Post on 03-Jan-2016
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
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
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
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
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
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
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
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
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
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%
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)
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
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