Biotechnology Flashcards
(129 cards)
1
Q
What is pharmaceutical biotechnology?
A
a field that uses micro and macro-organisms and genetically engineered cells to create pharmaceuticals
2
Q
What is the goal of pharmaceutical biotechnology?
A
create safer and more cost effective than conventionally produced pharmaceuticals
3
Q
What are some applications of biotechnology in pharmacy?
A
antibiotic production
antibody production
gene therapy and gene silencing
vaccines
personalized medicine
transgenic animals
4
Q
What is the pharmacists role in biotechnology?
A
product evaluation and selection
patient education and counselling
provision of drug information
assistance in patient monitoring
drug control and preparation
FACILITATING BETTER CARE
5
Q
Differentiate between eukaryotes and prokaryotes.
A
eukaryotes:
-nucleus and other organelles enclosed by a plasma membrane
-large (10-100um)
-most are multicellular, some are unicellular
-linear DNA
prokaryotes:
-unicellular
-lack membrane bound structures, even the nucleus
-nucleoid contains only one circular chromosome
-small (0.1-5um)
-extra chromosomal material: plasmids
6
Q
How many nucleotides are in a strand of DNA?
A
four
7
Q
What is the central dogma of molecular biology?
A
DNA–>RNA–>protein
transcription then translation
8
Q
What is a gene?
A
a portion of the DNA that codes for a mRNA that then translates into protein
include the coding sequence and adjacent sequence required for regulation of expression
9
Q
Differentiate gene organization of eukaryotes and prokaryotes.
A
prokaryotes:
-most of the DNA
-very little inter-genic DNA
-no introns
-1 gene=1 protein
eukaryotes:
-75% of the DNA is non-coding (regulatory)
-most of the DNA is inter-genic DNA in the genome
-genes are split: exons (1.5%) and introns (23.5%)
-1 gene=multiple proteins
10
Q
What are promoters?
A
DNA sequence that promote gene expression
required for DNA transcription
typically located upstream of the genes
RNA polymerase binding site
direct the exact place to initiate DNA transcription
determine when an how a gene is transcribe
11
Q
True or false: gene expression is highly regulated
A
true
12
Q
What is epigenetics?
A
the shape of chromatin influences gene expression
13
Q
What happens to gene expression in the following scenarios:
open DNA
compact DNA
highly compacted DNA
A
open DNA: easy to read, gene expression
compact DNA: difficult to read, some gene expression
highly compacted DNA: very hard to read, no gene expression
14
Q
What are housekeeping genes?
A
expressed in all cells all the time (almost)
responsible for routine metabolic functions common to all cells
15
Q
How are some other genes asides from housekeeping genes expressed?
A
some are expressed as a cell enters a particular pathway of differentiation
some are expressed all the time in only those cells that have differentiated in a particular way
some are expressed only as conditions around and in the cell change
16
Q
What does separation of transcription and translation in space and time allow eukaryotes to do?
A
greater control in regulating gene expression
17
Q
What are the four types of nucleotides in mRNA?
A
A, U, G, C
not thymine
pairs: A-U and G-C
multiple codons code for 1 amino acid
18
Q
What is recombinant DNA?
A
form of artificial DNA that is created by combining two sequences from different sources
allows proteins to be produced via artificial means
-engineer gene for more protein productivity
-produce desired proteins in vitro for therapeutic use
19
Q
What are some uses of rDNA?
A
insulin
growth hormone deficiency
20
Q
What are 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
21
Q
Which cells are used to mass produce proteins?
A
bacterial
yeast
mammalian
22
Q
How are clones typically produced for prokaryotes?
A
placing a DNA fragment of interest into a vector DNA molecule, which can replicate in a host cell
23
Q
What are the steps in DNA cloning?
A
- isolation of gene of interest
- isolation of plasmid DNA (cloning vector)
- manipulation of DNA sequence
a. cutting-restriction enzymes
b. joining-DNA ligase - transformation of bacteria
- selection of “correct” bacteria
- replication of the cells carrying rDNA molecules to get a genetically identical cells or clone
24
Q
What are cloning vectors?
