Role of Cell Culture Technology in new Vaccine Development

Transcription

1 Role of Cell Culture Technology in new Vaccine Development Asst. Prof. Dr.Kanokwan Poomputsa School of Bioresources and Technology King Mongkut s University of Technology Thonburi

2 Outline Introduction to cell culture Cell culture and vaccine production Cells as virus host JE vaccine, Influenza vaccine, Rabies vaccine Cells as protein factory Influenza vaccine (protein, VLP) Cells as antigen presenter Cancer vaccine (personalized vaccine)

3 Cell Culture Process by which animal cells are grown under controlled laboratory conditions.

4 Maintaining cell culture Basic environmental requirements Controlled Temperature Appropriate medium that maintains the correct ph and osmolarity Good substrate for cell attachment

5 Animal cell culture media is complex Animal is made up of hundreds of different cell types, each specialized to perform one or a few functions. Different cell types synthesize many different molecules needed, and these are secreted into the extracellular fluid and are circulated by the blood throughout the body. The mammalian cell medium is an attempt to recreate this extracellular fluid.

6 Growth factors are present in cell media

7 Culture Medium Culture medium should provide; basic nutrition requirements; Amino acids, vitamins, minerals and carbohydrates which cells used to build new proteins and other components and provide the energy necessary for metabolism Necessary growth factors and hormones 5-20% of animal blood such as calf serum Help to regulate and control the cell s growth rate and functional characteristics. Regulate the ph and osmolarity The medium control the ph range and buffers the cells from abrupt changes in ph.

8 Cell Culture Removal of cells, tissue or organ from an animal or plant and their subsequent placement into an artificial environment conducive to growth. Organ Culture/tissue culture A three dimensional culture of undisaggregated tissue retaining some or all of the features of the tissue in vivo. Cell Culture Single cells, no longer organised as tissues. Derived from dispersed cells taken from the original tissue.

9 Isolation of cells There are 2 basic methods; Explant Cultures : small pieces of tissues are attached to a glass or treated plastic culture vessel and bathed in culture medium. After a few days, individual cells will move from the tissue explant out onto the culture vessel surface or substrate where they will begin to divide and grow. Enzymatic Dissociation : addition of proteolytic enzyme e.g. Trypsin or collagenases, to the tissue fragment to dissolve the cement holding the cell together. This create a suspension of single cells.

10 Explant Enzymatic Dissociation Human foreskin explants in Culture dish (2 of 9 explants failed to grow) 1 week old Poeciliopsis lucida cells from embryo

11 Cell culture (Primary cell VS Continuous cell) When cells are surgically removed from an organism and placed into a suitable culture environment, they will attach, divide and grow. Most primary cell cultures have limited lifespan. After a certain number of population, doubling cells undergo the process of senescence and stop dividing, while generally retaining viability. Some cells may become immortal, continue to divide indefinitely, and are called continuous cell lines. They have undergone a fundamental irreversible changes or transformation.

12 Continuous Cell Lines An established or immortalized cell line has acquired the ability to proliferate indefinitely through; Spontaneously modification such as random mutation by drug, radiation or viruses. Deliberate modification such as artificial expression of the telomerase gene.

13 Continuous cell lines CHO (Chinese hamster ovary) cells Vero (Monkey kidney) cells MDCK (canine) cells BHK (Baby hamster kidney) cells NSO (mouse myeloma) cells HEK 293 (human embryonic kidney) cells PER.C6 (human retinoblasts) cells MRC-5 (fetal lung) cells

14 Cell Culture Systems Monolayer Culture Systems Adherent cells or Anchorage- Dependent cells cells attach to a glass or treated plastic substrate Cell growth is limited by available surface area on which cells can grow Suspension Culture Systems Anchorage-Independent - Cells grow floating free in suspension

15 Monolayer Culture Systems Dishes T-flaskes Roller bottles Microcarrier

16 Suspension Culture Systems Erlenmayer flasks Spinner vessels

17 Types of Cells Epithelial-like : Cells that are attached to a substrate and appear flattened and polygonal in shape. Lymphoblast-like : Cells that do not attach normally to a substrate but remain in suspension with a spherical shape. Fibroblast-like Cells that are attached to a substrate and appear elongated and bipolar, frequently forming swirls in heavy cultures.

