaceutical purposes and in particular suitable for administration to humans.

[0090] In addition to the absence of manual labor during the purification process (and thus reducing the overall time required to complete the purification process), the disclosed methods also reduce the amount of buffer used for purification, and the absence of a protein A chromatography step also reduces costs. In addition to allowing purification of a variety of mAbs, the disclosed purification methods also simplify the purification of mAbs, increase overall yields, and reduce raw materials, storage facilities, commodity costs, and processing time.

[0091] In contrast to conventional protein purification methods, as described above, the methods disclosed herein utilize a unique buffer for both the exchange step and for equilibrating the membrane adsorber during the polishing step.

[0092] As used herein, "buffer according to the present invention" refers to a buffer comprising Bis Tris, Tris, Tris-HCl, phosphate and / or citric acid. Bis Tris, Tris or Tris-HCl are compounds well known to those skilled in the art:

[0093] - For Bis Tris, its IUPAC name is 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol and its CAS number is 6976-37-0

[0094] - for Tris, the IUPAC name is 2-amino-2-(hydroxymethyl)propane-1,3-diol and the CAS number is 77-86-1, and

[0095] - For Tris-HCl, the IUPAC name is 2-amino-2-(hydroxymethyl)propane-1,3-diol hydrochloride and the CAS number is 1185-53-1.

[0096] Such buffers according to the invention may correspond to exchange buffers and equilibration buffers.

[0097] More specifically, this buffer according to the present invention may comprise or consist of the same chemical substance (one of which is Bis Tris, Tris, Tris-HCl, phosphate and / or citric acid) of varying concentrations. In a specific embodiment, the buffer comprises Bis Tris, Tris, Tris-HCl, phosphate and / or citric acid. In a specific embodiment, the buffer comprises or consists of the following items: (i) Bis Tris, Tris or Tris-HCl, (ii) acetic acid and (iii) water. In a more specific embodiment, the buffer comprises or consists of the following items: (i) Bis Tris, Tris or Tris-HCl, (ii) acetic acid, (iii) NaCl and (iv) water. In other aspects, this buffer comprises or consists of the following items: (i) Bis Tris, Tris or Tris-HCl of varying concentrations, (ii) acetic acid, (iii) NaCl and (iv) water.

[0098] For example, the exchange buffer may comprise or consist of 5 mM to 40 mM (eg 20 mM) Bis Tris and 15 mM to 150 mM (eg 75 mM) NaCl, pH adjusted to 6-9 (eg 7.5) with acetic acid.

[0099] Alternatively, the exchange buffer may comprise or consist of 5 mM to 40 mM (particularly 20 mM) Tris or Tris-HCl, 15 mM to 150 mM (particularly 75 mM) NaCl, pH adjusted to 6-9 (particularly 7.5) with acetic acid.

[0100] Alternatively, the exchange buffer may comprise or consist of 5 mM to 40 mM (particularly 20 mM) Tris or Tris-HCl, 15 mM to 150 mM (particularly 75 mM) NaCl, pH adjusted to 6-9 (particularly 7.5) with citric acid.

[0101] Alternatively, the exchange buffer may comprise or consist of 15 mM to 150 mM (particularly 75 mM) NaCl, pH adjusted to 6-9 (particularly 7.5) with 2HPO4 / NaH2PO4 (preferably 10 mM / 90 mM Na2HPO4 / NaH2PO4 to 90 mM / 10 mM Na2HPO4 / NaH2PO4).

[0102] Such exchange buffers are particularly suitable for use with exchange steps, in particular with TFF cassettes, but are also suitable for the equilibration of membrane adsorbers, in particular the equilibration of a combination of cation exchange membrane adsorbers and anion exchange membrane adsorbers.

[0103] The advantages of the above-mentioned buffer formulation include the ability to pass the mAb product through the three steps of the method, especially the exchange step and the polishing step, with greater compatibility, while minimizing undesirable interactions, limiting pH and conductivity drops, and promoting improved yields compared to traditional purification methods. The use of such a buffer formulation enables the method to be carried out without any intermediate storage between the three steps (a), (b) and (c).

[0104] Thus, in certain embodiments, the method does not include any intermediate storage between the three steps (a), (b) and (c).

[0105] When reusing the membrane adsorber of the polishing step, a sanitizing buffer may optionally be used. Such a sanitizing buffer may comprise or consist of at least NaOH, more preferably 0.05N to 1N (eg 0.5N) NaOH.

[0106] As used herein, the term "polypeptide" or "protein" refers to:

[0107] 1) a molecule having a native protein sequence, i.e. a) a protein produced by a naturally occurring and in particular non-recombinant cell, or b) a genetically engineered or recombinant cell, or

[0108] 2) Molecules that differ from the sequence of a native protein by deletion, addition and / or substitution of one or more amino acids and / or by at least one post-translational modification (eg glycosylation).

[0109] The molecules mentioned in paragraph 1) above may be referred to as natural proteins.

[0110] The molecules mentioned in paragraph 2) above are non-natural proteins.

[0111] In certain aspects, the protein to be purified is an antibody.

[0112] As used herein, the term "antibody" refers to an intact antibody, or a binding fragment thereof that competes with an intact antibody for specific binding. Binding fragments include, but are not limited to, F(ab), F(ab'), F(ab')2, Fv, single domain antibodies (such as VHH antibodies (nanobodies)), and single chain antibodies. The term "heavy chain" includes any immunoglobulin polypeptide having a variable region sequence sufficient to confer specificity to a target antigen.

[0113] As used herein, the term "heavy chain" encompasses full-length heavy chains and fragments thereof. A full-length heavy chain includes a variable region domain VH and three constant region domains CH1, CH2, and CH3. The VH domain is located at the amino terminus of the polypeptide, while the CH3 domain is located at the carboxyl terminus.

