Laetitia Malphettes

Macroscopic modeling of mammalian cell growth and metabolism

We review major modeling strategies and methods to understand and simulate the macroscopic behavior of mammalian cells. These strategies comprise two important steps: the first step is to identify stoichiometric relationships for the cultured cells connecting the extracellular inputs and outputs. In a second step, macroscopic kinetic models are introduced. These relationships together with bioreactor and metabolite balances provide a complete description of a system in the form of a set of differential equations. These can be used for the simulation of cell culture performance and further for optimization of production.AbstractWe review major modeling strategies and methods to understand and simulate the macroscopic behavior of mammalian cells. These strategies comprise two important steps: the first step is to identify stoichiometric relationships for the cultured cells connecting the extracellular inputs and outputs. In a second step, macroscopic kinetic models are introduced. These relationships together with bioreactor and metabolite balances provide a complete description of a system in the form of a set of differential equations. These can be used for the simulation of cell culture performance and further for optimization of production.

Evaluation of the advanced micro-scale bioreactor (ambr™) as a highthroughput tool for cell culture process development

Bio-pharmaceutical industries face an increasing demand to accelerate process development and reduce costs. This challenge requires high throughput tools to replace the traditional combination of shake flasks and small-scale stirred tank bioreactors. A conventional and widely used process development tool is the stirred tank reactor (STR) ranging from approximately 1L to 10L in working volume. Physical culture parameters such as pH, temperature and pO2 can be easily controlled in such systems. However preparation and operation of these systems are time and resource consuming. The ambr™ system from TAP Biosystems has the capabilities for automated sampling, feed addition, and control for pH, dissolved oxygen, gassing, agitation, and temperature. Here, through the evaluation of parameters including cell growth, viability, metabolite concentration and production titer during a fed-batch process using CHO cells producing a recombinant mAb, we assessed the reproducibility of the ambr™ system for standard conditions compared to 2L stirred tank bioreactors and the effects of parameter ranging between both culture systems, namely feed rate and pH ranging.

Medium and feed optimization for fed-batch production of a monoclonal antibody in CHO cells

Background Mammalian cells are used extensively in the production of recombinant proteins, and of monoclonal antibodies (MAbs) in particular. The trend towards avoiding animal-derived components in biopharmaceutical production processes has led to the extensive use of nonanimal origin hydrolysates such as plant hydrolysates or yeast hydrolysates. The source of hydrolysates affects cell growth and productivity and may also affect product quality. Accordingly, careful consideration should be given during process and cell culture media development, in order to determine the appropriate type and amount of hydrolysates to be added, for the cell and product at hand. In this study, we assessed the impact of several hydrolysate additives and chemically defined (CD) commercial feeds on MAb titers, MAb average specific productivity (average Qp), cell viabilities and metabolite profiles in suspension cultures of recombinant CHO cells expressing a monoclonal antibody in shake flasks and 2 L bioreactors.

ambr™ Mini-bioreactor as a high-throughput tool for culture process development to accelerate transfer to stainless steel manufacturing scale: comparability study from process performance to product quality attributes

