Manufacturing of Cervarix


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  • -Neoplasm: abnormal mass of tissue.
  • - Background of the vaccine
  • -Mainly targeted at young girls who havent started having sex.
  • Cervarix was EU approved in 2007 and the EU is currently its largest market. In 2009 Cervarix was approved by the FDA but sales forecasts anticipate limited market penetration, which is unsurprising given that Merck and Co’s Gardasil came to market 3 years earlier.However, entering the US market, world’s biggest drug market, is an important endorsement for a product that is judged to be Glaxo’s biggest individual sales growth driver over the next six years.Gardasil game to the global market a year before Cervarix, hence its higher annual sales.But the HPV vaccine market is predicted to almost double between 2008-2014 and because GSK is already established in emerging markets it is forecast to capture a larger segment
  • One dose of Cervarix, contains 20μg of HPV-16 L1 and 20μg of HPV-18 L1 proteins adjuvanted with AS04 and is presented as a sterile turbid liquid suspension for injection, filled as a 0.5ml monodose in either syringes or vials stored at 2-8°C. A shelf life of 3 years is proposed. The vaccination schedule includes 3 doses administered at 0, 1, and 6 months intramuscularly.
  • High-Five cells are egg cells from Trichoplusiani. These cells are less expensive to maintain since they may be grown without fetal calf serum.All three cells lines may be grown at room temperature (optimum = 25 - 27°C), and do not require CO2 incubators. Their doubling time is 18-24 hours
  • Several features make the Baculovirus Expression Vector System attractive for researchers: - High levels of heterologous gene expression are often achieved compared to other eukaryotic expression systems, particularly for intracellular proteins. In many cases, the recombinant proteins are soluble, post-translationally modified and easily recovered from infected cells late in infection when host protein synthesis is diminished.-The cell lines used for AcMNPV propagation grow well in suspension cultures, permitting the production of recombinant proteins in large-scale bioreactors.- Expression of hetero-oligomeric protein complexes can be achieved by simultaneously infecting cells with two or more viruses or by infecting cells with recombinant viruses containing two or more expression cassettes.- Baculoviruses have a restricted host range, limited to specific invertebrate species. They are safer to work with than most mammalian viruses since they are noninfectious to vertebrates.
  • HPV major capsid protein L1 can fold correctly and self-assemble into VLPs when expressed in eukaryotic cells. VLPs aim to protect against the development of cervical cancer; protection would be mediated by the induction of high titres of neutralizing antibodies against the HPV genotypes in the vaccine that prevent the virus infecting the transformation zone, a metaplastic area between the squamous and columnar epithelia in the cervix, where most cancers arise.The L1 proteins of HPV-16 and HPV-18 areseparately produced using a recombinant Baculovirus expression system and the insect cell line Hi-5Rix4446 derived from Trichoplusiani.The Baculovirus Expression Vector System is based on the introduction of a foreign gene into nonessential for viral replication genome region via of homologous recombination with a transfer vector containing target gene. The resulting recombinant Baculovirus lacks one of nonessential gene (polh, v-cath, chiA etc.) replaced with foreign gene encoding heterologous protein which can be expressed in cultured insect cells and insect larvae.
  • These proteins are produced in separate production runs using the Baculoviruses expression vector system (BEVS) using GSK´s insect cell line cloned from the Hi-5 Trichoplusianiinsect cell line and HPV-16 L1 or HPV-18 L1encoding recombinant Baculoviruses. Following expression the L1 proteins are purified by a series of chromatographic and filtration procedures and are finally assembled into (VLP) closely resembling the configuration of native HPV virus particles.First, recombinant baculovirusinocula of each serotype are prepared by amplification of the respective working seeds in the Hi-5 insect cell cultures.Next, these inocula are used for the infection of Hi-5 production cell culturesL1 protein is released from the cells by osmotic shockOsmotic shock or osmotic stress is a sudden change in the solute concentration around a cell, causing a rapid change in the movement of water across its cell membrane. Alternatively, at low concentrations of solutes, water enters the cell in large amounts, causing it to swell and either burst or undergo apoptosis
  • . Virtually any commercially available hydroxyapatite column material may be used in this invention. It is preferable to use a ceramic hydroxyapatite which a particle size of approximately 20-50 .mu.m and approximately an 800 .ANG. pore size. One such commercially available hydroxyapatite is sold by BioRad as "Ceramic hydroxyapatite, Type II". However, others are effective as well.In preparing the chromatography step of the purification process, it is recommended that the column feed be in a buffer with a pH of 6-8, and preferably approximately 7. A preferred buffer is 50 mM MOPS [3-(N-morpholino)propanesulfonic acid] ata pH of 7.0 and also containing 1.25 M NaCl.The partially purified VLPs in the buffered solution is allowed to come into contact with the hydroxyapatite medium under conditions which allow the VLPs to bind to the hydroxyapatite. These conditions include a broad temperature range; roomtemperature is preferred. Flow rate may also vary greatly, and a preferred range is approximately 90 cm/hour.After the VLPs are bound to the hydroxyapatite, the next step is recovery of the purified VLPs from the hydroxyapatite with an elution buffer. A preferred elution buffer solution contains a phosphate anion, such as a sodium or potassiumphosphate solution. Preferred molar ranges are from about 0.05 M to about 1 M, with approximately. 0.2 M being preferred. The pH of the elution buffer should range from about pH 6-8, with a pH of about 7 being preferred.Other advantages of the method of this invention include: (a) no requirement for special chromatography equipment and techniques; (b) the method is rapid, requiring no buffer changes; and (c) the method provides an excellent yield of HPV L1.
