Search

The implementation of single‐use technologies offers several major advantages, e.g. prevention of cross‐contamination, especially when spore‐forming microorganisms are present. This study investigated the application of a single‐use bioreactor in batch fermentation of filamentous fungus Penicillium sp. (IBWF 040‐09) from the Institute of Biotechnology and Drug Research (IBWF), which is capable of intracellular production of a protease inhibitor against parasitic proteases as a secondary metabolite. Several modifications to the SU bioreactor were suggested in this study to allow the fermentation in which the fungus forms pellets. Simultaneously, fermentations in conventional glass bioreactor were also conducted as reference. Although there are significant differences in the construction material and gassing system, the similarity of the two types of bioreactors in terms of fungal metabolic activity and the reproducibility of fermentations could be demonstrated using statistic methods. Under the selected cultivation conditions, growth rate, yield coefficient, substrate uptake rate, and formation of intracellular protease‐inhibiting substance in the single‐use bioreactor were similar to those in the glass bioreactor.

In this study, a hydrocyclone (HC) especially designed for mammalian cell separation was applied for the separation of Chinese hamster ovary cells. The effect of key features on the separation efficiency, such as type of pumphead in the peristaltic feed pump, use of an auxiliary pump to control the perfusate flow rate, and tubing size in the recirculation loop were evaluated in batch separation tests. Based on these preliminary batch tests, the HC was then integrated to 50-L disposable bioreactor bags. Three perfusion runs were performed, including one where perfusion was started from a low-viability late fed-batch culture, and viability was restored. The successive runs allowed optimization of the HC-bag configuration, and cultivations with 20-25 days duration at cell concentrations up to 50 × 106 cells/ml were performed. Separation efficiencies up to 96% were achieved at pressure drops up to 2.5 bar, with no issues of product retention. To our knowledge, this is the first report in literature of high cell densities obtained with a HC integrated to a disposable perfusion bioreactor.

Herein, we described a scale-up strategy focused on the dissolved carbon dioxide concentration (dCO2 ) during fed-batch cultivation of Chinese hamster ovary cells. A fed-batch culture process for a 2000-L scale stainless steel (SS) bioreactor was scaled-up from similarly shaped 200-L scale bioreactors based on power input per unit volume (P/V). However, during the 2000-L fed-batch culture, the dCO2 was higher compared with the 200-L scale bioreactor. Therefore, we developed an alternative approach by evaluating the kL a values of O2 (kL a[O2 ]) and CO2 [kL a(CO2 )] in the SS bioreactors as a scale-up factor for dCO2 reduction. The kL a ratios [kL a(CO2 )/kL a(O2 )] were different between the 200-L and 2000-L bioreactors under the same P/V condition. When the agitation conditions were changed, the kL a ratio of the 2000-L scale bioreactor became similar and the P/V value become smaller compared with those of the 200-L SS bioreactor. The dCO2 trends in fed-batch cultures performed in 2000-L scale bioreactors under the modified agitation conditions were similar to the control. This kL a ratio method was used for process development in single-use bioreactors (SUBs) with shapes different from those of the SS bioreactor. The kL a ratios for the SUBs were evaluated and conditions that provided kL a ratios similar to the 200-L scale SS bioreactors were determined. The cell culture performance and product quality at the end of the cultivation process were comparable for all tested SUBs. Therefore, we concluded that the kL a ratio is a powerful scale-up factor useful to control dCO2 during fed-batch cultures.

Stirred single-use bioreactors are present throughout the biopharmaceutical manufacturing of high-value cell products. Lots of work has been done determining relevant process engineering parameters to enable reliable scale-up on a physical foundation. However, heat transfer capabilities of such systems are not well described, yet. As processes are further intensified and microbial hosts offer interesting opportunities, heat transfer becomes increasingly relevant. The present work gives a comprehensive overview on the specific implications of a stirred, 50 L, single-use bioreactor by means of heat transfer. This includes the dependency of overall heat transfer coefficients on process parameters, i.e. stirring frequency and jacket flow, with a corresponding statistical analysis. Experimental results are compared against a theoretical approach, based on Nu numbers. Although a systematic discrepancy on the description of the jacket-side heat transfer is present, there already is a good agreement between experimental and literature derived values. The jacket-side impact on the overall performance is then analyzed in detail by the Wilson plot method, underlining the major influence of this aspect. Since the polymer-film layer represents a significant feature of single-use bioreactors, this effect has been investigated for two film types as well. Finally, a first comparison of the 50 L against a 200 L bioreactor was carried out to identify scale-dependencies.

