Systems and methods for use and regeneration of chromatography media

Abstract

Aspects of the present disclosure relate to a method of regenerating a hydrophobic interaction chromatography column into which a sample substance has been added, the method comprising passing one or more column volumes of an alkaline solution through a hydrophobic interaction medium inside the column, where the alkaline solution exhibits a pH of between about 10 and about 14 and an electrical conductivity of between 0.5 mS / cm and about 10 mS / cm, wherein substances bound to the hydrophobic interaction medium are removed. In some cases, the alkaline solution may comprise sodium hydroxide at a concentration of, for example, between about 0.1 mM and 10 mM.

Description

(Division of 202080074771.9) Cross - Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 905,033, filed September 24, 2019, and U.S. Provisional Patent Application No. 62 / 958,899, filed January 9, 2020, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This disclosure relates to systems, methods, and solutions for the use and regeneration of chromatographic media. Some aspects of this disclosure relate to systems and methods including a single-step hydrophobic interaction chromatographic media regeneration solution. Background Art

[0003] Chromatography is a widely used type of process that can be performed to separate the components of a mixture. Certain types of chromatography can be performed during the preparation of pharmaceutical products (e.g., in separating, collecting, isolating, purifying, polishing, etc. molecules for pharmaceutical products). Some molecules of interest (e.g., polypeptides, polynucleotides, etc.) need to be purified from substances such as the host cells in which they are produced. Separation or purification of molecules of interest using chromatography can reduce, remove, or separate host cell proteins (e.g., lipases), host cell substances (e.g., cell debris), and other impurities that can co-purify with the molecules of interest.

[0004] Chromatography can include the use of a stationary phase that contains a medium for facilitating the separation of the components of a mobile phase using the stationary phase. For example, hydrophobic interaction chromatography (HIC) can separate molecules (e.g., polypeptides, polynucleotides, etc.) based on differences in surface hydrophobicity by utilizing reversible interactions between the molecules and the hydrophobic surface of the HIC medium in the stationary phase. The interaction between the molecules and the hydrophobic surface of the HIC medium is affected by, for example, salts in the mobile buffer. A feedstock with a high salt concentration can be loaded into a HIC device, where the high salt concentration promotes the interaction between the hydrophobic interaction chromatography (HIC) medium inside the device and the molecules in the mixture. Subsequently, a solution with a reduced ionic strength (e.g., a buffer) can be flowed through the HIC device to reverse the hydrophobic interaction between the HIC medium and the molecules. The molecules with the lowest hydrophobicity can be eluted first and the molecules with the highest hydrophobicity can be eluted last, which requires a greater reduction in salt concentration to reverse their hydrophobic interaction with the HIC medium.

[0005] In some cases, the HIC medium can be reused for multiple chromatographic cycles. To maintain the efficacy, quality, and cleanliness of the HIC medium, prevent contamination between cycles, increase the service life of the HIC medium, prevent the accumulation of impurities, and / or meet or exceed operating standards (e.g., operating standards within a laboratory or institution or operating standards promulgated by a regulatory agency), methods for regenerating the HIC medium can be used to remove residual substances from the HIC medium after a HIC cycle has been run. SUMMARY OF THE INVENTION

[0006] Aspects of the present disclosure relate to regenerating a chromatographic column. In one aspect, the present disclosure relates to a method for regenerating a hydrophobic interaction chromatographic column to which an injection substance has been added. The method can include passing one or more column volumes of an alkaline solution through the hydrophobic interaction medium inside the chromatographic column, wherein the alkaline solution has a pH value between about 10 and about 14 and a conductivity between 0.5 mS / cm and about 10 mS / cm, and wherein substances bound to the hydrophobic interaction medium are removed. The alkaline solution can include one of sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or tris(hydroxymethyl)aminomethane (Tris); the alkaline solution can exhibit a conductivity between about 0.8 mS / cm and about 1.6 mS / cm, and / or the alkaline solution can include a total dissolved salt concentration between about 0.1 mM and about 10 mM.

[0007] After removal of the substances bound to the hydrophobic interaction medium, less than 1.0% of the injection substance will still remain bound to the hydrophobic interaction medium as a residue. Substances removed from the medium can include: host cell proteins, recombinant proteins, lipids, polypeptide fragments, biomolecules, or nucleic acids. In some embodiments, the substances removed from the medium may not include bacteria or fungi. In some embodiments, the method may not include contacting the hydrophobic interaction medium with a chaotropic agent or an organic solvent. The step of passing one or more column volumes of the alkaline solution through the hydrophobic interaction medium inside the chromatographic column takes between about 10 minutes and about 1 hour.

[0008] In another aspect, the present disclosure aims to provide a method for regenerating a chromatographic column into which a sample substance has been introduced; the method includes passing one or more column volumes of an alkaline solution through the medium inside the chromatographic column, where the alkaline solution contains sodium hydroxide at a total dissolved concentration between about 0.5 mM and about 50 mM, and the substances bound to the medium are removed. The medium may include a matrix that includes ligands of hydrocarbons between 2 and 10 in aliphatic or aromatic configurations. These ligands may be present in the medium at a density between about 20 and about 30 μmol / ml of the medium. In other embodiments, the medium does not have ligands containing 30 or more hydrocarbons; the chromatographic column is not used in a mixed-mode chromatography process; and / or the medium may include a matrix that includes cross-linked agarose and phenyl ligands. In other embodiments, the method does not include contacting the medium with alcohol, ethylene glycol, or sodium chloride. The method may further include passing one or more column volumes of a chaotropic agent through the chromatographic column after passing one or more column volumes of the alkaline solution through the chromatographic column, where the chaotropic agent is one of 6N guanidine hydrochloride or 8N urea. The method may further include contacting the chromatographic column with a stock buffer having a total dissolved concentration of sodium hydroxide between about 0.05 M and about 0.15 M. The method may also include adding a first sample substance to the chromatographic column and adding a second sample substance to the chromatographic column, where the method does not include cleaning the chromatographic column.

[0009] In another aspect, the present disclosure relates to a method for determining the concentration of an alkaline solution for a regeneration solution of a hydrophobic interaction chromatographic column; the method includes passing a volume of a first solution through the hydrophobic interaction medium inside the chromatographic column, where the first solution has water and an alkaline solution concentration that starts from about 0 N and increases at a substantially constant rate to a maximum concentration; passing a volume of a second solution through the hydrophobic interaction medium, where the second solution has water and an alkaline solution concentration that starts from the maximum concentration and decreases at a substantially constant rate to about 0 N; and determining a portion of the first or second solution that removes the substances bound to the hydrophobic interaction medium when passing through the hydrophobic interaction medium. The alkaline solution may contain sodium hydroxide and the maximum concentration may be about 1 N. In other embodiments, the volume of the first solution and the volume of the second solution are each about 20 column volumes.

[0010] In another aspect, the present disclosure includes predicting, evaluating, or comparing the availability and regeneration of various chromatographic resins. In some embodiments, a method for evaluating a chromatographic scheme includes: adding a sample substance containing a target molecule to a certain volume of chromatographic medium in a filter plate hole and collecting the flow-through from the filter plate hole, wherein the sample substance is at the scheme pH value; adding multiple aliquots of a buffer to the chromatographic medium to obtain an eluate from the chromatographic medium, wherein the buffer is at the buffer pH value and the concentration of kosmotropic salt linearly decreases in the multiple aliquots, and wherein a first amount of the target molecule is contained in the combined flow-through and eluate; and adding a second solution to the chromatographic medium to extract a second amount of the target molecule from the chromatographic medium; and adding a chaotropic agent to the chromatographic medium to extract a third amount of the target molecule from the chromatographic medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The drawings incorporated in and forming a part of this specification illustrate various exemplary embodiments and, together with the description, serve to explain the principles of the disclosed embodiments. Any feature of the embodiments or examples described herein (e.g., composition, formulation, method, etc.) may be combined with any other embodiment or example, and all such combinations are encompassed by the present disclosure. Additionally, the systems and methods described are not limited to any single aspect or its embodiments, nor to any combination or arrangement of the above aspects and embodiments. For the sake of brevity, certain permutations and combinations are not separately discussed and / or illustrated herein.

[0012] Figure 1 Exemplary methods in accordance with various aspects of the present disclosure are depicted in flowchart form.

[0013] Figure 2 Blot analysis of chromatographic columns in various states of use in accordance with various aspects of the present disclosure is depicted.

[0014] Figure 3 Blot analysis of chromatographic columns in various states of use in accordance with various aspects of the present disclosure is depicted.

[0015] Figure 4 Superposition of chromatographic data from multiple processes in accordance with various aspects of the present disclosure is depicted.

[0016] Figure 5 Blot analysis of chromatographic columns in various states of use in accordance with various aspects of the present disclosure is depicted.

[0017] Figure 6 Comparison of a column containing a hydrophobic interaction medium that has undergone multiple hydrophobic interaction chromatography cycles with a column containing unused hydrophobic interaction medium in accordance with various aspects of the present disclosure is depicted.

[0018] Figure 7A and Figure 7B depict chromatograms of regeneration processes after hydrophobic interaction chromatography, including the use of reverse osmosis deionized water, according to various aspects of the present disclosure.

[0019] Figure 8A and Figure 8B depict additional chromatograms of regeneration processes after hydrophobic interaction chromatography.

[0020] Figure 9 depict chromatograms of regeneration processes using guanidine hydrochloride solution according to various aspects of the present disclosure.

[0021] Figure 10A depict chromatograms of processes including multiple regeneration solutions according to various aspects of the present disclosure. Figure 10B depict Figure 10A an enlarged image of a portion of the chromatogram.

[0022] Figure 11 depict chromatograms of processes according to various aspects of the present disclosure in which sodium hydroxide solutions with gradually increasing / decreasing concentrations have been introduced into the chromatography column.

[0023] Figure 12 depict superimposed chromatograms of multiple two-solution column regeneration processes according to various aspects of the present disclosure.

[0024] Figure 13A and Figure 13B is a visual depiction of a statistical analysis of the various peaks of chromatograms depicting regeneration processes according to various aspects of the present disclosure.

[0025] Figure 14 depict superimposed chromatograms of multiple two-solution column regeneration processes containing sodium hydroxide or sodium chloride and guanidine hydrochloride according to various aspects of the present disclosure.

[0026] Figure 15 depict three chromatography columns into which solutions have been added according to various aspects of the present disclosure.

[0027] Figure 16 depict a graph of the peak area of the guanidine hydrochloride stripping solution as a function of sodium hydroxide concentration according to various aspects of the present disclosure.

[0028] Figure 17 and Figure 18 depict chromatograms of control regeneration processes and experimental regeneration processes each containing multiple regeneration solutions according to various aspects of the present disclosure.

[0029] Figure 19Depicts a series of chromatograms of hydrophobic interaction chromatography runs for purifying different monoclonal antibodies according to various aspects of the present disclosure.

[0030] Figures 20A - 20C Depicts chromatograms generated according to various aspects of the present disclosure using a protocol involving three different hydrophobic interaction chromatography media.

[0031] Figure 21A And 21B Depicts a boundary function graph formed according to various aspects of the present disclosure by analyzing data generated during high-throughput screening and full-scale chromatography runs.

[0032] Figure 22 Depicts an exemplary dynamic prediction model according to various aspects of the present disclosure.

[0033] Figures 23A - 26C Depicts a chromatogram of a first target molecule generated according to various aspects of the present disclosure using multiple hydrophobic interaction chromatography media, pH parameters, and stripping solution.

[0034] Figures 27A - 28D Depicts a chromatogram of a second target molecule generated according to various aspects of the present disclosure using multiple hydrophobic interaction chromatography media, pH parameters, and stripping solution. Detailed Description

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any suitable methods and materials (e.g., similar to or equivalent to those described herein) can be used in the practice or testing of this disclosure, specific methods are now described. All publications mentioned are incorporated herein by reference.

