Device for respiration activity measurement enables the determination of oxygen transfer rates of microbial cultures in shaken 96‐deepwell microtiter plates
Abstract:Mini-bioreactors with integrated online monitoring capabilities are well established in the early stages of process development. Mini-bioreactors fulfil the demand for highthroughput-applications and a simultaneous reduction of material costs and total experimental time. One of the most essential online monitored parameters is the oxygen transfer rate (OTR). OTR-monitoring allows fast characterization of bioprocesses and process transfer to larger scales. Currently, OTR-monitoring on a smallscale is limited to… Show more
“… Figure 1 Cultivation of P. pastoris Mut S reference strain in two types of microtiter plates with simultaneous online monitoring of the oxygen transfer rate (OTR). Cultivation was carried out ( a ) in a 48-round well microtiter plate (MTP), 0.8 mL filling volume per well, operated at 1000 rpm and 3 mm shaking diameter in a µRAMOS/BioLector 32 combination device and ( b ) in a 96-square well MTP, 0.6 mL filling volume per well, operated at 350 rpm and 50 mm shaking diameter in a µTOM device 34 . The OTR at both MTP scales is depicted without methanol addition (black line), and with 2% (v/v) of methanol added to the medium at the beginning (t = 0 h) of the cultivation to obtain auto-induction conditions (dark blue line).…”
Section: Resultsmentioning
confidence: 99%
“… Figure 2 Cultivation of P. pastoris Mut S reference strain at different glycerol and methanol concentrations in microtiter plates with simultaneous online monitoring of the oxygen transfer rate (OTR). Cultivation was carried out in a 96-square well microtiter plate, 0.6 mL filling volume per well, 350 rpm and 50 mm shaking diameter, in a µTOM device 34 . The OTR is depicted for cultivations with 5, 10 and 15 g/L glycerol, using auto-induction conditions, with ( a ) 1.5% v/v methanol and ( b ) 2% v/v methanol added at the beginning of the cultivation.…”
Section: Resultsmentioning
confidence: 99%
“…For cultivations at 96-well MTP scale, a micro‐scale Transfer rate Online Measurement device (µTOM) was used 34 . Cultivations were conducted in 96-deepwell MTP (riplate SW, 2.5 ml square deepwell plate; HJ‐Bioanalytik GmbH, Erkelenz, Germany), sealed with a gas‐permeable sealing film (AeraSeal™-film, Sigma Aldrich, MA, USA).…”
Section: Methodsmentioning
confidence: 99%
“…Cultivations were conducted in an in-house built combined µRAMOS/BioLector system that allows online-monitoring of the OTR, scattered light, and GFP fluorescence at 48-well plate microtiter plate (MTP) scale 32 , 33 . The RAMOS technology was also extended and applied to 96-deepwell microtiter plates 34 . 96-well MTP are the standard format for screening campaigns, because they allow the parallelization of cultivations at reasonable number and costs 35 , 36 .…”
The methylotrophic yeast Pichia pastoris (Komagataella phaffii) is a widely used host for recombinant protein production. In this study, a clonal library of P. pastoris MutS strains (S indicates slow methanol utilization) was screened for high green fluorescent protein (GFP) production. The expression cassette was under the control of the methanol inducible AOX promoter. The growth behavior was online-monitored in 48-well and 96-well microtiter plates by measuring the oxygen transfer rate (OTR). By comparing the different GFP producing strains, a correlation was established between the slope of the cumulative oxygen transfer during the methanol metabolization phase and the strain’s production performance. The correlation corresponds to metabolic burden during methanol induction. The findings were validated using a pre-selected strain library (7 strains) of high, medium, and low GFP producers. For those strains, the gene copy number was determined via Whole Genome Sequencing. The results were consistent with the described OTR correlation. Additionally, a larger clone library (45 strains) was tested to validate the applicability of the proposed method. The results from this study suggest that the cumulative oxygen transfer can be used as a screening criterion for protein production performance that allows for a simple primary screening process, facilitating the pre-selection of high producing strains.
