Application of Mammalian Cells Flashcards
What is an antibody?
A naturally occurring protein used by the immune system to identify and neutralise foreign objects (eg bacteria, viruses)
Each antibody recognises a specific antigen unique to its target
What is an antigen?
Any substance that causes the immune system to produce antibodies against it
What is an epitope?
The specific region in the antigen that is recognised by the immune cells
Name the two antibody types and explain them
Monoclonal (MAb)
- identical antibodies produced by one type of immune cell
- expensive can be produced in large quantities - infinite supply
- bind to same epitopes on taerget antigen
Polyclonal
- derived from different cell lines, mixture of antibodies
- cheaper but limited supply
- bind to same antigen but different epitopes
What are the 2 major functions of an antibodies?
1) to recognise and bind to antigens
2) to induce immune response after binding
What are the regions of an antibody and what do they do ?
The variable region mediates binding
The affinity for a given antigen is determined by the variable region
The constant region mediates the immune response after binding
What are the historical milestones associated with antibodies?
1975 - cesar milstein and George’s kohler : the hybridoma technique
1984 - milatein and kohler received Nobel prize
1986 - first mouse monoclonal antibody approved for human use ( murimonav - cd3- immunosuppressant for organ transplant)
2003 - first human monoclonal antibody (adalimumab = humira- for rheumatoid arthritis
Explain the invivo and in vitro method of the hybridoma technique
In vivo : injected in mice ( in peritoneal cavity, gut) produce tumours containing antibodies rich fluid (ascites fluid)
In vitro : grown indefinitely in cell culture media
Give the advantages and disadvantages of the in vivo method
ADV
- cheaper
- higher MAb production yield (5-20mg/ml)
DISADV
- unethical
- requires specialised personnel (specialised license for handling animals
Give the advantages and disadvantages of the in vitro method
ADV
- ethical
- doesn’t require specialised personnel
DISADV
- lower MAb productivity (5-10ug/ml)
- some hybridoma lines don’t grow in culture
Why are hybridoma cells micro encapsulated in alginate capsules?
This is because it significantly increases the MAb yields (10-100ug/ml) as a result of higher cell density
Damon biotech company and cell tech use encapsulated hybridoma cells for large scale production of MAbs. They employ 100 litre fermenters to yield about 100g of MAbs in about 2 week period
What are the disadvantages of the hybridoma technique?
- laborious
- expensive
- time consuming
- often caused immune reactions in patients
Suggest an alternative technology for MAb production
Recombinant DNA Technology
What is recombinant DNA technology ?
The insertion of DNA molecules/particles from a different species into a host organism (expression system) to produce useful products
Explain the basic principles of recombinant dna technology
When a fragment of dna from a donor cell or organism is isolated and then inserted into the dna of another cell or organism
This allows scientists Toni traduce a new characteristic into and organism by inserting a new gene into it
Recombinant dna tech involves 4 steps
1) a dna fragment containing the gene of interest is obtained from donor cell
2) a suitable plasmid is obtained from a bacterium
(Plasmids are commonly used as vectors to transfer the gene of interest into the host cell for expression
3) the dna fragment containing the gene of interest is cut using an enzym called restriction enzyme (enzyme recognises a specific base sequence and cures the dna at a specific point)
Same restriction enzyme is used to cut the plasmid
4) the dna fragment containing th green of interest is inserted into the open plasmid with the help of another enzyme called dna ligase (acts like a glue)
Calatlyses the jointing of dna fragment and plasmid
A recombinant plasmid is formed
The plasmid can be introduced to a host cell for different purposes for example producing protein or GM fruit and veg
Give 3 examples of expression vectors
- plasmid DNA
- virus vector
- bacterial vector
Give 2 examples of expression systems
Prokaryotic - bacteria ( ecoli)
Eukaryotic
- yeast ( sacharomyces verrvisiae)
- viral (baculovirus)
- mammalian ( CHO)
What expression system would be used for the following applications : large protein, small proteins, glycosylation, high yield low cost and post translational modifications?
Large proteins (>100 KD ) - eukaryote
Small protein (<30kD) - prokaryote
Glycosylation - baculovirus or mammalian cell culture
High yield low cost - E. coli
Post transitional modifications - yeast or baculovirus or mammalian cells
What are the advantages and disadvantages of mammalian cells (eg CHO, HEK293) as expression systems?
