Tech Review

Downstream processing tomorrow – Great Expectations?

16.11.2009

The biopharmaceutical industry has seen plenty of change over the past 20 years, with the yield of recombinant proteins increasing from tens of milligrams to more than 10 grams per liter for some monoclonal antibodies in early development [1]. As batch volumes increase in parallel, we face the real likelihood of 50- to 100-kg batches of protein in the not so distant future [2]. The majority of industry professionals now agree there are at least minor limitations in downstream processing, and in some cases major bottlenecks, with cost and capacity issues being most important [3]. Downstream processing is driven by mass rather than volume, and it is apparent that technological advances in downstream processing have failed to keep up with productivity increases upstream. This is because the rational approach, continuous upscaling, is no longer sufficient due to costs, technical limitations and the difficulties encountered in retrofitting existing facilities. Therefore the debate has turned to alternative solutions: streamlining operations, introducing novel high-technology solutions and revisiting older technologies to reduce the costs of large-scale processes.

The biopharmaceutical industry has seen plenty of change over the past 20 years, with the yield of recombinant proteins increasing from tens of milligrams to more than 10 grams per liter for some monoclonal antibodies in early development [1]. As batch volumes increase in parallel, we face the real likelihood of 50- to 100-kg batches of protein in the not so distant future [2]. The majority of industry professionals now agree there are at least minor limitations in downstream processing, and in some cases major bottlenecks, with cost and capacity issues being most important [3]. Downstream processing is driven by mass rather than volume, and it is apparent that technological advances in downstream processing have failed to keep up with productivity increases upstream. This is because the rational approach, continuous upscaling, is no longer sufficient due to costs, technical limitations and the difficulties encountered in retrofitting existing facilities. Therefore the debate has turned to alternative solutions: streamlining operations, introducing novel high-technology solutions and revisiting older technologies to reduce the costs of large-scale processes.


Most companies are actively streamlining their purification strategies to guarantee best practice under the restrictions we now see in the industry while reducing product cost as far as possible. Antibodies are at the forefront of this development, since they represent more than 50% of all biopharmaceuticals in the pipeline, yet have enough conserved properties to allow meaningful cross-platform comparisons. Significant benefits can be obtained by using generic platform processes that are adjusted rapidly to match the requirements of new products.

Streamlining operations –

Hard Times

The vast majority of companies employ a three-column platform comprising Protein A affinity chromatography for product capture, followed by a combination of cat­ion exchange (CEX), anion exchange (AEX) and/or hydrophobic interaction chromatography (HIC) for polishing and virus removal. As cell culture titers increase and pur­ification trains are challenged with larger amounts of antibody per fermentation cycle, costs increase in proportion to production scale – so there is no economy of scale effect. Rather than upscaling, companies are therefore fine-tuning their processes, as exemplified by Pfizer’s platform for antibody production, where depth filtration at the 500-litre scale was augmented with continuous flow centrifugation at the 1200-litre scale, the order of polishing steps was changed and the AEX column was replaced with a membrane adsorber, reducing the overall process time by almost 50%[4].

Revisiting old technologies –

the Ghost of Christmas Past

The implementation of inexpensive solutions that have been used successfully in industries with lower manufacturing costs (e.g. the food and chemical industries) is one low-technology approach that can be used in the manufacture of bio­pharmaceuticals[5]. Harvesting, which tends to involve complex filtration and/or centrifugation trains to remove small (sub-micrometer) particles, can be simplified by the use of flocculants to create larger particles that are easier to separate from the cell culture fluid. Flocculation has been used mainly for the removal of whole cells from fermentation broth, and more recently for the removal of cell debris and proteins.
Precipitation is among the simplest and least expensive fractionation methods, since it can be achieved by simple changes in solution conditions, e.g. the addition or removal of salt (salting out), metal ions, organic solvents, polymers/polyelectrolytes, or even by changes in temperature and pH. Precipitation can be used not only to remove impurities[6] but also to isolate a target antibody[7], which in the latter case also reduces the processing volume. Several groups have developed methods to precipitate antibodies in large-scale processes, and this could replace Protein A chromatography in the long term.
For the final purification steps, another traditional technology that is being considered for use in biopharmaceutical manufacturing is crystallisation, which involves the formation of a regularly-structured solid phase that impedes the incorporation of contaminants or solvent molecules, and therefore yields products of exceptional purity[8]. Protein crystallisation has been incorporated into several current manufacturing processes, including those for recombinant insulin, aprotinin and Apo2L[9].