[0036] As used herein, the words "comprising," "including," or any variation thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a series of elements does not include only those elements but also other elements not expressly listed or inherent to such process, method, article, or apparatus. The word "exemplary" is used in a sense that is "illustrative" rather than "ideal." With respect to the phrases "for example" and "such as" and their grammatical equivalents, the phrase "and not limited to" should be understood to follow, unless otherwise expressly stated.

[0037] As used herein, the term "about" is used to account for variations due to experimental error. When applied to a numerical value, the term "about" can represent a variation of + / - 5% relative to the disclosed numerical value, unless a different variation is specified. As used herein, the singular forms "a," "an," and "the" include plural referents unless the context dictates otherwise. Additionally, all ranges are understood to include endpoints; for example, a range from 1 centimeter (cm) to 5 cm will include 1 cm, 5 cm, and the lengths of all distances between 1 cm and 5 cm.

[0038] It should be noted that all numerical values disclosed herein (including all disclosed values, limits, and ranges) can have a variation of + / - 5% relative to the disclosed numerical value, unless a different variation is specified.

[0039] As used herein, the term "polypeptide" refers to a polymer of more than about 20 amino acids covalently linked via amide bonds. A protein contains one or more chains of amino acid polymers (e.g., polypeptides). Thus, a polypeptide can be a protein, and a protein can contain multiple polypeptides to form a single functional biomolecule.

[0040] Post-translational modifications can modify or alter the structure of a polypeptide. For example, in some proteins, disulfide bridges (e.g., S-S bonds between cysteine residues) can form post-translationally. Some disulfide bridges are essential for the proper structure, function, and interactions of polypeptides, immunoglobulins, proteins, cofactors, substrates, etc. In addition to disulfide bond formation, proteins can undergo other post-translational modifications such as lipidation (e.g., myristoylation, palmitoylation, farnesylation, geranylgeranylation, and glycosylphosphatidylinositol (GPI) anchor formation), alkylation (e.g., methylation), acylation, amidation, glycosylation (e.g., addition of sugars at arginine, asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine, and / or tryptophan), and phosphorylation (i.e., addition of a phosphate group to serine, threonine, tyrosine, and / or histidine). Post-translational modifications can affect hydrophobicity, electrostatic surface properties, or other properties that determine surface-surface interactions in which the polypeptide participates.

[0041] As used herein, the term "protein" includes biotherapeutic proteins, recombinant proteins for research or therapy, translocon-associated proteins, and other Fc-fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, human antibodies, bispecific antibodies, antibody fragments, antibody-like molecules, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, etc. A protein of interest (POI) can include any polypeptide or protein that is desired to be isolated, purified, or prepared. POIs can include polypeptides produced by cells, including antibodies.

[0042] As used herein, the term “antibody” includes immunoglobulins consisting of four polypeptide chains: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Generally, an antibody has a molecular weight of more than 100 kDa, such as between 130 kDa and 200 kDa, such as about 140 kDa, 145 kDa, 150 kDa, 155 kDa, or 160 kDa. Each heavy chain includes a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region includes three domains: CH1, CH2, and CH3. Each light chain includes a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region includes one domain: CL. The VH region and the VL region can be further subdivided into hypervariable regions (referred to as complementarity determining regions (CDRs)), interspersed with more conserved regions (referred to as framework regions (FRs)). Each VH and VL is composed of three CDRs and four FRs, which are arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs can be abbreviated as HCDR1, HCDR2, and HCDR3; light chain CDRs can be abbreviated as LCDR1, LCDR2, and LCDR3).

[0043] For example, a class of immunoglobulins called immunoglobulin G (IgG) is common in human serum and includes four polypeptide chains: two light chains and two heavy chains. Each light chain is linked to a heavy chain via a cystine disulfide bond, and the two heavy chains are bound to each other via two cystine disulfide bonds. Other classes of human immunoglobulins include IgA, IgM, IgD, and IgE. In the case of IgG, there are four subclasses: IgG1, IgG2, IgG3, and IgG4, each subclass differing in their constant regions and thus may have different effector functions. In some embodiments described herein, the POI may include a target polypeptide comprising IgG. In at least one embodiment, the target polypeptide comprises IgG4.

[0044] As used herein, the term "antibody" also encompasses antigen-binding fragments of whole antibody molecules. As used herein, terms such as "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc. include any naturally occurring, enzymatically obtained, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. For example, antigen-binding fragments of an antibody can be obtained from whole antibody molecules using any suitable standard techniques involving manipulation and expression of DNA encoding the variable and optionally constant domains of the antibody (such as protease cleavage or recombinant genetic engineering techniques). Such DNA is known and / or can be readily obtained from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated by chemical methods or by using molecular biotechnology, for example, to arrange one or more variable and / or constant domains in a suitable configuration, or to introduce codons, form cysteine residues, modify, add, or knock out amino acids, etc.

[0045] Target molecules (such as target polypeptides / antibodies) can be produced using recombinant cell-based production systems such as the insect baculovirus system, yeast systems (e.g., Pichia pastoris), or mammalian systems (e.g., CHO cells and CHO derivative-like CHO-K1 cells). The term "cell" includes any cell suitable for expressing a recombinant nucleic acid sequence. Cells include prokaryotic and eukaryotic cells (unicellular or multicellular), bacterial cells (e.g., cells of strains such as Escherichia coli, Bacillus, Streptomyces, etc.), mycobacterial cells, fungal cells, yeast cells (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Pichia methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells, or cell fusions (e.g., hybridomas or cell hybrids). In some embodiments, the cells can be cells of humans, monkeys, apes, hamsters, rats, or mice. In some embodiments, the cells can be eukaryotic cells and can be selected from the following cells: CHO (e.g., CHO K1, DXB-11CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, African green monkey kidney cells, CV1, kidney cells (e.g., HEK293, 293EBNA, MSR 293, MDCK, HaK, BHK), HeLa cells, HepG2, WI38, MRC5, Colo205 (human colon cancer cells), HB 8065 (human liver cancer cells), HL-60 (human promyelocytic acute leukemia cells, e.g., BHK21), Jurkat (human peripheral blood leukemia T cells), Daudi (human lymphoma cells), A431 (human epidermal cancer cells), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT 060562, Sertoli cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, and cell lines derived from the foregoing cells. In some embodiments, the cells can contain one or more viral genes, such as retinal cells expressing viral genes (e.g., PER.C6 TM cells).

[0046] In this disclosure, the term "target molecule" may be used to refer to a target polypeptide (e.g., an antibody, an antibody fragment, or other protein or protein fragment), or to other molecules to be produced, isolated, purified, and / or included in a pharmaceutical product (e.g., an adeno-associated virus (AAV) or other molecule for therapeutic use. Although the methods according to the present disclosure may relate to target polypeptides, they may be applicable to other target molecules. AAVs, for example, may be prepared according to suitable methods (e.g., depth filtration, affinity chromatography, etc.), and mixtures containing AAVs may be subjected to the methods according to the present disclosure. Before or after employing one or more methods of the present disclosure, mixtures containing AAVs may be subjected to additional processes (e.g., removal of "empty capsids" or AAVs that do not contain the target sequence).

[0047] In some embodiments, the target molecule is an antibody, a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecific antibody, a bispecific antibody, an antigen-binding antibody fragment, a single-chain antibody, a diabody, a triabody, or a tetrabody, a Fab fragment or an F(ab')2 fragment, an IgD antibody, an IgE antibody, an IgM antibody, an IgG antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In one embodiment, the antibody is an IgG1 antibody. In one embodiment, the antibody is an IgG2 antibody. In one embodiment, the antibody is an IgG4 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG4 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG1 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG1 / IgG4 antibody.

[0048] In some embodiments, the target molecule (e.g., an antibody) is selected from the group consisting of: anti-programmed cell death 1 antibody (e.g., anti-PD1 antibody, as described in U.S. Patent Application Publication No. US2015 / 0203579A1), anti-programmed cell death ligand-1 (e.g., anti-PD-L1 antibody, as described in U.S. Patent Application Publication No. US2015 / 0203580A1), anti-Dll4 antibody, anti-angiopoietin-2 antibody (e.g., anti-ANG2 antibody, as described in U.S. Patent No. 9,402,898), anti-angiopoietin-like 3 antibody (e.g., anti-AngPtl3 antibody, as described in U.S. Patent No. 9,018,356), anti-platelet-derived growth factor receptor antibody (e.g., anti-PDGFR antibody, as described in U.S. Patent No. 9,265,827), anti-prolactin receptor antibody (e.g., anti-PRLR antibody, as described in U.S. Patent No. 9,302,015), anti-complement 5 antibody (e.g., anti-C5 antibody, as described in U.S. Patent Application Publication No. US2015 / 0313194A1), anti-TNF antibody, anti-epidermal growth factor receptor antibody (e.g., anti-EGFR antibody, as described in U.S. Patent No. 9,132,192, or anti-EGFRvIII antibody, as described in U.S. Patent Application Publication US2015 / 0259423A1), anti-proprotein convertase subtilisin / kexin type 9 antibody (e.g., anti-PCSK9 antibody, as described in U.S. Patent No. 8,062,640 or U.S. Patent Application Publication No. US2014 / 0044730A1), anti-growth differentiation factor-8 antibody (e.g., anti-GDF8 antibody, also known as anti-myostatin antibody, as described in U.S. Patent Nos. 8,871,209 or 9,260,515), anti-glucagon receptor (e.g., anti-GCGR antibody, as described in U.S. Patent Application Publications US2015 / 0337045A1 or US2016 / 0075778A1), anti-VEGF antibody, anti-IL1R antibody, interleukin 4 receptor antibody (e.g., anti-IL4R antibody, as described in U.S. Patent Application Publication No. US2014 / 0271681A1 or U.S. Patent Nos. 8,735,095 or 8,945,559), anti-interleukin 6 receptor antibody (e.g., anti-IL6R antibody, as described in U.S. Patent Nos. 7,582,298, 8,043,617 or 9,173,as described in 880), anti - interleukin 33 (e.g., anti - IL33 antibody, such as those described in U.S. Patent Application Publication Nos. US2014 / 0271658A1 or US2014 / 0271642A1), anti - respiratory syncytial virus antibody (e.g., anti - RSV antibody, such as those described in U.S. Patent Application Publication No. US2014 / 0271653A1), anti - cluster of differentiation 3 (e.g., anti - CD3 antibody, such as those described in U.S. Patent Application Publication Nos. US2014 / 0088295A1 and US20150266966A1, and as described in U.S. Patent Application No. 62 / 222,605), anti - cluster of differentiation 20 (e.g., anti - CD20 antibody, such as those described in U.S. Patent Application Publication Nos. US2014 / 0088295A1 and US20150266966A1 and in U.S. Patent No. 7,879,984), anti - cluster of differentiation - 48 (e.g., anti - CD48 antibody, such as those described in U.S. Patent No. 9,228,014), anti - Feld1 antibody (e.g., as described in U.S. Patent No. 9,079,948), anti - Middle East respiratory syndrome virus (e.g., anti - MERS antibody), anti - Ebola virus antibody (e.g., REGN - EB3 from Regeneron), anti - CD19 antibody, anti - CD28 antibody, anti - IL1 antibody, anti - IL2 antibody, anti - IL3 antibody, anti - IL4 antibody, anti - IL5 antibody, anti - IL6 antibody, anti - IL7 antibody, anti - Erb3 antibody, anti - Zika virus antibody, anti - lymphocyte activation gene 3 (e.g., anti - LAG3 antibody or anti - CD223 antibody), and anti - activin A antibody. The entire contents of each of the U.S. patents and U.S. patent publications mentioned in this section are incorporated herein by reference.,

[0049] In some embodiments, the target molecule (e.g., a bispecific antibody) is selected from the group consisting of: anti - CD3x anti - CD20 bispecific antibody, anti - CD3x anti - Mucin 16 bispecific antibody, and anti - CD3 x anti - prostate - specific membrane antigen bispecific antibody. In some embodiments, the target molecule is selected from the group consisting of: alirocumab, sarelab, fasinumab, nevusolumab, dupilumab, tralokinumab, evesolumab, and linvosolumab.