[0114] As used herein, the term "light chain" encompasses full-length light chains and fragments thereof. Full-length light chains include variable region domains VL and constant region domains CL. Like heavy chains, the variable region domains of light chains are located at the amino terminus of polypeptides. As used herein, the term "light chain" includes any immunoglobulin polypeptide having a variable region sequence sufficient to confer specificity to a target antigen.

[0115] Naturally occurring antibody structural units generally comprise tetramers. Each such tetramer is generally composed of two identical polypeptide chain pairs, each pair having a full-length "light" chain (generally having a molecular weight of about 25 kDa) and a full-length "heavy" chain (generally having a molecular weight of about 50-70 kDa). The amino terminal portion of each light chain and heavy chain generally includes a variable region of about 100 to 110 or more amino acids, which is generally responsible for antigen recognition. The carboxyl terminal portion of each chain generally defines a constant region responsible for effector function. Human light chains are generally classified as κ and λ light chains. Heavy chains are generally classified as μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3 and IgG4. IgM has multiple subclasses, including but not limited to IgM1 and IgM2. IgA is similarly subdivided into multiple subclasses, including but not limited to IgA1 and IgA2. Within full-length light and heavy chains, the variable and constant regions are typically joined by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids.

[0116] The variable region of each light / heavy chain pair usually forms an antigen binding site. The variable region usually exhibits the same overall structure of a relatively conservative framework region (FR) joined by three hypervariable regions (also referred to as complementary determining regions or CDRs). The CDRs of the two chains from each pair are usually aligned by the framework region, which can enable binding to a specific epitope. From the N-terminal to the C-terminal, both the light chain and heavy chain variable regions usually include domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The amino acid distribution of each domain is usually based on the definition in the following publications: Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242. Bispecific or bifunctional antibodies are usually artificial hybrid antibodies with two different heavy chain / light chain pairs and two different binding sites.

[0117] The F(ab) fragment consists of a light chain and the CH1 and variable region of a heavy chain. The heavy chain of a F(ab) molecule cannot form a disulfide bond with another heavy chain molecule. The F(ab') fragment contains a light chain and a heavy chain containing more constant regions (between the CH1 and CH2 domains), so that an interchain disulfide bond can be formed between the two heavy chains to form a F(ab')2 molecule. The Fv region contains variable regions from both the heavy and light chains, but lacks the constant region. A single-chain antibody is an Fv molecule in which the heavy and light chain variable regions have been connected by a flexible linker to form a single polypeptide chain, thereby forming an antigen binding region. In certain embodiments, bivalent antibodies other than "multispecific" or "multifunctional" antibodies should be understood to contain binding sites with the same antigen specificity.

[0118] Monoclonal antibodies (mAbs) that can be purified by the disclosed method can be produced by various techniques, including conventional monoclonal antibody methods, such as standard somatic cell hybridization techniques well known in the art. Although somatic cell hybridization methods are preferred, other techniques for producing monoclonal antibodies can be used in principle, such as viral or oncogenic transformation of B lymphocytes. For example, monoclonal antibodies can correspond to mouse antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.

[0119] Non-limiting examples of antibodies that can be purified by the methods of the invention also include panitumumab, omalizumab, abavoumab, abciximab, actoxumab, adalimumab, adecatumumab, afelimomab, afutuzumab, alacizumab, alemtuzumab, alirocumab, atumomab, amatuximab, anatumomab, apolizumab, atinumab, tocilizumab, basilizimab, betumumab, Rimumab, bevacizumab, bicizumab, canakinumab, cetuximab, daclizumab, densumab, eculizumab, edrecolomab, efalizumab, ifengumab, ertumaxomab, etaracizumab, etanercept, golimumab, infliximab, natalizumab, palivizumab, panitumumab, pertuzumab, ranibizumab, rituximab, tocilizumab, trastuzumab, dupilumab, sarilumab, or fresolimumab.

[0120] In certain aspects, the protein to be purified is an enzyme.

[0121] Non-limiting examples of enzymes that can be purified by the methods of the present invention include: acid α-glucosidase, α-L-iduronidase, iduronate sulfatase, heparan N-sulfatase, galactose-6-sulfatase, acid β-galactosidase, β-glucuronidase, N-acetylglucosamine-1-phosphotransferase, α-N-acetylgalactosaminidase (α-galactosidase B), acid lipase, lysosomal acid ceramidase, acid sphingomyelinase, β-glucosidase, galactosylceramidase, α-galactosidase A, acid β-galactosidase, β-galactosidase, neuraminidase, hexosaminidase A or hexosaminidase B.

[0122] Other non-limiting examples of proteins that can be purified by the methods of the invention include human erythropoietin, tumor necrosis factor (eg, TNF-α, TNF-β, or TNF-κ), interferon α, or interferon β.

[0123] The solution comprising the protein to be purified may be a culture medium, preferably a clarified culture medium.The solution comprising the protein to be purified is, for example, a culture medium obtained in a perfusion bioreactor or a fed-batch bioreactor.

[0124] Examples of perfusion bioreactors or fed-batch bioreactors are disclosed in U.S. patent applications US 2014 / 255994, US 2015 / 232505, US 2015 / 183821 and US 2017 / 218012 and international application WO 2014 / 137903 (incorporated herein by reference in their entirety).

[0125] The term "clarified medium" means liquid culture medium obtained from a culture of mammalian, bacterial or yeast cells that is substantially free (e.g., at least 80%, 85%, 90%, 92%, 94%, 96%, 98% or 99% free) of mammalian, bacterial or yeast cells.

[0126] In a particular embodiment, the first filtration step of the method of the invention may be integrated in a clarification step for obtaining clarified culture medium in a cell culture recovery process, said filtration step thus becoming part of the clarification step.