Adv
- increased level of expression
- production of native structure proteins (correctly folded with appropriate bonding)
- easy to scale up by fermentation
Disadv
- expensive
- slower growing
What are the advantages and disadvantages of bacterial cells (eg E.coli) as expression systems?
Adv
- Simple, well understood genetics
- easy to manipulate genetically
- very cheap
- fast expression with short doubling time
- easy to scale up by fermentation
Disadv
- protein folding issue resulting in insoluble inclusion bodies
- low yield for large proteins
- glycoprotein modification issues
What is transformation in terms of DNA?
The alteration of genetic composition of a cell via the uptake of foreign DNA
- naturally I’m bacteria bu conjunction ( intra or inter species)
- naturally in bacteria by transduction ( viral- medicated DNA incorporation)
- artificially by chemical method and by electroporation
What is transfection?
The transformation and infection of cells to introduce foreign DNA
List 6 properties of transient transfection
- No genomic integration
- short term expression (24-48h)
- DNA is degraded by nucleuses or dilutes during cell division
- useful for rapid analysis of phenotype or for recombinant protein production
- works best with supercooled circular plasmids
- chemical method or electroporation
List 6 properties of stable transfection
DNA is integrated into the cells genome and is replicated with the host DNA
Permanent
Sustained expression for long periods of time
Works best with viral DNA
Viral or micronisation
Explain the process of transfection
Rely on masking the negatuve charges of the phosphate backbone of DNA to facilitate precipitation/ cell uptake
Particulate DNA complexes are taken up into cells by endocytosis
Naked DNA is digested within minute in the cytosol by nucleases
DNA is delivered to the nucleus by unknown mechanisms
Not clear whether DNA is delivered into the nucleus as a particulate (protected) or naked
Methods:
- calcium phosphate
- positively charged polymers
- liposome
What are the main 3 chemical methods of transfections?
Methods:
- calcium phosphate
- positively charged polymers
- liposome
Explain the calcium phosphate transfection
Use calcium phosphate to precipitate the DNA
Precipitates DNA added to cell media and eventually the cells will uptake it by endocytosis
What are the advantages and disadvantages of calcium phosphate transfection?
Advantages
- very simple
- inexpensive
- we’ll established
- works for most cell types
Disadvantages
- works for adherent cells only
- skill and experience required to get good precipitates
- not particularly efficient in some cell types
- many cells types do not like adherent precipitates
- difficult to automate or perform as a high throughput method
How is transfection done using positively charged polymers?
Polymer DIETHTLAMINOETHYL modified dextran used
DNA adheres to the DEAE dextran and remains soluble
The complex adheres to cells and is taken up by endocytosis
What are the advantages and disadvantages of transfection using positively charged polymers?
Advantages
- gentle, low toxicity
- works well for cells in suspension
- high volume transfection
Disadvantages
- doesn’t work well I many cell types
- doesn’t work well for stable transfection
- unclear mechanism of action makes optimisation difficult
- relatively expensive
- low throughput
What are liposomes?
Liposomes are vesicle composed of a lipid bilayer surrounding a hollow core into which drugs or other molecules can be loaded for delivery
How are unilamellar liposomes formed?
Can be formed from cationic lipids by :
Sonication - uneven liposome size, not reproducible
Microfluidisation - regular, small, stable liposomes, reproducible
How does liposomes mediated transfection work?
DNA is mixed with liposomes and binds electrostatically
Liposome/DNA complexes enter cells by fusion with plasma
Encapsulate gene in liposome which then fuses with plasma membrane and uptakes through endocytosis by cell
What are the advantages and disadvantages of liposome mediated transfection?
Advantages
- very simple to perform and optimise - anyone can do it
- easy to automate, high throughput
- reliable and reproducible
- stable and transient assays work well
- works well with many cell types and also in vivo, either adherent or non adherent cells
Disadvantages
- many formulations require use of serum free or serum reduce medium for good efficiency ( all types of neutral lipids)
- some formulations are unstable to oxygen
- variable toxicity necessitates carful optimisation for many types ( eg lypofectamine)
- very expensive to but ( but almost free to make)
What is electroporation?
Uses electrical field that transiently permeabilises the membrane during this period, the foreigner material can enter the cells
How is electroporation carried out?
Cells and DNA mixed and places into a cuvette between two electrode plates
High dc voltage applied as a pulse
Optimised by altering voltage, pulse length, wave form.
