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WO2009080781A1 - Procédé de clarification d'homogénats cellulaires - Google Patents

Procédé de clarification d'homogénats cellulaires Download PDF

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Publication number
WO2009080781A1
WO2009080781A1 PCT/EP2008/068087 EP2008068087W WO2009080781A1 WO 2009080781 A1 WO2009080781 A1 WO 2009080781A1 EP 2008068087 W EP2008068087 W EP 2008068087W WO 2009080781 A1 WO2009080781 A1 WO 2009080781A1
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WIPO (PCT)
Prior art keywords
homogenate
protein
bacteriophage
cell
acid
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PCT/EP2008/068087
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English (en)
Inventor
Christoph Peter
Karl Proba
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Cytos Biotechnology AG
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Cytos Biotechnology AG
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Priority to EP08863835A priority Critical patent/EP2235165A1/fr
Publication of WO2009080781A1 publication Critical patent/WO2009080781A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/18011—Details ssRNA Bacteriophages positive-sense
    • C12N2795/18111—Leviviridae
    • C12N2795/18123—Virus like particles [VLP]

Definitions

  • the invention relates to the filed of protein purification from cell homogenates.
  • a process for clarifying cell homogenates which allows the production of a cleared homogenate that is sufficiently clear to be filtrated through a sterile filter.
  • the process of the invention is useful for the large scale production of proteins or protein complexes, preferably of therapeutic proteins or protein complexes, where sterile filtration of the cleared homogenate is required.
  • the purification of proteins or protein complexes, and in particular of recombinant proteins or protein complexes, from a host cell typically comprises the steps of (a) homogenizing said cells in the medium or in a homogenization buffer, in order to release the target protein from the cells, and (b) clarifying the homogenate in order to remove cell debris.
  • the purpose of the clarification step is to obtain a homogenate which is sufficiently clear, i.e. free of cell debris, protein aggregates, etc., to allow subsequent processing.
  • the subsequent processing typically comprises filtration steps and chromatography steps.
  • sterile filtration of the cleared homogenate is a mandatory step which is required to remove infectious cells, pyrogenic cell components and the like.
  • Sufficient clarification of the homogenate is a prerequisite for efficient sterile filtration in order to avoid clogging of the filter membrane and / or in order to avoid the undesired increase of pressure during filtration.
  • WO2006/136566A1 discloses a clarification processes for homogenates of bacteria cells, wherein sufficient clearing of the homogenate is achieved by the combination of (a) centrifugation at high acceleration, typically at least 10 OOO x g, to efficiently remove cell debris; and / or (b) pre-filtration using a filter with a pore size which is slightly larger than the pore size of a sterile filter, i.e. typically about 0.45 ⁇ m, to remove aggregates of protein and small particles of cell debris, which would result in clogging of the sterile filter during the subsequent sterile filtration.
  • the cleared homogenate of WO2006/136566A1 is suitable for sterile filtration using a conventional filter membrane comprising a pore size of 0.22 ⁇ m. An improvement of the filterability of the cleared homogenate is however still desired.
  • the clarification process disclosed in WO2006/136566A1 requires centrifugation at high acceleration. The process is therefore limited to centrifugation in batch mode because continuous flow centrifugation typically does not achieve the required acceleration.
  • the process of WO2006/136566A1 is also limited with respect to the throughput of homogenate, because of the technical limitations for the bucket size in centrifugation and, thus, because of the limitation of the volume of homogenate that can be clarified per batch.
  • the clarification process disclosed in WO2006/136566A1 requires an additional filtration step which is generally undesired in a production process.
  • a process for the clarification of a cell homogenate which requires only centrifugation at low acceleration, preferably at an acceleration that can be achieved by continuous flow centrifugation, and which ideally does not require a filtration step prior to sterile filtration, is highly desired.
  • the invention provides a process for clarifying a cell homogenate, wherein said process is particularly useful in the context of the purification of a protein or protein complex from a cell homogenate.
  • the process of the invention provides cleared cell homogenates which are characterized by a high filterability, wherein preferably said filterability is at least 50 1 / m 2 of a conventional sterile filter membrane, wherein the pressure of said cleared homogenate before said filter membrane does not exceed 1 bar during the filtration process.
  • the process of the invention does not require a pre-filtration step in order to achieve said filterability of the cleared homogenate.
  • the main aspect of the invention is a process for clarifying a cell homogenate prior to the purification of a protein or of a protein complex from said homogenate, said process comprising the steps of: (a) providing a cell homogenate comprising said protein or protein complex; (b) adjusting the pH of said homogenate to an acidic pH, preferably to pH of 2.0 to 6.8, more preferably to pH 2.8 to 4.5, still more preferably to pH 3.5 to 4.5, wherein preferably the electrical conductivity of said homogenate during said incubating is 10 to 100 mS; (d) separating the cleared homogenate from the precipitated cell debris, wherein preferably said separating comprises the step of centrifuging said homogenate, preferably at an acceleration of less than 4'00O x g.