A
specifically modified DNA that are used to propagate foreign DNA in bacteria
25
What are the three essential features for a cloning vector?
origin of replication
dominant selectable marker
restriction sites
26
What are restriction endonucleases?
primarily bacterial enzymes that cut foreign DNA into fragments by recognizing specific nucleotide base pairs
27
What is DNA ligase?
unique ability to link or paste together DNA fragments that have been produced by exposure to restriciton endonucleases
28
What are methods of transformation?
heat shock
CaCl2 transformation
Lipofectin and similar molecules
electroporation
microinjection
29
What is the size, structure, modification, manufacturing, characterisation, stability, and immunogenicity of small molecule drugs.
size: small (single molecule), low molecular weight
structure: simple, well defined, independent of manufacturing
modification: well defined
manufacturing: chemical synthesis, predictable chemical process, identical copy can be made
characterisation: easy to characterise completely
immunogenicity: mostly non-immunogenic
30
What is the size, structure, modification, manufacturing, characterisation, stability, and immunogenicity of biological drugs.
size: large (mixture of related molecules), high molecular weight
structure: complex (heterogenous), defined by the exact manufacturing
modification: many options
manufacturing: produced in living cell culture, difficult to control from starting material to final API, impossible to ensure identical copy
characterisation: cannot be characterised completely the molecular composition and heterogenicity
stability: unstable, sensitive to external conditions
immunogenicity: immunogenic
31
What is a big issue with biologics?
cost, especially for chronic conditions such as rheumatoid arthritis
small molecule drugs cost on average US $1 per day, a biological costs on average US $22 per day
32
What is an example of an early use of biotechnology in the pharmaceutical industry?
insulin was made using E.coli cells in 1978
33
How did we used to make insulin? What was the issue?
previously isolated from the pancreas of cows, pigs, and other farm animals
could elicit an immune response
34
True or false: animal growth hormones have therapeutic value in humans
false
35
How was growth hormone extracted before the use of recombinant DNA? What was the issue?
extracted from the pituitary of cadavers
resulted in significant shortage in availability and transfer of diseases such as Creutzfeldt-Jacob disease
36
Differentiate between lag phase, log phase, stationary phase, and death phase.
lag phase: number of bacteria does not change with time
log phase: number of bacteria increases exponentially over time
stationary phase: there is no net change in number of bacteria with time, bacteria divide but also die at same rate
death phase: number of bacteria decreases with time
37
True or false: most important biological products such as antibiotics are secondary metabolites
true
38
At what phase do primary and secondary metabolites peak?
primary: log phase
secondary: late log phase and early stationary phase
39
Describe rifamycin B.
medically useful antibacterials
produced by Stretopmyces mediterranei
inhibit transcription by binding prokaryotic but not eukaryotic RNA polymerases
inhibit chain elongation, leaving inactivated RNA polymerase bound to the promoter
sterically blocks the path of RNA elongation when the transcript two or three nucleotides long
40
What are limitations with bacterial expression systems?
bacteria are not capable of producing glycoproteins because they lack the capacity to glycosylate
proteolytic cleavage may lead to degraded product
41
What is cell & tissue culture?
tissue culture is the general name for the removal of cells, tissues or organs from an animal or plant and their subsequent placement into artificial environment conductive to growth
the environment usually consists of a support medium that supplies nutrients
when the cells are removed from the organ fragments, disrupting their normal relationship with neighboring cells, it is called cell culture
42
What are the two classes of cultures of animal cells?
primary cells
cell lines
43
Describe primary culture.
cells are surgically removed from an organism and placed into a suitable culture environment they will attach, divide, and grow
primary culture cells have a lifespan of 5-10 divisions in vitro
due to lifespan, cannot do long term experiments
considered to be physiologically similar to in vivo cells
44
What is Hayflicks Phenomenon?
