WO2008071774A1 - Procédé de purification pour une protéine d'enveloppe de bactériophages à arn - Google Patents

Procédé de purification pour une protéine d'enveloppe de bactériophages à arn Download PDF

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Publication number
WO2008071774A1
WO2008071774A1 PCT/EP2007/063907 EP2007063907W WO2008071774A1 WO 2008071774 A1 WO2008071774 A1 WO 2008071774A1 EP 2007063907 W EP2007063907 W EP 2007063907W WO 2008071774 A1 WO2008071774 A1 WO 2008071774A1
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Prior art keywords
coat protein
bacteriophage
purified
seq
protein
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PCT/EP2007/063907
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English (en)
Inventor
Constanze GÜNTHER
Karl Proba
Susanne Richter
Simon Topell
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Cytos Biotechnology Ag
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Priority claimed from PCT/EP2006/069734 external-priority patent/WO2007068747A1/fr
Application filed by Cytos Biotechnology Ag filed Critical Cytos Biotechnology Ag
Publication of WO2008071774A1 publication Critical patent/WO2008071774A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C12N2795/18122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the invention relates to the filed of protein purification.
  • a process for purifying coat protein of an RNA bacteriophage comprises the steps of disassembling a virus-like particle of an RNA bacteriophage and subsequently purifying said coat protein.
  • expression of coat protein of an RNA bacteriophage in a bacterial host results in the formation of virus-like particle of said RNA bacteriophage by self assembly of the coat protein, wherein nucleic acids of the host cell, predominantly RNA, are packaged into said virus-like particle.
  • host cell RNA such recombinantly produced virus-like particles typically contain further host cell derived impurities, mainly host cell protein, host cell endotoxins, and host cell DNA.
  • the process described herein allows for the efficient removal of RNA and host cell derived impurities.
  • the process can be scaled up to produce up to 100 g coat protein and more per batch. It allows for the efficient removal of RNA and host cell derived impurities.
  • the invention further relates to purified coat protein of RNA bacteriophages, wherein host cell derived impurities remaining in the preparation are typically below the quantification level.
  • said purified coat protein typically and preferably comprises less than 0.5 ⁇ g RNA, less than 0.4 IU of endotoxin, less than 5 ng host cell protein, and less than 1.5 ng of host cell DNA per 100 ⁇ g of said coat protein.
  • Virus-like particles of RNA bacteriophages are potent stimulators of the immune system, in particular when packaged with immunostimulatory substances, e.g. oligonucleotides (WO2003/024481A2).
  • immunostimulatory substances e.g. oligonucleotides
  • the application of such virus-like particles in vaccination treatments requires a highly purified product which may comprise only minimal traces of host cell derived impurities.
  • Processes for producing compositions comprising a virus-like particle and an immunostimulatory substance, wherein said immunostimulatory substance is packaged into said virus-like particle have been described, for example, in WO2003/024481A2, WO2004/000351A1, WO2004/084940A1 and WO2004/007538A2.
  • WO2005/117963A1 Efficient and scalable processes for the production of recombinant virus-like particles of RNA bacteriophages are disclosed in WO2005/117963A1.
  • the virus-like particles obtained by the processes described therein are packaged with host cell RNA and further contain the above mentioned host cell derived impurities.
  • Processes for the large scale purification of endotoxin free, intact virus-like particles containing host cell RNA are disclosed in WO2007/039552A1.
  • Processes for the preparation of coat protein from recombinantly produced virus like particles (“disassembly") are disclosed, inter alia, in WO2003/024481A2 and WO2004/084940A1.
  • disassembly of the virus-like particle is, for example, achieved by contacting said virus-like particle with high concentrations of guanidinium hydrochloride, urea or magnesium chloride.
  • the invention relates to a process for purifying coat protein of an RNA bacteriophage, wherein said process comprises the steps of disassembling a virus-like particle of an RNA bacteriophage and subsequently purifying the released coat protein. It has surprisingly been found that coat protein of high purity can be obtained when said disassembly is performed at acidic pH in the presence of salt. Disassembly at a pH of 2.3 to 3.5 in the presence of 100 to 1000 mM of an inorganic salt results in the precipitation of host cell derived impurities, in particular of host cell derived RNA and host cell derived protein, while said coat protein remains in the solution.
  • Coat protein comprising less than 0.5 ⁇ g RNA, less than 0.3 IU of endotoxin, less than 5 ng host cell protein, and less than 1.5 ng of host cell DNA per 100 ⁇ g of said coat protein can be purified using the process of the invention, wherein the protein yield of said process typically is at least 25 %.
  • a pH below 2.3 will result in loss of coat protein by degradation and a pH above 3.5 will result in reduced purity.
  • the invention relates to a process for purifying coat protein of an RNA bacteriophage, said process comprising the steps of: (a) providing a virus-like particle of an RNA bacteriophage; (b) disassembling said virus-like particle, wherein said disassembling comprises the steps of (i) generating a disassembly mixture, wherein said disassembly mixture comprises said virus-like particle, 100 to 1000 mM of an inorganic salt; and a pH of 2.3 to 3.5; and (ii) incubating said virus-like particle in said disassembly mixture; and (c) purifying coat protein from said disassembly mixture, wherein preferably said steps are performed in the given order.
  • the disassembly of virus-like particles comprising intermolecular disulfide bonds can be significantly improved by reducing said disulfide bounds with a reducing agent.
  • the invention further relates to a process, wherein said disassembling comprises the steps of: (i) generating a reducing mixture, wherein said reducing mixture comprises said virus-like particle and a reducing agent, (ii) incubating said virus-like particle in said reducing mixture; (iii) generating a disassembly mixture, wherein said disassembly mixture comprises said virus-like particle, 100 to 1000 mM of an inorganic salt; and a pH of 2.3 to 3.5; and (iv) incubating said virus-like particle in said disassembly mixture.
  • a further aspect of the invention is purified coat protein obtainable by any one of the processes of the invention. Any one of the embodiments described herein also refers to this aspect.
  • the invention relates to purified coat protein obtainable by any one of the processes of the invention, wherein said coat protein comprises a purity of at least 98 %.
  • the invention further relates to purified coat protein obtainable by any one of the processes of the invention, wherein said purified coat protein comprises less than 0.5 ⁇ g RNA per 100 ⁇ g of said coat protein.
  • the invention further relates to purified coat protein obtainable by any one of the processes of the invention, wherein said purified coat protein comprises an endotoxin content of less than 0.3 IU per 100 ⁇ g of said coat protein.
  • the invention further relates to purified coat protein obtainable by any one of the processes of the invention, wherein said purified coat protein comprises less than 5 ng host cell protein per 100 ⁇ g of said coat protein.
