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Bioprocessing advances improve product yield, cut costs, and streamline integration between upstream and downstream processes.
Over the years, many advances have been made in bioprocessing as biomanufacturers strive to increase yield, improve product recovery, enhance product purity, and streamline manufacturing. Innovations in technology and equipment for both upstream and downstream processing have led to more integrated and efficient processes, but there still remain manufacturing challenges that drive the need for further innovation.
Successful upstream bioprocessing innovations have focused on key areas, including:
“These improvements have resulted in the ability to quickly advance fromcell-line generation to clinical currentgood manufacturing practices (cGMP) manufacturing with bioprocesses that are more productive and reproducible,” observes Brian Follstad, director, upstream process development, Catalent Biologics.
“There have certainly been a number of revolutionizing, innovative approaches in bioprocessing in different product areas,” says Vasily Medvedev, process development manager at Univercells. “If we consider monoclonal antibody (mAb) manufacture, one of the key bioprocessing advances seen over the years relates to the use of high cell density prefusion cell cultures. This technology reduces the size of the bioreactor needed, directly influencing the overall footprint of operations, capital expenditure (CAPEX), and, subsequently, cost of goods.”
Furthermore, Medvedev says the use of design of experiment (DoE) techniques is a promising trend in the industry, when these principles are correctly applied to process development and scale-up studies. “In order to fully benefit from the use of DoE studies, representative scale-down models are paramount to ensure identification of high-performing small-scale process design with seamless transfer to commercial manufacturing scale, reducing development timelines (time to market), and costs.”
Looking at the past five years in upstream bioprocessing, Thibaud Stoll, global head of operations, biologics, at Lonza Pharma & Biotech, notes three main innovations that have impacted bioprocessing operations:
“These innovations have contributed to improving process robustness in general, lowering cost of goods and services and enhancing flexibility to respond to fast-evolving demand,” Stoll states.
Other innovations, such as media development, cell line selection, improvements to host cell lines, optimized vectors, and the introduction of high-throughput screening technologies, have also revolutionized upstream bioprocessing, adds Atul Mohindra, senior director, research and development, Lonza Pharma & Biotech. Mohindra also counts improved processmodeling tools, which have enabled a better understanding of the cell culture process as well as the development of more advanced technical equipment (e.g., single-use technologies [SUT], inline testing technologies) as important upstream innovations. “This has enabled the industry to develop more complex molecules, to significantly shorten the time taken to manufacture a first-in-human batch as well as to reduce the costs of a development program,” Mohindra says.
“Besides the latest generation of single-use stirred tank and rocking motion (RM) bioreactors, one of the most significant recent introductions has been alternating tangential flow (ATF) filtration,” remarks Gerben Zijlstra, global technology consultant, continuous and intensified biomanufacturing, Sartorius Stedim Biotech, who notes that ATF provides for much more efficient and cost-effective perfusion culture than previous methods. “It is also gentler on cells, resulting in higher cell viability and lower levels of impurities to process downstream,” Zijlstra adds.
“This technology has enabled tremendous intensification of mammalian cell cultures by allowing 5–10-fold higher cell densities compared to traditional fed-batch processing,” Zijlstra explains. By applying different ATF filter pore sizes, continuous upstream manufacturing can be performed as either a dynamic or continuous perfusion, where the product passes the filter and is direct captured using continuous chromatography; or it can be performed as concentrated fed batch (CFB), where the product is retained in the bioreactor and is harvested batchwise, he says. “The productivity of these intensified cell culture processes greatly surpasses those of existing fed-batch platforms. For CFB, titers of around 30 g/L have been reported, while for dynamic perfusions, 60 g/L (equivalent) titers were reported,” Zijlstra says.
