High cell density cultivation and recombinant protein production with Escherichia coli in a rocking-motion-type bioreactor

Abstract

Background: Single-use rocking-motion-type bag bioreactors provide advantages compared to standard stirred tank bioreactors by decreased contamination risks, reduction of cleaning and sterilization time, lower investment costs, and simple and cheaper validation. Currently, they are widely used for cell cultures although their use for small and medium scale production of recombinant proteins with microbial hosts might be very attractive. However, the utilization of rocking- or wave-induced motion-type bioreactors for fast growing aerobic microbes is limited because of their lower oxygen mass transfer rate. A conventional approach to reduce the oxygen demand of a culture is the fed-batch technology. New developments, such as the BIOSTAT CultiBag RM system pave the way for applying advanced fed-batch control strategies also in rocking-motion-type bioreactors. Alternatively, internal substrate delivery systems such as EnBase Flo provide an opportunity for adopting simple to use fed-batch-type strategies to shaken cultures. Here, we investigate the possibilities which both strategies offer in view of high cell density cultivation of E. coli and recombinant protein production.

Results: Cultivation of E. coli in the BIOSTAT CultiBag RM system in a conventional batch mode without control yielded an optical density (OD(600)) of 3 to 4 which is comparable to shake flasks. The culture runs into oxygen limitation. In a glucose limited fed-batch culture with an exponential feed and oxygen pulsing, the culture grew fully aerobically to an OD(600) of 60 (20 g L(-1) cell dry weight). By the use of an internal controlled glucose delivery system, EnBase Flo, OD(600) of 30 (10 g L(-1) cell dry weight) is obtained without the demand of computer controlled external nutrient supply. EnBase Flo also worked well in the CultiBag RM system with a recombinant E. coli RB791 strain expressing a heterologous alcohol dehydrogenase (ADH) to very high levels, indicating that the enzyme based feed supply strategy functions well for recombinant protein production also in a rocking-motion-type bioreactor.

Conclusions: Rocking-motion-type bioreactors may provide an interesting alternative to standard cultivation in bioreactors for cultivation of bacteria and recombinant protein production. The BIOSTAT Cultibag RM system with the single-use sensors and advanced control system paves the way for the fed-batch technology also to rocking-motion-type bioreactors. It is possible to reach cell densities which are far above shake flasks and typical for stirred tank reactors with the improved oxygen transfer rate. For more simple applications the EnBase Flo method offers an easy and robust solution for rocking-motion-systems which do not have such advanced control possibilities.

Figure 1

Batch cultivation of E. coli BL21 in glucose mineral salt medium in (A) a shake flask with the Sensolux monitoring system and (B, C) with the CultiBag RM system. Cultivation conditions: 10 L bag with 5 L working volume, maximum rocking angle, 35 rocks per min, 37°C, air flow rate 1 L min^-1 in (B). In (C) the air flow rate was 1 L min^-1 at the start of the cultivation. After the pO₂ decreased to 50% a dual controller mode was started to maintain the pO₂ by (i) increasing the air flow rate to 6 L min^-1 and (ii) pulse addition of pure oxygen into the inlet air stream (up to 100%).

Figure 2

DO-stat based Fed-batch cultivation of E. coli BL21 in minimal medium with the CultiBag RM system. Cultivation conditions: 10 L bag with 4 L filling volume, maximum rocking angle, 35 rocks min^-1 during the batch phase and the maximum rocking speed (42 rocks min^-1) during the feeding phase, cultivation temperature 25°C during the batch phase and increase to 37°C after 11 hours. The pO₂ was controlled to 50% by a dual control of (i) the gas flow rate from 1 to 6 L min^-1 and (ii) pulsing of pure oxygen into the inlet air stream (up to 100%, maximum total flow rate of 6 L min^-1, named O₂ ratio).

Figure 3

Cultivation of E. coli BL21 in EnBase^® Flo with the CultiBag RM system. The cultivation was performed in a 2 L bag with 1 L EnBase^® Flo medium containing an amylase concentration of 1.5 U L^-1, which was increased up to 9 U L^-1. Boosting solution was added once after 23 hours.

Figure 4

Cultivation of E. coli RB791 pAdh in EnBase^® Flo with the CultiBag RM system. (A) Data of a cultivation in a 2 L bag with 1 L EnBase^® Flo medium containing 1.5 U L^-1 of amylase. Trace elements and MgSO₄ were added after 18 and 41 hours and ammonium sulfate was added after 26 hours. Protein expression was induced by 1 mM IPTG after 26 hours. (B) Coomassie Brilliant Blue stained SDS polyacrylamide gel with soluble (left lanes) and insoluble protein samples (right lanes). Same amounts of cells were loaded onto each lane. Lane 1) before induction; 2) at induction; 3) after 12 h; 4) after 17 h; 5) after 20 h. Most left lane - size standard.

Figure 5

Cultivation of E. coli RB791 pAdh expressing a heterologous alcohol dehydrogenase in the CultiBag RM system with 1 L of EnBase^® Flo medium with the addition of complex additives (EnBase^® Booster). (A) In this cultivation the amylase concentration was stepwise increased from 1.5 to 6 U L^-1; the boosting solution was added three times. Protein expression was induced with 1 mM IPTG after 27 hours. (B) Coomassie Brilliant Blue stained 12% SDS polyacrylamide gel of soluble and insoluble protein samples. Same amounts of cells were applied to each lane. Lane 1) before induction; 2) at induction; 3) 11 h; 4) 16 h; 5) 21 h after induction.