MXPA01001647A - Process for purifying human papillomavirus virus-like particles - Google Patents

Abstract

A process for purifying papillomavirus virus-like particles (VLPs) includes the step of passing a partially purified VLP-containing solution through a hydroxyapatite chromatography column. The VLPs are then eluted using a buffer containing phosphate anion. The advantages of this method include the recovery of a high yield of intact VLPs.

Description

PROCEDURE FOR PURIFYING PARTICLES OF VIRAL TYPE OF HUMAN PAPILLOMA VIRUSES FIELD OF THE INVENTION This invention relates to a method for making and purifying viral-like particles (VLPs) of human papilloma virus (HPV), which can be used as a component of a vaccine.

BACKGROUND OF THE INVENTION Papilloma virus infections occur in a variety of animals, including humans, sheep, dogs, cats, rabbits, monkeys, snakes and cows. The papilloma virus infects the epithelial cells, generally inducing benign epithelial or fibroepithelial tumors at the site of infection. Papilloma viruses are species-specific infectious agents; a human papilloma virus can not infect an animal that is not human. Papilloma viruses can be classified into different groups based on the host they infect. Human papilloma viruses (HPV) are further classified into more than 70 types that are based on the homology of the DNA sequence (for reference, see Papillomaviruses and Human Cancer, H. Pfister (ed.), CRC Press, Inc. , 1990). The virus types of ,. ? ¿S * ~ ^ * .sif * _ * a »x #? ** fcJ? Í? ___ L .. - m * ~ * ~ papilloma seem to be type-specific immunogens, since the neutralizing immunity to the infection for a type of Papilloma virus does not confer immunity against another type of papilloma virus. Papillomaviruses are small viruses (50-60nm), without cover, of icosahedral DNA that encode up to eight early genes and two late genes. The open reading frames (ORF) of virus genomes are designated E1 to E7 and L1 and L2, where "E" means early and "L" means late. L1 and L2 code for viral capsid proteins. The early genes (E) are associated with functions such as viral replication and cell transformation. The L1 protein is the major capsid protein and has a molecular weight of 55-60 kDa. The L2 protein is a secondary capsid protein having a predicted molecular weight of 55-60 kDa and an apparent molecular weight of 75-100 kDa determined by gel electrophoresis of polyacrylamide. Immunological data suggest that most of the L2 protein is internal to the L1 protein. The L2 proteins are conserved to a high degree among the different papilloma viruses, especially the 10 basic amino acids at the C-terminus. The L1 ORF is conserved to a high degree among the different papilloma viruses. A recombinant L1 protein has been made in a variety of hosts, and under suitable conditions it self-assembles into viral type particles, (VLPs), either alone or in combination with L2. They are candidates for a commercial vaccine. However, to be useful in a vaccine for the human being, the VLPs must be highly purified and free of contaminants from the host cells. In the past, cross-flow ultrafiltration has been used in a diafiltration mode to remove contaminating biomolecules. However, this method resulted in the proteolytic degradation of HPV L1. It would be desirable to have a purification process of the L1 protein that results in a highly pure product that is not degraded.

BRIEF DESCRIPTION OF THE INVENTION This invention relates to a method for purifying viral type particles (VLPs) of recombinant papilloma virus (HPV) comprising the following steps: Making contact between a lysate of cells containing partially purified VLPs with a hydroxyapatite medium in a column of chromatography, under conditions such that the VLPs bind to the hydroxyapatite medium; and eluting the bound VLPs with a solution consisting of phosphate anions; and recover the eluted VLPs. The purification procedure can be used with VLPs that consist substantially of the L1 protein, and can also be used with VLPs consisting of proteins L1 and L2. In addition, it can be used with VLPs that are chimeric, that is, they contain protein L1 and L2: fusion protein. In general, for use as a vaccine, VLPs containing only L1 proteins are preferred.

