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PY366 Type 1 Diabetes > Biotechnology > Flashcards

Biotechnology Flashcards

1
Q

outline the steps in producing a biologic

A
  1. isolate your gene from donor DNA (B pancreatic cell)
  2. insert the human insulin gene into a suitable cloning vector (eg. plasmid)
  3. Introduce the vector carrying the insulin gene into an expression host, such as bacteria or yeast, so it can transcribe this into mRNA and translate into human insulin
  4. Use the recombinant expression host to scale up production of recombinant insulin, purify, formulate and quality control test before therapeutic use
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2
Q

describe the flow of information in a cell

A
  1. DNA stores the instructions
  2. these instructions are executed chiefly by proteins
  3. DNA makes RNA makes protein
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3
Q

what is gene expression

A

the production of proteins from DNA via RNA intermediates

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4
Q

define cloning

A

the process of creating an exact copy of a biological unit. This unit can be:
1. a copy of a defined sequence of DNA (gene)
2. a genetically identical copy of a cell
3. a genetically identical copy of an organism

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5
Q

what is a gene

A

a sequence of DNA that codes for a specific protein in a living organism

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6
Q

describe the features of a eukaryotic (insulin) gene

A
  1. has an on switch (promotor) to start transcription
  2. but the actual DNA sequence that encodes the protein (exons) may be interspersed with introns that don’t encode the protein
  3. then, a Poly A sequence is found upstream of the exons to protect the mRNA
  4. an OFF switch (terminator) stops the transcription of mRNA
  5. this can make it difficult to find the gene you want and clone it without introns
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7
Q

what can be used to clone the gene without introns

A

use mRNA to make cDNA

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8
Q

describe the process of using mRNA to make cDNA

A
  1. in the cell, gene is transcribed into pre-mRNA (introns and exons)
  2. cellular factors cut out introns and splice the exons together, giving mRNA that just encodes the protein
  3. isolate mRNA
  4. use reverse transcriptase to convert mRNA to DNA (cloned DNA)
  5. but cDNA will contain gene and all the other active genes
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9
Q

what technique can be used to obtain a gene

A

Polymerase chain reaction can be used to isolate gene from cDNA

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10
Q

describe how PCR works

A
  1. can isolate a sequence of DNA by using a pair of primers to bracket the desired DNA sequence and copy it out using DNA polymerase
  2. the DNA polymerase enzyme was destroyed by heating the double stranded DNA to make the strands separate
  3. by using a polymerase isolated from a bacteria that lived in hot springs at 100 degrees, the process could be automated
  4. the process of denature, anneal, elongate is repeated 25-30 times using a thermal cycler to get enough cDNA to clone
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11
Q

what is PCR used for

A
  1. can be used to isolate gene from cDNA
  2. can add sequences to make cloning gene into a vector easier
  3. key method for modifying the insulin gene to produce the various insulin analogues
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12
Q

what are the 3 main steps of cloning genes into a plasmid vector

A
  1. insert gene into cloning vector to create chimeric vector
  2. transfect chimeric vector into prokaryotic cell
    - cell becomes transformed
  3. screen transformed cells to ensure correct gene and vector are present
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13
Q

describe the concept of a cloning vector

A
  1. all DNA that is to be copied needs to be part of a larger, stable structure that is capable of being copied
  2. normal cellular DNA in living organisms is organised into chromosomes (stable)
  3. but there are many naturally occurring smaller DNA structures that are capable of replication, used as cloning vectors
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14
Q

define cloning vector

A

a small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes

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15
Q

how is gene inserted into a vector

A
  1. gene inserted into plasmid
  2. plasmid put into bacterial cell
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16
Q

what are plasmids

A
  1. found in cytoplasm of bacteria and are small double stranded, self replicating circular DNA molecules
  2. can be transmitted from one bacterium to the next
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17
Q

outline the features of a classic plasmid vector

A
  1. Ampicillin resistance gene- allows transformed E coli to grow on medium containing ampicillin
  2. MCS (multiple cloning site)- where genes are inserted
  3. origin of replication- replication of plasmid is initiated here
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18
Q

describe the process of transforming the cells

A
  1. make bacterial cell temporarily permeable (competent) so that it takes up plasmid DNA
  2. then stop permeability so cell contents don’t leak out (transformation)
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19
Q

how can transformation be achieved by?

A
  1. chemical transformation/heat shock
  2. electrotransformation/electroporation
  3. lipofection
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20
Q

describe the process of screening transformed cells

A
  1. plate transformed E coli on agar plates containing appropriate antibiotic which vector confers resistance to
  2. only bacteria carrying plasmid will grow into colonies
  3. pick a single colony (Clone) then scale up in culture
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21
Q

describe the process of expressing the gene

A
  1. in order to copy the chimeric vector and produce recombinant protein from the inserted foreign gene, need cellular machinery required for DNA replication, transcription and translation
  2. this is provided by the expression host
  3. hosts can be prokaryotic or eukaryotic
  4. transgenic plants and animals can also express recombinant proteins
  5. each host has a specific range of vectors and ways of introducing them into the host
22
Q

give examples of common expression systems

A
  1. E coli
  2. yeast
  3. mammalian cells
23
Q

what is meant by protein processing

A

some proteins require additional modifications after they have been translated to become fully functional

