Application Note USD Microcarrier Mixing in PadReactor Mini Bioreactor

Transcription

1 Application Note USD 3012 Microcarrier Mixing in PadReactor Mini Bioreactor

2 Introduction The need for microcarriers in animal cell culture applications has been enhanced by the demand to increase production capacity. In many cases, the productivity yields per cell for recombinant proteins or viruses are low and require large volumes of production. Therefore, microcarriers have been used to allow the scale-up of anchorage-dependent cells in bioreactors. Cells attach to the carriers and can generate mono- or multi-layers depending on the geometry of beads (small spheres vs. macroporous structures). Some biotech companies are currently able to reach high cell densities depending the microcarrier concentrations and batch sizes. Hence, this increases the production capacities and eases the separation of the cells from the supernatant at harvest compared to suspension cell culture. Today, many applications using microcarriers have been established for the production of viral vaccines (e.g. Influenza, Poliovirus), recombinant proteins (e.g. Interferon) or monoclonal antibodies using different types of cell lines (CHO, MDCK, VERO, Hybridomas). Moreover, microcarriers appear to be the key solution for large scale production for cell therapy. Within the range of its PadReactor systems, Pall Life Sciences has introduced the PadReactor Mini bioreactor, a scaled-down model perfectly suited for feasibility studies, research, process development and small volume production of proteins, cells and viruses (Figure 1). The gentle mixing, linked to an efficient gas transfer, allows the PadReactor Mini system to host suspension and anchorage-dependent cell cultures (e.g. on microcarriers). Critical cell culture parameters such as ph, DO and temperature are controlled and monitored by the PadReactor Mini controller. For optimal use, it is recommended to put microcarriers in complete or homogeneous suspension (Figure 2). This ensures entire microcarrier surface availability for cell anchorage and growth. Stirring speed must be determined by considering: The complete suspension of microcarriers Low shear stress Good medium mixing Stirring speed impacts cell growth rate and productivity indirectly. Too low of a speed allows settling of microcarriers. Too high of a speed can damage cells or detach them from the microcarriers. Collagen-coated and Dextran-based microcarrier distributions and integrities inside the PadReactor Mini bioreactor have been assessed for several mixing speeds and microcarrier concentrations. Figure 1 PadReactor Mini system 2

3 Figure 2 Illustration of microcarrier suspension in PadReactor Mini bioreactor (A: partial suspension; B: complete suspension; C: homogeneous suspension) A B C Method This study was carried out in the PadReactor Mini bioreactor fitted with a 16 L biocontainer. Collagen-coated plastic (Solohill, cat #: C ; Diameter: µm; density: 1.026) and Dextran-based (Diameter: µm; density: 1.030) carriers were selected as microcarrier models. Working volumes and microcarrier concentrations were selected to screen the mixing efficiency of the system on both types of beads. Tested conditions are listed in Table 1. Bioreactor working volumes tested in this study were 5, 9 and 13 L, where 5 and 13 L represent the minimal and maximal working volumes in the PadReactor Mini biocontainer (equivalent to 20 to 80% of the total volume). The size of hydrated dextran microcarriers is related to osmolality. To control for size, the study was performed with a liquid having an osmolality close to a standard cell culture medium. Medium-like solution, composed of 96 mm NaCl, 42 mm NaHCO3, 2.5 mm NaH2PO4 and 0.04 mm Red Phenol, was identified as the best solution. Table 1 Conditions tested for the microcarrier homogeneity inside the PadReactor Mini bioreactor. Type of microcarrier Working volume (L) Microcarrier concentration (g/l) Dextran-based & , 6 & 12 Collagen-coated , & Stirring and Sampling At the beginning of each experiment, microcarriers were allowed to settle. Paddle rotation was started at the required speed and samples were taken after 15 minutes and at least 12 hours, to check that no settling over time occurred. For each sampling step, one 11 ml sample was taken at the top of the bag and a second one at the bottom, both with a graduated pipette. Sample Analysis The final goal was to compare microcarrier concentrations at the top and at the bottom of the PadReactor Mini biocontainer. To do so, each sample was inserted into a graduated cylinder. Once the microcarriers were settled, their volume was measured (Figure 3). Heterogeneity between the bottom and the top of the bag was calculated as follows: % heterogeneity (H) = 2 x (VolBottom VolTop) X 100 (VolBottom VolTop) 3

4 Precision of obtained data was not calculated. Heterogeneity <5% was identified as not significant. For this reason, conclusions were made as followed: % = 0 to 5%: Top and bottom concentrations are equivalent. Fully Homogeneous suspension state is reached (considering the precision range of 5%). (Figure 2). % = 5 to 10%: Top and concentrations are very close. Considering the precision, we can estimate the suspension as homogeneous. 100% > % > 10: The difference of beads from bottom and top is big and does not give a good suspension. % = 100%: All the microcarriers are settled down at the bottom of the bag. Microcarrier integrity was inspected under a microscope with a cell culture dish. Broken beads vs. total beads were counted for a specific area on the plate. Integrity of the microcarriers was calculated as followed: % Microcarriers integrity = (Total beads Broken beads) X 100 Total beads Figure 3 Graduated cylinder with 11 ml of microcarrier solution in DPBS Results and Interpretations Solohill Collagen-coated microcarriers (Figure 4) For a working volume of 9 L With g/l of beads, a suspension is obtained from (H = 5.4%). This suspension is fully homogeneous at (H 0% which is below the precision rate of 5%). With g/l of beads, microcarriers are in complete suspension at (H = 7.4%). Full homogeneity is achieved with a stirring speed of (H 0%). With g/l of beads, are in complete suspension at (H = 6.7%). Hence, homogeneous distribution of microcarriers is obtained at (H = 4.8%). Figure 4 Heterogeneity of Collagen-coated microcarriers after 15 minutes mixing from bottom and top of the PadReactor 4

