Download Separation Process Synthesis

Bioseparation Engineering
Young Je Yoo
Biotechnology
built on the genetic manipulation of organisms to produce
commercial products or processes
Biochemical Engineering
responsible for the implementation of the products and processes
Two Disciplines of Biochemical Engineering
Upstream Engineering (Fermentation/Cell Culture)
Downstream Engineering (Bioseparation or Purification)
Figure 1. Relation between starting product concentration in completed broth or medium,
and final selling price of the prepared product (Dwyer JL, 1984).
Figure 2. Primary factors affecting separation vs. particle size (Atkinson B and Mavituna F, 1983)
Separation Process Design
• mimic similar processes/cases
• new concept process
Separation Process Synthesis
Scale-up is to be considered.
*design software
*information from equipment vendors
Criteria for Process Design
Used in evaluating and designing bioseparation process
• product value, purity, impurities acceptable
• cost of production as related to yield
• scalability
• robustness with respect to process stream variables
• easy maintenance
Scale-up
• Filtration
• Distillation
• Electrophoresis
Stages of Bioseparation: An idealized process
(1)removal of solids (or recovery), (2)isolation of product, (3)purification,
and (4)polishing constitute a sequence of events applied to
nearly every product preparation
Table 1. Objectives and Typical Unit Operations of the Four Stages in Bioseparation (Harrison et al., 2003)
Stage
Objective(s)
Typical Unit Operations
Separation of
Remove or collect cells, cell debris, or other
Filtration, sedimentation,
insolubles
particulates
extraction, adsorption
Reduce volume (depends on unit operation)
Isolation of product
Remove materials having properties widely
different from those desired in product
Reduce volume (depends on unit operation)
Extraction, adsorption,
ultrafiltration, precipitation
Purification
Remove remaining impurities, which typically are
similar to the desired product in chemical
functionality and physical properties
Chromatography, affinity
methods, crystallization,
fractional precipitation
Polishing
Remove liquids
Convert the product to crystalline form
(not always possible)
Drying, crystallization
• Intracellular, extracellular product ?
• Fermentation – broth components ?
• In situ separation with fermentation
(Ex : ethanol, antibiotics, taxol)
• Separation for next step ?
(Ex : lactic acid for polymer)
Protein Purification
Protein Purification Processes
Final product requirement – define
(99% ~ 99.999%)
Process design approaches
• Follow examples
• Modify the process
(new concept can be introduced)
Rule 1. Based on different physical, chemical,
biochemical properties
2. Separate the most plentiful impurities, first
3. Differences in physicochemical properties
4. High resolution
5. Most difficult step, last
Protein Purification Processes
1. Cell separation
2. Cell disruption, debris separation
(for intracellular protein)
3. Concentration
4. High resolution purification
5. Polishing of final product
Efficiency: 0.9 for each step is assumed
after 6 ~ 7 steps → total yield ≈ 0.2
• increase yield/efficiency is very essential
• refolding is important
Protein
Extracellular protein (secreted): easy to separate
Inclusion body: cell disruption → centrifuge
solubilization → refolding - chromatography
Inclusion body is formed by overexpression in E. coli.
Advantages of Inclusion Body
• not breakable with protease
• not toxic to cells
• high expression
In general, economical
What to remove?
- cell debris, endotoxin, other proteins,
nucleic acid
- deaminated form, oxidized form, dimer
form of the product protein
- need more than 3 steps of chromatography
Protein Purification Processes
1. Cell Harvest by Centrifugation
- Continuous disk stack type or batch centrifuge
- tangential flow microfiltration (M/F)
2. Cell Disruption
- French press, lysozyme, ultrasonification or bead mill
E. coli: mainly high pressure homogenizer
2 – 3 times for high efficiency
Yeast: strong cell wall → bead mill
3. Solubilization of Inclusion Body
Inclusion body: can be obtained at 60 – 70% purity
Denaturing Agent (chaotropic agent)
Urea, guanidine HCL, NaOH (high pH)
In case of urea, it should have no cyanate.
protein + cyanate → carbamylation occurs
Guanidine: good but expensive
So, 7 – 8 M urea is widely used.
