Development of hyper osmotic resistant CHO host cells

We have developed a cell culture platform for monoclonal antibody (IgG) production by Chinese Hamster Ovary (CHO) cells. The platform feed used the continuous feeding method. This platform can maintain high cell density and produce high antibody titer. However because operation of continuous feed is complex, contract manufacturing organization (CMO) that can perform continuous feed is limited. Therefore, we tried to change the feeding method from continuous feed to bolus feed. However the previous studies showed that the rapid changes of osmolality by bolus feed and the hyper osmolality repressed the cell culture growth and the final titer. In this study, we developed hyper osmotic resistant CHO-S host cell A (resistant to 450mOsm). To establish osmotic resistant CHO-S host cells, original CHO-S cells were passaged on a hyper osmotic basal media with repetition for about 100 days. We demonstrated that there were obviously differences in the cell growth under osmotic pressure of iso- (328 mOsm) and hyper- (450 mOsm) osmolality between the two host cells. Metabolic analysis of cell culture supernatant on CHO-S host cell A and CHO-S host cells with/ without osmotic stress performed. Compared to original CHO-S host cells, the osmotic resistant CHO-S host cell A has a greater capacity to generate osmolytes (sorbitol and erthritol) and decreased level of oxidized glutathione (GSSG), which suggests the osmotic resistant CHO-S host cells A handles osmotic stress better. Moreover, the characteristic of osmotic resistant on hyper osmotic resistant CHO-S host cell A was maintained even after 7 passages on a basal medium (330 mOsm). We will establish hyper osmotic resistant antibody production CHO cell line by using the CHO-S host cell A

Genotype of CHO host cell line has higher impact on mAb production and quality than process strategy or cell culture medium

Chinese hamster ovary (CHO) cells comprise a variety of lineages, including CHO-DXB11, CHO-K1, CHO-DG44 and CHO-S. Despite the fact that CHO cell lines share a common ancestor, extensive mutagenesis and clonal selection have resulted in substantial genetic heterogeneity among them. Data from sequencing shows that different genes are lacking from individual CHO cell lines and that each cell line harbors a unique set of mutations that are relevant to the bioprocess. However, literature outlining how the observed genetic differences affect CHO cell performance during bioprocess operations remains scarce. In this study, we examined host cell-specific differences among three widely used CHO cell lines (CHO-K1, CHO-S and CHO-DG44) and recombinantly expressed the same monoclonal antibody (mAb) in an isogenic format in all cell lines by using bacterial artificial chromosomes (BACs) as transfer vector. Cell-specific growth, product formation and heavy and light chain mRNA levels were studied in batch, fed-batch and perfusion cultures. Furthermore, two different cell culture media were investigated. Product quality was studied through glycoprofiling, and the thermal denaturation was analyzed using differential scanning calorimetry (DSC). We found CHO cell line-specific preferences for mAb production or biomass synthesis that were determined by the host cell line rather than product-specific mRNA levels. Additionally, quality attributes of the expressed mAb were influenced by the host cell line and medium used

The effect of a carbohydrate mouth rinse on performance of the yo-yo intermittent recovery level 1 test with female university level hockey players