The optimization of operating conditions is vital for cell culture process development. In the present study, the pilot-scale single-use bioreactor XcellerexTM XDR-200, which is typically used in cell culture scale-up, is investigated. This unbaffled reactor is equipped with an off-centered, bottom mounted impeller, and has a maximum working volume of 200 L. The flow field for this sparsely investigated bioreactor configuration is studied with computational fluid dynamics. Moreover, numerical simulations of the mixing time and the volumetric oxygen mass transfer coefficient for seven different test conditions are validated against experimental data. Mixing times across all tested conditions are found to remain below 30 s, and the volumetric oxygen mass transfer coefficient is between 2.0 to 20.0 h−1, which is a typical range for animal cell culture processes. These values of the oxygen transfer coefficient are found for as low as 0.004 to 0.125 L L−1 min−1 sparging rates with the microporous sparger. The hydrodynamic stress remains below critical values for cell damage for all conditions investigated. Vortex formation is suppressed in the bioreactor even though it is unbaffled due to off-centered impeller. Across the test conditions, the highest investigated impeller speed of 200 rpm is the most favorable due to fast mixing and high oxygen transfer.

Stirred tank single-use bioreactors have proven their capability to successfully replace their stainless-steel counterparts in the biopharmaceutical industry. To date, however, only a five-fold volume expansion is achievable in a single-use stirred tank bioreactor, which in turn, necessitates intermediate equipment to scale up the culture to production volume. This study introduces a two-chamber single-use bioreactor that is capable of cell culture expansion from 1 to 50 L. The prototype is a proof of concept that can help users save costs of purchase and qualification of equipment, reduce factory footprint, and reduce the risk of contamination during culture transfer from one intermediate bag to another. The prototype is made of two chambers of different volumes, interconnected as a single, closed system. The design and construction of the prototype is described in detail and results from the engineering characterization (e.g., mixing time, power input per unit volume, and oxygen mass transfer coefficient) are reported for both chambers. The results are in good agreement with general criteria proposed in literature for bioreactor design and with published data for commercially available bioreactors. Further, the concept is not limited to the two-chamber design presented in this study; additional chambers can be integrated along with different volumes, geometries, mixing, sparging, and heating approaches.

Clostridium tetani, a spore-forming anaerobic bacterium, and a casein-based semisynthetic medium were used to produce tetanus toxin in this study. N-Z-Case TT (casein hydrolysate) solution and glucose stock media were mixed and autoclaved, which resulted in tetanus toxin expression. The toxin was expressed when the N-Z-Case TT solution reacted with the glucose stock at a high temperature, creating an adequate amount of Maillard reaction products (MRPs). After accumulating in C. tetani cells, tetanus toxin was secreted into the medium when cell lysis was induced by surface aeration. C. tetani was cultivated and tetanus toxin was expressed in a single-use bioreactor, which produced 80 Lf/mL of tetanus toxin in a medium with MRPs. While using the correct medium to induce tetanus toxin was important, other factors played a part in achieving the desired concentration of the toxin, including the medium processing and culture methods inside the bioreactor. Tetanus toxoid with a purity level greater than 2,500 Lf/mgPN was obtained by detoxifying and purifying the toxin recovered from the fermenter or single-use bioreactor. A single-use bioreactor could be used in a limited space without the need for constructing a large scale production facility, to produce the tetanus toxoid antigen for clinical trials.

