Single-Use Systems Bioreactors in the Biopharmaceutical Industry and its Use in SARS-CoV-2 Candidate Vaccine Production - A Review View PDF

According to the growing global demand, it is clear that nextgeneration vaccines will be produced using recombinant organisms and Single-Use Systems (SUSs) including SUSs bioreactors addressed to increase production capacities with reduced costs. SUSs devices, in general, refer to bioprocessing equipment designed to be used once or in a single production campaign and then discarded [1].

The description above can be evidenced in the SARS CoV- 2 pandemic. The worldwide presence of SARS-CoV-2 affected almost all industries and social functions globally, including the biopharmaceutical industry, and with it the bioprocesses for vaccines or its candidate’s production [2,3]. This fact has brought, promptly, a series of significant changes that include existing production practices and/or some preexisting trends aimed at an adaptation process to face this dramatic new situation.

Therefore, the new technology in this pandemic contributes to the rapid development and production of candidate vaccines, therapies, equipment, and diagnostics, all at the same time. Also, according to Kulkarni S (2019) [3], and Macdonald GJ (2020) [4], the changes that this global resource mobilization will bring to the biopharmaceutical industry include:

1. Single-use systems, there will be more SUSs in the new process lines and facilities;
2. Process intensification, faster bioprocessing with smaller facilities and less staff;
3. Greater automation, Plug and Play technology which allows switching between batches, with single-use bioprocessing, five times faster than the traditional process with a stainless steel environment; and
4. Greater biosecurity, security for both, production operations and research

Assuming only two or three pandemic-related vaccines or biotherapeutics are entering the market, each one will likely require the manufacture of hundreds of millions, if not billions, of doses per year. This will require multiple installations. For example, only vaccine manufacturing could involve 4,000 SUSs/year with a total production of eight million liters of culture media per year [4].

It is considered that SUSs demand, including SUSs bioreactors, will increase with the pandemic. It is also true that, since some years ago there was a significant increase of SUSs use in the biopharmaceutical industry, especially in the production of proteins from mammalian cell cultures [5].

It has also been proven that with the use of SUSs, for example in mammalian cell cultures titers, production can be increased from mg / L to g / L by improving the bio-manufacturing of SUSs, which has reduced goods cost during the last 30 years [6].

SUSs bioreactors versatility and potential capabilities positions them as an ideal system in the bioprocess industry as an alternative to conventional glass and stainless steel bioreactors, offering greater flexibility in the bioprocess and ease of operations [7-9].

This work provides, for the first time, an updated overview to academics and researchers, pointed to the use of single-use devices, specifically disposable bioreactors, in the production of viral vaccines, other biopharmaceutical applications and possible challenges of SUSs bioreactors in the race for the production of vaccine candidates in the fight against COVID -19 worldwide.

What are single-use systems (SUSs) devices?

SUSs include upstream or downstream devices such as bioreactors, membranes, mixing systems, connectors, buffer containers, etc. They are mainly composed of plastic material or other biodegradable biopolymers sealed and sterilized by radiation [10]. Figure 1 shows the trend in the use of different disposable components for bioprocessing between 2018 and 2019, including SUSs bioreactors.

Market behavior, as shown in Figure 2, shows a preference for SUSs use. SUSs bioreactors preference in bioprocesses has been increasing since 2013 with a growing projection to 2023 when compared with its stainless-steel counterparts (Figure 3).

Single-Use Bioreactors vs. Conventional Bioreactors

Among the indisputable operational and productive advantages of SUSs bioreactors we can find:

Production time saving, since SUSs eliminate the need for re-sterilization and validation of cleaning systems, which ensures a lower cross-contamination risk [11].

Low operational costs and reduced fixed capital investment because of the limited manpower and infrastructure needed [11].

The final product can enter the commercialization process more expeditiously compared to the products obtained using conventional bioreactors [11,12].

They allow fast and flexible manufacture. For that reason, SUSs are used to accelerate commercialization in the production of antibodies, vaccines, and therapeutic cells [13]. Consequently, SUSs have an advantage over stainless steel bioreactors for large-scale production of vaccines. Thereby, figure 4 shows some limitations of conventional bioreactors such as cross contamination, high water waste, a need for sterilization process, large production times and high cost and energy. These characteristics create a tendency for the biopharmaceutical industry to migrate towards a more flexible and agile production using SUSs.

Main Types of SUSs Used in Biopharmaceutical Applications

Bioreactor design has evolved to meet technical requirements for vaccine manufacturing since the early 1940s when the use of microorganisms in vaccine manufacturing was the main practice, until the use of mammalian cells. Mammalian cells had become a technical challenge for several companies, forcing them to continuously develop innovative products and workflows [14]. As a result, SUSs designs respond to the changing needs not only for the manufacture of vaccines but also for the production of cells, vectors, recombinant proteins, antibodies and secondary metabolites. In this regard, different systems are being designed to achieve these objectives, some of the most used in the biopharmaceutical industry are described below.

