Shortening Timelines for Upstream Bioprocessing of Protein-based Therapeutics

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1 Shortening Timelines for Upstream Bioprocessing of Protein-based Therapeutics Dr Jincai Li Executive Director, Biologics Process Development, Wuxi AppTec, China. Sarah Wang Technical Director, Bioprocess Solutions, Sartorius, China. Dr Barney Zoro ambr15 product manager, Sartorius Stedim Biotech, UK. Joerg Weyand Field Marketing Manager Fermentation Asia, Sartorius Stedim Biotech, Germany. The world-wide use of protein-based therapeutics has increased dramatically in the past 15 years. As a result, finding more cost-effective and efficient methods of improving upstream bioprocess production is a high priority for many biopharmaceutical firms. One strategy to improve upstream production is to use bioprocessing models that mimic large scale bioreactors to help choose the most appropriate clone and then optimise media, feed and process parameters. By identifying the best protein producing clone and optimising its culture process to maximise productivity, then manufacturing timescales and costs can be significantly reduced. This paper is a guide to the development of tools for increasing cell culture productivity and provides an overview of Sartorius Stedim Biotech s offering of automated micro and mini bioreactor platforms. An introduction to bioprocess development is given along with specific case studies of how automated bioreactor mimics can benefit upstream production. Introduction Worldwide sales of biologic drugs including monoclonal antibodies (mabs), fusion proteins and therapeutic enzymes exceed US $120 billion per year 1. With sales of around $50 billion, mabs are still the most widely used biologic therapy class and when the mab based drug, Humira, topped $10 billion dollars in sales it became one of the biggest selling drugs of all time. This growth demonstrates how mabs have become a highly popular choice for patients often because they are the only therapeutic option and induce fewer sideeffects than many small molecule drugs. Unfortunately, mab therapies can cost tens of thousands of dollars per patient per year 2. For example, the colorectal cancer treatments bevacizumab and cetuximab cost $20,000-30,000 for an eight-week course 3, well over 60 times more than comparable small molecule therapies. One reason for these high costs is the large dose volume needed to achieve clinical efficacy and hence the significant cell culture volumes needed for cgmp production. Coupled to the use of extensive purification steps, manufacturing costs are very high. Spiralling global healthcare costs have sometimes caused rationing of these drugs, with the antibody drug conjugate (ADC), Trastuzumab emtansine being judged as too expensive for use by the UK s National Health Service 4 for example. These factors are now driving the search for methods to reduce the cost of goods (COGs) of producing biologics. One method of reducing the COGs of biological is to increase upstream cell culture productivity by first choosing the most appropriate cell line and then optimising media, feed and process parameters to increase protein expression. If the most suitable protein-expressing cell line can be identified early in cell line development and its culture process optimised to maximise productivity, then manufacturing costs can be reduced. Upstream Bioprocess Development Clone Screening Traditionally, upstream process development begins with clone screening to find the most stable and 6 BioPharma Asia November/December 2015

2 productive clones. This is performed in small volumes of 0.1mL to 6mL and, because there is a need to conduct large numbers of experiments under bench top bioreactor conditions, this has resulted in the development and widespread use of shaking plate microscale models such as 96 well 5, 6 or more commonly 24 well microplate based 7 platforms. The 24-well shaken plate bioreactor mimics including the Micro-24 (Pall) and Micro-Matrix (Applikon) do not fully mimic the sparged, stirred action of a bioreactor. Additionally, they have a maximum working volume of 7mL which limits the quantity of analytical testing, particularly when multiple samples are required during a run. These shaken plate systems do not provide automation for all liquid handling operations (filling, feeding & sampling) and so require a lot of manual processing, resulting in reduced throughput, high FTE costs and greater scope for human error, compared to fully automated stirred, sparged bioreactors. Clone selection and early process development Clone selection and early process development to identify the most productive cell lines and define optimal media or bioprocessing conditions have historically been performed in shake flasks