The procedure is applicable to VLPs from virtually any strain of papillomavirus. It is preferred that a human papillomavirus (HPV) be used. Preferred strains of HPV are those that are known to cause the most serious diseases and conditions, including: HPV type 6a, HPV type 6b, HPV type 11, HPV type 16, HPV type 18 HPV type 31, HPV type 33, and HPV type 45. In general, a host cell is transformed with a vector that codes for L1 or L1 and L2 proteins, or for protein L1 and L2: fusion protein. As used throughout the detailed description and claims, the term "L2: fusion protein" means that the DNA encoding the L2 protein has been operably linked to another DNA encoding a desired protein, and preferably , another HPV protein, such as E1, E2, E3, E4, E5, E6, or E7. The L2 portion of the protein of The fusion may be full-length, or it may have deletions and / or truncations. Examples can be found in the co-pending US Provisional Patent application number S.N. 60 / 096,638, (Proxy case number 20276PV, which is incorporated in the present invention by reference) filed together with this document. The host cell can be any host cell that is easily cultured, as is known in the art, including yeast. { Saccharomyces cerevisiae), insect cells, bacterial or mammalian cells. Yeast cells are particularly preferred.

The vector may also contain other elements as are known in the art, such as transcriptional or translational control elements and / or marker genes. The expressed proteins L1, L1 and L2, or L1 and L2: fusion proteins will be assembled spontaneously in the VLPs. The host cells are typically used, and the cell lysate is then partially purified. The partial purification step may include the purification steps that are commonly used, and is not considered a critical step in this invention. For example, the cell lysate can be subjected to a microfiltration process and at least one step of chromatography, such as cation exchange chromatography. It has been found, in accordance with this invention, that the chromatography step using hydroxyapatite as the column medium, followed by elution with a pH-regulating solution containing phosphate anion, removes a large amount of contaminants from a cell lysate. partially purified. Specifically, it has been found that most contaminating biomolecules, including DNA, lipids and proteins, are removed from the lysate. According to this invention, the final preparation of the purified VLP is generally at least 75% pure, preferably at least 80% pure, and most preferably at least 90% pure, as measured using the test of SDS / PAGE. f1 | jg &? to? & Virtually, any commercially available hydroxyapatite column material can be used in this invention. It is preferred to use a ceramic hydroxyapatite whose particle size is about: 20.50 μm and with a pore size of about 800 5 A °. Said commercially available hydroxyapatite is sold by Bio Rad as "Ceramic hydroxyapatite, Type II" ("Ceramic hydroxyapatite, type II"). However, others are also effective. During the preparation of the chromatography step of the purification procedure, it is recommended that the feeding of the The column is a buffer solution with a pH of 6-8, and preferably around 7. A preferred buffer solution is MOPS [50-7MM 3- (N-morpholino) propanesulfonic acid] at a pH of 7.0 which also contains NaCl 1.25 M. Other pH regulation systems that can also be to use are obvious to those skilled in the art and include: MES [2- (N-morpholino) ethanesulfonic acid]; BIS-TRIS [b / s- (2-hydroxyethyl) -amino] fr / s- (hydroxymethyl) methane]; ADA [monosodium salt of N-2-acetamidoiminodiacetic acid,]; ACES [N-2-acetamido-2-aminoethanesulfonic acid]; PIPES [piperazin-N, N'-b / 's (2-ethanesulfonic acid)]; 20 MOPSO [(3-N-morpholino) -2-hydroxypropanesulfonic acid]; BIS-TRIS PROPANE [1, 3-b / 's [tris (hydroxymethyl) methylamino] propane]; BES [N, N-b / s- (2-hydroxyethyl) -2-amino-ethanesulfonic acid]; TES [N, .r / s (hydroxymethyl) methyl-2-aminoethanesulfonic acid and 2-2 ([2-hydroxy-1, 1- jb / 's (hydroxymethyl) ethyl) amino] ethanesulfonic acid]; HEPES [N-2-hydroxyethylpiperazin-N'-2-ethanesulfonic acid]; DIPSO [3- (N, N-b / s (2-hydroxyethyl) amino) -2-hydroxy-propanesulfonic acid]; TAPSO [3-N- [epsilon] (hydroxymethyl) methylamino] -2-hydroxy-propanesulfonic acid]; TRIS [tris- 5 (hydroxymethyl) -aminomethane]; HEPPSO [N- (2-hydroxyethyl) -piperazine-N '- [2-hydroxy-propanesulfonic acid]]; POPSO [acid (piperazin-N, N'-b / s [2-hydroxypropanesulfonic acid]]; EPPS [N- [2-Hydroxyethyl] piperazine-N '- [3-propanesulfonic acid and HEPPS]; TEA [triethanolamine]; TRICINE [N].? / S- (hydroxymethyl) methyl] glycine]; BICINE [N, N'-b / s- (2-hydroxyethyl] glycine]; TAPS [acid] 3-. { [.r / 's- (hydroxymethyl) methyl] amino} -propanesulfonic]; imidazole; HEPPS [N-2-hydroxyethylpiperazin-N, -3-propanesulfonic acid]; glycinamide hydrochloride; glycylglycine; citrate; acetate; and pH-regulating solutions based on succinate. It allows partially purified VLPs in the solution with regulated pH come into contact with the hydroxyapatite medium under conditions in which the VLPs are allowed to bind to the hydroxyapatite. These conditions include a wide temperature scale; and room temperature is preferred. The flow rate can also vary considerably, and the preferred scale is approximately 90 cm / hour. After the VLPs are bound to the hydroxyapatite, the next step is the recovery of the purified VLPs from the hydroxyapatite with a pH-regulating solution for elution. A pH buffer solution for preferred elution contains a phosphate anion, such as a solution of sodium or potassium phosphate. Preferred molar scales are from about 0.05 M to about 1 M, and what is most preferred is that it is about 0.2 M. The pH of the buffer solution for elution should vary from about 6 ^ 8, being a preferred pH of about 7. Other advantages of the method of this invention include: (a) neither special chromatography equipment nor techniques are required; (b) the method is rapid, which does not require changes of pH buffer, and (c) the method provides excellent performance of LI HPV. The following non-limiting examples are presented to better illustrate the invention.

