Jens H. Vogel

Isolation of recombinant protein drugs from cell culture supernatant is usually performed in batch mode, even if the fermentation process itself is continuous. As a novel approach, continuous separation techniques like continuous annular chromatography (CAC) can be used for continuous isolation. The potential of CAC for industrial application is demonstrated by continuous isolation of rFVIII from cell culture supernatant in pilot scale (i.e., 144–288 l/day). Thirty‐fold concentration can be achieved at 94% yield, while purity is increased 3–5‐fold. For this a batch direct feed ion exchange chromatography method was adapted to a commercial preparative CAC system (P‐CAC). A headspace loading technique was used to maximize the concentration factor, while buffer incompatibility problems were addressed by a specifically modified inlet geometry. To allow sterile on‐line coupling to FVIII‐producing perfusion fermenters, an autoclavable pilot scale P‐CAC prototype was developed. General characterization of P‐CAC revealed a current limitation of the technology, i.e., variations in the outlet flow rates of up to ±20%. These flow variations are shown to be caused mainly by a nonuniform annular resin bed and in turn result in “peak wobbling,” i.e., the slight variation of peak position (up to ±4°) and shape (e.g., A_s = 0.9–1.4) as a specific function of column position. Some additional peak broadening, although less significant, is caused by a “peak oscillation” effect that results from the necessary segmentation of flow into discrete outlets. Both effects are only measurable if peaks are either monitored continuously or at least measured at multiple column positions. For isolation processes, these nonideal flow phenomena mean that more outlet streams have to be collected in order to achieve maximum yield and thus the achievable concentration factor is somewhat lower than the theoretical maximum. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 559–568, 2002.

Controlled shear affinity filtration (CSAF) integrates animal cell separation and product isolation in a single unit operation through the use of a specifically designed rotating disk filter with incorporated membrane chromatography column. Because of the decoupling of shear force and pressure generation and the specific hydrodynamics of the system, shear rates can be easily optimized and precisely controlled to maximize filtration performance while viability of the shear sensitive animal cells is maintained. In this study, the general methodology is demonstrated using the integration of Chinese hamster ovary cell separation and isolation of recombinant tissue plasminogen activator (t‐PA) as a model example. Direct capture of t‐PA from cell culture broth was realized by using custom‐made affinity membranes with lysine as a robust, small molecular weight affinity ligand. Small‐scale t‐PA adsorption experiments, as well as microfiltration experiments, were used to design the integrated CSAF process. A Chinese hamster ovary batch culture was processed with a lab‐scale prototype, yielding 86% of the t‐PA in the concentrated, particle‐free eluate, whereas 95% of the bulk protein was removed. Because the viability of the cells is not significantly affected and high specific flux rates can be achieved, the CSAF technology should also be well suited for continuous perfusion with integrated product isolation. A truly continuous operation could be realized with two systems in tandem configuration. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 806–814, 2002.