Differential response in downstream processing of CHO cells grown under mild hypothermic conditions.

The manufacture of complex therapeutic proteins using mammalian cells is well established, with several strategies developed to improve productivity. The application of sustained mild hypothermic conditions during culture has been associated with increases in product titer and improved product quality. However, despite associated cell physiological effects, very few studies have investigated the impact on downstream processing (DSP). Characterization of cells grown under mild hypothermic conditions demonstrated that the stationary phase was prolonged by delaying the onset of apoptosis. This enabled cells to maintain viability for extended periods and increase volumetric productivity from 0.74 to 1.02 g L(-1) . However, host cell proteins, measured by ELISA, increased by ∼50%, attributed to the extended time course and higher peak and harvest cell densities. The individual components making up this impurity, as determined by SELDI-TOF MS and 2D-PAGE, were shown to be largely comparable. Under mild hypothermic conditions, cells were less shear sensitive than those maintained at 37°C, enhancing the preliminary primary recovery step. Adaptive changes in membrane fluidity were further investigated by adopting a pronounced temperature shift immediately prior to primary recovery and the improvement observed suggests that such a strategy may be implementable when shear sensitivity is of concern. Early and late apoptotic cells were particularly susceptible to shear, at either temperature, even under the lowest shear rate investigated. These findings demonstrate the importance of considering the impact of cell culture strategies and cell physiology on DSP, by implementing a range of experimental methods for process characterization. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 2013

High throughput automated microbial bioreactor system used for clone selection and rapid scale-down process optimization

High throughput automated fermentation systems have become a useful tool in early bioprocess development. In this study, we investigated a 24 x 15 mL single use microbioreactor system, ambr 15f, designed for microbial culture. We compared the fed-batch growth and production capabilities of this system for two Escherichia coli strains, BL21 (DE3) and MC4100, and two industrially relevant molecules, hGH and scFv. In addition, different carbon sources were tested using bolus, linear or exponential feeding strategies, showing the capacity of the ambr 15f system to handle automated feeding. We used power per unit volume (P/V) as a scale criterion to compare the ambr 15f with 1 L stirred bioreactors which were previously scaled-up to 20 L with a different biological system, thus showing a potential 1,300 fold scale comparability in terms of both growth and product yield. By exposing the cells grown in the ambr 15f system to a level of shear expected in an industrial centrifuge, we determined that the cells are as robust as those from a bench scale bioreactor. These results provide evidence that the ambr 15f system is an efficient high throughput microbial system that can be used for strain and molecule selection as well as rapid scale-up. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 2017

Holistic process development to mitigate proteolysis of a subunit rotavirus vaccine candidate produced in Pichia pastoris by means of an acid pH pulse during fed‐batch fermentation

To meet the challenges of global health, vaccine design and development must be reconsidered to achieve cost of goods as low as 15¢ per dose. A new recombinant protein‐based rotavirus vaccine candidate derived from non‐replicative viral subunits fused to a P2 tetanus toxoid CD4(+) T cell epitope is currently under clinical development. We have sought to simplify the existing manufacturing process to meet these aims. To this end, we have taken a holistic process development approach to reduce process complexity and costs while producing a product with the required characteristics. We have changed expression system from Escherichia coli to Pichia pastoris, to produce a secreted product, thereby reducing the number of purification steps. However, the presence of proteases poses challenges to product quality. To understand the effect of fermentation parameters on product quality small‐scale fermentations were carried out. Media pH and fermentation duration had the greatest impact on the proportion of full‐length product. A novel acidic pH pulse strategy was used to minimize proteolysis, and this combined with an early harvest time significantly increased the proportion of full‐length material (60–75%). An improved downstream process using a combination of CIEX and AIEX to further reduce proteases, resulted in maintaining product quality (95% yield)

Impact of Safety-Related Dose Reductions or Discontinuations on Sustained Virologic Response in HCV-Infected Patients: Results from the GUARD-C Cohort.

