WO2024017826A2 - Procédés de purification de polypeptides recombinants - Google Patents

Procédés de purification de polypeptides recombinants Download PDF

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WO2024017826A2
WO2024017826A2 PCT/EP2023/069769 EP2023069769W WO2024017826A2 WO 2024017826 A2 WO2024017826 A2 WO 2024017826A2 EP 2023069769 W EP2023069769 W EP 2023069769W WO 2024017826 A2 WO2024017826 A2 WO 2024017826A2
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cation
chromatography
anion
impurities
protein
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PCT/EP2023/069769
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WO2024017826A3 (fr
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Nicholas E. LEVY
Gerald J. Terfloth
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Glaxosmithkline Intellectual Property Development Limited
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation

Definitions

  • the present invention is directed to novel methods for purifying recombinant polypeptides from a solution comprising one or more impurities.
  • the methods include a chromatography step which uses a wash buffer containing a small radius cation and a large radius anion.
  • Recombinant polypeptides such as antibodies and other proteins, are used for the therapeutic treatment of a wide range of diseases.
  • the biopharmaceutical manufacture of these complex recombinant polypeptides typically requires the use of a biological host system, which through genetic engineering, is capable of expressing the product in a suitably active form.
  • Expression of recombinant polypeptides generally involves culturing prokaryotic or eukaryotic host cells under appropriate conditions. Once the recombinant polypeptide is expressed, intact host cells and cell debris can be separated from the cell culture media to provide a clarified unprocessed bulk (CUB) or clarified cell culture fluid (CCCF), which includes the recombinant polypeptide and other impurities.
  • CAB unprocessed bulk
  • CCCF clarified cell culture fluid
  • Recombinant polypeptides produced by biopharmaceutical manufacturing processes are typically associated with multiple undesirable impurities, including, but not limited to: host cell proteins (HCPs), DNA, viruses, high- and low-molecular weight species, and unwanted product and process variants, which can be difficult to remove and have the potential to significantly reduce the safety and efficacy of the biopharmaceuticals manufactured.
  • HCPs host cell proteins
  • the levels of the impurities therefore must be critically controlled to comply with regulatory guidelines, and the added complexity of contaminants with different physicochemical properties makes identification, quantification, and removal of them and their residual amounts even more challenging, particularly in the presence of large concentrations of the desired recombinant polypeptide product.
  • a method for purifying a recombinant polypeptide from a solution comprising one or more impurities wherein the method is a chromatography process comprising a wash buffer comprising a small ionic radius cation and a large ionic radius anion.
  • a recombinant polypeptide for use in therapy wherein the recombinant polypeptide is purified according to the methods defined herein.
  • a wash buffer comprising a small ionic radius cation and a large ionic radius anion in a chromatography process for purifying a recombinant polypeptide from a solution comprising one or more impurities.
  • a method for purifying an antigen binding protein from a solution comprising one or more impurities comprising the steps of:
  • wash buffer comprising about 0.1 M to about 2 M lithium bromide, about 0.1 M to about 1 M lithium bromide, or about 0.1 M to about IM lithium iodide, and optionally, wherein the wash buffer is at a pH of about 7.5;
  • Figure 1 Host cell protein concentration in the Protein A eluate following a wash buffer containing 0.5 M of the indicated salts using mAbl.
  • Figure 2 mAbl yield following a wash buffer containing 0.5 M of the indicated salts.
  • Figure 3 Host cell protein concentration in the Protein A eluate following a wash buffer containing 0.5 M of various bromide and iodide salts using mAb2.
  • Figure 4 PLBL2 concentration in the Protein A eluate following a wash buffer containing 0.5 M of various bromide and iodide salts using mAb2.
  • Figure 5 Non-reducing SDS-PAGE (left) and anti-CHO Western Blot (right) analysis of Protein A wash fractions for different wash buffers using mAbl.
  • Figure 6 Non-reducing SDS-PAGE (left), anti-CHO Western Blot (center), and anti- PLBL2 (right) western blot analysis of Protein A wash fractions for different wash buffers using mAb2.
  • Figure 7 Host cell protein concentration and step yield of mAbl for various concentration ranges of several bromide wash buffers.
  • D Host cell protein concentration (black) and step yield (gray) of a Protein A process following a MgBrz wash.
  • Figure 8 Host cell protein concentration and step yield of mAbl for various concentration ranges of several iodide wash buffers.
  • Figure 9 Percent fragmentation in the Protein A eluate and cation exchange eluate of mAb3 following either a caprylate or LiBr Protein A wash.
  • the present invention provides methods for purifying a recombinant polypeptide from a solution comprising one or more impurities, wherein the method comprises a wash buffer containing a small radius cation and a large radius anion.
  • the method is a chromatography process comprising a wash buffer comprising a small ionic radius cation and a large ionic radius anion.
  • Recombinant polypeptides are often generated using host cell lines, such as mammalian cells, yeast or E. coli, which are engineered to express the polypeptide of interest. Often, the engineered cells are grown in a bioreactor in appropriate culture media which allows for the expression and release of the polypeptide into the media. This process generates a cell culture solution containing cells, debris, impurities, and the expressed polypeptide. To prepare a product fit for pharmaceutical use, the polypeptide must be purified, and cells, debris, and impurities must be removed. In one embodiment, the recombinant polypeptide is produced intracellularly, and the cells must be lysed to release the polypeptide.
  • host cell lines such as mammalian cells, yeast or E. coli
  • the bioreactor may be a production bioreactor, or n-1 bioreactor, or n-2 bioreactor.
  • the bioreactor may operate in perfusion mode or fed-batch or batch or combinations thereof.
  • the bioreactor may be at a scale of 500 litres, 1000 litres, 2000 litres, 3000 litres, 4000 litres, or 5000 litres or greater.
  • the bioreactor may be at a scale of 10,000 litres, 15,000 litres, 20,000 litres, 25,000 litres, or 30,000 litres or greater.
  • the bioreactor may be single use or fixed.
  • the solution of the methods described herein is any liquid containing the recombinant polypeptide mixed with at least one impurity.
  • the solution is a cell culture feed stream. This may be a harvested feed stream or a continuous feed stream.
  • the solution may be a continuous feed stream from a bioreactor.
  • the solution may be a Clarified Unprocessed Bulk (CUB) (or clarified cell culture harvest/supernatant/fermentation/fluid).
  • CUB Clarified Unprocessed Bulk
  • the CUB is also known as a cell culture supernatant with cells and/or cellular debris removed by clarification.
  • the solution is a clarified cell culture supernatant.
  • Host cells and cell debris can be separated from the cell culture media by clarification, for example via sedimentation, centrifugation and/or filtration.
  • the solution may be a lysed preparation of cells expressing the recombinant polypeptide (e.g. a lysate).
  • the solution may be a clarified cell culture fluid (CCCF). Clarified cell culture fluid (CCCF) is equivalent to Clarified Unprocessed Bulk (CUB) and both terms can be used interchangeably.
  • the cell culture fees stream or clarified cell culture supernatant is from a culture of a mammalian cell line, yeast, or E. coli.
