downstream processing reports

8/10/2019 Downstream Processing Reports

1/13

Page 1 of

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).


2/13

Page 2 of

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


3/13

Page 3 of

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


4/13

Page 4 of

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


5/13

Page 5 of

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:


6/13

Page 6 of

Required: Annual Biomass Production

Solution:

Batch

[ ]

( )

Continuous


7/13

Page 7 of

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.


8/13


9/13

Page 9 of

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:


10/13

Page 10 of

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.


11/13

Page 11 of

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


12/13

Page 12 of

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 >


13/13

downstream processing reports

Documents