Sulfate limitation increases specific plasmid DNA yield and productivity in E. coli fed-batch processes.

Plasmid DNA (pDNA) is a key biotechnological product whose importance became apparent in the last years due to its role as a raw material in the messenger ribonucleic acid (mRNA) vaccine manufacturing process. In pharmaceutical production processes, cells need to grow in the defined medium in order to guarantee the highest standards of quality and repeatability. However, often these requirements result in low product titer, productivity, and yield. In this study, we used constraint-based metabolic modeling to optimize the average volumetric productivity of pDNA production in a fed-batch process. We identified a set of 13 nutrients in the growth medium that are essential for cell growth but not for pDNA replication. When these nutrients are depleted in the medium, cell growth is stalled and pDNA production is increased, raising the specific and volumetric yield and productivity. To exploit this effect we designed a three-stage process (1. batch, 2. fed-batch with cell growth, 3. fed-batch without cell growth). The transition between stage 2 and 3 is induced by sulfate starvation. Its onset can be easily controlled via the initial concentration of sulfate in the medium. We validated the decoupling behavior of sulfate and assessed pDNA quality attributes (supercoiled pDNA content) in E. coli with lab-scale bioreactor cultivations. The results showed an increase in supercoiled pDNA to biomass yield by 33% and an increase of supercoiled pDNA volumetric productivity by 13 % upon limitation of sulfate. In conclusion, even for routinely manufactured biotechnological products such as pDNA, simple changes in the growth medium can significantly improve the yield and quality.

Implementation of proton transfer reaction-mass spectrometry (PTR-MS) for advanced bioprocess monitoring.

We report on the implementation of proton transfer reaction-mass spectrometry (PTR-MS) technology for on-line monitoring of volatile organic compounds (VOCs) in the off-gas of bioreactors. The main part of the work was focused on the development of an interface between the bioreactor and an analyzer suitable for continuous sampling of VOCs emanating from the bioprocess. The permanently heated sampling line with an inert surface avoids condensation and interaction of volatiles during transfer to the PTR-MS. The interface is equipped with a sterile sinter filter unit directly connected to the bioreactor headspace, a condensate trap, and a series of valves allowing for dilution of the headspace gas, in-process calibration, and multiport operation. To assess the aptitude of the entire system, a case study was conducted comprising three identical cultivations with a recombinant E. coli strain, and the volatiles produced in the course of the experiments were monitored with the PTR-MS. The high reproducibility of the measurements proved that the established sampling interface allows for reproducible transfer of volatiles from the headspace to the PTR-MS analyzer. The set of volatile compounds monitored comprises metabolites of different pathways with diverse functions in cell physiology but also volatiles from the process matrix. The trends of individual compounds showed diverse patterns. The recorded signal levels covered a dynamic range of more than five orders of magnitude. It was possible to assign specific volatile compounds to distinctive events in the bioprocess. The presented results clearly show that PTR-MS was successfully implemented as a powerful bioprocess-monitoring tool and that access to volatiles emitted by the cells opens promising perspectives in terms of advanced process control.

High shear resistance of insect cells: the basis for substantial improvements in cell culture process design.

Multicellular organisms cultivated in continuous stirred tank reactors (CSTRs) are more sensitive to environmental conditions in the suspension culture than microbial cells. The hypothesis, that stirring induced shear stress is the main problem, persists, although it has been shown that these cells are not so sensitive to shear. As these results are largely based on Chinese Hamster Ovary (CHO) cell experiments the question remains if similar behavior is valid for insect cells with a higher specific oxygen demand. The requirement of higher oxygen transfer rates is associated with higher shear forces in the process. Consequently, we focused on the shear resistance of insect cells, using CHO cells as reference system. We applied a microfluidic device that allowed defined variations in shear rates. Both cell lines displayed high resistance to shear rates up to 8.73 × 105 s-1. Based on these results we used microbial CSTRs, operated at high revolution speeds and low aeration rates and found no negative impact on cell viability. Further, this cultivation approach led to substantially reduced gas flow rates, gas bubble and foam formation, while addition of pure oxygen was no longer necessary. Therefore, this study contributes to the development of more robust insect cell culture processes.

Accelerating HIV-1 VLP production using stable High Five insect cell pools.

Stable cell pools are receiving a renewed interest as a potential alternative system to clonal cell lines. The shorter development timelines and the capacity to achieve high product yields make them an interesting approach for recombinant protein production. In this study, stable High Five cell pools are assessed for the production of a simple protein, mCherry, and the more complex HIV-1 Gag-eGFP virus-like particles (VLPs). Random integration coupled to fluorescence-activated cell sorting (FACS) in suspension conditions is applied to accelerate the stable cell pool generation process and enrich it with high producer cells. This methodology is successfully transferred to a bioreactor for VLP production, resulting in a 2-fold increase in VLP yields with respect to shake flask cultures. In these conditions, maximum viable cell concentration improves by 1.5-fold, and by-product formation is significantly reduced. Remarkably, a global increase in the uptake of amino acids in the Gag-eGFP stable cell pool is observed when compared with parental High Five cells, reflecting the additional metabolic burden associated with VLP production. These results suggest that stable High Five cell pools are a robust and powerful approach to produce VLPs and other recombinant proteins, and put the basis for future studies aiming to scale up this system.

