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).

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 

). 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 

). However, a more diverse range of surface chemistries is now available (see 

). 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).

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  

’s editorial advisory board., uwe.gottschalk@sartorius.com. This is an updated version of an article previously published in the September 2011 issue of 

1. U. Gottschalk, K. Brorson, A.A. Shukla, 

4. U. Gottschalk, K. Brorson, A.A. Shukla, 

Pharmaceutical cGMPs For The 21st Century: A Risk-Based Approach

PAT Guidance for Industry--A Framework for Innovative Pharmaceutical Development, Manufacturing and Quality Assurance

13. S. Vunnum, G. Vedantham, B. Hubbard, “Protein A-based affinity chromatography,” In Gottschalk U (ed) 

15. J. Glynn et al., Development of a mAb harvest protocol. 

Biochemical Engineering XV: Engineering Biology from Biomolecules to Complex Systems

17. J. Thömmes and U. Gottschalk “Alternatives to packed-bed chromatography for antibody extraction and purification,” In: U. Gottschalk U (ed) 

18. A.A. Shulka, J.R. Kandula, “Harvest and recovery of monoclonal antibodies: cell removal and clarification,” In U. Gottschalk (ed) 

19. R. Shpritzer et al., “Calcium phosphate flocculation of antibody-producing mammalian cells at pilot scale,” 232nd ACS National Meeting, San Francisco CA, September 10-14, 2006.

20. J. Glynn, “Process scale precipitation of impurities in mammalian cell culture broth,” In U Gottschalk (ed), 

21. T. Przybycien, S. Narahari, L. Steele, 

23. M. Page, R. Thorpe, “Purification of IgG by precipitation with sodium sulfate or ammonium sulfate,” In: J.M. Walker (ed), 

, second edition. Humana Press, Inc., Totowa, NJ pp 983-984 (2002).

27. J. Peters, T. Minuth, and W. Schröder, 

Articles

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

The Future of Downstream Processing-2013

This article appears in a special supplement, A Renaissance in Biomanufacturing:The Art of Purification, published in August 2013.


 What are the challenges to developing a reliable, robust means of performing protein purification in a single-use unit? What properties would a single-use product for protein purification need to be economically viable?


: Industry is changing to a ‘market-pull’ scenario, mainly due to regulatory pressure to pro-actively provide best practice. Single-use manufacturing adds value in certain downstream unit operations. While such practice has never been questioned for steps such as virus or sterile filtration, we are in the middle of that shift in chromatography and X-Flow filtration. Although it can be demonstrated that single-use strategies provide better process economies, their main advantages stem from factors such as accelerated development timelines and risk mitigation.


: What recent developments in membrane adsorbers could lead to single-use technology for protein purification? Could membrane adsorbers replace packed-bed column chromatography?


: Membrane adsorbers offer two main advantages compared to packed-bed chromatography: the fluid dynamics of a convective media that can process large feed-stream volumes with extremely high flow rates, and large pore sizes that provide accessability and thus high dynamic binding capacities for large molecules such as DNA and viruses. 
As a result, single-use membrane chromatography devices are typically much smaller in size and require only about 5% of the original buffer volume. The sweet spot for membrane chromatography is related to these two stand-alone features and it shines in areas like contaminant removal (polishing in flow-through mode) and purification of viral vaccines. In these applications they start dominating the industry’s development platforms and will take over from resins completely. Recent developments include salt-tolerant chemistries on membranes that bind viruses under physiological conditions (no in-process dilution requirements).

: What technologies in development could make protein chromatography a continuous process?


: In general, continuous processing offers the advantage of higher productivities, from a smaller footprint to an advantageous process economy and chromatography. Technologies such as simulated moving-bed chromatography have the potential to decrease column sizes because they use the total binding capacity as well as the overall lifetime of the chromatography medium. In this setup, a single-use design is possible if, for example, the same sample of medium is recycled within the purification of just one batch of product. Although this scenario would cut costs during clinical manufacturing, it is probably less beneficial in routine manufacturing and questions of scale up remain.

Uwe Gottschalk, vice-president of purification technologies, Sartorius Stedim Biotech, discusses specific challenges in protein purification.

Protein Purification Using Single-Use Technology

The Renaissance sculptor Michelangelo described his work by taking blocks of marble and freeing a form already contained within by removing all that did not belong in the masterpiece. The same principles apply in downstream processing, where holistic concepts are used to remove everything that does not belong in the drug substance, and it helps to reframe this task by focusing on the contaminants rather than the product. 

