Multi-rate nonlinear state and parameter estimation in a bioreactor.

The monitoring and control of bioprocesses is of the utmost importance in order to provide a consistent, safe, and high-quality product for consumers. Current monitoring and control schemes rely on infrequent and time consuming offline sampling methods, which inherently leads to some variability in the process which may impact the product quality profile. As part of Lonza's dedication to process analytical technology (PAT) initiatives this study evaluated the ability to generate generic calibration models, which are independent of the cell line, using Raman probes to monitor changes in glucose, lactate, glutamate, ammonium, viable cell concentration (VCC), total cell concentration (TCC) and product concentration. Calibration models were developed from cell culture using two different CHOK1SV GS-KOTM cell lines producing different monoclonal antibodies (mAbs). Developed predictive models, measured changes in glucose, lactate, ammonium, VCC, and TCC with average prediction errors of 0.44, 0.23, 0.03 gL-1 , 1.90 × 106 cellsmL-1 , and 1.85 × 106 cellsmL-1 , respectively over the course of cell culture with minimal cell line dependence. The development of these generic models allows the application of spectroscopic PAT techniques in clinical and commercial manufacturing environments, where processes are typically run once or twice in GMP manufacturing based on a common platform process. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:730-737, 2018.

DEVELOPMENT OF A NEW PROBE FOR IN-SITU OXYGEN UPTAKE RATE (OUR) MEASUREMENT IN MAMMALIAN CELL CULTURE PROCESSES

Effective tPA production by BHK cells in nutrients-controlled culture using an on-line HPLC measuring system

The steady-state performance of a multistage fermentor with cell filtering through a membrane and recycling of concentrated cells is studied numerically for continuous production of acetic acid. The tanks-in-series model is used to describe the flow behaviour of culture broth in the multistage fermentor. Kinetic expressions and parameter values are taken from the literature. The effects of the total number of stages, the dilution rate, the bleed ratio, the recycle ratio and the feed concentrations as operating conditions on fermentor characteristics such as the concentrations of total and viable cells as well as substrate and product in each stage, the cell viability, the acetic acid productivity and the substrate conversion were examined under the conditions of equal tank volumes and a recycle ratio equal to or greater than one. An increase in the number of stages and decreases in the bleed and recycle ratios and in the feed concentration of product enhance the productivity. The relations between the maximum productivity and the corresponding optimum dilution rate and between the corresponding outlet concentrations of the four components and the optimum dilution rate are presented as figures. Equations for the average cell viability and the critical dilution rate causing cell washout are also obtained. Furthermore, the minimum feed concentration of substrate for its complete consumption is mentioned. Compared to a single tank fermentor, staging and cell recycle increase substrate utilisation and acetic acid productivity. The computed performance of the fermentor configuration proposed in this study is sufficiently promising for the continuous production of acetic acid that it should now be checked experimentally.

DNA, RNA, and protein concentrations from starved ANT-300 cell populations grown at different growth rates fluctuated corresponding to the three stages of starvation-survival on total and viable cell bases. During stage 1 of starvation-survival, two to three peaks in the concentration levels for all three macromolecules were characteristic. During stage 2, DNA per total cell dropped to between 4.2 and 8.3% of the original amount for all of the cell populations examined, and it stabilized throughout stage 3. The decrease in DNA per cell was also observed in electron micrographs of cellular DNA in unstarved compared with starved cells. The fluctuations of RNA and protein per total cell concentrations observed during stage 2 coincided in all cases, except for the cells from dilution rate (D) = 0.015 h. This ANT-300 cell population showed a decrease in RNA per total cell to only 29.2% and an increase in protein to 129.7% of the original amount after 98 days of starvation. During stage 3, DNA, RNA, and protein concentrations per total cell also stabilized to continuous levels. Cells from the faster-growth-rate cell populations of D = 0.170 h and batch culture had elevated protein per total cell concentrations, which remained primarily residual during the starvation period. Starved cells from D = 0.015 h had estimated nucleoid and cell volumes of 0.018 and 0.05 mum, respectively, yielding a nucleoid volume/cell volume ratio of 0.40. We consider these data to indicate that slow-growth-rate cells are better adapted for starvation-survival than their faster-growth-rate counterparts.

