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 to which an injection material has been added, the method comprising passing one or more column volumes of a basic solution through a hydrophobic interaction medium inside the column, wherein the basic solution exhibits a pH between about 10 and about 14, and a conductivity between 0.5 mS / cm and about 10 mS / cm, wherein materials bound to the hydrophobic interaction medium are removed. In some cases, the basic solution may comprise sodium hydroxide at a concentration of, for example, between about 0.1 mM and 10 mM.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 905,033, filed on September 24, 2019, and U.S. Provisional Patent Application No. 62 / 958,899, filed on January 9, 2020, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to systems, methods, and solutions for the use and regeneration of chromatography media.Some aspects of the present disclosure relate to systems and methods including single-step hydrophobic interaction chromatography media regeneration solutions. Background Art

[0004] 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 a drug product (e.g., in separating, collecting, isolating, purifying, polishing, etc., molecules for use in a drug product). Some molecules of interest (e.g., polypeptides, polynucleotides, etc.) may need to be purified from materials such as the host cells in which they are produced. Separation or purification of molecules of interest using chromatography can reduce, remove, or isolate host cell proteins (e.g., lipases), host cell materials (e.g., cell debris), and other impurities that may co-purify with the molecule of interest.

[0005] Chromatography can include the use of a stationary phase comprising a medium for assisting in the separation of components of a mobile phase using a 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 molecules in the stationary phase and the hydrophobic surface of the HIC medium. The interaction between the molecule and the hydrophobic surface of the HIC medium can be affected by, for example, salts in the flow buffer. An input material with a high salt concentration can be loaded into the HIC device, wherein the high salt concentration promotes the interaction between the hydrophobic interaction chromatography (HIC) medium and the molecules in the mixture inside the device. Subsequently, a solution with a reduced ionic strength (e.g., a buffer) can be flowed through the HIC device to reverse the hydrophobic interactions between the HIC medium and the molecules. The molecules with the lowest hydrophobicity can be eluted first and the molecules with the greatest hydrophobicity can be eluted last, which requires a larger reduction in salt concentration to reverse their hydrophobic interactions with the HIC medium.

[0006] In some cases, the HIC media can be reused for multiple chromatography cycles. In order to maintain the performance, quality, and cleanliness of the HIC media, prevent contamination between cycles, increase the useful life of the HIC media, prevent buildup of impurities, and / or meet or exceed operating standards (e.g., operating standards within a laboratory or institution or operating standards issued by a regulatory agency), methods for regenerating the HIC media can be used to remove residual materials from the HIC media after a HIC cycle has been run. Summary of the invention

[0007] Aspects of the present disclosure relate to regenerating a chromatographic column. In one aspect, the present disclosure relates to a method of regenerating a hydrophobic interaction chromatographic column to which an injection material has been added. The method may include passing one or more column volumes of an alkaline solution through a hydrophobic interaction medium inside the chromatographic column, wherein the alkaline solution exhibits a pH between about 10 and about 14 and a conductivity between 0.5 mS / cm and about 10 mS / cm, wherein substances bound to the hydrophobic interaction medium are removed. The alkaline solution may comprise one of sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or tris (Tris); the alkaline solution may exhibit a conductivity between about 0.8 mS / cm and about 1.6 mS / cm, and / or the alkaline solution may include a total dissolved salt concentration between about 0.1 mM and about 10 mM.

[0008] After removal of material bound to the hydrophobic interaction medium, less than 1.0% of the injected material remains bound to the hydrophobic interaction medium as residue. The material removed from the medium may include: host cell proteins, recommended proteins, lipids, polypeptide fragments, biomolecules, or nucleic acids. In some embodiments, the material 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 an alkaline solution through the hydrophobic interaction medium inside the chromatographic column takes between about 10 minutes and about 1 hour.

[0009] In another aspect, the present disclosure is directed to a method for regenerating a chromatographic column to which an injection substance has been added; the method comprises passing one or more column volumes of an alkaline solution through a medium inside the chromatographic column, wherein the alkaline solution comprises sodium hydroxide at a total dissolved concentration between about 0.5 mM and about 50 mM, wherein substances bound to the medium are removed. The medium may comprise a matrix comprising ligands of between 2 and 10 hydrocarbons in an aliphatic or aromatic configuration. These ligands may be present in the medium at a density between about 20 and about 30 μmol / ml of medium. In other embodiments, the medium does not have ligands comprising 30 or more hydrocarbons; the chromatographic column is not used in a mixed mode chromatography process; and / or the medium may comprise a matrix comprising 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 also comprise 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, wherein the chaotropic agent is one of 6N guanidine hydrochloride or 8N urea. The method may further include contacting the chromatography column with a stock buffer having a total dissolved sodium hydroxide concentration of between about 0.05 M and about 0.15 M. The method may also include adding a first feed material to the chromatography column, and adding a second feed material to the chromatography column, wherein the method does not include washing the chromatography column.

[0010] In another aspect, the present disclosure relates to a method for determining the concentration of a basic solution for a hydrophobic interaction chromatography column regeneration solution; the method includes passing a volume of a first solution through a hydrophobic interaction medium inside a chromatography column, wherein the first solution has water and a basic solution concentration that increases at a substantially constant rate from about 0N to a maximum concentration; passing a volume of a second solution through the hydrophobic interaction medium, wherein the second solution has water and a basic solution concentration that decreases at a substantially constant rate from the maximum concentration to about 0N; and determining a portion of the first or second solution that removes substances bound to the hydrophobic interaction medium when passing through the hydrophobic interaction medium. The basic solution may include sodium hydroxide and the maximum concentration may be about 1N. In other embodiments, the volume of the first solution and the volume of the second solution are each about 20 column volumes.

[0011] In another aspect, the disclosure includes predicting, evaluating or comparing the availability and regeneration of various chromatography resins. In some embodiments, a method for evaluating a chromatography protocol includes: adding an input material containing a target molecule to a volume of a chromatography medium in a filter plate well and collecting a flow-through from the filter plate well, wherein the input material is at a protocol pH; adding multiple aliquots of a buffer to the chromatography medium to obtain an eluent from the chromatography medium, wherein the buffer is at a buffered pH and the concentration of a kosmotropic salt decreases linearly in the multiple aliquots, and wherein a first amount of the target molecule is contained in the combined flow-through and the eluent; and 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate various exemplary embodiments and, together with the description, are used to explain the principles of the disclosed embodiments. Any features (e.g., composition, formulation, method, etc.) of the embodiments or embodiments described herein may be combined with any other embodiments or embodiments, and all such combinations are included by the present disclosure. In addition, the described systems and methods are neither limited to any single aspect nor to their embodiments, nor to any combination or arrangement of the above aspects and embodiments. For the sake of brevity, certain arrangements and combinations are not discussed and / or described separately herein.

[0013] Figure 1 Exemplary methods according to aspects of the present disclosure are depicted in flowchart form.

[0014] Figure 2 Depicted are blot analyses of chromatography columns in various states of use according to aspects of the present disclosure.

[0015] Figure 3 Depicted are blot analyses of chromatography columns in various states of use according to aspects of the present disclosure.

[0016] Figure 4 Depicted is an overlay of chromatographic data from multiple processes according to aspects of the present disclosure.

[0017] Figure 5 Depicted are blot analyses of chromatography columns in various states of use according to aspects of the present disclosure.

[0018] Figure 6 Depicted is a comparison of a column containing a hydrophobic interaction medium that has undergone multiple cycles of hydrophobic interaction chromatography in accordance with aspects of the present disclosure and a column containing unused hydrophobic interaction medium.

[0019] Fig. 7A and Figure 7B Depicted are chromatograms of regeneration processes following hydrophobic interaction chromatography, including the use of reverse osmosis deionized water, according to aspects of the present disclosure.

[0020] Fig. 8A and Figure 8B Additional chromatograms depicting the regeneration process after hydrophobic interaction chromatography.

[0021] Fig. 9 Depicted is a chromatogram of a regeneration process using a guanidine hydrochloride solution according to aspects of the present disclosure.

[0022] Fig. 10A A chromatogram depicting a process including multiple regeneration solutions according to aspects of the present disclosure. Fig. 10B Depicted Fig. 10A A magnified image of a portion of the chromatogram.

[0023] Fig.11 Depicted is a chromatogram of a process in which sodium hydroxide solutions having increasing / decreasing concentrations have been introduced into a chromatography column in accordance with aspects of the present disclosure.

[0024] Fig.12 Depicted are overlaid chromatograms of multiple two-solution column regeneration processes according to aspects of the present disclosure.

[0025] Fig.13A and Fig. 13B is a visual depiction of a statistical analysis of various peaks of a chromatogram depicting a regeneration process in accordance with aspects of the present disclosure.

[0026] Fig.14 Depicted are overlaid chromatograms of various two-solution column regeneration processes comprising sodium hydroxide or sodium chloride and guanidine hydrochloride according to aspects of the present disclosure.

[0027] Fig.15 Depicted are three chromatography columns into which solutions have been added according to aspects of the present disclosure.

[0028] Fig.16 A graph depicting guanidine hydrochloride stripping solution peak area as a function of sodium hydroxide concentration according to aspects of the present disclosure.

