PURIFICATION PROCESS FOR COAT PROTEIN OF RNA BACTERIOPHAGES

FIELD OF THE INVENTION

[0001] The invention relates to the filed of protein purification. Disclosed is 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 said coat protein. Typically, 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. Besides 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. I.e. said purified coat protein typically and preferably comprises less than 0.5 μg 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 μg of said coat protein.

BACKGROND AND RELATED ART

[0002] 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). 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. The cited documents disclose a variety of methods for the preparation of such compositions. Most commonly used are processes which are based on the assembly of purified coat protein in the presence of said immunostimulatory substance, e.g. of an oligonucleotide. This application

discloses a process which provides coat protein of improved purity which is particularly suited for the preparation of reassembled virus-like particles.

[0003] 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. In the prior art, 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. These disassembly methods are either not suitable for scale-up of the process and/or do not lead to a satisfying purity of the product.

SUMMARY OF THE INVENTION

[0004] 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 μg 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 μg 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.

[0005] Thus, in a first aspect, 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.

[0006] The disassembly of virus-like particles comprising intermolecular disulfide bonds can be significantly improved by reducing said disulfide bounds with a reducing agent. Thus, 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.

[0007] 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. In particular, 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 %. [0008] 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 μg RNA per 100 μg of said coat protein.

[0009] 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 μg of said coat protein.

[0010] 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 μg of said coat protein.

[0011] 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 μg of said coat protein.

[0012] 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.

[0013] In a further aspect, 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 μg RNA per 100 μg 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 μg of said coat protein; (d) wherein said purified coat protein comprises less than 5 ng host cell protein per 100 μg 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 μg of said coat protein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 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 μl for both samples. The protein concentration was 1.6 mg/ml Qbeta VLP (A) and 1 mg/ml Qbeta coat protein (B), respectively.

DETAILED DESCRIPTION OF THE INVENTION

[0014] The definitions and embodiments described in the following are, unless explicitly stated otherwise, applicable to any one of the aspects, and embodiments, processes, and purified coat proteins of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0015] "coat protein": As used herein, the term "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. Thus, the term coat protein refers to the protein forming the capsid of a RNA bacteriophage or a VLP of a RNA bacteriophage. Typically and preferably, coat protein of RNA bacteriophages has a dimeric structure. [0016] "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. Preferably the fragment is obtained by at

least one internal deletion, at least one truncation or at least one combination thereof. The term "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. The term "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.

[0017] "virus-like particle (VLP)", as used herein, 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. The term "non-replicative", as used herein, refers to being incapable of replicating the genome comprised by the VLP. The term "non- infectious", as used herein, refers to being incapable of entering the host cell. Preferably 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. In one embodiment, 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. Typically and more preferably 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. For example, the capsids of RNA bacteriophages have a spherical form of icosahedral symmetry.

[0018] "virus-like particle of an RNA bacteriophage": As used herein, the term "virus- like particle of a 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. In addition, 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. Preferred VLPs derived from RNA bacteriophages exhibit icosahedral symmetry and consist of 180 subunits. In the context of the invention the term 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.

[0019] "Tentacle anion exchange matrix": The expressions "tentacle anion exchange matrix" as used herein 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. Specifically preferred 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.

[0020] "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. Typically and preferably, the 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. [0021] "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. [0022] "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.

[0023] "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.

[0024] "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.

[0025] "one", "a/an": When the terms "one," "a," or "an" are used in this disclosure, they mean "at least one" or "one or more," unless otherwise indicated.

[0026] "about": within the meaning of the present application the expression about shall have the meaning of +/- 10 %. For example about 100 shall mean 90 to 110. [0027] 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.

[0028] In a preferred embodiment, 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. In a preferred embodiment said coat protein comprises or preferably consists of the amino acid sequence of SEQ ID NO:5. In a further preferred embodiment said RNA bacteriophage is bacteriophage GA.

[0029] 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 Qβ 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.

