Clear insight into complex multimodal resins and impurities to overcome recombinant protein purification challenges: A review.

Increasing attention has been paid to the purity of therapeutic proteins imposing extensive costs and challenges to the downstream processing of biopharmaceuticals. One of the efforts, that has been exerted to overcome such limitations, was developing multimodal or mixed-mode chromatography (MMC) resins for launching selective, orthogonal, non-affinity purification platforms. Despite relatively extensive usage of MMC resins, their real potential and fulfillment have not been extensively reviewed yet. In this work, the explanation of practical and key aspects of downstream processing of recombinant proteins with or without MMC resins was debated, as being useful for further purification process development. This review has been written as a step-by-step guide to deconvolute both inherent protein purification and MMC complexities. Here, after complete elucidation of the potential of MMC resins, the effects of frequently used additives (mobile phase modifiers) and their possible interactions during the purification process, the critical characteristics of common product-related impurities (e.g., aggregates, charge variants, fragments), host-related impurities (e.g., host cell protein and DNA) and process related impurities (e.g., endotoxin, and viruses) with solved or unsolved challenges of traditional and MMC resins have been discussed. Such collective experiences which are reported in this study could be considered as an applied guide for developing successful downstream processing in challenging conditions by providing a clear insight into complex MMC resins and impurities.

DOE-based process optimization for development of efficient methods for purification of recombinant hepatitis B surface antigen from Pichia pastoris feedstock using Capto adhere resin.

Background: The multimodal chromatography resins, such as Capto adhere, are considered good candidates to be utilized in downstream processing due to their high capacity and selectivity; however, their multimodal interactions lead to an intricacy in the adsorption-desorption patterns and systematic characterization of conditions for process steps is necessary. Methods: Capto adhere, a strong ion exchanger with multimodal functionality, was used in this study for the final aim of recombinant hepatitis B surface antigen (rHBsAg) purification from Pichia pastoris (P. pastoris) industrial feedstock. Optimization of various parameters was done using the design of experiments (DOE) approach to determine the best binding and non-binding conditions. Results: Maximum rHBsAg binding on Capto adhere occurred in 20 mM sodium acetate, pH 4.5, and a binding capacity of about 0.75 mg/ml was achieved, which was much higher than rHBsAg binding capacity of other resins reported so far. In elution optimization investigations, it was revealed that 1 M arginine (buffered in 50 mM sodium phosphate, pH 6.5) was the most efficient eluting agent. The binding and elution optimal conditions were utilized for further purification of rHBsAg from P. pastoris industrial feedstock in bind-elute mode, and the recovery and purity of the obtained rHBsAg were about 60% and 100%, respectively. Following optimization in the flow-through purification mode, the target protein recovery was significantly increased (up to 97%) and the target protein purity of more than 95% was achievable. SEC-HPLC analysis showed that the obtained retention times for the purified rHBsAg were similar to those reported previously. Conclusions: These results suggest that Capto adhere under such optimized conditions can be considered as a good candidate for efficient purification of rHBsAg from P. pastoris industrial feedstock in downstream processing.

Toxicity of Pesticides Applied in European Vineyards on Anagyrus vladimiri and Trichogramma evanescens, Parasitoids of Planococcus ficus and Lobesia botrana.

Risk assessments of chemical pesticides toward natural enemies are crucial for ensuring sustainable grapevine-integrated pest management. In this context, laboratory experiments were conducted to evaluate the toxicity of four insecticides (lambda-cyhalothrin, flupyradifurone, acetamiprid, and cyantraniliprole) and one fungicide (spiroxamine) commonly applied in German (European) vineyards on the pupae and adults of both Anagyrus vladimiri, a parasitoid of the vine mealybug Planococcus ficus, and Trichogramma evanescens, a parasitoid of the European grapevine moth, Lobesia botrana. The tested pesticides did not significantly affect the development of the pupal stage inside mealybug mummies or the emergence of the parasitoid A. vladimiri. The pesticides flupyradifurone, acetamiprid, and spiroxamine resulted in the highest mortality percentages for all emerged A. vladimiri parasitoids at 8 and 10 days after treatment compared with either in lambda-cyhalothrin or cyantraniliprole. However, all pesticides, except the diamide insecticide cyantraniliprole, significantly affected the development of the pupal stage and the emergence of the parasitoid T. evanescens. The percentages of T. evanescens emergence following the application of the fungicide spiroxamine or either lambda-cyhalothrin or flupyradifurone were significantly higher than those observed in the acetamiprid treatment. Regarding direct contact toxicity, the highest percentages (100%) of A. vladimiri adult parasitoid mortality were obtained in the flupyradifurone, acetamiprid, and spiroxamine treatments, while the lowest mortality percentages were observed in lambda-cyhalothrin, cyantraniliprole, and untreated control treatments. According to the IOBC classes of toxicity, flupyradifurone, acetamiprid, and spiroxamine were classified as harmful, while both lambda-cyhalothrin and cyantraniliprole were classified as slightly harmful to A. vladimiri adults. As such, all pesticides had a significant impact on the survival of exposed T. evanescens adults. The highest percentages of adult T. evanescens mortality were obtained in the flupyradifurone, acetamiprid, and spiroxamine treatments, with the fungicide spiroxamine resulting in significantly higher mortality percentages than either flupyradifurone or acetamiprid, while the lowest mortality percentages were found in the lambda-cyhalothrin and cyantraniliprole treatments. Therefore, applying the insecticides acetamiprid and/or flupyradifurone and the fungicide spiroxamine should be avoided when A. vladimiri and/or T. evanescens are naturally present or released in grapes. The insights gained from these two easy-to-rear parasitoid species allow analogous conclusions to be drawn for closely related species in vineyards belonging to either family Encyrtidae or Trichogrammatidae, which are not easy to rear. Interestingly, using the safer insecticides lambda-cyhalothrin and/or cyantraniliprole could be compatible with both parasitoid species, which could be sustainably exploited in either conservation or augmentative biological control in vineyards.

