Biotechnology Flashcards
What is recombinant DNA?
Recombinant DNA is a form of artificial DNA that is created by combining two sequences from different sources (generation of novel DNA sequence)
What is the application of recombinant DNA?
- Engineer gene for more protein productivity
- Produce desired proteins in vitro for therapeutic use
What are some examples of therapeutic agents made from recombinant DNA?
Insulin
Human Growth Hormone
What types of cells can be used to produce proteins from recombinant DNA?
Bacteria
Yeast
Mammalian
What is the advantages of engineering prokaryotes?
Cultivation of prokaryotes is easy (grow and maintain)
Gene manipulation is easy as plasmid DNA is easy to isolate and manipulate.
Prokaryotes do not have histones and can accommodate extra nuclear DNA (plasmids), both qualities improving their ease of use in producing therapeutic products
How are recombinant DNA sequences inserted into a cell?
To produce a large number of identical DNA sequences, the desired sequence is inserted into a vector DNA molecule. This new recombinant DNA molecule is then inserted into the host cell. This allows the recombinant DNA sequence to propagate
What are the steps of DNA cloning?
- Isolation of gene of interest
- Isolation of plasmid DNA
- Manipulation of DNA sequence (cutting via restriction enzymes, and joining via DNA ligase)
- Transformation of bacteria
- Selection of “correct” bacteria
- Replication of the cells carrying rDNA molecules to get a genetically identical cells or clone
Review slide 25 to see the process of producing protein products from recombinant DNA
What are cloning vectors?
Specifically modified DNA that are used to propagate foreign DNA in bacteria (E. coli)
What are the three essential features for a cloning vector?
Review slide 27 to view a diagram explaining the three features
- Origin of replication
- Dominant selectable marker
- Restriction sites
What are restriction endonucleases and their relevance to recombinant DNA?
Primarily bacterial enzymes that cut foreign DNA into fragments by recognizing specific nucleotide base pairs
Different DNA samples, but cut with the same restriction enzymes, can be mixed and joined together using ligase, thus creating recombinant molecules
What are some methods of transformation (getting recombinant DNA into the cell)?
Heat shock
CaCl2 Transformation
Lipofectin and similar molecules
Electroporation
Microinjection
What are differences between small molecule drugs and biological drugs?
Small molecule drugs:
Simple, well defined
Produced by chemical synthesis
Easy to characterize completely
Stable
Non-immunogenic
Less expensive
Biological drugs:
Complex (heterogenous), defined by the exact manufacturing process
Produced in living cell culture
Cannot be characterized completely
Unstable, sensitive to external conditions
Immunogenic
More expensive
What are the risks of obtaining biologics from other animals and cadavers?
Insulin used to be isolated from the pancreases of cows, pigs, and other farm animals. This non-human insulin was subject to immune response once injected
HGH used to be excreted from the pituitary of cadavers. This subjected living patients to a shortage of HGH and transfer of disease from the dead patient (Creutzfeldt-Jacob disease)
Describe the four stages of bacterial growth?
LAG Phase: Number of bacteria does not change with time in lag phase
LOG Phase: Number of bacteria increases exponentially in log phase
STATIONARY Phase: No net change in number of bacteria with time in stationary phase. Bacteria divide but also die at the same rate
DEATH Phase: Number of bacteria decreases with time
What is the difference between primary and secondary metabolites?
Primary metabolites like fats, carbs, and proteins are produced in the LOG phase
Secondary metabolites like antibiotics are not needed for growth, but for defence instead. These are produced in the late LOG phase and early STATIONARY phase
What are some limitations of using bacteria to produce biologic products for human use?
Bacteria do not do post-translational modifications (phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acerylation, lipidation)
50% of human proteins are glycosylated (post-translational mod.), these cannot be produced by bacteria
What is tissue culture?
It is the general name for the removal of cells, tissues or organs from an animal or plant and their subsequent placement into in vitro/petri dish conducive to growth
What are some requirements for tissue culture in terms of the artificial environment?
Usually consists of a suitable glass or plastic or plastic culture vessel contains a liquid or semi-solid support medium that supplies the nutrients essential for survival and growth
What is cell culture and why is it different from tissue culture?
When cells are removed from the tissue culture (organ fragments), thus disrupting their normal relationship with neighbouring cells, it is called cell culture
What are cell cultures used for?