[0050] In some embodiments, the target molecule is a recombinant protein (e.g., an Fc-fusion protein) that contains an Fc portion and another domain. In some embodiments, the Fc-fusion protein is a receptor Fc-fusion protein that contains one or more extracellular domains of a receptor coupled to the Fc portion. In some embodiments, the Fc portion includes a hinge region followed by the CH2 and CH3 domains of IgG. In some embodiments, the receptor Fc-fusion protein contains two or more separate receptor chains that bind to a single ligand or multiple ligands. For example, the Fc-fusion protein is a trap (TRAP) protein, such as an IL-1 trap (e.g., rilonacept, which contains the IL-1RAcP ligand-binding region fused to the extracellular region of Il-1R1, and the extracellular region of Il-1R1 is fused to the Fc of hIgG1; see U.S. Patent No. 6,927,004, the entire content of which is incorporated herein by reference), or a VEGF trap (e.g., aflibercept or ziv-aflibercept, which contains the Ig domain 2 of VEGF receptor Flt1 fused to the Ig domain 3 of VEGF receptor Flk1, and the VEGF receptor Flt1 is fused to the Fc of hIgG1; see U.S. Patents 7,087,411 and 7,279,159, the entire contents of which are incorporated herein by reference). In other embodiments, the Fc-fusion protein is a ScFv-Fc-fusion protein that contains one or more (such as variable heavy chain fragments and variable light chain fragments) of the antigen-binding domains of an antibody coupled to the Fc portion.

[0051] Embodiments of the present disclosure can be used for the preparation of various pharmaceutical products or for establishing methods for purifying various pharmaceutical products. In some embodiments, the present disclosure can be used for the preparation or purification of pharmaceutical products containing antigen-binding molecules or AAVs. In some aspects, embodiments of the present disclosure can be suitable for the preparation of pharmaceutical products containing various components (e.g., aflibercept, alirocumab, pegcetacoplan, bevacizumab, brodalumab, conbercept, dupilumab, evolocumab, tocilizumab, certolizumab, abatacept, rituximab, infliximab, ranibizumab, sarilumab, adalimumab, anakinra, trastuzumab, filgrastim, interferon beta-1a, insulin glargine [rDNA origin], alpha-erythropoietin, darbepoetin, recombinant human colony-stimulating factor, golimumab, etanercept, any of the above antigen-binding fragments, or a combination of such binding domains, such as a bispecific antibody against VEGF or angiopoietin-2, etc.).

[0052] The term "hydrophobic interaction medium" or "HIC medium" refers to a combination of a support structure and a hydrophobic moiety, where the hydrophobic moiety is attached to the support structure. The medium can be in the form of a chromatographic medium (e.g., beads or other particles held in a packed bed column), in the form of a membrane, or in any form that can hold a liquid containing a protein of interest and contaminants. Thus, the support structure includes agarose beads (e.g., agarose gel), silica beads, cellulose membranes, cellulose beads, hydrophilic polymer beads, resins, etc. The hydrophobic moiety binds to hydrophobic molecules and the hydrophobic surfaces of proteins. The degree of hydrophobicity of the medium can be controlled by selecting the hydrophobic moiety. A process called hydrophobic interaction chromatography (HIC) uses a hydrophobic interaction medium to separate a target molecule (such as a protein or other molecule of interest) from product- and process-related contaminants. Some product- and process-related components that should ultimately be separated out when manufacturing and / or purifying a target molecule in and / or from a host cell are referred to as host cell proteins (HCP) and cell debris. In some cases, a mixture containing the target molecule and other components is added to the HIC medium in a buffer that is designed to promote the binding of the hydrophobic groups in the target molecule to the hydrophobic moiety of the HIC medium. Such a mixture can be referred to as the "load mass". HIC takes advantage of the hydrophobicity differences between the target molecule and the impurities, which results in separation during the loading, washing, or regeneration stages. The target molecule is often separated into the flow-through, while the impurities bind to the HIC medium. HIC can also be operated in a mode where the target molecule binds to the HIC medium while the HCP and cell debris do not bind and flow through. The present disclosure is applicable to either situation (whether the target molecule binds to the hydrophobic interaction moiety or not).

[0053] The HIC medium can be periodically stripped or regenerated after it is used for purifying / collecting the target molecule. As used herein, the terms "stripping" and "regeneration" can be used interchangeably and / or in combination to refer to the process of removing any residual components of the sample substance from the HIC medium after a purification cycle and preparing the HIC medium for a subsequent purification cycle. For example, after a HIC device is used to separate or purify a molecule of interest from a sample substance containing host cell material (e.g., host cell debris, host cell proteins, etc.) and the molecule of interest is eluted from the HIC device, the HIC medium can be regenerated to remove residues (e.g., host cell material, host cell proteins, lipids, residual polypeptides, aggregated proteins, nucleic acids, biomolecules, etc.) from the HIC medium and prepare the HIC medium for use in purifying the molecule of interest from another sample substance. In some embodiments, regeneration of the HIC medium can include disrupting the hydrophobic interactions between the residual host cell material and / or the target molecule and the HIC medium and / or denaturing the residual host cell material. Regeneration can be performed between hydrophobic interaction chromatography cycles to "reset" the HIC medium without the need for a longer cleaning process. In some embodiments, regeneration can be performed between HIC cycles to prevent or reduce discoloration of the HIC medium over time. In some embodiments, the regeneration process according to the present disclosure can take between, for example, about 5 minutes and about 1 hour, such as between about 10 minutes and about 1 hour, between about 10 minutes and about 45 minutes, or between about 10 minutes and about 30 minutes. Preferably, regeneration of the HIC medium can be accomplished without subjecting the HIC medium to more aggressive cleaning solutions, which may have adverse effects or cause additional problems. The regeneration process can be configured without particular concern for the removal of bacteria, fungi, or other microorganisms from the chromatography medium.

[0054] "Stripping" and "regeneration" can be distinguished from, for example, "cleaning" the chromatography medium. Cleaning can include processes used to thoroughly disinfect and / or purify the chromatography medium, the chromatography device, and / or the laboratory environment. For example, the cleaning process can include using an antibacterial, antifungal, or antimicrobial solution, other disinfecting solutions, sterilization, etc. at a concentration and amount that disinfects and / or sterilizes the chromatography medium or the chromatography device. In contrast, while regeneration can include the use of solutions with antimicrobial properties in some cases, the primary purpose of regeneration can be to remove residual components of the sample substance from the chromatography medium after a purification process. In some embodiments, additional protective measures or processes may be required during and / or after the cleaning process to ensure that subsequent chromatography cycles are not affected by the disinfection, purification, antimicrobial or antibacterial solutions used during the cleaning process. In many cases, the cleaning process can be longer than the regeneration process (e.g., greater than about 1 hour).

[0055] The separation of molecules in a HIC medium can be accomplished, for example, by exposing the HIC medium to a feed substance having a high salt concentration to increase the hydrophobic interaction between the HIC medium and the target molecules in the feed substance and then passing a volume of a solution (e.g., a buffer) having an increasing or decreasing salt concentration through the HIC medium to reverse the hydrophobic interaction. Thus, it is generally understood that under low-salt or salt-free conditions, less substance will bind to the HIC medium. However, unexpectedly, it has been found that certain types of HIC media exhibit an increased binding (e.g., hydrophobic interaction) to residues (e.g., host cell proteins) under salt-free conditions. In addition, it has been found that some proteins (e.g., monoclonal antibodies) denature onto some types of HIC media (e.g., Capto TM Phenyl (High Sub) medium (GE Healthcare Life Sciences). Although the proteins can resume their native conformation once eluted from the HIC medium, such proteins in their denatured state are not removed from the HIC medium by a regeneration process comprising, for example, reverse osmosis deionized water (RODI), 1N sodium hydroxide, and / or 20% ethanol. It is hypothesized that in some cases, the elution of molecules from the HIC medium depends on the pH value and / or the conductivity.

[0056] Aspects of the present disclosure relate to a regeneration solution and an evaluation of the availability (including ease of regeneration) of a chromatographic medium.

[0057] In some embodiments of the present disclosure, the regeneration solution can be an alkaline solution. It is contemplated that in some embodiments of the present disclosure, high pH can be a driving factor for the effectiveness of the regeneration solution; however, it is also contemplated that in some cases, the effectiveness of high pH can be offset by high ionic strength. Thus, in some embodiments, the efficacy of the regeneration solution can be driven by a high pH value together with a low but non-zero conductivity (see, for example, Example 14 discussed below). For example, in some embodiments, the regeneration solution can exhibit a pH value between about 8 and about 14, such as between about 10 and about 14. In some embodiments, the regeneration solution can generally exhibit a low conductivity. For example, in some embodiments, the regeneration solution can exhibit a conductivity between about 0.5 mS / cm and about 10 mS / cm, such as between about 0.5 mS / cm and about 5 mS / cm, between about 5.0 mS / cm and about 10 mS / cm, between about 0.5 mS / cm and about 3 mS / cm, between about 0.5 mS and about 1.6 mS, between about 0.8 mS / cm and about 1.6 mS / cm, about 0.5 mS / cm, about 1.0 mS / cm, about 1.5 mS / cm, about 2.0 mS / cm, about 2.5 mS / cm, about 3 mS / cm, about 3.5 mS / cm, about 4.0 mS / cm, about 4.5 mS / cm, or about 5.0 mS / cm.

[0058] In some embodiments, the regeneration solution can be an alkaline solution comprising a certain concentration of, for example, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, tris(hydroxymethyl)aminomethane (Tris), other alkaline solutions, or combinations thereof. In some embodiments, the regeneration solution can include a total dissolved salt concentration between about 0.1 mM and about 50 mM, such as between about 0.1 mM and about 25 mM, between about 0.1 mM and about 20 mM, between about 0.1 mM and about 15 mM, between about 0.1 mM and about 10 mM, between about 0.1 mM and about 5 mM, between about 0.1 mM and about 2.5 mM, between about 1 mM and about 10 mM, between about 1 mM and about 7 mM, between about 2.5 mM and about 5 mM, or between about 2.5 mM and about 7 mM, such as about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM, about 7 mM, about 7.5 mM, about 8 mM, about 8.5 mM, about 9 mM, about 9.5 mM, about 10 mM, about 15 mM, about 20 mM, or about 25 mM.

[0059] In some embodiments, the regeneration solution according to the present disclosure may be suitable for use in a single-step regeneration process. That is, in some embodiments, a method of regenerating an HIC medium may include contacting the HIC medium with a single solution, where the solution exhibits one or more of the properties described herein, and where less than about 5% of the injected material remains bound to the HIC medium as a residue after contact with the single solution. For example, in some embodiments, a method of regenerating an HIC medium may include contacting the HIC medium with a solution having a pH value between about 10 and about 14 and a conductivity between about 0.5 mS / cm and about 10 mS / cm, after which less than about 5% of the injected material remains bound to the HIC medium as a residue. In some embodiments, the residue may be less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the injected material.