EXAMPLES 15 EXAMPLE 1 Preparation of the partially purified lysate The transformed yeast cells were harvested for express VLPs and were frozen for storage at -70 ° C. The suspension of frozen yeast cells was removed from storage and thawed for a period of about 3 hours at room temperature and then for 18 hours at 4 ° C. BENZONASE® was added (Nycomed Pharma AS, Copenhagen, Denmark) (2.8 x 105 units / rg ^ and 0.21 mg protein / ml) to the cell suspension at a final concentration of 750 units per gram of wet cell weight, and in one experiment was reduced to 335 units per gram of wet cell weight. The cells were shaken for 15 minutes, then dissolved by passing twice through a sanitized homogenizer type APV Gaulin 30CD with chamber pressures of 1.019.35 to 1, 124.8 kg / cm2, resulting in a dissolution of cells from the cell. 95% The remaining lysate was gently shaken for 18 hours at 4 ° C.

Clarification by microfiltration The cell lysate was clarified by cross-flow microfiltration in a diafiltration mode according to the following: the lysate was transferred to a sterile processing tank with an inlet of 2.54 cm in diameter and ports of exit. The microfilter was a hollow fiber filter cartridge with a pore size of 0.65 microns with a surface area of 46.45 m2 (A / G Technologies # CFP-6-D-8A, Needham, MA) housed in a fiber system Hollow type FlexStand® Benchtop Pilot from A / G Technologies. The retentate was diafiltered with 3 volumes of Diafiltration pH buffer (see below) to produce the clarified lysate. The diafiltration pH regulating solution was 0.2M (Na +) MOPS, pH 7.0 + 0.4M NaCl. ^ mm m Chromatography of clarified lysate Clarified lysate was fractionated by column chromatography using strong cation exchange chromatography resin: POROS® 50HS (PerSeptive Biosystems, Framingham, MA) packed in a chromatography column. The column was sanitized with 0.5 N NaOH before use. The column was equilibrated with pH-regulating solution of HPV diafiltration [(Na +) MOPS 0.2M, pH 7.0 + 0.4M NaCl] at room temperature. The cold clarified lysate (4 ° C) was pumped on the column at 125 ml / minute and the column was washed with 8 column volumes at room temperature with HPV column pH A buffer [(Na +) MOPS 0.05M pH 7.0 + NaCl 0.5M)] at 125 ml / minute with a linear gradient of 100% pH A buffer from HPV column at 100% buffer pH B buffer of HPV column [(Na +) MOPS 0.05M, pH 7.0 + NaCl 1.5 M]. The total linear gradient was 10 column volumes and was collected in equal fractions of 10 volumes. After the gradient, the column was washed with two column volumes of pH B buffer solution of HPV column at room temperature and at 125 ml / minutes which were collected in two additional fractions. The fractions were collected in 2 liter sterile plastic bottles and stored at 4 ° C. Fractions containing the last UV absorption peak (A280nm and A230 nm) in the gradient were combined and filtered using a MILLIPAK-200 disposable filter unit (Mlllipore, Bedford, MA) and stored at 4 ° C.