  • the invention further relates to purified coat protein obtainable by any one of the processes of the invention, wherein said purified coat protein comprises less than 1.5 ng host cell DNA per 100 ⁇ g of said coat protein.
  • the invention further relates to purified coat protein obtainable by any one of the processes of the invention, wherein said purified coat protein is a coat protein selected from the group consisting of: (a) coat protein of RNA bacteriophage Qbeta; (b) coat protein of RNA bacteriophage AP205; (b) coat protein of RNA bacteriophage fr; and (b) coat protein of RNA bacteriophage GA.
  • the invention relates to purified coat protein of an RNA bacteriophage, (a) wherein said purified coat protein comprises a purity of at least 95 %, preferably at least 98 %, and most preferably of at least 99 %; (b) wherein said purified coat - A - protein comprises less than 0.5 ⁇ g RNA per 100 ⁇ g of said coat protein; (c) wherein said purified coat protein comprises an endotoxin content of less than 0.4, preferably less than 0.3, more preferably less than 0.2, still more preferably less than 0.1 IU per 100 ⁇ g of said coat protein; (d) wherein said purified coat protein comprises less than 5 ng host cell protein per 100 ⁇ g of said coat protein; and/or (f) wherein said purified coat protein comprises less than 1.5, preferably less than 1.0, more preferably less than 0.5, and most preferably less than 0.25 ng host cell DNA per 100 ⁇ g of said coat protein.
  • FIG. 1 Analytic size exclusion chromatography of Qbeta VLP (A) and Qbeta coat protein (B).
  • HPLC was performed on BioSil SEC-250 (BioRad, Cat. No. 125-0062) as described in Example 5. Absorption at 260 nm is indicated by dashed lines; absorption at 280 nm is indicated by drawn through lines. The injection volume was 40 ⁇ l for both samples.
  • the protein concentration was 1.6 mg/ml Qbeta VLP (A) and 1 mg/ml Qbeta coat protein (B), respectively.
  • 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 macro molecular 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.
  • tentacle anion exchange matrix refers to an anion exchange matrix implementing the tentacle technology typically and preferably as disclosed in WO96/22316, WO97/49754, EP0337144, DE4334359 or WO95/09695.
  • Anion exchange matrices implementing the tentacle technology are resin particles comprising, preferably on their surface, spacers formed by linear polymer chains (tentacles), wherein said tentacles are substituted with functional groups having anion exchange activity.
  • Preferred tentacle anion exchange matrices are based on resins of copolymers on a methacrylate basis or on resins of vinyl polymers.
  • tentacle ion exchange matrices are Fractogel ® EMD TMAE ion exchangers and Fractoprep ® DEAE ion exchangers (Merck), the most preferred tentacle anion exchange matrix is Fractogel ® ion EMD TMAE.
  • protein yield the terms “protein yield” or “yield of a coat protein” as used herein refer to the percentage of purified coat protein obtained by the process of the invention relative to the amount of coat protein contained in the virus-like particle provided in the first step of the process.
  • concentration of said coat protein and/or said virus-like particle is determined by HPLC, wherein preferably said HPLC is performed with the parameters essentially as, preferably exactly as disclosed in Example 4.
  • purity of a virus-like particle The purity of a virus-like particle is determined as the percentage of the peak area of said virus-like particle relative to the total peak area in a chromatogram obtained by HPLC, wherein preferably said HPLC is performed with the parameters essentially as, preferably exactly as disclosed in Example 3.
  • purity of a coat protein The purity of a coat protein, preferably of a purified coat protein, is determined as the percentage of the peak area of said coat protein or purified coat protein relative to the total peak area in a chromatogram obtained by HPLC, wherein preferably said HPLC is performed with the parameters essentially as, preferably exactly as disclosed in Example 5.
  • concentration of host cell derived impurities The concentration of host cell derived impurities, namely of host cell RNA, host cell DNA, host cell protein, and endotoxins, in purified coat protein is determined essentially as, preferably exactly as disclosed in Examples 6 to 9.
  • 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. Very preferably, in all embodiment of the invention 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 relates to a process for purifying coat protein of an RNA bacteriophage, said process comprising the steps of: (a) providing a virus-like particle of an RNA bacteriophage; (b) disassembling said virus-like particle, wherein said disassembling comprises the steps of (i) generating a disassembly mixture, wherein said disassembly mixture comprises said virus- like particle, 100 to 1000 mM of an inorganic salt, and a pH of 2.3 to 3.5; and (ii) incubating said virus-like particle in said disassembly mixture; and (c) purifying coat protein from said disassembly mixture, wherein preferably said steps are performed in the given order.
  • said process comprising the steps of: (a) providing a virus-like particle of an RNA bacteriophage; (b) disassembling said virus-like particle, wherein said disassembling comprises the steps of (i) generating a disassembly mixture, wherein said disassembly mixture comprises said virus-like particle, 100 to 1000 mM of an inorganic salt, and a pH of 2.3 to 3.5; and (ii) incubating said virus-like particle in said disassembly mixture; and (c) purifying coat protein from said disassembly mixture, wherein preferably the amino acid sequence of the coat protein of said virus-like particle does not comprise cysteine.
  • said coat protein comprises or preferably consists of the amino acid sequence of SEQ ID NO:5.
  • said RNA bacteriophage is bacteriophage GA.
  • the coat proteins of many RNA bacteriophages comprise cysteine residues which are capable of forming intermolecular disulfide bonds. Furthermore, the virus-like particles of certain RNA bacteriophages, in particular of bacteriophage Q ⁇ and bacteriophage AP205, are stabilized by intermolecular disulfide bonds between the protein subunits forming the capsid or virus-like particle. The efficiency of the disassembly process can be significantly enhanced by incubating said virus-like particle with a reducing agent in order to reduce said disulfide bonds. Furthermore, the treatment with a reducing agent can prevent the formation of disulfide bonds in cysteine containing coat protein preparations.
  • said disassembling comprises the steps of: (i) generating a reducing mixture, wherein said reducing mixture comprises said virus-like particle and a reducing agent, (ii) incubating said virus-like particle in said reducing mixture; (iii) generating a disassembly mixture, wherein said disassembly mixture comprises said virus-like particle, 100 to 1000 mM of an inorganic salt; and a pH of 2.3 to 3.5; and (iv) incubating said virus-like particle in said disassembly mixture, wherein preferably the amino acid sequence of the coat protein of said virus-like particle comprises at least one cysteine residue.
