Cold ethanol precipitation andcalcium-phosphate flocculation of recombinant antibodiesUniversity of Natural Resources and Life Sciences Vienna, AustriaDepartment of BiotechnologyNikolaus Hammerschmidt, Ralf Sommer,Anne Tscheliessnig, Henk Schulz, Bernhard Helk, Alois JungbauerIntegrated Continuous BiomanufacturingBarcelona, 21.10.2013
Objectives of our project
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Development of different precipitation methods for proteins,with an emphesis on recombinant antibodies
Replacement of chromatography based process by a series ofselective precipitation steps
Implementation of the process in continuous mode
Status quo - Commercial mAb processes
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S. Sommerfeld, J. Strube,
Chem. Eng. Proc. 44 (2005) 1123–1137
AC
Virus inactivation
CEX
AEX
HIC
Sterile filtration
Virus clearance
SEC
Rituxan
Cell removal
AC
Virus inactivation
AEX
CEX
Sterile filtration
Virus clearance
MabCampathTM
Cell removal
AC
Virus inactivation
CEX
SEC
Sterile filtration
HerceptinTM
AEX
Virus clearance
SynagisTM
Virus inactivation
CEX
Sterile filtration
Virus clearance
Cell removal
AC
Virus inactivation
CEX
AEX
AEX
Sterile filtration
RemicadeTM
Virus clearance
Cell removal
Cell removal
AEX
Status quo - Commercial mAb processes
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S. Sommerfeld, J. Strube,
Chem. Eng. Proc. 44 (2005) 1123–1137
AC
Virus inactivation
CEX
AEX
HIC
Sterile filtration
Virus clearance
SEC
Rituxan
Cell removal
AC
Virus inactivation
AEX
CEX
Sterile filtration
Virus clearance
MabCampathTM
Cell removal
AC
Virus inactivation
CEX
SEC
Sterile filtration
HerceptinTM
AEX
Virus clearance
SynagisTM
Virus inactivation
CEX
Sterile filtration
Virus clearance
Cell removal
AC
Virus inactivation
CEX
AEX
AEX
Sterile filtration
RemicadeTM
Virus clearance
Cell removal
Cell removal
AEX
Design by solubility curve 1
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logS = logS0 – βω (1)
(1) Juckes I.R.M.: Fractionation of proteins and viruses withpolyethylene glycol. Biochim. Biophys. Acta 229: 535-546 (1971)
mAb → blue line
Impurities→ red line
Below solubility curve: protein insolution
Above solubility curve: proteinprecipitates
Solubility curves
Design by solubility curve 2
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Region 1: Impurities and mAb precipitate
Region 2: impurities precipitate, mAb insolution
Region 3: mAb precipitates
Region 4: mAb and impurities in solution
Solubility curves
Ethanol – effect on antibody
[1] V.P.M. Belousov, I.L.Vestn. St.-Peterb. Univ. Ser. 4Fiz. Khim., Vestn. St.-Peterb. Univ. Ser. 4 Fiz. Khim., 2222 (1970) 101.
