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Understanding the process and product goals is just the first step to a holistic approach to process development.
Advances in upstream processing of monoclonal antibodies (mAbs) have resulted in increasing titers, cell densities, and other properties that have challenged existing downstream processing operations. It is essential when developing downstream mAb purification processes to understand the product and process goals and focus on platform solutions that offer the greatest throughput.
Advances in both upstream and downstream technologies are helping to facilitate downstream processing of mAbs, but companies need to use a more holistic approach to process development and pursue various avenues of process intensification if real progress is to be made.
Before any process development work is completed, it is imperative, asserts Louise Duffy, senior VP and head of scientific project leaders at Abzena, to first understand exactly how the mAb is constructed and how it will work clinically.
Developers should start by identifying their product quality goals, driven by critical quality attributes, along with process requirements, facility capability, product requirements, and cost of goods, agrees Natraj Ram, vice-president of innovation, bioproduction at Thermo Fisher Scientific.
Considering the product stage (early or late) is also critical to ensure which aspects are prioritized, so that the appropriate level of process understanding and control strategies are achieved during development, according to Ram. Identifying the target scale for the commercial process will also help determine the scale and technology choice and transitions throughout development, he adds.
The next step, Duffy says, is to develop the downstream process to enhance the purity of the desired mAb while reducing product and process related impurities. That should be done, says Kevin Brower, global head of purification development–mammalian at Sanofi, with a defined platform set of raw materials and a platform approach for performing development.
“Using this approach is the most critical best practice because it provides structure for all process development scientists in their work at the bench as well as structure for interactions across the manufacturing network for tech transfer,” Brower states.
Understanding the tools needed to identify product-related impurities while developing purification is also key, according to Duffy. “These two activities must be done collectively with assay and downstream capability developed collaboratively,” she insists.
The ability to achieve high throughput is always an important objective in a production area. For downstream process development, that includes higher flow rate, more rapid turnaround, and more streamlined processes, according to Haiou Yang, director of downstream process development for Avid Bioservices.
For downstream process development, the appropriate choice of resins, filters, chemicals, and single-use components is critical to the success of scale up and manufacturability, adds Ram. Potential requirements for chemicals should be considered depending on the target markets for the mAb product, while resins selection should take into account scalability if a chromatography process will need to be scaled from 1-L to 200-L columns. Assurance of integrity and access to standardized connections are two important factors that should drive the selection of single-use (SU) components.
The strategy for downstream processing for a particular mAb is dependent on the specifics of the upstream process, the capabilities of the facility, and the available equipment for the various unit operations required for mAb purification and final formulation. In general, each unit operation is sized based on the amount of mAb to be processed, factoring in the recovery of each step as it goes down the sequence.
Overall, the downstream processing capacity is the amount of upstream material that can be processed in a certain amount of time. Sanofi currently uses a mix of in-house Excel calculations and external software to model downstream capacity, identify bottlenecks, and guide its tech transfer activities, according to Brower. He notes that these methods are pretty well-established in the industry.
There are two possible strategies for determining the overall downstream processing capacity, says Ram. The first involves determination of the process throughput for each unit operation, choosing technologies that provide the best throughput considering the operational constraints at scale.
The second option is particularly relevant for batch operations such as mixing and filtration, according to Ram. “In these cases, both throughput and the ease of transitioning from one step to another are both considered and the choice of [single-use] SU technologies thus makes a difference,” he observes.
Having appropriate mixing systems with the right bags, filters, and sensors can help with combining multiple steps in one, Ram explains. For example, he notes that performing the inactivation and neutralization steps with a single mixing system that has a pH sensor built in and is equipped with appropriate tubing to pump in and pump out the material will help with efficient processing.
High titers from upstream can either be processed through scaling up processing equipment, which may be limited for some facilities, or through multiple cycles, which can extend processing time, according to Gene Yoshioka, senior director of manufacturing at Avid Bioservices.
Assessing process capacity up front saves time and resources and establishes feasibility, Duffy says. “Performing upfront work to characterize maximum binding capacities, retention times, and equipment scales becomes critical to optimizing the downstream process to match with the upstream titer,” Yoshioka agrees. Designing a process with the capacity of the existing facility in mind also facilitates a smooth technology transfer and enables early production planning.
The typical approach to estimating capacity for a downstream process, explains Duffy, is to look at the titers in the bioreactor upstream, and then size up first steps to hit the sweet spot between not spending too much on resin while allowing for a reasonable number of sub-batches and managing time for the operations. The best strategy, she asserts, is to map the entire process, perform a full mass balance across the whole process, and determine where hold steps may be needed/wanted and then work out how to develop data to support those hold steps.
Internal collaboration is beneficial for this approach, Duffy adds. Process development teams can discuss the feasibility of the process and operational teams can look at more pragmatic issues such as fit with current facilities.
Titer is not the only aspect to consider, though. “During upstream production,” observes Ram, “if too much focus is placed on product titers, other factors such as cell concentration, cell viability, and various product quality characteristics may be impacted. These upstream factors will most likely impact the subsequent recovery and purification process steps.”
Indeed, in recent years there has been a greater focus placed on developing strategies to match downstream technologies with high-titer upstream processes, according to Yoshioka. He stresses once again that it is not just an issue of process capacity. With media and cell lines that are able to maintain higher cell densities, there is also an increase in process impurities (i.e., DNA, host-cell proteins, charge variants, etc.) that the downstream process needs to be designed to remove.