Role of glutamine in hybridoma cell culture: Effects on cell growth, antibody production, and cell metabolism

Aim. Optimization of culture conditions of the probiotic strain Lactobacillus acidophilus 55 in the medium with sodium selenite. Methods. Studies of the influence of sodium selenite, inoculum size and cultivation time on the concentration of viable cells of L. acidophilus 55 were performed using a three-level factorial experiment on Box-Behnken (BBD). Results. It has been found that the inoculum size is the factor that has the greatest effect on the concentration of viable cells. The optimum conditions to maximize the concentration of viable cells in the presence of selenium ions were: the content of sodium selenite in the medium of 8.3%, the amount of inoculum 5.3% and 18 hours of cultivation time. Conclusions. When L. acidophilus 55 was cultured at optimized conditions, the highest concentration of viable cells was 2.5×108 CFU/ml.

Mouse hybridoma cells were grown in suspension in continuous stirred bioreactors. Cell growth, substrate utilization, and monoclonal antibody (MAb) production were studied using serum-free medium. Steady-state data were obtained at different dilution rates, between 0.012 and 0.039 h(-1) Viability was profoundly affected by dilution rate, particularly near the lower end of the dilution-rate range investigated. MAb concentration and productivity went through a maximum with respect to dilution rate. Lactate yield on glucose declined with increasing dilution rate. Experiments were carried out to study the effects of medium glucose concentration on cell growth, product formation, and lactate yield on glucose. Reduction of glucose concentration in the feed medium did not considerably affect cell density and MAb concentration in the culture, but lactate levels dropped sharply; lactate yield on glucose declined substantially, indicating alterations in cell metabolic path ways for energy metabolism. Optimization strategy for continuous cell culture is discussed.

Monoclonal antibody production in semi-continuous serum- and protein-free culture: Effect of glutamine concentration and culture conditions on cell growth and antibody secretion

Major depressive disorder (MDD) is characterized by diverse metabolic and functional abnormalities that occur in, among other regions, the pregenual anterior cingulate cortex (pgACC), a cortical region linked to anhedonia. To contextualize metabolic, functional, and clinical parameters and thus to reveal cellular mechanisms related to anhedonia. The pgACC was investigated using a combined functional magnetic resonance imaging and magnetic resonance spectroscopic approach. Negative blood oxygenation level-dependent (BOLD) activations in the pgACC were assessed during emotional stimulation. Quantitative J-resolved magnetic resonance spectroscopy in the pgACC enabled simultaneous determination of glutamine, glutamate, N-acetylaspartate, glucose, and gamma-aminobutyric acid concentrations. Subjective emotional intensity ratings as well as various clinical parameters were determined. The patients were recruited and evaluated in the Department of Psychiatry, University of Zurich, while the measurements were performed in the Institute of Biomedical Engineering, University of Zurich and the Technical University Zurich. Nineteen unmedicated patients with MDD and 24 healthy subjects. Reduced glutamine levels and lower functional responses in pgACC in anhedonic depressed patients were expected to be the predominant effect of abnormal glutamatergic transmission. It was further tested if, among patients, the ratings of emotional intensity on visual stimulation predicted the amount of metabolic and functional alterations in terms of reduced relative metabolite concentrations and BOLD changes. Patients with highly anhedonic MDD show decreased glutamine but normal glutamate and gamma-aminobutyric acid concentrations, with glutamine concentrations being dissociated from glucose concentrations. Glutamate and N-acetylaspartate concentrations in pgACC correlate with negative BOLD responses induced by emotional stimulation in MDD; whereas in healthy subjects, negative BOLD responses correlate with gamma-aminobutyric acid instead. Negative BOLD responses as well as glutamate and N-acetylaspartate concentrations correlate with emotional intensity ratings, an anhedonia surrogate, in those with MDD but not in healthy subjects. Aberrant neuronal activation patterns of the pgACC in anhedonic depression are related to deficits of glutamatergic metabolism.

