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PHARM 124 > Pharmaceutical Biotechnology > Flashcards

Pharmaceutical Biotechnology Flashcards

1
Q

What is pharmaceutical biotechnology?

A

A field that uses micro- and macro-organisms and genetically engineered cells to create pharmaceuticals

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2
Q

What are primarily application of techniques of biotechnology?

A

discovery research
product development

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3
Q

List difference applications of biotechnology in pharmacy

A

Antibiotic production
Antibody production
Transgenic animals
Gene therapy and Gene silencing
Vaccines
Personalized medicine

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4
Q

What is the pharmacists role in biotechnology?

A

product evaluation and selection
pt education and counselling
provision of drug information
assistance in patient monitoring
drug control and preparation

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5
Q

Go through the central dogma

A

DNA - transcription –> RNA — translation–> protein

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6
Q

What are housekeeping genes?

A

they are responsible for the routine metabolic functions common to all cells

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7
Q

What is recombinant DNA?

A

form of artificial DNA that is created by combining two sequences from different sources

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8
Q

What is recombinant DNA technology?

A

tech which allows proteins to be produced via artificial means
engineer gene for more protein productivity
produce desired protein in vitro for therapeutic use

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9
Q

What is the most used recombinant cells used to mass produce proteins?

A

E. coli

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10
Q

List advantages of engineering prokaryotes

A

cultivation of prokaryotes is easy
gene manipulation is easy as plasmid DNA is easy to isolate and manipulate

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11
Q

List the steps in DNA cloning

A
  1. Isolation of gene of interest
  2. Isolation of plasmid DNA (Cloning vector)
  3. Manipulation of DNA sequence
    a. Cutting- Restriction enzymes
    b. Joining- 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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12
Q

What are the three essential features for cloning vector?

A

1) Origin of replication
2) Dominant selectable marker
3) Restriction sites

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13
Q

What is extrachromosomal self-replicating DNA molecules?

A

plasmids

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14
Q

Define restriction endonucleases

A

primarily bacterial enzymes that cut foreign DNA into fragments by recognizing specific nucleotide base pairs.

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15
Q

Define DNA ligase

A

have the unique ability to link or “paste” together DNA fragments that have been produced by exposure to restriction endonucleases.

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16
Q

List methods of transformation

A

Heat shock
CaCl2 Transformation
Lipofectin® and similar molecules
Electroporation
Microinjection

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17
Q

To make the recombinant plasmid permeable to DNA molecules, which of the chemicals is added?
a) MgCl2
b) CaCl2
c) NaCl
d) HCl

A

B) CaCl2

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18
Q

Generally a plasmid vector contains how many elements?a) 1b) 2c) 3d) 4

A

C) 3

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19
Q

Which of the following enzyme is required for end to end joining of DNA?a) DNA ligaseb) Restriction endonucleasec) RNA polymerased) DNA polymerase

A

A) DNA ligase

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20
Q

Which of the following enzyme is responsible for making a DNA copy from RNA?a) Reverse transcriptaseb) DNA polymerasec) RNA polId) RNA polII

A

A) reverse transcriptase

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21
Q

Can two DNAs cut with different restriction enzymes join together to form a recombinant plasmid?

A

Yes, if they are cut at the same place they will match

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22
Q

Compare small molecule drugs and biological drugs
Based on Size

A

small
low molecular weight

large
high molecular weight

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23
Q

Compare small molecule drugs and biological drugs
Based on structure

A

simple, well defined, independent of manufacturing process

complex defined by the exact manufacturing process

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24
Q

Compare small molecule drugs and biological drugs
Based on modification

A

well defined

many options

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25
Q

Compare small molecule drugs and biological drugs
Based on manufacturing

A

produced by chemical synthesis
predictable chemical process
identical copy can be made

produced in living cell culture
difficult to control from starting material to final API
impossible to ensure identical copy

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26
Q

Compare small molecule drugs and biological drugs
Based on characteristics

A

easy to characterise completely

cannot be characterised completely the molecular composition and heterogeneity

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27
Q

Compare small molecule drugs and biological drugs
Based on stability

A

stable

unstable, sensitive to external conditions

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28
Q

Compare small molecule drugs and biological drugs
Based on immunogenicity

A

mostly non-immunogenic

immunogenic

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29
Q

What was the first example of biotech products?

