WO2015128788A1 - Purification de la protéine l2 du papillomavirus - Google Patents

Purification de la protéine l2 du papillomavirus Download PDF

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WO2015128788A1
WO2015128788A1 PCT/IB2015/051347 IB2015051347W WO2015128788A1 WO 2015128788 A1 WO2015128788 A1 WO 2015128788A1 IB 2015051347 W IB2015051347 W IB 2015051347W WO 2015128788 A1 WO2015128788 A1 WO 2015128788A1
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protein
papillomavirus
urea
hpv
chromatography matrix
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Vijayarangam DAMOTHARAN
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Shantha Biotechnics Private Limited
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20051—Methods of production or purification of viral material

Definitions

  • the invention relates to the field of purification of papillomavirus L2 (minor capsid) protein using chromatography steps, preferably it relates to the method of purifying multitype or multimeric human papillomavirus L2 (minor capsid) protein.
  • HPVs Human papillomaviruses
  • HPVs are responsible for causing genital warts and intra epithelial neoplasia of the vulva, cervix or anus, which may lead to cervical cancer in women.
  • HPVs are increasingly found to infect men also, although the incidence of their infection has not been extensively studied compared to women.
  • Some oropharyngeal, anal, and penile cancers have been found to be etiologically related to HPV infection in men (Gillison, M. L. et al. Cancer 2008 113 :3036-3046).
  • Vaccines have been shown to prevent HPV infection.
  • Gardasil and Cervarix which both comprise virus like particles (VLPs) formed from LI (major capsid) protein.
  • VLPs virus like particles
  • LI major capsid protein
  • VLP based HPV vaccines have been demonstrated to be efficacious, they only raise a type specific immune response and hence do not confer protection against many of the HPV types that are known to cause cervical cancer. It is reported that the non vaccine HPV types currently account for about 30% of cervical cancers (Vincenzo, Rosa De et al. Gynecologic Oncology 2013 130:642-651).
  • L2 minor capsid protein
  • Fragments of L2 peptides or their fusion peptides are also suitable functional L2 protein derivatives capable of raising an immune response (WO2006083984 and WO2009059325).
  • L2 peptides or protein as vaccine candidates depends upon their availability in large quantities for immunizing large number of susceptible population. Recombinant expression of such protein yields sufficient amount of the protein. However, complexities associated with recombinant protein production, like the formation of inclusion bodies, usually results in highly heterogenous array of impurities or contaminants. Such impurities are highly undesirable in a product which is intended for use as a vaccine.
  • WO 199626277 discloses a method of purifying recombinantly expressed HPV L2E7 protein in E. coli using a combination of multiple chromatography steps viz., cation exchange, anion exchange followed by size exclusion chromatography and an anion exchange chromatography step for concentration. Such a process is complex owing to multiple chromatography steps and is not industrially feasible.
  • fusion proteins such as the multitype HPV L2 protein are generally constructed by splicing two or more proteins or their domains to obtain new non- natural polypeptide or synthetic fusion protein with combined functionalities of the parental proteins or peptides (WO2009059325).
  • Synthetic fusion proteins have been known to be potentially unstable because of the coming together of multiple domains and the absence of stabilizing structures, especially at the junctions (for example, peptide bonds joining two or more peptides or domains), as they have not been selected by nature through an evolutionary process (Fast, Jonas L. et al. Biochemistry 2009 48: 11724-11736).
  • Some such proteins are prone to aggregation, while others may have the propensity to produce polydisperse population of proteins or both, and sometimes may even give rise to several degradation products. Purifying such proteins from a mix of several aggregated, polydispersed and degradation products require considerable skills. Additional difficulties arise when the protein domains derived from one or more parental protein that has been used to construct the fusion protein contains conserved sequences. The aggregated and degraded products that results from such fusion proteins exhibit slight or minor differences in their charges and hydrophobic densities because of their conserved amino acid sequences making the purification of the target protein extremely difficult.
  • papillomavirus L2 protein for example multitype or multimeric FIPV L2 protein
  • production of significant quantities of papillomavirus L2 protein, for example multitype or multimeric FIPV L2 protein, for use in vaccines has been hindered due to the complexities discussed above. Therefore, there is a need to develop methods for purifying papillomavirus L2 protein from its associated impurities for use as a vaccine.
  • the invention is based on the discovery that purifying papillomavirus L2 protein using hydrophobic interaction chromatography in the presence of urea is efficient over prior art processes.
