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May 01, 2012 (Vol. 32, No. 9)

Integrating Clarification & Purification Steps

Simultaneous Automated Chromatography and Crossflow Filtration at the Pilot Scale

  • UNICORN Software and Method Setup

    Click Image To Enlarge +
    Figure 2. Graphical representation of the Method sequences for UniFlux 10 and ÄKTApilot

    Method creation using the method wizard for the ÄKTApilot and a method template set for UniFlux 10 saved at least 2–3 days of writing and testing time required when creating methods from scratch. The method queue feature in UNICORN allows the user to link methods together, enable optional start times for the entire queue, or start time separation between methods within the queue.

    For this application five methods for each system were incorporated into one method queue file (Figure 2).

    Wherever possible, the method queue sequence was configured such that both systems were run simultaneously to reduce wait times between process steps. All UniFlux methods, except for water flush, contained a Ready instruction to enable simultaneous processing by starting the ÄKTApilot system. On the chromatography side, the purification method contained a Ready instruction to start the postproduct method water flush of the filter. The timing of this signal was determined empirically.

  • Simultaneous Processing

    Figure 2 shows the method sequence between both the filtration and chromatography systems. Each method is numbered to the left of each box to illustrate the order in which it was executed.

    The preproduct equilibration of the filter automatically started the water flush of the ÄKTApilot system from storage, followed by equilibration of the chromatography column.

    The product section began with clarification/diafiltration of the yeast harvest with passage of the target protein into the permeate. An intermediate vessel between the two systems contained the permeate outlet as well as the chromatography inlet tubing. Protein was pumped onto the column from the intermediate vessel until detection of air terminated the load.

    The postproduct sequence began with a water flush of the hollow-fiber filter. This method was started via Ready signal from the purification method. It was followed by CIP of the filter. At this time the purification method was completed and a signal was sent to begin CIP of the chromatography column.

    CIP of the filter was completed first, initiating filter storage. At this time CIP of the column was completed. The ÄKTApilot system received a signal from the filter storage method to begin storage of the system and column.

    Some manual interaction was required to move the inlet line on UniFlux 10 to the next buffer or sample, and the operator was alerted via an on-screen message. The current UniFlux platform offers a standard option of a four-valve inlet panel for increased automation. The ÄKTApilot system only required manual interaction at the start of the equilibration method and for the inlets to storage method at the end of the process. Again, the operator was notified via on-screen messaging. The method was paused until the operator confirmed the correct configuration.

  • Results

    Click Image To Enlarge +
    Figure 3. ÄKTApilot result chromatogram showing the resultant purification profile.

    The chromatograph for the resultant purification profile (Figure 3) contains Set Mark notations to show the different segments of the method such as sample load, wash out unbound, etc. A mark is included to show the signal to UniFlux 10 to begin the postproduct filter water flush method. The eluted peak is collected via conditional programming to a dedicated outlet.

  • Conclusions

    Using standard UNICORN software to simultaneously control a UniFlux crossflow filtration system and an ÄKTA chromatography system allowed the clarification and purification unit operations to be completed in a reduced amount of time. By decreasing or eliminating long hold times between the two unit operations the in-process storage requirement can be completely eliminated.

    This type of process intensification step contributes to the robustness of the process by minimizing the risk of product degradation during storage and also provides economic benefit by reducing overall process time. This integration technology has been proven at pilot scale for the production of proteins or any other application requiring these unit operations.

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