Lecture 9a scaling up
- 1. Lecture 9 Animal Cell Biotechnology Scaling up the production process Scaling up animal cell/microbial processes • optimal physiological conditions obtained via small scale processes maintained for large scale operation • must consider: 1. fermentor/bioreactor system 2. control of agitation, pH, temperature, dissolved oxygen
- 2. Lecture 9 Animal Cell Biotechnology Scaling up the production process T-flasks
- 3. Lecture 9 Animal Cell Biotechnology Scaling up the production process Spinner bottle
- 4. Lecture 9 Animal Cell Biotechnology Scaling up the production process Multiple process vs. unit process • can have 1000 100 mL flasks or a single 100 litre fermentor (same volume) 1. Multiple process • easy to replicate samples and conditions, large surface area/volume ratio, tedious to take care of all samples, labor intensive 2. Unit process • good for large scale production of biological products, ease of handling, decreased possibility of contamination
- 5. Lecture 9 Animal Cell Biotechnology Scaling up the production process Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P203.
- 6. Lecture 9 Animal Cell Biotechnology Scaling up the production process Suspension vs. Anchorage-dependence • suspension cells are readily scaled up to production level using similar fermentation vessels as microbial cells (with modifications) • anchorage-dependent cells require a surface for attachment in order to grow → grow as static cultures (i.e. hollow fibres) → grow on microcarriers, in a stirred tank reactor (fermentor)
- 7. Cell culture processes • Anchorage-dependent systems Microcarrier (dextran, collagen or plastic) : 200 µm Advantages: -Higher cell yields - increased productivity. - ↑ surface ratio (100 -200 cells). - suspension cultures • Suspension systems: > Industrial culture
- 9. Cell Culture system • Vero cells •Why? Regulated by WHO • already licensed for vaccine production such as polio, influenza, rabies, others • Serum-free medium •Why? Safety and reproducibility • Microcarriers •Why? Scalability and ease of infection
- 11. Lecture 9 Animal Cell Biotechnology Scaling up the production process Usually scale up 10x at a time: 1 → 10 → 100 → 1000 → 10,000 L Factors limiting scale-up • supply of oxygen • shear damage from mixing • build up of toxic metabolites
- 12. Lecture 9 Animal Cell Biotechnology Scaling up the production process during scale up decreased product yields may be experienced due to the following: 1. inoculation – must consider quality and quantity of inoculum used to start process $ 2. choice of medium – cheaper materials often used for large $ $ scale production due to cost limitations - omission / reduction of serum, antibiotics 3. large scale sterilization – longer sterilization times may result in degradation of heat labile compounds, reducing quality of media 4. development of gradients - larger fermentors/bioreactors may be subject to development of nutrient, temperature, pH, and oxygen gradients 5. other factors -scaling up may also alter the generation of foam, shear forces
- 13. Lecture 9 Animal Cell Biotechnology Scaling up the production process: The Stirred Tank Reactor • bioreactor is another term for fermentor • stirred tank reactor simplest and most widely used system (pot and paddle) → small vessels (<20 litres) made of glass → large vessels (>20 litres) made of stainless steel
- 14. Bioreactor (Fermenter) • Vessel that allows the growth of cells. • Stirred tank bioreactor (STR). Growth: Bacteria, yeast, mammalian cells. • Homogeneous solution mixed by an impeller: -Laminar stirred: Just one dimension to lift the liquid. Cultures >1L: Uneven oxygen exchange and poor nutrient distribution www.corning.com
- 15. Stirred Tank Reactor • Shape: -curve bottom mammalian cells -square bottom poor mixing • Materials used: Vessel up to 10 L (glass) Head plate steel stainless • Heat control: (heat pad or jacket water) - Constant To -mammalian cell culture (37o C)
- 16. Lecture 9 Animal Cell Biotechnology Scaling up the production process: The Stirred Tank Reactor Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P156.
- 17. Fig. 9.2
- 18. Fig. 9.3