  • the process of the invention is operationally simple, easily scalable and offers good yield and product of consistent quality.
  • the present invention discloses a method of purifying papillomavirus L2 protein.
  • papillomavirus L2 protein or its fragment thereof of any papillomavirus type may be used.
  • the papillomavirus L2 protein comprises the N-terminal fragments of L2 protein from one or more papillomavirus types, optionally in the form of a fusion protein.
  • the invention relates to a method of purifying papillomavirus L2 protein comprising the steps of:
  • step b washing the hydrophobic chromatography matrix of step a with a wash buffer to collect purified papillomavirus L2 protein retarded onto the hydrophobic chromatography matrix
  • step b eluting partially bound purified papillomavirus L2 protein obtained from step b using a first elution buffer.
  • the invention relates to a method of purifying papillomavirus L2 protein comprising the steps of:
  • a contacting a crude papillomavirus L2 protein onto a hydrophobic chromatography matrix in the presence of urea at a concentration between 3.5 M to 4.2 M, b. washing the hydrophobic chromatography matrix of step a with a wash buffer to collect purified papillomavirus L2 protein retarded onto the hydrophobic chromatography matrix, and
  • step b eluting partially bound purified papillomavirus L2 protein obtained from step b using a first elution buffer.
  • the purified papillomavirus L2 protein is collected by washing the hydrophobic chromatography matrix with the wash buffer comprising similar concentration of urea as used in step (a) i.e, without changing the urea concentration.
  • the first elution buffer comprises higher concentration of urea compared to the urea concentration present in the wash buffer.
  • the papillomavirus L2 protein comprises N-terminal fragments of animal or human papillomavirus (HPV) L2 protein.
  • the papillomavirus L2 protein comprises N-terminal fragments of human papillomavirus (HPV) L2 protein belonging to one or more HPV types.
  • HPV human papillomavirus
  • the papillomavirus L2 protein comprises a multitype or multimeric human papillomavirus (HPV) L2 protein or polypeptide obtained by joining N-terminal fragments of HPV L2 protein belonging to one or more HPV types such as, but not limited to, HPV1, HPV2, HPV5, HPV6, HPV11, HPV16, HP VI 8, HPV31, HPV33, HPV34, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, or HPV73.
  • HPV human papillomavirus
  • the multitype or multimeric human papillomavirus (HPV) L2 protein or polypeptide is obtained by joining 11-88 amino acids from the N- terminus of HPV L2 protein of at least five different HPV types such as, but not limited to, HPV6, HPV16, HPV18, HPV31, and HPV39.
  • the invention relates to a method of purifying multitype or multimeric HPV L2 protein comprising the steps of:
  • step b washing the hydrophobic chromatography matrix of step a with a wash buffer to collect purified multitype or multimeric HPV L2 protein retarded onto the hydrophobic chromatography matrix
  • the papillomavirus L2 protein retarded onto hydrophobic chromatography matrix is collected by washing the chromatography matrix with a wash buffer post initial washes.
  • the invention relates to a method of purifying target protein, for example, papillomavirus L2 protein such as multitype or multimeric HPV L2 protein by hydrophobic interaction chromatography employing different strengths or concentrations of urea.
  • target protein for example, papillomavirus L2 protein such as multitype or multimeric HPV L2 protein by hydrophobic interaction chromatography employing different strengths or concentrations of urea.
  • the concentration of urea is selected such that some of the target protein, for example, papillomavirus L2 protein such as multitype or multimeric HPV L2 protein is partially bound to the hydrophobic chromatography matrix while remainder of the target protein is retarded onto the hydrophobic chromatography matrix.
  • papillomavirus L2 protein such as multitype or multimeric HPV L2 protein
  • the target protein for example papillomavirus L2 protein such as multitype or multimeric HPV L2 protein retarded onto the hydrophobic chromatography matrix is collected by washing the hydrophobic chromatography matrix with a buffer without changing the urea concentration, while the partially bound target protein is eluted by passing a buffer with higher concentration of urea.
  • FIG 1 Hydrophobic Interaction Chromatography (HIC) profile of papillomavirus L2 protein showing pure fractions represented as " ⁇ " (corresponding to lanes 4-8 of figure 2 representing post initial wash fractions and first elution fraction) and impure fractions represented as "I” (corresponding to lanes 2 & 3 of figure 2 representing flowthrough fraction and initial wash fractions) & "III” (corresponding to lane 9 of figure 2 representing second elution fraction).
