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

Addition of sodium alginate and pectin to a carbohydrate-electrolyte solution does not influence substrate oxidation, gastrointestinal comfort, or cycling performance

Eight well-trained cyclists ingested 68 g·h-1 of a carbohydrate-electrolyte solution with sodium alginate and pectin (CHO-ALG) or a taste and carbohydrate-type matched carbohydrate-electrolyte solution (CHO) during 120 min cycling at 55% Wmax followed by a ~20 min time trial. V̇O2, V̇CO2 blood glucose concentration, substrate oxidation, gastrointestinal symptoms and time trial performance (CHO-ALG: 1219 ± 84 s, CHO: 1267 ± 102 s; P = 0.185) were not different between trials. Novelty bullet: • Inclusion of sodium alginate and pectin in a carbohydrate drink does not influence blood glucose, substrate oxidation, gastrointestinal comfort or performance in cyclists

Lipidomic analysis to enhance the understanding of Chinese Hamster ovary cells

Chinese Hamster Ovary (CHO) cell lines are common hosts for the production of biotherapeutic proteins. Achieving high level of specific protein production by CHO cell lines remains a challenge. In order to address this issue, we are incorporating lipidomic analyses to study the role of lipids played in CHO-S cells. In our study, we have applied chromatography (TLC) methods for lipid analysis in terms of lipid polarity. For polar lipids, 2-D HPTLC (2-dimensional high performance TLC) was used instead of conventional 1D- TLC by virtue of its high separation capacity. The eluting solvent system was optimized for the 1st and 2nd dimension, respectively. Neutral lipids were separated on 1-D HPTLC with the optimal elution solvent of hexane-diethyl ether-acetic acid. The lipid spots on the TLC plates were stained by 0.2% of 2,7-dichlorofluorescein dissolved in ethanol solution and illuminated with UV. Multiple lipid standards were also run to correctly identify the lipid spots and the fluorescence of lipid spots was semi-quantitatively measured with ImageJ. By optimization of TLC conditions, the lipids of CHO-S cell line were separated successfully and the lipid contents were semi-quantified. From neutral lipids result, we observed high level of certain lipids in CHO-S cell lines. We will further investigate which lipid play a key role in various cell processes

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

Cre-loxP-controlled cell-cycle checkpoint engineering in Chinese Hamster ovary cells

The gene amplification system is widely used in Chinese hamster ovary (CHO) cells for the productive cell line construction of therapeutic proteins. To enhance the efficiency of conventional gene amplification systems, we previously presented a novel method using cell-cycle checkpoint engineering1). Here, we constructed high-producing and stable cells by the conditional expression of mutant cell division cycle 25 homolog B (CDC25B) using the Cre-loxP system2). A bispecific antibody-producing CHO DG44-derived cell line was transfected with floxed mutant CDC25B. After inducing gene amplification in the presence of 250 nM methotrexate, mutant CDC25B sequence was removed by Cre recombinase protein expression. Overexpression of the floxed mutant CDC25B significantly enhanced the efficiency of transgene amplification and productivity. Moreover, the specific production rate of the isolated clone CHO Cre-1 and Cre-2 were approximately 11-fold and 15-fold higher than that of mock-transfected clone CHO Mock-S. Chromosomal aneuploidy was increased by mutant CDC25B overexpression, but Cre-1 and Cre-2 did not show any changes in chromosome number during long-term cultivation, as is the case with CHO Mock-S. Our results suggest that high-producing and stable cells can be constructed by conditionally controlling a cell-cycle checkpoint integrated in conventional gene amplification systems

The effect of swilling carbohydrate, menthol or a combination on 40km cycling time trial in the heat.

Introduction: Both carbohydrate and menthol mouth swills have shown ergogenic effects under a variety of settings. The aim of the current study was to compare the effect of the aforementioned mouth swill solutions on 40 km time trial (TT) performance in the heat (32°C, 40% humidity, 300kw radiant load) and investigate associated subjective measures (thermal comfort, thermal sensation, thirst, and RPE) every 5km. Methods: Six (6) recreationally trained male cyclists (31.8 ± 5.9 years, 178.2 ± 6.0 cm, 75.7 ± 10.0 kg) completed 3 trials, swilling either menthol (MEN), carbohydrate (CHO), or a combination (BOTH) at 10km intervals (5, 15, 25, 35km). Results: There was no statistically significant difference in 40km TT performance between mouth swills (P = 1.00), with MEN producing slightly quicker times on average (MEN 65:43 ± 4:48, CHO 66:09 ± 4:13, BOTH 65:57 ± 3:58 min:sec). Subjective measures were not significantly different, however MEN showed small (0.2-0.6) and moderate (0.6-1.2) effect size increases on thermal comfort compared to CHO and BOTH 5km post swill. Discussion: The ability to activate receptors in the oral cavity may be responsible for improved athletic performance due to potential central activation. The ability to perceptually cool and or fuel an athlete while exercising, especially in the heat, may allow for improved levels of thermal comfort and subsequently enhanced performance Take Home Message: Results, however, indicate that while MEN showed a beneficial effect on making participants feel more comfortable while exercising in the heat compared to CHO or BOTH, 40km TT was not significantly difference between solutions

Problems of Double Charm Production in e+e−e^+e^- Annihilation at s=10.6\sqrt{s}=10.6 GeV

Using the nonrelativistic QCD(NRQCD) factorization formalism, we calculate the color-singlet cross sections for exclusive production processes ${e^++e^-\to J/\psi+\eta_c}$~ and ~$e^++e^-\to J/\psi + \chi_{cJ}$~$(J=0,1,2)$ at the center-of-mass energy $\sqrt{s}$=10.6 GeV. The cross sections are estimated to be 5.5fb, 6.7fb, 1.1fb, and 1.6fb for $\eta_c, \chi_{c0}, \chi_{c1}$ and $\chi_{c2}$, respectively. The calculated $J/\psi+\eta_c$ production rate is smaller than the recent Belle data by about an order of magnitude, which might indicate the failure of perturbative QCD calculation to explain the double-charmonium production data. The complete $\cal{O}$$(\alpha^2_{s})$ color-singlet cross section for ${e^++e^-\to \chi_{c0}+ c\bar {c}}$ is calculated. In addition, we also evaluate the ratio of exclusive to inclusive production cross sections. The ratio of $J/\psi\eta_c$ production to $J/\psi c\bar{c}$ production could be consistent with the experimental data.Comment: 10 pages, 4 figures. A few references added, errors and typoes correcte