Mosquito-borne pathogens are a global health burden. The realization of a scalable serum-free mosquito cell suspension cultivation method would enable the production of novel biotechnological products, including vaccines, recombinant proteins, and (modified) viruses for biological control. This study reports the adaptation of two Aedes-derived cell lines, C6/36 and U4.4, to serum-free suspension conditions using the chemically defined EX-CELL medium. Both cell lines achieved high cell concentrations (> 1.0 × 107 cells/mL) in shake flasks with minimal adaptation. The C6/36 suspension cultures were subsequently transferred to a 500 mL stirred tank bioreactor (STR), which likewise achieved high cell concentrations (1.3 × 107 cells/mL) and a doubling time of 31–36 h. As a proof of concept for the production of a virus in this system, C6/36 cells were cultivated in a STR and infected with the mosquito-borne flavivirus Usutu virus (USUV). These cultures produced high virus titers (> 3.6 × 108 TCID50/mL), with similar growth kinetics compared to adherent and shake flask cultures. This work is the first to show C6/36 mosquito cell cultivation in a bioreactor and demonstrates the potential of this serum-free suspension culture system for the efficient production of mosquito-infecting viruses.

In vivo gene therapy is rapidly advancing to treat patients with a wide range of genetic disorders. Recombinant adeno-associated virus (rAAV) vectors provide long-term gene expression, low immunogenicity, and adaptable tissue tropism. Production of rAAV in insect cells with the Baculovirus Expression Vector System currently depends on infection with multiple baculoviruses encoding AAV genes Rep, Cap, and the therapeutic gene. In this research article, we present simplified rAAV production using a single baculovirus vector with improved genetic stability, called BAC6Rep, with AAV Rep locked into the baculovirus genome. Robust AAV Rep and Cap expression was maintained upon serial passaging, and high rAAV yields (up to 3.5e11 genome copies/ml) were obtained at high and low multiplicities of infection. Next-generation sequencing revealed a significantly improved genetic stability of BAC6Rep, which can now be used as a plug-in hybrid vector for flexible insertion of different AAV Cap and gene of interest tailored to the therapeutic target.

The baculovirus expression vector system is an established platform for large-scale production of (glyco)proteins, subunit vaccines, virus-like particles, and recombinant adeno-associated virus (rAAV) vectors. We engineered a novel bacmid vector (BAC6) to improve genetic stability by deletion of the non-homologous repeat (hr) origin of DNA replication (ori) and to preserve product integrity and recovery through deletion of chitinase and cathepsin. Tn7-based transposition in E. coli and homologous recombination in insect cells were combined in BAC6 to drive expression from the odv-e56 and polyhedrin loci, respectively. Virus growth kinetics of BAC6 were similar to the parental bacmid, and genetic stability was investigated for at least eight serial passages at high multiplicity of infection. Next-generation sequencing was used to identify mutations, deletions, and defective interfering particle (DIP) formation, which became apparent only in later passages. With BAC6, the enrichment of DIPs originating from the non-hr ori was prevented. BAC6 versatility was demonstrated by high-yield (12 mg/L) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike production in suspension Sf9 insect cells. Finally, rAAV production with BAC6, simultaneously employing both transgene insertion sites, resulted in yields of 5.8e10 AAV capsids/mL and 2.3e10 genome copies/mL. BAC6 provides insertion of multiple transgenes at two different loci and is non-inferior to commercial baculovirus expression vectors with regard to genetic stability and productivity.

The baculovirus expression vector system (BEVS) is a scalable platform used to produce recombinant adeno-associated virus vectors (rAAV) in insect cells. A major challenge in this system is reducing the formation of empty rAAV capsids, which do not contain vector DNA and lack therapeutic value. The proportion of empty capsids is influenced by the balance between the two baculovirus constructs that coinfect the producer cells: Bac-Rep-Cap, which supplies AAV replication and capsid proteins, and Bac-GOI-ITR, which delivers the therapeutic gene of interest. Digital holographic microscopy (DHM) is a label-free imaging technique that allows real-time monitoring of cell morphology in suspension cultures. Previous studies have used DHM to track cell density and baculovirus infection; however, its ability to evaluate different coinfections has not been explored. In this study, we combined DHM with machine learning to identify morphological patterns associated with various coinfections for rAAV production. Shaker-flask experiments with different Bac-Rep-Cap: Bac-GOI-ITR ratios created a dataset of cell morphologies to train a predictive classification model. When applied to real-time bioreactor measurements, the model revealed shiftsin the classification patterns related to the initial multiplicity of infection (MOI). The integration of DHM with the classification model has the potential to produce a qualitative "process fingerprint," where deviations in morphological patterns can serve as early indicators of suboptimal coinfection. Such early warning signs enable timely batch termination, reducing downstream processing of inconsistent material. Overall, DHM combined with machine learning offers a non-invasive, real-time tool for process benchmarking and quality assurance in rAAV manufacturing.

