Changes in the biopharmaceuticals market are affecting all aspects of manufacturing, particularly downstream processes. The expected launch of numerous biosimilar monoclonal antibodies (mAbs) will create cost pressures for existing products, while increases in antibody titers are rapidly outpacing improvements in the productivity of downstream processes. At the same time, there is a shift toward smaller-volume, flexible manufacturing to meet local market needs, for which the cost equation is different than that of the conventional large-scale approach. In addition, many drug candidates are highly potent because they are designed for targeted delivery, and thus smaller doses are required, which equates to lower product volumes needed. To be competitive in this environment, manufacturers of both established products and new types of therapies must find ways to lower the cost of downstream processing while simultaneously increasing productivity.
Downstream processing of established products
Improvements in cell-culture techniques and widespread adoption of single-use technologies for upstream operations have led to dramatic increases in titers for mAbs, which represent the largest class of existing drugs (and drugs in development). As a result, chromatography unit operations are the largest bottleneck in the downstream processing of mAbs, according to Kimo Sanderson, vice-president of client services for Asahi Kasei Bioprocess. “Increased mAb titers are placing pressure on downstream processing time and cost, with Protein A resins and viral filtration membranes representing a significant portion of the cost,” says Sylvio Bengio, PhD Pall Life Sciences, Chromatography Group. They are, however, also resulting in increased contaminant levels (higher host-cell protein concentrations, aggregates), which further increases the downstream processing challenges, observes Matthias Jöhnck, head of Merck Millipore chromatography R&D. Jöhnck also notes that cost pressure from biosimilars entering the market is an issue for branded biologics manufacturers.
Advanced technologies required for many candidate drugs
The development of many new classes of therapeutics will have a significant impact on downstream biopharmaceutical processing operations, according to Bengio. “A significant number of new drug candidates are not full-length antibodies, but antibody fragments, engineered antibodies (diabodies, tribodies, multi-specific antibodies, and antibody-drug conjugates), and non-antibody scaffolds, and there are no ‘platform purification technologies’ (such as Protein A for mAbs), thus new purification methods using conventional or nonconventional tools must be found. Affinity chromatography with specific ligands could be an elegant capture solution, but the design of new protein-specific ligands and their immobilization on appropriate and scalable chromatography resins at a ‘reasonable’ cost is a challenge,” Bengio says.
Jöhnck adds that the driving forces for these new templates are the need to reduce side effects and achieve higher efficiencies by targeting more than one aspect of a disease simultaneously using one molecule. Generally, these new molecules also aim to achieve higher potency, which results in reduced processing volumes in manufacturing and lower manufacturing costs. “Most importantly,” asserts Uwe Gottschalk, vice-president of purification technologies at Sartorius Stedim Biotech, “the new candidates being developed are characterized by technical differentiation. The same can be said for biosimilars. Only those manufacturers that can develop a modern process will be able to compete and reach the commercialization stage. As a result, the biopharmaceutical companies developing new therapeutics and biosimilars are very interested in evaluating innovative technologies and prepared to take on the risk associated with adopting newer standards.”
The flexibility factor
Downstream processing technologies must also be adjusted to meet the needs of smaller, more flexible production facilities with adaptable capacities. “There is a real trend toward local manufacturing, so all aspects of the production process need to be competitive at smaller scales,” Gottschalk says. There is also the need for increased flexibility and the capability to adapt to multiple production lines in a single facility, according to Bengio. “Biopharmaceutical manufacturers are fundamentally trying to grapple with unknown demand expectations and how to run their operations at maximum efficiency while meeting variable demand,” observes Sanderson. He adds that plant capacity management is crucial, and as a result, there is increasing use of sophisticated scheduling and planning software that is designed to identify bottlenecks in the manufacturing process and determine the most effective way to mitigate then.
Smaller-scale harvesting solution
One aspect of biopharmaceutical manufacturing that is often overlooked, and in some cases considered part of upstream rather than downstream processing, is the harvesting step, according to Gottschalk. Centrifugation is typically used for large-scale processes, but it is not practical for smaller volumes. Depth filters are also not applicable. Sartorius Stedim has found that dynamic body feed (DBF), which is used in the blood, plasma, and food industries, is an effective solution. “DBF is similar to depth filtration, but diatomaceous earth (DE) is used with cell material to form a cake that acts as a very efficient filter layer that provides high productivity, even with high biomass,” says Gottschalk. To address concerns about dust handling associated with DE, Sartorius has developed a self-contained filter, which the company is currently beta testing.
Improvements in virus filtration
Virus filtration is an expensive downstream processing step that has been a focus of technology development. Much improvement has been made since virus filtration was first used approximately 15 years ago, according to Gottschalk. “Initial virus filters suffered from breakthrough and were very susceptible to blockage by aggregates if the feed was not ultrapure. The devices available today have 10 times greater productivity as a result of changes in the morphology of the filters and the use of surface modification to increase the hydrophilicity. I expect even further advances given that we are still in the very early stages of using this technology,” comments Gottschalk.
