1. Bioseparation Engineering
Young Je Yoo

2. Two Disciplines of Biochemical Engineering
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)

3. Figure 1. Relation between starting product concentration in completed broth or medium,
and final selling price of the prepared product (Dwyer JL, 1984).

4. Figure 2. Primary factors affecting separation vs
Figure 2. Primary factors affecting separation vs. particle size (Atkinson B and Mavituna F, 1983)

5. 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

6. 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

7. Scale-up
Filtration
Distillation
Electrophoresis

8. 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 insolubles
Remove or collect cells, cell debris, or other particulates
Reduce volume (depends on unit operation)
Filtration, sedimentation, extraction, adsorption
Isolation of product
Remove materials having properties widely different from those desired in product
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

9. Intracellular, extracellular product ?
Fermentation – broth components ?
In situ separation with fermentation
(Ex : ethanol, antibiotics, taxol)
Separation for next step ?
(Ex : lactic acid for polymer)

10. Protein Purification

11. Final product requirement – define
Protein Purification Processes
Final product requirement – define
(99% ~ %)
Process design approaches
Follow examples
Modify the process
(new concept can be introduced)
Rule
Based on different physical, chemical, biochemical properties
Separate the most plentiful impurities, first
Differences in physicochemical properties
High resolution
Most difficult step, last

12. Protein Purification Processes
Cell separation
Cell disruption, debris separation (for intracellular protein)
Concentration
High resolution purification
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

13. Protein In general, economical
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

14. 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

15. 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

16. 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

17. 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.

18. After this process, solution having the following is obtained.
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

19. 5. Refolding 6. Purification 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)

20. 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

21. Evaluation of Separation and Purification Processes in Antibiotic Industry

22. 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.
+ purification은 균이 없는 상태에서 .. Denaturation을 막기위해.
+ antibiotic은 soluble form에서 쉽게 degradation되므로 rapide processing.. Place time을 잘 조절해야됨.
+ 또, antibiotic은 온도에 민감하므로 온도 조절도 잘 해야함.

23. Penicillin Cephalosporin
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.

24. 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.
During biosynthesis of CPC, the formation of the synthesizing enzyme is
sequentially induced in the metabolic pathway (Fig. 1). It is therefore necessary
to separate/isolate the enzymes, particularly for analytical purposes and for
understanding the mechanisms of biosynthesis. They are usually isolated as
cell-free crude extract by preliminary recovery methods involving cell disruption
by sonication, and harvested by centrifugation and cross-flow filtration.
세팔로스포린은 hydrophilic하다. Adsorption 으로 분리시 좀 까다롭다.. The earlier
generation of adsorbents, i.e., activated carbon, molecular sieves, silica gel and
cellulose are no longer used because of regeneration problems, but are used with
some success in the development stages of cephalosporin antibiotics [50]. Direct
isolation by adsorption in a suitably designed treatment train can offer the
advantages of high selectivity and yield. In general, non-polar macroporous
resins are suited for the isolation of hydrophilic weak acids or bases as well as
amphoteric or neutral molecules [50].
(CPC)

25. 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

26. 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.

27. Isomerization
This is biologically inactive.

28. Difficult to isolate and purify due to a highly polar side-chain.

29. 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.

30. 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???

31. Figure 4. Possible alternative scheme for penicillin purification.
New concept for Separation of Pen-G
Microfiltration
Ultrafiltration
Reverse Osmosis
Product
(Penicillin G)
Broth
Biomass
Solvent
Figure 4. Possible alternative scheme for penicillin purification.

32. Protein Stability
and Formulation

33. Protein Formulation/Stability Test
→ 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

34. 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

35. N ↔ U → I Protein Stability: Model
unfolding
inactivation
N ↔ U → I
reversible
irreversible
N – native (folded)
U – unfolded
I – inactivated
where:
Thermodynamic (conformational) stability
Long-term (kinetic) stability

36. Protein Stability: Thermodynamics
Gibb’s Free Energy
relatively stable, when ∆Gu is big.

37. Folding Stability MeasurementUV
Optical
Fluorescence
CD (circular dichroism)
viscosity
Molecular
Size Change
light scattering
turbidity
Aggregation
Net Charge
Change
gel electrophoresis
HPLC

38. Stability: Experiment
Assume: A ↔ B (linear)

39. Stability: Experiment
N ↔ U
Equilibirum constant
ΔG in the absence of denaturant
Can be estimated by molecular modeling

40. 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)

41. Structure
Tertiary structure of hGH
3D structure of pGH

42. Characterization Spectroscopy Electrophoresis Immunoassays Bioassays
- 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

43. Degradation Deamidation :
Conversion of the side chain in aspargine and glutamine residues
to the carboxylate groups of aspartate and glutamate, respectively

44. Degradation Oxidation : Reduction / Interchange of disulfide bonds
Methionine, tryptophan, histidine and tyrosine residues
 corresponding sulfoxide in methionine
Reduction / Interchange of disulfide bonds
Aggregation
Proteolysis / Hydrolysis

45. 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(■).

46. Stability Stability in solid state
Plot of the percent dimer, as measured by a size-exclusion HPLC assay, for freeze-dried samples of hGH, as a function of storage time at 400C

48. Biopia.snu.ac.kr