Wave Bioreactor Research Articles

Fast and robust recombinant protein production utilizing episomal stable pools in WAVE bioreactors

Protein reagents are essential resources for several stages of drug discovery projects from structural biology and assay development through lead optimization. Depending on the aim of the project different amounts of pure protein are required. Small-scale expressions are initially used to determine the reachable levels of production and quality before scaling up protein reagent supply. Commonly, amounts of several hundreds of milligrams to grams are needed for different experiments, including structural investigations and activity evaluations, which require rather large cultivation volumes. This implies that cultivation of large volumes of either transiently transfected cells or stable pools/stable cell lines is needed. Hence, a production process that is scalable, speeds up the development projects, and increases the robustness of protein reagent quality throughout scales. Here we present a protein production pipeline with high scalability. We show that our protocols for protein production in Chinese hamster ovary cells allow for a seamless and efficient scale-up with robust product quality and high performance. The flexible scale of the production process, as shown here, allows for shorter lead times in drug discovery projects where there is a reagent demand for a specific protein or a set of target proteins.

Determination of Optimal Antigen Yield and Virus Inactivation Conditions for the Production of the Candidate Foot-and-Mouth Disease Recombinant Vaccine Strain Asia1 Shamir-R in a Bioreactor.

Since the foot-and-mouth disease (FMD) outbreak in South Korea in 2010-2011, vaccination policies utilizing inactivated FMD vaccines composed of types O and A have been implemented nationwide. However, because type Asia1 occurred in North Korea in 2007 and intermittently in neighboring countries, the risk of type Asia1 introduction cannot be ruled out. This study evaluated the antigen yield and viral inactivation kinetics of the recombinant Asia1 Shamir vaccine strain (Asia1 Shamir-R). When Asia1 Shamir-R was proliferated in shaking flasks (1 L), a 2 L bioreactor (1 L), and a wave bioreactor (25 L), the antigen yields were 7.5 μg/mL, 5.2 μg/mL, and 3.8 μg/mL, respectively. The optimal FMDV inactivation conditions were 2 mM BEI at 26 °C and 1.0 mM BEI at 37 °C. There was no antigen loss due to BEI treatment, and only a decrease in antigen levels was observed during storage. The sera from pigs immunized with antigen derived from a bioreactor exhibited a neutralizing antibody titer of approximately 1/1000 against Asia1 Shamir and Asia1/MOG/05 viruses; therefore, Asia1 Shamir-R is expected to provide sufficient protection against both viruses. If an FMD vaccine production facility is established, this Asia1 Shamir-R can be employed for domestic antigen banks in South Korea.

Application of Multivariate Data Analysis on Historical Recombinant Adenovirus Zoster Vaccine Production Data for Upstream Process Improvements

In recent years, multivariate data analysis (MVDA) has been widely used for process characterization and fault diagnosis in the biopharmaceutical industry. This study aims to investigate the feasibility of using MVDA for the development and scale-up of a perfusion process for HEK293 cell-based recombinant adenovirus zoster vaccine (Ad-HER) production. The Principal Component Analysis (PCA) results suggested comparable performance among the ATF, PATFP, and BFP perfusion systems in benchtop-scale stirred-tank bioreactor (STR). Then a Batch Evolution Model (BEM) was built using representative data from 10 L STR with a BFP system to assess the Ad-HER perfusion process performance at pilot-scale bioreactor (50 L STR and 50 L wave bioreactor). Furthermore, another BEM model and Batch Level Model (BLM) were built to monitor process parameters over time and predict the final adenovirus titer in 50 L wave bioreactor. The loading plot revealed that lactate dehydrogenase activity, viable cell diameter, and base-added during the virus production phase could be used as preliminary indicators of adenovirus yield. Finally, an adenovirus titer of 2.0±0.3×1010 IFU/mL was achieved in the 50 L wave bioreactor with BFP system, highlighting the robustness of the Ad-HER perfusion process at pilot-scale. Overall, this study emphasizes the effectiveness of MVDA as a tool for advancing the understanding of recombinant adenovirus vaccine perfusion production process development and scale-up.