cells will continue to grow and divide normally for a limited number of passages
when they get to a certain point even if they are given appropriate nutrients, they simply stop dividing and die
correlation between maximal number of passages and aging
number of passages decreases when cells are harvested from older individuals
45
Describe cell lines.
if the cells in a cell strain undergo a transformation process that makes them immortal they are called a cell line
it is unknown how a diploid cell strain becomes a cell line, although it may be mimicked with oncogenic viruses or chemical carcinogens
often have abnormal chromosome # and maybe tumorigenic
46
What are other techniques to transform cells?
chemical or gamma ray treated cells
viral infection with SV40 T antigen
*Result is a cell with altered function, morphological, and growth characteristics*
47
What is a suspension cell culture?
derived from cells which can divide and survive without being attached to a substrate
maintained in culture vessels that are not tissue-culture treated
requires agitation for gas exchange
easier to passage
48
What is an adherent cell culture?
must adhere to a surface to survive
form monolayers
growth is limited by surface area
dissociated enzymatically or mechanically from surface
49
Describe cell culture medium.
cells have complex nutritional requirements that must be met to permit propagation in vitro
different types of cells have different growth requirements and a number of chemically-defined formulations
some serum-free media are available and some cell lines have been adapted to growing in such a medium, most cell lines require the addition of 5-10% serum for multiplication
50
Describe culture vessels.
provide a contamination barrier to protect the cultures from external environment while maintaining the internal environment
for anchorage-dependent cells, surface for cell attachment
easy access to the cultures and optically clear viewing surfaces
51
List examples of culture vessels.
cell culture dishes:
-cheapest and provide access to the growth surface
-anchorage dependent cells
multiwell plates:
-savings in space, media, and reagents
flasks:
-wide range of growing areas, shapes, several neck designs
-anchorage dependent cells
surface coatings:
-vessel surface is treated to render hydrophilic
-coating surface with serum, collagen, laminin, gelatin, poly-L-lysine
52
What are contaminants of cell culture?
chemical (serum, water, endotoxins, ions, plasticizers)
biological (mycoplasma, yeast, bacteria, fungus)
53
What are the effects of biological contaminants?
compete for nutrients with host
secrete acidic or alkaline by-products
produce H2O2
54
What is the most important part of all animal cell culture?
prevention of contamination by different microorganisms
55
How can the risk of contamination be avoided?
adding antibiotics:
-penicillin
-streptomycin
-gentamycin
-nystatin for fungi and yeast
*routine use of antibiotics is generally not recommended because resistant organism may develop, biochemical alteration may be produced*
56
How are most biotherapeutic proteins produced?
Chinese hamster ovary and murine myeloma cell lines
there has been a recent shift toward the use of human cell lines
57
Why do we use CHO cells?
established safety profile
produce proteins with complex bioactive PTMs that are similar to those produced in humans
reduced susceptibility to certain viral infections and routine screening systems
grow in suspension culture
serum-free chemically defined media
allow gene amplification
creation of stronger expression units
highly tolerant to changes in pH, O2 levels, pressure or temperature during manufacturing
58
What is transgenesis?
process of introducing foreign or exogenous DNA into an animals genome
59
Why do we use transgenesis?
improve genetic features of domesticated animals
provide animal models for study of human diseases
pharming using farm animals for production of human pharmaceuticals
study gene regulation and development of animals
60
Describe retrovirus-mediated transgenics.
infecting mouse embryos with retroviruses before the embryos are implanted
size of transgene is limited
61
Describe pronuclear microinjection.
introduces the transgene DNA at the earliest possible stage of development of the zygote
DNA is injected directly into nucleus of egg or sperm
62
Why express rProtein in milk?
easy to purify-few other proteins in milk
doesnt harm transgenic animal
rProtein is authentically modified post-translationally
large quantities
renewable source
63
Describe atryn.
