Jun. 23, 2025
Previous installments of this series throughout have discussed the basics of bioreactor scaling, mixing, and rates of oxygen uptake and mass transfer, along with the most commonly applied dynamic method for mass-transfer measurement. This month, part 4 continues the discussion of the volumetric mass-transfer coefficient (kLa) by showing how different culture and bioreactor operating parameters can affect its value. The next installment of this series will begin by presenting some theoretical correlations described in published literature that are used to determine kLa and often used in bioreactor scale-up exercises.
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Bioreactors are used for suspension-culture–based manufacture of most modern biopharmaceuticals. In such processes, oxygen is a key nutrient for cell growth, culture maintenance, and protein production. The typical oxygen uptake rate value for mammalian cell cultures at a concentration of ≈106 cells/mL is 0.05–1.5 mmol O2 per liter of culture per hour. The kLa value is a key performance parameter, representing a given bioreactor’s capacity for oxygen supply and efficiency of transfer from introduced gases into liquid solution. Such measurement is important because living cells can metabolize oxygen only when it is dissolved in solution. To enable proper cell metabolism, oxygen must be supplied continuously to make sufficient dissolved oxygen (DO) available to every point in the vessel.
Gaseous oxygen in suspended air bubbles takes up to eight steps to transit through the gas, reach liquid medium, and become available intracellularly for energetic transformations (Figure 1). Resistance to mass transfer can be encountered at different points throughout this process:
• transfer from the bulk gas phase (gas-bubble interiors) to the gas–liquid interface
• transfer across the gas–liquid interface
•diffusion through relatively stagnant liquid films
• transport through bulk liquid
• diffusion through relatively stagnant liquid films around cells
• movement across the liquid–cell-surface interface
• for clumps (flocs) of cells or those attached to microcarriers, diffusion across the solid to an individual cell
• transport through the cytoplasm to the consumption site (mitochondria).
Figure 1: Mass-transfer steps (and resistances) encountered during oxygen transfer from a gas phase to an individual cell or a cellular floc (1).
Not all transport resistances encountered during those steps are equal in magnitude, and some can be neglected depending on conditions prevailing in a given culture. Because oxygen solubility in an aqueous solution is low, liquid films offer the most resistance to mass transfer and thus are key determinants of the value of kLa.
The most practical means of supplying oxygen to a suspension cell culture is by blowing air into the bioreactor through a sparger. The sparger generally is placed under the mixing impeller so that the blade’s rotation will break up bubbles and entrench them into the liquid medium. Those bubbles then take a tortuous path to move through the liquid layers and eventually burst after reaching the surface of the culture. The liquid height in a small-scale bioreactor with a height/diameter (H/D) ratio close to 1:1 generally will be small, so air bubbles reach the top and emerge rather quickly, making the efficiency of oxygen transfer for a given air-flow rate relatively low. However, as bioreactor scale increases, so do liquid height (H/D > 1) and the holding time of air in the culture. That in turn increases the efficiency of oxygen transfer.
In stirred-tank reactors, air supply is optimized to provide maximum gas transfer through the use of shear, power, and turbulence to maximize the ratio of gaseous surface area to liquid volume and gas velocity. The oxygen transfer rate (OTR) of a bioreactor is strongly influenced by the hydrodynamic conditions prevailing in the vessel. Those conditions are a function of turbulent kinetic energy dissipation. Thus, the kLa value is influenced by a number of factors: process parameters (agitator speed, temperature, pH, and gassing rate), physiochemical characteristics of the culture medium (viscosity, density, salt content, surface tension, and coalescence behavior), biochemical parameters (cell concentration and OUR), and equipment geometries (vessel, agitator, and sparger). Figure 2 illustrates the interrelationship of such factors. Their influence can change the value of either the liquid mass-transfer coefficient (kL), the interfacial area (a), and/or the driving force for mass transfer (C* – C, where C represents oxygen concentration and C* is the oxygen-saturation concentration).
Figure 2: Interrelationship of bioprocess variables with oxygen transfer rate (OTR) and kLa (11); OUR = oxygen uptake rate, μ = coefficient of friction, ρ = fluid density, σ = surface tension, L = length, D = diameter, N = impeller rotational speed, Vs = gas superficial velocity.
Gas Bubbles: The behavior of the gas bubbles inside a bioreactor exerts a strong influence on the overall value of kLa. Some properties of gas bubbles affect the magnitude of kL; others change a.
The most important property of the gas bubbles is their size. For a given volume of gas, more interfacial area will be created if it is dispersed into many small bubbles rather than a few large bubbles (1). Small bubbles have slow bubble-rise velocities and hence spend more time in the liquid, allowing more time for oxygen to dissolve (and CO2 to be adsorbed from the liquid). Therefore, small bubbles lead to higher gas hold-up (the fraction of the bioreactor working volume that is occupied by the gas).
During gas sparging in a bioreactor, bubbles either break up into smaller bubbles or merge together with others to form larger bubbles. Both processes — bubble break-up and coalescence — greatly influence the gas-bubble size distribution and hence the mass-transfer area. Some practical considerations, however, constrain the size of gas bubbles in a bioreactor. Those much smaller than 1 mm in diameter often cause foam formation on the liquid surface, which is undesirable for many reasons. In addition, the rising velocity of those very small bubbles is slow enough that oxygen (and CO2) concentration in them can equilibrate quickly with that of the liquid medium, eliminating their function for transferring gas into and out of the liquid.
