Review Questions from Lectures Flashcards
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)
How do you define the exponential feeding strategy?
- All the nutrients except for carbon and oxygen are in excess throughout the run. Culture medium with very low or no glucose
- Growth rate is set based on nutrient that will be added when it starts to enter stationary/highest biomass, growth rate slows down and get product after inducer is added.
- Exponential feeding has much faster biomass accumulation and is cheaper so it is a better option.
Explain feed-back feeding using glucose as an example.
- Dissolved oxygen rises after glucose is depleted then start nutrient addition.
- Glucose is added each time DO rises, causing it to go down again.
- Stop feeding when there is enough biomass, then induce to produce product and later harvest.
- Due to incomplete oxidation of glucose lactic acid can be produced which limits cell growth, can control this with the feeding designs (controlling the toxic metabolites).
Why is it so important to fine tune the feeding strategy for a bioprocess?
Important because want to make sure to increase biomass and get a high amount of product while making sure limiting metabolites are not a problem for the process.
How would you perform a primary product recovery if your product is found in an inclusion body?
- Mechanical breakage of the cell is the typical choice but non-mechanical methods can be used to.
- Done to separate the cells from the IB.
- IB are separated from cell debris after cell breakage by high-speed tubular centrifuge.
- Aggregated protein product in IB has to be resolubilized in denatured form using 6-8M urea or 6-7M guanidine HCL at pH 9-12.
- Refolding protein into active form by dilution into a refolding buffer.
- Large volume of material that must be concentrated and buffer exchanged using TFF into a buffer suitable for purification steps.
What are some reasons for a primary recovery hold point? *done because want to test it and check stability before commercializing
- Define hold conditions as a contingency.
- To do DSP2 repeatability studies using material from 1 bioreactor.
- Scheduling of upstream + PP + DSP2.
- Sizing of bioreactor vs. DSP.
What are some of the steps in the downstream purification process? *descending order
- Product capture – separation of product from impurities, column chromatography in packed bed format.
- Virus inactivation – only included in purification of proteins derived from mammalian culture, intended to inactivate enveloped viruses.
- Removal of product-related impurities – host cell protein, host DNA, endotoxins done by column chromatography ion exchange, hydrophobic interaction, or mixed mode chromatography in packed bed mode.
- Virus filtration – only included in purification of proteins from mammalian culture, intended to remove enveloped and non-enveloped viruses.
- Formulation – buffer exchange the product into liquid formation for final product, adjust product concentration to final level, sterile filtration of product in container, lyophilize if that is final storage format or store as liquid.
What are some important points in the overall viral clearance strategy?
- Reduce viral contamination – screen raw materials, test process intermediates and evaluates how effectively manufacturing steps collectively inactivate and remove viruses.
- Assess risk of carried viruses being introduced from personnel/other sources.
- Assess anticipated virus load into the process, incorporating additional clearance capability as assurance for safety (defined by spiking studies) - Each step is evaluated with these studies to assess if the cumulative viral inactivation/removal is sufficient.
- No viruses introduced to a patient by a biopharmaceutical, but they have been detected in in-process intermediate material and removed.
What are keys elements to consider during final drug product formulation?
Bulk drug substance (BDS) formulation:
- Minimize degradation during storage of the BDS.
- Typically a 2-year shelf life is required.
- Want the most cost efficient and stable product possible.
Key tasks/questions to develop drug formulation:
- Determine degradation & inactivation mechanisms for protein.
- Consider composition of the formulation – refrigeration or room temperature?
- pH? Bulking agent if lyophilized? Isotonic solution? Stability of excipients in solution?
- How is it administered to patient?
Formulation components:
•BDS – drug potency.
•Excipients:
-Buffers – control pH to control solubility.
-Salts, polyols – adjust osmolarity or bulking agent for lyophilized final drug product.
-Surfactants – minimize denaturation.
-Antioxidants – prevent degradation.
-Preservatives – allow multi-dose containers.
What are some applications for mAbs?
- Diagnostic – blood typing, detection of pathogenic microorganisms/viruses, levels of drugs in bloodstream, pregnancy, contaminants in food, Ag shed by tumors.
- Therapeutic – treatment of disease.
- Protein purification.
What is HAT medium and why do we use it during hybridoma development?
- HAT = hypoxanthing-aminopterin-thymidine medium.
- No growth in HAT medium – myelomas are Thymidine Kinase positive (TK+) and Hypoxanthine Guanine Phosphoribosyl Kinase negative (HGPRT-).
- Only hybrid cells will grow (fused B cells that are TK- and HGPRT+ with the myelomas that are TK+ and HGPRT-) – heterokaryon.