[0029] Fig.17 and Fig.18 Depicted are chromatograms of a control regeneration process and an experimental regeneration process, each comprising a plurality of regeneration solutions, according to aspects of the present disclosure.

[0030] Fig.19Depicted are a series of chromatograms of hydrophobic interaction chromatography runs for purification of different monoclonal antibodies according to aspects of the present disclosure.

[0031] Figure 20A-20C Depicted are chromatograms generated using a protocol comprising three different hydrophobic interaction chromatography media in accordance with aspects of the present disclosure.

[0032] Fig.21A and 21B Depicted are boundary function graphs formed from analysis of data generated during high throughput screening and full scale chromatography runs in accordance with aspects of the present disclosure.

[0033] Fig. 22 An exemplary dynamic prediction model according to aspects of the present disclosure is depicted.

[0034] Figure 23A-Figure 26C Depicted are chromatograms of a first target molecule generated using a variety of hydrophobic interaction chromatography media, pH parameters, and stripping solutions according to aspects of the present disclosure.

[0035] Figures 27A-28D Depicted are chromatograms of a second target molecule generated using a variety of hydrophobic interaction chromatography media, pH parameters, and stripping solutions according to aspects of the present disclosure. DETAILED DESCRIPTION

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present 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 the present disclosure, specific methods are now described. All publications mentioned are incorporated herein by reference.

[0037] As used herein, the words "include," "comprising," or any variation thereof are intended to cover a non-exclusive inclusion, whereby a process, method, article, or apparatus that includes a list of elements includes not only those elements, but also other elements not expressly listed or unique to such method, method, article, or apparatus. The word "exemplary" is used in an "exemplary" rather than an "ideal" sense. With respect to the words "for example" and "such as," and their grammatical equivalents, they should be read in accordance with the phrase "and, but not limited to," unless expressly stated otherwise.

[0038] As used herein, the word "about" is used to account for variations due to experimental error. When applied to numerical values, the word "about" can mean 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 indicates otherwise. In addition, all ranges are understood to include endpoints, for example, from 1 centimeter (cm) to 5 cm will include the length of 1 cm, 5 cm, and all distances between 1 cm and 5 cm.

[0039] It should be noted that all numerical values ​​disclosed herein (including all disclosed values, limits, and ranges) may have a + / - 5% variation relative to the disclosed numerical value unless a different variation is specified.

[0040] As used herein, the term "polypeptide" refers to a polymer of amino acids having more than about 20 covalently linked via amide bonds. Proteins contain one or more amino acid polymer chains (e.g., polypeptides). Thus, a polypeptide can be a protein, and a protein can contain multiple polypeptides to form a single functional biomolecule.

[0041] 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 be formed post-translationally. Some disulfide bridges are essential for the proper structure, function, and interaction of polypeptides, immunoglobulins, proteins, cofactors, substrates, and the like. In addition to disulfide bond formation, proteins can undergo other post-translational modifications, such as lipidation (e.g., myristoylation, palmitoylation, farnesoylation, geranylgeranylation, and glycosylphosphatidylinositol (GPI) anchor formation), alkylation (e.g., methylation), acylation, amidation, glycosylation (e.g., addition of sugar groups at arginine, asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine, and / or tryptophan), and phosphorylation (i.e., addition of phosphate groups to serine, threonine, tyrosine, and / or histidine). Post-translational modifications can affect hydrophobicity, electrostatic surface properties, or other properties that determine surface-surface interactions participated in by the polypeptide.

[0042] The term "protein" as used herein includes biotherapeutic proteins, recombinant proteins for research or treatment, translocon-related 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. Proteins of interest (POI) may include any polypeptide or protein that is desired to be isolated, purified, or prepared. POIs may include polypeptides produced by cells, including antibodies.

[0043] The term "antibody" as used herein includes immunoglobulins consisting of four polypeptide chains: two heavy (H) chains and two light (L) chains interconnected via disulfide bonds. Typically, antibodies have 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 (the heavy chain CDR can be abbreviated as HCDR1, HCDR2 and HCDR3; the light chain CDR can be abbreviated as LCDR1, LCDR2 and LCDR3.

[0044] For example, a class of immunoglobulins known as immunoglobulin G (IgG) is common in human serum and includes four polypeptide chains: two light chains and two heavy chains. Each light chain is connected 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 of which differs in their constant regions and therefore 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 includes IgG4.

[0045] The term "antibody" as used herein also includes antigen-binding fragments of whole antibody molecules. As used herein, the term "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc. include any naturally occurring, enzymatically obtained, synthesized, or genetically engineered polypeptides or glycoproteins that specifically bind to an antigen to form a complex. For example, the antigen-binding fragment of an antibody can be obtained from a whole antibody molecule using any suitable standard techniques (such as protease digestion or recombinant genetic engineering techniques) involving the manipulation and expression of DNA encoding variable and optionally constant domains of an antibody. Such DNA is known and / or can be easily obtained from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. DNA can be sorted and manipulated by chemical methods or by using molecular biological techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, form cysteine ​​residues, modify, add or knock out amino acids, etc.

[0046] The target molecule (such as target polypeptide / antibody) can be produced using a recombinant cell-based production system, such as an insect baculovirus system, a yeast system (e.g., Pichia pastoris), or a mammalian system (e.g., CHO cells and CHO derivatives-like CHO-K1 cells). The term "cell" includes any cell suitable for expressing a recombinant nucleic acid sequence. Cells include prokaryotic and eukaryotic cells (single 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 hybridomas). In some embodiments, the cell can be a cell of a human, monkey, ape, hamster, rat, or mouse. In some embodiments, the cell can be a eukaryotic cell and can be selected from the following cells: CHO (e.g., CHO K1, DXB-11 CHO, 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 hepatoma cells), HL-60 (human promyelocytic acute leukemia cells, such as 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 aforementioned cells. In some embodiments, the cells may contain one or more viral genes, such as retinal cells expressing viral genes (e.g., PER.C6 TM cell).

[0047] The term "target molecule" may be used herein to refer to target polypeptides (e.g., antibodies, antibody fragments, or other proteins or protein fragments), or to refer to other molecules that are used to be produced, isolated, purified, and / or included in a pharmaceutical product (e.g., adeno-associated virus (AAV) or other molecules for therapeutic use). Although the methods according to the present disclosure may involve target polypeptides, they are applicable to other target molecules. AAVs can be prepared, for example, according to suitable methods (e.g., depth filtration, affinity chromatography, etc.), and mixtures containing AAVs can be subjected to methods according to the present disclosure. Before or after using one or more methods of the present disclosure, the mixture containing AAVs can be subjected to additional processes (e.g., removal of "empty cassettes" or AAVs that do not contain target sequences).

[0048] 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 two-chain antibody, a three-chain antibody or a four-chain antibody, a Fab fragment or a 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.

[0049] In some embodiments, the target molecule (e.g., 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-D114 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. Pat. No. 9,018,356), anti-platelet derived growth factor receptor antibody (e.g., anti-PDGFR antibody, as described in U.S. Pat. No. 9,265,827), anti-prolactin receptor antibody (e.g., anti-PRLR antibody, as described in U.S. Pat. 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. Pat. No. 9,132 , as described in U.S. Patent No. 192, or anti-EGFRvIII antibodies, as described in U.S. Patent Application Publication No. US2015 / 0259423A1), anti-protein convertase subtilisin-9 antibodies (e.g., anti-PCSK9 antibodies, as described in U.S. Patent No. 8,062,640 or described in U.S. Patent Application Publication No. US2014 / 0044730A1), anti-growth factor and differentiation factor-8 antibodies (e.g., anti-GDF8 antibodies, also known as anti-myostatin antibodies, as described in U.S. Patent Nos. 8,871,209 or 9,260,515), anti-glucagon , interleukin 4 receptor antibodies (e.g., anti-GCGR antibodies, as described in U.S. Patent Application Publication No. US2015 / 0337045A1 or US2016 / 0075778A1), anti-VEGF antibodies, anti-IL1R antibodies, interleukin 4 receptor antibodies (e.g., anti-IL4R antibodies, 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 antibodies (e.g., anti-IL6R antibodies, as described in U.S. Patent Nos. 7,582,298, 8,043,617 or 9,173,880), anti-interleukin 33 (e.g., anti-IL33 antibodies, as described in U.S. Patent Application Publication No. US2014 / 0271658A1 or US2014 / 0271642A1), anti-respiratory syncytial virus antibodies (e.g., anti-RSV antibodies, as described in U.S. Patent Application Publication No. US2014 / 0271653A1), anti-cluster of differentiation 3 (e.g., anti-CD3 antibodies, as described in U.S. Patent Application Publication No. US2014 / 0088295A1 and US20150266966A1, and described in US Patent Application No. 62 / 222,605), anti-differentiation cluster 20 (e.g., anti-CD20 antibodies, as described in US Patent Application Publications US2014 / 0088295A1 and US20150266966A1 and in US Patent No. 7,879,984), anti-differentiation cluster-48 (e.g., anti-CD48 antibodies, as described in US Patent No. 9,228,014), anti-Fel d1 antibody (e.g., as described in U.S. Pat. No. 9,079,948), anti-Middle East respiratory syndrome virus (e.g., anti-MERS antibody), anti-Ebola virus antibody (e.g., REGN-EB3 of 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. ,

[0050] In some embodiments, the target molecule (e.g., bispecific antibody) is selected from the group consisting of: anti-CD3 x anti-CD20 bispecific antibody, anti-CD3 x 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, sarrelumab, fasinumab, nevacuzumab, dupilumab, trogeluzumab, evuximab, and linusumab.