Small bubbles generally are beneficial, however, providing higher gas hold-up and interfacial area than larger bubbles. The optimal bubble diameter for mass transfer in a bioreactor is 2–3 mm. Beyond 3-mm size, any further increase in kLa will be minimal.
Bubble Break-Up: With bioreactors in which turbulent flow generally prevails, four mechanisms have been identified to cause bubble break-up (2): turbulent fluctuations and collisions, viscous shear stress, shearing-off processes, and interfacial instability. Mathematical modeling of bubble break-up typically includes two submodels: one for the frequency of break-ups and the other for the resulting “daughter”-bubble size distribution. Most models assume a simple and equilateral binary break-up (3, 4).
Bubble Coalescence: Coalescence of small bubbles into larger ones is generally undesirable because it reduces the total interfacial area and gas hold-up, thus limiting oxygen mass transfer. The phenomenon of bubble coalescence is more complicated than that of bubble break-up, with several published theories and mathematical models describing the process (5–9). Pure water is a coalescing liquid, but the presence of solutes (such as salt or ions) in it renders it noncoalescing.
Because cell-culture media contain salts and other organic compounds, they behave to a certain extent as noncoalescing liquids, which is advantageous for oxygen transfer. The addition of ions to water in sparged vessels markedly reduces the average bubble size overall and increases the gas hold-up, leading to as much as 10× greater interfacial area than that obtained without salts (10). Because the composition — and therefore the coalescence properties — of a highly dense fed-batch cell-culture broth vary over time, kLa also can be expected to vary accordingly.
Sparger Design: The biopharmaceutical industry uses several different types of spargers in bioreactors for cell culture, from sintered spargers that generate μm-sized bubbles to drilled-hole or open-pipe (perforated) spargers that have defined numbers and sizes of holes to generate precisely sized bubbles. In a stirred-tank bioreactor, the bubbles leaving the sparger usually fall within a narrow size range. However, impeller rotation and the resulting turbulence cause dispersion, with continual break-up and coalescence of bubbles taking place. Hence, the actual bubble size in such a bioreactor is quite different from what the sparger emits.
Gas Dispersion: Two-phase gas–liquid flow in stirred tanks has been studied extensively and is well documented (11). Gas dispersion and bubble size — and the residence time of bubbles in a bioreactor — both depend on impeller type and location as well as stirring speed. The kLa values generally will increase along with impeller tip speed. Balancing impeller rotation and gas-flow rate determines the effectiveness of bubble break-up and dispersion. At sufficiently high stirrer speeds, gas will disperse and be recirculated around the tank and thus contribute to mass transfer. Similarly, impeller design has a strong influence on oxygen mass transfer, with Rushton turbines proven to be more effective in generating higher gas dispersion and bubble break-up than pitched-blade impellers (1, 11).
Physiochemical Properties of the Liquid: The physiochemical properties of a fluid affect oxygen transfer through it due to their significant effects on bubble size, gas hold-up, and hence kLa. Equation 1 below correlates kLa with fluid density (ρ) and viscosity (μ) (12):
kLa = 37.5 (ρ/μ)0.667
The inverse correlation of kLa with viscosity in Equation 1 is due to the fact that with increasing viscosity, the thickness of liquid film surrounding each gas bubble increases. High viscosity also dampens turbulence and reduces the effectiveness of gas dispersion. An increase in culture-broth density, on the other hand, positively influences kLa. Figure 3 clearly shows the effects of the coalescing behavior of liquids and of viscosity on kLa. A 5% Na2SO4 solution is noncoalescing, so it yields higher kLa values with the same power input than water (a coalescing liquid), even though both solutions have similar viscosity. Carboxymethyl cellulose is highly viscous, thus yielding very low kLa values even with very high stirrer power input.
Figure 3: Effect of solution behavior and viscosity on kLa in a stirred-tank reactor; ○ = 5% sodium sulfate in water (low viscosity, noncoalescing), ● = water (low viscosity, coalescing), = 0.7% carboxymethyl cellulose in water (viscous) (11).
Air-Flow Rate and Stirrer Speed: Increasing the air-flow rate also improves mass transfer and increases kLa values, but only to a certain extent (Figure 4, top panel). In most cases, the increase of kLa with higher gas-flow rates is limited as those flow rates overwhelm the impeller — a condition known as flooding — making it unable to distribute gas in the tank. In addition, very high gas-flow rates can blow liquid contents out of a bioreactor tank.
Figure 4: Effect of aeration rate (top) on the volumetric mass transfer coefficient (kLa) at different agitation speeds and of agitation speed (bottom) on kLa at different aeration rates
Source: Abdella A, Segato F, Wilkins MR. Optimization of Process Parameters and Fermentation Strategy for Xylanase Production in a Stirred Tank Reactor Using a Mutant Aspergillus nidulans Strain. Biotechnol. Rep. 26, : e; https://doi.
org/10./j.btre..e.
Alternatively, increasing the impeller stirring speed (power input) generally yields higher kLa values (Figure 4, bottom panel). Higher power input increases turbulence intensity, which in turn produces smaller bubbles, increases gas hold-up, and improves overall gas distribution. Those effects generally provide significant improvements in oxygen mass transfer and kLa, as discussed above.