[0030] Thus, in a preferred embodiment 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. In a further preferred embodiment said coat protein comprises ore preferably consists of SEQ ID NO:1 (Qβ CP); (b) a mixture of SEQ ID NO:1 and SEQ ID NO:2 (Qβ 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). In a further preferred embodiment said virus like particle comprises intermolecular disulfide bonds. In a still further preferred embodiment said virus-like particle is a virus-like particle of bacteriophage Qβ or bacteriophage AP205, most preferably of bacteriophage Qβ. [0031] In a preferred embodiment said reducing agent is selected from DTT (dithiothreitol), β-mecaptoethanol, TCEP and other reducing agents generally known in the art. In a very preferred embodiment said reducing agent is DTT, wherein preferably the concentration of said DTT in said reducing mixture is 2 to 25 mM, preferably 10 mM. In order to allow reduction of said disulfide bonds, in a very preferred embodiment said reducing mixture comprises a pH of 6.5 to 8.0, preferably of 6.8 to 7.5, most preferably of 7.2. In a further preferred embodiment, 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. Thus, in a preferred embodiment 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. In a further preferred embodiment 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.

[0032] In a further preferred embodiment 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.

[0033] In a further preferred embodiment said an inorganic salt is sodium chloride. [0034] In a further preferred embodiment 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. In a very preferred embodiment said disassembly mixture comprises 600 mM sodium chloride.

[0035] Under the salt conditions described above, high molecular RNA and host cell derived protein is efficiently precipitated at pH 2.3 to 3.5, while said coat protein remains dissolved. Thus, in a preferred embodiment 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. In a very preferred embodiment 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.

[0036] Buffer systems stabilizing the pH of said disassembly mixture at the desired value are generally known in the art. In one embodiment 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. In a preferred embodiment said buffer comprises dihydrogenphosphate, preferably sodium or potassium dihydrogenphosphate, most preferably sodium dihydrogenphosphate, and an organic acid, preferably citric acid. In a very preferred embodiment 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. In a still further preferred embodiment the concentration of said dihydrogenphosphate, preferably of said sodium dihydrogenphosphate, in said disassembly mixture is about 85 mM, and the concentration of said organic acid, preferably of said citric acid, in said disassembly mixture is about 64 mM. In a still further preferred embodiment the concentration of said sodium

dihydrogenphosphate in said disassembly mixture is 85 mM, and the concentration of said citric acid, in said disassembly mixture is 64 mM.

[0037] In a further preferred embodiment 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. [0038] 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). In a preferred embodiment 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. [0039] In a further preferred embodiment 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.

[0040] In a further preferred embodiment 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.

[0041] In a further preferred embodiment 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.

[0042] In a further preferred embodiment 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.

[0043] Any cation exchange matrix known in the art may be used for the purpose of the invention. In a preferred embodiment 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. In a very preferred embodiment said cation exchange matrix is cross-linked agarose, wherein said cross-linked agarose comprises sulfopropyl groups. In a still further preferred embodiment said cation exchange matrix is sepharose, preferably SP Sepharose FF (GE Healthcare, Cat No. 17-0729-01).

[0044] Removal of high molecular RNA significantly improves the binding of said coat protein to said cation exchange matrix. Thus, in a further preferred embodiment 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. In a very preferred embodiment 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.

[0045] In a further preferred embodiment 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.

[0046] It has surprisingly been found that host cell derived impurities, namely low molecular weight RNA, which are remaining in the coat protein preparation after said cation exchange chromatography, can efficiently be removed by a polishing step, wherein said polishing step is an anion exchange chromatography using an adsorber membrane. Thus, in a further preferred embodiment 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 μm.

[0047] In a preferred embodiment said polymer is selected from the group consisting of: (a) polyethersulfone; (b) polyethyleneimine; and (c) cellulose. In a further preferred embodiment said polymer is polyethersulfone, preferably hydrophilic polyethersulfone, wherein further preferably said membrane comprises a pore size of about 0.8 μm.

[0048] In a further preferred embodiment said filtrating is performed at pH 6 to 7.5, preferably at pH 7.0.

[0049] In a further preferred embodiment the concentration of said coat protein is adjusted to 2 to 6.5, preferably 5.5 mg/ml prior to said filtrating.