Single‐use technologies, in particular disposable bioreactor bags, have become integral within the biopharmaceutical community. However, safety concerns arose upon the identification of toxic leachable compounds derived from the plastic materials. Although the leachable bis(2,4‐di‐tert‐butylphenyl)‐phosphate (bDtBPP) has been previously shown to inhibit CHO cell growth, it is critical to determine if other compounds like this are still present in subsequent generations of films for industrial application. This study compares the performance of CHO cells, CHO‐K1, and CHO‐DP12, cultured in media conditioned in an older single‐use bioreactor (SUB) film (F‐1) and a newer generation film (F‐2) from the same vendor. CHO cells cultured in media conditioned for 7 days in the F‐1 film demonstrated significantly reduced growth and antibody productivity profiles when compared to controls and media conditioned for the same time period in the newer F‐2 film. Proteomic profiling of CHO cells cultured in the F‐1 conditioned media identified differentially expressed proteins involved in oxidative stress response as well as compromised ATP synthesis. These potentially metabolically compromised cells exhibited reduced oxidative phosphorylation activity as well as lower glycolytic metabolism, characteristic of slower growing cells. Nonvolatile and metal leachables analysis of film extracts by LC–MS revealed a reduction in the abundance of the analyzed leachates from F‐2 films when compared to F‐1 films including bDtBPP, potentially explaining improved CHO cell growth in F‐2 conditioned media. Furthermore, in vitro endocrine disruptor testing of the known leachable revealed this molecule to possess the potential to act as an androgen antagonist. This study demonstrates an improvement in the materials composition used in modern generations of SUBs for safe application in the bioprocess. Science Foundation Ireland Irish Industry Technical Group (Allergan Pharmaceuticals Ireland, BioMarin Manufacturing Ireland Ltd., Eli Lilly and Company, Genzyme Ireland Ltd. A Sanofi Company, Janssen Biologics, MSD and Pfizer Ireland Pharmaceuticals) Dublin City University

Erworben im Rahmen der Schweizer Nationallizenz (http://www.nationallizenzen.ch), Disposable bioreactors have increasingly been incorporated into preclinical, clinical, and production-scale biotechnological facilities over the last few years. Driven by market needs, and, in particular, by the developers and manufacturers of drugs, vaccines, and further biologicals, there has been a trend toward the use of disposable seed bioreactors as well as production bioreactors. Numerous studies documenting their advantages in use have contributed to further new developments and have resulted in the availability of a multitude of disposable bioreactor types which differ in power input, design, instrumentation, and scale of the cultivation container. In this review, the term “disposable bioreactor” is defined, the benefits and constraints of disposable bioreactors are discussed, and critical phases and milestones in the development of disposable bioreactors are summarized. An overview of the disposable bioreactors that are currently commercially available is provided, and the domination of wave-mixed, orbitally shaken, and, in particular, stirred disposable bioreactors in animal cell-derived productions at cubic meter scale is reported. The growth of this type of reactor system is attributed to the recent availability of stirred disposable benchtop systems such as the Mobius CellReady 3 L Bioreactor. Analysis of the data from computational fluid dynamic simulation studies and first cultivation runs confirms that this novel bioreactor system is a viable alternative to traditional cell culture bioreactors at benchtop scale.

The increasing application of regenerative medicine has generated a growing demand for stem cells and their derivatives. Single-use bioreactors offer an attractive platform for stem cell expansion owing to their scalability for large-scale production and feasibility of meeting clinical-grade standards. The current work evaluated the capacity of a single-use bioreactor system (1 L working volume) for expanding Meg01 cells, a megakaryocytic (MK) progenitor cell line. Oxygen supply was provided by surface aeration to minimize foaming and orbital shaking was used to promote oxygen transfer. Oxygen transfer rates (kL a) of shaking speeds 50, 100, and 125 rpm were estimated to be 0.39, 1.12, and 10.45 h-1 , respectively. Shaking speed was a critical factor for optimizing cell growth. At 50 rpm, Meg01 cells exhibited restricted growth due to insufficient mixing. A negative effect occurred when the shaking speed was increased to 125 rpm, likely caused by high hydrodynamic shear stress. The bioreactor culture achieved the highest growth profile when shaken at 100 rpm, achieving a total expansion rate up to 5.7-fold with a total cell number of 1.2 ± 0.2 × 109 cells L-1 . In addition, cells expanded using the bioreactor system could maintain their potency to differentiate following the MK lineage, as analyzed from specific surface protein and morphological similarity with the cells grown in the conventional culturing system. Our study reports the impact of operational variables such as shaking speed for growth profile and MK differentiation potential of a progenitor cell line in a single-use bioreactor. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:362-369, 2018.

