Interleukin-1 beta (IL-1 beta)-converting enzyme cleaves the IL-1 beta precursor to mature IL-1 beta, an important mediator of inflammation. The identification of the enzyme as a unique cysteine protease and the design of potent peptide aldehyde inhibitors are described. Purification and cloning of the complementary DNA indicates that IL-1 beta-converting enzyme is composed of two nonidentical subunits that are derived from a single proenzyme, possibly by autoproteolysis. Selective inhibition of the enzyme in human blood monocytes blocks production of mature IL-1 beta, indicating that it is a potential therapeutic target.
SER-109 successfully prevented CDI and had a favorable safety profile, supporting a novel microbiome-based intervention as a potential therapy for recurrent CDI.
An amplified NS0 cell line transfected with a vector expressing a humanized monoclonal antibody (MAb) against CD‐18 and glutamine synthetase (GS) was cultivated in a 1.5 L fed‐batch culture using a serum‐free, glutamine‐free medium. Concentrated solutions of key nutrient components were fed periodically using a simple feeding control strategy. Feeding amounts were adjusted daily based on the integral of viable cell concentration over time (IVC) and assumed constant specific nutrient consumption rates or yields to maintain concentrations of the key nutrient components around their initial levels. On‐line oxygen uptake rate (OUR) measurement was used to aid empirically the adjustment of the feeding time points and amounts by inferring time points of nutrient depletion. Through effective nutritional control, both cell growth phase and culture lifetime were prolonged significantly, resulting in a maximal viable cell concentration of 6.6 × 109 cells/L and a final IVC of 1.6 × 1012 cells‐h/L at 672 h. The final MAb concentration reached more than 2.7 g/L. In this fed‐batch culture, cellular metabolism shifts were repeatedly observed. Accompanying the culture phase transition from the exponential growth to the stationary phase, lactate, which was produced in the exponential growth phase, became consumed. The time point at which this metabolism shift occurred corresponded to that of rapid decrease of OUR, which most likely was caused by nutrient depletion. This transition coincided with the onset of ammonia, glutamate and glutamine accumulation. With removal of the nutrient depletion by increasing the daily nutrient feeding amount, OUR recovered and viable cell concentration increased, while cell metabolism shifted again. Instead of consumption, lactate became produced again. These results suggest close relationships among nutrient depletion, cell metabolism transition, and cell death. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 783–792, 1997.
Background Recurrent Clostridioides difficile infection (rCDI) is associated with loss of microbial diversity and microbe-derived secondary bile acids, which inhibit C. difficile germination and growth. SER-109, an investigational microbiome drug of donor-derived, purified spores, reduced recurrence in a dose-ranging, phase (P) 1 study in subjects with multiple rCDIs. Methods In a P2 double-blind trial, subjects with clinical resolution on standard-of-care antibiotics were stratified by age (< or ≥65 years) and randomized 2:1 to single-dose SER-109 or placebo. Subjects were diagnosed at study entry by PCR or toxin testing. Safety, C. difficile–positive diarrhea through week 8, SER-109 engraftment, and bile acid changes were assessed. Results 89 subjects enrolled (67% female; 80.9% diagnosed by PCR). rCDI rates were lower in the SER-109 arm than placebo (44.1% vs 53.3%) but did not meet statistical significance. In a preplanned analysis, rates were reduced among subjects ≥65 years (45.2% vs 80%, respectively; RR, 1.77; 95% CI, 1.11–2.81), while the <65 group showed no benefit. Early engraftment of SER-109 was associated with nonrecurrence (P < .05) and increased secondary bile acid concentrations (P < .0001). Whole-metagenomic sequencing from this study and the P1 study revealed previously unappreciated dose-dependent engraftment kinetics and confirmed an association between early engraftment and nonrecurrence. Engraftment kinetics suggest that P2 dosing was suboptimal. Adverse events were generally mild to moderate in severity. Conclusions Early SER-109 engraftment was associated with reduced CDI recurrence and favorable safety was observed. A higher dose of SER-109 and requirements for toxin testing were implemented in the current P3 trial. Clinical Trials Registration NCT02437487, https://clinicaltrials.gov/ct2/show/NCT02437487?term=SER-109&draw= 2&rank=4.
A fed-batch process using concentrated medium was evaluated for its ability to improve cell culture longevity and final monoclonal antibody (MAb) titers for two monoclonal antibody producing cell lines. It was found to result in up to 7-fold increases in final antibody titers compared to batch culture controls. Although the development cell line specific fed-batch protocols is critical to the development of cost-efficient large-scale production processes, the use of complete medium concentrates provided us with a quick and simple method for producing large quantities of antibodies in the early stages of process development, thus accelerating early work on purification process development, analytical development, biochemical characterization, and safety studies. Insights gained from the concentrated medium fed-batch approach were valuable for the development of refined, cell line specific feeding strategies yielding final MAb titers on the order of 1-2 g/L. Process development data on the effects of inhibitory growth byproducts, medium osmolarity, and the mode of nutrient feed addition on culture longevity and MAb production and information on culture metabolic behavior were successfully incorporated in the development of the optimized fed-batch protocols.
With increasing timeline pressures to get therapeutic and vaccine candidates into the clinic, resource intensive approaches such as the use of shake flasks and bench-top bioreactors may limit the design space for experimentation to yield highly productive processes. The need to conduct large numbers of experiments has resulted in the use of miniaturized high-throughput (HT) technology for process development. One such high-throughput system is the SimCell platform, a robotically driven, cell culture bioreactor system developed by BioProcessors Corp. This study describes the use of the SimCell micro-bioreactor technology for fed-batch cultivation of a GS-CHO transfectant expressing a model IgG4 monoclonal antibody. Cultivations were conducted in gas-permeable chambers based on a micro-fluidic design, with six micro-bioreactors (MBs) per micro-bioreactor array (MBA). Online, non-invasive measurement of total cell density, pH and dissolved oxygen (DO) was performed. One hundred fourteen parallel MBs (19 MBAs) were employed to examine process reproducibility and scalability at shake flask, 3- and 100-L bioreactor scales. The results of the study demonstrate that the SimCell platform operated under fed-batch conditions could support viable cell concentrations up to least 12 x 10(6) cells/mL. In addition, both intra-MB (MB to MB) as well as intra-MBA (MBA to MBA) culture performance was found to be highly reproducible. The intra-MB and -MBA variability was calculated for each measurement as the coefficient of variation defined as CV (%) = (standard deviation/mean) x 100. The % CV values for most intra-MB and intra-MBA measurements were generally under 10% and the intra-MBA values were slightly lower than those for intra-MB. Cell growth, process parameters, metabolic and protein titer profiles were also compared to those from shake flask, bench-top, and pilot scale bioreactor cultivations and found to be within +/-20% of the historical averages.
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