Method for disinfecting and / or regenerating chromatography media

Abstract

The present disclosure relates to a method for disinfecting chromatography media. The present disclosure also relates to the regeneration of chromatography media.

Description

[Technical field]

[0001] Related Application Data This application claims priority from European Patent Application No. EP22167454.2, filed April 8, 2022, entitled "Methods of sanitizing and regenerating a chromatography medium," the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application is filed with a Sequence Listing in electronic format, the entire contents of which are incorporated herein by reference.

[0003] The present disclosure relates to methods for disinfecting and / or regenerating chromatography media. [Background technology]

[0004] Purification of proteins (from natural sources, e.g., albumin from plasma, and proteins produced by recombinant techniques) is a critical step in the production of proteins, especially if the protein is for therapeutic purposes. To ensure that the protein is of suitable quality (e.g., purity and stability), several purification steps are typically involved in such production processes.

[0005] Chromatography-based purification methods are highly used for the purification of proteins. Existing chromatography-based methods of purifying proteins include affinity chromatography (e.g., Protein A chromatography), anion exchange chromatography, hydrophobic interaction chromatography, and cation exchange chromatography. An obstacle of existing methods used for commercial-scale purification of proteins is the high cost, considering the large amount of chromatography medium required. To address this issue, the chromatography medium is often reused. Between use cycles, the chromatography medium is treated to remove any non-specifically bound proteins that may reduce the ability of the medium to purify the protein of interest (i.e., the medium is regenerated), and the medium is sanitized to prevent contamination of future batches of the protein of interest (i.e., the medium is sanitized). However, the harsh methods used to regenerate and / or sanitize the chromatography medium may reduce the lifespan of the resin. Some protein-based chromatography resins are sensitive to harsh sanitization conditions. For example, aside from the new caustic-resistant ligands, Protein A ligands are sensitive to caustic cleaning conditions, such as high sodium hydroxide (NaOH) concentrations, which destroy the ligand over time and reduce its ability to be reused. Thus, there is a need in the art for methods to regenerate and / or sanitize chromatography media that maintain the ability of the media to purify a protein of interest for multiple cycles of purification. Summary of the Invention

[0006] In the work leading to the present invention, the inventors sought to identify a method for disinfecting and / or regenerating chromatography media used to purify proteins, e.g., immunoglobulins from serum or plasma. Specifically, the inventors recognized that chromatography media containing protein ligands are sensitive to harsh or caustic disinfection methods that reduce the lifespan of the resin. Thus, the inventors sought to identify a method for disinfecting chromatography media containing ligands (e.g., protein ligands) that are sensitive to caustic conditions. The inventors tested several options and identified a process for disinfecting chromatography resins that uses an alkaline detergent. Surprisingly, the inventors found that the use of an alkaline detergent, traditionally used as a surface disinfectant, was able to sufficiently inactivate and remove pathogens from the chromatography media without reducing the lifespan of the resin and / or without degrading the ligand and / or without causing significant loss of the binding activity of the ligand. The inventors additionally investigated options for regenerating chromatography media and found that a combination of ethanol and sodium hydroxide was most effective for removing remaining binding proteins from the media, e.g., when used in combination with an alkaline detergent to disinfect the resin. The inventors have also found that using low concentrations of sodium hydroxide in combination with ethanol for regeneration is particularly effective in removing residual bound proteins from the chromatography medium, and that this combination does not cause resin precipitation or clogging, a problem commonly observed with the use of standard high concentrations of sodium hydroxide for regeneration. Furthermore, the inventors have found that combining ethanol and sodium hydroxide into a single solution further improves the method by reducing the volume of solution and / or contact time required to regenerate the chromatography medium. The method produced by the inventors allows for the use of multiple cycles of chromatography medium. By increasing the number of cycles that the chromatography medium can be effectively used, the inventors have also achieved a significantly lower cost per use of the chromatography medium.

[0007] The findings of the present inventors provide a method of disinfecting a chromatography medium, the method comprising contacting the medium with an alkaline detergent. In one embodiment, the alkaline detergent comprises an alkaline salt, one or more surfactants, and / or a chelating agent. In one embodiment, the alkaline detergent comprises an alkaline salt, one or more surfactants, and optionally a chelating agent. In one embodiment, the alkaline detergent comprises an alkaline salt, one or more surfactants. In one embodiment, the alkaline detergent comprises an alkaline salt, one or more surfactants, and a chelating agent.

[0008] In one embodiment, the alkaline detergent is mixed in a solution. In one embodiment, the solution comprises 0.01% to 10%. For example, the solution comprises 0.1% to 10% alkaline detergent. In one embodiment, the solution comprises 0.01% to 5% alkaline detergent. In one embodiment, the solution comprises 0.05% to 5% alkaline detergent. In one embodiment, the solution comprises 0.01% to 5% alkaline detergent. For example, the solution comprises 0.01% to 2.5% alkaline detergent. In another embodiment, the solution comprises 0.01% to 0.1% alkaline detergent. For example, the solution comprises about 0.02% alkaline detergent. In one embodiment, the solution comprises about 0.02% to 0.1% alkaline detergent. In a further embodiment, the solution comprises 0.05% to 0.15% alkaline detergent. In one embodiment, the solution comprises 0.5% to 2.5% alkaline detergent. In one embodiment, the solution comprises 1% to 2.5% alkaline detergent. For example, the solution contains 2% alkaline detergent.

[0009] In one embodiment, the alkaline cleaner is CIP-100. For example, the solution includes 0.1% to 10% CIP-100. In one embodiment, the solution includes 0.1% to 5% CIP-100. In one embodiment, the solution includes 0.5% to 5% CIP-100. In one embodiment, the solution includes 1% to 5% CIP-100. For example, the solution includes 0.01% to 2.5% CIP-100. In another embodiment, the solution includes 1% to 2.5% CIP-100. For example, the solution includes about 2% CIP-100. In a further embodiment, the solution includes 1.5% to 2.5% CIP-100. In one embodiment, the solution includes 0.5% to 2.5% CIP-100.

[0010] In one embodiment, the alkaline salt is selected from the group consisting of potassium hydroxide, sodium hydroxide, sodium carbonate, sodium acetate, sodium sulfide, and sodium bicarbonate. For example, the alkaline salt is potassium hydroxide or sodium hydroxide. In one embodiment, the alkaline salt is potassium hydroxide. In another embodiment, the alkaline salt is sodium hydroxide.

[0011] In one embodiment, the alkaline detergent comprises between 10 mM and 150 mM of an alkali salt. For example, the alkaline detergent comprises between 20 mM and 150 mM of an alkali salt. In another embodiment, the alkaline detergent comprises between 30 mM and 140 mM of an alkali salt. In a further embodiment, the alkaline detergent comprises between 40 mM and 130 mM of an alkali salt. For example, the alkaline detergent comprises between 50 mM and 120 mM of an alkali salt. In one embodiment, the alkaline detergent comprises between 50 mM and 100 mM of an alkali salt. In another embodiment, the alkaline detergent comprises between 60 mM and 90 mM of an alkali salt. In a further embodiment, the alkaline detergent comprises between 60 mM and 80 mM of an alkali salt. For example, the alkaline detergent comprises between 70 mM and 80 mM of an alkali salt. In one embodiment, the alkaline detergent comprises about 75 mM of an alkali salt.

[0012] In one embodiment, the alkaline detergent comprises 10 mM, or 15 mM, or 20 mM, or 25 mM, or 30 mM, or 35 mM, or 40 mM, or 45 mM, or 50 mM, or 55 mM, or 60 mM, or 65 mM, or 70 mM, or 75 mM, or 80 mM, or 85 mM, or 90 mM, 95 mM, or 100 mM, or 105 mM, or 110 mM, or 115 mM, or 120 mM, or 125 mM, or 130 mM, or 135 mM, or 140 mM, or 145 mM, or 150 mM of an alkaline salt.

[0013] In one embodiment, the alkali salt is potassium hydroxide or sodium hydroxide, and the alkali salt is at a concentration of 20 mM to 150 mM. For example, the alkali salt is potassium hydroxide or sodium hydroxide, and the alkali salt is at a concentration of 50 mM to 100 mM. In one embodiment, the alkali salt is potassium hydroxide or sodium hydroxide, and the alkali salt is at a concentration of 60 mM to 80 mM. For example, the alkali salt is potassium hydroxide or sodium hydroxide, and the alkali salt is at a concentration of about 75 mM.

[0014] In one embodiment, the alkaline salt is potassium hydroxide at a concentration of 20 mM to 150 mM. For example, the alkaline salt is potassium hydroxide at a concentration of 50 mM to 100 mM. In one embodiment, the alkaline salt is potassium hydroxide at a concentration of 60 mM to 80 mM. For example, the alkaline salt is potassium hydroxide at a concentration of about 75 mM.

[0015] In one embodiment, the alkaline salt is sodium hydroxide at a concentration of 20 mM to 150 mM. For example, the alkaline salt is sodium hydroxide at a concentration of 50 mM to 100 mM. In one embodiment, the alkaline salt is sodium hydroxide at a concentration of 60 mM to 80 mM. For example, the alkaline salt is sodium hydroxide at a concentration of about 75 mM.

[0016] In one embodiment, the one or more surfactants are selected from the group consisting of amine ethoxylates, amphocarboxylates, triethanolamine, octyl-β-D glucopyranoside (OGP), polysorbates, poloxamers, lauryldimethylamine (LDAO), myristyldimethylamine-N-oxide (TDAO), sodium cholate, decyl-β-D-glucopyranoside, and dodecylmaltoside. For example, the polysorbate is polysorbate 80 or polysorbate 20.

[0017] In one embodiment, the surfactant is a polysorbate, such as polysorbate 20.

[0018] In one embodiment, the surfactant is TDAO. Commercially available TDAO solutions will be apparent to those skilled in the art and include, for example, Deviron® C16. In one embodiment, the surfactant is Deviron® C16.

[0019] In one embodiment, the surfactant is TDAO at a concentration of 0.1% to 10% (w / v). In one embodiment, the surfactant is TDAO at a concentration of 0.1% to 5% (w / v). For example, the surfactant is TDAO at a concentration of 0.01% to 2% (w / v). In another embodiment, the surfactant is TDAO at a concentration of 0.01% to 0.1% (w / v). In one embodiment, the surfactant is TDAO at a concentration of 0.02% to 0.1% (w / v). For example, the surfactant is TDAO at a concentration of 0.02% (w / v). In a further embodiment, the surfactant is TDAO at a concentration of 0.05% to 0.15% (w / v). In one embodiment, the surfactant is TDAO at a concentration of 0.5% to 2% (w / v).

[0020] In one embodiment, the alkaline detergent comprises TDAO and an alkaline salt selected from potassium hydroxide and sodium hydroxide.

[0021] In one embodiment, the alkaline detergent comprises TDAO and potassium hydroxide. In one embodiment, the alkaline detergent comprises 10 mM to 150 mM potassium hydroxide and 0.01% to 2% (w / v) TDAO. In one embodiment, the alkaline detergent comprises 60 mM to 80 mM potassium hydroxide and 0.01% to 0.1% (w / v) TDAO. In one embodiment, the alkaline detergent comprises 60 mM to 80 mM potassium hydroxide and 0.02% to 0.1% (w / v) TDAO. In one embodiment, the alkaline detergent comprises 75 mM potassium hydroxide and 0.02% to 0.1% (w / v) TDAO. For example, the alkaline detergent comprises about 0.02% TDAO and about 75 mM potassium hydroxide.

[0022] In one embodiment, the alkaline detergent comprises TDAO and sodium hydroxide. In one embodiment, the alkaline detergent comprises 10 mM to 150 mM sodium hydroxide and 0.01% to 2% (w / v) TDAO. In one embodiment, the alkaline detergent comprises 60 mM to 80 mM sodium hydroxide and 0.01% to 0.1% (w / v) TDAO. In one embodiment, the alkaline detergent comprises 60 mM to 80 mM sodium hydroxide and 0.02% to 0.1% (w / v) TDAO. In one embodiment, the alkaline detergent comprises 75 mM sodium hydroxide and 0.02% to 0.1% (w / v) TDAO. For example, the alkaline detergent comprises about 0.02% TDAO and about 75 mM sodium hydroxide.

[0023] In one embodiment, the surfactant and the alkaline salt are combined and mixed. For example, the surfactant and the alkaline salt are in the same solution (i.e., combined and mixed in the same solution). In another embodiment, the surfactant and the alkaline salt are in separate solutions. From the disclosure herein, it will be apparent to one skilled in the art that when the surfactant and the alkaline salt are in separate solutions, the medium is disinfected by contacting the medium with the surfactant and the alkaline salt simultaneously or sequentially. For example, the medium is disinfected by contacting the medium with an alkaline salt followed by a surfactant. In another embodiment, the medium is disinfected by contacting the medium with a surfactant followed by an alkaline salt. In the disclosed exemplary method, the medium is disinfected by contacting the medium with 0.02% to 0.1% surfactant followed by 75 mM of an alkaline salt selected from sodium hydroxide and potassium hydroxide.

[0024] In one embodiment, the chelating agent is ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), ethylenediamine-N,N'-disuccinic acid (EDDS), iminodisuccinic acid (IDS), methylglycine diacetic acid (MGDA), triethylenetetramine (Trien), iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), tripolyphosphate (TPP), diethylenetriaminepentaacetic acid (DTPA), sodium diethyldithiocarbamate (DDC), L-glutamic acid N,N-diacetic acid, tetrasodium salt (GLDA), and penicillamine, sodium gluconate, trisodium citrate, DMSA, DMPS, BAPTA, ALA, and salts thereof.

[0025] In one embodiment, the chelating agent is a biodegradable chelating agent such as ethylenediamine-N,N'-disuccinic acid (EDDS), iminodisuccinic acid (IDS), methylglycine diacetic acid (MGDA) or nitrilotriacetic acid (NTA).

[0026] In some embodiments, the chromatography medium is contacted with multiple column volumes (CV) of alkaline detergent to disinfect the medium. In this regard, a volume of alkaline detergent approximately equal to the volume of chromatography medium flowing through the medium is equal to 1 CV. In some embodiments, 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 column volumes of solution are flowed through the chromatography medium.

[0027] In some embodiments, the chromatography medium is contacted with an alkaline detergent to disinfect the medium for a defined period of time. For example, the period is 10 minutes, or 15 minutes, or 20 minutes, or 30 minutes, or 45 minutes, or 60 minutes. In one embodiment, the period is 10 minutes, or 15 minutes, or 20 minutes, or 30 minutes, or 45 minutes, or 60 minutes, or 75 minutes, or 90 minutes. In one embodiment, the period is 10 minutes. In one embodiment, the period is 15 minutes. In one embodiment, the period is 20 minutes. In one embodiment, the period is 30 minutes. In one embodiment, the period is 45 minutes. In one embodiment, the period is 60 minutes. In one embodiment, the period is 75 minutes. In one embodiment, the period is 90 minutes.

[0028] In one embodiment, the method includes contacting the medium with the alkaline detergent for a defined period of time. For example, the period is 10 minutes, or 15 minutes, or 20 minutes, or 30 minutes, or 45 minutes, or 60 minutes. In one embodiment, the method includes contacting the medium with the alkaline detergent for 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes. In one embodiment, the period is 10 minutes. In one embodiment, the method includes contacting the medium with the alkaline detergent for 15 minutes. In one embodiment, the method includes contacting the medium with the alkaline detergent for 20 minutes. In one embodiment, the method includes contacting the medium with the alkaline detergent for 30 minutes. In one embodiment, the method includes contacting the medium with the alkaline detergent for 45 minutes. In one embodiment, the method includes contacting the medium with the alkaline detergent for 60 minutes. In one embodiment, the method includes contacting the medium with the alkaline detergent for 75 minutes. In one embodiment, the method includes contacting the medium with the alkaline detergent for 90 minutes.

[0029] In some forms of the disclosure, the methods described herein additionally include regenerating the chromatography medium. For example, the methods include regenerating the chromatography medium prior to disinfecting the chromatography medium.

[0030] Methods for regenerating chromatography media are known in the art and will be apparent to one of ordinary skill in the art. For example, regenerating a chromatography medium includes contacting the chromatography medium with a solution including, but not limited to, a salt solution (e.g., NaCl), a caustic solution (e.g., up to 1.5 M sodium hydroxide), a solvent such as hydrochloric acid, acetic acid, ethanol, methanol, isopropanol, and acetonitrile, Tris or Tris EDTA, or a compound such as urea or guanidine hydrochloride. In one example, regenerating a chromatography medium includes contacting the chromatography medium with a polar solution.

