WO2024258734A2 - Procédé à ph élevé pour la purification de protéines hydrophobes - Google Patents

Procédé à ph élevé pour la purification de protéines hydrophobes Download PDF

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Publication number
WO2024258734A2
WO2024258734A2 PCT/US2024/032894 US2024032894W WO2024258734A2 WO 2024258734 A2 WO2024258734 A2 WO 2024258734A2 US 2024032894 W US2024032894 W US 2024032894W WO 2024258734 A2 WO2024258734 A2 WO 2024258734A2
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Prior art keywords
target protein
solution
combination
solids
absent
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WO2024258734A3 (fr
Inventor
Benjamin D. Allen
Huihun JUNG
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Tandem Repeat Technologies Inc
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Tandem Repeat Technologies Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types

Definitions

  • Protein materials are ubiquitous in nature, playing critical protective and structural roles in forms as familiar as our own skin, hair, and fingernails, as well as providing the basis for some of our oldest technologies: fibers and textiles based on animal-derived materials like silk and wool.
  • the development of modern biotechnology offers new possibilities for protein materials, including genetic engineering of a wide array of material properties, intrinsic biocompatibility and biodegradability, and sustainable, animal-free production in recombinant microbes.
  • the most mature recombinant technology for protein-material production has been achieved for sequences based on various types of silk.
  • the disclosure in one aspect, relates to a process for purification of hydrophobic proteins and/or proteins that have a tendency to aggregate when produced at high levels in cell culture.
  • the process involves subjecting intact cells to a strong base in a solution with a pH of greater than 12.5, with an optional initial pretreatment at a pH of greater than 13.
  • the process operates efficiently with a high biomass loading of up to or greater than about 20% (w/v) and does not require the use of external flocculants.
  • the target protein can purified by the disclosed process inexpensively and efficiently, producing a high-quality end product having few to no contaminants.
  • FIG. 1 is a flow chart showing a non-limiting, exemplary process for protein purification according to the present disclosure.
  • FIG. 2 is a flow chart showing an alternative non-limiting, exemplary process for protein purification according to the present disclosure.
  • Disclosed herein is a method for purifying proteins having high isoelectric points, proteins that are insoluble at low pH values, and/or proteins that are soluble under typical cellular conditions but that tend to aggregate under recombinant protein production conditions, such as, for example, inclusion bodies in proteins produced by E. coli.
  • a method for separating a target protein from an intact cell including at least the steps of:
  • the solution including the intact cell can be contacted with one or more additives prior to step (a) or step (b).
  • the first solution can be contacted with one or more additives prior to step (d).
  • the one or more additives can be sodium chloride, an organic solvent, calcium chloride, or any combination thereof.
  • the sodium chloride can have a concentration of from about 0.37 M to about 1 .23 M in the solution including the intact cell or in the first solution, or of about 0.37, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 1.1 , 1.2, or about 1.23 M, or a combination of any of the foregoing values, or a range encompassing any of the foregoing values.
  • the calcium chloride can have a concentration of from about 0.37 M to about 0.40 M in the solution including the intact cell or in the first solution, or about 0.37, 0.38, 0.39, or about 0.40 M, or a combination of any of the foregoing values, or a range encompassing any of the foregoing values.
  • the organic solvent can be ethanol or acetone and can be present at from about 8% (v/v) to about 50% (v/v) in the solution including the intact cell or in the first solution, or about 8, 10, 15, 20, 25, 30, 35, 40, 45, or about 50% (v/v), or a combination of any of the foregoing values, or a range encompassing any of the foregoing values.
  • a solution containing the intact cell can be pretreated at a pH greater than or equal to 13 for a period of time prior to performing step (b).
  • the pH higher than 13 can be at least 13.4.
  • the period of time can be from about 5 min to about 1 hour, or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or about 60 min, or a combination of any of the foregoing values, or a range encompassing any of the foregoing values.
  • performing this step results in a more efficient downstream process for purification of target proteins in terms of process speed, time and materials required, and other factors.
