Gas supply apparatus using rotational motion of shaking incubator for flask culture of aerobic microorganisms.

Shake flask cultivation, a cornerstone in bioprocess research encounters limitations in supplying sufficient oxygen and exchanging gases, restricting its accuracy in assessing microbial growth and metabolic activity. In this communication, we introduce an innovative gas supply apparatus that harnesses the rotational motion of a shaking incubator to facilitate continuous air delivery, effectively overcoming these limitations. We measured the mass transfer coefficient (kLa) and conducted batch cultures of Corynebacterium glutamicum H36LsGAD using various working volumes to assess its performance. Results demonstrated that the gas supply apparatus significantly outperforms conventional silicone stoppers regarding oxygen delivery, with kLa values of 2531.7 h-1 compared to 20.25 h-1 at 230 rpm. Moreover, in batch cultures, the gas supply apparatus enabled substantial improvements in microbial growth, maintaining exponential growth even at larger working volumes. Compared to the existing system, an increase in final cell mass by a factor of 3.4-fold was observed when utilizing 20% of the flask's volume, and a remarkable 9-fold increase was achieved when using 60%. Furthermore, the gas supply apparatus ensured consistent oxygen supply and efficient gas exchange within the flask, overcoming challenges associated with low working volumes. This approach offers a simple yet effective solution to enhance gas transfer in shake flask cultivation, bridging the gap between laboratory-scale experiments and industrial fermenters. Its broad applicability holds promise for advancing research in bioprocess optimization and scale-up endeavors.

Reinventing shake flask fermentation: The breathable flask.

Cultivating cells in shake flasks is a routine operation that is largely unchanged since its inception. A glass or plastic Erlenmeyer vessel with the primary gas exchange taking place across various porous plugs is used with media volumes typically ranging from 100 mL to 2 L. Oxygen limitation and carbon dioxide accumulation in the vessel is a major concern for studies involving shake flask cultures. In this study, we enhance mass transfer in a conventional shake flask by replacing the body wall with a permeable membrane. Naturally occurring concentration gradient across the permeable membrane walls facilitates the movement of oxygen and carbon dioxide between the flask and the external environment. The modified flask called the breathable flask, has shown a 40% improvement in mass transfer coefficient (kLa) determined using the static diffusion method. The prokaryotic cell culture studies performed with Escherichia coli showed an improvement of 28%-66% in biomass and 41%-56% in recombinant product yield. The eukaryotic cell culture study performed with Pichia pastoris expressing proinsulin exhibited a 40% improvement in biomass and 115% improvement in protein yield. The study demonstrates a novel approach to addressing the mass transfer limitations in conventional shake flask cultures. The proposed flask amplifies its value by providing a membrane-diffusion-based sensing platform for the integration of low-cost, noninvasive sensing capabilities for real-time monitoring of critical cell culture parameters like dissolved oxygen and dissolved carbon dioxide.

Pilot-Scale Fermentation of Pseudoalteromonas sp. Strain FDHY-MZ2: An Effective Strategy for Increasing Algicidal Activity.

The role of microorganisms in effectively terminating harmful algal blooms (HABs) is crucial for maintaining environmental stability. Recent studies have placed increased emphasis on bio-agents capable of inhibiting HABs. The bacterium Pseudoalteromonas sp. strain FDHY-MZ2 has exhibited impressive algicidal abilities against Karenia mikimotoi, a notorious global HAB-forming species. To augment this capability, cultures were progressively scaled from shake flask conditions to small-scale (5 L) and pilot-scale (50 L) fermentation. By employing a specifically tailored culture medium (2216E basal medium with 1.5% soluble starch and 0.5% peptone), under precise conditions (66 h, 20 °C, 450 rpm, 30 L/min ventilation, 3% seeding, and constant starch flow), a notable increase in algicidal bacterial biomass was observed; the bacterial dosage required to entirely wipe out K. mikimotoi within a day decreased from 1% to 0.025%. Compared to an unoptimized shake flask group, the optimized fermentation culture caused significant reductions in algal chlorophyll and protein levels (21.85% and 78.3%, respectively). Co-culturing induced increases in algal malondialdehyde and H2O2 by 5.98 and 5.38 times, respectively, leading to further disruption of algal photosynthesis. This study underscores the unexplored potential of systematically utilized microbial agents in mitigating HABs, providing a pathway for their wider application.