Another recent innovation is the introduction of several online process analytical technology (PAT) tools, such as cell density monitors that use capacitance sensors, adds Thomas Erdenberger, also a global technology consultant, continuous and intensified biomanufacturing, at Sartorius Stedim Biotech. “These sensors allow real-time cell density monitoring of viable cells without having to measure density using traditional methods by taking a sample, risking contaminating the culture. This new method allows the automation of bioreactor feeding as well as cell bleeding using the sensor coupled with a supervisory control system. At Sartorius, we can place these monitors in any of our single-use bioreactors and RM bioreactors to fully automate the entire seed train and main bioreactor,” Erdenberger says.
Another PAT tool gaining traction is Raman spectroscopy. Previously, the key difficulty of using this technology was the need to “train” the mathematical models, the correlation of individual metabolites with the complex Raman spectra. “To solve this challenge, Sartorius recently launched a scalable Raman probe interface so these models are already preset in the 15-mL ambr [Sartorius] high-throughput mini bioreactors. This allows deep process insight and improved process understanding from early development, onwards,” states Erdenberger.
Meanwhile, the latest generation ofdepth filters has also been a powerful innovation for upstream bioprocessing, emphasizes Peter Levison, executive director of business development at Pall Biotech. “As bioprocessing trends have continued to evolve, SUT have offered an alternative solution to drug manufacturers to accommodate shifting drug profiles. Yet, this presented a new challenge when looking at the clarification step,” according to Levison.
In traditional stainless-steel facilities, centrifugation has been a widely adopted solution for clarification, but when working in smaller facilities that deliver higher cell densities, such as the newer SUT installations, centrifugation is no longer as feasible, Levison explains. “Not only is it costly to implement, requiring large capital and process investments, it also has a larger footprint and does not scale down so easily. So, while centrifugation is well suited for 10,000-L stainless steel bioreactors and other large facilities, it is not an ideal solution for facilities based around 2000-L single-use bioreactors that many manufacturers use today,” Levison asserts.
Initially, depth filters offered an alternative to centrifugation with someperformance limitations-traditionally handling cell densities of up to around 20 million cells/mL. “With advances in cell culture and titer increases, we are routinely pushing cell densities up towards the 30 million cells/mL mark, and this is where advanced depth filters deliver the next generation of clarification. The high-performance platform is flexible to support semi- to fully continuous bioprocessing, allowing users to process more product per unit of bioreactor volume. To achieve this performance improvement, two dual-layered depth filtration stages are combined into one clarification step with a flexible chassis to accommodate the capsule configuration needed to deliver consistent filtrate quality in a significantly decreased footprint,” Levison states.
For gene therapy products, the traditional technologies for cell culture and virus production are not suited for commercial-scale manufacture, notes Tania Pereira Chilima, deputy technology officer at Univercells Technologies. “This is not only due to capacity constraints, as these flasks are only compatible with a scale-out approach, increasing costs, and CAPEX, but also due to the laborious nature of operations associated with these technologies and the lack of control over critical process parameters (e.g., pH, dissolved oxygen). This poses some regulatory concerns related to process reliability and reproducibility. Moreover, there is an industry-wide shortage of skilled labor, which means that labor-intensive processes are not as feasible due to resource constraints,” Chilima states.
The use of bioreactors for viral vector manufacture poses several benefits, making cell culture and virus production possible in a highly controlled microenvironment. Moreover, these systems are highly scalable and can benefit from the incorporation of PAT to increase the level of process controlwhile simplifying operations. The most advanced bioreactors for cell culture and virus production incorporate principles of process intensification to enable high-titer and low-footprint virus production, Chilima says.
“Technology improvements in process intensification and connection of unit operations have enabled manufacturing updates to run more efficiently and produce higher yields in less time and space, reducing the amount of capital investment,” concurs Darren Verlenden, head of bioprocessing, MilliporeSigma. “In upstream, we’ve seen that utilizing perfusion technology can increase cost efficiencies, decrease risk, and enhance manufacturing flexibility. Between 50– 60% of companies are already exploring or have implemented perfusion technologies for seed train or production bioreactor steps,” he explains.
