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JEAN RAYNELL S. BELLO November 14, 2014
5ChEA Quiz#4
I. Downstream Processing/Recovery and Purification of Products
a.
Similarities and differences in the separation processes of intracellular and extracellular
products.
i. Techniques Performed
The most crucial element that will affect downstream process is the availability of
specific assays for the target protein. If the product of interest is secreted
(extracellular), then the liquid part containing extracted proteins, metabolic
products, organic acids and alcohols, is kept and this step is generally called broth
clarification. On the other hand, if the product is intracellular, the solid fraction ofthe harvesting such as membrane proteins and lipids is kept. This step is known as
cell harvesting step (Rizvi et.al., 2008). The liquid phase of the fermentation broth
is separated by centrifuging after fermentation and the cell walls are broken down.
ii. Overall economic impact
Cost determinants in the operations generally depend on the number of unit
operations involved. Larger number of equipment and processes breeds higher
costs. Concentrating dilute products requires higher throughput and polishing steps
are often more expensive. Analytical grade chemicals and pharmaceutical products
requires strict purifying process, hence more activity is required. The figure below
summarizes the relationship between selling price and concentration before
downstream processing (Dwyer Plot).
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Figure 0:Dwyer Plot - Relationship between selling price and concentration before
downstream processing (Doran, 2013)
b.
Study focusing on the downstream processing of any biochemical reaction product
i. Citation
Title: Downstream processing of triple layered rotavirus like particles
C. Peixoto, M.F.Q. Sousa, A.C. Silva, M.J.T. Carrondo, P.M. Alves,Downstream
processing of triple layered rotavirus like particles, Journal of Biotechnology,
Volume 127, Issue 3, 10 January 2007, Pages 452-461, ISSN 0168-1656,
http://dx.doi.org/10.1016/j.jbiotec.2006.08.002.
(http://www.sciencedirect.com/science/article/pii/S016816560600647X)
Keywords: Rotavirus like particles; Purification; Scale-up; Rotavirus vaccine
ii. Downstream Process Summary
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1. Rotavirus CultivationSpodoptera frugiperda Sf-9cells were grown in
spinner flasks and used as inoculum for 21 bioreactors. Cells were infected
with baculovirus. At the time of infection and every 48 hours afterwards, the
culture was supplemented with protease inhibitors. The bioreactor was
harvested at 120 hour post-infection.
2. Rotavirus VLPs DeterminationVLPs were quantified using a sandwich
ELISA protocol developed by the authors.
3. Characterization of VLPssamples were collected during the purificaqtion
process and loaded onto NuPage precast, gradient 4012% acrylamide gels to
determine the protein content. Protein was visualized by Simply Blue Safe
Strain.
4.
VLP PurificationUltracentrifugation was used to purify the VLPs. Extraction
with Vertrel was performed to remove lipids from the sample.
5. Detection of Recombinant Baculovirus DNATo detect recombinant
baculovirus in final purified samples, DNA extraction of the collected samples
was performed and analyzed using real time PCR in a LightCycler system.
6. Determination of Protein and DNA contentBCA kit 23225 protein assay was
performed on the samples collected at the end of each downstream process
step.
7. Electron Microscope AnalysisThis step was performed to analyze the
presence, integrity and morphology (shape, size) of the VLPs.
8. Assessment of VLPs integrityThe stability of the VLPs was examined by
size exclusion chromatography.
II. Optimization of Oxygen Mass Transfer in a Multiphase Bioreactor With
Perfluorodecalin as a Second Liquid Phase
a.
Background
i. Perfluorodecalin
Perfluorodecalin (PFCs) are petroleum-based compounds that are synthesized by
replacing hydrogen in the decaline by fluorine atom. The molecular formula is
C10F18. They are considered to be good candidates as oxygen carriers in
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fermentation media because of its non-toxicity towards living cells. Strong C-F
bonds make the compound stable and chemically inert. The oxygen solubility in the
compound is 10-20 times higher than in water. PFCs have very low solubility in
water which makes it convenient for recovery and recycle processes; hence
commercially feasible. Another feature of PFCs is that it enhances mass-transfer in
gas-liquid systems with its very low surface tension (Amaral et.al 2007).
Perfluorodecalin has the following physical and chemical properties shown in Table
1 (F2 Chemicals Ltd, 2012):
Table 1:Perfluorodecalin Properties
Chemical name: Perfluorodecalin
CAS Registry Number: 306-94-5IUPAC Name:
1,1,2,2,3,3,4,4,4a,5,5,6,6,7,7,8,8,8a-
Octadecafluoronaphthalene
Formula: C10F18
Molecular Wt.: 462
Boiling Point: 142.0C
Pour Point: -5.0C
Density: 1.941 g/ml
Specific Heat: 1.05 kJ/kg/KDynamic Viscosity: 5.0999999 mPa s
Kinematic Viscosity: 2.63 mm2/s
Critical Temperature: 565.20001 K
(292.1C)
Vapour Pressure: 0.88 kPa
Vapour Density: 0.0149 g/ml
Refractive Index: 1.313
ii.