  • Figure 2 SDS-PAGE showing pure and impure fractions.
  • Lane 1 represents load
  • lanes 2 & 3 represents impure fractions collected from flowthrough and initial washes
  • lanes 4-7 represents retarded papillomavirus L2 protein collected from post initial washes
  • lane 8 represents partially bound papillomavirus L2 protein collected as eluate (with first elution buffer)
  • lane 9 represents bound impurities which are eluted (with second elution buffer)
  • lane 10 represents molecular weight marker
  • lane 11 represents purified papillomavirus L2 protein representing pooled fractions of post initial washes and first elution. Definitions
  • buffer includes those agents that maintain the pH of a solution in a desired range. Those skilled in the art will understand that much of the strength of the buffering capacity of a buffer will decrease outside of about one pH unit of its pKa and can easily determine whether the buffer is suitable for use in the process of the invention.
  • the term "equilibration buffer” as used herein means a buffer that is used to prepare or equilibrate the chromatography matrix for loading the sample onto the chromatography matrix.
  • the equilibration buffer comprises of urea concentration at about 3.5 M to about 4.2 M, at about 3.6 M to about 4.1 M, at about 3.7 M to about 4.0 M, or preferably 3.8 M.
  • an exemplary equilibration buffer comprises of 20 mM sodium acetate pH 4.0, 5.0 mM EDTA, 3.8 M urea and conductivity of about 11-12 mS/cm.
  • wash buffer means a buffer that is passed over the chromatography matrix following loading of the sample to be purified.
  • the wash buffer is used to carry away or remove impurities, for example during initial wash cycle.
  • the wash buffer is used to carry away or remove the retarded target protein, for example papillomavirus L2 protein such as multitype or multimeric HPV L2 protein from the chromatography matrix during post initial wash cycle.
  • wash buffer and equilibration buffer comprises similar or identical concentration of urea.
  • papillomavirus L2 protein such as multitype or multimeric HPV L2 protein
  • papillomavirus L2 protein retarded onto the chromatography matrix is collected during post initial washes (i.e., by passing wash buffer after completing initial wash cycle).
  • initial wash cycle or “initial wash” has been used interchangeably and means a wash cycle comprising washing the chromatography matrix with as many column volumes of wash buffer as the number of washes, for example if the chromatography matrix is washed twice, thrice, or more it requires two, three, or more column volumes of wash buffer respectively.
  • the number of initial washes may be determined by running the initial wash fraction on SDS gel and detecting the presence of bands representing impurities. If several bands appear in addition to the band representing target protein on the SDS-PAGE, it is advisable to continue initial wash cycle.
  • the initial wash cycle comprises a wash cycle comprising washing the chromatography matrix at least one or two times with one or two column volumes of wash buffer respectively.
  • post initial wash or “post initial wash cycle” or “later wash” or “later wash cycle” has been used interchangeably to mean a wash cycle comprising washing the chromatography matrix with as many column volumes of wash buffer as the number of washes after completing the initial wash cycle.
  • the post initial washes comprises a wash cycle comprising washing the chromatography matrix at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times with as many column volumes of wash buffer after completing the initial wash cycle.
  • the post initial washes comprises a wash cycle comprising washing the chromatography matrix at least about 2-6 times, at least about 2-5 times, at least about 2-4 times with at least about 2-6, at least about 2-5, at least about 2-4 column volumes of wash buffer respectively after completing the initial wash cycle.
  • fraction or "chromatography fraction” has been used interchangeably and means aliquots collected with same or different buffer after loading the sample onto the matrix during chromatography run, and includes fractions collected from flowthrough, initial wash, post initial wash, and elution in accordance with the invention.
  • Elution buffer refers to a buffer that is used to dissociate the target protein from the chromatography matrix after it has been washed with one or more column volumes of wash buffer.
  • Elution buffer generally contains substances, such as, but not limited to, appropriate concentration of salts, chelating agents, denaturing agents, free affinity ligands or analogs, or other chemicals or their combinations, that promote dissociation of the protein from the chromatography matrix or resin.
  • the partially bound papillomavirus L2 protein is dissociated from the matrix with first elution buffer, while the impurities are dissociated from the matrix with second elution buffer.
