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The author reviews the state of downstream processing and considers potential solutions, including the streamlining of full processes and borrowed technologies.
The biopharmaceutical industry relies on change to make progress in a commercial environment that simultaneously demands higher productivity, higher quality, and lower costs (1). Recombinant protein titers have improved from tens of milligrams to more than 10 grams per liter during the past 20 years, and at the same time, batch volumes have increased so that industry faces the real prospect of batch yields exceeding 100 kg of protein in the next decade (2). Over the same period, regulatory demands have become more onerous, and the pressure to reduce costs has increased as more biopharmaceuticals come off patent and overseas manufacturers begin to take an interest in Western markets (3, 4). Indeed, the unthinkable is increasingly becoming inevitable—biopharmaceuticals will at some point be regarded as commodities, and manufacturing on the ton scale will be necessary for certain products that are required in large, repetitive doses, such as topical antibody formulations.
There is no doubt that progress in the industry has been impressive, but the polished bodywork of bioprocessing hides an engine of despair that groans under the strain of current demands. Most of the increases in productivity that have been achieved in previous decades have resulted from improvements in the upstream production phase, with more efficient bioreactors and better media formulations sharing the limelight with cell lines that are intrinsically more productive due to the development of more effective screening technologies to identify the most productive clones (5). Downstream processing is now routinely found to be the bottleneck in biopharmaceutical manufacturing because its capacity has not kept pace with upstream production (1). In some cases, the lack of downstream processing capacity can seriously affect the profitability of a new pharmaceutical product and even result in its failure. Manufacturers facing shrinking margins and less bountiful product pipelines are therefore looking for opportunities to increase the efficiency of downstream processing without inflating the cost of goods sold.
SARTORIUS STEDIM BIOTECH
Running to stand still
Perhaps the major challenge facing the biomanufacturing industry today is that downstream processing has no economy of scale (i.e., higher titers translate linearly into higher manufacturing costs) (6). Looking back, we can almost consider the first 15 years of biomanufacturing as a golden era, where manufacturers had the luxury of using inefficient processes because the product itself was far more important (3). Most biopharmaceuticals were required in small doses, and demand was sufficiently low to allow plenty of slack in the system. It was also pointless investing in process efficiency when any tweaks and modifications would arouse the suspicious eye of regulators. It was better to let sleeping dogs lie and be satisfied with the status quo. In this environment, innovation was considered a burden rather than a bonus.
Inevitably, this relaxed attitude to process efficiency resulted in an immense amount of waste because up to 50% of product batches failed to meet specifications (3). To address this, FDA ordered that processes be designed with quality attributes taken into account (7, 8). The process was no longer a means to an end to achieve the product, but rather the process became part of the product. As the economic screws began to tighten and demand increased, manufacturers turned to the age-old strategy of scaling up their production to achieve cost savings. It was at this time that industry began to flounder.
Whereas upstream production can be scaled up almost indefinitely by increasing the productivity of cells growing in a bioreactor, downstream processing has limits imposed by physics and chemistry. Downstream processing is driven by the mass of product. If one makes more product per batch, one needs correspondingly larger volumes of buffer, larger storage tanks, preparation areas, larger filters, and most importantly, larger amounts of chromatography media. For the production of antibodies (where Protein A resin is typically used in the primary capture step), the costs of scaling up are in some cases greater than the extra revenue made possible by the increased upstream productivity. Manufacturers find themselves in the paradoxical situation that there is no longer an economy scale in manufacturing, but rather an economic depression reflecting the physical limits that constrain the size of the apparatus used in separations (e.g. chromatography columns and the associated piping, skids, and buffer reservoirs). So far, the extra demand has been absorbed by contract manufacturers offering their spare capacity to fulfill quotas, but this is a short-term measure that cannot cope with the predicted increases in demand as industry faces hundreds of products in clinical development, all requiring at least pilot-scale manufacture according to GMP (9).
How can this productivity dilemma be addressed? With constant scaling up no longer a viable approach, the sector must return to its roots and innovate to succeed. Three solutions are currently being considered by manufacturers, all inspired in some way by the more encouraging regulatory landscape that rewards rather than punishes innovation. This article examines several issues: the streamlining of existing processes, the revisiting of simple technology solutions currently employed in the bulk-chemical industry, and the use of innovative technologies from the biopharmaceutical research sector. These innovative technologies have the potential to introduce game-changing processing options into an industry still mired in technologies that were the state of the art 20 years ago. On a cautionary note, however, technologies from the research sector can fail, and the rash adoption of new and untested technology platforms can punish an eager company seeking innovative solutions. This is the new dilemma in downstream processing.