  • the mammalian host cell line may be selected from CHO, NS0, Sp2/0, COS, K562, BHK, PER.C6, and/or HEK cells.
  • the host cell is a Chinese Hamster Ovary cell line (CHO).
  • the present invention allows for methods for purifying a recombinant polypeptide from a solution comprising one or more impurities in which the method includes a chromatography step or process.
  • the chromatography step/process may include a wash buffer comprising a small ionic radius cation and a large ionic radius anion.
  • the chromatography step/process may comprise: (a) a loading step; (b) a washing step; and/or (c) an eluting step.
  • the purified recombinant polypeptide is (i) optionally further purified and (ii) formulated for therapeutic use.
  • the purified recombinant polypeptide is recovered from the eluate of step (c), and optionally formulated.
  • the chromatography process comprises one or more of affinity chromatography ⁇ e.g. dye-ligand chromatography); ion exchange chromatography e.g. anion exchange chromatography or cation exchange chromatography); size exclusion chromatography ⁇ e.g. gel-permeation or gel filtration chromatography); hydrophobic interaction chromatography (HIC); and/or mixed mode chromatography (MMC) ⁇ e.g. ceramic hydroxyapatite chromatography).
  • the operational mode of the chromatography process is bind- elute mode or flow-through mode.
  • the method comprises (i) any one or a combination of affinity chromatography e.g.
  • dye-ligand chromatography ion exchange chromatography e.g. anion exchange chromatography or cation exchange chromatography); size exclusion chromatography e.g. gel-permeation or gel filtration chromatography); hydrophobic interaction chromatography (HIC); and/or mixed mode chromatography (MMC) e.g. ceramic hydroxyapatite chromatography); (ii) an operational mode of bind-elute mode or flow-through mode; and (iii) any one or a combination of (a) a loading step, (b) a washing step and/or (c) an eluting step.
  • ion exchange chromatography e.g. anion exchange chromatography or cation exchange chromatography
  • size exclusion chromatography e.g. gel-permeation or gel filtration chromatography
  • MMC mixed mode chromatography
  • MMC mixed mode chromatography
  • the methods comprise (a) applying a solution comprising a recombinant polypeptide and one or more impurities to a chromatography solid support; (b) washing the chromatography solid support with a wash buffer comprising a small ionic radius cation and a large ionic radius anion; and (c) eluting the recombinant polypeptide from the chromatography solid support.
  • the method used is liquid chromatography.
  • the process used is: affinity chromatography; ion exchange chromatography; anion or cation exchange chromatography; gel-permeation or gel-filtration chromatography; dye-ligand chromatography; hydrophobic interaction chromatography (HIC); mixed mode chromatography (MMC); or ceramic hydroxyapatite chromatography.
  • the process used is affinity chromatography.
  • the one or more chromatography methods comprise Protein A affinity chromatography.
  • the chromatography process is carried out using a chromatography support and a mobile phase; wherein the chromatography support is either aqueous or nonaqueous.
  • the nonaqueous phase comprises: agarose, sepharose, glass, silica, polystyrene, collodion charcoal, sand, polymethacrylate, cross-linked poly(styrene-divinylbenzene), agarose with dextran surface extender, or any other suitable material.
  • the nonaqueous phase is MABSELECT SURE resin.
  • the nonaqueous phase is linked to an affinity ligand, for example Protein A, Protein G, Protein L, or Protein A/G.
  • the solid support comprises a nonaqueous chromatography support linked to an affinity ligand selected from at least one of Protein A, Protein G, or Protein L.
  • the affinity ligand is Protein A.
  • the nonaqueous phase is cation exchange chromatography.
  • the chromatography process comprises the partitioning of a recombinant polypeptide between two immiscible aqueous phases (e.g. counter current liquid extraction).
  • the recombinant polypeptide is partitioned between two mobile phases.
  • the chromatography process comprises partitioning the recombinant polypeptide between a stationary and a mobile phase, such as an aqueous phase and a nonaqueous support.
  • the affinity ligand may be from a native source or synthetic, or a synthetic variant thereof.
  • the Protein A used is from a native source or it is synthetic, or it is a synthetic variant thereof which has the ability to bind polypeptides with a CH2/CH3 region.
  • Protein A can bind to the Fc region and can also bind to the variable region of the heavy chain (VH3), the affinity of which is strengthened in the absence of an Fc region.
  • Protein L can bind to the variable region of the light chain.
  • Protein G can bind to the Fc region and can also bind to the variable region (Fab).
  • affinity chromatography using one or more of Protein A, Protein L, or Protein G can be used to purify a number of different antigen binding proteins such as IgG, scFv, dAb, Fab, diabody, nanobody, Fc-containing fusion protein, i.e. including those that do not contain Fc regions.
  • the use of Protein A, Protein L, and Protein G to purify such antigen binding proteins is known and is routine in the art.
  • the chromatography process comprises a washing step, wherein the washing step comprises a wash buffer comprising a small radius cation and a large radius anion.
  • Standard wash buffers are well known in the art, for example Holstein eta/., (2015) BioProcess International, 13(2): 56-62.
  • the wash buffer additionally comprises tris base, acetic acid, and/or sodium acetate.
  • the wash buffer additionally comprises tris base and acetic acid.
  • the wash buffer comprises an additive, for example: an aliphatic carboxylate or salt thereof such as caproate, heptanoate, caprylate, decanoate, and dodecanoate; arginine; lysine; and/or sodium chloride.
  • the washing step uses a wash buffer that does not comprise sodium chloride.
  • the additive concentration is about 1 mM to about 500 mM, or about 75 mM to about 300 mM. The additive concentration may be about 0.1 M to about 2 M.
  • the tris base concentration is about 55 mM.
  • the acetic acid concentration is about 45 mM acetic acid.
  • the buffer in the wash buffer is sodium acetate
  • the sodium acetate concentration is about 300 mM to about 1 M.
  • the additive in the wash buffer is caprylate
  • the caprylate concentration is about 250 mM, or about 100 mM.
  • the caprylate is sodium caprylate.
  • the additive in the wash buffer is arginine
  • the arginine concentration is about 1 mM to about 2 M, such as about 1.1 M.
  • the additive in the wash buffer is lysine
  • the lysine concentration is about 0.5 M to about 1 M lysine, for example about 0.75 M lysine.
  • the wash buffers of the present invention comprise a small ionic radius cation and a large ionic radius anion.
  • the cations and anions herein may represent "soft ions” and the term “soft ion” is used to refer to any compound with a low charge density. Ions may be ranked according to the Hofmeister series based on relative protein stabilization (chaotropic) or destabilization (kosmotropic). Chaotropic ions are thought to disrupt the hydrogen bonding network between water molecules and allow for increased solvation of protein molecules. Conversely, kosmotropic ions are thought to promote hydrogen bonding between water molecules and decrease protein solvation; kosmotropic salts promote salting-out behavior of proteins.
  • the wash buffers of the present invention have been shown herein to provide good removal of impurities while maintaining good yield of recombinant polypeptide, and this may at least in part be due to increased solvation and disruption of antibody-HCP interactions.