Stirred Tank SUSs Bioreactors

Single-use stirred tank bioreactors are a viable alternative for culturing animal cells because of their high throughput, flexibility, and low cross-contamination. They consist of a plastic container with a built-in impeller, contained within a steel tank that facilitates heat transfer [15].

Agitated tank SUSs have successfully replaced their stainless-steel counterparts. Kazemzadeh A, et al. (2018) [16], evaluated the effect of gas volumetric flow rate, impeller speed on shear rate, gas retention and bubble diameter for an angular-axis SUSs stirred tank bioreactor. This study also determined that the mixing system is similar to that of conventional stirred tank bioreactors and meets the requirements for culturing cells.

Currently, the two main manufacturers of this type of SUSs are Thermo-Fisher and Xcellerex and the main commercial brands are AllegroTM STR200, BioBLU®, bioReactor, BIOSTAT® Cultibag STR200, CerCell, iCELLis® fixed-bed bioreactor, Mobius® CellReady, Sartorius Ambr®, SmartVessel^TM / HyPerforma^TM, and Xcellerex^TM XDR [16].

Vaccine development, viral vectors and cell growth are the most recent application of this type of SUSs. Also, it has been proven that the combination with microcarriers in the system provides good results [17]. Lawson T, et al. (2017) [18], achieved a 43-fold expansion of human mesenchymal stromal cells (hMSC) by incorporating microcarriers in the bioreactor. The expanded cells showed inhibition of T cell growth and inducibility of indoleamine 2,3- dioxygenase. A similar study was conducted by Dufey V, et al. (2016) [19], who used the

BioBLU® 0.3c system and generated 1 ×10⁸ cells in a 250 mL working volume. On the other hand, Yang J, et al. (2019) [17], developed an expansion process with microcarriers for HEK293T and Vero cells growth, obtaining a maximum cell concentration of 1.5 × 10⁶ cells / mL and 3.1 × 10⁶ cells / mL with the XDR-50 and XDR-200 bioreactors respectively.

Other successful studies have shown the potential of these SUSs for the production of viral vaccines, and vectors. In 2019, Fedosyuk S, et al. (2019) [20], manufactured vaccine candidates against rabies, malaria and Rift Valley fever, using a type of adenovirus in a single-use stirred tank bioreactor.

Oscillating SUSs Bioreactors

Oscillating wave bioreactors are easy to use and mainly suitable for shear sensitive cell cultures, also displaying comprehensive options for process control and monitoring. Therefore, it has several useful applications for example in cell bank processes and seed inocula within the upstream bioprocess for various cell types: CHO cells, NS0 cells, Escherichia coli, stem cells in clinical applications, insect cells, Madin-Darby Canine Kidney cells (MDCK), HEK 293, algae and cyanobacteria [11,21, and 22].

These type of SUSs generally consist of a biocompatible polymer bag placed on a rocker unit which enables the medium of the bag to be gently mixed through rocking motion [23]. Some of the available platforms include: Biostat® RM, Flexsafe® RM Bags (Sartorius AG) and Wave Bioreactor™ (Cytiva, GE, US) [24].

However, there are certain limitations in the characterization of oscillating bioreactors, mainly from the lack of information. The mass transfer of gas-liquid oxygen in these systems depends on the surface aeration with the air drag of the breaking waves during the forward and backward rolling action [11]. The energy input on gas-liquid mass transfer and mixing in the bioreactor is unknown. Bai Y, et al. (2019) [11], determined that the gas-liquid mass transfer and mixing time decrease with increasing energy input. Still, a more detailed investigation is necessary to gain more technical information.

Due to its rapid process development and clinical manufacturing characteristics, the oscillating bioreactor can be used in cell-based immunotherapy. Meng Y, et al. (2018) [21], took advantage of this kind of bioreactor in the production of peripheral blood mononuclear cell (PBMC), Cytokine-induced killer cells (CIK), Natural Killers (NK), and Dendritic cells (DCs) for clinical trials [21]. The achieved results were promising for cell expansion, generating effective antineoplastic CIK and NK cells; which is an alternative for the production of mammalian cells on a large scale under good manufacturing practices conditions with useful therapeutic applications [21].

The baculovirus expression system is widely used for the production of recombinant proteins because of its versatility and high level of protein expression. Given the advantages of using this expression system, Ghasemi A, et al. (2019) [23], analyzed the implementation of a single-use wave bioreactor for the expression of human papillomavirus L1 protein in infected/uninfected insect cells, obtained from Spodoptera frugiperda (Sf9) and derived from Trichoplusia ni (Tn-5). The effect of the specific oxygen absorption rate (SOUR) was evaluated to scale the process. The results obtained the highest SOUR values after Tn-5 cell infection. The wave bioreactor system (GE, US) used did not use bubbles, which demonstrated minimal shear impact on cells [23].

