Intensified fed‐batch (IFB), a popular cell culture intensification strategy, has been widely used for productivity improvement through high density inoculation followed by fed‐batch cultivation. ...However, such an intensification strategy may counterproductively induce rapidly progressing cell apoptosis and difficult‐to‐sustain productivity. To improve culture performance, we developed a novel cell culture process intermittent‐perfusion fed‐batch (IPFB) which incorporates one single or multiple cycles of intermittent perfusion during an IFB process for better sustained cellular and metabolic behaviors and notably improved productivity. Unlike continuous perfusion or other semi‐continuous processes such as hybrid perfusion fed‐batch with only early‐stage perfusion, IPFB applies limited times of intermittent perfusion in the mid‐to‐late stage of production and still inherits bolus feedings on nonperfusion days as in a fed‐batch culture. Compared to IFB, an average titer increase of ~45% was obtained in eight recombinant CHO cell lines studied. Beyond IPFB, ultra‐intensified IPFB (UI‐IPFB) was designed with a markedly elevated seeding density of 20–80 × 106 cell/mL, achieved through the conventional alternating tangential flow filtration (ATF) perfusion expansion followed with a cell culture concentration step using the same ATF system. With UI‐IPFB, up to ~6 folds of traditional fed‐batch and ~3 folds of IFB productivity were achieved. Furthermore, the application grounded in these two novel processes showed broad‐based feasibility in multiple cell lines and products of interest, and was proven to be effective in cost of goods reduction and readily scalable to a larger scale in existing facilities.
Normally, the growth profile of a CHO cell fed-batch process can be divided into two main phases based on changes in cell concentration, being an exponential growth phase and a stationary ...(non-growth) phase. In this study, an additional phase is observed during which the cell division comes to a halt but the cell growth continues in the form of an increase in cell size. The cell size increase (SI) phase occurs between the exponential proliferation phase (also called the number increase or NI phase) and the stationary phase. During the SI phase, the average volume and dry weight per cell increase threefold linearly with time. The average mAb specific productivity per cell increases linearly with the cell volume and therefore is on average two times higher in the SI phase than in the NI phase. The specific essential amino acids consumption rates per cell remain fairly constant between the NI and the SI phase, which agrees with the similar biomass production rate per cell between these two phases. Accumulation of fatty acids and formation of lipid droplets in the cells are observed during the SI phase, indicating that the fatty acids synthesis rate exceeds the demand for the synthesis of membrane lipids. A metabolic comparison between NI and SI phase shows that the cells with a larger size produce more mAb per unit of O
2
and nutrient consumed, which can be used for further process optimization.
In the present report and for the first time in the international literature, the impact of the addition of NaCl upon growth and lipid production on the oleaginous yeast Rhodosporidium toruloides was ...studied. Moreover, equally for first time, lipid production by R. toruloides was performed under nonaseptic conditions. Therefore, the potentiality of R. toruloides DSM 4444 to produce lipid in media containing several initial concentrations of NaCl with glucose employed as carbon source was studied. Preliminary batch‐flask trials with increasing amounts of NaCl revealed the tolerance of the strain against NaCl content up to 6.0% w/v. However, 4.0% w/v of NaCl stimulated lipid accumulation for this strain, by enhancing lipid production up to 71.3% w/w per dry cell weight. The same amount of NaCl was employed in pasteurized batch‐flask cultures in order to investigate the role of the salt as bacterial inhibiting agent. The combination of NaCl and high glucose concentrations was found to satisfactorily suppress bacterial contamination of R. toruloides cultures under these conditions. Batch‐bioreactor trials of the yeast in the same media with high glucose content (up to 150 g/L) resulted in satisfactory substrate assimilation, with almost linear kinetic profile for lipid production, regardless of the initial glucose concentration imposed. Finally, fed‐batch bioreactor cultures led to the production of 37.2 g/L of biomass, accompanied by 64.5% w/w of lipid yield. Lipid yield per unit of glucose consumed received the very satisfactory value of 0.21 g/g, a value among the highest ones in the literature. The yeast lipid produced contained mainly oleic acid and to lesser extent palmitic and stearic acids, thus constituting a perfect starting material for “second generation” biodiesel.
