May 1, 2012 (Vol. 32, No. 9)

Simultaneous Automated Chromatography and Crossflow Filtration at the Pilot Scale

The production of biotherapeutics involves many unit operations, and it is common practice to store in-process material as dictated by the manufacturing setup and scheduling of unit operations.

The stability of in-process materials can vary widely, and it is important to maintain parameters such as bioactivity and stability throughout the entire process. When a target molecule is expressed as extracellular protein that is secreted into the bioreactor or fermentation broth, the unit operations of protein harvest and initial column capture can be combined to protect proteins with limited stability.

The elimination of storage requirements and susceptibility to any cooling or freeze/thaw cycles between these two unit operations can contribute to a more robust process. Already proven at bench-scale and now running at pilot-scale, scientists at GE Healthcare Life Sciences have used automated hardware and software to link two such processes together.

We found that both the initial clarification/diafiltration, using a microfilter, and the initial column capture step, using adsorptive chromatography, were completed in less than eight hours. If run traditionally as individual unit operations these steps would have required twice the time and storage of the target protein in between the two operations. In addition, we reduced the process time by 50% by using standard UNICORN™ software for both crossflow filtration on UniFlux™ to clarify and ÄKTApilot™ to purify a target protein from yeast in one day.

Process Description

The process to clarify/diafilter the target protein followed by column capture consisted of:

  1. Preproduct: Equilibrate both the filter and chromatography column.
  2. Product: Clarify/diafilter the sample while simultaneously loading the permeate onto the chromatography column. Terminate column load via air detection.
  3. Postproduct: At completion of clarification/diafiltration, run postproduct steps on the filter including water flush, clean-in-place (CIP), and storage.
  4. Postproduct: At completion of purification run postproduct steps on the column including CIP and storage.


Sample: BSA 1 g/L, 10% WCW baker’s yeast (locally sourced) in PBS buffer

Crossflow Filter: CFP2E5A hollow fiber (0.2 µm, 0.12 m2, 30 cm length)

Column: BPG™ 100/500 column with Q Sepharose™ Fast Flow media, 1 L

Normal Flow Filter: ULTA™ Cap CG 2″ capsule column guard

System Configuration

UniFlux 10 is a 10 liter per minute crossflow filtration system that can accommodate flat sheet cassettes or hollow-fiber filters for micro- or ultrafiltration applications. ÄKTApilot is a benchtop pilot-scale chromatography system capable of purifying 10 grams or more of product per cycle depending upon media capacity. Both systems are controlled by UNICORN software, a 21 CFR Part 11 compliant platform.

Figure 1 shows the configuration of the two systems for simultaneous processing. The outlet from the UniFlux 10 permeate is sent to an intermediate vessel where the inlet from the ÄKTApilot sample valve block is also placed. The intermediate vessel could be a single-use bag and tubing from ReadyToProcess™ products, or a cleanable container. Ports can also be configured to enable sampling during processing for analysis.

Figure 1. Flow scheme diagram with UniFlux 10 system and ÄKTApilot system configured for simultaneous processing: A column guard normal flow filter protects the chromatography media.

UNICORN Software and Method Setup

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.

Figure 2. Graphical representation of the Method sequences for UniFlux 10 and ÄKTApilot

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.


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.

Figure 3. ÄKTApilot result chromatogram showing the resultant purification profile.


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.

Deborah R. Cohen ([email protected]) is senior scientist, Fast Trak Biopharma Services at GE Healthcare, USA.

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