  • said coat protein comprises ore preferably consists of 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 (Rl 7 coat protein); (d) SEQ ID NO:4 (fr coat protein); (e) SEQ ID NO:6 (SP coat protein); (f) a mixture of SEQ ID NO:6 and SEQ ID NO:7; (g) SEQ ID NO:8 (MS2 coat protein); (h) SEQ ID NO:9 (Mi l coat protein); (i) SEQ ID NO:10 (MXl coat protein); 0) SEQ ID NO: 11 (NL95 coat protein); (k) SEQ ID NO: 12 (f2 coat protein); (1) SEQ ID NO: 13 (PP7 coat protein); and (m) SEQ ID NO: 19 (AP205 coat protein).
  • said virus like particle comprises intermolecular disulfide bonds.
  • said virus-like particle is a virus-like particle of bacteriophage Q ⁇ or bacteriophage AP205, most preferably of bacteriophage Q ⁇ .
  • said reducing agent is selected from DTT (dithiothreitol), ⁇ -mecaptoethanol, TCEP and other reducing agents generally known in the art.
  • said reducing agent is DTT, wherein preferably the concentration of said DTT in said reducing mixture is 2 to 25 mM, preferably 10 mM.
  • said reducing mixture comprises a pH of 6.5 to 8.0, preferably of 6.8 to 7.5, most preferably of 7.2.
  • said incubating said virus-like particle in said reducing mixture is performed at 4 to 30 0 C, preferably at 10 to 30 0 C, more preferably at 18 to 25 0 C, still more preferably at about 22 0 C and most preferably at 22 0 C. Said incubating is preferably performed until a complete reduction of all disulfide bounds of said virus-like particle is achieved.
  • said incubating said virus-like particle in said reducing mixture is performed for at least 5 min to at most 24 h, preferably for 5 min to 2 h, more preferably for 25 min to 35 min, and most preferably for 30 min.
  • said incubating said virus-like particle in said reducing mixture comprises stirring said reducing mixture, wherein preferably said stirring is performed at 50 to 500 rpm, preferably at 100 to 300 rpm, and most preferably at about 200 rpm.
  • the protein concentration in said reducing mixture is 0.5 to 3.5 mg/ml, preferably 2.0 to 3 mg/ml, more preferably about 2.5 mg/ml, and most preferably 2.5 mg/ml.
  • said an inorganic salt is sodium chloride.
  • said disassembly mixture comprises 200 to 800 mM, more preferably 300 to 700 mM, still more preferably 400 to 700 mM, still more preferably 500 to 700 mM, still more preferably 550 to 650 mM, still more preferably about 600 mM, and most preferably 600 mM of said inorganic salt, wherein preferably said inorganic salt is sodium chloride.
  • said disassembly mixture comprises 600 mM sodium chloride.
  • said disassembly mixture comprises a pH of 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, or 3.5.
  • said disassembly mixture comprises a pH of 2.3 to 3.0, preferably of 2.3 to 2.8, more preferably of 2.5 to 2.7, still more preferably of about 2.6, and most preferably of 2.6.
  • said disassembly mixture further comprises a buffer, wherein said buffer comprises a composition selected from the group consisting of: (a) dihydrogenphosphate / organic acid; (b) Glycine-HCl; (c) HCl-KCl; (d) phosphoric acid / phosphate; (e) formic acid; (f) sodium-acetate / formic acid; (g) pyridine / formic acid; (h) chloracetate; (i) maleate; and (J) malonic acid.
  • a buffer comprises a composition selected from the group consisting of: (a) dihydrogenphosphate / organic acid; (b) Glycine-HCl; (c) HCl-KCl; (d) phosphoric acid / phosphate; (e) formic acid; (f) sodium-acetate / formic acid; (g) pyridine / formic acid; (h) chloracetate; (i) maleate; and (J) malonic acid.
  • said buffer comprises dihydrogenphosphate, preferably sodium or potassium dihydrogenphosphate, most preferably sodium dihydrogenphosphate, and an organic acid, preferably citric acid.
  • said disassembly mixture comprises a pH of about 2.6, and said disassembly mixture further comprises a buffer, wherein said buffer comprises or preferably consists of sodium dihydrogenphosphate and citric acid.
  • the concentration of said dihydrogenphosphate, preferably of said sodium dihydrogenphosphate, in said disassembly mixture is about 85 mM
  • the concentration of said organic acid, preferably of said citric acid, in said disassembly mixture is about 64 mM.
  • the concentration of said sodium dihydrogenphosphate in said disassembly mixture is 85 mM
  • the concentration of said citric acid, in said disassembly mixture is 64 mM.
  • said incubating said virus-like particle in said disassembly mixture is performed at 4 to 30 0 C, preferably at 10 to 30 0 C, more preferably at 18 to 25 0 C, still more preferably at about 22 0 C and most preferably at 22 0 C.
  • Said incubating is preferably performed until a complete disassembly of said virus- like particle is achieved, i.e. no virus-like particle is detected while all protein is in the form of coat protein.
  • the progress of the disassembly reaction can, for example, be monitored by analyzing samples by analytic size exclusion chromatography as described in the Examples section (cf. Examples 3 to 5).
  • said incubating of said virus-like particle in said disassembly mixture is performed for at least 5 min to at most 24 h, preferably for 5 min to 2 h, more preferably for 25 min to 35 min, and most preferably for 30 min.
  • said incubating said virus-like particle in said disassembly mixture comprises stirring said disassembly mixture, wherein preferably said stirring is performed at 200 to 1000 rpm, preferably at 300 to 700 rpm, more preferably at 400 to 600 rpm, and most preferably at 500 rpm.
  • said purifying said coat protein from said disassembly mixture comprises cation exchange chromatography, wherein said cation exchange chromatography comprises the steps of: (i) binding said coat protein to an cation exchange matrix, wherein said binding is performed in the presence of 50 to 500 mM of an inorganic salt, and at a pH of 2.3 to 4; (ii) washing said cation exchange matrix, wherein said washing is performed in the presence of 10 to 500 mM of an inorganic salt, and at a pH of 2.3 to 8.5; and (iii) eluting said coat protein, wherein said eluting is performed in the presence of 350 to 1000 mM of an inorganic salt, and at a pH of 5 to 8.5, wherein preferably said inorganic salt is sodium chloride.
  • said washing comprises a first washing step, wherein said first washing step is performed in the presence of 10 to 500 mM, preferably 300 mM, of an inorganic salt, and at a pH of 2.3 to 4, preferably 3.3; and wherein preferably said washing further comprises a second washing step, wherein said second washing step is performed in the presence of 10 to 500 mM, preferably 300 mM of an inorganic salt, and at a pH of 4 to 8.5, preferably 7.2.
  • said purifying said coat protein from said disassembly mixture comprises cation exchange chromatography, wherein said cation exchange chromatography comprises the steps of: (i) binding said coat protein to an cation exchange matrix, wherein said binding is performed in the presence of 300 mM of an inorganic salt, and at a pH 3.3; (ii) washing said cation exchange matrix, wherein said washing is performed in the presence of 300 mM of an inorganic salt, and at a pH of 3.3 to 7.2; and (iii) eluting said coat protein, wherein said eluting is performed in the presence of 550 mM of an inorganic salt, and at a pH of 7.2, wherein preferably said inorganic salt is sodium chloride.