[0050] In a preferred embodiment 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 Qβ; (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. In a preferred embodiment said RNA bacteriophage is bacteriophage

Qβ-

[0051] In a further preferred embodiment said RNA bacteriophage is selected from the group consisting of (a) bacteriophage Qβ; (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.

[0052] In a further preferred embodiment said RNA bacteriophage is bacteriophage Qβ. 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.

[0053] In a further preferred embodiment said 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.

[0054] In a further preferred embodiment 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).

[0055] In a further preferred embodiment said 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. [0056] In one 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 (Qβ CP); (b) a mixture of SEQ ID NO:1 and SEQ ID NO:2 (Qβ 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 coat protein). In a further preferred embodiment, 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.

[0057] Furthermore, mutant coat protein of bacteriophage Qβ wherein exposed lysine residues are replaced by arginines can be purified by the process of the invention. Thus, in a further preferred embodiment said coat protein comprises, consists essentially of or alternatively consists of mutant Qβ coat proteins as disclosed WO02/056905 (cf. Example 18 therein). In a further preferred embodiment said mutant coat protein of bacteriophage Qβ comprise or preferably consist of any one of SEQ ID NO: 14 to SEQ ID NO: 18. [0058] Further RNA bacteriophage coat proteins have also been shown to self-assemble upon expression in a bacterial host (Kastelein, RA. et al., Gene 23:245-254 (1983), Kozlovskaya, TM. et al., Dokl. Akad. Nauk SSSR 287:452-455 (1986), Adhin, MR. et al., Virology 170:238-242 (1989), Priano, C. et al., J. MoI. Biol. 249:283-297 (1995)). In particular the biological and biochemical properties of GA (Ni, CZ., et al., Protein Sci. 5:2485-2493 (1996), Tars, K et al., J. Mol.Biol. 271 :759-773(1997)) and of fr (Pushko P. et al., Prot. Eng. 6:883-891 (1993), Liljas, L et al. J MoI. Biol. 244:279-290, (1994)) have been disclosed. The crystal structure of several RNA bacteriophages has been determined (Golmohammadi, R. et al., Structure 4:543-554 (1996)).

[0059] Processes and methods for the expression of virus-like particles of RNA bacteriophages, in particular of bacteriophage Qβ, 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. The 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. However, the starting material for the process of the invention may also be generated by an alternative process. Thus, in a further embodiment 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.

[0060] 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.

[0061] In a further preferred embodiment 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. Alternatively or additionally, preferably additionally, 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. In a very preferred embodiment 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 %. Alternatively or additionally, said homogenizing is performed by destabilizing the cell wall of said bacteria cells by exposure to a cell wall degrading enzyme, most preferably lysozyme.

[0062] The disruption of the bacteria cells is improved when the cell suspension is passed through a high pressure liquid homogenizer repeatedly. In a preferred embodiment 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 %. In a very preferred embodiment said buffer comprises a pH of 8.0, 5 mM EDTA and 0.1 % (w/w) Triton X-100. In a further preferred embodiment said buffer comprises a cell wall degrading enzyme, most preferably lysozyme.

[0063] 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. In one embodiment said clarifying comprises filtrating said homogenate through a membrane, wherein said membrane comprises a pore size of 0.2 to 1.0 μm, preferably of 0.3 to 0.6 μm, more preferably of about 0.45 μm, still more preferably about 0.45 μm, and most preferably 0.45 μm, wherein preferably said filtrating is performed by tangential flow filtration. In a further preferred embodiment 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.

[0064] In a further preferred embodiment 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. In a very preferred embodiment 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 linear gradient.

[0065] In a preferred embodiment said anion exchange matrix comprises trimethylaminoethyl groups, wherein preferably 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. In a very preferred embodiment said anion exchange matrix is Fractogel® EMD TMAE, preferably having a particle size of 40-90 μm.

[0066] In a further preferred embodiment 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.

[0067] In a further embodiment 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. In a preferred embodiment said purified coat protein comprises a purity of at least 95 %, preferably at least 98 %, and most preferably of at least 99 %.

[0068] In a further preferred embodiment said purified coat protein comprises less than 0.5 μg RNA per 100 μg of said coat protein, wherein preferably said RNA concentration is determined as described in Example 6.

