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BACKGROUND: The lifetime of chromatography resins typically averages between 10 and 300 cycles for the manufacture of a therapeutic protein. Developing and establishing the robustness of the method for each separation process represents a significant challenge and is subject to extensive regulatory oversight. This paper presents a novel fluorescence-based assay for residual aggregated proteins to aid the evaluation of the extent of resin regeneration. RESULTS: The versatility of this method was demonstrated by using strong anion and cation exchange agarose resins PraestoQ and SP in conjunction with bovine serum albumin and monoclonal antibody feed materials. The assay entails applying a molecular rotor dye to a sample of free resin and measuring the fluorescence intensity using a plate reader or visualizing under a confocal laser scanning microscope to gain a more detailed characterization. Following five consecutive chromatography cycles, both methods revealed a 10-fold increase in fluorescence intensity along with a proportional reduction in dynamic binding capacity. Furthermore, the use of the assay suggested that fouling was dependent on spatial bead position in the column, bead channel structure and cleaning conditions. CONCLUSION: This work presents a simple assay suitable for use in resin lifetime studies to enhance process understanding.

Expanded bed adsorption is a technique for recovery of biomolecules directly from unclarified feedstocks. The work described here demonstrates that expanded bed adsorption is a scaleable technique. The methods used to test scaleability were ''determination of degree of bed expansion'', ''determination of axial dispersion'' and ''determination of protein breakthrough capacity''. The performance of a production scale expanded bed column with 600 mm diameter was tested using these methods and the results were found to be consistent with the results obtained from lab scale and pilot scale expanded bed columns.The scaleability and function of the expanded bed technique was also tested by performing a ''process example'': a purification mimicking a real process using a yeast culture spiked with bovine serum albumin as feedstock. The results show that the 600 mm diameter production scale column was as efficient as a 25 mm diameter lab scale column in recovering bovine serum albumin from the unclarified yeast culture. The production scale runs were fully automated using a software controlled system containing an adaptor position sensor and an adsorbent sensor.A cleaning study was performed which showed that after use of a proper cleaning protocol, no surviving microorganisms could be detected in the column or in the adsorbent.

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