  • the cell is pretreated at a pH of greater than or equal to 13 in step (a), lysed in step (b), or both using a strong base such as, for example, NaOH, KOH, Ba(OH) 2 , Ca(OH) 2 , LiOH, RbOH, CsOH, Sr(OH) 2 , or any combination thereof.
  • a strong base such as, for example, NaOH, KOH, Ba(OH) 2 , Ca(OH) 2 , LiOH, RbOH, CsOH, Sr(OH) 2 , or any combination thereof.
  • the strong base is NaOH.
  • exposure to a strong base can function both to rupture cells and to dissolve the target proteins.
  • non-target molecules present can solidify under the high pH conditions of the disclosed method and can thus easily be separated from the dissolved target proteins by centrifugation and/or filtration.
  • effective separation of cell debris after NaOH treatment is important for product quality.
  • non-target molecules can be precipitated using a first precipitation agent, a first acid, or a combination thereof.
  • step (b) can be 12.5, 12.6, 12.7, 12.8, or greater.
  • step (d) can be accomplished by centrifugation, filtration, or any combination thereof.
  • centrifugation may be sufficient for removing cell debris, while in larger scale purifications, filtration may be more effective.
  • solubility of the target protein and non-target molecules can be decreased using a first precipitation agent, a first acid, or any combination thereof.
  • the first precipitation agent can be or include F, Cl’, Br, I’, CO 3 2 ’, HCO 3 ’, SO 4 2 ’, HSO 4 ’, PO 4 3 ’, HPO 4 2 ’, H 2 PO 4 ’, formate, acetate, Li + , Na + , K + , Zn 2+ , Al 3+ , Fe 3+ , Mg 2+ , Ca 2+ , NH 4 + , trehalose, glucose, proline, terf-butanol, trimethylamine N-oxide, ectoine, glycine betaine, 3-dimethylsulfoniopropionate, or any combination thereof.
  • the first acid can be sulfuric acid, phosphoric acid, carbonic acid, hydrochloric acid, or any combination thereof.
  • the pH of the first solution can be decreased to from about 10.9 to about 1 1.6, or to about 10.9, 1 1.0, 1 1.1 , 11.2, 1 1.3, 1 1.4, 11.5, or about 1 1.6, or a combination of any of the foregoing values, or a range encompassing any of the foregoing values.
  • the pH of the first solution in step (e), can be decreased to from about 10.2 to about 10.8, or to about 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, or about 10.8, or a combination of any of the foregoing values, or a range encompassing any of the foregoing values.
  • decreasing the pH of the first solution can be accomplished using ammonium sulfate, wherein the second precipitation agent and second acid are present as one ionic compound that dissociates in water.
  • various precipitation-agent/acid mixtures including the components described herein for use in optional step (c) of the disclosed process, are contemplated for use in the disclosed process as the second precipitation agent and/or second acid instead of or in addition to the ammonium sulfate.
  • the target protein solids can be resuspended in water in step (g).
  • the pH of the second solution can be decreased to about 8 in step (h).
  • the pH of the second solution in step (h) can be decreased using an aqueous solution of an acid such as, for example, 1 M HCI, although other aqueous acids at other concentrations are also contemplated and should be considered disclosed.
  • the first solution includes from about 5% to about 20%, or about 5, 10, or 20% (w/v) solids in an aqueous solution, wherein the solids include cell debris as well as the target protein.
  • a flocculant is not used during performance of the method.
  • previous protein purification research has proceeded with dilute solutions, sometimes as low as 1 % or less, of biomass (i.e., cell solids and the like), which required the use of flocculants to help aggregate the cell debris and separate it from solution, while with the disclosed process, it has been fortuitously discovered that when the biomass is first concentrated, higher purity and yield can be achieved in a lower working volume without the need for flocculants.
  • flocculants such as, for example, cationic polyethylenimines, chitosan, cationic poly(diallyldimethyl-ammonium chloride), cationic polyacrylamides, cationic polyamines, or any combination thereof can be used.
  • the present disclosure provides effective processes for protein purification without the need for these or other flocculants.
  • the pH value in step (b) is greater than the isoelectric point of the target protein.
  • the target protein solids separated in step (i) can be further washed with DI water, dried by a method such as lyophilization or spray-drying, or any combination thereof.
