Production system of biologics
•
2 gefällt mir•645 views
This presentation deals with different types of production system like bacterial,insect,fungal,animal etc.
Empfohlen
Weitere ähnliche Inhalte
Was ist angesagt?
Was ist angesagt? (20)
Ähnlich wie Production system of biologics
Ähnlich wie Production system of biologics (20)
Mehr von Erin Davis
Mehr von Erin Davis (18)
Kürzlich hochgeladen
Kürzlich hochgeladen (20)
Production system of biologics
- 2. Production Systems Bacteria Yeast Plant tissue culture Animal cells (CHO) Transgenic animals Insect Expression of recombinant proteins Adapted from www.cloud-clone.com/
- 3. Bacteria For e.g, Escherichia coli (Gram negative bacteria) strains in the production of recombinant Insulin Pros Well characterized system High yield and cost effective Well-established fermentation procedures Cons Poor secretion Inclusion bodies: costly to renature inactive product Others: Accumulation of toxic metabolites, Proteolysis
- 4. Fungal For e.g, Saccharomyces cerevisiae and Pichia pastoris in the production of recombinant Insulin Pros Post translational modifications: Disulphide bonds Secretion possible Cons Safety: use of Methanol Glycosylation of non-glycosylated proteins (Half-life and Immunogenicity)
- 5. Insect E.g. Trichoplusiani, Hi 5 insect cell line used to produce Cervarix HPV vaccine Baculovirus-insect cell system (BEVS) Pros Target gene transfer – high efficiency High recombinant protein yields Cons Complex, defined media; costly Construction & amplification of viral stocks – 2-3 weeks Clone into bacteria transfer vector Recombinant baculovirus generation Small scale expression test Optimization and large scale expression
- 6. Plant Tissue Culture E.g. human serum albumin, derived from Nicotiana tabacum suspensions culture Plant-cell based expression system Adapted from Singhabahu et al., 2016 Pros High efficiency expression technique and scalable Intrinsically safe – neither harbor human pathogens nor produce endotoxins Cons Reduction in genetic diversity Disease infection may continue through generation
- 7. Animal cell culture Animal cell cultures are the recent achievements and perspectives in the production of biopharmaceuticals Efficacy of several humanized monoclonal antibodies that are used for therapeutic use E.g. Trastuzumab, Bevacizumab, Alemtuzumab, Belimumab Production Fused cells, myeloma cells, B cells. HAT (Hypozanthine Aminopterine thymidine) medium Blocks the De Novo pathway of production of nucleotide bases Only the Hybrid cells are capable of producing HGPRT (Hypoxanthine-Guanine Phospho Ribosyl Transferase) HGPRT- Induction of the production of Purines and Pyrimidines by Salvage pathway Hybrid cells can survive the HAT medium
- 8. Animal cell culture Pros Physiological & Physio-chemical environment is controlled Easy maintenance of Cell Homogenecity Long term storage using liquid Nitrogen and DMSO Usage of animals for scientific study can be reduced Cons Sterility and aseptic maintenance Capital investment is high and effective maintenance required High chance of genomic variation due to rapid growth
- 9. Recombinant Biological Products • Insulin • Interferons • Erythroprotein • Plasminogen activators • Blood clotting factors
- 10. Steps to making recombinant biological products 1. Develop host 2. Establish cell bank 3. Protein production system 4. Purification 5. Analysis 6. Formulation
- 11. Recombinant Human Insulin Production Separate A and B chain production
- 12. Recombinant Human Insulin Production Working Cell Bank Vial Thaw Pre-culture (n 1) (shake flask) Seed Culture (n 1) (Fermentor) Main Fermentation 1. Cell Growth 2. Induction and Protein Expression Harvest (Centrifugation) Clarification (Depth filtration) Cation Exchange Chromatography Anion Exchange Chromatography Ultrafiltration / Diafiltration (Buffer Exchange) Drug Substance Formulation Bulk Drug Substorage Seed train Fermentation Primary Recovery Upstream Process Downstream Process Inoculum expansion
- 13. General considerations for Recombinant Protein Production Adapted from Mudassar Ahmad et al, 2014P.pastoris
- 14. Conclusion Bacteria: first choice for protein production; fact growth and simple culture procedures Yeast: Post-translational modifications and secretion of proteins Insect: High recombinant protein yield with high efficient target gene transfer Plant: High efficiently expression system for ease of production, produce safe edible vaccines Animal Cell Cultures are the recent achievement for the production of Biologics and Biosimilars Efficacy of several humanized monoclonal antibodies that are required for therapeutic use
