System and method for using and regenerating chromatography

Abstract

To provide a method for regenerating a hydrophobic interaction chromatography column on which a load mass is applied.SOLUTION: In the method for regenerating a hydrophobic interaction chromatography column, a material which combines with a hydrophobic interaction medium is removed by passing one or more column volumes of an alkaline solution having a pH of about 10 to about 14 and a conductivity of 0.5 mS / cm to about 10 mS / cm through a hydrophobic interaction medium in the column. The alkaline solution contains sodium hydroxide in the concentration of about 0.1 mM to about 10 mM.SELECTED DRAWING: Figure 12

Description

Technical Field

[0001] Cross - reference to Related Applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 905,0 33, filed on September 24, 2019, and U.S. Provisional Patent Application No. 62 / 958,899, filed on January 9, 2020, the entire contents of both of which are incorporated herein by reference .. .

Background Art

[0002] Field of Disclosure

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

[0003] Introduction

[0003] Chromatography is a widely used category of processes that can be performed to separate the components of a mixture. Certain types of chromatography can be performed in the pharmaceutical product preparation process (e.g., separation, collection, isolation, purification, polishing, etc. of molecules for use in pharmaceutical products). Some molecules of interest ( e.g., polypeptides, polynucleotides, etc.) may need to be produced from the material of the host cells in which they are generated. The separation or purification of molecules of interest using chromatography .. .. .. .. Purification reduces, removes, or separates host cell proteins (such as lipases), host cell materials (such as cell debris), and other impurities that can otherwise be purified with the molecule of interest.

[0004]

[0004] Chromatography can involve the use of a stationary phase that includes a medium configured to assist in the separation of components of a mobile phase that passes through the stationary phase. For example, hydrophobic interaction chromatography (HIC) can separate molecules (such as polypeptides, polynucleotides, etc.) according to differences in surface hydrophobicity by using a reversible interaction between the molecule and the hydrophobic surface of the HIC medium in the stationary phase. The interaction between the molecule and the hydrophobic surface of the HIC medium can be affected, for example, by salts in a running buffer. A load mass with a high salt concentration can be loaded into the HIC device, where the high salt concentration promotes the interaction between the HIC medium in the device and the molecules in the mixture. Subsequently, a solution (such as a buffer) with a decreasing ionic strength flows through the HIC device, and the hydrophobic interaction between the HIC medium and the molecule can be reversed. Molecules with the lowest hydrophobicity can be eluted first, and molecules with the highest hydrophobicity require a greater decrease in salt concentration to reverse the hydrophobic interaction with the HIC medium and can thus be eluted last.

[0005]

[0005] In some cases, the HIC medium is reusable for multiple chromatography cycles. To maintain the effectiveness, quality, and cleanliness of the HIC medium, prevent contamination between cycles, increase the lifespan of the HIC medium, prevent the accumulation of impurities, and / or otherwise meet or exceed operating standards (e.g., operating standards within a laboratory or organization, or operating standards mandated by a regulatory agency), a method for regenerating the HIC medium may be used to remove residual material from the HIC medium after performing an HIC cycle.

SUMMARY OF THE INVENTION

[0006]

[0006] Aspects of the present disclosure relate to the regeneration of chromatography columns. In one aspect, the present disclosure relates to a method for regenerating a hydrophobic interaction chromatography column to which a load mass has been applied. The method may include passing one or more column volumes of an alkaline solution through the hydrophobic interaction media within the column, the alkaline solution exhibiting a pH between about 10 and about 14 and a conductivity between 0.5 mS / cm and about 10 mS / cm, and substances bound to the hydrophobic interaction media being removed. The alkaline solution may include one of sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or Tris. It is possible; the alkaline solution exhibits a conductivity between about 0.8 mS / cm and about 1.6 mS / cm It is also possible that the alkaline solution contains a total dissolved salt concentration between about 0.1 mM and about 10 mM (a total dissolved salt concentration).

[0007]

[0007] After removal of the substances bound to the hydrophobic interaction medium, less than 1.0% of the loading mass may remain bound to the hydrophobic interaction medium as a residual mass. The substances removed from the medium may include host cell proteins, suggested proteins, lipids, polypeptide fragments, biomolecules, or nucleic acids. The substances removed from the medium may, in some embodiments, be free of bacteria or fungi. The method may, in some embodiments, not involve contacting a chaotropic agent or an organic solvent with the hydrophobic interaction medium. Passing one or more column volumes of the alkaline solution through the hydrophobic interaction medium in the column takes from about 10 minutes to about 1 hour. The substances removed from the medium may include host cell proteins, suggested proteins, lipids, polypeptide fragments, biomolecules, or nucleic acids. The substances removed from the medium may, in some embodiments, be free of bacteria or fungi. The method may, in some embodiments, not involve contacting a chaotropic agent or an organic solvent with the hydrophobic interaction medium. Passing one or more column volumes of the alkaline solution through the hydrophobic interaction medium in the column takes from about 10 minutes to about 1 hour. The substances removed from the medium may include host cell proteins, suggested proteins, lipids, polypeptide fragments, biomolecules, or nucleic acids. The substances removed from the medium may, in some embodiments, be free of bacteria or fungi. The method may, in some embodiments, not involve contacting a chaotropic agent or an organic solvent with the hydrophobic interaction medium. Passing one or more column volumes of the alkaline solution through the hydrophobic interaction medium in the column takes from about 10 minutes to about 1 hour. The substances removed from the medium may include host cell proteins, suggested proteins, lipids, polypeptide fragments, biomolecules, or nucleic acids. The substances removed from the medium may, in some embodiments, be free of bacteria or fungi. The method may, in some embodiments, not involve contacting a chaotropic agent or an organic solvent with the hydrophobic interaction medium. Passing one or more column volumes of the alkaline solution through the hydrophobic interaction medium in the column takes from about 10 minutes to about 1 hour. In some embodiments, it may be free of bacteria or fungi. The method may, in some embodiments, not involve contacting a chaotropic agent or an organic solvent with the hydrophobic interaction medium. Passing one or more column volumes of the alkaline solution through the hydrophobic interaction medium in the column takes from about 10 minutes to about 1 hour. In some embodiments, it may not involve contacting a chaotropic agent or an organic solvent with the hydrophobic interaction medium. Passing one or more column volumes of the alkaline solution through the hydrophobic interaction medium in the column takes from about 10 minutes to about 1 hour. In some embodiments, it may not involve contacting a chaotropic agent or an organic solvent with the hydrophobic interaction medium. Passing one or more column volumes of the alkaline solution through the hydrophobic interaction medium in the column takes from about 10 minutes to about 1 hour. Passing one or more column volumes of the alkaline solution through the hydrophobic interaction medium in the column takes from about 10 minutes to about 1 hour.

[0008]

[0008] In another aspect, the present disclosure is a method of regenerating a chromatography column to which a loading mass has been applied, the method including passing one or more column volumes of an alkaline solution through the hydrophobic interaction medium in the column, the alkaline solution containing sodium hydroxide at a total dissolved concentration between about 0.5 mM and about 50 mM, and the substances bound to the medium being removed. The medium has an aliphatic or aromatic configuration In another aspect, the present disclosure is a method of regenerating a chromatography column to which a loading mass has been applied, the method including passing one or more column volumes of an alkaline solution through the hydrophobic interaction medium in the column, the alkaline solution containing sodium hydroxide at a total dissolved concentration between about 0.5 mM and about 50 mM, and the substances bound to the medium being removed. The medium has an aliphatic or aromatic configuration In another aspect, the present disclosure is a method of regenerating a chromatography column to which a loading mass has been applied, the method including passing one or more column volumes of an alkaline solution through the hydrophobic interaction medium in the column, the alkaline solution containing sodium hydroxide at a total dissolved concentration between about 0.5 mM and about 50 mM, and the substances bound to the medium being removed. The medium has an aliphatic or aromatic configuration In another aspect, the present disclosure is a method of regenerating a chromatography column to which a loading mass has been applied, the method including passing one or more column volumes of an alkaline solution through the hydrophobic interaction medium in the column, the alkaline solution containing sodium hydroxide at a total dissolved concentration between about 0.5 mM and about 50 mM, and the substances bound to the medium being removed. The medium has an aliphatic or aromatic configuration In another aspect, the present disclosure is a method of regenerating a chromatography column to which a loading mass has been applied, the method including passing one or more column volumes of an alkaline solution through the hydrophobic interaction medium in the column, the alkaline solution containing sodium hydroxide at a total dissolved concentration between about 0.5 mM and about 50 mM, and the substances bound to the medium being removed. The medium has an aliphatic or aromatic configuration ​It may include a matrix containing a ligand having a hydrocarbon between 2 and 10. The ligand is It may be present in the medium at a density between about 20 and about 30 μmol per ml of the medium. In other examples, The medium may not contain a ligand containing 30 or more hydrocarbons; chromatography The column may not be used in a mixed-mode chromatography process; and / or the medium may include a matrix containing cross-linked agarose and a phenyl ligand . In other examples, the method may not include contacting the medium with alcohol, ethylene glycol, or sodium chloride. The method may include passing one or more column volumes of an alkaline solution through the column, and then passing one or more column volumes of a chaotropic agent through the column , and the chaotropic agent is one of 6N guanidine hydrochloride or 8N urea . The method may further include contacting the column with a storage buffer containing sodium hydroxide at a total dissolved concentration between about 0.05M and about 0.15M. The method may also further include applying a first loading mass to a chromatography column, and applying a second loading mass to the chromatography column , and does not include cleaning the chromatography column . .

[0009]

[0009] In another aspect, the present disclosure is a method of identifying the concentration of an alkaline solution for a hydrophobic interaction chromatography column regeneration solution, comprising passing a volume of a first solution through the hydrophobic interaction medium in the column, wherein the first solution is water and a concentration that starts from about 0N and increases to a maximum concentration at a substantially constant ratio . including an alkaline solution (wherein the first solution includes water and a concentrat ion of an alkaline solution beginning from about 0N and increasing at an approxi mately constant rate to a maximum concentration); passing a volume of a second solution through a hydrophobic interaction medium, wherein the second solution includes water and a concentration of an alkaline solution beginning from the maximum concentration and decreasing at an approximately constant rate to about 0N (wherein the second s olution includes water and a concentration of an alkaline solution beginning fro m the maximum concentration and decreasing at an approximately constant rate to about 0N); and identifying a portion of the first or second solution that removes a substance bound to the hydrophobic interaction medium when passing through the hydrophobic interaction medium. The alkaline solution may include sodium hydroxide

[0010]

[0010] In another aspect, the disclosure includes predicting, evaluating, or comparing the usefulness and regeneration of various chromatography resins. In some examples, a method for evaluating a chromatography protocol includes a filter plate well (a filter pla In a well, a charged mass containing a target molecule is added to a volume of chromatography medium, and a flow-through is collected from the filter plate well, where the charged mass exhibits a protocol pH; a plurality of aliquots of a buffer containing the chromatography medium are added to obtain an eluate from the chromatography medium, where the buffer exhibits a buffer pH and a concentration of a kosmotropic salt decreases linearly over the plurality of aliquots, and a first amount of the target molecule is contained in the combined flow-through and eluate; a second solution is added to the chromatography medium to extract a second amount of the target molecule from the chromatography medium; and a chaotropic agent is added to the chromatography medium to extract a third amount of the target molecule from the chromatography medium, related to a method. and a flow-through is collected from the filter plate well, where the charged mass exhibits a protocol pH; a plurality of aliquots of a buffer containing the chromatography medium are added to obtain an eluate from the chromatography medium, where the buffer exhibits a buffer pH and a concentration of a kosmotropic salt decreases linearly over the plurality of aliquots, and a first amount of the target molecule is contained in the combined flow-through and eluate; a second solution is added to the chromatography medium to extract a second amount of the target molecule from the chromatography medium; and a chaotropic agent is added to the chromatography medium to extract a third amount of the target molecule from the chromatography medium, related to a method. and a flow-through is collected from the filter plate well, where the charged mass exhibits a protocol pH; a plurality of aliquots of a buffer containing the chromatography medium are added to obtain an eluate from the chromatography medium, where the buffer exhibits a buffer pH and a concentration of a kosmotropic salt decreases linearly over the plurality of aliquots, and a first amount of the target molecule is contained in the combined flow-through and eluate; a second solution is added to the chromatography medium to extract a second amount of the target molecule from the chromatography medium; and a chaotropic agent is added to the chromatography medium to extract a third amount of the target molecule from the chromatography medium, related to a method. and a flow-through is collected from the filter plate well, where the charged mass exhibits a protocol pH; a plurality of aliquots of a buffer containing the chromatography medium are added to obtain an eluate from the chromatography medium, where the buffer exhibits a buffer pH and a concentration of a kosmotropic salt decreases linearly over the plurality of aliquots, and a first amount of the target molecule is contained in the combined flow-through and eluate; a second solution is added to the chromatography medium to extract a second amount of the target molecule from the chromatography medium; and a chaotropic agent is added to the chromatography medium to extract a third amount of the target molecule from the chromatography medium, related to a method. and a flow-through is collected from the filter plate well, where the charged mass exhibits a protocol pH; a plurality of aliquots of a buffer containing the chromatography medium are added to obtain an eluate from the chromatography medium, where the buffer exhibits a buffer pH and a concentration of a kosmotropic salt decreases linearly over the plurality of aliquots, and a first amount of the target molecule is contained in the combined flow-through and eluate; a second solution is added to the chromatography medium to extract a second amount of the target molecule from the chromatography medium; and a chaotropic agent is added to the chromatography medium to extract a third amount of the target molecule from the chromatography medium, related to a method. and a flow-through is collected from the filter plate well, where the charged mass exhibits a protocol pH; a plurality of aliquots of a buffer containing the chromatography medium are added to obtain an eluate from the chromatography medium, where the buffer exhibits a buffer pH and a concentration of a kosmotropic salt decreases linearly over the plurality of aliquots, and a first amount of the target molecule is contained in the combined flow-through and eluate; a second solution is added to the chromatography medium to extract a second amount of the target molecule from the chromatography medium; and a chaotropic agent is added to the chromatography medium to extract a third amount of the target molecule from the chromatography medium, related to a method. and a flow-through is collected from the filter plate well, where the charged mass exhibits a protocol pH; a plurality of aliquots of a buffer containing the chromatography medium are added to obtain an eluate from the chromatography medium, where the buffer exhibits a buffer pH and a concentration of a kosmotropic salt decreases linearly over the plurality of aliquots, and a first amount of the target molecule is contained in the combined flow-through and eluate; a second solution is added to the chromatography medium to extract a second amount of the target molecule from the chromatography medium; and a chaotropic agent is added to the chromatography medium to extract a third amount of the target molecule from the chromatography medium, related to a method. and a flow-through is collected from the filter plate well, where the charged mass exhibits a protocol pH; a plurality of aliquots of a buffer containing the chromatography medium are added to obtain an eluate from the chromatography medium, where the buffer exhibits a buffer pH and a concentration of a kosmotropic salt decreases linearly over the plurality of aliquots, and a first amount of the target molecule is contained in the combined flow-through and eluate; a second solution is added to the chromatography medium to extract a second amount of the target molecule from the chromatography medium; and a chaotropic agent is added to the chromatography medium to extract a third amount of the target molecule from the chromatography medium, related to a method. and a flow-through is collected from the filter plate well, where the charged mass exhibits a protocol pH; a plurality of aliquots of a buffer containing the chromatography medium are added to obtain an eluate from the chromatography medium, where the buffer exhibits a buffer pH and a concentration of a kosmotropic salt decreases linearly over the plurality of aliquots, and a first amount of the target molecule is contained in the combined flow-through and eluate; a second solution is added to the chromatography medium to extract a second amount of the target molecule from the chromatography medium; and a chaotropic agent is added to the chromatography medium to extract a third amount of the target molecule from the chromatography medium, related to a method. and a flow-through is collected from the filter plate well, where the charged mass exhibits a protocol pH; a plurality of aliquots of a buffer containing the chromatography medium are added to obtain an eluate from the chromatography medium, where the buffer exhibits a buffer pH and a concentration of a kosmotropic salt decreases linearly over the plurality of aliquots, and a first amount of the target molecule is contained in the combined flow-through and eluate; a second solution is added to the chromatography medium to extract a second amount of the target molecule from the chromatography medium; and a chaotropic agent is added to the chromatography medium to extract a third amount of the target molecule from the chromatography medium, related to a method. and a flow-through is collected from the filter plate well, where the charged mass exhibits a protocol pH; a plurality of aliquots of a buffer containing the chromatography medium are added to obtain an eluate from the chromatography medium, where the buffer exhibits a buffer pH and a concentration of a kosmotropic salt decreases linearly over the plurality of aliquots, and a first amount of the target molecule is contained in the combined flow-through and eluate; a second solution is added to the chromatography medium to extract a second amount of the target molecule from the chromatography medium; and a chaotropic agent is added to the chromatography medium to extract a third amount of the target molecule from the chromatography medium, related to a method. BRIEF DESCRIPTION OF THE DRAWINGS

[0011]

[0011] The accompanying drawings are incorporated herein and form a part hereof, illustrating various exemplary embodiments and, together with the specification, serve to explain the principles of the disclosed embodiments. Any feature (e.g., composition, formulation, method, etc.) of the embodiments or examples described herein can be combined with any other embodiment or example, and all such combinations are included in the present disclosure. Further, the described systems and methods are not limited to any one aspect or its embodiments, and any combination of such aspects and embodiments is

[0011] The accompanying drawings are incorporated herein and form a part hereof, illustrating various exemplary embodiments and, together with the specification, serve to explain the principles of the disclosed embodiments. Any feature (e.g., composition, formulation, method, etc.) of the embodiments or examples described herein can be combined with any other embodiment or example, and all such combinations are included in the present disclosure. Further, the described systems and methods are not limited to any one aspect or its embodiments, and any combination of such aspects and embodiments is

[0011] The accompanying drawings are incorporated herein and form a part hereof, illustrating various exemplary embodiments and, together with the specification, serve to explain the principles of the disclosed embodiments. Any feature (e.g., composition, formulation, method, etc.) of the embodiments or examples described herein can be combined with any other embodiment or example, and all such combinations are included in the present disclosure. Further, the described systems and methods are not limited to any one aspect or its embodiments, and any combination of such aspects and embodiments is

[0011] The accompanying drawings are incorporated herein and form a part hereof, illustrating various exemplary embodiments and, together with the specification, serve to explain the principles of the disclosed embodiments. Any feature (e.g., composition, formulation, method, etc.) of the embodiments or examples described herein can be combined with any other embodiment or example, and all such combinations are included in the present disclosure. Further, the described systems and methods are not limited to any one aspect or its embodiments, and any combination of such aspects and embodiments is

[0011] The accompanying drawings are incorporated herein and form a part hereof, illustrating various exemplary embodiments and, together with the specification, serve to explain the principles of the disclosed embodiments. Any feature (e.g., composition, formulation, method, etc.) of the embodiments or examples described herein can be combined with any other embodiment or example, and all such combinations are included in the present disclosure. Further, the described systems and methods are not limited to any one aspect or its embodiments, and any combination of such aspects and embodiments is also included in the present disclosure. Further, the described systems and methods are not limited to any one aspect or its embodiments, and any combination of such aspects and embodiments is or is not limited to a permutation. For the sake of brevity, specific permutations and combinations are not separately discussed and / or not exemplified in this specification.