Streamlining and redesigning existing manufacturing processes
Many processes for biopharmaceutical manufacturing were designed at a time where process efficiency was considered unimportant (3). More recently, manufacturers have sought to increase the efficiency of each unit operation, but they are only now starting to consider redesigning the entire process train to see whether cost savings can be made. The trend toward process streamlining owes a lot to FDA's quality-by-design (QbD) principles, which are derived from the design-of-experiments (DOE) concept. The design space of a manufacturing process is littered with efficiency peaks and troughs, but there is not always a simple path leading to the most efficient process. Therefore, process design incorporating efficiency and quality from first principles involves going back to the drawing board and evaluating the critical attributes that contribute to an efficient process.
Most companies are applying these principles and actively streamlining their processing strategies wherever possible. Antibodies take center stage because they represent more than half of all biopharmaceutical products in development, and their common properties make it possible for companies to share process efficiency data that are applicable across platforms (10, 11). It is for this reason that antibody manufacturing has benefitted from the development of so-called generic platform processes, which are broadly similar for all antibodies but can be tweaked to match the specific properties of individual products (12).
Antibody manufacturing provides an excellent example of the application of process redesign and streamlining principles to increase productivity, cut costs, and maintain product quality. Most manufacturers use three chromatography steps for antibody purification, starting with a very expensive Protein A capture step that is placed immediately after clarification, followed by anion exchange (AEX) chromatography in flow-through mode to extract negatively charged contaminants, such as host-cell protein (HCP), endotoxins, host DNA, and leached Protein A. Then, either cation exchange (CEX) chromatography or hydrophobic interaction chromatography (HIC) in retention mode is used to remove positively charged residual contaminants and product related impurities, such as aggregates and degradation products (13). Modern platform processes also serve as orthogonal strategies for virus removal.
Realizing that no further cost savings could be gained by scaling up the above process, Pfizer explored the design space around the standard process and found that certain modifications could reduce costs considerably without affecting the quality of the antibody (14). The company introduced two types of process modifications, one in which the order of the polishing steps was reversed and another in which different separation technologies were used to increase process capacity (i.e., using membrane absorbers for the flow-through chromatography step and replacing the depth filtration step with continuous centrifugation) (15). These changes increased the efficiency of purification to such an extent that, for some antibody products, the cation-exchange step became unnecessary reducing the process from three columns to two columns or even a single column. Not only did this save the direct costs of column resin and buffers, but also reduced the process time by >45% which doubled the productivity in terms of batch processing (14).
Eyeing simpler technologies
The capacity crunch in downstream processing has been avoided or overcome in other industries by adopting simple and inexpensive technologies (16). In bulk-chemical, conventional pharmaceutical and food and detergent industries, expensive processing solutions, such as chromatography, would never be considered because the costs of implementation would not be sustainable in these high-volume, low-margin processes. This simple approach could be applied to biopharmaceutical manufacturing.
Several recent developments suggest that simpler technologies could find a niche in biopharmaceutical manufacturing, particularly in the early processing steps where the complex mixture of particulates and solutes have the most potential to foul expensive membranes and resins (16, 17). Tangential flow microfiltration, depth filtration, and (continuous) centrifugation are the current methods of choice for the clarification of the feed stream, and one or more of these processes may be employed in series to remove larger particulates until finally a polishing depth filter or dead-end filter can be used to remove fines and thus reduce feed stream turbidity (18). Efficient and inexpensive clarification becomes more challenging with higher-titer cell culture processes because these are characterized by a greater cell density and often a longer process time, resulting in a higher solids content, more particle diversity (size and physical properties) and—most challenging of all—a greater proportion of fine particles that escape coarse filtration. A technology that is widely used in the beverage industry and in wastewater processing is the use flocculants to link small particles together and create aggregates that are easier to remove. Flocculation is achieved using polymers that bind simultaneously to the surfaces of several particles through electrostatic interactions, creating larger particles that may sink under gravity, or may be removed more easily by centrifugation or filtration.
In the bioprocessing industry, flocculation has been used to help remove whole cells from fermentation broth, but more recently it has also been used to remove fine cell debris and proteins. A simple and inexpensive strategy recently applied in antibody manufacturing is the creation of a calcium phosphate precipitate by adding calcium chloride to a final concentration of 30 mM and then potassium phosphate to a final concentration of 20 mM. Precipitation traps cell debris in larger particles, allowing removal by centrifugation for 10 min at 340 X g and yields a clear supernatant with the recovery of ~95% of the antibody (19). Interestingly, this strategy also removes some soluble host cell proteins and nucleic acids. Flocculation is that it does not introduce any additional impurities to the feed stream because the flocculant is removed along with the aggregated particles.