[0069] In a further preferred embodiment said purified coat protein comprises less than 5 ng host cell protein per 100 μg of said coat protein, wherein preferably the concentration of said host cell protein is determined as described in Example 7.

[0070] In a further preferred embodiment 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 μg of said coat protein, wherein preferably said endotoxin concentration is determined as described in Example 8.

[0071] In a further preferred embodiment 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 μg of said coat protein, wherein preferably the concentration of said host cell DNA is determined as described in Example 9.

[0072] In a further preferred embodiment 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. In a further preferred embodiment said purified coat protein is a coat protein of RNA bacteriophage Qbeta. In a further preferred embodiment said purified coat protein comprises or preferably consists of the sequence of SEQ ID NO: 1 (Qβ CP). [0073] Thus, in a very preferred embodiment said purified coat protein (a) comprises or preferably consists of the sequence of SEQ ID NO:1 (Qβ CP); (b) comprises less than 0.5 μg

RNA per 100 μg of said coat protein; (c) comprises less than 5 ng host cell protein per 100 μg of said coat protein; (d) comprises an endotoxin content of less than 0.3 IU per 100 μg of said coat protein; and (e) comprises less than 1.5 ng host cell DNA per 100 μg of said coat protein. [0074] In a further aspect, 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 μg RNA per 100 μg 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 μg of said coat protein; (d) wherein said purified coat protein comprises less than 5 ng host cell protein per 100 μg 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 μg of said coat protein. In a further preferred embodiment said purified coat protein comprises or preferably consists of the sequence of SEQ ID NO: 1 (Qβ CP).

EXAMPLES

Example 1

Purification of Qbeta VLPs as Starting Material for Disassembly

[0075] 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 microfϊltration. 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 μm 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. During this step the Qbeta VLP was transferred to the permeate by diafϊltration 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.

[0076] After 0.2 μm filtration of the permeate, 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. Elution of Qβ 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. Qβ 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%. The overall yield of this process for purification of Qbeta VLPs was 69% referring to a Qbeta VLP amount in the E. coli cell suspension used for homogenization of 12 g/L. An

overview over the process steps involved in the of Qbeta VLPs from E. coli cell suspension is provided in Table 1.

Table 1: Overview over the process for purifying Qbeta VLP from E. coli cell suspension as described in Example 1.

Output of the

Process Step Process Parameter Process step cellular wet weight ca 1 5 kg

Thawing of biomass cell suspension 200 g cellular wet weight/ L

Qbeta VLP concentration 12 g/L thawed cell suspension suspension volume 7 5 L suspension

Temperature 24±3°C

7.5 L disrupted cell

Cell Disruption suspension; 90 g released VLP

Clarification by

6.5 L clarified lysate centrifugation

Clarification by 6 L diafiltrate containing 80 g microfiltration clarified VLP resin Fractogel™ TMAE (M) equilibration in 20 mM Sodium Phosphate pH 7 2, 15O mM NaCl, step yield: 77%

Anion exchange column volume 7 4 L (62 g purified VLP) column diameter 180 mm chromatography elution by increase m conductivity (linear purity on SE-HPLC gradient or step) >98% collection of complete peak eluting between

43 and 68 mS/cm

Example 2

Disassembly of Qbeta VLPs and Purification of Qbeta Coat Protein

[0077] 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. 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 diafϊltration 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 μm filtrated and applied on a cation exchange chromatography column. 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. Elution was initiated in a step to 550 mM NaCl in 20 mM sodium phosphate pH 7.2 in 3.1 CV. Qbeta coat protein in sufficient purity for further processing eluted between 0.8 and 2.1 CV after the step to 550 mM NaCl in 20 mM sodium phosphate pH 7.2. The protein concentration of the eluate was 6.3 mg/ml and the purity on analytical size exclusion chromatography performed as described in Example 5 was higher than 98 %. 16.1 g Qbeta coat protein were obtained after this step.

