Trends in Biotechnology
Volume 31, Issue 8, August 2013, Pages 479-492
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Review
Feature Review
Continuous downstream processing of biopharmaceuticals

https://doi.org/10.1016/j.tibtech.2013.05.011Get rights and content

Highlights

  • Overview of continuous downstream processing.

  • Unit operations suited to continuous manufacturing.

  • Description of reactors used in continuous downstream processing.

Continuous manufacturing has been applied in many different industries but has been pursued reluctantly in biotechnology where the batchwise process is still the standard. A shift to continuous operation can improve productivity of a process and substantially reduce the footprint. Continuous operation also allows robust purification of labile biomolecules. A full set of unit operations is available to design continuous downstream processing of biopharmaceuticals. Chromatography, the central unit operation, is most advanced in respect to continuous operation. Here, the problem of ‘batch’ definition has been solved. This has also paved the way for implementation of continuous downstream processing from a regulatory viewpoint. Economic pressure, flexibility, and parametric release considerations will be the driving force to implement continuous manufacturing strategies in future.

Section snippets

Continuous manufacturing

Continuous manufacturing has been applied in many different industries but not in biotechnology, although bioethanol and microalgae may be the exceptions 1, 2. Continuous manufacturing of recombinant proteins is in its infancy, albeit a lot of companies are currently exploring the economy of continuous processes. One reason for the reluctance to implement these technologies rapidly may be the amount of product on demand in the market: annual market demand may be too low to justify a continuous

Flow schemes and connections between upstream and downstream processes

The entire process for manufacturing of a biotechnological product is divided into upstream and downstream processing. Upstream processing includes the generation of the cell line for overexpression of the protein, establishing the master and working cell bank, inoculum preparation and cultivation in the seed bioreactor, and full-scale cell cultivation. Thus, upstream processing includes more than only cell cultivation/fermentation. Downstream processing encompasses all process steps from cell

Continuous centrifuges

Continuous centrifuges are readily implemented in continuous processes for recovery of biomass, clarification of cell homogenates, washing and recovering inclusion bodies, or harvesting precipitates or protein crystals. In biotechnology, three common centrifuge configurations are used: (i) tube centrifuge; (ii) chamber centrifuge; and (iii) disc centrifuge (Figure 2). Only the disc stack centrifuge can be operated in a pseudocontinuous way [15]. Disc stack centrifuges are continuously fed with

Continuous filtration

Filtration, in the form of microfiltration and ultrafiltration, is a key unit operation in downstream processing of recombinant proteins. Microfiltration is mainly used for cell harvesting. Ultrafiltration is used for concentration of proteins and often in the form of diafiltration for buffer exchange. Ultrafiltration is also used to clear viruses from recombinant DNA produced in mammalian cells. This step is also named nanofiltration, although it is an ultrafiltration with a membrane with a

Continuous homogenization and cell lysis

High-pressure homogenizers are the industrial way to lyse cells in pilot and full scale. These machines consist of two main elements: a piston pump usually in the form of a triplex pump and a valve with a slit of around 100 μm to generate pressure up to 1500 bar. Cell lysis is caused by cavitation, which is generated at the valve and immediately behind the valve when the pressure is suddenly released. Modern high-pressure homogenizers can be operated continuously, because the cells are disrupted

Continuous precipitation/crystallization

Precipitation and crystallization can be used to capture a product from the culture supernatant (Figure 1). It is a relatively simple method that can be scaled up to the ton scale. The selectivity is lower compared with other methods such as chromatography. Precipitation or crystallization becomes an option when the feedstock has a high titer and fairly high purity; a prime target for this technology is antibodies [43]. The fundamental advantage of continuous precipitation/crystallization is

Continuous extraction

Extraction of proteins is also a relatively simple method for purifying and concentrating proteins and has been used a lot for extraction of proteins from natural sources such as plant and animal tissue, blood, and microorganisms. Two immiscible solvents must be found in which the impurities and product differentially partition. Water and organic solvent mixtures are not suitable, because proteins will denature in the organic phase. For proteins aqueous two-phase systems (ATPS) are usually

Continuous refolding

Expression of proteins as inclusion bodies allows production of protein at high titers with up to 30 g/l culture broth. Harvesting and initial concentration of the protein is only performed with mechanical unit operations such a centrifugation/microfiltration and high-pressure homogenization. Relatively high product purity, at least 80%, is obtained after inclusion body recovery by differential centrifugation. The drawback is the refolding of proteins, because inclusion bodies can only be

Continuous chromatography

The simplest way to operate chromatography in a continuous mode is to run several columns in parallel or concurrently [82]. While one column is loaded, the next ones are washed, eluted, regenerated, and finally re-equilibrated. In the simplest form, the number of columns must equal the number of individual steps required to perform the purification. Not all steps require the same time; thus, it may possible to connect two steps in one cycle. Often, loading is the most time-consuming step. Thus,

Concluding remarks

The main factors motivating a change in downstream processing from a batchwise to a continuous operation are the gain in productivity, the flexibility, and also the possibility to implement process-control strategies that may be required for a parametric/real-time release. The footprint for continuous operation will be smaller and, often, investment is substantially decreased. Especially in continuous chromatography, the operation becomes more complex but this is outweighed by the gain in

Acknowledgments

This work was supported by: the Federal Ministry of Economy, Family, and Youth (BMWFJ); the Federal Ministry of Traffic, Innovation, and Technology (BMVIT); the Styrian Business Promotion Agency; the Standortagentur Tirol; and the ZIT Technology Agency of the City of Vienna, through the COMET-funding Program, managed by the Austrian Research Promotion Agency, FFG.

Glossary

Baffles
static devices that regulate the flow of a fluid, in context of mixing they are built in to increase turbulence and/or shear.
Batchwise downstream processing
culture broth is processed in a batchwise manner, a predefined volume is subjected to the individual unit operations to concentrate and purify a product. The feed is usually from batch cultivation.
Camp number (NCa)
a dimensionless engineering parameter used to describe the influence of shear and ageing in precipitation and flocculation.

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