Determining buffer conditions for downstream processing of VLP-based recombinant hepatitis B surface antigen using multimodal resins in bind-elute and flow-through purification modes.

The difficulties in purification of VLP-based recombinant hepatitis B surface antigen (rHBsAg) are mainly emerged from inefficient semi-purification step plus proteins physicochemical properties and these issues make the downstream processing (DSP) very lengthy and expensive. In this study, optimization of rHBsAg (recombinantly-expressed in Pichia pastoris) DSP was performed using selection of buffering conditions in the semi-purification step. In the semi-purification optimization step, up to 73% of the protein impurities were eliminated and the utmost increase in rHBsAg purity (ca. 3.6-fold) was achieved using 20 mM sodium acetate, pH 4.5. By using rHBsAg binding and nonbinding situations obtained from the response surface plot in design of experiments (DOE), additional bind-elute and flow-through purification mode experiments were conducted and rHBsAg with high purity (near 100%) and recovery (> 83%) was achieved. Following assessment of critical quality attributes (i.e., purity, particle size distribution, host cell DNA, host cell protein, secondary structures, specific activity and relative potency), it was indicated that the characteristics of rHBsAg purified by the new DSP were similar or superior to the ones obtained from conventional DSP. The purification performance of the resin was constantly retained (97-100%) and no significant resin damage took place after 10 adsorption-elution-cleaning cycles. The new DSP developed for production of rHBsAg in this study can substitute the conventional one with granting satisfactory target protein quality, long-lasting resin efficacy, shorter and less expensive process. This process may be also employable for purification of both non-VLP- and VLP- based target proteins expressed in the yeast.

A Comprehensive Comparison of Rapid RNA Extraction Methods for Detection of SARS-CoV-2 as the Infectious Agent of the Upper Respiratory Tract using Direct RT-LAMP Assay.

Background: The current COVID-19 pandemic has highlighted the need for faster and more cost-effective diagnostic methods. The RNA extraction step in current diagnostic methods, such as real-time qPCR, increases the cost and time required for testing. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is a promising technique for developing diagnostic tests with desired sensitivity and specificity without the need for RNA extraction. Materials and Methods: An RT-LAMP assay was developed to detect SARS-CoV-2 with a sensitivity of 0.5 copies of positive control plasmid per microliter in 40 min. Several rapid RNA extraction protocols were evaluated using different reagents, including bovine serum albumin, Triton X-100, Tween 20, proteinase K, guanidine hydrochloride, guanidinium isothiocyanate (GITC), and thermal treatment. Finally, the sensitivity and specificity of the developed direct RT-LAMP were determined using 150 upper respiratory tract samples. Results: Method 10 was selected as the most efficient protocol for the RNA extraction step. The sensitivity and specificity of the developed direct RT-LAMP assay with clinical samples were estimated at 98.4% and 88.8%, respectively. Conclusion: These results suggest that the combination of GITC and Triton X-100 detergent is a highly efficient method for RNA extraction and direct RT-LAMP detection of SARS-CoV-2 in clinical samples, providing a valuable tool for the rapid and cost-effective diagnosis of COVID-19.

Intelligent Model of Nursing Shift in Tehran University of Medical Sciences, Tehran, Iran.