[0051] In some embodiments, the target molecule is a recombinant protein (e.g., Fc-fusion protein) containing an Fc portion and another domain. In some embodiments, the Fc-fusion protein is a receptor Fc-fusion protein containing 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., rilanacept, which contains an IL-1RAcP ligand binding region fused to the extracellular region of Il-1R1, which is fused to the Fc of hIgG1; see U.S. Pat. No. 6,927.004, the entire contents of which are incorporated herein by reference), or a VEGF trap (e.g., aflibercept or ziv-aflibercept, which contains an I fused to the VEGF receptor Flk1). g domain 3 of the VEGF receptor Flt1 fused to the Fc of hIgG1; see U.S. Pat. Nos. 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 containing one or more of the one or more antigen-binding domains of an antibody coupled to the Fc portion (e.g., a variable heavy chain fragment and a variable light chain fragment).

[0052] Embodiments of the present disclosure may be used in the preparation of various pharmaceutical products, or in establishing methods for purifying various pharmaceutical products. In some embodiments, the present disclosure may be used in the preparation or purification of pharmaceutical products comprising antigen-binding molecules or AAV. In some aspects, embodiments of the present disclosure may be suitable for use in the preparation of a pharmaceutical product comprising ingredients (e.g., aflibercept, alirocumab, pegavicept, bevacizumab, brolucizumab, conbercept, dupilumab, eloumab, tocilizumab, certolizumab, abatacept, rituximab, infliximab, ranibizumab, sarelumab, adalimumab, anakinra, trastuzumab, filgrastim, interferon beta-1a, insulin glargine [rDNA source], alpha-erythropoietin, dabepol, 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.).

[0053] The term "hydrophobic interaction medium" or "HIC medium" refers to a combination of a support structure and a hydrophobic portion, wherein the hydrophobic portion 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 manner that can accommodate a liquid containing a protein of interest and contaminants. Therefore, the support structure includes agarose beads (e.g., agarose gel), silica gel beads, cellulose membranes, cellulose beads, hydrophilic polymer beads, resins, etc. The hydrophobic portion is bound to the hydrophobic surface of hydrophobic molecules and proteins. The degree of hydrophobicity of the medium can be controlled by selecting the hydrophobic portion. The process known as hydrophobic interaction chromatography (HIC) uses a hydrophobic interaction medium for separating target molecules (such as proteins of interest or other molecules) from product and process-related contaminants. When manufacturing and / or purifying target molecules in and / or from host cells, some product and process-related components that should ultimately be separated therefrom are referred to as host cell proteins (HCPs) and cell debris. In some cases, a mixture containing the target molecule and other components is added to the HIC medium in a buffer designed to promote the binding of hydrophobic groups in the target molecule to the hydrophobic portion of the HIC medium. This mixture may be referred to as the "load mass". HIC exploits the difference in hydrophobicity between the target molecule and impurities, which results in separation during the injection, washing, or regeneration phases. The target molecule is often separated into the flow-through while the impurities bind to the HIC medium. HIC may also be run in a mode in which the target molecule binds to the HIC medium while HCPs and cell debris fail to bind and flow through. The present disclosure is applicable to either case (whether or not the target molecule binds to the hydrophobic interaction moiety).

[0054] The HIC medium can be periodically stripped or regenerated after it is used to purify / collect target molecules. As used herein, the terms "stripping" and "regeneration" can be used interchangeably and / or in combination to refer to a process for removing any residual components of an input material from the HIC medium after a purification cycle and preparing the HIC medium for a subsequent purification cycle. For example, after the HIC device is used to separate or purify molecules of interest from an input material containing host cell material (e.g., host cell fragments, host cell proteins, etc.) and the molecules of interest are 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 to be used to purify the molecules of interest from another input material. In some embodiments, regeneration of the HIC medium can include disrupting hydrophobic interactions between residual host cell material and / or target molecules and the HIC medium, and / or denaturing residual host cell material. Regeneration can be performed between hydrophobic interaction chromatography cycles. In some embodiments, regeneration may be performed between HIC cycles to prevent or reduce discoloration of the HIC medium over time. In some embodiments, a regeneration process according to the present disclosure may be used, for example, between 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 powerful cleaning solutions, which may have adverse effects or cause additional problems. The regeneration process can be configured without particular concern for, for example, the removal of bacteria, fungi, or other microorganisms from the chromatographic medium.

[0055] "Stripping" and "regeneration" can be distinguished from, for example, "cleaning" the chromatographic medium. Cleaning can include a process for thoroughly disinfecting and / or purifying the chromatographic medium, chromatographic device, and / or laboratory environment. For example, the cleaning process can include the use of antibacterial, antifungal, or antimicrobial solutions, other disinfecting solutions, sterilization, etc., at a concentration and amount that disinfects and / or sterilizes the chromatographic medium or chromatographic device. On the contrary, although regeneration may include the use of a solution with antimicrobial properties in some cases, the main purpose of regeneration may be to remove residual components of the injected material from the chromatographic medium after the purification process. In some embodiments, additional protective measures or processes may be required during and / or after the cleaning process to ensure that subsequent chromatographic cycles are not affected by disinfection, purification, the antimicrobial or antibacterial solutions used during the cleaning process. In many cases, the cleaning process may be longer (e.g., greater than about 1 hour) than the regeneration process.

[0056] Separation of molecules in HIC media can be accomplished by, for example, exposing the HIC media to an input material having a high salt concentration to increase hydrophobic interactions between the HIC media and target molecules in the input material and then passing a volume of a solution (e.g., a buffer) having an increasing or decreasing salt concentration through the HIC media to reverse the hydrophobic interactions. Thus, it is generally understood that under low salt or no salt conditions, less material will bind to the HIC media. However, unexpectedly, it has been discovered that certain types of HIC media exhibit increased binding (e.g., hydrophobic interactions) to residues (e.g., host cell proteins) under no salt conditions. In addition, it has been discovered that some proteins (e.g., monoclonal antibodies) can be mutated to certain types of HIC media (e.g., Capto TM Phenyl (High Sub) media (GE Healthcare Life Sciences). Although proteins can be restored to their native configuration once eluted from HIC media, such proteins in their denatured state are not removed from HIC media by regeneration processes containing, for example, reverse osmosis deionized water (RODI), 1N sodium hydroxide, and / or 20% ethanol. It is speculated that, in some cases, the elution of molecules from HIC media depends on pH and / or conductivity.

[0057] Aspects of the present disclosure relate to a regeneration solution and the evaluation of the usability of chromatographic media, including the ease of regeneration.

[0058] In some embodiments of the present disclosure, the regeneration solution can be an alkaline solution. It is conceivable 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 conceivable that in some cases, the effectiveness of high pH can be offset by high ionic strength. Therefore, in some embodiments, the effectiveness of the regeneration solution can be driven by high pH values ​​together with low but non-zero conductivity (see, for example, Example 14 discussed below). For example, in some embodiments, the regeneration solution can display a pH value between about 8 and about 14, such as between about 10 and about 14. In some embodiments, the regeneration solution may generally display low conductivity. For example, in some embodiments, the regeneration solution may exhibit a conductivity of 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.

[0059] In some embodiments, the regeneration solution can be an alkaline solution containing a certain concentration of, for example, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, tris (Tris), other alkaline solutions, or combinations thereof. In some embodiments, the regeneration solution can include 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. The total dissolved salt concentration may be between 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.

[0060] In some embodiments, a 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 a HIC medium may include contacting the HIC medium with a single solution, wherein the solution exhibits one or more properties described herein, and wherein after contact with the single solution, less than about 5% of the feed material remains bound to the HIC medium as a residue. For example, in some embodiments, a method of regenerating a HIC medium may include contacting the HIC medium with a solution exhibiting a pH 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 feed 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 feed material.

[0061] The volume of regeneration solution used in accordance with the present disclosure can be any suitable volume. For example, in some embodiments where an feed material is added to a HIC medium in a chromatography column, the volume of regeneration solution used in accordance with the present disclosure can be measured in column volumes (CV). For example, in some embodiments, a method of regenerating a HIC medium in a chromatography column can include passing at least 1 column volume (CV) of regeneration solution through the chromatography column. In some embodiments, the method can include passing between about 1 and about 20 column volumes, e.g., 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 a regeneration solution through the chromatography column.

[0062] The systems and methods according to the present disclosure may be applicable to a variety of separation media and / or processes. A single system, method, or scheme 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 may be applicable to media and / or processes in which the components of the sampled material 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 regeneration solutions and / or methods disclosed herein may be combined with other schemes and / or methods (e.g., implemented before or after). For example, a regeneration solution of the present disclosure may be added to the medium used in mixed mode chromatography to strip off residues that interact with the medium due to hydrophobicity. Another regeneration solution may be added to the medium to strip off residues that are bound to the medium due to charge.

