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

Objectives of our project

08.10.2013 2

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

Status quo - Commercial mAb processes

08.10.2013 3

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

08.10.2013 4

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 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

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

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

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

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

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 continuous

mode Automatisation GMP facilities already exist (blood

plasma 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 Toledousing built-in probes

Batch Continuous

Self-construction

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