[0012]

Figure 1

[0012] FIG. 1 shows an exemplary method in accordance with aspects of the present disclosure in flowchart form.

[0013]

Figure 2

[0013] FIG. 2 shows a blot analysis of chromatography columns in various states of use in accordance with aspects of the present disclosure.

[0014]

Figure 3

[0014] FIG. 3 shows a blot analysis of chromatography columns in various states of use in accordance with aspects of the present disclosure.

[0015]

Figure 4

[0015] FIG. 4 shows an overlay of chromatography data from a number of processes in accordance with aspects of the present disclosure.

[0016]

Figure 5

[0016] FIG. 5 shows a blot analysis of chromatography columns in various states of use in accordance with aspects of the present disclosure.

[0017]

Figure 6

[0017] FIG. 6 shows a comparison of a column containing a hydrophobic interaction medium that has been subjected to a number of hydrophobic interaction chromatography cycles and a column containing unused hydrophobic interaction medium in accordance with aspects of the present disclosure.

[0018]

Figure 7A

[0018] Figures 7A and 7B show chromatograms of a regeneration process following hydrophobic interaction chromatography, which process includes the use of reverse osmosis deionized water.

Figure 7B

[0018] Figures 7A and 7B show chromatograms of a regeneration process following hydrophobic interaction chromatography, which process includes the use of reverse osmosis deionized water.

[0019]

Figure 8A

[0019] Figures 8A and 8B show further chromatograms of a regeneration process following hydrophobic interaction chromatography.

Figure 8B

[0019] Figures 8A and 8B show further chromatograms of a regeneration process following hydrophobic interaction chromatography.

[0020]

Figure 9

[0020] Figure 9 shows a chromatogram of a regeneration process using a guanidine HCl solution, according to an aspect of the present disclosure.

[0021]

Figure 10A

[0021] Figure 10A shows a chromatogram of a process including a number of regeneration solutions, according to an aspect of the present disclosure.

Figure 10B

[0022]

Figure 11

[0022] Figure 11 shows a chromatogram of a process in which a sodium hydroxide solution having a gradually increasing / gradually decreasing concentration is introduced into a column, according to an aspect of the present disclosure.

[0023]

Figure 12

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

[0024]

Figure 13A

[0024] Figures 13A and 13B are visual depictions of statistical analyses of various peaks of chromatograms showing regeneration processes according to aspects of the present disclosure.

Figure 13B

[0024] Figures 13A and 13B are visual depictions of statistical analyses of various peaks of chromatograms showing regeneration processes according to aspects of the present disclosure.

[0025]

Figure 14

[0025] Figure 14 shows overlaid chromatograms of multiple two-solution column regeneration processes that include either sodium hydroxide or sodium chloride and guanidine HCl according to aspects of the present disclosure.

[0026]

Figure 15

[0026] Figure 15 shows three chromatography columns to which solutions are applied according to aspects of the present disclosure.

[0027]

Figure 16

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

[0028]

Figure 17

[0028] Figures 17 and 18 show chromatograms of a control regeneration process and an experimental regeneration process, respectively, each including multiple regeneration solutions according to aspects of the present disclosure.

Figure 18

[0028] Figures 17 and 18 show chromatograms of a control regeneration process and an experimental regeneration process, each containing a number of regeneration solutions, according to an embodiment of the present disclosure.

[0029]

Figure 19

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

[0030]

Figure 20A - B

[0030] Figures 20A - 20C show chromatograms generated using a protocol that includes three different hydrophobic interaction chromatography media according to an embodiment of the present disclosure.

Figure 20C

[0030] Figures 20A - 20C show chromatograms generated using a protocol that includes three different hydrophobic interaction chromatography media according to an embodiment of the present disclosure.

[0031]

Figure 21A - B

[0031] Figures 21A and 21B show plots of boundary functions developed through the analysis of data generated during high - throughput screening and full - scale chromatography runs according to an embodiment of the present disclosure.

[0032]

Figure 22

[0032] Figure 22 shows an exemplary dynamic prediction model according to an embodiment of the present disclosure.

[0033]

Figure 23A - B

[0033] ]Figures 23A - 26C show chromatograms for a first target molecule generated using a number of hydrophobic interaction chromatography media, pH parameters, and stripping solutions, in accordance with aspects of the present disclosure.

Figure 23C

[0033] ]Figures 23A - 26C show chromatograms for a first target molecule generated using a number of hydrophobic interaction chromatography media, pH parameters, and stripping solutions, in accordance with aspects of the present disclosure.

Figure 24A - B

[0033] ]Figures 23A - 26C show chromatograms for a first target molecule generated using a number of hydrophobic interaction chromatography media, pH parameters, and stripping solutions, in accordance with aspects of the present disclosure.

Figure 24C

[0033] ]Figures 23A - 26C show chromatograms for a first target molecule generated using a number of hydrophobic interaction chromatography media, pH parameters, and stripping solutions, in accordance with aspects of the present disclosure.

Figure 25A - B

[0033] ]Figures 23A - 26C show chromatograms for a first target molecule generated using a number of hydrophobic interaction chromatography media, pH parameters, and stripping solutions, in accordance with aspects of the present disclosure.

Figure 25C

[0033] ]Figures 23A - 26C show chromatograms for a first target molecule generated using a number of hydrophobic interaction chromatography media, pH parameters, and stripping solutions, in accordance with aspects of the present disclosure.

Figure 26A - B

[0033] ]Figures 23A - 26C show chromatograms for a first target molecule generated using a number of hydrophobic interaction chromatography media, pH parameters, and stripping solutions, in accordance with aspects of the present disclosure.

Figure 26C

[0033] ]Figures 23A - 26C show chromatograms for a first target molecule generated using a number of hydrophobic interaction chromatography media, pH parameters, and stripping solutions, in accordance with aspects of the present disclosure.

[0034]

Figure 27A - B

[0034] Figures 27A - 28D show chromatograms for a second target molecule generated using a number of hydrophobic interaction chromatography media, pH parameters, and stripping solutions, in accordance with aspects of the present disclosure.

Figure 27C - D

[0034] Figures 27A - 28D show chromatograms for a second target molecule generated using a number of hydrophobic interaction chromatography media, pH parameters, and stripping solutions, in accordance with aspects of the present disclosure.

Figure 28A - B

[0034] Figures 27A - 28D show chromatograms for a second target molecule generated using a number of hydrophobic interaction chromatography media, pH parameters, and stripping solutions, in accordance with aspects of the present disclosure.

Figure 28C - D

[0034] Figures 27A - 28D show chromatograms for a second target molecule generated using a number of hydrophobic interaction chromatography media, pH parameters, and stripping solutions, in accordance with aspects of the present disclosure.

DETAILED DESCRIPTION

[0035]

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

[0036]

[0036] As used herein, the term "comprises" “comprising” or any variant thereof refers to a process, method, article, or apparatus that includes a list of elements, and that the apparatus includes not only these elements but also other elements inherent to such a process, method, article, or apparatus that are not expressly listed or that are otherwise not explicitly stated. Thus, it is intended to include non-exclusive inclusion. The term “exemplary” is used in the sense of “example” rather than “ideal”. With respect to the terms “for example”, “such as”, and their grammatical equivalents, the phrase “and without limitation” is understood to follow unless expressly stated otherwise.

[0037]

[0037] As used herein, the term “about” means taking into account variations due to experimental error. When applied to a numerical value, the term “about” can indicate a variation of + / - 5% from the disclosed numerical value unless different variations are specified. As used herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly indicates otherwise. Further, all ranges are understood to include their endpoints, e.g., 1 centimeter (cm) to 5 cm includes the lengths of 1 cm, 5 cm, and all distances between 1 cm and 5 cm.

[0038]

[0038] All numerical values disclosed herein (including all disclosed values, limits, and ranges) can have a variation of + / - 5% from the disclosed numerical value unless different variations are specified.

[0039]

[0039] The term “polypeptide” as used herein refers to a covalent linkage Refers to any amino acid polymer having more than about 20 amino acids joined together. Protein contains one or more amino acid polymer chains (e.g., polypeptides). Thus, a polype ptide may be a protein, and a protein may contain a number of polypeptides to form one functional biomolecule.

[0040]

[0040] Post-translational modifications are capable of modifying or altering 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 polype ptides, immunoglobulins, proteins, cofactors, substrates, etc. In addition to disulfide bond formation, proteins can be subject to 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 a glycosyl group to arginine, asparagine, cysteine, hydroxylysine, serine, threonine, tyro sine, and / or tryptophan), and phosphorylation (i.e., addition of a phosphate group to serine, threonine, tyrosine, and / or histidine). Post-translational modifications can affect hydrophobicity, electrostatic surface properties, or other properties that determine surface -to-surface interactions in which the polypeptide is involved.

[0041]

[0041] As used herein, the term "protein" includes biotherapeutic proteins Proteins, recombinant proteins, trap proteins, and other F used in research or therapy c 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. are included. The protein of interest (POI) can include any polypeptide or protein that is desirably isolated, purified, or prepared in another manner. The POI can include polypeptides produced by cells, including antibodies.

[0042]

[0042] As used herein, the term "antibody" refers to an immunoglobulin composed of four polypeptide chains, two heavy (H) chains and two light (L) chains internally linked by disulfide bonds. Typically, an antibody has a molecular weight greater than 100 kDa, for example, between 130 kDa and 200 kDa, such as about 140 kDa, 145 kDa, 150 kDa, 15 5 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 and VL regions can be further divided into hypervariable regions called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxy terminus: FR1, C DR1, FR2, CDR2, FR3, CDR3, FR4. DR1, FR2, CDR2, FR3, CDR3, FR4, which are arranged in the following order from the amino terminus to the carboxy terminus: FR1, C DR1, FR2, CDR2, FR3, CDR3, FR4 (the heavy chain CDRs may be abbreviated as HCDR1, H CDR2 and HCDR3; the light chain CDRs may be abbreviated as LCDR1, LCDR2 and LCDR3).

[0043]

[0043] For example, the immunoglobulin class called immunoglobulin G (IgG) is common in human serum and contains four polypeptide chains, two light chains and two heavy chains. Each light chain is linked to one heavy chain through one cysteine disulfide bond, and the two heavy chains are linked to each other through two cysteine 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 sub class has a different constant region and, as a result, may have different effector functions. In some embodiments described herein, the POI may include a target polypeptide that includes IgG. In at least one embodiment, the target polypeptide includes IgG4.

[0044]

[0044] The term "antibody" also includes, as used herein, antigen-binding fragments of full-length antibody molecules. Terms such as "antigen-binding portion of an antibody", "antigen-binding fragment of an antibody", etc., as used herein, refer to any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of an antibody can be obtained, for example, by any suitable standard techniques, such as proteolytic digestion, or manipulation and expression of DNA It can be obtained from full-length antibody molecules using recombinant gene manipulation techniques involving the present. Such DNA is known and / or can be readily obtained, for example, from commercial sources, DNA libraries (e.g., phage -antibody libraries included), or can be synthesized, and it is possible. The DNA can be sequenced, chemically manipulated, or using molecular biology techniques to, for example, arrange one or more variable and / or constant domains in an appropriate conformation, or introduce codons, generate cysteine residues, modify amino acids, add or delete, etc., and can be manipulated.

[0045]

[0045] Production systems based on recombinant cells, such as insect baculovirus systems, yeast systems (e.g., Pichia species), or mammalian systems (e.g., CHO cells and CHO derivatives such as CHO-K1 cells) can be used to produce target molecules (e.g., target polype ptides / antibodies). The term "cell" includes any cell suitable for expressing a recombinant nucleic acid sequence. The cell can be prokaryotic and eukaryotic (unicellular or multicellular), bacterial cells (e.g., E. coli, Bacillus species, Streptomyces species strains), mycobacterial cells, fungal cells, yeast cells (e.g., S. cerevisia e, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Tric hoplusiani, etc.), non-human animal cells, human cells, or cell fusions, such as ha bridomas or quadromas. In some embodiments, the cell is human, ​​It may be a monkey, ape, hamster, rat, or mouse cell. In some embodiments the cell may be a eukaryotic cell and may be selected from the following cells, CH O (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g ., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI 38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21 ), Jurkat, Daudi, A431 (epithelial), CV-1, U937, 3T3, L cell , C127 cell, SP2 / 0, NS-0, MMT 060562, Sertoli cell, BR L 3A cell, HT1080 cell, myeloma cell, tumor cell, and cells derived from the aforementioned cells . In some embodiments, the cell may contain one or more viral genes , e.g., retinal cells that express viral genes (e.g., PER.C6 TM cells) may also be used .

[0046]

[0046] The term "target molecule" as used herein refers to a target polypeptide ptide (e.g., an antibody, antibody fragment, or other protein or protein fragment), or other molecules that are produced, isolated, purified, and / or intended to be included in a pharmaceutical product (e.g., adeno-associated virus (AAV) or other molecules for therapeutic use). The methods according to the present disclosure may refer to target polypeptides, but these may also be applicable to other target molecules. For example, AAV may be prepared according to appropriate methods (e.g., depth filtration, affinity chromatography, etc.), and a mixture containing AAV may be used . may be subjected to the methods according to the present disclosure. Before or after one or more of the methods of the present disclosure, the mixture containing AAV may be subjected to further treatment (e.g., removal of "empty cassettes" or AAV that does not contain the target sequence). After such methods, the mixture containing AAV may be subjected to further treatment (e.g., removal of "empty cassettes" or AAV that does not contain the target sequence). After such methods, the mixture containing AAV may be subjected to further treatment (e.g., removal of "empty cassettes" or AAV that does not contain the target sequence).

[0047]

[0047] In some embodiments, the target molecule is an antibody, a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecific antibody, a bispecific antibody, an antigen-binding antibody fragment, a single-chain antibody, a diabody, a triabody or a tetrabody, a Fab fragment or an F( ab’)2 fragment, an IgD antibody, an IgE antibody, an IgM antibody, an IgG antibody, an IgG1 antibody, I gG2 antibody, IgG3 antibody, or IgG4 antibody. In one embodiment, the antibody is I gG1 antibody. In one embodiment, the antibody is IgG2 antibody. In one embodiment, the antibody is IgG4 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG4 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG1 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG1 / IgG4 antibody.