Comparative Drug Solubility Studies Using Shake-Flask Versus a Laser-Based Robotic Method.

Drug solubility is of central importance to the pharmaceutical sciences, but reported values often show discrepancies. Various factors have been discussed in the literature to account for such differences, but the influence of manual testing in comparison to a robotic system has not been studied adequately before. In this study, four expert researchers were asked to measure the solubility of four drugs with various solubility behaviors (i.e., paracetamol, mesalazine, lamotrigine, and ketoconazole) in the same laboratory with the same instruments, method, and material sources and repeated their measurements after a time interval. In addition, the same solubility data were determined using an automated laser-based setup. The results suggest that manual testing leads to a handling influence on measured solubility values, and the results were discussed in more detail as compared to the automated laser-based system. Within the framework of unavoidable uncertainties of solubility testing, it is a possibility to combine minimal experimental testing that is preferably automated with mathematical modeling. That is a practical suggestion to support future pharmaceutical development in a more efficient way.

Quantification and modeling of macroparticle-induced mechanical stress for varying shake flask cultivation conditions.

In biotechnological processes, filamentous microorganisms are known for their broad product spectrum and complex cellular morphology. Product formation and cellular morphology are often closely linked, requiring a well-defined level of mechanical stress to achieve high product concentrations. Macroparticles were added to shake flask cultures of the filamentous actinomycete Lentzea aerocolonigenes to find these optimal cultivation conditions. However, there is currently no model concept for the dependence of the strength and frequency of the bead-induced stress on the process parameters. Therefore, shake flask simulations were performed for combinations of bead size, bead concentration, bead density and shaking frequency. Contact analysis showed that the highest shear stresses were caused by bead-bottom contacts. Based on this, a newly generated characteristic parameter, the stress area ratio (SAR), was defined, which relates the bead wall shear and normal stresses to the total shear area. Comparison of the SAR with previous cultivation results revealed an optimum pattern for product concentration and mean product-to-biomass related yield coefficient. Thus, this model is a suitable tool for future optimization, comparison and scaling up of shear-sensitive microorganism cultivation. Finally, the simulation results were validated using high-speed recordings of the bead motion on the bottom of the shake flask.

Workflow for shake flask and plate cultivations with fats for polyhydroxyalkanoate bioproduction.

Since natural resources for the bioproduction of commodity chemicals are scarce, waste animal fats (WAF) are an interesting alternative biogenic residual feedstock. They appear as by-product from meat production, but several challenges are related to their application: first, the high melting points (up to 60 °C); and second, the insolubility in the polar water phase of cultivations. This leads to film and clump formation in shake flasks and microwell plates, which inhibits microbial consumption. In this study, different flask and well designs were investigated to identify the most suitable experimental set-up and further to create an appropriate workflow to achieve the required reproducibility of growth and product synthesis. The dissolved oxygen concentration was measured in-line throughout experiments. It became obvious that the gas mass transfer differed strongly among the shake flask design variants in cultivations with the polyhydroxyalkanoate (PHA) accumulating organism Ralstonia eutropha. A high reproducibility was achieved for certain flask or well plate design variants together with tailored cultivation conditions. Best results were achieved with bottom baffled glass and bottom baffled single-use shake flasks with flat membranes, namely, >6 g L-1 of cell dry weight (CDW) with >80 wt% polyhydroxybutyrate (PHB) from 1 wt% WAF. Improved pre-emulsification conditions for round microwell plates resulted in a production of 14 g L-1 CDW with a PHA content of 70 wt% PHB from 3 wt% WAF. The proposed workflow allows the rapid examination of fat material as feedstock, in the microwell plate and shake flask scale, also beyond PHA production. KEY POINTS: • Evaluation of shake flask designs for cultivating with hydrophobic raw materials • Development of a workflow for microwell plate cultivations with hydrophobic raw materials • Production of polyhydroxyalkanoate in small scale experiments from waste animal fat.

Cascading Beta-oxidation Intermediates for the Polyhydroxyalkanoate Copolymer Biosynthesis by Metabolic Flux using Co-substrates and Inhibitors.