[0127] As used herein, the phrase "recovering the protein" refers to collecting the protein after using the disclosed purification methods.

[0128] In the context of the present invention, the expression "chelating agent" refers to any kind of particulate adsorption medium or immobilized ligand, such as activated carbon, diatomaceous earth, resin beads, which are used as absorbents in purification processes to separate the target molecule to be purified from the contaminant molecules present in the mixture. The expression "chelating agent matrix" does not include Protein A matrix.

[0129] In certain embodiments of the methods of the present invention, the at least one chelating agent matrix is ​​a particulate adsorption medium.

[0130] The at least one chelating agent matrix may be in the form of a column, a filter or added as a powder to the product to be purified.In a particular embodiment, the at least one chelating agent matrix used in the context of the present invention is added to the product to be purified in the form of a powder.

[0131] In certain embodiments of the disclosed methods, the at least one chelating agent matrix is ​​an activated carbon filter. In other certain embodiments of the disclosed methods, the at least one chelating agent is a resin.

[0132] The at least one chelating agent matrix, in particular an activated carbon filter or resin medium, interacts with the contaminants to achieve a high efficiency removal of the impurities.Another advantage of using a chelating agent matrix, in particular activated carbon or resin, is the lower affinity for monoclonal antibodies.

[0133] In a particular embodiment of the invention, the at least one chelating agent matrix in the filtering step is selected from activated carbon, diatomaceous earth, free cation exchange resins, free anion exchange resins and free mixed mode resins. In a particular embodiment, the at least one chelating agent matrix is ​​a combination of two different chelating agent matrices. In a more particular embodiment, the combination of two different chelating agent matrices is a combination of activated carbon, in particular an activated carbon filter and a free anion exchange resin.

[0134] In certain embodiments of the disclosed methods, the activated carbon filter is Zeta Plus 35SP (commercialized by 3M), Zeta Plus 53SP (commercialized by 3M), Millisak CR40 (commercialized by Millipore) or ZetaPlus 55SP grade (commercialized by 3M).

[0135] In another specific embodiment of the disclosed method, the free anion exchange resin is NH2-750F (commercialized by Tosoh) or Emphaze AEX (commercialized by 3M). The properties of the resin NH2-750F are summarized as follows.

[0136]

[0137] In the context of the present invention, the expression "diafiltration" refers to a technique for completely removing, replacing or reducing the concentration of salts or solvents from a solution using an ultrafiltration membrane.

[0138] As used herein, "ultrafiltration" or "UF" refers to a filtration technique that uses a semi-permeable membrane to physically and selectively remove particles and / or ions from a solution based on particle size and pore size in the UF membrane.

[0139] In one embodiment, the at least one diafiltration membrane of the exchanging step is a single pass tangential flow filtration (SPTFF) module or a tangential flow filtration (TFF) module. In a specific embodiment, the at least one diafiltration membrane of the exchanging step is in the form of a cartridge, a hollow fiber or a spiral wound.

[0140] In certain embodiments of the disclosed methods, at least one diafiltration membrane of the exchanging step is a Ready To Process hollow fiber cartridge 30 kDa (commercialized by GE).

[0141] In certain embodiments of the disclosed methods, at least one diafiltration membrane of the exchanging step is a Cadence in-line diafiltration device (commercialized by Pall).

[0142] In one embodiment, the at least one diafiltration membrane of the exchange step is preceded or followed by a Cadence in-line concentrator (commercialized by Pall).

[0143] In another specific embodiment of the disclosed method, at least one diafiltration membrane of the exchanging step is a Pellicon cassette (commercialized by Millipore) or a Sartocon cassette (commercialized by Sartorius).

[0144] Said exchange step advantageously allows purification and concentration of the filtered protein solution.In the present context, the expression "concentrating the filtered protein solution" means that the concentration of the protein in the partially purified retentate is increased compared to the protein concentration in the filtered protein solution.

[0145] In the context of the present invention, "membrane adsorber" refers to a planar layer of a polymer, especially an acrylic polymer, or a fiber or nonwoven medium with functional groups such as affinity groups and ion exchange groups. One of the differences between resins and membranes is the flow distribution: for resins it is by diffusion, and in membranes it is by convection.

[0146] The combination of membrane adsorbers used in the polishing step involves the use of at least two membrane adsorbers that are orthogonal in their mechanism of action.

[0147] In the context of the present invention, the expression "membrane adsorbers that are orthogonal in terms of their mechanism of action" means that the membrane adsorbers used have opposite or different mechanisms of action, such as cation exchange and anion exchange interactions, multimodal and anion exchange interactions, cation exchange and hydrophobic interactions, or anion exchange and hydrophobic interactions, and that the membrane adsorbers are a single integrated step, i.e. wherein they are processed together like a single filtration step.

[0148] In fact, the inventors have demonstrated that the use of this membrane adsorber combination enables the capture of impurities while minimizing the adsorption of the desired product.

[0149] In a preferred embodiment, more than two membrane adsorbers are used in the membrane adsorber combination of the fine purification step, that is, at least one additional membrane adsorber is a total of three, four or more membrane adsorbers, and the at least one additional membrane adsorber is a membrane with a different mechanism of action compared to the two membrane adsorbers that are orthogonal in terms of the mechanism of action; the total of more than two membrane adsorbers are still considered to be an integrated single step.

[0150] As a non-limiting example, if two membrane adsorbers that are orthogonal in terms of their mechanism of action have cation exchange and anion exchange interactions, respectively, the at least one additional membrane adsorber has a hydrophobic interaction or a multimodal interaction.

[0151] In the context of the present invention, the membrane adsorbers are specifically combined into a single integrated step. Thus, the number of steps and buffers is greatly reduced. Adjustments and manipulations are also removed, thereby simplifying the global process. In particular, there are no elution, adjustment or storage steps between one membrane adsorber of a membrane adsorber combination and another (multiple) membrane adsorber of this membrane adsorber combination.