Basic cell biology, effects of drugs on cells
Toxicity testing
Cancer research
Drug manufacturing
What are the classes of culture cells (not the same as cell culture)?
Primary cells
Cell lines
Describe primary cultures
These cells are surgically removed form an organism and are placed into a suitable culture environment. They will attach, divide, and grow
Most of there cells have a limited Hayflick’s limit (5-10 divisions)
Primary cells are considered to be more physiologically similar to in vivo cells vs. cell lines
Describe cell lines
These cells have undergone a transformation that effectively makes them immortal.
Immortality can be induced by oncogenes virus or chemical carcinogens
Cell lines often have abnormal chromosome numbers
Cell lines derive from tumours often do not exhibit contact-inhibition, they continue to pile up
What are the specifics for suspension cell culture?
They are derived from cells that can divide and survive without being attached to a substrate (ex. cells of hemopoietic lineage)
Can be maintained in culture vessels that are not tissue-culture treated
Requires agitation for gas exchange
Easier to process
What are the specifics of adherent cell culture?
Most adhere to a surface to survive
They form monolayers (cells derived from different tissues)
Growth is limited by surface area
These cells will stop proliferating once they fully cover the surface of cell culture vessel
Cells are dissociated enzymatically or mechanically from surface before being harvested
What is the most common cell culture medium for cell lines?
5-10% serum as a supplement to promote cellular multiplication (not 100% identical in composition, causing variation in end products)
What are some types of culture vessels?
Cell culture dishes (petri-dishes): great access to growth surface
Multi well plates: significant savings in space, media, and reagents
Flasks: range of growing areas, a variety of shapes, with different neck designs
What is the purpose of surface coatings on culture vesssels?
Vessels are treated to render the surfaces hydrophilic
What are the effects of biological contamination?
Contaminants compete for nutrients with host cells
Secrete acidic or alkaline by-products that cease the growth of the host cells
They also produce hydrogen peroxide (toxic to cells)
How can contamination be prevented?
Addition of antibiotics, be judicious in antibiotic use due to concerns about resistance
What are some commonly used cell lines?
Traditional cell lines:
Chinese hamster ovary (CHO) and murine myeloma (NS0, Sp2/0)
There has been a recent shift towards human cell lines, but still not as common as CHO or NS0.
Ex. Expi 203
Why are Chinese Hamster ovary cells used in some cell lines?
They have a long track record of safety
These cells are able to conduct post translational modifications that are similar those in humans
CHO cells also have reduced susceptibility to certain viral infections
CHO can grow in suspension culture, do not need serum media (increase reproducibility across different batches)
What is the utility of transgenic organisms in harvesting desired biologic products?
These animals can produce biologic products in things like milk or seeds
What is the definition of transgenesis?
The process of introducing foreign or exogenous DNA into an animal’s genome
Mice, cows, fish, birds, sheep are some animals that have undergone transgenesis
What is the medical benefit of transgenesis?
Provide animal models for study of human diseases
“Pharming” using animals for production of human pharmaceuticals
How is new genetic material inserted into animals?
Retrovirus-mediated transgenics (infect embryo before implantation)
Pro nuclear microinjection (DNA is injected directly into the nucleus)
Why is milk a good option to express desired protein expression?
Easy to purify (few other proteins in milk)
Large quantities
Renewable source
What is a bio pharmaceutical?
Any medically useful drug whose manufacturing involves microorganisms or substances that living organisms produce
Most bio pharmaceuticals employ recombinant DNA
What are some types of biologic products?
Hormones
Blood Products
Cytokines
Monoclonal antibodies
Growth factors
Gene and cellular therapies
Fusion proteins
What is cell banking?
When a cell line is used to be used over many manufacturing cycles, a two-tiered cell banking system consisting of:
- Master cell bank (MCB): storage
- Working cell bank (WCB) is utilized (few cells are taken from the MCB and multiplied)
What is a requirement for a cell line to become a master cell bank (MCB)?
The cell line needs to be stable and pure. MCBs need be characterized and extensively tested for contaminants such as bacteria, fungi, mycoplasma, and viruses
Review slide 84 for variation in biologics (will be on exam)
What are bioreactors?
Any device or system that supports a biologically active environment. The goal is to control, contain, and positively influence the biological reaction
These bioreactors are commonly cylindrical, ranging in size from litres to cubic meters
Made from stainless steel
What are the different types of processes that occur in a bioreactor?