Precipitation is widely used as a purification approach in the bulk-chemical industry, and given that precipitation can be induced by simple changes in the environment, such as varying the temperature or pH, increasing the salt concentration (i.e., salting out) or adding organic solvents, it should be easy to apply the same principles in bioprocessing (20). Precipitation has therefore been used to remove soluble impurities from the feed stream during antibody manufacturing, and these solids can then be trapped by filtration or pelleted by centrifugation leaving a clear feed stream relatively enriched for the target protein (20). In an innovative adaptation of this approach, the antibody can be precipitated under mild conditions and recovered from a collected pellet thus removing many contaminants in a single step (21). This is possible because the mild precipitation conditions allow the protein to be redissolved without loss of activity. Several groups have developed methods to precipitate antibodies in large-scale processes, and this method could replace Protein A chromatography in the long term (22, 23). Precipitation methods using n-octanoic acid are used for the removal of contaminants in at least two industrial antibody manufacturing processes (24, 25).
In the final purification steps, another traditional technology that is being considered for use in biopharmaceutical manufacturing is crystallization. Crystallization involves the separation of a solute from a supersaturated solution by encouraging the growth of crystals. The crystallization process involves the formation of a regularly structured solid phase, which impedes the incorporation of contaminants or solvent molecules, and therefore yields products of exceptional purity (26). It is this purity which makes crystallization particularly suitable for the preparation of pharmaceutical proteins, coupled with the realization that protein crystals enhance protein stability and provide a useful vehicle for drug delivery (27). Protein crystallization has been developed into a commercial technology for drug stabilization and delivery, and several current manufacturing processes involve crystallization including the production of recombinant insulin, aprotinin, and Apo2L (28).
Although process redesigns and traditional technologies can contribute to the development of downstream processes, they provide only incremental improvements that marginally increase process efficiency. Incremental or evolutionary technologies have been the mainstay of the bioprocessing industry for the past 20 years, and column chromatography provides one of the best examples of this phenomenon in action (29). However, these slow marginal gains are already beginning to decline, and it is becoming difficult to envisage how sustainable processing can continue without a major injection of downstream-processing capacity.
One way to address this concern is to embrace genuinely novel technological approaches that change the rules of the game. The fringes of the biopharmaceutical industry are populated by companies that survive on innovation, and some of these innovations are disruptive in the sense that their influence on the industry is unpredictable and could contribute to a radical change in bioprocessing.
Most technological innovations in bioprocessing have been incremental, but there are several recent examples of disruptive innovations that have challenged the established business model and caused real grass-roots change in the industry. Again, many of these changes have affected upstream productivity first (e.g., disposable bioreactors and buffer/media storage bags), but we are also seeing examples in downstream processing (e.g., the introduction of simulated moving bed chromatography, expanded bed chromatography, monoliths, and membrane adsorbers) (1). These innovations have taken hold in niche markets but are now beginning to adopt mainstream positions. Disposable modules for downstream processing occupy a more mature status in the development cycle (30). The use of disposable filter modules is now an industry standard and where filters first left their footprint, membrane adsorbers are set to follow (31).
Membrane technology. Disposable membranes adsorbers are beginning to replace traditional chromatography in a number of settings, just as disposable membrane filters replaced steel mesh filters. Indeed, membrane filters have evolved into charged filtration devices that use the principles of both sieving and chemical selection to improve filtration performance, thereby creating a precedent for the use of membrane chromatography in downstream processing. After a period of inertia, the benefits of membrane chromatography are now fairly well established, and manufacturers are willing to consider them as a genuine alternative to fixed columns rather than a step in the dark (32, 33). In contrast to resin-based flow through processes, membrane chromatography involves the use of thin, synthetic microporous or macroporous membranes stacked in layers within a disposable cartridge (34). The footprint of such devices is much smaller than columns with a similar output. A range of membranes is available with functional groups equivalent to the corresponding resins (e.g., membranes containing activated quaternary ammonium groups for anion exchange, or phenyl groups for hydrophobic interaction chromatography [HIC]), and a relatively new variant also allows salt tolerant interaction chromatography (STIC) in high-salt buffers (35, 36). The availability of STIC membranes is an important and innovative advance in biomanufacturing because even the most recent generation of membrane adsorbers fall short of some manufacturing requirements when challenged with the high-conductivity feed streams often produced in high-titer processes. STIC ensures more flexibility in process design and improves the clearance of host cell proteins and viruses in buffers containing high concentrations of salt (see Table I). These new adsorbers therefore allow polishing to be carried out at higher load densities without an interstitial dilution step after product capture, reducing process time and circumventing the need for additional buffer preparation and holding.
Table I: Broader polishing operation window with salt-tolerant membrane chromatography.
Table I demonstrates that Sartobind STIC provides higher binding capacities for BSA, DNA, and model viruses compared with a Q anion exchanger under high salt conditions (150 mM NaCl), thereby increasing the design space for polishing operating conditions.