Temperature and Pressure: Liquid temperatures in cell-culture bioreactors and aerobic fermentations have opposing effects on oxygen-saturation concentration (C*) and mass-transfer coefficient (kL) (1). Increasing temperatures lower oxygen solubility in water, thus reducing the driving force (C* – C). At the same time, oxygen becomes more diffusive in water, which increases kL. The net effect depends on the range of temperatures considered. Between 10 and 40 °C, an increase is likely to improve the rate of oxygen transfer; beyond 40 °C, the solubility of oxygen drops significantly, causing adverse effects on both driving force and mass transfer.
The total pressure at which a bioreactor (or fermentor) is operated also affects the solubility of gases in liquid culture broth. The partial pressure of a gas is related to its saturation concentration in liquid by means of Henry’s law, which states that the partial pressure of a gas is equal to the total pressure times the mole fraction of gas (Henry’s constant × saturation concentration) in liquid, or mathematically
pO2 = PT × yO2 = H C*O2
where pO2 is the partial pressure of oxygen, PT is total pressure, yO2 is the mole fraction of oxygen, and C*O2 is saturated oxygen concentration in liquid.
Depending on the oxygen demand of cells in bioreactor cultures, it may be necessary to provide them with oxygen-enriched air or pure oxygen rather than air. Equation 3 below expresses how using pure oxygen affects the concentration driving force for mass transfer. Regular air is 21% oxygen, so its mole fraction of oxygen is 0.21; if pure oxygen is used, then the mole fraction is 1.
Equation 3 above compares the concentration driving force (C*) for two sparged gases, one regular air and the other pure oxygen. Hence, sparging pure oxygen at the same temperature and pressure results in nearly fivefold higher oxygen concentration in liquid than regular air can provide. Similarly, oxygen solubility can be increased by using compressed air at higher total pressure (PT). However, using either pure oxygen or compressed air substantially increases the operating cost of a bioreactor.
Shear Protection and Antifoam Agents: Media additives for shear protection (e.g., Pluronic F-68 detergent, PF68, also known as poloxamer 168) and antifoam agents can have a profound effect on oxygen mass transfer and fluid dynamics in a bioreactor. The efficiency and effectiveness of PF68 as a shear-protective agent is well documented (13–17). Reports on its influence on mass-transfer characteristics in bioreactors have been mixed, with some studies showing a reduction (17, 18) and others reporting a significant increase (19, 20) in mass transfer. Media components and additives that can influence culture viscosity and surface-tension properties often are present as well, potentially masking the effects of PF68 in some studies.
Figure 5 illustrates results from a comprehensive study of how different concentrations of PF68 influence kLa in a 400-L stirred tank using different sparger systems (13). The experiments were conducted with a 60-W/m3 power input and an aeration rate of 20 L/min. The results clearly indicate that PF68 affects mass transfer significantly. At very low PF68 concentrations (0.02 g/L), kLa values immediately declined at least 50% compared with a reference (without PF68) with all tested sparger systems. That PF68 concentration is far below the accepted critical micelle concentration (CMC) of 0.33 g/L. Note that further increases in PF68 concentration significantly increased kLa with sintered spargers: Even at 1 g/L PF68, the kLa value for the sparger with the smallest pores (20–47 μm) was similar to that of the reference. No such initial decline and later recovery of kLa as a function of PF68 concentration was observed with either a ring sparger or an open tube.
Figure 5: kLa as a function of the Pluronic F-68 detergent concentrations in a 400-L stirred tank with different sparger systems (13).
Foam forms in bioprocesses through the introduction of gases into culture broth and is further stabilized by proteins from the cells in culture. Adding antifoam agents to culture media is the most common method of preventing foam build-up. Both positive and negative effects of antifoams on oxygen transfer have been observed in fermentation-based bioprocesses. In one study, a silicone-based antifoam negatively affected the mass transfer coefficient, gas hold-up, and gas velocity within media (21). Another report, however, showed that antifoams without silicone oil had no great effect on the rate of oxygen transfer, whereas those containing silicone oil did affect it significantly at the beginning of a process, an effect that decreased over the duration (22). Few studies have been published on the effects of antifoam addition on oxygen transfer or kLa of cell-culture systems. In one basic fed-batch study using Chinese hamster ovary (CHO) cells, researchers used bioreactor off-gas analysis by mass spectrometry to determine OUR and kLa values (23). They found that antifoam additions caused an increase in the former and a corresponding decrease in the latter.
Biomass and Macromolecules: Oxygen transfer to a culture broth also is influenced by the presence of cells themselves. Studies of microbial fermentations have shown the presence of microorganisms to enhance the OTRs in the cultures (2–5). Their consumption of oxygen increases the driving force by maintaining a low oxygen concentration in the medium. Microbe-derived variation in the OTR for a given driving force and interfacial area generally is characterized by means of an enhancement factor, E. It is defined as the ratio of mass-transfer flux of oxygen in the presence of microorganisms to the oxygen flux in their absence (11).