  • the invention provides a process for clarifying a cell homogenate prior to the purification of a protein complex from said homogenate, wherein said protein complex is a virus-like particle, preferably a virus-like particle of an RNA phage, more preferably a virus-like particle of RNA bacteriophage Q ⁇ or of RNA bacteriophage AP205, most preferably of RNA bacteriophage Q ⁇ , said process comprising the steps of: (a) providing a cell homogenate comprising (i) said protein complex; (ii) 50 to 150 mM, preferably about 70 mM of a phosphate buffer; and (iii) a pH of about 7.0; (b) adjusting the pH of said homogenate to pH 3.5 to 4.5; wherein said adjusting said pH is performed by the addition of citric acid to said homogenate to a final concentration of citric acid in said homogenate of 50 to 150 mM; (c) incubating said homogen
  • the invention provides a process for clarifying a cell homogenate prior to the purification of a protein from said homogenate, wherein said protein is a cytokine, preferably interleukin-1 ⁇ or a mutein thereof, most preferably IL- ⁇ D145K (SEQ ID NO:22), said process comprising the steps of: (a) providing a cell homogenate comprising (i) said protein; (ii) 50 to 150 mM, preferably about 70 mM of a phosphate buffer; and (iii) a pH of about 7.0; (b) adjusting the pH of said homogenate to pH 3.5 to 4.5; wherein said adjusting said pH is performed by the addition of citric acid to said homogenate to a final concentration of citric acid in said homogenate of 50 to 150 mM; (c) incubating said homogenate at said pH, preferably to allow precipitation of cell debris, wherein during said incubating said homogenate further comprises an in
  • electrical conductivity The salt composition of the homogenate can be characterized by the electrical conductivity of said homogenate.
  • the electrical conductivity of a solution is given in mS units. It is to be understood that the absolute value of the electrical conductivity strongly depends on the temperature of the solution.
  • electrical conductivity of a solution, preferably of a cell homogenate, as used herein is the electrical conductivity of said solution, preferably of said cell homogenate, that is measured at the actual temperature of said solution, preferably of said cell homogenate, i.e. the values for the electrical conductivity provided herein are not normalized to a standard temperature.
  • the electrical conductivity is measured at the temperature under which the incubation step of the process of the invention is performed, i.e. typically and preferably at 14 to 30 0 C, more preferably at 18 to 26 0 C, still more preferably at 20 to 24 0 C, and most preferably at 22 0 C.
  • turbidity The turbidity of a suspension or of a colloidal solution, preferably of a cell homogenate and most preferably of a clarified homogenate obtained by the process of the invention is typically and preferably measured in nephelometric turbidity units (NTU).
  • NTU nephelometric turbidity units
  • the turbidity of a cell homogenate or a cleared homogenate is preferably determined using a Turbidimeter, more preferably a 2100 AN Turbidimeter (Hach), wherein further preferably said turbidity is determined using Fiolax glass test tubes (10 mm, Hach).
  • protein complex refers to any complex comprising more than one protein chain, wherein said protein chains may comprise identical or different amino acid sequences.
  • protein complex includes complexes wherein the protein chains are bound to each other by non-covalent interactions or by covalent bonds, preferably by disulfide bonds.
  • Preferred embodiments of protein complexes are enzyme complexes, antibodies, preferably IgGs, and virus-like particles.
  • said protein complex is a virus-like particle, preferably a virus-like particle of an RNA bacteriophage.
  • coat protein refers to the protein(s) of a RNA bacteriophage capable of being incorporated within the capsid assembly of the bacteriophage or the RNA bacteriophage.
  • coat protein refers to the protein forming the capsid of a RNA bacteriophage or a VLP of a RNA bacteriophage.
  • coat protein of RNA bacteriophages has a dimeric structure.
  • fragment of a (recombinant) coat protein in particular fragment of a recombinant coat protein, as used herein, is defined as a polypeptide, which is of at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% the length of the wild-type coat protein, or wild type recombinant protein, respectively and which preferably retains the capability of forming VLP.
  • the fragment is obtained by at least one internal deletion, at least one truncation or at least one combination thereof.
  • fragment of a recombinant coat protein or “fragment of a coat protein” shall further encompass polypeptide, which has at least 80 %, preferably 90 %, even more preferably 95 % amino acid sequence identity with the wildtype coat protein, respectively, and which is preferably capable of assembling into a virus-like particle.
  • mutant coat protein refers to a polypeptide having an amino acid sequence derived from the wild type recombinant protein, or coat protein, respectively, wherein the amino acid sequence is at least 80 %, preferably at least 85 %, 90 %, 95 %, 97 %, or 99 % identical to the wild type sequence and preferably retains the ability to assemble into a VLP.
  • virus-like particle refers to a non-replicative or noninfectious, preferably a non-replicative and non-infectious virus particle, or refers to a non- replicative or non-infectious, preferably a non-replicative and non-infectious structure resembling a virus particle, preferably a capsid of a virus.
  • non-replicative refers to being incapable of replicating the genome comprised by the VLP.
  • non- infectious refers to being incapable of entering the host cell.
  • a virus-like particle in accordance with the invention is non-replicative and/or non-infectious since it lacks all or part of the viral genome or genome function.
  • a virus- like particle is a virus particle, in which the viral genome has been physically or chemically inactivated, removed by disassembly and reassembly, or by assembly of purified proteins into a VLP.
  • a virus-like particle lacks all or part of the replicative and infectious components of the viral genome.
  • a virus-like particle in accordance with the invention may contain nucleic acid distinct from their genome.
  • a typical and preferred embodiment of a virus-like particle in accordance with the present invention is a viral capsid such as the viral capsid of the corresponding virus, bacteriophage, preferably RNA bacteriophage.
  • the term "capsid”, refers to a macromolecular assembly composed of viral protein subunits. Typically, there are 60, 120, 180, 240, 300, 360 and more than 360 viral protein subunits. Typically and preferably, the interactions of these subunits lead to the formation of viral capsid with an inherent repetitive organization, wherein said structure typically and preferably is spherical.
  • the capsids of RNA bacteriophages have a spherical form of icosahedral symmetry.
  • virus-like particle of an RNA bacteriophage refers to a virus-like particle comprising, or preferably consisting essentially of or consisting of coat proteins, mutants or fragments thereof, of a RNA bacteriophage.