Single-use bioreactors continue to gain large interest in the biopharmaceutical industry. They are frequently used for mammalian cell cultivations, e.g. production of monoclonal antibodies and vaccines. This is motivated by several advantages of these bioreactors such as reduced risk of cross contaminations or short lead times. Single-use bioreactors differ in terms of shape, agitation principle, and gassing strategy. Hence, a direct process transfer or scale-up can be a challenge. Conventional stirred tank bioreactor designs are therefore still considered as the gold standard due to their well-defined and characterized properties. Based on this knowledge, a stirred single-use bioreactor family from 50 to 2000 L scale was developed with geometrical ratios similar to conventional reusable systems. To follow a quality by designapproach, the single-use bioreactor family evaluated here was characterized by using process engineering methods such as the power input per volume, the mixing time, and the kLa value. The process engineering characterization demonstrates that these systems are suitable for cultivations of mammalian cells, even for high cell density and high titer applications. Based on the data, a scale-up or process transfer is possible between this bioreactor family as well as reusable systems. Therefore, this bioreactor family represents a major progress for the single-use technology.

Power input is an important engineering and scale-up/down criterion in stirred bioreactors. However, reliably measuring power input in laboratory-scale systems is still challenging. Even though torque measurements have proven to be suitable in pilot scale systems, sensor accuracy, resolution, and errors from relatively high levels of friction inside bearings can become limiting factors at smaller scales. An experimental setup for power input measurements was developed in this study by focusing on stainless steel and single-use bioreactors in the single-digit volume range. The friction losses inside the air bearings were effectively reduced to less than 0.5% of themeasurement range of the torque meter. A comparison of dimensionless power numbers determined for a reference Rushton turbine stirrer (N-P = 4.17 +/- 0.14 for fully turbulent conditions) revealed good agreement with literature data. Hence, the power numbers of several reusable and single-use bioreactors could be determined over a wide range of Reynolds numbers between 100 and > 10(4). Power numbers of between 0.3 and 4.5 (for Re = 10(4)) were determined for the different systems. The rigid plastic vessels showed similar power characteristics to their reusable counterparts. Thus, it was demonstrated that the torque-based technique can be used to reliably measure power input in stirred reusable and single-use bioreactors at the laboratory scale.

In recent years, several automated scale-down bioreactor systems have been developed to increase efficiency in cell culture process development. ambr™ is an automated workstation that provides individual monitoring and control of culture dissolved oxygen and pH in single-use, stirred-tank bioreactors at a working volume of 10-15 mL. To evaluate the ambr™ system, we compared the performance of four recombinant Chinese hamster ovary cell lines in a fed-batch process in parallel ambr™, 2-L bench-top bioreactors, and shake flasks. Cultures in ambr™ matched 2-L bioreactors in controlling the environment (temperature, dissolved oxygen, and pH) and in culture performance (growth, viability, glucose, lactate, Na(+), osmolality, titer, and product quality). However, cultures in shake flasks did not show comparable performance to the ambr™ and 2-L bioreactors.

Anchorage-dependent cell cultures are used for the production of viruses, viral vectors, and vaccines, as well as for various cell therapies and tissue engineering applications. Most of these applications currently rely on planar technologies for the generation of biological products. However, as new cell therapy product candidates move from clinical trials towards potential commercialization, planar platforms have proven to be inadequate to meet large-scale manufacturing demand. Therefore, a new scalable platform for culturing anchorage-dependent cells at high cell volumetric concentrations is urgently needed. One promising solution is to grow cells on microcarriers suspended in single-use bioreactors. Toward this goal, a novel bioreactor system utilizing an innovative Vertical-Wheel™ technology was evaluated for its potential to support scalable cell culture process development. Two anchorage-dependent human cell types were used: human lung carcinoma cells (A549 cell line) and human bone marrow-derived mesenchymal stem cells (hMSC). Key hydrodynamic parameters such as power input, mixing time, Kolmogorov length scale, and shear stress were estimated. The performance of Vertical-Wheel bioreactors (PBS-VW) was then evaluated for A549 cell growth and oncolytic adenovirus type 5 production as well as for hMSC expansion. Regarding the first cell model, higher cell growth and number of infectious viruses per cell were achieved when compared with stirred tank (ST) bioreactors. For the hMSC model, although higher percentages of proliferative cells could be reached in the PBS-VW compared with ST bioreactors, no significant differences in the cell volumetric concentration and expansion factor were observed. Noteworthy, the hMSC population generated in the PBS-VW showed a significantly lower percentage of apoptotic cells as well as reduced levels of HLA-DR positive cells. Overall, these results showed that process transfer from ST bioreactor to PBS-VW, and scale-up was successfully carried out for two different microcarrier-based cell cultures. Ultimately, the data herein generated demonstrate the potential of Vertical-Wheel bioreactors as a new scalable biomanufacturing platform for microcarrier-based cell cultures of complex biopharmaceuticals.