Protein Expression via Transient Transfection of Mammalian Cells in a WAVE Bioreactor.

Large culture volumes are often required when expression constructs are particularly low-yielding or when end uses require significant amounts of material. In these cases, a single homogeneous culture is usually more convenient, in terms of both consistency of expression and labor/resource requirements, than multiple parallel cultures. Using a WAVE Bioreactor culture, volumes as high as 500L may be achieved in a single vessel. Here, we describe the transfection of Expi293F cells in a disposable 50L Cellbag on a WAVE Bioreactor platform to produce recombinant protein. The methods described herein may be adapted, with suitable optimizations, for other suspension-adapted mammalian cell lines.

Inducible HEK293 AAV packaging cell lines expressing Rep proteins

Packaging or producer cell lines for scalable recombinant adeno-associated virus (rAAV) production have been notoriously difficult to create due in part to the cytostatic nature of the Rep proteins required for AAV production. The most difficult challenge being creating AAV packaging cell lines using HEK293 parental cells, currently the best mammalian platform for rAAV production due to the constitutive expression of E1A in HEK293 cells, a key REP transcription activator. Using suspension and serum-free media adapted HEK293SF carrying a gene expression regulation system induced by addition of cumate and coumermycin, we were able to create REP-expressing AAV packaging cells. This was achieved by carefully choosing two of the AAV Rep proteins (Rep 40 and 68), using two inducible promoters with different expression levels and integrating into the cells through lentiviral vector transduction. Three of our best clones produced rAAV titers comparable to titers obtained by standard triple plasmid transfection of their parental cells. These clones were stable for up to 7weeks under continuous cultures condition. rAAV production from one clone was also validated at scale of 1L in a wave bioreactor using serum-free suspension culture.

Culture of EV71 virus in wave bioreactor system for production of hand, foot and mouth disease vaccine

The study was carried out to establish an EV71 virus culture system on Vero cells in a shake culture system (Wave bioreactor) incorporating microcarriers for the production of hand, foot and mouth (HFM) vaccine caused by the EV71 virus. The results of the investigation of 2 types of microcarriers (Cytodex 1 and Cytodex 3), showed that Cytodex 1 gave better efficiency when culturing Vero cells in suspension conditions. The combination with Cytodex 1 on the Wave bioreactor system gave Vero cell culture efficiency of over 3x106cells/ml. The optimal infectious dose of EV71 virus on Vero cells was determined to be 0.0001 TCID50/ml. The optimisation of EV71 virus culture conditions on the Wave bioreactor system has obtained suitable results for the production of vaccine with virus titres 108TCID50/ml on the fifth day after infection. The procedure of production of EV71 virus on Vero cell in Wave bioreactor system was applicable in vaccine production with virus titres 108 TCID50/ml on the fifth day after infection.

Development, analysis, and preclinical evaluation of inactivated vaccine candidate for prevention of Covid-19 disease

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in the sprawling capital of Central China’s Hubei province Wuhan in 2019. Since late 2019, SARS-CoV-2 has been responsible for a global pandemic that has resulted in 405,961,201 confirmed cases of infection and 5,789,567 deaths as of February 11, 2022, according to the World Health Organization (WHO). Here we report the development of an inactivated SARS-CoV-2 vaccine candidate and show that its efficacy and safety in preclinical studies warrant further clinical evaluation. Efficiency and possible side effects of vaccine candidate on different doses were evaluated using histopathology, K18 hACE2 transgenic mice challenge, T-cell response, and serum neutralization test in Balb/c mice. All pilot production and propagation experiments of SARS-CoV-2 were performed in the Level 3 Negative Pressure Biosafety Facility (BSL3/ABSL-3) according to COVID-19 interim guidance of WHO. All analytical studies have shown that the quality control parameters of the vaccine candidate meet the requirements for inactivated viral vaccines according to European Pharmacopeia. The vaccine candidate (KOCAK-19) has been authorized by the Turkish Medicines and Medical Devices Agency and has now entered phase 2 clinical development (NCT04838080). Abbreviations: SARS-CoV-2: Severe Acute Respiratory Syndrome Coronavirus 2.; MERS-CoV: Middle East Respiratory Syndrome Coronavirus.; qRT-PCR: Quantitative Real-Time Polymerase Chain Reaction.; TEM: Transmissible Electron Microscopy.; SEC: Size Exclusion.; AEX: Anion exchange.; BPL: β-Propiolactone.; CPE: Cytopathic Effect.; TCID50: 50% Tissue Culture Infectious Dose.; hACE-2: Human Angiotensin-Converting Enzyme 2.; mRNA: Messenger RNA.; RBD: Receptor binding domain.; Nab: Neutralizing antibody.; MOI: Multiply of Infection. HIGHLIGHTS A new inactivated vaccine candidate to protect against COVID −19 SARS-CoV-2 inactive vaccine development in disposable wave bioreactor systems with microcarrier technologies and analytical analysis Preclinical toxicity and immunity trials for COVID-19 inactive vaccine candidate in Balb/c mice, K18 transgenic mice and, ferrets.

Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System.

Adeno-associated viruses (AAV) are promising vectors for gene therapy applications. Here, the AAV2 vector is produced by co-culture of Spodoptera frugiperda (Sf9) cells with Sf9 cells infected with baculovirus (BV)-AAV2-GFP (or therapeutic gene) and BV-AAV2-rep-cap in serum-free suspension culture. Cells are cultured in a flask in an orbital shaker or Wave bioreactor. To release the AAV particles, producer cells are lysed in buffer containing detergent, which is subsequently clarified by low-speed centrifugation and filtration. AAV particles are purified from the cell lysate using AVB Sepharose column chromatography, which binds AAV particles. Bound particles are washed with PBS to remove contaminants and eluted from the resin using sodium citrate buffer at pH 3.0. The acidic eluate is neutralized with alkaline Tris-HCl buffer (pH 8.0), diluted with phosphate-buffered saline (PBS), and further concentrated with tangential flow filtration (TFF). The protocol describes small-scale pre-clinical vector production compatible with scale-up to large-scale clinical-grade AAV manufacturing for human gene therapy applications.

Stable lentiviral vector producer cell line for a scalable manufacture

<h3>Watch the video or read the poster to learn about:</h3> <ul><li>The benefits of using stable cell lines to produce lentivirus</li><li>The advantages the EuLV lentiviral production system offers as compared to plasmid transfection</li><li>An example 25L-scale LVV production process in WAVE Bioreactor</li></ul><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://bioinsights.blob.core.windows.net/uploads/2659927c4c9e47f9b3a3f2115bb4dda1.jpg" data-image="1"></div><div class="author-det"> <b>Mr Liu</b> graduated from the Hong Kong University of Science and Technology with a Master degree in Biotechnology. He previously worked in Bio-techne and InvivoGen. Mr Liu joined Eureka Bio in 2016 as COO, and he has extensive knowledge in Gene and Cell therapy industry, and is experienced in business development, project management, process development, cGMP etc. </div></div></div>

Numerical Optimization of Particle Dispersion in Wave Bioreactor for Static Cell Cultivation

Numerical Optimization of Particle Dispersion in Wave Bioreactor for Static Cell Cultivation

Expansion and activation of T cells in the wave bioreactor platform in chemically defined and serum free OptiPEAK T Lymphocyte media