[0063] In some embodiments, the regeneration solutions and / or methods disclosed herein may be applicable to HIC media having a high hydrophobicity. In some embodiments, chromatographic media applicable to the solutions and methods according to the present disclosure may include, for example, a hydrophobic matrix including cross-linked agarose, polystyrene divinyl benzene, or polymethacrylate. In some embodiments, the matrix may include ligands having between 2 and 10 hydrocarbons in an aliphatic or aromatic configuration. In some embodiments, the matrix does not include ligands containing 30 or more hydrocarbons. In some embodiments, for example, the ligands may include phenyl ligands, butyl ligands, or octyl ligands. In some embodiments, the ligands may be present in the medium at a density between about 20 and about 30 μmol / ml of medium. In some embodiments, the methods and regeneration solutions described herein may be specifically used to regenerate HIC media. In some embodiments, the methods and regeneration solutions described herein may be suitable for, for example, Capto TM Phenyl(High Sub), Capto TM Butyl, or Capto TM Octyl mediator (GE Healthcare LifeSciences), phenyl Medium (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 use in 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, a single-step regeneration solution according to the present disclosure can be used in a multi-column continuous hydrophobic interaction chromatography system, where the efficiency of the single-step regeneration solution can improve the overall efficiency of the multi-column system. In some embodiments, the methods and regeneration solutions described herein can be used in HIC systems and methods that include low or no drug-tropic conditions.

[0064] In some embodiments, the regeneration process according to the present disclosure can be performed without using reverse osmosis deionized water (RODI), organic solvents (e.g., ethanol or ethylene glycol), chaotropes (e.g., guanidine or urea), sodium chloride, and / or sodium hydroxide concentrations exceeding 50 mM. Advantageously, the regeneration process using the solution disclosed herein does not require additional processes to handle, for example, solvents such as ethanol (e.g., 20% ethanol) and chaotropes (e.g., guanidine and urea, such as 6N guanidine or 6N urea). However, in some embodiments, it is contemplated that the regeneration solution disclosed herein can be used before, after, or together with an organic solvent (e.g., 20% alcohol) or a chaotrope (e.g., 6N guanidine or 6N urea).

[0065] In some embodiments, methods according to the present disclosure may include passing a regeneration solution disclosed herein through a chromatography column prior to contacting the chromatography column with a reserve buffer for storage purposes. The reserve buffer may contain, for example, sodium hydroxide or another salt at a concentration between about 0.05M and about 0.15M.

[0066] In some embodiments, the method according to the present disclosure may include evaluating various chromatographic media to determine whether one or more chromatographic media are likely to challenge the use or regeneration of the media during the purification of the target molecule. The evaluation of the chromatographic media according to the present disclosure may include, for example, but not limited to: the use of one or more chromatographic media types, maintaining pH conditions, and target molecules. Advantageously, the method according to the present disclosure may include screening at a scale smaller than that typically performed for the purification of target molecules, thereby allowing significant savings in sample volume (e.g., using about 1 / 10, 1 / 100, 1 / 500, 1 / 700 or less of the sample volume required for evaluating the chromatographic media, such as HIC, according to conventional methods). In addition, multiple purification schemes with different variables (e.g., different combinations of media types, pH values, and / or target molecules) can be screened simultaneously using, for example, a high throughput screening (HTS) process. Alternatively, or outside of high throughput screening (HTS), elution assays can be used to exclude parameters of potential HIC schemes.

[0067] Advantageously, these screening techniques and assays can provide significant time savings for determining purification schemes suitable for use in large-scale purification processes, such as by optimizing HIC unit operations. For example, the 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 / usability issues in chromatography schemes for one or more unit operations (including hydrophobic interaction chromatography, ion exchange, affinity chromatography, etc.).

[0068] Evaluation method according to the present disclosure may not include filling a hole (such as filter plate hole) with a certain amount of chromatographic medium, wherein the chromatographic medium is intended to be used for potential purification scheme. The filter plate hole may have, for example, less than 5mL, such as less than 4mL, less than 3mL or less than 2mL capacity. In some embodiments, the filter plate hole may have a capacity of about 1mL, about 0.8mL, about 0.5mL, or any other suitable capacity. The filter plate hole may be equipped with a filter with a suitable mesh size (such as between about 0.5 and about 1.5 microns, such as about 0.8 micron, about 1.0 micron or 1.2 microns). The filter mesh size may depend on the size of the resin beads present in the chromatographic medium used in the scheme. The size of this chromatographic resin bead, for example, may be 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 wells can vary. For example, the amount of chromatographic medium can be in the range of, for example, from 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 comprising a plurality of wells can be filled with a plurality of different chromatographic media for simultaneous evaluation of a plurality of schemes in an array. This can be used for any chromatographic step in a purification scheme, including hydrophobic interaction chromatography, ion exchange chromatography, affinity chromatography, etc.

[0069] A certain volume of the load material can be loaded into the filled filter plate wells (or, in the case of evaluating multiple schemes at the same time, into each filled filter plate well). The load material may 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 a unit operation (such as HIC). In the case of evaluating multiple conditions for a single unit operation at the same time, the load materials used for different holes may contain different target molecules. The load material can be titrated to a specific pH value for the scheme. The approximate concentration of the target molecule in 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 the full-scale HIC scheme will use (e.g., the mass of the target molecule in the load material), such as 1 / 2, 1 / 5, 1 / 8, 1 / 10, 1 / 20, 1 / 50, 1 / 100 or less of the load material used in the full-scale scheme.

[0070] The method for evaluating a protocol for a unit operation may also include performing an elution and wash step suitable for a large-scale process (e.g., including the use of reverse osmosis deionized water (RODI), 1N sodium hydroxide, and / or 5mM sodium hydroxide), and then performing a stripping step using an alkaline agent or a chaotropic agent (e.g., 6N guanidine hydrochloride or 6N urea) or a solvent (e.g., 20% ethanol). The elution / wash step may be performed using, for example, an elution buffer titrated to a protocol-specific pH value. In the case where multiple protocols for a unit operation are evaluated, elution buffers at different protocol-specific pH values ​​may be used in different wells in a single array. The elution step may include, for example, exposing the chromatographic medium loaded in the filter well to a gradient elution buffer (starting at a relatively high concentration and ending at a concentration of 0). In another embodiment, the elution step may include exposing the loaded chromatographic medium to a buffer having an initial concentration of a lyophilic salt (e.g., 500mM, 400mM, 300mM, 200mM, etc., of citrate), and stepwise / linearly reducing the buffer concentration to 0. In some embodiments, a pseudo-gradient elution can be performed, wherein the loaded (injected) chromatographic medium is exposed to discrete volumes of elution buffer having a linearly decreasing concentration from a starting concentration (e.g., 300 mM) to 0 over multiple steps (e.g., 4, 5, 6, 7, 8, more or fewer steps).

[0071] The first step may include any process to be tested as a unit operation scheme. Typically, the first step may include the addition of a solution that is considered suitable for use in large-scale and repeated operations (e.g., a solution that does not present safety or toxicity issues). For example, the first step may include one or more washes of RODI and / or 1NaOH, which are implemented in series or in an alternating order, each implemented once or multiple times. The second step may include any solution intended to strip any residues that are bound to the chromatographic medium after the unit operation being evaluated is completed. This regeneration process is described elsewhere in this article, but typically may include a chaotropic agent, such as 6N guanidine hydrochloride or 6N urea, or a solvent, such as 20% ethanol.

[0072] Evaluation methods according to the present disclosure may include measuring the amount of target molecule recovered during, for example, a wash / elution step and a stripping step. These results may be compared across various chromatographic media. It is desirable to recover a relatively large percentage of the target molecule during the wash / elution step (i.e., during the period during which the HIC protocol is being tested) as opposed to during the stripping step (i.e., strict stripping of the chromatographic media).

[0073] Evaluation methods according to the present disclosure may also include determining the recovery of the target molecule during the stripping step, which is the percentage of all target molecules recovered during the unit operation scheme test. If the percentage recovery of the target molecule during the stripping step exceeds a predetermined threshold, the unit operation scheme can be predicted to potentially cause regeneration / reusability problems when amplified and / or when repeated many times. If the percentage recovery of the target molecule during the stripping step is at or below a predetermined threshold, it can be predicted that the unit operation scheme does not cause regeneration / reusability problems when amplified and / or repeated. The predetermined threshold can be any threshold determined by experiment, which is an indication of the amount of residues that are still attached to the chromatographic medium in the unit operation (such as after the HIC scheme). In some embodiments, the predetermined threshold can be, for example, between about 1% and about 10%, such as between about 3% and about 7%, such as about 4%, about 5%, or about 6%.

[0074] In embodiments where multiple chromatographic schemes are evaluated simultaneously, the above calculations of percent recovery of the target molecule can be determined for multiple chromatographic schemes at once, resulting in an initial impression of which schemes may be suitable or preferred for further use, testing, research, or development. The schemes for further study may include those wells having a normalized percent recovery attributable to the stripping step of 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%.

