Dr. Nandu Deorkar, vice president of research and development at Avantor, discusses the correct optimisation of chromatography technology for downstream bioprocessing production at Avantor.
Optimisation is critical for the continual improvement of manufacturing, automation, and business processes. Biologics manufacturers are investigating ways to significantly improve process efficiencies in downstream production in bioprocessing operations. The dramatically growing demand for biologics across multiple therapeutic applications and categories raises growing concerns about the cost and availability of these types of treatments.
Key challenges in downstream production
Downstream production currently encompasses about 60% of the total cost of biologic drug production. There are several challenges to remove bottlenecks and improve yields in downstream for more cost-effective production results.
Increased upstream yields: There have been significant investments in the technologies and processes used in upstream processing with the goal of improving yields. Efforts to optimise raw material characterisation, add single-use systems, perfusion systems and more precisely controlled bioreactors leading to detectable increases in upstream yields. However, improvements in downstream throughput have not kept a similar pace, leading to potential bottlenecks in the end-to-end process.
Loss from upstream to downstream: One of the fundamental structural challenges in biologic production is the approximate 30 to 40% loss as harvest material goes through downstream purification. Any percentage of improvement in downstream recovery can increase the ultimate process yield for the target biologic drug product.
Complexity of downstream production: Upstream productivity may be simpler to optimise due to a more straightforward process. Once the target molecule and raw materials are loaded into the bioreactor, the process runs to completion with the appropriate testing and quality control.
In contrast, downstream production involves multiple steps, where the biological material is moved from harvest, centrifugation and/or filtration to multiple chromatography steps before reaching final fill and finish. Each step requires a unique set of resins and buffers among other materials; storage and production systems at multiple steps; and parallel analytical and quality control sampling activities.
Finding efficiencies and economies of scale across downstream processing steps requires additional complex analysis and optimisation. Improvements may be reached after investigating key aspects of current purification steps and technologies, including:
- Expanding the use of high-performance affinity, mixed-mode, and multimode chromatography resins, including resins with targeted ligands that increase selectivity to process targeted molecules more efficiently.
- Exploring methods to make chromatographic buffers more effective by using novel additives, as well as pre-packaged single-use buffer materials to streamline buffer exchange steps.
Optimising process chromatography technology
The goal of downstream optimisation is to improve recovery and therefore normalise -and potentially reduce-the cost per gram of protein produced; thereby enabling the production of more drug product in less time, with the same amount of resin and buffer material. One of the most effective ways to do this is by optimising use of the newest generation of affinity, mixed-mode, and multimode resins.
Undesired glycosylated molecules and aggregates are likely to have limited differential binding to traditional affinity and ion exchangers and can co-elute, therefore presenting major challenges. Aggregates can impact dynamic binding capacity (DBC), where a false increase in signal from aggregates can lead to artificiallylow DBC data, in turn affecting yield. Thus, the combination of increased upstream yields and more complex molecules needs novel approaches to chromatography resins. In response, chemistry suppliers have focused their efforts on process chromatography selectivity and efficiency.
The traditional solution to this type of challenge is to utilise multiple downstream ion exchanges. While this approach lowers the yield of the targeted drug, the cost per gram can become prohibitive. More advanced methods of achieving effective selectivity are based on new ligand chemistries engineered to achieve precise selective interaction with the targeted protein. This approach increases selectivity while reducing the required number of steps, helping control process complexity and cost.
Targeted affinity chromatographic media are based on ligands tailored to interact with specific proteins, offering high selectivity for a target drug molecule. This can be a time-consuming approach if implemented for every new molecule; therefore, mixed-mode and multimode approaches should be considered. To overcome this obstacle and increase efficient throughput, new protein A resins with high DBC have been developed and have increased the protein amount that can undergo the purification process, compared to traditional resins.
Figure 1: Impact of DBC on theoretical number of cycles per batch –comparing advanced vs traditional affinity resins.
This results in decreased cycles, leading to savings in both cost and time. These new protein A resins can also purify IgG or Fc-Fusion proteins at both high and low molecular weight, increasing purity and decreasing pressure on subsequent purification steps.
Figure 2: Advanced affinity resins with improved Fc-specificity lead to higher quality of Fc-fusion protein.
Mixed-mode chromatography media are based on ligands that offer two or more interaction possibilities with the targeted drug molecule. The mixed-mode approach has proven to be effective and more productive in applications, such as intermediate and polishing steps, for purifying proteins based on differential salt-induced hydrophobicity. An advantage of the mixed-mode approach is that the same media can be used for different purification steps, and can be modulated by solution conditions, such as using multiple buffers or multiple elution steps. Newer mixed-mode resins have ligand chemistry that enable the use of multiple sequential interactions during the normal chromatographic process.
Figure 3: Mixed-mode ligand structure -mixed-mode media offer more interaction possibilities with the targeted drug molecule.
Multimode resins offer greater potential for efficiencies and improved yields. Rather than requiring multiple chromatographic purification steps, the simultaneous interaction canseparate closely related proteins in a single step. This concurrent purification can occur without requiring additional intermediate steps, such as buffer exchange, titration, or dilution.
Figure 4: Multimode ligand structure -multimode resins have the capacity to simultaneously interact with different sites or regions of the protein molecule.
Using a multimode or mixed-mode ligand with multiple interactions can boost chromatography yields while merging two process steps—as well as the ancillary time and costs associated with each step—ultimately impacting cost per gram. For example, typical chromatography processes may first use a separate cation exchange step, then an ion exchange step. The yield is approximately 80% pure after the first step, reaching upwards of 95% purity after the second step. With a multimode resin, it is possible to reach 95% purity in one step.
A multimode resin in the column is more efficient, processing 70 grams in one batch versus running 100 grams through separate cation and ion exchange steps. This is due to the overall higher throughput and the lower cost of materials, since this approach reduces the buffer consumption, types of filtration systems used, and ancillary costs associated with each chromatographic step. Each step typically takes up to two hours, cutting costly production time and labour in half.
Another method for optimising process chromatography is using the continuous chromatography method, in which the large column is split into several considerably smaller columns that operate in series over an increased number of cycles. While product is loaded onto some columns, other columns in the set are simultaneously going through the wash, elution, and regeneration phases. The combination of resin optimisation and merging two chromatography steps can potentially confer for a three-fold improvement over traditional processes.
New approaches to buffers
Optimising the resin chemistry presents significant opportunities to improve downstream production. Improving the ways buffers are formulated and delivered to the end user can also positively impact productivity. Additives in buffers can optimise performance of chromatography columns by increasing binding capacity, stability, and separation thereby increasing recovery and purity of the protein of interest. Established methods of buffer generation are for large volumes and need extensive infrastructure and space in large scale facilities. Novel approaches such as using single-use technology, or a hybrid approach of both outsourcing and in-house preparation enable small-and medium-scale facilities to implement different buffer preparation processes. Premade buffers or buffer concentrates enable cost-, time-, and resource-saving for downstream processing.
New potential for downstream optimisation
The goal for downstream optimisation is clear: managing and reducing the cost per gram of valuable biologic drugs. The more expensive elements of current downstream process steps, such as chromatography media, can be made more efficient by investigating how newer, state-of-the-art materials and methods offer ways to condense and streamline process steps