The application of single-use bioreactors studied in fungal cultures is of great interest due to the ability to synthesize secondary metabolites with biological activity. Aspergillus niger is a filamentous fungus with the ability to synthesize non-ribosomal peptides that show antibacterial, antifungal, insecticidal, anthelmintic, and anticancer activities [25]. Kurt T, et al. (2018) [25], evaluated the heterologous production of the commercial drug enniatin B and the hydrodynamic conditions of A. niger cultivated in a disposable two-dimensional rocking motion bioreactor (CELL-tainer®) compared to a conventional stirred bioreactor (BioFlo STR). It was observed that enniatin B titers obtained with BioFlo STR were higher than those obtained with CELL-tainer®. Although the values were not competitive with the conventional system, the use of these SUSs can be considered for the culture of mutant strains sensitive to shear, as well as for obtaining high cell density cultures or fast biomass acumulation [25].

In the pharmaceutical applications of oscillating SUSs using plant cells, Khojasteh A, et al. (2016) [26] studied the cell suspension cultures of Satureja khuzistanica, which were evaluated for the production of rosmarinic acid (RA), a secondary metabolite with biological activity in slowing down the development of Alzheimer's and as an anti-cancer agent. A mechanically driven single-use bioreactor (BIOSTAT RM 20/50 from Sartorius Stedim Biotech) was used for the study with a maximum RA production of 3.1 g / L and a biomass productivity of 18.7 g / L with methyl jasmonate as an inducer [26].

Also, Halgo D, et al. (2017) [27], evaluated the plant cell culture of Centella asiatica. This plant is used in traditional medicine for the treatment of dry skin conditions, leprosy, varicose ulcers, eczema and / or psoriasis due to its centelosides. The use of a 2L CellBag (GE Heathcare Bio - Sciences AB, Uppsala, Sweden) resulted in a successful growth of C. asiatica cell line, achieving a growth index of 4.8 with a final production of centeloside of 7.3 mg / g dry weight using combined treatments with inducers, crop feeding and natural sources of amyrins.

Perfusion SUSs Bioreactors

The implementation of continuous operations, also called perfusion systems, can obtain expensive biological products in small volumes. These systems are limited because they require continuous feeding of new media and disposal of the used media. In this SUSs model, cells are grown in a plastic bag where cells are retained in the bioreactor through the use of perfusion equipment such as alternate tangential flow devices, cross flow filters, centrifuges, settlers, rotating filters, hydrocyclones or by the use of hollow capillary fibers, flat plates, membranes and microcarriers. The advantages of this system are the ease of continuous culturing of cells due to the lower possibility of accumulation of waste products or filter clogging, flexibility, low cost, improved quality, speed, minimization of the probability of any product inhibition and high product yield [6,28, and 29].

These SUSs are used successfully in the production of complex proteins/enzymes [6]. They provide good nutrient distribution and excellent oxygen transfer without gas bubbles or shear forces, which make them suitable for anchorage-dependent cell line cultures and virus production [6]. There are many manufacturers in the development of these systems such as Pall Life Sciences, Applikon Biotechnology, Satorius, AcuSyt, GE Healthcare, PerfuseCell^TM, with trademarks like ambr® 250 perfusion, CellMembra^TM, CellRetention^TM, CellTernate^TM [28,31].

In one of its applications (2020), three processes for the manufacture of monoclonal antibodies produced by Chinese hamster ovary (CHO) cells were compared [6]. The used scheme achieved a substantial improvement in titration through intensified seed culture strategies. Besides, these systems can also achieve a rapid and economical process for the production of adenoviruses under good manufacturing practices conditions [30].

Also, Coronel J, et al. (2019) [32] evaluated influenza A virus production in a single-use orbital shaken bioreactor with perfusion systems. The study demonstrated that the cell line used, AGE1.CR1pIX, in suspension obtained large cell densities (50 ×10⁶ cells / ml), cell viability and short doubling time using tangential flow filtration (TFF) and alternating tangential flow filtration (ATF).

Fixed Bed SUSs Bioreactors

Fixed-bed SUSs are highly automated and allow process scaling [33]. Due to their low shear system, they have been used successfully to cultivate mammalian cells and have potential applications for vaccine manufacture [8,33, and 34]. Trademarks of this type of SUSs include: iCELLis®, developed by PALL; Bioreactor scale X, from Univercells; BelloCell® (laboratory scale) and TideCell002 (industrial scale), both from Cesco Bioengineering. The parts of Fixed Bed SUSs bioreactors are: thermocouple, feed line, preheating zone, electrical element, alumina jacket, grid, quartz beads, catalyst bed, asbestos, fire brick, thermocouple, furnace outer wall, and product line [35].

iCELLis® SUSs, for example, are compact, fixed-bed bioreactors with an integrated perfusion system. High yields of viral vaccines, recombinant proteins, adeno-associated viral vectors and retroviral vectors have been produced in these bioreactors [33,34]. Valkama AJ, et al. (2018) [34], tested clinical scaling with the PALL iCELLis® bioreactor, and 293T adherent cells for the production of lentiviral vectors, optimizing the production parameters.