[0075] In some embodiments, methods for evaluating chromatographic schemes according to the present disclosure (e.g., 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 a manner as described herein (e.g., high throughput screening and / or elution experiments), and HIC schemes predicted to cause regeneration / reusability problems can be eliminated or deprioritized for further study. Chromatographic schemes that are not predicted to cause regeneration / reusability problems 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 repeatability and longevity (e.g., whether they cause column discoloration or other adverse effects after 10, 25, 50, 75, 100 or more cycles).

[0076] In some embodiments, methods according to the present disclosure can advantageously prevent or reduce discoloration of regenerated columns, media, and / or equipment that may occur after one or more uses (see, e.g., Examples 6 and 9 discussed herein). In some embodiments, methods according to the present disclosure can advantageously help identify protocols for chromatography unit operations that will prevent regeneration / usability issues early on, thereby saving time and expense in developing a complete purification protocol only to discover that the protocol for a chromatography step later exhibits such problems.

[0077] Reference will now be made to the specific drawings. Figure 1 A method 100 of regenerating a chromatography column according to aspects of the present disclosure is depicted. According to step 102, a first feed material can be added to a HIC column. According to step 104, a protein of interest can be collected from the HIC column. According to step 106, a single alkaline regeneration solution can be added to the chromatography column to remove substances bound to the hydrophobic interaction medium in the chromatography column.

[0078] According to step 102, a first feed material may be added to the HIC column. The feed material may include a target molecule (e.g., a polypeptide) as well as residual components (e.g., host cell proteins, cell debris, etc.). According to step 104, the protein of interest may be collected from the hydrophobic interaction chromatography column. This may be done, for example, in a washing step or an elution step. According to step 106, a single alkaline regeneration solution may be added to the chromatographic column to remove substances bound to the hydrophobic interaction medium in the chromatographic column. The single alkaline regeneration solution may have one or more of the features described above.

[0079] Example

[0080] Example 1

[0081] The amount of non-esterified free fatty acids ("NEFA") in several sample solutions after incubation with polysorbate 20 ("PS20") was measured. The presence and amount of NEFA in the sample is considered to be an indication of PS20 degradation caused by, for example, impurities in the sample (such as host cell proteins). The first sample was taken out from the HIC injection material. Five additional samples were taken out from the HIC pool after 1, 2, 3, 5 and 10 subsequent cycles. All samples were incubated for the same amount of time. As shown in Table 1 below, from cycle 1 to cycle 5, PS20 degradation was calculated as a negative percentage, equivalent to a negative control. Negative PS20 degradation percentages are equivalent to no detectable NEFA, and therefore equivalent to no detectable lipase activity. NEFA is detectable between cycle 5 and cycle 10, thereby demonstrating an increase in lipase activity. The data show that lipase activity can increase as a function of the number of cycles.

[0082] Table 1

[0083] cycle PS20 degradation (%) Detection Injection material 1.83 Detectable Loop 1 -0.31 Equivalent to negative control Loop 2 -0.25 Equivalent to negative control Loop 3 -0.20 Equivalent to negative control Loop 5 -0.22 Equivalent to negative control Loop 10 0.47 Detectable

[0084] Example 2

[0085] Figure 2 Depicted are the HIC media (Capto TM Phenyl (High Sub) (GE Healthcare Life Sciences)) was analyzed by western blot as described in detail below.

[0086] Table 2

[0087]

[0088]

[0089] As in Figure 2 As can be seen in the 6N guanidine hydrochloride, dark areas are visible in each column that has been used, except for the column that has been exposed to 6N guanidine hydrochloride (column F). The column representing the native media (column A) also does not show dark areas. This data shows that 6N guanidine hydrochloride is able to remove residual media during regeneration of the chromatographic column compared to other solutions. Therefore, 6N guanidine hydrochloride can be used as a stripping agent and as a reagent that can be used after other stripping agents / regeneration agents to evaluate the performance of other reagents.

[0090] Example 3

[0091] Figure 3 Depicted are the HIC media (Capto TMPhenyl (High Sub)) blot analysis as described in detail below.

[0092] Table 3

[0093]

[0094] Table 3 lists the type of media used and the regenerants to which the media was exposed. Figure 3 As can be seen, the regeneration paradigm comprising RODI, 1.0 N NaOH, RODI, and 20% ethanol (used in media B and C) did not remove the residues from the media as completely as 6 N guanidine hydrochloride (D).

[0095] Example 4

[0096] Figure 4 Depicted are superimposed UV chromatograms depicting five cleanup runs following collection of monoclonal antibody mAb 1 during the process. Each cleanup run included a first 0.5N sodium hydroxide rinse of 2 column volumes (A), followed by a pause, and then a second 0.5N sodium hydroxide rinse of 1 column volume (B). Cleanup was considered complete at point C, after which a 2 column volume water for injection ("WFI") rinse was performed (D). As in Figure 4 As can be seen in the first sodium hydroxide rinse (A), high absorbance is produced in the early part of the rinse, which is associated with the removal of a large amount of impurities. The maximum absorbance seen during the HIC elution (regeneration) is 2.4 AU, while Figure 4 The maximum absorbance seen during the purification cycle was approximately 1.4 AU.

[0097] Example 5

[0098] Figure 5 Depicted are the HIC media (Capto TM Phenyl (High Sub)) blot analysis was performed as described in detail below. Specifically, the used HIC medium was exposed to decreasing concentrations of guanidine.

[0099] Table 4

[0100] mark Media Status * Reference Ladder A Indicates natural (unused) medium B Used media C Exposure of spent media to 20% ethanol D Spent medium exposed to 6N guanidine E Spent medium exposed to 3N guanidine F Spent medium exposed to 2N guanidine G Exposure to 1N guanidine in spent medium H Exposure to 0.5 N guanidine in the spent medium I Exposure to 0.1 N guanidine in the spent medium

[0101] As in Figure 5 As can be seen, the use of 6N guanidine hydrochloride (D) removed most of the residues from the HIC medium, while lower guanidine concentration solutions and 20% ethanol solutions were less effective in removing residues from the HIC medium.

[0102] Example 6

[0103] Figure 6 Depicts the presence of Capto TM Two columns of Phenyl (High Sub) HIC medium (GE Life Sciences). The left column was exposed to 49 cycles of HIC for purification of monoclonal antibody mAb2. Each cycle included regeneration of the 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. Discoloration of the left column may be an indication of insufficient regeneration.

[0104] Pools from cycles 1 and 49 were collected and tested for lipase activity and the presence of host cell proteins (HCPs). There was no clear trend in lipase activity or HCP values ​​between cycles 1 and 49.

[0105] Example 7

[0106] Fig. 7A and Figure 7B Depicted are the chromatograms of cycle 2 and cycle 49 performed on the left column as described in relation to Example 6, respectively. Fig. 7A and Figure 7B The following comments were made:

[0107] Table 5

[0108]

[0109] In both cycles, cycle 2 and cycle 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), thereby indicating that 1N NaOH was at least partially effective as a stripping solution. The introduction of a second RODI stripping removed some additional impurities and produced peaks P2 (cycle 2) and P4 (cycle 49). The addition of 20% ethanol solution did not produce additional peaks. This data from both cycles indicates that using RODI before sodium hydroxide is not an effective stripping solution.

[0110] Example 8

[0111] Two injection materials (each containing the target monoclonal antibody) were subjected to hydrophobic interaction chromatography processes. In the first process, after injection, washing, and elution of the target monoclonal antibody mAb 3 from the first injection material, the chromatography column was subjected to a 1N sodium hydroxide strip, followed by a RODI strip, and a 20% ethanol strip. In the second process, after injection, washing, and elution of the target monoclonal antibody mAb 4 from the second injection material, the chromatography column was subjected to a RODI strip, followed by a 1N sodium hydroxide strip, another RODI strip, and a 20% ethanol strip. Fig. 8A The chromatogram of the first process is depicted, Figure 8B The chromatograms for the second process are depicted. The chromatograms have been annotated as follows:

[0112] Table 6

[0113]

[0114] In by Figure 8B After the first RODI stripping (at X'), indicated by D' in FIG. , no peaks are present, which is equivalent to the peaks at Fig. 8A There are no peaks at the positions indicated by the X symbol (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 appreciable amount of material to be removed from the chromatographic column.

[0115] Example 9

[0116] Further Figure 6 The effectiveness of the 6N guanidine hydrochloride depicted in was evaluated as a stripping solution and as a potential solution for removing the discoloration on the left column. The column was subjected to the protocol described in the table below. The chromatogram generated during this protocol is shown in Fig. 9 .

[0117] Table 7

[0118] Solution Flow direction Chromatogram logo RODI Flow Downward A 6N Guanidine Hydrochloride Flow Downward B RODI Flow Downward C 6N Guanidine Hydrochloride* Upward mobility D RODI Upward mobility E

[0119] Each solution was moved through the column at a rate of 200 cm / hour in either a downward flow direction (i.e., in the same direction as the HIC purification process flow) or an upward flow direction (i.e., opposite to the downward flow, or "backward" through the chromatography column). The second addition of 6N guanidine hydrochloride (indicated by an asterisk) was maintained inside the column for approximately 16 hours.

