Gerhard Greller

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

BackgroundSingle-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.ResultsCultivation of E. coli in the BIOSTAT® CultiBag RM system in a conventional batch mode without control yielded an optical density (OD600) 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 OD600 of 60 (20 g L-1 cell dry weight). By the use of an internal controlled glucose delivery system, EnBase® Flo, OD600 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.ConclusionsRocking-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.

Verification of a New Biocompatible Single-Use Film Formulation with Optimized Additive Content for Multiple Bioprocess Applications

Single‐use bioprocessing bags and bioreactors gained significant importance in the industry as they offer a number of advantages over traditional stainless steel solutions. However, there is continued concern that the plastic materials might release potentially toxic substances negatively impacting cell growth and product titers, or even compromise drug safety when using single‐use bags for intermediate or drug substance storage. In this study, we have focused on the in vitro detection of potentially cytotoxic leachables originating from the recently developed new polyethylene (PE) multilayer film called S80. This new film was developed to guarantee biocompatibility for multiple bioprocess applications, for example, storage of process fluids, mixing, and cell culture bioreactors. For this purpose, we examined a protein‐free cell culture medium that had been used to extract leachables from freshly gamma‐irradiated sample bags in a standardized cell culture assay. We investigated sample bags from films generated to establish the operating ranges of the film extrusion process. Further, we studied sample bags of different age after gamma‐irradiation and finally, we performed extended media extraction trials at cold room conditions using sample bags. In contrast to a nonoptimized film formulation, our data demonstrate no cytotoxic effect of the S80 polymer film formulation under any of the investigated conditions. The S80 film formulation is based on an optimized PE polymer composition and additive package. Full traceability alongside specifications and controls of all critical raw materials, and process controls of the manufacturing process, that is, film extrusion and gamma‐irradiation, have been established to ensure lot‐to‐lot consistency.

Microbial High Cell Density Fermentations in a Stirred Single-Use Bioreactor

: Microbial fermentations are of major importance in the field of biotechnology. The range of applications is rather extensive, for example, the production of vaccines, recombinant proteins, and plasmids. During the past decades single-use bioreactors have become widely accepted in the biopharmaceutical industry. This acceptance is due to the several advantages these bioreactors offer, such as reduced operational and investment costs. Although this technology is attractive for microbial applications, its usage is rarely found. The main limitations are a relatively low oxygen transfer rate and cooling capacity. The aim of this study was to examine a stirred single-use bioreactor for its microbial suitability. Therefore, the important process engineering parameters volumetric mass transfer coefficient (k L a), mixing time, and the heat transfer coefficient were determined. Based on the k L a characteristics a mathematical model was established that was used with the other process engineering parameters to create a control space. For a further verification of the control space for microbial suitability, Escherichia coli and Pichia pastoris high cell density fermentations were carried out. The achieved cell density for the E. coli fermentation was OD600xa0=xa0175 (DCWxa0=xa060.8xa0g/L). For the P. pastoris cultivation a wet cell weight of 381xa0g/L was reached. The achieved cell densities were comparable to fermentations in stainless steel bioreactors. Furthermore, the expression of recombinant proteins with titers up to 9xa0g/L was guaranteed.

Single-use wave-mixed versus stirred bioreactors for insect-cell/BEVS-based protein expression at benchtop scale

Spodoptera frugiperda‐9 (Sf‐9) cells used in conjunction with the baculovirus expression vector system (BEVS) represent a promising platform for the rapid development and manufacture of protein complexes and virus‐like particle (VLP) products. Several studies have described the superiority of single‐use wave‐mixed bioreactors although reusable stirred and, more recently, single‐use stirred bioreactors have also been successfully applied. Due to their bioengineering characteristics (more homogeneous energy dissipation, reduced foam formation), wave‐mixed systems are often preferred. However, a direct comparison of the influence of single‐use wave‐mixed and single‐use stirred bioreactors on cell growth and protein expression in Sf‐9/BEVS‐based production processes was still lacking. We investigated Sf‐9 cell growth and expression of a recombinant secreted alkaline phosphatase (rSEAP) in the wave‐mixed BIOSTAT® RM as well as the stirred UniVessel® SU and a serum‐free culture medium. Irrespective of the bioreactor system, comparable growth, substrate, and metabolite courses as well as peak cell densities (>1.2 × 107 cells mL−1) were observed in Sf‐9 cell expansions performed in batch mode. Additionally, identical rSEAP quality and maximum rSEAP activities were found in biphasic productions in both bioreactor systems. Concluding, comparability of single‐use wave‐mixed and stirred bioreactors for insect cell culture processes was demonstrated for the first time.