[0048]

[0048] In some embodiments, the target molecule (e.g., an antibody) is an anti-programmed cell death 1 antibody (e.g., an anti-PD1 antibody as described in US Patent Application Publication No. US2015 / 0203579A1), an anti-programmed cell death ligand 1 (e.g., an anti-PD-L1 antibody as described in US Patent Application Publication No. US2015 / 0203580A1), an anti-Dll 4 antibody, an anti-angiopoietin-2 antibody (e.g., an anti-ANG2 antibody as described in US Patent No. 9,402,898), an anti-angiopoietin-like 3 antibody (e.g., US Patent No. 9,01 4 antibody, an anti-angiopoietin-2 antibody (e.g., an anti-ANG2 antibody as described in US Patent No. 9,402,898), an anti-angiopoietin-like 3 antibody (e.g., US Patent No. 9,01 4 antibody, an anti-angiopoietin-2 antibody (e.g., an anti-ANG2 antibody as described in US Patent No. 9,402,898), an anti-angiopoietin-like 3 antibody (e.g., US Patent No. 9,01 an anti-AngPtl3 antibody as described in No. 8,356, an anti-platelet-derived growth factor receptor antibody (e.g., an anti-PDGFR antibody as described in U.S. Patent No. 9,265,827), an anti-prolactin receptor antibody (e.g., an anti-PRLR antibody as described in U.S. Patent No. 9,302,015), an anti-complement 5 antibody (e.g., an anti-C5 antibody as described in U.S. Patent Application Publication No. US2015 / 03131 (e.g., an anti-EGFR antibody as described in U.S. Patent No. 9,132,192 or an anti-EGFRvIII antibody as described in U.S. Patent Application Publication No. US2015 / 0259423A1), an anti-proprotein convertase subtilisin / kexin type 9 antibody (e.g., an anti-PCSK9 antibody as described in U.S. Patent No. 8,062,640 or U.S. Patent Application Publication No. US2014 / 00447 30A1), an anti-growth and differentiation factor-8 antibody (e.g., an anti-GDF8 antibody, also known as an anti-myostatin antibody, as described in U.S. Patent No. 8,871,209 or 9,260,515), an anti-glucagon receptor (e.g., an anti-GCGR antibody as described in U.S. Patent Application Publication No. US2015 / 0337045A1 or No. US2016 / 007 5778A1), an anti-VEGF antibody, an anti-IL1R antibody , an interleukin 4 receptor antibody (e.g., an anti-IL-4R antibody as described in U.S. Patent Application Publication No. US2014 / 02716 81A1 or U.S. Patent No. 8,735,095 or 8,945,559), an anti-interleukin 6 receptor antibody (e.g., an anti-IL6R antibody as described in U.S. Patent No. 7,582,298, 8,043,617 or 9,173,880), an anti-interleukin 33 (e.g., in U.S. Patent Application Publication No. U S2014 / 0271681A1 or U.S. Patent No. 8,735,095 or 8,945,559), an anti-interleukin 6 receptor antibody (e.g., an anti-IL6R antibody as described in U.S. Patent No. 7,582,298, 8,043,617 or 9,173,880), an anti-interleukin 33 (e.g., in U.S. Patent Application Publication No. U S2014 / 0271681A1 or U.S. Patent No. 8,735,095 or 8,945,559), an anti-interleukin 6 receptor antibody (e.g., an anti-IL6R antibody as described in U.S. Patent No. 7,582,298, 8,043,617 or 9,173,880), an anti-interleukin 33 (e.g., in U.S. Patent Application Publication No. U as described in S2014 / 0271658A1 or US2014 / 0271642A1 such as an anti-IL33 antibody), an anti-respiratory syncytial virus antibody (e.g., as described in US Patent Application Publication an anti-RSV antibody as described in US2014 / 0271653A1), an anti-surface antigen class 3 (e.g., as described in US Patent Application Publications US2014 / 0088295A1 and US2 0150266966A1, and US Application No. 62 / 222,605 such as an anti-CD3 antibody), an anti-surface antigen class 20 (e.g., as described in US Patent Application Publication US2014 / 0088295A1 and US20150266966A1, and US Patent No. 7,879,984 such as an anti-CD20 antibody), an anti-surface antigen class 48 (e.g., as described in US Patent No. 9,228,014 such as an anti-CD48 antibody), an anti-Fel d 1 antibody (e.g., as described in US Patent No. 9,079,948), an anti-Middle East respiratory syndrome virus (e.g., an anti-MERS antibody), an anti-Ebola virus antibody (e.g., Regen eron's REGN-EB3), an anti-CD19 antibody, an anti-CD28 antibody, an anti-IL1 antibody, an anti-I L2 antibody, an anti-IL3 antibody, an anti-IL4 antibody, an anti-IL5 antibody, an anti-IL6 antibody an anti-Erb3 antibody, an anti-dengue virus antibody, an anti-lymphocyte activation gene 3 (e.g., an anti-LAG3 antibody or an anti-CD223 antibody) and an anti-activin A antibody. Each of the US patents and US patent publications referred to in this paragraph is hereby incorporated by reference in its entirety into this specification

[0049]

[0049] In some embodiments, the target molecule (e.g., a bispecific antibody) is an anti-C D3x anti-CD20 bispecific antibody, an anti-CD3x anti-mucin 16 bispecific antibody, and an anti-C​ It is selected from the group consisting of D3x anti-prostate specific membrane antigen bispecific antibodies. In some embodiments the target molecule is selected from the group consisting of alemtuzumab, sarilumab, fasinumab, nesvacumab, dupilumab, tregogumab, evinacumab, and linucumab.

[0050]

[0050] In some embodiments, the target molecule is a recombinant protein (e.g., an Fc fusion protein) containing an Fc portion and another domain. In some embodiments the Fc fusion protein is a receptor Fc fusion protein and contains one or more extracellular domains of a receptor coupled to the Fc portion. In some embodiments the Fc portion includes the CH2 and CH3 domains of IgG following the hinge region. In some embodiments, the receptor Fc fusion protein contains two or more distinct receptor chains that bind to either one ligand or multiple ligands. For example, the Fc fusion protein is a TRAP protein, such as an IL-1 trap (e.g., containing an IL-1RAcP ligand-binding region fused to the extracellular region of Il-1R1 fused to the Fc of hIgG1 rilonacept; see U.S. Patent No. 6,927,004, which is hereby incorporated by reference in its entirety), or a VEGF trap (e.g., containing the Ig domain 2 of VEGF receptor Flt1 fused to the Ig domain 3 of VEGF receptor Flk1 fused to the Fc of hIgG1 aflibercept or ziv-aflibercept; both see U.S. Patent Nos. 7,087,411 and 7,279,1 59, which are hereby incorporated by reference in their entirety). In other embodiments, the Fc fusion protein is a ScF ​​​​​​​​ v-Fc fusion protein, which comprises a variable heavy chain fragment of an antibody coupled to an Fc portion, and It contains one or more antigen binding domains, such as a variable light chain fragment.

[0051]

[0051] Embodiments of the present disclosure may be used in the preparation of various pharmaceutical products or in the purification of various pharmaceutical products. In some embodiments, the present disclosure may be used to develop methods for producing antigen-binding molecules. In some embodiments, the vector may be useful for the preparation or purification of pharmaceutical products that contain the vector or AAV. In particular, embodiments of the present disclosure are directed to, for example, aflibercept, alirocumab, abyssin, Palpegol, bevacizumab, brolucizumab, conbercept, duprimab, evolocum Mab, tocilizumab, certolizumab, abatacept, rituximab, infliximab , ranibizumab, sarilumab, adalimumab, anakinra, trastuzumab, pegfil Grastim, interferon beta-1a, insulin glargine [rDNA origin], Poetin alfa, darbepoetin, filigrastim, golimumab, etanercept, Any of the above antigen-binding fragments, or combinations of such binding domains, e.g., VE Preparation of pharmaceutical products containing components such as bispecific antibodies against GF or angiopoietin-2 may be suitable for use in manufacturing.

[0052] The term "hydrophobic interaction media" or " "HIC media" refers to a support structure and a hydrophobic portion. It refers to a combination of hydrophobic moieties, where the hydrophobic moieties are anchored to a supporting structure. The medium can be in the form of a chromatography medium, for example, in the form of beads or other particles held in a packed bed column format, in the form of a membrane, or in any form that can contain a liquid including the target protein and contaminants. Thus, the support structure includes agarose beads (such as Sepharose), silica beads, cellulose membranes, cellulose beads, hydrophilic polymer beads, resins, and the like. The hydrophobic moiety binds to hydrophobic molecules and the hydrophobic surfaces of proteins. The degree of hydrophobicity of the medium can be adjusted by selecting the hydrophobic moiety. Hydrophobic interaction media are used in a process known as hydrophobic interaction chromatography (HIC) to separate target molecules, such as the target protein or other molecules, from product and process related contaminants associated with the contaminants. When producing and / or purifying the target molecule from host cells, some products and process related components that must ultimately be separated from the target molecule are referred to as host cell proteins (HCP) and cellular debris. In some examples, a mixture containing the target molecule and other components is applied to the HIC medium in a buffer designed to facilitate the binding of hydrophobic groups in the target molecule to the hydrophobic moiety of the HIC medium. Such a mixture can be referred to as a "load mass". HIC is a negative load mass load mass load mass load mass load mass load mass load mass load mass load mass load mass load mass load mass load mass load mass load mass It should be noted that the text seems to be incomplete or have some formatting issues in the original, especially the repeated and somewhat unclear "load mass" statements. The translation has been done as accurately as possible based on the provided text.Target molecules that undergo separation during the load, wash, or regeneration phase It takes advantage of the hydrophobic differences between the target molecule(s) and the impurities. The impurities are bound to the HIC medium while the impurities are separated into the flow through. In addition, the target molecules bind to the HIC medium, whereas HCPs and cell debris cannot bind and flow through the medium. In either case, the present disclosure provides a method for determining whether a target molecule is a target of hydrophobic interaction. The present invention is applicable to the above-mentioned compounds (with or without being attached to a moiety).

[0053] After use in the purification / collection of target molecules, the HIC medium is periodically stripped ( As used herein, the term "stripped" or "regenerated" refers to a "Stripping" and "regenerating" are used interchangeably and / or or in combination to remove any residual components of the load mass from the HIC media after the purification cycle. Any residual components of a load mass were removed and H was used for the subsequent purification cycle. This refers to a process designed to prepare an IC medium. For example, The target fraction is separated from the payload mass, including the main cellular material (e.g., host cell debris, host cell proteins, etc.). After isolating or purifying the molecule of interest and eluting it from the HIC device, the HIC medium is reused. and remove residual material (e.g., host cell material, host cell proteins, lipids, residual Remove residual polypeptides, aggregated proteins, nucleic acids, biomolecules, etc. from different load masses and It is possible to prepare HIC media for use in purifying various molecules. In some embodiments, the regeneration of the HIC medium disrupts the hydrophobic interaction between the residual host cell material and / or target moiety and the HIC medium and / or denatures the residual host cell material. The regeneration can be performed between HIC cycles to "reset" the HIC medium without the need for a longer cleaning process. In some embodiments, performing the regeneration between HIC cycles can prevent or reduce discoloration of the HIC medium over time. In some embodiments, the regeneration process according to the present disclosure can be performed, for example, for about 5 minutes to about 1 hour, such as about 10 minutes to about 1 hour, about 10 minutes to about 45 minutes, or about 10 minutes to about 30 minutes. Preferably, the regeneration of the HIC medium can be completed without subjecting the HIC medium to a more robust cleaning solution that can have an undesirable effect or create further concerns. The regeneration process can be configured without particular emphasis on the removal of, for example, bacteria, fungi, or other microorganisms from the chromatography medium. The regeneration of the HIC medium can include disrupting the hydrophobic interaction between the residual host cell material and / or target moiety and the HIC medium and / or denaturing the residual host cell material. The regeneration is performed between HIC cycles and can "reset" the HIC medium without the need for a longer cleaning process. In some embodiments, performing the regeneration between HIC cycles can prevent or reduce discoloration of the HIC medium over time. In some embodiments, the regeneration process according to the present disclosure can be performed, for example, for about 5 minutes to about 1 hour, such as about 10 minutes to about 1 hour, about 10 minutes to about 45 minutes, or about 10 minutes to about 30 minutes. Preferably, the regeneration of the HIC medium can be completed without subjecting the HIC medium to a more robust cleaning solution that can have an undesirable effect or create further concerns. The regeneration process can be configured without particular emphasis on the removal of, for example, bacteria, fungi, or other microorganisms from the chromatography medium. "Stripping" and "regeneration" can be distinguished from, for example, "cleaning" of the chromatography medium. Cleaning can include a process intended to fully disinfect and / or decontaminate the chromatography medium, chromatography apparatus, and / or experimental setting. For example, the cleaning process can include concentrations of antibiotics, antifungal agents, or other forms of antimicrobial solutions, other disinfecting solutions, sterilization methods, etc., intended to sanitize and / or sterilize the chromatography medium or chromatography apparatus. The regeneration process can be configured without particular emphasis on the removal of, for example, bacteria, fungi, or other microorganisms from the chromatography medium.

[0054]

[0054] "Stripping" and "regeneration" can be distinguished from, for example, "cleaning" of the chromatography medium. Cleaning can include a process intended to fully disinfect and / or decontaminate the chromatography medium, chromatography apparatus, and / or experimental setting. For example, the cleaning process can include concentrations of antibiotics, antifungal agents, or other forms of antimicrobial solutions, other disinfecting solutions, sterilization methods, etc., intended to sanitize and / or sterilize the chromatography medium or chromatography apparatus. The regeneration process can be configured without particular emphasis on the removal of, for example, bacteria, fungi, or other microorganisms from the chromatography medium. ​​​It may include use in the amount of. In contrast, regeneration may, in some cases, include use of a solution having antimicrobial properties, but the main intention of regeneration may be to remove residual components of the loading mass from the chromatographic medium after the purification process. In some embodiments, additional protection or treatment may be required during and / or after the cleaning process to ensure that the subsequent chromatographic cycle is not affected by the disinfecting, sanitizing, antimicrobial, or antibiotic solution used during the cleaning process. In many cases, the cleaning process may be longer than the regeneration process (e.g., exceeding about 1 hour). The separation of molecules in the HIC medium can be achieved, for example, by exposing the HIC medium to a loading mass having a high salt concentration to increase the hydrophobic interaction between the HIC medium and the target molecules in the loading mass, and then passing a volume of a solution (e.g., a buffer) having a decreased or decreasing salt concentration through the HIC medium to reverse the hydrophobic interaction. Thus, it is conventionally understood that fewer substances bind to the HIC medium under low- or no-salt conditions. However, surprisingly, it has been discovered that certain types of HIC media exhibit increased binding (e.g., hydrophobic interaction) to residual substances (e.g., host cell proteins) under no-salt conditions. Furthermore, some proteins (e.g., monoclonal antibodies) bind to a certain type of HIC medium (e.g., Capto from the chromatographic medium. In some embodiments, additional protection or treatment may be required during and / or after the cleaning process to ensure that the subsequent chromatographic cycle is not affected by the disinfecting, sanitizing, antimicrobial, or antibiotic solution used during the cleaning process. In many cases, the cleaning process may be longer than the regeneration process (e.g., exceeding about 1 hour). The separation of molecules in the HIC medium can be achieved, for example, by exposing the HIC medium to a loading mass having a high salt concentration to increase the hydrophobic interaction between the HIC medium and the target molecules in the loading mass, and then passing a volume of a solution (e.g., a buffer) having a decreased or decreasing salt concentration through the HIC medium to reverse the hydrophobic interaction. Thus, it is conventionally understood that fewer substances bind to the HIC medium under low- or no-salt conditions. However, surprisingly, it has been discovered that certain types of HIC media exhibit increased binding (e.g., hydrophobic interaction) to residual substances (e.g., host cell proteins) under no-salt conditions. Furthermore, some proteins (e.g., monoclonal antibodies) bind to a certain type of HIC medium (e.g., Capto from the chromatographic medium. In some embodiments, additional protection or treatment may be required during and / or after the cleaning process to ensure that the subsequent chromatographic cycle is not affected by the disinfecting, sanitizing, antimicrobial, or antibiotic solution used during the cleaning process. In many cases, the cleaning process may be longer than the regeneration process (e.g., exceeding about 1 hour). from the chromatographic medium. In some embodiments, additional protection or treatment may be required during and / or after the cleaning process to ensure that the subsequent chromatographic cycle is not affected by the disinfecting, sanitizing, antimicrobial, or antibiotic solution used during the cleaning process. In many cases, the cleaning process may be longer than the regeneration process (e.g., exceeding about 1 hour). from the chromatographic medium. In some embodiments, additional protection or treatment may be required during and / or after the cleaning process to ensure that the subsequent chromatographic cycle is not affected by the disinfecting, sanitizing, antimicrobial, or antibiotic solution used during the cleaning process. In many cases, the cleaning process may be longer than the regeneration process (e.g., exceeding about 1 hour). from the chromatographic medium. In some embodiments, additional protection or treatment may be required during and / or after the cleaning process to ensure that the subsequent chromatographic cycle is not affected by the disinfecting, sanitizing, antimicrobial, or antibiotic solution used during the cleaning process. In many cases, the cleaning process may be longer than the regeneration process (e.g., exceeding about 1 hour).

[0055]

