Wolf Klöckner

Advances in shaking technologies

Shaking bioreactors are the most frequently used reactor system for screening and process optimization on a small scale. Their success can be attributed to their simple and functional design, which make shaking systems suitable for a large number of cost-efficient parallel experiments. Recently reported findings for oxygen transfer, power input, out-of-phase operation, hydromechanical stress and mixing in shaken bioreactors are summarized in this article. Novel monitoring techniques for the control of culture conditions in shake flasks and microtiter plates are described. The methods for characterizing culture conditions and the novel online measurement techniques that are summarized in this article can be utilized to tap the full potential of shaking reactor systems.

Scaled-up manufacturing of recombinant antibodies produced by plant cells in a 200-L orbitally-shaken disposable bioreactor.

Tobacco BY‐2 cells have emerged as a promising platform for the manufacture of biopharmaceutical proteins, offering efficient protein secretion, favourable growth characteristics and cultivation in containment under a controlled environment. The cultivation of BY‐2 cells in disposable bioreactors is a useful alternative to conventional stainless steel stirred‐tank reactors, and orbitally‐shaken bioreactors could provide further advantages such as simple bag geometry, scalability and predictable process settings. We carried out a scale‐up study, using a 200‐L orbitally‐shaken bioreactor holding disposable bags, and BY‐2 cells producing the human monoclonal antibody M12. We found that cell growth and recombinant protein accumulation were comparable to standard shake flask cultivation, despite a 200‐fold difference in cultivation volume. Final cell fresh weights of 300–387 g/L and M12 yields of ∼20 mg/L were achieved with both cultivation methods. Furthermore, we established an efficient downstream process for the recovery of M12 from the culture broth. The viscous spent medium prevented clarification using filtration devices, but we used expanded bed adsorption (EBA) chromatography with SP Sepharose as an alternative for the efficient capture of the M12 antibody. EBA was introduced as an initial purification step prior to protein A affinity chromatography, resulting in an overall M12 recovery of 75–85% and a purity of >95%. Our results demonstrate the suitability of orbitally‐shaken bioreactors for the scaled‐up cultivation of plant cell suspension cultures and provide a strategy for the efficient purification of antibodies from the BY‐2 culture medium. Biotechnol. Bioeng. 2015;112: 308–321.

Orbitally Shaken Single-Use Bioreactors

: Orbitally shaken single-use reactors are promising reactors for upstream processing, because they fulfill three general requirements for single-use equipment. First, the design of the disposable parts is inherently simple and cost-efficient, because no complex built-in elements such as baffles or rotating stirrers are required. Second, the liquid distribution induced by orbital shaking is well-defined and accurately predictable. Third, the scale-up from small-scale systems, where shaken bioreactors are commonly applied, is simple and has been successfully proven up to the cubic meter scale. However, orbitally shaken single-use reactors are only suitable for certain applications such as cultivating animal or plant cells with low oxygen demand. Thus, detailed knowledge about the performance of such systems on different scales is essential to exploit their full potential. This article presents an overview about opportunities and limitations of shaken single-use reactors.

Correlation between mass transfer coefficient kLa and relevant operating parameters in cylindrical disposable shaken bioreactors on a bench-to-pilot scale

BackgroundAmong disposable bioreactor systems, cylindrical orbitally shaken bioreactors show important advantages. They provide a well-defined hydrodynamic flow combined with excellent mixing and oxygen transfer for mammalian and plant cell cultivations. Since there is no known universal correlation between the volumetric mass transfer coefficient for oxygen kLa and relevant operating parameters in such bioreactor systems, the aim of this current study is to experimentally determine a universal kLa correlation.ResultsA Respiration Activity Monitoring System (RAMOS) was used to measure kLa values in cylindrical disposable shaken bioreactors and Buckingham’s π-Theorem was applied to define a dimensionless equation for kLa. In this way, a scale- and volume-independent kLa correlation was developed and validated in bioreactors with volumes from 2 L to 200 L. The final correlation was used to calculate cultivation parameters at different scales to allow a sufficient oxygen supply of tobacco BY-2 cell suspension cultures.ConclusionThe resulting equation can be universally applied to calculate the mass transfer coefficient for any of seven relevant cultivation parameters such as the reactor diameter, the shaking frequency, the filling volume, the viscosity, the oxygen diffusion coefficient, the gravitational acceleration or the shaking diameter within an accuracy range of +/− 30%. To our knowledge, this is the first kLa correlation that has been defined and validated for the cited bioreactor system on a bench-to-pilot scale.