  • any cation exchange matrix known in the art may be used for the purpose of the invention.
  • said cation exchange matrix is selected from the group consisting of: (a) cross-linked agarose; (b) cross-linked copolymer of allyl dextran and N 5 N- methylene bisacrylamide; (c) cross-linked polystyrene divinylbenzene; (d) methacrylate; and (e) silica, wherein preferably said cation exchange matrix comprises functional groups selected from the group consisting of (a) sulfopropyl; (b) sulfoethyl; (c) methyl sulfonate; and (d) sulfoisobutyl.
  • said cation exchange matrix is cross-linked agarose, wherein said cross-linked agarose comprises sulfopropyl groups.
  • said cation exchange matrix is sepharose, preferably SP Sepharose FF (GE Healthcare, Cat No. 17-0729-01).
  • said purifying said coat protein from said disassembly mixture comprises filtrating said disassembly mixture through a membrane, wherein said filtrating is performed prior to said cation exchange chromatography, and wherein said membrane comprises a molecular weight cut off of 100 to 500 kD, preferably of 300 kD.
  • Such membranes are capable of retaining high molecular RNA and allow the permeation of said coat protein.
  • said filtrating is performed by tangential flow filtration, wherein preferably said tangential flow filtration is performed against a buffer comprising 10 to 500, preferably 300 mM of an inorganic salt, preferably sodium chloride, and a pH of 2.3 to 4, preferably 3.3.
  • said membrane comprises or preferably consists of a material selected from the group consisting of (a) polyethersulfone; (b) hydrophilic polyethersulfone; (c) cellulose; and (d) polysulfone; wherein preferably said material is polyethersulfone.
  • RNA RNA
  • said polishing step is an anion exchange chromatography using an adsorber membrane.
  • said coat protein obtained by said eluting is further purified by anion exchange chromatography, wherein preferably said anion exchange chromatography is performed by filtrating said coat protein through a membrane, wherein said membrane comprises or preferably consists of a polymer, wherein said polymer preferably comprises quaternary amino groups, and wherein further preferably said membrane comprises a pore size of 0.2 to 3 ⁇ m.
  • said polymer is selected from the group consisting of: (a) polyethersulfone; (b) polyethyleneimine; and (c) cellulose.
  • said polymer is polyethersulfone, preferably hydrophilic polyethersulfone, wherein further preferably said membrane comprises a pore size of about 0.8 ⁇ m.
  • said filtrating is performed at pH 6 to 7.5, preferably at pH 7.0.
  • the concentration of said coat protein is adjusted to 2 to 6.5, preferably 5.5 mg/ml prior to said filtrating.
  • said coat protein comprises, or alternatively essentially consists of, or alternatively consists of recombinant proteins, or fragments thereof, of a RNA bacteriophage, wherein preferably said RNA bacteriophage is selected from the group consisting of: (a) bacteriophage Q ⁇ ; (b) bacteriophage Rl 7; (c) bacteriophage fr; (d) bacteriophage GA; (e) bacteriophage SP; (f) bacteriophage MS2; (g) bacteriophage Mi l; (h) bacteriophage MXl; (i) bacteriophage NL95; (J) bacteriophage f2; (k) bacteriophage PP7; and bacteriophage AP205.
  • said RNA bacteriophage is bacteriophage
  • RNA bacteriophage is selected from the group consisting of (a) bacteriophage Q ⁇ ; (b) bacteriophage Rl 7; (c) bacteriophage fr; (d) bacteriophage GA; (e) bacteriophage SP; (f) bacteriophage MS2; (g) bacteriophage Mi l; (h) bacteriophage MXl; (i) bacteriophage NL95; (j) bacteriophage f2; (k) bacteriophage PP7; and bacteriophage AP205.
  • RNA bacteriophage is bacteriophage Q ⁇ . In a further preferred embodiment said RNA bacteriophage is bacteriophage AP205. In a further preferred embodiment said RNA bacteriophage is bacteriophage fr. In a further preferred embodiment said RNA bacteriophage is bacteriophage GA.
  • RNA bacteriophage is bacteriophage AP205. Assembly-competent mutant forms of AP205 VLPs, including AP205 coat protein with the substitution of proline at amino acid 5 to threonine, may also be used in the practice of the invention.
  • WO2004/007538 describes, in particular in Example 1 and Example 2, how to obtain VLP comprising AP205 coat proteins, and hereby in particular their expression and purification.
  • WO 2004/007538 is incorporated herein by way of reference.
  • said RNA bacteriophage is bacteriophage fr. Recombinant fr VLP may be obtained as described by Pushko P et al. ((1993) Prot Engin 6:883-891).
  • RNA bacteriophage is bacteriophage GA.
  • GA VLP may be obtained by cloning GA coat protein cDNA isolated by reverse transcription from GA phage into pQbl85, which is described for example in WO2004/007538.
  • said coat protein comprises or preferably consists of an amino acid sequence 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 (£2 coat protein); (m) SEQ ID NO: 13 (PP7 coat protein); and (n) SEQ ID NO: 19 (AP205
  • said coat protein comprises or preferably consists of an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO:21. In a further very preferred embodiment 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.
  • mutant coat protein of bacteriophage Q ⁇ wherein exposed lysine residues are replaced by arginines can be purified by the process of the invention.
  • said coat protein comprises, consists essentially of or alternatively consists of mutant Q ⁇ coat proteins as disclosed WO02/056905 (cf. Example 18 therein).
  • said mutant coat protein of bacteriophage Q ⁇ comprise or preferably consist of any one of SEQ ID NO: 14 to SEQ ID NO: 18.
  • Further RNA bacteriophage coat proteins have also been shown to self-assemble upon expression in a bacterial host (Kastelein, RA.
  • RNA bacteriophages Processes and methods for the expression of virus-like particles of RNA bacteriophages, in particular of bacteriophage Q ⁇ , are disclosed in WO2006/125821A2.
  • WO2006/136566A1 discloses a process comprising two chromatography steps for purifying intact virus-like particles. Both applications are incorporated by reference.
  • virus-like particles obtained by the processes described in WO2006/136566A1 are essentially free of endotoxins but still contain host cell RNA packaged into the virus-like particle.
  • Such material is particularly suited as starting material for the process of the invention.
  • the starting material for the process of the invention may also be generated by an alternative process.
