High Throughput Cell Culture Experimentation with BioProcessors SimCell™ Platform Ache Stokelman1, Geoffrey Slaff, Ph.D.1, Andrey Zarur, Ph.D.2, Seth Rodgers, Ph.D.2, Duncan Low, Ph.D.1, and Thomas Seewoester, Ph.D.1

1 Amgen, Inc., Global Process Sciences, Thousand Oaks, CA 2BioProcessors Corp., Woburn, MA

BioProcessors SimCell™ platform is a highly automated miniaturized system for cell culture development. The platform is capable of running and monitoring large numbers (>1,000) of complex experiments. The system is designed to interface with downstream analysis equipment and offers intuitive experimental setup and control and a browsable structured database. This poster shows data from experiments which evaluate the systems reproducibility and comparability to conventional, bench-top scale cell culture systems.

Introduction

Project Background

Goals of the Project:

The purpose of this project was to verify the suitability of the SimCell™ System to perform process development of a typical monoclonal antibody system and generate results comparable to those obtained in typical bench-top bioreactors. The project also sought to evaluate the SimCell™ System in terms of statistical robustness, reproducibility and throughput. Rationale:

Traditional cell culture platforms are inadequate to perform high-quality cell culture optimization experiments in high-throughput. On one hand, inexpensive scale-down systems, such as well plates and shake flasks, cannot accurately reproduce operation in bioreactor systems. On the other hand bench-top bioreactors, while accurate and scalable are costly, labor and time intensive. Experimental Setup

A typical configuration of the BioProcessors SimCell™ Automated Management System capable of handling up to 126 micro bioreactor arrays was used for all experiments. This system is capable of inoculating cells into micro bioreactors, performing maintenance feeds and pH adjustments, and monitoring pH and biomass in a fully automated fashion. The system is also capable of maintaining three discrete temperature, O2 and CO2 settings

SimCell™ Fed-Batch Micro Bioreactor Arrays were used for all experiments. Each micro bioreactor has an operating volume of 600 ul and can be operated in batch, pH-controlled batch, or fed-batch mode. Each micro bioreactor was monitored twice-daily for pH using ratiometric fluorescence detection and biomass using optical density.

A total of 6 different experimental constructs were executed by Amgen and BioProcessors. Results from two constructs, including a media optimization study and a full-factorial process optimization for production of a typical monoclonal antibody in a CHO cell line are presented in this report.

6 SimCell micro bioreactors per array

Working volumes range from 30 - 800µl

Micro fluidic channels for inoculation. feeds, pH adjustment & sampling

• Mammalian Cell Cultures achieve higher than 5x107 Viable Cells/ml

• Simulates all standard production modes: Batch, Fed Batch and Perfusion

• Enables full factorial DoE execution

Proprietary gas-permeable materials facilitate gas transport without the use of traditional stirrers

Culture monitoring via external optical interrogation of in-chamber sensors

6 SimCell micro bioreactors per array

Working volumes range from 30 - 800µl

Micro fluidic channels for inoculation. feeds, pH adjustment & sampling

• Mammalian Cell Cultures achieve higher than 5x107 Viable Cells/ml

• Simulates all standard production modes: Batch, Fed Batch and Perfusion

• Enables full factorial DoE execution

Proprietary gas-permeable materials facilitate gas transport without the use of traditional stirrers

Culture monitoring via external optical interrogation of in-chamber sensors

SimCell™ Micro Bioreactor Arrays

The SimCell Automated Management System performs all experimental operations automatically, including fluidic operations, MBA transport, monitoring cell counts and culture progression in real time, performing measurements and control of experiment parameters. The system can be integrated with downstream analysis tools for product quantification and metabolite analysis. BioProcessors SimWare™ software allows for rapid and straightforward programming of experimental constructs and for powerful and intuitive data analysis.