  • the first elution buffer comprises urea at a concentration of about 4.3 M to about 4.9 M, preferably about 4.4 M to about 4.6 M, and more preferably about 4.5 M. In one of the embodiments, the first elution buffer comprises 20 mM sodium acetate, 5.0 mM EDTA, 4.5 M urea. In one of the embodiments, the bound impurities are eluted with second elution buffer having higher concentration of urea. In one of the embodiments, second elution buffer comprises urea at a concentration of about 5.5 M to about 8.0 M. In one of the preferred embodiments, second elution buffer comprises 6.0 M urea.
  • second elution buffer comprises 20 mM sodium acetate, 5.0 mM EDTA, and 6.0 M urea. It is preferable to use low conductivity elution buffer. In one of the embodiments, the elution buffers (first elution buffer and second elution buffer) has conductivity below 5 mS/cm, below 4 mS/cm, below 3 mS/cm, below 2 mS/cm, below 1 mS/cm.
  • partial binding or “partially bound” or variants thereof has been used interchangeably and is understood to mean a condition wherein the target protein, for example, papillomavirus L2 protein such as multitype or multimeric HPV L2 protein, does not strongly interacts (i.e., only interacts to a certain degree) with the ligands of the chromatography matrix such that it is possible to elute the target protein by varying the buffer composition compared to the buffer used for equilibration or washing of the chromatography matrix.
  • the partially bound target protein is eluted by passing first elution buffer.
  • the first elution buffer comprises higher concentration of urea compared to equilibration or wash buffer. In one of the embodiments, the first elution buffer comprises urea at a concentration of about 4.3 M to about 4.9 M, preferably about 4.4 M to about 4.7 M, more preferably about 4.5 M.
  • papillomavirus as used herein means any of the animal or human papillomaviruses such as, but not limited to, bovine papillomavirus (BPV), cottontail rabbit papillomavirus (CRPV), deer papillomavirus (DPV), European elk papillomavirus (EEPV), canine oral papillomavirus (COPV), Rhesus monkey papillomavirus (RhPV), rabbit oral papillomavirus (ROPV), or any of the human papillomavirus (HPV) types.
  • BBV bovine papillomavirus
  • CCPV cottontail rabbit papillomavirus
  • DDV deer papillomavirus
  • EEPV European elk papillomavirus
  • COV canine oral papillomavirus
  • RhPV Rhesus monkey papillomavirus
  • ROPV rabbit oral pap
  • human papillomavirus means any of the HPV type that infect human host and includes, but not limited to, HP VI, HPV2, HPV3, HPV4, HPV5, HPV6, HPV7, HPV8, HPV9, HPV10, HPV11, HPV12, HPV13, HPV14, HPV15, HPV16, HPV17, HPV18, HPV19, HPV20, HPV21, HPV22, HPV23, HPV24, HPV 25, HPV26, HPV27, HPV28, HPV29, HPV29, HPV30, HPV31, HPV32, HPV33, HPV34, HPV35, HPV36, HPV37, HPV38, HPV39, HPV40, HPV41, HPV42, HPV43, HPV44, HPV45, HPV46, HPV47, HPV48, HPV49, HPV50, HPV51, HPV
  • L2 protein or “minor capsid protein” has been used interchangeably herein to refer to the minor protein of the capsid of papillomaviruses that localizes along the inner surface of the virion within the central cavities beneath the LI (major capsid protein) capsomeres.
  • protein or "peptide” or “polypeptide” has been used interchangeably herein and means a polymer of amino acids linked through peptide bonds, but does not imply any limitation as to a specific length.
  • the term also includes fusion proteins, muteins, analogs or modified forms.
  • multitype L2 protein polypeptide has been used interchangeably to mean a fusion protein formed by joining fragments of L2 protein obtained from one or more papillomavirus types (animal and/or human papillomavirus types). Each fragment may be as short as ten amino acids in length and may extend up to any length of papillomavirus L2 polypeptide provided that it is not a full length papillomavirus L2 protein.
  • Such multitype or multimeric L2 protein may generally be represented as "peptide x(n)" wherein the "peptide” represents a fragment of L2 protein (generally beginning from 11 th amino acid of the full length L2 protein and extending up to the 300 th amino acid or less such that each such fragment is at least ten amino acids long) while the number "(n)” following "x” represents different "HPV types” (which may usually be two or more) from which the fragments of L2 protein are derived, or alternately the fragment of L2 protein may be repeated as many number of times as represented by the number "(n)” if belonging to only one HPV type (for example, 11-200 x3, 11-88 x5, 11-88 x8, 17-36 x22 and so on, wherein the number following "x” either represents as many different HPV types or number of times the fragment of L2 protein repeats if represented by only one HPV type).
