Vogel Jens H, Nguyen Huong, Pritschet Markus, Van Wegen Richard, Konstantinov Konstantin
Bayer Corporation, Biotechnology Unit, Cell Culture & Bioprocess Engineering Dept., Berkeley, CA, USA.
Biotechnol Bioeng. 2002 Dec 5;80(5):559-68. doi: 10.1002/bit.10411.
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 degrees ) 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.
从细胞培养上清液中分离重组蛋白药物通常采用分批模式进行，即便发酵过程本身是连续的。作为一种新方法，连续分离技术如连续环形色谱法（CAC）可用于连续分离。通过在中试规模（即每天144 - 288升）下从细胞培养上清液中连续分离重组凝血因子VIII（rFVIII），证明了CAC在工业应用中的潜力。可在94%的回收率下实现30倍的浓缩，同时纯度提高3至5倍。为此，将一种分批直接进料离子交换色谱方法应用于商业化制备型CAC系统（P - CAC）。采用顶空进样技术使浓缩因子最大化，同时通过专门改进的入口几何结构解决了缓冲液不相容问题。为了实现与生产FVIII的灌注发酵罐的无菌在线连接，开发了一种可高压灭菌的中试规模P - CAC原型。P - CAC的一般特性揭示了该技术目前的一个局限性，即出口流速变化高达±20%。这些流速变化主要是由环形树脂床不均匀引起的，进而导致“峰摆动”，即峰位置（高达±4度）和形状（例如，A(s)=0.9 - 1.4）作为柱位置的特定函数出现轻微变化。一些额外的峰展宽虽然不太显著，但由“峰振荡”效应引起，该效应是由于流体必须分割成离散出口所致。只有在连续监测峰或者至少在多个柱位置测量峰时，这两种效应才可以测量。对于分离过程而言，这些非理想的流动现象意味着必须收集更多的出口流以实现最大回收率，因此可实现的浓缩因子略低于理论最大值。