During electroporation electro pulse applied creating gaps in cell membrane, temporary, through pore DNA can pass through
Dna suspended in conductive solution
Close circuit
Short pulse lasting milliseconds applied distrusting bilayer
As electric potential across cell membrane rises due to the voltage applied allows charged particles to pass through
What are the advantages and disadvantages of electroporation ( non chemical transfection methods)?
Advantages
- high transfection efficiency
- effective for stable transfection
Disadvantages
- only works for cells in suspension
- devices for transferring adherent cells are tricky and cumbersome to clean
- required optimisation as cell permeability can lead to cell damages
- experienced users are required
- very sensitive to salt concentrations
- expensive
How is viral transfection or transduction carried out?
Happen naturally when virus transfers genetic material from one bacteria to another
Transferring foreign DNA into a cell using a virus
When and why is viral transfection used and talk about it’s efficiency
- used for hart to transfer cell types
- commonly used in clinical research
- often 100% transfection efficiency
- viruses evolved to efficiently introduce nuclei acid into cells
- can be used for in vivo transfection
- engineered for maximum infection but replication defective by mutation / deletion of gag, pol, Env
Give examples of viral transfection cells and why they’re used
Adenoviruses, retroviruses - used extensively for gene delivery in mammalian cell cultures
Baculovirus - expression in insect cells for Pepsi ting recombinant glycoproteins or membrane proteins
What is a vaccine?
biological preparation that provide active acquired immunity to a particular disease
Often made from weakened, inactivated or killed forms of microbes, toxins or one of its surface proteins
How is a virus produced?
Not very different from antibody production
The virus replicates inside cells and the cell environment is the ‘culture enviroment’
(Culture within a culture)
Viruses can be produced in eggs ( influenza) or in mammalian cell cultures
How are vaccines created in embryo ages eggs?
1) Fertilised eggs held in incubators (9-11 days)
2) inculcation with specific influenza virus
3) incubation for 48196 hours at 33-36*c
4) pre chilling in refrigerator
5) harvest of allantonic fluid
6) purification and virus inactivation
(Approx 15ug viral vaccine produces from 1 or 2 eggs ( single human dose))
Why are embryonated eggs used for vaccine production?
Eggs are perfect natural mini bioreactors ( nutrient rich, aseptic)
Many viruses grow very well in ebryonated chicken eggs
Robust yield enables use for reaserch and vaccine production
Give 5 examples of vaccines and what cells they use
Flucelvax (us) / optaflu(eu) (Novartis) - first mammalian cell based vaccine against the influenza virus (2012) produced in MDCK cells
Polio - in vero cells or human diploid fibroblasts
Rabies - in vero cells
Measles, mumps - in chicken embryo cells
Rubella, chicken pox - in human diploid lung fibroblasts
How are vaccines produced when creating it using a mammalian cell culture?
choose cell line of interest example - vero cells
Two phases
Growth phase - objective is to increase mammalian cell biomass once preferred cell density reached they are infected with viral particles
Production phase - objective is to maximise the conversion of the mammalian cell biomass to viable viral biomass
Disposable bioreactors usually used
2 types can be used either horizontal on orbital shakers or vertical bags have impeller in them to ensure good mixing
What cell types is most suitable for viral growth for large scale productions?
Anchorage dependant cell growth with micro-carrier cultures often used
Why use animal cell cultures for viral vaccine production?
Animal viruses propagated by cultured cells at relatively high density
How is virus production maximised?
Viral inoculation is accompanied by a feeder to keep cells alive and maximise virus production
What is polio?
Poliomyelitis or infantile paralysis
Caused by the polio virus
Transmission by oral faecal route
Multiplies in throat and intestine
Invaded blood and lymph
Sub clinical - mild symptoms in 95% of people
Clinical - non paralytic polio , paralytic polio 1% of cases
Explain the properties of the polio virus and it’s associated timeline
2 types of vaccine
- an inactivated poliovirus given by injection
- a weakened poliovirus given by mouth
Developed and tested in 1952 released 1955
Cultured live polio virus in monkey kidney epithelial cells (vero)
Virus harvested and killed by formaldehyde
Injected vaccine
- provides igG mediated immunity to prevent viremia
- 60-90% protection
I’m 1957 Albert Savin Sabin developed a live attenuate polio vaccine by passaging poliovirus through monkeys
- oral vaccine that given intestinal mucosal immunity
- occasionally mutates back and gives people polio
What’s an antibiotic?