30 goats are being used to make the anti-blood clotting protein, antithrombin
antithrombin is produced from the mammary glands of transgenic goats and harvested from their milk
prevention of blood clotting in antithrombin deficient patients
first drug made from genetically engineered animals approved by FDA
64
What is erythropoietin used to treat?
anemia or patients after chemotherapy as it increases RBCs
many athletes use it to increase # of RBC to increase oxygen capacity and produce improved physical activity
65
What does a two tiered cell banking system consist of?
master cell bank (MCB) and working cell bank (WCB)
66
What must a MCB be tested for?
contaminants such as bacteria, fungi, and mycoplasma
most cell lines also require testing for viruses
67
True or false: cells from the MCB are expanded to form the WCB, which is characterized for cell viability prior to use in the manufacturing process
true
68
What are bioreactors?
any device or system that supports a biologically active environment
device is meant for cells or tissue in the context of cell culture
69
In what form do most bioreactors come in?
cylindrical, ranging in size from liters to cubic meters, and are often made of stainless steel
70
What is the goal of an effective bioreactor?
control, contain, and positively influence the biological reaction
71
In general cells can be cultivated either in vessels containing an appropriate liquid growth medium in which the cells can be either: ____, ____, ____
free in suspension
attached to microspheres
immobilized state as monolayers
72
What are the different types of bioreactors?
liquid, also known as submerged (cells are submerged)
-most bioreactors (saves space and more amendable)
solid state, also known as surface (cells attached to the surface of a solid medium)
73
How are biopharmaceuticals synthesized/produced?
the original cell culture is started in small bottles
as the cell grows and multiply they are introduced into a small bioreactor
eventually they are grown in large bioreactors
74
What are the sources of variation in biologics?
cloning, protein expression, and production:
-different gene sequence
-different vector
-different expression
-different cell line
different bioreactor conditions
protein purification and formulation:
-different binding and elution conditions
-different filter supplier
-different methods, reagents, and reference standards
-different suppliers of excipients
75
What are the three different types of bioreactor systems for production of biotech products?
stirred-tank (most common)
airlift
microcarrier
76
What are the bioreaction parameters?
controlled temperature
sufficient substrate (usually a carbon source)
sugars, proteins, lipids
water availability
salts
vitamins
oxygen (for aerobic processes)
optimum pH
product addition and by-product removal
77
Describe the airlift bioreactor?
reaction medium is kept mixed and gassed by introduction of air or another gas (mixture) at the base of a column-like reactor
equipped with a tube by which the reactor volume is separated into a gassed and an un-gassed region thus generating a vertically circulating flow
78
Describe 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
79
Describe batch operation.
reactor is filled with medium and then inoculated with biocatalysts
reaction proceeds, substrate is consumed and products are formed, finally reactor is opened, product is taken and purified
once operation starts there is no inlet or outlet component, contents are mixed to provide homogeneity
between each batch run, operation stopped in order to harvest products (this is called down time)
80
Describe continuous operation.
there is a continuous medium flow through the reactor
incoming stream (feed) contains the substrate and leaving stem (effluent) contains the product
enzyme or cells can be immobilized inside the reactor, therefore they do not need to be added in the feed and also they dont leave the reactor in the effluent
81
What are the advantages and disadvantages of batch systems?
advantages:
-easy to operate and control
-genetic stability of organism could be controlled if it is genetically engineered biocatalyst
-lower contamination risk
disadvantages:
-lower contamination risk
-batch to batch variability
-accumulation of inhibitory problems
82
What are the advantages and disadvantages of continuous systems?
advantages:
-efficient, higher productivity
-product is obtained with uniform characteristics; quality of the product is almost same from time to time
-no accumulation of inhibitory products
disadvantages:
-degeneration of biocatalyst
-higher contamination risk
83
Describe fed-batch cultivation.
fed-batch cultivation is a modification of batch cultivation in which the nutrient is added intermittently to a batch culture
developed for the cultivation of yeasts on malt
84
Describe the production and recovery of proteins in E.coli and yeast.