It has been suggested that carbohydrate mouth rinse (CHO-MR) effects short duration, high intensity exercise by activation of sensory receptors on the tongue (Carter et al., 2004: Medicine and Science in Sports and Exercise, 36, 2107–2111). Research has predominately focused on the effects of CHO-MR on the performance of cycling and running time trials (Painelli et al., 2010: Nutritional Journal, 9, 1-4). Limited research has been conducted on the effectiveness of a CHO-MR on intermittent high-intensity field-based sports performance. The aim of this study was to analyse the effects of a CHO mouth rinse on performance of the Yo-yo Intermittent Recovery Level 1 Test (IR1T) with female University standard hockey players. Following ethical approval, twelve members (mean age 20 ± 0.98 years, stature 167 ± 7.09 cm, and body mass 64.7 ± 4.96 kg) of the University of Lincoln’s female 1st team volunteered for the study. The study used a single-blind counter-balanced design with repeated measures on two treatment conditions: 1) a CHO-MR, and 2) a placebo mouth rinse (PL-MR). Participants were instructed to maintain a normal diet and fasted for 12hr prior to testing. After a familiarisation test, twelve participants completed the IR1T twice, one week apart; rinsing with either a CHO-MR or PL-MR. Administration occurred before (20min prior) the IR1T and during the 10s active recovery periods, at intervals (IV) which corresponded to five level increments in speed (IV1-13.5km/h, IV2-14km/h, IV3-14.5km/h, IV4-15km/h, IV5-15km/h). At these points, rate of perceived exertion (RPE) was recorded using the traditional Borg scale. Total distance (m) achieved was recorded as the performance measure. A dependent t-test did not detect any performance improvement (P = >0.05) between CHO-MR (1060 ± 273m) and PL-MR (1127 ± 402m) trials. Multiple dependent t-tests revealed that at the first IV (SL 12.1), RPE scores were significantly different (P = 0.006) between CHO-MR (10.9 ± 0.79) and PL-MR (11.4 ± 1.08) trials. No differences were detected between CHO-MR and PL-MR trials during the rest of the protocol (IV2-5, all P = >0.05). A CHO-MR had no effect on IR1T test performance with female university level hockey players compared to a PL-MR. The participants did not experience any differences in the feeling of exertion between the two conditions as the IR1T progressed. Further research needs to illuminate any possible performance effects from CHO-MR with intermittent high intensity activity, revealing any plausible physiological mechanisms of action

Microfluidic accelerated evaluation of CHO cell clones by perfusion of fed-batch conditioned media

The generation of genetically engineered production CHO cell lines is normally the longest step in the race to scale-up protein manufacturing. This labor-intensive screening includes the expansion of hundreds of clonal cultures to sufficient numbers for their growth and productivity to be evaluated. Unfortunately, many clones that perform well when screened at the batch cloning stage display reduced performance under fed-batch conditions. Thus, screening potential clones under conditions more similar to the ultimate production cultures provides an opportunity for more effective clone selection. We combined the advantages of precise measurements in microfluidic nL volume cultures with our ability to modify the medium during cultures where clones are retained in thousands of isolated chambers. Our bead assay coupled with an automated image analysis pipeline quickly and reproducibly detected as little as 106 human IgG molecules in 4 nL chambers after 2 h of incubation. Thus, it is possible to evaluate the production of a single high performance CHO cell at the very start of the microfluidic culture. To further evaluate the performance of CHO-S clones under production conditions, we perfused media from untransfected parental CHO-S fed-batch cultures into the microfluidic device daily. This conditioned medium perfusion technique was developed using an automated robotic 24-well deep well plate culture system to demonstrate that clones in perfused fed-batch medium matched the growth profiles and specific productivities of those in larger scale fed-batches. This was replicated for multiple clonal CHO-S and CHO-K1 cell lines. Analysis of both the growth rate and productivity of the microfluidic cultures enabled the screening of hundreds of cultures in parallel under simulated fed-batch conditions. The dynamic nature of our microfluidic assay coupled with the perfusion of conditioned medium in nanoliter volumes enables more rapid and effective characterization of clonal CHO cell performance, thereby accelerating progress towards the manufacturing of valuable products

Adaptation to an MCFA-rich diet : effect on gastric tolerance, the capacity for MCFA oxidation, and performance while ingesting exogenous carbohydrate and structured oils during endurance exercise : a thesis presented in partial fulfilment of the requirements for the degree of Master of Sport Science at Massey University