A comprehensive antigen production and characterisation study for easy-to-implement, specific and quantitative SARS-CoV-2 serotests.

BACKGROUND: Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection or vaccination. While first-generation antibody tests have primarily provided qualitative results, accurate seroprevalence studies and tracking of antibody levels over time require highly specific, sensitive and quantitative test setups. METHODS: We have developed two quantitative, easy-to-implement SARS-CoV-2 antibody tests, based on the spike receptor binding domain and the nucleocapsid protein. Comprehensive evaluation of antigens from several biotechnological platforms enabled the identification of superior antigen designs for reliable serodiagnostic. Cut-off modelling based on unprecedented large and heterogeneous multicentric validation cohorts allowed us to define optimal thresholds for the tests' broad applications in different aspects of clinical use, such as seroprevalence studies and convalescent plasma donor qualification. FINDINGS: Both developed serotests individually performed similarly-well as fully-automated CE-marked test systems. Our described sensitivity-improved orthogonal test approach assures highest specificity (99.8%); thereby enabling robust serodiagnosis in low-prevalence settings with simple test formats. The inclusion of a calibrator permits accurate quantitative monitoring of antibody concentrations in samples collected at different time points during the acute and convalescent phase of COVID-19 and disclosed antibody level thresholds that correlate well with robust neutralization of authentic SARS-CoV-2 virus. INTERPRETATION: We demonstrate that antigen source and purity strongly impact serotest performance. Comprehensive biotechnology-assisted selection of antigens and in-depth characterisation of the assays allowed us to overcome limitations of simple ELISA-based antibody test formats based on chromometric reporters, to yield comparable assay performance as fully-automated platforms. FUNDING: WWTF, Project No. COV20-016; BOKU, LBI/LBG.

Evaluation of screening platforms for virus-like particle production with the baculovirus expression vector system in insect cells.

Recombinant protein and virus-like particle (VLP) production based on the baculovirus expression vector system is fast, flexible, and offers high yields. Independent from the product, a multitude of parameters are screened during process development/optimisation. Early development acceleration is a key requirement for economic efficiency, and µ-scale bioreactor systems represent an attractive solution for high-throughput (HTP) experimentation. However, limited practical knowledge is available on the relevance and transferability of screening data to pilot scales and manufacturing. The main goal of the present study was to evaluate a HTP µ-bioreactor platform with respect to its aptitude as a screening platform mainly based on transferability of results to benchtop bioreactors representing the conventional production regime. Second question was to investigate to what extent the online sensors of the µ-bioreactor contribute to process understanding and development. We demonstrated that transferability of infection screening results from the HTP µ-bioreactor scale to the benchtop bioreactor was equal or better than that from shaker cultivation. However, both experimental setups turned out to be sub-optimal solutions that only allowed for a first and rough ranking with low relevance in the case of absolute numbers. Bioreactor yields were up to one order of magnitude higher than the results of screening experiments.

PEI-Mediated Transient Transfection of High Five Cells at Bioreactor Scale for HIV-1 VLP Production.

High Five cells are an excellent host for the production of virus-like particles (VLPs) with the baculovirus expression vector system (BEVS). However, the concurrent production of high titers of baculovirus hinder the purification of these nanoparticles due to similarities in their physicochemical properties. In this study, first a transient gene expression (TGE) method based on the transfection reagent polyethylenimine (PEI) is optimized for the production of HIV-1 VLPs at shake flask level. Furthermore, VLP production by TGE in High Five cells is successfully demonstrated at bioreactor scale, resulting in a higher maximum viable cell concentration (5.1 × 106 cell/mL), the same transfection efficiency and a 1.8-fold increase in Gag-eGFP VLP production compared to shake flasks. Metabolism analysis of High Five cells indicates a reduction in the consumption of the main metabolites with respect to non-transfected cell cultures, and an increase in the uptake rate of several amino acids when asparagine is depleted. Quality assessment by nanoparticle tracking analysis and flow virometry of the VLPs produced shows an average size of 100-200 nm, in agreement with immature HIV-1 viruses reported in the literature. Overall, this work demonstrates that the High Five/TGE system is a suitable approach for the production of VLP-based vaccine candidates and other recombinant proteins.

Off-target effects of an insect cell-expressed influenza HA-pseudotyped Gag-VLP preparation in limiting postinfluenza Staphylococcus aureus infections.