EXAMPLE 2 Hydroxyapatite chromatography All steps were carried out at room temperature. A chromatography column (13 mm ID x 36 mm) packed with ceramic hydroxyapatite, type II (BioRad Cat. # 7320081, Hercules, CA), was pre-equilibrated in 50 mM MOPS, pH 7.0 + NaCl 1.25M. The partially purified HPV solution of Example 1 was applied to the column at a linear flow rate of 90 cm / hour. After the same application was completed of the sample, the column was washed with eight column volumes of pre-equilibrium pH buffer until the optical density of the column effluent was almost zero. The product of the HPV vaccine was eluted with a linear gradient of 0% up to 100% elution pH buffer (0.2 M sodium phosphate, pH 7.0 + 1.25M NaCl), also at a linear flow rate of 90 cm / hour. The total volume of the gradient was four column volumes. The fractions containing the vaccine product were identified by the RIA and Bradford protein analysis. The protein concentration of the product was 100 μg / ml.

Analysis: Bradford protein analyzes were performed using a Coomassie Plus assay reagent (Pierce, Rockford, IL), using bovine serum albumin (BSA) as standard. The analyzes of Lowry protein according to the procedure of Lowry et al 1951 J. Biol. Chem. 193: 265-270 using BSA as a calibration standard. The antigen was analyzed by multilayer ELISA using a monoclonal antibody recognizing a conformable epitope of the VLP. The microtiter plates were coated with goat polyclonal anti-HVP VLP antibodies. Standard and test samples were diluted with PBS containing 1% w / v BSA, 0.1% TWEEN-20, and 0.1% sodium azide and added to the wells where the antigen was captured by bound antibodies to the plate. The monoclonal anti-HPV VLP L1 antibody (Chemicon, Temecula, CA), was added to the wells to bind the captured antigen with the antibodies bound to the plate. Monoclonal anti-HPV VLP antibodies were detected with anti-mouse IgG antibodies conjugated with horseradish peroxidase. A chromogenic substrate for horseradish peroxidase, 3,3 ', 5,5'-15 tetramethylbenzidine (Pierce) was added and the absorbance at 450 nm was proportional to the VLP concentration of L1 in the sample. The dynamic capacity of the column for the product of the vaccine was 2.9 mg per milliliter of resin by Bradford, and 4.6 mg per milliliter of resin by RIA. The recovery through this step was 90% using the Bradford protein test or 82% using RIA when the column was loaded at 100% capacity. The recovery fell to 63% through Bradford and 50% through RIA when the column was loaded to 8% capacity.

^^^^^^^^^^ A ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^ A sample of L1 HPV 11 prepared essentially as described in Examples 1 and 2 was analyzed for the presence of DNA using a PCR-based test. The results, which are presented in the table below, indicate that this chromatography method is highly effective in removing contaminating DNA from the final product.