- 15. Jozala, A. F., et al. (2016). Biopharmaceuticals from microorganisms: from production to purification. Brazilian Journal of Microbiology. 47S:51–63 Baeshen, N. A., et al. (2014). Cell factories for insulin production. Microbial cell factories.13:141 Krammer, F. (2010). Grabherr, R. Alternative influenza vaccines made by insect cells. Trends in molecular medicine. 16: 313-320 Weber, W., & Fussenegger, M. (2009). Insect Cell-Based Recombinant Protein Production. Principles and Practice. 263–277 Singhabahu, S., Hefferon, K., & Makhzoum, A. (2016). Plant Molecular Pharming. In: Jha S. (eds). Transgenesis and Secondary Metabolism. 1–26 https://www.easybiologyclass.com/advantages-and-disadvantages-of-animal-cell-culture-short-lecture-notes/ https://www.abcam.com/protocols/a-comparison-between-polyclonal-and-monoclonal https://www.google.com/search?biw=1366&bih=606&tbm=isch&sa=1&ei=TxCmXJuIC9T-9QO- uLmYBA&q=animal+tissue+culture&oq=animal+tissue+culture&gs_l=img.3..35i39j0l9.44080.45251..46750...0.0.. 0.124.1551.9j7......1....1..gws-wiz-img.aZngU49oszY#imgrc=x7LroZ4NKeWa-M: References
Hinweis der Redaktion
- Recent years, insect cells have been widely used for biologically active recombinant proteins production. An example is the use of Hi5 insect cell line to produce HPV vaccine by Cervarix. Baculovirus-insect cell system is the most widely used insect cell-based expression system. In practice, the gene(s) of interest is first cloned into Baculovirus donor plasmid. In the following step, the constructed target gene-Baculovirus donor vector transform a special E.coli strain for generation of recombinant bacmid, which is then extracted and used for transfection of Hi5 insect cell to produce the first batch of recombinant baculovirus. Next, a small scale expression test is carried out. Once the cells are inspected to display signs of infection, the recombinant baculovirus is then harvested and amplified to produce high titer stock for protein expression and lastly purification to obtain purified recombinant protein. So, this system is advantages in target gene transfer with very high efficiency and high recombinant protein yield. However, the drawbacks of this system are it requires expensive complex media and the construction and amplification of viral stocks is necessary and takes up to 2-3 weeks. http://www.oppf.rc-harwell.ac.uk/OPPF/courses/oppf2015/talks/OPPFCourse2015__Arnuad_Poterszman.pdf https://www.profacgen.com/recombinant-protein-expression-in-insect-cells.htm https://biotop.boku.ac.at/Projects/2015/Grabherr2015c.html
- Next, Plants have also been increasingly researched as an alternative recombinant protein expression system. For example, human serum albumin derived from Nicotiana tabacum suspensions culture initiated from transgenic plant. In plant-cell based expression system, the candidate antigen can be expressed by stable integration, first method is by using natural plant pathogens like Agrobacterium tumefaciens as vector mediated carrier system for stable integration of antigenic DNA into plant nucleus at random loci. Another one is using ‘gene guns’ to integrate candidate gene into specific site in plant circular chloroplast DNA (cpDNA). For transient expression, candidate gene can be mediated by positive sense, single stranded plant RNA viruses (Tobacco etch virus, Cauliflower mosaic virus, Tobacco mosaic virus etc). In this method, the engineered viral genome will not become integrated into host plant genome and are expressed by infected generation only. Another is demonstrated by infiltration (agroinfiltration technique) of suspensions of Agrobacterium tumefaciens which harbouring a binary vector into leaf interstitial spaces. These expressions result in formation of stable transgenic plant, where the antigen can be extracted or directly used as edible plants. This system is benefited with high efficiency expression technique and easily scalable to production, and plant cells are intrinsically safe. The drawbacks are reduction in genetic diversity and may be disease infection throughout generation. __________ E.g. Eleyso (taliglucerase alpha), an enzyme with a human compatible glycan profile, produced by genetically modified carrot plant root cells. which is used in enzyme replacement therapy (ERT) to treat Gaucher’s disease, has been produced by Protalix Biotherapeutics. Stable integration: This method is benefited by the inherent capability of plant pathogens to infect and transfer its virulent genes to the nucleus of host cell. The main advantages of this method are ease of production, cost effectiveness, ability to introduce large DNA segments with higher efficiencies into the plant genome etc.