[0078] 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 μg/mg coat protein (determined as described in Example 6), a residual endotoxin content of 0.3 IU/100 μg coat protein, a residual host cell protein concentration lower than 5 ng/100 μg coat protein (limit of quantification of the assay) and a residual host cell DNA content of less than 1.5 ng hcDNA/100 μg coat protein (below limit of quantification if 1 μg

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. An overview over the process steps involved in the disassembly of Qbeta VLPs and the purification of Qbeta coat protein is provided in Table 2.

Table 2: Overview over the process for disassembly of Qbeta VLP and subsequent purification of Qbeta coat protein as described in Example 2.

Output of the

Process Step Process Parameter Process step start protein concentration 2 5 mg/1, final protein concentration 2 mg/ml DTT-concentration 10 mM (reduction of 30 L disassembled disulfide bonds at 7 2+0 2 for 30±5 mm, VLP-solution stirring at 200 rpm)

Disassembly NaCl-concentration 600 mM pH 2.6±0.2 disassembly under acidic conditions conductivity: 85 mM NaH ₂ PO ₄ , 64 mM citric acid pH 55±5 mSZcm 2 6±0 1 stirring at 500 rpm for 30± 5 mm

90 L clarified coat

Membrane 30O kD MWCO protein solution clarification by diafiltration volumes at least 3 (90 L) protein concentration: ultrafiltration diafiltration buffer 20 mM NaH ₂ PO ₄ / citrate, 0.25 mgZml, pH: 300 mM NaCl pH 3 3 3.1+0.2, conductivity:

39±4 mSZcm resin SP Sepharose Fast Flow equilibration buffer 20 mM NaH ₂ PO ₄ / citrate, protein concentration:

30O mM NaCI pH 3 3, 6.3 mg/ml

Cation Exchange washing step with 20 mM NaH ₂ PO ₄ / citrate, protein amount:

30O mM NaCI pH 3 3 chromatography washing step with 20 mM NaH ₂ PO ₄ ZNa ₂ HPO ₄ , 16.1 g coat protein

30O mM NaCI pH 7 2 purity on SE-HPLC > step elution with 20 mM NaH ₂ PO ₄ ZNa ₂ HPO ₄ , 98%

55O mM NaCI pH 7 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 cartridge anionic membrane adsorber residual RNA-content

Membrane adsorber Mustang Q < 5 μgZl mg coat bed volume 10 ml protein, Chromatography equilibration buffer 20 mM coat protein amount

NaH ₂ PO ₄ ZNa ₂ HPO ₄ , 150 mM NaCl pH 7 2 16j

%

Example 3 Characterization of Qbeta VLP by Analytical Size Exclusion Chromatography

[0079] Purified Qbeta VLP obtained as described in Example 1, i.e. after anion exchange chromatrography, was analysed by size exclusion high performance liquid chromatography (HPLC) using the following parameters:

Column: TSKgel G5000 PW _XL 7.8 mm x 300 mm (Tosoh

Bioscience, Cat. No. 08023)

Eluent: 20 mM sodium phosphate, 150 mM NaCl pH 7.2

Gradient: Isocratic

Column temperature: 25°C

Autosampler temperature: 8°C Flow rate: 0.8 ml/min

Sample concentration: 1 mg/ml

Injection volume: 40 μl (100 μg/ml to max. 4 mg/ml)

Evaluation wavelengths: 280 nm and 260 nm Bandwidth: 4 nm

Run time: 25 min

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.

[0080] The purity of the preparation was determined as the percentage of the peak area of Qbeta relative to the total peak area at 260 and 280 nm. The purity was determined at 260 nm as percentage of the peak area of Qbeta VLP relative to the total peak area. The purity was found to be at least 98 % (n=5).