- Not all cells seen will produce Ab though.
Why are human mAbs preferred?
- Blend in with human immune system.
- Indistinguishable from endogenous human Ab, human mAbs anticipated to have superior pharmacokinetic and pharmacodynamic properties compared to mAbs from non-human repertoires.
- Human & humanized mAbs less immunogenic than chimeric and non-human mAbs.
- Some human anti-human antibody (HAHA) responses can occur but it is likely fine tuning sequences/structures by engineering that can reduce HAHA.
What are the main steps in the humanization of a mAb?
- Construct 3D structural model of parental hybridoma murine mAb by homology modeling (based on homology & conformational search algorithms).
- Select acceptor human frameworks appropriate for Ag-binding activity, immunogenicity, expression, stability and pharmacokinetics.
- Choose cell line and preparation of expression vectors for multiple versions of humanized mAbs.
- Express + purify humanized mAbs.
- Evaluate humanized mAbs.
What are some ways to improve a mAb?
- Selection of amino-terminal Gln residues to force pyroglutamylation (decrease the number of charge variants).
- Mutation of instability hot spots in CDRs (to restore Ag binding).
- Removal of putative N-glycosylation sites in VH and VL (to prevent formation of N-glycoforms).
- N-glycosylation glycol-engineering (to decrease the number of glycoforms).
- Mutation of amino acids (to decrease susceptibility to aggregation).
- Introduction of Cys residues (for site-controlled cytotoxic dug conjugation).
- IgG4 hinge engineering (to avoid ‘half’ IgGs), IgG2 hinge engineering (to limit scrambling of disulphide bonds and the formation of structural isomers).
- Deletion of carboxy-terminal Lys residues (to decrease the number of charge variants).
Why is transient expression in mammalian cells for early production?
- Traditionally HEK293 (human embryonic kidney) cell lines.
- Extremely short timeframe from vector construction/verification to making mAb early material (~7-10 days).
- Several months or more to create stable CHO cell lines.
- Because of short time frame, genetic stability, and consistency likely higher.
- Suitable for screening multiple cell lines making different versions of a mAb at the same time.
- Expression vectors simpler.
- No need for selective pressure and integration into the host cell genome.
- mAb yield of ~ 100 – 400 mg/L is a good indicator of future high yields from stable transfected CHO cells.
What information is used to decide on the final mAb for product development?
- Compare biophysical and pharmacological properties.
- If only 1 mAb fits the requirements move forward with this mAb.
- Prioritize the key factors (antigen-binding activity, immunogenicity, stability/expression, pharmacokinetics) according to the properties required of the mAb, eg.:
- For subcutaneous formulations: stability and pharmacokinetics may have higher priority, since a stable, high concentration formulation is required (high concentration = potentially increased chance of aggregation).
- For oncology diseases: immunogenicity might not be a high priority since the immune response is often compromised in cancer patients and potency is more critical.
Explain how MSX and MTX are used to select for transfected cells
- There are different cell likes in the market with either the GS + or – ad for use with the GS+ vector or DHFR+ vector.
- MSX (methionine sulfoximine) and MTX (methotrexate) are the drugs used to select for the transfect the cell – inhibiting the functions of DHFR.
- DHFR is important for nucleotide production, in the presence of the inhibiting drugs (inhibiting DHFR) only the transfected cells can grow then.
- Transfected cells can grow because gene amplification of the DHFR gene will be amplified producing a large amount of DHFR that overcomes the inhibitory effect.
- GS also works like this because despite the inhibitory drug, the transfected cells can still grow.
Explain how neomycin is used in transfected cells
Neomycin is used to select for the non-transfected cells.
-It is a negative selection (done to ensure that there are no T cells that recognize and attack out own cells).
You have to develop a humanized antibody using the murine antibody as a blueprint. Why do you go through the development of a humanized antibody if you have a murine antibody that works?
Murine antibodies can illicit an immune response and get rejected that is why they must be humanized to bypass the rejected
You are planning to use an expression vector with matrix attachment regions. What are the advantages to using this type of expression system
- Matrix-attachment regions makes sure that the DNA is inserted and opens the loop (uncoils it) to allow the promoter region to be reached and makes sure that the vector is in the DNA for transcription to occur.
- Want the vector to create polycistronic mRNA to ensure the certain ratio of heavy to light chains are there to make fully functional Ab.
What would you have to consider if your final mAB is inconsistent with earlier Biophysical characteristics and functional tests?
Have to look at other clones from the 30 clone stage, or reassess humanization approach, or screen another of the humanized mAb versions.