Background Enhancing throughput of bioprocess development has become increasingly important to rapidly screen and optimize cell culture process parameters. With increasing timeline pressures to get therapeutic candidates into the clinic, resource intensive approaches such as the use of shake flasks and bench-top bioreactors may limit the design space for experimentation to yield highly productive processes. The need to conduct large numbers of experiments has resulted in the use of miniaturized high-throughput (HT) technology for bioprocess development. One such high-throughput system is the ambrTM platform, a robotically driven, mini-bioreactor system developed by TAP-Sartorius. In this study we assessed and compared the performance parameters of ambrTM mini-bioreactor runs to 2L glasses , 80L and 400L stainless steel bioreactors using a CHO cell line producing a recombinant monoclonal antibody. The daily parameters monitored during the cultures were cell growth and cell viability, offline pH and dissolved oxygen, metabolite profiles (glucose, lactate and ammonia) and monoclonal antibody titer. In addition, we compared the product quality attributes (high and low molecular weight species, charge variants) of the clarified cell culture fluid post Protein-A elution generated in the mini-bioreactor run to the larger manufacturing scales. Materials and methods A genetically engineered Dihydrofolate Reductase (DHFR)-/ DG44 Chinese Hamster Ovary (CHO) cell line with Methotrexate (MTX) as a selective agent, expressing a recombinant monoclonal antibody was used. Cells were cultivated for 14 days in a fed-batch mode in a chemically defined medium and fed according to process description. Culture systems: Different bioreactor scales were used in this study : ambrTM48 (TAP -Sartorius Biosystems), an automated system with 48 disposable microbioreactor vessels, 2L stirred tank glasses bioreactors with Biostat B-DCUII control systems (Sartorius Stedim), 80L and 400L stainless-steel bioreactors (Zeta). Data was analysed using JMP statistical (SAS) program. All the experiments were conducted using the same cell bank at the same cell age at bioreactor inoculation. pH (7.0 +/0.2) was controlled using CO2 and base addition. The scale independent factors (pH, DO set point, seeding density, temperature, culture duration, media and feed composition), were the same for all the scales. The scale dependent factors (culture start volume, feed volumes) were linearly adapted. Agitation speed and aeration that were determined theoretically or though experiment. Sampling plans and sample volumes were especially adapted to the ambrTM system to take into account the low bioreactor volume. * Correspondence: frederic.delouvroy@ucb.com Upstream Process Sciences, Biotech Sciences, UCB Pharma S.A., Chemin du Foriest, Braine l’Alleud, Belgium Delouvroy et al. BMC Proceedings 2015, 9(Suppl 9):P78 http://www.biomedcentral.com/1753-6561/9/S9/P78

Segmented linear modeling of CHO fed‐batch culture and its application to large scale production

We describe a systematic approach to model CHO metabolism during biopharmaceutical production across a wide range of cell culture conditions. To this end, we applied the metabolic steady state concept. We analyzed and modeled the production rates of metabolites as a function of the specific growth rate. First, the total number of metabolic steady state phases and the location of the breakpoints were determined by recursive partitioning. For this, the smoothed derivative of the metabolic rates with respect to the growth rate were used followed by hierarchical clustering of the obtained partition. We then applied a piecewise regression to the metabolic rates with the previously determined number of phases. This allowed identifying the growth rates at which the cells underwent a metabolic shift. The resulting model with piecewise linear relationships between metabolic rates and the growth rate did well describe cellular metabolism in the fed‐batch cultures. Using the model structure and parameter values from a small‐scale cell culture (2 L) training dataset, it was possible to predict metabolic rates of new fed‐batch cultures just using the experimental specific growth rates. Such prediction was successful both at the laboratory scale with 2 L bioreactors but also at the production scale of 2000 L. This type of modeling provides a flexible framework to set a solid foundation for metabolic flux analysis and mechanistic type of modeling. Biotechnol. Bioeng. 2017;114: 785–797.

Overcoming the clarification challenges of high cell density culture

Cell line engineering and process intensification efforts have led to high performing fed-batch cell culture processes where cell density can reach >30x106 cells/mL. These high performance processes also come with new challenges, in particular developing efficient and scalable primary recovery processes. Centrifugation coupled with depth filtration is a standard approach for clarification of mammalian cell culture broth. However, with high cell density cultures, it can become challenging to obtain low turbidity clarified cell culture fluid (CCCF), and the filtration surface area required for clarification may become limiting when scaling up.

Development and fine-tuning of a scale down model for process characterization studies of a monoclonal antibody upstream production process

Background It has always been an objective of process development and more recently it has also become a regulatory expectation to build robustness into and demonstrate proper control of a manufacturing process, thus ensuring that the biological product meets consistently its quality attributes and specifications. This is achieved mainly through systematic process development and understanding. Once a process is locked and ahead of consistency runs at the intended commercial scale, process characterization studies (PCS hereafter) further contribute to the demonstration of process robustness and the justification of process control ranges. These studies characterize the relationship between process parameters and process performance as well as product quality attributes. For practical reasons PCS are performed in a scale down model (SDM hereafter) of the manufacturing process studied. Therefore, it is essential to establish a SDM that is representative of the commercial scale. Here we describe a road map for developing a scale down model of a cell culture process for recombinant protein production. The cell culture process modeled was a 12,000 L scale fed batch process producing a monoclonal antibody.