A

insulin

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30
Q

List the phase of growth curve of bacteria

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:
There is no net change in number of bacteria with time in stationary phase. Bacteria divide but also die at same rate.

DEATH Phase:
Number of bacteria decreases with time

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31
Q

What is Rifamycin B?

A

a substance produced by bacteria
it both inhibit transcription by binding prokaryotic but not eukaryotic RNA polymerases

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32
Q

What does the structural model of rifampicin indicate?

A

that rifampicin sterically blocks the path of RNA elongation when the transcript becomes two or three nucleotides long

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33
Q

Why can’t bacteria always be used to make all drugs?

A

bacteria do not show some post translational modifcation

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34
Q

What is different about the expression system in bacteria?

A

they are not capable of producing glycoproteins because they lack the capacity to glycoylate

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35
Q

List some post tranlational modifications

A

phosporylation
glycosylation
ubiquitination
nitrosylation
methylation
acetylation
lipidation

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36
Q

list some areas where cell culture technology is currently playing a major role

A

model systems for basic cell biology
toxicity testing
cancer research
making drugs

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37
Q

What are the 2 classes of cultures of animal cells?

A

primary cells
cell lines

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38
Q

What is primary culture?

A

when cells are surgically removed from an organism and placed in a culture to grow, divide and live
most have a finite lifespan

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39
Q

Explain Hayflick’s Phenomenon

A

cell will continue to grow and divide normally for a limited number of passages

the number of passages decrease when cells are harvested from older individuals

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40
Q

What is a cell line?

A

if the cells in a cell strain undergo a process that makes them “immortal”

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41
Q

What are some processes used to make cells immortal?

A

chemical or gamma
viral infections with antigen

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42
Q

Define suspension cell culture

A

derived from cells which can divide and
survive without being attached to a substrate,
e.g. cells of haemopoietic lineage.
* Can be maintained in culture vessels that are
not tissue-culture treated.
* Requires agitation for adequate gas exchange.
* Easier to passage

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43
Q

Explain Adherent cell culture

A
  • Must adhere to a surface to survive
  • Form monolayers e.g. cells derived from
    different tissues (breast, liver)
  • Growth is limited by surface area
  • Will cease proliferating once they become
    confluent (completely cover the surface of cell
    culture vessel)
  • Cells are dissociated enzymatically or
    mechanically from surface
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44
Q

What is the purpose of culture vessels?

A

provide a contamination barrier to protect the cultures from the external environment while maintaining the proper internal environment

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45
Q

What does the cells vessels do for anchorage-dependent cells?

A

provide a suitable and consistent surface for cell attachment

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46
Q

List the types of culture vessels

A

cell culture dishes
multiwell plates
flaska

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47
Q

What is the main reasons for surface coatings?

A

it is to give something for the cell line to attach to
make the palate more hydrophilic

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48
Q

What are some effects of biological contamination?

A

They compete for nutrients with host cells
* Secret acidic or alkaline by-products that cease the
growth of the host cells
* They also produce H2O2 which is directly toxic to
cells

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49
Q

What is one way that is used to prevent contaminates by different microorganisms?
Why is this not always recommended?

A

adding different antibiotics

it may help develop resistant microorganisms

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50
Q

What are the two most common cell types used for biotherepeutic proteins?

A

CHO (hamster)
murine (mouse)

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51
Q

Why are CHO cells used?

A

PTMs are similar to humans (glycostylation)
reduced susceptibility to certain viral infections
grow in suspension cultures
serum free chemically defined media
allows gene amplification
high productivity
highly tolerant to changes in pH, Oxygen levels, pressure and temperature

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52
Q

How to get protein out?

A

plants –> seeds
cow –> Milk
Chicken –> eggs

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53
Q

Define transgenesis

A

process of introducing foreign or exogenous DNA into an animal’s genome

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54
Q

Why transgenesis?

A

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

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55
Q

Transgenic animals as bioreactors

A

Whole animals can serve as bioreactors to produce proteins
* Gene for a desired protein (Drug) is introduced via transgenics to the target cell
* By using cloning techniques, cell is raised to become an adult animal
* Produce milk or eggs that are rich in the desired prot

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56
Q

Explain retrovirus mediated transgenesis

A

infecting mouse embryos with retroviruses before the embryos are implanted
size of transgene is limited

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57
Q

Explain pronuclear microinjection

A

introduces the transgene DNA at the earliest possible stage of development of the zygote
DNA is injected directly into nucleus of egg or sperm

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58
Q

Why express rProtein in Milk?