“… Figure 1 Cultivation of P. pastoris Mut S reference strain in two types of microtiter plates with simultaneous online monitoring of the oxygen transfer rate (OTR). Cultivation was carried out ( a ) in a 48-round well microtiter plate (MTP), 0.8 mL filling volume per well, operated at 1000 rpm and 3 mm shaking diameter in a µRAMOS/BioLector 32 combination device and ( b ) in a 96-square well MTP, 0.6 mL filling volume per well, operated at 350 rpm and 50 mm shaking diameter in a µTOM device 34 . The OTR at both MTP scales is depicted without methanol addition (black line), and with 2% (v/v) of methanol added to the medium at the beginning (t = 0 h) of the cultivation to obtain auto-induction conditions (dark blue line).…”
Section: Resultsmentioning
confidence: 99%
“… Figure 2 Cultivation of P. pastoris Mut S reference strain at different glycerol and methanol concentrations in microtiter plates with simultaneous online monitoring of the oxygen transfer rate (OTR). Cultivation was carried out in a 96-square well microtiter plate, 0.6 mL filling volume per well, 350 rpm and 50 mm shaking diameter, in a µTOM device 34 . The OTR is depicted for cultivations with 5, 10 and 15 g/L glycerol, using auto-induction conditions, with ( a ) 1.5% v/v methanol and ( b ) 2% v/v methanol added at the beginning of the cultivation.…”
Section: Resultsmentioning
confidence: 99%
“…For cultivations at 96-well MTP scale, a micro‐scale Transfer rate Online Measurement device (µTOM) was used 34 . Cultivations were conducted in 96-deepwell MTP (riplate SW, 2.5 ml square deepwell plate; HJ‐Bioanalytik GmbH, Erkelenz, Germany), sealed with a gas‐permeable sealing film (AeraSeal™-film, Sigma Aldrich, MA, USA).…”
Section: Methodsmentioning
confidence: 99%
“…Cultivations were conducted in an in-house built combined µRAMOS/BioLector system that allows online-monitoring of the OTR, scattered light, and GFP fluorescence at 48-well plate microtiter plate (MTP) scale 32 , 33 . The RAMOS technology was also extended and applied to 96-deepwell microtiter plates 34 . 96-well MTP are the standard format for screening campaigns, because they allow the parallelization of cultivations at reasonable number and costs 35 , 36 .…”
The methylotrophic yeast Pichia pastoris (Komagataella phaffii) is a widely used host for recombinant protein production. In this study, a clonal library of P. pastoris MutS strains (S indicates slow methanol utilization) was screened for high green fluorescent protein (GFP) production. The expression cassette was under the control of the methanol inducible AOX promoter. The growth behavior was online-monitored in 48-well and 96-well microtiter plates by measuring the oxygen transfer rate (OTR). By comparing the different GFP producing strains, a correlation was established between the slope of the cumulative oxygen transfer during the methanol metabolization phase and the strain’s production performance. The correlation corresponds to metabolic burden during methanol induction. The findings were validated using a pre-selected strain library (7 strains) of high, medium, and low GFP producers. For those strains, the gene copy number was determined via Whole Genome Sequencing. The results were consistent with the described OTR correlation. Additionally, a larger clone library (45 strains) was tested to validate the applicability of the proposed method. The results from this study suggest that the cumulative oxygen transfer can be used as a screening criterion for protein production performance that allows for a simple primary screening process, facilitating the pre-selection of high producing strains.
“…Thus, it is well acknowledged, that a transfer to microtiter plate scale is very desirable to increase experimental throughput and to reduce the overall costs of the experiments. For measurement in MTPs, the µRAMOS, which enables monitoring of the OTR in 48-or 96-well microtiter plates [14,19], may be used. However, it has to the authors knowledge neither been applied for monitoring of mammalian cells in general nor CHO cells specifically, yet.…”
Section: Comparison Of Cytotoxicity Assessment By Otr Measurement Wit...mentioning
Background
Determining the cytotoxicity of test substances is essential for the safety assessment of chemicals. To quantify the cytotoxicity, dose–response curves are determined and the half-maximum inhibitory concentration (IC50) is subsequently calculated. Results often rely on a single endpoint evaluation which typically requires manual sampling and subsequent sample analysis to determine the IC50. Hence, no information on culture behavior are available during treatment. Here, measurement of the oxygen transfer rate (OTR) was applied as a method for time-resolved assessment of the cytotoxicity to (a) provide information on culture behavior during treatment and (b) quantitatively assess the cytotoxic effect of a test substance.
Results
To investigate the applicability of the method, different types and concentrations of test substances were added and the OTR was measured for two different CHO suspension cell lines in shake flasks: (a) For CHO cell line one, culture behavior was not affected when 0.1 mM ZnCl2 was added. However, for both cell lines, adding up to 0.2 mM CoCl2 led to a decrease in the OTR that was concentration dependent. For CHO cell line two, adding up to 5% DMSO led to a concentration-dependent decrease in the OTR and in the slope of the OTR; (b) From the measured OTR, dose–response curves were established and used to calculate the IC50. For cell line one, the IC50 for CoCl2 was 0.03 mM while the IC50 from a comparative measurement based on trypan blue exclusion was 0.06 mM. Cell line two was less susceptible to CoCl2 as the IC50 was not reached at the concentrations tested. For DMSO, single OTR values and determined slopes were used for evaluation. In both cases, the IC50 was calculated to 2.3%.
Conclusions
Our approach provides a method for analyzing the cytotoxicity of a test substance based on OTR measurements. This method provides (i) insights on culture behavior, (ii) information on the progression of cytotoxicity, (iii) dose–response curves, and (iv) a first indication of IC50 values. Quantitative assessment of cytotoxicity is possible non-invasively and in real-time during treatment. Compared to traditional endpoint evaluation, our method simplifies experimentations and enhances the comparison of assay results.
Cultivating microorganisms on solid agar media is a fundamental technique in microbiology and other related disciplines. For the evaluation, most often, a subjective visual examination is performed. Crucial information, such as metabolic activity, is not assessed. Thus, time‐resolved monitoring of the respiration activity in agar cultivations is presented to provide additional insightful data on the metabolism. A modified version of the Respiration Activity MOnitoring System (RAMOS) was used to determine area‐specific oxygen and carbon dioxide transfer rates and the resulting respiratory quotients of agar cultivations. Therewith, information on growth, substrate consumption, and product formation was obtained. The validity of the presented method was tested for different prokaryotic and eukaryotic organisms on agar, such as Escherichia coli BL21, Pseudomonas putida KT2440, Streptomyces coelicolor A3(2), Saccharomyces cerevisiae WT, Pichia pastoris WT, and Trichoderma reesei RUT‐C30. Furthermore, it is showcased that several potential applications, including the determination of colony forming units, antibiotic diffusion tests, quality control for spore production or for pre‐cultures and media optimization, can be quantitatively evaluated by interpretation of the respiration activity.
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