[0152] In certain embodiments, the specific combination of membrane adsorber and operating conditions (particularly pH, conductivity and / or buffer) of the polishing step is determined according to the properties of the protein to be purified (eg, pI, molecular weight, ...).

[0153] As will be appreciated by those skilled in the art, the optimal conditions for the polishing step should allow for obtaining the maximum yield of the protein of interest, i.e. should allow for minimal interaction of the protein of interest with the membrane adsorber combination, while allowing for maximum removal of contaminants, i.e. without any conditioning or elution steps between the membrane adsorbers of the membrane adsorber combination, while allowing for maximum interaction of the contaminants with the membrane adsorber combination.

[0154] Figure 3An example of determining the best combination of two membrane adsorbers for a fine purification step is illustrated. In the example, the best purification performance was obtained using a combination of Sartobind S and Sartobind STIC membrane adsorbers, with higher contaminant removal (lower HCP and HMW) while maximizing antibody yield. The figure also shows the effect of sample volume on yield and contaminant removal.

[0155] In the context of the present invention, in order to determine these optimal conditions, the membrane adsorbers of the membrane adsorber combination should be considered as a single entity or a single integrated step, i.e. they should be processed together like a single filtration step. For example, in the case of two membrane adsorbers, although the conventional method of determining the optimal conditions when using two membrane adsorbers involves determining the optimal conditions on the first membrane adsorber, then determining the optimal conditions on the second membrane adsorber, and then adjusting the product between the two optimal conditions (such as pH adjustment, conductivity adjustment or buffer adjustment), in the context of the present invention, the two membrane adsorbers are considered as only one, and a compromise should be determined for the separation performance of the two membrane adsorbers so that good purification can still be obtained.

[0156] Typically, when determining the optimized buffer for purification of a given protein, an exchange step can be performed with a neutral buffer, and the pH and conductivity of the solution containing the retentate can be manually adjusted to determine the optimal purification conditions in the polishing step. When the optimal conditions are determined, the process can be performed again using a buffer suitable for the exchange step, which will correspond to the equilibration buffer used in the polishing step.

[0157] Figure 4 An example of determining the optimal buffer (such as the conductivity or pH of the buffer) based on the combination of two membrane adsorbers and the pI of the target protein for the polishing step is illustrated. In this example, the pH and conductivity conditions that enable favorable purification (corresponding to HCP levels between 50 ng / ml and 500 ng / ml) correspond to the black area of ​​each figure.

[0158] In the context of the present invention, the one buffer used to equilibrate the at least two membrane adsorbers is the same as the buffer used for the exchange step. It allows direct flow-through of the protein to be purified to maximize yield while retaining most contaminants.

[0159] In certain embodiments, the polishing step is optimized by varying the pH and conductivity of the buffer used to condition the protein during the exchange step.

[0160] In certain embodiments, the polishing purification step comprises using a cation exchange membrane adsorber matrix in combination with an anion exchange membrane adsorber matrix. In other embodiments, the polishing purification step comprises using a multimodal (mixed mode) membrane adsorber matrix in combination with an anion exchange membrane adsorber matrix.

[0161] The combination of membrane adsorber matrices, in particular the combination of cation exchange membrane adsorbers and anion exchange membrane adsorbers, works through the interaction between the membrane adsorbers and the pollutants, resulting in efficient removal of impurities. The interaction with the pollutants is attributed to several mechanisms: ionic interactions, hydrophobic interactions, van der Waals interactions, and hydrogen bonding interactions.

[0162] In a specific embodiment, the cation exchange membrane adsorber is a Sartobind S membrane adsorber (Sartorius), a HD-C membrane adsorber (Natrix). In a specific embodiment, the cation exchange membrane adsorber is a Sartobind S membrane adsorber (Sartorius). In a specific embodiment, the anion exchange membrane adsorber is a Sartobind STIC membrane adsorber (Sartorius), a Sartobind Q membrane adsorber (Sartorius), or a HD-Q membrane adsorber (Natrix). In a specific embodiment, the anion exchange membrane adsorber is a Sartobind STIC membrane adsorber (Sartorius) or a Sartobind Q membrane adsorber (Sartorius). In other embodiments, the multimodal membrane adsorber is a HD-Sb membrane adsorber (Natrix). In other embodiments, the hydrophobic interaction membrane adsorber is a Sartobind phenyl membrane adsorber (Sartorius).

[0163] In a particular embodiment, the membrane adsorber combination is a combination of a Sartobind S membrane adsorber (Sartorius) and a Sartobind STIC membrane adsorber (Sartorius).

[0164] The main advantages of using a membrane adsorber rather than a column in the polishing step of the process of the present invention are summarized below:

[0165] - At a comparable scale, a membrane adsorber can be used at a flow rate 10 times higher than a column, thus greatly reducing the duration of the process. For example, a 5 mL column filled with resin will be used at a flow rate of 1 ml / min, while a corresponding 5 mL membrane adsorber will be used at a minimum flow rate of 10 ml / min. Thus, while a classical process using two chromatography steps for polishing can be performed in 2 hours and 30 minutes using two columns filled with resin, the classical process can be completed in less than 15 minutes using a membrane adsorber.

[0166] - Although membrane adsorbers are reusable, they are disposable devices and therefore can be discarded after a batch and do not require long-term storage. Therefore, there is no need to test them to ensure long-term stability.

[0167] - Use of membrane adsorbers has lower costs by avoiding column costs, column filling and column storage.

[0168] In a particular embodiment, when the membrane adsorber combination is a combination of a Sartobind S membrane adsorber (Sartorius) and a Startobind STIC membrane adsorber (Sartorius), the pH and conductivity of the one buffer are in the range Figure 4 The black area in the above figure is within the public range.