[0120] As in Fig. 9As shown in the chromatogram of , the first introduction of RODI produces peak P1, and the first stripping of 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 effectiveness of the first stripping of 6N guanidine hydrochloride in removing residues bound to the column. However, after the complete cleaning regimen, the chromatographic column still changed color (as shown in Figure 6 ).

[0121] Example 10

[0122] The regeneration paradigm was analyzed in detail. Fig. 10A Describes the use of Capto during TM Chromatogram of the HIC process for purification of monoclonal antibody mAb 4 using Phenyl (High Sub) media (GE Life Sciences). The HIC process including the regeneration paradigm included the following steps:

[0123] Table 8

[0124] Logo( Fig. 10A ) event A Pre-stripping B balance C Injection and cleaning D RODI Stripping E 1N NaOH stripping F RODI Stripping G 20% ethanol stripping H Pre-Guadinium Hydrochloride Regeneration RODI I 6N Guanidine Hydrochloride

[0125] Reference Fig. 10A , peak P1 follows the addition of 1N sodium hydroxide (E), and peak P2 is consistent with the second RODI stripping (F). Peak P3 follows the addition of 6N guanidine hydrochloride (I).

[0126] Fig. 10B A magnified image of peak P1 is shown. The first RODI stripping (D) resulted in no detectable absorbance. The 1 N NaOH stripping (E) appears to result in immediate and early removal of the residue from the column, as indicated by the presence of peak P1, which appears to reach the baseline as the conductivity increases. It is hypothesized that the initial RODI stripping (D) causes some residues to bind more tightly to the column, rather than promoting their removal. It is further hypothesized that the elution of the residue from the column caused by sodium hydroxide may be driven primarily by pH, but as the concentration of sodium hydroxide (a weak chaotropic agent) increases, as the conductivity of the solution increases, the residual protein may bind more tightly to the HIC medium.

[0127] Embodiment 11

[0128] Elution of the residue from the used HIC media was observed as a function of the NaOH concentration. Fig.11Describe the chromatogram of following process, wherein after HIC column is injected and cleaned to collect monoclonal antibody mAb 4 (during part A), 20CV (column volume) gradient is loaded into column, and the mixing of RODI and sodium hydroxide begins with single RODI and then the concentration of sodium hydroxide is gradually increased to the maximum concentration of 1N sodium hydroxide (part B). Single clear peak P1 is observed, and elution is carried out by passing ~ 5mM sodium hydroxide through column. Perform second 20CV gradient and load into column, start with RODI and 1N maximum concentration sodium hydroxide, and the concentration of sodium hydroxide is gradually reduced to 0 (part C). During this second gradient, no other peak is observed. Finally, at mark D, the solution of 6N guanidine hydrochloride is rinsed through column. During the passing through of 6N guanidine hydrochloride through column, small peak P2 is observed. The area under the curve (AUC) of each peak is calculated by the 280nm UV absorbance of integration. Peak P2 was calculated to have an AUC of 1,160 mL*mAU, compared to the AUC of 6N guanidine hydrochloride stripping during the control process (e.g., Table 8), which was calculated to be 13,305 mL*mAU. Thus, Peak P2 exhibited a 91.3% size reduction compared to the control.

[0129] This procedure showed that the bound material eluted from the column most when ~5 mM sodium hydroxide was passed through the column, leaving relatively little residue for elution with 6N guanidine hydrochloride.

[0130] Example 12

[0131] In separate two-solution regeneration processes, sodium hydroxide solutions of different concentrations (1000 mM, 500 mM, 100 mM, 50 mM, 25 mM, 10 mM, and 5 mM) were added, each performed after injection and washing of the HIC column. Each regeneration process included sodium hydroxide solution as the first regeneration solution and 6N guanidine hydrochloride solution as the second regeneration solution. Chromatograms of the regeneration processes were generated and superimposed on Fig.12. The sodium hydroxide solution in each regeneration process generated a first peak, represented by the peak group labeled A. The 6N guanidine hydrochloride solution in each regeneration process generated a second peak, represented by the peak group labeled B. It was determined that the regeneration process including 5mM NaOH generated the largest "A" peak (indicating the largest amount of residue eluted by the addition of sodium hydroxide solution) and the smallest "B" peak (indicating the smallest amount of residue eluted by the addition of guanidine hydrochloride). It was therefore determined that among the range of sodium hydroxide solutions tested, the 5mM sodium hydroxide solution was the most effective for regenerating the HIC column (i.e., it removed the majority of bound material from the column). The higher the sodium hydroxide concentration, the more residue remained on the chromatographic column for removal by the 6N guanidine hydrochloride solution. It is hypothesized that while the increase in pH drives the elution of residue from the HIC column, the increase in conductivity reduces the elution of residue by enhancing the binding between the residue and the HIC medium.

[0132] Embodiment 13

[0133] From Capto TM After mAb 4 was collected on a HIC column prepared with phenyl (High Sub) media (GE Healthcare Life Sciences), a one-way statistical analysis of variance was performed on the area under the curve (AUC) of the chromatogram peaks generated by the regeneration process. Fig.13A and Fig. 13B As shown in FIG, regeneration processes were performed using a control (A), RODI (B), and various concentrations of sodium hydroxide solution (CL). Each regeneration process included a stripping solution (in Fig.13A AUC analyzed in ) followed by 6N guanidine hydrochloride solution (in Fig. 13B All Pairs Turkey-Kramer test was performed for each analysis to describe statistical significance.

[0134] As in Fig.13A and Fig. 13B As shown in FIG. 1 , the processes associated with 0.5 mM NaOH and 1 mM NaOH regeneration solutions show that the peaks generated during the flow of these regeneration solutions have the highest AUC values ​​(circled in region 1300), indicating that these regeneration solutions result in more efficient removal of material from the HIC column. The processes associated with 0.5 mM NaOH, 1 mM NaOH, and 5 mM NaOH regeneration solutions show that the peaks generated during the flow of 6 N guanidine hydrochloride solution after the regeneration solution have the lowest AUC values ​​(circled in region 1300). Fig. 13B1350), thereby also supporting that these regeneration solutions are more effective in removing material from the HIC column, leaving less material for removal by the 6N guanidine hydrochloride. Therefore, solutions with sodium hydroxide concentrations ranging from 0.5 mM to 5 mM NaOH were demonstrated to be effective for regeneration of HIC columns.

[0135] Embodiment 14

[0136] The regeneration process using sodium hydroxide solution was compared to the regeneration process using sodium chloride solution. After collecting the monoclonal antibody mAb 5 from the HIC column, the column was regenerated using sodium hydroxide at a concentration of 3mM, 5mM, or 7mM, or using sodium chloride at a concentration of 5mM, 8mM, or 11mM. The pH 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, a solution of 6N guanidine hydrochloride was added to each column after the regeneration solution. Chromatograms were generated for each process, and all chromatograms were superimposed on Fig.14 The AUCs of the peaks associated with the flow through of the regeneration solution and the flow through of 6N guanidine hydrochloride were calculated. These solutions and AUCs are listed in the table below.

[0137] Table 9

[0138]

[0139] As depicted by the present data, the flow-through of the sodium hydroxide solution showed a much higher AUC value than the sodium chloride solution. Likewise, the flow-through of 6N guanidine hydrochloride after the sodium hydroxide solution showed a much lower AUC value than the flow-through of 6N guanidine hydrochloride after the sodium chloride solution. Fig.14 In FIG. 1 , the peak generated by the flow of sodium hydroxide is indicated by the symbol A, and the peak generated by the flow of sodium chloride (or the absence of such a peak) is indicated by the symbol B. Similarly, the peak generated by the flow of 6N guanidine hydrochloride after sodium hydroxide and the peak generated by the flow of 6N guanidine hydrochloride after sodium chloride are indicated by the symbols A' and B', respectively. Fig.14 As shown in , sodium hydroxide solution is a more effective stripping agent than sodium chloride solution.

[0140] Embodiment 15

[0141] Potential cleaning / regeneration protocols were tested on a column that had been subjected to 50 HIC cycles to purify the monoclonal antibody mAb 6. Fig.15As shown in (columns A, B, and C), the columns showed discoloration near the top of the column bed. Column A was washed with 2CV of 0.5M EDTA, column B was washed with 2CV of 0.5M acetic acid, and column C was used as a control. Neither solution was effective in reducing the browning / discoloration.

[0142] Example 16

[0143] Three sodium hydroxide solutions (2 mM, 5 mM, and 10 mM) were used as regeneration solutions after HIC purification of monoclonal antibody mAb 6. The regeneration potency (effect) of each sodium hydroxide solution on the HIC medium used during purification was characterized by the size of the chromatogram peak associated with 6N 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, whereas 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 therefore indicated a more effective sodium hydroxide regeneration solution. Fig.16 The curve shown shows the guanidine stripping peak AUC as a function of sodium hydroxide concentration. All three tested sodium hydroxide solutions showed more efficient regeneration (i.e., smaller guanidine stripping peaks) than the default cleaning mode (comprising RODI, 1N sodium hydroxide, RODI, 20% ethanol, and RODI sequentially). The 5mM sodium hydroxide solution had the lowest guanidine stripping peak area. Based on the curves extrapolated from these data points, the 7mM sodium hydroxide regeneration solution is likely to result in an even lower guanidine stripping peak area.