Examples of more traditional improvements helping to eliminate bottlenecks noted by Brower include higher capacity resins and membranes, as well as single-pass concentrators. Ram adds that adoption of higher capacity, lower cost resins, and membrane technologies is allowing manufacturers to increase facility throughput without retrofitting. However, he also notes that it is important to develop resins that provide better capacities, resolution, and throughput.
Advanced depth filtration technologies, meanwhile, enable the harvest of large volumes quickly and easily while providing the benefits of a more clarified feed stream going into the purification process, according to Ram. “Because these technologies combine chemistries with carefully designed matrices that provide size-based separations, these technologies go beyond improving throughput and provide higher quality product,” he says. They also allow for easier scale-down modeling compared to centrifuges, reduced cost of goods at scale up, and lower volume pools. The latest multi-mode versions under development make it possible to have smaller volumes, less dilution, and higher yields, Ram says.
Beyond advances in technology for specific unit operations, Ram points to the use of multi-column chromatography, integrated flow-through steps, continuous inactivation and neutralization, and in-line dilution of buffers as important downstream technologies that are enabling downstream processing to catch up to the higher levels of upstream productivity.
Brower also believes that high-throughput process development approaches allow mAb developers to better understand and characterize their processes than was possible five to 10 years ago. “Access to this greater knowledge leads to more robust process outcomes that can better handle the variability provided downstream by various forms of intensified upstream processes,” he explains.
Single-use technologies are having a major impact on how downstream processes are developed as well, according to Duffy. She notes that developers can more easily explore changing the order of process steps, or introducing new ones, depending on how a molecule is behaving and what the impurity profiles look like. “This greater flexibility allows for more rapid fine-tuning of processes,” Duffy says.
Yang cautions, though, that while the industry has been working to adapt to new devices and technologies, the process of converting to completely new/different techniques and instrumentation requires time and careful execution. “It is important to note that the easier a technology can be implemented, the faster the industry is able to adapt to it,” she comments.
Despite numerous advances in downstream processing technologies, there is still a disconnect between upstream and downstream processing, according to Duffy. “Firstly, in the last five to seven years, upstream titers have almost tripled, while downstream capacity has not necessarily matched pace. Secondly, some downstream technologies are still very manual and laborious to run,” she explains.
To address this issue, a holistic view of the entire manufacturing process is needed. Process developers, Duffy asserts, must proactively think about the impact of upstream decisions on downstream processing to fully understand what technologies will be required for downstream processing.
A holistic approach that considers both upstream and downstream processes is critical to success,” agrees Ram. “While these two manufacturing processes rely on each other to be successful, they are frequently developed separately. Developing them in parallel, however, can help meet aggressive program timelines and allow for inherent synergies. Harmonizing the processes enables greater understanding of the upstream challenges for the protein of interest being produced, which can then help negate possible problems encountered later in the development process,” he explains.
Given that most downstream steps are inherently mass based (process a certain amount of product), Ram asserts that efficiencies can only be gained through integration or intensification of these steps. “Many of the technology advances made to date have the potential to improve productivity or throughput, but until they are integrated to allow for synergistic benefits, much of the advantage cannot be realized,” he says.
While initial efforts have occurred, integration of unit operations from both operational and control standpoints has yet to be fully achieved and will require collaboration across the industry to bring harmonization of components, equipment controls, and software platforms, Ram comments.
In the near-term, agrees Brower, application of integrated and continuous (or connected) process operations will be the key to improving the productivity, long-term sustainability, and flexibility of downstream processing in the biopharma industry. “Such approaches,” he concludes, “have an impact on space requirements, increase the ability to apply single-use technologies, drive down cost of goods to increase patient access, and allow manufacturers to dynamically respond to changes in treatment approaches and reimbursement strategies.”
The need for process intensification and continuous processing will, in the long term, drive the development of process analytical technologies (PAT) beyond basic sensors that will allow for the enhancement of throughput with better process control for both continuous and batch processing, according to Yang.
“Novel purification formats (non-chromatography, flow-through technology) and the incorporation of PAT tools to enable real-time release and forward processing to drug product,” agrees Brower, “will lead to a further step change (leap) beyond the current standard approaches.”
Meanwhile, Ram notes that advances in single-use centrifugation or related technologies that reduce the burden on harvest filters could be a significant improvement to address challenges posed by increasing cell densities and higher titers in upstream processes. Integration of multicolumn chromatography skids with buffer blending capability will significantly improve the efficiency of this operation. There is also, Ram says, still great opportunity for the development of resins that can provide high resolution while also offering high capacity and throughput. He also notes that future single-use systems that can accommodate a wide range of scales will also help reduce overall capital costs for multiproduct facilities.
Another major opportunity for improvement in downstream processing, according to Brower, is less related to upstream advances and more related to the diversity of molecules in the biopharma pipeline. “This diversity of modalities presents unique purification challenges for which we are developing solutions and approaches across the industry,” he says.
Cynthia A. Challener, PhD, is a contributing editor to BioPharm International.
Vol. 34, No. 7
When referring to this article, please cite it as C. Challener, “Defining a Holistic Approach to mAb Purification Processes,” BioPharm International, 34(7) 2021.