Process development and optimization of fed-batch production processes for therapeutic proteins by CHO cells

Background In the biopharmaceutical industry, process development and optimization is key to produce high quality recombinant proteins at high yields. As technologies mature, pressure on cost and timelines becomes greater for delivering scalable and robust processes. Overall, process development should be viewed as a continuum from the early stages up to process validation. Here we outline a lean approach on upstream development during the initial phases to optimize yields while maintaining the desired product quality profiles. Early-stage process development was designed to lead to the establishment of a baseline process and to systematically include experiments with input parameters that have a high impact on performance and quality. At this stage, potential for pre-harvest titer and yield increases as well as product quality challenges were identified. Feed adjustments and systematic experiments with top, high, and medium impact parameters have then been performed to develop a robust and scalable process. This approach was applied to two early stage upstream processes.

Application of a curated genome-scale metabolic model of CHO DG44 to an industrial fed-batch process

CHO cells have become the favorite expression system for large scale production of complex biopharmaceuticals. However, industrial strategies for upstream process development are based on empirical results, due to a lack of fundamental understanding of intracellular activities. Genome scale models of CHO cells have been reconstructed to provide an economical way of analyzing and interpreting large-omics datasets, since they add cellular context to the data. Here the most recently available CHO-DG44 genome-scale specific model was manually curated and tailored to the metabolic profile of cell lines used for industrial protein production, by modifying 601 reactions. Generic changes were applied to simplify the model and cope with missing constraints related to regulatory effects as well as thermodynamic and osmotic forces. Cell line specific changes were related to the metabolism of high-yielding production cell lines. The model was semi-constrained with 24 metabolites measured on a daily basis in n = 4 independent industrial 2L fed batch cell culture processes for a therapeutic antibody production. This study is the first adaptation of a genome scale model for CHO cells to an industrial process, that successfully predicted cell phenotype. The tailored model predicted accurately both the exometabolomics data (r2 ≥ 0.8 for 96% of the considered metabolites) and growth rate (r2 = 0.91) of the industrial cell line. Flux distributions at different days of the process were analyzed for validation and suggestion of strategies for medium optimization. This study shows how to adapt a genome scale model to an industrial process and sheds light on the metabolic specificities of a high production process. The curated genome scale model is a great tool to gain insights into intracellular fluxes and to identify possible bottlenecks impacting cell performances during production process. The general use of genome scale models for modeling industrial recombinant cell lines is a long-term investment that will highly benefit process development and speed up time to market.

From Clone Selection to 80L Bioreactor Production: Better, Faster, Leaner

Materials and methods AmbrTM 48 and 80L stirred tank bioreactor were run for 14 days in a fed-batch mode in a chemically defined medium. A CHO cell line expressing a recombinant monoclonal antibody (MAb) was used. Feed was added daily from day 3 onwards. If required, antifoam C was added to the bioreactor. DO, pH, and temperature were controlled at setpoint. Viable cell concentration (VCC), cell viability, and average cell diameter were measured using a ViCell cell counter. Osmolality was obtained using an Osmometer (Advanced Instruments). On harvest day, MAb concentration of the supernatant samples was quantified using Protein A high performance liquid chromatography. For the minibioreactors run, triplicates were pooled on harvest day for product quality attributes analysis. For both scales cell culture fluid samples were centrifuged and filtered to remove cell debris. The monoclonal antibody was purified by AKTAXpress (GE Healthcare) Protein-A purification. The neutralized eluate was used for product quality analysis. Results High throughput technologies enable us to interconnect clone and process development thus reducing the risks associated with early-stage process development while reducing timelines and enabling us to achieve higher process robustness in a leaner manner. In one ambrTM48 run different feeding strategies were assessed on 4 preselected lead clones for selecting the final lead clone, the fed-batch feeding strategy and additional data with feed compositions all at once. The selected condition (clone, type of feed and feeding strategy) was based on the maximization of the MAb titer and target High Molecular Weight Species (HMWS) level and Acidic Peak Group (APG) level (data not shown). Scale up from ambrTM 48 to 80L stirred tank bioreactor was performed with the selected clone, feed and feeding strategy. Similar cell growth profiles were obtained at ambrTM scale and 80L scale for the selected condition (Figure 1). Monoclonal Antibody titers and percentages of Acidic Peak Group obtained at harvest day were comparable between scales (Table 1). Process robustness was shown by performing a number of 4 batches at 80L scale.