  • the wash buffer described herein reduces the interaction between the recombinant polypeptide and the impurities, such as HCP.
  • the large ionic radius anion is a soft ion with a low charge density.
  • Such large radius anions include halogen cations, sulphate cations, and/or inorganic counter anions. Therefore, in one embodiment the large radius anion is selected from a fluoride anion, a chlorine anion, a bromide anion, an iodide anion, an astatine anion, a tennessine anion, or a sulphate cation.
  • the identity of the anion has a greater effect on HCP clearance than that of the cation.
  • Such large radius anions are shown herein to improve HCP clearance compared to, for example a wash buffer containing 100 mM sodium caprylate.
  • the large radius anion is a bromide anion or an iodide anion.
  • bromide and iodide anions are shown herein to provide good clearance of total HCP as well as a specific HCP, PLBL2.
  • the large radius anion is iodide.
  • a wash buffer comprising an iodide anion is shown herein to provide very good clearance of PLBL2.
  • the small radius cation is selected from a hydrogen cation, an alkali metal cation, an alkaline earth metal cation, and/or an ammonium cation.
  • the small radius cation is a hydrogen cation, a lithium cation, a sodium cation, a potassium cation, a rubidium cation, a caesium cation, a francium cation, a beryllium cation, a magnesium cation, a calcium cation, a strontium cation, a barium cation, a radium cation, and/or an ammonium cation.
  • the wash buffer comprises a cation selected from a hydrogen cation, an alkali metal cation, an alkaline earth metal cation or an ammonium cation, together with either a bromide anion or an iodide anion.
  • the wash buffer may comprise hydrogen bromide, lithium bromide, sodium bromide, potassium bromide, rubidium bromide, caesium bromide, francium bromide, beryllium bromide, magnesium bromide, calcium bromide, strontium bromide, barium bromide, radium bromide, or ammonium bromide.
  • the wash buffer may comprise hydrogen iodide, lithium iodide, sodium iodide, potassium iodide, rubidium iodide, caesium iodide, francium iodide, beryllium iodide, magnesium iodide, calcium iodide, strontium iodide, barium iodide, radium iodide, or ammonium iodide.
  • the small radius cation is a lithium cation.
  • the small radius cation is a potassium or a sodium cation.
  • a wash buffer comprising a lithium, potassium or sodium cation is shown herein to provide good removal of HCP while maintaining recombinant protein yield, particularly when present with a bromide or iodide anion, such as in the form of lithium bromide, lithium iodide, potassium bromide, potassium iodide, sodium bromide, or sodium iodide.
  • the wash buffer comprises a lithium cation and a bromide anion, a sodium cation and a bromide anion, a potassium cation and a bromide anion, or a magnesium cation and a bromide anion.
  • the wash buffer comprises a lithium cation and a bromide anion, such as in the form of a lithium bromide salt.
  • the wash buffer comprises a lithium cation and an iodide anion, a sodium cation and an iodide anion, a potassium cation and an iodide anion, or a magnesium cation and an iodide anion.
  • the wash buffer comprises a lithium cation and an iodide anion, such as in the form of a lithium iodide salt.
  • the wash buffer comprises about 0.1 M to about 4 M lithium bromide. In one embodiment, the wash buffer comprises about 0.1 M to about 3 M lithium bromide. In a further embodiment, lithium bromide is comprised in the wash buffer at about 0.1 M to about 2M, such as wherein the wash buffer comprises about 0.1 M to about 1 M lithium bromide. In a particular embodiment, the wash buffer comprises about 0.5 M lithium bromide. In one embodiment, the wash buffer comprises about 0.5 M to about 4 M lithium bromide. In one embodiment, the wash buffer comprises about 0.5 M to about 3 M lithium bromide. In a further embodiment, lithium bromide is comprised in the wash buffer at about 0.5 M to about 2M.
  • the wash buffer comprises about 0.5 M to about 1 M lithium bromide. In another embodiment, the wash buffer comprises about 0.1 M to about 2 M lithium iodide. In a yet further embodiment, the wash buffer comprises about 0.1 M to about 1 M lithium iodide.
  • the wash buffer is at a pH of between about pH 4 to about pH 9, for example about pH 7 to about pH 9, for example from about pH 7.5 to about pH 8.5.
  • the pH is about pH 7.5.
  • the wash buffer further comprises an aliphatic carboxylate or salt thereof, arginine, lysine, and/or sodium acetate.
  • the wash buffer does not comprise sodium chloride.
  • a method for purifying an antigen binding protein from a solution comprising one or more impurities comprising the steps of:
  • wash buffer comprising about 0.1 M to about 2 M lithium bromide, about 0.1 M to about 1 M lithium bromide, 0.5 M lithium bromide, or about 0.1 M to about IM lithium iodide, and optionally, wherein the wash buffer is at a pH of about 7.5 and
  • the wash buffer in step (b) comprises 0.5 M lithium bromide.
  • elution buffers employed in step (c) will be readily apparent to those skilled in the art and will be appreciated to depend on the identity of the recombinant polypeptide, for example when the recombinant polypeptide is an antigen binding protein such as an antibody.
  • the elution buffer comprises sodium acetate.
  • the elution buffer comprises an acid, such as acetic acid.
  • the elution buffer is at a pH between about 3.0 to about 4.5.
  • the pH of the elution buffer is about 3.6 to about 3.9.
  • the pH of the elution buffer is about 3.6.
  • eluting according to step (c) above is performed using an elution buffer comprising sodium acetate and an acid, such as acetic acid.
  • elution is performed using an elution buffer with a pH between about 3.0 and about 4.5, in particular between about 3.6 to 3.9.
  • elution is performed using an elution buffer with a pH about 3.6.
  • the antigen binding protein purified according to the method is an antibody, scFv, dAb, Fab, diabody, nanobody, a bispecific antibody, or an Fc- containing fusion protein.
  • the antigen binding protein purified according to the method is a monoclonal antibody, such as an IgGl or an IgG4.
  • the recombinant polypeptide used in the method is an antigen binding protein.
  • the antigen binding protein is selected from the group consisting of an antibody, antibody fragment, bispecific antibody, immunoglobulin single variable domain (dAb), mAbdAb, Fab, F(ab')z, Fv, disulphide linked Fv, scFv, closed conformation multispecific antibody, disulphide-linked scFv, diabody or a soluble receptor.
  • the antigen binding protein is an antibody.
  • the antigen binding protein is a monoclonal antibody.
  • the recombinant polypeptide purified according to the methods described herein is an antigen binding protein, such as an antibody in particular a monoclonal antibody.
  • antibody is used herein in the broadest sense to refer to molecules with an immunoglobulin-like domain (for example IgG, IgM, IgA, IgD or IgE) and includes monoclonal, recombinant, polyclonal, chimeric, human, humanised, multispecific antibodies, including bispecific antibodies, and heteroconjugate antibodies; a single variable domain (e.g., a domain antibody (DAB)), antigen binding antibody fragments, Fab, F(abQ2, Fv, disulphide linked Fv, single chain Fv, d isu I phide-l i n ked scFv, diabodies, TANDABS, etc. and modified versions of any of the foregoing (for a summary of alternative "antibody” formats see Holliger and Hudson, Nature Biotechnology, 2005, Vol 23, No. 9, 1126-1136).