[0060] The volume of the regeneration solution used according to the present disclosure can be any suitable volume. For example, in some embodiments in which the injected material is added to the HIC medium in a chromatographic column, the volume of the regeneration solution used according to the present disclosure can be measured in column volumes (CV). For example, in some embodiments, a method of regenerating the HIC medium in a chromatographic column may include passing at least 1 column volume (CV) of the regeneration solution through the chromatographic column. In some embodiments, the method may include passing between about 1 and about 20 column volumes, such as between about 1 column volume and about 15 column volumes, between about 1 column volume and about 10 column volumes, between about 1 column volume and about 5 column volumes, between about 3 column volumes and about 17 column volumes, between about 5 column volumes and about 15 column volumes, or between about 5 column volumes and about 10 column volumes, such as about 1 column volume, about 2 column volumes, about 3 column volumes, about 4 column volumes, about 5 column volumes, about 6 column volumes, about 7 column volumes, about 8 column volumes, about 9 column volumes, about 10 column volumes, about 12 column volumes, about 14 column volumes, about 16 column volumes, about 18 column volumes, or about 20 column volumes, of the regeneration solution through the chromatographic column.

[0061] The systems and methods according to the present disclosure are applicable to a variety of separation media and / or processes. A single system, method, or aspect of the present disclosure may share some features with more than one embodiment described herein. In some exemplary embodiments, the systems and methods according to the present disclosure are applicable to media and / or processes in which the components of a sample substance are completely or partially separated based on their hydrophobicity, such as HIC, or to media / processes that utilize a combination of hydrophobicity and charge, such as ion exchange / hydrophobic interaction mixed-mode chromatography. In some embodiments, one or more of the regeneration solutions and / or methods disclosed herein may be combined with other aspects and / or methods (e.g., implemented before or after). For example, a regeneration solution according to the present disclosure may be added to the media used in mixed-mode chromatography to strip residues that interact with the media due to hydrophobicity. Another regeneration solution may be added to the media to strip residues that are bound to the media due to charge.

[0062] In some embodiments, the regeneration solutions and / or methods disclosed herein are applicable to HIC media with high hydrophobicity. In some embodiments, the chromatographic media applicable to the solutions and methods according to the present disclosure may include, for example, a hydrophobic matrix comprising crosslinked agarose, polystyrene divinylbenzene, or polymethacrylate. In some embodiments, the matrix may contain ligands having hydrocarbons in an aliphatic or aromatic configuration between 2 and 10. In some embodiments, the matrix does not include ligands containing 30 or more hydrocarbons. In some embodiments, the ligands may include, for example, phenyl ligands, butyl ligands, or octyl ligands. In some embodiments, the ligands may be present in the media at a density between about 20 and about 30 μmol / ml of media. In some embodiments, the methods and regeneration solutions described herein are specifically used to regenerate HIC media. In some embodiments, the methods and regeneration solutions described herein are suitable for, for example, Capto TM Phenyl (High Sub), Capto TM Butyl, or Capto TM Octyl media (GE Healthcare LifeSciences), Phenyl media (GE Healthcare Life Sciences), POROS TM Benzyl and POROS TM Ethyl HIC resin (Thermo Scientific TM ), or TOYOPEARL TMResin. In some embodiments, the methods and regeneration solutions described herein may be suitable for continuous (multi-column) HIC systems and methods, as disclosed in International Patent Application PCT / US2019 / 040148, filed on July 1, 2019, which is incorporated herein by reference. For example, the single-step regeneration solution according to the present disclosure may be used in a multi-column continuous hydrophobic interaction chromatography system, where the efficiency of the single-step regeneration solution may enhance the overall efficiency of the multi-column system. In some embodiments, the methods and regeneration solutions described herein may be used in HIC systems and methods that include low or no trending conditions.

[0063] In some embodiments, the regeneration process according to the present disclosure may be performed without using reverse osmosis deionized water (RODI), organic solvents (e.g., ethanol or ethylene glycol), chaotropic agents (e.g., guanidine or urea), sodium chloride, and / or a sodium hydroxide concentration of more than 50 mM. Advantageously, the regeneration process using the solutions disclosed herein does not require an additional process to handle solvents such as ethanol (e.g., 20% ethanol) and chaotropic agents (such as guanidine and urea, e.g., 6N guanidine or 6N urea). However, in some embodiments, it is contemplated that the regeneration solutions disclosed herein may be used before, after, or in combination with organic solvents (e.g., 20% alcohol) or chaotropic agents (e.g., 6N guanidine or 6N urea).

[0064] In some embodiments, the method according to the present disclosure may include passing the regeneration solution disclosed herein through the chromatography column for storage purposes before contacting the chromatography column with the storage buffer. The storage buffer may contain, for example, sodium hydroxide or another salt at a concentration between about 0.05 M and about 0.15 M.

[0065] In some embodiments, the method according to the present disclosure may include evaluating various chromatography media to determine whether one or more chromatography media are likely to pose challenges to the use or regeneration of the media during the purification of the target molecule. The evaluation of the chromatography media according to the present disclosure may include, for example but not limited to: the use of one or more chromatography media types, maintaining pH conditions, and the target molecule. Advantageously, the method according to the present disclosure may include screening at a scale smaller than that typically performed for purifying the target molecule, thereby allowing a significant savings in sample volume (e.g., about 1 / 10, 1 / 100, 1 / 500, 1 / 700 or less of the sample volume required to evaluate chromatography media, e.g., for HIC, according to conventional methods). Additionally, a high-throughput screening (HTS) process, for example, may be utilized to screen multiple purification protocols with different variables (e.g., different combinations of media types, pH values, and / or target molecules) simultaneously. Alternatively, or in addition to high-throughput screening (HTS), an elution assay may be used to exclude parameters of potential HIC protocols.

[0066] Advantageously, these screening techniques and assays can provide significant time savings in determining a purification protocol suitable for use in large-scale purification processes, such as by optimizing HIC unit operations. For example, an HTS process performed in accordance with the present disclosure can be about 10 times, 50 times, 60 times, 70 times, or even faster than conventional methods for determining regeneration / availability issues in chromatography protocols for one or more unit operations, including hydrophobic interaction chromatography, ion exchange, affinity chromatography, and the like.

[0067] The evaluation method according to the present disclosure may not include filling pores (such as filter plate pores) with a certain amount of chromatographic medium, where the chromatographic medium is intended for a potential purification protocol. The filter plate pores can have a volume of, for example, less than 5 mL, such as less than 4 mL, less than 3 mL, or less than 2 mL. In some embodiments, the filter plate pores can have a volume of about 1 mL, about 0.8 mL, about 0.5 mL, or any other suitable volume. The filter plate pores can be equipped with a filter having a suitable mesh size (such as between about 0.5 and about 1.5 microns, such as about 0.8 microns, about 1.0 microns, or 1.2 microns). The filter mesh size can depend on the size of the resin beads present in the chromatographic medium used in the protocol. The size of such chromatographic resin beads can be, for example, between about 40 microns and about 120 microns, such as between about 50 microns and about 100 microns, between about 60 microns and about 90 microns, between about 80 microns and about 90 microns, about 70 microns, about 80 microns, about 90 microns, about 100 microns, or about 110 microns. The amount of chromatographic medium filled into the pores can vary. For example, the amount of chromatographic medium can range from, for example, about 2.0 μL to about 50.0 μL, such as about 10 μL, about 20.0 μL, about 30.0 μL, or about 40.0 μL. In some methods according to the present disclosure, a filter plate including a plurality of pores can be filled with a plurality of different chromatographic media for simultaneous evaluation of a plurality of protocols in an array. This can be used for any chromatographic step in a purification protocol, including hydrophobic interaction chromatography, ion exchange chromatography, affinity chromatography, etc.

[0068] A volume of load material can be loaded into the pores of the packed filter plate (alternatively, in the case of evaluating multiple protocols simultaneously, into the pores of each packed filter plate). The load material can contain a target molecule that has undergone some initial purification process, such as affinity chromatography or ion exchange chromatography. According to the present disclosure, the load material can be used to test unit operations, such as HIC. In the case of evaluating multiple conditions for a single unit operation simultaneously, the load materials for different pores can contain different target molecules. The load material can be titrated to a protocol-specific pH value. The approximate concentration of the target molecule within the load material can be adjusted to any suitable unit-operation-specific concentration. In some embodiments, the volume of the load material can correspond to a fraction of the load mass that would be used in a full-scale HIC protocol (e.g., the mass of the target molecule within the load material), such as 1 / 2, 1 / 5, 1 / 8, 1 / 10, 1 / 20, 1 / 50, 1 / 100, or less of the load mass used in the full-scale protocol.

[0069] The method for evaluating a protocol for a unit operation can further include performing elution and cleaning steps suitable for a large-scale process (e.g., including the use of reverse osmosis deionized water (RODI), 1N sodium hydroxide, and / or 5 mM sodium hydroxide), and subsequently performing a stripping step using an alkaline agent or chaotropic agent (such as 6N guanidine hydrochloride or 6N urea) or a solvent (such as 20% ethanol). The elution / cleaning step can be performed using, for example, an elution buffer titrated to a protocol-specific pH value. In the case of evaluating multiple protocols for a unit operation, elution buffers at different protocol-specific pH values can be used in different pores within a single array. The elution step can include, for example, exposing the chromatographic medium loaded in the filter pores to a gradient elution buffer (starting at a relatively high concentration and ending at a concentration of 0). In another embodiment, the elution step can include exposing the loaded chromatographic medium to a buffer having an initial concentration of a lyotropic salt (e.g., citrate at 500 mM, 400 mM, 300 mM, 200 mM, etc.), and gradually / linearly reducing the buffer concentration to 0. In some embodiments, pseudo-gradient elution can be performed, where the loaded (injected) chromatographic medium is exposed to discrete volumes of elution buffer having a linearly decreasing concentration that is reduced from an initial concentration (e.g., 300 mM) to 0 over multiple steps (e.g., 4, 5, 6, 7, 8, more, or fewer steps).

[0070] The first step may include any process to be tested as part of a unit operation protocol. Generally, the first step may include adding solutions that are considered suitable for use in large-scale and repetitive operations (e.g., solutions that do not present safety or toxicity issues). For example, the first step may include one or more washes with RODI and / or 1N NaOH, which are performed in series or in an alternating sequence, each performed one or more times. The second step may include any solution intended to strip any residue that has bound to the chromatographic medium after the unit operation being evaluated is completed. Such a regeneration process is described elsewhere in this document, but generally may include chaotropic agents such as 6N guanidine hydrochloride or 6N urea, or solvents such as 20% ethanol.

[0071] The evaluation method according to the present disclosure may include measuring the amount of the target molecule recovered during, for example, the wash / elution step and the stripping step. These results may be compared across various chromatographic media. As opposed to during the stripping step (i.e., the stringent stripping of the chromatographic medium), it is desirable to recover a relatively large percentage of the target molecule during the wash / elution step (i.e., while the HIC protocol is being tested).

[0072] The evaluation method according to the present disclosure may also include determining the recovery rate of the target molecule during the stripping step, as a percentage of all the target molecules recovered during the unit operation protocol test. If the percentage recovery of the target molecule during the stripping step exceeds a predetermined threshold, then it can be predicted that the unit operation protocol may potentially cause regeneration / reusability issues when scaled up and / or when repeated many times. If the percentage recovery of the target molecule during the stripping step is at or below the predetermined threshold, then it can be predicted that the unit operation protocol will not cause regeneration / reusability issues when scaled up and / or repeated. The predetermined threshold can be any experimentally determined threshold that is indicative of the amount of residue that remains bound to the chromatographic medium after a unit operation (such as an HIC protocol). In some embodiments, the predetermined threshold may be between, for example, about 1% and about 10%, such as between about 3% and about 7%, about 4%, about 5%, or about 6%.

[0073] In embodiments where multiple chromatographic protocols are evaluated simultaneously, the above calculation of the percentage recovery of the target molecule can be determined for multiple chromatographic protocols at once, resulting in a preliminary impression of which protocol may be suitable for or preferred for further use, testing, research, or development. Protocols for further study may include those with a normalized percentage recovery due to the stripping step that is less than or equal to 1%, less than or equal to 3%, less than or equal to 5%, less than or equal to 7%, or less than or equal to 10%.