[0031] Some regeneration methods involve contacting the medium with a regeneration solution that includes ethylene glycol, sodium chloride, sodium hydroxide, or a combination of sodium hydroxide and ethanol.

[0032] In some embodiments, the medium is regenerated by contacting the medium with ethanol, e.g., a solution containing 5-30% ethanol (e.g., 20% ethanol). In some embodiments, the medium is regenerated by including one or more steps of washing the resin with one or more column volumes of ethanol, followed by washing the resin with one or more column volumes of ultrapure water.

[0033] In some embodiments, regenerating the chromatography medium comprises contacting the medium with a combination of solutions. In some embodiments, the medium is regenerated by including washing the resin with one or more column volumes of 1 M NaCl and 1 M sodium hydroxide, washing the resin with one or more column volumes of ultrapure water, washing the resin with one or more column volumes of 1 M acetic acid, followed by washing the resin with one or more column volumes of ultrapure water. Additional methods and reagents for regenerating chromatography resins are described herein.

[0034] In some examples, the medium is regenerated by using a combination of NaCl and sodium hydroxide, for example, by washing with a combination of one or more column volumes of 2.0 M NaCl and one or more column volumes of NaOH.

[0035] In some examples, the medium is regenerated by contacting the medium with ethanol, e.g., 5-30% ethanol (such as 20% ethanol), followed by sodium hydroxide, e.g., 5-30 mM sodium hydroxide (e.g., 20 mM sodium hydroxide).

[0036] In some embodiments, the ethanol and sodium hydroxide are applied sequentially. For example, the medium is regenerated by contacting the medium with ethanol followed by sodium hydroxide. In another embodiment, the medium is regenerated by contacting the medium with sodium hydroxide followed by ethanol. In some embodiments, the ethanol and sodium hydroxide are applied simultaneously as separate solutions. In one embodiment, the ethanol and sodium hydroxide are applied as a single solution.

[0037] In some embodiments, the medium is regenerated by contacting the resin with 0.2 M NaOH. In one embodiment, the medium is regenerated by contacting the resin with 5 mM NaOH.

[0038] In some embodiments, the chromatography medium is contacted with multiple column volumes (CV) of solution to regenerate the column. In this regard, a volume of solution approximately equal to the volume of chromatography medium flowing through the medium is equal to 1 CV. In some embodiments, 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 column volumes of solution are flowed through the chromatography medium.

[0039] In some embodiments, the chromatography medium is contacted with the solution to regenerate the column for a defined period of time, for example, 10 minutes, or 15 minutes, or 20 minutes, or 30 minutes, or 45 minutes, or 60 minutes.

[0040] The present disclosure additionally provides methods of disinfecting and regenerating chromatography media. For example, the method includes disinfecting the chromatography media, followed by regenerating the chromatography media. In another example, the method includes regenerating the chromatography media, followed by disinfecting the chromatography media.

[0041] The present disclosure provides a method for disinfecting and regenerating chromatography media, the method comprising: (i) disinfecting a chromatography medium by contacting the medium with an alkaline detergent; (ii) regenerating the chromatography medium by contacting the medium with a regenerating solution comprising ethylene glycol, sodium chloride, sodium hydroxide, or a combination of sodium hydroxide and ethanol.

[0042] In one embodiment, the method comprises: (i) disinfecting a chromatography medium by contacting the medium with an alkaline detergent comprising an alkaline salt, one or more surfactants, and optionally a chelating agent; (ii) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of sodium hydroxide and ethanol.

[0043] In one embodiment, the method comprises: (i) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.01% to 10% (w / v) of one or more surfactants and / or 10 mM to 150 mM of an alkali salt; (ii) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5-30 mM sodium hydroxide and 5-30% ethanol.

[0044] In one embodiment, the method comprises: (i) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.01% to 10% (w / v) of one or more surfactants and / or 10 mM to 150 mM of an alkali salt selected from sodium hydroxide and potassium hydroxide; (ii) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5-30 mM sodium hydroxide and 5-30% ethanol.

[0045] In one embodiment, the method comprises: (i) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.01% to 5% (w / v) of one or more surfactants and / or 50 mM to 100 mM of an alkali salt selected from sodium hydroxide and potassium hydroxide; (ii) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5-30 mM sodium hydroxide and 5-30% ethanol.

[0046] In one embodiment, the method comprises: (i) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.01% to 1% (w / v) of one or more surfactants and / or 60 mM to 80 mM of an alkali salt selected from sodium hydroxide and potassium hydroxide; (ii) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5-30 mM sodium hydroxide and 5-30% ethanol.

[0047] In one embodiment, the method comprises: (i) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.02% to 0.1% (w / v) of one or more surfactants and / or 60 mM to 80 mM of an alkali salt selected from sodium hydroxide and potassium hydroxide; (ii) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5-30 mM sodium hydroxide and 5-30% ethanol.

[0048] In one embodiment, the method comprises: (i) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.02% (w / v) of one or more surfactants and / or 75 mM of an alkali salt selected from sodium hydroxide and potassium hydroxide; (ii) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5 mM sodium hydroxide and 20% ethanol.

[0049] In one embodiment, the method comprises: (i) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.02% (w / v) of one or more surfactants and / or 75 mM sodium hydroxide; (ii) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5 mM sodium hydroxide and 20% ethanol.

[0050] In one embodiment, the method comprises: (i) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.02% (w / v) of one or more surfactants and / or 75 mM potassium hydroxide; (ii) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5 mM sodium hydroxide and 20% ethanol.

[0051] In one embodiment, the method comprises: (i) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.02% (w / v) of one or more surfactants and / or 75 mM sodium hydroxide; (ii) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 20 mM sodium hydroxide and 20% ethanol.

[0052] In one embodiment, the method comprises: (i) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.02% (w / v) of one or more surfactants and / or 75 mM potassium hydroxide; (ii) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 20 mM sodium hydroxide and 20% ethanol.

[0053] In one embodiment, the method comprises: (i) disinfecting a chromatography medium by contacting the medium with an alkaline detergent containing CIP-100; (ii) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of sodium hydroxide and ethanol.

[0054] In one embodiment, the method comprises: (i) disinfecting the chromatography medium by contacting the medium with an alkaline detergent containing 1% to 5% CIP-100; (ii) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5-30 mM sodium hydroxide and 5-30% ethanol.

[0055] In one embodiment, the method comprises: (i) disinfecting a chromatography medium by contacting the medium with an alkaline detergent containing 2% CIP-100; (ii) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5 mM or 20 mM sodium hydroxide and 20% ethanol.

[0056] In one embodiment, the method comprises: (i) disinfecting a chromatography medium by contacting the medium with an alkaline detergent containing 2% CIP-100; (ii) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5 mM sodium hydroxide and 20% ethanol.

[0057] In one embodiment, the method comprises: (i) disinfecting a chromatography medium by contacting the medium with an alkaline detergent containing 2% CIP-100; (ii) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 20 mM sodium hydroxide and 20% ethanol.

[0058] In one embodiment, the method comprises: (i) disinfecting a chromatography medium by contacting the medium with an alkaline detergent comprising myristyldimethylamine-N-oxide (TDAO) and an alkaline salt selected from potassium hydroxide and sodium hydroxide; (ii) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of sodium hydroxide and ethanol.

[0059] In one embodiment, the method comprises: (i) disinfecting a chromatography medium by contacting the medium with an alkaline detergent comprising 0.01% to 10% (w / v) myristyldimethylamine-N-oxide (TDAO) and an alkaline salt selected from 10 mM to 150 mM potassium hydroxide and sodium hydroxide; (ii) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5-30 mM sodium hydroxide and 5-30% ethanol.

[0060] In one embodiment, the method comprises: (i) disinfecting a chromatography medium by contacting the medium with an alkaline detergent comprising 0.01% to 1% (w / v) myristyldimethylamine-N-oxide (TDAO) and an alkaline salt selected from 60 mM to 80 mM potassium hydroxide and sodium hydroxide; (ii) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5 mM or 20 mM sodium hydroxide and 20% ethanol.

[0061] In one embodiment, the method comprises: (i) disinfecting a chromatography medium by contacting the medium with an alkaline detergent comprising 0.02% to 0.1% (w / v) myristyldimethylamine-N-oxide (TDAO) and 60 mM to 80 mM of an alkaline salt selected from potassium hydroxide and sodium hydroxide; (ii) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5 mM or 20 mM sodium hydroxide and 20% ethanol.

[0062] In one embodiment, the method comprises: (i) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.01% to 1% (w / v) myristyldimethylamine-N-oxide (TDAO) and 60 mM to 80 mM potassium hydroxide; (ii) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5 mM or 20 mM sodium hydroxide and 20% ethanol.

[0063] In one embodiment, the method comprises: (i) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.02% to 0.1% (w / v) myristyldimethylamine-N-oxide (TDAO) and 60 mM to 80 mM potassium hydroxide; (ii) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5 mM or 20 mM sodium hydroxide and 20% ethanol.

[0064] In one embodiment, the method comprises: (i) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.01% to 1% (w / v) myristyldimethylamine-N-oxide (TDAO) and 60 mM to 80 mM sodium hydroxide; (ii) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5 mM or 20 mM sodium hydroxide and 20% ethanol.

[0065] In one embodiment, the method comprises: (i) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.02% to 0.1% (w / v) myristyldimethylamine-N-oxide (TDAO) and 60 mM to 80 mM sodium hydroxide; (ii) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5 mM or 20 mM sodium hydroxide and 20% ethanol.

[0066] In one embodiment, the method comprises: (i) disinfecting a chromatography medium by contacting the medium with an alkaline detergent comprising 0.02% (w / v) myristyldimethylamine-N-oxide (TDAO) and 75 mM of an alkaline salt selected from potassium hydroxide and sodium hydroxide; (ii) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 20 mM sodium hydroxide and 20% ethanol.

[0067] In one embodiment, the method comprises: (i) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.02% (w / v) myristyldimethylamine-N-oxide (TDAO) and 75 mM potassium hydroxide; (ii) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 20 mM sodium hydroxide and 20% ethanol.

[0068] In one embodiment, the method comprises: (i) disinfecting the chromatography medium by contacting the medium with an alkaline detergent containing 0.02% (w / v) myristyldimethylamine-N-oxide (TDAO) and 75 mM sodium hydroxide; (ii) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 20 mM sodium hydroxide and 20% ethanol.

[0069] In one embodiment, the method comprises: (i) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.02% (w / v) myristyldimethylamine-N-oxide (TDAO) and 75 mM potassium hydroxide; (ii) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5 mM sodium hydroxide and 20% ethanol.

[0070] In one embodiment, the method comprises: (i) disinfecting the chromatography medium by contacting the medium with an alkaline detergent containing 0.02% (w / v) myristyldimethylamine-N-oxide (TDAO) and 75 mM sodium hydroxide; (ii) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5 mM sodium hydroxide and 20% ethanol.

[0071] The present disclosure provides a method for regenerating and disinfecting a chromatography medium, the method comprising: (i) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising ethylene glycol, sodium chloride, sodium hydroxide, or a combination of sodium hydroxide and ethanol; (ii) disinfecting the chromatography medium by contacting the medium with an alkaline detergent.

[0072] In one embodiment, the method comprises: (i) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of sodium hydroxide and ethanol; (ii) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising an alkaline salt, one or more surfactants, and optionally a chelating agent.

[0073] In one embodiment, the method comprises: (i) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5-30 mM sodium hydroxide and 5-30% ethanol; (ii) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.01% to 10% (w / v) of one or more surfactants and / or 10 mM to 150 mM of an alkali salt.

[0074] In one embodiment, the method comprises: (i) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5-30 mM sodium hydroxide and 5-30% ethanol; (ii) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.01% to 10% (w / v) of one or more surfactants and / or 10 mM to 150 mM of an alkali salt selected from sodium hydroxide and potassium hydroxide.

[0075] In one embodiment, the method comprises: (i) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5-30 mM sodium hydroxide and 5-30% ethanol; (ii) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.01% to 5% (w / v) of one or more surfactants and / or 50 mM to 100 mM of an alkali salt selected from sodium hydroxide and potassium hydroxide.

[0076] In one embodiment, the method comprises: (i) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5-30 mM sodium hydroxide and 5-30% ethanol; (ii) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.01% to 1% (w / v) of one or more surfactants and / or 60 mM to 80 mM of an alkali salt selected from sodium hydroxide and potassium hydroxide.

[0077] In one embodiment, the method comprises: (i) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5-30 mM sodium hydroxide and 5-30% ethanol; (ii) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.02% to 0.1% (w / v) of one or more surfactants and / or 60 mM to 80 mM of an alkali salt selected from sodium hydroxide and potassium hydroxide.

[0078] In one embodiment, the method comprises: (i) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5 mM sodium hydroxide and 20% ethanol; (ii) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.02% (w / v) of one or more surfactants and / or 75 mM of an alkali salt selected from sodium hydroxide and potassium hydroxide.

[0079] In one embodiment, the method comprises: (i) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5 mM sodium hydroxide and 20% ethanol; (ii) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.02% (w / v) of one or more surfactants and / or 75 mM sodium hydroxide.

[0080] In one embodiment, the method comprises: (i) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5 mM sodium hydroxide and 20% ethanol; (ii) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.02% (w / v) of one or more surfactants and / or 75 mM potassium hydroxide.

[0081] In one embodiment, the method comprises: (i) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 20 mM sodium hydroxide and 20% ethanol; (ii) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.02% (w / v) of one or more surfactants and / or 75 mM sodium hydroxide.

[0082] In one embodiment, the method comprises: (i) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 20 mM sodium hydroxide and 20% ethanol; (ii) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.02% (w / v) of one or more surfactants and / or 75 mM potassium hydroxide.

[0083] In one embodiment, the method comprises: (i) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of sodium hydroxide and ethanol; (ii) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising CIP-100.

[0084] In one embodiment, the method comprises: (i) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5-30 mM sodium hydroxide and 5-30% ethanol; (ii) disinfecting the chromatography medium by contacting the medium with an alkaline detergent containing 1% to 5% CIP-100.

[0085] In one embodiment, the method comprises: (i) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5 mM or 20 mM sodium hydroxide and 20% ethanol; (ii) disinfecting the chromatography medium by contacting the medium with an alkaline detergent containing 2% CIP-100.

[0086] In one embodiment, the method comprises: (i) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5 mM sodium hydroxide and 20% ethanol; (i) disinfecting the chromatography medium by contacting the medium with an alkaline detergent containing 2% CIP-100.

[0087] In one embodiment, the method comprises: (i) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 20 mM sodium hydroxide and 20% ethanol; (ii) disinfecting the chromatography medium by contacting the medium with an alkaline detergent containing 2% CIP-100.

[0088] In one embodiment, the method comprises: (i) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of sodium hydroxide and ethanol; (ii) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising myristyldimethylamine-N-oxide (TDAO) and an alkaline salt selected from potassium hydroxide and sodium hydroxide.

[0089] In one embodiment, the method comprises: (i) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5-30 mM sodium hydroxide and 5-30% ethanol; (ii) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.01% to 10% (w / v) myristyldimethylamine-N-oxide (TDAO) and 10 mM to 150 mM of an alkali salt selected from potassium hydroxide and sodium hydroxide.

[0090] In one embodiment, the method comprises: (i) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5 mM or 20 mM sodium hydroxide and 20% ethanol; (ii) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.01% to 1% (w / v) myristyldimethylamine-N-oxide (TDAO) and 60 mM to 80 mM of an alkali salt selected from potassium hydroxide and sodium hydroxide.

[0091] In one embodiment, the method comprises: (i) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5 mM or 20 mM sodium hydroxide and 20% ethanol; (ii) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.02% to 0.1% (w / v) myristyldimethylamine-N-oxide (TDAO) and 60 mM to 80 mM of an alkali salt selected from potassium hydroxide and sodium hydroxide.

[0092] In one embodiment, the method comprises: (i) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5 mM or 20 mM sodium hydroxide and 20% ethanol; (ii) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.01% to 1% (w / v) myristyldimethylamine-N-oxide (TDAO) and 60 mM to 80 mM potassium hydroxide.

[0093] In one embodiment, the method comprises: (i) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5 mM or 20 mM sodium hydroxide and 20% ethanol; (ii) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.02% to 0.1% (w / v) myristyldimethylamine-N-oxide (TDAO) and 60 mM to 80 mM potassium hydroxide.