[0055] The separation of molecules in the HIC medium can be achieved, for example, by exposing the HIC medium to a loading mass having a high salt concentration to increase the hydrophobic interaction between the HIC medium and the target molecules in the loading mass, and then passing a volume of a solution (e.g., a buffer) having a decreased or decreasing salt concentration through the HIC medium to reverse the hydrophobic interaction. Thus, it is conventionally understood that fewer substances bind to the HIC medium under low- or no-salt conditions. However, surprisingly, it has been discovered that certain types of HIC media exhibit increased binding (e.g., hydrophobic interaction) to residual substances (e.g., host cell proteins) under no-salt conditions. Furthermore, some proteins (e.g., monoclonal antibodies) bind to a certain type of HIC medium (e.g., Capto The separation of molecules in the HIC medium can be achieved, for example, by exposing the HIC medium to a loading mass having a high salt concentration to increase the hydrophobic interaction between the HIC medium and the target molecules in the loading mass, and then passing a volume of a solution (e.g., a buffer) having a decreased or decreasing salt concentration through the HIC medium to reverse the hydrophobic interaction. Thus, it is conventionally understood that fewer substances bind to the HIC medium under low- or no-salt conditions. However, surprisingly, it has been discovered that certain types of HIC media exhibit increased binding (e.g., hydrophobic interaction) to residual substances (e.g., host cell proteins) under no-salt conditions. Furthermore, some proteins (e.g., monoclonal antibodies) bind to a certain type of HIC medium (e.g., Capto The separation of molecules in the HIC medium can be achieved, for example, by exposing the HIC medium to a loading mass having a high salt concentration to increase the hydrophobic interaction between the HIC medium and the target molecules in the loading mass, and then passing a volume of a solution (e.g., a buffer) having a decreased or decreasing salt concentration through the HIC medium to reverse the hydrophobic interaction. Thus, it is conventionally understood that fewer substances bind to the HIC medium under low- or no-salt conditions. However, surprisingly, it has been discovered that certain types of HIC media exhibit increased binding (e.g., hydrophobic interaction) to residual substances (e.g., host cell proteins) under no-salt conditions. Furthermore, some proteins (e.g., monoclonal antibodies) bind to a certain type of HIC medium (e.g., Capto The separation of molecules in the HIC medium can be achieved, for example, by exposing the HIC medium to a loading mass having a high salt concentration to increase the hydrophobic interaction between the HIC medium and the target molecules in the loading mass, and then passing a volume of a solution (e.g., a buffer) having a decreased or decreasing salt concentration through the HIC medium to reverse the hydrophobic interaction. Thus, it is conventionally understood that fewer substances bind to the HIC medium under low- or no-salt conditions. However, surprisingly, it has been discovered that certain types of HIC media exhibit increased binding (e.g., hydrophobic interaction) to residual substances (e.g., host cell proteins) under no-salt conditions. Furthermore, some proteins (e.g., monoclonal antibodies) bind to a certain type of HIC medium (e.g., Capto The separation of molecules in the HIC medium can be achieved, for example, by exposing the HIC medium to a loading mass having a high salt concentration to increase the hydrophobic interaction between the HIC medium and the target molecules in the loading mass, and then passing a volume of a solution (e.g., a buffer) having a decreased or decreasing salt concentration through the HIC medium to reverse the hydrophobic interaction. Thus, it is conventionally understood that fewer substances bind to the HIC medium under low- or no-salt conditions. However, surprisingly, it has been discovered that certain types of HIC media exhibit increased binding (e.g., hydrophobic interaction) to residual substances (e.g., host cell proteins) under no-salt conditions. Furthermore, some proteins (e.g., monoclonal antibodies) bind to a certain type of HIC medium (e.g., Capto The separation of molecules in the HIC medium can be achieved, for example, by exposing the HIC medium to a loading mass having a high salt concentration to increase the hydrophobic interaction between the HIC medium and the target molecules in the loading mass, and then passing a volume of a solution (e.g., a buffer) having a decreased or decreasing salt concentration through the HIC medium to reverse the hydrophobic interaction. Thus, it is conventionally understood that fewer substances bind to the HIC medium under low- or no-salt conditions. However, surprisingly, it has been discovered that certain types of HIC media exhibit increased binding (e.g., hydrophobic interaction) to residual substances (e.g., host cell proteins) under no-salt conditions. Furthermore, some proteins (e.g., monoclonal antibodies) bind to a certain type of HIC medium (e.g., Capto The separation of molecules in the HIC medium can be achieved, for example, by exposing the HIC medium to a loading mass having a high salt concentration to increase the hydrophobic interaction between the HIC medium and the target molecules in the loading mass, and then passing a volume of a solution (e.g., a buffer) having a decreased or decreasing salt concentration through the HIC medium to reverse the hydrophobic interaction. Thus, it is conventionally understood that fewer substances bind to the HIC medium under low- or no-salt conditions. However, surprisingly, it has been discovered that certain types of HIC media exhibit increased binding (e.g., hydrophobic interaction) to residual substances (e.g., host cell proteins) under no-salt conditions. Furthermore, some proteins (e.g., monoclonal antibodies) bind to a certain type of HIC medium (e.g., Capto The separation of molecules in the HIC medium can be achieved, for example, by exposing the HIC medium to a loading mass having a high salt concentration to increase the hydrophobic interaction between the HIC medium and the target molecules in the loading mass, and then passing a volume of a solution (e.g., a buffer) having a decreased or decreasing salt concentration through the HIC medium to reverse the hydrophobic interaction. Thus, it is conventionally understood that fewer substances bind to the HIC medium under low- or no-salt conditions. However, surprisingly, it has been discovered that certain types of HIC media exhibit increased binding (e.g., hydrophobic interaction) to residual substances (e.g., host cell proteins) under no-salt conditions. Furthermore, some proteins (e.g., monoclonal antibodies) bind to a certain type of HIC medium (e.g., Capto The separation of molecules in the HIC medium can be achieved, for example, by exposing the HIC medium to a loading mass having a high salt concentration to increase the hydrophobic interaction between the HIC medium and the target molecules in the loading mass, and then passing a volume of a solution (e.g., a buffer) having a decreased or decreasing salt concentration through the HIC medium to reverse the hydrophobic interaction. Thus, it is conventionally understood that fewer substances bind to the HIC medium under low- or no-salt conditions. However, surprisingly, it has been discovered that certain types of HIC media exhibit increased binding (e.g., hydrophobic interaction) to residual substances (e.g., host cell proteins) under no-salt conditions. Furthermore, some proteins (e.g., monoclonal antibodies) bind to a certain type of HIC medium (e.g., Capto The separation of molecules in the HIC medium can be achieved, for example, by exposing the HIC medium to a loading mass having a high salt concentration to increase the hydrophobic interaction between the HIC medium and the target molecules in the loading mass, and then passing a volume of a solution (e.g., a buffer) having a decreased or decreasing salt concentration through the HIC medium to reverse the hydrophobic interaction. Thus, it is conventionally understood that fewer substances bind to the HIC medium under low- or no-salt conditions. However, surprisingly, it has been discovered that certain types of HIC media exhibit increased binding (e.g., hydrophobic interaction) to residual substances (e.g., host cell proteins) under no-salt conditions. Furthermore, some proteins (e.g., monoclonal antibodies) bind to a certain type of HIC medium (e.g., Capto TM It cannot be denatured on phenyl (High Sub) medium (GE Healthcare Life Sciences). It has been found that once eluted from the HIC medium, proteins are able to retain their native conformation. These proteins in the denatured state can revert to their native configurations. The quality may be, for example, reverse osmosis deionized water (RODI), 1N sodium hydroxide, and / or 2 It is possible that they are not removed from the HIC medium by the regeneration treatment with 0% ethanol. In this case, the elution of molecules from the HIC medium is assumed to be dependent on pH and / or conductivity. It is determined.

[0056] Aspects of the present disclosure include a regeneration solution and a clotting method for utility including ease of regeneration. Regarding evaluation of matographic media.

[0057] In some embodiments of the present disclosure, the regenerating solution may be an alkaline solution. In some embodiments of the present disclosure, the high pH is a contributing factor in the effectiveness of the regeneration solution. It is intended that this may be a driving factor; however, some It is further contemplated that in the case of, the effectiveness of high pH may be offset by high ionic strength. Thus, in some embodiments, the effectiveness of the regenerating solution is such that the conductivity is low but not zero. This can be driven by high pH in combination with high solubility (see, for example, the experimental (See Example 14.) For example, in some embodiments, the regeneration solution may be about 8 to 100 mL of water. In some embodiments, the pH may be about 14, for example, between about 10 and about 14. Raw solutions may generally exhibit low conductivity. For example, in some embodiments, regeneration The solution has a conductivity between about 0.5 mS / cm and about 10 mS / cm, for example 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 / cm and about 1.6 mS / cm, between about 0.8 mS / cm and about 1.6 mS / cm, about 0.5 mS / cm, about 1.0 mS / cm, about 1.5 mS / cm, about 2.0 mS / cm, about 2.5 mS / cm, about 3 mS / cm, about 3.5 mS / cm , about 4.0 mS / cm, about 4.5 mS / cm, or about 5.0 mS / cm.

[0058]

[0058] In some embodiments, the regeneration solution is an alkaline solution having a concentration of, for example, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, Tris, other alkaline solutions, or a combination thereof. In some embodiments, the regeneration solution is between about 0.1 mM and about 50 mM, for example 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, between about 0.1 mM and about 10 mM, between about 0.1 mM and about 5 mM, between about 0.1 mM and about 2 .5 mM, between about 1 mM and about 10 mM, between about 1 mM and about 7 mM, between about 2.5 mM and about 5 mM, or between about 2.5 mM and about 7 mM, for example 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 total dissolved salt concentration.

[0059] ​​​

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

[0060]

[0060] The volume of the regeneration solution used in accordance with the present disclosure can be any suitable volume. For example, in some embodiments where the loaded mass is applied to the HIC medium in a chromatography column, the volume of the regeneration solution used in accordance with the present disclosure can be measured in column volumes (CV). In some embodiments, for example, a method of regenerating a HIC medium in a chromatography column may include passing at least one CV of the regeneration solution through the column. In some embodiments, the method may be between about 1 and about 20 column volumes, such as between about 1 column volume and about 15 column volumes, about 1 column volume to about 10 column volumes, or about 2 column volumes to about 10 column volumes of the regeneration solution through the column. to about 20 column volumes of the regeneration solution through the column. 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 1 7 column volumes, between about 5 column volumes and about 15 column volumes, or between about 5 column volumes and about 10 column volumes, for example, about 1 column volume, about 2 column volumes, about 3 column volumes, about 4 column volumes, about 5 column volumes, about 6 column volumes, about 7 column volumes, about 8 column volumes, about 9 column volumes, about 10 column volumes, about 12 column volumes, about 14 column volumes, about 16 column volumes , about 18 column volumes, or about 20 column volumes of the regeneration solution may be passed through the column. It may be included.

[0061]

[0061] The systems and methods according to the present disclosure may be applicable to a variety of separation media and / or processes. One system, method, or solution of the present disclosure may share more than one aspect and feature described herein. In some exemplary embodiments, the systems and methods according to the present disclosure are media and / or processes that separate components of a loaded mass completely or partially based on their hydrophobicity, such as HIC, or media and / or processes that use a combination of hydrophobicity and charge, such as ion exchange / hydrophobic interaction mixed-mode chromatography. In some embodiments, one or more of the regeneration solutions and / or methods disclosed herein may be combined with other regeneration solutions and / or methods (e.g., performed before or after them). For example, the regeneration solution of the present disclosure is used in mixed-mode chromatography to strip the residual mass interacting with the medium due to hydrophobicity from the medium. It may be applicable. tem, method, or solution) may share more than one aspect and feature described herein. In some exemplary embodiments, the systems and methods according to the present disclosure are media and / or processes that separate components of a loaded mass completely or partially based on their hydrophobicity, such as HIC, or media and / or processes that use a combination of hydrophobicity and charge, such as ion exchange / hydrophobic interaction mixed-mode chromatography. In some embodiments, one or more of the regeneration solutions and / or methods disclosed herein may be combined with other regeneration solutions and / or methods (e.g., performed before or after them). For example, the regeneration solution of the present disclosure is used in mixed-mode chromatography to strip the residual mass interacting with the medium due to hydrophobicity from the medium. In some exemplary embodiments, the systems and methods according to the present disclosure are media and / or processes that separate components of a loaded mass completely or partially based on their hydrophobicity, such as HIC, or media and / or processes that use a combination of hydrophobicity and charge, such as ion exchange / hydrophobic interaction mixed-mode chromatography. In some embodiments, one or more of the regeneration solutions and / or methods disclosed herein may be combined with other regeneration solutions and / or methods (e.g., performed before or after them). For example, the regeneration solution of the present disclosure is used in mixed-mode chromatography to strip the residual mass interacting with the medium due to hydrophobicity from the medium. In some exemplary embodiments, the systems and methods according to the present disclosure are media and / or processes that separate components of a loaded mass completely or partially based on their hydrophobicity, such as HIC, or media and / or processes that use a combination of hydrophobicity and charge, such as ion exchange / hydrophobic interaction mixed-mode chromatography. In some embodiments, one or more of the regeneration solutions and / or methods disclosed herein may be combined with other regeneration solutions and / or methods (e.g., performed before or after them). For example, the regeneration solution of the present disclosure is used in mixed-mode chromatography to strip the residual mass interacting with the medium due to hydrophobicity from the medium. In some exemplary embodiments, the systems and methods according to the present disclosure are media and / or processes that separate components of a loaded mass completely or partially based on their hydrophobicity, such as HIC, or media and / or processes that use a combination of hydrophobicity and charge, such as ion exchange / hydrophobic interaction mixed-mode chromatography. In some embodiments, one or more of the regeneration solutions and / or methods disclosed herein may be combined with other regeneration solutions and / or methods (e.g., performed before or after them). For example, the regeneration solution of the present disclosure is used in mixed-mode chromatography to strip the residual mass interacting with the medium due to hydrophobicity from the medium. In some exemplary embodiments, the systems and methods according to the present disclosure are media and / or processes that separate components of a loaded mass completely or partially based on their hydrophobicity, such as HIC, or media and / or processes that use a combination of hydrophobicity and charge, such as ion exchange / hydrophobic interaction mixed-mode chromatography. In some embodiments, one or more of the regeneration solutions and / or methods disclosed herein may be combined with other regeneration solutions and / or methods (e.g., performed before or after them). For example, the regeneration solution of the present disclosure is used in mixed-mode chromatography to strip the residual mass interacting with the medium due to hydrophobicity from the medium. In some embodiments, one or more of the regeneration solutions and / or methods disclosed herein may be combined with other regeneration solutions and / or methods (e.g., performed before or after them). For example, the regeneration solution of the present disclosure is used in mixed-mode chromatography to strip the residual mass interacting with the medium due to hydrophobicity from the medium. In some embodiments, one or more of the regeneration solutions and / or methods disclosed herein may be combined with other regeneration solutions and / or methods (e.g., performed before or after them). For example, the regeneration solution of the present disclosure is used in mixed-mode chromatography to strip the residual mass interacting with the medium due to hydrophobicity from the medium. In some embodiments, one or more of the regeneration solutions and / or methods disclosed herein may be combined with other regeneration solutions and / or methods (e.g., performed before or after them). For example, the regeneration solution of the present disclosure is used in mixed-mode chromatography to strip the residual mass interacting with the medium due to hydrophobicity from the medium. In some embodiments, one or more of the regeneration solutions and / or methods disclosed herein may be combined with other regeneration solutions and / or methods (e.g., performed before or after them). For example, the regeneration solution of the present disclosure is used in mixed-mode chromatography to strip the residual mass interacting with the medium due to hydrophobicity from the medium. It may be applied to the body. To strip the residual mass bound to the medium by charge from the medium, another regeneration solution may also be applied to the medium. For this purpose, another regeneration solution may also be applied to the medium.

[0062]

[0062] In some embodiments, the regeneration solutions and / or methods disclosed herein may be applicable to HIC media having a high degree of hydrophobicity. In some embodiments the chromatographic media that can be used with the solutions and methods according to the present disclosure include, for example, hydrophobic matrices including cross-linked agarose, polystyrene divinylbenzene, or polymethacrylate. In some embodiments the matrix may include ligands having hydrocarbons between 2 and 10 in aliphatic or aromatic configurations. In some embodiments the matrix does not include ligands having 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 per ml of media. In some embodiments, the methods and regeneration solutions described herein may be particularly applied to regenerate HIC media. In some embodiments, the methods and regeneration solutions described herein are, for example, Capto TM Phenyl (High Sub), Capto TM Butyl, or Capto TM Octyl media (GE Healthcare Life Sciences), Phenyl Sepharose media (GE Healthcare Life Sciences), POROS (R) Benzyl and POR TM OS OSTM Ethyl HIC resin (Thermo Scientific TM ), or TOY OPEARL TM resin may be suitable. In some embodiments, the methods and regeneration solutions described herein are suitable for use in continuous (multi-column) HI C systems and methods, such as those disclosed in International Application No. PCT / US2019 / 040148, filed July 1, 2019, which is incorporated herein by reference . For example, a one-step regeneration solution according to the present disclosure may be used in a multi-column continuous HIC setup, where the efficiency of the one-step regeneration solution may enhance the overall efficiency of the multi-column setup. In some embodiments, the methods and regeneration solutions described herein may be useful in HIC systems and methods, including low- or no-kosmotrop e conditions. e conditions).

[0063]

[0063] In some embodiments, the regeneration process according to the present disclosure may be performed without the use of reverse osmosis deionized water (RODI), organic solvents ( such as ethanol or ethylene glycol), chaotropes (such as guanidine or urea), sodium chloride, and / or sodium hydroxide at a concentration greater than 50 mM . Preferably, the regeneration process using the solutions disclosed herein may not require additional processes to discard solvents, such as ethanol (such as 20% ethanol ), and chaotropes, such as guanidine and urea (such as 6N guanidine or 6N urea). However , in some embodiments, the regeneration solution disclosed herein may be used before, after, or in combination with an organic solvent (e.g., 20% ethanol) or a chaotropic agent (e.g., 6N guanidine or 6N urea). It is intended to be used before, after, or in combination with it.

[0064]

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

[0065]

[0065] In some embodiments, the method according to the present disclosure may include evaluating various chromatography media to determine whether one or more chromatography media present problems during the use or regeneration of the media during the purification of the target molecule. The evaluation of the chromatography media according to the present disclosure may include, for example and without limitation, the use of one or more chromatography media types, maintenance of pH conditions, and the target molecule. Preferably, the method according to the present disclosure includes cleaning on a smaller scale than that generally performed for purifying the target molecule, which may allow for a significant savings in the amount of sample (e.g., using approximately 1 / 10, 1 / 100, 1 / 500, 1 / 700, or less of the amount of sample required to evaluate a chromatography media, such as HIC, according to conventional methods). Further, for example, using a high-throughput screening (HTS) process, different variables (e.g., different combinations and types of media, pH, and / or temperature) can be evaluated. ​​​​​​​​​​A number of purification schemes having a target molecule) may be screened simultaneously. High-through Instead of, or in addition to, high-throughput screening (HTS), an elution assay may be used to eliminate potential HIC protocol parameters.

[0066]

[0066] Preferably, these screening techniques and assays can result in significant time savings when identifying a purification scheme suitable for use in large-scale purification processes, for example, by optimizing a HIC unit operation. For example the HTS process carried out in accordance with the present disclosure can be about 10 times faster, 50 times faster, 60 times faster than conventional means of identifying regeneration / usefulness issues in chromatography protocols for one or more unit operations, including, for example, HIC, ion exchange, affinity, and others and may be even faster.

[0067] The evaluation method according to the present disclosure may include filling a well, for example, a filter plate well, with a certain amount of chromatography medium intended for use in a potential purification scheme. The filter plate well may have a volume of less than, for example, 5 mL, less than, for example 4 mL, less than 3 mL, or less than 2 mL. In some embodiments the filter plate well may have a volume of about 1 mL, about 0.8 mL, about 0.5 mL

[0067] or any other suitable volume. A filter having a suitable mesh size, for example, between about 0.5 and about 1.5 microns, for example, about 0.8 microns, about 1.0 mic ron, or about 1.2 microns, may be attached to the filter plate well. ​​​​​​The filter mesh size is determined based on the chromatography media used in the protocol. The size of the chromatography resin beads may depend on the size of the resin beads present. The diameter is, for example, between about 40 microns and about 120 microns, for example, between about 50 microns and about 100 Between microns, between about 60 microns and about 90 microns, between about 80 microns and about 90 microns between about 70 microns, about 80 microns, about 90 microns, about 100 microns, or about The amount of chromatography media packed into the wells can vary. For example, the amount of chromatography medium can be, for example, about 2.0 μL to about 50.0 μL. In the μL range, for example about 10.0 μL, about 20.0 μL, about 30.0 μL, or about 40. In some methods according to the present disclosure, multiple protocols can be included in the array. To simultaneously evaluate multiple cloning, multiple wells in a filter plate were used to measure the number of different cloning sites. The chromatography media may be packed in a variety of ways, including HIC, ion exchange, affinity, and and others.

