As new targeted biologic modalities ramp up for commercialization, manufacturing methods must keep pace. If new efficacious therapeutics cannot be produced at reasonable costs they remain in restricted use or are shelved for economic reasons in lieu of other options.
For example, autologous CAR T-cell therapies have demonstrated remarkable success, but manufacturing costs remain extremely high due to the large footprint and substantial clean room costs. Miniaturizing manufacturing and bringing it to the point-of-care setting could, potentially, benefit more patients at a lower cost to the healthcare system.
CAR T cells are just one example. Many processes that produce recombinant proteins use either mammalian or microbial cells and could reap cost savings if development and optimization at small scale better reflected that at scale-up or scale-out volumes. A promising resource, microbioreactors are just beginning to show their might as they become more sophisticated with integrated sensors and automation instrumentation.
A microbioreactor is a way of “doing more with less” succinctly stated Wei-Xiang Sin, PhD, research scientist at SMART CAMP. As these tiny growth machines become even more sophisticated with machine learning and AI algorithms they can only positively impact the future of biologics production.
Point-of-care manufacturing
Over a decade ago, Kevin Lee, PhD, and Harry Lee, PhD, and their colleagues in the MIT laboratory of Professor Rajeev Ram, PhD, developed the 2 mL “Breez” microbioreactor platform technology, which was subsequently spun out as Erbi Biosystems. In 2020 Millipore-Sigma, the U.S. and Canada Life Science business of Merck KGaA, acquired Erbi Biosystems and expanded their Mobius bioreactor portfolio.
“We have been working with the Breez since its development,” said Sin. Various designs of the microfluidic chip, ranging from 100 µL to 1 and 2 mL working volumes, have been used for microbial and mammalian cell culture applications.
“We thought that automated, closed-system microfluidic bioreactors might also be a novel production approach for the personalized nature of autologous cell therapies,” said Sin. Autologous cell therapy manufacturing has lengthy processes, with large equipment footprints, low production throughputs, the need for centralized cleanroom facilities, and a high cost of goods. “In particular, the perfusion-capable, modular Breez can support extremely high viable cell densities in a small volume and footprint,” continued Sin. The parallelized format, with four “pods” per system, allows up to four simultaneous runs per system to raise production throughputs and enable efficient scale out.
The 2 mL design was used to test human CAR T-cell production. Various timelines of activation and transduction as well as two different perfusion schemes, were evaluated to determine optimal conditions.1 Minimal system modifications were made in this proof-of-concept study. Engineering improvements should help move the Breez closer to GMP compatibility on the way to potentially enabling decentralized, point-of-care manufacturing.
The small working volume could reduce the amount of GMP-grade viral vectors and reagents and thus the costs associated with CAR T-cell manufacturing. Importantly, the Breez has the smallest footprint (0.044 m2 per dose) compared to existing manufacturing methods, noted Sin. This attribute substantially decreases cleanroom fixed costs.
“The ability to make clinical-scale CAR T-cell doses in an extremely small form factor essentially means doing more with less—more production runs in parallel with less reagents, space, and manpower,” said Sin.
Better prediction and optimization
Many biological drugs are produced using mammalian cells. The process begins with cell line development (CLD) to determine which cell lines will produce the highest levels of recombinant proteins while maintaining stability during large-scale manufacturing.
According to Cheng-Han (Charles) Tsai, PhD, CEO at Cytena BPS, in the CLD workflow cell lines undergo incremental scaling of culture from static formats to shaker flask expansion and, eventually, to bioreactors. However, static formats lacking agitation technology face significant limitations. The size of multi-well plates and the cell numbers often restrict the ability to effectively agitate the culture, which in turn limits oxygen transfer and the overall culture environment. Microbioreactors offer small-scale, controlled culture environments that allow for better oxygen transfer and optimized cell growth conditions that can replicate the conditions needed for larger-scale bioreactors.
Currently focused on CLD applications, Cytena BPS’s microbioreactors provide precise control over culture conditions, enabling faster and more efficient cell line screening and optimization, according to Tsai, who adds that “additional applications include spheroid culture, stem cell/iPSC culture, long-term proliferation, metabolism and dynamic cellular behavior monitoring.”
The company’s C.NEST® microplate agitation culture system is designed for high-throughput screening in 96- and 24-well plate formats. Customizable mixing intensities allow adjustment of the agitation levels according to the specific cell type and concentration improving oxygen transfer and environmental conditions. In addition, the S.NEST™ system incorporates sensors that provide real-time measurement of dissolved oxygen (DO) and pH to provide more accurate assessments of cell growth conditions to accelerate cell line development, improve process optimization, and efficiently evaluate cell culture health.
“Customers report that introducing mixing early has accelerated their scale-up processes, increased cell concentrations at each stage, and reduced the number of passaging steps,” said Tsai. Notably, the Cytena BPS workflow not only reduced a client’s CLD process time but also significantly increased cell viability in later-stage selection. “Facilitating tests at smaller scales while allowing for precise control of production parameters can permit better prediction and optimization of results before scaling up to larger production volumes,” he added.
Expediting screening
When contemplating the addition of microbioreactors to a workflow, Cristina Martija-Harris, product manager, Beckman Coulter Life Sciences, recommends evaluating usability, scalability, reproducibility, and reliability, as well as compatibility with existing data management and analysis tools. Suppliers can assist with a cost-benefit analysis to determine the economic viability of adoption.
The automated high-throughput BioLector XT Microbioreactor expedites the screening process for different microbial strains/clones, explained Martija-Harris. Applications are diverse including food and beverage, microbiome studies, agriculture, and many aspects of academic, pharmaceutical, and biotech R&D.
The microbial screening platform allows users to design and execute sophisticated experiments that align with biological signals, enhancing scalability and reproducibility, Martija-Harris continued. Online measurements increase data reliability and robustness. In combination with the Biomek i5 Liquid Handler workstation the system permits individually triggered actions such as sampling, dosing of inducers or feed solutions, and inoculation of culture wells in a microtiter plate. “These actions are executed in response to real-time signals from the microbioreactor, including biomass, pH value, DO concentration, and experiment time without interruption to the shaking of the microtiter plate,” said Martija-Harris.
She pointed out that the BioLector XT Microbioreactor allows efficient clone selection along with problem solving during process development and optimization when there are many mutually influencing parameters.2 The system utilizes a standard 48-well microtiter plate format that operates with online, pre-calibrated optical sensors for real-time measurement of cultivation parameters. Patented microfluidic technology facilitates concurrent pH control and feeding processes per cultivation well.
An optional Light Array Module (LAM) provides customizable light settings of 400-700 nm within the photosynthetic spectrum. Sixteen different LED-types can be controlled individually to deliver maximum irradiances and photon flux densities up to 3500 µmol/m2/s to support work with light-dependent organisms that require photosynthesis to grow, said Martija-Harris. “One of the standout features of the BioLector XT system is its strict anaerobic module, which is specifically designed to cultivate microorganisms that require an oxygen-free environment,” she explained.
References
- Sin, WX, Jagannathan, NS, Teo, DBL et al. A high-density microfluidic bioreactor for the automated manufacturing of CAR T cells. Nat. Biomed. Eng (2024). DOI: 10.1038/s41551-024-01219-1
- Fink, M, Cserjan-Puschmann, M, Reinisch, D et al. High-throughput microbioreactor provides a capable tool for early stage bioprocess development. Sci Rep 11, 2056 (2021). DOI: 10.1038/s41598-021-81633-6