New chromatography media are enabling higher flow-rates of up to 1000 cm/h and higher protein binding capacities, thus contributing to higher productivities and lower manufacturing costs, according to Jöhnck. Matrices have been introduced for the capture phase that can tolerate more impurities and higher salt concentrations, as well as provide much greater pressure resistance. Mixed-mode resins also represent a powerful solution, as a “low-salt or no-salt” alternative to hydrophobic interaction chromatography (HIC) and offer unique selectivities for challenging purification steps, according to Bengio.
With so many choices today, Bengio adds that high throughput chromatography screening tools in 96-well plates or small prepacked columns are enabling the rapid screening of multiple chromatography resins with minimal sample consumption, and when conducted in conjunction with a design of experiment approach allow the determination of appropriate separation conditions (e.g., load, pH, and conductivity).
In-line dilution is also an important technique for boosting the productivity of chromatography processes, both for next-generation purification technologies and existing systems, according to Sanderson. “Most new chromatography equipment has built-in in-line dilution capability, but biologics manufacturers with legacy systems don’t have access to this technology. Asahi Kasei Bioprocess has addressed this gap by designing a plug-and-play system that produces buffer on-demand for existing equipment,” he says. The company has also taken into consideration the needs of smaller-volume manufacturers and offers a flexible in-line buffer solution with a very small footprint and a capacity of up to 1000 L/hr.
Protein A here to stay
With respect to chromatography advances, improvements in protein A capacities for mAb purification cannot be neglected. “With Protein A, it is really a cost issue,” says Sanderson. There are efforts to make it cheaper. Jöhnck points to Protein A resins launched in the last few years that provide not only higher flow rates of up to 500 cm/h and higher dynamic binding capacities exceeding 50 g/l, but also high cycle stabilities. “Cycle numbers of 200 are achievable, which has dramatically reduced the antibody capture costs per cycle down to $3-5 g of antibody. Therefore, I do not expect any change regarding the dominant role of Protein A resin used in antibody manfacturing,” he observes. Gottschalk adds that alternative technologies to Protein A purification, such as precipitation and extraction, are being investigated, but he doubts they will replace Protein A in the near future.
Membranes an ideal alternative for small-volume processes
Not surprisingly, the use of classical resin-based chromatography is expected to remain dominant for large-scale processing. For smaller volumes, however, Jöhnck notes that membranes can be advantageous, because they are ready-to-use devices (no qualification work) that can handle high flow rates and are potentially lower cost at small scale. The polishing step, according to Gottschalk, is where membrane separations have had a significant impact. “These membranes look like filters and are operated like filters, and they can remove many different impurities. Yet they are very small devices that use approximately 5% of the buffer volume of a traditional column and are disposable, making them very easy to use,” he explains. Bengio adds that such membrane tools can be used either in flowthrough mode to remove DNA or impurities (host cell proteins) or even in capture mode to purify large molecular weights species (e.g., factor VIII, plasmids, viruses, and virus-like particles).
Such membrane filters are just one example of single-use technologies being employed for downstream processing. Others include disposable bags for buffer preparation, single-use devices for clarification, and even prepacked columns and new chromatography systems, according to Jöhnck. “Resin manufacturers are introducing new disposable chromatography columns that are available in different formats up to 32 cm iD or larger,” he notes. “There is a lot of interest in disposable technologies for downstream processing because of the growing need for flexibility and the ability to adapt to changing capacity demands,” agrees Gottschalk. “Single-use technologies won’t be the solution for all issues, but they will be advantageous for many downstream unit operations,” Gottschalk adds.
Another trend in downstream processing is the establishment of more continuous operations. “Continuous processing in many other industrial areas has been the key to dramatically reducing the manufacturing cost of products,” says Jöhnck. For antibody processing, for example, he notes that the combination of a continuous operating capture step using a multi-column Protein A resin approach combined with anionic and cationic columns/devices has already been reported. “These approaches certainly will be introduced in drug manufacturing in the coming years in order to meet the necessary requirements for many of the new materials that are under development. One example in this context is the development of disposable chromatography devices that do not utilize classical membranes or resins, but rather other types of new cost-effective supports, theoretically enabling true single-use chromatography,” he explains.
Bengio agrees that continuous chromatography systems and tools may reduce operational costs due to the use of smaller disposable systems, less chromatography resin, and increases in productivity. “Continuous processing approaches are changing the rules and the definition of a batch, and continuous chromatography streamlines operations and decreases purification costs,” he says.
Exploring other approaches
Reorganization of processes is also being pursued, and may be achieved in advance of true continuous processing, according to Gottschalk. Such reorganization will involve the elimination of the holding steps that typically exist between downstream processing unit operations. The result will be a smaller overall process train that will be compensated for by running the process repeatedly. Such processes may also include alternative technologies, such as simulated moving bed chromatography. Bengio suggests that non-chromatography methods, such as aqueous two-phase separation and crystallization, may allow in some cases for further cost reductions in downstream processing.
Separately, Bengio believes that in-silico and modeling approaches to evaluating mixed-mode chromatography will also be powerful tools for effectively evaluating the complex interactions involved in these types of powerful but sometimes difficult to optimize systems.
About the Author
Cynthia A. Challener is a contributing editor to Pharmaceutical Technology.