If disulfide bond exists in protein, add mercaptoethanol,
DTT (dithiothreitol) or cysteine → reduce disulfide bond
to free thiol
Add 1-2 mM EDTA to reduce metalloprotease activity.
After solubilization, viscous solution
containing the following is obtained.
70% of target protein, cell debris, DNA,
endotoxin, 7 – 8 M urea, 50 – 100 mM
cystein, 1 – 2 mM EDTA
• Host which has no protease is used.
• Short processing time is required.
4. Capture Impurities
For large volume, short time → chromatography
If histidine tag exists → IMAC
(immobilized metal affinity chromatography)
If no histidine tag → urea → ion-exchange chromatography
After this process, solution having the
following is obtained.
target protein (1 – 5 mg/ml), small impurities,
7 – 8 M urea, 50 – 100 mM cystein,
1 – 2 mM EDTA, pH 7.9 – 9.0 buffer
5. Refolding
- Remove denaturant
method:
dialysis, diafiltration
- Refolding is being performed at low concentration
(0.1 – 0.5 g/L) to prevent protein aggregation
6. Purification
- IEC, IMAC, HIC
HIC: hydroxyapatite chromatography
- diafiltration or gel permeation chromatography for
desalting (to exchange buffer solution)
7. Polishing
- Remove dimer, polymer, endotoxin
- Use chromatography
- After polishing → filtration using 0.2μ filter to
remove bacteria
Protein from mammalian cell
- secreted form
- inactivate virus, virus removal process is required
To prevent protein oxidation
- operation under nitrogen gas
low temperature is preferred
Evaluation of Separation and Purification Processes
in Antibiotic Industry
 Conventional separation technologies
Two main processing segments;
• fermentation section
• separation and purification section
 All antibiotic fermentations use
similar equipment, while different
antibiotics are produced by using
different cultures and growth media.
 The separation and purification
sections of an antibiotic plant can
differ substantially depending on the
specific antibiotic that is being
produced
and
enduse
purity
requirements.
 Filtration, centrifugation, extraction,
and crystallization are generally
employed.
 Penicillin
 With proper strain selection penicillin can be
produced in concentrations up to 40 g/L, which is
one or two orders of magnitude greater than many
other antibiotics.
 Cephalosporin
 broad-spectrum antibiotics that have low toxicity.
 They are produced by the same process used for
the penicillins, utilizing a different growth medium
and organisms.
 The final fermentation broth concentrations are
one to two orders of magnitude lower than
penicillin, resulting in a more difficult separation
and purification process.
(CPC)
•
Recovery of cephalosporin from the
filtrate is difficult because of the low
product concentration and the need to
remove
high
molecular
weight
biological compounds.
•
During biosynthesis of CPC, the
formation of the synthesizing enzyme
is sequentially induced in the
metabolic pathway. It is therefore
necessary to separate/isolate the
enzymes.
 General production scheme for penicillin
fermentation
Penicillin, growth
media, cell, other
metabolites
filtration
Removal of cells
→penicillin rich
filtrate
cooling
Minimization of
degradation
Solvent extraction
Using aqueous
solution at pH2-2.5
and organic solvent
Penicillin is favor in
organic solvents
because of their
protonation form.
Carbon-treatment
Removal of pigments
and other impurities
Extraction using pH
7.5 aqueous solution
Back into an
aqueous solution
crystallization
Adding sodium or
potassium acetate
Washing, drying
Using vacuum or
warm air
 General production scheme for cephalosporin
•
Many different separation and purification schemes are employed, including
conventional solvent extraction, ion exchange resins, and salting out procedures.
Adding water and a polar organic solvent
Fermentation
Filtration
Adsorption
W/ active C
Anion
exchange
Adding salt solution
•
Frequently, the carbon adsorption steps are replaced with a precipitation step. Many
different precipitations are possible.
1. Crystallization of the potassium or sodium salt from purified aqueous solution of the
cephalosporin by concentration and/or addition of large volumes of a miscible solvent.
2. The zinc salt (also copper, nickel, lead, cadmium, cobalt, iron, and manganese) can be
crystallized from purified aqueous solutions.