Upstream actions (sterilization and raw materials)
Production (free cells, immobilized cells, or enzyme bioreactor)
Downstream (product recovery)
What are some types of bioreactors?
Stirred-tank (preferred)
Airlift
Micro carrier (fixed bed bioreactors)
What are some parameters that can be controlled in a bioreactor?
Temperature
Sufficient substrate (sugars, lipids, and proteins)
Water availability
Salts
Vitamins
Oxygen
pH
Product addition
What are some notable components in a stirred-tank bioreactor?
Baffle (jerks liquid contents, eliminated vortex formation)
Sparger (Maintains air bubbles within the contents of the bioreactor)
Impeller (mixes contents and allows for even distribution of inputs and outputs
Describe some of the details of an airlift bioreactor
The reaction medium is kept mixed and gassed by the introduction of air or another gas at the base of a column-like reactor
Review slide 93
Describe some of the details about micro carrier bioreactors?
- Can provide extremely high productivity within a compact size
- Has been used widely for culture of immobilized mammalian cells
- Use porous glass beads to provide a large surface area for cells
What are the different operation types of bioreactors?
Batch operation
Continuous operation
Fed-batch operation
What happens in batch operation?
The reactor is filled with medium and then inoculated with bio catalyst (enzyme or cells)
As the reaction occurs, substrate is consumed and products are formed. Finally reactor is opened, product is taken and purified. There are no inlet or outlets
After each batch is done, operation must be stopped to harvest, clean, sterilize, and refill bioreactor
What happens in continuous operation?
There is continuous medium flow through the reactor
Inlet contains the substrate, while the outlet contains the product
Enzymes and cells can become immobilized inside the reactor so they are neither added to to input nor do they leave via outlet
Compare batch and continuous bioreactor operation
Batch operation:
Easy to operate
Lower contamination risk
Batch-to-batch variability is an issue
Continuous operation:
HIgher contamination risk is a disadvantage
Product is obtained with uniform characteristics, quality is the same almost every time
What happens in fed batch cultivation?
It is modification of batch cultivation in which the nutrient (input) is added intermittently to a batch culture.
This operation is used when substrate cannot be added all at one, or substrate increases viscosity too high when added all at once
Review table on slide 105 to see the difference between different types of operation
How are proteins excreted from prokaryotic cells ?
Cell lysis is required and product is harvested rapidly to avoid breakdown of product
How are proteins recovered from mammalian cells?
Protein is secreted into media (easier recovery of pure product, no need to conduct cell lysis). Protein is isolated from medium
What are inclusion bodies?
In some cases, normally soluble proteins can form dense, finely granular inclusions within the cytoplasm (can settle out and can be recovered easily). These inclusions probably form due to the build up of protein aggregates
Often occur in bacterial cells that overproduce proteins through the use of plasmid expression
What are the advantages of protein inclusion bodies in harvesting product?
Inclusion body proteins can easily be recovered to yield proteins with over 50% purity (very high compared to 1%, a common purity seen in other biologics)
These inclusions are also more resistant to proteolysis because most molecules in an aggregated form are inaccessible to proteolytic enzymes
What are some disadvantages of protein inclusion bodies?
Proteins bound in inclusion bodies are biologically inactive. Therefore these highly insoluble protein aggregates have first to be denatured under extreme conditions in order to be solubilized and refined for biological activity
Inability to directly measure inclusion bodies (located in cytosol) = inaccurate assessment of recovery and yield
Recovery also requires cell breakage, protein sedimentation, and pellet washing
How is cell lysis induced when harvesting intracellular products?
Mechanical: sonication and liquid shear homogenization
Non-mechanical: autolysis, osmotic shock
What is the concern of proteases in harvesting and purification of product?
When a cell is lysed, proteases are released. These proteases can breakdown thee product and have enhanced activity at higher temperatures
If a serum was used, it may also contain proteases
Purification strategies should be designed such that all the step can be carried out under 4*C (limit protease activity)
What is the effect of glycosylation on a protein product?
Solubility
Stability
Serum half-life
Pharmacological function
Immunogenicity
How are media used in mammalian cell cultures prepared?
A premixed serum starter can contain sugars, amino acids, electrolytes, and vitamins
Proteins like fetal calf serum, growth factors, and hormones are later added by researchers
All of the components are dissolved in purified water before sterile filtration
What are some contaminants in serum?
