The need for scaledown experiments at some point during bioprocessing is a near-inevitability. For an established process, manufacturers use scale-down experiments to troubleshoot or de-bottleneck steps that are running sub-optimally. In this instance benchtop bioreactors with 3 L to 5 L working volumes predominate.
During process development scales shrink further, and radically. At this stage in a product’s lifecycle all process-related entities are up for grabs: media composition, feed strategies, culture temperatures and pH, culture duration, even cell lines. Given the number of parameters and the staggeringly large number of combinations, using benchtop reactors is out of the question. The desire to define process space through design-of-experiment—a fancy term for “lots of replicates” —precludes working at scales much larger than about 20 mL for early work and 250 mL for late-stage experimentation. For at larger volumes if the expenditure of costly media and reagents were not the issue, time certainly would be.
Hence the emergence of the microbioreactor as must-have equipment.
Microbioreactors serve a multitude of scale-down needs for bioprocesses based on mammalian cells.
Microbioreactors serve a multitude of scale-down needs for bioprocesses based on mammalian cells. Although these parallel systems are widely adopted, the knock against them is process monitoring that is less than adequate. That is changing.
In June, Nova Biomedical announced that it had combined its BioProfile® FLEX2 automated cell culture analyzer with the nearly ubiquitous ambr® 15 automated microbioreactor from Sartorius Stedim Biotech. FLEX2 allows, for the first time, rapid, at-line, automated sample collection and analysis of cell culture data from the ambr system.
ambr 15 holds 24 or 48 single-use vessels at working volumes of 10 to 15 mL. To obtain process data before integration with FLEX2, operators needed to take manual samples and readings or rely on automated liquid handlers and workstations designed for general biochemical assay workflows. Building on decades of experience in clinical chemistry analyzers, BioProfile FLEX2 measures total and viable cell density, cell viability and diameter, pH, pCO2, pO2, glucose, lactate, glutamine, glutamate, ammonium, Na+, K+, Ca++, and osmolality. Nova’s analyzer uses Microsensor cards specific for subsets of those analytes. Analysis of all parameters requires just 265 microliters of sample and takes less than five minutes, the company reports.
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FLEX2 addresses the major issue of microbioreactors: the number of analytes that can be measured during an experiment. “Before integration with FLEX2, all chemistries necessary for microbioreactor systems were accessed via off-line measurements requiring manual sample preparation, manual positioning of the sample for analysis, and manual data transcription or upload to a microbioreactor control system,” says Nova’s Matt McRae, product line manager for biotech.
Sampling, sample preparation, analysis, and data handling take up to eight hours of manual labor per day per experiment. Through an external sampling module FLEX2 automates all analytical processing steps, thus saving time and reducing errors arising from human intervention and small reactor volumes. Small starting volumes for microbioreactor cultures severely limits volumes available for off-line or at-line measurements of process parameters. According to McRae the volume requirements for offline analyzers often exceeds the entire reactor volume .
Since users can pick and choose process or quality attributes as they like, the FLEX2 analysis has applications to culture media development. The platform is, however, limited to mammalian cell cultures.
Sartorius advertises its ambr system as scalable to bench-sized bioreactors and beyond. The inline FLEX2 analysis and monitoring platform complements this scalability with automated sampling for lab-scale through commercial manufacturing systems.
Perfusion Cultures
One drawback of perfusion cell culture is lack of a suitable scale-down bioreactors for troubleshooting and process development. Several companies are channeling development dollars into scale-down perfusion systems, among them OmniBRx Biotechnologies. The company’s CellBRx 0.2 and 0.5 perfusion microbioreactor feature working volumes of 200 and 500 mL, respectively. A 50 mL version is under development.
The single-use CellBRx is loaded with online monitoring and control of process parameters like pH, dissolved oxygen, pCO2, temperature, and glucose with lactate, cell density, and culture microscopy optional. The device runs as a dynamic-bed reactor scales directly to 500 L volumetric capacity. The key to scalability, says Ravindra Patel, OmniBRx CEO, is accurate duplication of mixing time, nutritional homogeneity, and mass transfer index.
Optimization of production-scale perfusion processes using perfusion-enabled microbioreactors had been an unresolved, Patel says. “The most important challenge was developing a microscale perfusion system that replicates and mimics the processing conditions of production-scale perfusion bioreactors. For perfusion bioprocessing, culturing the cells at very high density is essential and obligatory.”
Translating results of microscale perfusion processes to production scale requires precise definition of mixing conditions and mass transfer capacities. In contrast with stirred tank bioreactors that impose direct shear forces on cells using spargers and impellers, CellBRx employs proprietary cell carriers that contain cells at very high densities, and eliminate the use of external cell separation devices.
“Because of these characteristics CellBRx systems operate in perfusion mode with established control strategies and processing steps appropriate for design-of-experiment and multivariate studies at micro-scale,” Patel says.
Fermentations
Most commercial microbioreactors are in fact suitable for mammalian cell cultures but not for microbial fermentations, which differ significantly from cell cultures in terms of agitation, mass transfer, and cell growth. For these same reasons optimizing single-use bioreactors for fermentations has not been a straightforward exercise.
The “Lector” series of microbioreactors (BioLector I, BioLector Pro, and RoboLector) from Mp2-labs address this deficiency. The microtiter plate-based system operate at volumes of 800-2400 μL, depending on the plate format used. Biolector I processes up to 48 samples, while Pro and Robo handle 32 samples (16 wells of the 48-well plate are reservoirs).
As microplate-based systems, the instruments rely heavily on liquid-handling robots, which means that critical liquid transfers are completely automated. The number of measurable process parameters depends on the system.
BioLector I measures the most common fermentation parameters such as biomass, pH value, DO, fluorescence, NAD(P)H, riboflavins, and fluorescent molecules. Fermentations occur under temperature and humidity control and also under controlled gas atmospheres (CO2, O2, and N2, depending on the cell) of any desired composition. Anaerobic cultivation is possible, with fluorescence reads between 365 and 800 nm.
Where small reaction volumes are considered a liability for mammalian culture systems of similar volume the Biolector format, with built-in liquid handling and plate reading, makes this less of an issue.
“From our point of view, small operating volumes are not a limitation but an advantage for screenings and bioprocess development,” says Sebastian Blum, European sales director at Mp2-labs. “With our microbioreactors you can receive real time kinetics out of 48 (BioLector I) or 32 (BioLector Pro) parallel fermentations. Additionally, the measurements are taken while the system is operating, and the user receives a large amount of data and its analysis during the running experiments. Users can therefore conduct high-throughput fermentations with full automation. For rational decisions, the results from the microbioreactors can reliably be scaled up to benchtop fermenters, and both batch and fed-batch cultivations are possible.”
Image: The CellBRx 0.2/0.5 system from OmniBRX, in which ten 0.5L single-use bioreactors can be controlled and monitored independently. The system is equipped with online monitoring and control of perfusion process parameters like Perfusion rate (Rv/day), pH, Dissolved Oxygen (%DO), Temperature etc. Media and Feed reservoirs are also aseptically connected to their respective culture vessels. Image courtesy of OmniBRX.