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Cold ethanol precipitation and
calcium-phosphate flocculation of recombinant antibodies
University of Natural Resources and Life Sciences Vienna, AustriaDepartment of Biotechnology
Nikolaus Hammerschmidt, Ralf Sommer, Anne Tscheliessnig, Henk Schulz, Bernhard Helk, Alois Jungbauer
Integrated Continuous BiomanufacturingBarcelona, 21.10.2013
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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 of selective precipitation steps
Implementation of the process in continuous mode
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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
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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
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Design by solubility curve 1
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logS = logS0 – βω (1)
(1) Juckes I.R.M.: Fractionation of proteins and viruses with polyethylene glycol. Biochim. Biophys. Acta 229: 535-546 (1971)
mAb → blue line
Impurities→ red line
Below solubility curve: protein in solution
Above solubility curve: protein precipitates
Solubility curves
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Design by solubility curve 2
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Region 1: Impurities and mAb precipitate
Region 2: impurities precipitate, mAb in solution
Region 3: mAb precipitates
Region 4: mAb and impurities in solution
Solubility curves
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Ethanol – effect on antibody
Time [min]
5 10 15 20 25 30
Nor
mal
ized
abs
orba
nce
[ -
]
0.0
0.2
0.4
0.6
0.8
1.0
1.2
50 min30 min5 min
IgG
Aggregates
[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.
Excess enthalpy of water-ethanol mixtures
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Wavenumber [cm-1]
1600 1620 1640 1660 1680 1700
Sec
ond
deriv
ativ
e
-0.02
-0.01
0.00
0.01
0.025 min30 min50 minpA purified
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Wavenumber [cm-1]
160016201640166016801700
No
rmalize
d S
ignal [-]
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Wavenumber [cm-1]
160016201640166016801700
No
rmalize
d S
ignal [-]
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Precipitation - effect on secondary structure
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ATR FT-IR spectra
Dissolved precipitate vs drug substance
Dissolved precipitate vs 4 month storage at -10°C
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Cold ethanol precipitation platform processClarified 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 normal
working conditions Highly volatile Chemically inert Cheap and easily available FDA: Ethanol is class 3 solvent (Solvents with
Low Toxic Potential)
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Purity data: mAb1
Dilution factor
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
Dilution factor
IgG IgG yield Monomer HCPHCP
Reduction
[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
Dilution factor
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 processClarified 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 normal
working conditions Highly volatile Chemically inert Cheap and easily available FDA: Ethanol is class 3 solvent (Solvents with
Low Toxic Potential)
IEX monolith
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Volume (ml)
0 20 40 60 80
mA
U
0
500
1000
1500
2000
2500
3000
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
[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 at constant linear velocity
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Economic evaluation (CoGs) – Gantt chartsClassical 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 commercial4 g/L, 20% batch-failure rate
70% DSP yield10 kg
Resins discardedMulti-product plant
4 g/L, 20% batch failure rate70% DSP yield
3 batches at comm. scaleResins discarded
Multi-product plant
4 g/L, 5% batch failure rate70% DSP yield
Target production: 500 kg/aMulti-product plant
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Economic evaluation – Increasing titer
Precipitation scales with processed volume, not titer!
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Diameter of pA column: > 2 m
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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 continuous
mode Automatisation GMP facilities already exist (blood
plasma industry)
Rapid mixing and cooling Adaptation to continuous mode New to the field
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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!
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Stirred tank reactor – Tubular reactor
from Mettler Toledousing built-in probes
Batch Continuous
Self-construction
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