[0144] Embodiment 17

[0145] A first regeneration process designed as a control was performed on the HIC column after the monoclonal antibody mAb 5 was collected from the column. A chromatogram was generated using this process. Multiple solutions were used in the following order: starting with 1N sodium hydroxide, followed by RODI, 20% ethanol, RODI, and 6N guanidine hydrochloride. The chromatogram peak corresponding to the flow-through of 6N guanidine hydrochloride was used to measure the effectiveness of the regeneration process. The chromatogram is plotted on Fig.17 The symbols on the chromatogram represent the following events:

[0146] Table 10

[0147] Logo event A Injection B Pool collection starts C Cleaning D Introduce 1N NaOH solution E Import RODI F Introduce 20% ethanol G Import RODI H Import 6N guanidine hydrochloride

[0148] The AUC of the peak corresponding to the flow-through of the 6N guanidine hydrochloride solution was calculated to be 7,390 mL*mAU.

[0149] A second regeneration process containing 5 mM sodium hydroxide was performed after collection of mAb 5 from the HIC column and a chromatogram was generated, which is depicted in Fig.18Multiple solutions were used in the following order: starting with 5 mM sodium hydroxide, followed by RODI, 20% ethanol, RODI, 5 mM sodium hydroxide, RODI, 6N 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 following table:

[0150] Table 11

[0151] order 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)

[0152] like Fig.18 As shown in and reflected by the AUC values ​​in the table above, the initial 5 mM NaOH flow-through exhibits the largest AUC (peak A) with a large margin. 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 6N guanidine hydrochloride, but its value was 889 mL*mAU, which is less than one-thirtieth of the AUC value associated with the initial 5 mM NaOH flow-through. In addition, compared with the Fig.17 The smaller AUC value of the 6N guanidine HCl peak in the control process (7,390 mL*mAU) shown in Figure 2 compared to the smaller AUC value of the 6N guanidine HCl peak in this regeneration process indicates that the initial 5 mM NaOH solution provides greatly improved HIC column regeneration compared to the control run. RODI, 20% ethanol, 6N guanidine HCl, and 0.1 N NaOH provide minimal additional benefit to HIC regeneration when used after the 5 mM NaOH solution.

[0153] Embodiment 18

[0154] In the presence of Capto TM Six mixtures, each containing a different target molecule (e.g., target monoclonal antibody) and different citrate concentrations in the injection buffer, were subjected to HIC in a column of Phenyl (High Sub) media (GE Life Sciences). After elution of each monoclonal antibody, the used HIC column was passed through a volume of RODI and sodium hydroxide, with a gradient from no sodium hydroxide (pure RODI) to 1N sodium hydroxide, followed by a gradient in the reverse direction (from 1N sodium hydroxide back to pure RODI). Finally, each process ended with the injection of 6N guanidine hydrochloride and flow-through collection. The pH and citrate concentrations in each injection mixture are shown in the table below.

[0155] Table 12

[0156]

[0157] Chromatograms were generated for each process; Fig.19These chromatograms are depicted in order in FIG. 1. As shown, each chromatogram AF includes peaks (A', B', C', D', E', F') corresponding to the elution of material bound to the HIC medium when about 5 mM NaOH is passed through. The subsequent guanidine hydrochloride flow-through peak is minimal or absent. Thus, it is demonstrated that the effectiveness of the regeneration solution is not limited to the use of one monoclonal antibody.

[0158] Embodiment 19

[0159] Multiple HIC protocols with different conditions were evaluated to determine if the protocol would cause regeneration / usability issues. A 96-well filter plate (AcroPrep TM The wells of Advance 1 mL filter plates, 1.2 μm Supor membrane, part number 8130, Pall Corporation) were filled with 0.02 μL of various HIC media:

[0160] Table 13

[0161]

[0162] Aliquots of the injected material were prepared containing one of the three target antibodies (mAb 1, mAb 2, mAb 3) and adjusted to a concentration of 0.33 g / L, with a target concentration of 5 g / L when mixed with the HIC medium. Aliquots of the injected material were titrated to different pH values ​​(4.5, 6.25, or 8.0) with 2 M acetic acid or 2 M Tris base for each of the three target antibodies to prepare a total of 9 aliquots, each containing one of the three target antibodies and displaying one of the three different pH values.

[0163] Each column of three wells filled with a single media type was subjected to a purification protocol at a different pH value (4.5, 6.25, or 8.0) to establish a series of protocols performed using various combinations of media and pH. Each column of wells was subdivided into multiple groups so that each group of these columns was subjected to a protocol for a different target antibody (mAb1, mAb 2, or mAb 3). In order to run each protocol at the respective pH values, for each target antibody, an aliquot of the injection material containing the target antibody of the corresponding pH value was used, and an equilibrium buffer of 40 mM Tris, 300 mM citrate of the corresponding pH was used.

[0164] Perform the following steps simultaneously on the array:

[0165] 1. Equilibrate the wells filled with various HIC resins three times with equilibration buffer at the respective pH values.

[0166] 2. Add the feed material containing the desired target antibody to each well at the respective pH and incubate for up to one hour.

[0167] 3. The plate was spun at 1100 rpm.

[0168] 4. Collect the flow-through (i.e., components that did not bind to the medium during the incubation period).

[0169] 5. Wash the wells twice with equilibration buffer at the appropriate pH for each test protocol.

[0170] 6. Perform pseudo gradient elution using equilibration buffer of corresponding pH values. Each well was exposed to equilibration buffer for seven iterations, where the citrate concentration was linearly decreased from 300 mM to 0 mM in all seven iterations (increment of 42.9 mM per iteration).

[0171] 7. Wash the wells twice with RODI and twice with 1N NaOH.

[0172] 8. Spin the plate at 1100 rpm.

[0173] 9. The holes were stripped twice with a 6N guanidine hydrochloride solution.

[0174] 10. The wells were spun at 1100 rpm.

[0175] For each well, a chromatogram of the target polypeptide in the material removed from the well (flow-through, eluent, wash, etc.) is generated. For analysis of the results, each chromatogram is subdivided into three regions, wherein 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). An example of three chromatograms generated from a protocol for purification of the target antibody mAb 1 at pH 6.25, and three different HIC media subdivided into three regions ( Hexyl-650C, Phenyl Sepharose 6Fast Flow (HighSub), and POROS TM Each medium of Ethyl is depicted in Figure 20A-20C middle.

[0176] The comparison showed that the protocol and analysis performed on the test wells were consistent with those performed on the large-scale columns with a statistically significant accuracy of 98%.

[0177] Embodiment 20

[0178] The collection of data using the high throughput screening techniques described herein enables the creation of predictive functions that aid in the creation of HIC protocols with improved efficiency and yield. For example, boundary functions can be drawn that describe the relationship between multiple parameters (e.g., pH, elution buffer citrate concentration, load mass, HIC media selection) and the efficiency or yield of the protocol.

[0179] Fig.21A is a boundary function graph based on a given antibody and Medium (Phenyl The HIC protocol was used to correlate the elution buffer citrate concentration and pH with the predicted yield of the HIC protocol. The shaded area represents the combination of HIC protocol parameters that results in a predicted yield less than 90% of the theoretical yield. This boundary function tells what citrate and pH parameters should be considered for a given antibody and The combinations of pH and citrate concentration outside this shaded area are those used for a given antibody and Variable HIC protocol parameters for the medium, while the combinations of pH and citrate concentration inside the shaded area are the excluded parameters.

[0180] Fig.21B Based on the given antibody and Capto TM A collection of high throughput screening data for phenyl (High Sub) chromatography media at 100 g / liter media injection, plot of a bounding function relating elution buffer citrate concentration and pH to predicted yields for HIC protocols. The shaded area represents combinations of HIC parameters for which the predicted yield is less than 90% of theoretical yield. This bounding function informs what citrate and pH parameters should be considered for a given antibody and Capto TM HIC protocol for phenyl (High Sub) media. The combinations of pH and citrate concentration outside the shaded area are the pH values ​​for a given antibody and Capto TM Variable HIC protocol parameters for Phenyl (HighSub) media, while the combinations of pH and citrate concentration inside the shaded area are the excluded parameters.

[0181] 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 aid in the design of HIC protocols. For example, a transfer function can be regressed to relate data from an elution assay, high throughput screening, or laboratory scale HIC protocol to a full scale HIC protocol. For example, for a group of HIC protocols that include predicted yields (e.g., yields predicted by a small scale experiment or high throughput screening), the actual yields of each HIC protocol can be determined by a full scale chromatogram. The relationship between predicted yields and actual yields can be regressed to improve the prediction model. Future predicted yields can be calculated based at least in part on a regression relationship (e.g., a transfer function) applied to data from an elution assay, high throughput screening, or laboratory scale HIC protocol.

[0182] One or more transfer functions, boundary functions, and / or prediction models may be combined to create a dynamic prediction model. Fig. 22 , which shows a dynamic prediction model. The dynamic prediction model calculates how changes to one or more HIC protocol parameters (e.g., pH, citrate concentration, loading mass, chromatographic resin) affect the quantitative properties of the HIC protocol. Fig. 22 In the example shown in , protocol parameters were then selected, including pH 6, 30 mM citrate concentration, 100 g / liter HIC medium loading mass, and resin C (e.g., Phenyl 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. Fig. 22 As shown in , the selected parameters resulted 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.