[0094] In one embodiment, the method comprises: (i) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5 mM or 20 mM sodium hydroxide and 20% ethanol; (ii) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.01% to 1% (w / v) myristyldimethylamine-N-oxide (TDAO) and 60 mM to 80 mM sodium hydroxide.

[0095] In one embodiment, the method comprises: (i) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5 mM or 20 mM sodium hydroxide and 20% ethanol; (ii) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.02% to 0.1% (w / v) myristyldimethylamine-N-oxide (TDAO) and 60 mM to 80 mM sodium hydroxide.

[0096] In one embodiment, the method comprises: (i) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 20 mM sodium hydroxide and 20% ethanol; (ii) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.02% (w / v) myristyldimethylamine-N-oxide (TDAO) and 75 mM of an alkali salt selected from potassium hydroxide and sodium hydroxide.

[0097] In one embodiment, the method comprises: (i) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 20 mM sodium hydroxide and 20% ethanol; (ii) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.02% (w / v) myristyldimethylamine-N-oxide (TDAO) and 75 mM potassium hydroxide.

[0098] In one embodiment, the method comprises: (i) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 20 mM sodium hydroxide and 20% ethanol; (ii) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.02% (w / v) myristyldimethylamine-N-oxide (TDAO) and 75 mM sodium hydroxide.

[0099] In one embodiment, the method comprises: (i) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5 mM sodium hydroxide and 20% ethanol; (ii) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.02% (w / v) myristyldimethylamine-N-oxide (TDAO) and 75 mM potassium hydroxide.

[0100] In one embodiment, the method comprises: (i) regenerating the chromatography medium by contacting the medium with a regeneration solution comprising a combination of 5 mM sodium hydroxide and 20% ethanol; (ii) disinfecting the chromatography medium by contacting the medium with an alkaline detergent comprising 0.02% (w / v) myristyldimethylamine-N-oxide (TDAO) and 75 mM sodium hydroxide.

[0101] In some examples, the chromatography medium is an ion exchange medium, an affinity medium, a hydrophobic interaction chromatography medium, a size exclusion column, or a mixed mode chromatography medium. For example, the medium is an affinity chromatography medium, e.g., an affinity chromatography medium that binds to the Fc region of an antibody. Exemplary affinity chromatography mediums include Protein A or an antibody binding fragment thereof, Protein G or an antibody binding fragment thereof, or a ligand capable of specifically binding to the CH1 or CH3 domain of human IgG. In one example, a ligand capable of specifically binding to the CH3 domain of human IgG comprises a single domain [VHH] antibody fragment from a camelid.

[0102] In some embodiments, the medium is not caustic or alkaline stable.

[0103] In some embodiments, the medium is an affinity chromatography medium that is not caustic-stable or alkaline-stable.

[0104] In one embodiment, the chromatography medium comprises a matrix selected from the group consisting of poly(styrene-divinylbenzene), cross-linked poly(styrene-divinylbenzene), silica, agarose, cross-linked agarose, controlled pore glass, polymethacrylate, and cellulose. In an exemplary embodiment of the present disclosure, the chromatography medium comprises cross-linked poly(styrene-divinylbenzene).

[0105] In one example, the chromatography medium comprises a VHH antibody fragment capable of specifically binding to the CH3 domain of human IgG conjugated to a cross-linked poly(styrene-divinylbenzene) matrix.

[0106] The present disclosure provides a method comprising contacting a chromatography medium comprising a VHH antibody fragment capable of specifically binding to the CH3 domain of human IgG conjugated to a cross-linked poly(styrene-divinylbenzene) matrix with an alkaline detergent comprising an alkaline salt, one or more surfactants, and / or chelating agents.

[0107] In one embodiment, the alkaline detergent comprises an alkaline salt, one or more surfactants, and optionally a chelating agent. In one embodiment, the alkaline detergent comprises an alkaline salt, one or more surfactants. In one embodiment, the alkaline detergent comprises an alkaline salt, one or more surfactants, and a chelating agent.

[0108] The present disclosure additionally provides a method comprising regenerating a chromatography medium comprising a VHH antibody fragment capable of specifically binding to a CH3 domain of human IgG conjugated to a cross-linked poly(styrene-divinylbenzene) matrix by contacting the medium with a solution comprising a combination of sodium hydroxide and ethanol, and disinfecting the medium by contacting it with an alkaline detergent comprising an alkaline salt, one or more surfactants, and optionally a chelating agent.

[0109] In one embodiment, the alkaline detergent comprises an alkaline salt, one or more surfactants, and optionally a chelating agent. In one embodiment, the alkaline detergent comprises an alkaline salt, one or more surfactants. In one embodiment, the alkaline detergent comprises an alkaline salt, one or more surfactants, and a chelating agent.

[0110] In one embodiment, the chromatography medium has been previously contacted with the sample, for example the sample being plasma or a fraction thereof.

[0111] In one embodiment, the plasma is clarified cryo-rich plasma.

[0112] In one embodiment, the plasma fraction is clarified cryopreserved plasma.

[0113] In some embodiments, the methods described herein are performed as part of a continuous chromatography process.

[0114] In one embodiment, the method described herein is repeated on the resin where multiple batches of plasma or a fraction thereof are applied to the resin, e.g., the method described herein is repeated on the resin after each batch of plasma or a fraction thereof.

[0115] It will be apparent to one of skill in the art from the disclosure herein that the number of cycles per batch of plasma or a fraction thereof will depend on the volume and / or weight of the plasma or a fraction thereof.

[0116] In one embodiment, the method described herein is performed on the resin after each batch of plasma or a fraction thereof, the batch comprises at least 1 cycle. In another embodiment, the batch comprises at least 5 cycles. For example, the batch comprises at least 10 cycles. In a further embodiment, the batch comprises at least 15 cycles. In another embodiment, the batch comprises at least 20 cycles. In one embodiment, the batch comprises at least 25 cycles. In one embodiment, the batch comprises at least 30 cycles. In another embodiment, the batch comprises at least 35 cycles. In a further embodiment, the batch comprises at least 40 cycles. In one embodiment, the batch comprises at least 45 cycles. In another embodiment, the batch comprises at least 50 cycles. In a further embodiment, the batch comprises at least 60 cycles, or at least 70 cycles, or at least 80 cycles, or at least 90 cycles, or at least 100 cycles. In one embodiment, the batch comprises between 1 and 100 cycles. For example, the batch comprises between 10 and 100 cycles. In another embodiment, the batch comprises between 20 and 80 cycles. In a further embodiment, the batch comprises between 30 and 60 cycles.

[0117] In one embodiment, the batch includes 1 cycle. In another embodiment, the batch includes at least 5 cycles. For example, the batch includes 10 cycles. In a further embodiment, the batch includes 15 cycles. In another embodiment, the batch includes 20 cycles. In one embodiment, the batch includes 25 cycles. In one embodiment, the batch includes 30 cycles. In another embodiment, the batch includes 35 cycles. In a further embodiment, the batch includes 40 cycles. In one embodiment, the batch includes 45 cycles. In another embodiment, the batch includes 50 cycles. In further embodiments, the batch includes 60 cycles, or 70 cycles, or 80 cycles, or 90 cycles, or 100 cycles.

[0118] In one embodiment, the method described herein is repeated on the resin after at least 50 cycles of plasma or a fraction thereof. For example, the method is repeated on the resin after at least 50 cycles per batch of plasma or a fraction thereof. In one embodiment, the method is repeated on the resin after 50-80 cycles, 60-80 cycles, or 70-80 cycles per batch of plasma or a fraction thereof. For example, the method is repeated on the resin after at least 60, or 65, or 70, or 75, or 80 cycles per batch of plasma or a fraction thereof.

[0119] In one embodiment, the method is repeated on the resin after each batch of plasma or a fraction thereof, where multiple batches of plasma or a fraction thereof are applied to the resin. In one embodiment, the method is repeated on the resin after each batch of plasma or a fraction thereof, where 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 batches of plasma or a fraction thereof are applied to the resin. In one embodiment, the method is repeated on the resin after each batch of plasma or a fraction thereof, where 4 to 10 batches of plasma or a fraction thereof are applied to the resin.

[0120] In one embodiment, the regeneration and / or disinfection methods described herein are performed on the resin after each individual cycle. In another embodiment, the regeneration and / or disinfection methods described herein are performed on the resin after multiple cycles. For example, the regeneration and / or disinfection methods described herein are performed on the resin after at least 50 cycles. In one embodiment, the regeneration and / or disinfection methods described herein are performed on the resin after at least 100 cycles. In another embodiment, the regeneration and / or disinfection methods described herein are performed on the resin after at least 150 cycles. In a further embodiment, the regeneration and / or disinfection methods described herein are performed on the resin after at least 200 cycles. In one embodiment, the regeneration and / or disinfection methods described herein are performed on the resin after each batch of plasma or a fraction thereof. For example, the regeneration and / or disinfection methods described herein are performed on the resin between each batch of plasma or a fraction thereof, i.e., before each batch of plasma or a fraction thereof is loaded onto the resin.

[0121] In one embodiment, a regeneration step is performed on the resin as part of each individual cycle, e.g., a regeneration step is performed on the resin after each individual cycle.

[0122] In one embodiment, the disinfection step is performed after each batch of plasma or a fraction thereof. For example, the disinfection step is performed after multiple cycles of plasma or a fraction thereof. In one embodiment, the disinfection step is performed after each batch of plasma or a fraction thereof, each batch comprising at least 50 cycles.

[0123] In one embodiment, a regeneration step is performed on the resin after each individual cycle and a disinfection step is performed after each batch of plasma or a fraction thereof.

[0124] It will be apparent to one of skill in the art from the disclosure herein that performing the disclosed method on the resin between each batch of plasma or a fraction thereof extends the lifespan of the resin. For example, the disclosed method extends the lifespan of the resin or allows the resin to be reused for up to a total of 800 cycles. In one embodiment, the disclosed method extends the lifespan of the resin or allows the resin to be reused for up to a total of 100, or 200, or 300, or 400, or 500, or 600, or 700 cycles. For example, the disclosed method extends the lifespan of the resin or allows the resin to be reused for up to a total of 100 cycles. For example, the disclosed method extends the lifespan of the resin or allows the resin to be reused for up to a total of 200 cycles. For example, the disclosed method extends the lifespan of the resin or allows the resin to be reused for up to a total of 300 cycles. For example, the disclosed method may extend the life of the resin or allow for reuse of the resin for up to a total of 400 cycles. For example, the disclosed method may extend the life of the resin or allow for reuse of the resin for up to a total of 500 cycles. For example, the disclosed method may extend the life of the resin or allow for reuse of the resin for up to a total of 600 cycles. For example, the disclosed method may extend the life of the resin or allow for reuse of the resin for up to a total of 700 cycles.

[0125] In one embodiment, the resin is reused with up to 10 batches of plasma or a fraction thereof for up to a total of 500 cycles, with the method of the disclosure being performed on the resin between each batch of plasma or a fraction thereof.

[0126] In one embodiment, the continuous chromatography is continuous affinity chromatography.

[0127] In one embodiment, the continuous affinity chromatography is selected from the group consisting of simulated moving bed (SMB) chromatography, cyclic countercurrent chromatography (PCC), continuous countercurrent tangential chromatography (CCTC), and continuous countercurrent spiral chromatography (CCSC).

[0128] In one embodiment, the continuous affinity chromatography is simulated moving bed (SMB) chromatography. In another embodiment, the continuous affinity chromatography is cyclic countercurrent chromatography (PCC). In a further embodiment, the continuous affinity chromatography is continuous countercurrent tangential chromatography (CCTC). In one embodiment, the continuous affinity chromatography is continuous countercurrent spiral chromatography (CCSC).

[0129] In one embodiment, the continuous affinity chromatography process is carried out at a pressure ranging from about 2 to about 5 bar. For example, the continuous affinity chromatography process is carried out at a pressure ranging from about 2 to about 4 bar. In one embodiment, the continuous affinity chromatography process is carried out at a pressure ranging from about 2 to about 3.5 bar.

[0130] In one example, the method performed herein is a method for purifying IgG from plasma or a fraction thereof, resulting in a yield of greater than 85%, or 90%, or 95% of the total IgG loaded onto the chromatography medium, and the chromatography medium has been exposed to the disinfection method described herein at least once, or five times, or ten times.

[0131] In one example, the method performed herein is a method for purifying IgG from plasma or a fraction thereof, resulting in a yield of greater than 85%, or 90%, or 95% of the total IgG loaded onto the chromatography medium, and the chromatography medium has been exposed to the regeneration and disinfection methods described herein at least once, or five times, or ten times.

[0132] In one embodiment, the method is performed on a large scale. For example, the method is performed on an industrial or commercial scale. Methods performed on an industrial or commercial scale will be apparent to one of skill in the art and / or described herein. For example, methods performed on an industrial scale include large scale purification of IgG from plasma or a fraction thereof.

[0133] In one embodiment, the large scale purification of IgG is carried out using at least 500 kg of plasma or a fraction thereof. For example, the large scale purification of IgG is carried out using 500 kg to 1000 kg, or 1000 kg to 2500 kg, or 2500 kg to 5000 kg, or 5000 kg to 7500 kg, or 7500 kg, or 10000 kg, or 10000 kg to 12500 kg, or 12500 kg to 15000 kg of plasma or a fraction thereof. In one embodiment, the large scale purification of IgG is carried out using at least 1000 kg, or 2500 kg, or 5000 kg, or 7500 kg, or 10000 kg, or 12500 kg, or 15000 kg of plasma or a fraction thereof. In one embodiment, the large scale purification of IgG is carried out using at least 1000 kg of plasma or a fraction thereof. In one embodiment, the large scale purification of IgG is carried out using at least 2500 kg of said fraction of plasma. In one embodiment, the large scale purification of IgG is carried out using at least 5000 kg of said fraction of plasma. In one embodiment, the large scale purification of IgG is carried out using at least 7500 kg of said fraction of plasma. In one embodiment, the large scale purification of IgG is carried out using at least 10000 kg of said fraction of plasma. In one embodiment, the large scale purification of IgG is carried out using at least 12500 kg of said fraction of plasma. In one embodiment, the large scale purification of IgG is carried out using at least 15000 kg of said fraction of plasma.

[0134] In one example, the method performed herein is for purifying IgG from plasma or a fraction thereof, resulting in a solution containing less than 9 mg albumin / g IgG and / or less than 5 mg IgM / g IgG and / or less than 7 mg IgA / g IgG.

[0135] In one example, the method performed herein is for purifying IgG from plasma or a fraction thereof, resulting in IgG having a purity of greater than 95%.

[0136] In one embodiment, the disinfection methods described herein reduce the levels of viruses or prions in a chromatography medium. In one embodiment, the disinfection methods described herein reduce the levels of viruses in a chromatography medium.

[0137] In one embodiment, the virus or prion levels are reduced by at least 1 log, or 2 logs, or 3 logs, or 4 logs, or 5 logs. For example, the virus or prion levels are reduced by at least 3 logs. For example, the virus or prion levels are reduced by at least 4 logs. For example, the virus or prion levels are reduced below the quantification level of the assay used to detect the virus or prion.

[0138] In one embodiment, the disclosed method allows for in-situ cleaning, i.e., can be or is performed on a chromatography medium from its location within a facility, for example, without removing the medium from the column or columns in which it is contained. [Brief description of the drawings]

[0139] [Figure 1] 1 is a graphical representation of a chromatogram showing the results of regenerating FcXP chromatography medium using 20% ​​ethanol followed by 20 mM sodium hydroxide and then sanitizing the medium using 2% CIP-100. [Diagram 2]Two graphical representations are included showing the binding capacity of two FcXP chromatography media (columns 1 and 2) after purification of eight batches of IgG from plasma, refolding with 20% ethanol, followed by disinfection with 20 mM sodium hydroxide and 2% CIP-100. [Diagram 3] FIG. 1 is a graphical representation showing impurity levels (albumin, IgM and IgA as indicated) per gram of IgG in the eluate from FcXP chromatography medium after purification of 10 batches of IgG from plasma, refolding with 20% ethanol followed by refolding with 20 mM sodium hydroxide and disinfection using 2% CIP-100. Data for batches 1, 5 and 10 are shown. [Figure 4] 1 is a graphical representation showing the % purity of IgG in the eluate from FcXP chromatography medium after purification of 10 batches of IgG from plasma, refolding with 20% ethanol followed by disinfection using 20 mM sodium hydroxide and 2% CIP-100. Data for batches 1, 5, and 10 are shown. [Diagram 5] 5A and 5B are graphical representations showing inactivation of BVDV following treatment with 2% CIP-100 at room temperature (RT). Samples are as indicated. Viral titers are shown. [Figure 6] FIG. 1 is a graphical representation showing inactivation of Hepatitis A virus following treatment with 2% CIP-100 at room temperature (RT). Samples are as indicated. Viral titers are shown. [Figure 7] FIG. 1 is a graphical representation showing feed pump pressure profiles from a continuous chromatography lifespan study of 10 batches (1 batch=50 production cycles) with regeneration (ethanol and sodium hydroxide) and sanitization performed between each batch.