Summary: Stirred-tank bioreactors are used widely in bioprocessing (e.g., aerobic fermentation and cell-culture–based production of therapeutic proteins, among others). Such bioreactors provide high rates of mass and heat transfer with excellent mixing. Many variables affect mass transfer and mixing in stirred-tank systems, but the most important among them are stirrer speed, type, and number as well as gas-flow rate. Hence, possible ways to optimize (increase) kLa and OTR include
• increasing power input and/or gassing rate
• optimizing bioreactor design and agitator geometry
• changing the composition of culture media.
Keeping kLa constant across scales has been used as a criterion for successful scale-up of many microbial fermentation bioprocesses. Their oxygen demands generally are high, and it is well known that oxygen gas–liquid mass transfer is the most significant factor for scale-up of aerobic fermentations. In fact, nearly a third (30%) of microbial fermentation scale-up operations are based on keeping kLa constant (24). The approach has been used in production of antibiotics (24), exopolysaccharides (26), and secondary metabolites (24, 27), as well as Escherichia coli–based production of recombinant proteins (28).
In one study, for example, a secondary-metabolite production process based on aerobic fermentation was scaled up from laboratory (5 L) to pilot scale (50 L) by keeping kLa constant (24). The goal was to provide nearly equivalent kLa values to each fermentor by manipulating agitation and aeration rates, thereby ensuring similar oxygen transfer at both scales. Figure 6 compares the kLa values obtained for each system. In the 5-L fermentor, the highest productivity was obtained with 200-rpm agitation and 1.5-vvm aeration, resulting in a kLa value of 0.02 s–1. To maintain a constant kLa, the authors chose a combination of 180 rpm and 0.5 vvm for the 50-L fermenter (indicated by arrows in Figure 6). Using those values in the 50-L fermentor provided very similar productivities and profiles of cell growth, substrate consumption, DO, and pH — thereby indicating a successful scale-up.
Figure 6: Comparing kLa at 5-L laboratory scale and 50-L pilot scale in stirred-tank bioreactor systems (24).
The Xcellerex (XDR) single-use bioreactor system for cell-culture–based bioprocessing is available from Cytiva in a range of sizes from 10-L to -L volumes. That makes it possible to develop a bioprocess at laboratory scale (10 L) and scale it up to pilot (500 L) then clinical and commercial manufacturing (– L) scales. The supplier has published engineering characterization documents (with mixing times, power inputs, and oxygen-transfer capabilities, including achievable kLa values) for each XDR bioreactor model (29–31). Figure 7 compares kLa values achievable using a microsparger (2-μm pore size) in XDR-10, XDR-500, and XDR- systems. Those bioreactors respectively represent laboratory, pilot, and clinical/commercial manufacturing scales for cell-culture processes.
Figure 7: Four-dimensional (4D) response contour plots of kLa representing different air-flow rates, working volumes, and agitation speeds for Cytiva Xcellerex XDR-10 (top row), XRD-500 (middle row), and XDR- (bottom row) bioreactor systems from Cytiva with 2-μm microsparger pore size (29–31).
It is evident that the value of kLa rises with increasing agitation and air-flow rates. However, the XDR bioreactor working volume affects the achievable kLa value negatively, with the achievable value decreasing substantially as working volume increases with a 2-μm pore-size microsparger installed. With a bottom mounted single impeller, however, increasing the working volume with a given agitation rate would lower the volumetric power input to the liquid, thus decreasing turbulence intensity and potentially increasing the frequency of bubble coalescence. All those factors combine to lower both kL and a and thus kLa . In a XDR-10 bioreactor, for example, a kLa value of 27 h–1 is achieved at a much lower combination of agitation and air-flow rate with a working volume of 4.5 L than would be required to achieve the same value at the full working volume of 10 L.
Process engineers applying equal kLa as a scale-up criterion across the XDR platform using the contour plots in Figure 7 from XDR-10 to XDR- systems with 2-μm pore-size microspargers would need to begin by choosing a working volume. In the figure, note that the kLa value that aligns with your needed agitation and aeration-flow rates. Next, choose a working volume for the large-scale bioreactor (XDR-), and from the contour plot that also contains the value of required kLa (from the XDR-10 scale), you can determine the necessary agitation and aeration flow rates for the XDR- culture. Then you can operate the large-scale bioreactor at your chosen agitation and aeration flow rates to check the reproducibility of cell-culture performance and product-quality attributes across scales.
Because working volume can have such a profoundly negative influence on the values of kLa in XDR bioreactors, it is possible that some values achievable at small scale might not work at large scale. For example, the kLa value of 18 h–1 achievable in a Cytiva Xcellerex XDR-10 bioreactor operating at its full working volume of 10 L cannot be attained in XDR- system at its full working volume with any combination of agitation and aeration rates.
Part 1 — Exploring Introductory Principles. BioProcess Int. 22(1–2) : 22–27; https://www.bioprocessintl.com/bioreactors/lessons-in-bioreactor-scale-up-part-1-mdash-exploring-introductory-principles.
Part 2: A Refresher on Fluid Flow and Mixing. BioProcess Int. 22(6) : 32–40; https://www.bioprocessintl.com/bioreactors/lessons-in-bioreactor-scale-uppart-2-a-refresher-on-fluid-flow-and-mixing.
