With the rapid development and intensifying competition in the biopharmaceutical industry, it has become particularly urgent to shorten the drug development cycle, develop efficient, robust, and high-quality production processes, and reduce production costs. Currently, most enterprises are leveraging high-throughput technologies, platform processes, and single-use production technologies to accelerate product development. Among these, high-throughput technologies enable the simultaneous execution of multiple sets of experiments for design of experiments (DOE), which can significantly shorten the process development time and yield better experimental results.

High-throughput micro-bioreactors come in a wide variety. Early products such as microplates and 24-well deep-well plates, with volumes in the microliter range, were primarily used for primary clone screening. However, due to their inability to monitor critical parameters during cell culture, their application scope was limited, and they have gradually been phased out. Currently, three widely used high-throughput micro-bioreactors are Simcell, Ambr 15, and Ambr 250, each with varying culture volume capacities and suited for different stages of cell culture process development.

The SimCell microreactor system comprises five culture chambers, each containing 42 cartridge-based microreactor modules. Each module houses six independently controlled microreactors with a total volume of less than 800μL (culture volume ranging from 300-700 μL). In theory, a single SimCell system can simultaneously operate up to 1,260 microreactors. Each culture chamber allows for independent control of temperature, relative humidity, and ventilation, enabling online measurement of parameters such as cell density, pH, and dissolved oxygen (DO). The SimCell microreactor is suitable for clone screening and early-stage process optimization.

The notable advantages of the SimCell system lie in its ultra-high throughput and ultra-micro volumes. However, the small volumes may not meet the requirements for conventional offline analysis, and the absence of stirring paddles may hinder the accurate simulation of large-scale reactors.

The Ambr system consists of three main components: disposable reactor vessels, an automated workstation, and operational software, encompassing both Ambr 15 and Ambr 250. The Ambr system boasts advantages such as high throughput, automation, and compact size. Its vessel geometry, including aspect ratios and impeller designs, is similar to classic stirred reactors, enabling real-time monitoring and control of various process parameters with excellent parallelism and scalability. The Ambr system software offers efficient data recording, management, and analysis capabilities, facilitating the execution of DOE and data analysis during process development and late-stage clinical phases. Currently, the Ambr system is widely applied in the biopharmaceutical industry.

The Ambr 15 operates with a working volume of 10-15mL, capable of running 24-48 bioreactors simultaneously. It is equipped with stirring impellers and independent gas supply lines. Apart from temperature and agitation speed, which are centrally controlled by the workstation, other parameters like pH and DO can be independently regulated. Leveraging a highly automated robotic arm, the Ambr 15 system can automatically perform sampling and media replenishment. However, due to its small culture volume, the Ambr 15 system limits sampling frequency and harvest volume during cultivation, unable to provide sufficient samples for downstream purification and formulation analysis. Additionally, operations such as sampling, feeding, and base addition during cell culture may cause fluctuations in online DO, pH, and temperature readings within the Ambr 15 system.

The Ambr250 is an upgraded version of the Ambr 15, capable of independently operating 12-24 disposable bioreactors with a working volume of 100-250mL. It meets the subsequent needs for purification and sample analysis. The Ambr 250 allows for independent control of temperature and agitation speed, and each reactor is connected to four feeding lines, enabling either batch feeding (10μL-10mL) or continuous feeding, thereby accommodating different process requirements for cell culture and microbial fermentation.

The Ambr 250 disposable bioreactors feature double-layer inclined three-blade impellers, coupled with four baffles to enhance liquid mixing. This design shares similar geometric dimensions and hydrodynamic properties with traditional bioreactors, ensuring that the microenvironment for cell culture in the Ambr 250 is, to a certain extent, closer to that of traditional reactors, facilitating easier process scale-up.

In recent years, high-throughput micro-bioreactors have gained widespread application in the biopharmaceutical industry, covering various stages such as cell line screening, media and process development, as well as process characterization.

1.Clone Screening and Media Development

Cell line screening involves integrating the target gene into the genome, a process that is inherently random. Consequently, each transfected cell exhibits unique characteristics, necessitating extensive clone screening to identify the final high-yielding and stable cell line. Early high-throughput clone screening typically employs microplates, followed by further screening in 24-well deep plates, shake tubes, or shake flasks through fed-batch cultivation. However, discrepancies may arise when transitioning from these smaller-scale cultures to reactors, as some highly expressing clones may not be scalable. Additionally, these cultivation containers lack the ability to monitor critical parameters like pH and DO, leading to a substantial discrepancy in the microenvironment compared to benchtop reactors, thus limiting the accuracy of predicting the reactor-scale performance of high-yielding clones. Traditional reactors, due to their low throughput and high operational costs, can only evaluate a limited number of clones, making them suboptimal for clone screening. To mitigate the risk of abnormalities during process scale-up, the high-throughput Ambr system can be employed for screening and evaluation, offering superior predictability and scalability as an efficient and reliable tool for clone screening. Furthermore, integrated clone screening software enables the ranking and scoring of clones, guiding the selection of candidate clones. Additionally, media development is crucial for enhancing yield, quality, and reducing production costs. High-throughput cell culture equipment facilitates rapid, high-throughput screening of media to optimize its key components.