The objectives of this study were to (1) estimate the clinical status of the mammary glands and (2) compare it with the bacteriological findings, the total cell content (TCC) and its percentage of polymorphonuclear leucocytes (PMNLs) and pH in colostrum and milk secretion of sows on 2 different feeding regimes, high versus low, during late pregnancy. The milk samples were collected from both agalactia post partum (APP) sows and clinically healthy sows. Sows with a rectal temperature exceeding 39.5 degrees C within 48 h after parturition were considered to be diseased in APP and treated medically. The sows were sampled on days 1, 3, 8 and 22 of lactation during 6 consecutive lactations. Irrespective of feeding regimes, 49 out of 77 lactations among the APP sows and 15 out of 96 lactations among the clinically healthy sows revealed E. coli in pure cultures with a concomitant TCC exceeding 10 x 10(6) cell/ml already on the first day of lactation. The healthy sows with E. coli infection were denominated as being subclinically infected sows. The intensity in growth of E. coli successively declined, and the bacteria were finally eliminated between days 3 and 8 of lactation. The TCC were 82 x 10(6) cells/ml and 157 x 10(6) cells/ml in the clinically and subclinically E. coli infected glands, respectively, on the first day of sampling. The TCC declined gradually in both groups of sows, but was still higher than in bacteriologically negative milk on day 22 of lactation. The percentages of PMNLs were 66% and 79% in clinically and subclinically infected glands, respectively, on day 1 of lactation, thereafter decreasing to approximately 50% on day 22 of lactation in both groups of sows. In APP sows, swelling, reddening and/or soreness were registered in 38 out of 87 mammary glands with E. coli mastitis on the first sampling occasion. The TCC in bacteriologically negative colostrum and milk collected from APP sows on day 1 of lactation was significantly higher, 2.27 x 10(6) cells/ml, when compared with the TCC in bacteriologically negative milk secretion from the clinically healthy or subclinically infected sows, 1.38 x 10(6) cells/ml versus 1.51 x 10(6) cells/ml, respectively. The PMNLs were higher on day 1 in clinically healthy sows, 59.6%, than in subclinically infected and APP sows (43.5% and 48.3% respectively). The pH in secretion from clinically or subclinically E. coli infected glands (6.57 versus 6.46) were higher than in bacteriologically negative colostrum samples (6.29) from clinically diseased sows on the first day of sampling. On day 22 of lactation, pH-values had stabilized on a level of approximately 7.00 in all milk samples from earlier bacteriologically positive or negative mammary glands. The 2 feeding regimes, low versus high, were not found to influence TCC, PMNLs or pH except for TCC in bacteriologically negative samples of APP sows (2.69 versus 3.62). The lactation number influenced the PMNLs in both groups of sows with E. coli infected mammary glands, and both the TCC and PMNLs in bacteriologically negative colostrum and milk.

Materials and methods AmbrTM 48 and 80L stirred tank bioreactor were run for 14 days in a fed-batch mode in a chemically defined medium. A CHO cell line expressing a recombinant monoclonal antibody (MAb) was used. Feed was added daily from day 3 onwards. If required, antifoam C was added to the bioreactor. DO, pH, and temperature were controlled at setpoint. Viable cell concentration (VCC), cell viability, and average cell diameter were measured using a ViCell cell counter. Osmolality was obtained using an Osmometer (Advanced Instruments). On harvest day, MAb concentration of the supernatant samples was quantified using Protein A high performance liquid chromatography. For the minibioreactors run, triplicates were pooled on harvest day for product quality attributes analysis. For both scales cell culture fluid samples were centrifuged and filtered to remove cell debris. The monoclonal antibody was purified by AKTAXpress (GE Healthcare) Protein-A purification. The neutralized eluate was used for product quality analysis. Results High throughput technologies enable us to interconnect clone and process development thus reducing the risks associated with early-stage process development while reducing timelines and enabling us to achieve higher process robustness in a leaner manner. In one ambrTM48 run different feeding strategies were assessed on 4 preselected lead clones for selecting the final lead clone, the fed-batch feeding strategy and additional data with feed compositions all at once. The selected condition (clone, type of feed and feeding strategy) was based on the maximization of the MAb titer and target High Molecular Weight Species (HMWS) level and Acidic Peak Group (APG) level (data not shown). Scale up from ambrTM 48 to 80L stirred tank bioreactor was performed with the selected clone, feed and feeding strategy. Similar cell growth profiles were obtained at ambrTM scale and 80L scale for the selected condition (Figure 1). Monoclonal Antibody titers and percentages of Acidic Peak Group obtained at harvest day were comparable between scales (Table 1). Process robustness was shown by performing a number of 4 batches at 80L scale.

Methods for quantification of biosorption in high-rate activated sludge systems

Kinetic study of hybridoma metabolism and antibody production in continuous culture using serum-free medium

Somatic Cell Content Variation in Fraction-Collected Milk