Part 3: Experimental Determination and Application of Oxygen Mass-Transfer Rate, Mass-Transfer Coefficient, and Oxygen- Uptake Rate. BioProcess Int. 22(9) : 26–33; https://www.bioprocessintl.com/bioreactors/lessons-in-bioreactor-scaleup-part-3-experimental-determinationand-application-of-oxygen-mass-transferrate-mass-transfer-coefficient-andoxygen-uptake-rate.
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For more sparger ringinformation, please contact us. We will provide professional answers.
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Gene therapy has made significant advances over the past two decades. Gene transfer therapy involves the administration of specific genetic material (i.e., DNA or RNA) via a carrier, known as a ‘vector’. Viral vectors offer a new class of biologics which facilitates gene transfer and modification in living cells, potentially treating many conditions with genetic causes. Currently, the most used viral vectors for gene transfer therapy include gamma retrovirus (RV), adenovirus (AV), adeno-associated virus (AAV), and lentivirus (LV) [1]Sayed N, Allawadhi P, Khurana A, Singh V et al. Gene therapy: Comprehensive overview and therapeutic applications. Life Sci. ; 294(), . .
Previously, gene therapy mainly addressed rare or very rare diseases and therefore the manufacture of gene therapy viral vectors were only set out to meet the market demands of a relatively small group of patients within the orphan disease market space, where meeting demand has not always been a big problem. Advancement in gene transfer therapy-based treatments and its inevitable extension to common indications such as cancer, Parkinson’s and Alzheimer’s means that gene therapy viral vectors must be manufactured in larger scales. This production gap is one of the main challenges in the gene transfer therapy field.
According to Precedence Research, the global gene therapy market is expected to be valued at over US$15 billion by [2]Gene Therapy Market Size, Growth, Trends, Report –. . This expected growth has generated a huge pressure on biomanufacturing companies to develop new technologies to be able to satisfy the high demand of gene therapy products / technologies.
This trend is also driving a greater need for the scalable production of viral vectors for gene therapy. Traditionally, viral vector production is mostly based on adherent cell lines using systems such as multi-trays, that can only be scaled out. Adherent bioreactors such as Pall’s iCELLis® bioreactor have been developed during the past decade allowing to scale up of such processes up to a certain surface (500 m2 for the iCELLis 500+ bioreactor). Over recent years, more manufacturers have investigated adapting their cells for viral vector manufacturing to suspension culture to reach higher volume vector-producing batches, that can be required for large dose/large population applications. Pall developed the Allegro Stirred-Tank (STR) Bioreactor for suspension cells which can be easily scaled up to L (Figure 1Pall Allegro STR 50, 200, 500, and L bioreactors.). Pall’s expertise and understanding of process scaling technology has enabled large scale manufacturing of gene therapy products to meet the ever-increasing demand.
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The success of meeting the increasing demand of gene therapy heavily depends on the provision of more bioreactor manufacturing capacity. The COVID-19 pandemic has added to the strained capacity as some of the vaccine’s programs are also using viral vectors. Pall’s Allegro STR bioreactors are perfect candidates to reduce this capacity crunch in producing viral vectors for gene transfer therapy.
The Allegro STR bioreactor family combines Pall’s bioprocess engineering expertise, cell culture know-how and our drive for quality into a series of single-use bioreactors that deliver consistent and scalable cell culture performance for cell culture and viral vector production across the Allegro STR bioreactor range. From the outset of the design, Pall placed strong emphasis on providing compact, ergonomic, and intuitive turnkey bioreactor design concepts to maximize usability and process assurance, while maintaining optimal performance and reliability needed in a cell culture and viral vector production environment through several easy and intuitive operation features such as [3]Nienow AW, Isailovic B, Barrett TA et al. Design and Performance of Single-Use, Stirred-Tank Bioreactors. BioProcess Int. ; 14(10), 12–21.:
Scalable manufacturing is one of the critical processes required to be able to provide the quantities of vector needed to bring these potentially life-saving treatments to waiting patient populations. Many gene therapy manufacturing processes rely on culturing HEK293 cell lines (or derivative AAV293 cell lines), and several early and current forms of production culture these cells on an adherent substrate [4]Sanderson T, Erlandson T, Hazi N et al. Scalability comparison between 50 and 500 liter stirred tank bioreactor for production of rAAV viral vector. Cell Gene Ther. Ins. ; 7(9), –.
Pall’s expert knowledge of process scaling, and critical scaling parameters ensures that processes are easily scaled up or down across all sizes of the Allegro STR bioreactors with working volumes ranging from 10 to L with focus on critical scaling parameters such as specific power input, kLa (volumetric oxygen mass transfer coefficient), mixing time, and aspect ratio so that cell culture environment and conditions are as similar as possible regardless of size [4]Sanderson T, Erlandson T, Hazi N et al. Scalability comparison between 50 and 500 liter stirred tank bioreactor for production of rAAV viral vector. Cell Gene Ther. Ins. ; 7(9), –.