On the other hand, scale-X bioreactors have a variety of growth surfaces: scale-X 'hydro' (< 3 m2), 'carbo' (10-30 m2) and 'nitro' (200-600 m2) [33]. Nogueira DES, et al. (2019) [36], used the scale- X^TM carbo system to produce a recombinant vaccine against vesicular stomatitis virus using Vero cells. The results detailed a 4-log increase in virus production when using the bioreactor, demonstrating higher virus production per surface area compared to classical production. In addition, the scale-X carbo bioreactor avoids the limitation of suffering a decrease in production efficiency in scaling up, that other fixed-bed bioreactors have, by increasing the height of the bed design while maintaining the diameter to limit the impact.

Also, BelloCell and TideCell002 SUSs, were used in the cultivation of MDCK and Vero cells for the production of vaccines against H5N1 and H7N9 viruses [20]. The data showed that TideCell002 can produce 10-20 L of vaccine per practice and that the process is 20 times higher in volume compared to the BelloCell-500A® system. Due to the linear scaling of the TideCell002 system, researchers consider it easy for scaling to 500-1000L bioreactors.

Vertical Wheel Sus Bioreactors

Single Use Vertical Wheel Bioreactors (SUVW) are available in a wide range of scales, from 100 mL to 80 L. Agitation in these bioreactors is provided by a large vertical impeller, which when combined with a U-shaped bottom allows better homogenization of the content with a reduced energy input [36].

SUVWs have been successful in the production of various cells such as human mesenchymal stem cells (MSCs), embryonic stem cells (ESC), pluripotent stem cells (PSC), and chondrocytes. As a very significant advantage of SUVWs, compared to traditional bioreactors, cells are exposed to lower shear levels [36,37].

Besides, SUVWs have been successfully used for the expansion of human-induced pluripotent stem cells (hiPSC) as floating aggregates because they show a promising alternative for drug industry and cell-based therapies. To carry out this experience, Nogueira DES, et al. (2019) [36], filled a PBS MINI 0.1 (PBS Biotech) bioreactor with 60 mL of working volume. Two different culture media, mTeSR1 and mTeSR3D, were tested, demonstrating that mTeSR3D supports the expansion of hiPSC stem cells, although with lower cell density than when mTeSR1 was used. In addition, media supplemented with dextran sulfate allowed to obtain a higher cell density with one less day of culture. Therefore, according to the obtained results, SUVWs can be considered as a promising technology for the bioprocessing of hiPSC.

General Parameters to be Considered for Vaccine Production and Other Biopharmaceutical Applications Using SUSs

The steady increase in the manufacture of biologics, the patent expiration for the use of highly successful drugs / molecules, as well as the limited number of potential products in the pipeline, have motivated companies to adopt new techniques and technologies. SUSs are a viable solution for which, different parameters, such as: type of host cells to be used, type of SUSs and operational variables, have been established by different authors to meet the desired results as shown in Table 1 and Table 2:

Table 1: General parameters and variables to take into account for Single-Use Systems bioreactors use.

SUSs update in the production of vaccine candidates against SARS-CoV-2

By the end of 2019, China reported the presence of the severe acute respiratory syndrome SARS-CoV-2 [48]. Therefore, in March 2020, due to its rapid worldwide spread, it was declared a pandemic by the World Health Organization (WHO).

Since then, several vaccine formulations against COVID-19 have been developed and registered mainly in Asia, Europe and North America since August 2020 [49]. Also Latin America joined the race of developing an appropriate vaccine candidate [50,54]. In all such research and production efforts, SUSs devices, including SUSs bioreactors are being applied as a cutting edge technology in the clinical production of biopharmaceuticals [55].

To generate rapid results against the COVID-19 pandemic, the U.S Department of Health and Human services through the Advanced Biomedical Research and Development Authorization (BARDA) created a public-private partnerships called Centers for Innovation in Advanced Development and Manufacturing (CIADM) in order to develop countermeasures to medical emergencies [56].

The Bayview campus of Emergent Biosolutions was selected as one of the three CIADMs due to its experience in the employment of single-use technology for rapid, high-volume manufacturing of vaccines and therapeutics including those related to SARS-CoV-2 [56]. Emergent Biosolutions announced the development and manufacture of its COVID-19 vaccine candidates in collaboration with the Johnson & Johnson Company, which investigated genetically modified adenovirus-based vaccines against SARS-CoV-2 [57]. They also settled an agreement with Novavax for the manufacture of a vaccine candidate and with Vaxart in the development of an oral vaccine for COVID-19 [58]. On June 11, 2020, Emergent BioSolutions declared the provision of its services by using single use technology for the large-scale development and the manufacturing of AstraZeneca's AZD1222 vaccine candidate, a viral vector-based vaccine that contains genetic material from the SARS-CoV-2 Spike (S) protein [57].