  • FIG. 1 One exemplary protein purification process is shown in FIG. 1. Biomass is washed in DI water 100 and resuspended in DI water 102. The resuspended biomass is then brought to pH 12.8 using an NaOH solution 104, under which conditions the desired protein is soluble but cell debris remains solid. The cell debris is removed 106 and the supernatant containing the protein of interest is brought to pH 10.2 using ammonium sulfate solution 108. Desired protein solids are separated from the remaining solution 110 and the solids are resuspended in DI water 112. The solids are then brought to pH 8 114 and separated from the remaining solution again 116. The solids are washed with DI water 118 and lyophilized to dry 120. The depicted process is nonlimiting; other steps and combinations of steps as described herein should also be considered disclosed.
  • a second exemplary protein purification process is shown in FIG. 2.
  • this process may be especially useful for large-scale protein purifications.
  • Harvested biomass is diluted in DI water 202.
  • the biomass is directly harvested by disc-stack centrifugation, which results in a slurry of 10-15% biomass solids in residual broth, and the dilution brings the concentration down to about 5% before proceeding.
  • the diluted biomass is then brought to pH 12.8 using an NaOH solution 204, under which conditions the desired protein is soluble but cell debris remains solid.
  • the cell debris is removed 206 and the supernatant containing the protein of interest is brought to pH 10.2 using ammonium sulfate solution 208.
  • Desired protein solids are separated from the remaining solution 210 and the solids are resuspended in DI water 212. The solids are then brought to pH 8 214 and separated from the remaining solution again 216. The solids are washed with DI water 218 and lyophilized to dry 220.
  • the biomass can be contacted with one or more additives as described herein (203, 205) either before raising the pH to 12.8 using an NaOH solution 204 or before removal of cell debris 206.
  • the depicted process is non-limiting; other steps and combinations of steps as described herein should also be considered disclosed.
  • the disclosed method offers numerous benefits and improvements over known protein purification processes.
  • the disclosed method takes place in aqueous solution and does not require any extraction with volatile, hazardous, or expensive organic solvents, thus it is more environmentally benign and offers a cost savings over many known processes.
  • volumes of water required at various stages are lower.
  • a lower volume of water directly results in a lower materials cost since smaller amounts of reagents are needed for pH changes and the like.
  • large-scale, larger-volume processes can still be carried out, but will result in much higher amounts of target protein compared to equivalent volumes of previously known processes.
  • the water used in various steps of the process can be recycled, optionally after one or more purification steps.
  • the disclosed process can offer higher recovery of the target protein in a shorter amount of time and at lower cost compared to known processes.
  • a typical protein purification process can be surprisingly difficult due to particle size and solution conditions, requiring the use of a flocculant to aggregate cell debris and/or other particles.
  • the disclosed process does not require the use of any external flocculants.
  • conducting the process in the absence of external flocculants represents yet another reduction in cost and increase in efficiency when the disclosed process is compared to known processes.
  • both target protein yield and quality are improved over those seen with known processes, when using the disclosed process.
  • Process Scale-Up it may be desirable to purify target proteins on an industrial scale.
  • large-scale production of proteins can require modification of certain process steps in order to achieve the desired purity and yield levels.
  • large-scale production of target proteins can benefit from milder conditions.
  • scaled-up separation of proteins can be challenging with 20% biomass solids and may improve with a lower solids loading such as, for example, less than 10%, about 6% or less, about 5% or less, or the like.
  • scaled-up separation can be accomplished using lower centrifugation speeds and shorter centrifugation times.
  • bench-scale separations may take up to 1 hour of centrifugation time, the same centrifugation can be accomplished in less than 10 minutes, or in about 7 minutes, on a larger scale.
  • bench-scale separations may require centrifugation speeds up to 17,000 ref, large scale separations can be accomplished at less than 10,000 ref, or at about 6,000 ref.
  • optional additives such as sodium chloride, calcium chloride, and/or organic solvents as discussed above can be incorporated in order to enhance the separation process and increase product yields.
  • proteins purified by the disclosed method can be particularly useful for purification of certain target proteins.
  • any protein that is found in a solid phase or as a solid aggregate after production can be subjected to the disclosed process including, but not limited to, inclusion bodies.