[0169] In another specific embodiment, when the membrane adsorber combination is a combination of a Sartobind S membrane adsorber (Sartorius) and a Startobind Q membrane adsorber (Sartorius), the pH and conductivity of the one buffer are in the range Figure 4 The black area in the figure below is within the public range.

[0170] The purification method according to the present invention is a complete flow-through purification method.

[0171] In the present context, "complete flow-through purification method" means that the different purification steps of the method are meant to only bind impurities while passing the protein of interest through the purification steps.

[0172] In one embodiment, the method according to the invention does not comprise adjusting the pH of the filtered protein solution at the end of the filtration step and / or adjusting the pH of the retentate at the end of the exchange step.

[0173] In a specific embodiment, the filtered protein solution obtained at the end of the filtering step is directly passed through a diafiltration membrane. More specifically, there is no subsequent processing (such as pH adjustment, buffer exchange or dilution) between two steps. In this method, the diafiltration membrane can for example correspond to the Ready To Process 30kD hollow fiber module or the Cadence online diafiltration module before or after the Cadence online concentrator. In addition, in a specific embodiment, the retentate obtained at the end of the exchange step is directly passed through the membrane adsorber combination of fine purification step. More specifically, there is no subsequent processing (such as pH adjustment, buffer exchange or dilution) between two steps. In this method, the diafiltration membrane can for example correspond to the Ready To Process 30kD hollow fiber module and / or the membrane adsorber combination can for example correspond to the combination of SartobindS membrane adsorber and Sartobind STIC membrane adsorber.

[0174] In this method, inter-step processing (eg, dilution, conductivity adjustment, and pH adjustment) requiring manual intervention and opening of the purification system is completely absent.

[0175] Therefore, the method of the present invention can be performed in a flexible automated chromatography system comprising multiple pumps and sensors, as well as interconnecting valves and switching valves for sequential or continuous operation of the three purification steps.

[0176] A non-limiting example of a multi-handling system is a multi-column chromatography system (MCCS) with appropriate adaptations.

[0177] The method of the present invention may be run in a continuous manner. In other words, the method of the present invention may be a continuous method for purifying a protein from a solution.

[0178] The term "continuous process" or "process in a continuous manner" means a process in which a fluid is continuously fed through at least a portion of a system.

[0179] As used herein, the term "fluid" refers to any liquid, such as a solution containing a protein to be purified, a buffer, or a low pH or acidic pH solution for virus inactivation.

[0180] In certain embodiments, the first, second and third purification steps are continuously fed with fluid.

[0181] The term "integrated process" means a process performed using structural elements that act cooperatively to achieve a specific result (eg, production of a purified protein from a liquid culture medium).

[0182] In addition, the method of the present invention can be run from the first step to the last step of the method in a closed system. In other words, the method of the present invention preferably has a functionally closed flow path. In particular, three purification steps and optional one or more filtration steps (e.g., nanofiltration step and / or final ultrafiltration and / or diafiltration step) can be performed in a closed system. In a specific embodiment of the method of the present invention, the solution containing the protein is passed through the three purification steps one by one, and each passage of a part of the solution corresponds to a run. Then collect and merge the protein recovered at the end of each run. In this method, the membrane adsorber of the purification step is used many times, and optionally disinfected using, for example, a disinfection buffer as described above, so that the volume of the membrane adsorber device and the required buffer can be reduced. For example, a series of 3 to 50 runs (e.g., 3 to 30 runs, 5 to 25 runs, 10 to 20 runs, or 15 runs) can be performed continuously. More specifically, 3, 4, 5, 6, 7 or 8 runs can be performed in a continuous manner, and then the membrane adsorber is disinfected (e.g., using a disinfection buffer). This can be repeated, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times, such as Figure 2 shown.

[0183] The methods disclosed herein can be used to recover purified proteins. As used herein, "purified" refers to a purity that allows effective use of the protein in vitro, in vitro, or in vivo. In order for a protein to be effective for in vitro, in vitro, or in vivo applications, the protein should be substantially free of contaminants, other proteins, and / or chemicals that may interfere with the use of the protein in such applications, or at least chemicals that are not desired to be included in the target protein. Such applications include the preparation of therapeutic compositions, administration of proteins in therapeutic compositions, and other methods disclosed herein. Preferably, as referred to herein, a "purified" protein is a protein that can be produced by any method (i.e., by direct purification from a natural source, recombinant or synthetic) and has been purified from other protein components such that the protein comprises at least about 70% weight / weight of the total protein in a given composition, and more preferably at least about 80% or at least about 85%, and more preferably at least about 90%, and more preferably at least about 91%, and more preferably at least about 92%, and more preferably at least about 93%, and more preferably at least about 94%, and more preferably at least about 95%, and more preferably at least about 96%, and more preferably at least about 97%, and more preferably at least about 98%, and more preferably at least about 99% weight / weight of the total protein in a given composition.

[0184] In certain embodiments, the methods of the present invention comprise:

[0185] (a) a filtering step, comprising:

[0186] (i) passing said solution through said at least one chelating agent matrix in said flow-through manner,

[0187] (ii) recovering a filtered protein solution from the flow-through of the at least one chelator matrix,

[0188] (b) an exchanging step, comprising:

[0189] (i) passing the filtered protein solution obtained from step (a) and the one buffer through the at least one diafiltration membrane,

[0190] (ii) recovering the retentate containing the partially purified protein from the at least one diafiltration membrane;

[0191] as well as

[0192] (c) a fine purification step, which comprises:

[0193] (i) passing an equilibration buffer through the membrane adsorber assembly, wherein the equilibration buffer is the same as the one buffer used for exchange in step (b),

[0194] (ii) passing the retentate obtained from step (b) through the membrane adsorber assembly in said flow-through mode,