Proteins
Variable composition
Viruses, bacteria, prions, and fungi
Endotoxin
Proteases
What is the most common source of virus introduction to a biologic product?
Animal serum, therefore there is a trend toward using more defined growth media (use less serum)
What is the effect of pyrogens on biologic products?
Pyrogens (gram -ve endotoxins) are potentially hazardous substances
Simple sterile filtration does not remove endotoxins, need to use ion-exchange chromatography
What is the purpose of purification processes in biologic product processing?
It yields proteins with well-defined characteristics for human use from which “all” contaminants have been removed
Isolating a single protein from a complex mixture is a very labourites process (rate limiting step in biologic manufacturing)
What is chromatography?
The most common method of achieving a pure sample exploits chromatography
The protein mixture solution is flowed through a column packed with various materials. Proteins interact with the column material and are separated based on their properties
What are the phases of chromatography?
Stationary: insoluble matrix twitch which components of the mixture interact with during separation
Mobile: Solution that is passed through or over the stationary phase, carrying with it the components of the mixture (liquid or gas)
What are some factors monitored in biologic product quality control?
Characterization of plasmid and host cells (gene stability, cell growth, contaminants)
Protein Stability (amino acid sequencing, chromatography, Western blot analysis)
Process validation
Final product batch
Describe how Western Blot/ELISA test work
This assay employs antibodies to tag proteins.
In direct detection, a primary antibody has a fluorescent tag and binds directly to the protein
In indirect detection, a secondary antibody has a fluorescent tag and will bind to primary antibodies that are directly attached to protein product
The fluorescence can be used to measure protein product
What is the difference between Western blotting and ELISA?
Western blotting is time consuming and requires well trained and skilled personnel
Describe what happens in the process validation step of quality control
It is the continuous in-process testing and revaluation of the purification process
Final yield of a protein is closely watched
Purification process is retaliated every year
Describe what happens to the final product in quality control
Protein stability tests are repeated (freezing, heating, denaturing), can take years of fine tuning
Ensure purified protein has maintained its activity
New purity and stability tests
Protein concentration and potency are performed
What is amino terminal heterogeneity?
This means that all produced proteins do not have a NH2-terminus, unlike like authentic human proteins
The final product is a mixture of several methionyl variants of the protein in question
When is quality control performed?
At every step of the manufacturing process
Can proteins be sterilized by the standard process?
No, cannot be sterilized normally due to denaturation at high temperatures
What factors effect excipient choice in biologic drugs?
The nature of the protein and its therapeutic use can make formulations quite complex
What are some excipients used in parenteral biologic products?
Solubility Enhnacers
Anti-adsorption and anti-aggregation agents
Buffer components
Anti-oxidants
Preservatives
Active ingredients
Describe solubility enhancers?
At high concentrations of proteins, they can aggregate and precipitate out of the solution (lowers potency)
Once precipitated, these protein particles cannot be administered to a patient parenterally
Approaches that can be used to enhance solubility:
Select proper pH and ionic strength
Addition of amino acids (Lysine and Arginine)
Addition of surfactants
Sugars (glucose and sucrose)
Describe the utility of anti-adsorption and anti-aggregation agents
Some proteins tend to expose hydrophobic cites, normally present in the core of the native protein structure when an interface is present (water/air, water/container, aqueous solution/utensil)
Once the adsorbed protein returns to the aqueous solution, it’s exposed hydrophobic sites bind to other proteins (causing aggregation)
Anti-adsorption agents are added to reduce adsorption of the active protein to interfaces. This ultimately reduces aggregation
Example of a protein that can be adsorbed (insulin)
Example of an anti-adsorption agent (albumin)
What factors impact protein solution stability?
- pH
- Ionic strength
- Temperature
- The presence of stabilizers
What are some common buffer systems encountered in biotechnology formulations?
Buffer election is important because protein solubility; physical and chemical stability depend on pH.
Ex. Phosphate, Citrate, and Acetate
How are oxidation reactions limited in biologic products
Replacement of oxygen by inert gasses in the vials helps reduce oxidative stress. This requires an airtight vial.
Other antioxidants:
Vitamin A, C, and E
Acetylcysteine
Acetic acid, sodium citrate, or selenium
What is the purpose of preservatives in biologic products?
Natural or synthetic chemicals can be added to prevent decomposition
Preservatives are survival to prevent bacterial growth
Ex. Phenylmercuric nitrate, thimersol, phenol, benzyl alcohol, chlorobutanol
