Application note RAMbio 1 Introduction The production of recombinant proteins in various expression systems is a common and well described method. Most of these strategies are based on suspension cultures. Small scale systems, such as standard orbital shakers are widely used. Despite the widespread use, the oxygen limitation is often a major problem during the cultivation/fermentation. The aim of this application note is the description of growth characteristics of different expression systems. Therefore, a standard shaked incubator is compared to the new fermentation system RAMbio, introduced by Applicon Biotechnology. 2 Theory In fermentation and cultivation processes, the feed of nutrients and oxygen is of major importance for aerobic suspension cultures. Thereby, oxygen is often the limiting factor in orbital shaken systems, as the oxygen transfer into the medium is only diffusive and limited by the exchange surface and the mixing charactistics of the applied fermentation system. The oxygen transfer can be increased by different methods. An increase in the pressure within the fermentation system increases the oxygen solubility and therefore the transfer rate. Furthermore, the partial pressure in the inlet-air can be maximized using pure oxygen. The first method requires a pressure resistant unit, whereas the second method demands a gas mixing system as a peripheral unit to the reaction system. To further increase the oxygen transfer rate, the exchange surface may be increased. This method is the most frequently applied and supported by several published successful studies. Standardized wide spread orbital shaken incubators are well studied. In these systems, the mixing of the culture is realized using a two-dimensional, horizontal movement. Difficulties arise with the development of laminar flow. This can be diminished by varying orbital frequencies or with vial integrated baffles. Such baffles are located in the lower part of the flask and allow eddy-formation. This causes an increase in the exchange surface and therefore the rate of oxygen transfer into the medium. The system of interest, the RAMbio, introduced by the company applikon Biotechnology, works with eddy-formation through a one-dimensional, vertical movement. The movement is induced by a resonance system, which is located under the mixing platform and runs at a frequency of 58-66 Hz. The oscillating platform transfers the energy to the flask, which then oscillate as well and the culture suspension in the flask is being mixed. At the same time, gas bubbles being transported in the flask due to the vibration are further split. This results in increase of the exchange surface by the gas bubbles and the oxygen transfer rate is raised. Accompanying the oscillating movement, the need to seal the flask is present. Therefore, so called “OxyPump” stopper made of flexible silicone are used. The medium is remained within the flask and the filter membrane decreases the risk of contamination. The vertical movement of the flasks causes the flexible Stopper to pump the carbon dioxide containing air inside the flask which is replaced by fresh air. Application Note RAMbio 1 3 Material and Methods 3.1 Media To analyze the system behaviour and influence on different expression systems, different organisms were used and cultured in each two different media (tab. 1) Table 1: Expressionsystems and used media for cultivation Expression system Medium YPD-Medium Escherichia coli BL21 LB-Medium YPD-Medium Pichia pastoris pink LB-Medium The media ingredients are listed in the following: Table 2: Media and media compostions Medium LB‐Medium YPD‐ Medium Contents Concentration [g/L] Peptone 10 Yeast extract 5 Sodiumchloride 5 Glucose 5 Peptone 20 Yeast extract 10 Glucose 20 The production of the media for the fermentation with E.coli and P.pastoris is exemplary described for YPD media: Yeast extract and peptone were solved in distilled water using ¾ of the total volume and autoclaved. Glucose was solved in ¼ of the total volume and autoclaved separately. The cold sterile solutions were combined. In each flask a total of 150 ppm antifoam-solution (Struktol J647) was added. 3.2 Inoculation To start the fermentation, an inoculation culture was prepared. For each media, a preculture in a 1 L shake flask with 200 ml media was started with 1 ml working seed. The culture was incubated over night in a orbital shaken incubator (INOFRS HT Multitron Standard) at 300 rpm and 30 °C (P.pastoris) respectively 37 °C (E. coli). After the culture ran over night, the optical density was determined using a photometer ((Thermo SpectronicHeios ) at 600 nm. Application Note RAMbio 2 3.3 Cultivation For the cultivation in the orbital shaker 1L-baffled flasks filled with 200 mL media were used. For the cultivation in the RAMbio 500mL-Erlenmeyer flasks filled with 200 mL media were used. Starting from the pre-culture, a optical density of 0.1 was each inoculated in the flasks for the fermentation studies in the different systems. The cultivation were run parallel in the INFORS HT Multitron Standard and in the RAMbio by applikon Biotechnology. The INFORS was set to 300 rpm being equivalent to 15xg set for the RAMbio. The cultivations were run at the culture temperature specific for the organism each for 25 h and were stopped thereafter. 