Example 4

Quantification of Qbeta Coat Protein by Reversed Phase HPLC

[0081] 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 μg coat protein. The analysis was performed using the following parameters:

Column: Jupiter C ₄ -RP-HPLC 150 x 4.60 mm, 5 micron

(Phenomenex, Cat. No. 00F-4167-E0)

Guard column: Widepore C4, 4x3.0 mm (Phenomenex, Cat. No. AJO-

4330-S) in cartridge holder (Phenomenex, Cat. No. KJO-

4282)

Eluent A: 0.12 % trifluoracidic acid in water Eluent B: 0.10 % trifluoracidic acid in acetonitrile Gradient: isocratic 40 % B for 2 min; linear gradient from 40 % B to 50 %B in 6 min; isocratic 50% B for 2 min; step to

100 % B for 2 min; step to 40 % B for 2 min

Column temperature: 50 ⁰ C Autosampler temperature: 8 ⁰ C Flow rate: 1 ml/min

Sample concentration: ca. 0.3 mg/ml Injection volume: 50 μl

Injection volumes for calibration (standard concentration: 0.3 mg/ml): 5, 10, 20, 40, and 80 μl

Evaluation wavelength: 215 nm

Bandwidth: 4 nm

Run time: 14 min

Example 5

Characterization of Qbeta Coat Protein by Analytical Size Exclusion Chromatography

[0082] 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:

Column: Bio-Sil SEC 250, 7.8 x 300 mm, BioRad

(Cat. No. 125-0062)

Eluent: 50 mM sodium phosphate pH 6.5, 150 mM NaCl

Gradient: Isocratic

Column temperature: 25°C

Autosampler temperature: 8°C Flow rate: 1 ml/min

Sample concentration: 1 mg/ml

Injection volume: 40 μl (0.5 mg/ml to 2.5 mg/ml, preferably 1.0 mg/ml)

Evaluation wavelengths: 280 nm and 260 nm Bandwidth: 4 nm

Run time: 20 min

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.

[0083] 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 absorbance ratio at 260 to 280 nm of Qbeta VLP was determined as 1.87 (1.85-1.90, n = 10) indicating the high content of ribonucleic acid in the core of the particle, whereas that of the purified Qbeta coat protein was 0.48 (0.45-0.57, n = 5). 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 %.

Example 6

Determination of Residual Ribonucleic Acids

[0084] 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 μg 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 μg/ml (tRNA from E.coli MRE 600, Roche, Cat. No. 109 541); addition of TCEP (Tris(2-carboxyethyl)phosphine) for reduction of potential disulfide bonds to a final concentration of 138 μM and incubation for 15 min at room temperature; addition of NaCl to a final concentration of 1 M and incubation at 60 ⁰ C for 15 min; removal of precipitated protein by centrifugation (20O00 g for 2 minutes at 20 ⁰ C);

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.

[0085] The tRNA-content of samples of the Qbeta coat protein obtained in Example 2 was below the quantification limit of this method, i.e. lower than 5 μg RNA/mg coat protein.

Example 7

Determination of residual host cell protein

[0086] 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. After sample application, the 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. The 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 μg Qbeta coat protein.

Example 8

Analysis of endotoxin activity in Qbeta coat protein solutions

[0087] Testing for endotoxin contamination of Qβ coat protein containing solutions was performed as laid out in Pharm Eur 2.6.14. Method E using either Biowhittaker Kinetic- QCL® chromogenic assay or Charles River Endochrome-K™ kits. The endotoxin concentration of Qbeat coat protein preparations obtained in Example 2 were regularly found to be in the range of 0.5-5 EU/100 μg Qbeta coat protein.

Example 9

Determination of residual host cell DNA

[0088] 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 μg 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 μg Qbeta coat protein.

Example 10 Disassembly of AP205 VLPs and Purification of AP205 Coat Protein

[0089] 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. 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. By diafϊltration of the disassembly mixture against 3 volumes of 20 mM sodium dihydrogen phosphate/citrate, 300 mM NaCl pH 3.3, 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 μm filtrated and applied on a cation exchange chromatography column. Purification of AP205 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. AP205 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.

[0090] 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.

Example 11

Disassembly of GA VLPs and Purification of GA Coat Protein

[0091] 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. By diafiltration of the disassembly mixture against 3 volumes of 20 mM sodium dihydrogen phosphate/citrate, 300 mM NaCl pH 3.3, 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 μm 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.

[0092] 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.

Example 12

Disassembly of fr VLPs and Purification of fr Coat Protein

[0093] 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. By diafiltration of the disassembly mixture against 3 volumes of 20 mM sodium dihydrogen phosphate/citrate, 300 mM NaCl pH 3.3, the 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 μm 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.

[0094] 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.