  • DAB domain antibody
  • the five classes of antibodies IgM, IgA, IgG, IgE and IgD are defined by distinct heavy chain amino acid sequences which are called p, a, y, E and 6 respectively, each heavy chain can pair with either a K or A light chain.
  • the majority of antibodies in the serum belong to the IgG class, there are four isotypes of human IgG, IgGl, IgG2, IgG3 and IgG4, the sequences of which differ mainly in their hinge region.
  • multi-specific antigen binding protein refers to antigen binding proteins which comprise at least two different antigen binding sites. Each of these antigen-binding sites will be capable of binding to a different epitope, which may be present on the same antigen or different antigens.
  • the multi-specific antigen binding protein may have specificity for more than one antigen, for example two antigens, or for three antigens, or for four antigens.
  • Bispecifics may be generally classified as having a symmetric or asymmetric architecture. Bispecifics may have an Fc or may be fragmentbased (lacking an Fc). Fragment based bispecifics combine multiple antigen-binding antibody fragments in one molecule without an Fc region e.g. Fab-scFv, Fab-scFv2, orthoganol Fab-Fab, Fab-Fv, tandem scFc (e.g. BiTE and BiKE molecules), Diabody, DART, TandAb, scDiabody, tandem dAb etc.
  • the antibody is humanised or chimeric.
  • the recombinant polypeptide is an antibody, wherein the antibody is an IgGl, IgG4 or mAbdAb.
  • the term mAbdAb refers to a monoclonal antibody linked to a further binding domain, in particular a single variable domain such as a domain antibody.
  • a mAbdAb has at least two antigen binding sites, at least one of which is from a domain antibody, and at least one is from a paired VH/VL domain.
  • the antibody is a monoclonal antibody (mAb), such as, for example, an IgGl, or an IgG4.
  • the antibody is a bispecific antibody, for example a mAbdAb.
  • Impurity is defined by the European Agency for the Evaluation of Medicinal Products (the EMA) as "any component of the new drug substance which is not the chemical entity defined as the new drug substance" (see also ICH Harmonised Tripartite Guideline Topic Q3A(R2): Impurities in New Drug Substances). Impurities fall into two categories: product related which includes starting materials, by-products, intermediates, nucleic acids, fragments and degradation products; and process related which includes salts, endotoxins and microbes.
  • the one or more impurities of the method are one or more of: host cell proteins (HCPs), nucleic acids, endotoxins, product variants, process variants, cell culture media associated impurities, and/or fragmented polypeptide.
  • the one or more impurities are host cell proteins (HCPs).
  • the nucleic acid is host cell DNA.
  • the one or more impurities present in the process are produced by or derived from a host cell, which is a eukaryotic cell.
  • the eukaryotic cell is a mammalian cell; a fungal cell; or a yeast cell.
  • the one or more impurities are produced by or derived from a mammalian cell.
  • the mammalian cell is selected from: a human or rodent (such as a hamster or mouse) cell.
  • the mammalian cell is selected from: CHO, NSO, Sp2/0, COS, K562, BHK, PER.C6, and/or HEK cells.
  • the host cell is a HEK, CHO, PER.C6, Sp2/0, and/or NSO cell.
  • the yeast cell is Pichia pastoris, Saccharomyces cerevisiae, or Schizosaccharomyces pombe.
  • the fungal cell is Aspergillus sp. or Neurospora crassa.
  • the one or more impurities present in the process are produced by or derived from a host cell, which is a prokaryotic cell, for example a bacterial cell.
  • a host cell which is a prokaryotic cell, for example a bacterial cell.
  • the bacterial cell is: E. collec example, W3110, BL21), B. subtih's, and/or other suitable bacteria.
  • the host cell protein is selected from: PLBL2 (Phospholipase B- Like 2 protein), cathepsin L, cathepsin D, thyrodoxin, neural cell adhesion molecule, renin receptor, lipoprotein lipase, chondroitin sulfate protoglycan 4, alpha-enolase, galectin-3-binding protein, G- protein coupled receptor 56, V-type proton ATPase subunit SI, Nidogen-1, ATP synthase subunit beta, mitochondrial, Vimentin, Heat shock protein, Actin, Peroxirodoxin 1, SPARC, Clusterin, Complement Clr-a sub-component, Metalloproteinase inhibitor 1, insulin, sulphated glycoprotein 1, and/or Lysosomal protective protein.
  • PLBL2 Phospholipase B- Like 2 protein
  • cathepsin L cathepsin D
  • thyrodoxin thyrodoxin
  • the HCP is phospholipase B-Like 2 protein (PLBL2).
  • PLBL2 has been found to be an HCP impurity that is difficult to remove during the downstream processing of antibodies due to its apparent binding to the recombinant polypeptide.
  • the recombinant polypeptide is an antibody, such as an IgG antibody, in particular an IgG4 antibody.
  • the PLBL2 amount can be measured using methods known in the art, such as by ELISA (enzyme-linked immunosorbent assay), for example the PLBL2-specific ELISA disclosed in WO2015/038884.
  • the wash buffer described herein reduces the interaction between PLBL2 and the recombinant polypeptide, such as an antibody, and/or reduces the binding of PLBL2 to the recombinant polypeptide.
  • the HCP is cathepsin L.
  • Cathepsin L is a protease produced during CHO cell culture which can potentially degrade recombinant polypeptides that are antibodies.
  • the recombinant polypeptide is an antibody, such as an IgG antibody, in particular an IgGl antibody.
  • the purification of the recombinant polypeptide from cathepsin L can be measured by a reduced cathepsin L activity (for example with PROMOKINE PK-CA577-K142, cathepsin L activity assay kit) in the eluate of step (c).
  • the wash buffer described herein reduces the interaction between cathepsin L and the recombinant polypeptide, such as an antibody, and/or reduces the binding of cathepsin L to the recombinant polypeptide.
  • the methods described herein remove about or greater than 80% impurities, about or greater than 85% impurities, about or greater than 90% impurities, or about or greater than 95% impurities from the solution, such as about 96%, 97%, 98% or 99% impurities from the solution. In one embodiment, the methods described herein remove substantially all impurities from the solution. Thus, in a further embodiment the purified recombinant polypeptide contains less than about 20% impurities, less than about 15% impurities, less than about 10% impurities, or less than about 5% impurities. In a particular embodiment, the purified recombinant polypeptide contains less than about 5% impurities. In a yet further embodiment, the purified recombinant polypeptide contains less than about 4%, such as less than about 3%, less than about 2%, or less than about 1% impurities.
  • the purified recombinant polypeptide contains less than 400 ppm host cell proteins, less than 200 ppm host cell proteins, less than 100 ppm host cell proteins, or less than 50 ppm host cell proteins. In other embodiments, the purified recombinant polypeptide contains less than 1300 ppm host cell proteins, less than 1100 ppm host cell proteins, less than 900 ppm host cell proteins, less than 700 ppm host cell proteins, or less than 500 ppm host cell proteins. In a particular embodiment, the purified recombinant polypeptide contains less than 700 ppm host cell proteins, such as about 600 ppm host cell proteins.