Polyhydroxyalkanoates (PHAs) are biopolymers that are produced within the microbial cells in the presence of excess carbon and nutrient limitation. Different strategies have been studied to increase the quality and quantity of this biopolymer which in turn can be utilized as biodegradable polymers replacing conventional petrochemical plastics. In the present study, Bacillus endophyticus, a gram-positive PHA-producing bacterium, was cultivated in the presence of fatty acids along with beta-oxidation inhibitor acrylic acid. A novel approach for incorporating different hydroxyacyl groups provided using fatty acids as co-substrate and beta-oxidation inhibitors to direct the intermediates to co-polymer synthesis was experimented. It was observed that higher fatty acids and inhibitors had a greater influence on PHA production. The addition of acrylic acid along with propionic acid had a positive impact, giving 56.49% of PHA along with sucrose which was 1.2-fold more than the control devoid of fatty acids and inhibitors. Along with the copolymer production, the possible PHA pathway functional leading to the copolymer biosynthesis was hypothetically interpreted in this study. The obtained PHA was analyzed by FTIR and 1H NMR to confirm the copolymer production, which indicated the presence of poly3hydroxybutyrate-co-hydroxyvalerate (PHB-co-PHV), poly3hydroxybutyrate-co-hydroxyhexanoate (PHB-co-PHx).

Modeling the effect of oxidative stress on Bordetella pertussis fermentations.

A mathematical model is proposed for Bordetella pertussis with the main goal to better understand and describe the relation between cell growth, oxidative stress and NADPH levels under different oxidative conditions. The model is validated with flask experiments conducted under different conditions of oxidative stress induced by high initial glutamate concentrations, low initial inoculum and secondary culturing following exposure to starvation. The model exhibited good accuracy when calibrated and validated for the different experimental conditions. From comparisons of model predictions to data with different model mechanisms, it was concluded that intracellular reactive oxidative species only have an indirect effect on growth rate by reacting with NADPH and thereby reducing the amount of NADPH that is available for growth.

Effect of temperature and propylene glycol as a cosolvent on dissolution of clotrimazole.

Herein, the solubility study of clotrimazole was performed in a propylene glycol+water system. The solubility values were fitted to various cosolvency equations. The model accuracies were studied with the computation of the mean relative deviations. The thermodynamic behavior was investigated according to the van't Hoff and Gibbs equations for clotrimazole in the propylene glycol+water system. Furthermore, the density data for clotrimazole were determined in mixtures of propylene glycol+water and fitted to the Jouyban-Acree equation.

Optimization of Escherichia coli shake flask culture medium by artificial neural network / 中国生物制品学杂志

@#Objective To optimize a shake flask culture medium for Escherichia coli(E.coli)with high biomass and viability using artificial neural networks(ANN). Methods Using the proportion of glucose(Glu),yeast extract(YE),yeast peptone(YP),soy peptone(SP)and yeast nitrogen base(YNB)as the mixture component,and the A_(600)(A1)value of cell suspension,wet bacterial weight(G,g/L)of culture and cell viability(A2,A_(460))as the response values,the mixture design was used to screen the mixture components that had a significant effect on the response value. The ANN model was constructed with the test results of mixture design as training and verification data samples. The input variables were mixture components and restricted the upper and lower limits of the mixture components,and the output variables were mixture design response values. The optimized medium formula and reference values were obtained by the constructed ANN. The medium formula was further adjusted by Monte Carlo simulation to obtain the shake flask medium formula of E.coli,which was then verified for 10 times. Results The shake flask culture medium of E.coli was composed of Glu 26 g/L,SP 26 g/L,YNB13 g/L with the total concentration of 65 g/L. The verification results showed that the probability of A1 ≥ 14 was 60%,the probability of G ≥ 77 g/L was 50% and the probability of A2 ≥ 11 was 40%. The mean values of the incubation result data were equivalent to the reference values. Conclusion The shake flask culture medium of E.coli optimized in this study can obtain E.coli with high biomass and bacterial activity.

Accessing Lipophilicity and Biomimetic Chromatography Profile of Biologically Active Ingredients of Botanicals Used in the Treatment of Inflammatory Bowel Disease.