and modified 50 ml centrifuge tubes (TubeSpin). However, similar to microplates, these provide only very limited control over ph and DO and the mixing environment is unlike that in a bioreactor. Additionally, shake flasks are often manipulated by hand and so it is difficult to perform nutrient feeding or sampling without introducing variability. As a result, the use of shake flasks can often result in different cell growth and protein expression profiles, compared to those seen after scale-up in bioreactors. Published data demonstrate that when cultured in shake flasks, Chinese Hamster Ovary (CHO) cell lines expressing mabs do not show comparable culture performance with those grown in 2L bench top bioreactors 8. This shows that clones selected using shake flask data may not produce equivalent results when scaled up to pilot or larger-scale bioreactors 9, introducing significant risks to project timelines, process productivity and product quality. Process Optimisation Process optimisation traditionally takes place in bench top bioreactors at around 1-5 L working volume. This is then scaled up to 10-50L and then, to provide sufficient quantities of biologics for use in pre-clinical (toxicity) and clinical studies, bioprocess runs of L (or larger) are required. Due to issues of cost, throughput and resource requirements, it is difficult to evaluate many candidate clones and process strategies in bench top bioreactors. Therefore, the current clone selection Fig 1. ambr15 micro scale bioreactor system process typically involves several rounds of batch and fed-batch shake flasks to reduce an initially large numbers of cell lines down to a sensible number that can be tested in bench top bioreactors. If the chosen clones perform sub-optimally upon scale-up, then final yield or product quality may be affected. This often leaves biopharmaceutical companies with the dilemma that if a larger number of clones had been tested under conditions representative of the bioreactor environment, then a better performing clone may have been identified, saving thousands of dollars in manufacturing costs. Fully automated microscale bioreactor systems such as ambr 15 and ambr250 remove the bottlenecks and resource limitations around bioreactor experiments, and have been shown to provide consistent, scalable results to a range of scales including benchtop 8, 9 pilot and production scale bioreactors 10. Automated bioreactor mimics To meet the need for a bioreactor model that provides comparable mixing, gassing and sampling parameters and can be used in place of microplate models, shake flasks, spin tubes and bench top bioreactors, the advanced micro and mini scale bioreactor systems, ambr 15 and ambr 250 (Sartorius Stedim Biotech) (see Figure 1) are available. Both systems have three components: the single use bioreactor, the automated workstation and the software. Key to the success of these systems as bioreactor mimics is that the culture is stirred by an impeller and gases are supplied by sparging just as they are in a manufacturing scale single-use bioreactor. Each workstation is installed inside a biological safety cabinet for sterile operation and provides independent parallel control of 12, 24 or 48 bioreactors. The workstation controls the stir speed, gas supply November/December 2015 BioPharma Asia 7

3 and temperature and also provides liquid handling automation functions for the bioreactors, each of which can have its own medium, feed, inoculum and sampling strategy. Each bioreactor also incorporates sensors for real-time measurement and automated control of DO and ph, and settings for these can again be bioreactor-specific. The ph is regulated by CO 2 and liquid reagent additions, while DO is regulated by oxygen sparging. Samples can be delivered into a range of vessels, including Vi-Cell sample cups or 24 and 96 well plates. Both automated mini bioreactor systems are supported by software in which a user-defined work list is used to specify operating parameters such as DO/pH values or agitation speeds along with activities such as inoculation, medium addition and sample removal. They also have integrated BioPAT MODDE Software for Design of Experiments (DoE), powered by Umetrics. This allows implementation of DoE into the work flow for simpler process optimisation and scale-up to larger single-use BIOSTAT pilot and manufacturing scale bioreactors. Real-time data such as culture volumes and DO/pH values are logged continuously and external analytical values such as titres or cell counts can be also be imported into the DoE software to identify critical process parameters, optimise bioprocessing conditions and define a robust design space. The combination of high throughput bioreactors and integrated DoE software provides breakthrough and very