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