Introduction: Elevating the availability of fatty-acids to the muscle can potentially benefit endurance exercise performance by reducing intramuscular-glycogen utilisation. Digestion of triglycerides containing long-chain fatty acids (LCFAs) is slow, and fatty acids must pass through the carnitine palmityl transferase (CPT) transport system to enter the mitochondria, which potentially limits fat oxidation during prolonged-heavy exercise. Conversely, medium-chain triglycerides (MCTs) are rapidly digested and their constituent fatty acids (MCFAs) by-pass the CPT transport system. Ingestion of MCFAs may therefore supply mitochondrial acetyl-CoA, potentially reducing the requirement for glycolytic flux during exercise. However, studies comparing carbohydrate (CHO) with CHO-containing MCFA-rich exercise supplements have revealed inconsistent results, probably because of the variation in gastrointestinal (GI) distress suffered by participants associated with MCT ingestion. Purpose: To investigate whether 2-weeks of dietary adaptation to MCFA-rich supplements reduces the severity of gastrointestinal (Gl) distress, or increases the rate of MCFA oxidation during endurance exercise. A decrease in ratings of GI distress, or an increase in MCFA oxidation was anticipated to lead to performance benefits. Method: Nine well-trained male endurance cyclists participated in a double-blind, pseudo-randomised. triple-crossover protocol. Participants were 37 ± 7.26 years, 81.36 ± 7.67 kg. training at least 8-10 h per week and riding competitively. Mean VO2 max and peak power output (PPO) were 4.84 ± 0.46 L-min-1 and 357.33 ± 20.55 W respectively. The effects of a 2-week MCFA-rich diet +13 C-enriched MCFA+CHO exercise supplement (MC-MC) on GI distress, MCFA-oxidation rate and sprint performance variables were compared against a 2-week LCFA-rich diet with either: (a) a13 C-enriched MCFA+CHO exercise supplement (LC-MC), or (b) a CMO-only supplement (LC-CHO). Dietary and exercise MCFA-rich supplements were consumed in the form of randomised-structured triacylglycerols made with a 3:1 molar ratio of MC- and LCFAs randomly esterified to glycerol backbones. Participants followed a controlled training regime whilst on the diets. The performance test consisted of a 3-h ride at 50% PPO followed by 10 maximal sprints. At rest and every 20-min throughout the ride, participant ratings of GI and exertion sensations were recorded, followed by external respiratory-gas analysis, collection of a breath sample for breathl3 C-enrichment analysis, a venous blood sample and ingestion of a supplement. Similarly, after sprints 1, 4, 7 and 10 participants recorded their GI ratings followed by a blood sample. Results: Peak MCFA-oxidation rates were 0.38 g-min-1(95% Cl 0.31-0.47) and 0.43 g-min-1(0.30-0.61, p-value = 0.21) in the MC-MC and LC-MC conditions respectively, but there was no evidence for CHO sparing following MCFA adaptation. Participant ratings of GI distress decreased slightly during exercise with 2-weeks of a diet high in MCFAs relative to LCFAs. Ratings of reflux, bloatedness, nausea, and urge to vomit were, respectively, 1.34 (0.88-3.14), 1.03 (0.74-2.27), 0.81 (0.62-1.69) and 0.93 (0.64-245) scale units lower in the MC-MC condition relative to LC-MC. The attenuation in GI distress corresponded with a tendency toward increased sprint mean power, which was 3.4% (± 5.9%, 0.25) higher in the MC-MC condition relative to LC-MC. However, sprint mean power was still lower in both the MC-MC (6.8% ± 2.8%, <0.0001) and LC-MC (10.4% ± 5.5%, 0.0004) conditions relative to LC-CHO. Mechanism covariate analysis illustrated a negative effect of the GI distress marker nausea on sprint performance. For every 1 unit increase in nausea for the MC-MC and LC- MC conditions, sprint power decreased by 6 W (± 3.8,0.004) relative to LC-CHO. Conclusion: No clear metabolic adaptation was evident with high dietary MCFA relative to LCFA. In addition, MCFA-rich exercise supplements caused a decrement in performance relative to CHO ingestion in both MC-MC and LC-MC conditions, suggesting that light- moderate GI distress still causes substantial performance detriments. There was little evidence to support the ingestion of randomised structured triglycerides high in MCFA with the intention of enhancing endurance performance

Cho and Pak reply to Lamm et al. comment on "A Convergent Series for the QED Effective Action"

Cho and Pak reply to Lamm et al. [hep-th/0007108] comment on "A Convergent Series for the Effective Action of QED" [hep-th/0006057].Comment: 1 pag

A community genome-scale model of Chinese Hamster ovary cell metabolism identifies differences in the efficiency of resource utilization for various bioprocesses