  • said providing a virus-like particle of an RNA bacteriophage comprises: (a) homogenizing bacteria cells expressing said virus-like particle; (b) clarifying the homogenate obtained by said homogenizing; and (c) purifying said virus- like particle by anion exchange chromatography.
  • Said homomogenizing may be performed by any method known in the art, in particular by those disclosed in WO2006/136566A1. In a preferred embodiment said homogenizing is performed 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 the bacteria cells have been disrupted by physical and/or enzymatic means.
  • said homogenizing is performed by disrupting the cell wall of said bacteria cells by sonication, by passage through a high pressure liquid homogenizer like, for example, APV LAB 1000, by passage through a French press, or by grinding with aluminium oxide.
  • said homogenizing is performed by destabilizing the cell wall of said bacteria cells by detergent, preferably by sodium dodecyl sulphate (SDS), or, more preferably, by non-ionic detergents, preferably selected from Triton ® X-100, Triton ® X-114, Tween ® 20, Igepal ® CA 630, Brij ® 35 and mixtures thereof.
  • SDS sodium dodecyl sulphate
  • said detergent is Triton ® X-100.
  • Said detergent is preferably applied in a concentration of 0.01 to 30 %, more preferably 0.01 to 5 %, most preferably about 0.1 %.
  • said homogenizing is performed by destabilizing the cell wall of said bacteria cells by exposure to a cell wall degrading enzyme, most preferably lysozyme.
  • the disruption of the bacteria cells is improved when the cell suspension is passed through a high pressure liquid homogenizer repeatedly.
  • said homogenizing said bacterial host is performed by passing said bacterial host through a high pressure liquid homogenizer at least once, preferably at least twice, more preferably at least three times, most preferably three times.
  • Said homogenizing is preferably performed in a buffer, wherein said buffer preferably comprises an alkaline pH of about 8, an agent capable of forming complexes with metal ions, preferably EDTA, most preferably 1-50 mM EDTA, and a detergent, preferably selected from SDS, Tween-20 or Triton X-IOO, 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, 5 mM EDTA and 0.1 % (w/w) Triton X-100.
  • said buffer comprises a cell wall degrading enzyme, most preferably lysozyme.
  • Said purifying further comprises the step of clarifying the homogenate obtained by said homogenizing, wherein e.g. cell debris is removed from the homogenate by either filtration or centrifugation.
  • said clarifying comprises filtrating said homogenate through a membrane, wherein said membrane comprises a pore size of 0.2 to 1.0 ⁇ m, preferably of 0.3 to 0.6 ⁇ m, more preferably of about 0.45 ⁇ m, still more preferably about 0.45 ⁇ m, and most preferably 0.45 ⁇ m, wherein preferably said filtrating is performed by tangential flow filtration.
  • said clarifying of said homogenate comprises clarifying said homogenate by centrifugation, wherein preferably said centrifugation is performed prior to said filtrating of said homogenate, and wherein further preferably said homogenate is exposed to an acceleration of at least 7,000 x g, more preferably at least 10,000 x g for a period of time which is sufficient for the complete sedimentation of the cell debris.
  • said purifying said virus-like particle by anion exchange chromatography comprises: (a) binding said virus-like particle contained in the clarified homogenate obtained by said clarifying to an anion exchange matrix, wherein said binding is performed in the presence of 50 to 200 mM, preferably 150 mM of an inorganic salt, preferably sodium chloride, and at a pH of about 7.2; (b) washing said anion exchange matrix in the presence of 150 to 500, preferably of 150 to 425 mM of an inorganic salt, preferably sodium chloride, and at a pH of about 7.2; and (c) eluting said virus-like particle from said anion exchange matrix in the presence of 425 to 650 mM of an inorganic salt, preferably sodium chloride and at a pH of about 7.2.
  • said washing and / or said eluting are performed by applying a concentration gradient of said inorganic salt, preferably of said sodium chloride, wherein preferably said concentration gradient is a
  • said anion exchange matrix comprises trimethylaminoethyl groups
  • said anion exchange matrix is a tentacle anion exchange matrix comprising (i) resin particles of cross-linked methacrylate polymer or cross- linked vinyl polymer (ii) acrylamide tentacles, wherein said acrylamide tentacles are attached to the surface of said resin particles, and wherein said acrylamide tentacles are substituted with TMAE (trimethylaminoethyl-) groups.
  • said anion exchange matrix is Fractogel® EMD TMAE, preferably having a particle size of 40-90 ⁇ m.
  • the process of the invention comprises a yield of said coat protein, wherein said yield is at least 15 %, preferably at least 20 %, more preferably at least 25 %, still more preferably at least 26 %, and most preferably at least 35 % of the protein content of said virus-like particle.
  • the invention provides purified coat protein obtainable by any one of the processes of the invention, wherein said processes may implement any one of the embodiment as described above.
  • said purified coat protein comprises a purity of at least 95 %, preferably at least 98 %, and most preferably of at least 99 %.
  • said purified coat protein comprises less than 0.5 ⁇ g RNA per 100 ⁇ g of said coat protein, wherein preferably said RNA concentration is determined as described in Example 6.
  • said purified coat protein comprises less than 5 ng host cell protein per 100 ⁇ g of said coat protein, wherein preferably the concentration of said host cell protein is determined as described in Example 7.
  • said purified coat protein comprises an endotoxin content of less than 0.3, more preferably less than 0.2, still more preferably less than 0.1 IU per 100 ⁇ g of said coat protein, wherein preferably said endotoxin concentration is determined as described in Example 8.
  • said purified coat protein comprises less than 1.5, preferably less than 1.0, more preferably less than 0.5, and most preferably less than 0.25 ng host cell DNA per 100 ⁇ g of said coat protein, wherein preferably the concentration of said host cell DNA is determined as described in Example 9.
  • said purified coat protein is a coat protein selected from the group consisting of: (a) coat protein of RNA bacteriophage Qbeta; (b) coat protein of RNA bacteriophage AP205; (b) coat protein of RNA bacteriophage fr; and (b) coat protein of RNA bacteriophage GA.
  • said purified coat protein is a coat protein of RNA bacteriophage Qbeta.
  • said purified coat protein comprises or preferably consists of the sequence of SEQ ID NO: 1 (Q ⁇ CP).
  • said purified coat protein (a) comprises or preferably consists of the sequence of SEQ ID NO:1 (Q ⁇ CP); (b) comprises less than 0.5 ⁇ g RNA per 100 ⁇ g of said coat protein; (c) comprises less than 5 ng host cell protein per 100 ⁇ g of said coat protein; (d) comprises an endotoxin content of less than 0.3 IU per 100 ⁇ g of said coat protein; and (e) comprises less than 1.5 ng host cell DNA per 100 ⁇ g of said coat protein.