On the other hand, CanSinBio Biologics Inc. and the Institute of Bioengineering of the Academy of Military Medical Sciences used the single-use bioreactor BIOSTAT® STR for the production of the recombinant adenovirus vaccine Ad5-nCoV due to its advantages in bioprocess development time, scalability, bioprocess flexibility and cross contamination risks reduction [59]. The Biostat STR® system includes a BioPAT® toolbox that monitors the process and the Flexsafe®STR single-use bags that range from 50 L to 2000 L [60,61]. The process of production for the Ad5-nCoV vaccine is described in figure 5.

The process begins with the optimization of the peak protein gene with UpGene software. This gene is subsequently cloned into a shuttle plasmid of AdMax adenovirus system (Microbix Biosystem, Canada) and co-transfected into HEK293 cells. Finally, for the high production of recombinant particles, cells are amplified using the single-use bioreactor BIOSTAT® STR from Sartorius.

Additionally, the Virology laboratory and the Bioprocess Engineering Group at Wageningen University use SUSs from Applikon Biotechnology for SARS-CoV-2 vaccine production. Their vaccine candidate is based on the production of the viral S protein using baculovirus and Sf9 insect cells as hosts. Their studies consisted of determining the optimal conditions for cell growth and protein production in Sf9 cells. The micro-Matrix system (Applikon Biotechnology, Netherlands) was used for the control of DO, pH and temperature because it offers a total control of 24 independent bioreactors. Optimal conditions were also verified in the miniBio (single-use autoclavable bioreactor) of 500 mL and the AppliFlex ST SUSs bioreactor of 500 mL, which presents scalability characteristics (scalable to 20 L), is customizable, data generates in less time, has simple configuration of its operation and its flexible to the demands of the process [62,63]. However, in some studies, SUSs have been scaled up to 2000 L, but it could be a limitation if a volume greater than 2000 L is required [5,13]. Reactor brands that work with volumes of 2000 L are BIOSTAT ® Cultibag STR200, Mobius ® CellReady and Xcellerex XDR^TM [5]. The problems for achieving a scale-up greater than 2000 L focus on the structural soundness of the reactors and the leaks, but this approach is still under discussion.

However, ABEC, a leading global provider of pharmaceutical product development solutions and services, delivered SUSs for the vaccine manufacturing of Novavax's COVID-19 candidate, NVX-CoV2373, which consisted of six 4000 L Custom Single Run (CSR) bioreactors donated to the Serum Institute of India. The aim of the application of these SUSs was to promote a supply of NVX-CoV2373 vaccine throughout India and low/middle income countries doubling its productivity at a lower cost [64].

The expected market for SUSs increased by 17.8% between 2016 and 2021, but it will continuously grow at a rate of nearly 15.5% over the forecast period, 2018 to 2023 [65]. This increment in SUSs systems has gained popularity because they have demonstrated that can provide an excellent option to adapt the production capacity to both, research and production demands [66]. This fact makes them a viable solution for mass production of different biological reagents in sensitive cells, antibodies or vaccine candidates to face pandemic-type situations such as COVID-19.

In the biopharmaceutical industry, the use of SUSs influences the production process with cost reduction, flexibility, time reduction when executing by eliminating additional steps and consequently with a reduction of cross contamination risks [67,68]. Also, its use significantly reduces process fluid waste, labor costs, and quality of in-situ validation requirements.

However, there are certain limitations in the use of these innovative SUSs mainly related to personnel training to deal with these new technologies, the challenges regarding the scaling up process due to the mechanical resistance of the bag’s materials, as well as the optimization to determine appropriate working conditions at higher scales. Even so, the aforementioned limitations related to scaling up can be quickly overcome by the increasing automatization and the use of adequate biodegradable materials for their design.

Other possible limitations of SUSs are the risk of rupturing or leaking of disposable bags caused by their chemical, physical and biological properties; also, the migration of pollutants from the plastic material to the environment; and, the possible increase in operating costs due to the consumption of single-use materials such as bags, pipe filters, etc., resulting in high production of waste [69].

Despite the possible limitations, the use of SUSs is still a viable solution to the need for mass production of vaccines to face pandemic-type situations such as COVID-19. This is due to the advantages that these systems have over conventional stainless-steel bioreactors in terms of production time and costs. Especially, the manageable parameters of the SUSs could be adjusted, benefiting the cultivation of mammalian and insect cells necessary for the production of vaccines and other applications such as clinical cell treatment, production of therapeutic proteins, among others.

Continuous research is still needed to establish a guide so that we could determine which production way is the most appropriate between conventional stainless-steel equipment and SUSs. The use of conventional or SUSs bioreactors will strongly depend on three main items: the purpose to be carried out, the bioprocess and the client specifications. These studies will be necessary to overcome as soon as possible any SUSs limitations in order to take advantage of its indisputable productivity advantages.

The authors thank the Universidad de las Fuerzas Armadas - ESPE and the Industrial Biotechnology and Bioproducts Research Group, Center for Nanoscience and Nanotechnology (CENCINAT), for the opportunity provided to us, as well as for their invaluable help and guidance during the development of this work.