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized medicine innovations. However, translating these advances into therapeutic applications will require in vitro systems that allow for robust, flexible, and cost effective bioreactor systems. There are several bioreactor systems currently utilized in research and commercial settings; however, many of these systems are not optimal for establishing, expanding, and monitoring the growth of different cell types. The culture parameters most challenging to control in these systems include, minimizing hydrodynamic shear, preventing nutrient gradient formation, establishing uniform culture medium aeration, preventing microbial contamination, and monitoring and adjusting culture conditions in real-time. Using a pneumatic single-use bioreactor system, we demonstrate the assembly and operation of this novel bioreactor for mammalian cells grown on micro-carriers. This bioreactor system eliminates many of the challenges associated with currently available systems by minimizing hydrodynamic shear and nutrient gradient formation, and allowing for uniform culture medium aeration. Moreover, the bioreactor’s software allows for remote real-time monitoring and adjusting of the bioreactor run parameters. This bioreactor system also has tremendous potential for scale-up of adherent and suspension mammalian cells for production of a variety therapeutic proteins, monoclonal antibodies, stem cells, biosimilars, and vaccines.

This work describes a novel bioreactor system for the cultivation of animal, insect, and plant cells using wave agitation induced by a rocking motion. This agitation system provides good nutrient distribution, off-bottom suspension, and excellent oxygen transfer without damaging fluid shear or gas bubbles. Unlike other cell culture systems, such as spinners, hollow-fiber bioreactors, and roller bottles, scale-up is simple, and has been demonstrated up to 100 L of culture volume. The bioreactor is disposable, and therefore requires no cleaning or sterilization. Additions and sampling are possible without the need for a laminar flow cabinet. The unit can be placed in an incubator requiring minimal instrumentation. These features dramatically lower the purchase cost, and operating expenses of this laboratory/pilot scale cell cultivation system. Results are presented for various model systems: 1) recombinant NS0 cells in suspension; 2) adenovirus production using human 293 cells in suspension; 3) Sf9 insect cell/baculovirus system; and 4) human 293 cells on microcarrier. These examples show the general suitability of the system for cells in suspension, anchorage-dependent culture, and virus production in research and GMP applications.

Erlenmeyer flask or conical flask, usually made of glass, is widely used for laboratory scale suspension culture of microorganism, such as Escherichia Coli and yeast. Due to being non-disposable culture vessel, it has to be cleaned, packaged and sterilized prior to use, which are time, labor and energy consuming work, and has the potential risk of cross-contamination. Despite the rigid plastic conical flasks are possible for single use, they are not economically effective and produce more waste. To overcome these drawbacks, here we successfully developed a novel disposable flexible bioreactor with a plastic film through a thermo-fusion technique. With a triangular pyramid shape, the bioreactor enabled itself to keep a three-dimensional internal space without needing air inflation and well adapted to the commercial available orbital shaker. Unlike the conventional rigid conical flasks and other reported flexible flasks, which had to be fixed in the shaker, the flexible bioreactor could keep sitting on the silicone pad-carpeted platform of the orbital shaker steadily without any fixation needed at the shaking speeds below 250 rpm, thus making it simple to handle. Compared with the traditional conical glass flasks, the innovative flexible bioreactors achieved a significant higher efficiency in bacteria growth and oxygen transfer rates. In conclusion, the novel flexible bioreactor is an ideal disposable culture vessel for microorganism suspension culture at laboratory scale and holds a promising potential to replace the glass flask and rigid plastic flask in the future.

Stirred single-use bioreactors can be used as substitutes for their conventional counterparts made of glass or stainless steel in the development and production of biopharmaceuticals wherever possible. Various studies have confirmed their comparability in cell growth as well as in product quantity and quality. However, information about their engineering characteristics is still rare. This study focuses on the stirred Mobius® CellReady 3L bioreactor. The main engineering parameters for typical operation conditions used in animal cell cultivations are presented for the first time. Numerical simulations with a commercial CFD package (Fluent 6.3) were accomplished to obtain data on the single- and multi-phase fluid flow, power input, mixing time and oxygen mass transfer. The results, which were compared with data from experiments and from the literature, reveal the suitability of the Mobius® CellReady 3L bioreactor for cell expansion and protein production with animal cell cultures. Furthermore, the data enable comparisons with other single-use and reusable cell culture bioreactors at bench-scale.