  • the invention relates to purified coat protein of an RNA bacteriophage, wherein preferably said RNA bacteriophage is bacteriophage Qbeta, (a) wherein said purified coat protein comprises a purity of at least 95 %, preferably at least 98 %, and most preferably of at least 99 %; (b) wherein said purified coat protein comprises less than 0.5 ⁇ g RNA per 100 ⁇ g of said coat protein; (c) wherein said purified coat protein comprises an endotoxin content of less than 0.4, preferably less than 0.3, more preferably less than 0.2, still more preferably less than 0.1 IU per 100 ⁇ g of said coat protein; (d) wherein said purified coat protein comprises less than 5 ng host cell protein per 100 ⁇ g of said coat protein; and/or (f) wherein said purified coat protein comprises less than 1.5, preferably less than 1.0, more preferably less than 0.5, and most preferably less than 0.25 ng host
  • 7.5 L of a Qbeta VLP containing E. coli cell suspension were prepared for cell disruption.
  • the concentration of cellular wet weight was adjusted to 200 g/L by addition of 50 mM Tris-HCl, 5 mM EDTA pH 8.0 (4°C) and the Triton X-100 concentration was adjusted to 0.1% (v/v).
  • Cell disruption was performed at 700 bar for 3 cycles using a high pressure liquid homogenizer APVlOOO.
  • the homogenized cell suspension was cooled to values below 10 0 C in between the homogenization cycles. 90 g Qbeta- VLP was released from the 7.5 L cell suspension.
  • the homogenized cells were clarified in a two step procedure: first by centrifugation and afterwards by microf ⁇ ltration.
  • the first clarification step was performed at 10 OOO g and 4°C for 120 minutes using a Sorvall Evolution RC centrifuge and a fixed angle rotor SLC-6000.
  • the supernatant was further clarified using tangential flow filtration and a 0.45 ⁇ m micro filtration membrane, either composed of regenerated cellulose (Pellicon, Millipore), stabilized cellulose (Sartocon, Sartorius) or polyethersulfone (SUPOR Pall).
  • An effective membrane area of 0.1 m 2 was used for clarification of up to 6.5 L centrifuged E. coli cell lysate.
  • the Qbeta VLP was transferred to the permeate by diaf ⁇ ltration using 50 mM Tris-HCl, 5 mM EDTA pH 8.0 (4 0 C). Starting the process with 1.5 kg cellular wet weight results in 6 L of diafiltrate containing 80 g Qbeta VLP.
  • the diafiltrate was applied on an anion exchange chromatography column (Fractogel TMAE, column diameter: 180 mm, bed height: 30 cm) equilibrated in 20 mM sodium phosphate, 150 mM NaCl pH 7.2. Unbound proteins were removed by washing with 3 column volumes of 20 mM sodium phosphate, 150 mM NaCl pH 7.2. Weakly bound impurities were eluted with 5 column volumes 20 mM sodium phosphate, 425 mM NaCl pH 7.2.
  • Q ⁇ VLP Elution of Q ⁇ VLP was initiated in a linear gradient from 425 mM NaCl to 650 mM NaCl in 20 mM sodium phosphate pH 7.2 in 3 column volumes and a continuation of elution at 650 mM NaCl in 20 mM sodium phosphate pH 7.2 for another 3 column volumes.
  • Q ⁇ VLP of sufficient purity for further processing eluted between 1.0 and 4.3 column volumes after start of the linear gradient.
  • the yield of the anion exchange chromatography step was 77 % resulting in 62 g purified Qbeta VLP which was characterized by a relative Qbeta peak area on analytical size exclusion chromatography of more than 98.0%.
  • Table 1 Overview over the process for purifying Qbeta VLP from E. coli cell suspension as described in Example 1.
  • Disassembly was started with 25 L of a protein solution containing 62 g purified Qbeta VLPs as obtained in Example 1. Reduction of disulfide bonds was initiated in the presence of 10 mM dithiotreitol (DTT) at pH 7.2. The reduction was performed at room temperature for 30 minutes. The solution was stirred at 200 rpm. Afterwards sodium chloride was added to a final concentration of 600 mM. The disassembly was initiated under acidic conditions by addition of a stock solution of 1 M sodium dihydrogen phosphate and 0.75 M citric acid pH 2.3 to result in a final concentration of 85 mM sodium dihydrogen phosphate, 64 mM citric acid and a final pH of 2.6.
  • DTT dithiotreitol
  • the disassembly mixture was stirred at ca. 500 rpm for 30 minutes. Clarification of the disassembly mixture was performed by ultrafiltration using a polyethersulfone membrane with a nominal molecular weight cut off of 300 kD. By diaf ⁇ ltration of the disassembly mixture against 3 volumes of 20 mM sodium dihydrogen phosphate/citrate, 300 mM NaCl pH 3.3, the Qbeta coat protein was transferred to the permeate whereas the majority of ribonucleic acids and associated impurities were retained by the membrane. The permeate was 0.2 ⁇ m filtrated and applied on a cation exchange chromatography column.
  • Qbeta coat protein Purification of Qbeta coat protein was performed on SP Sepharose FF (GE Healthcare, Cat. No. 17-0729-01) equilibrated in 20 mM sodium dihydrogen phosphate/citrate, 300 mM NaCl pH 3.3. 90 L of a Qbeta coat protein containing solution (protein concentration: 0.25 mg/ml) were applied on a 1.9 L column (column diameter: 130 mm; bed height: 15 cm). Unbound material was removed by washing with 20 mM sodium dihydrogen phosphate/citrate, 300 mM NaCl pH 3.3 for 5 CV, followed by elution of impurities in the presence of 300 mM NaCl in 20 mM sodium phosphate pH 7.2 for 10 CV.
  • SP Sepharose FF GE Healthcare, Cat. No. 17-0729-01
  • the protein concentration was adjusted to 5.5 mg/ml using 20 mM sodium phosphate pH 7.0 in order to reduce the conductivity of the sample before the polishing step.
  • Polishing was performed using a membrane adsorber decorated with quaternary ammonium groups as ligands (Pall, Mustang Q, Cat. No. CLM05MSTGQP1; bed volume: 10 ml) equilibrated in 20 mM sodium phosphate, 150 mM NaCl pH 7.2. Whereas the Qbeta coat protein passes the membrane adsorber without interaction, residual ribonucleic acids interact with the functional groups of the adsorber and are removed.
  • the yield of this non-adsorptive step with respect to Qbeta coat protein was almost quantitative (> 99 %).
