Cold ethanol precipitation and
calcium-phosphate flocculation of recombinant antibodies
University of Natural Resources and Life Sciences Vienna, Austria
Department of Biotechnology
Nikolaus Hammerschmidt, Ralf Sommer,
Anne Tscheliessnig, Henk Schulz, Bernhard Helk, Alois Jungbauer
Integrated Continuous Biomanufacturing
Barcelona, 21.10.2013
Objectives of our project
 Development of different precipitation methods for proteins,
with an emphesis on recombinant antibodies
 Replacement of chromatography based process by a series of
selective precipitation steps
 Implementation of the process in continuous mode
08.10.2013
2
Status quo - Commercial mAb processes
SynagisTM
RemicadeTM
Cell removal
Cell removal
Cell removal
AC
AC
AEX
AC
Virus inactivation
Virus inactivation
Virus inactivation
CEX
Virus inactivation
CEX
AEX
CEX
Virus inactivation
CEX
AEX
CEX
SEC
Virus clearance
Virus clearance
HIC
Virus clearance
Virus clearance
AEX
AEX
Sterile filtration
Sterile filtration
Sterile filtration
Virus clearance
AEX
SEC
Sterile filtration
HerceptinTM
Rituxan
Cell removal
Cell removal
AC
S. Sommerfeld, J. Strube,
MabCampathTM
Sterile filtration
Chem. Eng. Proc. 44 (2005) 1123–1137
08.10.2013
3
Status quo - Commercial mAb processes
SynagisTM
RemicadeTM
Cell removal
Cell removal
Cell removal
AC
AC
AEX
AC
Virus inactivation
Virus inactivation
Virus inactivation
CEX
Virus inactivation
CEX
AEX
CEX
Virus inactivation
CEX
AEX
CEX
SEC
Virus clearance
Virus clearance
HIC
Virus clearance
Virus clearance
AEX
AEX
Sterile filtration
Sterile filtration
Sterile filtration
Virus clearance
AEX
SEC
Sterile filtration
HerceptinTM
Rituxan
Cell removal
Cell removal
AC
S. Sommerfeld, J. Strube,
MabCampathTM
Sterile filtration
Chem. Eng. Proc. 44 (2005) 1123–1137
08.10.2013
4
Design by solubility curve 1
Solubility curves

mAb → blue line

Impurities→ red line

Below solubility curve: protein in
solution

Above solubility curve: protein
precipitates
logS = logS0 – βω (1)
(1) Juckes I.R.M.: Fractionation of proteins and viruses with
polyethylene glycol. Biochim. Biophys. Acta 229: 535-546 (1971)
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5
Design by solubility curve 2
Solubility curves
08.10.2013

Region 1: Impurities and mAb precipitate

Region 2: impurities precipitate, mAb in
solution

Region 3: mAb precipitates

Region 4: mAb and impurities in solution
6
Ethanol – effect on antibody
Excess enthalpy of water-ethanol mixtures
Normalized absorbance [ - ]
1.2
50 min
30 min
5 min
1.0
IgG
0.8
0.6
0.4
Aggregates
0.2
0.0
5
10
15
20
25
30
Time [min]
[1] V.P.M. Belousov, I.L.Vestn. St.-Peterb. Univ. Ser. 4
Fiz. Khim., Vestn. St.-Peterb. Univ. Ser. 4 Fiz. Khim., 22
22 (1970) 101.
Second derivative
0.02
0.01
0.00
-0.01
-0.02
1600
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5 min
30 min
50 min
pA purified
1620
1640
1660
Wavenumber [cm-1]
1680
1700
7
Precipitation - effect on secondary structure
1.2
1.2
1.0
1.0
Normalized Signal [-]
Normalized Signal [-]
ATR FT-IR spectra
0.8
0.6
0.4
0.2
0.8
0.6
0.4
0.2
0.0
0.0
1700
1680
1660
1640
Wavenumber [cm -1 ]
Dissolved precipitate vs
drug substance
08.10.2013
1620
1600
1700
1680
1660
1640
1620
1600
Wavenumber [cm -1 ]
Dissolved precipitate vs 4
month storage at -10°C
8
Cold ethanol precipitation platform process
Clarified supernatant
 4-step process
1st CaCl2 precipitation
~4 mM phosphate, pH 8.5,
250 mM CaCl2, 20°C
1st ethanol precipitation
pH 6.5, -10°C, 25%(v/v) EtOH
2nd CaCl2 precipitation
~4 mM phosphate, pH 8.5,
250 mM CaCl2, 20°C
 Advantages of ethanol:
 Low toxicity
 Miscible with water
 No explosive gaseous mixtures under normal