[0068] The volume of the loaded material is determined by the volume of the filled filter plate wells (or multiple protocols). If multiple samples are to be evaluated simultaneously, they may be loaded into each packed filter plate well. The load material is purified by some initial purification process, e.g. affinity chromatography or The loading material may include a target molecule that has undergone chromatography or ion exchange chromatography. One unit operation (a) may be used in accordance with the present disclosure to test a cellular function, such as HIC. When multiple conditions are evaluated simultaneously for single unit operation, different wells are used. The loading substance used may contain different target molecules. The loading substance may be titrated against a protocol-specific pH (a protocol-specific pH). The approximate concentration of the target molecule in the loading substance may be adjusted to any suitable unit operation-specific concentration. In some embodiments, the volume of the loading substance is a fraction of the loading mass used in the full-scale protocol , for example, 1 / 2, 1 / 5, 1 / 8, 1 / 10, 1 / 20, 1 / 50, 1 / 100 of the loading mass used in the full-scale protocol or less of the loading mass (e.g., the mass of the target molecule in the loading substance).

[0069]

[0069] A method for evaluating a protocol for a unit operation is to perform elution and wash steps (e.g., including the use of RODI , 1N sodium hydroxide, and / or 5mM sodium hydroxide) suitable for use in a large-scale process, followed by further including a stripping step using a corrosive or chaotropic agent, such as 6N guanidine HCl or 6N urea, or a solvent, such as 20% ethanol. The elution / wash step may be performed using an elution buffer titrated against a protocol-specific pH . When evaluating multiple protocols for a unit operation, elution buffers showing different protocol-specific pH values may be used in different wells in a single array . The elution step may involve, for example, eluting the chromatography medium loaded in a filter plate well with a gradient of elution buffer (relatively high concentration ). in the elution buffer) (relatively high concentration ​​​​may include exposing it to (starting at a temperature of 0 degrees and ending at a concentration of 0). In another embodiment, the elution step involves exposing the loaded chromatography medium to a buffer having an initial concentration of a cosmotropic salt (e.g., citrate at 500 mM, 400 mM, 300 mM, 200 mM, etc.) and gradually / linearly decreasing the buffer concentration to 0. This may include exposing it to a buffer where the buffer concentration gradually / linearly decreases to 0. In some embodiments, a pseudo-gradient elution may be performed, where the loaded chromatography medium is exposed to separate volumes of elution buffer having linearly decreasing concentrations from a starting concentration (e.g., 300 mM) to 0 over a number of steps (e.g., 4, 5, 6, 7, 8, more or fewer steps).

[0070]

[0070] The first step may include any process intended to be tested as a unit operation protocol. Generally, the first step may include applying a solution (e.g., a solution that does not present concerns regarding safety or toxicity) considered suitable for use in large-scale and repetitive operations. For example, the first step may include one or more washings of RODI and / or 1N NaOH applied one or multiple times, continuously or alternately. The second step may include any solution intended to strip any residual substances bound to the chromatography medium after the unit operation protocol being evaluated has been completed. Such regeneration processes are described elsewhere in this specification and generally may include chaotropic agents, such as 6N guanidine HCl or 6N urea, or solvents, such as 20% ethanol.

[0071]

[0071] The evaluation method according to the present disclosure may include, for example, a step of measuring the amount of the target molecule recovered during the washing / elution step and the stripping step. These results may be compared among various chromatography media. A relatively large percentage of the target molecule may preferably be recovered during the washing / elution step (i.e., during the HIC protocol being tested) rather than during the stripping step (i.e., rigorous stripping of the chromatography media). It may include a step of measuring the amount of the target molecule recovered during the washing / elution step and the stripping step. These results may be compared among various chromatography media. A relatively large percentage of the target molecule may preferably be recovered during the washing / elution step (i.e., during the HIC protocol being tested) rather than during the stripping step (i.e., rigorous stripping of the chromatography media). It may include a step of measuring the amount of the target molecule recovered during the washing / elution step and the stripping step. These results may be compared among various chromatography media. A relatively large percentage of the target molecule may preferably be recovered during the washing / elution step (i.e., during the HIC protocol being tested) rather than during the stripping step (i.e., rigorous stripping of the chromatography media). It may include a step of measuring the amount of the target molecule recovered during the washing / elution step and the stripping step. These results may be compared among various chromatography media. A relatively large percentage of the target molecule may preferably be recovered during the washing / elution step (i.e., during the HIC protocol being tested) rather than during the stripping step (i.e., rigorous stripping of the chromatography media). It may include a step of measuring the amount of the target molecule recovered during the washing / elution step and the stripping step. These results may be compared among various chromatography media. A relatively large percentage of the target molecule may preferably be recovered during the washing / elution step (i.e., during the HIC protocol being tested) rather than during the stripping step (i.e., rigorous stripping of the chromatography media). It may include a step of measuring the amount of the target molecule recovered during the washing / elution step and the stripping step. These results may be compared among various chromatography media. A relatively large percentage of the target molecule may preferably be recovered during the washing / elution step (i.e., during the HIC protocol being tested) rather than during the stripping step (i.e., rigorous stripping of the chromatography media).

[0072]

[0072] The evaluation method according to the present disclosure may further include a step of determining the recovery of the target molecule during the stripping step as a percentage of the total target molecule recovered during the unit operation protocol being tested. If the percentage of the target molecule recovered during the stripping step exceeds a predetermined threshold, it can be predicted that the unit operation protocol may cause potential problems with regeneration / reusability when scaled up and / or repeated multiple times. If the percentage of the target molecule recovered during the stripping step is at or below a predetermined threshold, it can be predicted that the unit operation protocol will not cause problems with regeneration / reusability when scaled up and / or repeated multiple times. The predetermined threshold may be any experimentally determined threshold that serves as an indicator of the amount of residue remaining bound to the chromatography media after a unit operation, such as an HIC protocol. In some embodiments, the predetermined threshold is, for example, from about 1% to about 10%. It may further include a step of determining the recovery of the target molecule during the stripping step as a percentage of the total target molecule recovered during the unit operation protocol being tested. If the percentage of the target molecule recovered during the stripping step exceeds a predetermined threshold, it can be predicted that the unit operation protocol may cause potential problems with regeneration / reusability when scaled up and / or repeated multiple times. If the percentage of the target molecule recovered during the stripping step is at or below a predetermined threshold, it can be predicted that the unit operation protocol will not cause problems with regeneration / reusability when scaled up and / or repeated multiple times. The predetermined threshold may be any experimentally determined threshold that serves as an indicator of the amount of residue remaining bound to the chromatography media after a unit operation, such as an HIC protocol. In some embodiments, the predetermined threshold is, for example, from about 1% to about 10%. It may further include a step of determining the recovery of the target molecule during the stripping step as a percentage of the total target molecule recovered during the unit operation protocol being tested. If the percentage of the target molecule recovered during the stripping step exceeds a predetermined threshold, it can be predicted that the unit operation protocol may cause potential problems with regeneration / reusability when scaled up and / or repeated multiple times. If the percentage of the target molecule recovered during the stripping step is at or below a predetermined threshold, it can be predicted that the unit operation protocol will not cause problems with regeneration / reusability when scaled up and / or repeated multiple times. The predetermined threshold may be any experimentally determined threshold that serves as an indicator of the amount of residue remaining bound to the chromatography media after a unit operation, such as an HIC protocol. In some embodiments, the predetermined threshold is, for example, from about 1% to about 10%. It may further include a step of determining the recovery of the target molecule during the stripping step as a percentage of the total target molecule recovered during the unit operation protocol being tested. If the percentage of the target molecule recovered during the stripping step exceeds a predetermined threshold, it can be predicted that the unit operation protocol may cause potential problems with regeneration / reusability when scaled up and / or repeated multiple times. If the percentage of the target molecule recovered during the stripping step is at or below a predetermined threshold, it can be predicted that the unit operation protocol will not cause problems with regeneration / reusability when scaled up and / or repeated multiple times. The predetermined threshold may be any experimentally determined threshold that serves as an indicator of the amount of residue remaining bound to the chromatography media after a unit operation, such as an HIC protocol. In some embodiments, the predetermined threshold is, for example, from about 1% to about 10%. It may further include a step of determining the recovery of the target molecule during the stripping step as a percentage of the total target molecule recovered during the unit operation protocol being tested. If the percentage of the target molecule recovered during the stripping step exceeds a predetermined threshold, it can be predicted that the unit operation protocol may cause potential problems with regeneration / reusability when scaled up and / or repeated multiple times. If the percentage of the target molecule recovered during the stripping step is at or below a predetermined threshold, it can be predicted that the unit operation protocol will not cause problems with regeneration / reusability when scaled up and / or repeated multiple times. The predetermined threshold may be any experimentally determined threshold that serves as an indicator of the amount of residue remaining bound to the chromatography media after a unit operation, such as an HIC protocol. In some embodiments, the predetermined threshold is, for example, from about 1% to about 10%. It may further include a step of determining the recovery of the target molecule during the stripping step as a percentage of the total target molecule recovered during the unit operation protocol being tested. If the percentage of the target molecule recovered during the stripping step exceeds a predetermined threshold, it can be predicted that the unit operation protocol may cause potential problems with regeneration / reusability when scaled up and / or repeated multiple times. If the percentage of the target molecule recovered during the stripping step is at or below a predetermined threshold, it can be predicted that the unit operation protocol will not cause problems with regeneration / reusability when scaled up and / or repeated multiple times. The predetermined threshold may be any experimentally determined threshold that serves as an indicator of the amount of residue remaining bound to the chromatography media after a unit operation, such as an HIC protocol. In some embodiments, the predetermined threshold is, for example, from about 1% to about 10%. It may further include a step of determining the recovery of the target molecule during the stripping step as a percentage of the total target molecule recovered during the unit operation protocol being tested. If the percentage of the target molecule recovered during the stripping step exceeds a predetermined threshold, it can be predicted that the unit operation protocol may cause potential problems with regeneration / reusability when scaled up and / or repeated multiple times. If the percentage of the target molecule recovered during the stripping step is at or below a predetermined threshold, it can be predicted that the unit operation protocol will not cause problems with regeneration / reusability when scaled up and / or repeated multiple times. The predetermined threshold may be any experimentally determined threshold that serves as an indicator of the amount of residue remaining bound to the chromatography media after a unit operation, such as an HIC protocol. In some embodiments, the predetermined threshold is, for example, from about 1% to about 10%. It may further include a step of determining the recovery of the target molecule during the stripping step as a percentage of the total target molecule recovered during the unit operation protocol being tested. If the percentage of the target molecule recovered during the stripping step exceeds a predetermined threshold, it can be predicted that the unit operation protocol may cause potential problems with regeneration / reusability when scaled up and / or repeated multiple times. If the percentage of the target molecule recovered during the stripping step is at or below a predetermined threshold, it can be predicted that the unit operation protocol will not cause problems with regeneration / reusability when scaled up and / or repeated multiple times. The predetermined threshold may be any experimentally determined threshold that serves as an indicator of the amount of residue remaining bound to the chromatography media after a unit operation, such as an HIC protocol. In some embodiments, the predetermined threshold is, for example, from about 1% to about 10%. It may further include a step of determining the recovery of the target molecule during the stripping step as a percentage of the total target molecule recovered during the unit operation protocol being tested. If the percentage of the target molecule recovered during the stripping step exceeds a predetermined threshold, it can be predicted that the unit operation protocol may cause potential problems with regeneration / reusability when scaled up and / or repeated multiple times. If the percentage of the target molecule recovered during the stripping step is at or below a predetermined threshold, it can be predicted that the unit operation protocol will not cause problems with regeneration / reusability when scaled up and / or repeated multiple times. The predetermined threshold may be any experimentally determined threshold that serves as an indicator of the amount of residue remaining bound to the chromatography media after a unit operation, such as an HIC protocol. In some embodiments, the predetermined threshold is, for example, from about 1% to about 10%. The predetermined threshold may be any experimentally determined threshold that serves as an indicator of the amount of residue remaining bound to the chromatography media after a unit operation, such as an HIC protocol. In some embodiments, the predetermined threshold is, for example, from about 1% to about 10%. The predetermined threshold may be any experimentally determined threshold that serves as an indicator of the amount of residue remaining bound to the chromatography media after a unit operation, such as an HIC protocol. In some embodiments, the predetermined threshold is, for example, from about 1% to about 10%. The predetermined threshold may be any experimentally determined threshold that serves as an indicator of the amount of residue remaining bound to the chromatography media after a unit operation, such as an HIC protocol. In some embodiments, the predetermined threshold is, for example, from about 1% to about 10%. It can be, for example, between about 3% and about 7%, such as about 4%, about 5%, or about 6%.

[0073]

[0073] In embodiments where multiple chromatography protocols are evaluated simultaneously, the above calculation of the percentage of target molecule recovery can be determined at once for multiple chromatographies, and can give an initial impression of which protocol may be suitable or preferred for further use, testing, investigation, or development. Protocols for further study can include wells where the normalized percentage recovery due to the stripping step can be 1% or less, 3% or less, 5% or less, 7% or less, or 10% or less.

[0074]

[0074] In some embodiments, a method for evaluating a chromatography protocol according to the present disclosure, such as a protocol for an HIC unit operation, may be performed at an early stage of developing a purification process. For example, multiple HIC protocols can be evaluated as described herein (e.g., high-throughput screening and / or elution assays), and HIC protocols predicted to pose regeneration / reusability issues can be excluded from further study or deprioritized. Chromatography protocols not predicted to pose regeneration / reusability issues can be subjected to further testing (e.g., full-scale testing) or study to, for example, maximize yield and minimize impurities and confirm that they meet internal and external quality control guidelines, and reproducibility and lifespan (e.g., if they are 10, 25, 50, 75, 100​​​​​​​​​​​​ or whether it will cause column discoloration or other undesirable effects after one or more cycles can be evaluated.

[0075]

[0075] In some embodiments, the method according to the present disclosure preferably prevents or reduces discoloration of the regeneration column, medium, and / or device that might otherwise occur after one or more uses (see, for example, Examples 6 and 9 discussed herein). In some embodiments, the method according to the present disclosure can preferably assist in early identification of a protocol for a chromatographic unit operation that might present a regeneration / usefulness issue, thus saving time and cost that might otherwise be involved in developing a complete purification scheme and only later finding that the protocol for that chromatographic step presents such problems. In some embodiments, the method according to the present disclosure can preferably assist in early identification of a protocol for a chromatographic unit operation that might present a regeneration / usefulness issue, thus saving the time and cost that might otherwise be involved in developing a complete purification scheme and only later finding that the protocol for that chromatographic step presents such problems. issues.

[0076]

[0076] Now, reference is made to specific figures. FIG. 1 shows a method 100 for regenerating a chromatography column in accordance with an aspect of the present disclosure. According to step 102, a first load mass can be applied to a hydrophobic interaction chromatography column. According to step 104, the target protein can be collected from the hydrophobic interaction chromatography column. According to step 106, thus, a single alkaline regeneration solution can be applied to the chromatography column to remove substances bound to the hydrophobic interaction medium in the column.

[0077]

[0077] According to step 102, a first load mass can be applied to a hydrophobic interaction chromatography column. The load mass includes the target molecule (e.g., polypeptide) as well as residual components, For example, it may contain host cell proteins, cell fragments, etc. According to step 104, the target protein can be collected from the hydrophobic interaction chromatography column. This can be done, for example, in a washing step or an elution step. According to step 106, a single alkaline regeneration solution is applied to the chromatography column to remove substances bound to the hydrophobic interaction medium in the column. The single alkaline regeneration solution may have one or more of the above-described characteristics. The target protein can be collected from the hydrophobic interaction chromatography column. This can be done, for example, in a washing step or an elution step. According to step 106, a single alkaline regeneration solution is applied to the chromatography column to remove substances bound to the hydrophobic interaction medium in the column. The single alkaline regeneration solution may have one or more of the above-described characteristics.

Example

[0078]

[0078] Example 1

[0079]

[0079] After incubation with polysorbate 20 (“PS20”), the amount of non-esterified free fatty acids (“NEFA”) in several sample solutions was measured. The presence and amount of NEFA in the sample are interpreted as an indicator of PS20 degradation caused by impurities in the sample, for example, host cell proteins. A first sample was taken from the HIC load mass. Five additional samples were taken from the HIC pools after subsequent cycles 1, 2, 3, 5, and 10. All samples were incubated for the same time. As shown in Table 1 below, from cycle 1 to cycle 5, PS20 degradation was calculated to be a negative rate equivalent to the negative control. A negative PS20 degradation rate corresponds to the absence of detectable NEFA and, as a result, the absence of detectable lipase activity. NEFA was detectable between cycles 5 and 10, indicating an increase in lipase activity. This data shows that lipase activity can increase as a function of cycling. A first sample was taken from the HIC load mass. Five additional samples were taken from the HIC pools after subsequent cycles 1, 2, 3, 5, and 10. All samples were incubated for the same time. As shown in Table 1 below, from cycle 1 to cycle 5, PS20 degradation was calculated to be a negative rate equivalent to the negative control. A negative PS20 degradation rate corresponds to the absence of detectable NEFA and, as a result, the absence of detectable lipase activity. NEFA was detectable between cycles 5 and 10, indicating an increase in lipase activity. This data shows that lipase activity can increase as a function of cycling.

Table 1

[0080]

[0080] Example 2

[0081]

[0081] Figure 2 shows the blot analysis of various states of a HIC medium (Capto Phenyl (High Sub) (GE Healthcare Life S TM ciences)) after exposure to the use or regeneration solution.