The emergence of new viruses and the spread of existing pathogens necessitate efficient vaccine production methods. The baculovirus expression vector system (BEVS) is an efficient and scalable system for subunit and virus-like particle vaccine production and gene therapy vectors. However, current production processes are often limited to low cell concentrations (1-4 × 106 cells/mL) in fed-batch mode. To improve the volumetric productivity of the BEVS, a medium exchange strategy was investigated. Screening experiments were performed to test baculovirus (expressing green fluorescent protein; GFP) infection and productivity of insect cell cultures infected at high cell concentration (1-2 × 107 cells/mL), showing that infection at high cell concentrations was possible with medium exchange. Next, duplicate perfusion runs with baculovirus infection were performed using a cell concentration upon infection (CCI) of 1.2 × 107 cells/mL and a multiplicity of infection (MOI) of 0.01, reaching a maximum viable cell concentration of 2.8 × 107 cells/mL and a maximum GFP production of 263 mg/L. The volumetric productivity of these perfusion runs was 4.8 times higher than for reference batch processes with a CCI of 3 × 106 cells/mL and an MOI of 1. These results demonstrate that process intensification can be achieved for the BEVS by implementing perfusion, resulting in a higher volumetric productivity.

Current lab-scale purification methods for ADDomer are labour-intensive, time-consuming and poorly scalable. The intracellular nature of ADDomer production further complicates downstream processing, requiring robust, scalable solutions for cell lysis and clarification. In the present work we focus on developing a scalable, GMP-compliant process for ADDomer purification. The workflow combines tangential microfiltration (TMF) using a 0.4 μm hollow fiber for cell retention, chemical lysis with 0.06

After more than four decades, the field of sponge biotechnology has finally overcome its most formidable obstacle: establishing a sponge cell line. Cells from the deep water marine sponge Geodia barretti divide rapidly and continuously in optimized nutrient media, clearing the path for new lines of research--whether to uncover how multicellular life evolved from unicellular organisms, study the origins of animal-microbe symbiosis, or develop production strains to industrialize new sponge-derived drugs and exploit the untapped biotechnological potential of the Porifera. Now is an exciting time in sponge biotechnology, so what's next?

In biopharmaceutical manufacturing, continuous perfusion cultivation enables high space-time yields and increased plant utilization, which are critical targets for modern upstream process intensification. However, filter-based cell retention devices, utilized in these processes, have significant disadvantages: Significant sieving effects and the risk of filter blockage alongside the retention of harmful substances and non-viable cells, represent a major challenge and often reduce the viability of the culture. To enable the next-generation of continuous processes, novel cell retention strategies are required. Therefore, the aim of this study was to develop an approach for large-scale sorting of viable and non-viable cells and to investigate its applicability for novel continuous cultivation strategies. To remove non-viable cells and thus to enrich viable cells in the culture, a single-use fluidized bed centrifuge (FBC) was used, which is usually applied for concentration and washing of mammalian cells. A novel FBC method was introduced by overloading the centrifuge chambers that allows high throughput sorting depending on the culture´s viability. The impact of the sorting on the subsequent cultivation and productivity of the cells was investigated in a multi-parallel 15 mL bioreactor setup. Cell sorting after regular fed-batch cultivation showed +14% increase of viability, continued cell growth, and thus +13% higher titers. Thereafter, periodic cell sorting was tested on a 5-L scale bioreactor, combining the advantageous characteristics of fed-batch and perfusion cultivation. The feasibility was successfully demonstrated for 20 days, achieving a high average space-time yield of 0.75 g/L/d. In both cultivation trials, up to +38% higher cell specific antibody productivities were found after cell sorting. Overall, the FBC sorting method in combination with innovative cultivation concepts addresses current limitations and challenges of continuous biopharmaceutical manufacturing and has great potential to further advance modern process intensification.