  • the purified Qbeta coat protein was characterized by a protein concentration of 5 mg/ml, a purity on analytical size exclusion chromatography of > 99 % (see Example 5), a residual content of ribonucleic acids of less than 5 ⁇ g/mg coat protein (determined as described in Example 6), a residual endotoxin content of 0.3 IU/100 ⁇ g coat protein, a residual host cell protein concentration lower than 5 ng/100 ⁇ g coat protein (limit of quantification of the assay) and a residual host cell DNA content of less than 1.5 ng hcDNA/100 ⁇ g coat protein (below limit of quantification if 1 ⁇ g coat protein is used for analysis).
  • the overall yield of the disassembly process, the cation exchange chromatography and the polishing step is 26 % referring to a Qbeta VLP amount of 62 g used for disassembly.
  • Table 2 Overview over the process for disassembly of Qbeta VLP and subsequent purification of Qbeta coat protein as described in Example 2.
  • dilution buffer 20 mM NaH 2 PO + ZNa 2 HPO 4 conductivity adjusted dilution of eluate pH 7 0 for membrane target protein concentration 5 5 mg/ml adsorber
  • Membrane adsorber Mustang Q ⁇ 5 ⁇ gZl mg coat bed volume 10 ml protein, Chromatography equilibration buffer 20 mM coat protein amount
  • Injection volume 40 ⁇ l (100 ⁇ g/ml to max. 4 mg/ml)
  • Sample preparation The samples were diluted in eluent to 1.0 mg/ml, mixed, centrifuged at 16'0OO g for 10 minutes at 4 0 C.
  • the concentration of Qbeta coat protein was determined by separating the Qbeta coat protein from impurities by reversed phase HPLC and comparing the area of the Qbeta peak with areas of a Qbeta coat protein reference with known concentration (determined by amino acid analysis).
  • Samples were prepared in 50 mM Tris-HCl pH 7.5, 50 mM DTT and 2 M guanidinium hydrochloride. After incubation for 40 min at room temperature, the samples were centrifuged at 18O00 g for 10 minutes at 4 0 C and the supernatant was used for analysis. A calibration curve was recorded in a range from 1.5 to 24 ⁇ g coat protein. The analysis was performed using the following parameters:
  • Eluent A 0.12 % trifluoracidic acid in water
  • Eluent B 0.10 % trifluoracidic acid in acetonitrile
  • Injection volumes for calibration (standard concentration: 0.3 mg/ml): 5, 10, 20, 40, and 80 ⁇ l
  • the starting material for disassembly i.e. the purified Qbeta VLP obtained in Example 1, and the purified coat protein obtained in Example 2 were analysed by size exclusion high performance liquid chromatography (HPLC) using the following parameters:
  • Injection volume 40 ⁇ l (0.5 mg/ml to 2.5 mg/ml, preferably 1.0 mg/ml)
  • Sample preparation The samples were diluted in eluent to 1.0 mg/ml, mixed, centrifuged at 16O00 g for 10 minutes at 4 0 C.
  • Typical chromatograms of Qbeta VLP obtained in Example 1 and of purified Qbeta coat protein obtained in Example 2 are shown in Figure 1.
  • the purity was determined at 280 nm as percentage of the peak area of Qbeta coat protein relative to the total peak area. Purity of purified Qbeta coat protein as obtained in Example 2 was generally found to be at least 98 %.
  • the content of residual ribonucleic acid was determined spectrophotometrically at 260 nm after extraction of the ribonucleic acid from the purified coat protein.
  • the assay validity was controlled by equivalent treatment of reference solutions of tRNA containing 5 ⁇ g tRNA/ml and by a Qbeta VLP standard as positive control. The procedure was performed as follows: dilution of all samples and Qbeta VLP standard to 1 mg/ml using 20 mM sodium phosphate, 150 mM NaCl pH 7.2; and dilution of the tRNA-standard to 5 ⁇ g/ml (tRNA from E.coli MRE 600, Roche, Cat. No.
  • TCEP Tris(2-carboxyethyl)phosphine
  • VLP standard 20-fold dilution of the positive control (VLP standard); all other samples were processed without dilution; heat denaturation at 95 0 C for 5 minutes, incubation on ice until spectroscopic analysis; measurement of UV-absorbance of the samples and standards at 260 and 340 nm; blank is the equivalently treated 20 mM sodium phosphate, 150 mM NaCl pH 7.2 solution; correction of the absorbance at 260 nm by subtracting the absorbance at 340 nm; the absorbance of the tRNA standards is used for calculation of the actual RNA concentration of the samples considering the respective dilution factors of the samples.
  • the content of residual host cell protein in Qbeta coat protein solutions was determined using a host cell protein ELISA (enzyme- linked immunosorbent assay) specific for products derived from the E. coli expression strain used for the recombinant Expression of the coat protein.
  • the host cell protein standard was produced from a cleared lysate of the E. coli strain used for production containing a plasmid in which the sequence coding the Qbeta coat protein had been deleted. This standard was also used for immunization of goats for production of polyclonal antisera. Affinity-purified anti-host cell protein antibodies from goat antiserum were used as primary antibodies for coating of ELISA plates.
  • biotinylated variant of these anti-host cell protein antibodies (secondary antibodies) were applied, followed by the addition of alkaline-phosphatase-conjugated Streptavidin (Jackson ImmunoResearch; Cat. No. 016-050-084).
  • the conversion of the pam- NitroPhenyl Phosphate (pNPP) substrate was measured at 405 nm.
  • the host cell protein concentration of the samples is calculated from a host cell protein standard curve in the range from 5 to 60 ng/ml.
  • concentration of host cell protein in Qbeta coat protein preparations obtained in Example 2 was regularly found to be below the quantification limit of this method, i.e. below 5 ng host cell protein / 100 ⁇ g Qbeta coat protein.
  • the content of residual host cell DNA was determined using the total DNA assay (Molecular Devices, Cat. No. R9009) and the Threshold system provided by Molecular Devices. Before starting the labelling reaction samples were incubated in the presence of proteinase K (e.g. Roche, Cat. No. 3115887) for degradation of protein. DNA was extracted using the sodium iodide method (e.g using the DNA extractor kit provided by WAKO, Cat. No. 295-50201). When 1 ⁇ g protein was used in the assay, the concentration of host cell DNA in coat protein preparations obtained in Example 2 were regularly found to be below the limit of quantification of this method, i.e. below 1.5 ng host cell DNA / 100 ⁇ g Qbeta coat protein.
  • Example 10 Disassembly of AP205 VLPs and Purification of AP205 Coat Protein
  • Disassembly is started with a protein solution containing AP205 VLPs obtained essentially as described in Example 1. Reduction of disulfide bonds is initiated in the presence of 10 mM dithiotreitol (DTT) at pH 7.2. The reduction is performed at room temperature for 30 minutes. Afterwards sodium chloride is added to a final concentration of 600 mM. The disassembly is initiated under acidic conditions by addition of a stock solution of 1 M sodium dihydrogen phosphate and 0.75 M citric acid pH 2.3 to result in a final concentration of 85 mM sodium dihydrogen phosphate, 64 mM citric acid and a final pH of 2.6. The disassembly mixture is stirred at ca.
