Advancing upstream biomanufacturing: continuous perfusion cell culture
Christopher Brau, R&D process development manager - Thermo Fisher Scientific, explains how continuous perfusion can support small molecule manufacturers and achieve fully intensified biomanufacturing.
Establishing continuous manufacturing (CM) workflows is becoming increasingly popular for intensifying manufacturing processes within the pharmaceutical industry. This is particularly true for small molecule drug manufacturing, following the successful regulatory approval of several CM processes. However, for biopharmaceutical manufacturers, implementation of CM is more complex.
Because biotherapeutics are produced using living cells, manufacturing requires highly specific process conditions. This is essential to maximise productivity and, most importantly, ensure the molecules produced fulfil the critical quality attributes (CQAs) which validate their safety and efficacy. A solution to meeting these required process parameters while intensifying production is a type of bioprocessing known as perfusion cell culture.
By using a cell retention device and a cell culture media exchange system, this method enables manufacturers to maintain a high cell density under constant conditions for an extended period. This can improve productivity and allow for a smaller footprint. There are a number of different types of perfusion process; the most suitable for integration into upstream CM workflows is continuous perfusion.
Advantages of continuous perfusion
As media exchange replenishes nutrients and removes waste products, a cell bleed prevents excess cell density. This allows continuous perfusion to maintain a steady-state bioreactor environment. This state enables high cell densities and viabilities to be preserved for long periods of time—often for around 30–90 days—and product of consistent quality to be produced.
By enabling manufacturers to intensify production, continuous perfusion can provide significant productivity benefits. Additionally, by removing the product of interest throughout the manufacturing process, improvements in overall product quality can also be achieved. These advantages can reduce overall cost of goods compared to traditional batch or fed-batch processes over the lifetime of the process.
Where is continuous perfusion now?
Although there is significant interest focused on implementation of continuous perfusion processes, challenges remain.
One of the primary obstacles currently facing manufacturers is the lack of comprehensive regulatory guidance. Although many regulatory groups are actively supporting the transition to CM and are continuing to issue advice to support its implementation, there is not yet a full set of “default” standards to which manufacturers can refer. This uncertainty makes many manufacturers hesitant to fully commit to implementing a continuous process.
Outside of regulatory challenges, there are also limitations with existing bioprocessing equipment, as many installed fed-batch workflows are not capable of facilitating continuous perfusion. There has been significant innovation to help support manufacturers, however, that requires installing new equipment in some cases.
Innovations to support continuous perfusion
Process development solutions
One of the primary challenges for manufacturers looking to establish continuous perfusion processes is the limited ability of current small-scale development tools. Specifically, existing small-scale bioreactors and high-throughput solutions are often unable to support the mass transfer needs of high-density cell cultures as well as continuous media exchange.
To overcome this challenge, R&D teams across the industry have developed approaches to simulate perfusion processes. Although many of these remain labour intensive and have limitations around the conditions that can be achieved, they are playing a vital role in developing the knowledge needed for progression towards continuous perfusion processes. There are also novel equipment solutions becoming available, including microbioreactors that are specifically designed for intensified and continuous process development, which could have a transformative effect.
Single-use technology
Compared to batch and fed-batch processes, initial continuous perfusion process development is often more time consuming. However, once the process has been established, this can be offset by productivity advantages. These advantages include allowing for smaller scale-up production operating volume targets, enabling manufacturers to reach clinical production volumes more easily.
These benefits can be amplified using single-use bioreactors. By using lower volume single-use equipment, manufacturers can reduce their capital expenditure costs compared to stainless-steel alternatives. Additionally, the process itself can become easier to replicate. Consequently, parallel small-scale set-ups can be established in multiple locations rather than needing a large central facility, lowering costs and possibly lead times, in the long term.
Bioreactor design
Due to the high cell densities that perfusion achieves, existing equipment can struggle to meet the necessary mass transfer and agitation requirements of the cells.
To help support perfusion processes, equipment manufacturers now offer bioreactors with specific features that can overcome these challenges. This includes appropriately sized mass flow controllers to regulate gas flow at higher rates and better designed spargers to optimise oxygenation. Agitation design is also a critical factor as agitators need to provide increased movement without causing cavitation, sheer stress issues, or excessively influencing sparge behaviour.
Process analytics
Advances in process analytical technology (PAT) are also enhancing perfusion workflows due to their crucial role in helping to achieve, and then maintain, an optimal steady-state environment. In particular, spectroscopy and capacitance-based solutions are continuing to see increased use.
One recent PAT development that has simplified the monitoring of perfusion processes is increased use of autosamplers. These can play a major role as, due to the relatively slow changes in steady-state cell culture conditions, they are able to make pseudo, real-time process adjustments. Feedback from at-line instruments can enable manufacturers to automate identification of any deviations using the same at-line instrumentation they already use to qualify their processes.
Media formulation
In addition to the process equipment, another crucial element of perfusion workflows is the cell culture medium used. This is important as the medium provides the essential compounds and nutrients required for maintaining high cell density and achieving maximum productivity throughout the process. Crucially, the requirements for a cell culture medium used for perfusion are distinctly different to those used in batch and fed-batch processes.
For example, a perfusion medium must be formulated with a higher osmolality to offset the drop associated with typical perfusion workflows. It must also balance running richly on highly consumed components and lean on components that risk build-up in cells in a way that minimally inhibits cell growth rate.
To simplify process development, it is also optimal for the same medium to be used throughout the workflow—from initial seed-train steps through to steady-state perfusion. To meet these requirements, media manufacturers have developed commercially available media products specifically designed for use in perfusion processes.
Future of continuous perfusion cell culture
Although there remain several key challenges preventing widespread uptake of CM within the biopharmaceutical industry, significant progress has been made over recent years. By combining the latest technological innovations with increasing process development knowledge and regulatory guidance, implementation of continuous perfusion is becoming increasingly achievable for manufacturers.
Through continued development in these areas, the biopharmaceutical industry has the opportunity to embrace the CM revolution alongside small molecule manufacturers and achieve fully intensified biologic manufacturing.