In the present study, various procedures have been compared for the determination of lipophilicity, hydrophobicity, and plasma protein binding of curcuminoids, boswellic acids, andrographolides, and piperine as biologically active ingredients of botanicals used in IBD treatment. Our results have shown that IAM-HPLC assay is the most suitable one for lipophilicity determination of all analytes regardless of their class and botanical source. HSA-HPAC and AGP-HPAC assays revealed that all investigated compounds have a higher affinity for HSA which is the most abundant protein in human plasma. The high affinity of biologically active compounds to all biological structures (phospholipids and proteins) admonishes that their small portion is available for therapeutic effects in IBD patients. Our experimental research is complemented by various theoretical approaches based on different algorithms for pharmacokinetic properties prediction. The similarities between experimental and calculated values were evaluated using PCA and CA as a statistical tool. The statistical analysis implies that plasma protein binding is a complex process, and theoretical approaches still cannot fully replace experimental ones.

Synergistic effects on itaconic acid production in engineered Aspergillus niger expressing the two distinct biosynthesis clusters from Aspergillus terreus and Ustilago maydis.

BACKGROUND: Itaconic acid (IA) is a versatile platform chemical widely used for the synthesis of various polymers and current methods for IA production based on Aspergillus terreus fermentation are limited in terms of process efficiency and productivity. To construct more efficient IA production strains, A. niger was used as a chassis for engineering IA production by assembling the key components of IA biosynthesis pathways from both A. terreus and Ustilago maydis. RESULTS: Recombinant A. niger S1596 overexpressing the A. terreus IA biosynthesis genes cadA, mttA, mfsA produced IA of 4.32 g/L, while A. niger S2120 overexpressing the U. maydis IA gene cluster adi1, tad1, mtt1, itp1 achieved IA of 3.02 g/L. Integration of the two IA production pathways led to the construction of A. niger S2083 with IA titers of 5.58 g/L. Increasing cadA copy number in strain S2083 created strain S2209 with titers of 7.99 g/L and deleting ictA to block IA degradation in S2209 created strain S2288 with IA titers of 8.70 g/L. Overexpressing acoA to enhance the supply of IA precursor in strain S2288 generated strain S2444 with IA titers of 9.08 g/L in shake flask. CONCLUSION: Recombinant A. niger overexpressing the U. maydis IA biosynthesis pathway was capable of IA accumulation. Combined expression of the two IA biosynthesis pathways from A. terreus and U. maydis in A. niger resulted in much higher IA titers. Furthermore, increasing cadA copy number, deleting ictA to block IA degradation and overexpressing acoA to enhance IA precursor supply all showed beneficial effects on IA accumulation.

Improved Time Resolved KPI and Strain Characterization of Multiple Hosts in Shake Flasks Using Advanced Online Analytics and Data Science.

Shake flasks remain one of the most widely used cultivation systems in biotechnology, especially for process development (cell line and parameter screening). This can be justified by their ease of use as well as their low investment and running costs. A disadvantage, however, is that cultivations in shake flasks are black box processes with reduced possibilities for recording online data, resulting in a lack of control and time-consuming, manual data analysis. Although different measurement methods have been developed for shake flasks, they lack comparability, especially when changing production organisms. In this study, the use of online backscattered light, dissolved oxygen, and pH data for characterization of animal, plant, and microbial cell culture processes in shake flasks are evaluated and compared. The application of these different online measurement techniques allows key performance indicators (KPIs) to be determined based on online data. This paper evaluates a novel data science workflow to automatically determine KPIs using online data from early development stages without human bias. This enables standardized and cost-effective process-oriented cell line characterization of shake flask cultivations to be performed in accordance with the process analytical technology (PAT) initiative. The comparison showed very good agreement between KPIs determined using offline data, manual techniques, and automatic calculations based on multiple signals of varying strengths with respect to the selected measurement signal.

Control of carbon dioxide concentration in headspace of multiple flasks using both non-electric bellows pump and shaking incubator.

Current methods of controlling gas in the headspace involve constant speed aeration and proportional-integral-differential (PID) controlled aeration using improved monitoring devices or gas cylinders. However, these approaches are restricted and inconvenient to use. In this study, we propose a method to control the CO2 concentration in the headspace while maintaining the convenience of shake-flask culture. A combination of a non-electric bellows pump for shake-flask (NeBP-sf) and a CO2 incubator was used to control the flask gas phase by shaking without additional external power. The CO2 half-life, as an indicator of the ventilation ability of the system, was measured using a circulation direct monitoring and sampling system, and the NeBP-sf was optimised. The ventilation capacity varied depending on the shaking speed, and under optimal conditions, was 10 min compared with 45 min when only a breathable culture plug was used. In conventional microbial shaking culture, the CO2 concentration in the flask gas phase remained higher than the 5% set-value with a maximum of 9%, resulting in a large concentration difference with the set point. Therefore, the ventilation capacity of the conventional shake-flask culture was insufficient for aerobic culture. Cultivation of Escherichia coli and Lactiplantibacillus plantarum using the system showed no significant difference between the set point and real point values. Thus, the system combined an NeBP-sf and a gas incubator built-in shaking table to achieve the reproducibility of gas control while maintaining a high level of convenience.