working conditions
Highly volatile
Chemically inert
Cheap and easily available
FDA: Ethanol is class 3 solvent (Solvents with
Low Toxic Potential)
2nd ethanol precipitation
pH 6.5, -10°C, 25%(v/v) EtOH
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Purity data: mAb1
Dilution
factor
IgG
[µg/ml]
IgG yield
step
overall
HCP
[ppm]
2563.4
Supernatant
HCP Reduction
step
overall
109230
1st CaCl2 precipitation
1.06
2379.4
98%
1st EtOH precipitation - SN
1.35
38.8
2%
1st EtOH precipitation - PP
1.00
2172.4
2nd CaCl2 precipitation
1.07
2nd EtOH precipitation - SN
2nd EtOH precipitation - PP
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Monomer
98%
66462
1.6
1.6
91%
89%
15224
4.4
7.0
1991.7
98%
88%
3863
3.9
28.3
1.35
22.5
2%
1.00
1816.4
91%
1201.6
3.2
90.9
80%
99.9%
10
Purity data: mAb2
Dilution
factor
IgG
IgG yield
Monomer
HCP
[µg/ml]
step
overall
[ppm]
HCP
Reduction
step
overall
Supernatant
1.00
1952.9
0%
0%
180099
1st CaCl2 precipitation
1.07
1807.6
99%
99%
66462
2.7
2.7
1st EtOH precipitation - SN
1.35
34.8
3%
1st EtOH precipitation - PP
1.00
1649.0
89%
88%
31648
2.1
5.7
2nd CaCl2 precipitation
1.07
1487.3
100%
88%
13760
2.3
13.1
2nd EtOH precipitation - SN
1.35
19.2
2%
2nd EtOH precipitation - PP
1.00
1390.9
94%
8276
1.7
21.8
08.10.2013
83%
90%
11
Purity data: mAb3
IgG
IgG yield
Monomer
HCP
Dilution
factor
[µg/ml]
step
overall
[ppm]
Supernatant
0.00
3322.2
0%
0%
81752
1st CaCl2 precipitation
1.09
2825.6
92%
92%
1st EtOH precipitation - SN
1.35
7.3
1st EtOH precipitation - PP
1.00
n.a.
n.a.
2nd CaCl2 precipitation
1.08
2336.6
2nd EtOH precipitation - SN
1.35
3.3
2nd EtOH precipitation - PP
1.00
2162.4
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HCP Reduction
step
overall
64212
1.2
1.2
n.a.
n.a.
n.a.
n.a.
89%
82%
3863
2.3
17.2
93%
76%
3701
22
48.7
99%
12
Cold ethanol precipitation platform process
Clarified supernatant
 Currently 5-step process
1st CaCl2 precipitation
~4 mM phosphate, pH 8.5,
250 mM CaCl2, 20°C
1st ethanol precipitation
pH 6.5, -10°C, 25%(v/v) EtOH
2nd CaCl2 precipitation
~4 mM phosphate, pH 8.5,
250 mM CaCl2, 20°C
 Advantages of ethanol:
 Low toxicity
 Miscible with water
 No explosive gaseous mixtures under normal