Zika virus (ZIKV) caused unprecedented outbreaks in South America and the Caribbean in 2015-2016, leading primarily to a series of abnormalities in neonates termed congenital Zika syndrome. The threat of ZIKV reemergence has seen the development of multiple ZIKV vaccines that are at the preclinical stage or in early-stage clinical trials. Herein, we describe a pathway to the development of ZIKV vaccines generated using a baculovirus-insect cell expression system, which is widely applied for the manufacture of biologics for human use. Virus-like particle (VLP) vaccines comprising CprME and subviral particle (SVP) vaccines comprising prME were evaluated for their ability to mediate protection against ZIKV challenge in Ifnar1-/- mice. Initial attempts resulted in VLP and SVP vaccines that failed to present quaternary epitopes and did not provide effective protection. To improve the SVP vaccine, two modifications were introduced: firstly, an alanine to cysteine substitution (A264C) in the E domain II region to promote the formation of stabilized E homodimers and, secondly, the use of Spodoptera frugiperda Sf9 insect cells that had been adapted to grow and produce vaccine at a neutral pH of 7. E homodimers largely retain their pre-fusion conformation at pH 7, which is a requirement for the induction of effective neutralizing antibody responses. The stabilized SVP-A26C vaccine induced high levels of neutralizing antibodies and protected male Ifnar1-/- mice against viremia and testicular damage. Our study reiterates the need to present the immune system with E dimers arranged in authentic quaternary conformations and provides a scalable production method for this novel ZIKV vaccine.IMPORTANCEWe describe the generation of a subviral particle (SVP) vaccine comprising prME proteins of ZIKV, with an envelope protein substitution, A264C, that stabilizes E dimer formation. The SVP vaccine was produced in a novel Sf9 insect cell line adapted to grow in suspension at pH 7. The study highlights the importance of challenge experiments to ascertain whether the responses induced by an experimental vaccine actually mediate protection against virus infection and disease. The study also reiterates the contention that effective flavivirus vaccines need to present the immunogen in an authentic tertiary and quaternary structure with a pre-fusion conformation.

Recombinant adeno-associated virus vectors (rAAVs) play an important role in gene therapy for the effective delivery of therapeutic genes into target cells. The Baculovirus Expression Vector System (BEVS) has gained significant attention for its versatility and scalability as an rAAV production platform. The existing Dual-Bac system uses two separate baculovirus constructs (Bac-GOI-ITR and Bac-Rep-Cap), each carrying essential genetic elements for rAAV production in insect cells. This study investigated how two infection parameters of the Dual-Bac system, the total Multiplicity of Infection (MOI) and the baculovirus co-infection ratio, influence rAAV production efficiency. Different budded virus (BV) concentrations were used to explore the effects on assembled rAAV capsid and encapsidated transgenic genome yields. An excess of Bac-Rep-Cap in synchronous co-infection produced high-quality rAAVs, whereas increasing the ratio of Bac-GOI-ITR to Bac-Rep-Cap resulted in more empty rAAV capsids. The optimal BV ratio varied with the total MOI used for co-infection, and when applying a BV ratio of one, MOI variations had a minimal impact on rAAV quality. These findings highlight the importance of optimizing the MOI and BV ratio to enhance rAAV yield and quality, contributing to more robust gene therapy production.

Therapeutic proteins such as monoclonal antibodies are usually manufactured either in continuous processes such as steady-state perfusion or in discontinuous processes like fed-batch (FB). Thereby, both process formats have fundamentally different requirements for the utilized cell factories. This poses a problem as common cell line development programs are designed to select cell clones to perform well in FB cultivation. The aim of this study was to identify critical and easy to access cell line attributes for each process format and with that for the transfer of clones from fed-batch to perfusion. As a result, increased cell-volume specific productivity could be identified as beneficial within the FB not only impacting performance but as well diminishing media utilization and host-cell-proteins level. Within the perfusion, a stable cell diameter was identified as major beneficial characteristic, positively influencing cell viability, metabolite and glycan profile, as well as equipment utilization. Consistent with this, our data suggest that the inclusion of cell volume in the screening parameters is important for clone selection for both process formats and can lead to improved process design and robust cell line process transfer. Overall, this work gives valuable new insights in cell behaviour across discontinuous and continuous process formats to improve clone cell selection for both process strategies and streamline the process transfer from discontinuous towards continuous.

