Identification of the critical cultivation parameters for scaling up the upstream process of recombinant human Follicle Stimulating Hormone production from Chinese Hamster Ovary cells

Biotechnology industry has experienced a massive development in the production of recombinant protein therapeutics over the past few decades. Stainless steel bioreactors have been considered the gold standards for the upstream processing for a long time. However, over the past decade, the single use technology has gained a big interest in the field especially with the mammalian and insect cell cultures. The single use based processes offered many advantages over the conventional ones. Disposable systems eliminated the need for cleaning in place (CIP), sterilization in place (SIP) and cleaning validation, reduced the risks of cross contamination, and they also offered less production turnaround times. In 2010, Rhein Minapharm Biogenetics, a leading company in biopharmaceutical production in the Middle East, presented the first single use technology based upstream process for recombinant human- follicle stimulating hormone (rh-FSH) production from Chinese hamster ovary (CHO) cells on the HyClone 50L single use bioreactor (SUB). The process was then scaled up to the BIOSTAT STR 200L bioreactor. However, although the BIOSTAT STR 200L bioreactor offered a higher cell density, the product concentration measured by ELISA was only two thirds, the concentration on HPLC was about 75% and the specific cell productivity was only about 50% when compared to the HyClone 50L SUB process. In the current study, the operational parameters that were observed to be different in the two processes were examined. The aim was to determine the critical cultivation parameters to be considered during the scaling of the process. Those parameters were tested at different operating values covering a wide but reasonable range. The experiments were run on the DASGIP lab scale bioreactor at a 750mL working volume. The tested parameters were the culture cell density, the carbon dioxide partial pressure (pCO2) and osmolality levels in the culture, and the effect of operating at as much as 10 folds higher power input per unit volume (P/V), the parameter which was also tested on the 200L scale. The experiments on the lab scale showed that the lower cell density and the higher pCO2 and osmolality levels, being comparable to those of the 50L process yielded results comparable to the HyClone 50L SUB process. On the other hand, operating at higher cell density and lower pCO2 and osmolality levels, being comparable to those of the 200L process, yielded results comparable to the BIOSTAT STR 200L process. No remarkable differences were observed either in the cell growth rate, the product titer or in the cell productivity when operating at P/V of ten folds difference. It was concluded that the cell density and the pCO2 and/or osmolality levels are the critical cultivation parameters in the lab scale experiments. These parameters are to be on focus and to be carefully optimized during scaling up of the process

Development, Characterization, and Optimization of a Multi-Chamber, Single-Use Bioreactor

Stirred-tank single-use bioreactors have proven their capability to successfully replace their stainless-steel counterparts in the biopharmaceutical industry. To date, however, only a five-fold volume expansion is achievable in a single-use stirred tank bioreactor, which in turn necessitates intermediate equipment to scale-up the culture to production volume. This work introduces a novel multi-chamber single-use bioreactor design that can help users save costs of purchase and qualification of equipment, reduce factory footprint, and reduce the risk of contamination. A bioreactor prototype is presented in this work that is made of two chambers of different volumes, interconnected as a single, closed system. The design and construction of the prototype is described in detail, and engineering characterization results are reported for both chambers and compared with published data for commercially available bioreactors. The results help identify some areas of potential improvements in the bioreactor design. Optimization of a bioreactor design can be an especially challenging process and can lead to an exceedingly large number of configurations and necessary experiments. Therefore, a computational fluid dynamics model is used in this work to model the multiphase flow in the 50 L chamber of the multi-chamber bioreactor prototype. The model results are validated with oxygen mass transfer coefficient (kLa) measurements within the prototype. The validated model is projected to predict the effect of using different sparger types and sizes and the effect of varying the impeller diameter on kLa. The simulations show that ring spargers result in a superior kLa compared to pipe spargers, with an optimum sparger-to-impeller diameter ratio of 0.8. Also, larger impellers are shown to improve kLa. A correlation of kLa is presented as a function of both the reactor geometry and operating conditions. The resulting correlation can be used to predict kLa in a bioreactor and to optimize its design and operating parameters. Finally, a full commercialization plan for the multi-chamber bioreactor is presented. The plan proves that the proposed design not only offers a solution to a variety of industry problems but also presents an attractive investment opportunity to further develop the technology to a market ready product

