Connecting several unit operations to remove non-value-adding steps is one method of intensifying your bioprocess to achieve better operational efficiency. In this study, we integrated an inline conditioning (IC) system that served as a buffer preparation skid with chromatography and filtration systems. This automated the buffer preparation process and reduced the holdup volumes and tanks needed for buffers by feeding different downstream systems with buffers on demand, saving time, space, and costs.

Our setup consisted of a BioProcess™ IC system connected to ÄKTAprocess™ and ÄKTA ready™ chromatography systems. The BioProcess™ IC system can be connected to a total of five different systems. The IC system automatically prepared and delivered the requested buffers to the connected systems directly. During the run, only the pumps on the IC system were running, pushing the buffer through the pump on the ÄKTAprocess™ system or bypassing the peristaltic pump in the ÄKTA ready™ system.

With this setup, only one skid was driving the flow, making longer distances between the systems possible. The receiving system determined the flow rate in the IC system by using the external flow setpoint function in the UNICORN™ software.

Process engineers with ÄKTA process chromatography system with ILD

Fig 1. Schematic picture of the setup with ÄKTA ready™ XL (ARX in the figure) and ÄKTAprocess™ (AP in the figure) chromatography systems.

With this setup, you can place the IC system in a clean non-classified area with a transfer hose passing through the wall, connected to the receiving downstream systems placed in the cleanroom. This saves space in the process area and intensifies the buffer preparation areas. Using a setup like this also enables inline conditioning in combination with single-use technologies for the product contact parts of the process line. With inline conditioning, you can reduce the buffer bottleneck that comes with increased titers and batch sizes, while taking advantage of the benefits of single use.

One limitation is that the IC system can only deliver a buffer to one system at a time. Two IC systems, on the other hand, can be used in parallel to give faster processing, easier scheduling, and utilization of the downstream processing suites. An example of such a production layout is shown in Figure 2. This setup reduces the number of bins to be moved around and tubing passing through the wall even further. In the production setup, only one hose per downstream system with IC is needed compared to one hose per buffer with traditionally prepared buffers. In the IC-connected example below, most of the stock solutions are prepared for two to five batches, while in traditional buffer preparation all solutions are prepared for only one batch at a time.

Process engineer working with ÄKTA process chromatography system

Process engineers working with ÄKTA process chromatography system HMI

Fig 2. (A) Example production layout with two BioProcess™ IC systems connected to three downstream processing suites, including ÄKTA ready™ and ÄKTA ready™ XL chromatography systems, and a buffer preparation area. (B) Here, the IC systems are replaced with single-use vessels for buffer hold and a large mixer in a more traditional setup.

IC — delivering solutions on request

When using UNICORN™ software in a connected buffer preparation setup, the downstream systems connect to the IC system and send a buffer request. The IC system then finds the requested buffer and starts preparing it. Once the buffer is within the required specification, the IC system opens the outlet connected to the downstream system and starts delivering the buffer. The flow rate is determined by a flow setpoint to the IC system. Each downstream system has a defined outlet on the IC system (for example, Fig 1).

When the downstream system does not need more buffer, it signals the IC system, which then stops the buffer preparation and delivery. When the downstream run is completed, the IC system is released and can then be connected to another system. The method for IC can contain several different buffers, that is, all the buffers needed for one or several purification processes. When it comes to other automation solutions than UNICORN™, for example, DeltaV™ automation solution, the process for controlling and automating the connected systems may work differently.


Figure 3 shows an overlay of the pH, conductivity, and flow rate curves from the BioProcess™ IC system and ÄKTA process™ system. The run mimicked a capture step. The Figure shows that the flow rates were the same in both systems. The pH and the conductivity were also aligned in both systems as expected. The drift seen in pH during load in the downstream system is due to that water was used instead of product.

Overlay of result files from BioProcess™ IC and ÄKTAprocess™ system capture step.

Fig 3. Overlay of result files from BioProcess™ IC and ÄKTAprocess™ system capture step.

Replacing manual buffer preparation, using the BioProcess™ IC System

With IC, buffers can be produced just in time from single component stock concentrates of acid, base, and salt, diluted to the correct proportions. IC systems have been designed to prepare buffers inline consistently and accurately, replacing manual preparation.

For accurate buffer formulation and consistency between buffer preparation runs, the feedback mode controls the set of critical process parameters. There are three modes of feedback control that can be used: recipe and flow; pH and flow; and pH and conductivity. UNICORN™ software has functions that are used to release and monitor the buffer to ensure that only the buffer within the set specifications of pH and/or conductivity is sent to the receiving system.

The receiving system can be used to verify that the pH is stable, and/or that the conductivity is within limits before putting the column inline. Single-use probes do not have the same accuracy as reusable probes.

Using an IC system to prepare buffers inline and on-demand minimizes the time needed for manual preparation of solutions and the number of hold vessels can be reduced. Figure 4 compares the footprint and number of solutions needed for a process line with manual buffer preparation with one utilizing the IC-connected setup for a batch size of 10 kg mAb from a 2000 L bioreactor using about 12 000 L buffer.