  • virus-like particle of a RNA bacteriophage resembling the structure of a RNA bacteriophage, being non replicative and/or non- infectious, and lacking at least the gene or genes encoding for the replication machinery of the RNA bacteriophage, and typically also lacking the gene or genes encoding the protein or proteins responsible for viral attachment to or entry into the host.
  • VLPs derived from RNA bacteriophages exhibit icosahedral symmetry and consist of 180 subunits.
  • virus-like particle of an RNA bacteriophage preferably relates to a macromolecular structure obtained by the self-assembly of recombinant coat protein of an RNA bacteriophage, or fragments or mutants thereof, wherein preferably said self-assembly took place in the presence of and oligonucleotide.
  • yield the term “yield” with respect to the process of the invention refers to the percentage (w/w) of said protein or protein complex contained in the cleared homogenate obtained by the process of the invention relative to the amount of said protein or protein complex contained in the homogenate provided in step (a) of said process.
  • inorganic salt As used herein, the term “inorganic salt” relates to any inorganic salt of an alkaline metal or earth alkaline metal, preferably to a halogenide of an alkaline metal or earth alkaline metal, more preferably to a chloride of an alkaline metal or earth alkaline metal, most preferably to a chloride of an alkaline metal.
  • said inorganic salt is potassium chloride or sodium chloride, or a mixture of both. Still more preferably, said inorganic salt is sodium chloride in all embodiments of the invention.
  • the invention provides a process for clarifying a cell homogenate prior to the purification of a protein or of a protein complex from said homogenate, said process comprising the steps of: (a) providing a cell homogenate comprising said protein or protein complex; (b) adjusting the pH of said homogenate to an acidic pH, preferably to pH 2.0 to 6.8, more preferably to pH 2.8 to 4.5, still more preferably to pH 3.5 to 4.5; (c) incubating said homogenate at said pH, preferably to allow precipitation of cell debris, wherein further preferably the electrical conductivity of said homogenate during said incubating is 10 to 100 mS; (d) separating the cleared homogenate from the precipitated cell debris.
  • said process comprises the steps of: (a) providing a cell homogenate comprising said protein or protein complex; (b) adjusting the pH of said homogenate to an acidic pH, preferably to pH 2.0 to 6.8, more preferably to pH 2.8 to 4.5, still more preferably to pH 3.5 to 4.5; (c) incubating said homogenate at said pH, preferably to allow precipitation of cell debris, wherein further preferably the electrical conductivity of said homogenate during said incubating is 10 to 100 mS; (d) centrifuging said homogenate; and (e) separating the cleared homogenate from the precipitated cell debris.
  • said process comprises the steps of: (a) providing a cell homogenate comprising said protein or protein complex; (b) adjusting the pH of said homogenate to an acidic pH, preferably to pH 2.0 to 6.8, more preferably to pH 2.8 to 4.5, still more preferably to pH 3.5 to 4.5; (c) incubating said homogenate at said pH, preferably to allow precipitation of cell debris, wherein further preferably the electrical conductivity of said homogenate during said incubating is 10 to 100 mS; (d) separating the cleared homogenate from the precipitated cell debris, wherein preferably said separating comprises the step of centrifuging said homogenate.
  • the acidic pH of said homogenate during said incubating results in the precipitation of host cell derived impurities such as host cell derived RNA, DNA and protein.
  • host cell derived impurities such as host cell derived RNA, DNA and protein.
  • the efficiency of the clarification process is higher at lower pH.
  • low pH also facilitates the precipitation of said protein or protein complex. Too low pH may therefore result in reduced yield.
  • the optimal pH may also depend on the nature of the purifies protein or protein complex.
  • said pH of said homogenate is adjusted to pH 2.0 to 6.8, more preferably to pH 2.5 to 6, still more preferably to pH 2.8 to 4.5, still more preferably to pH 3.0 to 4.5, still more preferably to pH 3.8 to 4.2, and most preferably to pH 4.0.
  • said pH is 3.8 to 4.2, wherein most preferably said pH is 4.0.
  • said process is a process for clarifying a cell homogenate prior to the purification of a protein complex from said homogenate, wherein preferably said protein complex is a VLP, and wherein said process comprises the steps of: (a) providing a cell homogenate comprising said protein complex, preferably said VLP; (b) adjusting the pH of said homogenate to an acidic pH, preferably to pH 2.0 to 6.8, more preferably to pH 2.8 to 4.5, still more preferably to pH 3.5 to 4.5; (c) incubating said homogenate at said pH, preferably to allow precipitation of cell debris, wherein further preferably the electrical conductivity of said homogenate during said incubating is 10 to 100 mS; (d) separating the cleared homogenate from the precipitated cell debris, wherein preferably said separating comprises the step of centrifuging said homogenate.
  • said protein complex is a VLP of an RNA bacteriophage, preferably of RNA bacteriophage Q ⁇ or of RNA bacteriophage AP205, most preferably of RNA bacteriophage Q ⁇ , wherein said pH is 3.8 to 4.2, and wherein preferably said pH is about 4.0, and wherein most preferably said pH is 4.0.
  • said process is a process for clarifying a cell homogenate prior to the purification of a protein from said homogenate, wherein preferably said protein is a cytokine, and wherein said process comprises the steps of: (a) providing a cell homogenate comprising said protein, preferably said cytokine; (b) adjusting the pH of said homogenate to an acidic pH, preferably to pH 2.0 to 6.8, more preferably to pH 2.8 to 4.5, still more preferably to pH 3.5 to 4.5; (c) incubating said homogenate at said pH, preferably to allow precipitation of cell debris, wherein further preferably the electrical conductivity of said homogenate during said incubating is 10 to 100 mS; (d) separating the cleared homogenate from the precipitated cell debris, wherein preferably said separating comprises the step of centrifuging said homogenate.