The majority of influenza vaccines are manufactured using embryonated hens' eggs. The potential occurrence of a pandemic outbreak of avian influenza might reduce or even eliminate the supply of eggs, leaving the human population at risk. Also, the egg-based production technology is intrinsically cumbersome and not easily scalable to provide a rapid worldwide supply of vaccine. In this communication, the production of a cell culture (Madin-Darby canine kidney (MDCK)) derived live attenuated influenza vaccine (LAIV) in a fully disposable platform process using a novel Single Use Bioreactor (SUB) is presented. The cell culture and virus infection was maintained in a disposable stirred tank reactor with PID control of pH, DO, agitation, and temperature, similar to traditional glass or stainless steel bioreactors. The application of this technology was tested using MDCK cells grown on microcarriers in proprietary serum free medium and infection with 2006/2007 seasonal LAIV strains at 25-30 L scale. The MDCK cell growth was optimal at the agitation rate of 100 rpm. Optimization of this parameter allowed the cells to grow at a rate similar to that achieved in the conventional 3 L glass stirred tank bioreactors. Influenza vaccine virus strains, A/New Caledonia/20/99 (H1N1 strain), A/Wisconsin/67/05 (H3N2 strain), and B/Malaysia/2506/04 (B strain) were all successfully produced in SUB with peak virus titers > or =8.6 log(10) FFU/mL. This result demonstrated that more than 1 million doses of vaccine can be produced through one single run of a small bioreactor at the scale of 30 L and thus provided an alternative to the current vaccine production platform with fast turn-around and low upfront facility investment, features that are particularly useful for emerging and developing countries and clinical trial material production.

A single-pass, plug-flow bioreactor has been developed in which oxygen is supplied to entrapped hybridoma cells via silicone tubes threaded through the square channels of a macroporous ceramic monolith. Oxygen diffuses from the gas phase, through the silicone tubing, across the open square channel, and into the pores of the ceramic wall where it is consumed by entrapped cells. Advantages of such a reactor include higher product yields, protection of cells from detrimental hydrodynamic effects, no internal moving parts to compromise asepsis, and simplicity of operation. A prototype bioreactor was constructed and operated over a range of residence times. A side-by-side experimental comparison with a conventional recycle bioreactor was performed by inoculating both bioreactors with cells from the same stock culture and feeding medium from the same reservoir. Final antibody titers were 80% higher in the single-pass bioreactor at a residence time of 200 minutes compared with those of the recycle bioreactor at a residence time of 800 minutes. A theoretical analysis of oxygen transport in this bioreactor is developed to highlight important design criteria and operating strategies for scale-up.

A concentric-cylinder airlift reactor, in which the annulus is a packed bed of glass fibers, has been developed in order to facilitate the scaleup and enhance the volumetric productivity of anchorage-dependent animal cell cultures. In this bio-reactor, oxygen-containing gas is sparged through the inner draft tube, causing bubble-free medium to flow through the fiber bed in the outer cylinder and providing both oxygenation and convective nutrient transfer to the cells. Several other desirable features for reactor operation are also provided by this design. Cell cultivations in this bioreactor have been successfully carried out and provide data for the feasibility of the large-scale cell cultivation.

Background During the development of processes for high production CHO fed batch cultures, there is usually insufficient time for thorough process optimization. Once defined, these processes often have ranges rather than specific values for many parameters, resulting in minor variations in the runs. These small process variations, combined with resultant differences in performance, can be used to further optimize the process. Over the course of one year, eleven 20 L fed batch cultures of an antibody producing CHO-S cell line were generated in the Allegro XRS 20 bioreactor. In addition to normal minor process variation, small process changes occurred due to method optimization efforts or culture timing requirements. An analysis of the combined historical data was undertaken to better understand the specific culture performance drivers, leading to further improvement in culture outputs, specifically final antibody concentration and maximum viable cell density (VCD).