  • the amount of impurities, for example HCPs, present in the solution or eluate may be determined by ELISA, OCTET (assay system), or other suitable methods.
  • the HCP level is determined by ELISA and/or Western Blot. A reduction in HCP content may be shown when compared to a control wash step without the wash buffers described herein, and/or when compared to, for example, clarified unprocessed bulk (CUB) (CCCF) prior to purification.
  • CAB clarified unprocessed bulk
  • the solution or eluate has an HCP content which is reduced by more than half of the HCP content in the initial load; for example the HCP content is reduced by 60% or more, 70% or more, 80% or more, or 90% or more.
  • the eluate containing the purified recombinant polypeptide has an HCP content which is reduced by greater than about 90%, such as by about 95%, by about 96%, by about 97%, by about 98%, or by about 99%.
  • the solution or eluate has an HCP content which is ⁇ 1100ppm, ⁇ 1000ppm, ⁇ 900 ppm, ⁇ 700 ppm, ⁇ 500 ppm, ⁇ 400 ppm, ⁇ 300 ppm, ⁇ 250 ppm, ⁇ 200 ppm, ⁇ 150 ppm, ⁇ 100 ppm, ⁇ 75 ppm, or ⁇ 50 ppm.
  • the content of the impurity which is HCP is ⁇ 700 ppm.
  • the content of the impurity which is HCP is ⁇ 200 ppm.
  • the HCP content is ⁇ 195 ppm, ⁇ 190 ppm, ⁇ 185 ppm, ⁇ 180 ppm, ⁇ 175 ppm, ⁇ 170 ppm, ⁇ 165 ppm, ⁇ 160 ppm, ⁇ 155 ppm, or ⁇ 150 ppm.
  • the HCP content is ⁇ 120 ppm, such as about 115 ppm.
  • the HCP present is ⁇ 5ppm, such as about 3ppm.
  • the amount of host cell nucleic acid for example DNA, e.g. residual genomic DNA (rgDNA) can be determined by Polymerase Chain Reaction (PCR), such as by quantitative PCR (qPCR).
  • PCR Polymerase Chain Reaction
  • qPCR quantitative PCR
  • a reduction in rgDNA content may be shown when compared to a control process without the wash buffers described herein.
  • the solution or eluate has a rgDNA content which is reduced compared to the initial sample; for example the rgDNA content is reduced by 10-fold, 20- fold, 50-fold, 100-fold or more.
  • the rgDNA is about 50,000pg/mg or less, about 30,000pg/mg or less, about 25,000pg/mg or less, about 10,000pg/mg or less, about 5,000pg/mg or less, about l,000pg/mg or less, about 500pg/mg or less, about 250pg/mg or less, or about lOOpg/mg or less following the addition of the wash buffers described herein.
  • the amount of PLBL2 can be determined by ELISA.
  • the PLBL2 level is determined by ELISA and expressed as an amount of PLBL2 in the eluate compared to the amount of purified recombinant polypeptide (e.g. as ng/mg or ppm).
  • a reduction in PLBL2 content may be shown when compared to a control process without the wash buffers described herein.
  • the solution or eluate has a PLBL2 content which is reduced by more than half of the PLBL2 content in the initial load; for example the PLBL2 content is reduced by 60% or more, 70% or more, 80% or more, or 90% or more compared to the PLBL2 content in the initial solution comprising one or more impurities.
  • the eluate may have a PLBL2 content 40% or less, 30% or less, 20% or less, or 10% or less than the PLBL2 content in the initial solution comprising one or more impurities.
  • the PLBL2 is about 70 ppm or less, 60 ppm or less, 50 ppm or less, about 25 ppm or less, about 20 ppm or less, about 15 ppm or less, about 10 ppm or less, or about 5 ppm or less following the addition of the wash buffers described herein. In a particular embodiment, the PLBL2 is between about 70 ppm to about 60 ppm or less following addition of the wash buffers described herein. In a further embodiment, the PLBL2 is about 5 ppm or less following addition of the wash buffers described herein.
  • the eluate has a PLBL2 content compared to the amount of purified recombinant polypeptide which is less than that achieved with a control process without the wash buffers described herein.
  • the eluate has a PLBL2 content of about 120 ng/mg or less, such as about 110 ng/mg or less, 100 ng/mg or less, 80 ng/mg or less, 60 ng/mg or less, 40 ng/mg or less, or 20 ng/mg or less.
  • the eluate has a PLBL2 content of about 5 ng/mg or less.
  • the eluate has a PLBL2 content of about 70 ng/mg or less, such as between 60 ng/mg to 70 ng/mg.
  • the monomer content of the purified recombinant polypeptide may be 80% or more, 85% or more, 90% or more, or 95% or more. Monomer is distinguished relative to the product-related impurities aggregates and fragments.
  • the monomer purity of the purified recombinant polypeptide in the eluate is measured by SEC-HPLC in the Examples herein, alternative suitable methods may also be used.
  • the purified recombinant polypeptide in the eluate has a monomer content ranging from about 80% to about 100%, such as about 90% or about 95% to about 100%.
  • the purified recombinant polypeptide in the eluate has a monomer content of >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, or >99%.
  • the monomer content is >96%. In another embodiment, the monomer content is >97%.
  • the amount of aggregation of the purified recombinant polypeptide is ⁇ 5% of the total purified polypeptide, for example ⁇ 2%. In some embodiments, the amount of aggregation is ⁇ 3%, such as about 2% or less or about 1% or less. In a further embodiment, the amount of aggregation is about 1% or less, such as about 1%. In a yet further embodiment, the amount of aggregation is less than 1%, such as about 0.9%, about 0.8%, about 0.7%, about 0.6%, or about 0.5% In a still further embodiment, the amount of aggregation is 0%.
  • the purified recombinant polypeptide is an antibody.
  • the yield can be measured as the percentage of recombinant polypeptide resulting from the purification process as compared to the start of the process. It is known that purification methods can remove both impurities and the recombinant polypeptide, and so a balance must be struck.
  • the yield of recombinant polypeptide may be 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more. In one embodiment, the yield of purified recombinant polypeptide is >90%, such as about 92% or greater, 94% or greater, 96% or greater, or 98% or greater. In a further embodiment, the yield of purified recombinant polypeptide is about 97% or greater. In a yet further embodiment, the yield of purified recombinant polypeptide is about 99% or greater.
  • the purified recombinant polypeptide in the eluate has a monomer content of >95% and the eluate has an HCP content of ⁇ 200 ng/mg or ⁇ 200 ppm.
  • the purified recombinant polypeptide in the eluate has a monomer content of >99% and the eluate has an HCP content of about 120 ng/mg or less or 120 ppm or less, such as about 115 ng/mg or about 115 ppm.
  • the purified recombinant polypeptide in the eluate has a monomer content of >97% and the eluate has an HCP content of about 3 ng/mg or about 3 ppm.
  • the HCP content is further reduced by subsequent downstream processing.
  • the purified recombinant polypeptide contains less than about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or about ⁇ 1% fragmented recombinant polypeptide.
