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WO1998018819A1 - Purification d'antigenes du virus respiratoire syncytial - Google Patents

Purification d'antigenes du virus respiratoire syncytial Download PDF

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Publication number
WO1998018819A1
WO1998018819A1 PCT/EP1997/006016 EP9706016W WO9818819A1 WO 1998018819 A1 WO1998018819 A1 WO 1998018819A1 EP 9706016 W EP9706016 W EP 9706016W WO 9818819 A1 WO9818819 A1 WO 9818819A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
rsv
resin
exchange chromatography
chromatography
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP1997/006016
Other languages
English (en)
Inventor
Alex Bollen
Dirk Gheysen
Sophie Houard
Jean-Paul Prieels
Moncef Mohamed Slaoui
Omar Van Opstal
Christophe Jacques Robert Andre
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
GlaxoSmithKline Biologicals SA
Original Assignee
SmithKline Beecham Biologicals SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB9622438.1A external-priority patent/GB9622438D0/en
Priority claimed from GBGB9622439.9A external-priority patent/GB9622439D0/en
Application filed by SmithKline Beecham Biologicals SA filed Critical SmithKline Beecham Biologicals SA
Priority to AU69085/98A priority Critical patent/AU6908598A/en
Publication of WO1998018819A1 publication Critical patent/WO1998018819A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18522—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to purification of respiratory syncytial virus (RSV) proteins and for use in vaccines that are able to clear the lungs from a subsequent viral infection without inducing significant pulmonary pathology.
  • RSV respiratory syncytial virus
  • U.S. patent 5,194,595 (Upjohn) describes chimeric glycoproteins containing immunogemc segments of the F and G glycoproteins of RSV and suggests that such proteins can be expressed from a variety of systems including bacterial, yeast, mammalian (e.g., CHO cells) and insect cells (using for example a baculovirus).
  • Wathen et al (J. Gen. Virol. 1989, 70, 2625-2635) describes a particular RSV F-G chimeric glycoprotein expressed using a baculovirus vector consisting of amino acids 1-489 of the F protein linked to amino acids 97-279 of the G protein.
  • the present invention is an improved method to purifiy RSV
  • F-G chimeric proteins which comprises contacting an impure solution containing said protein with a filter to remove viruses (viral clearance); passing the filtered solution through a cation exchange, a metal chelate and anion exchange resin
  • the present invention is an improved method to purifiy RSV F-G chimeric proteins, wherein the viral clearance step occurs after one or more of the following steps: cation exchange, metal chelate and/or anion exchange chromatography.
  • the present invetion comprises RSV F-G chimeric proteins obtained by the methods of the invention and a method to prepare a pharmaceutical composition comprising purification of RSV F-G chimeric proteins obtained by the methods of the invention and admixing said proteins with an adjuvant.
  • Figure 1 is the study on the induction of enhanced pulmonary pathology upon vaccination of RSV F-G.
  • Figure 2 is an outline of the Cotton rat experiment.
  • the present invention comprises an improved method to purify proteins that aggregate or form viral-like particles.
  • the present invention relates to RSV F-G chimeric proteins where it was discovered that such molecules readily aggregate in a purified state.
  • the present invention relates to a method to purify RSV F-G chimeric proteins, suitable for clinical use, by conducting a viral clearance step very early in the purification scheme, rather than as the last step, or one of the last steps, as is typically done.
  • the early viral clearance step can also be useful for purification of other antigens that aggregate. For example, many viral surface proteins (e.g., Hepatitis B surface antigen) tend to form particles.
  • the chimeric proteins of the instant invention comprise at least one immunogenic fragment from both human respiratory syncytial virus glycoproteins F and G.
  • the chimeric protein contains 80% (contiguous sequence) or more of the F and G extracellular domains, while lacking their respective cytoplasmic domains.
  • the G protein is lacking the signal sequence region.
  • the respective F and G protein transmembrane regions are also missing.
  • a preferred example is a RSV F-G chimeric protein comprising amino acids 1-526 of the F protein fused to amino acids 69-298 of the G protein.
  • the purification scheme of the instant invention involves the following steps. Starting with an impure solution (e.g., cell culture supernatant) of RSV F-G, a viral clearance step is conducted, followed by (in any order) a cation exchange, metal chelate, and anion exchange chromatography. As a regulatory guideline, the solution containing RSV F-G is then treated (e.g., by altering the pH, temperature, etc.) to inactivate any potential bacterial or viral contaminants. Lastly, the solution is filter sterilized. As noted in the preceding paragraph, the viral clearance step is a requisite for ensuring safety of materials for clinical use.
  • an impure solution e.g., cell culture supernatant
  • a viral clearance step is conducted, followed by (in any order) a cation exchange, metal chelate, and anion exchange chromatography.
  • the solution containing RSV F-G is then treated (e.g., by altering the pH, temperature, etc.) to inactivate any potential bacterial
  • Planova 15 is a preferred membrane because it removes small non-enveloped viruses. It has the advantage over Planova 35 in that it flters smaller particle sizes. However, unlike Planova 35, this membrane (PLANOVA 15) cannot be used at the end of the purification scheme as the chimeric F-G protein aggregates are the same size as small viruses that the membrane is intended to remove. Preferably, Planova 15 is loaded less than 1 mg protem per cm
  • clarifying filters or prefilters are used prior to the viral clearance step to remove contaminants that could otherwise plug the membrane and thus reduce the membrane's effectiveness and service life.
  • Ion exchange resins are well known in the art.
  • a preferable resin for cation exchange chromatography is SP Sepaharose (Pharmacia).
  • a preferable resin for anion exchange chromatography is Q Sepaharose (Pharmacia).
  • the solution containing the chimeric RSV F-G protein can undergo an adsorption/desorption/gel filtration step prior to the inactivation step.
