AIR2-CT93-0911 The Development, Investigation and Assessment of A Novel Continuous Affinity Separations Technique

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AIR Cluster III - Bioconversion : Fine Chemicals : Pharmaceuticals/Cosmetics : Protein/Amino Acid : Separation/Fractionation

	Contractl No:	AIR2-CT93-0911
	Date Prepared:	September 1999
	Source:	Consolidated report Year 3 October 1996

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Consolidated report Year 3 October 1996

Summary

Objectives The aim of this project was to develop an affinity separation method in which a mobile solid phase adsorbent is recycled through feedstock and eluent streams in a continuous process. This is different from conventional approaches to adsorptive separations in the use of a non-porous, non-particulate solid phase. As the rates of the adsorption and desorption processes were not limited by the diffusion of the solute into the matrix, faster ligand cycle times could be used that would compensate for the low surface area. The potential advantages were lower costs through the use of small amounts of affinity ligand, inexpensive support materials, and continuous operation.

Activities Over the first two years of the project, prototype apparatus had been constructed and several different affinity systems were investigated. The main challenge was to overcome the low recoveries and throughputs that were obtained during continuous separations. The causes of poor performance that were identified were inefficient binding and elution processes, caused by operational conditions and non-specific binding of proteins, and limitations in the design of the apparatus that resulted in a compromise between process rate and efficiency.

Three different approaches were adopted during to try and solve these problems. A novel potential belt material became available through an association between Biometra and Whatman Ltd that may increase ligand density and reduce non-specific binding. This was adapted for use in the continuous separation apparatus. The conditions used in the binding and elution with IgG/Protein A and NS3-(His)/metal chelation were inefficient and could be improved. Thirdly, preliminary work had suggested that the formation of a concentration gradient in the adsorption chamber could alleviate the problem of low recoveries at higher purification rates. Several problems were encountered with the manufacture of the new belt material in the conformation required for the continuous separation application. These were finally solved at the end of this year, but insufficient time remained for the new belts to be tested in the apparatus. This would require apparatus specifically designed around this material.

New adsorption and elution conditions were developed for the purification of IgG that resulted in improved recoveries. The best achieved with pure rabbit IgG was 40% recovery at 4 micrograms per minute. Although higher throughput was obtained in experiments using rabbit serum as feedstock, this was at the expense of recovery.

Metal chelation affinity separations had not been possible using apparatus with only four chambers as additional regeneration steps were required to recharge the belt with metal. With the availability of the six-chambered Prototype B, it was again possible to consider this type of separation. Using a crude preparation of the recombinant protein NS3-(His) as feedstock, highly purified product was obtained, although throughput rates and recoveries were again low.

A method was devised for creating concentration gradients in the adsorption and elution chambers, thus reducing losses and increasing recoveries. Both chambers were replaced by three sub-chambers. These were constructed such that the liquid entering the first section could flow freely by gravity to the outlet in the third section where it was removed by peristaltic pump. This arrangement effectively partitioned the adsorption chamber into three sections which limited the mixing of the chamber contents into a uniform concentration. A concentration gradient could thus be established as the target protein was removed by adsorption onto the belt. It was demonstrated that this design could produce significantly more efficient operation. Using the trypsin/SBTI system, a throughput of 2 mg per hour at over 75% recovery was achieved.

The new apparatus design and the new belt material with IgG and metal chelation separations require further testing. However, it is apparent that this continuous separation method is capable of efficient operation, as shown with the trypsin purification.