In the current competitive biopharmaceutical

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1 Image Courtesy of Sartorius Stedim Biotech single-use Bioreactors for the rapid Production of Preclinical and clinical Biopharmaceuticals Rüdiger Heidemann, Christopher R. Cruz, Paul Wu, Mikal Sherman, Jessica Martin, and Christel Fenge ABSTRACT The article describes successful incorporation of single-use bioreactors as part of a fed-batch platform technology for the production of clinical biopharmaceuticals. By matching general reactor characteristics, the authors were able to scale-up fed-batch processes from traditional bench-scale bioreactors to the large-scale single-use systems without any significant operational differences or changes in critical product quality attributes. Rüdiger Heidemann, PhD, is senior staff development scientist, Christopher R. Cruz is senior associate development scientist, and Paul Wu, PhD, is director, Upstream development, all three at cell culture development, Global Biological development, Bayer healthcare llc, Berkeley, ca 9471; Mikal Sherman is application specialist, fermentation technologies, and Jessica Martin is field marketing manager, single-use Bioreactors, both at sartorius stedim north america, Bohemia, ny 11716, Usa; and Christel Fenge, PhD, is vice-president of marketing for fermentation technologies at sartorius stedim Biotech, 3779 Göttingen, Germany. PEER-REVIEWED article submitted: april 14, 214. article accepted: august 29, 214. In the current competitive biopharmaceutical production landscape, the substantially higher process development costs mean that new candidate molecules are increasingly under pressure to advance to clinical proof-of-concept stage in the shortest possible time with a minimum commitment of capital resources. Single-use bioreactors have the potential to alleviate both of these constraints because they confer specific advantages over conventional stainless-steel bioreactors. Acquisition and implementation costs of disposable bioreactors tend to be much lower compared to traditional stainless-steel bioreactor systems. Some of these advantages can be attributed to their modular nature, which enables the use of existing manufacturing facilities with minimal modifications to the current infrastructure, allowing execution of a straightforward roll in-roll out concept. Furthermore, because the product contact surface is changed with each experiment/campaign, carry over between fermentations is nonexistent, thus removing the need to perform cleaning verification/validation and enabling a more rapid turnaround between products. As a result, single-use systems from various manufacturers are now available on the market, and pharmaceutical development groups have increased the frequency with which these systems are used to produce biopharmaceuticals (1, 2). Although the benefits concerning this technology are fairly clear, one of the primary unresolved concerns focuses on performance comparability between single-use and conventional stainless-steel systems. In this study, the authors present data from two different fed-batch processes producing monoclonal antibodies (mabs) using both system types. To achieve good correlations between bench-scale development reactors and the single- 22 BioPharm International October 214

2 Figure 1: Tip speed and power input values for different bioreactors. Two different types of impellers were used in the 2 L reactors. Tip speed ( m /s) Tip speed and power input 2L a 2L b L 1L 2L SUB 1L SUB Power input ( W /m3) Tip speed = N i dπ Power input = nn pρ N i 3d V N i - agitation rate N p - impeller power number n - number of impellers ρ - fuid density d - impeller diameter V - reactor volume ALL FIGURES ARE COURTESY OF THE AUTHORS use bioreactors, a continuous stirred tank reactors (CSTR) design was used throughout the study as described by De Wilde et al. (3). One major outcome of this work was the development of an entirely single-use upstream platform for the manufacture of biopharmaceuticals from vial thaw to harvest clarification. Materials and Methods Recombinant Chinese hamster ovary (CHO) cell lines were used to produce two different fully human mabs in fed-batch processes. Process A was used to produce an IgG 2 and Process B to produce an IgG1. Both processes used a combination of commercially available off the shelf and proprietary basal media and feed solutions. Initial process development was performed in traditional bioreactor designs at the 2-L, -L, and/or 1-L scale (Applikon Biotechnology, Netherlands). Preclinical and clinical material was generated in the singleuse Biostat STR (Sartorius Biotech, Germany) at the 2-L and 1-L scales. A standard bag design consisting of two marine-type impellers, a macro-sparger and optical sensors (PreSens, Germany) for ph and dissolved oxygen was used. Stirrer geometry, tip speed, and overall power input were matched as closely as possible among all bioreactors, similar to the approach taken by De Wilde and Adams (4) and Noack et al. (). Figure 1 illustrates tip speed and power input values among the different bioreactors. Although the power input of the 1-L system is slightly higher than the rest, the overall tip speed remains comparable to that of the disposable units. Standard shaker flasks were used during the seed train expansion process. Cells were cultivated in a CO 2 incubator maintained at 37. o C and 7% CO 2. Scale-up passaging occurred every two to three days until sufficient cell mass had accumulated to directly inoculate the bioreactors regardless of scale, with the exception of the 1 L. The 2-L bioreactor was inoculated at an initial volume of 8 L operation range due to variation in the accumulated cell mass of the seed train. Basal medium was added to the reactor to passage the cells by dilution and considered in scale-up mode until a predefined total cell number was reached; this time point was defined as fed-batch day zero. If required, the 2-L unit was used as part of the seed train expansion process and culture was directly transferred to the 1-L reactor to achieve a target starting cell density on inoculation day. Fermenter process parameters (e.g., ph, temperature, po 2 ) were maintained by biocontrollers manufactured by either Sartorius (DCU 2/3/Biostat STR, Sartorius Biotech, Germany) or Applikon (icontrol, Applikon Biotechnology, Netherlands). All bioreactors were configured with a macro-sparger to deliver mixed gas consisting of air, N 2, CO 2, Contin. on page 26 October BioPharm International 23

