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A sensitive monitoring system for mammalian cell cultivation processes: a PAT approach

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Abstract

Biopharmaceuticals such as antibodies are produced in cultivated mammalian cells, which must be monitored to comply with good manufacturing practice. We, therefore, developed a fully automated system comprising a specific exhaust gas analyzer, inline analytics and a corresponding algorithm to precisely determine the oxygen uptake rate, carbon dioxide evolution rate, carbon dioxide transfer rate, transfer quotient and respiratory quotient without interrupting the ongoing cultivation, in order to assess its reproducibility. The system was verified using chemical simulation experiments and was able to measure the respiratory activity of hybridoma cells and DG44 cells (derived from Chinese hamster ovary cells) with satisfactory results at a minimum viable cell density of ~2.0 × 105 cells ml−1. The system was suitable for both batch and fed-batch cultivations in bubble-aerated and membrane-aerated reactors, with and without the control of pH and dissolved oxygen.

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Abbreviations

\(a_{{{\text{co}}_{ 2} }}\) :

Activity of carbon dioxide (–)

\(a_{{{\text{H}}^{ + } }}\) :

Activity of hydrogen ion (–)

\(a_{{{\text{HCO}}_{ 3}^{ - } }}\) :

Activity of hydrogen carbonate (–)

CER:

Carbon dioxide evolution rate (mol l−1 h−1)

c i :

Molar concentration of an ion (mol l−1)

\(c_{{{\text{CO}}_{2} , 0}}\) :

Saturation concentration of carbon dioxide at the boundary layer of the gas phase (mol l−1)

\(c_{{{\text{CO}}_{2} , {\text{g}}}}\) :

Molar concentration of carbon dioxide in the gas phase (mol l−1)

\(c_{{{\text{CO}}_{2} , {\text{l}}}}\) :

Molar concentration of carbon dioxide in the liquid phase (mol l−1)

\(c_{{{\text{HCO}}_{3}^{ - } }}\) :

Molar concentration of hydrogen carbonate (mol l−1)

\(c_{\text{IC}}\) :

Total molar concentration of inorganic carbon (mol l−1)

\(c_{\text{IC, t1}}\) :

Molar concentration of inorganic carbon at time one (mol l−1)

\(c_{\text{IC, t2}}\) :

Molar concentration of inorganic carbon at time two (mol l−1)

CT:

Total carbon dioxide transfer (mol l−1)

CTR:

Carbon dioxide transfer rate (mol l−1 h−1)

c*:

Saturation concentration of carbon dioxide (mol l−1)

\(\frac{{{\text{dc}}_{\text{IC}} }}{{{\text{d}}_{t} }}\) :

Variation of inorganic carbon concentration in one period (mol l−1 h−1)

\(H_{{{\text{CO}}_{2} }}\) :

Henry constant for carbon dioxide (mol l−1 bar−1)

I:

Ionic strength (mol l−1)

\({\text{k}}_{\text{L}} a_{{{\text{CO}}_{2} }}\) :

Volume-specific carbon dioxide mass transfer coefficient (h−1)

\(K_{\text{S}}\) :

Mass action constant (mol l−1)

OTR:

Oxygen transfer rate (mol l−1 h−1)

OUR:

Oxygen uptake rate (mol l−1 h−1)

p ambient :

Ambient pressure (bar)

\(p_{{{\text{CO}}_{2} , {\text{Ferm}}}}\) :

Partial pressure of carbon dioxide in the liquid phase of the bioreactor (bar)

RQ:

Respiratory quotient (–)

T :

Temperature (K)

T1 headspace :

Time delay caused by headspace of the bioreactor (h)

TQ:

Transfer quotient (–)

t :

Time (h)

t P :

Process time (h)

τ :

Delay time (h)

\(V_{\text{culture volume}}\) :

Culture volume in the bioreactor (l)

\(\dot{V}_{\text{inlet, gas}}\) :

Inlet gas flow rate (l h−1)

\(V_{\text{m}}\) :

Molar gas volume (mol l−1)

\(x_{{{\text{CO}}_{2} , {\text{in}}}}\) :

Molar fraction of carbon dioxide in the inlet gas (%)

\(x_{{{\text{CO}}_{2} , {\text{out}}}}\) :

Molar fraction of carbon dioxide in the off-gas (%)

\(x_{{{\text{O}}_{2} , {\text{ in}}}}\) :

Molar fraction of oxygen in the inlet gas (%)

\(x_{{{\text{O}}_{2} , {\text{out}}}}\) :

Molar fraction of oxygen in the off-gas (%)

\(x_{{{\text{O}}_{2} , {\text{ out, averaged}}}}\) :

Average molar fraction of oxygen in the off-gas (%)

\(X_{{{\text{PT}}1,{\text{n}}}}\) :

Value of time point n (depends on step response)

\(X_{{{\text{PT}}1,{\text{n}} - 1}}\) :

Value of time point n-1 (depends on step response)

\(X_{\text{step}}\) :

Current value of step response (depends on step response)

\(\delta_{{{\text{HCO}}_{3}^{ - } }}\) :

Activity coefficient of hydrogen carbonate (–)

\(\xi\) :

Flow rate correction factor (–)

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Acknowledgments

This project was funded by MWEIMH of Germany with encouragement of the European Union.

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Authors and Affiliations

  1. Institute of Nano and Biotechnologies (INB), FH Aachen University of Applied Sciences, Campus Jülich, Heinrich-Mubmann-Str.1, 52428, Jülich, Germany

    Silvia Winckler, Thomas Schnitzler & Manfred Biselli

  2. HiTec Zang GmbH, Ebertstr. 30-32, 52134, Herzogenrath, Germany

    Rolf Krueger & Werner Zang

  3. Fraunhofer Institute for Molecular Biology and Applied Ecology, Forckenbeckstr. 6, 52074, Aachen, Germany

    Rainer Fischer

  4. Institute for Molecular Biotechnology, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany

    Rainer Fischer

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  1. Silvia Winckler
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  2. Rolf Krueger
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  3. Thomas Schnitzler
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  4. Werner Zang
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  5. Rainer Fischer
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Correspondence to Manfred Biselli.

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Winckler, S., Krueger, R., Schnitzler, T. et al. A sensitive monitoring system for mammalian cell cultivation processes: a PAT approach. Bioprocess Biosyst Eng 37, 901–912 (2014). https://doi.org/10.1007/s00449-013-1062-8

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  • DOI: https://doi.org/10.1007/s00449-013-1062-8

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