[0140] Key to the array table SEQ ID NO:1 is the amino acid sequence of the VHH fragment SEQ ID NO:2 is the amino acid sequence of CDR1 of the VHH fragment SEQ ID NO:3 is the amino acid sequence of CDR2 of the VHH fragment SEQ ID NO: 4 is the amino acid sequence of the CDR3 of the VHH fragment DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0141] general Throughout this specification, unless specifically stated otherwise or the context requires otherwise, references to a single step, composition of matter, group of steps or group of compositions of matter shall be interpreted as encompassing one and more (i.e., one or more) of that step, composition of matter, group of steps or group of compositions of matter.

[0142] Those skilled in the art will understand that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The present disclosure also includes all of the steps, features, compositions, and compounds referred to or shown herein, individually or collectively, and any and all combinations, or any two or more of such steps or features.

[0143] The present disclosure is not to be limited in scope by the specific examples described herein, which are for the purpose of illustration only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure.

[0144] Any embodiment of the present disclosure herein is intended to apply mutatis mutandis to any other embodiment of the present disclosure, unless expressly stated otherwise.

[0145] Unless specifically defined otherwise, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0146] The term "and / or," e.g., "X and / or Y," shall be understood to mean either "X and Y" or "X or Y," and shall be interpreted as providing explicit support for both meanings or for either meaning.

[0147] Throughout this specification the term "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0148] As used herein, the term "derived from" shall be interpreted as indicating that the specified integer may be obtained from a particular source, although not necessarily directly from that source.

[0149] Additionally, as used herein, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise.

[0150] Selected Definitions The term "disinfection" in the context of chromatography media shall be taken to mean a process that substantially inactivates and / or removes microorganisms (including pathogenic and other bacteria, vegetative forms, and spores) and / or prions on and / or within the chromatography media. In one embodiment, disinfection of a chromatography medium reduces microorganisms on and / or within the chromatography medium by 5%, 10%, 15%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to a chromatography medium that has not been disinfected by the methods of the present disclosure.

[0151] In one embodiment, the disinfection of the chromatography medium inactivates 5%, 10%, 15%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the microorganisms on and / or within the chromatography medium compared to a chromatography medium that has not been disinfected by the method of the present disclosure. In one embodiment, the disinfection includes sterilization. Exemplary microorganisms that may be disinfected from the chromatography medium by the method of the present disclosure include fungi, bacteria, viruses, protozoa, mildew, mold, and mold spores. For example, microorganisms include viruses (Parvovirus B19, Human Immunodeficiency Virus (HIV), Hepatitis viruses, Human Herpes viruses, Cytomegalovirus, Epstein-Barr virus, West Nile virus), or bacteria (Treponema pallidum, Neisseria gonorrhoea, Chlamydia trachomatis, Streptococcus pyogenes, Mycobacterium tuberculosis, Brucella melitensis, Ehrlichia, Staphylococci, Pseudomonas aeruginos), or parasites (Plasmodium, Trypanosoma cruzi, Babesia microti). As discussed above, "disinfection" also includes the inactivation and / or removal of prions.

[0152] The term "regeneration" in the context of a chromatography medium shall be taken to mean a process in which the strength and / or properties of the chromatography medium are partially or substantially restored to allow the use of the chromatography medium in one or more further chromatography cycles. Regeneration can increase the effectiveness of the chromatography medium while extending its useful life. In one example, regeneration of a chromatography medium involves the removal of adsorbed proteins from the chromatography medium.

[0153] The term "chromatography medium" shall be construed to mean a solid or semi-solid phase for use in chromatography. In one embodiment, the chromatography medium is comprised of a porous or non-porous support to which multiple ligands are attached, an embodiment of which is described herein. For example, the chromatography medium is a chromatography resin. Exemplary chromatography resins include MabSelect® SuRe® (Cytiva), MabSelect® SuRe® LX (Cytiva), POROS® PI50 (Thermo Fisher), CaptureSelect® FcXP affinity chromatography resin (ThermoFisher), CaptureSelect® FcXL affinity resin (Thermo Fisher), CaptureSelect® IgG-CH1 affinity resin (Thermo Fisher), and CaptureSelect® FcXP agarose affinity resin (Thermo Fisher). Further exemplary affinity chromatography resins include IgSelect® affinity resin (Cytiva), HiTrap® IgSelect® affinity resin (Cytiva), Pierce® Protein G agarose affinity resin (Thermo Fisher), and Protein G Sepharose 4 fast flow affinity resin (Cytiva). For example, the chromatography medium is a chromatography membrane. Exemplary chromatography membranes include Purexa® A (Purilogics), Mustang Q (Pall Corporation).

[0154] The term "ligand" shall be taken to mean a molecule immobilized on the matrix of a chromatography medium, e.g. an affinity chromatography resin, which specifically binds to the CH3 domain of human IgG. For example, the ligand is a single domain [VHH] antibody fragment of camelid origin.

[0155] The term "matrix" is intended to mean a support to which a ligand is immobilized. Exemplary matrices are cross-linked poly(styrene-divinylbenzene) matrices and agarose-based matrices.

[0156] The term "alkaline detergent" shall be taken to mean a water-soluble agent capable of dissolving fats, oils, and greases. Alkaline detergents typically include an alkaline salt (e.g., potassium hydroxide and sodium hydroxide), one or more surfactants and / or chelating agents. Exemplary alkaline detergents include CIP-100 (Steris Life Sciences), ProKlenz One (Steris Life Sciences), ProKlenz-100 (Steris Life Sciences), and COSA CIP-92 (Ecolab). In one embodiment, the alkaline detergent includes 2% CIP-100.

[0157] The term "contacting" is intended to include, but is not limited to, methods of washing, immersing, dipping, bathing, soaking, storing, spraying, or rinsing the chromatography medium with an alkaline detergent.

[0158] The term "alkali salt" shall be taken to mean a basic ionic salt of an alkali metal or alkaline earth metal element. Exemplary alkali salts include potassium hydroxide or sodium hydroxide.

[0159] The term "surfactant" shall be taken to mean a substance that tends to reduce the surface tension of the liquid it is dissolved in. Surfactants may act as dispersing agents, foaming agents, emulsifying agents, wetting agents or detergents.

[0160] The term "chelating agent" is intended to refer to compounds that form stable water-soluble complexes with metal ions.

[0161] The term "ion exchange medium" shall be taken to mean a chromatography medium that contains negatively or positively charged functional groups. For example, ion exchange medium includes anion exchange chromatography medium and cation exchange chromatography medium. Anion exchange chromatography medium is a positively charged medium that has an affinity for molecules with a net negative surface charge (e.g., proteins). Exemplary anion exchange chromatography media include POROS® PI50 anion resin (ThermoFisher), POROS® XQ anion resin (ThermoFisher), POROS® HQ 50 anion resin (ThermoFisher), and POROS® D50 anion resin (ThermoFisher). Cation exchange chromatography medium is a negatively charged medium that has an affinity for molecules with a net positive surface charge (e.g., proteins). Exemplary cation exchange chromatography media include POROS® XS strong cation exchange resin (ThermoFisher) and POROS® HS strong cation exchange resin (ThermoFisher).

[0162] The term "affinity chromatography medium" shall be taken to mean a chromatography medium comprising an affinity chromatography ligand (e.g., a camelid-derived single domain [VHH] antibody fragment, or Protein A or an antibody fragment thereof, or Protein G or an antibody fragment thereof) bound to a matrix, such as, for example, those described herein. Exemplary affinity chromatography media for use in the methods described herein include POROS® CaptureSelect® FcXP affinity resin (Thermo Fisher), and CaptureSelect® FcXP agarose affinity resin (Thermo Fisher). Further exemplary affinity chromatography media include media having an amino acid sequence encoded by SEQ ID NO: 1, or a variant thereof that specifically binds to the CH3 domain of human IgG. In one example, an exemplary affinity chromatography medium comprises a VHH antigen binding protein comprising a complementarity determining region (CDR) 1, CDR2, and / or CDR3 having the amino acid sequence of SEQ ID NO: 2, 3, or 4, respectively. Exemplary affinity chromatography resins are also described in US10259886. Exemplary Protein A chromatography media include MabSelect® Prism A Protein A resin (Cytiva), Praesto® Jetted A50 Protein A resin (Purolite Corp.), and Amsphere® A3 (JSR Corp.), MabSelect® SuRe® Protein A resin (Cytiva), and MabSelect® SuRe® LX Protein A resin (Cytiva).Exemplary protein G chromatography media include Protein G Sepharose 4 Fast Flow Resin (Cytiva), Protein G Resin (abbexa), Dynabeads® Protein G Magnetic Beads (ThermoFisher), Pierce® Protein G Agarose (ThermoFisher), Pierce® Protein G Plus Agarose (ThermoFisher), POROS® MabCapture® G Select (ThermoFisher), PROTEINDEX® Protein G Agarose (Margvelgent Biosciences), and ProteinIso® Protein G Resin (TransGen Biotech Co., LTD).

[0163] The term "hydrophobic interaction chromatography medium" shall be taken to mean a chromatography medium comprising a hydrophobic ligand bound to a matrix, such as those described herein. Hydrophobic interaction chromatography (HIC), comprising a hydrophobic interaction chromatography medium, separates molecules (e.g., proteins) based on their hydrophobicity (i.e., aversion to water). Exemplary hydrophobic interaction chromatography media include POROS® Ethyl Hydrophobic Interaction Chromatography (HIC) Resin (ThermoFisher) and POROS® Benzyl Superhydrophobic Interaction Chromatography (HIC) Resin (ThermoFisher).

[0164] The term "mixed-mode chromatography medium" shall be taken to mean a chromatography medium that allows for the separation of molecules (e.g., proteins) based on two or more types of interactions between the molecules and the medium. For example, a mixed-mode chromatography medium may include affinity chromatography ligands and ion-exchange functional groups bound to a matrix. Exemplary mixed-mode chromatography media include CMM HyperCel (Satorius), MEP HyperCel (Satorius), HEA HyperCel (Sartorius), PPA HyperCel (Sartorius), and HA Ultragel® (Satorius).

[0165] The terms "specifically binds," "specifically binding," or "binds specifically" shall be taken to mean that a protein of the disclosure reacts or associates more frequently, more rapidly, with a longer duration, and / or with a higher affinity with a particular antigen or cell expressing the same, as compared to alternative antigens or cells. For example, a ligand capable of specifically binding to the CH3 domain of human IgG with a substantially higher affinity (e.g., 1.5-fold or 2-fold or 5-fold or 10-fold or 20-fold or 40-fold or 60-fold or 80-fold to 100-fold or 150-fold or 200-fold) than other antigens. Generally, but not necessarily, reference to binding shall mean specific binding, and each term shall be understood to explicitly support the other term.

[0166] The term "immunoglobulin G (IgG)", also known as "gamma globulin" or "immunoglobulin", shall be taken to mean an antibody of the isotype G. There are several subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4.

[0167] The term "camelid-derived single domain [VHH] antibody fragment" shall be taken to mean the VHH domain of a camelid antibody. Camelid antibodies are antibodies generated from camels and llamas that do not have the CH1 domain normally present in human immunoglobulins and have only one VHH domain. Exemplary affinity chromatography resins comprising camelid-derived [VHH] antibody fragments include CaptureSelect® antibody affinity chromatography resins (Thermo Fisher). For example, CaptureSelect® FcXL affinity resin, POROS® CaptureSelect® FcXP affinity resin, CaptureSelect IgG-CH1 affinity resin, and CaptureSelect FcXP agarose affinity resin.

[0168] The term "dynamic binding capacity" or "DBC" of a chromatography medium shall be interpreted as referring to the maximum amount of protein that the medium will bind under operating conditions before significant breakthrough of unbound protein occurs.

[0169] The term "plasma" refers to the straw-colored / light yellow component of blood obtained from one or more blood donors. Methods for obtaining plasma from a donor will be apparent to one of skill in the art and / or are described herein. For example, plasma may be obtained by removing red blood cells from donated blood. For example, plasma may be obtained by plasma substitution methods.

[0170] The term "plasma fraction" or "fraction thereof" refers to plasma that has been fractionated to isolate one or more desired protein components from the plasma. For example, plasma may be fractionated to isolate a cryoprecipitate (proteins that precipitate out of solution when a unit of fresh frozen plasma is slowly thawed in the cold) and a cryosupernatant (also known as cryoprecipitate plasma). For example, plasma may be fractionated by ethanol precipitation to produce an IgG-containing Oncley fraction, a Cohn fraction, an ammonium sulfate precipitate, or a precipitate A (KN A) from the plasma, as described in U.S. Pat. No. 3,301,842. Plasma fractions include the II+III precipitate produced by the Cohn method, e.g., Method 6, Cohn et al. J. Am; Chem. Soc., 68(3), 459-475 (1946), Method 9, Oncley et al. J. Am; Chem. Soc., 71, 541-550 (1946), or the I+II+III precipitate, Method 10, Cohn et al. J. Am; Chem. Soc., 72, 465-474 (1950), as well as Precipitate-A, Helv. Chim. Acta 37, 866-873 (1954), of Deutsch et al. J. Biol. Chem. 164, 109-118 (1946) or Nitschmann and Kistler Vox Sang. 7, 414-424 (1962). For example, plasma can be fractionated by octanoic acid fractionation as described in European Application No. 893450. Typically, the Cohn fraction, Kistler / Nitschmann Precipitate A (KN A), exists as a suspended paste. Other purification techniques, including chromatography, can be used.

[0171] The term "cryo-precipitate" or "cryo-precipitates" refers to proteins in plasma that precipitate out of solution when a unit of fresh frozen plasma is slowly thawed in the cold. Cryoprecipitates include factor VIII, fibrinogen, von Willebrand factor, factor XIII, and platelet membrane microparticles.

[0172] The term "cryoprecipitate plasma" shall be taken to mean plasma from which the cryoprecipitate has been removed.

[0173] The term "cryo-rich plasma" shall be taken to mean plasma that contains components typically found in cryoprecipitates.

[0174] disinfection The present disclosure provides a method for disinfecting a chromatography medium. Suitable chromatography media for use in the method will be apparent to one of skill in the art and / or described herein. In one embodiment, the chromatography medium is used in ion exchange chromatography (e.g., anion or cation exchange chromatography), affinity chromatography (e.g., protein A or protein G chromatography), mixed mode chromatography, or size exclusion chromatography. For example, the chromatography medium is disinfected after a chromatography cycle in which the chromatography medium is used in one or more equilibration, sample loading, binding, washing, and / or elution. Disinfecting the chromatography medium inactivates and / or removes microorganisms from the medium. In one embodiment, disinfecting the chromatography medium inactivates microorganisms on the medium. In one embodiment, disinfecting the chromatography medium removes microorganisms from the medium.