  • DTT dithiotreitol
  • Clarification of the disassembly mixture is performed by ultrafiltration using a polyethersulfone membrane with a nominal molecular weight cut off of 300 kD.
  • the AP205 coat protein is transferred to the permeate whereas the majority of ribonucleic acids and associated impurities are retained by the membrane.
  • the permeate is 0.2 ⁇ m filtrated and applied on a cation exchange chromatography column.
  • Purification of AP205 coat protein is performed on SP Sepharose FF (GE Healthcare, Cat. No.
  • Polishing is performed using a membrane adsorber decorated with quaternary ammonium groups as ligands (Pall, Mustang Q, Cat. No. CLM05MSTGQP1; bed volume: 10 ml) equilibrated in 20 mM sodium phosphate, 150 mM NaCl pH 7.2. Whereas the AP205 coat protein passes the membrane adsorber without interaction, residual ribonucleic acids interact with the functional groups of the adsorber and are removed.
  • quaternary ammonium groups as ligands Pall, Mustang Q, Cat. No. CLM05MSTGQP1; bed volume: 10 ml
  • Disassembly is started with a protein solution containing GA VLPs obtained essentially as described in Example 1. Sodium chloride is added to a final concentration of 600 mM. The disassembly is initiated under acidic conditions by addition of a stock solution of 1 M sodium dihydrogen phosphate and 0.75 M citric acid pH 2.3 to result in a final concentration of 85 mM sodium dihydrogen phosphate, 64 mM citric acid and a final pH of 2.6. The disassembly mixture is stirred at ca. 500 rpm for 30 minutes. Clarification of the disassembly mixture is performed by ultrafiltration using a polyethersulfone membrane with a nominal molecular weight cut off of 300 kD.
  • the GA coat protein is transferred to the permeate whereas the majority of ribonucleic acids and associated impurities are retained by the membrane.
  • the permeate is 0.2 ⁇ m filtrated and applied on a cation exchange chromatography column.
  • Purification of GA coat protein is performed on SP Sepharose FF (GE Healthcare, Cat. No. 17-0729-01) equilibrated in 20 mM sodium dihydrogen phosphate/citrate, 300 mM NaCl pH 3.3.
  • Unbound material is removed by washing with 20 mM sodium dihydrogen phosphate/citrate, 300 mM NaCl pH 3.3, followed by elution of impurities in the presence of 300 mM NaCl in 20 mM sodium phosphate pH 7.2. Elution is initiated in a step to 550 mM NaCl in 20 mM sodium phosphate pH 7.2.
  • GA coat protein in sufficient purity for further processing elutes between 0.8 and 2.1 CV after the step to 550 mM NaCl in 20 mM sodium phosphate pH 7.2.
  • Polishing is performed using a membrane adsorber decorated with quaternary ammonium groups as ligands (Pall, Mustang Q, Cat. No. CLM05MSTGQP1; bed volume: 10 ml) equilibrated in 20 mM sodium phosphate, 150 mM NaCl pH 7.2. Whereas the GA coat protein passes the membrane adsorber without interaction, residual ribonucleic acids interact with the functional groups of the adsorber and are removed.
  • quaternary ammonium groups as ligands Pall, Mustang Q, Cat. No. CLM05MSTGQP1; bed volume: 10 ml
  • Disassembly is started with a protein solution containing fr VLPs obtained essentially as described in Example 1. Sodium chloride is added to a final concentration of 600 mM. The disassembly is initiated under acidic conditions by addition of a stock solution of 1 M sodium dihydrogen phosphate and 0.75 M citric acid pH 2.3 to result in a final concentration of 85 mM sodium dihydrogen phosphate, 64 mM citric acid and a final pH of 2.6. The disassembly mixture is stirred at ca. 500 rpm for 30 minutes. Clarification of the disassembly mixture is performed by ultrafiltration using a polyethersulfone membrane with a nominal molecular weight cut off of 300 kD.
  • fr coat protein is transferred to the permeate whereas the majority of ribonucleic acids and associated impurities are retained by the membrane.
  • the permeate is 0.2 ⁇ m filtrated and applied on a cation exchange chromatography column. Purification of fr coat protein is performed on SP Sepharose FF (GE Healthcare, Cat. No. 17-0729-01) equilibrated in 20 mM sodium dihydrogen phosphate/citrate, 300 mM NaCl pH 3.3.
  • Unbound material is removed by washing with 20 mM sodium dihydrogen phosphate/citrate, 300 mM NaCl pH 3.3, followed by elution of impurities in the presence of 300 mM NaCl in 20 mM sodium phosphate pH 7.2. Elution is initiated in a step to 550 mM NaCl in 20 mM sodium phosphate pH 7.2. fr coat protein in sufficient purity for further processing elutes between 0.8 and 2.1 CV after the step to 550 mM NaCl in 20 mM sodium phosphate pH 7.2.
  • Polishing is performed using a membrane adsorber decorated with quaternary ammonium groups as ligands (Pall, Mustang Q, Cat. No. CLM05MSTGQP1; bed volume: 10 ml) equilibrated in 20 mM sodium phosphate, 150 mM NaCl pH 7.2. Whereas the fr coat protein passes the membrane adsorber without interaction, residual ribonucleic acids interact with the functional groups of the adsorber and are removed.
  • quaternary ammonium groups as ligands Pall, Mustang Q, Cat. No. CLM05MSTGQP1; bed volume: 10 ml

Abstract

L'invention porte sur un procédé destiné à purifier une protéine d'enveloppe d'un bactériophage à ARN, ledit procédé comprenant les étapes consistant à désassembler une particule de type virus d'un bactériophage à ARN, et, par la suite, à purifier ladite protéine d'enveloppe. Le procédé décrit ici est mis au point pour être mis à l'échelle d'une production de jusqu'à 100 g de protéine d'enveloppe et plus par lot, et il permet le retrait efficace de l'ARN et des impuretés issues de la cellule hôte, typiquement au-dessous du niveau de quantification. L'invention porte en outre sur une protéine d'enveloppe purifiée de bactériophages à ARN, ladite protéine d'enveloppe purifiée comprenant typiquement moins de 0,5 µg d'ARN, moins de 0,4 UI d'endotoxine, moins de 5 ng de protéine de cellule hôte, et moins de 1,5 ng d'ADN de cellule hôte pour 100 µg de ladite protéine d'enveloppe.
PCT/EP2007/063907 2006-12-14 2007-12-13 Procédé de purification pour une protéine d'enveloppe de bactériophages à arn WO2008071774A1 (fr)

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