Recent estimates place the value of the biopharmaceutical market upwards of $75 billion with a predicted annual growth of 12 per cent. Escalating production of biotherapeutic molecules is fuelling a requirement for innovative filtration and separation solutions. In particular, processes such as the clarification and purification of viral vaccines, recombinant proteins and monoclonal antibodies expressed by yeast, bacterial and mammalian cells, demand new technologies if they are to operate successfully on a commercial scale. New technologies will have to overcome the requirement for the extensive sterilisation and cleaning validation that is associated with non-disposable hardware. The reasons for this will be explained throughout the article.
Challenges
In the highly competitive biotechnology arena, manufacturing processes have to be high throughput, high yielding and highly efficient to maximise the potential from existing facilities. Devices that can improve yield, reduce the risk of batch-to-batch contamination and, therefore, reduce the time and costs associated with meeting Good Manufacturing Practice (GMP) regulations on cleaning validation are eagerly received.
The challenge for commercial-scale filtration is that feedstocks containing the target protein are derived from cell culture, which means inherent variation in product content and composition. Achieving the high level of purity required for large scale manufacture of biopharmaceuticals demands highly selective and robust technologies.
Fermentation capacity poses a major challenge, too. In the past, this was frequently the rate limiting step in the production of biotherapeutic molecules, especially when manufacturing monoclonal antibodies (MAbs). With MAb production accounting for a significant proportion of the total market value, the industry has responded by developing new and novel expression systems, increasing cell densities and optimising growth media – the net result being MAb fermentation yields in excess of 5g/L. This in turn creates a greater challenge in terms of separating product from the host cell.
There is also the issue of process validation, as any source of variation that can affect or contribute to changes in the product needs to be identified, monitored and controlled during process development [1]. Critical variables should be defined and fixed at Phase II or Phase III of process development.
Historically, re-usable stainless steel equipment would have been implemented throughout the fermentation, clarification and purification processes. This generates a need to validate that clean-in-place (CIP) and steam-in-place (SIP) procedures are effective at contamination removal and that batch-to-batch carryover is eliminated.
Contract Manufacturing Organisations (CMOs) with multi-functional plants have to pay particular attention to this area, as cleaning validation must be developed for each process and the equipment is often dedicated to the type of host used (for example, bacterial, mammalian and yeast).
Analysis of cleaning costs is therefore a key factor when evaluating the cost of ownership between disposable and re-usable technology. It is well documented that a high proportion of the capital cost associated with a stainless steel manufacturing process relates to the infrastructure (media feeds, buffers, utilities CIP and SIP). This is estimated to be as high as 52% of the total, which compares unfavourably with the 32% contribution of bioreactors and 16% in downstream processing (DSP). Up to 85% of the water usage in a stainless steel biotech facility relates to cleaning of non disposable equipment [2].
Lastly, CMOs need to maximise plant efficiency, as this has a significant impact on the cost of products they produce and, therefore, the on the longevity and success of the organisation. Facility downtime, reduced batch failure and the number of successful campaigns have a much greater impact than raw material or labour costs [3].
Disposables
In the past five years [4] single use disposable systems and components have become increasingly popular, offering process flexibility, robustness, and ergonomic and economic advantages. Some types of disposables have been used for years yet they tend to be associated with the fermentation process – bioreactors, media feeds and buffer bags, for example. The use of disposables in clarification and purification related duties is relatively new [5].
The top reasons cited by biopharmaceutical manufacturers and CMOs for the increasing trend towards the use of disposable systems, are:
• elimination of cleaning requirements; • decreased risk of product cross-contamination; and • reduced time to get the facility operational.
Disposable filtration
Disposable filtration is well established late in the downstream process (DSP) with sterilising grade membranes [6]. However, use of disposable technology during clarification (depth filtration stages) is increasingly common. Historically, this stage would have comprised of single use lenticular filters in reusable stainless steel housings.
Single-use filters were first established in laboratories; in the early stages they were used for buffers and other ‘clean’ process fluids, and later for general use at pilot scale. Today, manufacturers tend to specify disposable filtration wherever feasible. The benefits of disposable technologies include: reduced validation resource, lower hardware costs, elimination of cleaning in place and sterilisation in place.
Robert Conway, Ph.D., a consultant at Bioprocess Technology Consultants, has noted that completely disposable filtration systems, already established for late downstream sterilisation, have moved upstream into clarification and depth filtration. However, ultrafiltration remains a challenge for single-use filters.
Dr. Conway explained: “Because of issues related to materials of construction and the challenges of ultrafiltration, which is done either with cassettes or hollow fibres, the move toward complete disposable systems in this area has been much slower than for sterilisation.”