Background & Aim Expansion of human T cells has become a critical tool in the development of immunotherapies to treat a wide spectrum of diseases. As both autologous and allogeneic immunotherapies continue to show success in the clinic, the importance of having an optimized T cell expansion platform has been made even more apparent. Standard components used to grow these cells often include human or bovine serum which not only risk the introduction of harmful pathogens but also have significant limitations on the ability to deliver robust and consistent cell culture performance. Methods, Results & Conclusion To circumvent these issues, we have developed a chemically defined and animal-component free media, OptiPEAK T Lymphocyte, that demonstrates sufficient growth and expansion of human primary T cells in the wave bioreactor without requiring the addition of serum or serum-derived proteins. Expanded cells in OptiPEAK T Lymphocyte also exhibit a less differentiated phenotypic profile as proven by the presence of increased T memory stem cell subsets. In conclusion, T cell expansion in the wave bioreactor with OptiPEAK T Lymphocyte chemically defined media ensures robust performance without compromising safety, growth, or differentiation to deliver an effective dose for these immunotherapies. Expansion of human T cells has become a critical tool in the development of immunotherapies to treat a wide spectrum of diseases. As both autologous and allogeneic immunotherapies continue to show success in the clinic, the importance of having an optimized T cell expansion platform has been made even more apparent. Standard components used to grow these cells often include human or bovine serum which not only risk the introduction of harmful pathogens but also have significant limitations on the ability to deliver robust and consistent cell culture performance. To circumvent these issues, we have developed a chemically defined and animal-component free media, OptiPEAK T Lymphocyte, that demonstrates sufficient growth and expansion of human primary T cells in the wave bioreactor without requiring the addition of serum or serum-derived proteins. Expanded cells in OptiPEAK T Lymphocyte also exhibit a less differentiated phenotypic profile as proven by the presence of increased T memory stem cell subsets. In conclusion, T cell expansion in the wave bioreactor with OptiPEAK T Lymphocyte chemically defined media ensures robust performance without compromising safety, growth, or differentiation to deliver an effective dose for these immunotherapies.

Trends in Monoclonal Antibody Production Using Various Bioreactor Syst.

Monoclonal antibodies are widely used as diagnostic reagents and for therapeutic purposes, and their demand is increasing extensively. To produce these proteins in sufficient quantities for commercial use, it is necessary to raise the output by scaling up the production processes. This review describes recent trends in high-density cell culture systems established for monoclonal antibody production that are excellent methods to scale up from the lab-scale cell culture. Among the reactors, hollow fiber bioreactors contribute to a major part of high-density cell culture as they can provide a tremendous amount of surface area in a small volume for cell growth. As an alternative to hollow fiber reactors, a novel disposable bioreactor has been developed, which consists of a polymer-based supermacroporous material, cryogel, as a matrix for cell growth. Packed bed systems and disposable wave bioreactors have also been introduced for high cell density culture. These developments in high-density cell culture systems have led to the monoclonal antibody production in an economically favourable manner and made monoclonal antibodies one of the dominant therapeutic and diagnostic proteins in biopharmaceutical industry.

An efficient bioreactor platform for scaling up the proliferation of Nordmann fir’s (Abies nordmanniana) somatic embryos

Increased market demand for high-quality Nordmann fir (Abies nordmanniana) Christmas trees, achieving reduced production costs and improved pest resistance, necessitates the establishment of efficient mass micropropagation platforms for the species. Somatic embryogenesis is a powerful tool for clonal propagation of woody plants. Utilizing the advantages of liquid culture media over solid media, the scalability of Nordmann fir somatic embryo proliferation was investigated through developing a batch culture using the disposable WAVE bioreactor (DWB) system, without feedback controls for pH and dissolved oxygen. This study revealed that the inoculation of a 10-L cellbag with 1 g L−1 FW (fresh weight) of A. nordmanniana embryogenic callus can produce ~ 70,000–111,000 vigorous and synchronized somatic embryos. Such outcomes were achieved with the rock speeds of 25 and 35 rpm and rock angle of 6° for 25 days at 24 ± 1 °C, respectively. The gas mix was a combination of O2 (21%) and a stepwise reduction of CO2 ended by 2% (v/v), at a flow rate of 0.2 L min−1. The application of 500 mg L−1 of 2-(N-morpholino)ethanesulfonic acid (MES) effectively stabilized the pH between 5.3 and 5.4. Furthermore, DWB was found an efficient system for accelerating and scaling up the proliferation of A. nordmanniana somatic embryos, as it is a high-yielding, user-friendly, functional, and rapid alternative to conventional methods with solid media.