[0074] In some embodiments, methods for evaluating chromatography schemes according to the present disclosure (such as for HIC unit operations) can be performed in the early stages of establishing a purification process. For example, multiple HIC schemes can be evaluated in the manner described herein (e.g., high-throughput screening and / or elution tests), and HIC schemes predicted to cause regeneration / reusability issues can be excluded or deprioritized for further study. Chromatography schemes not predicted to cause regeneration / reusability issues can undergo further testing (e.g., full-scale testing) or study to, for example, maximize yield and minimize impurities, confirm that they meet internal and external quality control criteria, and evaluate reproducibility and lifetime (e.g., after 10, 25, 50, 75, 100 or more cycles, whether they result in column discoloration or other adverse effects).

[0075] In some embodiments, the methods according to the present disclosure can advantageously prevent or reduce discoloration of the regenerated column, media, and / or equipment that may occur after one or more uses (see, e.g., Examples 6 and 9 discussed herein). In some embodiments, the methods according to the present disclosure can advantageously help identify, early on, schemes for chromatography unit operations that prevent regeneration / availability issues, thus saving time and cost in developing a complete purification scheme that would otherwise be spent discovering such issues for the chromatography steps later on.

[0076] Reference will now be made to specific drawings. Figure 1 A method 100 for regenerating a chromatography column according to various aspects of the present disclosure is depicted. According to step 102, a first injection substance can be added to the HIC column. According to step 104, a protein of interest can be collected from the HIC column. According to step 106, a single basic regeneration solution can be added to the chromatography column to remove substances bound to the hydrophobic interaction media in the chromatography column.

[0077] According to step 102, a first injection substance can be added to the HIC column. The injection substance can include a target molecule (e.g., a polypeptide) and residual components (such as host cell proteins, cell debris, etc.). According to step 104, a protein of interest can be collected from the hydrophobic interaction chromatography column. This can be done, for example, in a washing step or an elution step. According to step 106, a single basic regeneration solution can be added to the chromatography column to remove substances bound to the hydrophobic interaction media in the chromatography column. The single basic regeneration solution can have one or more of the characteristics described above. Examples

[0078] Example 1

[0079] Measure the amount of non-esterified free fatty acids (“NEFA”) in several sample solutions after incubation with polysorbate 20 (“PS20”). The presence and amount of NEFA in the samples are considered to be indicators of PS20 degradation caused by impurities in the samples, such as host cell proteins. A first sample is taken from the HIC feed material. Five additional samples are taken from the HIC pool after 1, 2, 3, 5, and 10 subsequent cycles. All samples are incubated for the same amount of time. As shown in Table 1 below, from cycle 1 to cycle 5, PS20 degradation is calculated as a negative percentage, equivalent to the negative control. A negative PS20 degradation percentage is equivalent to no detectable NEFA and thus equivalent to no detectable lipase activity. NEFA is detectable between cycles 5 and 10, demonstrating an increase in lipase activity. This data indicates that lipase activity can increase as a function of the number of cycles. Table 1 Cycle PS20 Degradation (%) Detection Injected Substance 1.83 Detectable Cycle 1 -0.31 Equivalent to Negative Control Cycle 2 -0.25 Equivalent to Negative Control Cycle 3 -0.20 Equivalent to Negative Control Cycle 5 -0.22 Equivalent to Negative Control Cycle 10 0.47 Detectable

[0080] Example 2

[0081] Figure 2 Depicts an imprint analysis of the HIC medium (Capto TM Phenyl (High Sub) (GE Healthcare Life Sciences)) in various states of use or after exposure to the regeneration solution, as detailed below. Table 2

[0082] As can be seen Figure 2 in, in each of the used columns there are visible dark areas, except for the column (column F) that has been in contact with 6N guanidine hydrochloride. The column representing the native medium (column A) also does not show dark areas. This data indicates that 6N guanidine hydrochloride is capable of removing residual medium during column regeneration compared to other solutions. Thus, 6N guanidine hydrochloride can be used as a stripping agent and as a reagent that can be used after other stripping agents / regenerants to evaluate the performance of other reagents.

[0083] Example 3

[0084] Figure 3 Depicts an imprint analysis of the HIC medium (Capto TM Phenyl (High Sub)) in various states of use or after exposure to the regeneration solution, as described in detail below. Table 3

[0085] Table 3 lists the types of media used and what regenerant the media was exposed to. As can be seen in Figure 3 , the regeneration examples containing RODI, 1.0N NaOH, RODI, and 20% ethanol (used for media B and C) are not as effective in removing residues from the media as 6N guanidine hydrochloride (D) is in removing residues from the media.

[0086] Example 4

[0087] Figure 4 depicts the superimposed UV chromatograms that depict five purification processes after the collection of monoclonal antibody mAb 1 during the process. Each purification process includes a first 0.5N sodium hydroxide rinse (A) of 2 column volumes, followed by a pause, and then a second 0.5N sodium hydroxide rinse (B) of 1 column volume. Purification is considered complete at point C, after which a 2-column volume rinse with water for injection ("WFI") (D) is performed. As can be seen in Figure 4 , the first sodium hydroxide rinse (A) produces a high absorbance in the early part of the rinse, which is associated with the removal of a large amount of impurities. The maximum absorbance seen during HIC elution (regeneration) is 2.4 AU, while the maximum absorbance seen during the purification cycle of Figure 4 is approximately 1.4 AU.

[0088] Example 5

[0089] Figure 5 depicts the blot analysis of HIC media (Capto TM Phenyl (High Sub)) in various usage states or after exposure to stripping solution, as described in detail below. In particular, the used HIC media is exposed to decreasing concentrations of guanidine. Table 4 Label Medium State * Reference Ladder A Indicates Natural (Unused) Medium B Used Medium C Used Medium Exposed to 20% Ethanol D Used Medium Exposed to 6N Guanidine Hydrochloride E Used Medium Exposed to 3N Guanidine Hydrochloride F Used Medium Exposed to 2N Guanidine Hydrochloride G Used Medium Exposed to 1N Guanidine Hydrochloride H Used Medium Exposed to 0.5N Guanidine Hydrochloride I Used Medium Exposed to 0.1N Guanidine Hydrochloride

[0090] As can be seen in Figure 5 , the use of 6N guanidine hydrochloride (D) removes most of the residues from the HIC media, while solutions of lower guanidine concentrations and 20% ethanol solution are less effective in removing residues from the HIC media.

[0091] Example 6

[0092] Figure 6 depicts containing Capto TMTwo columns of Phenyl (High Sub) HIC media (GE Life Sciences). The left column was exposed to 49 cycles of HIC for the purification of monoclonal antibody mAb2. Each cycle included regeneration of the chromatography column in the order of RODI, 1N sodium hydroxide, RODI, and 20% ethanol. After the 40th cycle, a yellow band was confirmed at the bottom of the column. The right column (for comparison) was native Capto TM Representative of Phenyl (High Sub) HIC media. The discoloration of the left column can be an indication of insufficient regeneration.

[0093] Pools from cycles 1 and 49 were collected and assayed for lipase activity and the presence of host cell protein (HCP). There was no clear trend of change in lipase activity or HCP values between cycle 1 and cycle 49.

[0094] Example 7

[0095] Figure 7A and Figure 7B Chromatograms of cycle 2 and cycle 49 performed on the left column as described in Example 6 are depicted respectively. For Figure 7A and Figure 7B the following annotations were made: Table 5

[0096] In both cycles 2 and 49, RODI was not an effective stripping solution, as no peaks were shown after C or C’ (at markers X and X’). The addition of 1N NaOH produced peaks P1 (cycle 2) and P3 (cycle 49), indicating that 1N NaOH was at least partially effective as a stripping solution. The introduction of a second RODI strip removed some additional impurities and produced peaks P2 (cycle 2) and P4 (cycle 49). The addition of the 20% ethanol solution did not produce additional peaks. This data from both cycles indicates that RODI used before sodium hydroxide was not an effective stripping solution.

[0097] Example 8

[0098] Two injected substances (each containing the target monoclonal antibody) were subjected to a hydrophobic interaction chromatography process. In the first process, after injection, washing, and elution of the target monoclonal antibody mAb 3 from the first injected substance, the chromatography column was subjected to a 1N sodium hydroxide stripping solution, followed by RODI stripping, and 20% ethanol stripping. In the second process, after injection, washing, and elution of the target monoclonal antibody mAb 4 from the second injected substance, the chromatography column was subjected to RODI stripping, followed by 1N sodium hydroxide stripping, another RODI stripping, and 20% ethanol stripping.Figure 8A depicts the chromatogram of the first process, Figure 8B depicts the chromatogram of the second process. Each chromatogram has been annotated as follows: Table 6

[0099] After the first RODI stripping (at marker X') indicated by marker D' in Figure 8B , there are no peaks, which is equivalent to no peaks at the position indicated by marker X of Figure 8A (where the first RODI stripping was not performed). Therefore, the conductivity reduction of the first RODI stripping applied in the first process does not cause any perceptible amount of substance to be removed from the chromatographic column.

[0100] Example 9

[0101] Furthermore, the effectiveness of 6N guanidine hydrochloride as a stripping solution and as a potential solution for removing discoloration on the left column, as depicted in Figure 6 , was evaluated. The column was subjected to the protocol described in the following table. The chromatogram generated during this protocol is shown in Figure 9 . Table 7 Solution Flow Direction Chromatogram Marker RODI Downward Flow A 6N Guanidine Hydrochloride Downward Flow B RODI Downward Flow C 6N Guanidine Hydrochloride * Upward Flow D RODI Upward Flow E At a rate of 200 cm / hour, each solution was passed through the column in the downward flow direction (i.e., in the same direction as the flow direction of the HIC purification process) or in the upward flow direction (i.e., opposite to the downward flow, or "backward" flow through the chromatographic column). The second addition of 6N guanidine hydrochloride (indicated by an asterisk) was maintained inside the column for approximately 16 hours.

[0102] As shown in the chromatogram of Figure 9 , the first introduction of RODI produces peak P1, and the first stripping with 6N guanidine hydrochloride after the first introduction of RODI produces a high peak P2. There is no peak associated with the second stripping of 6N guanidine hydrochloride, which was kept in the column overnight before flowing through. This may indicate the efficacy of the first stripping of 6N guanidine hydrochloride in removing residues bound to the column. However, after the complete cleaning protocol, the chromatographic column still shows discoloration (as shown in Figure 6 ).

[0103] Example 10

[0104] The regeneration example was analyzed in detail. Figure 10A depicts the use of Capto during TMChromatogram of the HIC process for the purification of monoclonal antibody mAb 4 using Phenyl(High Sub) media (GE Life Sciences). The HIC process, including the regeneration example, consists of the following steps: Table 8 Mark ( Figure 10A ) Event A Pre - Stripping B Equilibration C Injection and Cleaning D RODI Stripping E 1N NaOH Stripping F RODI Stripping G 20% Ethanol Stripping H Pre - Hydrochloride Guanidine RODI Regeneration I 6N Guanidine Hydrochloride

[0105] Reference Figure 10A , peak P1 follows the addition of 1N sodium hydroxide (E), and peak P2 coincides with the second RODI stripping (F). Peak P3 follows the addition of 6N guanidine hydrochloride (I).

[0106] Figure 10B An enlarged image of peak P1 is shown. The first RODI stripping (D) did not result in detectable absorbance. The 1N NaOH stripping (E) seems to cause the immediate and early removal of residues from the column, as indicated by the presence of peak P1, which seems to reach the baseline as the conductivity increases. It is assumed that initially the RODI stripping (D) would cause some residues to bind more tightly to the column rather than facilitate their removal. It is further assumed that the elution of residues from the column caused by sodium hydroxide may be mainly driven by the pH value, but as the concentration of sodium hydroxide (a weak chaotropic agent) increases and the conductivity of the solution increases, residual proteins may bind more tightly to the HIC medium.