Clinical and historical data underscore the ability of influenza viruses to ally with Staphylococcus aureus and predispose the host for secondary bacterial pneumonia, which is a leading cause of influenza-associated mortality. This is fundamental because no vaccine for S. aureus is available and the number of antibiotic-resistant strains is alarmingly rising. Hence, this leaves influenza vaccination the only strategy to prevent postinfluenza staphylococcal infections. In the present work, we assessed the off-target effects of a Tnms42 insect cell-expressed BEI-treated Gag-VLP preparation expressing the HA of A/Puerto Rico/8/1934 (H1N1) in preventing S. aureus superinfection in mice pre-infected with a homologous or heterologous H1N1 viral challenge strain. Our results demonstrate that matched anti-hemagglutinin immunity elicited by a VLP preparation may suffice to prevent morbidity and mortality caused by lethal secondary bacterial infection. This effect was observed even when employing a single low antigen dose of 50 ng HA per animal. However, induction of anti-hemagglutinin immunity alone was not helpful in inhibiting heterologous viral replication and subsequent bacterial infection. Our results indicate the potential of the VLP vaccine approach in terms of immunogenicity but suggest that anti-HA immunity should not be considered as the sole preventive method for combatting influenza and postinfluenza bacterial infections.

Exploring the Limits of Cell Adhesion under Shear Stress within Physiological Conditions and beyond on a Chip.

Cell adhesion processes are of ubiquitous importance for biomedical applications such as optimization of implant materials. Here, not only physiological conditions such as temperature or pH, but also topographical structures play crucial roles, as inflammatory reactions after surgery can diminish osseointegration. In this study, we systematically investigate cell adhesion under static, dynamic and physiologically relevant conditions employing a lab-on-a-chip system. We screen adhesion of the bone osteosarcoma cell line SaOs-2 on a titanium implant material for pH and temperature values in the physiological range and beyond, to explore the limits of cell adhesion, e.g., for feverish and acidic conditions. A detailed study of different surface roughness Rq gives insight into the correlation between the cells' abilities to adhere and withstand shear flow and the topography of the substrates, finding a local optimum at Rq = 22 nm. We use shear stress induced by acoustic streaming to determine a measure for the ability of cell adhesion under an external force for various conditions. We find an optimum of cell adhesion for T = 37 °C and pH = 7.4 with decreasing cell adhesion outside the physiological range, especially for high T and low pH. We find constant detachment rates in the physiological regime, but this behavior tends to collapse at the limits of 41 °C and pH 4.

A microscale method of protein extraction from bacteria: Interaction of Escherichia coli with cationic microparticles.

We developed a simple, highly selective, efficient method for extracting recombinant proteins from Escherichia coli. Our recombinant protein yield was equivalent to those obtained with high pressure homogenization, and did not require exposure to harsh thermal, chemical, or other potentially denaturing factors. We first ground conventional resin, designed for the exchange of small anions, into microparticles about 1µm in size. Then, these cationic microparticles were brought convectively into close contact with bacteria, and cell membranes were rapidly perforated, but solid cell structures were not disrupted. The released soluble components were adsorbed onto the cell wall associated microparticles or diffused directly into the supernatant. Consequently, the selective adsorption and desorption of acidic molecules is built into our extraction method, and replaces the equally effective subsequent capture on anion exchange media. Simultaneously to cell perforation flocculation was induced by the microparticles facilitating separation of cells yet after desorption of proteins with NaCl. Relative to high pressure homogenization, endogenous component release was reduced by up to three orders of magnitude, including DNA, endotoxins, and host cell proteins, particularly outer membrane protein, which indicates the presence of cell debris.

A surface acoustic wave-driven micropump for particle uptake investigation under physiological flow conditions in very small volumes.

Static conditions represent an important shortcoming of many in vitro experiments on the cellular uptake of nanoparticles. Here, we present a versatile microfluidic device based on acoustic streaming induced by surface acoustic waves (SAWs). The device offers a convenient method for introducing fluid motion in standard cell culture chambers and for mimicking capillary blood flow. We show that shear rates over the whole physiological range in sample volumes as small as 200 µL can be achieved. A precise characterization method for the induced flow profile is presented and the influence of flow on the uptake of Pt-decorated CeO2 particles by endothelial cells (HMEC-1) is demonstrated. Under physiological flow conditions the particle uptake rates for this system are significantly lower than at low shear conditions. This underlines the vital importance of the fluidic environment for cellular uptake mechanisms.

Intake of silica nanoparticles by giant lipid vesicles: influence of particle size and thermodynamic membrane state.

The uptake of nanoparticles into cells often involves their engulfment by the plasma membrane and a fission of the latter. Understanding the physical mechanisms underlying these uptake processes may be achieved by the investigation of simple model systems that can be compared to theoretical models. Here, we present experiments on a massive uptake of silica nanoparticles by giant unilamellar lipid vesicles (GUVs). We find that this uptake process depends on the size of the particles as well as on the thermodynamic state of the lipid membrane. Our findings are discussed in the light of several theoretical models and indicate that these models have to be extended in order to capture the interaction between nanomaterials and biological membranes correctly.