24
Q

what does post translational modifications include

A
  1. cleavage of precursor proteins (insulin)
  2. amino acid modifications- acylation, methylation, phosphorylation, glycosylation
25
Q

what is meant by glycosylation

A

addition of carbohydrate chains to form glycoproteins

26
Q

what are the 3 components in an expression system

A
  1. an expression host
  2. an expression vector
  3. a transfection reagent or device
27
Q

what are the advantages of prokaryotic (E coli) expression systems

A
  1. very fast growth kinetics
  2. high cell densities achieved- good for scale up
  3. rich, complex media relatively cheap
  4. transformation with plasmids cheap and easy
  5. many high copy number expression plasmids available
  6. high yield of product
  7. product bioactive
28
Q

what are the disadvantages of prokaryotic (E coli) expression systems

A
  1. can lead to lower yield
  2. product can reduce rate of cell growth
  3. don’t integrate into genome
  4. only for simple monomeric proteins
    - limited post translational modifications
29
Q

give examples of recombinant protein biologics produced in E coli

A
  1. short acting insulin (humulin)
  2. somatotropin (human growth hormone)
  3. tissue plasminogen activator
30
Q

what are the advantages of yeast expression systems

A
  1. well characterised, fast growing, cheap and safe
  2. performs some post translational modifications
  3. secretes some proteins
31
Q

what are the disadvantages of yeast expression systems

A
  1. expression levels less than 5% of total protein
  2. when growing large culture volumes, yeast plasmids can be lost
  3. compared to mammalian cells, yeast cells tend to overglycosylate
  4. secreted proteins usually trapped in periplasmic space
32
Q

give examples of recombinant protein biologics made in yeast

A
  1. short acting insulin
  2. hepatitis B surface antigen (vaccine)
  3. hirudin anticoagulant
33
Q

what can be used in a mammalian cell expression system

A
  1. fibroblast based line, Chinese hamster ovary cells (CHO) is mostly used
  2. can grow cells in adherent culture
  3. can also use conventional bioreactor or a disposable wave bag
34
Q

describe the features of mammalian cell expression vectors

A
  1. mammalian cell expression plasmids are shuttle vectors- have E coli components to allow cloning of foreign genes to be undertaken in E coli
  2. chimeric plasmid- CHO cells by complexing with calcium phosphate, PEI or lipofection
    - mammalian cells don’t need to be made competent
  3. plasmid also contains a strong viral promotor, regulatory elements and a selectable marker to verify successful transformation and optimise gene expression
35
Q

what are the advantages of mammalian cell expression systems

A
  1. correct post translational modification
  2. correctly folded quaternary structure
  3. can create recombinant cell lines for consistent batch to batch product
  4. protein can be secreted- simpler purification
  5. can adapt cells to suspension culture for scale up
36
Q

what are the disadvantages of mammalian cell expression systems

A
  1. slow culture growth
  2. expensive media required
  3. long development time to provide high yielding stable cell lines
  4. relatively low yields
  5. risk of viral contamination of product
37
Q

give examples of proteins made in mammalian cells

A
  1. Factor VIII and IX
  2. therapeutic monoclonal antibodies
  3. interferons
38
Q

what are the 3 stages in the production process of manufacturing your recombinant protein

A
  1. upstream processing
  2. fermentation
  3. downstream processing
39
Q

explain what is involved in upstream processing

A
  1. medium preparation- formulated and optimised for host organisms to result in optimum product yield at minimum costs
    - seperation of particulate and inhibitory chemicals from medium
    - medium sterilisation
  2. sterilisation of bioreactor
  3. purification of air
  4. adequate supply of producer cells
    - cell banking, validation and maintenance
40
Q

describe the use of cell banking systems

A
  1. production cell lines are stored at -170 degrees in a 2 tiered cell bank of master and working cell banks
  2. master cell banks cells must be extensively validated before use
  3. this ensures long term availability of producer cell lines
41
Q

describe the process of fermentation

A
  1. working bank vial removed from storage
  2. generation of starter cultures
  3. production scale cell culture
42
Q

what are stirred tank bioreactors

A
  1. high grade stainless steel, fully sterilisable
  2. jacketed for temperature control
  3. impeller ensures even distribution of nutrients and cells
  4. air/gas sparger
  5. ports:
    - probes for pH, temperatures
    - pH adjustment and addition of nutrients
43
Q

where can fermentations be carried out

A
  1. can only be carried out in cell types that grow in suspension (most prokaryotes and some simple eukaryotes)
  2. most mammalian cell lines are anchorage dependent
    - they need to grow attached to solid surfaces
  3. many suspension adapted CHO lines are available but require expensive specialised media
44
Q

what is used in mammalian suspension cultures

A
  1. mammalian cell walls are thin and fragile, so use
    - air mixing
    - a range of adapted impellers for low shear and high aeration
  2. other bioreactors such as wave bags are gentler
  3. if only small amounts of product are required, adherent cells produced in Roller bottles or cell factories may be just as economic and don’t require expensive media
45
Q

what are anchorage dependent cultures

A
  1. need to be attached to solid substrates
  2. lab or medium production scale, can use roller bottles or cell factories
  3. reactor scale, can use hollow fibre reactors or a fluidised bed of micro carriers
46
Q

where is downstream processing carried out

A
  1. normally carried out in a pharmaceutical clean room
    - must be sterile
  2. operators use a Grade A laminar flow cabinet
  3. the filling process is automated to prevent contamination between the operator and the product
47
Q

what are the 3 main steps involved in downstream processing

A
  1. cell harvesting and recovery of crude product
  2. purification
    - concentration and initial purification
    - main purification (chromatography)
  3. final formulation
    - final product formulation
    - product filling, freeze drying and sealing
    - labelling and packaging
48
Q

describe the analysis of the final product

A

a range of potential impurities present in pharmaceutical products may have medical consequences and requires testing:
- microorganisms
- viral particles
- pyrogenic substances
- DNA
- contaminating proteins

49
Q

what is the device used for most fermentations

A

a bioreactor

50
Q

why must alternative devices be considered for the fermentation of mammalian cells

A

due to their fragility in suspension culture

PY366 Type 1 Diabetes (12 decks)

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