2. Process Development

Utilizing the Ambr 15 reactor to simultaneously run 24 experimental conditions, investigating key process parameters such as inoculum density, pH, and DO, the results demonstrated a high degree of consistency in cell growth, metabolism, and protein expression among reactors under identical conditions, while significant differences were observed under varying conditions. This significantly enhanced the efficiency of process optimization. When testing a newly developed media in both shake flasks and the Ambr 15 reactor, it was found that the cell viability and protein yield in the Ambr 15 reactor were notably lower compared to the shake flasks. Further analysis revealed that the decrease in protein yield and cell viability in the Ambr 15 reactor could be attributed to increased lactic acid concentration.

To investigate the cause of elevated lactic acid concentration in the Ambr 15 reactor, the effects of agitation speed, aeration, and pH on lactic acid accumulation were examined. The results indicated that both aeration and pH control strategies had statistically significant impacts on lactic acid accumulation, with pH being the most influential factor. By employing optimized conditions in the Ambr 15 reactor, consistent cell viability and expression levels were achieved comparable to shake flask cultures.

As process optimization conducted in high-throughput micro-bioreactors needs to be scaled up to production-scale reactors, assessing the scalability of both equipment and processes is crucial. Studies have shown that cell culture processes developed in the Ambr 15 reactor could be successfully scaled up to 2L reactors, 80L, and even 400L Zeta stainless steel bioreactors, with consistent results in cell growth, metabolism, protein expression, and product quality attributes across different scales. This outcome demonstrates the excellent scalability of the Ambr 15 reactor.

3. Process Characterization

The objective of cell culture process characterization is to investigate the relationship between critical process parameters (CPPs) and key performance indicators (KPIs) of cell culture, such as cell growth, product expression, and critical quality attributes (CQAs) of the final product. This process aims to establish acceptable ranges for each CPP and demonstrate the robustness of the process. Traditionally, extensive Design of Experiments (DOE) utilizing benchtop reactors ranging from 1 to 10 liters in scale have been conducted to evaluate the main effects and interactions of various parameters, which requires significant time, manpower, and resources. However, there have been numerous studies utilizing the high-throughput Ambr system as a scaled-down model for both process development and process characterization.

The Ambr system serves as an efficient scaled-down model for both process development and process characterization, significantly accelerating these processes. However, utilizing the Ambr system as a scaled-down model for process characterization in commercial production processes still faces several challenges. Firstly, the scaled-down model standards are not directly comparable between the Ambr system and large-scale bioreactors, necessitating more detailed and targeted characterization studies of the Ambr system and the adoption of novel scaled-down model strategies. Secondly, more suitable risk assessment and statistical analysis software are required to address the bottlenecks in high-throughput experimental design and data analysis. Lastly, it is crucial to recognize that not all processes can be modeled in a scaled-down manner using the Ambr system, and a thorough understanding of the equipment's limitations, as well as the relevant critical process parameters and critical quality attributes, is necessary.

High-throughput micro-bioreactor systems can be employed in the early stages of process development to conduct DOE (Design of Experiments) for high-throughput process research. This approach shortens the process development time, reduces manpower and material costs, accelerates process optimization and characterization in the late clinical stages, and thereby promotes the rapid development of biopharmaceuticals. High-throughput micro-reactors offer advantages such as simple operation, high throughput, and cost reduction, significantly enhancing the efficiency of process development while lowering R&D costs and shortening the R&D cycle. Additionally, these reactors can effectively integrate with DOE designs for process development, improving process stability and controllability of process quality, further broadening the application prospects of high-throughput micro-reactors.

References:

[1] Yao Xiaoyuan, Pei Xin, Zha Xinhua, Zhang Lei, Ji Yu. Application of High-throughput Micro-bioreactors in the Biopharmaceutical Industry [J]. Chinese Journal of New Drugs, 2021, 30(22): 2075-2082.

[2] Guo Yulei, Tang Liang, Sun Ruiqiang, Li You, Chen Yijun. Research Progress of High-throughput Micro-bioreactors [J]. China Biotechnology, 2018, 38(08): 69-75. DOI: 10.13523/j.cb.20180809.

About the Author

Xiaonisha, a food technology professional holding a Master's degree in Food Science, is currently employed at a prominent domestic pharmaceutical research and development company. Her primary focus lies in the development and research of nutritional foods, where she contributes her expertise and passion to create innovative products.