Characterization of power input and its impact on mass transfer in a rocking disposable bioreactor

The impact of specific power input on gas-liquid mass transfer and mixing has not been extensively reported for rocking disposable bioreactors. An electrical method was applied for measuring the specific power input into a disposable rocking (Wave) bioreactor at benchtop scales. The peak power input was shown to be suitable for characterizing the impact of operational parameters including rocking frequency, rocking angle and liquid volume. The average power inputs ranged from 66.5 W/m3 to 680.1 W/m3 which were comparable to those for stirred tank and orbitally-shaken disposable bioreactors. The gas-liquid oxygen mass transfer coefficient was shown to correlate with the peak power input in a power law model, confirming that the mass transfer capacity rapidly increased with power input, especially at 600 W/m3 and greater. The correlation between mixing time and power input indicated that power input at higher than 400 W/m3 was sufficient to induce relatively rapid mixing.

Comprehensive study on Wave bioreactor system to scale up the cultivation of and recombinant protein expression in baculovirus-infected insect cells

Although the baculovirus expression vector system has been widely used to produce a variety of recombinant proteins, the shear sensitivity of insect cells besides their relatively high oxygen uptake rates have limited their large scale applicability. To address such issues, Wave bioreactor systems have been developed to provide the insect cells with sufficient dissolved oxygen in a bubble free medium of minimal shear stress. However, selection of rocking parameters, and the methods used to control the processes have not been well described in literature. Herein, oxygen transfer capacity of Wave bioreactor has been investigated at different combinations of rocking parameters. Considering the specific oxygen uptake rates (SOUR) and the assessed oxygen transfer capacity of the Wave bioreactor, rocking parameters were set to cultivate non-infected Spodoptera frugiperda 9 cells and infected/non-infected BTI-TN-5B1-4 cells in 2 L Cellbags. The obtained results were exploited to estimate the SOUR of the insect cells under actual condition of Wave bioreactor system. Furthermore, although Express Five medium was recommended by the supplier, the yield of the active soluble human papillomavirus (HPV) 16L1 protein expression was improved in Sf900 II medium. Using the obtained results, protein expression in BTI-TN-5B1-4 cells was finally scaled up to 50 L confirming that the obtained information could be successfully used to scale the Wave bioreactor by specifying the rocking parameters.

Study of hydrodynamics in wave bioreactors by computational fluid dynamics reveals a resonance phenomenon

Culture of mammalian or human cells in Wave bioreactor is widely used for cell expansion or for biologics manufacturing. Wave bioreactor cultivation of sensitive cells such as stem cells, immune cells or anchorage-dependent cells, is recognized as an attractive option for culture in suspension or adherently on microcarriers. A systematic optimization of the mixing, oxygen transfer rate and shear stress, most favorable for the cells requires a deep understanding of the hydrodynamics inside the Wave bioreactor bag, i.e. cellbag. Numerical simulation by Computation Fluid Dynamics (CFD), is considered as an inexpensive and efficient tool for predicting the fluid behavior in many fields. In the present study, we perform numerical simulations by Ansys-FLUENT to characterize the flow conditions in a 10 L cellbag. The numerical simulations are carried out to investigate the fluid structures for nine different operating conditions of rocking speed and angle. The influence of these operating parameters on the mixing and the shear stress induced by the liquid motion are studied. We find that the mixing and shear stress increase with the cellbag angle from 4° to 7° but that increasing rocking speeds are not systematically associated with increasing mixing and shear stress. It is concluded that a resonance phenomenon is responsible for the fact that the lowest studied rocking speed, 15 rpm, generates the highest fluid velocity, mixing and shear stress compared to the higher speeds of 22 and 30 rpm.

A bioreactor system for the manufacture of a genetically modified Plasmodium falciparum blood stage malaria cell bank for use in a clinical trial