The Renaissance was a time of great innovation in Europe, a time when new ideas spread and took hold, and accepted paradigms were challenged. In the biomanufacturing industry, innovation is required. We need to adapt to a changing commercial environment shaped by diverse forces including the demands of the market, which try to drive down costs; the emergence of new industry players, particularly those seeking to profit from generics; and, of course, the regulatory authorities, which apply ever more stringent quality requirements to ensure patient safety. As the pincers close around us, we need to innovate to move forward, to take advantage of new technologies, processes, and strategies. We need to negotiate the changing landscape of our industry by thinking smarter and using new ideas to create new opportunities.

In seeking ways to improve our fortune, it is tempting to reach for the low-hanging fruit and thus make small and incremental improvements to our processes. But most of the easy pickings are already gone. We must look further than before and shine the light of change into every corner of biomanufacturing, no matter how well established and safe we feel the paradigms may be. Perhaps the best example to illustrate this change is polishing, the final phase of downstream processing, which is essential to remove impurities, and contaminants, and especially for the clearance of pathogenic agents such as endogenous and adventitious viruses. 

The 
        Art 
        of Purification

A New Scale-Down Membrane Adsorber Device for Process Development and Validation"

Polishing Complex Therapeutic Proteins: A New Downstream Purification Platform Using a Salt-Tolerant Membrane Adsorber"

Development of an Alternative Monoclonal Antibody Polishing Step

Virus Filtration Using a High-Throughput Parvovirus-Retentive Membrane

Today, technology platforms are implemented early on in process development, and at least two orthogonal steps are assigned for the robust and efficient clearance of impurities and contaminants, ensuring that manufacturing processes meet the specifications established during validation. We need to harness the power of innovation to improve the efficiency and robustness of polishing without driving up the cost of goods.

Recent innovations in polishing that have started to address these goals include the use of membrane chromatography that can target all critical contaminants under a wide range of process conditions, but boost productivity at the same time; and the development of novel dead-end filters with tapered pores to increase virus retention without blocking. These developments come with new, integrated and (if appropriate) single-use concepts that meet the requirements of modern process trains.

 collects together a number of ground-breaking articles by authors at the cutting edge of downstream processing, focusing on polishing and virus removal. The supplement begins with a report by Nathalie Frau et al. on the development of a new scale-down device for polishing and virus removal. This is complemented by an article by Min Lin and colleagues describing the development of a salt-tolerant membrane adsorber that can be used for polishing during the production of complex proteins in high-conductivity feed streams. Another report by Yun (Kenneth) Kang and colleagues outlines a novel approach to antibody polishing involving a combination of high-throughput screening and design of experiments optimization to establish a salt-tolerant interaction chromatography step for the efficient clearance of impurities from four antibodies under high-conductivity conditions. The final article by Thom et al. highlights the exciting development of a new high-throughput parvovirus-retentive membrane with an optional adsorptive pre-filter as an integrated concept. These research articles are followed by a broader outlook discussing the future of downstream processing to demonstrate the need for continuous innovation in an industry that is running to stand still. The supplement closes with a short interview about the options of single-use technology in protein purification.

Fortune favors the brave, and thus to ensure future progress, we need to prepare courageously for a commercial environment where change and adaptation is a way of life. We need to strive to be better, but also to be smarter. Like the Renaissance masters that came before, we must not resist this season of change but instead we must embrace it!

The industry needs to embrace innovation to improve the efficiency and robustness of biopharmaceutical purification without driving up the cost of goods.

A Renaissance in Biomanufacturing: The Art of Purification

Photo courtesy of Sartorius Stedim BiotechAll biotechnology products derived from animal sources carry a risk of contamination with viruses, including those endogenous to the source material, such as retroviruses (1) and those introduced adventitiously during manufacturing by personnel or contaminated raw materials. Viruses in biopharmaceutical products could potentially be transmitted to patients with dire consequences, particularly if the patient is immunocompromised (2). However, no such events have been reported in the context of recombinant proteins produced by fermentation because of the rigorous safety standards applied during manufacturing, including dedicated steps for virus removal and/or inactivation and a program of tests to ensure these steps are efficient, based on the guidelines set out in ICH Q5A (2).

Current regulatory guidelines require at least two orthogonal steps for the inactivation and/or removal of viruses, thus different principles of separation/inactivation must be used in each method (2,3). Because viruses vary in size, charge, and the presence or absence of an envelope, the available methods differ in their effectiveness against particular types of virus; these factors must be integrated into the design space during the development of a process ahead of Phase I clinical trials. Manufacturers are also expected to deliver a virus clearance strategy that has been optimized for each product because biopharmaceuticals are often large, complex proteins that resemble smaller viruses in their physical and chemical properties. Virus clearance strategies must, therefore, be tailored to avoid product loss. The virus removal steps must then be tested against at least two model viruses representing those most likely to be present in the process stream (2-3) and should include an endogenous virus if relevant to the process. Ideally, an adventitious virus such as minute virus of mice (MVM) or porcine parvovirus (PPV) should also be tested, because these are the gold standards for size-dependent clearance steps using 20-nm filters (2-4). Removal/inactivation is usually demonstrated in spiking studies, where specific viruses are added deliberately to the process stream ahead of the relevant unit operations. Before Phase III clinical trials can be authorized, two further viruses must be tested if specific contaminants are likely in the process stream. The two testing schemes have different aims: general virus clearance is designed to test process robustness, whereas specific virus clearance using anticipated contaminants aims to ensure product safety (4).