  • the recombinant polypeptide is an antibody and the eluted antibody contains less than about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or about ⁇ 1% fragmented antibody.
  • the purified recombinant polypeptide is about 1% fragmented or less, such as about 1% fragmented.
  • the purified recombinant polypeptide has ⁇ 1% fragmentation, such as 0% fragmentation.
  • HCP host cell proteins
  • the host cell protein is PLBL2. In a further embodiment, the host cell protein is cathepsin L.
  • a method for reducing host cell DNA from a solution comprising a recombinant polypeptide and one or more impurities wherein the method is a purification process which comprises a chromatography wash buffer containing a small radius cation and a large radius anion as described herein.
  • a method for increasing yield and reducing the level of one or more impurities from a solution comprising a recombinant polypeptide and one or more impurities wherein the method is a purification process which comprises a chromatography wash buffer containing a small radius cation and a large radius anion as described herein.
  • the yield of the purified recombinant protein in the eluate is increased.
  • a method for increasing monomer content and reducing the level of one or more impurities from a solution comprising a recombinant polypeptide and one or more impurities wherein the method is a purification process which comprises a chromatography wash buffer containing a small radius cation and a large radius anion as described herein.
  • the monomer content of the purified recombinant protein in the eluate is increased.
  • a method for reducing the amount of aggregates and/or fragments and reducing the level of one or more impurities from a solution comprising a recombinant polypeptide and one or more impurities wherein the method is a purification process which comprises a chromatography wash buffer containing a small radius cation and a large radius anion as described herein.
  • the amount of aggregate and/or fragmented purified recombinant polypeptide in the eluate is reduced.
  • a wash buffer comprising a small ionic radius cation and a large ionic radius anion in a chromatography process for purifying a recombinant polypeptide from a solution comprising one or more impurities.
  • the use comprises a wash buffer comprising a small ionic radius cation and a large ionic radius anion as described herein.
  • the use comprises a chromatography process as described herein.
  • the use comprises purifying a recombinant polypeptide as described herein, and/or wherein the solution and/or impurities are as described herein.
  • a wash buffer comprising a small ionic radius cation and a large ionic radius anion in a chromatography process for reducing host cell proteins (HCP) from a solution comprising a recombinant polypeptide and one or more impurities.
  • HCP host cell proteins
  • the host cell protein is PLBL2. In a further embodiment, the host cell protein is cathepsin L.
  • a wash buffer comprising a small ionic radius cation and a large ionic radius anion in a chromatography process for reducing host cell DNA from a solution comprising a recombinant polypeptide and one or more impurities.
  • the reduction in host cell DNA is as compared to a chromatography process using wash buffer that does not comprise a small ionic radius cation and a large ionic radius anion.
  • a wash buffer comprising a small ionic radius cation and a large ionic radius anion in a chromatography process for increasing yield and reducing the level of one or more impurities from a solution comprising a recombinant polypeptide and one or more impurities.
  • the increased yield and reduction in the level of one or more impurities are as compared to a chromatography process using wash buffer that does not comprise a small ionic radius cation and a large ionic radius anion.
  • the yield of the purified recombinant protein in the eluate is increased.
  • a wash buffer comprising a small ionic radius cation and a large ionic radius anion in a chromatography process for increasing monomer content and reducing the level of one or more impurities from a solution comprising a recombinant polypeptide and one or more impurities.
  • the increased monomer content and reduction in the level of one or more impurities is as compared to a chromatography process using a wash buffer that does not comprise a small ionic radius cation and a large ionic radius anion.
  • the monomer content of the purified recombinant protein in the eluate is increased.
  • a wash buffer comprising a small radius cation and a large radius anion in a chromatography process for reducing the amount of aggregates and/or fragments and reducing the level of one or more impurities from a solution comprising a recombinant polypeptide and one or more impurities.
  • the reduced amount of aggregates and/or fragments and reduction in the level of one or more impurities are as compared to a chromatography process using a wash buffer that does not comprise a small ionic radius cation and a large ionic radius anion.
  • the amount of aggregate and/or fragmented purified recombinant polypeptide in the eluate is reduced.
  • HCPs host cell proteins
  • HCPs host cell proteins
  • HCPs host cell proteins
  • HCPs host cell proteins
  • the recombinant polypeptide is an antibody.
  • affinity chromatography is a chromatographic method that makes use of the specific, reversible interactions between biomolecules rather than general properties of the biomolecule such as isoelectric point, hydrophobicity, or size, to effect chromatographic separation.
  • a “buffer” is a buffered solution that resists changes in pH by the action of its acid-base conjugate components.
  • An “equilibration buffer” refers to a solution used to prepare the chromatography support for chromatography.
  • a “loading buffer” refers to a solution used to load the solution of the recombinant polypeptide and impurities onto the support. The equilibration and loading buffers can be the same. The equilibration, load and wash buffers can be the same.
  • a “wash buffer” refers to a solution used to remove impurities from the chromatography support after loading is completed. The “elution buffer” is used to remove the target recombinant polypeptide from the chromatography support, thus resulting in purification of the recombinant polypeptide.
  • a “salt” is a compound formed by the interaction of an acid and a base.
  • the "aliphatic carboxylate” can be either straight chained or branched.
  • the aliphatic carboxylate can be an aliphatic carboxylic acid or salt thereof, or the source of the aliphatic carboxylate can be an aliphatic carboxylic acid or salt thereof.
  • the aliphatic carboxylate is straight chained and selected from the group consisting of: methanoic (formic) acid, ethanoic (acetic) acid, propanoic (propionic) acid, butanoic (butyric) acid, pentanoic (valeric) acid, hexanoic (caproic) acid, heptanoic (enanthic) acid, octanoic (caprylic) acid, nonanoic (pelargonic) acid, decanoic (capric) acid, undecanoic (undecylic) acid, dodecanoic (lauric) acid, tridecanoic (tridecylic) acid, tetradecanoic (myristic) acid, pentadecanoic acid, hexadecanoic (palmitic) acid, heptadecanoic (margaric) acid, octadecanoic (stearic) acid, and icosanoic
  • the aliphatic carboxylate can comprise a carbon backbone of 1-20 carbons in length.
  • an aliphatic carboxylate comprises a 6-12 carbon backbone.
  • the aliphatic carboxylate is selected from the group consisting of: caproate, heptanoate, caprylate, decanoate, and dodecanoate.
  • the source of the aliphatic carboxylate is selected from the group consisting of an aliphatic carboxylic acid, such as a sodium salt of an aliphatic carboxylic acid, a potassium salt of an aliphatic carboxylic acid, and an ammonium salt of an aliphatic carboxylic acid.
  • the "recombinant polypeptide comprising one or more impurities” may be a solution which is a cell culture medium, for example a cell culture feedstream.
  • the feedstream may be filtered.
  • the solution may be a Clarified Unprocessed Bulk (CUB) (or clarified cell culture harvest/supernatant/ fermentation broth).
  • CUB is also known as a cell culture supernatant with any cells and/or cellular debris removed by clarification.
  • the solution may be a lysed preparation of cells expressing the recombinant polypeptide (e.g. a lysate).