  • fragments are typically joined by peptide bonds.
  • the multitype L2 protein or polypeptides may be prepared by the methods described in WO2009059325; Jagu, Subhashini et al. (Journal of National Cancer Institute 2009 101 :782-792; and Journal of Virology 2013 87(11):6127-6136).
  • a "fragment" of a reference polypeptide refers to a contiguous stretch of amino acids from any portion of the reference polypeptide.
  • a fragment may be of any length that is less than the length of the full length reference polypeptide.
  • HIC Hydrophobic Interaction Chromatography
  • resin or matrix or medium have been interchangeably used to denote chromatography stationary phase.
  • papillomavirus L2 protein for example, multitype or multimeric HPV L2 protein, which has not been purified or partially purified to render it free from one or more of its associated contaminants or impurities.
  • Such crude papillomavirus L2 protein often contain impurities, such as, but not limited to, host cell proteins, host cell DNA, aggregated or polydispersed forms of papillomavirus L2 protein, or degraded products of papillomavirus L2 protein.
  • the process of the invention is suitable to purify papillomavirus L2 protein from one or more of its associated contaminants.
  • Papillomavirus L2 protein is normally associated with contaminants such as, but not limited to host cell protein, host cell DNA, aggregated or polydispersed forms of papillomavirus L2 protein, or degraded products of papillomavirus L2 protein.
  • purified has been used interchangeably to mean papillomavirus L2 protein, for example multitype or multimeric HPV L2 protein which has been rendered free from its associated contaminants or impurities such that the papillomavirus L2 protein is at least about 60 % pure or more, or at least about 70 % pure or more, at least about 80 % pure or more, at least about 90 % pure or more.
  • a purified papillomavirus L2 protein is also understood to mean a papillomavirus L2 protein which has been rendered free from its associated contaminants to a degree specified by pharmacopoeal specification, or regulatory authority or established by relevant immunological correlate for the papillomavirus L2 protein. Purity of the protein may be measured by techniques known in the art.
  • vaccine refers to a pharmaceutically acceptable formulation of at least one antigen, for example, papillomavirus L2 protein. Such pharmaceutically acceptable formulations of an antigen may also include adjuvants, excipients, diluents, etc., that enhance the activity, stability, etc., of a formulation or administration.
  • antigen as used herein means a component of the formulation which is responsible for stimulating an immune response i.e., generation of antibodies against it when administered to an animal or human, such as papillomavirus L2 protein as described herein. Description
  • any fragment of the papillomavirus L2 protein may be purified by the process of the invention, preferably a multitype or multimeric HPV L2 protein.
  • the papillomavirus L2 protein of the invention may conveniently be produced by recombinant means.
  • Bacterial cells genetically engineered to express truncated papillomavirus L2 protein may be constructed using N-terminal fragments of papillomavirus L2 protein from any papillomavirus types using recombinant techniques known in the art (Sambrook, J. et al, Molecular Cloning: A Laboratory Manual, 2 nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel, Frederick M.
  • papillomavirus L2 protein or peptides are described in PCT Publication Nos. WO2006083984 and WO2009059325. Any length of papillomavirus L2 peptide from any papillomavirus type from N-terminus may be used provided that it is not a full length papillomavirus L2 protein.
  • papillomavirus L2 protein fragments that may at least be about 10 amino acids residues in length having a sequence corresponding to the amino acid sequence from the N-terminal amino acids 11-300 of full length papillomavirus L2 protein, or at least about 10 amino acids residues in length having a sequence corresponding to the amino acid sequence from the N-terminal amino acids 11-200 of full length papillomavirus L2 protein, or at least about 10 amino acids residues in length having a sequence corresponding to the amino acid sequence from the N-terminal amino acids 1 1-100 of full length papillomavirus L2 protein, or at least about 10 amino acids residues in length having a sequence corresponding to a sequence from the N-terminal amino acids 11-88 of full length papillomavirus L2 protein.
  • Fragments of L2 protein from two or more papillomavirus types may be cloned in expression vectors and transformed into a suitable host to express as a fusion protein or polypeptide of papillomavirus L2 protein for purification in accordance with the invention. It is preferred that a multitype HPV L2 polypeptide is expressed as a fusion polypeptide or protein such as those described in WO2009059325 and Jagu, Subhasini et al. (Journal of National Cancer Institute 2009 101 :782-792). Such fusion proteins may also be expressed in insect, yeast or mammalian cells currently used to express heterologous genes.