  • hydrophobic proteins that are insoluble or form in aggregates in water or aqueous solutions, or inside intact cells in which they are expressed can be purified by the disclosed process.
  • target proteins produced by the disclosed method.
  • the target protein produces a transparent, uniform, and freely flowing solution in DMSO.
  • the target protein includes at least 5% tyrosine residues.
  • the target protein can have a sequence of Formula I:
  • Ai is absent, is a methionine, or is an amino acid sequence 1 to 4 residues in length;
  • Ei a GLY-rich amino acid sequence 8 to 58 residues in length including amino acids selected from the group consisting of glycine, leucine, tyrosine, phenylalanine, and proline, or any combination thereof;
  • Bi is an ASTVH-rich sequence amino acid sequence 6 to 17 residues in length including amino acids selected from the group consisting of alanine, serine, threonine, valine, histidine, glycine, glutamine, and proline, or any combination thereof.
  • Li is absent or is an amino acid sequence 1 to 7 residues in length including amino acids selected from the group consisting of proline, glycine, leucine, serine, and threonine, or any combination thereof;
  • E 2 is absent or is E
  • E3 is absent or is E1;
  • Pi is absent or is proline
  • P 2 is absent or is P
  • P 3 is absent or is P l_3 is absent or is Li
  • G1 is absent or is an amino acid sequence 1 to 4 residues in length; and wherein n is 4 to 100.
  • ASTVH-rich sequence refers to a sequence that can comprise additional sequences and in a different order than a peptide of ASTVH.
  • the ASTVH-rich sequence includes at least one alanine, at least one serine, at least one threonine, at least one valine, and at least one histidine.
  • the ASTVH-rich sequence includes two or more alanines.
  • the ASTVH-rich sequence includes two or more serines.
  • the ASTVH-rich sequence includes two or more threonines.
  • the ASTVH-rich sequence includes two or more valines.
  • the ASTVH-rich sequence includes two or more histidines.
  • Bi can be selected from one of SEQ ID NOs. 1-103.
  • Li is absent or is an amino acid sequence 1 to 7 residues in length including amino acids selected from the group consisting of glycine, leucine, serine, and threonine, or any combination thereof, or Li can be or can be PST, PS, P, ST, or S or can have one of SEQ ID NOs. 236-242.
  • the GLY-rich sequence can include at least one glycine, at least one leucine, and at least one tyrosine.
  • Ei is an GLY-rich amino acid sequence 8 to 58 residues in length comprising amino acids selected from the group consisting of glycine, leucine, tyrosine, phenylalanine, and proline, or any combination thereof.
  • Ei is a third amino sequence comprising a combination of two or more of glycine, leucine, tyrosine, phenylalanine, and proline, or can be selected from one of SEQ ID NOs. 119- 235.
  • Gi is absent or is an amino acid sequence 1 to 4 residues in length. In some embodiments, Gi is an amino acid sequence including serine and/or threonine. In some embodiments, Gi is absent.
  • n is in a range between 4-100, 4-90, 4-80, 4-70, 4-60, 4-50, 4-40, 4-30, 4-20, 4-10, 6-20, 8-20, 10-20, 10-30, 4-16, 6-16, 8-16, 10-16, 12-16, 4-12, 6-12, 8-12, or 10-12. In one aspect, n is 4. In another aspect, n is 12.
  • the target protein can be an aggregation-prone hydrophobic protein, a protein occurring in an inclusion body, or a natural or recombinant squid ring tooth protein.
  • the target protein can have a sequence selected from SEQ ID NOs. 104-118, 243, and 244.
  • the target protein can be or include any one of the recombinant squid ring tooth proteins identified by SEQ ID NOs. 104, 111 , 112, 113, 117, or 118.
  • exemplary aggregation-prone hydrophobic proteins and/or proteins occurring in inclusion bodies can be selected from VEGF165 (examples include, but are not limited to, those disclosed in US Patent No. 9,994,612); IGF-1 (examples include, but are not limited to, those disclosed in US Patent No. 5,288,931); neublastin (examples include, but are not limited to, those disclosed in US Patent No. 8,969,042); lgG2 antibody fragment (examples include, but are not limited to, those disclosed in European Patent No. 1805320); transforming growth factor type p (TGF-P)-like (examples include, but are not limited to, those disclosed in US Patent No.