EXAMPLE 4 Purification of a Chimeric VLP / Purification of HPV Type 16 L1 / Chimeric VLPs of L2min / E2 Construction of the modified L2 gene Vector YP3 (minimal L2) This vector retains the coding sequences for the amino-terminal amino acids 69 and carboxy-terminal amino acids 84 (aa) of HPV16 L2 that are fused in a frame by a pollinator * < v - * t, "*» * "'t5 _.» í8 r ra. "r .___ _SI__ffv j¡_i_.v synthetic that introduces unique restriction enzyme sites Not I, Sac I, and Xho I and that result in the insertion of a glutamic acid residue and the mutation of a serine residue to Glutamic acid PCR primers (Midland Certified Reagents) 5 were designated to amplify the L2 sequences from the native L2 gene contained within the vector, pGal110 HPV16L1 + L2 The primers I (5'-CTT CCC CCC GGG CAC AAA ACÁ AAA TGC-3 '; SEQ.ID.NO.1) and C (5'-CTC GAG CTC GCG GCC GCC TGT ACC CGA CCC-3'; SEQ ID NO.2) amplified a 265 bp sequence encoding for the aminoterminal 69 aa and 23 bp of the upstream untranslated sequence including a Sma I restriction enzyme site. The C primer modified and extended the coding region for the aminoterminal L2 and annexed the restriction enzyme sites Not I , Sac I and Xho I downstream of the sequences coding for L2. 15 Initiators A (5'-GCG GCC GCG AGC TCG AGG GTT ATA TTC CTG CAA ATA CAA-3 '; SEQ.ID.NO.3), C and D (5'-CCC TCC AGA TCT CTA GGC AGC CAA AGA GAC ATC TG-3 '; SEQ.ID.NO.4) amplified a sequence of 285 bp coding for carboxy -terminal 84 aa of L2 plus 6 bp that added a Bgl II restriction enzyme site. The initiator A also annexed a 17 bp sequence containing sites of Not I, Sac I and Xho I upstream of the sequence encoding L2. The minimal L2 expression construct was assembled through the complementary sequences added by primers A and C. Both DNA products isolated from the above l / C and A / D amplification reactions were used in a PCR reaction that included oligos I and D as the amplification primers. To facilitate the binding of the fragments through their complementary sequence 17 bp, three cycles of 5 PCR were developed with the fixation temperature at 37 ° C, then by 15 cycles at 57 ° C. The resulting amplification product was a shaved end ligated into pcrScript (Stratagene, La Jolla) and transformed into XL-1 Blue MRF 'cells (Stratagene, La Jolla). Positive clones were identified by PCR using primers I and D, and confirmed by the digestion analysis of restriction. The construction was then verified by automated sequence analysis (Perkin Elmer, Inc., Foster City, CA). The plasmid DNA of the suitable isolated material was then digested with Sma I and Bgl II; A fragment of approximately 0.5 kilobase pairs (kb) was gel purified and ligated with the fragment of the L1 HPV16 vector of 14 kb Sma I and Bgl II pGAL110. The competent E. coli DH5 cells (Gibco BRL, Rockville, MD) were transformed with the ligation mixture and with the selected transformants on LB ampicillin plates (Remel, Lenexa, KS). Clones were detected initially by PCR whose primers D and I were used to amplify portions of L2; Subsequently, the appropriate clones were confirmed by restriction digestion analysis. The candidate YP3 # 1 clone was verified by sequence analysis as explained above. r | ^ aafc ^ & ^^^ «» A ^ aijri ^ gj ^ efe ^ - »^ __ __K_r _'____ i _______ í YP3 # 1 was then used as the structure construction within which the genes that code for open reading frames HPV16E1, E2 or E7.

Insertion of genes encoding the HPV E protein The gene encoding HPV16 E2 was obtained by PCR amplification of a positive clinical sample of HPV16 that was subsequently inserted directly into the subcloning vector pCRIl (Stratagene, La Jolla, CA ) and the sequence was verified as in the previous one. Subsequently the sequence of the E2 gene was modified in the following manner: 1) Within frame, the DNA sequences containing Xho I, Nae I, Not I, were added to the amino terminal portion of E2. Additionally, sequences containing Not I, Nae I, and Xho I were added to the carboxy-terminal portion of E2 to facilitate insertion into E2 at Not I and Xho I sites. 2) DNA sequences were altered by PCR mutagenesis to encode the alanine residues encoding glutamic acid residues 39 and solucin 73. This was designed to inactivate the function of the E2 protein. The modified HPV16 E2 gene described above was digested with Not I, Xho I, and ligated with a similarly digested YP3 # 1 vector. Transformants containing the E2 sequences suitably inserted by PCR were selected and the sequence verified.