3 Contin. from page 23 Figure 2: Schematic overview of the upstream fed-batch process using disposable bioreactors. Duration ~ 14 days Duration ~ 2 days Duration ~ 1 day 1mL cryo-vial shaker asks (1L required for inoculation) L min working volume in 2L 2L min working volume in 1L Single-Use / Disposable 3lters already implemented for downstream processes Seed-Train Expansion 2L BIOSTAT STR 1L BIOSTAT STR Cell Sep: Dead-End Filtration Clari3ed Harvest Clari3ed Harvest and O 2. Dissolved oxygen was controlled at 4% air saturation, ph at , and the initial process temperature was set to 36. o C. For Process B, a temperature shift to 33. o C was performed once the cell density reached 8 x 1 6 cells/ml. The bioreactors were sampled daily to determine viable cell density and viability (Cedex cell counter, Roche Diagnostics, Germany) as well as glucose and lactate values (YSI, Yellow Springs Instruments, OH, USA). Dissolved oxygen, ph, and levels were measured offline using a blood gas analyzer (Siemens Diagnostics, NJ, USA) and used to verify the ph and dissolved oxygen probes of the bioreactors. mab titers were measured using a protein A high-performance liquid chromatography (HPLC) method. The fed-batch process was terminated after reaching predefined harvest criteria (viability and/or time based), and the crude harvest was clarified by dead-end filtration. The 2-L Biostat, in addition to being the seed source for the 1-L system, was also used to verify the bench-scale fed-batch process and to produce preclinical material. The 1-L reactor was used to produce GMP material for clinical trials. The entire clinical upstream process consisting of the 2-L and 1-L bioreactors as depicted in Figure 2, uses disposable materials throughout, including bags for basal media, feeds, and clarified harvest. results and discussion To compare the performance of the singleuse bioreactors to traditional fermenters, critical process parameters for each vessel type are plotted together. Data from multi-use reactors are plotted as grey dots to define a point cloud of expected values and singleuse disposable runs are shown as continuous lines. Figure 3 and Figure 4 summarize the data obtained in Process A and B, respectively. As shown for Process A, cell growth, viability, glucose, and lactate concentrations obtained with the single-use systems follow the general trends defined by the bench-scale reusable bioreactors. Lactate values were slightly higher in the bench-scale reactor cultures. These cultures were used for the initial process development, specifically to optimize the feeding process. The slightly higher viability can be attributed to slight differences in the Cedex cell counter used during these cultures. Offline ph and po 2 measurements are well aligned, dem- 26 BioPharm International October 214

4 Figure 3: Process A parameters. Grey squares: bench-scale data ( and 1 L), blue lines: 2 L Biostat STR, green lines: 1 L Biostat STR. 3 Viable cell density 11 Cell viability 2 1 VCD (1 6 cells /ml) Viability (%) Glucose 2. Lactate Glucose (g/l) Lactate ( g /L) ph offine ph po offine po Titer (relative units) Titer onstrating that single-use bioreactor control is comparable and was not affected by vessel type or the measurement technology (i.e., conventional glass electrodes and Clark oxygen probes vs. single-use fluorescence patches). As a result, most Biostat STR data Contin. on page 3 October BioPharm International 27

5 Contin. from page 27 Figure 4: Process B parameters. Grey squares: bench-scale data ( and 1 L), blue lines: 2 L Biostat STR, green lines: 1 L Biostat STR. 14 Viable cell density 11 Cell viability 12 1 VCD (1 6 cells /ml) Viability (%) Glucose 3. Lactate Glucose (g/l) Lactate ( g /L) ph offine ph po offine po Titer (relative units) Titer were highly comparable to the data obtained at small scale, and certain parameters shifted towards more desirable profiles (i.e., reduced lactate concentrations and increased titer per unit time). Process B in Figure 4 shows similar behavior, with tighter clustering of 3 BioPharm International October 214

