Review Questions from Lectures Flashcards
(44 cards)
What and when was the first historical realization about living cells in fermentations?
the early realization that yeast cells are living
10,000 BC – 1877 AD
What and when was the first discovery of antibiotics?
the discover that molds secrete antibiotics that kill bacteria
1881 – 1940s AD
Antibiotics are not just crucial to medicine, what are they also used for?
used as selective markers on plasmid expression vector
What and when did microbial techniques come into play in history?
using systematic and detailed culture techniques lead to the birth of the fermentation industry
1881 – 1940s AD
What and when did the discovery of DNA play in history?
Darwin’s theory stimulated researchers to search for “the genetic molecule”
1953 – 1973 (Watson + Crick)
What did the discovery of DNA lead to?
this led to DNA, which then lead to discovery of plasmids, which then converged with antibiotic selection to create expression vectors & rDNA tech (cloning).
What and when did DNA sequencing play in history?
DNA sequencing then improved the study of genes and led to whole genome sequencing, which led to systematic study of cellular metabolism to improve cell lines, and gene therapy
1976-1984
What and when did cell therapies play in history?
the use of stem cells in regenerative medicine is one of the next big steps forward
1985 – 1996
What and when did personalized medicine play in history?
using personalized medicine; eg. CAT-T cells, in which a person’s own T-cells are removed, modified to express receptors for disease target, then reintroduced to the person for precise and potent therapy
1990s - now
How can you possibly reduce production costs in a bioprocess?
- Using inexpensive materials.
- Materials that are easier to handle to reduce storage and transport costs.
- In-house development of an optimized cell line saves you licencing fees but takes longer.
- Use of a low-cost media.
- Reduce amount of downstream processing needed.
What are targets in improving a chosen cell line?
- Growth rate – optimizing growth rate by cell type or clone screening or culture conditions.
- Genetic stability – plasmid expression vectors that are maintained efficiently during culture in bacterial cells or integrate in a stable manner into transcription hot spots in genomic DNA of mammalian cells.
- Non-toxicity to humans – cannot use a product that even has a little bit of toxicity to humans.
- Cell size – larger cells are more easily separated from culture fluid at the end of culture, mammalian cells are easier to separate from broths than bacterial cells.
- Ability to use cheaper substrates – cells that use minimal medium are best for processing efficiency (process control, analysis).
- Modification of submerged morphology – concern mostly for fungi.
- Elimination of production of compounds that interfere with downstream processing
- Permeability alterations to improve product secretion – intracellular products need easy lysis.
- Tagging protein products – tagging protein products make them able to be purified more efficiently and increase the yield of the initial protein capture in downstream processing.
What influences your final choice for a culture medium?
- Cost and availability – should be inexpensive and available year-round.
- Ease of handling – solid or liquid, transport and storage costs (eg. temp.).
- Sterilization requirements/denaturation problems.
- Formulation, mixing, complexing and viscosity characteristics – influence agitation, aeration and foaming during fermentation as well as yield/g of substrate.
- Levels and rage of impurities – potential for generating undesired products during process.
- Health and safety implications.
- Thermal damage – reduces the level of specific ingredients and also can produce inhibitory by-products that interfere with downstream processing.
Why is industry trying to go away from raw materials of animal origin?
Animal-sourced materials can be a source of viruses, mycoplasmas, prions, so require much more testing to use.
What are advantages and disadvantages of complex media components?
Advantages:
Complex media are cheaper.
Can enhance growth significantly when added to minimal/defined media without much optimization (Good choice for early R&D work or where development time window is tight)
Disadvantages:
Can contain animal-sourced materials that can be a source of viruses, mycoplasmas and prions.
Variability can be subtle or significant between lots due to origin and time of production of source biological material.
Analysis can be difficult, especially where most abundant components mask detection of trace/lesser components. Also, difficult when one or more components vary.
Regulatory scrutiny is higher during process licencing.
Don’t necessarily know the exact components because you can’t analyze that.