Implementation of Perforated Concentric Ring Walls Considerably Improves Gas-Liquid Mass Transfer of Shaken Bioreactors.

Since their first use in the 1930s, shake flasks have been a widely used bioreactor type for screening and process development due to a number of advantages. However, the limited gas-liquid mass transfer capacities-resulting from practical operation limits regarding shaking frequency and filling volumes-are a major drawback. The common way to increase the gas-liquid mass transfer in shake flasks with the implementation of baffles is generally not recommended as it comes along with several severe disadvantages. Thus, a new design principle for shaken bioreactors that aims for improving the gas-liquid mass transfer without losing the positive characteristics of unbaffled shake flasks is introduced. The flasks consist of cylindrical glass vessels with implemented perforated concentric ring walls. The ring walls improve the gas-liquid mass transfer via the formation of additional liquid films on both of its sides, whereas the perforations allow for mixing between the compartments. Sulfite oxidation experiments revealed over 200% higher maximum oxygen transfer capacities (OTRmax) compared to conventional shake flasks. In batch cultivations of Escherichia coli BL21 in mineral media, unlimited growth until glucose depletion and oxygen transfer rates (OTR) of up to 138 mmol/L/h instead of an oxygen limitation at 57 mmol/L/h as in normal shake flasks under comparable conditions could be achieved. Even overflow metabolism could be prevented due to sufficient oxygen supply without the use of unconventional shaking conditions or oxygen enrichment. Therefore, we believe that the new perforated ring flask principle has a high potential to considerably improve biotechnological screening and process development steps.

Optimization of the production process for the anticancer lead compound illudin M: improving titers in shake-flasks.

BACKGROUND: The fungal sesquiterpenes Illudin M and S are important base molecules for the development of new anticancer agents due to their strong activity against some resistant tumor cell lines. Due to nonspecific toxicity of the natural compounds, improvement of the pharmacophore is required. A semisynthetic derivative of illudin S (Irofulven) entered phase II clinical trials for the treatment of castration-resistant metastatic prostate cancer. Several semisynthetic illudin M derivatives showed increased in vitro selectivity and improved therapeutic index against certain tumor cell lines, encouraging further investigation. This requires a sustainable supply of the natural compound, which is produced by Basidiomycota of the genus Omphalotus. We aimed to develop a robust biotechnological process to deliver illudin M in quantities sufficient to support medicinal chemistry studies and future preclinical and clinical development. In this study, we report the initial steps towards this goal. RESULTS: After establishing analytical workflows, different culture media and commercially available Omphalotus strains were screened for the production of illudin M.Omphalotus nidiformis cultivated in a medium containing corn steep solids reached ~ 38 mg L-1 setting the starting point for optimization. Improved seed preparation in combination with a simplified medium (glucose 13.5 g L-1; corn steep solids 7.0 g L- 1; Dox broth modified 35 mL), reduced cultivation time and enhanced titers significantly (~ 400 mg L-1). Based on a reproducible cultivation method, a feeding strategy was developed considering potential biosynthetic bottlenecks. Acetate and glucose were fed at 96 h (8.0 g L-1) and 120 h (6.0 g L-1) respectively, which resulted in final illudin M titer of ~ 940 mg L-1 after eight days. This is a 25 fold increase compared to the initial titer. CONCLUSION: After strict standardization of seed-preparation and cultivation parameters, a combination of experimental design, empirical trials and additional supply of limiting biosynthetic precursors, led to a highly reproducible process in shake flasks with high titers of illudin M. These findings are the base for further work towards a scalable biotechnological process for a stable illudin M supply.

AlphaLogD determination: An optimized Reversed-Phase Liquid Chromatography method to measure lipophilicity on neutral and basic small and Beyond-Rule-of-Five compounds.