Although virus clearance should be built into the design space on a product-by-product basis, several robust and effective strategies have become established in the industry (5-6). Appropriate methods for virus inactivation include heating/pasteurization or solvent/detergent treatments (7), although these may also have a significant negative impact on some recombinant proteins and are only effective against enveloped viruses. More often, a low-pH hold (8) is used for enveloped viruses if this is compatible with the buffer conditions in the process (e.g., during the production of monoclonal antibodies); exposure to ultraviolet light in the UVC range is used to inactivate all viruses by cross-linking the nucleic acids at 254 nm (9). Virus removal methods physically separate virus particles from the feed stream, and the most suitable methods are chromatography, where virus particles are captured by adsorption (10), and retentive filtration using 20-nm filters, which eliminates even the smallest viruses by size exclusion (2,4). However, there are currently no common standards for virus filtration. Instead, it is left to manufacturers to show, on a case-by-case basis, that their virus clearance steps are acceptable and efficient.

Figure 1a: Hollow fiber cross section. (All figures courtesy of the authors.)To overcome current limitations in virus filtration—and as a step toward the development of common standards—a high-performance, hollow-fiber parvovirus filter has been developed, which demonstrates robust retention at high transmembrane pressures. The unique structure of the membrane and its chemically modified surface address many of the limitations of current retentive filters.

Figure 1b: Comparison of a 0.8/2.4-mÂ² process module and a 5-cmÂ²  laboratory module with vent filter for contained flushing.The Virosart HF filter (Sartorius-Stedim) features a surface-modified asymmetric polyethersulfone (PES) hollow-fiber membrane optimized for the manufacture of monoclonal antibodies (see 

). The membrane is characterized by a funnel-like pore size gradient designed to achieve the robust retention of parvoviruses under challenging conditions (such as high blockage or pressure release) without impeding the efficient transfer of high-molecular-weight proteins such as monoclonal antibodies. The membrane is surface-modified with a hydrogel-forming, low-binding polymer, to reduce the adsorption of soluble proteins and protein aggregates. The pore size gradient and the hydrogel are unique aspects of the membrane that contribute to its high performance. The hollow fibers can be packed densely into modules ranging in capacity from 5 cm² to 2.4 m², the latter presented as a presterilized 10-in single-use device (see Figure 1b). The capacity of the filter can be extended by combining it with the Virosart MAX adsorptive pre-filter, featuring an optimized polyamide microfiltration flat-sheet membrane in a homogeneous triple-layer configuration, with a nominal pore size of 0.1 µm.

Figure 2: Water flow rate in Virosart HF capsules taken from three individual lots of the 2.4-mÂ² module. Each capsule lot was built from a different membrane lot. The average permeability is 170 liter/mÂ²h bar.Validation of Virosart HF has proven the consistent performance of the product family. 

 shows selected validation data (permeability of 2.4-m² process modules) for illustration. However, measures have been taken to ensure future product quality from lot-to-lot. Validated in-process as well as release tests are performed during the manufacture and release of Virosart HF membranes and modules according to pre-defined sampling plans to measure and monitor critical performance attributes of all product components.

Membrane testing includes in-process and lot release tests. Membrane performance release tests are executed on laboratory modules that have experienced the same manufacturing steps as laboratory or process modules that would be shipped to customers. Virosart HF modules are in-house integrity tested by air-diffusion as well as gamma irradiated. Three membrane release tests—bacteriophage PP7 retention in buffer, bacteriophage PP7 retention in human IgG (grab sample at 75% flux decay), and water permeability—are consequently performed on lab modules, which have also been flushed with water, dried, and then exposed to gamma irradiation. Protein filtration capacity is monitored while PP7 retention in buffered human IgG solution is determined. These release tests ensure that membrane performance items meet expected and validated levels. 

In addition, Virosart HF modules are released based on a 100% inspection scheme. Water flow rate and integrity of each module is tested prior to shipment. Integrity testing is based on an air-diffusion test at 4.5 bar and modules subsequently released based on a correlation between diffusive flow rate and PP7 retention.