5 Mini bioreactor (represents the stirring speed where the microcarriers are in suspension (H 5%)). Dextran-based microcarriers (Figure 5) 30.0 Collagen-coated microcarriers heterogeneity in PadReactor Mini Percentage of concentration diference between bottom and top (%) L g/l 8L g/l 8L g/l Tested conditions 30 RPM 35 RPM 70 RPM 80 RPM For a working volume of 13 L (maximum working volume) With 10 g/l of carriers, a suspension is obtained from 70 RPM (H = 5.2%). This suspension is fully homogeneous at 90 RPM (H = 1.1% which is below the precision rate of 5%). With 6 g/l of carriers, microcarriers are fully homogeneous starting from (H = 4.7%) and seems to improve with increase of stirring speed of (H = 1.3%). With 3 g/l of carriers, are in complete suspension at (H = 8.0%). Hence, homogeneous distribution of microcarriers is obtained at 80 RPM (H = 3.2%). Working volume: 9 L With 6 g/l of carriers, full homogeneity is achieved with a stirring speed of (H = 2.5%). With 3 g/l of carriers, microcarriers are in suspension from 30 RPM (H = 5.3%), while the distribution of microcarriers is fully homogeneous at (H = 0.6%). Working volume: 5 L (minimum working volume) With 3 g/l of carriers, homogeneous distribution of microcarriers is obtained at (H = 0.6%). Figure 5 Heterogeneity of Dextran-based microcarriers after 15 minutes mixing from bottom and top of the PadReactor Mini bioreactor (represents the stirring speed where the microcarriers are in suspension (H 5%)). Microcarrier integrity 30.0 Dextran-based microcarriers heterogeneity in PadReactor Mini Percentage of concentration diference between bottom and top (%) RPM 13L - 10 g/l 90RPM 13L - 6 g/l 80 RPM 13L - 3 g/l 9L - 6 g/l Tested conditions 30 RPM 9L - 3 g/l 5L - 3 g/l 30 RPM 70 RPM 80 RPM 90 RPM 5

6 During these settlement tests, both types of microcarriers were sampled and analyzed under a microscope to quantify their integrity. After more than 15 days of mixing of Dextran-based carriers (agitation speed set to keep carrier in homogenous suspension), less than 2% of the microcarriers were broken. This observation was even lower (<1%) for the Collagen-coated plastic carriers. These data demonstrate PadReactor Mini bioreactor delivers a good mixing efficiency while reducing the shear stress effect. Conclusions This study describes the mixing efficiency of diverse concentrations of Collagen-coated plastic and Dextranbased microcarriers in the PadReactor Mini bioreactor at different working volumes (Figure 4 and 5). It showed that the system is able to get a complete suspension of microcarriers within its stirring speed range (from 0 to 100 rpm), which is the right suspension state for efficient cell anchorage and growth. It also demonstrates the specificity of each microcarrier regarding the stirring speed to apply to maintain these beads in suspension. Using two types of microcarriers having similar diameters and densities does not imply similar suspension behavior. Indeed, identical stirring speed was used to maintain wide concentrations of the two carriers, depicting the important dependence of these results on the type of microcarrier in use. For this reason, Pall Life Sciences recommends operators characterize mixing efficiency of the PadReactor Mini bioreactor with their specific type of microcarrier used for their application. Finally, it is important to highlight that the impact of cell growth and sparger bubbling on microcarrier mixing was not considered in this study. Depending on its importance, stirring speed should be adjusted to maintain microcarriers in suspension. The study results show that the PadReactor Mini system is suitable for adherent cell culture on microcarriers. In addition, the study provides PadReactor Mini system users with tools for microcarrier cultures for different working volumes and microcarrier concentrations. Information contained in this document was used to perform a 13 L working volume adherent cell culture in the PadReactor Mini system. Visit us on the Web at us at bioreactors@pall.com Corporate Headquarters Port Washington, NY, USA toll free (USA) phone biopharm@pall.com European Headquarters Fribourg, Switzerland +41 (0) phone LifeSciences.EU@pall.com Asia-Pacific Headquarters Singapore phone sgcustomerservice@pall.com International Offices Pall Corporation has offices and plants throughout the world in locations such as: Argentina, Australia, Austria, Belgium, Brazil, Canada, China, France, Germany, India, Indonesia, Ireland, Italy, Japan, Korea, Malaysia, Mexico, the Netherlands, New Zealand, Norway, Poland, Puerto Rico, Russia, Singapore, South Africa, Spain, Sweden, Switzerland, Taiwan, Thailand, the United Kingdom, the United States, and Venezuela. Distributors in all major industrial areas of the world. To locate the Pall office or distributor nearest you, visit The information provided in this literature was reviewed for accuracy at the time of publication. Product data may be subject to change without notice. For current information consult your local Pall distributor or contact Pall directly. 2015, Pall Corporation. Pall,, PadReactor and Solohill are trademarks of Pall Corporation. indicates a trademark registered in the USA and TM indicates a common law trademark. Filtration.Separation.Solution. is a service mark of Pall Corporation. 2/15, PDF, GN USD3012