Compositions and methods for cleaning

Abstract

The present technology relates to a novel cleaning method for chromatography media and / or support devices, which involves treatment with a novel cleaning solution comprising sodium hydroxide and hexylene glycol.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Provisional Patent Application No. 63 / 382,985, filed November 9, 2022, the disclosure of which is incorporated herein by reference in its entirety. [Background technology]

[0002] High commercial demand for biopharmaceuticals has led pharmaceutical companies to focus on maximizing productivity and product quality while controlling costs associated with production. This drive for improved product quality has enabled hydrophobic interaction chromatography (HIC) to come to the forefront as a versatile technique for purifying analytes of interest (e.g., proteins) from crude mixtures while maintaining biological activity through the use of conditions and matrices that operate under minimally denaturing conditions. HIC combines the non-denaturing properties of salt precipitation with the precision of chromatography to provide high resolution with good recovery of biological activity. HIC is commonly used to separate the desired monomeric form of a target analyte from less desirable aggregated forms. When the target analyte is a protein, HIC can also provide excellent selectivity for the removal of misfolded or truncated forms of the protein.

[0003] All bioprocess chromatography applications, including HIC, require advanced control over contaminant and impurity removal. These contaminants include, but are not limited to, proteins, carbohydrates, lipids, lipopolysaccharides (e.g., endotoxins), lipoproteins, and / or nucleic acids. Macromolecular impurities, such as proteins, carbohydrates, lipids, and nucleic acids, are often addressed by utilizing various intermolecular forces to separate target analytes from the impurities during the wash and elution phases of a chromatography run. Traditionally, HIC resin decontamination methods involve treatment with a wash solution containing sodium hydroxide, ethylene glycol, and ethanol. However, ethylene glycol is highly toxic, and ethanol is highly flammable. Summary of the Invention [Means for solving the problem]

[0004] The present disclosure provides a cleaning solution consisting essentially of a strong base and hexylene glycol. In some embodiments, the strong base is an alkali metal hydroxide. In some embodiments, the strong base is sodium hydroxide. In some embodiments, the concentration of sodium hydroxide is about 0.1 M to about 0.5 M. In some embodiments, the concentration of hexylene glycol is about 35% to about 55%. In some embodiments, the cleaning solution consists essentially of about 0.1 M sodium hydroxide and about 50% hexylene glycol. In some embodiments, the pH of the cleaning solution is about 12.

[0005] In some embodiments, the cleaning solutions of the present disclosure are for use in methods to functionally clean chromatography media and / or support devices.

[0006] In some embodiments, the present disclosure provides a method for functionally cleaning a chromatography medium and / or support device, the method comprising contacting the chromatography medium and / or support device with a cleaning solution of the present disclosure.

[0007] In some embodiments, the chromatography medium is a hydrophobic interaction chromatography (HIC) resin. In some embodiments, the HIC resin is selected from Capto MMC, Capto Butyl, Capto Phenyl, and Toyopearl Hexyl-650C. In some embodiments, the HIC resin is Capto Butyl.

[0008] In some embodiments, the present disclosure provides a purification process comprising a washing step according to the washing methods disclosed herein. In some embodiments, the washing step comprises a step within an integrated, continuous biomanufacturing process for the purification of a polypeptide. In some embodiments, the polypeptide is an antibody or a recombinant enzyme. In some embodiments, the recombinant enzyme is a human recombinant enzyme. In some embodiments, the human recombinant enzyme is β-glucocerebrosidase (β-D-glucosyl-N-acylsphingosine monocohydrolase). In some embodiments, the recombinant enzyme is Cerezyme®. [Brief explanation of the drawings]

[0009] [Figure 1A] 1 shows a HiScreen™ Capto™ Butyl column after washing with a standard wash solution containing 0.1 M sodium hydroxide, 50% ethylene glycol, and 10% ethanol. [Figure 1B] 1 shows a HiScreen™ Capto™ Butyl column after washing with a solution containing 0.1 M sodium hydroxide and 50% hexylene glycol. [Figure 2] Chromatograms for screening various wash solutions - CIP1 (100 mM sodium acetate, 700 mM arginine, pH 3); CIP2 (1.0 M sodium hydroxide); CIP3 (0.1 M sodium hydroxide, 50% ethylene glycol and 10% ethanol); and CIP4 (0.5 M sodium hydroxide and 50% hexylene glycol). [Figure 3] 1 is a plot showing the change in specific activity (U / mg) of an exemplary human recombinant enzyme eluted from a Capto™ Butyl column over 40 cycles. [Figure 4] 1 is a plot showing the change in % activity yield of an exemplary human recombinant enzyme eluted from a Capto™ Butyl column over 40 cycles. DETAILED DESCRIPTION OF THE INVENTION

[0010] The features, objects, and advantages of the present technology will be apparent from the following detailed description. However, it should be understood that the detailed description, while illustrating embodiments and aspects of the present technology, is given by way of example only, not limitation. Various changes and modifications within the scope of the present technology will become apparent to those skilled in the art from the detailed description.