Background: Nurses play a key role in increasing the efficiency of healthcare systems. Given the 24-hour performance of hospitals and the small number of nurses in the field of treatment, it is quintessential to re-shift them in the hospital. This study set out to achieve coherence in nursing shift planning and justice in the order of shifts in hospital. Methods: This applied and a developmental study was performed from 2019 to 2020. We used genetic algorithm to provide operational solutions and define flexible shifts and plan nurses' working hours in Yas Hospital, Tehran University of Medical Sciences Hospital, Tehran, Iran. Results: Based on the selection of each nurse and determining the approved shifts of each ward, the possibility of appropriate planning was provided to determine the required shifts per month and to estimate the needs of each department. Conclusion: Using genetic algorithm and nursing shift in office automation console provides useful tools for managers at all organizational levels, according to which a good balance between the hospital's need for nurse and nurses' demands in different time periods.

Design and Psychometrics of the Assessment Instrument for Innovation Capabilities of Medical Sciences Universities Using the Cube Model Approach.

BACKGROUND: To investigate any subject, a scholar needs a suitable instrument to collect the required information with the utmost accuracy and the least amount of error. Therefore, this study aimed at designing and conducting a psychometric analysis of an assessment instrument for innovation capabilities of Medical Sciences Universities using the Cube Model Approach. METHODS: This study began by searching in questionnaires in the fields of input and process, considering innovation outcomes. Accordingly, a preliminary questionnaire was developed, and in the second stage, to determine the validity of the designed instrument, the face validity, content validity, and construct validity of the instrument were approved, and in the third stage, using Cronbach's alpha, its reliability was assessed. At first 200 phrases were obtained, finally, 25 questions were initially approved in three areas of structure (input), innovation processes, and output. All the phrases were retained in the face validity and content validity carried out quantitatively and qualitatively. RESULTS: Exploratory Factor Analysis was performed on 25 items, and finally the terms were set in six factors. These factors explained 53.19% of the total variance. The rotated factor loading for all questions was obtained more than 0.3, and therefore, no questions were eliminated. Calculation of the Cronbach's alpha coefficient confirmed the high internal consistency of the questionnaire (0.762). CONCLUSION: This instrument was designed for the first time in the context of Iranian academic culture and seems to be a suitable instrument for the assessment of innovation capabilities, considering its adequate validity and reliability, simplicity, and practicality.

Innovation Network Development Model in Telemedicine: A Change in Participation.

OBJECTIVES: This paper introduces a telemedicine innovation network and reports its implementation in Tehran University of Medical Sciences. The required conditions for the development of future projects in the field of telemedicine are also discussed; such projects should be based on the common needs and opportunities in the areas of healthcare, education, and technology. METHODS: The development of the telemedicine innovation network in Tehran University of Medical Sciences was carried out in two phases: identifying the beneficiaries of telemedicine, and codification of the innovation network memorandum; and brainstorming of three workgroup members, and completion and clustering ideas. The present study employed a qualitative survey by using brain storming method. Thus, the ideas of the innovation network members were gathered, and by using Freeplane software, all of them were clustered and innovation projects were defined. RESULTS: In the services workgroup, 87 and 25 ideas were confirmed in phase 1 and phase 2, respectively. In the education workgroup, 8 new programs in the areas of telemedicine, tele-education and teleconsultation were codified. In the technology workgroup, 101 and 11 ideas were registered in phase 1 and phase 2, respectively. CONCLUSIONS: Today, innovation is considered a major infrastructural element of any change or progress. Thus, the successful implementation of a telemedicine project not only needs funding, human resources, and full equipment. It also requires the use of innovation models to cover several different aspects of change and progress. The results of the study can provide a basis for the implementation of future telemedicine projects using new participatory, creative, and innovative models.

The simulation model of teleradiology in telemedicine project.

Telemedicine projects are aimed at offering medical services to people who do not have access to direct diagnosis and treatment services. As a powerful tool for analyzing the performance of complex systems and taking probable events into consideration, systemic simulation can facilitate the analysis of implementation processes of telemedicine projects in real-life-like situations. The aim of the present study was to propose a model for planning resource capacities and allocating human and operational resources to promote the efficiency of telemedicine project by investigating the process of teleradiology. In this article, after verification of the conceptual model by the experts of this field, the computerized simulation model is developed using simulation software Arena. After specifying the required data, different improvement scenarios are run using the computerized model by feeding the data into the software and validation and verification of the model. Fixing input data of the system such as the number of patients, their waiting time, and process time of each function, for example, magnetic resonance imaging or scan, has been compared with the current radiology process. Implementing the teleradiology model resulted in reduction of time of patients in the system (current: 1.84 ± 0.00, tele: 0.81 ± 0.00). Furthermore, through this process, they can allocate the lower resources to perform better functions of staff. The use of computerized simulation is essential for designing processes, optimal allocation of resources, planning, and making appropriate decisions for providing timely services to patients.