Design space definition for a stirred single‐use bioreactor family from 50 to 2000 L scale

Single‐use bioreactors continue to gain large interest in the biopharmaceutical industry. They are frequently used for mammalian cell cultivations, e.g. production of monoclonal antibodies and vaccines. This is motivated by several advantages of these bioreactors such as reduced risk of cross contaminations or short lead times. Single‐use bioreactors differ in terms of shape, agitation principle, and gassing strategy. Hence, a direct process transfer or scale‐up can be a challenge. Conventional stirred tank bioreactor designs are therefore still considered as the gold standard due to their well‐defined and characterized properties. Based on this knowledge, a stirred single‐use bioreactor family from 50 to 2000 L scale was developed with geometrical ratios similar to conventional reusable systems. To follow a quality by designapproach, the single‐use bioreactor family evaluated here was characterized by using process engineering methods such as the power input per volume, the mixing time, and the kLa value. The process engineering characterization demonstrates that these systems are suitable for cultivations of mammalian cells, even for high cell density and high titer applications. Based on the data, a scale‐up or process transfer is possible between this bioreactor family as well as reusable systems. Therefore, this bioreactor family represents a major progress for the single‐use technology.

High cell density growth of High Five suspension cells in DO-controlled wave-mixed bioreactors

Insect cells such as High Five cells used in the manufacture of biopharmaceuticals are best grown in wave-mixed bioreactors (1). This is due to the continual blending of foam with the culture medium which results from the wave-induced mixing and permanent renewal of the medium surface. Even the addition of an antifoam agent is not required. n nProcess conditions which ensure maximum High Five cell densities and which have been reported to be about 8 x 106 cells x mL-1 (2) were determined in Biostat CultiBag RM50 optical experiments for batch mode and 1 L culture volume. Seed inoculum for these experiments was generated in single-use shake flasks (Corning) incubated in an Infors`Multitron shaker (27°C, 100 rpm, 25 mm shaking diameter). Biostat CultiBag RM50 optical was controlled in four different modes: non-pH- and non-DO-controlled, DO- controlled, pH-controlled, DO- as well as pH-controlled. The DO level was guaranteed by increasing the rocking rate up to 28 rpm and, if required by addition of pure oxygen. In process control was supplemented with off-line analyses of cell density, viability, metabolites (glucose, glutamine, glutamate, lactate, ammonium) and pH. n nWhile the influence of the type of bioreactor`s control on the maximum growth rate (0.041-0.044 h-1) and doubling time (15.6-17.7 h) was negligible, maximum cell densities were achieved with DO regulation (set point 50%). Maximum cell densities ranged between 8.2 and 9.4 x 106 cells x mL-1 and represent the highest values described for High Five cells so far in the literature. They are 35% higher compared to those seen in pH-controlled and non-controlled experiments. Controlling both DO and pH level did not lead to any further improvement of cell growth i.e. the range of growth parameter values was the same as that observed in the previous experiments. For High Five cell-based biopharmaceuticals this knowledge enables optimized seed inoculum/seed train production in wave-mixed bag bioreactors.