SimCell™ Management System

Cell Growth in SimCell™ Micro Bioreactors

A series of preliminary experiments were conducted to verify the suitability of the SimCell™ MBAs to sustain mammalian cell growth for expended periods under realistic production conditions. Specifically the availability of oxygen for sustaining high-density cell growth was investigated. Results:

It was found that growth of production CHO cell lines in SimCell™ MBAs is comparable to that observed in traditional bench-top bioreactors. Furthermore, it was found that oxygen is not limiting even at relatively high cell concentrations. In general, the growth behavior and total antibody production observed in SimCell™ MBAs was very similar to those observed in 2 liter bench-top bioreactors operated under equivalent conditions. Additionally, the variance of the results obtained in SimCell™ MBAs was significantly lower than that observed in traditional bioreactors.

Growth and monoclonal antibody volumetric productivity in 600 ul MBAs compared to a 2 Liter bench-top bioreactor. Cells were inoculated at 400,000 cells/ml. Temperature was controlled at 36°C and CO2 was kept at 5% for both cases. Cultures were fed on days 3 and 9. In general, growth and productivity in MBAs matched extremely well with those observed in bench-top bioreactors. Data collected in MBAs was found to have significantly less scatter than that obtained in bioreactors. Slight deviations in biomass were observed during certain periods of the growth phase. However those deviations were small and usually within the scatter of the bioreactor data.

Cell Growth in SimCell™ MBAs

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Screening of Production Medium in SimCell™ MBAs

A total of 10 different production media were screened in SimCell™ MBAs to assess the suitability of the platform to identify compositions resulting in rapid cell growth and maximized antibody productivity. The results obtained from MBAs were compared quantitatively to those obtained in bench-top bioreactors in terms of growth rate and specific productivity, and qualitatively in terms of relative ranking of the different media.

A total of 120 discrete micro bioreactors were operated. Results:

The results from SimCell™ MBAs matched well what was observed in bench-top bioreactors both in terms of doubling times and specific productivities, as well as relative ranking of different media according to growth and antibody output. It was observed that different media induced varying degrees of clumping within the culture. Clumping was not found to affect growth in a significant manner, however cultures where aggregation was severe exhibited in general lower antibody output than those where cells were well dispersed.

Cultures were inoculated at an initial densities of 4x105 cells/ml into MBAs containing the 10 different media formulations. Cultures were kept at 36°C, monitored twice daily for pH and optical density, and were harvested after 12 days and analyzed for protein productivity. During the course of the experiment microphotographs of cultures were collected directly from MBAs to characterize clumping of cells under different media formulations. Doubling times were obtained by fitting an exponential growth model to growth curves between 24 and 72 hours. Observations of doubling time and productivity matched well with what was observed in bench-top bioreactors. Media formulations 3 and 4 resulted in highest volumetric productivity which agreed well with bioreactor results. It was also found that these media resulted in well-dispersed cell suspensions. In contrast, medias 122 132, and 135 which resulted in the lowest observed productivity both in bioreactors and MBAs, exhibited significant clumping in MBAs.

Media Selection in SimCell™ MBAs

Medium Productivity Initial doubling time

1 223 18 2 289 26 3 410 21 4 444 >63 122 171 34 125 271 16.5 135 215 16 137 234 19 132 150 >100 127 217 >100

Process Development Full Factorial Experimental Construct

One of the most appealing features of BioProcessors SimCell™ technology is its ability to carry out full-factorial design of experiments (DoE) constructs in a fully automated fashion with minimum resource load.

In this experimental construct, then production process of a typical monoclonal antibody produced in a CHO cell line was optimized in terms of cell growth, total biomass yield, and ultimately specific volumetric productivity according to the following table:

The full factorial consisted of 54 discrete protocols. For each protocol 12 replicate micro bioreactors were run for statistical analysis purposes. The target outputs were optimized in terms of the processing variables using response surfaces. A total of 648 individual micro bioreactors were operated. Results:

The factorial construct yielded well-behaved response surfaces. Good correlation with response models was obtained for the target outputs in terms of the environmental variables. For those states where bench-top bioreactor data was available, growth and productivity matched well with those observed in SimCell™ micro bioreactors. The SimCell™ data showed low scatter compared to bench-top bioreactor runs. The optimized state of the system obtained in SimCell™ matched well with that observed in conventional bioreactors.