[0195] (iii) recovering the purified protein from the flow-through of the membrane adsorber assembly,

[0196] wherein the purification method does not include Protein A chromatography,

[0197] wherein said buffer comprises or consists of:

[0198] - 5mM to 40mM (e.g. 20mM) Bis Tris and 15mM to 150mM (e.g. 75mM) NaCl, pH adjusted to 6-9 (e.g. 7.5) with acetic acid,

[0199] -5 mM to 40 mM (e.g. 20 mM) Tris or Tris-HCl, 15 mM to 150 mM (e.g. 75 mM) NaCl, pH adjusted to 6-9 (e.g. 7.5) with acetic acid,

[0200] -5 mM to 40 mM (e.g. 20 mM) Tris or Tris-HCl, 15 mM to 150 mM (e.g. 75 mM) NaCl, pH adjusted to 6-9 (e.g. 7.5) with citric acid, or

[0201] -15 mM to 150 mM (e.g. 75 mM) NaCl, pH adjusted to 6-9 (e.g. 7.5) with 2HPO4 / NaH2PO4 (preferably 10 mM / 90 mM Na2HPO4 / NaH2PO4 to 90 mM / 10 mM Na2HPO4 / NaH2PO4).

[0202] The method for purifying protein from solution may include at least one additional final filtration step after the fine purification step, such as a nanofiltration step, an ultrafiltration step and / or a diafiltration step. When purifying recombinant proteins for pharmaceutical purposes, an additional final filtration step is usually performed after the fine purification step. Therefore, the method of the present invention may further include a nanofiltration step after step (c). After the nanofiltration step, ultrafiltration and diafiltration steps may be further performed. As used herein, "ultrafiltration" or "UF" refers to a filtration technique that uses a semi-permeable membrane to physically and selectively remove particles and / or ions from a solution based on the pore size in the particle size and UF membrane. As used herein, "nanofiltration" refers to filtering a solution through a nanofilter for removing, for example, viral particles. As used herein, "diafiltration" refers to a technique for completely removing, replacing, or reducing the concentration of salts or solvents from a solution using an ultrafiltration membrane.

[0203] The method of the present invention can also further include at least one virus inactivation step. The at least one virus inactivation step can be carried out at any stage of the method of the present invention, for example, before step (a), after step (a), after step (b), after step (c), after nanofiltration step and / or after ultrafiltration and / or diafiltration step. This virus inactivation step can generally be a low pH or acidic pH inactivation step. As used herein, "low pH or acidic pH inactivation" refers to a virus inactivation technique that uses acidic pH to denature viruses, particularly enveloped viruses. Generally, the low pH or acidic pH inactivation step is by culturing the protein recovered at a pH of at least 15 minutes (for example, a time period between 15 minutes and 1 hour, a time period between about 30 minutes and 2 hours, or a time period between about 45 minutes and 2 hours) between about 3.0 and 5.0 (for example, between about 3.5 and about 4.5, between about 3.5 and about 4.25, between about 3.5 and about 4.0, for example 4.0) for a time period of at least 15 minutes. For example, the low pH or acidic pH inactivation step is performed by incubating the recovered protein at pH 4 for, for example, 30 minutes to 2 hours.

[0204] Prior to step (a), the method of the present invention may further comprise the step of providing a liquid culture medium containing the protein to be purified which has been clarified to remove cells and is substantially free of cells, wherein the liquid culture medium is fed through the at least one chelating agent matrix.

[0205] For example, a method of the invention for purifying a protein from a solution may comprise:

[0206] (a) providing a liquid culture medium containing the protein to be purified which has been clarified to remove cells and is substantially free of cells,

[0207] (a) a filtering step, comprising:

[0208] - passing the liquid culture medium of step (a) through at least one chelating agent matrix in a flow-through manner;

[0209] - recovering a filtered protein solution from the flow-through of the at least one chelator matrix;

[0210] (b) an exchanging step, comprising:

[0211] - passing said filtered protein solution obtained at the end of step (a) through at least one diafiltration membrane using only one buffer for said exchange;

[0212] - recovering the retentate of said at least one diafiltration membrane containing the partially purified protein; and

[0213] (c) a fine purification step, which comprises:

[0214] - passing the retentate obtained at the end of step (b) in the flow-through manner through a membrane adsorber assembly, wherein the two membrane adsorbers of the membrane adsorber assembly are orthogonal in terms of the mechanism of action and the membrane adsorber assembly has been previously equilibrated with the same equilibration buffer as the one buffer used for the exchange in step (b);

[0215] - recovering the purified protein from the flow-through of the membrane adsorber assembly;

[0216] wherein the purification method does not include a Protein A chromatography step.

[0217] Finally, the purified protein can be formulated into a composition suitable for storage, and / or into a pharmaceutical composition particularly suitable for administration to animals and / or humans.

[0218] One of the many advantages of the disclosed method is that it allows obtaining high-purity proteins in high yields. For example, the purified protein recovered by the method of the present invention can exhibit a purity of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5% or 99.9%. More particularly, one of the many advantages of the disclosed method is that it allows obtaining a high-purity protein solution containing a reduced amount of contaminating DNA, high molecular weight (HMW) substances (which correspond to protein aggregates) and / or host cell proteins (HCP). For example, a solution comprising a purified protein recovered by the method of the present invention can exhibit an amount of contaminating DNA less than 0.4 ppb, less than 0.3 ppb, less than 0.2 ppb or less than 0.1 ppb. For example, a solution comprising a purified protein recovered by the methods of the invention may also exhibit a concentration of HMW species of less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1%. For example, a solution comprising a purified protein recovered by the methods of the invention may also exhibit a HCP concentration of less than 500 ng / ml, less than 100 ng / ml, less than 90 ng / ml, less than 85 ng / ml, less than 80 ng / ml, less than 75 ng / ml, or less than 70 ng / ml. In addition, the methods of the invention may allow the recovery of purified protein in a yield of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0219] The present invention further relates to a kit comprising or consisting of:

