Chromatography systems 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]. 

The remainder of this introductory chapter focuses on downstream processing and bioseparation relevant to the chapters presented in this book. Thus, the following topics are covered multiphase systems, membrane separation, centrifugation and adsorption techniques, electrophoresis, chromatography, and affinity separations. 

The separation tasks for ion-exchange chromatography differ mainly by the type of expression system used. The most abundant systems are mammalian cells, microbial cells (yeast), and the bacterial organism E. coli. All expression systems have a common feature that they not only generate the target protein but also process related impurities, such as host cell proteins, DNA, viruses, endotoxins. In addition the downstream process has to isolate the active form of the target molecule from all its derivatives, for example, oxidated, deamidated, acetylated forms, dimers, aggregates, and unfolded proteins. 

Another promising downstream technique that could be used for ISPR systems is counter-current chromatography [I]. This technique uses two immiscible phases to separate solutes on the basis of their relative solubility in the two solvents. It is essentially an intensive Hquid-Hquid extraction process and allows... 

Bearing in mind the general requirements, the next stage in designing a system is to produce a functional or block flow diagram of the process. This will usually also take into account not only the chromatography step but also the upstream and downstream requirements of the process. This identifies the number and volumes of solvents, sample, and fractions, together with column size. 

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