Genome-scale models of metabolism have successfully been employed in many microbial and eukaryotic metabolic engineering efforts by guiding pathway engineering and media optimization. They have also been used to explore the genotype-phenotype relationship in mammalian cells. The publication of the genomic sequence for Chinese hamster ovary (CHO) cells has allowed generation of genome-scale metabolic models (GeMs) for this organism. Here we have developed a high-quality community CHO GeM via careful reconciliation and manual curation of three independently developed CHO GeMs. This metabolic model, consisting of over 4000 metabolites and 6000 reactions, is capable of integrating proteomic, transcriptomic, and metabolomic data and can accurately simulate experimentally measured growth rates. Integration of transcriptomic and proteomic data from CHO-K1 and CHO-S shed light on the enzymatic basis for various amino acid auxotrophies characteristic of the cell lines. We show that experimental arginine and cysteine auxotrophies are recapitulated by model predictions (via reaction inactivation) while the characteristic proline auxotrophy is not, due to detectable levels of expression in biosynthetic pathways for this amino acid. We additionally used the model to assess the metabolic limitations on recombinant protein producing lines subject to different cell line and process modifications and found that some alterations result in specific productivities up to 20-fold lower than computational predictions of metabolically feasible production rates. The results indicate a possible secretory bottleneck and implicate engineering the secretory pathway as a lucrative target to pursue in future CHO cell line engineering

Engineering CHO cells for the production of Hard-To-Produce proteins

Over the past decades, the CHO cell has become increasingly popular as the favorite host cell line for the production of protein based therapeutic drugs. In comparison with the popularity of the CHO cells and the frequent use of these cells to produce a large part of the bestselling blockbuster drugs, less intensive efforts have been done to understand the machinery used by the CHO cells during growth and production. The main approach has (broadly speaking) been to approach the CHO cell as a “black box” where one could insert the gene of interest, perform a number of amplifying steps, like gene amplification, selection for stable clones, intense screening for stably expressing high producers, and massive efforts to optimize a specific bioprocess for the selected cell line(s). Since 2013, the Novo Nordisk Foundation Center for Biosustainablity at the Technical University of Denmark has embarked on a large CHO program to open up the “black box”, to get a deeper understanding of the available machinery inside the protein producing “cell factory” that is CHO cells. We are using this understanding to engineer new CHO cell lines having significantly improved features for the production of therapeutic proteins. We are not only doing this by improving the titer, quality, downstream processing and speed of development for already well-known proteins (e.g. Ab), but also for the production of therapeutic proteins that cannot be produced in CHO cells today, due low titer, wrong post translational modifications, and/or low activity. By combining the competences embedded in the CHO program, we are able to exploit the combination of genome scale modelling, high throughput protein expression, deep understanding of both the glycosylation machinery as well as the secretory and metabolic pathways involved in the expression of secreted proteins. This knowledge is being used as input to a high throughput CHO cell line engineering pipeline, able to engineer up to 10 cell lines and 25 gene targets in parallel. This has resulted in a large number of new CHO cell lines enabling the production of proteins with specific tailor-made glycoprofiles, higher quality, less degradation, improved bioprocess, higher viable cell density and better cell viability. We have made a cell lines where we have removed a number of naturally expressed host cell proteins (HCP) from CHO, which has resulted in higher titer and higher VCD, cell lines showing increased resistance to viral infections, cell lines displaying homogenous glycoprofiles, reduced degradation, and drastically changed cell lines that does not produce lactate. These features are currently being combined to engineer CHO cells able to produce proteins that have not been possible to produce with adequate product quality and titer using CHO cells to date

Nature sẽ ủng hộ kế hoạch Plan S

Tại thị trường Châu Âu, Springer Nature cho biết có đến 4 quốc gia có hơn 70% tác giả đang lựa chọn công bố mở. Tuy nhiên, để đạt được 30% còn lại thì các nhà xuất bản như Springer Nature cần đưa ra các lựa chọn hấp dẫn và thuyết phục hơn là ép buộc các tác giả phải lựa chọn Open Access như yêu cầu hiện nay

A Note on the Hybrid Equilibrium in the Besley-Smart Model

This note shows that there is always a non-empty set of parameter values for which the hybrid equilibrium in the Besley and Smart(2003) model is unstable in the sense of Cho and Kreps. This set may include all the parameter values for which a hybrid equilibrium exists. For these parameter values, it is shown that a fully separating equilibrium always exists, which is Cho-Kreps stable. In this equilibrium, the good incumbent distorts ?scal policy to signal his type. An implication is that equilibrium in their model is not (generically) unique.