A comprehensive study of glucose and oxygen gradients in a scaled-down model of recombinant HuGM-CSF production in thermoinduced Escherichia coli fed-batch cultures

Abstract

The effect of gradients of elevated glucose and low dissolved oxygen in the addition zone of fed-batch E. coli thermoinduced recombinant high cell density cultures can be evaluated through two-compartment scale-down models. Here, glucose was fed in the inlet of a plug flow bioreactor (PFB) connected to a stirred tank bioreactor (STB). E. coli cells diminished growth from 48.2 ± 2.2 g/L in the stage of RP production if compared to control (STB) with STB-PFB experiments, when residence time inside the PFB was 25 s (34.1 ± 3.5 g/L) and 40 s (25.6 ± 5.1 g/L), respectively. The recombinant granulocyte-macrophage colony-stimulating factor (rHuGM-CSF) production decreased from 34 ± 7% of RP in inclusion bodies (IB) in control cultures to 21 ± 8%, and 7 ± 4% during the thermoinduction production phase when increasing residence time inside the PFB to 25 s and 40 s, respectively. This, along with the accumulation of acetic and formic acid (up to 4 g/L), indicates metabolic redirection of central carbon routes through metabolic flow and mixed acid fermentation. Special care must be taken when producing a recombinant protein in heat-induced E. coli, because the yield and productivity of the protein decreases as the size of the bioreactors increases, especially if they are carried at high cell density.

HIGHLIGHTS

• Thermoinduced recombinant E. coli grew less in a two-compartment scale-down model.

• Heat-inducible E. coli cultures at a large scale significantly decrease recombinant protein production.

• The accumulation of acetic and formic acid increases when E. coli is exposed to glucose and oxygen gradients.

• The axial flow pattern inside the PFB mimics glucose and dissolved oxygen gradients at the industrial scale.

Keywords:

• Escherichia coli
• fed-batch
• inclusion bodies
• recombinant proteins
• scale-down
• thermoinduction

Acknowledgments

Greta Isabel Reynoso Cereceda is a doctoral student from Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM), and received a fellowship from Consejo Nacional de Humanidades, Ciencias y Tecnologías (CONAHCYT, CVU 549681). NAV-C and MAT-R thanks the sabbatical to “Programa de Apoyos para la Superación del Personal Académico” PASPA-DGAPA- UNAM. We thank M. Eng. Martha Elena Carrasco Fuentes, and Eng. Abel Blancas-Cabrera for technical support. Technical assistance on bioreactor controllability by Dusstthon Llorente, Eng. is also appreciated. This project was developed under the Institutional Program of the Instituto de Investigaciones Biomédicas UNAM: “La producción de biomoléculas de interés biomédico en bacterias y hongos.”

CRediT authorship statement

GIR-C: Conceptualization, methodology, investigation, writing – original draft. NOP: Supervision, writing – review and editing. NAV-C: Supervision, writing – review and editing. MAT-R: Conceptualization, supervision, resources, writing – review and editing, funding acquisition.

Disclosure statement

NOP works in Probiomed S.A. de C.V., which manufactures recombinant human therapeutic proteins. No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by “Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica, Universidad Nacional Autónoma de México” (NAVC: PAPIIT-UNAM IN-210822. MATR: IN211422). The funders had no role in data collection and analysis, publication decisions, or manuscript preparation.