In the calculation, we assumed running 30 batches/yr and preparing small-volume stock solutions for more than one batch at a time. We assumed that all solutions were prepared using single-use mixers and stored in single-use bags with containers. The results show that the preparation time can be reduced by 40% and the return on investment is less than 2 yr.

Normalized comparison of footprint and number of solutions needed for a batch size of 10 kg mAb from a 2000 L bioreactor.

Fig 4. Normalized comparison of footprint and number of solutions needed for a batch size of 10 kg mAb from a 2000 L bioreactor.

Because IC systems can use single-component solutions, one stock solution can be used to produce several buffers with different concentrations, pH, or conductivities, reducing the number of solutions that need to be prepared in advance. Another benefit of using single-stock components is that higher concentrations can be achieved. As higher concentrations can be used, the stock solution volumes are significantly smaller compared to the final volume of buffer. IC technology also enables making stock solutions for more than one batch at a time, reducing labor even further.


Here we have shown that it is possible to prepare buffers using BioProcess™ IC system and to deliver them directly to different connected downstream systems. The buffers were delivered on demand without the use of buffer hold vessels reducing the space needed for buffer preparation. We have shown how this IC connected solution can be used to reduce non-value-adding steps like buffer preparation and storage of manually prepared buffers, which will intensify your process. In addition, IC systems in combination with single-use systems can reduce change-over time between batches.

Want to learn more about large-scale buffer management? Find the resources you need on our webpage dedicated to buffer management for downstream bioprocessing.


The IC system was connected to two downstream systems — one ÄKTAprocess™ system and one ÄKTA ready™ system. The tubing used between the BioProcess™ IC system and ÄKTAprocess™ system was 4 m (16 mm i.d.) with a hold-up volume of 0.8 L. The tubing for the BioProcess™ IC system to ÄKTA ready™ system was 6 m (12 mm i.d.) to the junction after pump A with a hold-up volume of 0.7 L.

The IC method contained all the buffers needed for running the chromatography steps with each buffer having a unique buffer ID. The first step was run on the ÄKTA process™ system mimicking a capture step on a Protein A resin (Table 1). The second was an ion exchange bind/elute step run on the ÄKTA ready™ system (Table 2). The column was replaced with a short piece of tubing, and water was used as a product during load and applied using the pump on the downstream system. All stock solutions needed were connected to the IC system from start (Table 3).

The clean-in-place (CIP) solution during capture was applied with the downstream system pump. When this step was complete, the system then requested buffer ID 255 from Table 1. When this happens, the IC does not run a flow rate but will wait for the load or CIP step to finish.

Once a buffer was released and delivered to the downstream system the minimum time to rinse the downstream system, as programmed in the method, was 1 min before checking for a stable signal. This required 5 to 10 L of buffer depending on flow rate. The total rinse time before release of the buffer to the column was approximately 1.5 min. This step could be optimized to reduce the buffer volume required when compared to the 3 to 4 L needed to rinse the ÄKTAprocess™ system from the inlet (no AirTrap) or approximately 4 L needed to rinse the ÄKTA ready™ system from the inlet including AirTrap.

Table 1. Buffers prepared by the IC system for the ÄKTAprocess™ system capture step

Step Buffer Buffer ID CV Flow rate (L/h)
Equilibration 20 mM sodium phosphate, 150 mM NaCl, pH 7.4 1 2 440
Load Water 255 3 250
Wash 1 20 mM sodium phosphate, 500 mM NaCl, pH 7.0 2 3 440
Wash 2 50 mM acetate, pH 6.0 3 2 440
Elution 50 mM acetate, pH 3.5 4 4 250
Strip 100 mM acetic acid, pH 2.9 5 2 440
CIP 0.5 M NaOH 255 1 300
Re-equilibration 20 mM sodium phosphate, 150 mM NaCl, pH 7.4 1 2 440

Table 2.
Buffers prepared by the IC system for ÄKTA ready™ system polishing step

Step Buffer Buffer ID CV Flow rate (L/h)
Equilibration 50 mM acetate, 50 mM NaCl, pH 5.0 8 3 350
Load Water 255 6 350
Wash 50 mM acetate, 50 mM NaCl, pH 5.0 8 3 300
Elution 50 mM acetate, 400 mM NaCl, pH 5.0 9 4 190
Re-Equilibration 50 mM acetate, 50 mM NaCl, pH 5.0 8 3 350

Table 3.
Stock solutions used by the IC system to prepare buffers

Concentration (M) Component Type
0.35 Dibasic sodium phosphate Base
1.00 Sodium acetate Base
0.10 Sodium acetate Base
0.20 Monobasic sodium phosphate Acid
0.20 Acetic acid Acid
1.00 Acetic acid Acid
4.00 Sodium chloride Salt