work carried out during my tenure

1. Recombinant protein purification using
gradient assisted Simulated Moving Bed-
Hydrophobic Interaction Chromatography
(SMB-HIC)
Sivakumar P.
BT04D006
PhD viva-voce
MAX-PLANCK-INSTITUT
Research Advisors: DYNAMIK KOMPLEXER
TECHNISCHER
Prof. Guhan Jayaraman
SYSTEME
MAGDEBURG
Prof.Andreas Seidel-Morgenstern

2. Overview
 Background and objectives
 Scheme for the work
 Preparative work
 Production and purification of recombinant streptokinase (rec-stk)
 Adsorption isotherm estimation
 SMB theory
 Design of SMB
 Scanning the separation zone
 Simulation profiles
 Experimental realization
 Conclusion and Discussion
2

3. Back ground and Objective
 Background
work at IIT-Madras
purification of rec-stk (batch chromatography*)
 Objectives
 Developing a continuous gradient assisted
HIC-SMB process for the purification of rec- stk
3
*B. Balagurunathan et al. / Biochemical Engineering Journal 39 (2008) 84–90

4. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Scheme for the work
Preparative work for SMB (batch)
Adsorption isotherm estimation
SMB Design
SMB Experiments
Data analysis
100L reactor SMB
HZI MPI
4

5. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Preparative work for SMB
rec-stk was produced in 100 L reactor and used as a feed for
HIC Matrices screening
Pure rec-stk production for adsorption isotherm estimation
SMB experiments
HIC matrices screening- Butyl sepharose
Selectivity
Binding capacity
Ease of regeneration
Preparative batch purification of rec-stk
5

6. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Adsorption equilibrium constants
(PULSE experiments)
14
STK
(NH4)2SO4 Contaminants STK STK+Degraded product
12
[mM] (Target)
KH,con1 KH,con2 KH,STK 10
50 2.23 2.23 8
H,i
100 1.59 3.69
˜ 0 6
K
150 1.52 7.76
4
200 1.57 11.74
2
qi K H ,i (Csalt )Ci i= degraded STK+STK,STK 0
0 50 100 150 200 250
C (NH [M]
4)2SO4
KH,i depend on Csalt approximately linearly between 100 and 200mM
6
Hi (Csalt ) a1,i a2,i (Csalt ) a1,im 1.59; a2,im 0.0002 a1, sk 4.34; a2, sk 0.081

7. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Introduction to continuous chromatography
Batch Chromatography
Species have different affinity towards adsorbent:
migrate with different velocities
leave column at different time points
S A A, B B True Moving Bed Chromatography
Analogy
Liquid and solid phase move in countercurrent:
Continuous operation.
Solid Phase Two outlets. Good for binary separations.
Difficult to realise due to the movement of the solid
phase.
Simulated Moving Bed
S
Raffinate Feed Extract
Practical realisation of the TMB concept: A A, B B
 periodic switching of the columns
1 2 3 4 5 6
(or ports) 7
Periodic behaviour (cyclic steady state). 7

8. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
SMB theory
8

9. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Isocratic separation of binary mixture
(four zone SMB)
Lets consider
A+B mixture
Zone IV
A is weakly retained, B is strongly retained
&
mIV VIV Raffinate
A & follows linear Henrys isotherm model
, VR
Zone III Keys for separation
&
mIII , VIII
Feed &
&
Vi
&
VF mi
Zone II &
VS
A+B
&
mII , VII Extract i 1, 2, 3, 4
B
&
VE
Zone I
&
mI , VI Flow rate ratios mi
&
Vsolid m4 HA < H B m1
Desorbent
H A m2 H 9
B Triangle
H A m3 HB
Morbidelli et al., 1997

10. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Four zones closed loop SMB
Illustration of Triangle theory
HA Flow rate ratios mi
QI Safety value
mI HB
QS
4 2 6 HB QII
mII HA
QS
1 HB / H A 1
3 QIII
mIII HB /
QS
QIV
mIV HA /
QS
5 H A m2 H
B Triangle
H A m3 HB
HA After considering dead volume
10
Morbidelli et al., 1997

11. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Separation region (gradient SMB)
Linear isotherm
HA 1. Complete separation
2. Pure extract and raffinate polluted with species B
3. Pure raffinate and extract polluted with species A
2 HB 4. Both raffinate extract containing A and B
4 6
5. Extract flooded with desorbent (A&B at raffinate)
1 6. Raffinate flooded with desorbent (A&B at extract)
3
7. Extract flooded with desorbent (A accumulates)
8. Raffinate flooded with desorbent (B accumulates)
9. Raffinate and extract flooded and (A& B accumulates)
5
HA
After considering dead volume
11
Mazzotti et al., 1999, Abel et al., 2002

12. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Design of continuous separation process
 3-zone open-loop two step-gradient SMB
12

13. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Design of 3-zone open-loop
two-step gradient process (TMB Model)
Raffinate: Two-step salt
contaminants, gradient
degraded &
VZ
streptokinase mz Z I , II , III
&
VS
Feed: E.coli cell
salt concentration
homogenate
containing
streptokinase
Extract:
streptokinase
Vcol (1 )
t* D R F
C salt Csalt C salt
&
VS 13
Gueorguieva L et al. J. Chromatogr. A, 1176,(2007) 69.

14. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Design of 3-zone open-loop gradient process
Parameters set: F D &
Csalt , Csalt , VF
&
VZ
Design parameters: mz Z I , II , III
&
VS
Inequalities for complete separation under gradient conditions
Henrys constants
D D
mI K H , STK
(C salt
) K H ,i K H ,i (Csalt ) i con, STK
D D D D D D
K H ,con (C salt ) 1.55
K H , con ( C salt ) m II K H , STK ( C salt )
D D
R R R R K H , STK (C salt ) 3.69
K H , con ( C salt ) m III K H , STK ( C salt ) R R
K H ,con (C salt ) 1.55
Feed D R R
R ( mIII mII )C salt mII C salt K H , STK (C salt ) 11.74
C salt
mIII 14
Rhee et al. (1970), Mazzotti et al. (1997)

15. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Design of 3-zone open-loop gradient TMB
Calculation of separation regions
(scanning program MPI)
1) Equilibrium theory Nz→ , Purity=99.99%
2) Equilibrium stage model NI= NII=NIII=20 , Purity=99%
16
F
C
1 . salt
Csalt
200mM , F
III
Csalt , D
D
m m II 14
C salt C
100mMsalt , F Csalt , R
12
& P2
VF 1mL/ min
2. H sk2R (Csalt , R )
L
,
*
10 L2
Value in zone 1 *
Csalt , R Csalt , D
PurityDconstraint H sk , D )
H sk , ( H sk , F
III
8
m
Csalt , F Csalt , D L1
6 lower
D D bound
K H ,con (C ) 1.55
salt C salt ,F C salt , R
*
II , L 2 *
3. m
D (C ) H sk2R
D
L 4
K H , STK (C ) 3.69 , C salt ,F C salt ,D
salt , R
salt P3
2
R R
K H ,con (C salt ) 1.55 Csalt ,F Csalt ,D P1
III , L 2 *
4. mR (CR ,R
salt ) m II ,L 2 * 0 15
K H , STK (C salt ) 11.74 Csalt ,F Csalt ,R 0 1 Him,D 2 3 Hsk,D4 5
II
m

16. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Separation region predictions from
scanning program
16
Separation regions
14
Thick lines: equilibrium theory
12 Symbols: predictions of
R2
equilibrium stage model
10
8 NI N II N III 50 I
2
III
m
R3 R1
6
R1: (○), Pusk ,E Puim,R 99%
4
R2: (∆), Pusk ,E 99%, Puim,R 70%
2
R3: (x), Pusk ,E 70%, Puim,R 99%
0
0 1 2 3 4 5
II
m 16

17. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Experimental operating points
16
14
14
Operation line 175mM
b
14
b 1
12
12
12
P2
10
10
b
2 140mM
10 L2
88
Separation zone a
Operation
m III
8 b
3
III
m
L1
III
points lower
m
a
66
6 1
bound a
b
4 2
4 a
44 3 P3
a
2 4 Diagonal line
22 P1
0
00
0 1 Him,D 2 3 Hsk,D4 5
II
0,0
0,0 0,5
0,5 1,0
1,0 1,5
1,5 2,0
2,0 m2,5
2,5 3,0
3,0 3,5
3,5 4,0
4,0 4,5
4,5 5,0
5,0
II
II
m
m
17

18. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Parameters used for
simulation studies
Point I & & & &
Csalt , R m II m III t* VD VE VR VF
Nr. [mM] [-] [-] [-] [s] [mL/min] [mL/min] [mL/min] [mL/min]
1b 175 3.30 13.2 2 84 0.75 0.41 1.33 1
2b 175 2.64 10.6 2 125 0.93 0.60 1.33 1
3b 175 1.98 7.95 2 167 1.24 0.91 1.33 1
4b 175 1.32 5.3 2 208 1.86 1.53 1.33 1
1a 140 3.30 5.5 1.1 37 1.85 0.35 2.50 1
2a 140 2.64 4.4 1.1 28 2.32 0.82 2.50 1
3a 140 1.98 3.3 1.1 37 3.09 1.59 2.50 1
18
4a 140 1.32 2.2 1.1 46 2.32 1.57 1.25 0.5

19. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Illustration of internal profiles
(rec stk enriched) at 175mM raffinate salt concentration
R
C salt
= 175mM STK
30 b
CSTK= CIM = 5mg/mL 1
b
C
F
= 200mM 2
25 salt
b
C
D
salt
= 100mM 3
C STK, C IM [mg/ml]
VF = 1mL/Min
20
15
10
IM
Feed
concentration5
0
0.00 0.33 0.66 0.99
Desorbent Extract Feed Raffinate 19
Normalised Length

20. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Experimental Validation
 3-zone open-loop two-step gradient SMB
20

21. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
KNAUER (moving columns) precission valve from smb.mp4
CSEP C916 Multi-function valve Configuration
stator
rotor
Stationary phase Butyl HP
Mobile phase
Configuration
Desorbent
Three zones 1-1-1-(1)
phosphate buffer +100 mM (NH4)2 SO4
Each zone one 2 x1mL column
Feed
20 mM phosphate buffer+200 mM (NH4)2 SO4 UV product profile
Regeneration Internal UV profile and conductivity profile
Milli Q at flow rate of 3 mL/min. Cleaning zone conductivity and UV profile
21
SEC, SDS(PAGE) analysis

22. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Experimental operating points
14
b
Operation line
b
1
12 175mM
b
10 2
8
140mM a
Separation zone
3
b
III
m
Operation a
6
points 1
b a
4 2
a
4 3
a
4
2 Diagonal line
0
0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0
II
m 22

23. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Parameters used for experiments
Point I & & & &
Csalt , R m II m III t* VD VE VR VF
Nr. [mM] [-] [-] [-] [s] [mL/min]
1b 175 3.30 13.2 2 84 0.83 0.39 1.42 1
2b 175 2.64 10.6 2 125 1.03 0.57 1.44 1
3b 175 1.98 7.95 2 167 1.37 0.87 1.48 1
4b 175 1.32 5.3 2 208 - - - 1
1a 140 3.30 5.5 1.1 37 2.21 0.24 2.89 1
2a 140 2.64 4.4 1.1 28 2.76 0.67 2.99 1
3a 140 1.98 3.3 1.1 37 3.68 1.40 3.15 1
23
4a 140 1.32 2.2 1.1 46 2.76 1.42 1.74 0.5

24. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
SDS-PAGE analysis of the
extract and raffinate outlet..
14
b
b 1
175mM
12
b
10 2
8
a 140mM
b
3
III
m
a
6 1
b a
4 2
a
4 3
a
4
Raffinate 2 Raffinate
salt concentration 140mM 0 salt concentration 175mM
0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0
II
4a 3a 2a 1a m
L 3b 2b 1b R-Raffinate
R E R E R E R E R E R E R E
E-Extract
L-Load
rec-stk
24
purity 73% 84% 78% 70% 77% 57% 56%

25. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Product trends
Closed symbols (175mM) 140mM (open symbols)
mII vs Extract purity mII vs Extract concentration
100 2.0
95
90 1.8
Extract concentraiton (mg/mL)
85
1.6
80
Extract purity (%)
75 3b 1.4
70
65 2b 1.2
60
55 1b 1.0
50
45
4a 0.8
0.6
40 3a
35 0.4
30 2a
25 0.2
20
1a
0.0
15
10 -0.2
5
0 -0.4
1.0 1.5 2.0 2.5 3.0 3.5 4.0
-0.6
1.0 1.5 2.0 2.5 3.0 3.5 4.0
25
mII mII

26. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Experiments Vs simulation prediction
Point exp th exp th Yield
Csalt , R Csk ,E Csk ,E Pusk ,E Pusk ,E
Nr. [mM] [mg/mL] [mg/mL] [%] [%] [%]
1a 140 0.34 13.6 70 100 2
2a 140 0.50 6.07 78 100 8
3a 140 0.98 3.14 84 99.88 31
4a 140 1.42 1.60 73 78.83 93
1b 175 0.33 2.97 56.0 100 4
2b 175 0.31 6.15 57.0 100 6
3b 175 0.44 5.51 77 99.96 12
4b* 175 - 3.29 - 94.38 -
26
* Operation point 4b is not feasible because of the flow rate constraints

27. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Extract purity
Experimental Theoretical
100 100
Extract purity [%]
Extract purity [%]
80 80
60 60
40 40
20 20
0
0
1.0 1.5 2.0 2.5 3.0 3.5 4.0
1.0 1.5 2.0 2.5 3.0 3.5 4.0
II
m
II m
Closed squares for raffinate salt concentration 140mM
a) b) 27
Closed triangles for raffinate salt concentration 175mM

28. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Extract concentrations
Experimental Theoretical
2.0 14
Extract concentraiton [mg/mL]
Extract concentraiton [mg/mL]
12
1.5
10
8
1.0
6
4
0.5
2
0.0 0
1.0 1.5 2.0 2.5 3.0 3.5 4.0 1.0 1.5 2.0 2.5 3.0 3.5 4.0
II II
m m
Closed squares for raffinate salt concentration 140mM
Closed triangles for raffinate salt concentration 175mMb)
a) 28

29. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Enhancing the yield of the process
Effect of safety values (β factor)
14 SDA-PAGE gel analysis
12
b 175mM
1
b
b
10 2
8
a 140mM
b
III
m 3
a
6 1
b a
4 2
a
4 3
a
4
2
0
0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 R E
β =1.1
II
m
S. Operational β =1.1 β =2 R- Raffinate
NO. point 2a E- Extract
1 Load (mg/mL) 17.2 17.2
2 Raffinate (mg/mL) 5.94 6.65
3 Extract (mg/mL) 0.34 0.20
4 Purity of stk (%) 69.8 69.4 29
R E
5 Recovery (%) 1.5% 6.7% β =2

30. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Reprocessing the Raffinate
(isocratic run at 140mM raffinate salt concentration )
14 14
13 13
12 12
11 11
10 10
9 9
8 8
mIII 7 7
6 6
5 5
4 4
3 3 β =1.1 β =2
2 2
1 1 R E R E L
0 0
0 1 2 3 4 5 6 7 8 9 10 11 12
mII
S.NO β =1.1 β =2
1 Load (mg/mL) 6.76 6.76
2 Raffinate (mg/mL) 2.69 3.12 R- Raffinate
3 Extract (mg/mL) 0.38 0.20 E- Extract
L- Load (raffinate from SMB)
4 Purity of stk (%) 79 84 30
5 Recovery (%) 38 42

31. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Conclusions..
 Butyl sepharose matrice found to be suitable for the continuous HIC-SMB
purification of rec-stk.
 Adsorption isotherms for rec-stk with butyl sepharose matrice found to be:
 linear adsorption isotherm, parameters depend on the salt
concentration.
 perturbation method applied to evaluate isotherm linearity.
 3 zone open- loop two- step gradient SMB design was proposed and
designed.
31

32. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Conclusions
 Scanning program based on the equilibrium model and the equilibrium stage
model was employed to predict the separation zone.
 Experimental realisation of the continuous purification of rec-stk.
 Simulation predicts for more idealistic conditions and correlate with product
purity trends.
 Highest product purity=84%(3a), and concentration of =1.4mg/mL with a mass
&
m flux of =2mg/min for operation point 4a
 Recycling the raffinate will increase the yield of the process
32

33. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Discussion
 Demonstrate the potential of SMB for the continuous recombinant protein
purification.
 SMB could be placed for the high throughput capture and intermediate
purification of the recombinant protein from complex mixtures.
 Fractionation and Feed back SMB or Tandem chromatography could be
proposed for increasing the yield of the purification process
33

34. presentations
Palani S., Jayaraman G., Gueorguieva L., Rinas U., Seidel-Morgenstern A., “Determination of adsorption isotherm
parameters from total protein mixtures” PREP 2009 meeting, July 19-22, 2009, Loews philadelphia, USA(oral).
Palani S., Jayaraman G., Gueorguieva L., Rinas U., Seidel-Morgenstern A., “Determination of adsorption isotherm
parameters for recombinant streptokinase using perturbation method” 5th Doktorandenseminar Präparative
chromatographie, March 1-3,2009, Wetter Ruhr, Germany(oral).
Palani S., Gueorguieva L., Rinas U., Jayaraman G., Seidel-Morgenstern A., “Simulated Moving Bed Chromatography
(SMBC) current status and applications for protein purification”(oral presentation), International symposium Gene to vial
concept for the biotechnology based health care molecules, Feb 7-10, 2010, VIT, Vellore, T.N, India.(oral).
Palani S., Jayaraman G., Gueorguieva L., Kessler L C., Rinas U., Seidel-Morgenstern A., “Continuous separation of
Recombinant streptokinase using hic gradient simulated moving bed chromatography”, 28th International Symposium on the
separation of Proteins, Peptides and Polynucleotide’s (ISPPP 2008), September 21-24, 2008, Baden-Baden,
Germany(poster).
Palani S., Jayaraman G., Gueorguieva L., Rinas U., Seidel-Morgenstern A., “Continuous simulated moving bed (SMB)
purification of recombinant streptokinase”, 34th International Symposium on High Performance Liquid Phase Separations
and Related techniques, HPLC 2009,June 28-July 2, 2008, Dresden, Germany(poster).
Palani S., Jayaraman G., Gueorguieva L., Rinas U., Seidel-Morgenstern A., “Kontinuierliche aufreinigung der
rekombinanten streptokinase mittels simulated moving bed (SMB) chromatographie”, 27th DECHEMA Jahrestagung der
34
Biotechnologen, gemeinsam mit International workshop on downstream processing. September 8-10, 2009, Mannheim,
Germany(poster).

35. Publications
Palani S., Gueorguieva L, Rinas U., Seidel-Morgenstern A., Jayaraman G., “ Continuous
purification of recombinant streptokinase using Hydrophobic Interaction- Gradient assisted
Simulated moving Bed Chromatography. part I. Determination of adsorption isotherms
applying perturbation method ”(Journal of chromatography A., manuscript submitted).
Gueorguieva L., Palani S., Rinas U., Jayaraman G.,Seidel-Morgenstern A.,“ Continuous
purification of recombinant streptokinase using Hydrophobic Interaction- Gradient assisted
Simulated moving Bed Chromatography. Part II. SMB experimental design analysis for the
operating conditions”(Journal of chromatography A., manuscript submitted).
35

36. Acknowledgement
Prof Guhan Jayaraman
Herr.Prof Andreas Seidel-Morgenstern, MPI,Magdeburg
Frau Ludmila Guorguieva, Dr.Christian Kessler,MPI
Frau Dr. Rinas, HZI, Braunschweig
Herr Dr. Wilko, IFN, Magdeburg
Collegues at IITM,MPI,IFN,HZI
IITM,Deutscher Academic Austausch Dienst (DAAD),
GDCh, Deutsche Forschungemeinschaft (SFG-578)
still lots needs to be explored with SMB!!!
36

37. 37

38. Back up slides

39. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Steady state profiles
Cleaning profile Outlet (Raffinate ,Extract) profile
1200 180 1200
Extract concentration
160
Raffinate concentration
1000
Detector signal 280nm [mAu]
1000
Detector signal 280nm [mAu]
140
Conductivity [mS/cM]
800 120 800
100
600 600
80
400 60 400
40
200 200
20
0 0 0
322 324 326 328 330 322 324 326 328 330
1200 180
Time [min] Time [min]
160
1000
Detector signal 280nm [mAu]
140
Conductivity [mS/cM]
800 120
100
600
80
Internal profile
400 60
40
200
20
39
0 0
322 324 326 328 330
Time [min]

40. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Classical True Moving Bed (TMB) chromatography
B
C Zone IV
S Zone IV
M
mIV V&IV
&
mIV VIV Raffinate
Raffinate
A &
A &
,, VR
VR
C
S
M
B
Zone III
Zone III
mIII ,,V&III
&
mIII VIII&&
Feed
Feed
&
&
VF
VF S
M
B
C
A+B
A+B Zone II
Zone II
mII ,,V&II
mII VII&
Extract
Extract
B
B
M
B
C
S
Zone II
Zone
V&E
&
VE
mII,,V&I
m VI &
&
&
Vsolid
Vsolid
Desorbent
Desorbent
40

41. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Adsorption isotherm estimation
Dynamic methods
Method and Special favorable feature Special Applicable for
characterization unfavorable more than one
feature solute
Perturbation No detector calibration Isotherm Difficult
(Dynamic, small samples) required model required
Dispersed front analysis Low sample amount, High column No
(ECP) small number of efficiency
(dynamic, intermediate experiments required
samples)
Chromatogram fitting Low sample amount, Models for the Difficult
(dynamic) small number of isotherms and
experiments to simulate the
chromatogram
41
required
Seidel-Morgenstern A, J. Chromatogr. A 1037,(2004) 255

42. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Adsorption isotherms
• Relationship between the
equilibrium protein
concentration in the
stationary phase and the
protein concentration in the
mobile phase
• Slope gives the information
about the affinity
• Plateau gives the information
about the capacity
42

43. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
SMB under linear and non-linear conditions
Regions 1 to 4 correspond to higher and higher feed concentrations
43

44. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Effect of feed concentration over flow rate ratios
44

45. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Hydrophobic chromatographic purification of
streptokinase
 Equilibration 5CV
 Washing 15CV
 75% step gradient 10CV
 85% step gradient 12CV
 100% step gradient 10CV
 Resin Phenyl Sepharose
 Buffer A: 20mM sodium phosphate buffer pH 7.2
+1M Ammonium sulphate
 Buffer B : 20mM sodium phosphate buffer pH 7.2 45
 Flow rate 1ml/min

46. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
SDS-PAGE analysis (silver staining)
1 2 3 4 5 6
rec-stk
 lane 1: molecular weight marker
 lane 2: inlet feed sample
 lane 3: Standard streptokinase
46
 lane 4, 5, 6: 85% step gradient fraction number 28, 29, 40, respectively

47. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Procedure Total Activity Specific Purific Yield %
Protein (units) activity ation
(mg) Units/mg factor
Crude 35 5,30,833 15,166 0 100
cellular
extract
HIC 1.4 3,60,966 2,57,883 17 68
47
B. Balagurunathan et al. / Biochemical Engineering Journal 39 (2008) 84–90

48. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Pulse experiment fraction analysis
2 3 4 5
18
17 Vinj=100 µl 1 2 3 4 5 6
16 Vinj=50 µl
15
14
13
12
Signal [mAu]
11
10
9
8
7
6
5
4
3
2
1
2 3 4
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
VR [mL]
1 Fraction 2 (contaminant )
Butyl HP (1mL) column 2 Fraction 3 (stk degraded product)
3 Fraction 4 (stk + stk degraded product)
V F =0.5mL /Min
4 Fraction 5( stk)
CSTK =2mg/ML 5 load 48
Mobile phase 150mM (NH4)2 SO4 6 Molecular weight marker

49. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Motivation for perturbation method
• Commercial preparations
• Presence of Bovine Serum Albumin (BSA) as stabiliser
• Standards from NIBSC
• Smaller in quantity
• Over expression and purification
•Storage and degradation problem
• Perturbation method
• Crude homogenate as the feed material
49
Blumel C, Hugo p, Seidel Morgenstern P, (1999) J. Chromatogr. A 865 (1999)51
Heuer C, Kusers E, Plattner T, Seidel-Morgenstern A, J. Chromatogr. A 827 (1998)175.

50. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Retention time and adsorption isotherm
parameters
6
8
STK+degraded product
5
STK
Streptokinase
4 6
100 mM
tR, i [min]
150 mM
3 200 mM
Contaminants 4
KH,i
2 100 mM
150 mM
200 mM
1 2
0
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0
Ci [mg/ml] 0 50 100 150 200 250
C(NH )2SO4
[M]
4
linear isotherms; KHi depend on Csalt
qi K H ,i (Csalt )Ci
50
i= degraded STK+STK,STK

51. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
True Moving bed chromatography-Analogy
Stationary phase
Mobile Phase
51

52. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Illustration of binary separation
52

53. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Illustration of binary separation
(Fast solid flow)
53

54. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Illustration of binary separation
Slow solid flow
54

55. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Bioreactor production of rec-stk
(HZI, Braunschweig)
Preparation of the 10L Inoculums preparation Preparation for the Reactor
(100L) reactor
Inoculation
Optical Density(OD) 4
Product analysis
Induction Fermentation Harvesting and Storage of cell pellet
55
with IPTG (4 hours) centrifugation in -80 ˚C

56. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Bioreactor Production of rec-stk
10 L Bioreactor
S.No Parameters Fermentor SDS PAGE analysis
1 Agitator speed 400-1000 in cascade rec-stk
mode 1 2 3 4 5
2 Temperature 37 Degree Celsius
3 Aeration 5 LPM 1 un-induced sample
4 Inoculum volume 2%
to fermentation 2 after induction (1.20 hours)
volume
3 after induction (2.20 hours)
5 IPTG 0.1mM
concentration
4 after induction (3.30 hours)
6 Induction OD 4 5 after induction (5 hours)
7 Fermentation 11 hours
duration (total)
8 Wet biomass 260 grams (10L)
produced 2.8kG (100L)
56

57. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
HIC matrices screening
Screening criteria
• binding conditions at 250,500,750mM
•Selective binding for the target protein
•Step (or) linear elution
Positive candidates
•Ease of regeneration
Phenyl sepharose 57
Butyl sepharose

58. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
HIC matrices screening
 Phenyl sepharose – more hydrophobic streptokinase tails all over the gradients
 Butyl sepharose - weakly hydrophobic
 Binding capacity is higher for stk at low salt concentration
 More selective binding of stk and easy regeneration
Manufacturers
recommendations
1 Recommended flow rate 1mL/Min
2 Maximum flow rate 4mL/Min
3 Column dimensions 0.7X2.5 cm
4 Column volume 1mL
5 Maximum backpressure 3 bar, 42psi, 0.3Mpa 58

59. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Preparative purification of recombinant
streptokinase
2 46 9 1115 21 23 35
3500
100
Absorbance at 280 nm (mAu)
Modifier Concentration (%B)
3000
80
2500
2000 60
1500
40
1000
20
500
0 0
0 20 40 60 80 100 120 140 160 180
Volume (mL) Absorbance at 280 nm (mAu)
Conductivity (mS/cm)
Modifier concentration (%B)
Feed : Total protein mixture from E.coli homogenate
Solid Phase : Butyl sepharose HP, GE Biosciences
L= 12cm; D= 0.5 cm,
Vcol= 9.5mL; dpart= 34 µm
Ligand : Butyl, 10 μmol/mL,
Mobile Phase : Buffer A: 20 mM sodium phosphate buffer + 0.2M (NH4)2S04 (pH 7.2)
59
Elution buffer : Buffer B: 20 mM sodium phosphate buffer (pH 7.2)

60. Scheme Prep. purification Est. Adsorption SMB SMB SMB Conclusion &
of rec-Stk isotherm Theory Design Experiments discussion
Preparative batch product analysis
SDS-PAGE analysis (qualitative)
9 13 17 21 35
1 2 3 4 5 6 7 8 9 10 MW Marker
100
100
117
Absorbance at 280 nm (mAu)
Modifier Concentration (%B)
80
85
80
3500
100
60 3000
2500
80 60 48
2000 60
1500
40 40
40
1000
20
34
500
0 0
0 20 40 60 80 100 120 140 160 180
20 20 26
0 0
40 60 80 100 120 140 160 180 1-3. unbound (fraction 2,4,6)
Volume (mL)
Absorbance at 280 nM (mAu)
4-6,9. elution at 25% B,150mM (NH4)2SO4)
Conductivity (mS/cm)
Modifier concentration (%B) 7 .Molecular weight marker
8. Load
60
10. Regeneration fractions