working conditions
Highly volatile
Chemically inert
Cheap and easily available
FDA: Ethanol is class 3 solvent (Solvents with
Low Toxic Potential)
2nd ethanol precipitation
pH 6.5, -10°C, 25%(v/v) EtOH
IEX monolith
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13
Polishing by IEX flowthrough
 Negative purification
 High pI of therapeutic mAbs exploited
 Impurities bound (DNA, HCPs), product in
flow through
 Monolith – mass transfer by convection
3
0
0
0
[1] A. Jungbauer, R. Hahn, Journal of Chromatography A 1184
(2008) 62.
2
5
0
0
2
0
0
0
mAU
1
5
0
0
1
0
0
0
5
0
0
0
0
2
0
4
0
6
0
8
0
From: http://www.biaseparations.com/pr/1702/cimmultus-qa-8advanced-composite-column
V
o
l
u
m
e
(
m
l
)
08.10.2013
14
Purity data: mAb1
mAb1
IgG
Yield
[µg/ml]
step
IgG monomer
overall
DNA
HCP
[ppm]
[ppm]
2583 ± 0
136424 ± 0
HCP Reduction
step
overall
Supernatant
2509 ± 0
1st CaCl2
2272 ± 28
96% ± 1% 96% ± 1%
30 ± 1
107010 ± 3387
1.3 ± 0.0
1.3 ± 0.0
1st CEP
2161 ± 41
95% ± 2% 91% ± 2%
166 ± 58
28350 ± 2559
3.8 ± 0.4
4.8 ± 0.5
2nd CaCl2
1845 ± 27
91% ± 2% 83% ± 1%
<LLOQ
6406 ± 801
4.5 ± 0.5
21.5 ± 2.5
2nd CEP
1743 ± 3
96% ± 1% 79% ± 2%
99.92% ± 0.02%
136 ± 37
1254 ± 182
5.1 ± 0.3
110.3 ± 15.4
DEAE AEX
1715 ± 49
99% ± 1% 78% ± 2%
99.95% ± 0.01%
121 ± 21
80 ± 14
15.7 ± 1.1
1739.2 ± 326.7
Carified cell culture supernatant
Relative Abs @ 210 nm
1
Purified antibody
0.8
0.6
0.4
0.2
0
0
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20
30
Time [min]
40
50
60
15
Continuous reactor – Scale up and throughput
Diameter [cm]
Throughput
L/min
L/24h
kg/24h
1
0.1
136
0.44
2
0.4
543
1.74
5
2.4
3393
10.86
10
9.6
13572
43.43
Assumption: Linear flow rate: 2 cm/s; titer: 4 g/L; yield: 80%
Reactor diameter doubled  throughput inceases 4x at
constant linear velocity
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Economic evaluation (CoGs) – Gantt charts
Classical process: Fed-batch + chromatography
Hybrid: Fed-batch + continuous precipitation
Fully continuous: Perfusion + continuous precipitation
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17
Economic evaluation – 3 scenarios
Assumptions
Phase I, II
Phase III
Very large commercial
4 g/L, 20% batch-failure rate
70% DSP yield
10 kg
Resins discarded
Multi-product plant
4 g/L, 20% batch failure rate
70% DSP yield
3 batches at comm. scale
Resins discarded
Multi-product plant
4 g/L, 5% batch failure rate
70% DSP yield
Target production: 500 kg/a
Multi-product plant
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18
Economic evaluation – Increasing titer
Diameter of pA column: > 2 m
Precipitation scales with processed volume, not titer!
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Advantages and challenges of new process
Advantages







Suitable for high titer processes
Disposable format possible
Reduction of footprint
Platform process
Can be run in batch AND continuous
mode
Automatisation
GMP facilities already exist (blood
plasma industry)
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Challenges



Rapid mixing and cooling
Adaptation to continuous mode
New to the field
20
Acknowledgments





Alois Jungbauer
Anne Tscheließnig
Ralf Sommer
Novartis AG – Bernhard Helk
Novartis AG – Henk Schulz
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Questions???
Thank you!
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22
Stirred tank reactor – Tubular reactor
Batch
from Mettler Toledo
using built-in probes
08.10.2013
Continuous
Self-construction
24