18 Cell Culture and Vaccine Manufacturing Control of product properties e.g. glycosylation Cost efficient manufacturing process especially for high volume products Speed in development, especially for early phase

19 Cell cultures can be scaled-up for biomanufacturing using bioreactors

20 Cell Culture and Vaccine Production Viral vaccines Cultivation of virus for vaccine production e.g. polio, rabies, chicken pox, hepatitis B & measles Recombinant protein vaccines Production of recombinant proteins from animal cells using different expression system

21 Viral Vaccine Production Cell-Culture-Based Vaccine Production virus is propagated in animal cells and harvested downstream processing parameters for purification filling, and packaging of the vaccine cells Infection with seed virus Splitting, inactivation and purification

22 Cells for virus production Propagation of cells Virus- Infection Seed cells Preculture Virus Propagation Production fermenter Cell Viral Particles

23 Vaccine Production Plant Fermenter 4 Virus Production Fermenter 3 Cell propagation High Cell Density Fermenter 2 Cell propagation Fermenter 1 Cell propagation

24 Purification Virus- Infection Virus Propagation Seed cells Preculture Harvest Ultrapurification Centrifuge (Cells/Debris) Ultracentrifugation; BPL-Inactivation/ + Virus splitting Chromatography + Ultra-/Diafiltration Filter (small particles)

25 Japanese Encephalitis Vaccines JE virus (JEV), a mosquito-borne flavivirus, is the most common vaccine-preventable cause of encephalitis in Asia Two JE vaccines are licensed in the United States. An inactivated mouse brain derived JE vaccine (JE-VAX [JE-MB]) An inactivated Vero cell culture-derived vaccine (IXIARO [JE-VC])

26 Japanese Encephalitis Vaccines An inactivated mouse brain derived JE vaccine [JE-MB]) It has been licensed since 1992 to prevent JE in persons aged 1 year traveling to JE-endemic countries. Use of mouse brains as the substrate for virus growth has raised concerns about the possibility of neurologic side effects associated with the JE vaccine. Moderate to severe neurologic symptoms including encephalitis, seizures, gait disturbances, and parkinsonism, have been reported at a rate of cases per 100,000 vaccinees with variation by country. Supplies of this vaccine are limited because production has ceased.

27 Japanese Encephalitis Vaccines An inactivated Vero cell culture-derived vaccine (IXIARO [JE-VC]) It was licensed in March 2009 for use in persons aged 17 years. JE-VC does not contain gelatin or murine proteins, it might be associated with fewer hypersensitivity or neurologic adverse events than the mouse brain derived vaccine.

28 Japanese Encephalitis Vaccines MMWR Recommendations and Reports March 12, 2010 / Vol. 59 / No. RR-1

29 Influenza Vaccines Influenza Vaccine Manufacturing Today Vast majority (90%) of licensed capacity is in egg-based products Reliable process for seasonal production but the eggbased process has significant limitations and lacks flexibility (embryonated eggs require~6 months from order to delivery) Egg Lead Time ~6 Months Production Time ~6 Months 1x ~1x No Chickens No Eggs No Vaccine

30 Influenza Vaccines Cell Culture: An alternative to Egg-based production No egg lead time involved, production can start as soon as seed virus is available Cell culture is a practical alternative Industrializable can be run routinely and cost effectively Established processes can be run at practical scale to provide sufficient vaccine for interpandemic and pandemic needs

31 Cell-based vaccine production: critical points Cell line Virus production Regulatory path Industrialization capacity Growth in suspension or on microcarriers Synthetic or complex medium Vaccine manufacturing and formulation Whole virus, split, subunit vaccines Removal of DNA Adjuvants to enhance immunogenicity and dose sparing

32 Influenza Vaccines Cell lines PerC.6(Crucell) EBx : a stem cell line derived from chicken embryos (Sigma-Aldrich Group) VERO: a kidney cell from the African green monkey (GSK) MDCK : Madin-Darby canine kidney cells used by Chiron Requirements Good cell-virus interaction Broadly and highly permissive for a wide variety of flu strains Restricted growth of non-flu human pathogens that may be present in the viral seed Scalable, industrializable high yield, high volume production Cell growth in chemically defined medium (no animal-derived components)

33 Cell-based vaccine production: regulations There must be documentation to support the complete removal of the cells from the final product and documentation to show that the cell line does not bring any transforming agent (oncogenic transformation) into the final product Documentation must show that no genetic material is left from the cell line in the final product that can cause tumors to be formed. All residual DNA must be removed or inactivated so it cannot give rise to tumors in animal models Documentation must show the removal and/or inactivation of infectious and/or oncogenic agents from the final product, regardless of whether they originated in the media or the cell line.

34 Influenza Vaccines EU approves Novartis's cell-based flu vaccine Jun 13, 2007 (CIDRAP News) The European Union (EU) has approved Novartis's seasonal influenza vaccine, Optaflu, the first flu vaccine grown in cell culture rather than eggs. The vaccine has been approved for use in all 27 EU member states plus Iceland and Norway.