  • the multitype HPV L2 fusion protein may be expressed as inclusion bodies m E. coli as described by Jagu, Subhashini (Journal of Virology 2013 87(11):6127-6136).
  • the harvested cells may be pelleted by centrifugation before being washed with suitable buffers.
  • suitable buffers such as glycine phosphate buffer, Tris, MES, HEPES, etc. may be employed.
  • the buffer solution may additionally be supplemented with metal chelators such as EDTA, EGTA.
  • Cell lysis may be achieved by either mechanical or non-mechanical means. A mechanical means is preferred which may be achieved by agitation with abrasives or by liquid shear lysis methods.
  • Multitype or multimeric HPV L2 protein in the form of inclusion bodies may be isolated and solubilized by techniques known to the person skilled in the art, for example, inclusion bodies may be solubilized in buffer containing various additives such as detergents, denaturants, ionic concentrations, reducing agents chaotropic agents, polyols, chelating agents, protease inhibitors or combinations thereof (WO 199626277).
  • the solubilized inclusion bodies may be purified on hydrophobic interaction chromatography in accordance with the invention. Hydrophobic Interaction Chromatography (HIC)
  • HIC separates proteins according to differences in their surface hydrophobicity by utilizing a reversible interaction between the protein and the hydrophobic surface of the HIC medium or matrix.
  • the protein in a high salt buffer is loaded on the HIC column to promote interaction between hydrophobic moieties of the protein and insoluble, immobilized hydrophobic groups (ligands) on the matrix.
  • a decreasing salt gradient is used to elute proteins from the matrix.
  • the ligands commonly used with the hydrophobic chromatography matrix are those having alkyl groups with a carbon chain length of about 2 to about 10 carbon atoms, such as butyl, propyl, or octyl, or aryl groups such as phenyl functional groups attached to a resin derived from cellulose, agarose, or dextran.
  • HIC supports for gels and columns may be obtained commercially from suppliers such as GE Healthcare under the product names Butyl Sepharose 4 fast flow (FF), Butyl Sepharose 6 fast flow (FF), Octyl Sepharose 4 FF, Phenyl Sepharose 6 FF (High sub), Phenyl Sepharose 6 FF (low sub); Tosoh Corporation under the product names Butyl 650 M, TSK-Gel Phenyl 5PW; or Merck Millipore under the product name Fractogel.
  • FF Butyl Sepharose 4 fast flow
  • FF Butyl Sepharose 6 fast flow
  • Octyl Sepharose 4 FF Phenyl Sepharose 6 FF (High sub)
  • Phenyl Sepharose 6 FF Low sub
  • Tosoh Corporation under the product names Butyl 650 M, TSK-Gel Phenyl 5PW
  • Merck Millipore under the product name Fractogel.
  • Sodium, potassium or ammonium sulfates produce relatively high precipitation. It is these salts that effectively promote hydrophobic interaction and have a stabilizing influence on protein structure. Urea has been found to interfere with hydrophobic binding or interactions and is therefore not preferred to purify proteins.
  • hydrophobic chromatography medium has been used as a solid support to minimize the degree of misfolded proteins by passing a buffer containing urea in pulses to enhance recovery of refolded protein (Su, Z., Lu, D. and Liu, Z. (2011) Refolding of Inclusion Body Proteins from E. coli, in Protein Purification: Principles, High Resolution Methods, and Applications, Third Edition (ed J.-C. Janson), John Wiley & Sons, Inc.).
  • urea is used as an agent for regeneration or cleaning of hydrophobic medium or matrix after chromatography runs (Deutscher, Murray P. Methods in Enzymology 1990 187:339-343).
  • papillomavirus L2 protein such as multitype or multimeric HPV L2 protein
  • purify proteins for example, papillomavirus L2 protein such as multitype or multimeric HPV L2 protein
  • the method comprises purifying papillomavirus L2 protein such as multitype or multimeric HPV L2 protein by hydrophobic interaction chromatography using different concentrations of urea.
  • the method comprises using the urea concentration such that some of the papillomavirus L2 protein, preferably multitype or multimeric HPV L2 protein is partially bound to the hydrophobic chromatography matrix while the remainder of the papillomavirus L2 protein remain retarded onto the hydrophobic chromatography matrix.