The same strategy was used for the genes coding for HPV16E1 and HPV16E7. For E1, glycine 482 was altered to aspartic acid; for E7, both cysteine 24 and glutamic acid 26 were changed to glycine to deactivate the function of the protein. Then the resulting 5 constructs were used to transform the yeast for expression analysis.

EXAMPLE 5 Identification and growth of chimeric VLPs expressing yeast The YP3 # 1 plasmid DNA and its derivatives were used as described above, to transform Saccharomyces cerevisiae (MATa, leu2-04, prb1 :: HIS3, mnn9.:URA3, °) by the spheroplasts method (Hinnen et al., 1978, Proc. Nati. Acad. Sci. USA 75 :: 1929-1933). The transformed spheroplasts were plated on a selective medium (less leucine) (Remel, Lenexa, KS). The clones were isolated through two rounds of a simple selection of colonies. Small liquid cultures of candidate clones were cultured to obtain a high cell density in the medium containing galactose. The crude extracts were prepared by vigorous stirring with glass beads and then centrifugation. The clarified extracts were analyzed to detect the expression of the L1, and L2 component, and of VLPs by various methods, including SDS PAGE, ELISA, immuno hybridization and EIA, using - «** ^^^^^^^^^^^? S ^ s ^ ejjgfei ^^ Stó & i ^^^^^ A ^^^^^^^^^^ monoclonal antibodies or monospecific polyclonal antiserum that he recognized L1, or L2, or the amino or carboxy termini of L2, or VLPs of L1, or E1, or E2, or E7, or any other protein or peptide fused to the modified L2. Clones expressing the L2 component and forming VLPs were selected for further characterization. One liter or 16 liter cultures of selected clones were grown in a medium containing galactose for the large scale preparation of chimeric VLPs. Frozen cell pellets were stored at -70CC. The frozen cells (wet weight = 148 g) were thawed and resuspended in 740 ml "Dissolution pH regulator" (200 mM MOPS, pH 7, 1 mM CaCl 2) to give approximately 20% (w / v) suspension . The nuclease BENZONASE® (Nycomed Pharma) was added at 750 units / cell weight wet. The cell suspension was dissolved at a pressure of approximately 1, 335.7 kg / cm2 by 5 steps in an M110-Y microfluidizer (Microfluidics Corp., Newton, MA). The cell suspension was collected and held on ice during its dissolution. The hematocrit test indicated a solution >80% The mature cell lysate was clarified by microfiltration through a hollow fiber cartridge with a pore size of 0.65 microns (A / G Technologies) using a tangential flow microfiltration apparatus that was run in diafiltration mode. The lysate was diafiltered with three volumes of 0.25 M sodium citrate, 0.2 M MOPS and pH 7.0. The antigen passed through the membrane and was collected in the permeate.