Olive OilOlive oil was another compound used in the bioreactor design as second liquid
phase with higher oxygen solubility. It was selected for it also induces the lipase
production from Yarrowia lipolyticathe cell being cultivated in the study - due to
the presence of triglycerides in olive oil (Amaral et.al, 2007). However, olive oil
caused a reduction in the oxygen mass-transfer coefficient, kLa. This problem is due
to the fact that olive oil is more viscous than PFC and less dense than water. Hence
it stays at the top of the bioreactor which makes the dispersion of the second phase
more difficult requiring very high agitation rates. Yet with high agitation rates, it
may lead to cell inactivation due to high shear rates. Table 2 shows the physical
properties of olive oil (Kiritsakis, 1998):
Table 2:Physical Properties of Olive Oil
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Density or Specific Gravity 0.9150-0.9180 @ 15.5C
Viscosity 84 mPa.s (84 cP) at 20C
Specific Heat 2.0 J/(g.)( C) or .47Btu/(lb.)(F)
Thermal Conductivity 0.17 @ 20C
Dielectric Constant, e 3.1 @ 20C
Density 920 kg/m3 @ 20C or 7.8 lbs/U.S. GallonVolumetric Heat Capacity 1.650 10
6J/m3 @ 20C
Thermal Diffusivity 10 x 10-8m2/s @ 20C
Boiling Point 570 degrees Fahrenheit
Calories per Tablespoon About 120 calories
b.Critique on Methodology
Their design of the bioreactor has considered too many parameters including the
influence of olive oil as a second liquid phase. Although the observation regarding the
performance of olive oil proved to be informative, I believe it is not necessary be
included in the study. PFC has been proven feasible and effective, and should have been
the focus of the dissertation. As for the biomass, it was not stated why the strain Y.
lipolyticawas selected. Overall, the writersmethodology is understandable; the
knowledge of PFCs performance is truly a great contribution to the industry.
c.
Significance of the Results
It was clearly concluded that PFCs generally enhance oxygen mass-transfer. Limitations
are always present in every controlling parameter of the bioreactor but the study has
significantly provided the design and good correlation model for this type of reactor.
Moreover, it has once again proved PFCs to be a good oxygen carrier in fermentation
bioreactors, cost-effective and commercially feasible to overcome the main problem of
oxygen limitation in aqueous aerobic fermentations.
III.Problem Solving
Given:
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Required: Annual Biomass Production
Solution:
Batch
[ ]
( )
Continuous
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Analysis: Continuous operation produces 12.96 times more than that of batch operations
every year.
IV.Mixing/Agitation
a. Criteria in Selecting Agitators
The following factors must be taken into consideration when selecting equipment for
mixing liquids (Towler and Sinnott, 2007):
1.Batch or continuous operation;
2.
Nature of the process; miscible liquids, preparation of solutions, or dispersion of
immiscible liquids;
3.Degree of mixing required;
4.Physical properties of the liquids, particularly the viscosity;
5.Whether the mixing is associated with other operations: reaction, heat transfer.
Inline mixers are used for continuous mixing of low-viscosity fluids. Stirred vessels or
proprietary equipment will be used for other mixing operations.
The most suitable agitator depends on the following:
1.Type of mixing required;
2.The capacity of the vessel;
3.Fluid properties, specifically the viscosityof the substance.
Figure 1 shows the selection chart which can be used to make a preliminary selection of
the agitator type, based on the liquid viscosity and tank volume.
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Figure 2: Inline Mixers: a) Tee. b) Injection. c) Annular
Stirred Tanksthese vessels are equipped with agitators for blending liquids and
preparing solutions. Bulk flow is the dominant mixing mechanism required for the
blending of miscible liquids. Shear-controlled processes are important in heat- and mass-
transfer applications which can be considered turbulent mixing.
The power required to drive an agitator is provided by the generalized dimensionless
equation (Eq. 10.12 Towler and Sinnott, 2007):
Where:
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Power curves for propeller and turbine agitators are shown in Figures 3 and 4 (Towler
and Sinnott, 2007). An estimate of power requirements for various application can be
obtained from Table 3.
Figure 3: Power correlation for single three-bladed propellers baffled. p = D blade pitch,
D = impeller diameter, DT = tank diameter.
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Figure 4:Power correlation for baffled turbine impellers, for tank with 4 baffles. w =
impeller width, D = impeller diameter
Table 3: Power RequirementsBaffled Agitated Tanks
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REFERENCES
P. F. F. Amaral, M. G. Freire, M. H. M. Rocha-Leo, I. M. Marrucho, J. A. P. Coutinho, and M. A. Z. Coelho,
Optimization of oxygen mass transfer in a multiphase bioreactor with perfluorodecalin as a second
liquid phase, Biotechnology and Bioengineering, vol. 99, no. 3, pp. 588598, 2008.
Apostolos (Paul) K. Kiritsakis: Olive Oil, From the Tree to the Table, Second Edition
C. Peixoto, M.F.Q. Sousa, A.C. Silva, M.J.T. Carrondo, P.M. Alves, Downstream processing of triple
layered rotavirus like particles, Journal of Biotechnology, Volume 127, Issue 3, 10 January 2007, Pages
452-461, ISSN 0168-1656, http://dx.doi.org/10.1016/j.jbiotec.2006.08.002.
Doran, P. M. (2013). Bioprocess engineering principles. Waltham, MA: Academic Press.
Pabby, A. K., Rizvi, S. S. H., & Sastre, A. M. (2009). Handbook of membrane separations: Chemical,
pharmaceutical, food, and biotechnological applications. Boca Raton: CRC Press.
Towler, G. P., Sinnott, R. K. (2013). Chemical engineering design: Principles, practice and economics of
plant and process design, second edition(2nd Ed.). Kidlington, Oxford, U.K.; Waltham, Mass.:
Butterworth-Heinemann.
F2 Chemicals Ltd.(2012). Flutec PP6.< http://f2chemicals.com/perfluorodecalin.html>
GEA Westfalia Separator Group. (2014). Enzymes.< http://www.westfalia-
separator.com/applications/chemical-pharmaceutical-technology/pharmaceutical-
biotechnology/enzymes.html >
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