[0220] (a) at least one chelating agent matrix, at least one diafiltration membrane and a membrane adsorber combination, wherein the two membrane adsorbers of the membrane adsorber combination are orthogonal in terms of the mechanism of action; and

[0221] (b) a buffer comprising Tris, Tris-HCl, Bis Tris, phosphate and / or citric acid, in particular comprising or consisting of: (i) Bis Tris, Tris or Tris-HCl, (ii) acetic acid, (iii) water and (iv) optionally NaCl; and / or instructions for preparing a buffer comprising Tris, Tris-HCl, Bis Tris, phosphate and / or citric acid, in particular comprising or consisting of: (i) Bis Tris, Tris or Tris-HCl, (ii) acetic acid, (iii) water and (iv) optionally NaCl.

[0222] The present invention will be further described below by means of drawings and examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0223] Figure 1 A scheme representing the 3-step complete flow-through process of the present invention is shown.

[0224] Figure 2 A scheme representing a continuous version of the 3-step complete flow-through process of the present invention as used in Example 4 is shown.

[0225] Figure 3 Shown are histograms showing a comparison of the yield (%), HMW (%) and HCP (ng / ml) obtained using four membrane adsorber combinations (HD-C+HD-Q; HD-Sb+HD-Q; Sartobind S+Q; Sartobind S+STIC) according to the capacity of the membrane (mg / ml).

[0226] Figure 4 The optimum point diagrams of the two membrane adsorber combinations according to pH and conductivity (mS / cm) of the equilibrium buffer (upper diagram: Sartobind S+STIC; lower diagram: Sartobind S+Q) and 3 pIs of the protein to be purified (6; 7.5 and 9) are shown. The pH and conductivity conditions that enable a favorable purification (corresponding to HCP levels between 50 and 500 ng / ml) correspond to the black areas of each diagram.

[0227] Figure 5 A partial chromatogram (UV 280 nm) of the purification performed on the membrane during a continuous bench-scale process experiment is shown.

[0228] Example

[0229] Example 1 : Laboratory scale process according to the present invention

[0230] The method of the present invention was used to carry out batch purification of humanized monoclonal antibody mAb1 on a laboratory scale.

[0231] This experiment shows the impurity removal results obtained at laboratory scale after three steps. The goal was to remove aggregates (HMW) and host cell proteins (HCP), HMW below 1% and HCP below 500 ng / ml.

[0232] The inventors succeeded in achieving this goal using 2 different chelating agents, an exchange step and a pair of membranes (Sartobind S and STIC or Sartobind S and Q).

[0233]

[0234] Example 2: Laboratory scale process according to the invention

[0235] The method of the present invention was used to carry out laboratory-scale batch purification of humanized monoclonal antibody mAb2.

[0236] The experiment shows the results of impurity removal obtained on a laboratory scale after three steps. The purpose is to remove aggregates (HMW) and host cell proteins (HCP), HMW is less than 1% and HCP is less than 500ng / ml. The inventors successfully achieved the purpose using 2 chelating agents, an exchange step and a pair of membranes (Sartobind S and STIC or Sartobind S and Q). The best results were obtained with Sartobind S+STIC when the injection conditions were set to pH 8.5 and 3ms / cm.

[0237]

[0238]

[0239] Example 3: Pilot scale process according to the invention

[0240] mAbl was purified in a pilot scale batch using the method of the present invention.

[0241] The same process as disclosed in Example 2 was scaled up on a pilot scale. The aim was to purify 10 g through these three steps and a subsequent nanofiltration step.

[0242] 8 L of clarified cell culture supernatant was purified by the three steps of the method of the present invention and nanofiltration. 8 L was introduced into a 20 L mixer, and the following items were introduced successively: 250 ml Tosoh NH2-750F resin, followed by stirring for 13 min; 160 g activated carbon (Norit SA2 grade), followed by stirring for 14 min; and 30 g activated carbon (Norit SA2 grade), followed by stirring for 10 min.

[0243] Before starting the exchange step, the product was filtered (Filtrox filter). Approximately 9 L of product was recovered after filtration (1 L of sterile water was used to push the product out of the filter). UF / DF was performed on a GE Uniflux using a hollow fiber (790 cm2-50KD - Spectrumlabs). The buffer used for diafiltration of the product through the hollow fiber was 20 mM Bis Tris buffer (adjusted to pH 7.2 with appropriate amount of acetic acid).

[0244] 2.64 kg (18.2 g monoclonal antibody) were recovered at 6.9 g / L. 2 L (14 g) of the recovered product were then pushed through two membrane adsorbers: 75 ml Sartobind S and 200 ml Sartobind Q. The two membranes were connected in series and used in flow-through mode using GE AktaProcess.

[0245] Finally, the product was nanofiltered through a prefilter (XOHC–Millipore) and a Viresolve Pro filter (Millipore) to obtain the final quality.

[0246] The following table shows a comparison between the conventional process and the method of the present invention.

[0247]

[0248] Example 4: Fully continuous laboratory scale process according to the invention

[0249] The humanized monoclonal antibody mAb1 was purified in a continuous manner using the method of the present invention in a laboratory scale batch manner.

[0250] The aim of the experiment was to purify mAb1 from a clarified bulk harvest using a continuous approach, meaning no interruptions, storage or adjustments between each step. The inventors succeeded in achieving this using filtration on chelating agents (immobilized AEX, activated carbon CA), one exchange step (single pass diafiltration) and a pair of membranes (Sartobind S and STIC).