[0175] For example, disinfection of a chromatography medium reduces microorganisms on and / or within the chromatography medium by 5% compared to a chromatography medium not disinfected by the disclosed method. In one example, disinfection of a chromatography medium reduces microorganisms on and / or within the chromatography medium by 10% compared to a chromatography medium not disinfected by the disclosed method. In one example, disinfection of a chromatography medium reduces microorganisms on and / or within the chromatography medium by 15% compared to a chromatography medium not disinfected by the disclosed method. In one example, disinfection of a chromatography medium reduces microorganisms on and / or within the chromatography medium by 20% compared to a chromatography medium not disinfected by the disclosed method. In one example, disinfection of a chromatography medium reduces microorganisms on and / or within the chromatography medium by 25% compared to a chromatography medium not disinfected by the disclosed method. In one example, disinfection of a chromatography medium reduces microorganisms on and / or within the chromatography medium by 30% compared to a chromatography medium not disinfected by the disclosed method. In one example, disinfection of a chromatography medium reduces microorganisms on and / or within the chromatography medium by 35% compared to a chromatography medium not disinfected by the disclosed method. In one example, disinfection of a chromatography medium reduces microorganisms on and / or within the chromatography medium by 40% compared to a chromatography medium not disinfected by the disclosed method. In one example, disinfection of a chromatography medium reduces microorganisms on and / or within the chromatography medium by 50% compared to a chromatography medium not disinfected by the disclosed method. In one example, disinfection of a chromatography medium reduces microorganisms on and / or within the chromatography medium by 60% compared to a chromatography medium not disinfected by the disclosed method.In one example, disinfection of a chromatography medium reduces microorganisms on and / or within the chromatography medium by 70% compared to a chromatography medium not disinfected by the disclosed method. In one example, disinfection of a chromatography medium reduces microorganisms on and / or within the chromatography medium by 80% compared to a chromatography medium not disinfected by the disclosed method. In one example, disinfection of a chromatography medium reduces microorganisms on and / or within the chromatography medium by 90% compared to a chromatography medium not disinfected by the disclosed method. In one example, disinfection of a chromatography medium reduces microorganisms on and / or within the chromatography medium by 100% compared to a chromatography medium not disinfected by the disclosed method.

[0176] In another example, the disinfection of the chromatography medium inactivates 5% of the microorganisms on and / or within the chromatography medium compared to a chromatography medium not disinfected by the method of the present disclosure. In one example, the disinfection of the chromatography medium inactivates 10% of the microorganisms on and / or within the chromatography medium compared to a chromatography medium not disinfected by the method of the present disclosure. In one example, the disinfection of the chromatography medium inactivates 15% of the microorganisms on and / or within the chromatography medium compared to a chromatography medium not disinfected by the method of the present disclosure. In one example, the disinfection of the chromatography medium inactivates 20% of the microorganisms on and / or within the chromatography medium compared to a chromatography medium not disinfected by the method of the present disclosure. In one example, the disinfection of the chromatography medium inactivates 25% of the microorganisms on and / or within the chromatography medium compared to a chromatography medium not disinfected by the method of the present disclosure. In one example, the disinfection of the chromatography medium inactivates 30% of the microorganisms on and / or within the chromatography medium compared to a chromatography medium not disinfected by the method of the present disclosure. In one embodiment, disinfection of the chromatography medium inactivates 35% of the microorganisms on and / or within the chromatography medium compared to a chromatography medium not disinfected by the method of the present disclosure. In one embodiment, disinfection of the chromatography medium inactivates 40% of the microorganisms on and / or within the chromatography medium compared to a chromatography medium not disinfected by the method of the present disclosure. In one embodiment, disinfection of the chromatography medium inactivates 50% of the microorganisms on and / or within the chromatography medium compared to a chromatography medium not disinfected by the method of the present disclosure.In one embodiment, the disinfection of the chromatography medium inactivates 60% of the microorganisms on and / or within the chromatography medium compared to a chromatography medium not disinfected by the method of the present disclosure. In one embodiment, the disinfection of the chromatography medium inactivates 70% of the microorganisms on and / or within the chromatography medium compared to a chromatography medium not disinfected by the method of the present disclosure. In one embodiment, the disinfection of the chromatography medium inactivates 80% of the microorganisms on and / or within the chromatography medium compared to a chromatography medium not disinfected by the method of the present disclosure. In one embodiment, the disinfection of the chromatography medium inactivates 90% of the microorganisms on and / or within the chromatography medium compared to a chromatography medium not disinfected by the method of the present disclosure. In one embodiment, the disinfection of the chromatography medium inactivates 100% of the microorganisms on and / or within the chromatography medium compared to a chromatography medium not disinfected by the method of the present disclosure.

[0177] Suitable alkaline detergents for use in the methods of the present disclosure will be apparent to those skilled in the art and / or described herein. Typically, alkaline detergents include an alkaline salt, one or more surfactants and / or chelating agents. Exemplary commercially available alkaline detergents include CIP-100 (Steris Life Sciences), ProKlenz One (Steris Life Sciences), ProKlenz-100 (Steris Life Sciences), and COSA CIP-92 (Ecolab). For example, a method of disinfection may include contacting a chromatography medium with 2% CIP-100 (Steris Life Sciences).

[0178] In one embodiment, the alkaline detergent is mixed into the solution. For example, the solution includes 0.1% to 10% alkaline detergent. For example, the solution includes 0.2% to 9%, or 0.3% to 8%, or 0.4% to 7% alkaline detergent. In one embodiment, the solution includes 0.5% to 5% alkaline detergent. In one embodiment, the solution includes 1%, or 2%, or 3%, or 4%, or 5% alkaline detergent. In one embodiment, the solution includes 1% alkaline detergent. For example, the solution includes 2% alkaline detergent. In one embodiment, the solution includes 3% alkaline detergent. In one embodiment, the solution includes 4% alkaline detergent. In one embodiment, the solution includes 5% alkaline detergent.

[0179] In one embodiment, the alkaline detergent comprises an alkaline salt, one or more surfactants, and / or a chelating agent. In one embodiment, the alkaline detergent comprises an alkaline salt and one or more surfactants. In one embodiment, the alkaline detergent comprises an alkaline salt, one or more surfactants, and a chelating agent.

[0180] In one embodiment, the alkali salt is selected from the group consisting of potassium hydroxide, sodium hydroxide, sodium carbonate, sodium acetate, sodium sulfide, and sodium bicarbonate. For example, the alkali salt is potassium hydroxide. For example, the alkali salt is sodium hydroxide. For example, the alkali salt is sodium carbonate. For example, the alkali salt is sodium acetate. For example, the alkali salt is sodium sulfide. For example, the alkali salt is sodium bicarbonate.

[0181] In one embodiment, the alkaline salt is potassium hydroxide or sodium hydroxide.

[0182] In one embodiment, the alkaline detergent comprises 5% to 45%, or 5% to 10%, or 10% to 15%, or 15% to 20%, or 20% to 25%, or 25% to 30%, or 30% to 35%, or 35% to 40%, or 40% to 45% (w / v) of an alkaline salt. For example, the alkaline detergent comprises 5% to 10% (w / v) of an alkaline salt. For example, the alkaline detergent comprises 10% to 15% (w / v) of an alkaline salt. For example, the alkaline detergent comprises 15% to 20% (w / v) of an alkaline salt. For example, the alkaline detergent comprises 20% to 25% (w / v) of an alkaline salt. For example, the alkaline detergent comprises 25% to 30% (w / v) of an alkaline salt. For example, the alkaline detergent comprises 30% to 35% (w / v) of an alkaline salt. For example, the alkaline detergent contains 35% to 40% (w / v) of an alkaline salt, for example, 40% to 45% (w / v) of an alkaline salt.

[0183] In one embodiment, the alkaline detergent comprises at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45% (w / v) of an alkaline salt. For example, the alkaline detergent comprises at least 5% (w / v) of an alkaline salt. For example, the alkaline detergent comprises at least 10% (w / v) of an alkaline salt. For example, the alkaline detergent comprises at least 15% (w / v) of an alkaline salt. For example, the alkaline detergent comprises at least 20% (w / v) of an alkaline salt. For example, the alkaline detergent comprises at least 25% (w / v) of an alkaline salt. For example, the alkaline detergent comprises at least 30% (w / v) of an alkaline salt. For example, the alkaline detergent comprises at least 35% (w / v) of an alkaline salt. For example, the alkaline detergent comprises at least 40% (w / v) of an alkaline salt. For example, the alkaline detergent comprises at least 45% (w / v) of an alkaline salt.

[0184] In one embodiment, the alkaline detergent comprises 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% (w / v) of an alkaline salt. For example, the alkaline detergent comprises 5% (w / v) of an alkaline salt. For example, the alkaline detergent comprises 10% (w / v) of an alkaline salt. For example, the alkaline detergent comprises 15% (w / v) of an alkaline salt. For example, the alkaline detergent comprises 20% (w / v) of an alkaline salt. For example, the alkaline detergent comprises 25% (w / v) of an alkaline salt. For example, the alkaline detergent comprises 30% (w / v) of an alkaline salt. For example, the alkaline detergent comprises 35% (w / v) of an alkaline salt. For example, the alkaline detergent comprises 40% (w / v) of an alkaline salt. For example, the alkaline detergent comprises 45% (w / v) of an alkaline salt.

[0185] In one embodiment, the alkaline detergent comprises between 10 mM and 15 mM, or between 15 mM and 20 mM, or between 20 mM and 30 mM, or between 30 mM and 40 mM, or between 40 mM and 50 mM, or between 50 mM and 60 mM, or between 60 mM and 70 mM, or between 70 mM and 80 mM, or between 80 mM and 90 mM of an alkaline salt.

[0186] In one embodiment, the alkaline detergent comprises 10 mM to 15 mM, or 15 mM to 20 mM, or 20 mM to 30 mM, or 30 mM to 40 mM, or 40 mM to 50 mM, or 50 mM to 60 mM, or 60 mM to 70 mM, or 70 mM to 80 mM, or 80 mM to 90 mM, or 90 mM to 100 mM, or 100 mM to 110 mM, or 110 mM to 120 mM, or 120 mM to 130 mM, or 130 mM to 140 mM, or 140 mM to 150 mM of an alkaline salt. For example, the alkaline detergent comprises 10 mM to 15 mM of an alkaline salt. For example, the alkaline detergent comprises 15 mM to 20 mM of an alkaline salt. For example, the alkaline detergent comprises 20 mM to 30 mM of an alkaline salt. For example, the alkaline detergent contains 30 mM to 40 mM of an alkaline salt. For example, the alkaline detergent contains 40 mM to 50 mM of an alkaline salt. For example, the alkaline detergent contains 50 mM to 60 mM of an alkaline salt. For example, the alkaline detergent contains 60 mM to 70 mM of an alkaline salt. For example, the alkaline detergent contains 70 mM to 80 mM of an alkaline salt. For example, the alkaline detergent contains 80 mM to 90 mM of an alkaline salt. For example, the alkaline detergent contains 90 mM to 100 mM of an alkaline salt. For example, the alkaline detergent contains 100 mM to 110 mM of an alkaline salt. For example, the alkaline detergent contains 110 mM to 120 mM of an alkaline salt. For example, the alkaline detergent contains 120 mM to 130 mM of an alkaline salt. For example, the alkaline detergent contains 130 mM to 140 mM of an alkaline salt. For example, the alkaline detergent contains 140 mM to 150 mM of an alkaline salt.

[0187] In one embodiment, the alkaline detergent comprises at least 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, or 90 mM of an alkaline salt. In one embodiment, the alkaline detergent comprises at least 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, or 90 mM, or 100 mM, or 110 mM, or 120 mM, or 130 mM, or 140 mM of an alkaline salt. For example, the alkaline detergent comprises at least 10 mM of an alkaline salt. For example, the alkaline detergent comprises at least 15 mM of an alkaline salt. For example, the alkaline detergent comprises at least 20 mM of an alkaline salt. For example, the alkaline detergent comprises at least 30 mM of an alkaline salt. For example, the alkaline detergent comprises at least 40 mM of an alkaline salt. For example, the alkaline detergent comprises at least 50 mM of an alkaline salt. For example, the alkaline detergent comprises at least 60 mM of an alkaline salt. For example, the alkaline detergent comprises at least 70 mM of an alkaline salt. For example, the alkaline detergent comprises at least 80 mM of an alkaline salt. For example, the alkaline detergent comprises at least 90 mM of an alkaline salt. For example, the alkaline detergent comprises at least 100 mM of an alkaline salt. For example, the alkaline detergent comprises at least 110 mM of an alkaline salt. For example, the alkaline detergent comprises at least 120 mM of an alkaline salt. For example, the alkaline detergent comprises at least 130 mM of an alkaline salt. For example, the alkaline detergent comprises at least 140 mM of an alkaline salt.

[0188] In one embodiment, the alkaline detergent comprises 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, or 90 mM of an alkaline salt. In one embodiment, the alkaline detergent comprises 10 mM, or 15 mM, or 20 mM, or 25 mM, or 30 mM, or 35 mM, or 40 mM, or 45 mM, or 50 mM, or 55 mM, or 60 mM, or 65 mM, or 70 mM, or 75 mM, or 80 mM, or 85 mM, or 90 mM, 95 mM, or 100 mM, or 105 mM, or 110 mM, or 115 mM, or 120 mM, or 125 mM, or 130 mM, or 135 mM, or 140 mM, or 145 mM, or 150 mM of an alkaline salt. For example, the alkaline detergent comprises 10 mM of an alkaline salt. For example, the alkaline detergent comprises 15 mM of an alkaline salt. For example, the alkaline detergent comprises 20 mM of an alkaline salt. For example, the alkaline detergent comprises 25 mM of an alkaline salt. For example, the alkaline detergent comprises 30 mM of an alkaline salt. For example, the alkaline detergent comprises 35 mM of an alkaline salt. For example, the alkaline detergent comprises 40 mM of an alkaline salt. For example, the alkaline detergent comprises 45 mM of an alkaline salt. For example, the alkaline detergent comprises 50 mM of an alkaline salt. For example, the alkaline detergent comprises 55 mM of an alkaline salt. For example, the alkaline detergent comprises 60 mM of an alkaline salt. For example, the alkaline detergent comprises 65 mM of an alkaline salt. For example, the alkaline detergent comprises 70 mM of an alkaline salt. For example, the alkaline detergent comprises 75 mM of an alkaline salt. For example, the alkaline detergent comprises 80 mM of an alkaline salt. For example, the alkaline detergent comprises 85 mM of an alkaline salt. For example, the alkaline detergent comprises 90 mM of an alkaline salt. For example, the alkaline detergent comprises 95 mM of an alkaline salt. For example, the alkaline detergent comprises 100 mM of an alkaline salt. For example, the alkaline detergent comprises 105 mM of an alkaline salt. For example, the alkaline detergent comprises 110 mM of an alkaline salt. For example, the alkaline detergent comprises 115 mM of an alkaline salt. For example, the alkaline detergent comprises 120 mM of an alkaline salt. For example, the alkaline detergent comprises 125 mM of an alkaline salt. For example, the alkaline detergent comprises 130 mM of an alkaline salt. For example, the alkaline detergent comprises 135 mM of an alkaline salt.For example, the alkaline detergent contains 140 mM of an alkaline salt. For example, the alkaline detergent contains 145 mM of an alkaline salt. For example, the alkaline detergent contains 150 mM of an alkaline salt.

[0189] In one embodiment, the one or more surfactants are selected from the group consisting of amine ethoxylates, amphocarboxylates, triethanolamine, octyl-β-D glucopyranoside (OGP), polysorbates, poloxamers, lauryl dimethyl-amine (LDAO), myristyl dimethylamine-N-oxide (TDAO), sodium cholate, decyl-β-D-glucopyranoside, and dodecyl maltoside. For example, the one or more surfactants are amine ethoxylates. For example, the one or more surfactants are amphocarboxylates. For example, the one or more surfactants are triethanolamine. For example, the one or more surfactants are OGP (octyl-β-D glucopyranoside). For example, the one or more surfactants are polysorbate 80. For example, the one or more surfactants are polysorbate 20. For example, the one or more surfactants are LDAO (lauryl dimethyl-amine). For example, the one or more surfactants are TDAO (myristyl dimethylamine-N-oxide). For example, one or more of the surfactants is sodium cholate. For example, one or more of the surfactants is decyl-β-D-glucopyranoside. For example, one or more of the surfactants is dodecyl maltoside.

[0190] In one embodiment, the alkaline detergent comprises one or more surfactants at 0.01% to 10% (w / v). For example, the alkaline detergent comprises one or more surfactants at 0.01% to 5% (w / v). In one embodiment, the alkaline detergent comprises one or more surfactants at 0.01% to 1.0% (w / v). For example, the alkaline detergent comprises one or more surfactants at 0.01% to 1% (w / v), or 0.01% to 0.5% (w / v), or 0.01% to 0.1% (w / v), or 0.01% to 0.05% (w / v). For example, the alkaline detergent comprises one or more surfactants at about 0.02% (w / v). In one embodiment, the alkaline detergent comprises one or more surfactants at 0.5% to 1% (w / v).

