The insect cell-baculovirus expression vector system (IC-BEVS) has shown to be a powerful platform to produce complex biopharmaceutical products, such as recombinant proteins and VLPs. More recently IC-BEVS has been also used as an alternative to produce adeno-associated virus (AAV). However, little is known about the variability of insect cell populations and the potential effect of heterogeneity on product titer and/or quality. In this study, transcriptomics analysis of Sf9 insect cells during the production of recombinant AAV using a low multiplicity of infection, dual-baculovirus system was performed via single-cell RNA-seq (scRNA-seq). Before infection, the principal source of variability in Sf9 insect cells was associated to cell cycle. Over the course of infection, an increase in transcriptional heterogeneity was detected, this being linked to the expression of baculovirus genes as well as to differences in AAV transgenes ( rep, cap and gfp) expression. Noteworthy, at 24 hours post-infection (hpi) only 29 % of cells showed to enclose all three necessary AAV transgenes to produce packed AAV particles, indicating limitations of the dual baculovirus system. In addition, the trajectory analysis herein performed highlighted biological processes such as protein folding, metabolic processes, translation and stress response has been significantly altered upon infection. Overall, this work reports the first application of scRNA-seq to the IC-BEVS and highlights significant variations in individual cells within the population, providing insight for rational cell and process engineering towards improved AAV production in IC-BEVS.

SARS-CoV-2 is an RNA coronavirus that causes severe acute pneumonia, also known as COVID 19 disease. The World Health Organization declared the COVID-19 outbreak in January 2020 and a pandemic 2 months later. Serological assays are valuable tools to study virus spread among the population and, importantly, to identify individuals that were already infected and would be potentially immune to a virus re-infection. SARS-CoV-2 Spike protein and its Receptor Binding Domain (RBD) are the antigens with higher potential to develop SARS-CoV-2 serological assays. Moreover, structural studies of these antigens are key to understand the molecular basis for Spike interaction with angiotensin converting enzyme 2 receptor, hopefully enabling the discovery and development of COVID-19 therapeutics. Thus, it is urgent that significant amounts of this protein became available at the highest quality. In this work we evaluated the impact of different and scalable bioprocessing approaches on Spike and RBD production yields and, more importantly, in these antigens’ quality attributes. Using negative and positive sera collected from human donors, we show an excellent performance of the produced antigens, assessed in serologic ELISA tests, as denoted by the high specificity and sensitivity of the test. We have shown that, despite of the human cell host and the cell culture strategy used, for production scales ranging from 1 L to up to 30 L, final yields of approx. 2 mg and 90 mg per liter of purified bulk for Spike and RBD, respectively, could be obtained. To the best of our knowledge these are the highest yields for RBD production reported to date. An in-depth characterization of SARS CoV-2 Spike and RBD proteins was also performed, namely the antigens oligomeric state, glycosylation profiles and thermal stability during storage. The correlation of these quality attributes with ELISA performance show equivalent reactivity to SARS CoV 2 positive serum, for all Spike and RBD produced, and for all the storage conditions tested. Overall, we provide herein straightforward protocols to produce high-quality SARS CoV-2 Spike and RBD antigens, that can be easily adapted to both academic and industrial settings; and integrate, for the first time, studies on the impact of bioprocess with an in-deep characterization of these proteins, correlating antigens glycosylation and biophysical attributes to performance of COVID-19 serologic tests. We strongly believe that our work will contribute to advance the current and recent knowledge on SARS-CoV-2 proteins and support the scientific society that is persistently searching for solutions for COVID-19 pandemics.