BACKGROUND: Despite the introduction of direct-acting antiviral agents for chronic hepatitis C virus (HCV) infection, peginterferon alfa/ribavirin remains relevant in many resource-constrained settings. The non-randomized GUARD-C cohort investigated baseline predictors of safety-related dose reductions or discontinuations (sr-RD) and their impact on sustained virologic response (SVR) in patients receiving peginterferon alfa/ribavirin in routine practice. METHODS: A total of 3181 HCV-mono-infected treatment-naive patients were assigned to 24 or 48 weeks of peginterferon alfa/ribavirin by their physician. Patients were categorized by time-to-first sr-RD (Week 4/12). Detailed analyses of the impact of sr-RD on SVR24 (HCV RNA <50 IU/mL) were conducted in 951 Caucasian, noncirrhotic genotype (G)1 patients assigned to peginterferon alfa-2a/ribavirin for 48 weeks. The probability of SVR24 was identified by a baseline scoring system (range: 0-9 points) on which scores of 5 to 9 and <5 represent high and low probability of SVR24, respectively. RESULTS: SVR24 rates were 46.1% (754/1634), 77.1% (279/362), 68.0% (514/756), and 51.3% (203/396), respectively, in G1, 2, 3, and 4 patients. Overall, 16.9% and 21.8% patients experienced ≥1 sr-RD for peginterferon alfa and ribavirin, respectively. Among Caucasian noncirrhotic G1 patients: female sex, lower body mass index, pre-existing cardiovascular/pulmonary disease, and low hematological indices were prognostic factors of sr-RD; SVR24 was lower in patients with ≥1 vs. no sr-RD by Week 4 (37.9% vs. 54.4%; P = 0.0046) and Week 12 (41.7% vs. 55.3%; P = 0.0016); sr-RD by Week 4/12 significantly reduced SVR24 in patients with scores <5 but not ≥5. CONCLUSIONS: In conclusion, sr-RD to peginterferon alfa-2a/ribavirin significantly impacts on SVR24 rates in treatment-naive G1 noncirrhotic Caucasian patients. Baseline characteristics can help select patients with a high probability of SVR24 and a low probability of sr-RD with peginterferon alfa-2a/ribavirin.This study was sponsored by F. Hoffmann-La Roche Ltd, Basel, Switzerland. Support for third-party writing assistance for this manuscript, furnished by Blair Jarvis MSc, ELS, of Health Interactions, was provided by F. Hoffmann-La Roche Ltd, Basel, Switzerland

Multi-messenger observations of a binary neutron star merger

On 2017 August 17 a binary neutron star coalescence candidate (later designated GW170817) with merger time 12:41:04 UTC was observed through gravitational waves by the Advanced LIGO and Advanced Virgo detectors. The Fermi Gamma-ray Burst Monitor independently detected a gamma-ray burst (GRB 170817A) with a time delay of ~1.7 s with respect to the merger time. From the gravitational-wave signal, the source was initially localized to a sky region of 31 deg2 at a luminosity distance of 40+8-8 Mpc and with component masses consistent with neutron stars. The component masses were later measured to be in the range 0.86 to 2.26 Mo. An extensive observing campaign was launched across the electromagnetic spectrum leading to the discovery of a bright optical transient (SSS17a, now with the IAU identification of AT 2017gfo) in NGC 4993 (at ~40 Mpc) less than 11 hours after the merger by the One- Meter, Two Hemisphere (1M2H) team using the 1 m Swope Telescope. The optical transient was independently detected by multiple teams within an hour. Subsequent observations targeted the object and its environment. Early ultraviolet observations revealed a blue transient that faded within 48 hours. Optical and infrared observations showed a redward evolution over ~10 days. Following early non-detections, X-ray and radio emission were discovered at the transient’s position ~9 and ~16 days, respectively, after the merger. Both the X-ray and radio emission likely arise from a physical process that is distinct from the one that generates the UV/optical/near-infrared emission. No ultra-high-energy gamma-rays and no neutrino candidates consistent with the source were found in follow-up searches. These observations support the hypothesis that GW170817 was produced by the merger of two neutron stars in NGC4993 followed by a short gamma-ray burst (GRB 170817A) and a kilonova/macronova powered by the radioactive decay of r-process nuclei synthesized in the ejecta

The Effect of Macular Hole Duration on Surgical Outcomes: An Individual Participant Data Study of Randomized Controlled Trials