[0191] In one embodiment, the alkaline detergent comprises 0.01%-0.10% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 0.01%-0.05%, or 0.05%-0.1% (w / v) of one or more surfactants. In one embodiment, the alkaline detergent comprises 0.10%-0.15%, or 0.15%-0.20%, or 0.20%-0.25%, or 0.25%-0.30%, or 0.30%-0.35%, or 0.35%-0.40%, or 0.40%-0.45%, or 0.45%-0.50%, or 0.50%-0.55%, or 0.55%-0.60%, or 0.60%-0. 65%, or 0.65% to 0.70%, or 0.70% to 0.75%, or 0.75% to 0.80%, or 0.80% to 0.85%, or 0.85% to 0.90%, or 0.90% to 0.95%, or 0.95% to 1.00%, or 1.00% to 1.05%, or 1.05% to 1.10%, or 1.10% to 1.20% (w / v) of one or more surfactants. For example, an alkaline detergent contains one or more surfactants at 0.10% to 0.15% (w / v). For example, an alkaline detergent contains one or more surfactants at 0.15% to 0.20% (w / v). For example, an alkaline detergent contains one or more surfactants at 0.20% to 0.25% (w / v). For example, the alkaline detergent contains one or more surfactants at 0.25% to 0.30% (w / v). For example, the alkaline detergent contains one or more surfactants at 0.30% to 0.35% (w / v). For example, the alkaline detergent contains one or more surfactants at 0.35% to 0.40% (w / v). For example, the alkaline detergent contains one or more surfactants at 0.40% to 0.45% (w / v). For example, the alkaline detergent contains one or more surfactants at 0.45% to 0.50% (w / v). For example, the alkaline detergent contains one or more surfactants at 0.50% to 0.55% (w / v). For example, the alkaline detergent contains one or more surfactants at 0.55% to 0.60% (w / v). For example, the alkaline detergent contains one or more surfactants at 0.60% to 0.65% (w / v). For example, the alkaline detergent contains 0.65% to 0.70% (w / v) of one or more surfactants. For example, the alkaline detergent contains 0.70% to 0.75% (w / v) of one or more surfactants.For example, the alkaline detergent contains one or more surfactants at 0.75% to 0.80% (w / v). For example, the alkaline detergent contains one or more surfactants at 0.80% to 0.85% (w / v). For example, the alkaline detergent contains one or more surfactants at 0.85% to 0.90% (w / v). For example, the alkaline detergent contains one or more surfactants at 0.90% to 0.95% (w / v). For example, the alkaline detergent contains one or more surfactants at 0.95% to 1.00% (w / v). For example, the alkaline detergent contains one or more detergents at 1.00% to 1.05% (w / v). For example, the alkaline detergent contains one or more surfactants at 1.05% to 1.10% (w / v). For example, the alkaline detergent contains one or more surfactants at 1.10% to 1.20% (w / v).

[0192] In one embodiment, the alkaline detergent comprises at least 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% (w / v) of one or more surfactants. In one embodiment, the alkaline detergent comprises at least 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%, 0.50%, 0.55%, 0.60%, 0.65%, 0.70%, 0.75%, 0.80%, 0.85%, 0.90%, 0.95%, 1.00%, 1.05%, 1.10%, or 1.20% (w / v) of one or more surfactants. For example, the alkaline detergent comprises at least 0.01% (w / v) of one or more surfactants. For example, the alkaline detergent comprises at least 0.02% (w / v) of one or more surfactants. For example, the alkaline detergent comprises at least 0.03% (w / v) of one or more surfactants. For example, the alkaline detergent comprises at least 0.04% (w / v) of one or more surfactants. For example, the alkaline detergent comprises at least 0.05% (w / v) of one or more surfactants. For example, the alkaline detergent comprises at least 0.06% (w / v) of one or more surfactants. For example, the alkaline detergent comprises at least 0.07% (w / v) of one or more surfactants. For example, the alkaline detergent comprises at least 0.08% (w / v) of one or more surfactants. For example, the alkaline detergent comprises at least 0.09% (w / v) of one or more surfactants. For example, the alkaline detergent comprises at least 0.10% (w / v) of one or more surfactants. For example, the alkaline detergent comprises at least 0.15% (w / v) of one or more surfactants. For example, the alkaline detergent comprises at least 0.20% (w / v) of one or more surfactants. For example, the alkaline detergent comprises at least 0.25% (w / v) of one or more surfactants. For example, the alkaline detergent comprises at least 0.30% (w / v) of one or more surfactants. For example, the alkaline detergent comprises at least 0.35% (w / v) of one or more surfactants. For example, the alkaline detergent comprises at least 0.40% (w / v) of one or more surfactants. For example, the alkaline detergent comprises at least 0.45% (w / v) of one or more surfactants.For example, the alkaline detergent comprises at least 0.50% (w / v) of one or more surfactants. For example, the alkaline detergent comprises at least 0.55% (w / v) of one or more surfactants. For example, the alkaline detergent comprises at least 0.60% (w / v) of one or more surfactants. For example, the alkaline detergent comprises at least 0.65% (w / v) of one or more surfactants. For example, the alkaline detergent comprises at least 0.70% (w / v) of one or more surfactants. For example, the alkaline detergent comprises at least 0.75% (w / v) of one or more surfactants. For example, the alkaline detergent comprises at least 0.80% (w / v) of one or more surfactants. For example, the alkaline detergent comprises at least 0.85% (w / v) of one or more surfactants. For example, the alkaline detergent comprises at least 0.90% (w / v) of one or more surfactants. For example, the alkaline detergent comprises at least 0.95% (w / v) of one or more surfactants. For example, the alkaline detergent comprises at least 1.00% (w / v) of one or more surfactants. For example, the alkaline detergent comprises at least 1.05% (w / v) of one or more surfactants. For example, the alkaline detergent comprises at least 1.10% (w / v) of one or more surfactants. For example, the alkaline detergent comprises at least 1.20% (w / v) of one or more surfactants.

[0193] In one embodiment, the alkaline detergent comprises 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% (w / v) of one or more surfactants. In one embodiment, the alkaline detergent comprises 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%, 0.50%, 0.55%, 0.60%, 0.65%, 0.70%, 0.75%, 0.80%, 0.85%, 0.90%, 0.95%, 1.00%, 1.05%, 1.10%, or 1.20% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 0.01% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 0.02% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 0.03% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 0.04% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 0.05% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 0.06% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 0.07% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 0.08% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 0.09% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 0.10% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 0.15% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 0.20% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 0.25% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 0.30% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 0.35% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 0.40% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 0.45% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 0.50% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 0.55% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 0.60% (w / v) of one or more surfactants.For example, the alkaline detergent comprises 0.65% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 0.70% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 0.75% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 0.80% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 0.85% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 0.90% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 0.95% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 1.00% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 1.05% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 1.10% (w / v) of one or more surfactants. For example, the alkaline detergent comprises 1.20% (w / v) of one or more surfactants.

[0194] In one embodiment, the chelating agent is ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), sodium gluconate, trisodium citrate, diethylenetriaminepentaacetic acid (DTPA), meso-2,3-dimercaptosuccinic acid (DMSA), 2,3-dimercaptopropane-1-sulfonic acid (DMPS), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), alpha-lipoic acid (ALA), ethylenediamine-N,N'-disuccinic acid. (EDDS), iminodisuccinic acid (IDS), methylglycine diacetate (MGDA), triethylenetetramine (Trien, iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), tripolyphosphate (TPP), sodium diethyldithiocarbamate (DDC), L-glutamic acid N,N-diacetate (GLDA), tetrasodium salt, penicillamine, or any salt thereof, including calcium, or a sodium salt thereof. For example, the chelating agent is ethylenediaminetetraacetic acid (EDTA). For example, the chelating agent is ethyleneglycol-bis(β- For example, the chelating agent is 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (EGTA). For example, the chelating agent is sodium gluconate. For example, the chelating agent is trisodium citrate. For example, the chelating agent is diethylenetriaminepentaacetic acid (DTPA). For example, the chelating agent is meso-2,3-dimercaptosuccinic acid (DMSA). For example, the chelating agent is 2,3 dimercaptopropane-1-sulfonic acid (DMPS). For example, the chelating agent is 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAP For example, the chelating agent is alpha-lipoic acid (ALA). For example, the chelating agent is ethylenediamine-N,N'-disuccinic acid (EDDS). For example, the chelating agent is iminodisuccinic acid (IDS). For example, the chelating agent is methylglycine diacetate (MGDA). For example, the chelating agent is triethylenetetramine (Trien). For example, the chelating agent is iminodiacetic acid (IDA). For example, the chelating agent is nitrilotriacetic acid (NTA). For example, the chelating agent is tripolyphosphate (TPP).For example, the chelating agent is sodium diethyldithiocarbamate (DDC). For example, the chelating agent is L-glutamic acid N,N-diacetyl acid (GLDA). For example, the chelating agent is the tetrasodium salt. For example, the chelating agent is penicillamine.

[0195] In one embodiment, the chelating agent is EDTA or sodium gluconate.

[0196] In one embodiment, the chelating agent is a biodegradable chelating agent.

[0197] In one embodiment, the biodegradable chelating agent is selected from the group consisting of ethylenediamine-N,N'-disuccinic acid (EDDS), iminodisuccinic acid (IDS), methylglycine diacetic acid (MGDA), and nitrilotriacetic acid (NTA).

[0198] In one embodiment, the alkaline detergent comprises 0.5% to 1%, or 1% to 1.5%, or 1.5% to 2.0%, or 2.0% to 2.5%, or 2.5% to 3%, or 3% to 3.5%, or 3.5% to 4%, or 4% to 4.5%, or 4.5% to 5%, or 5% to 5.5%, or 5.5% to 6%, or 6% to 6.5%, or 6.5% to 7.0%, or 7.0% to 7.5%, or 7.5% to 8.0%, or 8.0% to 8.5% (w / v) of the chelating agent. For example, the alkaline detergent comprises 0.5% to 1% (w / v) of the chelating agent. For example, the alkaline detergent comprises 1% to 1.5% (w / v) of the chelating agent. For example, the alkaline detergent comprises 1.5% to 2.0% (w / v) of the chelating agent. For example, the alkaline detergent contains 2.0% to 2.5% (w / v) of the chelating agent. For example, the alkaline detergent contains 2.5% to 3% (w / v) of the chelating agent. For example, the alkaline detergent contains 3% to 3.5% (w / v) of the chelating agent. For example, the alkaline detergent contains 3.5% to 4% (w / v) of the chelating agent. For example, the alkaline detergent contains 4% to 4.5% (w / v) of the chelating agent. For example, the alkaline detergent contains 4.5% to 5% (w / v) of the chelating agent. For example, the alkaline detergent contains 5% to 5.5% (w / v) of the chelating agent. For example, the alkaline detergent contains 5.5% to 6% (w / v) of the chelating agent. For example, the alkaline detergent contains 6% to 6.5% (w / v) of the chelating agent. For example, the alkaline detergent contains 6.5% to 7.0% (w / v) of the chelating agent. For example, the alkaline detergent contains 7.0% to 7.5% (w / v) of the chelating agent. For example, the alkaline detergent contains 7.5% to 8.0% (w / v) of the chelating agent. For example, the alkaline detergent contains 8.0% to 8.5% (w / v) of the chelating agent.

[0199] In one embodiment, the alkaline detergent comprises at least 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5% (w / v) of the chelating agent. For example, the alkaline detergent comprises at least 0.5% (w / v) of the chelating agent. For example, the alkaline detergent comprises at least 1% (w / v) of the chelating agent. For example, the alkaline detergent comprises at least 1.5% (w / v) of the chelating agent. For example, the alkaline detergent comprises at least 2% (w / v) of the chelating agent. For example, the alkaline detergent comprises at least 2.5% (w / v) of the chelating agent. For example, the alkaline detergent comprises at least 3% (w / v) of the chelating agent. For example, the alkaline detergent comprises at least 3.5% (w / v) of the chelating agent. For example, the alkaline detergent comprises at least 4% (w / v) chelating agent. For example, the alkaline detergent comprises at least 4.5% (w / v) chelating agent. For example, the alkaline detergent comprises at least 5% (w / v) chelating agent. For example, the alkaline detergent comprises at least 5.5% (w / v) chelating agent. For example, the alkaline detergent comprises at least 6% (w / v) chelating agent. For example, the alkaline detergent comprises at least 6.5% (w / v) chelating agent. For example, the alkaline detergent comprises at least 7% (w / v) chelating agent. For example, the alkaline detergent comprises at least 7.5% (w / v) chelating agent. For example, the alkaline detergent comprises at least 8% (w / v) chelating agent. For example, the alkaline detergent comprises at least 8.5% (w / v) chelating agent.

[0200] In one embodiment, the alkaline detergent comprises 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5% (w / v) chelating agent. For example, the alkaline detergent comprises 0.5% (w / v) chelating agent. For example, the alkaline detergent comprises 1% (w / v) chelating agent. For example, the alkaline detergent comprises 1.5% (w / v) chelating agent. For example, the alkaline detergent comprises 2% (w / v) chelating agent. For example, the alkaline detergent comprises 2.5% (w / v) chelating agent. For example, the alkaline detergent comprises 3% (w / v) chelating agent. For example, the alkaline detergent comprises 3.5% (w / v) chelating agent. For example, the alkaline detergent comprises 4% (w / v) chelating agent. For example, the alkaline detergent comprises 4.5% (w / v) chelating agent. For example, the alkaline detergent comprises 5% (w / v) chelating agent. For example, the alkaline detergent comprises 5.5% (w / v) chelating agent. For example, the alkaline detergent comprises 6% (w / v) chelating agent. For example, the alkaline detergent comprises 6.5% (w / v) chelating agent. For example, the alkaline detergent comprises 7% (w / v) chelating agent. For example, the alkaline detergent comprises 7.5% (w / v) chelating agent. For example, the alkaline detergent comprises 8% (w / v) chelating agent. For example, the alkaline detergent comprises 8.5% (w / v) chelating agent.

[0201] In one embodiment, the alkaline detergent is diluted, for example in water. For example, the chromatography resin is contacted with a solution containing 0.1-5% alkaline detergent, for example 0.5-4% or 1-3% alkaline detergent. In one embodiment, the chromatography resin is contacted with a solution containing 0.5% alkaline detergent. In another embodiment, the chromatography resin is contacted with a solution containing 1% alkaline detergent. For example, the chromatography resin is contacted with a solution containing 2% alkaline detergent.

[0202] In one embodiment, multiple column volumes of alkaline detergent are passed through the chromatography medium to disinfect the medium. The alkaline detergent can be passed through the chromatography medium in a downflow direction and / or an upflow direction. For example, multiple column volumes are passed through the chromatography medium in an upflow direction, and then multiple column volumes are passed through the chromatography medium in a downflow direction. For example, at least three or four or five or six column volumes are passed through the chromatography medium in an upflow direction. For example, at least three or four or five or six column volumes are passed through the chromatography medium in a downflow direction.

[0203] In one embodiment, the alkaline detergent is contacted with the chromatography medium for at least 10 minutes to disinfect the medium. For example, the alkaline detergent is contacted with the chromatography medium for at least 15 minutes, or 30 minutes, or 40 minutes, or 50 minutes, or 60 minutes. In one embodiment, the alkaline detergent is contacted with the chromatography medium for 120 minutes or less. For example, the alkaline detergent is contacted with the chromatography medium for 15 to 120 minutes, or 30 to 100 minutes, or 40 to 90 minutes, or 50 to 70 minutes. For example, the alkaline detergent is contacted with the chromatography medium for about 60 minutes.

[0204] reproduction The present disclosure further provides a method of regenerating a chromatography medium, which may be performed before or after disinfecting the chromatography medium. For example, the chromatography medium is regenerated before disinfection. Regeneration of the chromatography medium restores the binding capacity and / or properties of the chromatography medium, allowing its use in one or more additional chromatography cycles.

[0205] Suitable solutions for regenerating chromatography media will be apparent to those of skill in the art and / or are described herein.

[0206] Methods for regenerating chromatography media are known in the art and will be apparent to those skilled in the art. For example, regenerating chromatography media includes contacting the chromatography media with solutions including, but not limited to, salt solutions (NaCl), caustic solutions of up to 1.5 molar sodium hydroxide, solvents such as acetic acid, ethanol, isopropanol and acetonitrile, Tris or Tris EDTA. Regeneration methods also include contacting the media with a regeneration solution including ethylene glycol, sodium chloride, sodium hydroxide, or a combination of sodium hydroxide and ethanol.

[0207] In some examples, regenerating the chromatography medium comprises contacting the medium with a combination of solutions, which can be mixed prior to contacting the medium or can be contacted sequentially with the medium, and optionally separated by washing, e.g., with water.

[0208] As will be apparent to one of skill in the art, the method for regenerating the chromatography medium may depend on the type of chromatography medium.