growth in large fermenters at high cell density
cell lysis required (protein secreted inside the cell)
protein is harvested and purified
rapid isolation of protein is necessary
85
Describe the production and recovery of proteins in mammalian cells.
grown in large scale cell culture roller bottles
protein secreted into media
isolation of protein from medium
86
What are bacterial inclusion bodies?
normally soluble proteins can form dense, finely granular inclusions within cytoplasm
these inclusion bodies overproduce proteins through the use of plasmid expression
appear in electron micrographs as large, dense bodies often spanning the entire diameter of the cell
formed by build-up of amorphous protein aggregates
87
What are the advantages of protein inclusion bodies?
easily recovered to yield proteins with >50% purity (improvement over purity of soluble proteins)
aggregated forms of the proteins are more resistant to proteolysis
88
What are the disadvantages of protein inclusion bodies?
proteins bound in inclusion bodies are biologically inactive
-need to be denatured in order to be solubilized and then refolded for biological activity
sometimes interlinked by disulfide bonds
inaccurate assessment of recovery
recovery of proteins from inclusion bodies requires cell breakage
dissolution and refolding of inclusion proteins
89
What are the denaturing agents for protein inclusion bodies?
sodium dodecyl sulfate
urea
guanidine hydrochloride
90
Describe cell disruption.
some products are intracellular and need to disrupt the cell and release products
achieved by mechanical and non-mechanical methods
91
What is released when cells die or break?
proteases
important to control growth and harvest conditions in order to minimize this effect
more susceptible to proteases at elevated temps (carried out at <4C)
92
What is another source of proteolytic attacks?
components of the medium
ex: serum contains a number of proteases
use protease inhibitors to control proteolysis
93
What is the effect of glycosylation in proteins?
affects a number of important characteristics:
-solubility
-stability
-serum half life
-pharmacological function
-immunogenicity
*determines therapeutic profile*
94
True or false: glycosylation occurs after transcription and is easy to control
false
after translation
hard to control
95
What are the conditions we must control for optimal growth in cell culture media?
pH
oxygen
temperature
proper nutrients
96
The media used for mammalian cell culture are complex and consist of a mixture of diverse components such as?
sugars
amino acids
electrolytes
vitamins
fetal calf serum (proteins)
growth factors (proteins)
hormones (proteins)
97
What are the issues with animal serum?
contribute to the presence of contaminating proteins (especially fetal calf serum)
composition is variable
may introduce viruses, bacteria, prions, fungi
may contain substantial quantities of endotoxins
proteases in serum
98
What is the most frequent source of virus introduction?
animal serum
99
How are viruses introduced into serum?
by nutrients or generated by an infected production cell line
100
What do bacteria introduce?
pyrogens
101
Describe bacterial pyrogens.
potentially hazardous substances
removal is complicated further because pyrogens vary in size and chemical composition
simple sterile filtration does not remove pyrogens
removed by ion-exchange chromatography
102
What is protein purification?
isolating a single protein from a complex mixture (usually bacterial or animal cells)
separation steps exploit differences in protein size, physicochemical properties and binding affinity
often the rate limiting step in the development of a biotech pharmaceutical
103
What is chromatography?
most common method of achieving a pure sample exploits chromatography
the basic procedure is to flow the protein mixture solution through a column packed with various materials
different proteins interact differently with the column material, and can thus be separated by the time required to pass the column or the conditions required to elute the protein from the column
104
What are the phases of chromatography?
stationary phase: insoluble matrix with which components interact
mobile phase: carrying components of the mixture
*separation is based on differences in distribution between the two phases*
105
What are the steps in quality control of biologic drugs?
1. plasmids and host cells
-stability of inserted gene
-cell growth monitored
-remove contaminants
2. protein stability
-aa sequencing, peptide mapping, HPLC, western blot
3. process validation
-spiking the pre-purification mixture with endotoxins
-final yield of protein is a closely watched indicator
-purification process is revalidated every year
4. final product batch
-many of the tests done when checking protein stability are repeated
-ensure that the purified protein has maintained its activity
-new tests for purity and sterility
-tests for protein concetntrations and potency
106
What are the similarities between Elisa and Western Blot?