Bag-based rapid and safe seed-train expansion method for Trichoplusia ni suspension cells

Trichoplusia ni suspension cells (High Five™) used in conjunction with the baculovirus vector expression system (BEVS) are regarded as potential product system of new, recombinant virus-like particle (VLP) vaccines. In order to push vaccine development and production, biomanufacturers use single-use technology when- and wherever possible. This applies to upstream processing and in particular seed-train expansion ranging from cryopreserved vials via t-flasks, spinners (respectively shake flasks) to stirred stainless steel bioreactors. The stainless steel bioreactors deliver inoculum for seed bioreactors and have been increasingly replaced by wave-mixed single-use bag bioreactors during the last 5 years [1]. n nThe approach presented for seed-train cell expansion of High Five suspension cells is based on the Biostat CultiBag RM50 optical (Sartorius Stedim Biotech). It was used for the production of cells for long-term storage and for the expansion of cells for subsequent production experiments. For long-term storage the cells were frozen at high cell concentrations (20 - 40 x 106 cells x mL-1) in 60 mL Cryobags and stored in nitrogen at -196 °C in vapour phase. n nInitial experiments were aimed at the growth characterization of High Five suspension cells from a vial working cell bank (WCB). The High Five cells were grown in batch mode and in 250 mL single-use shake flasks (Corning and Sartorius Stedim Biotech) on a Certomat® CT Plus shaker (Sartorius Stedim Biotech) during six days (triplicates, 27 °C, 100 rpm, 25 mm shaking diameter). Afterwards a procedure was developed in which thawed cells from a Cryobag were directly transferred into and expanded in a Biostat CultiBag RM. Under optimal process conditions (500 mL starting volume, a starting cell density of 1 x 106 cells x mL-1, 27 °C, rocking angle of 6 °, 20 - 30 rpm, 0.2 vvm, DO set point 50%) growth rate (0.039 - 0.042 h-1), doubling time (18 - 20 h) and maximal cell density (7.8 - 8.9 x 106 cells x mL-1) showed good correlation with results arising from CultiBags which were inoculated with cells from shake flasks. This bag-based seed-train expansion allows time saving of about one week and reduces cross-contamination, both advantages being due to omitted intermediate cultivation steps in shake flasks.

Application of heat compensation calorimetry to an E. coli fed-batch process

The application of biocalorimetry to fermentation processes offers advantageous insights, while being less complex compared to other, sophisticated PAT solutions. Although the general concept is established, calorimetric methods vary in detail. In this work, a special approach, called heat compensation calorimetry, was applied to an E. coli fed-batch process. Much work has been done for batch processes, proving the validity and accuracy of this calorimetric mode. However, the adaption of this strategy to fed-batch processes has some implications. In the first section of this work, batch fermentations were performed, comparing heat capacity calorimetry to the compensation mode. Both processes showed very good agreement by means of growth behavior. The heat related differences, e.g. temperature profiles, were obvious. In addition, the impact of the chosen mode on the calculation of in-process heat transfer coefficients was shown. Finally, a fed-batch fermentation was performed. The compensation mode was kept sufficiently, up to the point where the metabolic heat production accelerated strongly. Controller tuning was a neuralgic point, which would have needed further optimization under these conditions. Nevertheless, in the present work it was possible to realize a working compensation process while demonstrating critical aspects that must be considered when establishing such approach.

Measurement of heat transfer coefficients in stirred single-use bioreactors by the decay of hydrogen peroxide

Single‐use bioreactors are barely described by means of their heat transfer characteristics, although some of their properties might affect this process. Steady‐state methods that use external heat sources enable precise investigations. One option, commonly present in stirred, stainless steel tanks, is to use adjustable electrical heaters. An alternative are exothermic chemical reactions that offer a higher flexibility and scalability. Here, the catalytic decay of hydrogen peroxide was considered a possible reaction, because of the high reaction enthalpy of –98.2 kJ/mole and its uncritical reaction products. To establish the reaction, a proper catalyst needed to be determined upfront. Three candidates were screened: catalase, iron(III)‐nitrate and manganese(IV)‐oxide. Whilst catalase showed strong inactivation kinetic and general instability and iron(III)‐nitrate solution has a pH of 2, it was decided to use manganese(IV)‐oxide for the bioreactor studies. First, a comparison between electrical and chemical power input in a benchtop glass bioreactor of 3.5 L showed good agreement. Afterwards the method was transferred to a 50 L stirred single‐use bioreactor. The deviation in the final results was acceptable. The heat transfer coefficient for the electrical method was 242 W/m2/K, while the value achieved with the chemical differed by less than 5%. Finally, experiments were carried out in a 200 L single‐use bioreactor proving the applicability of the chemical power input at technical relevant scales.