Bioprocessing

Over the past decade, biopharmaceuticals haveconsistently been the fastest growing segment of thepharmaceutical market, showing 19 per centcompound annual growth (CAG) since 1998. This –coupled with pricing pressures and the advent ofcompetition from biosimilars – has resulted ingrowing pressure to increase manufacturingproductivity and maximise the potential of existingfacilities. In 2008, the global biopharmaceuticalmarket was estimated at $75 billion (1), with anexpected growth rate of 12 per cent per annum for thenext decade (2).

Historically, the major bottleneck in bio-pharmaceutical production has been fermentationcapacity. The industry has responded by producingever-higher yields as a result of using both new andnovel expression systems, developing and optimisingnew and existing growth media, and increasing celldensities. All of these improvements have helped toincrease the productivity of upstream fermentationprocesses, with the best examples of optimisedmonoclonal antibody fermentation achieving yields ofover 27g/L (3). This has meant that, over the past fewyears, an increasing emphasis has been placed ondeveloping effective downstream purification processesto cope with these higher yields. At the same time, in-process impurities have increased, mainly due to morecomplex growth media and higher cell densities withlower cell viabilities. This introduces elevated levels ofhost cell proteins, lipids and nucleic acids into theprocess and offers a greater challenge to thepurification process (4).

With these advances in fermentation technology, thebottleneck has been pushed further downstream wherefilter and column sizes, and larger processing volumesand times, are becoming an issue. There are two mainsolutions to this problem. The first is to invest in greaterinfrastructure, which can be a costly and risky ventureand, ultimately, is limited by column sizes. The secondis to use current capacity more efficiently – this beingthe preferred option to meet current and futurechallenges. Seeking advice early in the developmentprogramme, and gaining a sound knowledge ofmanufacturing capabilities and regulatory requirements,can pay dividends and reduce the requirement foradditional process development further down the line.Coupled with the general trend within the industrytowards disposable technologies as opposed to fixedequipment, this makes the integration and combiningof unit operations, with a view to reducing the numberof overall processing steps, an increasingly attractiveprospect. Increasing the efficiency of any process has anumber of other cost benefits too, including a reductionin raw material requirements, buffer consumption and waste disposal considerations, and downstreamprocessing facility requirements.

DOWNSTREAM PROCESS DEVELOPMENT

Typically, during the development of any purificationstrategy, processes can be split into a number of definedsteps or unit operations, each mainly dependent on theactivity to be performed at each stage. An understandingof how each operational step affects the performance ofthe subsequent downstream step is fundamental to

Integrating and Streamlining Biopharm Purification ProcessesAdvances in fermentation technology have pushed the bottleneck inbiopharmaceutical production further downstream and the emphasis is nowon developing effective purification processes to cope with higher yields.

By Andrew Clutterbuck at Eden Biodesign Ltd

Andrew Clutterbuck is responsible for the Downstream Process Development Team at Eden Biodesign Ltd, a companythat develops and optimises purification strategies for recombinant proteins, antibodies, viruses and virus-like particles.He also plays a key role in the subsequent scale up and tech transfer of processes into the cGMP processing facilities atthe National Biomanufacturing Centre (Liverpool, UK) and in providing technical support during manufacturing activities.Previously Andrew was a Senior Research Scientist for Avecia Biotechnology in Billingham, working within both thedevelopment laboratories and cGMP production facilities developing and running purification processes over a widerange of scales.

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commercial success. The strategy should aimto achieve the highest yield and level ofcontaminant removal with the fewestnumber of unit operations.

Taking the model of a therapeutic proteinsecreted from cells into the growth medium,a simplistic view of the downstream processcan be seen as:

� Primary separations – removal of cells and cellular debris from the process stream

� Primary capture – initial productcapture and concentration

� Intermediate purification – removal of bulk in-process impurities

� Polishing chromatography – removal of specific, low level in-process impurities

� Final formulation – final dosageconcentration and buffer formulationalso includes bio-burden reductionfiltration

PRIMARY SEPARATIONS

The primary separation of cells from thefermentation medium is a key area ofconsideration during process developmentand scale up. Advances in cell technology –which have led to increased yields – in somecases have also led to increased cell densitiesfrom both mammalian and microbialsystems, both of which place an increaseddemand on the initial separation step. Theprimary separation can be the mostsignificant bottleneck in any process becausecentrifugation – the classical cell and cell debris removaltechnique – is effective at small scales but becomes moreproblematic when dealing with increased volumes.

Centrifugation has very high operating costs in terms ofenergy consumption and processing times, whichcontributes to decreasing productivity. At volumes above100L batch, centrifugation becomes impractical andcontinuous centrifugation becomes the preferred option.This requires further, complicated process development,which may change the impurity profile of the process

material. The high shear forces and stresses involved maydamage the cells and release contaminants – such as hostcell protein and DNA – into the process material whichmay, in turn, adversely affect the subsequent downstreamprocess. This also makes microfiltration an increasinglyattractive prospect at larger scales, but it also has largeenergy requirements, as well as a need for expensive,specialist equipment.