3. Insoluble derivatives such as the n-2,4-dichlorobenzoyl cephalosporin and
tetrabromocarboxybenzoyl cephalosporin are crystallized as the acid from solution.
4. Sodium-2-ethyl hexanoate will precipitate the sodium salt of N-derivatized
cephalosporins from solvents.
Isomerization
This is biologically inactive.
Difficult to isolate and purify due to
a highly polar side-chain.
Separation Process Synthesis
Ex : Penicillin Production
Figure 3. Penicillin production. After fermentation the biomass is separated by filtration. The antibiotic,
which is in the filtrate, is isolated and purified by extraction. It is then polished by crystallization and dried.
Separation Process Synthesis
Increasing amount of washing water increases recovery
but thus the amount of wastewater generated
Vacuum Filter
Temperature: material degradation and yield
Solvent: type of solvent, solvent to water ratio
Mode of Operation: Single or multiple stage
Extraction
Optimization of operating condition is essential!!
Are there other novel concepts of separation???
New concept for Separation of Pen-G
Broth
Microfiltration
Ultrafiltration
Biomass
Solvent
Reverse
Osmosis
Figure 4. Possible alternative scheme for penicillin purification.
Product
(Penicillin G)
Protein Stability
and Formulation
Protein Formulation/Stability Test
Formulation:
→ Storage stability before use (1.5 ~ 2 years)
→ Add stabilizer and bulking agent
→ 0.22 μ filter (for sterilization)
→ Packing , or
→ Freeze drying (lyophilization) → powder packing
Stable Protein → liquid–form product
Unstable Protein → solid–form product
Protein Formulation/Stability Test
Stabilizer:
→ human serum albumin
lowers glass wall attachment
→ amino acid
• lowers lysozyme attachment to glass wall
• lowers globulin aggregation
→ polyol (sorbitol, glycerol, mannitol)
use for lyophilization
→ antioxidant, salt and surfactant
Protein Stability: Model
unfolding
inactivation
N↔U→I
reversible irreversible
where: N – native (folded)
U – unfolded
I – inactivated
Thermodynamic (conformational) stability
Long-term (kinetic) stability
Protein Stability: Thermodynamics
Gibb’s Free Energy
Gu  Gu  G f
relatively stable, when ∆Gu is big.
G  Gu wild  Gu mutant  0
Folding Stability Measurement
Optical
UV
Fluorescence
CD (circular dichroism)
Molecular
Size Change
viscosity
light scattering
turbidity
Net Charge
Change
gel electrophoresis
HPLC
Aggregation
Stability: Experiment
Assume: A ↔ B (linear)
Stability: Experiment
N↔U
Equilibirum constant
U
Kf 
N
G f   RT ln K f
ΔG in the absence of denaturant
Can be estimated by molecular modeling
Case study
 Human Growth Hormone
Ref : “Directed expression in Escherichia coli of a DNA
sequence coding for human growth hormone”,
Goeddel, D.V. et al., Nature 281:544 (1979)
Structure
Tertiary structure of hGH
3D structure of pGH
Characterization
 Spectroscopy
- UV absorption
- CD (Circular dichroism)
- Fluorescence
 Electrophoresis
- SDS-PAGE
- IEF (Isoelectric focusing) gel electrophoresis
 Immunoassays
 Bioassays
 Chromatographic methods
- Reversed – phase HPLC
- Size – exclusion chromatography
- Ion - exchange chromatography
Degradation
 Deamidation :
Conversion of the side chain in aspargine and glutamine residues
to the carboxylate groups of aspartate and glutamate, respectively
Degradation
 Oxidation :
Methionine, tryptophan, histidine and tyrosine residues
 corresponding sulfoxide in methionine
 Reduction / Interchange of disulfide bonds
 Aggregation
 Proteolysis / Hydrolysis
Stability
 Solution stability
Plot of the first – order rate constants in days for deamidation of hGH
in solution as a function of pH at 250C(•), 400C(■).
Stability
 Stability in solid state
Plot of the percent dimer, as measured by a size-exclusion HPLC assay, for freezedried samples of hGH, as a function of storage time at 400C
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