A

Easy to purify - few other proteins in milk
* Doesn’t harm transgenic animal- no change in physiology
* rProtein is authentically modified post-translationally
* Large quantities
* Renewable source

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59
Q

What is Atryn?

A

it is an anti blood clotting protein derived from goats on this farm (antithrombin)
it is produced from the mammary glands of transgenic goats and harvested from their milk
it is the first drug made from genetically engineered animals approved by FDA

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60
Q

What are some concerns with transference of diseases?

A

There is also an added concern with cows & chickens, because the fear of
transference of diseases through drugs, such as mad cow disease and bird
flu.
* Leakage of transgene.
* Ethical concerns on using animals as machines for making drugs.

61
Q

What is EPO?

A

it is used to treat anemia
it has been genetic engineered to be produced from mammals like sheep or cows
this is a replacement of erythropoietin

62
Q

Define biotech pharmaceutical

A

is simplu any medically useful drug whose manufacture involves microorganisms or substances that living organisms produce

63
Q

List the types of biologics

A

hormones
blood products
monoclonal antibodies
cytokines
growth factors
vaccines
gene and cellular therapies
targeted therapy
fusion proteins

64
Q

What are the two-tiered cell banking?

A

master cell bank
working cell bank

65
Q

What needs to be verified for MCB?

A

that you have the cell line you wanted to produce
it is not contaminated (bacteria & viruses)

66
Q

Explain the produce of commercial production of recombinant human erythropoietin product

A

recombinant host cells from master cell bank is mixed with specially formulated media
these cells are grown to very high numbers in media
growth continued until there are enough cells to inoculated thousands of roller bottles
cells grow in roller botles and secrete r-HuEPO into media
then it is containg media is harvested
prufied
solution
packaging

67
Q

What are some sources of variation in biologics?

A

cloning
protein expression
production

68
Q

Define bioreactors

A

any device or system that supports a biologically active environment

69
Q

Explain cloning of specific gene into DNA vector

A

gene seq should be same

70
Q

Explain recombinant DNA plasmid

A

vector diff
possibility of variation -should be same vector
–> company may not disclose which vector they use

71
Q

Explain cell expansion

A

same - diff cell line
diff expression

72
Q

What is the goal of an effective bioreactor?

A

to control, contain and positively influence the biological rxn
(cells are growing and dividing)

73
Q

What are the steps of process design of bioreactor?

A

upstream steps
production
downstream

74
Q

In general cells can be cultivated either in vessels containing an appropriate liquid growth medium in which the cells can be either:

A

free in suspension
attached to microspheres or entrapped in matrices
immobilized state as monolayers

75
Q

What are the types of bioreactors?

A

liquid (submerged) –>more common
solid state (surface)

76
Q

What are the types of bioreactors products?

A

stirred tank - most preferred
airlift
microcarrier

77
Q

List the bioreaction parameters (influence growth)

A
  • Controlled temperature
  • Sufficient substrate (usually a carbon source)
  • Sugars, proteins and lipids
  • Water availability
  • Salts (for nutrition)
  • Vitamins
  • Oxygen (for aerobic processes)
  • Optimum pH
  • Product addition and by-product removal
78
Q

List some of the parts of the stirred tank

A

motor
controller
nutrient feed
oxygen probe
sparger
baffle
temperature probe

79
Q

What is the purpose of the motor?

A

movement
impeller mix nutrients - distributes

80
Q

What is the purpose of the controller?

A

control pH

81
Q

What is the purpose of baffle?

A

stops from spilling –> disturbs vortex made from stirring –> maintain homogeneity

82
Q

What is the sparger?