Filter technology has improved significantly overrecent years, with a number of companies offering arange of process options to allow direct clarification of

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Figure 1: 30L Fermenter in PD laboratory

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the whole cell medium, and negating the requirementfor centrifugation or microfiltration. The techniqueinvolves minimal equipment and is directly scalable tomultiple thousand-litre volumes. Charged Lenticular-type filters, available from a number of suppliers, mayalso reduce other in-process impurities such as DNA and endotoxin. The downside of this techniqueis that the fermentation process must be robust, well defined and reproducible, as any minor changesupstream may adversely affect filter performance orlead to premature filter fouling. However, smallvariations in the feed material can be compensated for if, during the development phase, a certain

amount of redundant filter capacity is engineered intothe process.

CLASSICAL CHROMATOGRAPHY

At present, monoclonal antibodies or Fc fusion proteinsdominate the biopharmaceutical industry pipeline. Assuch, the use of affinity chromatography resin – now amainstay of antibody purification – offers a simple andeffective way of reducing the number of processing stepsby incorporating both a capture and concentration stepwith a high degree of purification. The major drawback ofthis approach is that the resin is expensive which, in thelong term, may be offset against the cost of plant time asa result of the overall reduction in processing time. Asecondary drawback is that the ligand may potentiallyleach from the column and need to be cleared from thefinal product. And, finally, for certain mAb-basedproducts, affinity chromatography simply isn’t an optionand more classical methods must be employed. High-capacity, high-throughput ‘classical’ chromatographyresins are being offered on the market from a number ofcompanies and are becoming increasingly popular andincorporated more frequently into processes.

MEMBRANE CHROMATOGRAPHY

Within the industry, increasing attention has been given to the developing area of membrane-basedchromatography. Membrane adsorbers, as they are oftenknown, work on exactly the same principle as resin-based chromatography systems, often with the samebasic ligand chemistry available. When clearing specificin-process impurities – such as endotoxins, host cellproteins and DNA – membrane adsorbers used in‘flowthrough’ are being used in a growing number ofmanufacturing processes (5). These easy-to-use, single-use, disposable capsules have significant advantages interms of throughput due to increased operating flowrates and minimal hardware requirements.

CONCENTRATION AND BUFFER EXCHANGE

Concentration and buffer exchange, both in process and asa final formulation step, are mainly performed by anultrafiltration and dia-filtration (UF/DF) process. TheUF/DF process works on the principle that the process feedstream flows parallel to the surface of the membrane face,creating a sweeping motion, thereby reducing fouling ofthe pores and the build-up of a gel layer on the surface ofthe membrane. This is in contrast to normal flow filtration,where the fluid flow path is dead-ended onto the filter surface. This technique is ideally suited for the

44 Innovations in Pharmaceutical Technology

At present, monoclonal antibodies or Fc fusion proteins dominate the

biopharmaceutical industry pipeline. Assuch, the use of affinity chromatography

resin – now a mainstay of antibodypurification – offers a simple and

effective way of reducing the number ofprocessing steps by incorporating both a capture and concentration step with

a high degree of purification.

Figure 2: 6mm bioprocess skid in GMP unit

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concentration and buffer exchange of larger volumes, as themembrane area can be directly scaled. Advances in filterdesign and construction materials have been made over thepast few years, and each of the main suppliers has furtherdeveloped and expanded their existing product lines – butno specific breakthroughs have been made, with theexception of fully disposable systems. Selecting the correctmembrane and fully optimising the step may take a largeportion of the development time, but will ultimatelycontribute significantly to overall product yields.

INCREASED THROUGHPUT UTILISINGDISPOSABLE OPTIONS

Increasing overall facility throughput in a multi-productfacility can also be achieved by using disposable systems.This eliminates the requirement for costly and time-consuming cleaning validation and CIP cycles betweenbatches, and is especially attractive for contractmanufacturers where cross-contamination may be aproblem. Reducing the need for fixed equipment alsohelps to build flexibility into and minimise the spacerequirements for each unit operation. The trend withinbiotechnology over the last few years towards disposablesystems will continue to grow with the introduction ofdisposable fermenters, columns and ultrafiltrationsystems – making it eventually possible to develop acompletely disposable process.

CONCLUSION

The growth of high titre expression technology can beexpected to slow, since current cell lines can only bepushed so far and, as downstream processing technologycontinues to advance, there will be a gradual uncorkingof the downstream bottleneck. It is inevitable that onceone problem is solved, emphasis will be placed onanother aspect of processing. But, with the current andprojected growth in the marketplace, the industry will beready to rise to the challenge.

The author can be contacted at andrew.clutterbuck@edenbiodesign.com

References

1. Biopharmaceuticals – Current Market Dynamics

and Future Outlook, AS Insights, May 2005

2. Jakovcic K, Biomanufacturing Strategies, Business

Insights Ltd, 2007, www.globalbusinessinsights.com

3. Crucell website

http://investors.crucell.com/C/132631/PR/200806/12

27870_5_5.html

4. Roush DJ and Lu Y, Biotechnol Prog, 24(3),

pp488-495, 2008

5. Clutterbuck A, Biopharm International, 20(5),

pp44-54, 2007

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Figure 3: Sartoflow®

beta crossflow filtration unit in GMP unit

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