Expanded-bed affinity

FIGURE I Expanded-bed affinity chromatography (EBAC) setup Tl and T2, tanks for homogenate, washing, elution, and reequilibration buffers P, peristaltic pump C, expanded-bed column W, wastes collector E, eluent collector VI, V2, V3, and V4, three-way valves V5, four-way valve K, knitted silicone tubing used to reduce flow pulsation. 

FIGURE 3 Breakthrough and elution profile for total protein ( ) and L-lactate dehydrogenase activity (O) on an expanded-bed affinity column. (From Lali et al.20 with permission.)... 

Lali, A., Kaul, R., Galaev, I. Yu., and Mattiasson, B. (1997). Purification of L-lactate dehydrogenase from crude homogenate of porcine muscle by expanded bed affinity chromatography (EBAC). Iso. Purif. 2, 289-300. 

Nilesh A., Kamat, M., Arvind, L. (2004). Expanded bed affinity purification of bacterial a-amylase and Cellulase on composite substrate analogue-Cellulose matrices. Process Biochemistry, 39, 565-570. 

Clemmit, R.H. and Chase, H.A., Immobilized metal affinity chromatography of P-galactosidase from unclarified Escherichia coli homogenates using expanded bed adsorption, /. Chromatogr. A, 874, 27, 2000. 

Porous supports like agarose, pol3mrethacrylate, or silica beads are generally used in current applications of affinity chromatography. However, in the past several years other types of supports have also become available commercially. Many of these newer materials have properties that give them superior performance in certain applications. Materials that fall in this category include nonporous supports, membranes, flow-through beads, continuous beds and expanded-bed particles. 

Note that so far the expanded bed mainly has been used with rather uncomplicated systems such as for purification of monoclonal antibodies from culture broth, isolation of extracellular substances from microbial cultivations, harvesting of fusion proteins from Escherichia coli cell homogenates, affinity isolation of certain enzymes from microbial homogenates, and separation of serum proteins from serum. To our knowledge there are very few reports on the isolation from homogenates of mammalian tissues or plant material. 

TABLE 3 STREAMLINE Matrices Available for Affinity Expanded-Bed Adsorption... 

Zapata, G. (1999). Recovery of recombinant antibodies from unclarified Chinese hamster ovary cell culture fluid by expanded bed Protein A affinity chromatography. Proc. Waterside Monoclonal Conf. Norfolk, VA. 

John Pedersen (Unizyme Laboratories) demonstrated how multienzyme systems can be used along with His affinity tags and subtractive IMAC to produce industrial enzymes in both packed or expanded bed adsorption systems. The efficiency of the approach was demonstrated with the produc-... 

Chase, H A. and Draeger, N. M. (1992) Affinity punfication of proteins using expanded beds J Chromatogr 597, 129-145. 

Willoughby N.A., Kirschner T., Smith M.P., Hjorth R. and Titchener-Hooker N.J. 1999. Immobilised metal ion affinity chromatography purification of alcohol dehydrogenase from baker s yeast using an expanded bed adsorption system, J. Chromatogr. A, 40, 195-204. 

Clemmitt R.H. and Chase H.A. 2000. Facilitated downstream processing of a histidine-tagged protein from unclarified E. coli homogenates using immobilized metal affinity expanded-bed adsorption, Biotechnol. Bioeng., 67, 206. 

Garg N, Galaev lY, Mattiasson B. Polymer-shielded dye affinity chromatography of lactate dehydrogenase from porcine muscle in an expanded bed system. Bioseparation 1996 6 193-9. 

A useful literature relating to polypeptide and protein adsorption kinetics and equilibrium behavior in finite bath systems for both affinity and ion-ex-change HPLC sorbents is now available160,169,171-174,228,234 319 323 402"405 and various mathematical models have been developed, incorporating data on the adsorption behavior of proteins in a finite bath.8,160 167-169 171-174 400 403-405 406 One such model, the so-called combined-batch adsorption model (BAMcomb), initially developed for nonporous particles, takes into account the dynamic adsorption behavior of polypeptides and proteins in a finite bath. Due to the absence of pore diffusion, analytical solutions for nonporous HPLC sorbents can be readily developed using this model and its two simplified cases, and the effects of both surface interaction and film mass transfer can be independently addressed. Based on this knowledge, extension of the BAMcomb approach to porous sorbents in bath systems, and subsequently to packed-, expanded-, and fluidized-bed systems, can then be achieved. 

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