In a recent example described by the Italian biopharmaceutical company Philogen, membrane adsorbers were substituted for the flow through and bind-and-elute steps for the polishing of a new monoclonal antibody fusion protein in Phase I–II clinical development, achieving 90% recovery and 99.9% purity (37). The performance benefits of membranes provide value for the user, but the complete elimination of cleaning and validation requirements is often cited as the major advantage because this avoids the costs of the chemicals, personnel, and record-keeping, and more importantly avoids the inevitable process down time while cleaning takes place. Spent modules are simply replaced with prevalidated new ones, available in a range of sizes and configurations for maximum flexibility (32).
The performance advantage of membranes over resins reflects the transport of solutes to their binding sites mainly by convection, while pore diffusion is minimal (see Figure 1a). Because of these hydrodynamic benefits, membrane adsorbers can operate at much greater flow rates than columns, thereby considerably reducing buffer consumption and shortening the overall process time by up to 100-fold. The use of membrane adsorbers can be viewed as the equivalent of shortening traditional columns to near zero length to allow large-scale processes to run with only a small pressure drop at high flow rates. For example, polishing with an anion exchange membrane can be conducted with a bed height of 4 mm at flow rates of more than 600 cm/h, and provide a high frontal surface area-to-bed height ratio (see Figure 1b). Small-volume disposable membrane chromatography devices can now handle up to 50 L /min/bar/m2. Even at these high flow rates, the membrane pores provide adequate binding capacity for large molecules such as viruses and DNA, so they can play an important role in the overall viral clearance strategy for antibody purification (38, 39).
Figure 1: (a) Mechanistic comparison of solute transport in bead resins (left) and membrane adsorbers (right), where thicker arrows represent bulk convection, thinner arrows represent film diffusion and curved arrows represent pore diffusion. (b) Comparison of bed height in columns (left) and membrane adsorbers (right). Using membrane adsorbers is functionally equivalent to shortening columns to near-zero length, resulting in a similarly small pressure drop that allows extremely high flow rates, thereby reducing overall process times up to a 100-fold. In this example, both formats have a 1350 cm2 frontal surface; the column has a bed height of 15 cm; and the membrane adsorber has a bed height of 0.4 cm. The height to frontal surface ratio is approximately 100 for the column and nearer to 3500 for the membrane device. (ALL FIGURES ARE COURTESY OF THE AUTHOR)
Disposable modules. The flexibility of disposable modules is arguably the most important benefit in the context of the whole process, and this reflects the broad industry perspective that manufacturing flexibility is now perhaps at least as important as capacity considering the large numbers of products in clinical development. Process development can be streamlined and expedited because different modules can be tested in various combinations to arrive quickly at the best overall set of process options, and the absence of cleaning and validation requirements can shorten the time required to develop a finalized process by months or years. The ability to replace each module completely also makes it easier to assemble process trains for new products in existing premises without cross-contamination. The flexibility is most noticeable during scale-up because disposable devices are generally modular and available in various sizes, and scaling up simply involves swapping one module for another with a higher capacity. It is thus apparent that membrane devices can be scaled up with none of the attendant disadvantages of column resins, thereby making the goal of polishing 100-kg batches of antibody entirely possible without oversizing.
Figure 2: Selection guide for convective media, such as membrane adsorbers. HIC is hydrophobic interaction chromatography. STIC is salt tolerant interaction chromatography.
Innovations that take into account the current state of the industry as well as potential challenges and demands are likely to be the most successful in the long term. At the same time, technologies borrowed from the edge of research always come with risks that must be evaluated by manufacturers looking at major investments into capacity. The perceived bottleneck in downstream processing can be addressed with lower-risk approaches, such as streamlining current production processes, with moderate-risk approaches, such as introducing technologies that have already proven suitable in other industry settings, or with higher-risk approaches involving the incorporation of novel technologies.
In several cases, these novel technologies have already proven their credentials in several processes and companies following the paths set by the first adopters, the trailblazers of the industry, can be assured that the technologies involved now have established their credibility.
The future of biomanufacturing is likely to rely more on innovation and flexibility than on traditional strengths, such as large facilities and the financial muscle to invest in them. Disposable manufacturing is likely to play an increasingly important role as companies maneuver in a crowded market to protect their R&D investments while more and more generics become available. The ability to scale up or down quickly, to switch to new campaigns rapidly and to produce multiple products in the same facility will be a key metric of success. Ultimately, the future of bioprocessing will require industry players to embrace the need to change.
Uwe Gottschalk, PhD, is vice-president of purification technologies at Sartorius Stedim Biotech GmbH, email@example.com.
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