In a scaling study using Allegro STR 50 and 500 bioreactors for production of rAAV viral vector published by Sanderson et al., productivity between the two scales was compared [4]Sanderson T, Erlandson T, Hazi N et al. Scalability comparison between 50 and 500 liter stirred tank bioreactor for production of rAAV viral vector. Cell Gene Ther. Ins. ; 7(9), –. Both STRs were inoculated from the same cell culture bolus at half capacity and expanded to the full working volume after 24 hours. The operational parameters were matched throughout the process. The results showed near identical cell growth and viability between both the Allegro STR 50 and STR 500 bioreactor cultures up until transfection on day 3. After transfection, there was a drop in viability in the two STRs while the viable cell density continued to increase. Both cultures reached a maximum viable cell density of ∼1.8 x 106 cells/mL (Figure 3Cell growth and viability profiles in Allegro STR 50 and STR 500 bioreactors.). The data shows rAAV titer increases throughout the culture with maximum titer being observed at harvest. The final rAAV titers were 4.3 x gc/mL and 4.8 x gc/mL for the Allegro STR 500 and STR 50 bioreactors respectively (Figure 4rAAV titers in gc/mL in Allegro STR 50 and STR 500 bioreactors.), comparable in range to those reported in the literature [5]Chahal PS, Schulze E, Tran R, Montes J, Kamen AA. Production of adeno-associated virus (AAV) serotypes by transient transfection of HEK293 cell suspension cultures for gene delivery. J. Virol. Methods ; 196: 163–73. , [6]Trivedi PD, Yu C, Chaudhuri P et al. Comparison of highly pure rAAV9 vector stocks produced in suspension by PEI transfection or HSV infection reveals striking quantitative and qualitative differences. Mol. Ther. Methods Clin. Dev. ; 24: 154–170. .
The nutrient and metabolites were also analyzed daily throughout the production run, and they were comparable. Figure 5Glucose and lactate profiles in Allegro STR 50 and STR 500 bioreactors. shows a comparison of the glucose and lactate measurements.
The result from this comparative study demonstrates that the Allegro STR 50 and STR 500 bioreactors are appropriate for rAAV production and that they provide similar bioreactor cell culture conditions at both the 50 and 500 L scales. This scalability is realized when utilizing Pall’s recommended scale up strategy across the Allegro STR family [4]Sanderson T, Erlandson T, Hazi N et al. Scalability comparison between 50 and 500 liter stirred tank bioreactor for production of rAAV viral vector. Cell Gene Ther. Ins. ; 7(9), –.
Cell damage caused by agitation is a topic that is commonly discussed in the industry but the design features of the Allegro STR bioreactor are such that they mitigate damage from shear. As discussed previously [3]Nienow AW, Isailovic B, Barrett TA et al. Design and Performance of Single-Use, Stirred-Tank Bioreactors. BioProcess Int. ; 14(10), 12–21., a modern theory for damage to a range of cell types, including those on microcarriers, suggests that it occurs when cells are larger than the Kolmogorov eddy size, λK:
λK = (ν3 / ΦεT)1/4
where ν is the kinematic viscosity, Φ is the ratio of the maximum local energy dissipation rate compared to the average, and εT is the specific power input in W/kg (1 W/kg = ~103 W/m3 for fluids of a density similar to cell culture media). In the case of the Allegro STR bioreactors, Φ is ~15 [3]Nienow AW, Isailovic B, Barrett TA et al. Design and Performance of Single-Use, Stirred-Tank Bioreactors. BioProcess Int. ; 14(10), 12–21.. To avoid cell damage, clearly λK must be >~20 µm (average HEK293 cell size). However, at the maximum speed available, εT = 0.4 W/kg and λK = ~35 µm. Thus, cell damage should not occur [7]Nienow AW. Reactor Engineering in Large Scale Animal Cell Culture. Cytotechnology ; 50, 9–33..
For most animal cell cultures, the Allegro STR bioreactors would be programmed for up-flow pumping, even if the system can do both directions. The Allegro STR 200 bioreactor impeller drive system is designed for agitation speeds of up to 150 rpm. In the qualification studies, this bioreactor achieved a specific power output of 0.31 W/kg at 150 rpm in the up-flow mode. This specific power level is significantly higher than what is used generally to meet the mass-transfer requirements of currently achievable cell densities [8]USD . Application Note: Characterization and Engineering Performance of the Allegro™ STR 200 Single-Use Stirred Tank Bioreactor System. Pall Corporation: East Hills, NY, . The ratio of impeller diameter to bioreactor side length (D/T) is an important parameter (Figure 6Allegro STR Impeller. & Figure 7Dimensional representation – Allegro STR bioreactor.) that affects both flow pattern and power input. For the 200 L Allegro STR systems, the D/T ratio was set to 0.5, which for a given specific power input (W/kg) ensures that mixing times are shorter than for smaller impellers [7]Nienow AW. Reactor Engineering in Large Scale Animal Cell Culture. Cytotechnology ; 50, 9–33., [9]Isailovic B, Kradolfer M, Rees B. Fluid Dynamics of a Single-Use Stirred Tank Bioreactor for Mammalian Cell Culture. BioProcess Int. ; 13(8), 60–66..
Shear can often be perceived as being a potential cause of damage to the viral vectors once produced. Indeed, once the cells are transfected (or induced in the case of stable cell lines), they start expressing the viral genes and produce and package the vector [4]Sanderson T, Erlandson T, Hazi N et al. Scalability comparison between 50 and 500 liter stirred tank bioreactor for production of rAAV viral vector. Cell Gene Ther. Ins. ; 7(9), –. In some cases, the vector remains mostly intracellular (AAV2, AAV5 for example), but it can also be completely or partially excreted by the cells into the cell culture media, either through exocytosis or cell lysis caused by the vector production cycle.