[0011] Definitions of certain terms used herein are provided below: Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

[0012] I. Definition The moieties described below can be substituted or unsubstituted. "Substituted" refers to the replacement of a hydrogen atom of a molecule or R group with one or more additional R groups, such as deuterium, halogen, alkyl, haloalkyl, alkenyl, alkoxy, alkoxyalkyl, alkylthio, trifluoromethyl, acyloxy, hydroxy, hydroxyalkyl, mercapto, carboxy, cyano, acyl, aryloxy, aryl, arylalkyl, heteroaryl, amino, aminoalkyl, alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-1-yl, piperazin-1-yl, nitro, phosphine, phosphinate, phosphonate, sulfate, =0, =S, or other R groups. Unless otherwise indicated, an optionally substituted group may have a substituent at each substitutable position of the group. Combinations of substituents contemplated herein are preferably stable (e.g., substantially unchanged for more than one week when maintained at a temperature of 40°C or less in the absence of moisture or other chemically reactive conditions) or result in the formation of chemically feasible compounds.

[0013] Unless otherwise specified, the term "strong base" as used herein refers to a hydroxide of an alkali metal or alkaline earth metal. Examples of strong bases include, but are not limited to, sodium hydroxide (NaOH), potassium hydroxide (KOH), lithium hydroxide (LiOH), rubidium hydroxide (RbOH), cesium hydroxide (CsOH), calcium hydroxide (Ca(OH)), barium hydroxide (Ba(OH)), and strontium hydroxide (Sr(OH)).

[0014] Unless otherwise specified, the term "glycol" as used herein refers to a group of compounds of the formula ((R 2 )(R 3 )-C(OH)-C(OH)-(R 4 )(R 5 ) in which R 2 , R 3 , R 4 and R 5 are each independently hydrogen, deuterium, halo, amino, hydroxy, cyano, formyl, furyl, nitro, alkyl, haloalkyl, alkenyl, haloalkenyl, alkynyl, haloalkynyl, acyloxy, alkoxy, haloalkoxy, thioalkoxy, halothioalkoxy, alkanoyl, haloalkanoyl, thioalkanoyl, halothioalkanoyl, carboxy, carbonyloxy, halocarbonyloxy, carbonylthio, halocarbonylthio, thiocarbonyloxy, halothiocarbonyloxy, thiocarbonylthio, halothiocarbonylthio, and -S(O) n R 11 (n=0-2, R 11 is directly bonded to S), where R 11is selected from the group consisting of hydrogen, deuterium, halo, amino, hydroxy, thiol, cyano, formyl, alkyl, haloalkyl, alkenyl, haloalkenyl, alkynyl, haloalkynyl, acyloxy, alkoxy, haloalkoxy, thioalkoxy, halothioalkoxy, alkanoyl, haloalkanoyl, thioalkanoyl, halothioalkanoyl, carboxy, carbonyloxy, halocarbonyloxy, carbonylthio, halocarbonylthio, thiocarbonyloxy, halothiocarbonyloxy, thiocarbonylthio, and halothiocarbonylthio.

[0015] As used herein, the term "functional cleaning" refers to a cleaning process in which there is no measurable carryover of impurities (e.g., host cell proteins, protein aggregates, DNA, lipids, cellular debris, etc.) from one purification cycle to the next, and / or the accumulation of impurities is so low that the life of the chromatography media and / or supporting equipment typically exceeds 100 useful cycles.

[0016] As used herein, the term "variant" encompasses any form of a particular protein that is recombinantly expressed in a host cell or a non-native host cell. In some embodiments, the term "variant" refers to a protein that is recombinantly expressed from its native DNA sequence. In some embodiments, the term "variant" refers to a protein that is recombinantly expressed from a codon-optimized DNA sequence. In some embodiments, the term "variant" refers to a recombinantly expressed full-length protein. In some embodiments, the term "variant" refers to a recombinantly expressed truncated form of a protein. In some embodiments, the term "variant" refers to a recombinantly expressed mutant protein. In some embodiments, a mutant protein contains point mutations at one or more sites in its amino acid sequence. In some embodiments, the term "variant" refers to a recombinantly expressed engineered protein, e.g., a genetically engineered protein. In some embodiments, the term "variant" refers to a recombinantly expressed engineered protein.

[0017] II. Cleaning Solution In some embodiments, compositions and methods are provided herein for cleaning chromatography media and / or support equipment for reuse. Chromatography reuse is a changeover procedure in which chromatography materials are cleaned so that they can be reused to purify the same or different target analytes. Significant cost savings can be achieved when chromatography resins, such as HIC resins, are reused for the purification of multiple analytes or multiple batches of the same analyte. In some embodiments, the cleaning solutions of the present disclosure are suitable for functional cleaning of chromatography media and / or support equipment such that there is no measurable carryover of impurities from one elution cycle to the next, or the accumulation of impurities is so low that the column life exceeds 100 useful cycles.