Topic: To define the effect of symptom duration on outcomes in people undergoing surgery for idiopathic full-thickness macular holes (iFTMHs) by means of an individual participant data (IPD) study of randomized controlled trials (RCTs). The outcomes assessed were primary iFTMH closure and postoperative best-corrected visual acuity (BCVA). Clinical Relevance: Idiopathic full-thickness macular holes are visually disabling with a prevalence of up to 0.5%. Untreated BCVA is typically reduced to 20/200. Surgery can close holes and improve vision. Symptom duration is thought to affect outcomes with surgery, but the effect is unclear. Methods: A systematic review identified eligible RCTs that included adults with iFTMH undergoing vitrectomy with gas tamponade in which symptom duration, primary iFTMH closure, and postoperative BCVA were recorded. Bibliographic databases were searched for articles published between 2000 and 2020. Individual participant data were requested from eligible studies. Results: Twenty eligible RCTs were identified. Data were requested from all studies and obtained from 12, representing 940 eyes in total. Median symptom duration was 6 months (interquartile range, 3–10). Primary closure was achieved in 81.5% of eyes. There was a linear relationship between predicted probability of closure and symptom duration. Multilevel logistic regression showed each additional month of duration was associated with 0.965 times lower odds of closure (95% confidence interval [CI], 0.935–0.996, P = 0.026). Internal limiting membrane (ILM) peeling, ILM flap use, better preoperative BCVA, face-down positioning, and smaller iFTMH size were associated with increased odds of primary closure. Median postoperative BCVA in eyes achieving primary closure was 0.48 logarithm of the minimum angle of resolution (logMAR) (20/60). Multilevel logistic regression showed for eyes achieving primary iFTMH closure, each additional month of symptom duration was associated with worsening BCVA by 0.008 logMAR units (95% CI, 0.005–0.011, P < 0.001) (i.e., ∼1 Early Treatment Diabetic Retinopathy Study letter loss per 2 months). ILM flaps, intraocular tamponade using long-acting gas, better preoperative BCVA, smaller iFTMH size, and phakic status were also associated with improved postoperative BCVA. Conclusions: Symptom duration was independently associated with both anatomic and visual outcomes in persons undergoing surgery for iFTMH. Time to surgery should be minimized and care pathways designed to enable this

Epidemiology and clinical features of vivax malaria imported to Europe: Sentinel surveillance data from TropNetEurop

BACKGROUND: Plasmodium vivax is the second most common species among malaria patients diagnosed in Europe, but epidemiological and clinical data on imported P. vivax malaria are limited. The TropNetEurop surveillance network has monitored the importation of vivax malaria into Europe since 1999. OBJECTIVES: To present epidemiological and clinical data on imported P. vivax malaria collected at European level. MATERIAL AND METHODS: Data of primary cases of P. vivax malaria reported between January 1999 and September 2003 were analysed, focusing on disease frequency, patient characteristics, place of infection, course of disease, treatment and differences between network-member countries. RESULTS: Within the surveillance period 4,801 cases of imported malaria were reported. 618 (12.9%) were attributed to P. vivax. European travellers and immigrants were the largest patient groups, but their proportion varied among the reporting countries. The main regions of infection in descending order were the Indian subcontinent, Indonesia, South America and Western and Eastern Africa, as a group accounting for more than 60% of the cases. Regular use of malaria chemoprophylaxis was reported by 118 patients. With 86 (inter-quartile range 41–158) versus 31 days (inter-quartile range 4–133) the median symptom onset was significantly delayed in patients with chemoprophylaxis (p < 0.0001). Common complaints were fever, headache, fatigue, and musculo-skeletal symptoms. All patients survived and severe clinical complications were rare. Hospitalization was provided for 60% and primaquine treatment administered to 83.8% of the patients, but frequencies varied strongly among reporting countries. CONCLUSIONS: TropNetEurop data can contribute to the harmonization of European treatment policies

Lattice Boltzmann method for the fractional advection-diffusion equation

Mass transport, such as movement of phosphorus in soils and solutes in rivers, is a natural phenomenon and its study plays an important role in science and engineering. It is found that there are numerous practical diffusion phenomena that do not obey the classical advection-diffusion equation (ADE). Such diffusion is called abnormal or superdiffusion, and it is well described using a fractional advection-diffusion equation (FADE). The FADE finds a wide range of applications in various areas with great potential for studying complex mass transport in real hydrological systems. However, solution to the FADE is difficult, and the existing numerical methods are complicated and inefficient. In this study, a fresh lattice Boltzmann method is developed for solving the fractional advection-diffusion equation (LabFADE). The FADE is transformed into an equation similar to an advection-diffusion equation and solved using the lattice Boltzmann method. The LabFADE has all the advantages of the conventional lattice Boltzmann method and avoids a complex solution procedure, unlike other existing numerical methods. The method has been validated through simulations of several benchmark tests: a point-source diffusion, a boundary-value problem of steady diffusion, and an initial-boundary-value problem of unsteady diffusion with the coexistence of source and sink terms. In addition, by including the effects of the skewness β , the fractional order α , and the single relaxation time τ , the accuracy and convergence of the method have been assessed. The numerical predictions are compared with the analytical solutions, and they indicate that the method is second-order accurate. The method presented will allow the FADE to be more widely applied to complex mass transport problems in science and engineering