Innovations in technology and equipment have also benefitted downstream bioprocessing. Recent innovations, which include acoustophoresis (ultrasonics) cell separation and high-precision microfluidics for label-free cell selection, in-line cell washing, and rapid gene delivery, have resulted in significant productivity gains, states Jenna Balestrini, head of precision medicine and cell bioprocessing at Draper, a Cambridge, MA-based not-for-profit engineering firm.
To accomplish cell separation on a clinical blood sample, for example, Draper developed a system that performs acoustophoresis in a high-performance microfluidic device compatible with a range of patient materials and input volumes. This system bypasses the need for centrifugation. “The module continuously and rapidly removes interfering cell contaminants without compromising cell health. With less handling than conventional approaches, acoustophoresis improves end-to-end yield of cells and accelerates delivery to downstream steps in the process,” according to Balestrini.
In the gene therapy space, Draper has developed a microfluidic transduction module that can co-localize viral vector around cells, increasing viral-cell interaction while using about half the viral vector typically needed to achieve high transduction efficiency. This allows for more controlled viral gene delivery. The system can transduce at standard efficiency levels in 90 minutes using a wide range of vector sources, Balestrini says.
And finally, to allow for a variety of payloads, such as ribonucleoprotein, mRNA, or DNA to be introduced into the cell without the need for viral vectors or even activation steps, Draper has engineered a practical continuous-flow electroporation module and in-line buffer exchanger that uses high-precision microfluidics to tightly control cells exposure to electrical signal, increase throughput, reduce manual-touch labor, and allow for in-line wash steps, Balestrini explains.
“Groundbreaking innovation is seen across different product classes in downstream processing,” adds Medvedev. “In mAb manufacture, the advent of continuous purification processes- namely, chromatography-has enabled significant improvements in process productivities, yields, and utilization of key materials (e.g., protein A resin).”
Chilima further adds that in the gene therapy field, the use of alternative media (e.g., monolithic or membrane chromatography, as opposed to traditional bead-based separation) has proven to increase the dynamic binding capacity (DBC) and reduce processing times as this alternative media can be operated at higher flowrates. “Moreover, the use of membrane and monolithic chromatography systems with advanced separation modalities has enabled a consistent increase of resolution in the separation of empty and full capsids to be achieved with this type of media, which is critical in gene therapy manufacture,” Chilima says.
The innovations in downstream processing have had similar impacts as in upstream processing innovations, namely the development of continuous manufacturing-in particular, continuous chromatography-that can increase facility throughput while reducing costs and the further development of disposable equipment and digital tools, Stoll says.
“Improved in-silico and in-vitro modeling tools, which, when combined with high-throughput screening technologies, can increase our capabilities and reduce time,” says Mohindra. Further downstream processing innovations he identifies are the introduction of end-to-end, single-use solutions for clinical manufacturing (e.g., prepacked columns, single-use flow paths) and the development of new, high-binding capacity resins.
Erdenberger further highlights the development of newer resins as a particularly beneficial innovation. “Firstly, for the capture step, improved (Protein A) resins with higher binding capacity and improved caustic compatibility have allowed for substantially improved downstream processing productivity, reduced cost of goods, and improved bioburden control,” Erdenberger observes. “Currently, even ‘closed, sterile’ chromatography seems to be within reach with gamma-irradiated pre-packed columns, enabling continuous, no/low bioburden chromatography operations. In parallel, continuous downstream technologies such as multi-column and simulated moving-bed chromatography have matured and are increasingly implemented to intensify downstream processing,” Erdenberger adds.
For the polishing steps, substantial improvements in mixed mode resins, membrane adsorbers, and associated equipment are now, and increasingly, allowing for flow-through polishing as a highly efficient mode of operation, Erdenberger states. He further explains that, for the virus removal steps, methods that allow for continuous virus inactivation and virus filtration are being introduced and that continuous methodologies are even available for crossflow filtration.