[0018] Conventional chromatography media can be cleaned and disinfected to sufficiently reduce tissue culture impurities (proteins, DNA, lipids, cellular debris, etc.) with a combination of sodium hydroxide and salt. Hydrophobic interaction and multimodal interaction chromatography typically require solvents in addition to sodium hydroxide and salt to clean. In some embodiments, provided herein are compositions for cleaning chromatography media and / or supporting devices (e.g., chromatography columns, such as hydrophobic interaction columns (HIC)).

[0019] In some embodiments, the present technology relates to a cleaning solution comprising a strong base and a glycol.

[0020] In some embodiments, wash solutions of the present disclosure comprising a strong base and a glycol (e.g., sodium hydroxide and glycol) are effective in eluting and washing HIC columns, such as those used in processes for preparing recombinant enzymes.

[0021] In some embodiments, the present technology relates to a cleaning solution comprising sodium hydroxide. In some embodiments, the solution comprises about 0.05-0.5 M sodium hydroxide. In some embodiments, the solution comprises about 0.05 M, 0.08 M, 0.1 M, 0.15 M, 0.2 M, 0.25 M, 0.3 M, 0.35 M, 0.4 M, 0.45 M, or 0.5 M sodium hydroxide. In some embodiments, the solution comprises about 0.1 M sodium hydroxide.

[0022] In some embodiments, the present technology relates to a cleaning solution comprising hexylene glycol. In some embodiments, the solution comprises about 8-80% hexylene glycol. In some embodiments, the solution comprises about 10-70%, about 12-60%, about 14-50%, about 16-40%, about 18-30%, or about 20-25% hexylene glycol. In some embodiments, the solution comprises about 8%, 10%, 12%, 14%, 16%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, or 80% hexylene glycol. In some embodiments, the solution comprises about 50% hexylene glycol.

[0023] In some embodiments, the present technology relates to a wash solution comprising sodium hydroxide and hexylene glycol. In some embodiments, the wash solution comprising sodium hydroxide and hexylene glycol is superior for washing and eluting an HIC column compared to a wash solution comprising ethylene or propylene glycol and sodium hydroxide without ethanol or some other co-solvent. In some embodiments, when the wash solution comprising sodium hydroxide and hexylene glycol is used in a biomanufacturing process (e.g., an integrated continuous biomanufacturing process), the product yield and peak sharpness of a target analyte, such as a recombinant protein (such as a recombinant enzyme), are improved.

[0024] In some embodiments, the present technology relates to a cleaning solution comprising about 0.05M to 0.5M sodium hydroxide and about 8 to 80% hexylene glycol. In some embodiments, the solution comprises about 0.05M sodium hydroxide and 35-55% hexylene glycol, 0.08M sodium hydroxide and 35-55% hexylene glycol, 0.1M sodium hydroxide and 35-55% hexylene glycol, 0.15M sodium hydroxide and 35-55% hexylene glycol, 0.2M sodium hydroxide and 35-55% hexylene glycol, 0.25M sodium hydroxide and 35-55% hexylene glycol, 0.3M sodium hydroxide and 35-55% hexylene glycol, 0.35M sodium hydroxide and 35-55% hexylene glycol, 0.4M sodium hydroxide and 35-55% hexylene glycol, 0.45M sodium hydroxide and 35-55% hexylene glycol, or 0.5M sodium hydroxide and 35-55% hexylene glycol. In some embodiments, the solution comprises about 0.1 M sodium hydroxide and 50% hexylene glycol.

[0025] In some embodiments, the present technology relates to a cleaning solution comprising about 0.05 M to 0.5 M sodium hydroxide and about 50% hexylene glycol. In some embodiments, the solution comprises about 0.05 M sodium hydroxide and 50% hexylene glycol, 0.08 M sodium hydroxide and 50% hexylene glycol, 0.1 M sodium hydroxide and 50% hexylene glycol, 0.15 M sodium hydroxide and 50% hexylene glycol, 0.2 M sodium hydroxide and 50% hexylene glycol, 0.25 M sodium hydroxide and 50% hexylene glycol, 0.3 M sodium hydroxide and 50% hexylene glycol, 0.35 M sodium hydroxide and 50% hexylene glycol, 0.4 M sodium hydroxide and 50% hexylene glycol, 0.45 M sodium hydroxide and 50% hexylene glycol, or 0.5 M sodium hydroxide and 50% hexylene glycol. In some embodiments, the solution comprises about 0.1 M sodium hydroxide and 50% hexylene glycol.

[0026] In some embodiments, the present technology relates to a cleaning solution having a pH of about 12. In some embodiments, the solution has a pH of about 10, about 10.5, about 11, about 11.5, about 11.7, about 11.9, about 12.1, about 12.3, about 12.5, or about 13. In some embodiments, the solution has a pH of about 12.0.

[0027] In some embodiments, the cleaning solution comprises about 0.1 M sodium hydroxide and about 50% hexylene glycol and has a pH of about 12.0.