Disposable filtration is also becoming established in specialised areas such as in viral clearance and in membrane adsorption processes. Bioprocessors increasingly add a filtration step to attain higher viral clearance than would be expected from other separations. A hybrid of filtration and chromatography, called membrane adsorption, is routinely applied to the removal of DNA during polishing and to virus capture for gene therapy and vaccine work, as well as for viral clearance.
Achieving higher capacities remains a challenge with membranes, as Dr. Conway notes: “For some applications they compete quite well with gel- or bead-based chromatography resins by providing higher flow. They are also cost-competitive and, since they are mostly disposable, they require no cleaning.”
Paul Miraglia, director of biotechnology at Integrated Project Services (IPS), has also remarked on the trend for disposables to take over in hitherto uncharted applications. “This includes large filters for polishing bioreactor broth that has been clarified with centrifugation,” he said, noting the success of multi-layer formatted charged depth filters, which are said to require fewer housings and to compress a multiple stage filtration process into a single step. Advantages for end users include enhanced ease-of use and savings in labour time and cost.
Miraglia observes that the trickle of ultrafiltration and nanofiltration products now available as fully disposable flow-paths offer advantages similar to those for other single-use equipment including segregation and a reduction in batch-to-batch or product-to-product cross-contamination. But single-use filtration products will probably not work at large (10–20 kL) scales. “Cartridge filters just don't have that kind of capacity,” notes Miraglia.
That is not to say that disposable filtration is limited to low volumes. The industry has already developed the first user-friendly, ergonomically designed, disposable lenticular depth filter in a fully encapsulated format.
Historically, lenticular filters were offered as single or multiple-use media cartridges supported within stainless steel holders. Filters are stacked in the same orientation in which they are used, vertically, which makes loading difficult for some operators. The new disposable line will be stacked horizontally at waist height, and then rotated to a vertical position for operation. When set up, it will utilise the same flow path as current depth filters, but have the convenience of better ergonomics and complete disposability.
Meeting this demand are new Encapsulated Filter Systems, designed to work with advanced dual zone depth media that provide optimal clarification of bioprocess, biological and pharmaceutical fluids.
Encapsulated filter system
Linear scale-up is ensured by providing two devices: a small system that is suited to laboratory or pilot-scale cell culture clarification or downstream impurity removal processes; and a large disposable depth filtration system designed for production-scale manufacturing. Maximum versatility is offered by providing a filter holder, a set of disposable manifolds and a flexible number of disposable filter capsules.
The ability to pivot large filter holders between the horizontal and vertical position allows convenient loading and unloading at waist height, as well as minimal footprint during filtration and reduced fluid spills. The CAM locking mechanism makes for a fast, easy and robust capsule-to-capsule connection.
Applications
To date, such disposable depth filtration systems have been used primarily for post fermentation clarification processes, where they can be employed alone, or in combination with centrifugation or Tangential Flow Filtration (TFF). However, the same disposable filter system has been successfully utilised globally in downstream impurity removal. And the filter media, with positive charge capacity, have been shown to be effective in removing contaminants such as host cell proteins (HCP), viruses, DNA, protein aggregates and endotoxins.
A further application is in monoclonal antibody production, where disposable media are increasingly used as a polishing step after Protein A column. Lower cost, reduced set-up times and improved efficiency are reported compared with traditional anion exchange chromatography. These are compelling drivers: factors that have already influenced one major UK contract manufacturing organisation in implementing disposable dual zone depth filters as a platform technology for MAb production from a yeast expression system.
Conclusions
Disposable filtration technologies offer a robust, flexible and economic solution for the manufacture of therapeutic proteins in today's challenging biopharmaceutical market. Implementation of scalable disposable platforms accelerate the clinical manufacturing process and meet the varied challenges of: process variation, a demand for increased yield, cleaning and process validation, cleaning costs and plant efficiency – which are all vital to being a competitive manufacturer of biotherapeutic products.
References
1 Considerations When Outsourcing the Production of Clinical Material. Biopharm International, (June 2008), pp. 22–23.
2 Andrew Sinclair, How to Evaluate the Cost Impact of Using Disposables in Biomanufacturing. Biopharm International, (June 2008), pp. 26–29.
3 Disposables Cost Contributions: A Sensitivity Analysis. Biopharm International, (April 2009), pp. 28–32.
4 Eric S. Langer, Users Are Sold on Single-Use Systems. Survey of Adoption Trends Shows Burgeoning Usage Across All Sectors of Industry. Genetic Engineering & Biotechnology News, 28 16 (September 15 2008).
5 Judy Glynn, et al. Advances on Monoclonal Antibody Purification. Supplement to Biopharm International, (March 2010), pp. 4–10.
6 Angelo DePalma, Single-Use Filtration Hits the Mainstream. Vendors Introduce New Products to Meet Demand for Non traditional Applications. Genetic Engineering & Biotechnology News, 28 20 (November 15 2008).