  • Clarified Unprocessed Bulk (CUB) is equivalent to clarified cell culture fluid (CCCF), and both terms can be used interchangeably.
  • impurity refers to any product that does not share the same nature as the recombinant polypeptide of interest.
  • impurity may be defined as per the EMA's definition provided hereinbefore.
  • impurity refers to any foreign or undesirable molecule that is present in the load sample prior to chromatography or after chromatography, in the eluate.
  • process-related impurities present. These are impurities that are present as a result of the process in which the polypeptide of interest is produced. For example, these include host cell proteins (HCPs), RNA, and DNA.
  • HCP refers to proteins, not related to the polypeptide of interest, produced by the host cell during cell culture or fermentation, including intracellular and/or secreted proteins.
  • An example of a host cell protein is a protease, which can cause damage to the recombinant polypeptide of interest if it is still present during and after purification. For example, if a protease remains in the sample comprising the polypeptide of interest, it can create "product-related" substances or impurities which were not originally present and are not desired. The presence of proteases can cause decay, e.g. fragmentation, of the polypeptide of interest over time during the purification process, and/or in the final formulation.
  • impurities also include components used to grow the cells or to ensure expression of the polypeptide of interest, for example, solvents (e.g. methanol used to culture yeast cells), antibiotics, methotrexate (MTX), media components, flocculants, etc. Also included are molecules that are part of the chromatography support that leach into the sample during, for example, Protein A, Protein G, or Protein L chromatography.
  • Impurities also include "product-related variants" which include proteins that retain their activity but are different in their structure, and proteins that have lost their activity because of their difference in structure.
  • product-related variants include, for example, high molecular weight species (HMWs), low molecular weight species (LMWs), aggregated proteins, precursors, degraded proteins, misfolded proteins, underdisulfide-bonded proteins, fragments, and deamidated species.
  • HMWs high molecular weight species
  • LMWs low molecular weight species
  • aggregated proteins precursors, degraded proteins, misfolded proteins, underdisulfide-bonded proteins, fragments, and deamidated species.
  • HCP detected in "ppm” is equivalent to ng/mg
  • ppb parts per billion
  • rgDNA residual genomic DNA
  • PLBL2 parts per million
  • Protein A encompasses Protein A recovered from a native source (e.g. the cell wall of Staphylococcus aureus), Protein A produced synthetically (e.g. by peptide synthesis or by recombinant techniques), and variants thereof which retain the ability to bind proteins which have a CH2/CH3 region. Protein A can also bind to the variable region of the heavy chain (VH3), the affinity of which is strengthened in the absence of an Fc region. Protein A can be purchased commercially, for example from Repligen or Pharmacia or GE Healthcare.
  • VH3 variable region of the heavy chain
  • Protein A affinity chromatography or “Protein A chromatography” refers to a specific affinity chromatographic method that makes use of the affinity of the IgG binding domains of Protein A for the Fc portion and/or variable region of an immunoglobulin molecule.
  • This Fc portion comprises human or animal immunoglobulin constant domains CH2 and CH3 or immunoglobulin domains substantially similar to these.
  • Protein A chromatography involves using Protein A immobilized to a chromatography support which is a solid support (see Gagnon, Protein A Affinity Chromatography, Purification Tools for Monoclonal Antibodies, pp. 155-198, Validated Biosystems, (1996)). Protein G and Protein L may also be used for affinity chromatography.
  • Any suitable method can be used to affix the Protein A to the chromatography support.
  • Methods for affixing proteins are well known in the art (see e.g. Ostrove, in Guide to Protein Purification, Methods in Enzymology, (1990) 182: 357-371).
  • Such chromatography supports, with and without immobilized Protein A or Protein L, are readily available from many commercial sources such as Vector Laboratory (Burlingame, Calif.), Santa Cruz Biotechnology (Santa Cruz, Calif.), BioRad (Hercules, Calif.), Amersham Biosciences (part of GE Healthcare, Uppsala, Sweden) and Millipore (Billerica, Mass.).
  • polypeptide and protein are interchangeable and refer to a polymer of amino acid residues and does not refer to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide.
  • This term also does not refer to or exclude post-expression modifications of the polypeptide although chemical or post-expression modifications of these polypeptides may be included or excluded as specific embodiments. Therefore, for example, modifications to polypeptides that include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide. Further, polypeptides with these modifications may be specified as individual species to be included or excluded from the present disclosure.
  • the molecule is a polypeptide or their related analogs or derivatives thereof.
  • a polypeptide can be of natural (tissue-derived) origins, recombinant or natural expression from prokaryotic or eukaryotic cellular preparations, or produced chemically via synthetic methods.
  • Recombinant when used with reference to a polypeptide indicates that the cell has been modified by the introduction of a heterologous nucleic acid or polypeptide or the alteration of a native nucleic acid or polypeptide.
  • References to "arginine” not only refer to the natural amino acids, but also encompass arginine derivatives or salts thereof, such as arginine HCI, acetyl arginine, agmatine, arginic acid, N-alpha-butyroyl-L-arginine, or N-alpha-pyvaloyl arginine.
  • column volume refers to the total volume in a packed column.
  • chromatography support is interchangeable with “media”; “solid support”; “stationary phase” ; “resin”; “matrix”; “bead”; “gel”; or any other term that can be used to describe the material used to pack a chromatography column.
  • MSS refers to MABSELECT SURE resin, which is affinity chromatography media used for the capture of monoclonal antibodies (mAbs) at process scale.
  • HCP impurities are often difficult to remove due to direct interaction with an antibody (mAb) product during protein A purification (Levy et al., 2014; Aboulaich et al., 2014).
  • Solution conditions that disrupt host cell protein (HCP)-mAb interactions can provide improved HCP clearance during the protein A wash step.
  • mAb antibody
  • Wash effectiveness was determined by measuring impurity clearance (total HCP concentration and PLBL2 concentration in the protein A eluate) and step yield.
  • mAbl and mAb2 CUB was cultured in a production bioreactor using methods known in the art for producing monoclonal antibodies.
  • MabSelect SuRe Protein A resin (GE-Healthcare) was packed in 0.5 cm diameter columns to a final bed height of 25 cm; the resin was gravity-settled in the column and then flow packed in 0.4 M NaCI at a maximum linear flow rate of 475 cm/hr for 2 hours on an AKTA Avant 25. Packing quality was confirmed by measuring the height equivalent to a theoretical plate (HETP) and the asymmetry - with target values of at least 1,000 plates per meter and 1.0 +/- 0.2, respectively - by injecting lOOpI of 2 M NaCI and analyzing the conductivity trace. All experiments used a load ratio of 35mg mAb/ml of resin and 300 cm/hr flow rates unless otherwise specified.
  • the Protein A chromatography method and buffers are described in Table 1 below:
  • Protein A yield was determined by measuring mAb concentration in the eluate using a Nanodrop 2000c (Thermo Scientific). Three Nanodrop readings for each eluate sample were averaged to determine protein concentration; total mAb content in the Protein A eluate was calculated by multiplying mAb concentration by eluate volume (determined from chromatogram). The mAb concentration in the load was determined by analytical Protein A chromatography on an Agilent 1100 series HPLC. The raw data for each CUB sample on analytical Protein A was compared to a standard with known concentration for each particular mAb to calculate a titer.