[0028] III. Cleaning Method In some embodiments, the present disclosure provides a method for cleaning chromatography media and / or support devices, the method comprising treatment with a cleaning solution comprising a strong base and a glycol. Potential contaminants addressed by the present technology include, but are not limited to, carryover impurities, proteins, DNA, lipids, cellular debris, etc. In some embodiments, the present method provides functionally cleaned chromatography media and / or support devices with no measurable carryover of impurities from one purification cycle to the next and / or with very low impurity accumulation such that the chromatography media and / or support devices have an extended lifespan.

[0029] In some embodiments, the present disclosure relates to methods of cleaning chromatography media and / or support devices by contacting the media and / or devices with a cleaning solution described herein. In some embodiments, the cleaning solution comprises a strong base and a glycol.

[0030] In some embodiments, the present technology relates to a method for cleaning a chromatography medium and / or support device by contacting the medium and / or device with a cleaning solution comprising sodium hydroxide. In some embodiments, the solution comprises about 0.05-0.5 M sodium hydroxide. In some embodiments, the solution comprises about 0.05 M, 0.08 M, 0.1 M, 0.15 M, 0.2 M, 0.25 M, 0.3 M, 0.35 M, 0.4 M, 0.45 M, or 0.5 M sodium hydroxide. In some embodiments, the solution comprises about 0.1 M sodium hydroxide.

[0031] In some embodiments, the present technology relates to a method for cleaning chromatography media and / or support devices by contacting the media and / or devices with a cleaning solution comprising hexylene glycol. In some embodiments, the solution comprises about 8-80% hexylene glycol. In some embodiments, the solution comprises about 10-70%, about 12-60%, about 14-50%, about 16-40%, about 18-30%, or about 20-25% hexylene glycol. In some embodiments, the solution comprises about 8%, 10%, 12%, 14%, 16%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, or 80% hexylene glycol. In some embodiments, the solution comprises about 50% hexylene glycol.

[0032] In some embodiments, the present technology provides for the preparation of a medium and / or device using a solution of about 0.05M sodium hydroxide and 35-55% hexylene glycol, 0.08M sodium hydroxide and 35-55% hexylene glycol, 0.1M sodium hydroxide and 35-55% hexylene glycol, 0.15M sodium hydroxide and 35-55% hexylene glycol, 0.2M sodium hydroxide and 35-55% hexylene glycol, 0.25M sodium hydroxide and 35-55% hexylene glycol, The present invention relates to a method for cleaning a chromatography medium and / or support device by contacting the medium and / or support device with a cleaning solution comprising 0.3 M sodium hydroxide and 35-55% hexylene glycol, 0.35 M sodium hydroxide and 35-55% hexylene glycol, 0.4 M sodium hydroxide and 35-55% hexylene glycol, 0.45 M sodium hydroxide and 35-55% hexylene glycol, or 0.5 M sodium hydroxide and 35-55% hexylene glycol. In some embodiments, the solution comprises about 0.1 M sodium hydroxide and 35-55% hexylene glycol.

[0033] In some embodiments, the technology relates to methods of cleaning chromatography media and / or support devices by contacting the media and / or device with a cleaning solution comprising about 0.05M sodium hydroxide and 50% hexylene glycol, 0.08M sodium hydroxide and 50% hexylene glycol, 0.1M sodium hydroxide and 50% hexylene glycol, 0.15M sodium hydroxide and 50% hexylene glycol, 0.2M sodium hydroxide and 50% hexylene glycol, 0.25M sodium hydroxide and 50% hexylene glycol, 0.3M sodium hydroxide and 50% hexylene glycol, 0.35M sodium hydroxide and 50% hexylene glycol, 0.4M sodium hydroxide and 50% hexylene glycol, 0.45M sodium hydroxide and 50% hexylene glycol, or 0.5M sodium hydroxide and 50% hexylene glycol. In some embodiments, the cleaning solution comprises about 0.1 M sodium hydroxide and 50% hexylene glycol.

[0034] In some embodiments, the present technology relates to methods of washing chromatography media and / or support devices by contacting the media and / or device with a wash solution having a pH of about 12. In some embodiments, the wash solution has a pH of about 10, about 10.5, about 11, about 11.5, about 11.7, about 11.9, about 12.1, about 12.3, about 12.5, or about 13. In some embodiments, the wash solution has a pH of about 12.0.

[0035] In some embodiments, the chromatography medium and / or support device is treated with a cleaning solution of the present disclosure at a temperature of about 0°C to about 40°C. In some embodiments, the chromatography medium and / or support device is treated with a cleaning solution at a temperature of about 0°C to about 25°C. In some embodiments, the chromatography medium and / or support device is treated with a cleaning solution at a temperature of about 5°C to about 25°C. In some embodiments, the chromatography medium and / or support device is treated with a cleaning solution at a temperature of about 10°C to about 25°C. In some embodiments, the temperature is about 5°C, about 10°C, about 15°C, about 20°C, about 22°C, about 25°C, about 30°C, about 35°C, or about 40°C. In some embodiments, the temperature is about 20°C. In some embodiments, the temperature is about 22°C.