The authors declare that they have no conflicts of interest, that the work has been approved by the ethics committee responsible in the workplace, and do not declare means of financing of the work carried out.

1. Cappia JM, Langlois C, Hogreve M, Hauk A, Wormuth K (2019) Single-use systems for vaccine manufacturing addressing biocompatibility, integrity and supply chain issues. Sartorius Stedim Biotech
2. World Health Organization (2020) Novel coronavirus (2019-nCoV): situation report, 22. World Health Organization Geneva, Switzerland.
3. Kulkarni S (2019) Paving the way to a bright future for single-use bioprocessing. Eur Pharm Rev.
4. Macdonald GJ (2020) COVID-19 increases demand for next-generation bioprocessing technology. Genet Eng Biotechnol News.
5. Junne S, Neubauer P (2018) How scalable and suitable are single-use bioreactors? Curr Opin Biotechnol 53: 240-247. https://doi.org/10.1016/j.copbio.2018.04.003
6. Xu J, Xu X, Huang C, Angelo J, Oliveira CL, et al. (2020) Biomanufacturing evolution from conventional to intensified processes for productivity improvement: a case MAbs 12: 1770669. https://doi.org/10.1080/19420862.2020.1770669
7. Amer M, Ramsey JD (2018) Multi-chamber single-use bioreactor– A proof of concept prototype. Biochem Eng J 130: 113-120. https://doi.org/10.1016/j.bej.2017.12.002
8. Lai CC, Weng TC, Tseng YF, Chiang JR, Lee MS, et al. (2019) Evaluation of novel disposable bioreactors on pandemic influenza virus production. PLoS One 14: e0220803. https://doi.org/10.1371/journal.pone.0220803
9. Maltby R, Tian S, Chew YMJ (2018) Computational studies of a novel magnetically driven single-use-technology bioreactor: A comparison of mass transfer models. Chem Eng Sci 187: 157-173. https://doi.org/10.1016/j.ces.2018.05.006
10. Langer R, Rader E (2018) Fifteen years of progress: biopharmaceutical industry survey results. BioPharm Int.
11. Bai Y, Moo-Young M, Anderson WA (2019) Characterization of power input and its impact on mass transfer in a rocking disposable bioreactor. Chem Eng Sci 209: https://doi.org/10.1016/j.ces.2019.115183
12. Eibl D, Eibl R (2019) Single?use equipment in biopharmaceutical manufacture: a brief introduc 5: 1-11. https://doi.org/10.1002/9781119477891.ch1
13. Pollard D, Kistler C (2017) Disposable bioreactors. Curr Develop Biotechnol Bioeng 2017: 353-379. https://doi.org/10.1016/B978-0-444-63663-8.00012-4
14. Tay A (2020) Bioreactor designs respond to evolving needs in vaccine Lab Manag.
15. Kazemzadeh A, Elias C, Tamer M, Lohi A, Ein-Mozaffari F (2020) Mass transfer in a single- use angled-shaft aerated stirred bioreactor applicable for animal cell culture. Chem Eng Sci 219: 115606. https://doi.org/10.1016/j.ces.2020.115606
16. Kazemzadeh A, Elias C, Tamer M, Ein-Mozaffari F (2018) Hydrodynamic performance of a single-use aerated stirred bioreactor in animal cell culture: applications of tomography, dynamic gas disengagement (DGD), and CFD. Bioprocess Biosyst Eng 41: 679-695. https://doi.org/10.1007/s00449-018-1902-7
17. Yang J, Guertin P, Jia G, Lv Z, Yang H, et al. (2019) Large-scale microcarrier culture of HEK293T cells and Vero cells in single-use bioreactors. AMB Express 9: https://doi.org/10.1186/s13568-019-0794-5
18. Lawson T, Kehoe DE, Schnitzler AC, Rapiejko PJ, Der KA, et al. (2017) Process development for expansion of human mesenchymal stromal cells in a 50L single-use stirred tank bioreactor. Biochem Eng J 120: 49-62. https://doi.org/10.1016/j.bej.2016.11.020
19. Dufey V, Tacheny A, Art M, Becken U, Longueville FD (2016) Expansion of human bone marrow-derived mesenchymal stem cells in BioBLU® 3c single-use bioreactors. Appl Note 305: 1-8.
20. Fedosyuk S, Merritt T, Peralta-Alvarez MP, Morris SJ, Lam A, et (2019) Simian adenovirus vector production for early-phase clinical trials: A simple method applicable to multiple serotypes and using entirely disposable product-contact components. Vaccine 37: 6951-6961. https://doi.org/10.1016/j.vaccine.2019.04.056
21. Meng Y, Sun J, Hu T, Ma Y, Du T, et al. (2018) Rapid expansion in the WAVE bioreactor of clinical scale cells for tumor Hum Vaccines Immunother 14: 2516-2526. https://doi.org/10.1080/21645515.2018.1480241