Elisa and western blot are two types of techniques used in diagnostics
both methods are based on immuno-detection
they are based on the formation of the antibody-protein complex
both methods can analyze proteins
western blotting is a time-consuming technique
well trained and skilled personnel are required to carry out western blotting
107
What is included in the stability testing of the final labeled product?
1. freezing
2. heating
3. denaturing
long term integrity and sterility are also monitored
entire manufacturing process for one biological can take years of testing and fine-tuning
108
What is the amino terminal heterogeneity?
important to geneterate proteins with an NH2 terminus
when the proteins are not produced in the correct way, the final product may be a mixture of several methyionyl variants of the protein in question that contain proteins lacking one or more residues from the amino terminus
109
Why cant most proteins be sterilized by the standard methods?
due to denaturation at high temperature
*all equipment and excipients are treated and sterilized prior to drug formulation*
110
What are the issues that need to be addressed when formulating a biotech drug?
route of administration
delivery system used
ability for target specific delivery
stability of the protein
111
What are the components of parenteral formulations?
solubility enhancers
anti-adsorption and anti-aggregation agents
buffer components
anti-oxidants
preservatives
active ingredient
112
What do proteins tend to do at high concentrations?
aggregate and precipitate out of solution
*aggregation can lower the potency of the drug and increase the chance of immunogenicity*
113
When does aggregation occur?
when there are hydrophobic or electrostatic interactions between different protein molecules
114
What are approaches to enhance the solubility of proteins in parenterals?
selection of the proper pH and ionic strength conditions
addition of amino acids such as lysine or arginine
addition of surfactants such as SDS
sugars such as glucose and sucrose
115
What do proteins tend to expose in the core of the native protein structure when an interface is present?
hydrophobic sites
these interfaces can be water/air, water/container wall or interfaces formed between the aqueous phase and utensils used to administer the drug
116
Why are anti-adsorption agents added to parenterals?
reduce adsorption of the active proteins to interfaces
117
What are the equilibrium states that insulin exists in?
monomeric, dimeric, tetrameric, and hexameric
the relative abundance of the different states depends on:
-pH
-insulin concentration
-ionic strength
-specific excipients
*dimeric form adsorbs to hydrophobic interfaces and subsequently forms larger aggregates*
118
How is the adsorption of insulin prevented?
other molecules are added that will cover the hydrophobic surfaces
often it is albumin
119
True or false: anti-adhesion agents can also act as anti-aggregation agents
true
120
Low concentrations of ___ and ___ have been to shown to exert a fibrillation-inhibitory effect
phospholipids
surfactants
121
What are the factors affecting stability of protein solutions?
pH
ionic strength
temperature
presence of stabilizers
122
What are the buffer systems regularly encountereded in biotech formulations?
phosphate
citrate
acetate
123
Which amino acids are readily oxidized?
methionine
cysteine (capable of stabilizing protein structure with covalent bonds)
tryptophan
tyrosine
histidine
124
What can help reduce oxidative stress?
replacement of oxygen by inert gases
air tight vials
antioxidants:
-vitamins: A, C, E
-amino acids: acetylcysteine
-chemicals: acetic acid, sodium citrate, selenium
125
What are preservatives?
natural or synthetic chemicals added to prevent decomposition by microbial growth or by undesirable chemical changes
crucial to prevent bacterial growth
126
What are commonly used preservatives?
phenylmercuric nitrate
thimersol
p-hydroxybenzoic acids
phenol
benzyl alcohol
chlorobutanol
127
Why should preservatives be kept at the lowest effective concentration?
to reduce side effects
128
Primary Ab binds ______, secondary Ab binds _____.
antigen
primary Ab
129
During direct assay, the _____ carries the tag/enzyme. During indirect assay, the ____ carries the tag/enzyme.
direct: primary Ab
indirect: secondary Ab