The use of Raman models for glucose and phenylalanine concentrations to provide the signal for a control algorithm to continuously adjust the feed rate of two separate supplemental feeds during the ...fed-batch culture of a CHOK1SV GS-KO® cell line in a platform process was evaluated. Automated feed rate adjustment of the glucose feed using a Raman model for glucose concentration, maintained the glucose concentration within the desired target (average deviation ± 0.49 g/L). Automated feed rate adjustment of the nutrient feed using a Raman model for phenylalanine concentration, maintained phenylalanine concentrations within the target (average deviation ± 29.97 mg/L). The novel use of a Raman model for phenylalanine concentration, combined with a Raman model for glucose concentration, to maintain target glucose and phenylalanine concentrations through feed-rate adjustments, reduced the average cumulative glucose and nutrient feed additions (19% and 27% respectively) compared to manually adjusted cultures. Additionally, the proposed automation strategy led to lower osmolality during culture, maintained the nutrient environment more consistently, and achieved higher harvest product concentration (≈ 20% higher) compared to typical fed-batch process control for the cell line and platform process evaluated. Furthermore, the proposed feeding strategy yielded similar glycosylation and charge variant profiles compared to manually adjusted fed-batch process control. The ability to continuously adjust the feed rate addition of two separate feeds in this manner helps enable a shift away from the current daily offline sampling needed to control fed-batch mammalian cell culture during clinical and commercial manufacturing on platform processes.
The demand for 1,3-propanediol (1,3-PDO) has increased sharply due to its role as a monomer for the synthesis of polytrimethylene terephthalate (PTT). Although
Clostridium butyricum
is considered to ...be one of the most promising bioproducers for 1,3-PDO, its low productivity hinders its application on industrial scale because of the longer time needed for anaerobic cultivation. In this study, an excellent
C. butyricum
(DL07) strain was obtained with high-level titer and productivity of 1,3-PDO, i.e., 104.8 g/L and 3.38 g/(L•h) vs. 94.2 g/L and 3.04 g/(L•h) using pure or crude glycerol as substrate in fed-batch fermentation, respectively. Furthermore, a novel sequential fed-batch fermentation was investigated, in which the next bioreactor was inoculated by
C. butyricum
DL07 cells growing at exponential phase in the prior bioreactor. It could run steadily for at least eight cycles. The average concentration of 1,3-PDO in eight cycles was 85 g/L with the average productivity of 3.1 g/(L•h). The sequential fed-batch fermentation could achieve semi-continuous production of 1,3-PDO with higher productivity than repeated fed-batch fermentation and would greatly contribute to the industrial production of 1,3-PDO by
C. butyricum
.
Key points
•
A novel C. butyricum strain was screened to produce 104.8 g/L 1,3-PDO from glycerol.
• Corn steep liquor powder was used as a cheap nitrogen source for 1,3-PDO production.
• A sequential fed-batch fermentation process was established for 1,3-PDO production.
• An automatic glycerol feeding strategy was applied in the production of 1,3-PDO.
Demand for high-quality plasmid DNA (pDNA) has greatly increased in recent years due to its myriad applications across therapeutic modalities – including gene therapies, vaccines, and mRNA. This ...increased demand poses a challenge to pDNA manufacturers, which must continue to increase yield and productivity without compromising on rigorous standards for purity, and ultimately, safety. Productivity in pDNA manufacturing is heavily influenced by host strain, plasmid type and size, growth medium and growth conditions.
A widely used culture medium, TB (Terrific Broth) offers high cell density cultures but lacks the capacity to generate large quantities of pDNA (>60 ng/µl). In understanding the significance and effect of peptones in TB a proprietary chemically defined media was designed to increase pDNA titers. This abstract captures efforts to create a high yielding and scalable fermentation process, involving the evaluation of various strategies in Escherichia coli DH5α transformed cells. The development of the process was driven by a three-level single-factor experimental design focused on media composition, temperature, concentration of peptone and carbon source. Improvements in small- and large-scale pDNA yield during fermentation, resulting from media selection and optimization are summarized. The results revealed that a chemically defined media used in conjunction with a nutrient-dense feed supported high cell density cultures and increased pDNA yield and specific productivity when compared to conventional exponential fed batch cultures in TB media. The feed approach developed was optimal for the three plasmids tested and constitute a robust pDNA yield optimization platform established at Akron enabling the production of cost effective pDNA for advanced therapies. This feed-based optimization platform is being tested with various E. Coli Stains and plasmid sizes for fitness and possible strain specific optimizations.
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