[0036] In some embodiments, the present disclosure relates to a method of cleaning a chromatography medium and / or support device by contacting the medium and / or support device with a cleaning solution comprising about 0.1 M sodium hydroxide and about 50% hexylene glycol, at a pH of about 12, at a temperature of about 20-22°C.

[0037] By way of example and not limitation, in some embodiments, chromatography media refers to any material packed into a column. In some embodiments, the material is a resin or particles. In some embodiments, the resin is a polymeric support or base matrix. In some embodiments, the polymeric support or base matrix can include, but is not limited to, agarose, cellulose, sepharose, polymethacrylate, or polyvinyl ether. In some embodiments, the polymeric support or base matrix is ​​bound to a ligand. In some embodiments, the ligand is an affinity ligand or a hydrophobic ligand. In some embodiments, the hydrophobic ligand includes an alkyl group (e.g., a straight-chain alkyl group such as butyl, octyl, etc.) or an aryl group (e.g., phenyl).

[0038] In some embodiments, the chromatographic media is designed for use in immobilized metal affinity chromatography (IMAC), ion exchange chromatography (IEX), such as cation exchange chromatography (CEX) or anion exchange chromatography (AEX), gel filtration chromatography (also known as size exclusion chromatography (SEC)), hydrophobic interaction chromatography (HIC), supercritical fluid chromatography (SFC), high performance liquid chromatography (HPLC), ultra high performance liquid chromatography (UHPLC), high turbulence liquid chromatography (HTLC), normal phase chromatography (NPC), reversed phase chromatography (RPC), capillary liquid chromatography, electrochromatography, membrane chromatography, monolith chromatography, and nano- or capillary liquid chromatography.

[0039] By way of example and not limitation, in some embodiments, the support device is one or more selected from a chromatography column, a pump, an injector, interconnecting tubing, a detector, a sample collector, a mixer, a flow restrictor, an in-line filter, a valve, a bubble trap, and any other liquid contacting surface.

[0040] In some embodiments, the present technology does not impair the functionality of the chromatography media / resin or shorten the media / resin's lifetime performance. Additionally or alternatively, in some embodiments, the disclosed cleaning solutions are low in toxicity, non-flammable, and / or do not cause protein aggregation. In some embodiments, the disclosed cleaning solutions have high wetting properties that allow for efficient distribution throughout the chromatography media itself, i.e., the chromatography beads and bead pores.

[0041] IV. Sample preparation In some embodiments, the present technology can be applied to purify and / or detect one or more analytes of interest from any source sample, such as a biological or environmental sample. In some embodiments, the biological sample can be derived from humans, animals, plants, microorganisms, or any living organelle, such as cell and tissue cultures, tissue biopsies, whole blood, dried blood spots, plasma, deproteinized plasma, serum, deproteinized serum, ascites, semen, sputum, urine, stool, sweat, saliva, bile, tears, cerebrospinal fluid, swabs of body parts, skin, and hair. In some embodiments, the environmental sample can be an air sample, soil sample, water sample, food sample, or any material sample. In some embodiments, the source sample is obtained from a cell / tissue culture. In some embodiments, the source sample is obtained from a cell / tissue culture supernatant. In some embodiments, the source sample is obtained from a cell lysate.

[0042] In some embodiments, the analyte of interest can be a small molecule, such as a drug substance, or a macromolecule, such as a polypeptide, peptide, nucleic acid, lipid or fatty acid, carbohydrate, lipoprotein, lipopolysaccharide (e.g., endotoxin), hormone, vitamin, steroid, or metabolite. In some embodiments, the analyte of interest is a polypeptide. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the polypeptide is an enzyme or a recombinant enzyme. In some embodiments, the recombinant enzyme is a human recombinant enzyme. In some embodiments, the polypeptide is a non-enzymatic protein, such as a structural protein (e.g., collagen), a transport protein (e.g., hemoglobin), a regulatory protein (e.g., peptide hormone), a motor protein (e.g., myosin), or an immune protein (e.g., antibody). In some embodiments, the polypeptide is an antibody, such as a monoclonal antibody (mAb), polyclonal antibody (pAb), bispecific antibody (BsAb), trispecific antibody (TsAb), antigen-binding fragment thereof, or antibody fusion protein. In some embodiments, the antibody is a recombinant monoclonal antibody. As used herein, the term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to the same antigen as the whole antibody from which that fragment is derived. Examples of "antigen-binding fragments" include, but are not limited to, Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, dAb fragments, isolated complementarity-determining regions (CDRs), scFvs, and diabodies.

[0043] In some embodiments, the source sample (e.g., clarified cell culture fluid from a bioreactor harvest) is loaded directly onto a chromatography column without further conditioning (e.g., depth filtration, pH adjustment, etc.), thereby providing continuous manufacturing capacity for the purification process.

