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Application of adaptive DO-stat feeding control to Pichia pastoris X33 cultures expressing a single chain antibody fragment (scFv)

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Abstract

In this study, fed-batch cultures of a Pichia pastoris strain constitutively expressing a single chain antibody fragment (scFv) under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter were performed in a pilot 50 L bioreactor. Due to the very high cell density achieved within the first 75 h, typically between 140 and 160 g-DCW/L of dry cell weight (DCW), most of the scFv is produced under hard oxygen transfer limitation. To improve scFv productivity, a direct adaptive dissolved oxygen (DO)-stat feeding controller that maximizes glycerol feeding under the constraint of available oxygen transfer capacity was developed and applied to this process. The developed adaptive controller enabled to maximize glycerol feeding through the regulation of DO concentration between 3 and 5 % of saturation, thereby improving process productivity. Set-point convergence dynamics are characterized by a fast response upon large perturbations to DO, followed by a slower but very robust convergence in the vicinity of the boundary with almost imperceptible overshoot. Such control performance enabled operating closer to the 0 % boundary for longer periods of time when compared to a traditional proportional–integral–derivative algorithm, which tends to destabilize with increasing cell density.

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Abbreviations

DO:

Dissolved oxygen

PID:

Proportional–integral–derivative

Y PX :

Yield of product on biomass (w/w)

PI:

Proportional–integral

WCW:

Wet cell weight

DCW:

Dry cell weight

GB:

Glycerol batch (phase)

GFB:

Glycerol fed-batch (phase)

OTL:

Oxygen transfer limitation (phase)

F :

Glycerol feeding rate (L/h)

F 0 :

Initial glycerol feeding rate (L/h)

t :

Time (h)

C 0 :

Dissolved oxygen concentration (g/L)

\( C_{0}^{*} \) :

Oxygen saturation concentration (g/L)

S 0 :

Glycerol concentration in the feed solution (g/L)

Y OS :

Observed yield of oxygen on glycerol (w/w)

k L a :

Oxygen mass transfer coefficient (h−1)

x :

100 − DOT (%)

K :

Time-varying ‘parameter’

q O :

Oxygen flux [g/(L h)]

q S,an :

Glycerol flux for anabolism [g/(L h)]

q S,en :

Glycerol flux for catabolism [g/(L h)]

Y OS,an :

Yield of oxygen on glycerol for anabolism (w/w)

Y OS,en :

Yield of oxygen on glycerol for catabolism (w/w)

P :

Product titer (g/L)

V :

Liquid volume in the bioreactor (L)

X :

Biomass concentration (g/L)

e :

Deviation to set-point

DOT:

Dissolved oxygen tension (% saturation)

a p, k p :

Adaptive controller parameter

\( \dot{y} \) :

Variable derivative

\( \hat{y} \) :

Variable estimates

y * :

Variable set-point

τ c :

Controller time constant (s)

γ :

Adaptation gain

μ :

Desired specific growth rate (h−1)

θ :

Time-varying ‘parameter’

β :

Specific protein synthesis rate [AU/(g-WCW h)]

φ :

θ estimation error

ψ :

K estimation error

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Acknowledgments

This work was supported by the Portuguese Fundação para a Ciência e Tecnologia through PhD Grant SFRH/BD/36285/2007 (Ana R Ferreira), PhD Grant SFRH/BD/43956/2008 (Filipe Ataide), PhD Grant SFRH/BD/36990/2007 (Moritz von Stosch), Post-doc Grant SFRH/BPD/46277/2008 (João Dias) and Project PTDC/EBB-EBI/103761/2008.

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Authors and Affiliations

  1. REQUIMTE, Systems Biology and Engineering Group, DQ/FCT, Universidade Nova de Lisboa, Campus Caparica, Caparica, Portugal

    A. R. Ferreira, F. Ataíde, J. M. L. Dias & R. Oliveira

  2. Instituto de Biologia Experimental e Tecnológica (IBET), Apartado 12, 2781-901, Oeiras, Portugal

    J. J. Clemente, A. E. Cunha & R. Oliveira

  3. LEPAE, Departamento de Engenharia Química, Faculdade de Engenharia, Universidade do Porto, Rua Dr. Roberto Frias s/n, 4200-465, Porto, Portugal

    M. von Stosch

  4. Chemistry Department, Faculty of Sciences and Technology, Universidade Nova de Lisboa, 2829-516, Caparica, Portugal

    R. Oliveira

Authors
  1. A. R. Ferreira
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  2. F. Ataíde
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  3. M. von Stosch
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  4. J. M. L. Dias
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  6. A. E. Cunha
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Correspondence to R. Oliveira.

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Ferreira, A.R., Ataíde, F., von Stosch, M. et al. Application of adaptive DO-stat feeding control to Pichia pastoris X33 cultures expressing a single chain antibody fragment (scFv). Bioprocess Biosyst Eng 35, 1603–1614 (2012). https://doi.org/10.1007/s00449-012-0751-z

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