3.4 Growth kinetics The growth rate determination is essential for the culture comparison. Therefore, the optical density as well as the glucose concentration was measured twice every hour using 3 ml samples. If the value of the optical density was higher than 0.9 a dilution with medium was required. Glucose analysis was performed using the EKF Diagnostic C-Line according to the SOP by the manufacturer. The maximum growth rate was calculated using the following formula: μ , [1] whereas the maximum growth rate was determined only in the exponential growth phase. Application Note RAMbio 3 4 Results 4.1 Pichia pastoris pink The optical density as a function of the culture time for pichia pastoris pink is shown in the following figure. For all four culture types, the same starting density was used. A short lag-phase was followed by strong exponential growth. Pichia pastoris pink 15 LB-Media Orbital Shaker LB-Media RAMbio YPD-Media Orbital Shaker YPD-Media RAMbio Optical Densitiy 10 5 0 0 5 10 15 20 25 Time (h) Figure 1: Arithmetic average of the optical density as a function of the culture time for Pichia pastoris pink in different shake flask cultures. The glucose concentrations of these cultures are shown in figure 2. The decrease in the glucose concentration over time is obvious and correlates with the optical density. Pichia pastoris pink 20 Glucose Concentration (mg/mL) 18 LB-Media Orbital Shaker LB-Media RAMbio YPD-Media Orbital Shaker YPD-Media RAMbio 16 14 12 10 8 6 4 2 0 -2 0 5 10 15 20 25 Time (h) Figure 1: Arithmetic average of the glucose concentration over time for Pichia pastoris pink in different shake flask cultures Application Note RAMbio 4 The following table shows the optical density values at the end of the culture (at 25 h) and the maximum growth rate in the exponential growth phase. Table 3: Optical density at the of the culture as well as the maximum growth rate µmax during the exponential growth phase for Pichia pastoris pink Incubator Media OD600 (final) µmax [1/h] RamBio LB 11.540 1.017 INFORS LB 4.065 0.975 RamBio YPD 12.850 1.208 INFORS YPD 5.700 1.184 Application Note RAMbio 5 4.2 Escherichia coli BL21 The following figures 3 and 4 show the optical density and glucose concentration each as a function of time for the E. coli cultures. The progress is analogous to those of Pichia pastoris pink. Escherichia coli BL21 10 Optical Densitiy 8 6 4 2 LB-Media Orbital Shaker LB-Media RAMbio YPD-Media Orbital Shaker YPD-Media RAMbio 0 0 5 10 15 20 25 Time (h) Figure 2: Arithmetic average of the optical density as a function of the culture time for Escherichia coli BL21 in different shake flask cultures. Escherichia coli BL21 Glucose Concentration (mg/mL) 20 LB-Media Orbital Shaker LB-Media RAMbio YPD-Media Orbital Shaker YPD-Media RAMbio 15 10 5 0 -5 0 5 10 15 20 25 Time (h) Figure 3: Arithmetic average of the Glucose concentration over time for Escherichia coli BL21 in different shake flask cultures Application Note RAMbio 6 The following table shows the optical density values at the end of the culture (at 25 h) and the maximum growth rate in the exponential growth phase. Table 4: Optical density at the of the culture as well as the maximum growth rate µmax for Escherichia coli BL21 Incubator Media OD600 (final) µmax [1/h] RamBio LB 9.99 1.03 INFORS LB 6.58 1.05 RamBio YPD 10.17 1.16 INFORS YPD 5.7 1.18 Application Note RAMbio 7 5 Discussion A closer look on figure 1 and 3 showed that the optical density at the start was equal for all cultures. Fermentation differences were the choice of the medium as well as the cultivation device. For all cultures, the growth phases were similar within the first 5 hours. As the results from the glucose measurement (figure 2 and 4) show, no significant influence of the cultivation device on the glucose consumption can be seen, whereas a major difference in the optical density measurement can be determined (figure 1 and 3). The optical density in YPD-media increases slower than in LB-media although higher concentrations of nutrients are available for the microorganisms in the first media. An Inhibition of growth by the substrate can be excluded as figure 1 shows similar curves in the optical densities within the first 5 hours. The major difference in the optical densities after 5 hours can be explained by oxygen-limitation in the orbital shaker than in the RAMbio. This assumption is supported by the results of applikon as the RAMbio is capable of supplying more dissolved oxygen into the media. The maximum optical density in the RAMbio has been increased by a factor of up to 3. 6 Outlook The described experiments showed that the two expression systems with each different media can be cultured in different fermentation systems. An increase in the oxygen transfer rate can be achieved by the choice of the apparatus used. The mixing principle of the RamBio showed promising results, allowing thrice the optical density in comparison to standard orbital shaken systems. The application of such systems in small scale productions or for scientific needs may be of high benefit. Studies on the oxygen transfer rate including partial pressure measurements could help to increase the process understanding. Application Note RAMbio 8