Time efficient way to calculate oxygen transfer areas and power input in cylindrical disposable shaken bioreactors

Disposable orbitally shaken bioreactors are a promising alternative to stirred or wave agitated systems for mammalian and plant cell cultivation, because they provide a homogeneous and well‐defined liquid distribution together with a simple and cost‐efficient design. Cultivation conditions in the surface‐aerated bioreactors are mainly affected by the size of the volumetric oxygen transfer area (a) and the volumetric power input (P∕VL) that both result from the liquid distribution during shaking. Since Computational Fluid Dynamics (CFD)—commonly applied to simulate the liquid distribution in such bioreactors—needs high computing power, this technique is poorly suited to investigate the influence of many different operating conditions in various scales. Thus, the aim of this paper is to introduce a new mathematical model for calculating the values of a and P∕VL for liquids with water‐like viscosities. The model equations were derived from the balance of centrifugal and gravitational forces exerted during shaking. A good agreement was found among calculated values for a and P∕VL, CFD simulation values and empirical results. The newly proposed model enables a time efficient way to calculate the oxygen transfer areas and power input for various shaking frequencies, filling volumes and shaking and reactor diameters. All these parameters can be calculated fast and with little computing power.

Shake-Flask Bioreactors

Shake flasks are the most popular reaction vessels for screening and process development in bioprocess engineering. The simplicity of the system allows its application for a considerable variety of different tasks. In spite of its large practical relevance, the importance of engineering aspects for the cultivation in shake flasks has been underestimated for a long time. In this article, the important physical characteristics for an optimized use of shake flasks as cultivation vessels are described. Mathematical correlations based on experimental findings for important scale-up parameters as, for example, the power input and the gas/liquid mass transfer are discussed in detail. It is described how the flow conditions in shake flasks can be defined as ‘in-phase’ and ‘out-of-phase’ operation conditions and how these conditions affect the cultivation of microorganisms. Important process variables such as the mixing time, the hydromechanical stress, and the maximum energy dissipation rate are discussed for the optimization of culture conditions. Finally, important future trends as online measuring techniques, for example, oxygen and carbon dioxide transfer rate, optical density, pH, and dissolved oxygen tension as well as systems for fed-batch and continuous operation of shaken bioreactors are described.

Oxygen supply controls the onset of pristinamycins production by Streptomyces pristinaespiralis in shaking flasks

Antibiotics are secondary metabolites, generally produced during stationary phase of growth under different nutritional and hydrodynamic stresses. However, the exact mechanisms of the induction of antibiotics production are still not clearly established. In a previous study, the induction of pristinamycins production by Streptomyces pristinaespiralis as well as product concentrations were correlated with power dissipation per unit of volume (P/V) in shaking flasks. In this study, detailed kinetics of growth, substrate consumption, oxygen transfer rate and pristinamycins production under varying P/V conditions have been obtained and analyzed. Our results showed that higher P/V resulted in a higher concentration of biomass and promoted an earlier nutrient limitation and ultimately an earlier induction of pristinamycins production. The maximal specific growth rate, specific oxygen consumption rate and specific consumption rate of glutamate increased with P/V while influence was less marked with specific consumption rate of glucose, arginine, ammonium ions and phosphate. When oxygen uptake rate (OUR) was limited by free‐surface oxygen transfer, pristinamycins production was not detected despite the occurrence of nitrogen and/or phosphate sources limitation. The threshold value for OUR observed was around 25 mmol L−1 h−1. This suggested that a limitation in nitrogen and/or phosphate alone was not sufficient to induce pristinamycins production by S. pristinaespiralis pr11. To induce this production, the oxygen transfer had to be non‐limiting. Biotechnol. Bioeng. 2011;108:2151–2161.