[0044] In some embodiments, the majority of contaminants and interfering materials are removed before applying chromatographic methods to the original sample. In some embodiments, the analytes of interest are concentrated and isolated by filtration, precipitation, centrifugation, extraction, dilution, or a combination thereof. In some embodiments, the analytes of interest are concentrated from the original sample by solid phase extraction (SPE). SPE concentrates the analytes of interest by using a sample preparation cartridge. The SPE extract containing the analytes can be dried and reconstituted in a solvent system compatible with the chromatographic system.

[0045] In some embodiments, analytes of interest are extracted from the original sample by liquid-liquid extraction (LLE). LLE is used to separate analytes based on their relative solubility in two immiscible or partially miscible liquids, usually a polar solvent such as water and a non-polar organic solvent. The target analytes are first partitioned by the solvent, and then extracted, concentrated, and diluted.

[0046] In some embodiments, the analytes of interest are extracted from the original sample by solid-supported liquid-liquid extraction (SLE). In SLE, an aqueous solution of the original sample is loaded onto a support composed of diatomaceous earth. After sample absorption onto the support, it is washed several times with an organic extraction solvent, such as methyl tert-butyl ether. After the analytes of interest partition into the organic phase, it is concentrated by drying and then reconstituted with a solvent compatible with the chromatography system.

[0047] In some embodiments, where the analytes of interest are proteins, they are concentrated from the original sample by protein precipitation extraction (PPE). Protein precipitation methods can include desalting, isoelectric precipitation, and organic solvent extraction. In one example, the original sample is prepared for loading onto a chromatography system by desalting. This protein precipitation technique relies on proteins "salting out" from solution in response to increasing concentrations of neutral salts, such as ammonium sulfate. In some embodiments, the original sample is prepared by isoelectric precipitation, which can be used to precipitate contaminating proteins rather than the target protein. The isoelectric point (pI) is the pH at which a protein's net primary charge is zero. For most proteins, the pI is in the pH range of 4 to 6. In some embodiments, inorganic acids, such as hydrochloric acid and sulfuric acid, are used as precipitants. A potential drawback of isoelectric precipitation is irreversible denaturation caused by inorganic acids.

[0048] V. Chromatography In some embodiments, once the original sample has been processed, for example by centrifugation and / or filtration, the clarified, unconditioned sample is loaded onto a chromatography system, for example a liquid chromatography system.

[0049] Liquid chromatography (LC) is a process that selectively retains one or more components of a fluid solution (mobile phase) as it passes through a column of finely divided material (stationary phase) by pumping, pressure, or gravity to achieve diffusion into and through the pores of the chromatographic medium. Retention of selected components in the fluid solution by the stationary phase results from the component's greater affinity for the stationary phase than for the mobile phase. In some embodiments, the liquid chromatography used is hydrophobic interaction chromatography (HIC), affinity chromatography (AC), ion exchange chromatography (IEX), size exclusion chromatography (SEC), supercritical fluid chromatography (SFC), high performance liquid chromatography (HPLC), ultra-high performance liquid chromatography (UHPLC), high turbulence liquid chromatography (HTLC), normal phase chromatography (NPC), reversed phase chromatography (RPC), capillary liquid chromatography, electrochromatography, membrane chromatography, monolith chromatography, nano- or capillary liquid chromatography. In some embodiments, the liquid chromatography system used in this technique is hydrophobic interaction chromatography (HIC).

[0050] In some embodiments, the analytes of interest are retained by the stationary phase and then eluted. In some embodiments, the analytes of interest flow through the stationary phase without being retained. In some embodiments, the analytes in the eluate or effluent are monitored based on retention time, peak intensity, peak area, by various means including UV, fluorescence, refractive index, light scattering, and conductivity. In some embodiments, further detailed analysis of the analytes is performed using techniques such as mass spectrometry.

[0051] In some embodiments, LC solvents include, but are not limited to, water, methanol, ethanol, acetonitrile, trifluoroacetic acid, heptafluorobutyric acid, ether, hexane, hexylene glycol, propylene glycol, ethylene glycol, ethyl acetate, and organic solvents such as hydrocarbon solvents (e.g., aliphatic and aromatic solvents), oxygenated solvents (e.g., alcohols, glycols, ketones, aldehydes, glycol ethers, esters, and glycol ether esters), and halogenated solvents (e.g., chlorinated and brominated hydrocarbons). In some embodiments, LC solvents are buffered and can include various salts and buffering agents routinely used in the art, such as sodium hydroxide, sodium acetate, sodium phosphate, ammonium acetate, ammonium formate, ammonium bicarbonate, acetic acid, trifluoroacetic acid, formic acid, trimethylamine, triethylamine, and the like. In some embodiments, LC solvents also include detergents such as Tween, SDS, and the like.