6 Figure : Pressure drop profles for all STR bags. A pressure profle from a defective bag is shown for comparison Pressure drop (psi) L bag 1 L bag defective bag Time (min) 2 data among the reactor types and scales. For some of the bench-scale reactors as well as the 1-L disposable unit the temperature shift was programed into the control unit at a rate of. o C per 3 minutes. The temperature decline was linear over the 3. h time span with less than. o C under shoot. The 2-L disposable unit used a heating blanket, therefore, active cooling like in the 1-L unit was not possible. The temperature shift here took approximately 11 h with a slightly larger under shoot (data not shown). Nevertheless, this second process also demonstrates the feasibility of implementing temperature shift control schemes with single-use bioreactors. Overall, the temperature shift for Process B was necessary to obtain the correct product quality attributes. In addition, it increased the space-time productivity of the antibody. In addition to similar in-process culture performance, critical product quality attributes of the harvest like protein aggregation rates, charge heterogeneity, and glycosylation profiles remained consistent across all bioreactor systems (data not shown). Overall, the data show good comparability between the bench-scale cultures and the large-scale fermentations, demonstrating both the scalability of each processes and the successful integration of single-use bioreactor technology into the authors clinical manufacturing platform for mab fed-batch processes. One of the primary concerns surrounding the adoption of single-use reactor technology for full commercial manufacturing is bag robustness. Due to the nature of the materials employed, defects can potentially be introduced at any point during the life of the bag; at manufacture, shipping, unpacking/setup, or operation. During the preliminary implementation at Bayer HealthCare, a 2-L culture was terminated one day after inoculation because a 24 h medium sterility-hold at L did not reveal a pinhole defect. That incident triggered an in-house integrity test of all single-use reactors prior to use which consisted of pressurizing a mounted bag to.2.3 psi (17 24 mbar) and monitoring the pressure for 1 min (2 L) or 2 min (1 L), shown in Figure. An intact bag will only lose ~.2 psi (1.4 mbar) during the test period whereas a defective bag will rapidly depressurize during the test procedure. Unfortunately, this practice does not guarantee success, as the metal wall of the bag Contin. on page 34 October BioPharm International 31

7 Contin. from page 31 holder can mask potential defects during the pressure hold and despite a passing test. Bag failure can result in contamination and lost cultures. To mitigate this risk, Sartorius Stedim Biotech developed and qualified a pressure test comparable to the one described by the authors but utilizing a patented fleece that inserts a mesh gap between the bag and holder surfaces to prevent these potential masking effects. The approach allows post bag installation and pre-use testing of the entire single-use bioreactor assembly (6). The fleece is designed to be easily removed prior to the start of a run, to ensure normal bioreactor temperature control. This method has been qualified for reliable defect detection for various bag sizes (i.e., a correlation between reliably detectable defect size and pressure decay has been established) (7). This method is, therefore, a more reliable detection method and fully encompasses any typical defects that might be incurred during transportation, storage, unpacking, and installation that cannot be identified by simple visual inspection. In essence, this approach is similar to conventional stainlesssteel bioreactor practices, where the sterilization method is qualified during installation qualification/operation qualification (IQ/ OQ) and a pressure hold test is often performed for risk mitigation purposes to detect any miss-assemblies during regular operation. The sterilization IQ/OQ can be compared to the vendor assembly and sterilization qualification of single-use bags and the pressure decay testing serves in a comparable way as a risk mitigation tool. conclusion As more and more single-use bioreactors and bags are used in late-phase and commercial production, especially with the availability of 2-L single-use stirred tank bioreactors, the need for consistent bag quality, robustness, improved assurance of supply, change management, and business continuity planning become crucial. To satisfy these requirements, Sartorius Stedim Biotech developed a new polyethylene film in close collaboration with resin and film suppliers to meet future industry needs and to further improve bag consistency, robustness, and performance for single-use bioprocessing applications. During development, attention was paid to working with vendors to ensure a stable supply chain, clearly defining and controlling the resin and additive packages, establishing acceptable film extrusion ranges with design space studies, and optimizing of the bag welding process. The result was a system with significantly improved strength and flexibility characteristics, alleviating many of the potential avenues to introduce defects during the manufacture and handling of these singleuse bioreactors (8). Furthermore, cell culture and leachable studies were performed during all stages of the new film development to ensure the new formulation is not toxic and does not impede cell growth (9). All these aspects help to pave the way towards wide implementation of commercial scale single-use biomanufacturing, benefitting from the initially mentioned advantages of reduced upfront investment, flexibility, quick change-over, and minimal validation effort. references 1. C. Heath and R. Kiss, Biotechnol. Prog. 23, 46 1 (27). 2. M. George et al., Biotechnol. Bioeng. 16, (21). 3. D. De Wilde et al., BioProcess Int. 7 (Suppl 4) (29). 4. D. De Wilde and T. Adams, Eur. J. Parent. Pharm. Sci. 1, (21).. U. Noack et al., Single-Use Stirred Tank Reactor BIOSTAT CultiBag STR: Characterization and Applications, in Single-Use Technology in Biopharmaceutical Manufacture (Eds. R. Eibl and D. Eibl), John Wiley & Sons, Inc. (Hoboken, NJ, USA, 21). 6. M. Stering et al., Genetic Engineering & Biotechnology News, 33 (11) (213). 7. M. Stering et al., BioProcess Int. 12 (Suppl ) 8 61 (214). 8. E. Vachette et al., BioProcess Int. 12 (Suppl ) (214). 9. E. Jurkiewicz et al., Verification of a new biocompatible single-use film formulation with optimized additive content for multiple bioprocess applications. Biotechn. Prog., Epub ahead of print, accepted: 21 May 214, DOI: 1.12/btpr BioPharm International October 214