ABSTRACT
Nucleic acid vaccines have been proven to be a revolutionary technology to induce an efficient, safe and rapid response against pandemics, like the coronavirus disease (COVID-19). Minicircle DNA (mcDNA) is an innovative vector more stable than messenger RNA and more efficient in cell transfection and transgene expression than conventional plasmid DNA. This work describes the construction of a parental plasmid (PP) vector encoding the receptor-binding domain (RBD) of the S protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the use of the Design of Experiments (DoE) to optimize PP recombination into mcDNA vector in an orbital shaker. First, the results revealed that host cells should be grown at 42 °C and the Terrific Broth (TB) medium should be replaced by Luria Broth (LB) medium containing 0.01% L-arabinose for the induction step. The antibiotic concentration, the induction time, and the induction temperature were used as DoE inputs to maximize the % of recombined mcDNA. The quadratic model was statistically significant (p-value < 0.05) and presented a non-significant lack of fit (p-value > 0.05) with a suitable coefficient of determination. The optimal point was validated using 1 h of induction, at 30 °C, without the presence of antibiotics, obtaining 93.87% of recombined mcDNA. Based on these conditions, the production of mcDNA was then maximized in a mini-bioreactor platform. The most favorable condition obtained in the bioreactor was obtained by applying 60% pO2 in the fermentation step during 5 h and 30% pO2 in the induction step, with 0.01% L-arabinose throughout 5 h. The yield of mcDNA-RBD was increased to a concentration of 1.15 g/L, when compared to the orbital shaker studies (16.48 mg/L). These data revealed that the bioreactor application strongly incremented the host biomass yield and simultaneously improved the recombination levels of PP into mcDNA. Altogether, these results contributed to improving mcDNA-RBD biosynthesis to make the scale-up of mcDNA manufacture simpler, cost-effective, and attractive for the biotechnology industry.
ABSTRACT
IMPORTANCE: Pharmacists are among the healthcare professionals involved in the response to the COVID-19 pandemic, maintaining essential services. In the context of restrictions and reorganization of human resources, as a result of policies recently applied to the health sector, following international guidelines, and given the scarcity of data on burnout in pharmaceutical activity, it was considered highly relevant to promote a nationwide survey aiming to collect more complete evidence on the burnout syndrome and to understand how pharmacists have viewed their work and the people they worked closely with. OBJECTIVES: The study aimed to assess the occurrence of pharmacist burnout and determine outcomes for each of its dimensions; identify potentially associated characteristics; and determine profiles and critical limits. DESIGN: A cross-sectional observational study conducted by a multidisciplinary panel from the Portuguese Pharmaceutical Society (PPS). Professionals from community and hospital pharmacies who were at the forefront of the COVID-19 response were involved in (i) confirming the need and pertinence for conducting this research, (ii) identifying the main factors leading to pharmaceutical emotional distress ("burnout"), and (iii) disseminating the survey. The questionnaire was designed for digital voluntary, confidential, and anonymous participation and divided into four segments of data collection: (i) demographics, (ii) employment and workplace characterization, (iii) pandemic impact on labor activity, and (iv) burnout assessment (as described ahead). SETTING: An electronic survey was addressed to all PPS members, and an account was created solely to manage the questionnaire data for the research team. The web-based and user-friendly platform Google Forms supported the data capture and provided an intuitive interface for validated data entry. PARTICIPANTS: In a population of 15,565 pharmacists (members of the PPS), the minimum recommended sample size (Epi Info software), with a 5% margin of error and a 99.9% confidence interval, should be 1,012 individuals. A total of 1,362 pharmacists participated in the study. Of these, 91.4% (n = 1,246) were involved in direct patient care activity and 7.7% (n = 106) in non-direct patient care activity.