Figure 3: The performance of the Virosart HF module family was tested for scalability using the same batch of a buffered human lgG model protein stream (highly blocking) until 95% flux decay was achieved.The performance of the Virosart HF module family was tested for scalability using the same batch of a buffered human IgG at 2 g/L. Three different 5-cm² laboratory modules and one 0.8-m² process module were challenged at 2 bar differential pressure until 95% flux decay was achieved (see 

). The volume vs. time filtration data for the three laboratory modules was averaged and compared to the corresponding data for the process module according to the filtered protein mass per filtration area. Figure 3 confirms that (based on the performance data gathered using 5-cm² devices) laboratory modules can be scaled-up to larger feed stream volumes and filter areas.

Figure 4: Virosart HF laboratory modules were challenged with a 10-20-g/L monoclonal antibody solution (pH 6-7, conductivity 4-8 mS/cm) spiked with 0.5% MMV. Experiments were carried out at constant flux at 120 L/m2h. The membrane was challenged with up to 7.4-kg antibody/m2 resulting in a permeability decay of more than 70%. A log reduction value of greater than 5 was achieved in both spike trials (Run B and Run C).The retentive capabilities of the Virosart HF filter were tested under worst-case conditions, by challenging with a 10-20-g/L monoclonal antibody solution (pH 6-7, conductivity 4-8 mS/cm) spiked with 0.5% MMV. Experiments were carried out at constant flux at 120 L/m²h. To implement worst-case load conditions, the membrane was challenged with more than 5.5-kg antibody/m² resulting in a permeability decay of more than 70%. Two spike trials and one control trial were conducted for comparison.

In all three trials, the transmembrane pressure increased over time but the pressure profiles varied slightly from run to run with the mass throughput ranging from 5.8 to 7.4 kg/m² (see 

). The transmembrane pressure did not increase above 2.7 bar in any of the trials and thus remained below operating pressure. Breakthrough was observed in one of the spike trials but the other achieved complete retention. The log reduction values for the pooled permeate were 5.19 and 5.21, respectively (see 

Table I: Summary of the filtration and MMV retention data for the control trial (Run A) and the two spike trials (Run B and Run C).These data show that Virosart HF achieves robust log reduction values of greater than 5 even under challenging conditions, thus meeting the retentive requirements of a high-performance parvovirus filter with minimal lot-to-lot variability.

Figure 5: PPV retention in a human lgG model protein feed stream at different degrees of flux decay, during post wash and in the final filtrate and wash pool executed at two different spike levels.The Virosart HF was next tested with a feed stream comprising PPV spiked buffered human IgG solution at a IgG concentration of 0.1 g/L. Two spike trials were carried out with different amounts of virus, the first containing 5x105 pfu/mL and the second 5x106 pfu/mL. The log reduction values were determined using permeate grab fractions taken at 25% and 90% flux decay. The log reduction value was also determined in the post-wash fraction and the overall filtrate and wash pool. As shown in 

, Virosart HF achieved robust log reduction values for human viruses regardless of the extent of blockage. The log reduction value for PPV was also high in the post-wash fraction, resulting in an overall pool log reduction value >6 in both tests.

Figure 6: Analysis of the recovery flush of a 0.8-mÂ² Virosart HF process module. The data shows that, already after 3 liter flushing per mÂ² membrane area, 99% of protein is recovered.Finally, the flush volume required to achieve the recovery of the target protein was determined by monitoring the permeate stream using an in-line UV-detector at 280 nm (see 

). A 0.8-m² Virosart HF module was challenged with a 2-g/L buffered human IgG model solution until ~40% flow decay was observed, and then the module was flushed with buffer at 2 bar differential pressure. The protein concentration in the permeate stream was calculated from the UV measurement based on a previously determined calibration curve. Flushing the membrane with 3 L/m² achieved 99% protein recovery. 

The Virosart HF is a novel, high-performance parvovirus filter based on a surface-modified asymmetric PES hollow fiber membrane. Rigorous testing confirmed its consistently robust retentive properties and high filtration capacity under challenging conditions, including an inlet pressure of up to 5 bar, up to 90% blockage, loading of up to 7.4-kg antibody/m² and spiking with 0.5% PPV. The hollow fiber membrane can be packed densely into scalable modules with a small footprint ideal for single-use campaigns.

The authors would like to thank Amit Mehta, Alexander Seay, and John Salvador from Genentech, a member of the Roche Group, for their contributions and support of this development. 

Susanne Roederstein is director purification technologies Europe and Volkmar Thom is director precipitation technologies, Sartorius Stedim Biotech GmbH, August Spindler Str. 11, 37079 Göttingen, Germany.

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