  • the step can be performed with one resin (e.g., CM EMD TSK (Merck) or via a combination of resins (e.g., Ether Toyopearl (ToyoHaas) followed by a gel filtration (sizing) step with, for example, Sephacryl S resin (such as S-200 HR) or G25 (Pharmacia).
  • one resin e.g., CM EMD TSK (Merck) or via a combination of resins (e.g., Ether Toyopearl (ToyoHaas) followed by a gel filtration (sizing) step with, for example, Sephacryl S resin (such as S-200 HR) or G25 (Pharmacia).
  • Suitable antigens include an inactivated RSV virus, such as a formalin inactivated RSV virus, or antigens derived from the RSV virus such as the RSV F or G protein or immunogemc fragments thereof as disclosed for example in U.S. Patent 5,149,650, or a chimeric polypeptide comprising at least one immunogenic fragment from both RSV F and G proteins, advantageously an RSV F-G chimeric protein as disclosed, for example, in U.S. Patent 5,194,595, preferably expressed from CHO cells.
  • the CHO-expressed polypeptide is preferably a RSV F-G chimeric protein comprising amino acids 1-526 of the F protein fused to the amino acids 69-298 of the G protein.
  • each dose will comprise 1-1000 ⁇ g of protein, preferably 2-100 ⁇ g, most preferably 5-50 ⁇ g.
  • An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of appropriate immune responses in subjects. Following an initial vaccination, subjects may receive one or several booster immunizations adequately spaced.
  • the vaccine used in the present invention may comprise an additional adjuvant, preferably a saponin adjuvant such as QS21 as described for example in WO 9517210, optionally in the presence of a sterol, such as cholesterol as described for example in PCT/EP96/01464.
  • a saponin adjuvant such as QS21 as described for example in WO 9517210
  • a sterol such as cholesterol as described for example in PCT/EP96/01464.
  • CHO Kl cells derived from MCB O24M were transfected with 20 ug of pEE14-FG plasmid DNA twice CsCl purified using the Ca-phosphate - glycerol transfection procedure.
  • Cell clones were selected according to the procedure of the GS (glutamine synthetase ) expression system (Crocett et al BioTechn., 1990, Vol8, 662) and amplified in the presence of 25 micro molar mettoonine sulphoximine (MSX) in G MEM medium containing no glutamine and supplemented with 10% dialyzed FBS (Foetal Bovine Serum).
  • GS glutamine synthetase
  • MSX micro molar mettoonine sulphoximine
  • the expression level of CHO-FG 13.1 is 5-12 ug of FG/ml after treatment with butyrate. Under accumulation conditions and medium replacements (3 to 5 days ) yields of 16 to 28 ug of secreted FG protein /ml were obtained.
  • CHOK1 FG 13.1 cell line was subsequently adapted to grow in suspension and serum-free (S/SF) conditions using a proprietary growth medium.
  • S/SF serum-free
  • the elimination of sodium butyrate may be desirable as butyrate is suspected to affect glycosylation patterns of glycoproteins.
  • FG protein produced in CHO appears to be more stable than the baculovirus produced FG.
  • Both recombinant RSV surface glycoproteins have been included in an immunological evaluation. Both antigens are fusion proteins between F and G but differ in primary amino acid structure, in expression system (Baculovirus versus rec. CHO cells) and in the purification procedure.
  • a final polishing step is performed on a S-Sepharose column in the presence of Tween 80.
  • the purified antigen is heavily glycosylated (83% O- linked; 17% N-linked) and migrates on SDS-PAGE as a broad doublet around 100- 130 kDa with additional antigen related bands of lower MM.
  • the expression level at 20L scale is relatively low, ranging from 0.08 to 0.85 mg FG/L culture and the antigen is purified to 95% homogeneity with an overall yield of 21 % . This material was used in cotton rat experiment described below.
  • the purification process includes very harsh treatments such as reversed phase chromatography (organic solvent in strongly acidic conditions).
  • a refolding step by dialysis out of GuHCl in the presence of detergent (Tween 80) is included into the scheme.
  • This treatment leads, according to the authors, to native antigen as it is recognized in-vitro by a panel of 14 monoclonal antibodies directed against linear and conformational epitopes on the F and G sequences of the molecule
  • FG antigen is eluted with 20 mM TRIS-Cl pH 7.5, 3M NaCl, 1 % Tween 80. FG positive fractions are pooled and dialyzed at 4 °C against buffer B.
  • the dialyzed eluate from step II is loaded onto a column packed with Pharmacia SP Sepharose HP or fast flow resin.
  • the SP Sepharose HP or fast flow resin is equilibrated with 20 mM MES pH 6.5, 1 % thesit (buffer D).
  • the column is washed with buffer D until the absorbance at 280 nm returns to baseline.
  • the column is eluted successively with buffer D containing 150 mM NaCl, than 300 mM NaCl and finally 1 M NaCl.
  • FG is eluted with buffer D containing 300 mM NaCl.
  • the eluate could be, if necessary, concentrated on a Filtron OMEGA 30 or 50 kDa membrane.
  • the pool containing the antigen will be loaded onto 40 ml of Zn-chelate column (Pharmacia).
  • the flow-through fraction contains the antigen.
  • the pool containing the antigen will be loaded onto 40 ml of Zn-chelate column (Pharmacia).
  • the flow-through fraction contains the antigen.
  • Tween 20 0.1 to 1% V/V
  • This solution will then be filtered through PLANOVA 15 membrane.
  • the cotton rat model (see Example III) was chosen for evaluation of protection against the enhanced pulmonary irvflammatory pathology occurring in the presence of an inadequate immune response.
  • the pathology observed upon RSV challenge of animals vaccinated with Fi RSV Al(OH) 3 closely resembles what was observed in the infants described previously.
  • the animals were challenged under anesthesia by the intranasal route on day 49 using 10 6 pfu of live RSV/Long strain.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Virology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Cette invention concerne la purification de protéines du virus respiratoire syncytial (VRS) et l'utilisation de ces dernières dans des vaccins.
PCT/EP1997/006016 1996-10-29 1997-10-27 Purification d'antigenes du virus respiratoire syncytial Ceased WO1998018819A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU69085/98A AU6908598A (en) 1996-10-29 1997-10-27 Purification of respiratory syncytial virus antigens