[0209] Exemplary methods for regenerating a cation exchange chromatography medium include contacting the medium with 1 M sodium hydroxide or 1 M NaCl, or a combination of an amino acid such as arginine and sodium chloride, or a combination of an alcohol and sodium hydroxide, or a combination of an alcohol and NaCl.

[0210] An exemplary method for regenerating anion exchange chromatography medium includes contacting the medium with 1 M sodium hydroxide or 1 M NaCl, or a combination of sodium hydroxide or NaCl and urea, and optionally, EDTA or citric acid.

[0211] Exemplary methods for regenerating hydrophobic interaction or mixed-mode chromatography medium include contacting the medium with citric acid (10-200 mM), or hydrochloric acid (10 mM), or sodium hydroxide (0.5-1 M), or guanidine hydrochloride (6 M), or urea (2-8 M), or propanol (40%), or 20-100% ethanol or methanol, or a combination of Tris, EDTA, and NaCl.

[0212] An exemplary method for regenerating affinity chromatography medium, e.g., Protein A chromatography medium, includes contacting the medium with a combination of sodium hydroxide (10-50 mM) and NaCl (1 M), or with urea, or guanidine hydrochloride, or citric acid.

[0213] Exemplary methods for regenerating affinity chromatography media such as FcXP are included in the following paragraphs. These methods are applicable to other chromatography media.

[0214] In one example, the method includes regenerating the chromatography medium by contacting the medium with a solution comprising sodium chloride, arginine, ethanol, a polymer, e.g., ethylene glycol, sodium hydroxide, or a combination of sodium hydroxide and ethanol. For example, the method includes regenerating the chromatography medium by contacting the medium with ethylene glycol, e.g., 20% or 40% ethylene glycol. For example, the method includes regenerating the chromatography medium by contacting the medium with a solution comprising sodium hydroxide. For example, the method includes regenerating the chromatography medium by contacting the medium with a combination of sodium hydroxide and ethanol. For example, the method includes regenerating the chromatography medium by contacting the medium with ethanol.

[0215] In one example, the method includes regenerating the chromatography medium by contacting the medium with ethanol and then contacting the resin with sodium hydroxide.

[0216] In one embodiment, the resin is contacted with 5% to 50% ethanol, for example, 10% to 40% ethanol, for example, 15% to 30% ethanol. For example, the resin is contacted with 20% ethanol. In one embodiment, the resin is contacted with 5% ethanol. In one embodiment, the resin is contacted with 10% ethanol. In one embodiment, the resin is contacted with 15% ethanol. In one embodiment, the resin is contacted with 20% ethanol. In one embodiment, the resin is contacted with 25% ethanol. In one embodiment, the resin is contacted with 30% ethanol. In one embodiment, the resin is contacted with 35% ethanol. In one embodiment, the resin is contacted with 40% ethanol. In one embodiment, the resin is contacted with 45% ethanol. In one embodiment, the resin is contacted with 50% ethanol.

[0217] In one embodiment, the resin is contacted with 5 mM to 50 mM sodium hydroxide, such as 10 mM to 40 mM sodium hydroxide, such as 15 mM to 30 mM sodium hydroxide. For example, the resin is contacted with 20 mM sodium hydroxide. In one embodiment, the resin is contacted with 5 mM sodium hydroxide. In one embodiment, the resin is contacted with 10 mM sodium hydroxide. In one embodiment, the resin is contacted with 15 mM sodium hydroxide. In one embodiment, the resin is contacted with 25 mM sodium hydroxide. In one embodiment, the resin is contacted with 30 mM sodium hydroxide. In one embodiment, the resin is contacted with 35 mM sodium hydroxide. In one embodiment, the resin is contacted with 40 mM sodium hydroxide. In one embodiment, the resin is contacted with 45 mM sodium hydroxide. In one embodiment, the resin is contacted with 50 mM sodium hydroxide.

[0218] In one embodiment, the medium is contacted with a solution comprising 20% ​​ethanol followed by a solution comprising 5 mM or 10 mM or 10 mM or 15 mM or 20 mM sodium hydroxide. In one embodiment, the medium is contacted with a solution comprising 20% ​​ethanol followed by a solution comprising 5 mM sodium hydroxide. In one embodiment, the medium is contacted with a solution comprising 20% ​​ethanol followed by a solution comprising 20 mM sodium hydroxide.

[0219] In one embodiment, the ethanol and sodium hydroxide are included in the same solution. Suitable amounts or concentrations of ethanol and sodium hydroxide are described herein. For example, the medium is contacted with a solution containing 20% ​​ethanol and 5 mM sodium hydroxide. In one embodiment, the medium is contacted with a solution containing 20% ​​ethanol and 20 mM sodium hydroxide.

[0220] In one embodiment, the chromatography medium is regenerated using ethylene glycol. For example, the chromatography medium is contacted with a solution containing 20% ​​ethylene glycol and / or a solution containing 40% ethylene glycol. For example, the medium is contacted with a solution containing 20% ​​ethylene glycol and then with a solution containing 40% ethylene glycol.

[0221] In one embodiment, multiple column volumes of a solution for regenerating the chromatography medium are passed through the chromatography medium to regenerate the medium. The solution can be passed through the chromatography medium in a downflow direction and / or an upflow direction. For example, multiple column volumes are passed through the chromatography medium in an upflow direction, and then multiple column volumes are passed through the chromatography medium in a downflow direction. For example, at least one or two or three or four or five or six column volumes are passed through the chromatography medium in an upflow direction. For example, at least one or two or three or four or five or six column volumes are passed through the chromatography medium in a downflow direction.

[0222] In one embodiment, the method of the present disclosure comprises: (i) regenerating a chromatography medium as described herein; and (ii) disinfecting the chromatography medium of (i) as described herein.

[0223] In one embodiment, the method of the present disclosure comprises: (i) regenerating the chromatography medium by contacting the chromatography medium with a solution comprising ethanol and a solution comprising sodium hydroxide, e.g., as described herein; (ii) disinfecting the chromatography medium of (i) by contacting it with an alkaline detergent or a solution comprising those described herein.

[0224] In one embodiment, the method of the present disclosure comprises: (i) regenerating the chromatography medium by contacting the medium with a solution comprising ethanol and sodium hydroxide as described herein; (ii) disinfecting the chromatography medium of (i) by contacting it with an alkaline detergent or a solution comprising those described herein.

[0225] The advantage of disinfecting and regenerating chromatography media using the methods of the present disclosure is that the useful life of the chromatography media may be increased compared to conventional disinfection and / or regeneration methods. For example, the methods of the present disclosure may allow the chromatography media to be used at least 1, 2, 3, 4, 5, 10, 25, or more times more frequently compared to chromatography media that have not been disinfected and / or regenerated using the methods of the present disclosure. Adding one or two purification batches to the life of the chromatography media may provide advantages such as, for example, cost and / or time-saving advantages in purifying a desired protein from a sample.

[0226] Continuous Chromatography The present disclosure of disinfection (and optionally regeneration) of chromatography media may be performed as part of a continuous chromatography process.

[0227] The term "continuous chromatography" is intended to mean a chromatography method comprising one or more columns packed with a chromatography medium, each column comprising one or more zones. A zone is a column or a region of a column containing a chromatography medium in which one or more chromatography steps can be performed. For example, the zone is selected from the group consisting of an equilibration zone, a binding zone, a wash zone, an elution zone, a stripping zone, or a combination thereof.

[0228] Sequential chromatography involving two or more columns involves columns connected in an arrangement that allows the columns to be operated in series and / or in parallel. In principle, a sample containing the desired protein can be loaded onto a first column and / or onto a subsequent column while other columns (or other zones of columns) are simultaneously passing through equilibration, washing, elution, disinfection, and / or regeneration. Examples of sequential chromatography will be apparent to those skilled in the art and / or are described herein.

[0229] Examples of columns that can be used to perform the methods of the present disclosure will be apparent to those of skill in the art and / or are described herein. For example, the continuous chromatography method can be performed using a Tricorn 5 / 100 column (Cytiva) or an Eco plus 1 cm column (YMC). In another example, the continuous chromatography method can be performed using a BioSMB PD System (Sartorius).

[0230] In one embodiment, the continuous chromatography is continuous affinity chromatography.

[0231] The term "sequential affinity chromatography" is intended to mean a chromatography method comprising one or more columns packed with the same affinity chromatography medium, each column comprising one or more zones.

[0232] Simulated Moving Bed (SMB) Chromatography In one example, the continuous chromatography is simulated moving bed (SMB) chromatography. The term "simulated moving bed chromatography" or "SMB chromatography" refers to a chromatography method first described in U.S. Pat. No. 2,985,589. Examples of SMB chromatography set-ups and / or equipment will be apparent to those skilled in the art and / or are described herein. The simulated moving bed concept involves using multiple smaller columns (rather than one large column) containing a solid sorbent (e.g., affinity chromatography medium) and simultaneously performing one or more continuous chromatography steps (i.e., equilibration, binding, washing, elution, or removal) on different columns in a continuous loop.

[0233] An example of an SMB chromatography setup has columns arranged in four sections with one or more columns per section. Two inlet streams (feed and eluate) and two outlet streams (extract and raffinate) are alternately directed between the column rings. The inlet and outlet positions are switched at regular time intervals in the direction of liquid flow, thus simulating countercurrent movement of the columns. The feed (containing the adsorbable components (extract)) is loaded into one or more columns of the SMB chromatography setup, and the extract is allowed to bind to the chromatography medium in the column. Meanwhile, less adsorbable components (raffinate) in the feed pass through the column. The raffinate can be loaded into one or more subsequent columns or removed from the SMB chromatography system as waste. The eluate is loaded into the column and the extract is collected. For example, the eluate can be collected from the first column while more feed is loaded into one or more subsequent columns.

[0234] Chromatography media in SMB chromatography may undergo multiple cycles (e.g., 50 cycles) of media equilibration, loading, binding, and / or elution of the protein of interest along with stripping, sanitizing, and / or regeneration of the chromatography media. Multiple batch runs (e.g., 4-10 batches) may be performed using SMB chromatography. The total lifespan of chromatography media in SMB chromatography may range from 200-500 cycles (if not more) before the chromatography media becomes unusable.

[0235] Periodic Countercurrent Chromatography (PCC) In one embodiment, the continuous chromatography is cyclic countercurrent chromatography (PCC). Examples of PCC set-ups and / or equipment will be apparent to those skilled in the art and / or are described herein. The concept of PCC involves using multiple columns containing solid sorbents (e.g., affinity chromatography media) to perform chromatographic steps in parallel in a quasi-continuous manner.

[0236] An example of a PCC setup involves the use of two columns. In a first step, the sample is loaded onto the first column above the DBC of the chromatography medium such that unbound products (e.g., IgG) pass through the first column and are captured by the second column. In a second step, the first column is washed, eluted, clarified, and / or re-equilibrated independently of the second column, which is loaded with the additional sample. In a third step, additional sample is loaded onto the second column above the DBC of the chromatography medium such that unbound products pass through the second column and are captured by the first column. In a fourth step, the second column is washed, eluted, clarified, and / or re-equilibrated independently of the first column, which is loaded with the additional sample. The process steps are cycled continuously between the two columns.

[0237] Another example of a PCC setup involves the use of multiple columns. For example, a variation of the PCC setup described above may involve the use of multiple columns to capture unbound products, simulating the use of a large column.

[0238] Continuous Countercurrent Tangential Chromatography (CCTC) In one embodiment, the continuous chromatography is continuous countercurrent tangential chromatography (CCTC). Examples of CCTC set-ups and / or equipment will be apparent to those skilled in the art and / or are described herein. The CCTC concept involves using a chromatography medium in the form of a slurry, where the slurry is continuously guided through several static mixers and hollow fiber membranes that separate the fluid phase from the chromatography medium. CCTC is usually performed at low pressure (e.g., less than 70 kPa).

[0239] An example of a CCTC process involves the steps of binding, first wash, second wash, elution, disinfection, regeneration and / or equilibration. The sample (e.g., plasma or a fraction thereof) and chromatography medium pass through a static mixer and hollow fiber membrane in the binding step. Impurities in the flow-through of the hollow fiber membrane are removed in the washing step, while the chromatography medium-bound product is retained by the membrane. The hollow fiber retains the chromatography medium and allows the product to flow through in the elution step. The chromatography medium is disinfected, regenerated and / or equilibrated, and the process is repeated.

[0240] Continuous Countercurrent Spiral Chromatography (CCSC) In one embodiment, the continuous chromatography is continuous countercurrent spiral chromatography (CCSC). Examples of CCSC setups and / or equipment will be apparent to those skilled in the art and / or are described herein. The CCSC concept involves the use of a compact rotating coil separation column mounted on the centrifuge rotary frame. There are two separation column designs currently available: the spiral disk assembly and the spiral tube support assembly.

[0241] An exemplary CCSC process involves rotating a coiled separation column around the central axis of a centrifuge while it rotates synchronously around its own axis (e.g., at 1,000-1,200 rpm). The mobile phase can be passed through the centrifuge rotor without a rotating seal, and a large amount of the stationary phase is retained while the two phases are mixed along the length of the column, resulting in highly efficient solute separation.

[0242] Analysis of chromatographic media disinfection Methods for determining disinfection of chromatography media will be apparent to those of skill in the art and / or are described herein.

[0243] In one embodiment, disinfection is determined by measuring the amount of microorganisms before and after disinfection using a microbial challenge test. 6 The chromatography medium is loaded with more than CFU / ml of microorganisms (e.g., Pseudomonas aeruginosa), and one or more samples following disinfection are collected to determine the number of viable microorganisms (CFU) per mL.

[0244] In another embodiment, disinfection is determined by measuring the amount of virus before and after disinfection using a virus spiking challenge. Briefly, a sample is spiked with a known amount of virus and the sample is passed through a chromatography matrix. The chromatography matrix is ​​disinfected using the method of the present disclosure and one or more samples collected after disinfection. The amount of virus after disinfection is determined and compared to the amount before disinfection. For example, a reduction in the amount of virus to a level below the detection level of the assay indicates that the disinfection method is effective.

[0245] reproduction Methods for determining regeneration of a chromatography medium will be apparent to one of skill in the art and / or are described herein.

[0246] In one example, regeneration is determined by assessing the dynamic binding capacity of the resin using a known amount of a pure protein of interest (e.g., IgG) before and after regeneration using the methods of the present disclosure, e.g., the similar dynamic binding capacity is determined before and after regeneration.

[0247] In another example, regeneration is determined by monitoring the change in protein content of the chromatography medium using spectrophotometry. Briefly, the area under the UV(A280) peak is measured before, during, and / or after regeneration of the chromatography medium to determine the change in the level of protein (e.g., host cell proteins or other protein-based contaminants) of the medium. EXAMPLES

[0248] Example 1: Materials and Methods sample Cryo-rich plasma (CRP) was prepared from pooled blood donations and stored at −20° C. CRP was thawed at 37° C. and filtered through 1.2 μm Sartopure® (Sartorius) and 0.45+0.2 μm Sartobran® P (Sartorius) capsule filters at less than 0.5 bar pressure and provided at room temperature for use in the chromatographic methods described herein.

[0249] The samples used to determine the binding capacity of the chromatography medium after disinfection and regeneration of the chromatography medium were prepared from the eluate containing IgG from the batch chromatography run of CRP on the AKTA system. Before being used to determine the binding capacity of the chromatography medium, the volume of the solution containing IgG was adjusted with EQB buffer (20 mM sodium phosphate, 500 mM NaCl, pH 7.4) to an optical density (OD) of approximately 7, a pH of about 7.4, and a conductivity of about 21 mS / cm, and filtered through a 0.45 μm syringe filter (Sterives, SVGV010RS) and a 0.22 μm syringe filter (Sterive, SVH010RS).

[0250] Chromatography Media The method described herein was evaluated using affinity chromatography medium CaptureSelect® FcXP (Thermo Fisher). The affinity chromatography medium was resuspended and packed into a Cytiva Tricorn column (column volume 1.31 ml, column height 6.7 cm) with 0.1 M sodium chloride (NaCl) at a flow rate of 20 ml / min until a chromatography medium bed was formed. Affinity chromatography using columns packed with CaptureSelect® FcXP (Thermo Fisher) was performed in simulated moving bed mode on a BioSMB PD system (Sartorius).

[0251] binding capacity In a longevity study of the chromatography media of the present disclosure (eg, as described in Example 3), the binding capacity of the chromatography media was determined.

