Protein Pharmaceuticals (II)
“Recombination and Production”
Dr. Aws Alshamsan
Department of Pharmaceutics
Office: AA87
Tel: 4677363
aalshamsan@ksu.edu.sa
Objectives of this lecture
By the end of this lecture you will be able to:
1. Recognize the advantages of recombinant
over naturally-isolated proteins
2. Choose proper vehicle for recombinant
protein production
3. Compare different methods of protein
production
Polypeptides and Proteins
• Most protein pharmaceuticals today are
recombinant products
– Cheaper, safer, abundant supply
Six-Step Process
1.
2.
3.
4.
5.
6.
Isolation of gene of interest
Introduction of gene to expression vector
Transformation into host cells
Growth of cells through fermentation
Isolation & purification of protein
Formulation of protein product
Requirements for recombinant protein
production
Vector
Plasmid
Gene
Insulin
Host
E. coli
pUC18
Selectable marker
a gene (antibiotic resistance)
when expressed on plasmid will
allow host cells to survive
Polylinker (multiple cloning site MSC) is a
many
short segment of DNA which contains
(up to ~20) restriction sites
Promoter is a short DNA
sequence
which enhances expression of
adjacent gene
Ori origin of replication is a particular
sequence where replication is
initiated
Restriction Nucleases
Enzymes that cut double-stranded DNA at
specific recognition nucleotide sequences
known as restriction sites (palindrome)
‫مودته تدوم لكل هول *** وهل كل مودته تدوم‬
Ligases
• DNA ligation is the act of joining together DNA strands with
covalent bonds with the aim of making new viable DNA or
plasmid
• T4 DNA ligase has the unique ability to join sticky and blunt
ended fragments
DNA Recombination
Choice of production vehicle
• In principle, any protein can be produced
using any genetically engineered organism
Bacteria
Yeast
Mammalian
Recombinant Protein Expression
Systems
•
•
•
•
•
•
•
Escherichia coli and other bacteria
Pichia pastoris and other yeasts
Baculovirus
Mammalian cell culture
Plants
Animals
Cell free
Choice of Expression System
• Choice depends on size and character of protein
– Large proteins (>100 kD)? Choose eukaryote
– Small proteins (<30 kD)? Choose prokaryote
– Glycosylation essential? Choose baculovirus or
mammalian cell culture
– High yields, low cost? Choose E. coli
– Post-translational modifications essential? Choose
yeast, baculovirus or other eukaryote
Choice of Expression System
• Not every type of protein can be produced
by any cell type
Prokaryotic
Eukaryotic
Eukaryotic
Bacteria
Yeast
Mammalian
Concentration
High
High
Low
Molecular weight
Low
High
High
Secretion
No
Yes/No
Yes
Incorrect
Correct
Correct
Glycosylation
No
Incorrect
Correct
Retrovirus
No
No
Possible
Endotoxin
Low risk
Viruses
Production Quality
Low
Moderate
High
Scale-Up Capacity
High
High
Low
Protein Feature
Folding
Contamination
Cultivation Method
• The culture method will determine the
separation and purification methods.
• For research (small scale):
– Cell culture flasks
• For productions (large scale):
– Fermentors and Bioreactors
Growth of Microorganisms
• Culture medium
• Oxygen
• Temperature control
Culture Media
• It is important to provide nutritional conditions that
exist in bacterial natural habitat.
• Common components:
•
•
•
•
•
•
Water
Source of carbon and energy
Source of nitrogen
Trace elements
Growth factors
Buffer
Terrific Broth
• Formulation per one liter:
• 12 g Peptone (peptic digest of casein) or Tryptone (tryptic
digest of casein)
• 24 g Yeast Extract
• 9.4 g dipotassium hydrogen phosphate
• 2.2 g potassium dihydrogen phosphate
• 4 g Glycerol
• Water
• Phenol Red
Common Media
• Commonly used bacterial E.coli culture media:
•
•
•
•
•
•
•
LB (Luria Bertani) Miller broth (1%NaCl): 1% peptone, 0.5% yeast extract, and 1%
NaCl
LB (Luria Bertani) Lennox Broth (0.5% NaCl): 1% peptone, 0.5% yeast extract, and
0.5% NaCl
SOB medium (Super Optimal Broth): 2% peptone, 0.5% Yeast extract, 10mM
NaCl, 2.5mM KCl, 10mM MgCl2 , 10mM MgSO4
SOC medium (Super Optimal broth with Catabolic repressor): SOB + 20mM
glucose
2x YT broth (2x Yeast extract and Tryptone): 1.6% peptone, 1% yeast extract, and
0.5% NaCl
TB (Terrific Broth) medium: 1.2% peptone, 2.4% yeast extract, 72 mM K2HPO4, 17
mM KH2PO4 and 0.4% glycerol
SB (Super Broth) medium: 3.2% peptone, 2% yeast extract, and 0.5% NaCl
Choice of Medium
Medium Name
Applications
LB Miller Broth
E.coli growth, culture and propagation; plasmid DNA and protein
production
LB Lennox Broth
E.coli growth, culture and propagation; plasmid DNA and protein
production
SOB Medium
Prepare high efficiency competent cells; plasmid DNA and protein
production
SOC Medium
Plasmid transformation and growth of competent cells
2x YT Medium
Phage DNA production
TB
High yield protein and plasmid DNA production
SB
High yield plasmid DNA and protein production
Fetal Bovine Serum (Fetal Calf Serum)
• Fetal bovine serum comes from the blood drawn
from a bovine fetus via a closed system of collection
at the slaughterhouse.