Monoclonal antibodies (mAb) are commonly manufactured by either discontinuous operations like fed-batch (FB) or continuous processes such as steady-state perfusion. Both process types comprise opposing advantages and disadvantages in areas such as plant utilization, feasible cell densities, media consumption and process monitoring effort. In this study, we show feasibility of a promising novel hybrid process strategy that combines beneficial attributes of both process formats. In detail, our strategy comprises a short duration FB, followed by a fast media exchange and cell density readjustment, marking the start of the next FB cycle. Utilizing a small-scale screening tool, we were able to identify beneficial process parameters, including FB interval duration and reinoculation cell density, that allow for multiple cycles of the outlined process in a reproducible manner. In addition, we could demonstrate scalability of the process to a 5L benchtop system, using a fluidized-bed centrifuge as scalable media exchange system. The novel process showed increased productivity (+217%) as well as longer cultivation duration, in comparison to a standard FB with a significantly lower media consumption per produced product (−50%) and a decreased need for process monitoring, in comparison to a perfusion cultivation. Further, the process revealed constant glycosylation pattern in comparison to the perfusion cultivation and has strong potential for further scale-up, due to the use of fully scalable cultivation and media exchange platforms. In summary, we have developed a novel hybrid process strategy that tackles the key challenges of current biomanufacturing of either low productivity or high media consumption, representing a new and innovative approach for future process intensification efforts.

Monoclonal antibodies are the workhorse of the pharmaceutical industry due to their potential to treat a variety of different diseases while providing high specificity and efficiency. As a consequence, a variety of production processes have been established within the biomanufacturing industry. However, the rapidly increasing demand for therapeutic molecules amid the recent COVID-19 pandemic demonstrated that there still is a clear need to establish novel, highly productive, and flexible production processes. Within this work, we designed a novel discontinuous process by combining two intensification strategies, thus increasing inoculation density and media exchange via a fluidized bed centrifuge, to fulfill the need for a flexible and highly productive production process for therapeutic molecules. To establish this new process, firstly, a small-scale experiment was conducted to verify synergies between both intensification strategies, followed by a process transfer towards the proof-of-concept scale. The combination of these two-process intensification measures revealed overall synergies resulting in decreased process duration (-37%) and strongly enhanced product formation (+116%) in comparison to the not-intensified standard operation. This led to an impressive threefold increase in space-time yield, while only negligible differences in product quality could be observed. Overall, this novel process not only increases the ways to react to emergency situations thanks to its flexibility and possible short development times, but also represents a possible alternative to the current established processes due to high increases in productivity, in comparison to standard fed-batch operations.

The potential of sponge-derived chemicals for pharmaceutical applications remains largely unexploited due to limited available biomass. Although many have attempted to culture marine sponge cells in vitro to create a scalable production platform for such biopharmaceuticals, these efforts have been mostly unsuccessful. We recently showed that Geodia barretti sponge cells could divide rapidly in M1 medium. In this study we established the first continuous marine sponge cell line, originating from G. barretti. G. barretti cells cultured in OpM1 medium, a modification of M1, grew more rapidly and to a higher density than in M1. Cells in OpM1 reached 1.74 population doublings after 30 min, more than twofold higher than the already rapid growth rate of 0.74 population doublings in 30 min in M1. The maximum number of population doublings increased from 5 doublings in M1 to at least 98 doublings in OpM1. Subcultured cells could be cryopreserved and used to inoculate new cultures. With these results, we have overcome a major obstacle that has blocked the path to producing biopharmaceuticals with sponge cells at industrial scale for decades.

Monoclonal antibodies (mAb) have gained enormous therapeutic application during the last decade as highly efficient and flexible tools for the treatment of various diseases. Despite this success, there remain opportunities to drive down the manufacturing costs of antibody-based therapies through cost efficiency measures. To reduce production costs, novel process intensification methods based on state-of-the-art fed-batch and perfusion have been implemented during the last few years. Building on process intensification, we demonstrate the feasibility and benefits of a novel, innovative hybrid process that combines the robustness of a fed-batch operation with the benefits of a complete media exchange enabled through a fluidized bed centrifuge (FBC). In an initial small-scale FBC-mimic screening, we investigated multiple process parameters, resulting in increased cell proliferation and an elongated viability profile. Consecutively, the most productive process scenario was transferred to the 5-L scale, further optimized and compared to a standard fed-batch process. Our data show that the novel hybrid process enables significantly higher peak cell densities (163%) and an impressive increase in mAb amount of approximately 254% while utilizing the same reactor size and process duration of the standard fed-batch operation. Furthermore, our data show comparable critical quality attributes (CQAs) between the processes and reveal scale-up possibilities and no need for extensive additional process monitoring. Therefore, this novel process intensification strategy yields strong potential for transfer into future industrial manufacturing processes.