[0107] Example 11

[0108] As a function of the sodium hydroxide concentration, the elution of residues from the used HIC medium was observed. Figure 11Depicts a chromatogram of the following process, where after injecting and washing the HIC column to collect monoclonal antibody mAb 4 (during part A), a 20 CV (column volume) gradient is loaded into the column, and the mixing of RODI with sodium hydroxide starts with separate RODI and then the concentration of sodium hydroxide is gradually increased to a maximum concentration of 1 N sodium hydroxide (part B). A single distinct peak P1 is observed and eluted by passing ~5 mM sodium hydroxide through the column. A second 20 CV gradient is performed and loaded into the column, starting with RODI and the maximum concentration of 1 N sodium hydroxide, and the concentration of sodium hydroxide is gradually decreased to 0 (part C). During this second gradient, no additional peaks are observed. Finally, at label D, a solution of 6 N guanidine hydrochloride is flushed through the column. During the passage of 6 N guanidine hydrochloride through the column, a small peak P2 is observed. The area under the curve (AUC) of each peak is calculated from the integrated 280 nm UV absorbance. The peak P2 is calculated to have an AUC of 1,160 mL*mAU, and as a comparison, for the AUC of 6 N guanidine hydrochloride stripping in a control process (e.g., Table 8), this AUC is calculated to be 13,305 mL*mAU. Thus, compared to the control, peak P2 shows a 91.3% size reduction.

[0109] This process demonstrates that the bound material eluted from the column is maximal when passing ~5 mM sodium hydroxide through the column, leaving relatively little residue to be eluted with 6 N guanidine hydrochloride.

[0110] Example 12

[0111] In an independent two-solution regeneration process, sodium hydroxide solutions of different concentrations (1000 mM, 500 mM, 100 mM, 50 mM, 25 mM, 10 mM, and 5 mM) are added, each performed after injection and washing of the HIC column. Each regeneration process includes a sodium hydroxide solution as the first regeneration solution and a 6 N guanidine hydrochloride solution as the second regeneration solution. Chromatograms of the regeneration processes are generated and overlaid on Figure 12。In each regeneration process, a sodium hydroxide solution generated a first peak, represented by the peak group labeled A. In each regeneration process, a 6N guanidine hydrochloride solution generated a second peak, represented by the peak group labeled B. It has been determined that the regeneration process including 5 mM NaOH generated the largest "A" peak (indicating the largest amount of residue eluted by adding a sodium hydroxide solution) and the smallest "B" peak (indicating the smallest amount of residue eluted by adding guanidine hydrochloride). Therefore, it is determined that within the range of sodium hydroxide solutions tested, the 5 mM sodium hydroxide solution is the most effective for HIC column regeneration (i.e., it removed most of the bound substances from the column). The higher the sodium hydroxide concentration, the more residue remains on the chromatographic column for the 6N guanidine hydrochloride solution to remove. It is assumed that although the increase in pH drives the elution of residues from the HIC column, the increase in conductivity reduces the elution of residues by strengthening the binding between the residues and the HIC medium.

[0112] Example 13

[0113] After collecting mAb 4 from an HIC column prepared with Capto TM Phenyl (High Sub) medium (GE Healthcare Life Sciences), a one-way statistical analysis of variance was performed on the area under the curve (AUC) of the chromatographic peaks generated by the regeneration process. As shown in Figure 13A and Figure 13B , regeneration processes were carried out using control (A), RODI (B), and various concentrations of sodium hydroxide solutions (C-L). Each regeneration process included a stripping solution (AUC analyzed in Figure 13A ) followed by a 6N guanidine hydrochloride solution (AUC analyzed in Figure 13B ). An AllPairs Turkey-Kramer test was performed on each analysis to describe the statistical significance.

[0114] As shown in Figure 13A and Figure 13B , the processes associated with the 0.5 mM NaOH and 1 mM NaOH regeneration solutions showed that the peaks generated during the flow of these regeneration solutions had the highest AUC values (circled in region 1300), indicating that these regeneration solutions enabled more effective removal of substances from the HIC column. The processes associated with the 0.5 mM NaOH, 1 mM NaOH, and 5 mM NaOH regeneration solutions showed that the peaks generated during the flow of the 6N guanidine hydrochloride solution after the regeneration solution had the lowest AUC values (circled in Figure 13Bin region 1350), thus also supporting more effective removal of substances from the HIC column by these regeneration solutions, leaving less substance for removal by 6N guanidine hydrochloride. Therefore, a solution having a sodium hydroxide concentration in the range of 0.5 mM to 5 mM NaOH has been shown to be effective for regeneration of the HIC column.

[0115] Example 14

[0116] The regeneration process using a sodium hydroxide solution was compared with the regeneration process using a sodium chloride solution. After collecting monoclonal antibody mAb 5 from the HIC column, the column was regenerated using sodium hydroxide at a concentration of 3 mM, 5 mM or 7 mM, or using sodium chloride at a concentration of 5 mM, 8 mM, or 11 mM. The pH value and conductivity of each regeneration solution were also recorded. The sodium hydroxide solutions all showed a pH value greater than 11, while the sodium chloride solutions all showed a pH value between 5.5 and 6.5. The conductivity of these solutions was similar. For each process, after the regeneration solution, a solution of 6N guanidine hydrochloride was added to each column. Chromatograms of each process were generated and all the chromatograms were superimposed Figure 14 in. The AUC of the peaks related to the flow-through of the regeneration solution and the flow-through of 6N guanidine hydrochloride was calculated. These solutions and AUC are listed in the table below. Table 9

[0117] As depicted by this data, the flow-through of the sodium hydroxide solution showed a much higher AUC value compared to the sodium chloride solution. Similarly, the flow-through of 6N guanidine hydrochloride after the sodium hydroxide solution showed a much lower AUC value compared to the flow-through of 6N guanidine hydrochloride after the sodium chloride solution. In Figure 14 the peaks generated by the flow-through of sodium hydroxide are labeled with the symbol A, and the peaks generated by the flow-through of sodium chloride (or the absence of such a peak) are labeled with the symbol B. Similarly, the peaks generated by the flow-through of 6N guanidine hydrochloride after sodium hydroxide and the flow-through of 6N guanidine hydrochloride after sodium chloride are labeled with the symbols A' and B', respectively. As shown in Figure 14 the figure, the sodium hydroxide solution is a more effective stripping agent than the sodium chloride solution.

[0118] Example 15

[0119] Potential cleaning / regeneration protocols were tested on columns that had undergone 50 HIC cycles to purify monoclonal antibody mAb 6. As Figure 15 (Columns A, B, and C) shown, discoloration occurred near the top of the column bed in the columns. Column A was rinsed with 2 CV of 0.5 M EDTA, column B was rinsed with 2 CV of 0.5 M acetic acid, and column C was used as a control. Neither solution was effective in reducing the browning / discoloration phenomenon.

[0120] Example 16

[0121] After the HIC purification of monoclonal antibody mAb 6, three sodium hydroxide solutions (2 mM, 5 mM, and 10 mM) were used as regeneration solutions. The regeneration efficacy (effect) of each sodium hydroxide solution on the HIC medium used in the purification process was characterized by the size of the chromatographic peaks associated with the 6 N guanidine hydrochloride stripping after the addition of each sodium hydroxide solution. A larger AUC associated with the guanidine stripping peak indicates that the sodium hydroxide solution used before stripping left a larger amount of residue, and conversely, a smaller AUC associated with the guanidine stripping peak indicates that the sodium hydroxide solution used before stripping left a smaller amount of residue and thus indicates a more effective sodium hydroxide regeneration solution. Figure 16 The curve shown displays the AUC of the guanidine stripping peak as a function of sodium hydroxide concentration. All three tested sodium hydroxide solutions showed more effective regeneration (i.e., smaller guanidine stripping peaks) compared to the default wash mode (comprising the sequence of RODI, 1 N sodium hydroxide, RODI, 20% ethanol, and RODI). The 5 mM sodium hydroxide solution had the lowest guanidine stripping peak area. Based on the curve extrapolated from these data points, a 7 mM sodium hydroxide regeneration solution has the potential to result in an even lower guanidine stripping peak area.

[0122] Example 17

[0123] After collecting monoclonal antibody mAb 5 from the chromatography column, a first regeneration process designed as a control was implemented on the HIC column. A chromatogram was generated using this process. Multiple solutions were used in the following order: starting with 1 N sodium hydroxide, followed by RODI, 20% ethanol, RODI, and 6 N guanidine hydrochloride. The chromatographic peak corresponding to the flow-through of 6 N guanidine hydrochloride was used to measure the effectiveness of the regeneration process. The chromatogram is depicted in Figure 17 . The markers on the chromatogram represent the following events: Table 10 Marker Event A Injection B Pool Collection Start C Cleaning D Introduction of 1N NaOH Solution E Introduction of RODI F Introduction of 20% Ethanol G Introduction of RODI H Introduction of 6N Guanidine Hydrochloride The AUC of the peak corresponding to the flow-through of the 6 N guanidine hydrochloride solution was calculated to be 7,390 mL*mAU.

[0124] After collecting mAb 5 from the HIC column, a second regeneration process containing 5 mM sodium hydroxide was implemented, and a chromatogram was generated, which is depicted in Figure 18 . Multiple solutions were used in the following order: starting with 5 mM sodium hydroxide, followed by RODI, 20% ethanol, RODI, 5 mM sodium hydroxide, RODI, 6 N guanidine hydrochloride, and 0.1 N sodium hydroxide. The AUC corresponding to the flow-through of each solution was calculated from the chromatogram, and the results are listed in the table below: Table 11 Sequence Solution AUC (mL * mAU) 1 5mM NaOH 29,119 2 RODI 196 3 20% EtOH 4 4 RODI 1 5 5mM NaOH 125 6 RODI 57 7 6N Guanidine Hydrochloride 889 8 0.1N NaOH N / A (Not Applicable)

[0125] As Figure 18 shown and as reflected by the AUC values in the table above, the initial 5 mM NaOH flow-through showed the largest AUC (peak A) with a large margin. The solutions added to the column after the initial 5 mM NaOH solution removed very little additional material from the HIC column, as indicated by their relatively small corresponding AUC values. The second largest AUC value was associated with the flow-through of 6 N guanidine hydrochloride, but its value of 889 mL*mAU was less than one-thirtieth of the AUC value associated with the initial 5 mM NaOH flow-through. Additionally, the smaller AUC value of the 6 N guanidine hydrochloride peak in this regeneration process compared to the AUC value of the 6 N guanidine hydrochloride peak in the control process (7,390 mL*mAU) shown in Figure 17 indicates that the initial 5 mM NaOH solution provides a significantly improved HIC column regeneration compared to the control run. When used after the 5 mM NaOH solution, RODI, 20% ethanol, 6 N guanidine hydrochloride, and 0.1 N NaOH provide very little additional benefit for HIC regeneration.

[0126] Example 18

[0127] In columns containing Capto TM Phenyl (High Sub) medium (GE Life Sciences), six mixtures, each containing a different target molecule (e.g., a target monoclonal antibody) and having different citrate concentrations in the injection buffer, were subjected to HIC. After the elution of each monoclonal antibody, the used HIC column was passed through a certain volume of RODI and sodium hydroxide, with a gradient from no sodium hydroxide (pure RODI) to 1 N sodium hydroxide, followed by a gradient in the reverse direction (from 1 N sodium hydroxide back to pure RODI). Finally, each process ended with an injection of 6 N guanidine hydrochloride and collection of the flow-through. The pH values and citrate concentrations in each injection mixture are shown in the table below. Table 12

[0128] Chromatograms were generated for each process; these chromatograms are depicted in order in Figure 19 . As shown, each chromatogram A - F includes peaks (A’, B’, C’, D’, E’, F’) corresponding to the elution of material bound to the HIC medium upon the flow-through of approximately 5 mM NaOH. The subsequent guanidine hydrochloride flow-through peaks are minimal or absent. Thus, the effectiveness of the regeneration solution is not limited to the use of one monoclonal antibody.