Chemical non toxic compounds used to treat infections caused primarily by bacteria.
What’s a natural antibiotic?
True antibiotic - produced naturally by fungi or bacteria to selectively inhibit the growth of others ( chrysogenum produces penicillin
What’s a semi synthetic antibiotic?
Chemically modified natural antibiotics ( ampicillin)
What’s a totally synthetic antibiotic?
Manufactured antibiotic like trimethoprim
Describe the timeline for the discovery of penicillin
1928 : Alexander Fleming noticed a secreted anti microbial produced by the penicillium fungus. This compound was penicillin
Between 1928 and 1945 Howard Florey and Ernst chain devised methods to produce large quantities of penicillin - the first B-road spectrum antibiotic with low toxicity and very good activity
1940s : penicillin was first introduced at large scale (20l) and purified by Howard Florey and Ernst chain
Fleming chain and Florey shared the Nobel prize for medicine in 1945
How do antibiotics work?
They work in one of 2 ways :
Bactericidal: kills the bacteria by preventing it to make a cell wall
Bacteriostatic : prevents bacteria from dividing by interfering with processing like DNA replication, metabolism ( enzyme activity) and protein production
What are broad and narrow spectrum antibiotics?
Broad spectrum - kill a wide range of bacteria ( penicillin)
Narrow spectrum - kill a specific types or group of bacteria ( isoniazid)
What are the 4 mechanism of action of antibiotics?
Cell wall synthesis inhibitors
- b lactams
- glycopeptides
- cycloserine, bacitracin
Targeting cell membrane
- polymyxin
Inhibit nucleic acid synthesis (DNA replication)
- sulphinamides
- inhibits cell division not kill
Inhibit protein synthesis
- stops replication
- tetracyclines
What was antibiotic production like in 1945?
Mass production was possible
Yield was 1g/l of penicillin
Not great yield
Biggest discovery I’m medicine
What does antibiotic production look like currently?
Same fungus used
Yields 50g/L of penicillin
Culture medium - lactose, other sugars, source of nitrogen - yeast extract
Fermented in fed batch mode ( to prolong stationary phase of the cells and increase production)
What phase of the growth cycle does penicillin production occure?
Penicillin is a secondary metabolite so is produced during the stationary phase of cell growth
Why is penicillin modified?
Penicillin G can be enzymatically modified to make a variety of other penicillins with slightly different properties ( penicillin O, ampicillin, amoxicillin)
Depending on what properties are required the penicillin is adjusted accordingly
Explain the downstream bioprocessing of antibiotics m
Required to increase purity
1) involves filtration to remove solids
2) the filtered broths is acidified and extracted with an organic solvent
Extract is then stripped with buffer to isolate a concentrate
3) pH of concentrate is adjusted with acid and product is purified by second extraction into organic solvent
4) final polishing consists of vaccine concentration, crystallisation, and drying of crystalline penicillin
What is cephalosporin?
A b lectim antibiotic used to treat wide range of infections from grim positive to grim negative bacteria
Broad spectrum antibiotic
What are the culture conditions for celphalosporin?
Media - source carbon ( glucose, starch, sucrose), source of nitrogen (meat extract, peptone)
Growth conditions - pH 6-7 temp 24-28, for 4-10 days
High oxygen consumption - compressed filter sterilised air is supplied
Fermentation - fed batch mode to prolong stationary phase
Downstream
RIPP
How does antibiotic resistance occur?
High number of bacteria
A few of them are resistant to antibiotics
Antibiotics kill bacteria causing illness as well as good bacteria protecting body from infection
The resistant bacteria now have preferred conditions to grow and take over
Bacteria can transfer their drug resistance to other bacteria causing more problems
How long can it take to bring a new antibiotic onto the market?
Up to 15 years
Explain the steps of the hybridoma technique for producing monoclonal antibodies
1) immunisation of mouse
2) B cell isolation from spleen
3) in parallel, the cultivation of myeloma cells ( lack the HGRT gene)
4) myeloma and B cells fusion ( electro fusion or chemically
5) culture fused in HAT medium for 10-14 days. Unfused myeloma cells will die in the HAT medium. Unfused B cells die because they have a short life span
6) dilution of the hybridoma suspension until one cell per well
7) cell line screening for antibodies of appropriate specificity ( by using elsa)
8) in vitro (a) or in vivo (b) multiplication
9) harvesting of MAbs