22. Bai Y, Moo?Young M, Anderson WA (2019) A mechanistic model for gas–liquid mass transfer prediction in a rocking disposable bioreactor. Biotechnol Bioeng 116: 1986-1998. https://doi.org/10.1002/bit.27000
23. Ghasemi A, Bozorg A, Rahmati F, Mirhassani R, Hosseininasab S (2019) Comprehensive study on Wave bioreactor system to scale up the cultivation of and recombinant protein expression in baculovirus-infected insect cells. Biochem Eng J 143: 121-130. https://doi.org/10.1016/j.bej.2018.12.011
24. Eibl R, Werner S, Eibl D (2009) Bag bioreactor based on wave-induced motion: characteristics and applications. Adv Biochem Eng Biotechnol 115: 55-87.
25. Kurt T, Marbà-Ardébol AM, Turan Z, Neubauer P, Junne S, et al. (2018) Rocking Aspergillus: morphology-controlled cultivation of Aspergillus niger in a wave-mixed bioreactor for the production of secondary Microb Cell Factories 17: 128. https://doi.org/10.1186/s12934-018-0975-y
26. Khojasteh A, Mirjalili MH, Palazon J, Eibl R, Cusido RM (2016) Methyl jasmonate enhanced production of rosmarinic acid in cell cultures of Satureja khuzistanica in a bioreactor. Eng Life Sci 16: 740-749. https://doi.org/10.1002/elsc.201600064
27. Halgo D, Steinmetz V, Brossat M, Tournier?Couturier L, Cusido RM, et (2017) An optimized biotechnological system for the production of centellosides based on elicitation and bioconversion of Centella asiatica cell cultures. Eng Life Sci 17: 413- 419. https://doi.org/10.1002/elsc.201600167
28. Ravindran S, Singh P, Nene S, Rale V, Mhetras N, et al. (2019) Microbioreactors and perfusion bioreactors for microbial and mammalian cell culture. Biotechnol Bioeng 6: 91. https://doi.org/10.5772/intechopen.83825
29. Thermo Fisher Scientific (2020) Gibco cell culture for bioprocessing. Thermo Fisher Scientific.
30. Chen KD, Wu XX, Yu DS, Ou HL, Li YH, et al. (2018) Process optimization for the rapid production of adenoviral vectors for clinical trials in a disposable bioreactor system. Appl Microbiol Biotechnol 102: 6469-6477. https://doi.org/10.1007/s00253-018-9091-5
31. PerfuseCell (2021) Perfusion bioreactor’s for any R&D setup.
32. Coronel J, Behrendt I, Bürgin T, Anderlei T, Sandig V, et (2019) Influenza A virus production in a single-use orbital shaken bioreactor with ATF or TFF perfusion systems. Vaccine 37: 7011-7018. https://doi.org/10.1016/j.vaccine.2019.06.005
33. Berrie DM, Waters RC, Montoya C, Chatel A, Vela EM (2020) Development of a high-yield live-virus vaccine production platform using a novel fixed-bed Vaccine 38: 3639-3645. https://doi.org/10.1016/j.vaccine.2020.03.041
34. Valkama AJ, Leinonen HM, Lipponen EM, Turkki V, Malinen J, et al. (2018) Optimization of lentiviral vector production for scale-up in fixed-bed bioreactor. Gene Ther 25: 39-46. https://doi.org/10.1038/gt.2017.91
35. Eshraghi A, Mirzaei AA, Atashi H (2015) Kinetics of the Fischer–Tropsch reaction in fixed- bed reactor over a nano-structured Fe–Co–Ce catalyst supported with SiO2. J Nat Gas Sci Eng 26: 940-947. https://doi.org/10.1016/j.jngse.2015.06.036
36. Nogueira DES, Rodrigues CAV, Carvalho MS, Miranda CC, Hashimura Y, et al. (2019) Strategies for the expansion of human induced pluripotent stem cells as aggregates in single-use Vertical-Wheel^TM J Biol Eng 13: 74. https://doi.org/10.1186/s13036-019-0204-1
37. Kelly PS, Dorival?García N, Paré S, Carillo S, Ta C, et al. (2019) Improvements in single?use bioreactor film material composition leads to robust and reliable Chinese hamster ovary cell performance. Biotechnol Prog 35: e2824. https://doi.org/10.1002/btpr.2824
38. Wierzchowski K, Grabowska I, Pilarek M (2020) Efficient propagation of suspended HL-60 cells in a disposable bioreactor supporting wave-induced agitation at various Reynolds number. Bioprocess Biosyst Eng 43: 1973-1985. https://doi.org/10.1007/s00449-020-02386-6
39. Galliher PM, Guertin P, Clapp K, Tuohey C, Damren R, et al. (2019) Single?use bioreactor platform for microbial fermentation. Single?Use Technology in Biopharmaceutical Manufacture. 5: 247-258. https://doi.org/10.1002/9781119477891.ch21