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB9622438.1 1996-10-29
GB9622439.9 1996-10-29
GBGB9622438.1A GB9622438D0 (en) 1996-10-29 1996-10-29 Vaccine composition
GBGB9622439.9A GB9622439D0 (en) 1996-10-29 1996-10-29 Vaccine composition

Publications (1)

Publication Number Publication Date
WO1998018819A1 true WO1998018819A1 (fr) 1998-05-07

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1997/006016 Ceased WO1998018819A1 (fr) 1996-10-29 1997-10-27 Purification d'antigenes du virus respiratoire syncytial

Country Status (3)

Country Link
AR (1) AR011270A1 (fr)
AU (1) AU6908598A (fr)
WO (1) WO1998018819A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000062802A3 (fr) * 1999-04-20 2001-01-11 Smithkline Beecham Biolog Vaccin
WO2000062801A3 (fr) * 1999-04-20 2001-01-11 Smithkline Beecham Biolog Nouvelles compositions

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989005823A1 (fr) * 1987-12-23 1989-06-29 The Upjohn Company Glycoproteines chimeriques contenant des segments immunogeniques des glycoproteines du virus syncytial respiratoire humain
EP0593339A1 (fr) * 1992-10-14 1994-04-20 PASTEUR MERIEUX SERUMS ET VACCINS, Société Anonyme : Procédé de préparation d'antigènes et de vaccins de l'hépatite A (HAV)
WO1994015968A1 (fr) * 1993-01-08 1994-07-21 The Upjohn Company Procede de purification et de repliement de la glycoproteine fg du virus syncytial respiratoire humain

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989005823A1 (fr) * 1987-12-23 1989-06-29 The Upjohn Company Glycoproteines chimeriques contenant des segments immunogeniques des glycoproteines du virus syncytial respiratoire humain
EP0593339A1 (fr) * 1992-10-14 1994-04-20 PASTEUR MERIEUX SERUMS ET VACCINS, Société Anonyme : Procédé de préparation d'antigènes et de vaccins de l'hépatite A (HAV)
WO1994015968A1 (fr) * 1993-01-08 1994-07-21 The Upjohn Company Procede de purification et de repliement de la glycoproteine fg du virus syncytial respiratoire humain

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000062802A3 (fr) * 1999-04-20 2001-01-11 Smithkline Beecham Biolog Vaccin
WO2000062801A3 (fr) * 1999-04-20 2001-01-11 Smithkline Beecham Biolog Nouvelles compositions
EP1927366A1 (fr) * 1999-04-20 2008-06-04 GlaxoSmithKline Biologicals S.A. Vaccin combiné contre Streptococcus pneumoniae et le virus respiratoire syncytial

Also Published As

Publication number Publication date
AR011270A1 (es) 2000-08-16
AU6908598A (en) 1998-05-22

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