New technologies – the Ghost of Christmas Yet to Come

The development of new, high-tech solutions can also help to reduce the cost of downstream processing, and disposable membrane adsorbers are at the forefront of this field. Disposable modules are already integral to many processes, particularly for filtration and media/buffer storage, but interest in membrane chromatography is growing because of the many advantages of membrane adsorbers over equivalent resin-packed columns, including the elimination of cleaning and validation costs and the much smaller footprint of membrane adsorbers.
A range of different membranes is available with functional groups equivalent to the corresponding resins, e.g. membranes containing quaternary ammonium groups for anion exchange, polyallylic ligands for the removal of contaminants under high salt conditions (Salt Tolerant Interaction Chromatography, STIC), or phenyl groups for hydrophobic interaction chromatography. Another significant functional advantage of membranes over resins is that the transport of solutes to their binding sites occurs mainly by convection, while pore diffusion is minimal. Because of these hydrodynamic benefits, membrane adsorbers can operate at much greater flow rates than columns, considerably reducing buffer consumption and shortening the overall process time up to 100-fold. The use of membrane adsorbers can be viewed as the equivalent of shortening traditional columns to near zero length, allowing large scale processes to run with only a small pressure drop at very high flow rates [10]. Even so, they continue to provide adequate binding capacity for large biomolecules such as

viruses and DNA, so they can play an important role in the overall viral clearance strategy for antibody purification [11].
For example, the process capacity of multi-layer Q membranes is much higher than equivalent volumes of resin with no loss of performance in contaminant and virus removal [12]. For smaller molecules, resins offer a higher capacity, but membrane absorbers offer speedier processing. The availability of STIC closes another gap in antibody purification as it enables the direct processing of CEX-eluates (cation exchange) without dilution[13].

Conclusions – A Tale of

Two Technologies

The perceived bottleneck in downstream processing can be addressed by streamlining today’s production processes, and to achieve this there are two technological approaches which can be used alone or in combination. The first is to replace the most expensive and time-consuming unit operations with new, high-tech solutions that offer more capacity, more efficiency and a smaller footprint, both in terms of the equipment itself and the room required for buffer preparation and storage. Disposable membrane adsorbers provide a key example of this approach. The second approach is to revisit older and more established technologies that are already widely used in the food and chemical industries. More and more processes are now benefiting from low-cost, low-technology solutions such as precipitation and crystallization in place of expensive chromatography trains. The combination of these two approaches on a case-by-case basis should allow downstream processing to cope with the increasing titers we can now see on the horizon. D


References
[1] Gottschalk, U. New and unknown challenges facing biomanufacturing. BioPharm International 2005 March, 24-28.
[2] Kelley, B. Designing a 10 ton antibody process: Is conventional chromatography limiting? 232nd American Chemical Society National Meeting, Sept 10-14, 2006, SanFrancisco, CA, BIOT division, paper 133.
[3] 6th Annual Survey of Biopharmaceutical Manufacturing. Eric S. Langer, BioPlan Associates Inc. 2009
[4] Glynn J et al. (2009) The development and application of a monoclonal antibody purification platform. Biopharm Intl Supplement Oct. 2009 (15-19)
[5] Thömmes J, Gottschalk U (2009) Alternatives to packed-bed chromatography for antibody extraction and purification. In: Gottschalk U (ed) Process-scale Purification of Antibodies. Wiley, NY.
[6] Wang M., Diehl T., Aguiar D., Dai X.-P., Arunakumari, A., (2009). Precipitation of Process-derived Impurities in non-Protein A Purification Schemes for Antibodies. Biopharm Intl Supplement Oct. 2009 (4-10)
[7] Przybycien, T., Narahari, S., Steele, L. (2004). Alternative bioseparation operations: life beyond packed-bed chromatography. Current Opinion in Biotechnology 15, 469–478.
[8] Klyushnichenko, V. (2003). Protein crystallization: from HTS to kilogram-scale. Current Opinion in Drug Discovery and Development 6, 848-854.
[9] Peters, J., Minuth, T., Schröder, W. (2005). Implementation of a crystallization step into the purification process of a recombinant protein. Protein Expression and Purification 39, 43-53.
[10] Gottschalk U (2009) Bioseparation in antibody manufacturing: the good, the bad and the ugly. Biotechnol Prog. 24:496-503
[11] Zhou, J.; Tressel, T. Basic concepts in Q membrane chromatography for large-scale antibody production. Biotechnol. Prog. 2006, 22, 341-349.
[12] Zhou, J. Orthogonal Virus Clearance Applications in Monoclonal Antibody. In: Process Scale Purification of Antibodies. Gottschalk U, ed. John Wiley & Sons
[13] Faber, R., Yang, Y., Gottschalk, U., Development of Salt Tolerant Interaction Chromatography (STIC) for Large Scale Polishing with convective Media. BioPharm Intl. Oct. 2009 (11-14)


Contact
Dr Uwe Gottschalk
Vice President Purification Technologies
Sartorius Stedim Biotech GmbH
August-Spindler-Str. 11
37079 Göttingen
uwe.gottschalk@sartorius-stedim.com
www. sartorius-stedim.com


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