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As constant scale up grows out of favor in the biopharmaceutical industry, new-and old-approaches are required. The author reviews the state of downstream processing and considers potential solutions, including the streamlining of full processes and borrowed technologies
This article is an updated version of a previously published article. This version was published in a special supplement to BioPharm International in August 2013.
Photo Credit: Sartorius Stedim Biotech The biopharmaceutical industry is becoming increasingly dependent on innovation and 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 over the past 25 years, and at the same time, batch volumes have increased so that we face the prospect of batch yields exceeding 100 kg of protein in the next decade (2). Over the same period, regulatory demands have become more onerous (3) 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 (4). It is inevitable that 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.
Progress in the industry has been impressive, but most of the increases in productivity 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 because of 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). This is largely due to the incremental nature of technological improvements in downstream processing, which do nothing to address the absence of economy of scale. Unlike upstream production, where a more productive cell line generates more of the product without increasing costs, the costs of upscaling downstream production are linear because a feed stream containing more of the product requires larger amounts of materials such as buffers and chromatography resins (i.e., higher titers), which translates linearly into higher manufacturing costs (6). The future success of downstream processing, therefore, depends on disruptive, game-changing innovations rather than incremental ones (1, 4). This need for innovation reflects the increased demand for biopharmaceutical products, the regulatory focus on quality in the manufacturing process, and the stratification of the market due to the advent of biosimilars or follow-on biologics (3).
Running to stand still
The first 15 years of biomanufacturing can be considered 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 wastage because up to 50% of product batches failed to come up to specifications (3). To address this waste, FDA ordered that processes should be designed with quality attributes taken into account (7, 8). The process was no longer simply a means to an end to generate the product, but became part of the product. As the economic screws began to tighten and demand increased, so manufacturers turned to the age old strategy of scaling up their production to achieve cost savings, and this is where the 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; therefore, increased productivity requires corresponding larger volumes of buffer, larger storage tanks and preparation areas, larger filters, and most importantly larger amounts of chromatography media. For the production of antibodies (i.e., 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 from hundreds of products currently in clinical development, all requiring at least pilot-scale manufacture according to GMPs (9).
How can this productivity dilemma be addressed? With constant scaling up no longer a viable approach, the industry must return to its roots and innovate to succeed. Manufacturers are currently considering three solutions, all inspired in some way by the more encouraging regulatory landscape that rewards rather than punishes innovation. These solutions are 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 bleeding edge of biopharmaceutical research. These latter technologies have the potential to introduce game-changing processing options into an industry still mired in technologies that were state-of-the-art 20 years ago. On a cautionary note, however, technologies from the bleeding edge can fail, and the rash adoption of new and untested technology platforms can punish the eager company seeking innovative solutions. This is the new dilemma in downstream processing.
Streamlining and redesigning an 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 if cost savings can be made through streamlining the process as a whole. The trend towards process streamlining owes a lot to FDA’s quality-by-design (QbD) principles, which themselves derive from the design-of-experiments (DOE) concept. QbD considers experimental design as a landscape with peaks of efficiency and troughs of inefficiency. Similarly, the design space of a manufacturing process is littered with efficiency peaks and troughs, but there is not always a simple path leading upwards 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 now 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 mean that it is 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, and then either cation exchange (CEX) chromatography or hydrophobic interaction chromatography (HIC) in retention mode to remove positively-charged residual contaminants and also 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 aforementioned process, Pfizer explored the design space around the standard process and found that certain modifications could reduce costs considerably without impacting on the quality of the antibody (14). They 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).
Looking with a fresh eye at older technologies
The capacity crunch in downstream processing has been avoided or overcome in other industries by adopting simple and inexpensive technologies (16). In the bulk chemical industry, the conventional pharmaceutical industry, and the 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. Is it possible for this simple approach to be applied also in biopharmaceutical manufacturing?
Several recent developments suggest that simpler technologies could indeed 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 (i.e., size and physical properties), and most challenging of all, a greater proportion of fine particles that escape coarse filtration. A technology that is widely employed in the beverage industry and also in wastewater processing is the use flocculants to link small particles together and create easier-to-remove aggregates. 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.
The beauty of 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 (e.g., varying the temperature or pH, increasing the salt concentration [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 itself 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 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 being considered for use in biopharmaceutical manufacturing is crystallization. This technology 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 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 (26). 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 (27).
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 (28). These slow marginal gains, however, are already beginning to decline and [the industry is] reaching the stage where it is becoming difficult to envisage how sustainable processing can continue without a major injection of downstream processing capacity. One way this can be addressed is to embrace genuinely novel technological approaches that change the rules of the game. Companies that survive on innovation populate the fringes of the biopharmaceutical industry, 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.
Figure 1a: 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. (All figures are courtesy of the author.)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 grassroots change in the industry. Again, many of these changes have affected upstream productivity first (e.g., disposable bioreactors and buffer/media storage bags), but there are examples in downstream processing (e.g., the introduction of simulated moving bed chromatography, expanded bed chromatography, monoliths, and membrane adsorbers) (1, 29). 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, but these are being complemented in more and more processes by disposable membrane adsorbers and innovative combinations that exploit both adsorption and size exclusion as orthogonal separative principles (31, 32).
Figure 1b: 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 cmÂ² 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. Disposable anion-exchange membrane adsorbers are replacing traditional flow-through chromatography steps for polishing, particularly the removal of host-cell proteins, nucleic acids, and viruses, because of their high flow rates compared to packed resins and the absence of cleaning and validation requirements (32-34). 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). These hydrodynamic benefits increase the flow rates and reduce buffer consumption compared to columns, thus shortening the overall process time by up to 100-fold. 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, providing a high frontal surface area to bed height ratio (see Figure 1b). However, a more diverse range of surface chemistries is now available (see Figure 2). Membrane adsorbers, therefore, are also challenging the hegemony of column chromatography in other biomanufacturing steps, such as bind-and-elute capture steps (35), hydrophobic interaction chromatography (36), and even salt-tolerant chromatography in high-conductivity buffers (37), which broadens the polishing window as shown in Table I. Membrane absorbers have been substituted for both flow through and bind-and-elute polishing steps during the manufacture of various commercial products. These devices are also increasingly viewed as ideal for virus clearance because they interact with both large and small, and both enveloped and non-enveloped viruses, and can easily be combined with other concepts such as irradiation with ultraviolet light (UVc) and dead-end filtration (38,39).
Figure 2. Selection guide for convective media, such as membrane adsorbers. HIC is hydrophobic interaction chromatography. STIC is salt tolerant interaction chromatography.The flexibility of disposable modules and their capacity to integrate into any stage of the production process is arguably their most important benefit. 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 (1,4). 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 and to achieve the ideal concept of continuous integrated bioprocessing (40). Continuous integrated bioprocessing has been implemented in upstream production using profusion cultures (4143) and, more recently, in a series of linked downstream operations (4446). Only in the past two years, however, have serious efforts been developed to link upstream and downstream components into a single unified continuous process (40, 47).
What does the future hold?
Table 1. Broader polishing operation window with salt-tolerant membrane chromatography.Innovations that take into account not only the current state of the industry but also future challenges and demands are likely to be the most successful in the long term, but bleeding-edge technologies 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. 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. The future of bioprocessing will require the industry players to embrace the need to change. In the words of US Congressman Bruce Fairchild Barton, “When you are through changing, you are through.”
Uwe Gottschalk, PhD, is vice-president of purification technologies at Sartorius Stedim Biotech GmbH and a member of BioPharm International’s editorial advisory board., firstname.lastname@example.org. This is an updated version of an article previously published in the September 2011 issue of BioPharm International.
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