  • said protein is a cytokine, wherein preferably said cytokine is inter leukine-1 (IL-I), and wherein said pH is 2.0 to 4.5.
  • said protein is interleukine-1 alpha (IL-I ⁇ ), and said pH is 2.0 to 3.5, more preferably 2.6 to 3.0.
  • said protein is IL-I ⁇ or a mutein thereof, and said pH is 3.5 to 4.5, preferably 3.7 to 4.1, most preferably 4.0.
  • said pH is preferably stabilized by a buffer.
  • Suitable buffers for the pH range of 3.5 to 4.5 are generally known in the art. Any buffer which is generally used in the filed of protein purification and which is capable of stabilizing the pH of a solution in said range may be used.
  • a buffer component may already be comprised in the homogenate provided in step (a) of the process and the pH of the buffered homogenate may be adjusted by the addition of acid, preferably of an organic acid comprising a pKa of 3.5 to 4.5.
  • said providing a cell homogenate comprises providing said cell homogenate, wherein said cell homogenate comprises a buffer, wherein preferably the pH of said buffer is 6.5 to 7.5, more preferably about 7.0.
  • the concentration of said buffer in said homogenate is 10 to 300 mM, preferably 20 to 200 mM, more preferably 50 to 150 mM, and most preferably about 100 mM.
  • said buffer is selected from the group consisting of: (a) sodium phosphate buffer; (b) potassium phosphate buffer; (c) a mixture of sodium phosphate buffer and potassium phosphate buffer; (d) Bis(2-hydroxyethyl)amino- tris(hydroxymethyl)methan (Bis-Tris); (e) N-(2-acetamido)iminodiacetic acid (ADA); (f) N- (2-acetamido)-2-aminoethanesulfonic acid (ACES); (g) 1,4 piperazine Bis (2-ethanosulfonic acid) (PIPES); (h) 3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO); (i) 1,3- bis(tris(hydroxymethyl)methylamino)propane (Bis-Tri s prop ane) ; (j ) N,N-Bis(2- hydroxyethyl)-2-aminoethane
  • said providing a cell homogenate comprises providing said cell homogenate, wherein said cell homogenate comprises 50 to 150 mM, preferably about 70 mM of a phosphate buffer, preferably of a mixture of sodium phosphate buffer and potassium phosphate buffer; wherein further preferably the pH of said cell homogenate is 6.5 to 7.5, most preferably about 7.0.
  • said adjusting the pH of said homogenate comprises adding acid to said homogenate, wherein preferably said acid is an organic acid, and wherein further preferably said organic acid comprises a pKa of 3.0 to 4.5.
  • said organic acid is added to a final concentration of 10 to 300 mM, preferably 20 to 200 mM, more preferably 50 to 150 mM, and most preferably of about 100 mM.
  • said organic acid is selected from the group consisting of: (a) citric acid; (b) acetic acid; (c) formic acid; (d) malonic acid; (e) succhinic acid; (f) glyoxylic acid; (g) glycolic acid; (h) propanoic acid; (i) lactic acid; (j) glyceric acid; (k) barbituric acid; (1) trans-fumaric acid; (m) acetoacetic acid; (n) methymalonic acid; (o) meso-tartaric acid; (p) N-glycylglycine; (q) glutaric acid; (r) L-glutamic acid; (s) ascorbic acid; and (t) isocitric acid.
  • said adjusting the pH is performed by the addition of citric acid to said homogenate to a final concentration of citric acid in said homogenate of 20 to 200 mM, more preferably 50 to 150 mM, and most preferably of about 80 mM.
  • said providing a cell homogenate comprises providing said cell homogenate, wherein said cell homogenate comprises 50 to 150 mM, preferably about 70 mM of a phosphate buffer, preferably of a mixture of sodium phosphate buffer and potassium phosphate buffer; wherein further preferably the pH of said cell homogenate is 6.5 to 7.5, most preferably about 7.0; and (b) said adjusting the pH is performed by the addition of citric acid to said homogenate to a final concentration of citric acid in said homogenate of 20 to 200 mM, more preferably 50 to 150 mM, and most preferably of about 80 mM.
  • said adjusting the pH may be performed by the addition of a salt, preferably of a sodium or potassium salt, of an organic acid to said homogenate, wherein preferably said organic acid comprises a pKa of 3.5 to 4.5; and by further adding a strong organic acid until the desired pH is reached.
  • a salt preferably of a sodium or potassium salt
  • said adjusting the pH is performed (i) by the addition of salt, preferably of a sodium or a potassium salt, of an organic acid, wherein preferably said organic acid comprises a pKa of 3.0 to 4.5; and wherein further preferably said salt of an organic acid is a salt of an organic acid selected from the group consisting of: (a) citric acid; (b) acetic acid; (c) formic acid; (d) malonic acid; (e) succhinic acid; (f) glyoxylic acid; (g) glycolic acid; (h) propanoic acid; (i) lactic acid; (j) glyceric acid; (k) barbituric acid; (1) trans-fumaric acid; (m) acetoacetic acid; (n) methymalonic acid; (o) meso-tartaric acid; (p) N-glycylglycine; (q) glutaric acid; (r) L-glutamic acid; (
  • said adjusting the pH is performed by the addition of acid or by the addition of an acidic buffer.
  • the addition of said acid or of said acidic buffer is performed by the addition of a concentrated solution of said acid or of said acidic buffer in order to minimize the dilution of said homogenate.
  • Said incubating is preferably performed under conditions under which said protein or protein complex remains in solution. It is apparent for the artisan that the yield of the process can be influenced by choosing a salt composition of said homogenate during said incubating which is optimized in order to achieve high solubility of said protein or protein complex in said homogenate during said incubating step.