  • The Hi-5 Rix4446 insect cell line is examined for the presence of adventitious agents.
  • The capacity of the individual steps of the purification cascade to efficiently eliminate viral contaminants has been demonstrated in viral clearance studies.
  • Manufacturing of Cervarix

    1. 1. Group 6
    2. 2. • Cervical Cancer & HPV• Cervarix (HPV vaccine)• The Manufacturing process• Quality Control• Conclusion
    3. 3. • Cervical cancer is a malignant neoplasm of the cervix uteri or cervical area.• Worldwide, it is the twelfth most common and fifth most deadly cancer in women.• It affects about 16 per 100,000 per year and kills about 9 per 100,000 per year globally.
    4. 4. • Cervarix is a preventive vaccine against certain types of cancer causing human papillomavirus.• Manufactured at GlaxoSmithKline Biologicals S.A. in Belgium.• Aims to prevent infection from HPV types 16 & 18 that cause about 70% of cervical cancer cases.• Developed, in parallel, by researchers at Georgetown University Medical Center, the University of Rochester, the University of Queensland Australia and the US National Cancer Institute
    5. 5. • Approved for use in females 10-25 years of age.• Consists of 3 doses of 0.5 mL each, by intramuscular injection at 0, 1 and 6 months.• The vaccine is available in 0.5 mL single dose vials and prefilled TIP-LOK syringes• Cervarix has shown to be 92% effective and effective for more than 4 years.• Shelf life: 3 years.
    6. 6. Cervical cancer vaccine Annual Sales World Wide ($m)salesProduct Company 2008 2010 2012 2014Gardasil Sanofi-Pasteur 865 1,222 1,686 1,984 MSDGardasil Merck & Co 1,403 1,227 1,399 1,372Cervarix GlaxoSmithKlin 231 797 1,246 1,353 eTotal HPV vaccine market 2,499 3,246 4,331 4,709 Adapted from GSK: 2010 Annual Report, Sanofi 2009 Annual Report
    7. 7. 2000 1800HPV sales ($m) 1600 1400 1200 1000 800 600 400 200 0 2014 2012 2010 2008
    8. 8. • Cervarix, HPV-16/18 L1 AS04 vaccine contains recombinant C-terminally truncated major capsid L1 proteins of HPV types 16 and 18 as active ingredients• The first vaccines for humans from Baculovirus Expression System
    9. 9. • The L1 proteins of HPV-16 and HPV-18 are separately produced• Usage of a:  recombinant Baculovirus expression system  insect cell line Hi-5 Rix4446 derived from Trichoplusia ni
    10. 10. • High levels of heterologous gene expression• Grows well in suspension cultures, easy for large- scale production• Safe (restricted to infection of invertebrate species)• Cheaper as Hi-5 Rix4446 can be cultured in serum free medium
    11. 11. Amplification of Seed Recombinant Formulation, filling and packaging Baculovirus Extraction of Recombinant Combination of HPV 16 and 18 L1 Baculovirus inocula proteins Infection of Trichoplusia ni Hi-5 Sterile filtration of L1 VLPproduction cell lines in fermenter Purification by L1 proteins are extracted by chromatography, nanometric methods of osmotic shock filtration and ultrafiltration
    12. 12. • The cell lysate may be subjected to a microfiltration and ultra-filtration process• Cation- exchange chromatogrophy• Hydroxyapatite Chromatography - hydroxyapatite as the column medium - elution with a buffer solution containing phosphate anion - removes a large amount of contaminants from a partially purified cellular lysate - Removes contaminating biomolecules, including DNA, lipids and proteins are removed from the lysate.
    13. 13. • The L1 proteins• AS04 adjuvant system composed of- aluminium hydroxide- 3-O-desacyl-4.- monophosphoryl lipid A (MPL)• The MPL immunostimulant- a detoxified derivative of the lipopolysaccharide ofthe gram negative bacterium Salmonella minnesotaR595 strain.
    14. 14. CELL SEED AND BANKING• The MCB, WCB, MS and WS and End of Production Cells (EPC) are tested for identity, purity and safety• The Assays are: – Tumourogenic Potential – Adventitious Agents – Genetic Stability – Specific Viral Contaminants
    15. 15. PROCESS CONTROL & VALIDATION• The 3 unit-steps of the drug substance production process are tested for consistency: – The recombinant HPV-16/18 baculovirus inoculum production process – The L1 single harvest production and L1 extraction – The L1 antigen purification process. MANUFACTURING PROCESS DEVELOPMENT• the final process purified bulks, are assessed by subjecting each type of materials to a series of physico-chemical analyses and immunogenic property tests.
    16. 16. Physico-Chemical Analyses Immunogenic Properties• SDS-PAGE • Antigenic activity• Western blotting• Capillary electrophoresis • Binding to polyclonal sera• Mass spectrometry • Immune response elicited in• Amino acid analysis mice• Peptide mapping• N-and C-terminal analysis• Infra-Red and circular dichromism spectroscopy• Size exclusion chromatography• Electron microscopy• Disc centrifugation size analysis
    17. 17. • Cervarix is a vaccine that prevents infection from HPV types 16 and 18• Market• Manufacured and purified from expression of L1 proteins in the Hi-5 Rix4446 insect cells
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