BackgroundAlthough the use of induced blood stage malaria infection has proven to be a valuable tool for testing the efficacy of vaccines and drugs against Plasmodium falciparum, a limiting factor has been the availability of Good Manufacturing Practice (GMP)—compliant defined P. falciparum strains for in vivo use. The aim of this study was to develop a cost-effective method for the large-scale production of P. falciparum cell banks suitable for use in clinical trials.MethodsGenetically-attenuated parasites (GAP) were produced by targeted deletion of the gene encoding the knob associated histidine rich protein (kahrp) from P. falciparum strain 3D7. A GAP master cell bank (MCB) was manufactured by culturing parasites in an FDA approved single use, closed system sterile plastic bioreactor. All components used to manufacture the MCB were screened to comply with standards appropriate for in vivo use. The cryopreserved MCB was subjected to extensive testing to ensure GMP compliance for a phase 1 investigational product.ResultsTwo hundred vials of the GAP MCB were successfully manufactured. At harvest, the GAP MCB had a parasitaemia of 6.3%, with 96% of parasites at ring stage. Testing confirmed that all release criteria were met (sterility, absence of viral contaminants and endotoxins, parasite viability following cryopreservation, identity and anti-malarial drug sensitivity of parasites).ConclusionLarge-scale in vitro culture of P. falciparum parasites using a wave bioreactor can be achieved under GMP-compliant conditions. This provides a cost-effective methodology for the production of malaria parasites suitable for administration in clinical trials.

Rapid expansion in the WAVE bioreactor of clinical scale cells for tumor immunotherapy

ABSTRACTCell-based immunotherapy using natural killer (NK) cells, cytokine-induced killer (CIK) cells and dendritic cells (DCs) is emerging as a potential novel approach in the auxiliary treatment of a tumor. However, non-standard operation procedure, small-scale cell number, or human error may limit the clinical development of cell-based immunotherapy. To simplify clinical scale NK cells, CIK cells and DCs expansions, we investigated the use of the WAVE bioreactor, a closed system bioreactor that utilizes active perfusion to generate high cell numbers in minimal volumes. We developed an optimized rapid expansion protocol for the WAVE bioreactor that produces clinically relevant number of cells for our adoptive cell transfer clinical protocols. The high proliferative rate, surface phenotypes, and cytotoxicity of these immune cells, as well as the safety of cultivation were analyzed to illuminate the effect of WAVE bioreactor. The results demonstrated that the benefit of utilizing modern WAVE bioreactors in cancer immunotherapy was simple, safe, and flexible production.

DROSOPHILA S2 cell culture in a WAVE Bioreactor: potential for scaling up the production of the recombinant rabies virus glycoprotein.

The transmembrane rabies virus glycoprotein (RVGP) is the main antigen of vaccine formulations used around the world to prevent rabies, the most lethal preventable infectious disease known. The objective of this work was to evaluate the potential of a bioreactor using wave-induced agitation in the initial steps of scaling up the rRVGP production process by a Drosophila melanogaster S2 cell line to produce rRVGP in sufficient quantities for immunization and characterization studies. Taking advantage of some remarkable features recognized in Drosophila S2 cells for scaling the culture process, a robust recombinant lineage (S2MtRVGPH-His) engineered by our group for the expression of rRVGP using a copper-inducible promoter was used in the bioreactor cultures. The WAVE Bioreactor was chosen because it represents an innovative approach to the cultivation of animal cells using single-use technology. For that purpose, we firstly established a procedure for culturing the S2MtRVGPH-His lineage in 100 mL Schott flasks. Using an inoculum of 5 × 105 cells/mL in culture medium (Sf900-III) induced with solution of CuSO4 (0.7mM) and a convenient pH range (6.2-7.0), optimal parameter values such as time of induction (72h) and temperature (28°C) to increase rRVGP production could be defined. This procedure was reproduced in culture experiments conducted in a WAVE Bioreactor™ 2/10 using a 2 L Cellbag. The results in Schott flasks and in WAVE Bioreactor™ were very similar, yielding a maximum titer of rRVGP above of 1 mg.L-1. The immunization study showed that the rRVGP produced in the bioreactor was of high immunogenic quality.

Protein Expression via Transient Transfection of Mammalian Cells in a WAVE Bioreactor.

Large culture volumes are often required when expression constructs are particularly low-yielding or when end-uses require significant amounts of material. In these cases, a single homogenous culture is usually more convenient, in terms of both consistency of expression and labour/resource requirements, than multiple parallel cultures. Using a WAVE Bioreactor culture volumes as high as 500L may be achieved in a single vessel. Here we describe the transfection of 293-6E cells in a disposable 50L Cellbag on a WAVE Bioreactor platform to produce recombinant protein. The methods described herein may be adapted, with suitable optimizations, for other suspension-adapted mammalian cell lines.