[0052] VI. Hydrophobic Interaction Chromatography (HIC) In some embodiments, the liquid chromatography used is hydrophobic interaction chromatography (HIC). HIC separates analytes according to differences in their surface hydrophobicity. HIC utilizes the reversible interaction between hydrophobic analytes and hydrophobic ligands immobilized on the HIC matrix. The interaction between the hydrophobic analyte and the HIC resin is significantly affected by the salt concentration of the chromatography buffer. High salt concentrations enhance the interaction between the analyte and the HIC resin, while lower salt concentrations weaken the interaction. The main advantage of HIC is the preservation of the biological activity of the target analytes through the use of conditions and matrices that operate under less denaturing conditions. HIC is primarily used for protein purification. However, HIC can also be applied to the separation of nucleic acids, viruses, cells, and carbohydrates. Retained analytes are eluted in order of increasing hydrophobicity.

[0053] The adsorption behavior of analytes on HIC resins is determined by the type of immobilized ligand. Generally, linear alkyl ligands exhibit hydrophobic properties, while aryl ligands exhibit mixed-mode behavior, allowing for both aromatic and hydrophobic interactions. The binding capacity of HIC resins has been observed to increase with increasing degree of substitution of the immobilized ligand. The most widely used HIC matrices are hydrophilic carbohydrates, such as cross-linked agarose and synthetic copolymer materials. The most commonly used hydrophobic ligands immobilized on HIC matrices include linear alkyl ligands (e.g., C4-C10 alkyl ligands such as butyl and octyl) and aryl ligands (e.g., phenyl).

[0054] In some embodiments, the wash solutions and methods of use disclosed herein include washing the resin of an HIC column. Examples of HIC resins include, but are not limited to, phenyl-, butyl-, octyl-Sepharose, Butyl-Sepharose® 4 Fast Flow, Phenyl Sepharose™ High Performance, Phenyl Sepharose™ 6 Fast Flow (low substitution), and Phenyl Sepharose™ 6 Fast Flow (high substitution).

[0055] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure. In the case of conflict, the present specification, including definitions, will control. Generally, the terms used in connection with and techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications as commonly accomplished in the art or as described herein. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and the embodiments, the words "have" and "comprise" or variations thereof, such as "has," "having," "comprises," or "including," are understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. Although several documents are cited herein, this citation does not constitute an admission that any of these documents form part of the common general knowledge in the art. [Example]

[0056] In order that the present technology may be better understood, the following examples are set forth, which are for illustrative purposes only and should not be construed as limiting the scope of the technology in any way.

[0057] Example 1 Optimization of cleaning solution This example describes a study to screen wash solutions for purifying human recombinant enzymes captured by HIC.

[0058] method A HiScreen™ Capto™ Butyl column used to capture recombinant enzymes from clarified, unconditioned harvests was observed to discolor even after washing with a buffer containing 0.1 M sodium hydroxide, 50% ethylene glycol, and 10% ethanol. After storing the column in a storage buffer containing 0.1 M sodium hydroxide for 3 weeks, a storage buffer rinse showed an A280 absorbance of over 3000 mAU. The presence of this peak after storage indicated that the column was not completely cleaned, suggesting the poor cleaning ability of the 0.1 M sodium hydroxide, 50% ethylene glycol, and 10% ethanol solution. Figures 1A and 1B show the discoloration of the HIC column before washing (pre-CIP) and its regeneration to a typical white resin after treatment with an optimized cleaning solution (post-CIP).

[0059] Several alternative CIP / wash solutions were screened using a HiScreen™ Capto™ column. Wash solutions were selected based on the A280 absorbance of the peaks generated during each wash phase. To avoid unexpected interactions between different wash solutions and isolate any UV peaks to a single wash solution, an equilibration buffer containing 40 mM sodium phosphate, 150 mM NaCl, pH 6.7 was used before and after each wash solution. Table 1 summarizes the CIP screen run on the Capto™ column.

[0060] [Table 1]

[0061] result The chromatograms used to determine the relative effectiveness of the screened wash solutions are shown in Figure 2. The CIP2 solution, containing 1.0 M sodium hydroxide, had a slight absorbance peak but was much more effective after a 1-hour retention rinse with reverse osmosis deionized (RODI) water. The CIP3 solution, containing 0.1 M sodium hydroxide, 50% ethylene glycol, and 10% ethanol, removed a small peak, but most of the impurities were still retained on the column, as indicated by the size of the A280 peak after treatment with the CIP4 solution. The peak after washing with the CIP4 solution, containing 50% hexylene glycol and 0.5 M sodium hydroxide, was also close to 3000 mAU; after this period, the resin appeared visually clean, as shown in Figure 1B.

[0062] The standard wash solution for the HIC column, containing 0.1 M sodium hydroxide, 50% ethylene glycol, and 10% ethanol (CIP3), produced a slight absorbance peak in the CIP screen. The CIP4 wash solution, containing 50% hexylene glycol and 0.5 M sodium hydroxide, significantly cleared the column, resulting in a visibly cleaner resin (Figure 1B). The wash solution containing 0.1 M sodium hydroxide and 50% hexylene glycol solution was chosen for further analysis because 0.1 M sodium hydroxide appeared to decrease the conductivity of the wash solution, thus improving the hydrophobicity of the solution.