Besides being considered pathogens, viruses are important drivers of evolution and they can shape large ecological and biogeochemical processes, by influencing host fitness, population dynamics, and community structures. Moreover, they are simple systems that can be used and manipulated to be beneficial and useful for biotechnological applications. In this context, microalgae biotechnology is a growing field of research, which investigated the usage of photosynthetic microorganisms for the sustainable production of food, fuel, chemical, and pharmaceutical sectors. Viruses infecting microalgae have become important subject of ecological studies related to marine and aquatic environments only four decades ago when virus-like-particles associated with bloom-forming algae were discovered. These first findings have opened new questions on evolution and identity. To date, 63 viruses that infect eukaryotic microalgae have been isolated and cultured. In this short review we briefly summarize what is known about viruses infecting eukaryotic microalgae, and how acknowledging their importance can shape future research focussed not only on marine ecology and evolutionary biology but also on biotechnological applications related to microalgae cell factories.

Chikungunya virus (CHIKV) is a rapidly emerging mosquito-borne virus that causes a severe febrile illness with long-lasting arthralgia in humans. As there is no vaccine to protect humans and limit CHIKV epidemics, the virus continues to be a global public health concern. The CHIKV envelope glycoproteins E1 and E2 are important immunogens; therefore, the aim of this study is to produce trimeric CHIKV spikes in insect cells using the baculovirus expression system. The CHIKV E1 and E2 ectodomains were covalently coupled by a flexible linker that replaces the 6K transmembrane protein. The C-terminal E1 transmembrane was replaced by a Strep-tag II for the purification of secreted spikes from the culture fluid. After production in Sf9 suspension cells (product yields of 5.8–7.6 mg/L), the CHIKV spikes were purified by Strep-Tactin affinity chromatography, which successfully cleared the co-produced baculoviruses. Bis(sulfosuccinimidyl)suberate cross-linking demonstrated that the spikes are secreted as trimers. PNGase F treatment showed that the spikes are glycosylated. LC–MS/MS-based glycoproteomic analysis confirmed the glycosylation and revealed that the majority are of the mannose- or hybrid-type N-glycans and <2% have complex-type N-glycans. The LC –MS/MS analysis also revealed three O-glycosylation sites in E1. In conclusion, the trimeric, glycosylated CHIKV spikes have been successfully produced in insect cells and are now available for vaccination studies.

Real-time, detailed online information on cell cultures is essential for understanding modern biopharmaceutical production processes. The determination of key parameters, such as cell density and viability, is usually based on the offline sampling of bioreactors. Gathering offline samples is invasive, has a low time resolution, and risks altering or contaminating the production process. In contrast, measuring process parameters online provides more safety for the process, has a high time resolution, and thus can aid in timely process control actions. We used online double differential digital holographic microscopy (D3HM) and machine learning to perform non-invasive online cell concentration and viability monitoring of insect cell cultures in bioreactors. The performance of D3HM and the machine learning model was tested for a selected variety of baculovirus constructs, products, and multiplicities of infection (MOI). The results show that with online holographic microscopy insect cell proliferation and baculovirus infection can be monitored effectively in real time with high resolution for a broad range of process parameters and baculovirus constructs. The high-resolution data generated by D3HM showed the exact moment of peak cell densities and temporary events caused by feeding. Furthermore, D3HM allowed us to obtain information on the state of the cell culture at the individual cell level. Combining this detailed, real-time information about cell cultures with methodical machine learning models can increase process understanding, aid in decision-making, and allow for timely process control actions during bioreactor production of recombinant proteins.

Process intensification is increasingly used in the mammalian biomanufacturing industry. The key driver of this trend is the need for more efficient and flexible production strategies to cope with the increased demand for biotherapeutics predicted in the next years. Therefore, such intensified production strategies should be designed, established, and characterized. We established a CHO cell process consisting of an intensified fed-batch (iFB), which is inoculated by an N-1 perfusion process that reaches high cell concentrations (100 × 106  c ml-1 ). We investigated the impact of butyric acid (BA) supplementation in this iFB process. Most prominently, higher cellular productivities of more than 33% were achieved, thus 3.5 g L-1 of immunoglobulin G (IgG) was produced in 6.5 days. Impacts on critical product quality attributes were small. To understand the biological mechanisms of BA in the iFB process, we performed a detailed transcriptomic analysis. Affected gene sets reflected concurrent inhibition of cell proliferation and impact on histone modification. These translate into subsequently enhanced mechanisms of protein biosynthesis: enriched regulation of transcription, messenger RNA processing and transport, ribosomal translation, and cellular trafficking of IgG intermediates. Furthermore, we identified mutual tackling points for optimization by gene engineering. The presented strategy can contribute to meet future requirements in the continuously demanding field of biotherapeutics production.