Downstream processing chromatography

In the development of new products, optimization of the fermentation medium for titer only often ignores the consequences of the medium properties on subsequent downstream processing steps such as filtration and chromatography. It is imperative, therefore, that there be effective communication and understanding between workers on the upstream and downstream phases of the produc t development if rational trade-offs are to be made to ensure overall optimahty of the process. One example is to make the conscious decision, in collaboration with those responsible for the downstream operations, whether to produce a protein in an unfolded form or in its native folded form the purification of the aggregated unfolded proteins is simpler than that of the native protein, but the refolding process itself to obtain the product in its final form may lack scalabihty. 

Chromatography, which is finding increasing applications in the downstream processing of biochemical products, is covered in Chapter 19 of Volume 2. 

E Ruckenstein, V Lesins. Classification of liquid chromatographic methods based on the interaction forces The niche of potential barrier chromatography. In A Mizrahi, ed. Advances in Biotechnological Processes, Vol 8 Downstream Processes Equipment and Techniques. New York Alan R. Liss, 1988, pp 241-314. 

High-resolution chromatography normally yields a protein that is 98-99 per cent pure. The next phase of downstream processing entails formulation into final product format. This generally involves ... 

Product manufacture entails viral vector propagation in a suitable animal packing cell line (known as HEK 293). After cell recovery and lysis, the crude product is clarified by filtration and concentrated by ultrafiltration. The product is then treated with a nuclease preparation in order to degrade contaminant DNA and further downstream processing entails multi-step high-resolution column chromatography (see also Figure 14.7). 

For the implementation of techniques such as step-elution, gradient-elution, displacement-elution, as well as for the wash and sanitation steps needed for ion exchange chromatography and continuous downstream processing in general. 

Process Residuals A source of potential impurities can be process residuals as well as host cells. Process residuals include fermentation media components as well as raw materials used in chromatography (immunoglobulin affinity ligands), etc. According to the ICH Guidelines,2 process-related impurities are impurities that are derived from the manufacturing process. They may be derived from cell substrates, cell culture, or downstream processing. 

Cruz and co-workers have used MOI s of 2 to 5 for the production of HIV-VLPs and HIV-CLPs [69]. They found that, for their system, the peak volumetric particle titre did not coincide with maximum particle quality, due to the increase of proteolytic activity in the supernatant, which is coincident with cell lysis. Nevertheless, that issue was surpassed by correct integration with downstream processing, where low weight particles were removed by gel filtration chromatography [32]. 

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