[0063] Example 2 Capto Butyl Cycling Test method After optimizing the column wash protocol, a 40 cycle study was performed to determine the effect of run number on the quality and activity yield of the target protein.

[0064] result A 40-cycle run was completed using daily collected harvests. All samples were initially tested for activity and high molecular weight (HMW) species, but samples were only tested intermittently after run 10. Table 2 summarizes the average activity and HMW results for samples tested in the run. The eluate averaged 1.2 CV volume with a standard deviation of 0.2 CV, indicating that a narrow elution peak can be expected over 40 cycles at large scale.

[0065] [Table 2]

[0066] The average specific activity of the eluate was 33.2 U / mg, a 22% increase in purity compared to the Capto™ MMC mean value of 27 U / mg. The specific activity of the eluate pool remained stable over the course of the study. The final run tested was 33.2 U / mg. Figure 3 shows the specific activity of the target recombinant enzyme over the course of the study. Overall, the specific activity was robust with a standard deviation of only 1.4 U / mg.

[0067] HMW species averaged 5.2% with a high standard deviation of 3.1%. However, HMW did not trend with cycle number; instead, the highest HMW samples were observed in the middle of the reactor run.

[0068] Activity yields varied significantly over the course of the study. The average specific activity yield was 90%, but the standard deviation for the samples tested was 10.3%. Figure 4 shows activity yields over 40 cycles. Variability in activity assays is expected to be ±5%, which may have contributed to the high standard deviation observed over the course of the study. Additionally, multiple runs on the same harvest pool were performed in one day, and activity loss during loading may have contributed to the yield decline. No significant decline in yield was observed, as later run cycles still resulted in activity yields of +90%.

[0069] Blank runs were performed for the ATTO-Tag test. The blank after 40 cycles was assumed to be a worst-case sample and most likely to contain protein carryover. None of the runs tested contained significant protein carryover (data not shown). Samples were above the detection limit of the assay but below accurate quantitation. None of the previous blank runs resulted in no protein carryover.

[0070] A wash solution of 50% hexylene glycol and 0.1 M sodium hydroxide not only provided excellent cleaning but also had no adverse effect on resin performance over 40 cycles. The average specific activity of the samples was 33.1 U / mg, and the activity yield was 90.1%. Furthermore, ATTO-tag testing showed that there was no significant protein carryover for later cycle runs.

Claims

1. A cleaning solution consisting essentially of a strong base and hexylene glycol.

2. 10. The cleaning solution of claim 1, wherein the strong base is an alkali metal hydroxide.

3. 3. The cleaning solution of claim 2, wherein the strong base is sodium hydroxide.

4. 4. The cleaning solution of claim 3, wherein the concentration of sodium hydroxide is from about 0.1M to about 0.5M.

5. The cleaning solution of any one of claims 1 to 4, wherein the concentration of hexylene glycol is from about 35% to about 55%.

6. 10. The cleaning solution of claim 1 consisting essentially of about 0.1 M sodium hydroxide and about 50% hexylene glycol.

7. A cleaning solution according to any one of claims 1 to 6, wherein the pH of the solution is about 12.

8. A cleaning solution according to any one of claims 1 to 7 for use in a method for functionally cleaning a chromatography medium and / or a support device.

9. 1. A method for functionally cleaning a chromatographic medium and / or support device, the method comprising contacting the chromatographic medium and / or support device with a cleaning solution comprising a strong base and hexylene glycol.

10. 10. The method of claim 9, wherein the strong base is an alkali metal hydroxide.

11. 11. The method of claim 10, wherein the strong base is sodium hydroxide.

12. 12. The method of claim 11, wherein the concentration of sodium hydroxide is from about 0.1 M to about 0.5 M.

13. 13. The method according to any one of claims 9 to 12, wherein the concentration of hexylene glycol is about 50%.

14. 10. The method of claim 9, wherein the solution comprises about 0.1 M sodium hydroxide and about 50% hexylene glycol.

15. The method of any one of claims 9 to 14, wherein the pH of the solution is about 12.

16. The method of any one of claims 9 to 15, wherein the chromatographic medium is a hydrophobic interaction chromatography (HIC) resin.

17. 17. The method of claim 16, wherein the HIC resin is selected from Capto MMC, Capto Butyl, Capto Phenyl, and Toyopearl Hexyl-650C.

18. 18. The method of claim 17, wherein the HIC resin is Capto Butyl.

19. A purification process comprising a washing step according to any one of claims 9 to 18.

20. 20. The purification process of claim 19, wherein the washing step comprises a step within an integrated, continuous biomanufacturing process for the purification of a polypeptide.

21. 21. The purification process of claim 19 or 20, wherein the polypeptide is an antibody or a recombinant enzyme.

22. 22. The purification process of claim 21, wherein the recombinant enzyme is a human recombinant enzyme.

23. 23. The purification process of claim 22, wherein the human recombinant enzyme is β-glucocerebrosidase (β-D-glucosyl-N-acylsphingosine glucohydrolase).

24. 22. The purification process of claim 21, wherein the recombinant enzyme is Cerezyme®.

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