[0183] In some embodiments, an overall normalized value can also be calculated as a combination of the quantitative properties of the scheme (e.g., a combination of predicted yield, high molecular weight fraction, and pool host cell protein concentration). The normalized value calculation can be based on the weighting of the quantitative properties of the HIC scheme. For example, changes to the HIC scheme that affect the yield of the scheme will be more important than changes that affect efficiency. The dynamic prediction model can take into account the relative importance of quantitative measurements and assign weights to different measurements so that they do not affect the overall normalized value equally. In some embodiments, the dynamic prediction model can be updated when additional chromatographic data (e.g., data from small-scale experiments, high-throughput screening, and full-scale chromatographic runs) are collected and assembled.

[0184] Embodiment 21

[0185] In gradient elution experiments, multiple HIC protocols including different combinations of target molecules and HIC media were tested at various pH values ​​to determine whether these combinations would cause regeneration / usability issues and whether some combinations of target molecules and HIC media should be excluded in further HIC protocol development.

[0186] 96-well filter plates (AcroPrep TM The wells of Advance 1 mL filter plates, 1.2 μm Supor membrane, part #8130, Pall Corporation) were filled with 0.02 μL of various HIC media. Aliquots of the injected material containing the target molecule were prepared and adjusted to a concentration of 0.33 g / L, with a target concentration of 5 g / L when mixed with the HIC media. For each of the three target antibodies, aliquots of the injected 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 containing the target antibody and exhibiting a different pH.

[0187] Each well of a 96-well plate filled with a single media type was subjected to the HIC protocol at a different pH (4.5, 6.25, or 8.0) to generate array protocols performed with various combinations of media and pH.

[0188] Perform the following steps simultaneously on the array:

[0189] 1. Wells filled with various HIC resins were equilibrated three times with equilibration buffers of respective pH.

[0190] 2. Add the injection material containing the desired target molecule to each well at the respective pH and incubate for up to one hour.

[0191] 3. The plate was spun at 1100 rpm.

[0192] 4. Collect the flow-through (containing, for example, components that were not bound to the medium during the incubation period).

[0193] 5. Wash the wells three times with the equilibration buffer at the pH corresponding to each test protocol.

[0194] 6. Perform a pseudo elution. Each well was exposed to elution buffer for seven iterations, where the plate was spun at 1100 rpm after each iteration and the eluate was collected.

[0195] 7. Wash the wells twice with NaOH. Different concentrations of NaOH may be used in different wells of the array. For example, two wells may contain the same chromatographic media and be injected at the same pH, but wash with 5 mM NaOH in one well and 1 N NaOH in the other. After each wash, spin the plate at 1100 rpm and collect the washes.

[0196] 8. The wells were stripped twice with a 6N guanidine hydrochloride solution and the stripped material was collected.

[0197] For each well, a chromatogram of the target molecule in material removed from the well (flow through, eluent, wash, etc.) was generated. To analyze the results, each chromatogram was subdivided into three regions, wherein the first region contained the mass of the target molecule observed in material removed during steps 2, 3, and 4 (flow through, wash, and elution), the second region contained the mass of the target molecule observed in material removed during steps 5, 6, 7 (using RODI and NaOH), and the third region contained the mass of the target molecule observed in material removed during step 8 (using 6N guanidine hydrochloride).

[0198] An example of a chromatogram generated from an elution experiment for a first target molecule is shown in Figure 23A-Figure 26C middle. Figure 23A-23C Capto TM Chromatogram of the elution test containing the first target molecule on Phenyl (High Sub) media, Figure 24A-24C Capto TM Chromatogram of the elution test on butyl media containing the first target molecule, Figure 25A-Figure 24C POROS TM Chromatogram of the elution test containing the first target molecule on benzyl medium, Figure 26A-26C Phenyl A chromatogram of the elution test containing the first target molecule on the medium. Fig.23A , Fig.24A , Fig.25A and Fig.26A The chromatogram for the elution test run at pH 4.5 is shown. Fig. 23B , Fig. 24B , Fig.25B and Fig.26B shows a chromatogram of an elution test run at a pH of 6.25, and Fig.23C , Fig.24C , Fig.25C and Fig.26C A chromatogram of an elution test run at pH 8 is shown. Figure 23A-Figure 26C In FIG. 5 , the dashed line represents the chromatogram from the elution experiment containing 5 mM NaOH as a washing solution, and the solid line represents the chromatogram from the elution experiment containing 1 N NaOH as a washing solution.

[0199] An example of a chromatogram generated from an elution experiment for a second target molecule is shown in Figures 27A-28D middle. Figure 27A-Figure 2 Each of Figures 8 shows three chromatograms, each from an elution test run at a different pH. Fig.27A Shown is a graph from Capto TM Chromatogram of an elution test run on Phenyl (High Sub) media, Fig.27B Shown is a graph from Capto TM Chromatogram of a test run of elution on butyl media, Fig.27C Shown from TOYOPEARL TM Chromatogram of an elution test run on Phenyl-650C media, Fig.27D Shown is the POROS TM Chromatogram of a test run of elution on ethyl medium, Fig.28A Shown from TOYOPEARL TM Chromatogram of an elution test run on Hexyl-650C media, Fig.28B Shown from TOYOPEARL TM Chromatogram of an elution test run on Butyl-650C media, Fig.28C Shown is the POROS TM Chromatogram of an elution test run on benzyl media, Fig.28D Shown in Phenyl Chromatogram of a test run of elution on the media. Figures 27A-28D In FIG. 5 , the dashed line represents the chromatogram from the elution experiment containing 5 mM NaOH as a washing solution, and the solid line represents the chromatogram from the elution experiment containing 1 N NaOH as a washing solution.

[0200] As can be seen from the chromatograms, all tested solutions containing a 1 N NaOH stripping solution had a greater percentage of target molecular mass than similar solutions containing a 5 mM NaOH stripping solution. That is, more target molecules remained bound to the column after the 1 N NaOH stripping solution compared to the 5 mM NaOH stripping solution.

[0201] By comparing chromatograms of different HIC protocols for a given target molecule, certain HIC media and / or pHs can be eliminated from consideration. The speed with which HIC protocols are established and tested can be increased by using a series of elution experiments to eliminate undesirable target molecule, HIC media, and pH combinations from consideration. For example, a chromatogram showing that more than 5% (e.g., more than 10%) of the target molecule is eluted in region 3 can indicate that a given HIC media, pH value, and target molecule combination is not suitable for incorporation into the HIC protocol.

[0202] 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 calculated Zone 3 mass from the chromatogram to predict the mass of the target molecule eluted in Zone 3 during a full-scale chromatography run. 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 over a range of HIC protocols using various combinations of HIC media and pH is shown in Table 14.

[0203] Table 14

[0204]

[0205] As can be seen in Table 14, including at a pH of 4.5 in Capto TM The elution test for a given target molecule on Phenyl (High Sub) media gave a normalized percent recovery of 38.2%. This result, when converted through the transfer function, gave a predicted full-size region 3 recovery of 10.6%. In embodiments where the predetermined threshold is 10%, this will be greater than the threshold, so the target molecule, Capto TM The combination of phenyl (High Sub) medium, and a pH of 4.5 will be excluded from further HIC protocol development. In some embodiments, if exclusion of a medium and pH combination is necessary for a given target molecule, that medium and pH combination may also be excluded from further protocol development involving other target molecules.

[0206] Those skilled in the art will appreciate that the concepts upon which this disclosure is based may readily be used as a basis for designing other methods and systems for carrying out the several purposes of this disclosure.Accordingly, the claims are not to be considered limited by the foregoing description.

Claims

1. A method for regenerating a hydrophobic interaction chromatography column to which an injection substance has been applied, the method comprising: passing one or more column volumes of a basic solution through the hydrophobic interaction medium inside the column, wherein the basic solution exhibits a pH value between 10 and 14 and a conductivity between 0.5 mS / cm and 10 mS / cm, Therein, substances bound to the hydrophobic interaction medium are removed.

2. The method of claim 1, wherein the alkaline solution comprises one of sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or tris (hydroxymethylaminomethane) (Tris).

3. The method of claim 1, wherein the alkaline solution exhibits a conductivity between 0.8 mS / cm and 1.6 mS / cm.

4. The method of claim 1, wherein the alkaline solution comprises a total dissolved salt concentration between 0.1 mM and 10 mM.

5. The method of claim 1, wherein after removing the material bound to the hydrophobic interaction medium, less than 1.0% of the input material remains bound to the hydrophobic interaction medium as residue.

6. The method of claim 1, wherein the substances removed from the medium include host cell proteins, aggregated proteins, lipids, polypeptide fragments, biomolecules or nucleic acids.

7. The method of claim 1, wherein the material removed from the medium does not include bacteria or fungi.

8. The method of claim 1, wherein the method does not comprise contacting the hydrophobic interaction medium with a chaotropic agent or an organic solvent.

9. The method of claim 1, wherein the step of passing the one or more column volumes of basic solution through the hydrophobic interaction medium inside the column takes between 10 minutes and 1 hour.

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