A

air bubbles
make sure homogeneity

83
Q

Define airlift bioreactor

A
  • A bioreactor in which the reaction medium
    is kept mixed and gassed by introduction
    of air or another gas (mixture) at the base
    of a column-like reactor.
84
Q

List some characteristics of micro-carrier bioreactors

A

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

85
Q
  1. Spargers are used in fermenters for
    A) Monitoring temperature
    B) Introducing air-bubbles of optimal size
    C) Increasing frothing
    D) Increasing pH
    E) Increasing vortexing
A

answer is B

86
Q
  1. Which is the most widely used bioreactor
    A) Fixed bed bioreactor
    B) Fluidised bed bioreactor
    C) Stir tank bioreactor
    D) Air-lift bioreactor
    E) Surface bioreactors
A

C

87
Q
  1. The most important function of Baffles in a bioreactor
    A) Prevent vortexing and ensure homogeneity of the culture media
    B) Prevent infection by bacteria and viruses
    C) Ensure that the products produced are not of high viscosity
    D) Optimal pH of the culture media is maintained
    E) Optimal temperature of the culture media is maintained
A

A

88
Q

What is the modes of bioreactors operations?

A

batch operation
continuous operation
fed-batch operation

89
Q

What are some advantages and disadvantages of batch ?

A

Easy to operate and control
Genetic stability of organism could be controlled if it is genetically engineered biocatalyst.
Lower contamination risk
Non-productive downtime is a disadvantage
Batch to batch variability is problem
Accumulation of inhibitory products is problem

90
Q

What are some advantages and disadvantages of continuous?

A

Degeneration of biocatalyst
Higher contamination risk is a disadvantage
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

91
Q

What is fed batch cultivation?

A

a modification of batch cultivation in which the nutrient is added intermittently to a batch culture

92
Q

List how many times you add media, product harvest, culture/cells for batch

A

once
once
once - constant

93
Q

List how many times you add media, product harvest, culture/cells for fed batch

A

> 1
once
once - constant

94
Q

List how many times you add media, product harvest, culture/cells for continuous

A

continuous > 1
continuous > 1
once - constant

95
Q

What is downstream processing

A

any treatment of culture broth after fermentation to concentrate and purify products

96
Q

Why is product recovery important?

A

economical
usually the product is available in very dilute form
removal of contaminants
scaling up should be considered

97
Q

What is needed for e.coli and yeast in order to product and recover?

A

grown in large fermenters at high cell density
cell lysis required
protein is harvested and purified
rapid isolation of protein necessary

98
Q

What is needed for mammalian cells in order to product and recover?

A

grown in large scale cell culture roller bottles
protein secreted into media
isolation of protein from medium
product in media

99
Q

What are inclusion bodies?

A

protein aggregates
overproduce proteins through the use of plasmid expression

100
Q

What are some advantages of protein inclusion bodies?

A

can easily be recovered to yield protein with >50% purity
aggregated forms of protein are more resistant to proteolysis

101
Q

What are some disadvantages of protein inclusion bodies?

A

inaccurate assessment of recovery
recovery requires cell breakage, protein sedimentation and pellet washing
dissolution and refolding

102
Q

What is intracellular product?

A

breaking of cells and removal of cell debris using centrifugation - generation of crude protein solution

103
Q

What is extracellular product?

A

recovery of cell culture medium by removal of cells using centrifugation or filtration

104
Q

What are some factors that are determined by glycosylation?

A

(1) Solubility
(2) Stability
(3) Serum half life
(4) Pharmacological function
(5) Immunogenicity

105
Q

What is quality of bioprocess design?

A

measured of product purity and product consistency

106
Q

In order to achieve optimal growth of cells it is important that conditions such as:

A

pH
O2
temperature
proper nutrients

107
Q

What are some media used for mammalian cell cultures?

A

fetal calf serum
growth factors
hormones

sugars
amino acids
electrolytes
vitamins

108
Q

What are the potential contaminants?

A

host related
product related
process related

109
Q

What is the most frequent source of virus introducted?

A

animal serum

110
Q

List some methods for reducing viral contaminants

A

heat treatment
radiation
dehydration
neutralization
chromatography
filtration
precipitation

111
Q

What are pyrogens?

A

are potentially hazardous substances (normally from gram negative bacteria)

112
Q

What are some general sources of protein contaminants?

A

(a) growth medium used
(b) the host proteins of the cells and
(c) ligands from affinity columns used in the purification process.

113
Q

What is the 3 steps of biopharm -eutical production?

A
  1. upstream (sterilize, get chemicals + media ready)
  2. Bioreactor (produce drug)
  3. Product recovery
114
Q

What does purification processes yield?