powerful tool for biosimilars development and QbD programs. The ambr15 is designed for clone screening, clone selection and early process development, whereas the ambr250 is primarily for process optimisation and scale up of cell culture and microbial fermentation. ambr 15 Feature ambr15 ambr250 working volume of bioreactor Number of vessels per system Primary workstation controls for each single use bioreactor Feed type ph range Gassing capability A summary of the different features of the two systems are shown in Table 1 and the two different single use bioreactors for each system in Figure 2. Case studies ml 24 or 48 vessels ph, DO, gas supply, liquid additions, sampling Bolus via pipette headspace or sparge ml 12 or 24 vessels ph, DO, stir speed, gas supply, temperature, liquid additions, sampling Continuous via four displacement pumps per bioreactor and bolus via pipette headspace and/or sparge Table 1. Features of two different automated mini bioreactor models Clone screening & early process development For the automated micro scale bioreactor to be a viable clone selection and early process development model, the process parameters and cell growth and viability results from micro bioreactor cultures have to be comparable to bench top bioreactors. To determine this, comparisons were carried out with the ambr15 ambr 250 Fig 2. Single-use microscale bioreactors with impellers 8 BioPharma Asia November/December 2015

4 Fig 3. Comparison of growth and protein expression profiles of three CHO clones cultured in an ambr15 and a 3L glass bioreactor. automated micro scale bioreactor (3 replicates), 1L and 3L glass bench top bioreactors. To test viable cell counts (VCCs) three CHO clones designated K1-3 expressing mabs were cultured in a fed-batch process in the micro bioreactors, a 3L glass bioreactor (Applikon) or a 1L DASGIP bioreactor (Eppendorf). The cell lines were cultured for days using proprietary cell culture conditions. Cultures were inoculated at 0.5x10 6 viable cells/ml and were sampled every 24 hours for the 1L and 3L bioreactors, and every hours for the ambr bioreactors, for offline ph, cell count and metabolites measurement. Viable cell counts (VCCs) were measured with a Vi-CELL Cell Viability Analyser (Beckman Coulter) and cells from the micro bioreactor (0.25mL) were diluted (1:3 vol/vol) with PBS buffer prior to Vi-CELL measurement to save sampling volume. Titer was measured and offline measurements for ph, pco 2, and DO were measured using a Blood Gas Analyzer (Siemens), and metabolites such as glucose, lactate, glutamine, glutamate were measured using NOVA 400 (Nova Biomedical). The cell growth and productivity results show (Figures 3 and 4) that all three CHO clones have similar growth and titre profiles when cultured in a 1L, 3L bench top bioreactor or an ambr 15 automated micro bioreactor. The cell metabolism results (Figure 5) show that all three CHO clones have comparable ph and pco 2 profiles when cultured in a 1L bench top bioreactor or an automated micro bioreactor. Fig 4. Comparison of growth and protein expression profiles of three CHO clones cultured in ambr 15 and a 1L glass bioreactor. November/December 2015 BioPharma Asia 9

5 Fig 5. Comparison of cell metabolism profiles of three CHO clones cultured in an automated micro bioreactor and 1L glass bioreactor. and after taking the final harvest, the amount of total protein was also analysed after final cell harvest and the results (Table 2) show a similar range of titres across all the bioreactor types used. Growth profiles from the ambr15, 7L glass bioreactor, a 10L single use bioreactor and the 200L single-use bioreactor also showed good comparability (Figure 6). Using the combination of automated micro and Bioreactor ambr15 ambr15 mini bioreactor systems, traditional shake flasks and bench top bioreactors can be replaced resulting in multiple benefits: improved data quality and reduced risk versus manually intensive shaker-based systems; higher bioreactor throughput; significantly reduced process development timeline (Figure 7); an increased number of bioreactor conditions examined in process development (Figure 7) which is likely to result in improved process productivity and/or product quality. 