Viruses are smaller than cells, and the size difference can have an impact on how these cells or viruses are subject to turbulence in the bioreactor. Lentiviruses are traditionally the most sensitive of the viruses used for gene therapy applications, due to their enveloped nature. They are known to be sensitive to not only shear, but also pH, temperature, salt, and foam generation [10]Perry C, Rayat ACME. Lentiviral Vector Bioprocessing. Viruses ; 13(2): 268. .
Through the data illustrated above and a numerous amounts of case studies comparing the Allegro STR bioreactor to other types of STRs, it has been shown there is no damage to AAV integrity [4]Sanderson T, Erlandson T, Hazi N et al. Scalability comparison between 50 and 500 liter stirred tank bioreactor for production of rAAV viral vector. Cell Gene Ther. Ins. ; 7(9), –, [11]Collignon M-L, Mainwaring D, Pedregal A et al. Targeting Cost-effective Large-scale Manufacturing for rAAV Vectors by Transient Transfection using Pall’s Allegro STR Bioreactor. Poster published at ESGCT ., [12]Mainwaring D, Joshi A, Coronel J, Niehus C. Transfer and scale-up from 10L BioBLU® to Allegro™ STR 50 and STR200 Bioreactors. Cell Gene Ther. Ins. ; 7(9), –..
In a recent Pall study performed with a customer, LV have also been cultured successfully in the Allegro STR bioreactor at 50 L scale, without any specific damage to vector integrity, suggesting the system to be gentle enough to successfully produce these very delicate vectors at any scale. Process performance in the Allegro STR 50 bioreactor was compared to a validated 5 L scale down model. Through replicate runs, metabolite profiles and product physical titer and quality (functional titer and impurities) were reproducible between the two scales [13]Heshmatifar S, Collignon M-L; Kingwood M et al. Evaluation of Pall’s Allegro™ STR Bioreactor Production For Use in Cultivation of Mammalian Suspension Cells and Lentiviral Vector Production. Poster published at ESACT ..
A constant and adequate supply of oxygen is crucial in cell culture. Allegro STR bioreactor spargers have been designed for optimal gas bubbles generation and distribution allowing good oxygen mass transfer (kLa) with reduced foaming. The most common efficient method for oxygen transfer and carbon dioxide (CO2) stripping across all bioreactor scales is sparging through a ring sparger which was designed with holes of suitable size and number to achieve high flow rates (0.2 vvm) without excessive linear velocities. The system produces relatively large bubbles, which are less likely to damage cells than are small bubbles [7]Nienow AW. Reactor Engineering in Large Scale Animal Cell Culture. Cytotechnology ; 50, 9–33. while maintaining high oxygen transfer through adequate specific power and sparge rate. Pall Corporation document reference USD outlines scalable gas transfer coefficients (kLa) and scalable CO2 stripping rates for all bioreactors scales [14]USD – Characterization and Engineering Performance of the Allegro™ STR Single-Use Stirred Tank Bioreactor Family Range. Pall Corporation: East Hills, NY, .
Overall, the Allegro STR spargers have been designed and aligned with the ‘elephant ear’ impeller for optimal gas bubbles generation and distribution allowing good oxygen mass transfer and carbon dioxide strip rates across all STR bioreactor scales.
Pall’s Allegro STR range of single use bioreactors are designed for biotechnology manufacturing. The Allegro STR bioreactors are tested and proven bioreactors in mAb manufacturing [15]USD – Scalability Between the Allegro™ STR 50 and STR 500 Bioreactors in a CHO-S Fed-Batch Process. Pall Corporation: East Hills, NY, . With Pall’s excellent customer support and bioprocess expertise the effective and successful transfer of any gene therapy processes into Pall’s Allegro STR bioreactors is assured. The ability to effectively scale enables speed to clinic in the gene therapy space.
Attention to system design for excellent scalable manufacturing, and usability makes Pall’s range of Allegro STR bioreactors a good choice for viral vector production. Successful testing and adoption by several companies have shown its effectiveness in the gene therapy space.
Collignon et al. transferred an r-AAV process from a competitor 50 L SUB to Pall’s Allegro STR 50 bioreactor, with the objective to scale up to L. The user friendliness of the system and software, with close support from Pall scientific teams, was noted along with a favorable increase in production yields obtained through a change in dissolved oxygen strategy, thus reducing the overall gas consumption and foam formation [11]Collignon M-L, Mainwaring D, Pedregal A et al. Targeting Cost-effective Large-scale Manufacturing for rAAV Vectors by Transient Transfection using Pall’s Allegro STR Bioreactor. Poster published at ESGCT ..
In another study published by Mainwaring et al., a stable AAV producer cell line was successfully transferred from bench scale BioBLU® 10c to the 50 L Allegro STR bioreactor, and further scaled up to the Allegro STR 200 bioreactor. They also demonstrated good capacity, yield, and scalability for the initial unit operations of the downstream process. As a result, processes developed with other manufacturers’ bioreactor can readily be transferred to Allegro STR bioreactors based off known scaling process parameters [12]Mainwaring D, Joshi A, Coronel J, Niehus C. Transfer and scale-up from 10L BioBLU® to Allegro™ STR 50 and STR200 Bioreactors. Cell Gene Ther. Ins. ; 7(9), –..