Table 2

[0082]

[0082] As can be seen from Figure 2, for each used column except for the column (Column F) contacted with 6N guanidine HCl, a dark area can be seen. The column (Column A) representing the untreated medium also does not show a dark area. This data indicates that 6N guanidine HCl can remove the residual medium during the regeneration of the chromatography column when compared with other solutions. 6N guanidine HCl can, therefore, be used as a stripping agent and as an agent to evaluate the effectiveness of other stripping / regeneration agents when used following these other agents.

[0083]

[0083] Example 3

[0084]

[0084] Figure 3 shows the blot analysis of various states of a HIC medium (Capto Phenyl (High Sub)) after exposure to the use or regeneration solution. TM

Table 3

[0085] ​​​​​​

[0085] Table 3 lists the type of media used and, if a regenerant was used, to which regenerant the media was exposed. As can be seen from Figure 3, the regeneration paradigm (used with Media B and C) that included RODI, 1.0N NaOH, RODI, and 20% ethanol was not as complete in its ability to remove residues from the media as 6N guanidine HCl (D). As can be seen from Figure 3, the regeneration paradigm (used with Media B and C) that included RODI, 1.0N NaOH, RODI, and 20% ethanol was not as complete in its ability to remove residues from the media as 6N guanidine HCl (D). ) was not as complete in its ability to remove residues from the media as 6N guanidine HCl (D). was not as complete in its ability to remove residues from the media as 6N guanidine HCl (D).

[0086]

[0086] Example 4

[0087]

[0087] Figure 4 shows an overlaid UV chromatogram showing five sanitization procedures after collection of monoclonal antibody mAb 1 during the process. Each sanitization procedure included a first 0.5N sodium hydroxide flush (A) of two column volumes, followed by an interruption, and then a second 0.5N sodium hydroxide flush (B) of one column volume. Sanitization was considered complete at point C, after which a two-column volume flush of water for injection ("WFI") was performed (D). As can be seen from Figure 4, the first sodium hydroxide flush (A) produced a high absorbance in the initial portion of the flush, which correlates with the removal of a large amount of impurities. The maximum absorbance seen during HIC elution (regeneration) was 2.4 AU, while the maximum absorbance seen during the sanitization cycle in Figure 4 was approximately 1.4 AU. Figure 4 shows an overlaid UV chromatogram showing five sanitization procedures after collection of monoclonal antibody mAb 1 during the process. Each sanitization procedure included a first 0.5N sodium hydroxide flush (A) of two column volumes, followed by an interruption, and then a second 0.5N sodium hydroxide flush (B) of one column volume. flush)(A), followed by an interruption, and then a second 0.5N sodium hydroxide flush (B) of one column volume. Sanitization was considered complete at point C, after which a two-column volume flush of water for injection ("WFI") was performed (D). Sanitization was considered complete at point C, after which a two-column volume flush of water for injection ("WFI") was performed (D) . As can be seen from Figure 4, the first sodium hydroxide flush (A) produced a high absorbance in the initial portion of the flush, which correlates with the removal of a large amount of impurities. The maximum absorbance seen during HIC elution (regeneration) was 2.4 AU, while the maximum absorbance seen during the sanitization cycle in Figure 4 was approximately 1.4 AU. The maximum absorbance seen during the sanitization cycle in Figure 4 was approximately 1.4 AU.

[0088]

[0088] Example 5

[0089]

[0089] Figure 5 shows, as detailed below, after use or exposure to the stripping solution HIC media (Capto TM phenyl (High Sub)) in various states are shown. In particular, the HIC media used were exposed to decreasing concentrations of guanidine.

Table 4

[0090]

[0090] As can be seen in Figure 5, the use of 6N guanidine HCl (D) removes most of the residues from the HIC media, while solutions with lower concentrations of guanidine and a 20 % ethanol solution were not as effective in removing residues from the HIC media. .

[0091]

[0091] Example 6

[0092]

[0092] Figure 6 shows two columns containing Capto TM phenyl (High Sub) (GE Healthcare Li fe Sciences). The left column was exposed to 49 cycles of HIC to purify the monoclonal antibody m Ab 2. Each cycle included column regeneration in the order of RODI , 1N sodium hydroxide, RODI, and 20% ethanol. After the 40th cycle, a yellow band was identified at the bottom of the column. As a comparison the right column is a representative of the untreated Capto phenyl (High Sub) HIC media. The discoloration of the left column could be an indication of insufficient regeneration. TM

[0093]

[0093] Pools were collected from cycles 1 and 49 and analyzed for lipase activity and the presence of host cell proteins (HCP). The lipase between cycles 1 and 49 There was no significant trend in the enzyme activity or HCP value.

[0094]

[0094] Example 7

[0095]

[0095] Figures 7A and 7B respectively show the chromatograms of cycles 2 and 49 performed on the left column described for Example 6. They are annotated as follows.

Table 5

[0096]

[0096] In both cycle 2 and cycle 49, RODI was not an effective stripping solution as indicated by the absence of peaks after C or C' (markers X and X'). The introduction of 1N NaOH resulted in the emergence of peaks P1 (cycle 2) and P3 (cycle 49), which means that 1N NaOH was effective as at least a partial stripping solution. The introduction of a second RODI strip removed some additional impurities and resulted in peaks P2 (cycle 2) and P4 (cycle 49). The introduction of a 20% ethanol solution did not result in additional peaks. This data from both cycles shows that RODI is not an effective stripping solution when used before sodium hydroxide.

[0097]

[0097] Example 8

[0098]

[0098] Two loaded masses, each containing a target monoclonal antibody, were subjected to a hydrophobic interaction chromatography process. In the first process, the target from the first loaded mass ​​​​​​​​​​After loading, washing, and eluting the monoclonal antibody mAb 3, the chromatography column was subjected to 1N sodium hydroxide stripping solution, then RODI strip, and 20% ethanol strip. In the second process, after loading, washing, and eluting the target monoclonal antibody mAb 4 from the second loading mass, the chromatography column was subjected to ROD I strip, then 1N sodium hydroxide strip, another RODI strip, and 20% ethanol strip. Figure 8A shows the chromatogram for the first process, and Figure 8B shows the chromatogram for the second process. Each chromatogram is annotated as follows. is annotated as follows.

Table 6

[0099]

[0099] After the first RODI strip shown by marker D’ in Figure 8B (marker X ’), there is no peak, which is comparable to the absence of a peak at the position indicated by marker X in Figure 8A (where the first RODI strip is not performed). Therefore, the decrease in the conductivity of the first RODI strip applied in the first process did not cause a recognizable amount of removal of substances from the chromatography column.

[0100]

[0100] Example 9

[0101]

[0101] The effectiveness of 6N guanidine HCl as a stripping solution and as a potential solution for removing discoloration on the left column shown in Figure 6 was further evaluated. The column was subjected to the protocol described in the following table. During the protocol, the chromatogram shown in Figure 9 was generated.

Table 7

[0102]

[0102] As shown in the chromatogram of FIG. 9, the first introduction of RODI resulted in peak P1, and the first strip of 6N guanidine HCl after the first introduction of RODI resulted in a high peak P2. There was no peak associated with the second strip of 6N guanidine HCl that was held overnight in the column before the flow-through. This likely indicates the effectiveness of the first strip of 6N guanidine HCl in removing the residue bound to the column. However, after the entire cleaning protocol, the column remained discolored (as shown in FIG. 6). and the first strip of 6N guanidine HCl after the first introduction of RODI resulted in a high peak P2. There was no peak associated with the second strip of 6N guanidine HCl that was held overnight in the column before the flow-through. This likely indicates the effectiveness of the first strip of 6N guanidine HCl in removing the residue bound to the column. However, after the entire cleaning protocol, the column remained discolored (as shown in FIG. 6). However, after the entire cleaning protocol, the column remained discolored (as shown in FIG. 6).

[0103]

[0103] Example 10

[0104]

[0104] The regeneration paradigm was analyzed in detail. FIG. 10A shows the chromatogram of an HIC treatment for purifying monoclonal antibody mAb 4 using a Capto TM Phenyl (High Sub) medium (GE Life Sciences). The HIC treatment including the regeneration paradigm included the following steps. The HIC treatment including the regeneration paradigm included the following steps. The HIC treatment including the regeneration paradigm included the following steps.

Table 8

[0105] Referring to Figure 10A, peak P1 follows the introduction of 1N sodium hydroxide (E), and peak P2 coincides with the second RODI strip (F). Peak P3 followed the introduction of 6 N guanidine HCl (I).

[0106]

[0106] Figure 10B shows an enlarged image of peak P1. The first RODI strip (D ) did not produce a detectable absorbance. The 1N NaOH strip (E) caused immediate and early removal of the residue from the column, as indicated by the presence of peak P 1, and peak P1 appeared to become the baseline as the conductivity increased. The first RODI strip (D) was assumed not to facilitate its removal, but rather to bind some residue more tightly to the column. It is further assumed that the elution of the residue from the column caused by sodium hydroxide is mainly promoted by pH, but as the concentration of sodium hydroxide (a weak cosmotrope) increases, the residual protein can bind more tightly to the HIC medium as the solution conductivity increases.

[0107]

[0107] Example 11

[0108]

[0108] The elution of the residue from the used HIC medium was observed as a function of sodium hydroxide concentration. Figure 11 shows a process in which, after loading and washing a HIC column for collecting monoclonal antibody mAb 4 (during section A), starting with RODI only and gradually increasing the concentration of sodium hydroxide to a maximum concentration of 1N sodium hydroxide (section B), a 20 CV gradient of a mixture of RODI and sodium hydroxide was loaded into the column. ​​​​​​shows the chromatogram. When ~5 mM sodium hydroxide is passed through the column, it is eluted , and one distinct peak P1 was observed. Starting with RODI and 1 N maximum concentration of sodium hydroxide and gradually decreasing the concentration of sodium hydroxide to 0 (section C), a second 20 CV gradient was performed and loaded onto the column. No additional peaks were observed during this second gradient. Finally, a 6 N guanidine HCl solution was flushed through the column at mark D. A small peak P2 was observed while passing 6 N guanidine HCl through the column. By integrating the 280 nm UV absorbance, the area under the curve (AUC) of each peak was calculated. Compared to the AUC of the 6 N guanidine HCl strip in the control treatment, which was calculated to be 13,305 mL*mAU, peak P2 was calculated to have 1,160 mL*mAU. Thus, peak P2 showed a 91.3% decrease in size compared to the control.

[0109]

[0109] This process showed that when ~5 mM sodium hydroxide was passed through the column, the bound substance eluting from the column was at its maximum, and relatively little residual medium eluted with 6 N guanidine HCl.

[0110]

[0110] Example 12

[0111]

[0111] Sodium hydroxide solutions with various concentrations (1000 mM, 500 mM, 100 mM, 50 mM, 25 mM, 10 mM, and 5 mM) were each applied in separate 2-solution regeneration processes performed after loading and washing the HIC column. Each regeneration process Seth included a sodium hydroxide solution as the first regeneration solution and a 6N guanidine HCl solution as the second regeneration solution. A chromatogram regarding the regeneration process was generated and overlaid in Figure 12. The sodium hydroxide solution in each regeneration process produced a first peak shown in the peak group labeled A . The 6N guanidine HCl solution in each regeneration process produced a second peak shown in the peak group labeled B . The regeneration process containing 5 mM Na OH produced the largest "A" peak (indicating the maximum residual amount eluted by the application of the sodium hydroxide solution) and the smallest "B" peak (indicating the minimum residual amount eluted by the application of guanidine HCl). Therefore, within the range of the sodium hydroxide solutions tested, the 5 mM sodium hydroxide solution was the most effective in regenerating the HIC column (i.e., removing the most bound substances from the column). The higher the sodium hydroxide concentration, the more residual mass remained on the column due to the removal by the 6N guanidine HCl solution. It was hypothesized that an increase in pH promoted the elution of residues from the HIC column, while an increase in conductivity decreased the elution of residues by strengthening the binding between the residues and the HIC medium .

[0112]

[0112] Example 13

[0113]

[0113] Capto TM phenyl (High Sub) medium (GE Healthcare Life Sciences). For the area under the curve (AUC) of the chromatogram peaks resulting from the regeneration treatment after the collection of mAb 4 from an HIC column prepared with this medium, one-way analysis of variance (ANOVA) was performed One-way statistical analysis of variance) was performed. As shown in FIGS. 13A and 13B using control (A), RODI (B), and sodium hydroxide solutions at various concentrations (C-L) a regeneration treatment was performed. Each regeneration treatment included a stripping solution (AUC analyzed in FIG. 13A ), followed by a 6N guanidine HCl solution (AUC analyzed in FIG. 13B). In addition, a full pair-wise Turkey-Kramer test was performed for each analysis to show statistical significance .

[0114]

[0114] As shown in FIGS. 13A and 13B, the treatments associated with the 0.5 mM NaOH and 1 mM NaOH regeneration solutions showed the highest AUC values (enclosed in region 1300) for the peaks occurring during the flow-through of these regeneration solutions, indicating that these regeneration solutions cause more effective removal of substances from the HI C column. The treatments associated with the 0.5 mM NaOH , 1 mM NaOH, and 5 mM NaOH regeneration solutions showed the lowest AUC values ( enclosed in region 1350 of FIG. 13B) for the peaks occurring during the flow-through of the 6N guanidine HCl solution following the regeneration solution, also indicating that these regeneration solutions cause more efficient removal of substances from the HIC column and supporting that less of what should be removed by 6N guanidine HCl remains. Therefore, solutions having a sodium hydroxide concentration in the range of 0.5 mM to 5 mM NaOH were shown to be effective for the regeneration of the HIC column.

[0115]

[0115] Example 14

[0116]

[0116] The regeneration process using a sodium hydroxide solution was replaced with a sodium chloride solution​​​​ was compared with the elution process. Collection of monoclonal antibody mAb 5 from the HIC column Subsequently, the column was regenerated using sodium hydroxide having a concentration of 3 mM, 5 mM, or 7 mM, or using sodium chloride having a concentration of 5 mM, 8 mM, or 11 mM. Attention was also paid to the pH and conductivity of each regeneration solution. The sodium hydroxide solution showed a pH greater than 11 for all, while the sodium chloride solution showed a pH between 5.5 and 6.5 for all. The solution conductivities were equivalent. For each process, a 6N guanidine H Cl solution was applied to each column after the regeneration solution. Chromatograms were generated for each process and all of them were overlaid in Figure 14. AUCs were calculated for the peaks associated with the flow-through of the regeneration solution and the flow-through of 6N guanidine HCl. The solutions and AUCs are listed in the following table. Cl solution was applied to each column after the regeneration solution. Chromatograms were generated for each process and all of them were overlaid in Figure 14. AUCs were calculated for the peaks associated with the flow-through of the regeneration solution and the flow-through of 6N guanidine Cl solution was applied to each column after the regeneration solution. Chromatograms were generated for each process and all of them were overlaid in Figure 14. AUCs were calculated for the peaks associated with the flow-through of the regeneration solution and the flow-through of 6N guanidine Cl solution was applied to each column after the regeneration solution. Chromatograms were generated for each process and all of them were overlaid in Figure 14. AUCs were calculated for the peaks associated with the flow-through of the regeneration solution and the flow-through of 6N guanidine HCl. The solutions and AUCs are listed in the following table. HCl. The solutions and AUCs are listed in the following table. [Table 9]

[0117]

[0117] As shown by this data, the flow-through of the sodium hydroxide solution showed a substantially higher AUC value than that of the sodium chloride solution. Similarly, the flow-through of 6N guanidine HCl after the sodium hydroxide solution showed a substantially lower AUC value than that of the flow-through of 6N guanidine HCl after the sodium chloride solution. In Figure 14, the peak caused by the flow-through of sodium hydroxide is indicated by marker A, and the peak (or absence of peak) caused by the flow-through of sodium chloride is indicated by marker B As shown by this data, the flow-through of the sodium hydroxide solution showed a substantially higher AUC value than that of the sodium chloride solution. Similarly, the flow-through of 6N guanidine HCl after the sodium hydroxide solution showed a substantially lower AUC value than that of the flow-through of 6N guanidine HCl after the sodium chloride solution. In Figure 14, the peak caused by the flow-through of sodium hydroxide is indicated by marker A, and the peak (or absence of peak) caused by the flow-through of sodium chloride is indicated by marker B As shown by this data, the flow-through of the sodium hydroxide solution showed a substantially higher AUC value than that of the sodium chloride solution. Similarly, the flow-through of 6N guanidine HCl after the sodium hydroxide solution showed a substantially lower AUC value than that of the flow-through of 6N guanidine HCl after the sodium chloride solution. In Figure 14, the peak caused by the flow-through of sodium hydroxide is indicated by marker A, and the peak (or absence of peak) caused by the flow-through of sodium chloride is indicated by marker B As shown by this data, the flow-through of the sodium hydroxide solution showed a substantially higher AUC value than that of the sodium chloride solution. Similarly, the flow-through of 6N guanidine HCl after the sodium hydroxide solution showed a substantially lower AUC value than that of the flow-through of 6N guanidine HCl after the sodium chloride solution. In Figure 14, the peak caused by the flow-through of sodium hydroxide is indicated by marker A, and the peak (or absence of peak) caused by the flow-through of sodium chloride is indicated by marker B As shown by this data, the flow-through of the sodium hydroxide solution showed a substantially higher AUC value than that of the sodium chloride solution. Similarly, the flow-through of 6N guanidine HCl after the sodium hydroxide solution showed a substantially lower AUC value than that of the flow-through of 6N guanidine HCl after the sodium chloride solution. In Figure 14, the peak caused by the flow-through of sodium hydroxide is indicated by marker A, and the peak (or absence of peak) caused by the flow-through of sodium chloride is indicated by marker B As shown by this data, the flow-through of the sodium hydroxide solution showed a substantially higher AUC value than that of the sodium chloride solution. Similarly, the flow-through of 6N guanidine HCl after the sodium hydroxide solution showed a substantially lower AUC value than that of the flow-through of 6N guanidine HCl after the sodium chloride solution. In Figure 14, the peak caused by the flow-through of sodium hydroxide is indicated by marker A, and the peak (or absence of peak) caused by the flow-through of sodium chloride is indicated by marker B As shown by this data, the flow-through of the sodium hydroxide solution showed a substantially higher AUC value than that of the sodium chloride solution. Similarly, the flow-through of 6N guanidine HCl after the sodium hydroxide solution showed a substantially lower AUC value than that of the flow-through of 6N guanidine HCl after the sodium chloride solution. In Figure 14, the peak caused by the flow-through of sodium hydroxide is indicated by marker A, and the peak (or absence of peak) caused by the flow-through of sodium chloride is indicated by marker B The peaks that occur, and by the flow-through of 6N guanidine HCl after sodium chloride The peaks that occur are indicated by marker A’ and B’, respectively. As shown in Figure 14 The sodium hydroxide solution was a more effective stripping agent than the sodium chloride solution.