Name 2 types of vessels each that can be used for suspended and attached cells?
Suspension:
- Shake-flask.
- Stirred tank reactor.
- Single-use (disposable) multi-bioreactor.
- Single-use bag-style stirred tank bioreactor.
- Airlift (batch) fermenter.
Supported:
- Fluidized bed bioreactor.
- Packed-bed bioreactor.
- Roller bottles.
What are some advantages to using a single-use multi-bioreactor system for R&D?
- Reduces cleaning and sterilization needs.
- Plant set up, space and operational cost are lower than classic fixed ones (~60% savings).
- Smaller footprint, less utilities/piping, can be changed quickly.
- Reduces risk of cross-contamination = biological/process safety.
- Cheaper & easier complex validation and quality control b/c no testing after each run.
- Contain fewer parts than conventional bioreactor so initial/maintenance costs lower.
- Flexibility in product output because of small max size so easy to add more bioreactors to increase production.
- Technology transfer from ss bioreactors to SUBs easy because they perform similarly.
- Disposable sensors and probes.
- Some suppliers can customize ports and sensor array.
What are some disadvantages to using a single-use multi-bioreactor system for R&D?
- Because small max size, best suited to high yield producing high value products.
- Limiting factor is achievable oxygen transfer into solution, not suitable for bacterial processes (better for mammalian because oxygen transfer is lower).
- Cost of the disposable bag bioreactors is high & quality concerns require testing.
- Generate lots of plastic wastes (environmental concern).
What is a seed-train?
Generation of an adequate number of cells for the inoculation of a production bioreactor.
Name 3 modes of operation for cell culture/fermentation.
- Batch – closed system, definite beginning and end.
- Fed-batch – closed system, extra nutrients added (continuously, intermittently).
- Continuous/perfusion – open system, fresh medium added while culture removed -System reaches steady state (concentration of nutrients and cell number do not vary).
What are advantages and disadvantages of a continuous operation mode?
Advantages:
- One medium for production & growth (no feeds), simpler.
- High cell density.
- Highest specific productivity (g/L/day).
- Best control of by-products which affect product yield and quality.
- Reduced down-time.
- Low running costs.
Disadvantages:
- High initial investment.
- Sterility must be maintained through 20-50 days or more ( a lost run more costly than Batch or Fed-batch).
- Larger tanks to store a supply of medium for continuous feeding.
- Long runs increase risk of low-yielding mutants developing in the culture.
- More technically demanding than fed-batch.
- Low volumetric production (g/L), makes downstream processing more difficult.
Name 3 parameters that are controlled/measured during a bioprocess?
Physical:
- Temperature (electrode).
- Airflow (meter).
- Agitation/speed of agitation (meter).
- Pressure (transducer).
- Liquid flow (transducer).
Chemical:
- Dissolved O2 (electrode).
- Dissolved CO2 (electrode).
- Nutrients eg. glucose (electrodes).
- pH (electrode).
- Metal ions (electrode).
- Foam level/detection (electrode).
- Acid/alkali addition (meter).
- On-line or off-line nutrient inflow and exhaust gas (mass spectra).
Biological:
- Biosensors for biologically active products (electrodes).
- Products (mass spec).
- Biomass (spectrophotometers - on-line and off-line).
Define off-line and in-line in regard to process monitoring?
- Off-line → taken to an instrument at a different lab bench (not close).
- In-line → sensor directly attached.
What is trypan blue used for and why?
-Typan blue – high molecular weight exclusion dye – used to identify dead cells from viable cells.
•Dead cells turn blue.
•Clear cells are viable.
-Based on viable cells you can calculate their productivity.
Name the basic requirements for a feeding strategy.
-Supply carbon + energy source.
Replenishing specific nutritional requirements.
-Controlling growth rate + length of culture.
-Control of inhibitory by-products formation
Eg. bacterial cell culture want to keep acetic acid low (because limits growth)
Eg. mammalian cell culture want to keep lactic acid (because limits growth), NH3+ (affects glycosylating pattern)