  • TGF-P transforming growth factor type p
  • IL-29 examples include, but are not limited to, those disclosed in US Patent No. 8,211 ,670
  • interleukin-2 examples include, but are not limited to, those disclosed in US Patent No. 11 ,091 ,525
  • GDNF examples include, but are not limited to, those disclosed in US Patent No. 7,226,758
  • memapsin examples include, but are not limited to, those disclosed in US Patent No. 7,829,669
  • proNGF examples include, but are not limited to, those disclosed in US Pre-Grant Publication No.
  • pro-EP-B2 examples include, but are not limited to, those disclosed in US Patent No. 8,148,105
  • IFN-alpha examples include, but are not limited to, those disclosed in Japanese Patent No. 5861223
  • T-cell receptor examples include, but are not limited to, those disclosed in Japanese Patent No. 6186412
  • antibody Fc region examples include, but are not limited to, those disclosed in US Patent No. 11 ,345,722
  • tenth fibronectin type III (10Fn3) domain examples include, but are not limited to, those disclosed in US Patent No. 8,067,201
  • human growth hormone examples include, but are not limited to, those disclosed in US Patent No. 8,178,494).
  • a strong base As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
  • reference to “a strong base,” “a target protein,” or “a precipitation agent,” include, but are not limited to, mixtures or combinations of two or more such strong bases, target proteins, or precipitation agents, and the like.
  • ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.
  • a further aspect includes from the one particular value and/or to the other particular value.
  • ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’.
  • the range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’.
  • the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’.
  • the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.
  • a numerical range of “about 0.1 % to 5%” should be interpreted to include not only the explicitly recited values of about 0.1 % to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.
  • the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined.
  • an “effective amount” refers to an amount that is sufficient to achieve the desired modification of a physical property of the composition or material.
  • an “effective amount” of a strong base refers to an amount that is sufficient to achieve the desired improvement in the method step modulated by the strong base, e.g. achieving the desired level of cell lysis accompanied by increased solubility for the target protein.
  • the specific level in terms of wt% in a composition required as an effective amount will depend upon a variety of factors including the amount and type of protein component, amount and type of cells being lysed, total biomass in solution, and scale of the purification reaction.
  • temperatures referred to herein are based on atmospheric pressure (i.e. one atmosphere).
  • Solids loading is defined as the ratio of biomass solids on a dry basis to aqueous alkaline solution. Solids loading percentages (w/v) of 5%, 10%, and 20% were tested. pH 13.3 Step
  • Mass yield is defined as the dry-basis yield of product based on the input dry biomass.
  • NaCI applied at final concentrations in the range of 0.6-1.3 M prior to alkaline extraction.
  • Solvents including acetone and ethanol, at concentrations in the range of 8-50% v/v just prior to cell-debris separation

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Abstract

Selon un aspect, la divulgation concerne un processus de purification de protéines et/ou de protéines hydrophobes qui ont tendance à s'agréger lorsqu'elles sont produites à des niveaux élevés dans la culture cellulaire. Dans un autre aspect, le processus consiste à soumettre des cellules intactes à une base forte dans une solution présentant un pH supérieur à 12,5, avec un prétraitement initial facultatif à un pH supérieur à 13. Le processus fonctionne efficacement avec un chargement de biomasse élevé allant jusqu'à environ 20 % (p/v) ou supérieur à environ 20 % et ne nécessite pas l'utilisation de floculants externes. La protéine cible peut être purifiée par le processus divulgué de manière peu coûteuse et efficace, ce qui permet de produire un produit final de haute qualité présentant peu ou pas de contaminants. Le présent abrégé est destiné à être utilisé comme outil d'exploration à des fins de recherche dans ce domaine technique particulier et ne se limite pas à la présente divulgation.
PCT/US2024/032894 2023-06-14 2024-06-07 Procédé à ph élevé pour la purification de protéines hydrophobes Pending WO2024258734A2 (fr)

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