A

should yield potent protein with well-defined characteristics for human use from which all contaminants have been removed

115
Q

What process can be used to further purify the proteins?

A

conventional chromatographic separations

116
Q

How does chromatography work?

A

basic prodcedure is to flow the protein mixture solution through a column packed with various materials

117
Q

List the phases of chromatography

A

stationary phase
mobile phase

118
Q

Define the stationary phase

A

insoluble matrix with which components of the mixture interact with during separation

119
Q

Define the mobile phase

A

solution that is passed through or over the stationary phase, carrying with it the components of the mixture

120
Q

If your protein is positive which ion exchange do you use?

A

cation exchange chromatography

121
Q

What is charge on resin in cation exchange?

A

negative

122
Q

What are the steps in quality control of biologic drugs?

A

plasmids and host cells
protein stability
process validation
final product batch

123
Q

Explain characterization of plasmids and host cells

A

Stability of the inserted gene must be monitored for host cells
cell growth is monitored (know when to extract bacteria)

124
Q

Explain protein stability

A

it is determining the protein content
AA sequencing, peptide mapping and high performance liquid chromatography (HPLC)
also use western blot analysis/ELISA test

125
Q

What does 1a/b bind to?

A

antigen

126
Q

what does 2 a/b bind to?

A

1 a/b

127
Q

What has fluorescent tag in indirect delection?

A

2 a/b

128
Q

What has fluorescent tag in direct detection?

A

1 a/b

129
Q

What does immunofluorescence detect?

A

detection molecule gives off fluorescence once attached to the specific protein
this is also know as western blot analysis

130
Q

What does ELISA detect?

A

detection molecule is an enzyme that converts substrate to product

131
Q

What are similarities between ELISA and western blot?

A

techniques used in diagnostics
both methods are based on immuno detection
they are based on the formation of the antibody-protein complex
can analyze proteins

132
Q

What are differences between ELISA and western blot?

A

western is time consuming
also takes well trained and skilled personnel to carry out western blotting

133
Q

Explain process validation

A

it is a revalidation of purification process
spiking the pre-purification mixture with endotoxings and ensuring they are removed during the purification process

134
Q

Explain the final product batch package

A

many of the tests are done and then repeated
ensure the purified protein has maintained its activity
tests for concentraion and potency are performed

135
Q

What are some stability testing done?

A

freezing
heating
denaturing

136
Q

What is amino terminal heterogeneity?

A

it is verifying that the protein have a authentic NH2 terminus to make sure it will be treated right in the body

137
Q

Why can most proteins not be sterilized by standard methods?

A

it would denature at high temperatures

138
Q

When formulating a biotech a number of issues need to be addressed including …

A

route of administration
delivery system used
ability for target specific delivery
stability of the protein

139
Q

List some types of excipients used

A
  1. Solubility Enhancers
  2. Anti-adsorption and anti-aggregation agents
  3. Buffer components
  4. Anti-oxidants
  5. Preservatives
  6. Active Ingredient
140
Q

Approaches that can be used to enhance solubility include:

A

proper pH and ionic strength conditions
addition of AA
Addition of surfactants
adding sugars as well

141
Q

What are anti-adsorption agents?

A

added to reduce adsorption of the active protein to interfaces

142
Q

What is surface phenomenon?

A

a protein adsorbed to a surface will return to the aqueous solution

143
Q

What can cause aggregates?

A

exposed AA results in the formation of aggregates as they interact with hydrophobic residues on other proteins

144
Q

What is the purpose of insulin adsorption?

A

to prevent adsorption of insulin to the surfaces other molecules
this is often albumin which has a high affinity for these surfaces

145
Q

List some common buffer agents

A

phosphate
citrate
acetate

146
Q

Why is buffer selection important part of formulation process?

A

because of the pH dependence of protein solubility and physical and chemical stability

147
Q

List some AA that are readily oxidized

A

methionine
cysteine
tryptophan
tyrosine
histidine

148
Q

List some antioxidants

A

vitamins A, C and E
AA - acetylcyteine
chemicals - Acetic acid, sodium citrate and selenium

149
Q

Define perservatives

A

natural or synthetic chemicals that are added to prevent decomposition by microbial growth or by undesirable chemical changes
prevent bacterial growth

PHARM 124 (14 decks)

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