Effect of Rosemary and Sweet Marjoram on three Predacious Mites of the Family Phytoseiidae (Acari: Phytoseiidae)

The direct toxicity of two essential oils, Majorana hortensis, Moench and Rosmarinus officinalis L. to adult females of the predacious mites, Amblyseius zaheri Yousef and El-Borolossy, Amblyseius barkeri (Hughes) and Typhlodromus athiasae Porath and Swirski were tested. Rosemary oil was the most toxic to females of A. barkeri and the least to A. zaheri. In contrast,sweet marjoram oil was relatively toxic to T. athiasae and slightly toxic to A. barkeri. Both essential oils,decreased the food consumption rate at the concentration used for A.barkeri and A. zaheri. Females of A. barkeri and A. zaheri suffered a depression in reproduction when treated with 1% of rosemary oil.Both material used seems to be harmless to T. athiasae at 1%

Repellent and Oviposition-Deterring Activity of Rosemary and Sweet Marjoram on the Spider Mites Tetranychus urticae and Eutetranychus orientalis (Acari: Tetranychidae)

The deterrent and toxic effect of two essential oils, Majorana hortensis. Moench and Rosma- rinus officinalis L. on the two tetranychid mites Tetranychus urticae Koch and Eutetranychus orientalis (Klein) were studied under laboratory conditions. Both materials used were more potent for E. orientalis than against T. urticae with a significant increase in repellency. Leaf discs treated with increasing concentrations of the two oils showed increased mortality of both spider mites and reduction in the total numbers of eggs laid. This result could be due to the oil of the higher oxygenated compounds content that was more effective in this respect

A Comparative Study of the Effect of Some Mineral and Plant Oils on the Two Spotted Spider Mite Tetranychus urticae Koch (Acari: Tetranychidae)

The direct toxicity of some mineral and plant oils to the eggs and females of the two spotted spider mite Tetranychus urticae Koch was tested. KZ oil was toxic to the egg stage compared to adult female. In contrast, Natur'l oil has a close toxic effect for both stages of T. urticae. Bio-dux oil was proved to be toxic to adult female and relatively in toxic to egg stage. Females of T. urticae suffered a depression in reproduction and shortened longevity when kept on plant leaves previously treated with different oils. Laboratory studies indicated that the vegetable oil Natur'l oil was effective on all biological aspects of T. urticae studied

Influence of Mint and Peppermint on Tetranychus urticae and Some Predacious Mites of the Family Phytoseiidae (Acari: Tetranychidae: Phytoseiidae)

The deterrent and toxicity effects of mint, Mentha virdis L. and peppermint, Mentha piperita L. on Tetranychus urticae Koch were studied under laboratory conditions. M. virdis was more potent for T. urticae than M. piperita, with a significant increase in repellency. Leaf discs treated with increasing concentrations of both materials showed reduction in the total numbers of eggs laid. A high percentage of T. urticae mortality was recorded in case of M. virdis. The direct toxicity of both essential oils to the female of the predacious mites namely Typhlodromus athiase Porath and Swirski Phytoseius finitimus Ribaga, Amblyseius barkeri (Hughes), Amblyseius zaheri Yousef and El-Borolossy, Amblyseius yousefi Zaher and El-Borolossy and Amblyseius deleoni (Muma and Denmark) were tested. At LC 50 level, M. virdis was the most toxic to females A. yousefi and the least to females T. athiasae. With the exception of A. zaheri, M. piperita proved to be more toxic to the predacious mites tested than M. virdis. The results obtained chemically and biologically, may suggest that the higher percentage of the hydrocarbons of M. virdis were responsible for the toxic effect