35 Rabies Vaccine Rabies is a zoonotic disease caused by RNA viruses in the Family Rhabdoviridae, Genus Lyssavirus. Virus is typically present in the saliva of clinically ill mammals and is transmitted through a bite. After entering the central nervous system of the next host, the virus causes an acute, progressive encephalomyelitis that is almost always fatal. Cell-based rabies vaccines which are licensed in the United States: human diploid cell vaccine, MRC-5 (HDCV, Imovax Rabies, sanofi pasteur), purified chick embryo cell vaccine, primary cultures of chicken fibroblasts (PCECV, RabAvert, Novartis Vaccines and Diagnostics)

36 Cell Culture and Vaccine Production Viral vaccines Cultivation of virus for vaccine production e.g. polio, rabies, chicken pox, hepatitis B & measles Recombinant protein vaccines Production of recombinant proteins from animal cells using different expression system

37 Established & Novel Expression Established Systems Systems Slow SPEED Fast transgenic plant mammalian cells COST E. coli baculovirus yeast $$$$ $ transgenic mammalian cells plant Unlikely baculovirus FDA Approval yeast E. coli Likely transgenic plant baculovirus Promising New Platforms yeast E. coli mammalian cells Cell free Nucleic acid duckweed crustacean

38 Animal Cell Expression System A. Transient transfection B. Stable transfection C. Viral vector-mediated protein expression (e.g. adenovirus, baculovirus)

39 Influenza Virus Vaccine based on the Baculovirus-Insect Cell Expression System FluBlok First recombinant influenza vaccine First cell-based influenza vaccine in U.S. FDA licensure in 2010 No additional safety or efficacy studies required The pandemic solution Only pandemic vaccine that can be quickly manufactured and/or transferred to and manufactured in other countries

40 HA (Hemagglutinin) Coat of the influenza virus Antibodies against HA protect against influenza Changes in HA require annual update of vaccine

41 Recombinant HA protein from Baculovirus-Insect Cell Expression System Baculovirus with influenza gene Days post infection Recombinant Influenza HA protein Produced in infected Insect Cells Purification of recombinant HA protein Formulate With PBS To Vaccine

42 Safety & Immunogenicity of FluBlok Potential Benefits rha antigens are produced in insect cells protein based vaccine with low endotoxin content highly purified and does not contain egg protein or other contaminants from eggs Selection or adaptation of influenza virus strains that produce at high levels in eggs is not required FluBlok can be prepared within 2 months FluBlok does not require large amounts of embryonated chicken eggs Manufacturing of FluBlok does not require biocontainment facilities Manufacture of rha does not include formalin inactivation or organic extraction procedures

43 Pandemic Preparedness Development Timeline recombinant HA1 vaccine

44 Virus-Like Particle (VLP)-based Influenza Vaccines What are VLPs? Made of recombinantly-expressed viral proteins that spontaneously assemble into 3-D structures similar to parent virus By electron microscopy, VLPs mimic the parent virus Immune response is similar to that which would be seen if exposed to the parent virus Do not contain genetic material Cannot replicate

45 How are Influenza VLPs Constructed?

46 Potential Immunologic Advantages of Influenza VLPs

47 Possible Limitations of Vaccine Production using cell culture Not all infectious agents can be grown in culture Animal/human cell culture is expensive Yield of viruses from cultures can be low Safety precautions for culture of live agents

48 Personalized Cancer Vaccine Use of material from a patient s own tumors to induce immune response Cells harvested from patients are fused with immune cells and expanded in the special instrument--

49 Antigen-Presenting Cells and cancer

50 DC-based cancer vaccine Dendritic cells (DC), a leukocyte population, represent unique antigen-producing cells capable of sensitizing T cells to both new and recall antigens. DCs are the most potent antigen-presenting cells (APS). Tumor antigens in different forms (DNA, RNA, proteins, peptides, viruses, cell lysates) become immunogenic when presented to T-lymphocytes by DCs. Immunization with ex vivo generated DC has proven feasible and permits the enhancement as well as the dampening of antigen-specific immune responses in man. Several DC-based clinical trials have demonstrated potent immunological and some clinical responses.

51 DC loading and activation Ex vivo generated dendritic cells(dc) can be loaded with antigens and re-infuse to the patients, or they can be used for ex vivo expansion of anti-tumor lymphocytes.

52 DC loading and activation Ex vivo generated dendritic cells(dc) can be loaded with antigens and re-infuse to the patients, or they can be used for ex vivo expansion of anti-tumor lymphocytes. scale up of patient Derived cells

53 Ex vivo expansion of loaded APCs AutovaxID GMP and GTP regulations

54 Personalized Cancer Vaccine More than 150 DC-based clinical studies for the treatment of solid or hematological malignancies have been reported in 2008 including; Melanoma ( 40 published clinical studies) prostrate cancer (20) Renal cell carcinoma (16) Breast cancer (12) Multiple myeloma (9) Leukemia (9) Colorectal cancer (9) Glioma (9) Nencioni, A. et.al. Critical Reviews in Oncology/Hematology 65(2008),

55 Role of Cell Culture Technology in new Vaccine Development Host for viral vaccine production Factory for gene expression for vaccine recombinant proteins VLP Personalized vaccine

56 Thank you คณะทร พยากรช วภาพและเทคโนโลย School of Bioresources and Technology Bangkhuntien Campus Established in