  • the papillomavirus L2 protein retarded onto the hydrophobic chromatography matrix is collected by washing the hydrophobic chromatography matrix while the partially bound papillomavirus L2 protein is eluted. In one of the embodiments, the papillomavirus L2 protein retarded onto the hydrophobic chromatography matrix is collected by washing the hydrophobic chromatography matrix with a wash buffer post initial wash. In one of the embodiments, the partially bound papillomavirus L2 protein is eluted with first elution buffer.
  • the column before loading is equilibrated with equilibration buffer.
  • equilibration buffer Any suitable buffer known in the art, such as, e.g., acetate or MES, may be used. It is preferable to use buffering agents that are suitable for maintaining a pH range of at least about pH 3.5 to 4.5.
  • the pH of the equilibration buffer is between about 3.5 to 4.5, between about 3.6 to 4.4, between about 3.7 to 4.3, between about 3.8 to 4.2, between about 3.9 to 4.1 or about 4.0.
  • the conductivity of the equilibration buffer may be adjusted by varying ionic concentration of the buffer composition.
  • the equilibration buffer has the conductivity between about 10-17 mS/cm, between about 10-15 mS/cm, between about 10-13 mS/cm, between about 11-12 mS/cm.
  • an exemplary equilibration buffer contains 20 mM sodium acetate pH 4.0, 5.0 mM EDTA, 3.8 M urea with a conductivity of approximately I l12 mS/cm.
  • the papillomavirus L2 protein is loaded onto the column in equilibration buffer at a flow rate of about 10-80 cm/h. In one of the embodiments, the papillomavirus L2 protein is loaded onto the column at the flow rate of about 10 cm/h, 20 cm/h, 30 cm/h, 40 cm/h, 50 cm/h, 60 cm/h, 70 cm/h, or 80 cm/h. In one of the preferred embodiments, the papillomavirus L2 protein is loaded onto the column at the flow rate of about 50-70 cm/h, more preferably at about 60 cm/h. A person skilled in the art understand that the flow rate may be adjusted so as to allow sufficient time for the contaminants as well as target protein to interact with the matrix. For example, when the sample is concentrated a slow flow rate is preferred, while the flow rate may be increased when the sample is dilute.
  • the hydrophobic chromatography matrix may be washed with several column volumes (CV) of wash buffer after loading the papillomavirus L2 protein.
  • the chromatography matrix is washed at least about three CV, at least about four CV, at least about five CV, at least about six CV, at least about seven CV, at least about eight CV, or at least about nine CV of wash buffer.
  • Initial washes (for example, one or two column volumes of wash buffer) carry away or remove impurities which are collected as initial wash fractions.
  • Post initial washes removes or dissociates the papillomavirus L2 protein retarded onto the chromatography matrix which is usually pure and such fractions are collected as post initial wash fractions.
  • the papillomavirus L2 protein retarded onto chromatography matrix is collected by washing the hydrophobic chromatography matrix using a wash buffer comprising urea concentration similar or identical to the equilibration buffer.
  • the wash buffer comprises urea concentration at about 3.5 M to about 4.2 M, at about 3.6 M to about 4.1 M, at about 3.7 M to about 4.0 M, or preferably 3.8 M.
  • an exemplary wash buffer comprises of 20 mM sodium acetate, 5.0 mM EDTA, 3.8 M urea and conductivity between about 11 mS/cm to about 12 mS/cm.
  • the papillomavirus L2 protein retarded onto chromatography matrix is collected by washing the hydrophobic chromatography matrix post initial washes i.e., after completing the initial washes.
  • the post initial washes comprises a wash cycle comprising washing the chromatography matrix at least two times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, or at least 8 times with as many column volumes of wash buffer after completing the initial wash cycle.
  • the post initial washes comprises a wash cycle comprising washing the chromatography matrix at least about 2-6 times, at least 3-5 times with as many column volumes of wash buffer after completing the initial wash cycle.
  • the partially bound papillomavirus L2 protein may be eluted from the matrix with first elution buffer comprising higher concentration of urea compared to the wash buffer.
  • the first elution buffer comprises about 4.3 M to about 4.9 M urea, about 4.4 M to about 4.6 M urea, or preferably about 4.5 M urea.
  • the partially bound papillomavirus L2 protein is eluted by passing first elution buffer comprising 20 mM sodium acetate pH 4.0 containing 5.0 mM EDTA, 4.5 M urea and conductivity about 2-3 mS/cm.
  • the impurities bound to the matrix are eluted with second elution buffer containing high concentration of urea.