7L 10L 200L 200L Titre (g/l) Table 2. Total protein titre from a CHO cell line cultured in ambr15, a 7L glass bioreactor, a 10L single use bioreactor and a 200L Sartorius single-use bioreactor. The data supports previous literature reports 8, 9 that the ambr 15 micro bioreactor system, with its high throughput capability to utilise 24 micro bioreactors per run, provides the ability to conduct many identical parallel cultures reproducibly. This throughput would be extremely difficult to achieve using manually-controlled shake flasks or bench top vessels without investing significant time and resources, as well as risking introducing variability into the results. Process scale-up For early process development, the ambr15 system is suitable as a mimic of bench top bioreactor conditions and published data indicates that the ambr15 mimics bench top bioreactors at 2L to 7L scale 8, 9, 11. For scaleup the ambr15 can also be used as a comparable model for key parameters such as cell growth and productivity in much larger reactors as well. In one study reported here, a mab-expressing CHO cell line was cultured in a fed-batch process in micro scale bioreactors alongside, a 7L glass bioreactor (Applikon), a 10L single use bioreactor (GE Healthcare Lifesciences) and a 200L single-use BIOSTAT STR bioreactor (Sartorius). Cells were inoculated at 0.5 x 10 5 viable cells/ml and cultured using proprietary cell culture conditions. Glucose was added as required and the cultures were run for 11 days. Cell counts were taken periodically Fig 6: Growth profiles of a CHO cell line cultured in ambr 15, a 7L glass bioreactor, a 10Lsingle use bioreactor and a 200L Sartorius single-use bioreactor. 10 BioPharma Asia November/December 2015

6 Fig 7: Process development using automated micro and mini bioreactors Conclusion The ambr15 automated micro bioreactor system accurately mimics the culture environment of benchtop bioreactors and produces comparable results in terms of cell growth, cell metabolism and protein production. The ambr 15 has also been shown here to provide highly scalable cell growth and viability to production scale single use Sartorius bioreactors. Therefore, this micro scale bioreactor could be used instead of shake flask models or conventional bench top reactors, saving considerable time and money in setup, operation, shut down, cleaning and sterilising tasks while retaining scalability to larger bioreactors. Since this micro scale system is fully automated and utilises smaller cell culture volumes, scientists can perform many more experiments in the same amount of time and using less resources such as media or feeds, making this a costeffective and high throughput method for early process development. Using the combination of automated micro and mini bioreactor systems, traditional shake flasks and bench top bioreactors can be replaced. Consequently, bioprocess optimisation is no longer limited by availability of bench top bioreactors, operator time or facility infrastructure and so the use of ambr micro and mini scale bioreactor technology is currently making a significant contribution in reducing development timelines and manufacturing costs of protein based therapies. 3. Biot, J., Fasano, C., Dos Santos, C. From orthoclone to denosumab, the fast growing market of monoclonal antibodies. Med Sci (Paris). 25 (12): , (2009) 4. White, C., NICE confirms advanced breast cancer drug is too expensive for NHS. British Medical Journal.349: g5078 (2014) 5. Amanullah, A., Otero, J.M., Mikola, M.,Hsu, A., Zhang, J., Aunins, J.,Schreyer, H.B., Hope, J.A., Russo, A.P. Novel micro-bioreactor high throughput technology for cell culture process development: Reproducibility and scalability assessment of fed-batch CHO cultures Biotechnol. Bioeng. 1, 106 (1):57-67 (2010). 6. Chen, A., Chitta, R., Chang, D., Amanullah, A. Twenty-four well plate miniature bioreactor system as a scale-down model for cell culture process development Biotechnol. Bioeng. 1, 102(1): (2009). 7. Warr, S., Patel,J., Ho,R, Newell, K. Use of Micro Bioreactor systems to streamline cell line evaluation and upstream process development for monoclonal antibody production. BMC Proc. 5 (Suppl 8):14 (2011) 8. Hsu, W.T., Aulakh, R.P., Traul, D.L., Yuk, I.H. Advanced microscale bioreactor system: a representative scale-down model for bench-top bioreactors. Cytotechnology 64, (6), (2012) 9. Nienow, A.W., Rielly, C.D., Brosnan, K., Lee, K., Coopman, K., Hewitt, C.J., Bargh, N. The physical characterisation of a microscale parallel bioreactor platform with an industrial CHO cell line expressing an IgG4. Biochemical Engineering Journal, 76, (2013) 10. Bareither, R., Bargh N., Oakeshott, R., Watts, K., Pollard, D. Automated disposable small scale reactor for high throughput bioprocess development: a proof of concept study. Biotechnol. Bioeng. 110 (12): (2013) 11. Lewis, G., Lugg, R., Lee, K. Wales, R. Novel Automated Micro-Scale Bioreactor Technology: A Qualitative and Quantitative Mimic for Early Process Development. BioProcess J. 9(1): 22-25, (2010) References: 1. Bandaranayake, A.D., Almo, S.C. Recent advances in mammalian protein production. FEBS Lett. 588, (2), (2014) 2. Cornes, P. The economic pressures for biosimilar drug use in cancer medicine. Target Oncol. 7, (Suppl 1), (2012) For more information visit: November/December 2015 BioPharma Asia 11