Lipophilicity can be measured with different methods, such as Shake-Flask or liquid chromatography. HPLC presents the advantage of overcoming solubility issues and therefore extending the range of lipophilicity to high values. A specific HPLC method, called ELogD, had been developed 20 years ago on a C16-amide stationary phase, enhancing hydrophobic and hydrogen bond interactions to mimic octanol-water partition. The emergence of novel stationary phases and the need for a less complex mobile phase have led to the development of a new HPLC assay called alphaLogD, applicable to neutral and basic compounds at pH 7.4, that combines superficially porous particles with a high number of equilibriums between solutes and stationary phase, leading to a lower number of isocratic methods to determine the logk'w at a higher throughput. Statistical studies have been run to successfully evaluate the alphaLogD method compared to the Shake-Flask method and to allow this lipophilicity measurement into the so-called Beyond-Rule-of-5-molecules space.

Exopolysaccharides production from marine Bacillus strains and their antioxidant and bio-flocculant capacities.

Microbial exopolysaccharides (EPS) have gained high scientific concern due to their exceptional physicochemical features and high industrial applicability. Owing to their biotechnological importance, the present study was designed to screen and isolate the EPS-producing Bacillus strains based on their growth potential on specific media and colony morphologies. The bacterial isolates Bacillus subtilis Bs1-01, Bacillus licheniformis Bl1-02, and Bacillus brevis Bb1-04 showed excellent EPS production due to their shortened lag phase and abundant biomass production. Shake-flask fermentation valued the maximum production yield of 50.19 ± 1.14 g/L by Bl1-02 after 72 h incubation (about 3.40 times higher than that of Bacillus thuringiensis Bt1-05). The basic component analysis revealed the improved amount of total carbohydrate, reducing sugar ends, and protein contents by Bl1-02 strain. Structural characteristics and functional groups of the EPS characterized by Fourier transform infrared spectroscopy demonstrated that all EPS were in close agreement to each other due to the presence of similar chemical bonds and functional groups. EPS from Bl1-02 strain showed stronger and more stable bio-emulsifying and hygroscopicity activities (12.23%). The crude EPS exhibited potent antioxidant properties which were examined against reducing potential (H2O2 scavenging) and total antioxidant tests. Among bio-flocculation activities of EPS at different concentrations, Bs1-01 strain produced EPS at a concentration of 60 mg/mL was observed to show the maximum value of 79.20%. In conclusion, the EPS from marine Bacillus strains showed excellent functional properties suggesting potential industrial applications that demand separate investigations.

Small-Scale Secretory VHH Expression in Saccharomyces cerevisiae.

After isolation of a single-domain antibody (VHH) binding to an antigen of interest, the soluble VHH is often produced in Escherichia coli. However, targeting VHH expression to the secretory pathway of Saccharomyces cerevisiae (baker's yeast) enables the secretion of correctly folded, soluble, disulfide-bonded, and N-glycosylated VHHs into the culture medium. Here, we describe the small-scale production of VHHs in baker's yeast in shaker flasks using both an episomal vector and a vector requiring genomic integration for higher VHH expression levels. This expression system results in the production of VHHs linked to the natural llama long hinge region including a single cysteine residue for partial dimerization. This format is especially suitable for the development of double antibody sandwich ELISAs by passive adsorption of unlabeled VHHs to polystyrene ELISA plates, antigen capture, and detection of the antigen of interest using a second biotinylated VHH. The procedures described here for detection of foot-and-mouth disease virus can also be applied to other antigens for which suitable VHHs are available.

Method to transfer Chinese hamster ovary (CHO) batch shake flask experiments to large-scale, computer-controlled fed-batch bioreactors.

Chinese hamster ovary (CHO) cell cultures in industry are most commonly conducted as fed-batch cultures in computer-controlled bioreactors, though most preliminary studies are conducted in fed-batch shake flasks. To improve comparability between bioreactor studies and shake flask studies, shake flask studies should be conducted as fed-batch. However, the smaller volumes and reduced control in shake flasks can impact pH and aeration, which leads to performance differences. Planning and awareness of these vessel and control differences can assist with experimental design as well as troubleshooting. This method will highlight several of the configuration and control issues that should be considered during the transitions from batch to fed-batch and shake flasks to bioreactors, as well as approaches to mitigate the differences. Furthermore, if significant differences occur between bioreactor and shake flask studies, approaches will be presented to isolate the main contributors for these differences.