The fraction (3.9 I) of the 0.65 mm diafiltered permeate material was loaded onto a 325 ml column (11.2 cm ID x 3.3 cm) of POROS® 50HS resin (Perseptive Biosystems, Cambridge, MA) equilibrated in 200 mM MOPS, pH 7, 250 mM sodium citrate. The column was washed with 8 5 volumes of 50 mM MOPS, 0.5 M NaCl, 5 mM sodium phosphate, pH 7 and eluted with a linear gradient of 10 volumes of 0.5 to 1.5 M NaCl in the same pH buffer. Both the fluid material and the fractions were collected in the overall amount while fractions of 1 volume were collected during the elution. The column fractions were analyzed by hybridization (Western Blott) and SDS-PAGE with colloidal Coomassie detection. Fractions containing predominantly p55 protein were mixed. The 50HS mixture was analyzed for total protein by the BCA test (Pierce). Based on the total protein (168 mg), a ceramic hydroxyapatite (HA) Type II column was poured (Bio-Rad) to give 1 mL of resin / 2 mg of protein. This column was 2.6 cm ID x 15.7 cm. The column was equilibrated in 50 mM MOPS, pH 7, 1.25 M NaCl, 5 mM sodium phosphate. The 50HS (770 mL) mixture was filtered at 0.22 mm and applied to the HA column at a flow rate of 113 cm / hr. The fluid material was collected globally. The HA column was washed with 5 volumes of The equilibrium pH buffer solution was eluted with pH 7 in 1.25 M NaCl. Fractions were collected during elution and analyzed by Western blot and SDS-PAGE with colloidal Coomassie detection. Fractions that showed purity and enrichment were mixed comparable of the L1 protein. The fractions were mixed aseptically and mixed through a 0.22 mm membrane and stored at 4 ° C. Both the retained material and the product resulting from the procedure were analyzed to detect the HPV 16 of L1 using specific EIA and 5 to detect the protein by means of the BCA test. The yield of the final purified product was 27 mg of protein with a specific activity of L1 1.00 mg / mg protein. Electron microscopy confirmed the presence of intact VLP particles with a mean diameter of 32 nm. For the purity analysis of SDS-PAGE, an aliquot of the final product was concentrated by TCA precipitation and analyzed by western blotting and SDS-PAGE with colloidal Coomassie detection. Quantification of L1 was performed using a 2.5 mg load and the yeast contaminants were quantified with a loading of 20.0 mg. It was shown by densitometry that the L1 protein was > 94% homogeneous. The co-purification of L1 and L2 was demonstrated m? N¡ / E2 by specific immunohybridization analysis of the fractions obtained from the procedure.

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Claims (9)

NOVELTY PEOPLE INVENTION CLAIMS

1. A method for purifying viral type particles of recombinant papilloma virus (VLPs) comprising: (a) making contact between a lysate of cells containing a partially purified VLP with a hydroxyapatite medium on a chromatography column, under such conditions that the VLPs bind to the hydroxyapatite medium; (b) eluting the bound VLPs with a solution comprising phosphate anions; (c) recover the eluted VLPs. 2. The method according to claim 1, further characterized in that the VLPs consist essentially of protein

L1.

3. The method according to claim 1, further characterized in that the VLPs are VLPs of human papillomavirus (HPV).

4. The method according to claim 3, further characterized in that the VLPs are selected from the group consisting of: HPV type 6a, HPV type 6b, HPV type 11, HPV type 16, HPV type 18, HPV type 31, HPV type 33, and HPV type 45.

5. The method according to claim 4, further characterized in that the eluted VLPs are at least 75% pure.

6. - The method according to claim 4, further characterized in that the VLPs are at least 90% pure.

7. The method according to claim 1, further characterized in that the VLPs comprise protein L1 and L2: fusion protein.

8. The method according to claim 7, further characterized in that L2: fusion protein has a portion L2 that is less than the total length.

9. A method for manufacturing a purified human papilloma virus VLP product, suitable for use in a human vaccine comprising: (a) partially purifying a cell lysate, wherein the cell lysate is obtained from yeast cells that have been transformed to express VLPs of L1 HPV; (b) contacting the lysate of cells containing partially purified VLP with a medium of hydroxyapatite on a chromatography column, under conditions such that the VLPs bind to the hydroxyapatite medium; (c) eluting the bound VLPs with a solution comprising phosphate anions; and (d) recover the eluted VLPs. ^ a ^ & ^^^^^^^^ * rf ^^^ a ^^^^ & ^^^.! ¿.. "_ ^ T fff¡¡ ¿^^ A method for purifying viral-like particles of the papilloma virus (VLPs) including the step of passing a partially purified VLP-containing solution through a hydroxyapatite chromatography column; the VLPs are eluted using a pH-regulating solution containing phosphate anion; The advantages of this method include the recovery of a large capacity of intact VLPs. RM / ald * sff * eos * jtc * mmt * P01 / 146F