40. Shen CF, Guilbault C, Li X, Elahi SM, Ansorge S, et al. (2019) Development of suspension adapted Vero cell culture process technology for production of viral vaccines. Vaccine 37: 6996-7002. https://doi.org/10.1016/j.vaccine.2019.07.003
41. Cytiva (2020) Viral vectors for use in gene therapy, cell therapy, and in the development of vaccines.
42. World Health Organization (2021) AZD1222 vaccine against COVID-19 developed by Oxford University and Astra Zeneca: background paper. World Health Organization, Geneva, Switzerland.
43. Petiot E, Cuperlovic-Culf M, Shen CF, Kamen A (2015) Influence of HEK293 metabolism on the production of viral vectors and vaccines. Vaccine 33: 5974-5981. https://doi.org/10.1016/j.vaccine.2015.05.097
44. Lohr V, Hädicke O, Genzel Y, Jordan I, Büntemeyer H, et al. (2014) The avian cell line AGE1.CR.pIX characterized by metabolic flux analysis. BMC Biotechnol 14: 1-4. https://doi.org/10.1186/1472-6750-14-72
45. Nurhayati RW, Ojima Y, Dohda T, Kino-Oka M (2018) Large-scale culture of a megakaryocytic progenitor cell line with a single-use bioreactor system. Biotechnol Prog 34: 362-369. https://doi.org/10.1002/btpr.2595
46. Dukes J, Whitley P, Chalmers A (2011) The MDCK variety pack: choosing the right BMC Cell Biol 12: 1-4. https://doi.org/10.1186/1471-2121-12-43
47. Barnes S, Bundy M, Thompson K, Yovcheva M (2020) Scalable protein and AAV production in the ExpiSf^TM Expression System.
48. Tu YF, Chien CS, Yarmishyn AA, Lin YY, Luo YH, et al. (2020) A review of SARS-CoV-2 and the ongoing clinical trials. Int J Mol Sci 21: https://doi.org/10.3390/ijms21072657
49. Ferbeyre G, Vispo N (2020) The race for a coronavirus vaccine. Bionatura. http://dx.doi.org/10.21931/RB/2020.05.04.1
50. Arboleya T (2020) The Sovereign Cuban Vaccine 01.              Cuba.
51. Concina N (2020) Científicos de la Universidad de San Martín se suman a la búsqueda de la vacuna. Télam SE Agencia Nac Not.
52. Corum J, Grady D, Wee SL, Zimmer C (2020) Coronavirus vaccine tracker. N Y Times 31: 1-17.
53. León-Velarde F (2020) COVID-19: La carrera peruana por la vacuna. Cons Nac Cienc Tecnol E Innov Tecnológica Perú.
54. World Health Organization (2020) Draft landscape of COVID-19 candidate vaccines. World Health Organization, Geneva, Switzerland.
55. Sartorius Stedim Biotech (2017) Advances in Single-Use Platforms for Commercial Manufacturing.
56. Markarian J (2020) Preparing pandemic vaccine capacity Pharmaceut Technol 44: 16-20.
57. BioSolutions E (2020) Emergent BioSolutions signs agreement to be U.S. manufacturing partner for AstraZeneca’s COVID-19 vaccine candidate.
58. BioSolutions E (2020) Our Innovation | Emergent BioSolutions.
59. Wu S, Zhong G, Zhang J, Shuai L, Zhang Z, et al. (2020) A single dose of an adenovirus-vectored vaccine provides protection against SARS-CoV-2 challenge. Nat Commun 11: https://doi.org/10.1038/s41467-020-17972-1
60. Atkinson S (2020) Sartorius supports coronavirus vaccine research in China. Membr Technol 2020: 4.
61. Sartorius AG (2020) The New Biostat STR® Generation 3 and Biobrain® Automation Platform. SARTORIUS.
62. Applikon Biotechnology (2020) Applikon bioreactors used in search for COVID-19 vaccine. Applikon Biotechnol.
63. Oudshoorn A (2020) Applikon Biotechnology joins the race to develop a COVID-19 vaccine. Genet Eng Biotechnol News 40: 17. https://doi.org/10.1089/gen.40.06.17
64. Businesswire (2020) Serum Institute selects ABEC for large-scale, single-use COVID-19 vaccine manufacturing.
65. Saxena P (2018) 6 benefits of single-use bioprocessing. Mark Res.
66. Market Updates (2020) Global single-use bioreactor market top countries data 2020: Industry trends, growth, size, segmentation, future demands, latest innovation, sales revenue by regional forecast.
67. Lopes AG (2015) Single-use in the biopharmaceutical industry: A review of current technology impact, challenges and limitations. Food Bioprod Process 93: 98-114. https://doi.org/10.1016/j.fbp.2013.12.002
68. Bou JR, De Wilde D (2014) Comparing multiuse and single-use bioreactors for virus production. BioProcess Int Suppl 12: 28-32.
69. Lütke-Eversloh DT, Rogge P (2018) Biopharmaceutical manufacturing in single-use bioreactors. Pharm Ind 80: 281-284.