[0118]

[0118] Example 15

[0119]

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

[0120]

[0120] Example 16

[0121]

[0121] 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 effectiveness of each sodium hydroxide solution in the regeneration of the HIC medium used during the purification process was characterized by the size of the chromatogram peaks associated with the 6N guanidine HCl strip after the application of each sodium hydroxide solution. A larger AUC associated with the guanidine strip peak indicated more remaining residue by the sodium hydroxide solution preceding the strip, and conversely, a smaller AUC associated with the guanidine strip peak indicated less The residual amount remaining by the sodium hydroxide solution preceding the trip is less, and as a result, the sodium hydroxide regeneration solution was shown to be more effective. Figure 16 shows a graph showing the guanidine strip peak AUC as a function of sodium hydroxide concentration. All three sodium hydroxide solutions tested showed more effective regeneration (i.e., smaller guanidine strip peaks) than the default cleaning paradigm including the order of RODI, 1N sodium hydroxide, RODI, 20% ethanol, and RODI. The 5 mM sodium hydroxide solution had the lowest guanidine strip peak area. Based on the curve extrapolated from the data points, a 7 mM sodium hydroxide regeneration solution may result in an even lower guanidine strip peak area.

[0122]

[0122] Example 17

[0123]

[0123] A first regeneration process shown as a control was used for the HIC column after collection of the monoclonal antibody mAb 5 from the column. The chromatogram was purified using this process. A number of solutions were used in the order starting with 1N sodium hydroxide, followed by RODI, 20% ethanol, RODI, and 6N guanidine HCl. The chromatogram peak corresponding to the flow-through of 6N guanidine HCl was used as a measure of the effectiveness of the regeneration process The chromatogram is shown in Figure 17. The markers on the chromatogram indicate the following events

Table 10

[0124]

[0124] After collection of mAb 5 from the HIC column, containing 5 mM sodium hydroxide A second regeneration process was used to generate the chromatogram shown in Figure 18. Starting with 5 mM sodium hydroxide, RODI, 20% ethanol, RODI, 5 mM sodium hydroxide, RODI, 6 N guanidine HCl, and 0.1 N sodium hydroxide followed in that order, using a number of solutions. The AUC corresponding to the flow-through of each solution was calculated from the chromatogram and the results are listed in the table below.

[0125] As shown in Figure 18 and reflected in the AUC values in the table above, the first 5 mM

Table 11

[0125]

[0125] As shown in Figure 18 and as reflected in the AUC values in the table above, the first 5 mM NaOH flow-through showed the highest AUC with the largest difference (peak A). The solutions applied to the column after the first 5 mM NaOH solution provided a minimal amount of material further removed from the HIC column, as indicated by the relatively small corresponding AUC values. The second largest AUC value was associated with the flow-through of 6 N guanidine HCl, but at 889 mL *mAU, less than one-thirtieth of the AUC value associated with the first 5 mM NaOH flow-through. Furthermore, when compared to the AUC value (7,390 mL*mAU) of the 6 N guanidine HCl peak during the control process shown in Figure 17, the smaller AUC value of the 6 N guanidine H Cl peak during this regeneration process indicated that the first 5 mM NaOH solution provided a substantially improved regeneration of the HIC column when compared to the control run. RODI, 20% ethanol, 6 N guanidine HCl, and 0.1 N NaOH were added to 5 mM Cl peak during this regeneration process indicated that the first 5 mM NaOH solution provided a substantially improved regeneration of the HIC column when compared to the control run. RODI, 20% ethanol, 6 N guanidine HCl, and 0.1 N NaOH were added to 5 mM Cl peak during this regeneration process indicated that the first 5 mM NaOH solution provided a substantially improved regeneration of the HIC column when compared to the control run. RODI, 20% ethanol, 6 N guanidine HCl, and 0.1 N NaOH were added to 5 mM Cl peak during this regeneration process indicated that the first 5 mM NaOH solution provided a substantially improved regeneration of the HIC column when compared to the control run. RODI, 20% ethanol, 6 N guanidine HCl, and 0.1 N NaOH were added to 5 mM DI, 20% ethanol, 6 N guanidine HCl, and 0.1 N NaOH were added to 5 mM The additional benefits provided for HIC regeneration when used after the NaOH solution were minimal at all.

[0126]

[0126] Example 18

[0127]

[0127] Six mixtures, each containing a different target molecule (e.g., a target monoclonal antibody) and different concentrations of citrate in the loading buffer, were subjected to HIC in a column containing Capto Phenyl (Hi TM gh Sub) medium (GE Life Sciences). After elution of each monoclonal antibody, the HIC column used was subjected to a gradient starting from a solution containing no sodium hydroxide (pure RODI) to 1N sodium hydroxide and then in the reverse direction (from 1N sodium hydroxide back to pure RODI) with a certain volume of RODI and sodium hydroxide. Finally, each process was terminated with the loading of 6N guanidine HCl and the collection of the flow-through. The following table shows the pH and citrate concentration in each loading mixture

Table 12

[0128]

[0128] For each process, chromatograms were generated; a series of chromatograms are shown in Figure 19. As shown, each chromatogram A - F corresponds to a peak (A’, B’, C’, D’, E’, F’) for the elution of the substances bound to the HIC medium at a flow-through of approximately 5 mM NaOH . The subsequent guanidine HCl flow-through peaks were extremely small or absent. Thus, it was shown that the effectiveness of the regeneration solution is not limited to the use of one monoclonal antibody

[0129] ​​

[0129] Example 19

[0130]

[0130] A number of HIC protocols with different conditions were evaluated to assess whether such protocols might pose a regeneration / usability problem. A 96-well filter plate (AcroPrep TM Advance 1mL filter plate, 1. 2μm Supor membrane, part number 8130, Pall Corporation) wells were filled with 0 .02 μL of various HIC media as follows.

Table 13

[0131]

[0131] An aliquot of the loading substance containing one of three target antibodies (mAb 1, mAb 2, mAb 3) was prepared and, when mixed with the HIC medium, was adjusted to a concentration of 0.33 g / L with a target concentration of 5 g / L. For each of the three target antibodies, aliquots of the loading substance were titrated to different pHs (4.5, 6.25, or 8.0) using 2M acetic acid or 2 M Tris base, yielding a total of nine aliquots, each containing one of the three target antibodies and showing one of three different pH values.

[0132]

[0132] Each of three columns of wells filled with one media type was subjected to a purification protocol at different pHs (4.5, 6.25, or 8.0), resulting in a series of protocols using various combinations of media and pH. The wells in each column were grouped by row, and each group of rows was loaded with a different target antibody (mAb 1, mAb 2, or mAb 3 ). ) was subjected to the protocol for each. Each protocol was run at its respective pH so that, for each target antibody, an aliquot of the loading substance containing the target antibody at the corresponding pH, as well as an equilibration buffer of 40 mM Tris and 300 mM citrate at the corresponding pH, was used. For each target antibody, an aliquot of the loading substance containing the target antibody at the corresponding pH, as well as an equilibration buffer of 40 mM Tris and 300 mM citrate at the corresponding pH, was used. was used.

[0133]

[0133] The following steps are performed simultaneously on the array. 1. Wells filled with various HIC resins are equilibrated 3 times with the equilibration buffer at each pH. 3 times equilibrated. 2. A loading substance containing the desired target antibody at each pH is added to each well and incubated for 1 hour. incubated for 1 hour. 3. The plate is rotated at 1100 rpm. 4. Flow-through (e.g., components not bound to the medium during incubation) is collected. collected. 5. For each protocol being tested, the wells are washed 2 times with the equilibration buffer at each pH. The wells are washed 2 times. 6. Pseudo-gradient elution is performed using the equilibration buffer at each pH. Each well is exposed to the equilibration buffer 7 times in repetition, and the citrate concentration linearly decreases from 300 mM to 0 mM over all 7 repetitions (an increment of 42.9 mM per repetition). The wells are repeatedly exposed to the equilibration buffer 7 times, and the citrate concentration linearly decreases from 300 mM to 0 mM over all 7 repetitions (an increment of 42.9 mM per repetition). mM to 0 mM linearly (an increment of 42.9 mM per repetition). 7. The wells are washed 2 times with RODI and 2 times with 1N sodium hydroxide. 8. The plate is rotated at 1100 rpm. 9. The wells are stripped 2 times with 6N guanidine HCl solution. 10. The plate is rotated at 1100 rpm.

[0134]

[0134] For each well, the substances removed from the well (flow-through, eluate, wash Chromatograms of the target polypeptide in the purified solution etc. were generated. To analyze the results, each chromatogram was divided into three zones. The first zone contains the mass of the target polypeptide observed in the substances removed during steps 2, 3, and 4 (flow through, washing, and elution). The second zone contains the mass of the target polypeptide observed in the substances removed during steps 5, 6, 7, 8 (using RODI and 1N sodium hydroxide). The third zone contains the mass of the target polypeptide observed in the substances removed during steps 9, 10 (using 6N guanidine HCl). Examples of three chromatograms generated from tocopherol and divided into three zones and purified with three different HIC media (TOY OPearl Hexyl-650C, Phenyl Sepharose 6 High Flow (High TM Sub), and POROS Ethyl) for the target antibody mAb 1 are shown in FIGS. 20 TM A - 20C. The comparison shows agreement with 98% statistically significant accuracy between the protocol and analysis performed on the test wells and the protocol and analysis performed on the large-scale column.

[0135]

[0135] The comparison shows agreement with 98% statistically significant accuracy between the protocol and analysis performed on the test wells and the protocol and analysis performed on the large-scale column.

[0136]

[0136] Example 20

[0137]

[0137] The collection totals of data using the high-throughput screening techniques described herein can provide information for the development of improved efficiency and yield HIC protocols, enabling the development of predictive functions. For example, a number of parameters (e.g., pH, elution buffer citrate concentration, loading mass, HIC media selection) and the protocol A boundary function may be plotted to describe the relationship between any of the efficiency or yield of

[0138]

[0138] Figure 21A is a plot of the boundary function with respect to the elution buffer citrate concentration and phenyl sepharose TM media, based on the total collection of high-throughput screening data for a given antibody and loading of 100 g per liter of media, against the predicted yield of the HIC protocol. The shaded area represents combinations of HIC protocol parameters that result in predicted yields of less than 90% of the theoretical yield. This boundary function gives information on which citrate and pH parameters should be considered for a given antibody and phenyl sepharose TM media HIC protocol. Combinations of pH and citrate concentrations outside the shaded area are usable HIC protocol parameters for a given antibody and phenyl sepharose media, while combinations of pH and citrate concentrations within the shaded area are parameters to be excluded. TM

[0139]

[0139] Figure 21B is a plot of the boundary function with respect to the elution buffer citrate concentration and pH against the predicted yield of the HIC protocol, based on the total collection of high-throughput TM screening data for a given antibody and Capto Phenyl (High Sub) chromatography media with a loading of 100 g per liter of media. The shaded area represents combinations of HIC protocol parameters that result in predicted yields of less than 90% of the theoretical yield. ​​corresponds to a combination. This boundary function provides information on which citrate and pH parameters TM should be considered for an HIC protocol involving a given antibody and Capto Phenyl (High Sub) medium. Combinations of pH and citrate concentrations outside the shaded region are available HIC protocol parameters for a given antibody and Capto Phenyl (High Sub) medium, while combinations of pH and citrate concentrations within the shaded region TM are parameters to be excluded for a given antibody and Capto Phenyl (High Sub) medium.

[0140]

[0140] In addition to the boundary function described above, data collected from high-throughput screening can enable the development of a transfer function or other mathematical model that can assist in the design of an HIC protocol. For example, a transfer function can be regressed to relate data from elution assays, high- throughput screens, or laboratory-scale HIC protocols to full-scale HIC protocols. For example, for an HIC protocol set including the predicted yield (e.g., the yield predicted by a small-scale assay or high-throughput screening), the actual yield for each HIC protocol can be determined through full-scale chromatography. It is possible to regress the relationship between the predicted yield and the actual yield to improve the prediction model. Based on the regression relationship (e.g., a transfer function) applied to data from elution assays, high- throughput screens, or laboratory-scale HIC protocols, it is possible to at least partially calculate the future-predicted yield.

[0141] Combine one or more transfer functions, boundary functions, and / or prediction models to develop a dynamic prediction model. Referring to Figure 22, a dynamic prediction model is shown . The dynamic prediction model calculates how changes to one or more HIC protocol parameters (e.g., pH, citrate concentration, loading mass, chromatography resin) affect the quantified properties of the HIC protocol . For example, in the example shown in Figure 22, as a result, the selected protocol parameters are a pH of 6, a citrate concentration of 30 mM, a loading mass of 100 g per liter of HIC medium, and resin C (e.g., phenylsepharose medium). The model then shows how each of these parameters affects the predicted yield, a high molecular weight fraction, and the pool host cell protein concentration of the eluate collected using the HIC protocol with the selected parameters TM . As shown in Figure 22, the selected parameters result in an approximate 95% predicted yield, an approximate high molecular weight fraction of 1.3, and an approximate pool host cell protein concentration of 22.5 ppm. These quantified properties may be used in the evaluation of the HIC protocol and can provide information on how protocol parameters affect the product of the HIC protocol. Values for the quantified properties calculated by the dynamic model can be updated in real time as the protocol parameters are modified. These quantified properties may be used in the evaluation of the HIC protocol and can provide information on how protocol parameters affect the product of the HIC protocol. Values for the quantified properties calculated by the dynamic model can be updated in real time as the protocol parameters are modified. Values for the quantified properties calculated by the dynamic model can be updated in real time as the protocol

[0142]

[0142] In some embodiments, a composite of the quantified properties of the protocol (e.g., a composite of the predicted yield, high molecular weight fraction, and pool host cell protein concentration) can be used Moreover, the overall desirability can also be calculated. The calculation of desirability may be based on the weighting of the quantitative characteristics of the HIC protocol. For example, changes to the HIC protocol that affect the yield of the protocol may be more important than changes that affect efficiency. The dynamic prediction model may take into account the relative importance of quantitative measurements and assign weights to different measurements so that they affect the overall desirability unevenly. In some embodiments, as additional chromatographic data (e.g., data from small-scale assays, high-throughput screening, and full-scale chromatography runs) is collected and integrated, the dynamic prediction model may be updated. It may be based on the weighting of the quantitative characteristics of the HIC protocol. For example, changes to the HIC protocol that affect the yield of the protocol may be more important than changes that affect efficiency. Moreover, the overall desirability can also be calculated. The calculation of desirability may be based on the weighting of the quantitative characteristics of the HIC protocol. For example, changes to the HIC protocol that affect the yield of the protocol may be more important than changes that affect efficiency. The dynamic prediction model may take into account the relative importance of quantitative measurements and assign weights to different measurements so that they affect the overall desirability unevenly. In some embodiments, as additional chromatographic data (e.g., data from small-scale assays, high-throughput screening, and full-scale chromatography runs) is collected and integrated, the dynamic prediction model may be updated. The dynamic prediction model may take into account the relative importance of quantitative measurements and assign weights to different measurements so that they affect the overall desirability unevenly. Moreover, the overall desirability can also be calculated. The calculation of desirability may be based on the weighting of the quantitative characteristics of the HIC protocol. For example, changes to the HIC protocol that affect the yield of the protocol may be more important than changes that affect efficiency. The dynamic prediction model may take into account the relative importance of quantitative measurements and assign weights to different measurements so that they affect the overall desirability unevenly. In some embodiments, as additional chromatographic data (e.g., data from small-scale assays, high-throughput screening, and full-scale chromatography runs) is collected and integrated, the dynamic prediction model may be updated. In some embodiments, as additional chromatographic data (e.g., data from small-scale assays, high-throughput screening, and full-scale chromatography runs) is collected and integrated, the dynamic prediction model may be updated. It may be based on the weighting of the quantitative characteristics of the HIC protocol. For example, changes to the HIC protocol that affect the yield of the protocol may be more important than changes that affect efficiency. The dynamic prediction model may take into account the relative importance of quantitative measurements and assign weights to different measurements so that they affect the overall desirability unevenly. In some embodiments, as additional chromatographic data (e.g., data from small-scale assays, high-throughput screening, and full-scale chromatography runs) is collected and integrated, the dynamic prediction model may be updated. Moreover, the overall desirability can also be calculated. The calculation of desirability may be based on the weighting of the quantitative characteristics of the HIC protocol. For example, changes to the HIC protocol that affect the yield of the protocol may be more important than changes that affect efficiency. The dynamic prediction model may take into account the relative importance of quantitative measurements and assign weights to different measurements so that they affect the overall desirability unevenly. In some embodiments, as additional chromatographic data (e.g., data from small-scale assays, high-throughput screening, and full-scale chromatography runs) is collected and integrated, the dynamic prediction model may be updated.

