Protein separation using membrane chromatography 2.docx

1、Protein separation using membrane chromatography 2Protein separation using membrane chromatography: opportunities and challenges Raja Ghosh, Department of Engineering Science, University of Oxford, Parks Road, Oxford OX1 3PJ, UK http:/dx.doi.org/10.1016/S0021-9673(02)00057-2, How to Cite or Link Usi

2、ng DOIPermissions & ReprintsAbstractSome of the problems associated with packed bed chromatography can be overcome by using synthetic macroporous and microporous membranes as chromatographic media. This paper reviews the current state of development in the area of membrane chromatographic separation

3、 of proteins. The transport phenomenon of membrane chromatography is briefly discussed and work done in this area is reviewed. The various separation chemistries which have been utilised for protein separation, along with different applications, are also reviewed. The technical challenges facing mem

4、brane chromatography are highlighted and the scope for future work is discussed.Keywords Reviews; Membrane chromatography; Stationary phases, LCKeywords Proteins1. IntroductionChromatography is by far the most widely used technique for high-resolution separation and analysis of proteins. These proce

5、sses are traditionally carried out using packed beds, which have several major limitations. The pressure drop across a packed bed is generally high and tends to increase during a process due to the combined effects of bed consolidation (caused by media deformation), and column blinding caused by acc

6、umulated colloidal material. Another major limitation with conventional chromatographic bioseparation processes, particularly those employing soft chromatographic media, is the dependence on intra-particle diffusion for the transport of solute molecules to their binding sites within the pores of suc

7、h media (see Fig. 1). This increases the process time since transport of macromolecules by diffusion is slow, and particularly so when it is hindered. Consequently, the recovery liquid volume (needed for elution) also increases. Channelling, i.e. the formation of flow passages due to cracking of the

8、 packed bed, is a major problem. This results in short-circuiting of material flow, leading to poor bed utilisation. Other problems include radial and axial dispersion limitations arising from the use of conventional polydisperse media. Some of these factors and the fact that the transport phenomeno

9、n is complicated make scale-up of packed bed chromatographic processes difficult.Fig. 1. Solute transport in packed bed chromatography and membrane chromatography.Figure optionsSome of the limitations of packed bed chromatography have been overcome by using newly developed monodisperse, non-porous,

10、rigid chromatographic media (e.g. Refs. 1 and 2). However, these media are generally expensive and the solute binding capacity is greatly reduced since binding can now only take place on the external surfaces. Also with these materials, the problem of high-pressure drop still persists.A radically di

11、fferent approach to overcome the limitations associated with packed beds is to use synthetic microporous or macroporous membranes as chromatographic media (e.g. Refs. 3, 4, 5 and 6). In membrane chromatographic processes the transport of solutes to their binding sites takes place predominantly by co

12、nvection (see Fig. 1), thereby reducing both process time and recovery liquid volume. The binding efficiency is generally independent of the feed flow-rate over a wide range and therefore very high flow-rates may be used. The pressure drop is also significantly lower than with packed beds. Another m

13、ajor advantage of membrane adsorbers is the relative ease of scale-up when compared with packed beds. However, this potential has not been fully utilised as yet in the bioprocess industry. Membrane chromatography is particularly suitable for larger proteins (i.e. Mr250000). Such proteins rarely ente

14、r pores present in particulate chromatographic media and only bind on the externally available surface area of such media. Therefore, for larger proteins, the surface area available for binding is significantly greater with membranes. The binding capacity of membrane adsorbers for smaller proteins i

15、s generally lower than with conventional gel-based media, but significantly higher than with monodisperse, non-porous, rigid media.Membrane chromatographic devices are generally easier and cheaper to mass-produce. This makes it possible to have disposable membrane adsorbers. These devices can be use

16、d until the desirable properties (i.e. hydraulic permeability, binding capacity, selectivity and resolving power) are maintained. Once they cease to function properly these devices can be replaced. This type of flexibility eliminates the requirement for cleaning and equipment revalidation.Different

17、separation chemistries are utilised in membrane chromatography of proteins. Some membranes already in use for other types of membrane processes (e.g. microfiltration) have been found to be suitable as chromatographic media. However, in most cases these available membranes have been modified to make

18、them more suitable for use as membrane adsorbers. Novel synthetic membranes have also been developed. Another alternative to packed bed chromatography, which has certain similarities with membrane chromatography, is based on the use of monolith columns. These columns are prepared using rod-shaped po

19、rous structures through which convective flow of mobile phase can take place. The main advantages of monolith columns are similar to those for membrane chromatography. However, monoliths differ from membranes in terms of material of construction and morphology. While a membrane by definition is a ba

20、rrier in which the lateral dimension far exceeds the longitudinal dimension, the converse is probably true with monoliths. Monoliths are perhaps more similar to packed bed chromatographic columns than to membranes.In this review article, the current state of development in the area of membrane chrom

21、atography is discussed. Published literature in the area of membrane chromatography of proteins is reviewed (e.g. Refs. 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50

22、, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,108 and 109). Potential limitations of membrane chromatography ar

23、e also highlighted. The wider acceptance of this technology depends largely on finding solutions to these limitations.2. Transport phenomena of membrane chromatographyThe advantage of membrane chromatography lies in the predominance of convective material transport. However, as evident from Fig. 1,

24、diffusive transport is not totally absent. The predominance of convection alone does not necessarily guarantee efficiency. Convective flow of inappropriate type can be a serious disadvantage. Flow distribution is a major concern in chromatographic and indeed most types of separation processes. Ratio

25、nal design of the membrane chromatographic process and equipment is possible only when the transport phenomena involved are properly understood. However, it may be worth mentioning that, in many chromatographic processes, particularly those relying on affinity-type interactions, the binding kinetics

26、 may be limiting. In such processes, improvement in transport phenomena is not likely to result in significant improvement in process efficiency.Generally speaking, three types of membrane adsorbers are used for protein bioseparation: flat sheet, hollow fibre and radial flow. Single flat sheets are

27、rarely used. More often, stacks of several flat sheets are housed within membrane modules. In addition to providing more adsorbent volume, the use of membrane stacks has certain other benefits which are discussed below. A hollow fibre membrane has a tubular geometry with the tubes typically ranging

28、from 0.25 to 2.5 mm in diameter. A hollow fibre membrane adsorber usually consists of a bundle of several hundred fibres potted together within a module in a shell and tube heat-exchanger-type configuration. Radial flow adsorbers are prepared by spirally winding a flat sheet membrane over a porous c

29、ylindrical core. Fig. 2 summarises the relative reported usage of the three major types of membrane adsorbers (based on the papers reviewed in this article). Flat sheet membranes are by far most widely used. Hollow fibres, even though advantageous in other types of membrane based technologies (e.g.

30、microfiltration, ultrafiltration, and dialysis), are perhaps not so well suited for membrane chromatography. The reasons for this are explained in the next paragraph. Most of the reports on the use of hollow fibres are from research groups actively engaged in development of hollow fibre membranes fo

31、r different uses. The use of radial flow devices is also not that widely reported in the published literature even though several adsorbers of this type are available on the market. Table 1 lists some of the commercially available membrane adsorbers. The fact that there are relatively few manufactur

32、ers of membrane adsorbers indicates the newness of the technology.Fig. 2. Membrane adsorber types (geometry).Figure optionsTable 1. Commercially available membrane adsorbersProduct nameMembrane material/typeConfigurationManufacturerSartobind MA5,Reinforced stabilised cellulose,Flat sheet, readySartorius MA15 and MA100strong cation exchange (S type),to use adsorbersstrong