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PHAR 124* > Biotechnology > Flashcards

Biotechnology Flashcards

1
Q

What is recombinant DNA?

A

Recombinant DNA is a form of artificial DNA that is created by combining two sequences from different sources (generation of novel DNA sequence)

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2
Q

What is the application of recombinant DNA?

A
  1. Engineer gene for more protein productivity
  2. Produce desired proteins in vitro for therapeutic use
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3
Q

What are some examples of therapeutic agents made from recombinant DNA?

A

Insulin

Human Growth Hormone

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4
Q

What types of cells can be used to produce proteins from recombinant DNA?

A

Bacteria

Yeast

Mammalian

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5
Q

What is the advantages of engineering prokaryotes?

A

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

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6
Q

How are recombinant DNA sequences inserted into a cell?

A

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

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7
Q

What are the steps of DNA cloning?

A
  1. Isolation of gene of interest
  2. Isolation of plasmid DNA
  3. Manipulation of DNA sequence (cutting via restriction enzymes, and joining via DNA ligase)
  4. Transformation of bacteria
  5. Selection of “correct” bacteria
  6. Replication of the cells carrying rDNA molecules to get a genetically identical cells or clone
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8
Q

Review slide 25 to see the process of producing protein products from recombinant DNA

A
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9
Q

What are cloning vectors?

A

Specifically modified DNA that are used to propagate foreign DNA in bacteria (E. coli)

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10
Q

What are the three essential features for a cloning vector?

A

Review slide 27 to view a diagram explaining the three features

  1. Origin of replication
  2. Dominant selectable marker
  3. Restriction sites
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11
Q

What are restriction endonucleases and their relevance to recombinant DNA?

A

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

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12
Q

What are some methods of transformation (getting recombinant DNA into the cell)?

A

Heat shock
CaCl2 Transformation
Lipofectin and similar molecules
Electroporation
Microinjection

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13
Q

What are differences between small molecule drugs and biological drugs?

A

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

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14
Q

What are the risks of obtaining biologics from other animals and cadavers?

A

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)

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15
Q

Describe the four stages of bacterial growth?

A

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

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16
Q

What is the difference between primary and secondary metabolites?

A

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

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17
Q

What are some limitations of using bacteria to produce biologic products for human use?

A

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

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18
Q

What is tissue culture?

A

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

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19
Q

What are some requirements for tissue culture in terms of the artificial environment?

A

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

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20
Q

What is cell culture and why is it different from tissue culture?

A

When cells are removed from the tissue culture (organ fragments), thus disrupting their normal relationship with neighbouring cells, it is called cell culture

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21
Q

What are cell cultures used for?

A

Basic cell biology, effects of drugs on cells

Toxicity testing

Cancer research

Drug manufacturing

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22
Q

What are the classes of culture cells (not the same as cell culture)?

A

Primary cells

Cell lines

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23
Q

Describe primary cultures

A

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

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24
Q

Describe cell lines

A

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

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25
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
26
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
27
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)
28
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
29
What is the purpose of surface coatings on culture vesssels?
Vessels are treated to render the surfaces hydrophilic
30
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)
31
How can contamination be prevented?
Addition of antibiotics, be judicious in antibiotic use due to concerns about resistance
32
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
33
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)
34
What is the utility of transgenic organisms in harvesting desired biologic products?
These animals can produce biologic products in things like milk or seeds
35
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
36
What is the medical benefit of transgenesis?
Provide animal models for study of human diseases “Pharming” using animals for production of human pharmaceuticals
37
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)
38
Why is milk a good option to express desired protein expression?
Easy to purify (few other proteins in milk) Large quantities Renewable source
39
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
40
What are some types of biologic products?
Hormones Blood Products Cytokines Monoclonal antibodies Growth factors Gene and cellular therapies Fusion proteins
41
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: 1. Master cell bank (MCB): storage 2. Working cell bank (WCB) is utilized (few cells are taken from the MCB and multiplied)
42
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
43
Review slide 84 for variation in biologics (will be on exam)
44
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
45
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)
46
What are some types of bioreactors?
Stirred-tank (preferred) Airlift Micro carrier (fixed bed bioreactors)
47
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
48
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
49
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
50
Describe some of the details about micro carrier bioreactors?
1. Can provide extremely high productivity within a compact size 2. Has been used widely for culture of immobilized mammalian cells 3. Use porous glass beads to provide a large surface area for cells
51
What are the different operation types of bioreactors?
Batch operation Continuous operation Fed-batch operation
52
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
53
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
54
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
55
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
56
Review table on slide 105 to see the difference between different types of operation
57
How are proteins excreted from prokaryotic cells ?
Cell lysis is required and product is harvested rapidly to avoid breakdown of product
58
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
59
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
60
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
61
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
62
How is cell lysis induced when harvesting intracellular products?
Mechanical: sonication and liquid shear homogenization Non-mechanical: autolysis, osmotic shock
63
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)
64
What is the effect of glycosylation on a protein product?
Solubility Stability Serum half-life Pharmacological function Immunogenicity
65
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
66
What are some contaminants in serum?
Proteins Variable composition Viruses, bacteria, prions, and fungi Endotoxin Proteases
67
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)
68
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
69
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)
70
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
71
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)
72
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
73
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
74
What is the difference between Western blotting and ELISA?
Western blotting is time consuming and requires well trained and skilled personnel
75
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
76
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
77
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
78
When is quality control performed?
At every step of the manufacturing process
79
Can proteins be sterilized by the standard process?
No, cannot be sterilized normally due to denaturation at high temperatures
80
What factors effect excipient choice in biologic drugs?
The nature of the protein and its therapeutic use can make formulations quite complex
81
What are some excipients used in parenteral biologic products?
Solubility Enhnacers Anti-adsorption and anti-aggregation agents Buffer components Anti-oxidants Preservatives Active ingredients
82
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)
83
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)
84
What factors impact protein solution stability?
1. pH 2. Ionic strength 3. Temperature 4. The presence of stabilizers
85
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
86
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
87
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

PHAR 124* (12 decks)

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