  • Host cell protein concentration was measured using an HCP ELISA that was developed inhouse to quantify the total amount of immunogenic HCP in CHO-derived product samples (Mihara et al., 2015).
  • This HCP ELISA was developed using custom goat anti-CHO HCP polyclonal antibodies and an in-house produced HCP reference standard for multi-product use across CHO- derived products. It is currently used as a platform method for measuring HCP concentration in all downstream process intermediates of mAb products.
  • an ELISA assay for Phospholipase B-Like 2 was also developed in house. This assay uses recombinant hamster PLBL2 as a reference standard.
  • the anti-PLBL2 antibodies are polyclonal, produced in rabbits, and were protein G purified.
  • the membranes were then incubated with mouse anti-Rabbit cy3 conjugate (Jackson Immunoresearch) at 1 pg/mL for one hour at room temperature. After incubation, the membranes were washed three times for 10 minutes with TBST. After washing, the membranes were allowed to dry for 30 minutes. The dried membranes were imaged on an FLA-3000 Fluorescent Image Analyzer.
  • FIG. 1 shows the HCP concentration measured in the Protein A eluate following a 5 CV wash containing 0.5 M of the specified wash buffer.
  • FIG. 2 shows the measured step yield for each trial.
  • the measured HCP concentrations vary from ⁇ 400 ng/mg up to ⁇ 1300 ng/mg. All bromide and iodide salts provided improved HCP clearance compared to a wash buffer containing 100 mM sodium caprylate (caprylate wash buffer produced eluate containing 1023.6 ng/ml HCP and step yield of 95.8% - data not shown).
  • HCP clearance improved when paired with larger anions.
  • Mg salts were much more successful than Ca salts - but for Group 1 salts the effect was minor compared to that of the anion.
  • step yields presented in FIG. 2 do not have the same trend as the HCP removal data.
  • Mg salts appear to follow the Hofmeister series, with more chaotropic anions resulting in decreased step yield.
  • the results with mAb2 are presented in FIG. 3 and FIG. 4.
  • the HCP concentration measured in Protein A eluates for mAb2 is presented in FIG. 3.
  • a 100 mM sodium caprylate Protein A wash buffer produced an eluate containing 72.4 ng/mg HCP (data not shown).
  • iodide salts provide better HCP clearance than bromide salts.
  • the majority of bromide and iodide salts provided improved HCP clearance compared to a wash containing 100 mM sodium caprylate (data not shown).
  • the PLBL2 concentration measured in Protein A eluates for mAb2 are presented in FIG. 4.
  • a 100 mM sodium caprylate Protein A wash buffer produced an eluate containing 277.6 ng/mg HCP (data not shown).
  • the PLBL2 clearance shows a slightly different trend than the total HCP clearance for mAb2 or mAbl.
  • the cation and anion were both important factors for PLBL2 clearance.
  • Iodide salts generally performed better than bromide, but bromide salts were found to have a very strong cation effect. This demonstrates that the clearance of any specific HCP impurity may not follow the same trend as the 'total HCP'. All bromide and iodide salts provided improved PLBL2 clearance compared to a wash containing 100 mM sodium caprylate (data not shown).
  • the prominent band ⁇ 150 kDa is product that is lost during the wash. While the different wash buffers appear to be removing the same population of HCPs that are removed by the 100 mM caprylate platform wash, the major differences observed are the relative abundance of specific HCPs in the different washes, and the wash buffer containing lithium removes a high MW impurity.
  • the HCP levels for mAb2 are even lower than mAbl, as indicated by fewer visible bands on each gel.
  • the mAb2 results also indicate that the iodide and bromide containing wash buffers are mostly removing the same HCP impurities as the platform wash, although in different relative amounts.
  • the PLBL2 western blot demonstrates - consistent with the ELISA results - that some of the iodide and bromide containing wash buffers remove significant amounts of PLBL2, whereas the platform wash removes very little PLBL2.
  • the lithium washes remove a high MW impurity similar to the mAbl result.
  • FIG. 7A-7D depict HCP concentration (black) and step yield (gray) of a Protein A process following a NaBr wash.
  • FIG. 7B depicts HCP concentration (black) and step yield (gray) of a Protein A process following a KBr wash.
  • FIG. 7C depicts HCP concentration (black) and step yield (gray) of a Protein A process following a LiBr wash.
  • FIG. 7D depicts HCP concentration (black) and step yield (gray) of a Protein A process following a MgBrz wash.
  • HCP clearance increases, and yield decreases with increasing salt concentration.
  • the yield is relatively constant until the wash concentration is increased above 1 M.
  • the potassium and sodium salts do not achieve additional HCP clearance above 1 M, while the lithium and magnesium salts continue to clear more HCP above 1 M.
  • FIG. 8A-8C depict HCP concentration (black) and step yield (gray) of Protein A process following a Nal wash.
  • FIG. 8B depicts HCP concentration (black) and step yield (gray) of Protein A process following a KI wash.
  • FIG. 8C depicts HCP concentration (black) and step yield (gray) of Protein A process following a Lil wash.
  • the iodide salts had similar behaviour with increasing concentration as the bromide salts; HCP concentration and step yield decrease with increasing salt concentration. Similarly to bromide, yield tends to gradually decrease up to 1 M and rapidly decrease at greater concentrations.
  • EXAMPLE 4 Removal of Cathepsin L During mAb3 Production
  • Lithium bromide was also tested for its capability to remove cathepsin L during production of a monoclonal antibody, mAb3.
  • mAb3 drug substance exhibits significant fragmentation during process intermediate holds.
  • cathepsin L a CHO HCP protease
  • Cathepsin L is present in an inactive proform during cell culture and the initial downstream unit operations, and it activates during the cation exchange (CEX) chromatography polishing step.
  • the presence of cathepsin L in mAb3 feed streams can be confirmed with an activity assay, or by observing an increase in fragment after the CEX step.
  • FIG. 9 shows the percent fragmentmenation (measured by SEC HPLC) in the Protein A eluate and the CEX eluate for 150 mM caprylate wash and the 0.5 M LiBr Protein A wash.
  • the lithium bromide Protein A wash buffer was tested with a monoclonal antibody, mAb4 that was harvested using flocculation methods. Specifically, the material was either flocculated by adding PEI, or it was flocculated in two sequential steps: (1) flocculation with PEI and depth filtration, (2) flocculation with caprylic acid and depth filtration. These two flocculation-based harvests were then processed through Protein A with either a sodium caprylate wash buffer or a lithium bromide wash buffer.
  • the HCP, SEC, and step yield results are presented in Table 3.

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Abstract

La présente invention concerne un nouveau procédé de purification d'un polypeptide recombinant à partir d'une solution contenant une ou plusieurs impuretés. Le procédé peut comprendre un processus de chromatographie qui comprend un tampon de lavage contenant un cation à faible rayon ionique et un anion à rayon ionique élevé.
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