Temperature Seeding Density Feeding Strategy

Lower Level

Higher Level

34°C 2x105 cells/ml 6.0% TV 4x105 cells/ml

35°C 7.5% TV 6x105 cells/ml 8x105 cells/ml

36°C 9.0% TV 1x106 cells/ml

Cultures were inoculated at an initial densities ranging from 2x105 to 1x106 cells/ml into MBAs containing standard growth media. Cultures were monitored twice daily for pH and optical density. Cultures were fed with production media on days 3 and 9 and were harvested after 14 days and analyzed for protein productivity. Integral biomass was calculated by deriving cell concentration from optical density measurements and integrating over the 14-day period. As was expected, integral biomass was found to be a strong function of seeding density and culture temperature. Percent of volume fed was found to have a less pronounced effect. Maximum cell density was achieved at the high-level for all variables of 36°C, 1x106 cells/ml seeding density and 9% of total volume fed. The model for predicting integral biomass was well behaved and exhibited high correlation of R squared higher than 0.9.

Optimization of Integral Biomass

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P<.0001 RSq=0.94 RMSE=5.27e6

Predicted Model (above) and response surface (below) showing the effect of seeding density and temperature on integral biomass.

Antibody productivity was found to be strongly dependent on temperature and seeding density. The effect of all three variables on productivity showed high degrees of convolution, causing the optimal seeding density and feeding strategy to shift as temperature was varied. In general, productivity levels in SimCell™ MBAs corresponded extremely well with those observed in traditional bioreactors, ranging between 0.8-1.15 g/l in both cases.

In particular, the center-point of the factorial study at 35°C, seeding density of 6x105 cells/ml and 7.5% of total volume fed resulted in productivity of approximately 1 g/l for both SimCell™ and conventional bioreactors. The SimCell™ model predicted a slightly higher productivity could be achieved by increasing the seeding density to 8x105 cells/ml. However, this condition has not yet been verified in scaled-up systems.

Optimization of Antibody Output

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Response surfaces showing the effect of seeding density and feeding strategy on protein productivity at different temperatures.

Optimization of monoclonal antibody productivity as a simultaneous function of temperature, seeding density and feed strategy

Conclusions

Growth of mammalian production system for typical monoclonal antibody in SimCell™ micro bioreactors was found to be comparable to bench-top bioreactors. Excellent scalability in terms of cell growth and protein production was demonstrated for a media screening experiment and a full-factorial process optimization construct. The SimCell™ platform was found to be suitable to conduct high-throughput cell culture experiments for process development. The system was able to perform all tasks common in bioreactor operation, such as cell inoculation, culture monitoring and control. The SimCell™ system showed extremely high reproducibility and low data scatter. Based on the success of this preliminary investigation BioProcessors and Amgen intend to continue evaluation of SimCell™ applications.

The SimCell™ system showed extremely high reproducibility between replicates and very low data scatter. Furthermore all micro bioreactors in the system operated under identical conditions were found to be equivalent, as no difference was observed in %CV in micro bioreactors located in different MBAs or within the same MBA. In addition, any single measurement within a micro bioreactor was found to be equivalent to any other single measurement on a second micro bioreactor operated under identical conditions. Finally, no systematic error accumulation was found, indicating all variation within the system is likely due to random errors within individual cultures.

Reproducibility

Analysis of variance for integral bio-mass in all 54 independent sub-protocols across MBAs and individual micro bioreactors

Growth of CHO cells at 36°C in media formulation 135 after 144 hours. Magnification 200x.

Growth of CHO cells at 36°C in media formulation 3 after 144 hours. Magnification 200x

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Integral Biomass (above) over 12 days of CHO cultures cultured at 36°C under different media compositions and table (below) summarizing total antibody output and initial doubling time.

Growth curve (above) and final monoclonal antibody productivity (below) of cultures in micro bioreactors compared to bench-top 2-liter Applikon® bioreactors.