[0143]

[0143] Example 21

[0144]

[0144] A number of HIC protocols, including combinations of different target molecules and HIC media at various pHs, were tested in a gradient elution assay to determine whether such combinations might pose a problem of regeneration / usefulness and whether any of the target molecule and HIC media combinations should be excluded from the development of further HIC protocols. A number of HIC protocols, including combinations of different target molecules and HIC media at various pHs, were tested in a gradient elution assay to determine whether such combinations might pose a problem of regeneration / usefulness and whether any of the target molecule and HIC media combinations should be excluded from the development of further HIC protocols. A number of HIC protocols, including combinations of different target molecules and HIC media at various pHs, were tested in a gradient elution assay to determine whether such combinations might pose a problem of regeneration / usefulness and whether any of the target molecule and HIC media combinations should be excluded from the development of further HIC protocols. A number of HIC protocols, including combinations of different target molecules and HIC media at various pHs, were tested in a gradient elution assay to determine whether such combinations might pose a problem of regeneration / usefulness and whether any of the target molecule and HIC media combinations should be excluded from the development of further HIC protocols. 。

[0145]

[0145] The wells of a 96-well filter plate (AcroPrep Advance 1 mL filter plate, 1.2 μm Supor membrane, part number 8130, Pall Corporation) were filled with 0.02 μL of various HIC media. An aliquot of the loading substance containing the target molecule was prepared and mixed with the HIC media at a concentration of 5 g / L as the target. TM Advance 1 mL filter plate, 1.2 μm Supor membrane, part number 8130, Pall Corporation The wells of a 96-well filter plate (AcroPrep Advance 1 mL filter plate, 1.2 μm Supor membrane, part number 8130, Pall Corporation) were filled with 0.02 μL of various HIC media. An aliquot of the loading substance containing the target molecule was prepared and mixed with the HIC media at a concentration of 5 g / L as the target. An aliquot of the loading substance containing the target molecule was prepared and mixed with the HIC media at a concentration of 5 g / L as the target. As a standard, it was adjusted to a concentration of 0.33 g / L. For each of the three target antibodies, aliquots of the loading substance were titrated against 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 showing different pH values.

[0146]

[0146] Each well of a 96-well plate filled with one media type was subjected to an HIC protocol at different pHs (4.5, 6.25, or 8.0), resulting in a series of protocols carried out using various combinations of media and pH.

[0147]

[0147] The following steps are performed simultaneously on the array. 1. Wells filled with various HIC resins are equilibrated 3 times using the equilibration buffer at each pH. 2. The loading substance containing the desired target molecule at each pH is added to each well and incubated for 1 hour. 3. The plate is rotated at 1100 rpm. 4. Flow-through (e.g., containing components not bound to the media during incubation) is collected. 5. For each protocol being tested, the wells are washed 3 times using the equilibration buffer at each pH. 6. Mock elution is performed. Each well is exposed to the elution buffer 7 times in succession, the plate is rotated at 1100 rpm, and the eluate is collected after each iteration. 7. The wells are washed twice with NaOH. Different wells of the array may use different concentrations of NaOH. For example, two wells may contain the same chromatography media and be loaded at the same pH, but one well uses a 5 mM NaOH wash while the other ​​​​​​​​​​ In the wells, 1N NaOH washing is used. After each washing, the plate is rotated at 1100 rpm to collect the washing solution. 8. Strip the wells twice with 6N guanidine HCl solution and collect the stripped material (the stripped material).

[0148]

[0148] For each well, a chromatogram of the target molecule in the material removed from the well (flow-through, eluate, washing solution, etc.) was generated. To analyze the results, each chromatogram was divided into three zones. The first zone included the mass of the target molecule observed in the material removed during steps 2, 3, and 4 (flow-through, washing, and elution). The second zone included the mass of the target molecule observed in the material removed during steps 5, 6, and 7 (using RODI and NaOH). The third zone included the mass of the target molecule observed in the material removed during step 8 (using 6N guanidine HCl). )

[0149]

[0149] Examples of chromatograms generated from the elution assay for the first target molecule are shown in FIGS. 23A - 26C. FIGS. 23A - 23C show the chromatogram of the elution assay containing the first target molecule on the Capto Phenyl (High TM Sub) medium. FIGS. 24A - 24C show the chromatogram of the elution assay containing the first target molecule on the Capto Butyl medium. FIGS. 25A - C show the chromatogram of the elution assay containing the first target molecule on the POROS TM Benzyl medium. FIGS. 26A - C show the chromatogram of the elution assay containing the first target molecule on the Phenyl Sepharose TM medium. FIGS. 23 (R) ​​​​​​ A, 24A, 25A, and 26A show the chromatograms of the elution assay run at a pH of 4.5 and FIGS. 23B, 24B, 25B, and 26B show the chromatograms of the elution assay run at a pH of 6.25 and FIGS. 23C, 24C, 25C, and 26C show the chromatograms of the elution assay run at a pH of 8. In FIGS. 23A - 26C, the dotted lines show the chromatograms from the elution assay containing a 5 mM NaOH wash, and the solid lines show the chromatograms from the elution assay containing a 1N NaOH wash.

[0150]

[0150] Examples of chromatograms generated from elution assays for a second target molecule are shown in FIGS. 27A - 28D. Each of FIGS. 27A - 28 shows three chromatograms, each derived from an elution assay run at a different pH. FIG. 27A shows the chromatogram from the elution assay run on a Capto TM Phenyl (High Sub) medium, FIG. 27 B shows the chromatogram from the elution assay run on a Capto TM Butyl medium, FIG. 27C shows the chromatogram from the elution assay run on a TOYOPEARL TM Phenyl - 650C medium, FIG. 27D shows the chromatogram from the elution assay run on a POROS TM Ethyl medium, FIG. 28A shows the chromatogram from the elution assay run on a TOYOPEARL TM Hexyl - 65 0C medium, FIG. 28B shows the chromatogram from the elution assay run on a TOYOP EARL TM Butyl - 650C medium, FIG. 28C shows the chromatogram from the elution assay run on a POROS TM Benzyl medium, shows the mu, and FIG. 28D shows phenyl sepharose (R) from the execution of the elution assay on the medium shows the chromatogram. In FIGS. 27A - 28D, the dotted line shows the chromatogram from the elution assay including a 5 mM NaOH wash, and the solid line shows the chromatogram from the elution assay including a 1N NaOH wash

[0151]

[0151] As can be seen from the chromatogram, all of the tested protocols including a 1N NaOH strip contained a larger percentage of the target molecular mass than comparable protocols including a 5 mM NaOH strip. That is, more target molecules remained bound to the column after a 1N NaOH strip compared to a 5 mM NaOH strip

[0152]

[0152] By comparing the chromatograms of different HIC protocols for a given target molecule, specific HIC media and / or pH can be excluded from consideration. Using a series of elution assays to exclude undesirable combinations of target molecules, HIC media, and pH can improve the speed of developing and testing HIC protocols For example, a chromatogram showing that more than 5% (e.g., more than 10%) of the target molecule elutes in zone 3 can indicate that a given combination of HIC media, pH, and target molecule is not suitable for integration into an HIC protocol

[0153]

[0153] In addition to using elution assays to exclude HIC media, pH, and target molecules from consideration, the zone 3 mass calculated from the chromatogram is passed through a transfer function ​​​​​​​​​​​​Transform to enable prediction of the mass of the target molecule eluting in zone 3 during full-scale chromatography. Using this predicted mass, it is also possible to exclude combinations of HIC media, pH, and the target molecule from consideration. An exemplary list of the calculated zone 3 target molecule recoveries for a given target molecule during a series of HIC protocols using various combinations of HIC media and pH is shown in Table 14. As can be seen from Table 14, an elution assay containing a given target molecule on a Capto phenyl (High Sub) medium at a pH of 4.5 resulted in a normalized recovery percentage of 38.2%. When this result is transformed through the transfer function, it results in a 10.6% recovery in the full-scale zone 3. In embodiments where the predetermined threshold is 10%, this is higher than the threshold, and thus the combination of the target molecule, Capto phenyl (High Sub) medium, and a pH of 4.5 is excluded from further HIC protocol development. In some embodiments, when the exclusion of combinations of media and pH for a given target molecule is guaranteed, that combination of media and pH can also be excluded from the development of further protocols containing other target molecules. Those skilled in the art will recognize that the concepts upon which this disclosure is based can be readily used as a basis for designing other methods and systems for carrying out some of the purposes of this disclosure. Accordingly, the claims are not to be regarded as limited by the foregoing description.

Table 14

Table 14

[0154]

[0154] As shown in Table 14, an elution assay containing a given target molecule on a Capto phenyl (High Sub) medium at a pH of 4.5 resulted in a normalized recovery percentage of 38.2%. When this result is transformed through the transfer function, it results in a 10.6% recovery in the full-scale zone 3. In embodiments where the predetermined threshold is 10%, this is higher than the threshold, and thus the combination of the target molecule, Capto phenyl (High Sub) medium, and a pH of 4.5 is excluded from further HIC protocol development. In some embodiments, when the exclusion of combinations of media and pH for a given target molecule is guaranteed, that combination of media and pH can also be excluded from the development of further protocols containing other target molecules. TM Phenyl (High Sub) As shown in Table 14, an elution assay containing a given target molecule on a Capto phenyl (High Sub) medium at a pH of 4.5 resulted in a normalized recovery percentage of 38.2%. When this result is transformed through the transfer function, it results in a 10.6% recovery in the full-scale zone 3. In embodiments where the predetermined threshold is 10%, this is higher than the threshold, and thus the combination of the target molecule, Capto phenyl (High Sub) medium, and a pH of 4.5 is excluded from further HIC protocol development. In some embodiments, when the exclusion of combinations of media and pH for a given target molecule is guaranteed, that combination of media and pH can also be excluded from the development of further protocols containing other target molecules. As shown in Table 14, an elution assay containing a given target molecule on a Capto phenyl (High Sub) medium at a pH of 4.5 resulted in a normalized recovery percentage of 38.2%. When this result is transformed through the transfer function, it results in a 10.6% recovery in the full-scale zone 3. In embodiments where the predetermined threshold is 10%, this is higher than the threshold, and thus the combination of the target molecule, Capto phenyl (High Sub) medium, and a pH of 4.5 is excluded from further HIC protocol development. In some embodiments, when the exclusion of combinations of media and pH for a given target molecule is guaranteed, that combination of media and pH can also be excluded from the development of further protocols containing other target molecules. As shown in Table 14, an elution assay containing a given target molecule on a Capto phenyl (High Sub) medium at a pH of 4.5 resulted in a normalized recovery percentage of 38.2%. When this result is transformed through the transfer function, it results in a 10.6% recovery in the full-scale zone 3. In embodiments where the predetermined threshold is 10%, this is higher than the threshold, and thus the combination of the target molecule, Capto phenyl (High Sub) medium, and a pH of 4.5 is excluded from further HIC protocol development. In some embodiments, when the exclusion of combinations of media and pH for a given target molecule is guaranteed, that combination of media and pH can also be excluded from the development of further protocols containing other target molecules. As shown in Table 14, an elution assay containing a given target molecule on a Capto phenyl (High Sub) medium at a pH of 4.5 resulted in a normalized recovery percentage of 38.2%. When this result is transformed through the transfer function, it results in a 10.6% recovery in the full-scale zone 3. In embodiments where the predetermined threshold is 10%, this is higher than the threshold, and thus the combination of the target molecule, Capto phenyl (High Sub) medium, and a pH of 4.5 is excluded from further HIC protocol development. In some embodiments, when the exclusion of combinations of media and pH for a given target molecule is guaranteed, that combination of media and pH can also be excluded from the development of further protocols containing other target molecules. TM Phenyl (High Sub) As shown in Table 14, an elution assay containing a given target molecule on a Capto phenyl (High Sub) medium at a pH of 4.5 resulted in a normalized recovery percentage of 38.2%. When this result is transformed through the transfer function, it results in a 10.6% recovery in the full-scale zone 3. In embodiments where the predetermined threshold is 10%, this is higher than the threshold, and thus the combination of the target molecule, Capto phenyl (High Sub) medium, and a pH of 4.5 is excluded from further HIC protocol development. In some embodiments, when the exclusion of combinations of media and pH for a given target molecule is guaranteed, that combination of media and pH can also be excluded from the development of further protocols containing other target molecules. In some embodiments, when the exclusion of combinations of media and pH for a given target molecule is guaranteed, that combination of media and pH can also be excluded from the development of further protocols containing other target molecules. In some embodiments, when the exclusion of combinations of media and pH for a given target molecule is guaranteed, that combination of media and pH can also be excluded from the development of further protocols containing other target molecules. In some embodiments, when the exclusion of combinations of media and pH for a given target molecule is guaranteed, that combination of media and pH can also be excluded from the development of further protocols containing other target molecules.

[0155]

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

Claims

1. A method for regenerating a hydrophobic interaction chromatography column to which a loading mass has been applied. There was, Passing one or more column volumes of an alkaline solution through a hydrophobic interaction medium within the column. wherein the alkaline solution has a pH between about 10 and about 14 and a pH of about 0.5 mS / cm exhibiting a conductivity between about 10 mS / cm and about 10 mS / cm; The method wherein substances bound to the hydrophobic interaction medium are removed.

2. The alkaline solution is sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide.

2. The method of claim 1 , further comprising one of the following: NaCl, ...

3. The alkaline solution exhibits a conductivity between about 0.8 mS / cm and about 1.6 mS / cm.

2. The method according to claim 1.

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

5. After removal of material bound to the hydrophobic interaction medium, less than about 1.0% of the loaded mass is the residual mass. The method of claim 1, wherein the hydrophobic interaction medium is bound to the hydrophobic interaction medium.

6. The substances removed from the medium include host cell proteins, aggregated proteins, lipids, polypeptides, etc. The method of claim 1, comprising administering to the subject a nucleic acid fragment, a biomolecule, or a nucleic acid.

7. The method of claim 1 , wherein the material removed from the medium is free of bacteria or fungi.

8. The method includes contacting the hydrophobic interaction medium with a chaotropic agent or an organic solvent. The method of claim 1 , wherein the

9. passing one or more column volumes of an alkaline solution through a hydrophobic interaction medium within the column.

10. The method of claim 1, wherein the step takes from about 10 minutes to about 1 hour.

10. 1. A method of regenerating a chromatography column to which a loading mass has been applied, comprising the steps of: passing one or more column volumes of an alkaline solution through the medium in the column; The alkaline solution contains sodium hydroxide at a total dissolved concentration of between about 0.5 mM and about 50 mM. 、 The method wherein substances bound to the medium are removed.

11. The medium comprises a ligand having between 2 and 10 hydrocarbons in an aliphatic or aromatic configuration. The method of claim 10, further comprising a matrix comprising:

12. the ligand is present in the medium at a density of between about 20 and about 30 μmol per ml of medium; The method of claim 11.

13. The method of claim 10, wherein the medium does not contain ligands containing 30 or more hydrocarbons.

14. The chromatography column is not used in a mixed-mode chromatography process The method according to claim 10.

15. 11. The medium of claim 10, wherein the medium comprises a matrix comprising cross-linked agarose and a phenyl ligand. The method described above.

16. The method includes contacting the medium with an alcohol, ethylene glycol, or sodium chloride. The method of claim 10, wherein the method does not include

17. After passing one or more column volumes of alkaline solution through the column, one or more of the chaotropic agent is added. The method further comprises passing at least one column volume through the column, wherein the chaotropic agent 11. The method of claim 10, wherein the acid is one of 6N guanidine hydrochloride or 8N urea.

18. 1. A method of preparing a chromatography column for storage, comprising: Carrying out the method of claim 10; and a storage buffer comprising sodium hydroxide at a total dissolved concentration of between about 0.05 M and about 0.15 M; , contacting the column A method comprising:

19. 1. A method for reusing a chromatography column, comprising: applying a first load mass to a chromatography column; performing the method of claim 10 on said chromatography column; and applying a second load mass to the chromatography column. Including, The method, wherein the method does not include cleaning the chromatography column.

20. Identify the concentration of alkaline solution for hydrophobic interaction chromatography column regeneration solution 1. A method for producing a pharmaceutical composition comprising the steps of: passing a volume of a first solution through a hydrophobic interaction medium in said column; The first solution is made of water and a solution of 0% water starting at a concentration that increases at a substantially constant rate up to a maximum concentration. and an alkaline solution of 0.5%; Passing a volume of a second solution through a hydrophobic interaction medium, said second The solution is water and alkali with a concentration that starts at a maximum and decreases at a nearly constant rate to about 0N. a solution; and removing substances bound to the hydrophobic interaction medium as they pass through the hydrophobic interaction medium; Identifying a portion of the first or second solution A method comprising:

21. 21. The method of claim 20, wherein the alkaline solution comprises sodium hydroxide and has a maximum concentration of about 1N. How to.

22. The volume of the first solution and the volume of the second solution are each about 20 column volumes.

18. The method according to claim 18.

23. A method for evaluating a plurality of chromatography protocols, comprising: the chromatography protocol includes a chromatography medium, a protocol pH, and a target molecule; For each chromatography protocol: The loaded mass containing the target molecule is then transferred to a volume of chromatophores in a filter plate well. Add to HPLC medium and collect flow-through from filter plate wells. wherein the loading mass indicates a protocol pH; Multiple aliquots of a buffer containing a chromatography medium are added to the chromatograph. Obtaining an eluate from a feed medium, said buffer exhibiting a buffer pH and a cosmotropic The concentration of Pick's salt is linearly decreased over the plurality of aliquots, and a first amount of the target molecule is was contained in the combined flow-through and eluent; A second solution is added to the chromatography medium to remove the target extracting a second amount of the molecule; and Chaotropic agents are added to the chromatography media to remove extracting a third amount of the target molecule from the A method comprising:

24. Using one of the above protocols, a chromatography method including hydrophobic interaction chromatography can be performed.

24. The method of claim 23, further comprising providing a roughy unit operation.

25. Use filter plates to synchronize methods for multiple chromatography protocols 24. The method of claim 23, comprising performing the step of:

26. 24. The method of claim 23, wherein the loaded mass comprises less than about 20 mg of target molecule.

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