As part of the Covid vaccine consortium in , Pall supported the rapid development and scale up of the ChAdOx1 vaccine, an adenovirus-based vaccine. The process was scaled up to the Pall Allegro STR 50 and Allegro STR 200 bioreactors in record time. Pall Allegro STR bioreactors up to L are consequently being used at various manufacturing sites to successfully produce the vaccine [16]Joe CCD, Jiang J, Linke T et al. Manufacturing a chimpanzee adenovirus-vectored SARA-CoV-2 vaccine to meet global needs. Biotechnol. Bioeng. ;1–11..
Ultimately, these case studies show that the Allegro STR bioreactor portfolio leverages decades of process engineering expertise to support successful cell culture in the gene therapy space. The availability of cost-effective gene therapies is critical for wider success of these novel medicines. Platform processes can contribute to that, especially where fully disposable or hybrid manufacturing is adopted.
The Allegro STR’s proven delivery of consistent and scalable cell culture performance can easily be part of the solution.
1. Sayed N, Allawadhi P, Khurana A, Singh V et al. Gene therapy: Comprehensive overview and therapeutic applications. Life Sci. ; 294(), . Crossref
2. Gene Therapy Market Size, Growth, Trends, Report –. Crossref
3. Nienow AW, Isailovic B, Barrett TA et al. Design and Performance of Single-Use, Stirred-Tank Bioreactors. BioProcess Int. ; 14(10), 12–21. Crossref
4. Sanderson T, Erlandson T, Hazi N et al. Scalability comparison between 50 and 500 liter stirred tank bioreactor for production of rAAV viral vector. Cell Gene Ther. Ins. ; 7(9), – Crossref
5. Chahal PS, Schulze E, Tran R, Montes J, Kamen AA. Production of adeno-associated virus (AAV) serotypes by transient transfection of HEK293 cell suspension cultures for gene delivery. J. Virol. Methods ; 196: 163–73. Crossref
6. Trivedi PD, Yu C, Chaudhuri P et al. Comparison of highly pure rAAV9 vector stocks produced in suspension by PEI transfection or HSV infection reveals striking quantitative and qualitative differences. Mol. Ther. Methods Clin. Dev. ; 24: 154–170. Crossref
7. Nienow AW. Reactor Engineering in Large Scale Animal Cell Culture. Cytotechnology ; 50, 9–33. Crossref
8. USD . Application Note: Characterization and Engineering Performance of the Allegro™ STR 200 Single-Use Stirred Tank Bioreactor System. Pall Corporation: East Hills, NY, Crossref
9. Isailovic B, Kradolfer M, Rees B. Fluid Dynamics of a Single-Use Stirred Tank Bioreactor for Mammalian Cell Culture. BioProcess Int. ; 13(8), 60–66. Crossref
10. Perry C, Rayat ACME. Lentiviral Vector Bioprocessing. Viruses ; 13(2): 268. Crossref
11. Collignon M-L, Mainwaring D, Pedregal A et al. Targeting Cost-effective Large-scale Manufacturing for rAAV Vectors by Transient Transfection using Pall’s Allegro STR Bioreactor. Poster published at ESGCT . Crossref
12. Mainwaring D, Joshi A, Coronel J, Niehus C. Transfer and scale-up from 10L BioBLU® to Allegro™ STR 50 and STR200 Bioreactors. Cell Gene Ther. Ins. ; 7(9), –. Crossref
13. Heshmatifar S, Collignon M-L; Kingwood M et al. Evaluation of Pall’s Allegro™ STR Bioreactor Production For Use in Cultivation of Mammalian Suspension Cells and Lentiviral Vector Production. Poster published at ESACT . Crossref
14. USD – Characterization and Engineering Performance of the Allegro™ STR Single-Use Stirred Tank Bioreactor Family Range. Pall Corporation: East Hills, NY, Crossref
15. USD – Scalability Between the Allegro™ STR 50 and STR 500 Bioreactors in a CHO-S Fed-Batch Process. Pall Corporation: East Hills, NY, Crossref
16. Joe CCD, Jiang J, Linke T et al. Manufacturing a chimpanzee adenovirus-vectored SARA-CoV-2 vaccine to meet global needs. Biotechnol. Bioeng. ;1–11. Crossref
Emmanuelle Cameau
Pall Corporation
Ernest Asilonu
Pall Corporation
Sheriff Bah
Pall Corporation
Pauline Nicholson
Pall Corporation
Timothy Barrett
Pall Corporation
Contributions: All named authors take responsibility for the integrity of the work as a whole, and have given their approval for this version to be published.
Acknowledgements: None.
Disclosure and potential conflicts of interest: The authors disclose that Pall Corporation owns patents relevant to the Bio-tech Industry.
Funding declaration: The authors received no financial support for the research, authorship and/or publication of this article.
Copyright: Published by Cell and Gene Therapy Insights under Creative Commons License Deed CC BY NC ND 4.0 which allows anyone to copy, distribute, and transmit the article provided it is properly attributed in the manner specified below. No commercial use without permission.
Attribution: Copyright © Pall Corporation, Pall Europe Limited, Pall France SAS. Published by Cell and Gene Therapy Insights under Creative Commons License Deed CC BY NC ND 4.0.
Article source: Invited; externally peer reviewed.
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