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A comprehensive comparison of mixing and mass transfer in shake flasks and their relationship with MAb productivity of CHO cells

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Abstract

The selection of highly recombinant protein (RP)-productive Chinese hamster ovary (CHO) cell lines is widely carried out in shake flasks. It is assumed that increases in the operating parameters in shake flasks lead to impairments in cell growth and RP production. These effects in cells metabolism are widely associated with high mass transfers and hydrodynamic stress. This study examined the impact of commonly used operational parameters on growth and specific productivity (qP) of two CHO cell lines differentially secreting a humanized anti-hIL8 monoclonal antibody (mAb) and cultured in 250 ml flasks. The evaluated parameters are filling volume (10, 15, and 20%), shaking frequency (60 and 120 revolutions per minute -rpm-), and orbital diameter (25.4 and 19 mm). The analysis of the oxygen transfer was done in terms of the measured volumetric mass transfer coefficient (kLa) and of the hydrodynamics in terms of power input per unit volume of liquid (P/V), the turbulent eddy length scale measured by the Kolmogorov’s microscale of turbulence, the energy dissipation rate, the average shear stress, and the shear rate. Though almost all measured kinetic and stoichiometric parameters remained unchanged, mAb titer included, significant differences were found in maximum cell concentration, 10–45% higher in conditions with lower values of kLa and P/V. Changes in glucose metabolism contributing to qP were only shown in the higher producer cell line. Non-lethal responses to elevated oxygen transfer and shear stress might be present and must be considered when evaluating CHO cell cultures in shake flasks.

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Data availability

The datasets generated during and analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

C L*:

Oxygen concentration at the interfacial saturation

C L :

Oxygen concentration in the liquid bulk

D :

Maximum inner flask diameter, m

d 0 :

Shaking diameter, Mm

d p :

Average cell diameter, m

DOT:

Dissolved oxygen tension, % air saturation

IVCC:

Integral viable cell concentration, 106 cells ml−1 day

k L a :

Volumetric oxygen transfer coefficient, h−1

Lac/Glc:

Lactate/glucose ratio

mAb:

Anti-hIL8 monoclonal antibody

n :

Shaking frequency, Rpm

Ne′:

Modified power number

Ph:

Phase number

P/V:

Power consumption per unit volume, W m−3

q Ca2 + :

Specific calcium uptake rate, nmol calcium 10–6 cells day−1

q Glc :

Specific glucose uptake rate, μMol glucose 10–6 cells day−1

q Lac :

Specific lactate production rate, μMol lactate 10–6 cells day−1

q Glu :

Specific glutamate production rate, μMol glutamate 10–6 cells day−1

q NH4 + :

Specific ammonium production rate, μMol ammonium 10–6 cells day−1

q P :

Specific IgG production rate, Pcd 10–3

Re:

Reynolds number

RP:

Recombinant protein

t D :

Doubling time, h

v :

Dynamic viscosity, Pa s

V N :

Nominal volume, ml

V F :

Filling volume, % liquid volume/nominal flask volume

V :

Filling volume, ml

X max :

Maximum cell concentration 106 cells ml−1

Ɛ :

Energy dissipation rate W kg−1

Ɛ 0 :

Average energy dissipation rate, W kg−1

ρ :

Density, kg m−3

μ:

Specific growth rate, h−1

λ :

Turbulent eddy length scale, μM

τ :

Average shear stress, N m−2

γ :

Shear rate, s−1

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Acknowledgements

Greta I. Reynoso-Cereceda is a doctoral student from Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM) and received the fellowship number 478675, from “Consejo Nacional de Ciencia y Tecnología” CONACYT. We want to extend our gratitude to Sartorius de México, S.A. de C.V. and Biosystems S.A. de C.V. Also, we thank to Nova Biomedicals for providing access to BioProfile FLEX2 Automated Cell Culture Analyzer.

Funding

This work was supported by “Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica, Universidad Nacional Autónoma de México” (PAPIIT-UNAM IN210419, IN210822, IN211422, IV201220). Funding sources had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Author notes

  1. Saumel Pérez-Rodriguez and Greta I. Reynoso-Cereceda contributed equally and should be considered co-first authors.    This manuscript is dedicated to Dr. Alfredo Torres-Larios an expert on structural and biophysical studies of biological macromolecules. 

Authors and Affiliations

  1. Unidad de Bioprocesos, Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Cd. Universitaria, Coyoacán, 04510, Ciudad de México, Mexico

    Saumel Pérez-Rodriguez, Greta I. Reynoso-Cereceda, Norma A. Valdez-Cruz & Mauricio A. Trujillo-Roldán

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  1. Saumel Pérez-Rodriguez
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  3. Norma A. Valdez-Cruz
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  4. Mauricio A. Trujillo-Roldán
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Contributions

Conceptualization: SP-R, GIR-C, NAV-C, and MAT-R; methodology: SP-R and GIR-C; formal analysis and investigation: SP-R, GIR-C, NAV-C, and MAT-R; writing—original draft preparation: SP-R and GIR-C; writing—review and editing: SP-R, GIR-C, NAV-C, and MAT-R; funding acquisition: NAV-C and MAT-R; resources: NAV-C and MAT-R.

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Correspondence to Mauricio A. Trujillo-Roldán.

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Pérez-Rodriguez, S., Reynoso-Cereceda, G.I., Valdez-Cruz, N.A. et al. A comprehensive comparison of mixing and mass transfer in shake flasks and their relationship with MAb productivity of CHO cells. Bioprocess Biosyst Eng 45, 1033–1045 (2022). https://doi.org/10.1007/s00449-022-02722-y

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