[0251] The cell culture supernatant of 3L clarification by three steps of the inventive method is purified. The product is filtered by immobilized anion exchanger (Emphaze AEX BV120) and activated carbon filter (Millipore CR40 270cm2) subsequently. Then the product is directly flowed into a single-pass diafiltration membrane (0.2m2) and concentrated and diafiltered (exchange step). The buffer used for the diafiltration product is 20mM Bis Tris, 20mM NaCl buffer (being adjusted to pH 7.5 with an appropriate amount of acetic acid).

[0252] The diafiltered product, which is directly recovered from the retentate side, is passed through an intermediate buffer bag to accommodate flow differences in subsequent steps. The product is then further pushed through two fine purification membrane adsorbers; 1 ml Sartobind S and 1 ml Sartobind STIC. The two membranes are connected in series and used in flow-through mode using GE Akta Pure. Each unit step is connected to another directly or through a buffer bag and is processed continuously.

[0253] Several purification cycles were performed on the membrane adsorber to process the entire volume of the product, e.g. Figure 5 As shown (extracts from 50 purification cycles on the membrane). At the end of the process, the entire product pool was recovered through a sterile filter. Purifying 40 mg of mAb every 5 minutes, a productivity of 240 g mAb1 per liter of membrane per hour (240 g / L / h) was achieved.

Claims

1. A complete flow-through method for purifying a protein from a solution, the method comprising: (a) a filtering step, comprising: - passing the solution through at least one chelating agent matrix in a flow-through manner, - recovering a filtered protein solution from the flow-through of the at least one chelator matrix; (b) an exchanging step comprising: - passing said filtered protein solution obtained at the end of step (a) through at least one diafiltration membrane using only one buffer for said exchange, - recovering the retentate of the at least one diafiltration membrane containing the partially purified protein; (c) a polishing step comprising: - passing the retentate obtained at the end of step (b) in the flow-through manner through a membrane adsorber combination as a single integrated step, wherein the two membrane adsorbers of the membrane adsorber combination are orthogonal in terms of the mechanism of action and the membrane adsorber combination has been previously equilibrated with the same equilibration buffer as the one buffer used for the exchange in step (b), and wherein no elution, conditioning or storage step is performed between one membrane adsorber in the membrane adsorber combination and one or more other membrane adsorbers in the membrane adsorber combination; - recovering the purified protein from the flow-through of the membrane adsorber assembly; wherein the method does not include a Protein A chromatography step, The method described herein uses only one buffer, wherein one of the buffers comprises Tris, Tris-HCl, Bis Tris, phosphate and / or citric acid, wherein at least one chelating agent matrix is ​​a combination of two different chelating agent matrices, the combination of the two different chelating agent matrices comprising activated carbon, The membrane adsorber in the fine purification step includes a cation exchange membrane adsorber and an anion exchange membrane adsorber, and Wherein the protein is an antibody.

2. The process according to claim 1, wherein the filtered protein solution obtained at the end of step (a) is passed directly through the at least one diafiltration membrane without any treatment.

3. The process according to claim 1 or 2, wherein the retentate obtained at the end of step (b) is passed directly through the membrane adsorber combination without any treatment.

4. The method according to claim 1 or 2, wherein the method does not include any intermediate storage between the three steps (a), (b) and (c).

5. The method of claim 1 or 2, wherein the method has a functionally closed flow path.

6. The method of claim 1 or 2, wherein the combination of two different chelating agent matrices is a combination of (i) activated carbon and (ii) free anion exchange resin, free cation exchange resin or free mixed mode resin.

7. The method according to claim 1 or 2, wherein the at least one diafiltration membrane in the exchanging step is a single pass tangential flow filtration (SPTFF) module or a tangential flow filtration (TFF) module.

8. The method of claim 1 or 2, wherein the at least one diafiltration membrane of the exchanging step is in the form of a cartridge, a hollow fiber or a spiral wound.

9. The method of claim 1 or 2, wherein the filtered protein solution is concentrated in the exchanging step.

10. The method according to claim 1 or 2, wherein the membrane adsorber combination of the polishing step further comprises a membrane adsorber selected from the group consisting of a multimodal membrane adsorber and a hydrophobic interaction membrane adsorber.

11. The method according to claim 1 or 2, wherein the membrane adsorber combination of the polishing step is a combination of a cation exchange membrane adsorber and an anion exchange membrane adsorber.

12. The method according to claim 1 or 2, further comprising a nanofiltration step after step (c).

13. The method according to claim 12, further comprising a final ultrafiltration and / or diafiltration step after the nanofiltration step.

14. The method of claim 1 or 2, wherein the protein is a monoclonal antibody.

15. The method of claim 1 or 2, wherein the one buffer comprises: - 5 mM to 40 mM Bis Tris, 15 mM to 150 mM NaCl, pH adjusted to 6-9 with acetic acid, - 5 mM to 40 mM Tris or Tris-HCl, 15 mM to 150 mM NaCl, pH adjusted to 6-9 with acetic acid, - 5 mM to 40 mM Tris or Tris-HCl, 15 mM to 150 mM NaCl, pH adjusted to 6-9 with citric acid, or - 15 mM to 150 mM NaCl, adjust pH to 6-9 with Na2HPO4 / NaH2PO4.

16. The method according to claim 1 or 2, further comprising the step of formulating the recovered purified protein into a pharmaceutical composition.

17. The method according to claim 1 or 2, wherein said one buffer comprises or consists of: (i) Bis Tris, Tris or Tris-HCl, (ii) acetic acid, (iii) water and (iv) optional salt.

18. The process according to claim 1 or 2, wherein the filtered protein solution obtained at the end of step (a) is directly passed through the at least one diafiltration membrane without any pH adjustment, buffer exchange or dilution.

19. The process according to claim 1 or 2, wherein the retentate obtained at the end of step (b) is passed directly through the membrane adsorber combination without any pH adjustment, buffer exchange or dilution.

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