  • optimal salt conditions for the purpose of the invention are characterized by an electrical conductivity of said homogenate during said incubating of 10 to 100 mS, preferably 10 to 50 mS, more preferably of 10 to 30 mS, and most preferably of about 20 mS.
  • the salt contributing to the desired electrical conductivity may either be already contained in the culture medium and / or in the homogenization buffer and, thus, may be already contained in the homogenate as provided in step (a) of the process.
  • said process further comprises the step of adding an inorganic salt to said homogenate, wherein preferably said inorganic salt is added to a concentration of 25 to 1000 mM, preferably about 100 mM, in said homogenate.
  • said homogenate further comprises an inorganic salt in a concentration of 25 to 1000 mM, preferably of 25 to 500 mM, more preferably of 50 to 250 mM, still more preferably of 75 to 150 mM, still more preferably of about 100 mM, and most preferably of 100 mM.
  • said inorganic salt is a halogenide of an alkaline metal, preferably a chloride of an alkaline metal, more preferably sodium chloride or potassium chloride or a mixture thereof, most preferably sodium chloride.
  • said homogenate comprises about 100 mM sodium chloride, wherein preferably the electrical conductivity of said homogenate is about 20 mS.
  • Said cells comprising said protein or said protein complex may be but do not need to be harvested from the culture medium, e.g. by centrifugation, and optionally stored at -80 0 C.
  • Homogenates of the cells can be produced by disrupting the cells by physical, chemical or enzymatic means or by a combination thereof.
  • the cells can be disrupted by sonication, by passage through a high pressure liquid homogenizer like APV LAB 1000, by passage through a French press, or by grinding with aluminium oxide.
  • cells can be lysed by detergents such as sodium dodecyl sulphate (SDS) or, preferably, non-ionic detergents like Triton ® X-100, Triton ® X-114, Tween ® 20 or Igepal ® CA 630 or mixtures thereof, most preferably Triton ® X-100.
  • SDS sodium dodecyl sulphate
  • non-ionic detergents like Triton ® X-100, Triton ® X-114, Tween ® 20 or Igepal ® CA 630 or mixtures thereof, most preferably Triton ® X-100.
  • Said detergents are preferably applied in a concentration of 0.01 to 30 %, more preferably 0.01 to 5 %, most preferably about 0.1 %.
  • the cell wall of bacteria can be digested by enzymes such as lysozyme.
  • said disruption of cells can be improved when the cell suspension is passed through the high pressure liquid homogenizer repeatedly.
  • the usage of a high pressure liquid homogenizer significantly improves the scalability of the process as it can be operated in a continuous mode.
  • said providing a cell homogenate comprises the homogenization of cells, preferably of bacteria cells, by suspending said cells in a homogenization buffer and passing the suspension at least once, preferably at least twice, more preferably at least three times, most preferably three times through a high pressure liquid homogenizer, e.g. APV LAB 1000, at a pressure of a bout 300 to 1200 bar, preferably 500 to 900 bar, more preferably 600 to 800 bar and most preferably about 700 bar.
  • a high pressure liquid homogenizer e.g. APV LAB 1000
  • said providing a cell homogenate comprises homogenizing said cells until at least 50 %, preferably at least 75 %, more preferably at least 90 %, still more preferably at least 95 %, most preferably at least 99 % of said cells are disrupted by physical and/or enzymatic means.
  • said homogenization buffer for suspending said cells comprises an alkaline pH of about 8, an agent, such as EDTA, capable of forming complexes with metal ions, preferably at a concentration of about 1-50 mM, and a detergent, preferably SDS, Tween-20 or Triton X-100, most preferably Triton X-100, wherein the concentration of the detergent is about 0.01 to 1.0 %, more preferably about 0.05 to 0.5 %, most preferably about 0.1 %.
  • said buffer comprises a pH of 8.0, 0.5 mM EDTA and 0.1 % Triton X-100.
  • said buffer additionally comprises lysozyme.
  • said providing said cell homogenate comprises homogenizing said cells in the culture medium, wherein preferably a buffer, most preferably a phosphate buffer is added to said medium.
  • said incubating is performed at a temperature of 14 to 30 0 C, preferably at 18 to 26 0 C, more preferably at 20 to 24 0 C, and most preferably at 22 0 C.
  • said incubating is performed for 30 min to 30 h, preferably for 1 h to 24 h, most preferably for 1 h to 2 h.
  • said incubating comprises agitating said homogenate during said incubating.
  • said centrifuging is performed at an acceleration of 10 OOO or less, preferably less than 5'00O x g, more preferably less than 4'00O x g, still more preferably of less than 3'500 x g, and most preferably of less than 3'00O x g.
  • said centrifuging is performed at an acceleration of 10'0OO or less, preferably less than 5'00O x g, more preferably less than 4'00O x g, still more preferably of less than 3'500 x g, and most preferably of less than 3'00O x g, wherein said acceleration is at least 500
  • centrifuging is performed in a continuous mode. In a further preferred embodiment said centrifuging and said separating are performed in a continuous mode.
  • said cell homogenate is a homogenate of cells selected from (a) bacteria cells; and (b) eukaryotic cells.
  • said cell homogenate is a homogenate of eukaryotic cells, wherein preferably said eukaryotic cells are selected from (a) fungus cells, preferably yeast cells, wherein further preferably said yeast cells are cells of Saccharomyces, preferably of Sa. cerevisiae or of Schizosaccharomyces, preferably of Sc. pombe ; (b) insect cells, and (c) mammalian cells.
  • said cell homogenate is a homogenate of prokaryotic cells, wherein preferably said eukaryotic cells are bacteria cells, more preferably gram negative bacteria cells, and most preferably enterobacteria cells.
