2009 with my group

1. DEVELOPMENT OF AN
INTEGRATED DOWNSTREAM
BIOPROCESS
CHIA WE YONG 0620673
STANLEY HOW 0651914
TEY MU FENG 0656683

2. •Protein purification
•Involve multistage of unit operation
•Very complicated, challenging and interesting
•Problems:
-Yield
-Cost
-Time
•Solution : Integrated the step
Introduction
Downstream Bioprocess

3. Bioprocess
Homogenisation
Integrated Recovery Operation
Fine Purification
Purified Protein
Introduction
Cell Harvesting
Bioprocess
Homogenisation
Solid-Liquid Separation
Concentration
Initial Purification
Fine Purification
Purified Protein
Cell Harvesting
Comparison of traditional and integrated downstream bioprocess
Traditional Integrated

4. Introduction
• Development of integrated downstream bioprocess
* Simple
* Cheap
* High Recovery and Purity
• Reference for large-commercial scale protein
purification
•Reference for others researcher
Project Objective

5. Recombinant System Selection
Recombinant Protein
• Green Fluorescence Protein ( GFP )
• Glow green light
• Genetic marker or reporter molecule
• Detect cancer cell
Recombinant Host
• Escherichia coli ( E.coli )
• Well understood of physiology and its genetics
• High level expressing
• Easy, fast, scalable, cheap.
Introduction

6. Introduction

7. Literature Review
• Traditional downstream bioprocess
 Chromatographic separation
• Integrated downstream bioprocess
 Expanded bed adsorption (EBA)
 Aqueous two-phase system
• Conclusion: Column separation technique
is common and
expensive

8. Material and Method
Fermentation
Homogenisation
Integrated Recovery Operation
Fine Purification
Cell Harvesting
Ultrasonicator
Centrifugal
Dead End Filter
SDS PAGE
Purified Protein

9. Integrated Operation - Dead End Filtration
• Selecting the best sequence of different pore size
filter between 10 kDa, 30 kDa, and 50 kDa.
1. Performing a 50 kDa filtration
2. Performing a 30 kDa filtration
3. Performing a 2nd
30 kDa filtration
• Picture taken
• Samples for SDS-PAGE
Material and Method

10. Results
 Supernatant (left) and
GFP Supernatant (right)
 Supernatant with GFP
glows green in UV
 GFP detectable with UV
to confirm E. coli cell
walls are broken

11.  GFP passes through 50 kDa but not 10 and 30 kDa
filters
 50 kDa > Molecular size of GFP > 30 kDa
 Eliminate 10 kDa filtration, first perform 50 kDa then 30
kDa filtration
Results
 Filtrates of 10 kDa
(left), 30 kDa (middle)
and 50 kDa (right)
filtrations

12.  Upper picture: 30 (left)
and 50 (right) kDa filtrates
 Lower picture: 50 kDa
filtrate (left) and 30 kDa
residue (right)
 Colour of 50 kDa filtrate
same as 30 kDa residue
 No significant loss of GFP
in 2nd filtration
Results

13. Results
 Another 30 kDa filtration
 Results named 30k
residue 2 & filtrate 2
 30k residue 2 after
calibration looks the
same as 50k filtrate 1
30 k Residue 2 30 k Filtrate 2
30 k
Filtrate
2
30 k
Residue
2
50 k
Filtrate

14. SDS-PAGE Analysis
GFP
250
150
100
75
50
37
25
20
15
10
kDa
1 2 3 4 5 6 7 8 9
Lane Sample loaded in gel well
1 Protein Marker
2 GFP Supernatant
3 Supernatant
4 50 k filtrate
5 30 k filtrate 1
6 30 k residue 1
7 30 k filtrate 2
8 30 k residue 2 after calibration
9 30 k residue 2 before calibration
Results

15. Discussion and Recommendations
Concentration of samples before SDS run
 Concentrated samples able to detect more detailed
information, thus better analysis
 Carried out by filtration in small filter size such as 10 kDa,
than centrifugation to remove excess water
Optimise filter size for filtration
 Carry out filtration by using filter size tubes such as 31
kDa and 33 kDa
 Remove larger amount of impurities, leaving a higher
purity of GFP

16. Use of cross flow filtration
Feed, directed parallel to the membrane surface, will help to
keep particle build-up to a minimum
Permeate flux is constant over time
Reduce the problem of unwanted protein retention
Discussion and Recommendations

17. Suggested Integrated downstream bioprocess
Discussion and Recommendations
Centrifugal
Cell Disruption Cross Filtration
50 kDa
Cross Filtration
31 ~ 35 kDa
Cross Filtration
31 ~ 35 kDa
Pure GFP
Fermenter
Pump
Filtrate
Residue
Residue

18. Conclusion
Size of GFP used ≈ 31 to 35 kDa
GFP able to pass through 50 kDa filter but not 30 kDa filter
Performing a 50 kDa followed by two 30 kDa filtrations
 large removal of contaminants
 high yield
Successfully developed integrated downstream bioprocess
Cross filtration only, no chromatography
Simple
Cheap
GFP obtained will be faster, purer and have minimum yield
loss