Fetal Bovine Serum (Fetal Calf Serum)
• Advantage:
– Nutrition
• Disadvantages:
–
–
–
–
Contaminating proteins
Variability among animals
Microbial contamination (virsues / bacteria / mycoplasma / fungi)
Endotoxin (even with sterile serum)
• Serum-free media are preferable in large scale
production
Contaminants
• Contamination does not always mean
“dirtiness” but it may refer to the presence of
unwanted materials
• Contaminants can be:
– Host related
– Product related
– Process related
Contaminants
Host Related
Product Related
Process Related
Viruses, Bacteria
Aminoacid substitution or
deletion
Growth medium
components
Host-derived proteins and
DNA
Denatured protein
Purification reagents
Glycosylation variants
Conformational isomer
Metals
N- and C-terminal varians
Dimers and aggregates
Column materials
Endotoxin
Disulfied pairing variants
Deamidated species
Protein fragments
Shake Flask Incubator
Shaking increases oxygen
transfer
Growth of Cells
Shake Flask Incubator
Shake Flask Incubator
• Heavily insulated, heated with
thermoregulation to keep temperature within
0.5 oC
• Rotatable platform to spin up to 500 rpm to
facilitate aeration (dissolves N2 and O2 needed
for growth)
• Designed for small-scale growth
Microorganisms Growth Curve
Fermentors and Bioreactors
• Larger scale, sustained growth requires
bioreactors & fermentors
• Fermentors have been used for centuries –
primarily for brewing alcohol and making
vinegar
• Modern technology and chemical engineering
principles continue to improve fermentor
design
Fermentor vs. Fermenter
Fermentor
Fermenter
Bioreactors
• Stirred tank reactors (mechanical agitation for
aeration)
• Bubble column reactors (bubbling air into media for
aeration)
• Airlift reactors (air and media circulate together)
• Microcarrier (fixed bed) reactor (cell immobilization
on macroporous bed)
• Membrane bioreactor: (combination of membrane
filtration process with a suspended growth
bioreactor)
Stirred Tank
Bubble Column and Airlift
Airlift reactor
Bubble column reactor
Fixed-bed reactor
Membrane Bioreactors
Nutrition
• Combine conventional
biological treatment processes
with membrane filtration
• Used widely in municipal and
industrial wastewater
treatment
Product
Waste
Fermentor Scale up
Stock culture Shaker flask
(5-10 mL)
(200-500 mL)
Seed fermentor
(10L to 100 L)
Production fermentor
(1000 L to 100,000 L)
Series of fixed-bed reactor systems in different scales
Production Process
• The most common growth processes are:
1. Batch
2. Continuous
3. Fed Batch
• The mode of feeding determines the
classification of the bioreactor
Batch Process
• The bioreactor is only fed once
• The bioreactor will be allowed to run till
completion:
– In aerobic batch reactors, stopping oxygen supply will turn
the system anaerobic and the will be the end of the batch
• Very difficult to achieve in real life because
there should be no input to or withdrawal
from the bioreactor even for sampling
Cells Retention in Batch Bioreactor
• Cells are not lost throughout the fermentation
• They have time to adapt and multiply to its maximum rate in
the batch bioreactor
• They will completely biotransform the substrate to products
provided other nutrient and environmental parameters are
sustained
• They will enter the stationary phase either due to limiting
nutrients or accumulation of toxic products
Continuous Process
• The bioreactor is fed continuously
• The amount of feed introduced into the
bioreactor equals the removed volume
• The process is sensitive and subjected to
influence from various factors
Cells Retention in Continuous Bioreactor
• Continuous wash of cells
• Cells always need to continually adapt to the input of
nutrients
• This is reduced if the cells are immobilized or
recycled
Fed Batch Process
• The addition of nutrients is controlled to
allow temporal variations in the supply
nutrients
• Possible to control the rate of growth of the
microorganisms or the concentration of the
biomass by controlling the feed parameters
• The most common process in industry
Cells Retention in Fed Batch Bioreactor
• Cells are exposed to intermittence of new
input of nutrients
• But the cells are maintained in healthy state
Feed Dosing of The Bioreactors
• The stability of the composition of the microbial population
depends on:
1.
2.
The impact of the changes
The rapidity of the microorganisms to adjust to the changes forced
upon the microorganisms
• Concentration, stable composition of the feed and how the
feed is introduced will affect the performance of the
bioreactor especially with:
1.
2.
3.
4.
Very sensitive and fastidious microorganisms
Very slow growers
Very small bioreactors
Bioreactors operated on very short retention time
Now you are able to:
 Recognize the advantages of recombinant
over naturally-isolated proteins
 Choose proper vehicle for recombinant
protein production
 Compare different methods of protein
production
Next Lecture
Purification of protein pharmaceuticals