[0129] Example 19

[0130] Multiple HIC protocols with different conditions were evaluated to determine whether such protocols would cause regeneration / availability issues. In the following manner, the wells of a 96-well filter plate (AcroPrep TM Advance 1mL filter plate, 1.2μm Supor membrane, part number 8130, Pall Corporation) were filled with 0.02 μL of various HIC media: Table 13

[0131] Aliquots of the sample material were prepared, each containing one of three target antibodies (mAb 1, mAb 2, mAb 3) and adjusted to a concentration of 0.33 g / L, with the goal of a concentration of 5 g / L when mixed with the HIC media. For each of the three target antibodies, aliquots of the sample material were titrated to different pH values (4.5, 6.25, or 8.0) with 2M acetic acid or 2M tris(hydroxymethyl)aminomethane (Tris) base to prepare a total of 9 aliquots, each containing one of the three target antibodies and exhibiting one of the three different pH values.

[0132] Each column of three wells filled with a single media type underwent a purification protocol at different pH values (4.5, 6.25, or 8.0) to establish a series of protocols performed with various combinations of media and pH. Each column of wells was subdivided into groups such that each group of the columns underwent a protocol for a different target antibody (mAb1, mAb 2, or mAb 3). To run each protocol at its respective pH value, for each target antibody, an aliquot of the sample material containing the target antibody at the corresponding pH value was used, and a equilibration buffer of 40 mM Tris, 300 mM citrate at the corresponding pH was used.

[0133] The following steps were performed simultaneously on the array: 1. Equilibrate the wells filled with various HIC resins three times with the equilibration buffer at their respective pH values. 2. Add the sample material containing the desired target antibody to each well at its respective pH and incubate for up to one hour. 3. Spin the plate at 1100 rpm. 4. Collect the flow-through (the components that did not bind to the media during incubation). 5. Wash the wells twice with the equilibration buffer at the corresponding pH value for each test protocol. 6. Perform pseudo-gradient elution using a balanced buffer at the corresponding pH. Expose each well to the balanced buffer for seven iterations, where the citrate concentration is linearly decreased from 300 mM to 0 mM (increments of 42.9 mM per iteration) over all seven iterations. 7. Wash the wells twice with RODI and then twice with 1N sodium hydroxide. 8. Spin the plate at 1100 rpm. 9. Strip the wells twice with a solution of 6N guanidine hydrochloride. 10. Spin the wells at 1100 rpm.

[0134] For each well, generate a chromatogram of the target polypeptide in the material removed from the well (flow-through, eluate, washes, etc.). For result analysis, each chromatogram is subdivided into three regions, where the first region contains the mass of the target polypeptide observed in the material removed during steps 2, 3, and 4 (flow-through, wash, and elution), the second region contains the mass of the target polypeptide observed in the material removed during steps 5, 6, 7, 8 (using RODI and 1N sodium hydroxide), and the third region contains the mass of the target polypeptide observed in the material removed during steps 9, 10 (using 6N guanidine hydrochloride). Examples of the three chromatograms generated from the protocol for purifying the target antibody mAb 1 at a pH of 6.25, and the subdivision into three regions for three different HIC media ( Hexyl-650C, Phenyl Sepharose 6Fast Flow (HighSub), and POROS TM Ethyl) are depicted in Figures 20A - 20C ).

[0135] Comparison shows that the protocol and analysis performed on the test wells are consistent with the protocol performed on a large-scale column, with 98% statistically significant accuracy.

[0136] Example 20

[0137] The aggregation of data using the high-throughput screening techniques described herein enables the establishment of predictive functions that assist in establishing HIC protocols with improved efficiency and yield. For example, boundary functions can be plotted that describe the relationship between multiple parameters (e.g., pH, citrate concentration of the elution buffer, loading mass, HIC media selection) and the efficiency or yield of the protocol.

[0138] Figure 21A is a boundary function plot based on a given antibody and media (Phenyl A set of high-throughput screening data at a sample load of 100 g / L of the medium, correlating the citrate concentration and pH of the elution buffer with the predicted yield of the HIC protocol. The shaded area represents the combination of HIC protocol parameters that result in a predicted yield less than 90% of the theoretical yield. This boundary function informs what citrate and pH parameters should be considered for the HIC protocol containing a given antibody and the medium. Combinations of pH and citrate concentration outside this shaded area are variable HIC protocol parameters for the given antibody and the medium, while combinations of pH and citrate concentration inside the shaded area are excluded parameters.

[0139] Figure 21B is based on a set of high-throughput screening data at a sample load of 100 g / L of the medium for a given antibody and Capto TM Phenyl (High Sub) chromatography medium, a graph of the boundary function that relates the citrate concentration and pH of the elution buffer to the predicted yield of the HIC protocol. The shaded area represents the combination of HIC parameters that result in a predicted yield less than 90% of the theoretical yield. This boundary function informs what citrate and pH parameters should be considered for the HIC protocol containing a given antibody and Capto TM Phenyl (High Sub) medium. Combinations of pH and citrate concentration outside the shaded area are variable HIC protocol parameters for the given antibody and Capto TM Phenyl(HighSub) medium, while combinations of pH and citrate concentration inside the shaded area are excluded parameters.

[0140] In addition to the boundary functions described above, data collected from high-throughput screening can be used to establish transfer functions or other mathematical models that assist in HIC protocol design. For example, a regression transfer function can relate data from elution assays, high-throughput screening, or laboratory-scale HIC protocols to full-scale HIC protocols. For example, for a set of HIC protocols that include predicted yields (e.g., yields predicted by small-scale experiments or high-throughput screening), the actual yield of each HIC protocol can be determined by full-scale chromatography. The relationship between the predicted yield and the actual yield can be regressed to improve the prediction model. Future predicted yields can be calculated based at least in part on the regression relationship (e.g., transfer function) applied to data from elution assays, high-throughput screening, or laboratory-scale HIC protocols.

[0141] One or more transfer functions, boundary functions, and / or prediction models can be combined to establish a dynamic prediction model. Refer to Figure 22, a dynamic prediction model is shown in the figure. The dynamic prediction model calculates how changes to one or more HIC protocol parameters (e.g., pH, citrate concentration, load mass, chromatography resin) affect the quantitative properties of the HIC protocol. For example, in Figure 22 in the illustrated embodiment, protocol parameters are then selected that include: a pH value of 6, a citrate concentration of 30 mM, a load mass of 100 g / liter of HIC medium, and resin C (e.g., Phenyl medium (phenyl agarose gel medium)). The model then shows how each of these parameters affects the predicted yield, high molecular weight fraction, and host cell protein concentration of the eluate collected using the HIC protocol with the selected parameters. As shown in Figure 22 in the figure, the selected parameters result in a predicted yield of approximately 95%, a high molecular weight fraction of approximately 1.3, and a pool host cell protein concentration of approximately 22.5 ppm. These quantitative properties can be used to evaluate the HIC chromatography protocol and can inform how these protocol parameters affect the product of the HIC protocol. When the protocol parameters are changed, the values of the quantitative properties calculated by the dynamic model can be updated in real time.

[0142] In some embodiments, an overall normalized value can also be calculated as a composite of the quantitative properties of the protocol (e.g., a composite of predicted yield, high molecular weight fraction, and pool host cell protein concentration). The normalization value calculation can be based on weighting of the quantitative properties of the HIC protocol. For example, a change to the HIC protocol that affects the yield of the protocol will be more important than a change that affects efficiency. The dynamic prediction model can take into account the relative importance of the quantitative measurements and assign weights to different measurements so that they do not equally affect the overall normalized value. In some embodiments, the dynamic prediction model can be updated when additional chromatography data (e.g., data from small scale trials, high throughput screening, and full scale chromatography runs) are collected and aggregated.

[0143] Example 21

[0144] In gradient elution experiments, multiple HIC protocols including different target molecule and HIC medium combinations were tested at various pH values to determine whether these combinations would cause regeneration / availability issues and whether some target molecule and HIC medium combinations should be excluded in further HIC protocol development.

[0145] A 96-well filter plate (AcroPrep TMThe wells of an Advance 1 mL filter plate, 1.2 μm Supor membrane, part number #8130, Pall Corporation) were filled with 0.02 μL of various HIC media. Aliquots of the sample material containing the target molecule were prepared and adjusted to a concentration of 0.33 g / L, with a concentration of 5 g / L as the target when mixed with the HIC media. For each of the three target antibodies, aliquots of the sample material were titrated to several different pHs (e.g., 4.5, 6.25, or 8.0) using 2 M acetic acid or 2 M Tris base to prepare several aliquots, each aliquot containing the target antibody and showing a different pH.

[0146] Each well of a 96-well plate filled with a single media type was subjected to an HIC protocol at different pHs (4.5, 6.25, or 8.0) to form an array protocol performed with various combinations of media and pH.

[0147] The following steps were performed simultaneously on the array: 1. Equilibrate the wells filled with various HIC resins three times with the equilibration buffer of their respective pH. 2. Add the sample material containing the desired target molecule to each well at its respective pH and incubate for up to one hour. 3. Spin the plate at 1100 rpm. 4. Collect the flow-through (containing components that did not bind to the media during incubation). 5. Wash the wells three times with the equilibration buffer of the pH corresponding to each test protocol. 6. Perform a pseudo-elution. Expose each well to the elution buffer for seven iterations, where after each iteration, spin the plate at 1100 rpm and collect the eluate. 7. Wash the wells twice with NaOH. Different wells of the array can use different concentrations of NaOH. For example, two wells can contain the same chromatographic media and be loaded at the same pH value, but one well is washed with 5 mM NaOH while the other well is washed with 1 N NaOH solution. After each wash, spin the plate at 1100 rpm and collect the wash solution. 8. Strip the wells twice with a solution of 6 N guanidine hydrochloride and collect the stripped material.

[0148] For each well, a chromatogram of the target molecule in the material removed (flowed, eluted, washed, etc.) from the well is generated. For the analysis of the results, each chromatogram is subdivided into three regions, where the first region contains the mass of the target molecule observed in the material removed during steps 2, 3, and 4 (flow-through, wash, and elution), the second region contains the mass of the target molecule observed in the material removed during steps 5, 6, 7 (using RODI and NaOH), and the third region contains the mass of the target molecule observed in the material removed during step 8 (using 6N guanidine hydrochloride).

[0149] Examples of chromatograms generated from the elution experiments for the first target molecule are shown in Figures 23A - 26C . Figures 23A - 23C Shows the chromatogram of the elution experiment containing the first target molecule on Capto TM Phenyl (High Sub) medium, Figures 24A - 24C Shows the chromatogram of the elution experiment containing the first target molecule on Capto TM Butyl medium, Figures 25A - 24C Shows the chromatogram of the elution experiment containing the first target molecule on POROS TM Benzyl medium, Figures 26A - 26C Shows the chromatogram of the elution experiment containing the first target molecule on Phenyl medium. Figure 23A , Figure 24A , Figure 25A and Figure 26A Show the chromatograms of the elution experiment runs at a pH of 4.5, Figure 23B , Figure 24B , Figure 25B and Figure 26B Show the chromatograms of the elution experiment runs at a pH value of 6.25, and Figure 23C , Figure 24C , Figure 25C and Figure 26C Show the chromatograms of the elution experiment runs at a pH value of 8. In Figures 23A - 26C , the dashed line represents the chromatogram from the elution experiment containing 5 mM NaOH wash solution, and the solid line represents the chromatogram from the elution experiment containing 1N NaOH wash solution.