  • said cell homogenate is a homogenate of
  • Escherichia coli cells wherein preferably said E. coli cells are cells of E. coli BL21 or, more preferably, cells of E. coli K12.
  • said homogenate is a homogenate of cells of an E. coli strain selected from the group consisting of: (a) E. coli RB791; (b) E. coli JMlOl;
  • said homogenate is a homogenate of E. coli RB791 cells.
  • said providing a cell homogenate comprises providing said homogenate, wherein the concentration of said protein or protein complex in said homogenate is at least 1 g / 1, and wherein preferably the concentration of said protein or protein complex in said homogenate is 1 to 20 g / 1, preferably 8 to 15 g / 1, and most preferably about 12 g / 1.
  • said protein or protein complex is a recombinant protein or a recombinant protein complex.
  • Said protein or protein complex may be a protein or protein complex which is naturally produced by said cell, or a protein or protein complex which is expressed from a recombinant gene, wherein said recombinant gene may be comprised in the genome of said cell, on a plasmid contained in said cell, or on DNA or RNA of a virus infecting said cell.
  • said protein or protein complex is expressed in said cell, wherein preferably said protein or protein complex is expressed from a DNA selected from (a) plasmid DNA; (b) genomic DNA; (c) viral DNA; and (d) viral RNA; wherein preferably said protein or protein complex is expressed from plasmid DNA.
  • said protein or protein complex comprises a solubility in said homogenate, preferably in said homogenate during said incubation step, of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 g / 1.
  • said solubility is 8 to 20 g / 1, more preferably 10 to 15 g / 1 and most preferably about 12 g / 1.
  • said process optionally comprises the step of oxidizing said protein or protein complex, wherein said oxidizing is preferably performed prior to said adjusting said pH, and wherein further preferably said oxidizing is performed by exposing said protein or protein complex to oxygen, e.g. by agitating said homogenate in an open vessel.
  • said oxidizing may be performed by the addition of an oxidizing agent such as, for example, hydrogen peroxide, ascorbate or glutathione.
  • the oxidation step may improve the stability of proteins or protein complexes which are stabilized by disulfide bridges, such as, for example, virus-like particles of RNA bacteriophage Q ⁇ and virus-like particles of RNA bacteriophage AP205.
  • said protein or protein complex is an enzyme, an antibody, preferably a monoclonal antibody, a hormone or a cytokine.
  • said process is a process for clarifying a cell homogenate prior to the purification of a protein from said homogenate, wherein said protein is a cytokine, wherein preferably said cytokine is selected from the group consisting of (a) IL-I ⁇ or a mutein thereof, and (b) IL-I ⁇ or a mutein thereof.
  • IL-I ⁇ and IL-I ⁇ proteins and muteins thereof are disclosed, for example in WO2008/037504A1.
  • said protein is IL-I (IL-I), more preferably IL-I ⁇ , or a mutant or fragment thereof.
  • said protein is a mutein of IL-I ⁇ , wherein most preferably said protein is the protein of SEQ ID NO:22 (IL-1 ⁇ D145K).
  • said protein or protein complex is a virus-like particle.
  • said protein or protein complex comprises or consists of a coat protein of a virus-like particle, preferably of a virus-like particle of an RNA bacteriophage, and most preferably of RNA bacteriophage Q ⁇ .
  • said protein or protein complex is a virus-like particle, wherein preferably said virus-like particle is a virus-like particle of an RNA virus, preferably of an RNA bacteriophage, most preferably of an RNA bacteriophage selected from the group consisting of: (a) bacteriophage Q ⁇ ; (b) bacteriophage Rl 7; (c) bacteriophage fr; (d) bacteriophage GA; (d) bacteriophage SP; (e) bacteriophage MS2; (f) bacteriophage Mi l; (g) bacteriophage MXl; (h) bacteriophage NL95; (i) bacteriophage f2; (j) bacteriophage PP7; and (k) bacteriophage AP205.
  • said protein or protein complex is a virus-like particle an RNA bacteriophage, wherein said RNA bacteriophage is bacter
  • said protein or protein complex comprises or consists of a protein, preferably of a coat protein, of a virus, preferably of an RNA virus, preferably of an RNA bacteriophage.
  • said protein or protein complex comprises or consists of a coat protein of an RNA bacteriophage, of a fragment or mutant thereof, wherein said RNA bacteriophage is selected from the group consisting of: (a) bacteriophage Q ⁇ ; (b) bacteriophage Rl 7; (c) bacteriophage fr; (d) bacteriophage GA; (d) bacteriophage SP; (e) bacteriophage MS2; (f) bacteriophage Mi l; (g) bacteriophage MXl; (h) bacteriophage NL95; (i) bacteriophage f2; (j) bacteriophage PP7; and (k) bacteriophage AP205.
  • said protein or protein complex comprises or consists of a coat protein or of a fragment or mutant thereof, wherein said coat protein is selected from the group consisting of: (a) SEQ ID NO:1 (Q ⁇ CP); (b) a mixture of SEQ ID NO:1 and SEQ ID NO:2 (Q ⁇ Al protein); (c) SEQ ID NO:3 (R17 coat protein); (d) SEQ ID NO:4 (fr coat protein); (e) SEQ ID NO:5 (GA coat protein); (f) SEQ ID NO:6 (SP coat protein); (g) a mixture of SEQ ID NO:6 and SEQ ID NO:7; (h) SEQ ID NO:8 (MS2 coat protein); (i) SEQ ID NO:9 (Mi l coat protein); (j) SEQ ID NO: 10 (MXl coat protein); (k) SEQ ID NO: 11 (NL95 coat protein); (1) SEQ ID NO: 12 (f2 coat protein); (m) SEQ ID NO: 13 (PP7
  • said protein or protein complex comprises or preferably consists of a coat protein selected from any one of SEQ ID NO: 1 to SEQ ID NO:21.