Excess enthalpy of water-ethanol mixtures
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Precipitation - effect on secondary structure
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ATR FT-IR spectra
Dissolved precipitate vsdrug substance
Dissolved precipitate vs 4month storage at -10°C
Cold ethanol precipitation platform process
Clarified supernatant
1st CaCl2 precipitation
1st ethanol precipitation
pH 6.5, -10°C, 25%(v/v) EtOH
~4 mM phosphate, pH 8.5,
250 mM CaCl2, 20°C
2nd CaCl2 precipitation
2nd ethanol precipitation
pH 6.5, -10°C, 25%(v/v) EtOH
~4 mM phosphate, pH 8.5,
250 mM CaCl2, 20°C
4-step process
Advantages of ethanol:
Low toxicity
Miscible with water
No explosive gaseous mixtures under normalworking conditions
Highly volatile
Chemically inert
Cheap and easily available
FDA: Ethanol is class 3 solvent (Solvents withLow Toxic Potential)
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Purity data: mAb1
Dilutionfactor
IgG
IgG yield
Monomer
HCP
HCP Reduction
[µg/ml]
step
overall
[ppm]
step
overall
Supernatant
2563.4
109230
1st CaCl2 precipitation
1.06
2379.4
98%
98%
66462
1.6
1.6
1st EtOH precipitation - SN
1.35
38.8
2%
1st EtOH precipitation - PP
1.00
2172.4
91%
89%
15224
4.4
7.0
2nd CaCl2 precipitation
1.07
1991.7
98%
88%
3863
3.9
28.3
2nd EtOH precipitation - SN
1.35
22.5
2%
2nd EtOH precipitation - PP
1.00
1816.4
91%
80%
99.9%
1201.6
3.2
90.9
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Purity data: mAb2
Dilutionfactor
IgG
IgG yield
Monomer
HCP
HCPReduction
[µg/ml]
step
overall
[ppm]
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%
83%
90%
8276
1.7
21.8
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Purity data: mAb3
Dilutionfactor
IgG
IgG yield
Monomer
HCP
HCP Reduction
[µg/ml]
step
overall
[ppm]
step
overall
Supernatant
0.00
3322.2
0%
0%
81752
1st CaCl2 precipitation
1.09
2825.6
92%
92%
64212
1.2
1.2
1st EtOH precipitation - SN
1.35
7.3
1st EtOH precipitation - PP
1.00
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
2nd CaCl2 precipitation
1.08
2336.6
89%
82%
3863
2.3
17.2
2nd EtOH precipitation - SN
1.35
3.3
2nd EtOH precipitation - PP
1.00
2162.4
93%
76%
99%
3701
22
48.7
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Cold ethanol precipitation platform process
Clarified supernatant
1st CaCl2 precipitation
1st ethanol precipitation
pH 6.5, -10°C, 25%(v/v) EtOH
~4 mM phosphate, pH 8.5,
250 mM CaCl2, 20°C
2nd CaCl2 precipitation
2nd ethanol precipitation
pH 6.5, -10°C, 25%(v/v) EtOH
~4 mM phosphate, pH 8.5,
250 mM CaCl2, 20°C
Currently 5-step process
Advantages of ethanol:
Low toxicity
Miscible with water
No explosive gaseous mixtures under normalworking conditions
Highly volatile
Chemically inert
Cheap and easily available
FDA: Ethanol is class 3 solvent (Solvents withLow Toxic Potential)
IEX monolith
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Polishing by IEX flowthrough
Negative purification
High pI of therapeutic mAbs exploited
Impurities bound (DNA, HCPs), product inflow through
Monolith – mass transfer by convection
[1] A. Jungbauer, R. Hahn, Journal of Chromatography A 1184(2008) 62.
From: http://www.biaseparations.com/pr/1702/cimmultus-qa-8-advanced-composite-column
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mAb1
IgG
Yield
IgG monomer
DNA
HCP
HCP Reduction
[µg/ml]
step
overall
[ppm]
[ppm]
step
overall
Supernatant
2509 ± 0
2583 ± 0
136424 ± 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
Purity data: mAb1
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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 atconstant 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
Processing constraint: 5 days
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Economic evaluation – 3 scenarios
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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
Economic evaluation – Increasing titer
Precipitation scales with processed volume, not titer!
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Diameter of pA column: > 2 m
Advantages and challenges of new process
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Advantages Challenges
Suitable for high titer processes
Disposable format possible
Reduction of footprint
Platform process
Can be run in batch AND continuousmode
Automatisation
GMP facilities already exist (bloodplasma industry)
Rapid mixing and cooling
Adaptation to continuous mode
New to the field
Acknowledgments
Alois Jungbauer
Anne Tscheließnig
Ralf Sommer
Novartis AG – Bernhard Helk
Novartis AG – Henk Schulz
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Questions???
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Thank you!
Stirred tank reactor – Tubular reactor
from Mettler Toledo
using built-in probes
Batch Continuous
Self-construction
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