  • the second elution buffer comprises about 5.5 M to about 8.0 M urea.
  • the second elution buffer comprises about 5.5 M to about 6.5 M urea, preferably 6.0 M urea.
  • an exemplary second elution buffer comprises 20 mM sodium acetate, 5.0 mM EDTA, 6.0 M urea and a conductivity of 2-3 mS/cm.
  • the present invention is further exemplified by the following examples.
  • Example 1 purification of papillomavirus L2 protein by Hydrophobic Interaction Chromatography
  • the papillomavirus L2 protein comprising 11-88 amino acids from the N-terminus of five different HPV types (11-88 x5 representing HPV types HPV6, HPV16, HPV18, HPV31, and HPV39) was prepared according to the methods described by Jagu, Subhashini et al. (Journal of Virology 2013 87(11):6127-6136; Journal of National Cancer Institute 2009 101 :782-792; WO2009529325).
  • the papillomavirus L2 protein was expressed in Escherichia coli as inclusion bodies. Harvested cells were lysed and centrifuged to collect the pellet. The pelleted inclusion bodies were solubilized using urea and DTT (WO 199626277).
  • the solubilized inclusion bodies were centrifuged and the supernatant is collected and filtered (0.22 micron filtration).
  • the supernatant was diluted using equilibration buffer (20 mM sodium acetate pH 4.0, conductivity 11-12 mS/cm, 5.0 mM EDTA and 3.8 M urea).
  • equilibration buffer (20 mM sodium acetate pH 4.0, conductivity 11-12 mS/cm, 5.0 mM EDTA and 3.8 M urea.
  • the chromatography column (BPG, GE Healthcare) packed with Phenyl Sepharose 6 FF (High Sub) matrix is equilibrated with equilibration buffer (20 mM sodium acetate, pH 4.0, 5.0 mM EDTA, 3.8 M urea, conductivity 11-12 mS/cm).
  • the papillomavirus L2 protein is loaded at the rate of 60 cm/h.
  • the column is washed with up to six column volumes (CV) of wash buffer.
  • the fractions collected from the initial washes (with one or two column volumes of wash buffer) are discarded (initial wash fractions) while the rest of the fractions obtained from the later washes which contained papillomavirus L2 protein retarded onto the matrix were collected (post initial wash fractions).
  • the partially bound papillomavirus L2 protein was eluted with first elution buffer (20 mM sodium acetate pH 4.0, 5.0 mM EDTA, 4.5 M urea, and conductivity 2-3 mS/cm).
  • the wash fractions containing retarded papillomavirus L2 protein, and first elution fraction (represented as "II” in Figure-1) were pooled and purity was assessed by SDS-PAGE and found to be 90% ( Figure -2).
  • Second elution buffer (20 mM sodium acetate pH 4.0, 5.0 mM EDTA, 6 M urea, and conductivity 2-3 mS/cm) was used to remove the bound impurities from the chromatography matrix (represented as "HI” in Figure-1).

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Abstract

L'invention concerne un procédé de purification de la protéine L2 du papillomavirus à l'aide d'une matrice de chromatographie hydrophobe. Le procédé consiste à mettre en contact une protéine L2 du papillomavirus brut avec une matrice de chromatographie hydrophobe en présence d'urée et à collecter la protéine L2 du papillomavirus retardée et partiellement purifiée et liée par des étapes de lavage et d'élution.
PCT/IB2015/051347 2014-02-27 2015-02-23 Purification de la protéine l2 du papillomavirus WO2015128788A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000059927A1 (fr) * 1999-04-01 2000-10-12 Cantab Pharmaceuticals Research Limited Purification de preparations biologiques
WO2009059325A2 (fr) * 2007-11-02 2009-05-07 The Johns Hopkins University Compositions de peptide hpv multitype et procédés de traitement ou de prévention d'une infection par papillomavirus humain

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000059927A1 (fr) * 1999-04-01 2000-10-12 Cantab Pharmaceuticals Research Limited Purification de preparations biologiques
WO2009059325A2 (fr) * 2007-11-02 2009-05-07 The Johns Hopkins University Compositions de peptide hpv multitype et procédés de traitement ou de prévention d'une infection par papillomavirus humain

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MJ WILSON: "Removal of tightly bound endotoxin from biological products", JOURNAL OF BIOTECHNOLOGY, vol. 88, no. 1, 2001, pages 67 - 75, XP027295919, ISSN: 0168-1656 *

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