“These technologies result in continuous or semi-continuous product streams to the next unit operation in a process and in much more efficient chromatography at reduced media cost. Processing is also more rapid. When connected with certain upstream operations such as perfusion, the downstream process can be directly connected for a continuous transition from upstream to downstream,” Erdenberger notes.
These advancements in both upstream and downstream processing have had a mixed impact, but overall a beneficial one. While creating the requirement for more well-thought-out and nuanced processes, they have also allowed for closer integration between upstream and downstream. “Although these innovations have increased bioprocess complexity, they have contributed to substantially reduced overall costs, time, and risk in generating drug substance and product. In some bioprocesses, for example, initiating culture harvest while the production culture is still running over the course of a few days (or weeks in continuous) allows purification to begin earlier when compared to historical approaches. Furthermore, on-line analytics and product attribute control strategies permit the measurement and real-time adjustment of CPQAs during a batch, allowing for a more efficient method of reproducibly producing drug substance,” remarks Follstad.
“Continuous improvement and interplay between bioreactor productivity and advancements in downstream unit operations are increasing efficiency and streamlining manufacturing,” adds Verlenden. “An initial response to upstream intensification includes single-pass tangential flow filtration for downstream debottlenecking. In new facilities, implementation of continuous capture and flow-through polishing can remove process constraints while enhancing facility fit by reducing buffer requirements by up to 47%,” Verlenden states.
Continuous biomanufacturing will play an integral role in better integrating upstream and downstream operations over the next five years as manufacturers look to suppliers for integrated solutions because they are thinking holistically about their processes while visualizing future scale up, Verlenden explains. “Our customers expect that, in five years, 40–50% of their processes will incorporate continuous capture and flow through polishing technologies, though adoption of fully continuous processes from end-to-end is likely to be further out,” he estimates.
Hearkening back to traditional batch processes, Levison notes that traditional processes are inherently more disconnected step by step. In the upstream, more effective clarification with advanced depth filtration offers a flexible solution. “Users can work towards a semi- or fully continuous processing approach with continuous streams of feedstock, which also impacts the ability to integrate downstream processes.
With the ability to integrate the downstream, manufacturers can create more efficient processes, increasing product quality, saving time and money, and maximizing overall productivity and facility utilization,” Levison says.
Another development of note on the cell-therapy biomanufacturing front, meanwhile, is the integration of a complicated multistep process into a closed, modular, automated benchtop system that enables effective, safe biomanufacturing that can be used in a hospital (point of care) or in a central manufacturing facility, says Balestrini. Current instrumentation and methods for manufacturing cellular therapies are expensive, time consuming to use, difficult to scale, and limited in their ability to effectively deliver genetic material, Balestrini notes. “A modular system is emerging as an industry gold standard,” she states.
Yet, despite these advances in bioprocessing, additional work is still required to ensure that upstream and downstream processing are smoothly integrated, interjects Medvedev. He points to the struggle that current single-use downstream processing technologies for antibody purification are coping with-namely the increasing titers achieved in upstream processing.In gene therapy, on the other hand, the high performance of membrane, beads, and monolithic chromatography systems has caused a mismatch between what upstream processing is able to deliver and what downstream operations are able to purify.
“This is caused by the fact that current technologies for viral vector purification were adapted from the protein industry, which makes them extremely oversized for viral vector applications.The DBC achieved using the current resins available is high with respect to the product harvested in upstream processing. This may cause manufacturers to pull different batches together with intermediate freeze steps, which has disadvantages in terms of yield loss, cold storage space, batch-to-batch variability concerns, and overall processcomplexity,” Medvedev explains.
Vol. 44, No. 5
When referring to this article, please cite it as F. Mirasol, “Technology Advances Streamline Bioprocessing,” Pharmaceutical Technology 44 (5) 2020.