  • said coat protein comprises or preferably consists of an amino acid sequence selected from the group consisting of: (a) SEQ ID NO:1; and (b) a mixture of SEQ ID NO: 1 and SEQ ID NO:2.
  • said process is a process for clarifying a cell homogenate prior to the purification of a protein complex from said homogenate, wherein preferably said protein complex comprises a coat protein of a virus, preferably of an RNA virus, more preferably of an RNA bacteriophage, most preferably of an RNA bacteriophage selected from the group consisting of: (a) bacteriophage Q ⁇ ; (b) bacteriophage R17; (c) bacteriophage fr; (d) bacteriophage GA; (d) bacteriophage SP; (e) bacteriophage MS2; (f) bacteriophage Ml 1; (g) bacteriophage MXl; (h) bacteriophage NL95; (i) bacteriophage f2; (j) bacteriophage PP7; and (k) bacteriophage AP205.
  • said protein is a coat protein of RNA bacteri
  • the yield of said protein or protein complex contained in the cleared homogenate obtained after said centrifuging is at least 30 % (w/w), preferably at least 50 % (w/w), more preferably at least 75 % (w/w), still more preferably at least 80 % (w/w), still more preferably at least 90 % (w/w), and most preferably at least 95 % (w/w) of the amount of said protein or protein complex contained in said homogenate provided in step (a).
  • Particles which remain in the cleared homogenate obtained by the process of the invention may result in filter clogging during subsequent sterile filtration.
  • the efficiency of the clarification process can therefore be assessed, for example by a measurement of the optical density of the cleared homogenate at 600 nm (OD600). Low OD600 of the homogenate indicates the efficient removal of particles.
  • the particle content of the cleared homogenate is assessed by the determination of the turbidity of the cleared homogenate in nephelometric turbidity units.
  • the cleared homogenate obtained by the process of the invention comprises a turbidity of 10 to 50 NTU, preferably of 10 to 20 NTU.
  • the ultimate goal of the clarification process of the invention is providing a cleared homogenate of high filterability.
  • the cleared homogenate obtained after said centrifuging comprises a filterability of at least 30 1 / m 2 , preferably at least 40 1 / m 2 , more preferably at least 50 1 / m 2 , still more preferably at least 70 1 / m 2 , and most preferably at least 80 1 / m 2 , when a filter membrane with a pore size of 0.18 to 0.25 ⁇ m, preferably 0.20 or 0.22 ⁇ m, most preferably 0.22 ⁇ m is used, and wherein the pressure of said cleared homogenate before said filter membrane does not exceed 1 bar during the filtration process.
  • said filter membrane is composed of a material selected from the group consisting of: (a) polyvinylidenfluoride; (b) polyethersulfone; (c) nylon; (d) cellulose; (e) cellulose esters; (f) nitrocellulose; (g) polytetrafluorethylen; (h) polysulfone; (i) polypropylene; and (j) acrylic copolymers; wherein preferably said filter membrane is composed of polyvinylidenfluoride.
  • the cleared homogenate obtained by the process of the invention may be neutralized by the addition of alkali, preferably by the addition of 0.5 M NaOH.
  • said process is a process for clarifying a cell homogenate prior to the purification of a protein complex from said homogenate, wherein said protein complex is a VLP of RNA bacteriophage Q ⁇ , and wherein preferably said VLP comprises the coat protein of SEQ ID NO:1, and wherein said process comprises the steps of: (a) providing a cell homogenate comprising said protein complex; (b) adjusting the pH of said homogenate to pH 3.5 to 4.5, preferably to pH 3.8 to 4.2, preferably to pH 4.0; (c) incubating said homogenate at said pH to allow precipitation of cell debris, wherein the electrical conductivity of said homogenate during said incubating is 10 to 100 mS; (d) separating the cleared homogenate from the precipitated cell debris, wherein said separating comprises the step of centrifuging said homogenate, preferably at an acceleration of at most
  • said process is a process for clarifying a cell homogenate prior to the purification of a protein from said homogenate, wherein said protein complex is IL-I ⁇ or a mutein thereof, and wherein preferably said protein is IL-I ⁇ D145K (SEQ ID NO:22), and wherein said process comprises the steps of: (a) providing a cell homogenate comprising said protein, preferably IL-I ⁇ D 145K (SEQ ID NO:22); (b) adjusting the pH of said homogenate to pH 3.5 to 4.5, preferably to pH 3.8 to 4.2, preferably to pH 4.0; (c) incubating said homogenate at said pH to allow precipitation of cell debris, wherein the electrical conductivity of said homogenate during said incubating is 10 to 100 mS; (d) separating the cleared homogenate from the precipitated cell debris, wherein said separating comprises the step of centrifuging said homogenate, preferably at an acceleration of at most
  • RB791 cell pellet was dispersed in 100 rnM sodium phosphate pH 7.5 buffer thereby adjusting a concentration of 200 g cell wet weight per litre.
  • the resulting dispersion was processed 3 times repeatedly using a high pressure homogenizer (APV LAB 1000) at a pressure of approx. 700 bar.
  • Acidic treatment and clarification 700 ml of the resulting "oxidized homogenate" was mixed with 28 ml of 5 M NaCl stock solution and 672 ml of water. The diluted homogenate was stirred rigorously and approx. 54 ml of 1 M citric acid stock solution was added resulting in a pH value of approx. 4.0. The mixture was further incubated for 1.5 h at room temperature with gentle stirring. Then, the mixture was centrifuged for 30 min at room temperature (Sorvall RC 5C Plus with SLA-3000 rotor, 3'200 g), resulting in a clear, yellow supernatant and a compact, light grey pellet. Approx. 1 '310 ml of the clear supernatant was mixed with 270 ml of 0.5 M NaOH stock solution resulting in a neutralized solution of approx. pH 7.0, applicable for subsequent purification steps.
