ELUTION DIAGRAM OF CYTOCHROME- C FROM ION-EXCHANGE MAGNETIC NANOPARTICLES.
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Transcript of ELUTION DIAGRAM OF CYTOCHROME- C FROM ION-EXCHANGE MAGNETIC NANOPARTICLES.
![Page 1: ELUTION DIAGRAM OF CYTOCHROME- C FROM ION-EXCHANGE MAGNETIC NANOPARTICLES.](https://reader033.fdocuments.net/reader033/viewer/2022051516/56649f085503460f94c1d373/html5/thumbnails/1.jpg)
0
0.2
0.4
0.6
0 0.2 0.4 0.6
Bound Protein (g/gparticles)
Elu
ted
Pro
tein
(g
/gpa
rtic
les)
completeelution
ELUTION DIAGRAM OF CYTOCHROME-C FROM ION-EXCHANGE MAGNETIC
NANOPARTICLES
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0
200
400
600
800
1000
0 20 40 60 80
0
100
200
300
400
500
Fermentation time (hr)
OD
600
Wet
Cel
l Den
sity
(g/L
)
glycerolbatchphase
methanolfed-batch
phase
glycerolfed-batch
phase
0
100
200
300
400
0 20 40 60 80
0
500
1000
1500
Fermentation time (hr)
Ext
race
llula
r Pro
tein
(mg/
L)
Met
hano
l Add
ed (g
)
FERMENTATION PROFILE OF Pichia pastoris: PRODUCTION OF RECOMBINANT DROSOMYCIN
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Unbound Drosomycin (g/mL)
Bo
und
Dro
smyc
in(m
g/g
pa
rtic
les)
2x diluted
undiluted
0
20
40
0 50 100 150
ADSORPTION ISOTHERM OF DROSOMYCIN FERMENTATION BROTH
ON MAGNETIC NANOPARTICLES
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Ionic Strength Drosomycin DrosomycinpH Eluted Purity Use
(M NaCl) (%) (%)
3 0 ~0 (-) column wash
7 0.5 79.7 90.0 drosomycin elution
10 0.5 99.3 46.3 particle regeneration
SUMMARY OF DROSOMYCIN PURIFICATION USING
MAGNETIC NANOPARTICLES
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d rosom yc in
s tanda rds
fe rm en ta tion
b ro th
unbound
p ro te in
e lu ted pH = 7
0 .5M N aC l
e lu ted pH = 10
0 .5M N aC l
SDS GEL ELECTROPHORESIS USING MAGNETIC NANOPARATICLES: ELUTION
PROFILES OF DROSOMYCIN
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1
10
100
1000
100 1000 10000 100000
Linear Flow Rate (cm/h)
(Pro
duc
tivity
) x
(Cap
acity
)(c
m/h
) x
(g/m
L)
magnetic nanoclusters
magnetoliposomes
expanded bed (zirconium core)
packed column
expanded bed
COMPARISON OF MAGNETIC NANOPARTICLES
WITH OTHER PURIFICATION SCHEMES
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NEW CONCEPT IN INCREASING OXYGENTRANSFER RATE USING MAGNETIC
NANOPARTICLES
20 nm
30-50 nm
Magneticparticle Oleic Acid
• Low amount of coating • Very high interfacial areas• Readily recovered by magnetic filtration
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SYNTHESIS OF MAGNETIC NANO-PARTICLES
• Fast (30 min) simple synthesis (stirred tank)
• Inexpensive, readily available materials
Aqueous solution of FeCl2 and FeCl3
NH4OH80ºC
Oleic acidcoating
Hitenolcoating
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42Cu
232 SONaO2
1SONa
2
[SO32-] = 0.67M
[Cu2+] = 1x10-3 M
Dtank = 22cm
HL
= 1
4.5c
m
Di = 10cm
VTOTAL = 20L
VWORKING = 5.5L
air to mass spec
MASS TRANSFER CHARACTERIZATION IN BIOREACTORS: SULFITE OXIDATION
V
C
C
C
CF
RateUptakeOxygen outN
O
inN
O
inN
2
2
2
2
2
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100
1000
1 10 100
k La
(m
mo
l/(at
m L
hr)
)
Power Input per Unit Volume, PG/VL (HP/1000L)
0.01
0.005
0.0025
0 (control)
100
1000
1 10 100
k La
(m
mo
l/(at
m L
hr)
)
Power Input per Unit Volume, PG/VL (HP/1000L)
0.01
0.005
0.0025
0 (control)
OXYGEN MASS TRANSFER COEFFICIENT VERSUSGASSED POWER PER UNIT VOLUME IN 20-LITER
BIOREACTOR
Ф: particle wt%
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OXYGEN MASS TRANSFER IN E. coli FERMENTATIONS
• Seed culture- 100 ml LB in 500 ml shake flask
- overnight culture at 37 oC, 220rpm
• Fermentation culture (7.5 L fermentor) - inoculation volume: 10% (v/v)
- initial fermentation volume: 3 L
- temperature: 37 oC
- agitation speed: 600 rpm
- pH = 6.8-6.9, adjusted by 4 M (NH4OH:NaOH = 2:2)
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SUMMARY OF OXYGEN TRANSFER COEFFICIENT INE. COLI FERMEMENTATION WITH
AND WITHOUT MAGNETIC NANOPARTICLES
Particles
(w/v)
Air-flow rate
(L/min)
Oxygen Transfer
(from 7 to 10 hrs)
[mmol O2/(L-h)]
kLa
(from 7 to 10 hrs)
[mmol O2/(L-h-Atm O2]
Normalized kLa
(air-flow rate = 2 L/min)
[mmol O2/(L-h-Atm O2]
Fermentation III
none 2.0 26.68 127.0 127.0
Fermentation
Inone 3.0 36.30 172.9 131.8
Fermentation II
2% 1.0 74.04 352.6 561.0
Fermentation IV
4% 2.0 122.84 585.0 585.0
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SUMMARY AND CONCLUSIONS
• Micro-Bioreactors Will Began to Have Impacts in Biotechnology Processes Reducing Time in Process Development
Strain Selection Medium Development Product Quality in Mammalian Cell Culture
• Nano-Technology Has Definite Future Product Purification Over-Coming Transport Barriers
Oxygen Transfer Biocatalysis and Co-Factor Regeneration