Bioreactor scale-down studies of temperature-inducible recombinant protein production in E. coli

Title: Bioreactor Scale-Down Studies of Temperature-Inducible Recombinant Protein Production in E. coli

Speaker: Octavio T. Ramírez

Affiliation: Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología Universidad Nacional Autónoma de México

Host: Paula Alves, Animal Cell Technology Unit

Abstract
A major focus of our research group during the past 14 years has been the study of spatial gradients in fundamental culture parameters, such as dissolved oxygen, dissolved carbon dioxide, pH and concentration of substrates, that can commonly occur in large-scale bioreactors. Such a problem is due to a deficient mixing that results from inherent limitations in the traditional scaling-up methods as well as practical constraints of the design and operation of large-scale bioreactors [1]. The approach taken by our group has been to simulate in scale-down systems various environmental gradients relevant to a variety of industrially important organisms, including bacteria, insect, and mammalian cells [2]. In this presentation, we will discuss a scale-down study that simulates the typical heating rates that can be achieved in large-scale bioreactors during expression of recombinant proteins by an E. coli thermo-inducible expression system [3]. At the laboratory scale, sudden step increases from 30 to 42 °C can be readily accomplished for attaining full induction conditions. However, for large scale-cultures only slow ramp-type increases in temperature are possible due to heat transfer limitations, where the heating rate decreases as the scale increases. In this presentation we will show how heating rate affects the transcriptional and metabolic responses of recombinant an E. coli strain during temperature-induced synthesis of pre-proinsulin in fed-batch high cell density cultures. Heating rates between 6 and 0.4 °C/min were tested in a scale-down approach to mimic fermentor scales between 0.1 and 100 m3. As the heating rate increased, the yield and maximum recombinant protein concentration decreased, whereas a larger fraction of carbon skeletons was lost as acetate, lactate, and formate. Such results, in addition to the transcriptional profiles from six relevant heat shock genes (as measured by RT-PCR), three genes from the transcriptional and translational machinery, five stress control genes, and the gene coding for the heterologous protein, revealed that cells subjected to slow temperature increases can adapt to stress. The results that will be discussed indicate that slow heating rates, such as those likely to occur in conventional large-scale fermentors, favor heterologous protein synthesis by the thermo-inducible expression system used in this report. Furthermore, based on the knowledge of the effect of heating rate on bacterial physiology and product formation, a novel strategy for temperature induction of recombinant protein will be presented. Altogether, the present study should be useful for the rational design of scale-down and scale-up strategies and optimum recombinant protein induction schemes.

Short CV
In 1985 he earned his B. Sc. in Chemical Engineering from the National Autonomous University of Mexico, Mexico. In 1987 he earned his M. Sc. and in 1990 his Ph. D., both degrees in Chemical and Biochemical Engineering from Drexel University, Philadelphia, PA, U.S.A. In 1990 he joined the Institute of Biotechnology of the National Autonomous University of Mexico, where he is now Professor and Head of the Department of Molecular Medicine and Bioprocesses. His present research interest include: bioengineering issues in animal cell culture, glycosylation of recombinant proteins, production of virus-like particles for vaccine and nanobiotechnology applications, computerized control of bioprocesses with emphasis on scale-up, scale-down and integration of fermentations with recombinant microorganisms (E. coli), mammalian and insect cells - baculovirus for production of recombinant proteins and vaccines. Active as lecturer, at the graduate level, in various fields of Biochemical Engineering. His academic activity has resulted in more than 100 publications, and has supervised ca 30 graduate students. He was a key participant in the foundation of Probiomed, the first Mexican company dedicated to modern biotechnology (recombinant pharmaceutical proteins and vaccines). Since 1995 he has a joint appointment as Head of the Department of Research and Development, Probiomed, where he has been responsible of research and development of new biopharmaceutical processes and products, and has participated in the design, construction, start-up, and operation of cGMP facilities for the production of r-DNA technology products used in prophylaxis and therapy. In addition, he is consultant to various pharmaceutical companies, and has received multiple national and international awards.