  • Homogenization An E. coli RB791 culture expressing Q ⁇ coat protein was grown to a cell density of approx. 200 g cell wet weight per litre. The resulting cell suspension was processed 2 times repeatedly using a high pressure homogenizer (APV LAB 1000) at a pressure of approx. 700 bar. The pH value of the resulting homogenate was adjusted to 7.0 by addition of 0.5 M Na 2 HPO 4 stock solution. Approx. 2'500 ml of the pH-adjusted homogenate was transferred into a 10-L glass bottle and stirred for approx. 44 h at 4 0 C using a magnetic stirrer in order to allow formation of the disulfide bond network in the VLP protein shell.
  • AMV LAB 1000 high pressure homogenizer
  • Homogenization An E. coli RB791 culture expressing Q ⁇ coat protein is grown to a cell density of approx. 200 g cell wet weight per litre in industry standard E. coli fermenters of up to 5'0OO L. The resulting cell suspension is processed once or twice repeatedly using a high pressure homogenizer (any industry standard model) at a pressure of approx. 700 bar. The pressure is adjusted according to the overall homogenization efficiency of the actual equipment to achieve optimal cell disruption with minimal pressure and cycle number. The pH value of the resulting homogenate is adjusted to 7.0 by addition of a Na 2 HPO 4 stock solution. The pH-adjusted homogenate is transferred into a stirred tank with active aeration and stirred until in-process controls shows sufficient formation of the disulfide bond network in the VLP protein shell.
  • Acidic treatment and clarification The "oxidized homogenate" is mixed with a sodium chloride stock solution to achieve a NaCl concentration of 100 mM. Under vigorous stirring a concentrated solution of citric acid is added until a pH of approx. 4.0 is reached. The mixture is further incubated for 1 to 12 h at ambient temperature at reduced stirrer speed. Then, the precipitate is separated from the supernatant by continuous centrifugation, e.g. in a standard disc stack separator, also at ambient temperature. The solids content is approx. 25 % and the flow rate and discharge intervals are adapted accordingly. The cleared phase is yellow and the solids phase is a light grey sludge.
  • BL21DE3 cell pellet was dispersed in 100 rnM sodium phosphate pH 7.5 buffer thereby adjusting a concentration of 200 g cell wet weight per litre. The resulting dispersion was processed 3 times repeatedly using a high pressure homogenizer (APV) at a pressure of approx. 700 bar.
  • AAV high pressure homogenizer
  • Acidic treatment and clarification 60 ml of the resulting homogenate was mixed with 50 ml of water and 6 ml of 5 M NaCl stock solution. The diluted homogenate was stirred rigorously and 1 M citric acid stock solution was added (approx. 4 ml) until pH 4.0 was reached. The mixture was further incubated for 30 min at room temperature with gentle stirring. Then, the mixture was centrifuged for 15 min at room temperature using approx. 3'200 g force, resulting in a clear supernatant and a compact, solid pellet. The clear supernatant was separated from the pellet and neutralized by addition of 0.5 M NaOH stock solution, prior further chromatographic processing.

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Abstract

L'invention concerne le domaine de la purification des protéines à partir d'homogénats cellulaires. L'invention porte sur un procédé de clarification d'homogénats cellulaires qui permet la production d'un homogénat clarifié qui est suffisamment clarifié pour être filtré à travers un filtre stérile. Le procédé de l'invention est utile pour la production à grande échelle de protéines ou de complexes de protéine, de préférence de protéines ou complexes de protéine thérapeutiques, où une filtration stérile de l'homogénat clarifié est requise.
PCT/EP2008/068087 2007-12-21 2008-12-19 Procédé de clarification d'homogénats cellulaires Ceased WO2009080781A1 (fr)

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CN102260651A (zh) * 2011-06-24 2011-11-30 华中科技大学同济医学院附属协和医院 一种纯化病毒样颗粒蛋白的方法及其应用
US10167320B2 (en) 2012-06-22 2019-01-01 Takeda Vaccines, Inc. Purification of virus like particles
US10172930B2 (en) 2007-03-14 2019-01-08 Takeda Vaccines, Inc. Virus like particle purification
CN110041397A (zh) * 2011-03-23 2019-07-23 麦迪卡格公司 回收植物来源的蛋白质的方法

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Cited By (8)

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Publication number Priority date Publication date Assignee Title
US10172930B2 (en) 2007-03-14 2019-01-08 Takeda Vaccines, Inc. Virus like particle purification
US10792353B2 (en) 2007-03-14 2020-10-06 Takeda Vaccines, Inc. Virus like particle purification
CN110041397A (zh) * 2011-03-23 2019-07-23 麦迪卡格公司 回收植物来源的蛋白质的方法
CN102260651A (zh) * 2011-06-24 2011-11-30 华中科技大学同济医学院附属协和医院 一种纯化病毒样颗粒蛋白的方法及其应用
US10167320B2 (en) 2012-06-22 2019-01-01 Takeda Vaccines, Inc. Purification of virus like particles
AU2013277959B2 (en) * 2012-06-22 2019-05-16 Takeda Vaccines, Inc. Purification of virus like particles
US11091519B2 (en) 2012-06-22 2021-08-17 Takeda Vaccines, Inc. Purification of virus like particles
US12286456B2 (en) 2012-06-22 2025-04-29 Takeda Vaccines, Inc. Purification of virus like particles

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