Burnout among surgeons before and during the SARS-CoV-2 pandemic: an international survey

Background: SARS-CoV-2 pandemic has had many significant impacts within the surgical realm, and surgeons have been obligated to reconsider almost every aspect of daily clinical practice. Methods: This is a cross-sectional study reported in compliance with the CHERRIES guidelines and conducted through an online platform from June 14th to July 15th, 2020. The primary outcome was the burden of burnout during the pandemic indicated by the validated Shirom-Melamed Burnout Measure. Results: Nine hundred fifty-four surgeons completed the survey. The median length of practice was 10 years; 78.2% included were male with a median age of 37 years old, 39.5% were consultants, 68.9% were general surgeons, and 55.7% were affiliated with an academic institution. Overall, there was a significant increase in the mean burnout score during the pandemic; longer years of practice and older age were significantly associated with less burnout. There were significant reductions in the median number of outpatient visits, operated cases, on-call hours, emergency visits, and research work, so, 48.2% of respondents felt that the training resources were insufficient. The majority (81.3%) of respondents reported that their hospitals were included in the management of COVID-19, 66.5% felt their roles had been minimized; 41% were asked to assist in non-surgical medical practices, and 37.6% of respondents were included in COVID-19 management. Conclusions: There was a significant burnout among trainees. Almost all aspects of clinical and research activities were affected with a significant reduction in the volume of research, outpatient clinic visits, surgical procedures, on-call hours, and emergency cases hindering the training. Trial registration: The study was registered on clicaltrials.gov "NCT04433286" on 16/06/2020

Influence of the Sweet Basil, Ocimum basilicum L. on Some Predacious Mites of the Family Phytoseiidae (Acari: Phytoseiidae)

The direct toxicity of the essential oil, Ocimum basilicum L. to females of six species of predacious mites of the family phytoseiidae was tested. The phytoseiid mites tested namely, Typhlodromus athiasae Porath and Swirski, Euseius yousefi Zaher and El-Borolossy, Amblyseius zaheri Yousef and El-Borolossy, Amblyseius deleoni (Muma and Denmark), Amblyseius swirskii Athias-Henriot and Amblyseius barkeri (Hughes). Sweet basil oil was highly toxic to females E. yousefi and was relatively intoxic to females A. swirskii. The essential oil has a close toxic effect for predator species, T. athiasae and A. barkeri. With the exception of A. zaheri, females of all predacious mites tested suffered a depression in reproduction and food consumption when treated with sweet basil oil at conc. 2%

Effect of some essential oils on the predacious mite Amblyseius swirskii A. H. (Acari: Phytoseiidae)

The direct toxicity of four essential oils, Majorana hortensis Moench, Ros­marinus officinalis L., Mentha piperita L. and Lavandula officinalis Chaix, to adult females of the predacious mite Amblyseius swirskii Athias-Henriot were tested. Peppermint oil was the most toxic to females A. swiriskii while the French lavender oil was the least toxic to the pre­dator. All essential oils, at the two concentrations used, decreased the food consumption rate as well as egg laying. All four materials tested, at con­cen­tration 0.25% were considered to be safe for the predator since no mortalities had been recorded

Effect of French lavender essential oil on some predacious mites of the family Phytoseiidae (Acari: Phytoseiidae)

The direct toxicity of the essential oil Lavendula officinalis Chais to egg stage and adult females of the predacious mites, Euseius yousefi Zaher and El-Borolossy, Neoseiulus barkeri (hughes), Amblyseius zaheri Yousef and El-Borolossy and Typhlodromus athiase Porth and Swirski was studied. French Lavender oil was highly toxic to both stages of E. yousefi and relatively in toxic to T. athiase. A depression on consumption was recorded on A. zaheri and E. yousefi, while a depression on reproduction was reported only in case of E. yousefi, when females treated with LC25 concentration. French Lavender oil, at concentration of (LC25) was considered to be safe for A. zaheri, N. barkeri and T. athiase, since no mortalities had been recorded