[0252] Breakthrough curve (BTC) measurements of the chromatography media in contact with EQB buffer or after the regeneration and disinfection procedure (at the beginning of the lifetime study) were determined using an Akta avant 25 system (Cytiva) at UV absorbance at 280 nm.

[0253] Commercially available software (e.g., UNICORN Evaluation Classic) was used to calculate the binding capacity of the chromatography medium in contact with EQB buffer or alkaline detergent based on the BTC measurements. The binding capacity of the chromatography medium was expressed as a percentage (%) compared to the binding capacity of the chromatography medium at the start of the longevity study (e.g., BTC measurements of the chromatography medium in contact with EQB buffer).

[0254] yield The concentration of IgG in the FcXP eluate, column flow-through, and waste fraction (EQB phase) after purification of immunoglobulins from plasma and subsequent renaturation and disinfection was determined using an immunospecific densitometry method. Briefly, the light scattering properties of IgG in the test samples were determined and compared with those of a series of standard solutions containing known concentrations of IgG. The yield was expressed as a percentage (%) of the IgG content in the starting material (i.e., the sample loaded onto the chromatography medium).

[0255] Immunoglobulin Class Distribution Quantification of immunoglobulin subclasses and albumin in the FcXP eluate after purification of immunoglobulins from plasma and subsequent renaturation and disinfection was determined using immunospecific densitometry. Briefly, the light scattering properties of immunoglobulins (i.e., IgG, IgM, IgA) and albumin in the FcXP eluate were determined by comparing them to the light scattering properties of a series of standard solutions containing known concentrations of the particular immunoglobulin or albumin.

[0256] purity Purification of immunoglobulins from plasma and the purity of the FcXP eluate after subsequent renaturation and disinfection were determined by LabChip® electrophoresis (Perkin Elmer). LabChip is based on traditional gel electrophoresis principles and has been transferred to a microfluidic chip format to ensure high throughput analysis. The chip format can dramatically reduce separation times from 90 minutes to about 1 minute and provide automated molecular weight size, purity, and concentration of protein samples in digital format. Perkin Elmer (manufacturer) Protein Exact Assay was used to determine the purity of the samples.

[0257] Briefly, the FcXP eluate was loaded into individual wells of a chip used for LabChip® electrophoresis (Perkin Elmer), the chip was loaded into a characterization system (e.g., LabChip GXII Touch Protein Characterization System), and purity was determined using the LabChip Reviewer software.

[0258] SDS-PAGE analysis The purity of the FcXP eluate after purification of immunoglobulins from plasma and subsequent renaturation and disinfection was qualitatively determined by SDS-PAGE. Briefly, one or more FcXP eluates after purification of immunoglobulins from plasma and subsequent renaturation and disinfection as described herein were loaded onto a suitable SDS-PAGE gel (e.g., 8-16% TRIS-glycine) under reducing and non-reducing conditions along with protein size markers and a positive control for IgG (e.g., Privigen). Proteins were separated based on size and bands visualized by Coomassie Blue staining. Purity was determined by analyzing the bands of the FcXP eluate after purification of immunoglobulins from plasma and subsequent renaturation and disinfection in comparison to the bands of the positive control for IgG (e.g., Privigen).

[0259] Example 2: Identification of a suitable regeneration and disinfection solution To identify a suitable solution for regenerating FcXP chromatography medium after purification of immunoglobulins from serum or plasma, the following solutions were tested: ●EQB buffer containing 1% Tween 80 300mM arginine 0.5% Caprylate ●20% ethanol ●40% ethanol ●20% and 40% ethylene glycol 20mM sodium hydroxide 10mM DTT

[0260] The results showed that 20% ethanol, 20% or 40% ethylene glycol, and 20 mM sodium hydroxide were most effective for regenerating the FcXP resin.

[0261] A sequence of 20% ethanol followed by 20 mM sodium hydroxide was then tested and shown to be effective in regenerating the FcXP resin. An exemplary chromatogram showing the regeneration of FcXP resin with 20% ethanol and 20 mM sodium hydroxide is shown in FIG.

[0262] Analysis of the flow-through from the FcXP chromatography medium during renaturation using reducing or nonreducing polyacrylamide gel electrophoresis showed that ethanol primarily removed the protein clusterin, whereas sodium hydroxide removed the remaining nonspecifically bound proteins.

[0263] The alkaline detergent CIP-100 diluted to 2% was used for disinfection. The results showing the disinfection of FcXP chromatography medium by 2% CIP-100 are also shown in Figure 1.

[0264] Example 3: FcXP Lifespan Study A study was performed to determine the effect of 20% ethanol and 20 mM sodium hydroxide regeneration and 2% CIP-100 disinfection on the longevity of FcXP. After each batch of successive chromatography, the resin was exposed to the following regeneration and disinfection conditions: 20% EtOH (total contact time: 62 min) 1) 5CV, 1.3ml / min, upflow 2) 4CV, 0.1ml / min, upflow 3) 5CV, 1.3ml / min, downflow 20 mM NaOH (total contact time: 62 min) 1) 5CV, 1.3ml / min, upflow 2) 4CV, 0.1ml / min, upflow 3) 5CV, 1.3ml / min, downflow 2% CIP-100 (steps 2-5 were performed for 61 min) 1) EQB, 5CV, 1.3ml / min, upflow 2) CIP, 3CV, 1.3ml / min, upflow 3) CIP, 3CV, 1.3ml / min, downflow 4) CIP, 3CV, 1.3ml / min, upflow 5) CIP, 4CV, 0.1ml / min, upflow 6) EQB, 20CV, 1.3ml / min, upflow 7) EQB, 20CV, 1.3ml / min, downflow

[0265] Table 1 and Figure 2 show the effect of regeneration and sanitization on the binding capacity of FcXP chromatography medium over eight batches of purification of immunoglobulins from plasma. [Table 1]

[0266] The data in Table 1 and FIG. 2 show a slight decrease in binding capacity of about 1.6% per batch and regeneration / disinfection cycle.

[0267] Table 2 and Figures 3-4 show the analysis of the purification of immunoglobulins from 10 purification batches from two plasma pools (pool A from 25 donors and pool B from 10 donors). The recovered immunoglobulins were also generally more than 95% pure. Data from the BCA assay measured on blank run eluates additionally show that the renaturation sequence is effective in avoiding protein carryover. [Table 2]

[0268] FIG. 7 shows the effect of regeneration and disinfection on the pressure profile of FcXP chromatography medium over 10 batches of purification of immunoglobulins from plasma, where regeneration and disinfection were performed after each batch (1 batch = 50 product cycles).

[0269] Example 4: CIP-100 as a regenerating and disinfecting solution Experiments were performed to compare the efficiency of 2% CIP-100 alone as a regeneration and disinfection solution compared to 20% ethanol, 20 mM sodium hydroxide for regeneration, and 2% CIP-100 for disinfection. Results showed that the loss of protein binding capacity of the resin was similar for both strategies for regeneration / disinfection, but there was a slightly higher pressure increase when using CIP-100 alone (15.7%) compared to ethanol / sodium hydroxide / CIP-100 (11.2%). Estimated protein carryover was similar for both strategies, but SDS-PAGE showed that slightly more protein remained bound to the resin after regeneration / disinfection using CIP-100 alone.

[0270] Example 5: Optimization of regeneration To optimize the regeneration of FcXP chromatography medium following purification of immunoglobulins from plasma, studies were performed to investigate the effect of each regeneration step in reducing the CV.

[0271] Additionally, studies will be conducted to evaluate the effect of combining ethanol and sodium hydroxide into a single solution. These studies will be conducted at a constant 20% ethanol and varying concentrations of sodium hydroxide from 5 mM to 20 mM.

[0272] Example 6: Application of regeneration and disinfection to additional chromatography media The affinity chromatography resins MabSelect® SuRe and MabSelect® SuRe LX (Thermo Fisher) are used to evaluate the suitability of alkaline detergents in disinfecting chromatography media. Each chromatography medium is packed into a Tricorn column of Cytiva (5 mm diameter) and chromatographed on an Akta avant 25 system (Cytiva). After one or more chromatography cycles in the purification of the protein of interest, the microbial load of the chromatography medium is determined.

[0273] Each chromatography medium is then regenerated using 20% ​​ethanol followed by 20 mM sodium hydroxide, and then disinfected by contacting the medium with an alkaline detergent (0.5% to 4.5%). The alkaline detergent used for disinfection is CIP-100 (Steris Life Sciences) at concentrations of 0.5%, 2% and 4.5%.

[0274] Example 7: Evaluation of Additional Alkaline Detergents The sanitization and regeneration of the chromatography medium as detailed in Examples 2 and 5 is repeated using additional alkaline detergent. The regeneration step is maintained as 20% ethanol followed by 20 mM sodium hydroxide. Specifically, alkaline detergents ProKlenz One (Steris Life Sciences), ProKlenz-100 (Steris Life Sciences) and COSA CIP-92 (Ecolab) at concentrations of 0.5%, 2% and 4.5% are tested, as well as alkaline detergents containing potassium hydroxide (alkaline salt), EDTA (chelating agent) and surfactants.

[0275] Example 8: Viral inactivation by CIP-100 To test the effectiveness of CIP-100 for viral inactivation, samples spiked with either Bovine viral diarrhea virus (BVDV, a model for Hepatitis C virus) or Hepatitis A virus (HAV) were loaded onto FcXP resin. Chromatography was performed and the column was sanitized with 2% CIP-100. As shown in Figures 5 and 6, CIP-100 inactivates BVDV to levels below the limit of detection (i.e., complete inactivation) and inactivates HAV by approximately 3 logs.

[0276] Example 9: Virus and prion inactivation by CIP-100 To test the effectiveness of CIP-100 for inactivating viruses and prions, a sample spiked with virus or prion is loaded onto FcXP resin. Chromatography is performed and the column is disinfected with 2% CIP-100. The eluate is then subjected to a second, unspiked chromatography and the eluate is analyzed. If the sample from the second chromatography does not contain infectious virus or prion or the levels are substantially reduced, the disinfection is effective.

Claims

1. A method for disinfecting a chromatography medium, comprising contacting the medium with an alkaline detergent.

2. The method according to claim 1, wherein the alkaline detergent comprises an alkaline salt and optionally a chelating agent.

3. The method according to claim 1, wherein the alkaline detergent comprises an alkaline salt, one or more surfactants, and optionally a chelating agent.

4. (i) The alkali salt is selected from the group consisting of potassium hydroxide, sodium hydroxide, sodium carbonate, sodium acetate, sodium sulfide, and sodium bicarbonate, and optionally the alkali salt is potassium hydroxide or sodium hydroxide, and optionally the alkali salt has a concentration of 75 mM. (ii) The one or more surfactants are selected from the group consisting of amine ethoxylates, amphocarboxylates, triethanolamine, octyl-β-D glucopyranoside (OGP), polysorbate, poloxamer, lauryldimethylamine (LDAO), myristyldimethylamine-N-oxide (TDAO), sodium cholate, decyl-β-D glucopyranoside, and dodecyl maltoside, and / or (iii) The chelating agent is ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), ethylenediamine-N,N'-disuccinic acid (EDDS), iminodisuccinic acid (IDS), methylglycinediacetic acid (MGDA), triethylenetetramine (Trien), iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), tripolyphosphate (TPP), diethylenetriaminepentaacetic acid (DTPA), di The chelating agent is selected from the group consisting of ethylenediamine-N,N'-disuccinic acid (EDDS), iminodisuccinic acid (IDS), methylglycine diacetic acid (MGDA), nitrilotriacetic acid (NTA), and salts thereof, and optionally the chelating agent is selected from the group consisting of ethylenediamine-N,N'-disuccinic acid (EDDS), iminodisuccinic acid (IDS), methylglycine diacetic acid (MGDA), nitrilotriacetic acid (NTA), and salts thereof. The method according to claim 2 or 3, wherein the biodegradable chelating agent is a biodegradable chelating agent such as a biodegradable chelating agent.

5. The aforementioned alkaline detergent, (i) 0.01% to 10% (w / v) of one or more surfactants, (ii) One or more surfactants in an amount of 0.01% to 1% (w / v), (iii) 10 mM to 150 mM alkali salts, and / or (iv) 50 mM to 100 mM alkali salt The method according to claim 1 or 2, including the method described in claim 1 or 2.

6. The method according to claim 1 or 2, wherein the alkaline detergent comprises TDAO and an alkali salt selected from sodium hydroxide and potassium hydroxide, optionally comprising 10 mM to 150 mM potassium hydroxide or sodium hydroxide and 0.01% to 0.1% (w / v) TDAO, and further optionally comprising 75 mM potassium hydroxide or sodium hydroxide and 0.02% (w / v) TDAO.

7. The method described above is (i) Contact the culture medium with the alkaline detergent for 60 minutes, and / or (ii) Regenerating the chromatography medium, optionally, regenerating the chromatography medium before disinfecting it. The method according to claim 1 or 2, including the method described in claim 1 or 2.

8. The method according to claim 7, wherein the chromatography medium is regenerated, and the method includes regenerating the chromatography medium by contacting the medium with a solution containing ethylene glycol, sodium chloride, sodium hydroxide, or a combination of sodium hydroxide and ethanol.

9. The chromatography medium is (i) an ion exchange medium, affinity medium, hydrophobic interaction chromatography medium, size exclusion column, or mixed-mode chromatography medium, and / or The method according to claim 1 or 2, comprising a matrix selected from the group consisting of poly(styrene-divinylbenzene), crosslinked poly(styrene-divinylbenzene), silica, agarose, crosslinked agarose, controlled-pore glass, polymethacrylate, and cellulose, wherein optionally the chromatography medium comprises crosslinked poly(styrene-divinylbenzene), and optionally the chromatography medium comprises a VHH antibody fragment that can specifically bind to the CH3 domain of human IgG conjugated to the crosslinked poly(styrene-divinylbenzene) matrix.

10. The method according to claim 9, wherein the culture medium is an affinity chromatography medium, preferably the affinity chromatography medium is bound to the Fc region of the antibody, and optionally the affinity chromatography medium contains protein A or an antibody-bound fragment thereof, protein G or an antibody-bound fragment thereof, or a ligand that can specifically bind to the CH1 or CH3 domain of human IgG, and optionally the ligand that can specifically bind to the CH3 domain of human IgG contains a single-domain [VHH] antibody fragment derived from a camelid animal.

11. The method according to claim 1 or 2, comprising contacting a chromatography medium containing a VHH antibody fragment that can specifically bind to the CH3 domain of human IgG conjugated to a crosslinked poly(styrene-divinylbenzene) matrix with an alkaline detergent comprising an alkaline salt, one or more surfactants, and optionally a chelating agent.

12. The method according to claim 1 or 2, comprising: regenerating a chromatography medium containing a VHH antibody fragment that can specifically bind to the CH3 domain of human IgG conjugated to a crosslinked poly(styrene-divinylbenzene) matrix by contacting the medium with a solution containing a combination of sodium hydroxide and ethanol; and disinfecting the medium by contacting it with an alkaline detergent containing an alkali salt, one or more surfactants, and optionally a chelating agent.

13. The method according to claim 1 or 2, wherein the chromatography medium has been in contact with the sample beforehand, and optionally the sample is plasma or a fraction thereof.

14. The method according to claim 1 or 2, wherein the method is carried out as part of a continuous chromatography process.

15. A method for regenerating and disinfecting chromatography media, (i) Regenerating the chromatography medium by contacting the medium with a regeneration solution containing ethylene glycol, sodium chloride, sodium hydroxide, or a combination of sodium hydroxide and ethanol, (ii) A method comprising disinfecting the chromatography medium by contacting the medium with an alkaline detergent.

16. The method described above is (i) Regenerating the chromatography medium by contacting the medium with a regeneration solution containing a combination of 5-30 mM sodium hydroxide and 5-30% ethanol, (ii) The method according to claim 15, comprising disinfecting the chromatography medium by contacting the medium with an alkaline detergent containing 0.01% to 1% (w / v) of one or more surfactants and / or an alkaline salt selected from 60 mM to 80 mM sodium hydroxide and potassium hydroxide.

17. (i) The alkaline detergent, (a) 75 mM potassium hydroxide or sodium hydroxide, and 0.02% (w / v) TDAO, (b) 2% CIP-100 including and / or (ii) The method according to claim 15 or 16, wherein the regeneration solution comprises a combination of 5 mM or 20 mM sodium hydroxide and 20% ethanol.

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