[0150] Examples of chromatograms generated from the elution experiments for the second target molecule are shown in Figures 27A - 28D . Figures 27A - 2 Each of the 8 figures shows three chromatograms, each from an elution experiment run at a different pH. Figure 27A Shows the chromatogram from the elution experiment run on Capto TM Phenyl (High Sub) medium,Figure 27B shows a chromatogram from an elution test run on Capto TM butyl media, Figure 27C shows a chromatogram from an elution test run on TOYOPEARL TM phenyl-650C media, Figure 27D shows a chromatogram from an elution test run on POROS TM ethyl media, Figure 28A shows a chromatogram from an elution test run on TOYOPEARL TM hexyl-650C media, Figure 28B shows a chromatogram from an elution test run on TOYOPEARL TM butyl-650C media, Figure 28C shows a chromatogram from an elution test run on POROS TM benzyl media, Figure 28D shows a chromatogram from an elution test run on Phenyl media. In Figures 27A - 28D , the dashed line represents the chromatogram from an elution test containing 5 mM NaOH wash solution, and the solid line represents the chromatogram from an elution test containing 1 N NaOH wash solution.

[0151] As can be seen from the chromatograms, all test scenarios containing 1 N NaOH stripping solution have a greater percentage of the target molecular mass compared to similar scenarios containing 5 mM NaOH stripping solution. That is, more of the target molecules remain bound to the column after 1 N NaOH stripping compared to 5 mM NaOH stripping.

[0152] By comparing the chromatograms of different HIC scenarios for a given target molecule, certain HIC media and / or pH can be excluded from consideration. Using a series of elution tests to remove unwanted target molecules, HIC media, and pH combinations from consideration can improve the speed of establishing and testing HIC scenarios. For example, a chromatogram showing that more than 5% (e.g., more than 10%) of the target molecules are eluted in region 3 can indicate that a given combination of HIC media, pH value, and target molecule is not suitable for incorporation into an HIC scenario.

[0153] In addition to using elution experiments to exclude HIC media, pH, and target molecules from consideration, a transfer function can be used to transform the mass of zone 3 calculated from the chromatogram to predict the mass of the target molecule eluted in zone 3 during full-scale chromatography runs. This predicted mass can also be used to exclude combinations of HIC media, pH, and target molecules from consideration. An exemplary list of zone 3 target molecule recoveries calculated for a given target molecule in a series of HIC protocols using various combinations of HIC media and pH is shown in Table 14. Table 14

[0154] As can be seen in Table 14, an elution experiment of a given target molecule on Capto TM Phenyl (High Sub) media at a pH of 4.5 gave a normalized percent recovery of 38.2%. This result, when transformed through the transfer function, gave a predicted full-scale zone 3 recovery of 10.6%. In an embodiment where the predetermined threshold is 10%, this would be greater than the threshold, and thus the combination of the target molecule, Capto TM Phenyl (High Sub) media, and a pH of 4.5 would be excluded from further HIC protocol development. In some embodiments, if the exclusion of a media and pH combination for a given target molecule is necessary, the media and pH combination can also be excluded from further protocol development for other target molecules.

[0155] Those skilled in the art will understand that the concepts upon which this disclosure is based can readily be used as a basis for designing other methods and systems for carrying out several purposes of this disclosure. Accordingly, the claims are not to be regarded as limited by the foregoing description.

Claims

1. A method for regenerating a chromatographic column that has been applied with a sample substance, the method comprising: passing an alkaline solution through the medium in the chromatographic column, wherein the alkaline solution contains sodium hydroxide with a total dissolved concentration between about 0.5 mM and about 50 mM, wherein the substances bound to the medium are removed.

2. The method according to claim 1, wherein the medium comprises a matrix containing ligands, and the ligands have 2 to 10 hydrocarbons in an aliphatic or aromatic configuration.

3. The method according to claim 2, wherein the ligands are present in the medium at a density of about 20 to about 30 μmol / ml of the medium.

4. The method according to claim 1, wherein the medium comprises a matrix containing cross-linked agarose and phenyl ligands.

5. A method for preparing a chromatographic column for storage, comprising: performing the method according to claim 1; and contacting the chromatographic column with a storage buffer containing sodium hydroxide with a total dissolved concentration between about 0.05 M and about 0.15 M.

6. A method for reusing a chromatographic column, the method comprising: adding a first sample substance to the chromatographic column; performing the method according to claim 1 on the chromatographic column; and adding a second sample substance to the chromatographic column, wherein the method does not include cleaning the chromatographic column.

7. The method according to claim 1, wherein the sample substance comprises dupilumab.

8. The method according to claim 1, wherein the sample substance comprises an anti-interleukin-4 receptor antibody.

9. The method according to claim 1, wherein the substances bound to the medium include a target molecule, and the target molecule is dupilumab.

10. The method according to claim 1, wherein the substances bound to the medium include a target molecule, and the target molecule is an anti-interleukin-4 receptor antibody.

11. A method for determining the concentration of an alkaline solution for a hydrophobic interaction chromatographic column regeneration solution, the method comprising: passing a certain volume of a first solution through a hydrophobic interaction medium in a hydrophobic interaction chromatographic column, wherein the first solution contains water and an alkaline solution concentration that starts from about 0 N and increases at a substantially constant rate to a maximum concentration; passing a certain volume of a second solution through the hydrophobic interaction medium, wherein the second solution contains water and an alkaline solution concentration that starts from the maximum concentration and decreases at a substantially constant rate to about 0 N; and determining a portion of the first solution or the second solution that, when passed through the hydrophobic interaction medium, removes the substances bound to the hydrophobic interaction medium.

12. The method according to claim 11, wherein the alkaline solution contains sodium hydroxide, and the maximum concentration is about 1 N.

13. The method according to claim 11, wherein the volume of the first solution and the volume of the second solution are each about 20 column volumes.

14. The method according to claim 11, further comprising adding a sample substance to the hydrophobic interaction chromatography column before passing the certain volume of the first solution through the hydrophobic interaction medium in the hydrophobic interaction chromatography column, wherein the sample substance comprises dupilumab.

15. The method according to claim 11, further comprising adding a sample substance to the hydrophobic interaction chromatography column before passing the certain volume of the first solution through the hydrophobic interaction medium in the hydrophobic interaction chromatography column, wherein the sample substance comprises an anti-interleukin-4 receptor antibody.

16. The method according to claim 11, wherein the substance bound to the hydrophobic interaction medium comprises a target molecule, and the target molecule is dupilumab.

17. The method according to claim 11, wherein the substance bound to the hydrophobic interaction medium comprises a target molecule, and the target molecule is an anti-interleukin-4 receptor antibody.

18. A method for evaluating a plurality of chromatography schemes, each chromatography scheme comprising a chromatography medium, a scheme pH value, and a target molecule, the method comprising: For each chromatography scheme: In a filtration plate well, adding a sample substance containing the target molecule to a certain volume of the chromatography medium and collecting the effluent from the filtration plate well, wherein the sample substance has the pH value of the scheme; Adding a plurality of buffer aliquots to the chromatography medium to obtain an eluate from the chromatography medium, wherein the buffer has a buffer pH value and a concentration of a lyotropic salt, the concentration of the lyotropic salt linearly decreasing in the plurality of aliquots, and wherein a first amount of the target molecule is contained in the combined effluent and eluate; Adding a second solution to the chromatography medium to extract a second amount of the target molecule from the chromatography medium; And Adding a chaotropic agent to the chromatography medium to extract a third amount of the target molecule from the chromatography medium.

19. The method according to claim 18, further comprising: Preparing a chromatography unit operation using one of the chromatography schemes, wherein the chromatography unit operation comprises hydrophobic interaction chromatography.

20. The method according to claim 18, comprising performing the method on a plurality of chromatography schemes simultaneously using a filtration plate.

21. The method according to claim 18, wherein the sample substance comprises less than about 20 mg of the target molecule.

22. The method according to claim 18, wherein the target molecule is dupilumab.

23. The method according to claim 18, wherein the target molecule is an anti-interleukin-4 receptor antibody.

24. A method for regenerating a hydrophobic interaction chromatography column, the method comprising: Adding a sample substance to the hydrophobic interaction chromatography column; And Passing an alkaline solution through the hydrophobic interaction chromatography column, wherein less than about 5% of the sample substance remains bound to the hydrophobic interaction chromatography column after the alkaline solution passes through the hydrophobic interaction chromatography column.

25. The method according to claim 24, wherein the pH value of the alkaline solution is from about 10 to about 14.

26. The method according to claim 24, wherein the total dissolved salt concentration of the alkaline solution is from about 0.1 mM to about 50 mM.

27. The method according to claim 24, passing the alkaline solution through the hydrophobic interaction chromatography column includes passing from about 1 column volume to about 20 column volumes of the alkaline solution through the hydrophobic interaction chromatography column.

28. The method according to claim 24, wherein the conductivity of the alkaline solution is from about 0.5 mS / cm to about 10 mS / cm.

29. The method according to claim 24, after the alkaline solution passes through the hydrophobic interaction chromatography column, from about 0.0% to about 1.0% of the injected substance remains bound to the hydrophobic interaction chromatography column.

30. The method according to claim 24, after adding the injected substance and before passing the alkaline solution, the target molecule remains bound to the hydrophobic interaction chromatography column.

31. The method according to claim 30, after adding the injected substance and before passing the alkaline solution, further includes introducing an elution buffer into the hydrophobic interaction chromatography column.

32. The method according to claim 24, further includes introducing a storage buffer into the hydrophobic interaction chromatography column, wherein the storage buffer contains from about 0.05 M to about 0.15 M of sodium hydroxide.

33. The method according to claim 24, wherein the injected substance includes dupilumab.

34. The method according to claim 24, wherein the injected substance includes an anti - interleukin - 4 receptor antibody.

35. The method according to claim 24, the less than about 5% of the injected substance that remains bound to the hydrophobic interaction chromatography column includes the target molecule, and the target molecule is dupilumab.

36. The method according to claim 24, the less than about 5% of the injected substance that remains bound to the hydrophobic interaction chromatography column includes the target molecule, and the target molecule is an anti - interleukin - 4 receptor antibody.

37. A method for regenerating a chromatography column, the method includes: passing an alkaline solution through the chromatography column, wherein the conductivity of the alkaline solution is about 0.5 mS / cm to about 10 mS / cm; and after passing the alkaline solution through the chromatography column, introducing a storage buffer into the chromatography column, wherein the storage buffer contains from about 0.05 M to about 0.15 M of sodium hydroxide.

38. The method according to claim 37, wherein the total dissolved salt concentration of the alkaline solution is from about 0.1 mM to about 10 mM.

39. The method according to claim 37, before passing the alkaline solution through the chromatography column, further includes adding an injected substance to the chromatography column, wherein the injected substance includes dupilumab.

40. The method according to claim 37, before passing the alkaline solution through the chromatography column, further includes adding an injected substance to the chromatography column, wherein the injected substance includes an anti - interleukin - 4 receptor antibody.

41. The method according to claim 37, further comprising introducing a sample substance into the chromatographic column before passing the alkaline solution through the chromatographic column; and wherein from about 0% to about 3% of the sample substance is bound to the chromatographic column after passing the alkaline solution through the chromatographic column and before introducing a storage buffer into the chromatographic column.

42. The method according to claim 41, wherein the sample substance bound to the chromatographic column comprises a target molecule, and the target molecule is dupilumab.

43. The method according to claim 41, wherein the sample substance bound to the chromatographic column comprises a target molecule, and the target molecule is an anti-interleukin-4 receptor antibody.

44. The method according to claim 37, wherein the chromatographic column comprises a chromatographic medium containing a ligand having 2 to 10 carbons, and wherein the ligand is aliphatic or aromatic.

45. The method according to claim 37, wherein the chromatographic column comprises a chromatographic medium containing a ligand having a density of about 20 μmol / ml to about 30 μmol / ml.

46. The method according to claim 37, wherein the chromatographic column comprises a chromatographic medium containing crosslinked agarose, polystyrene divinylbenzene, or polymethacrylate.

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