Production of biopharmaceuticals often involves many different unit operations, such as cell culture, filtration, and chromatography. Connecting these individual steps into a fully continuous process can be challenging. Most studies on process intensification focus on single unit operations—like perfusion cell culture or continuous chromatography—rather than the full sequence. Examples where two or more unit-operations are integrated are especially valuable, as they provide insights into both the technical solutions and the overall process control.
In this article, we show how we can digitally and physically connect process scale downstream equipment to create an intensified process. The capture step and virus inactivation step for mAb downstream purification were connected using UNICORN™ software to control a large-scale ÄKTA ready™ chromatography system linked to a pair of automated single-use mixers.
Introduction
The biopharmaceutical industry continues to accelerate the adoption of automated solutions to support process intensification and enhanced process control. Advances in cell culture technology have shifted the primary biomanufacturing bottleneck from upstream to downstream operations, driving the need for more efficient and robust downstream purification strategies.
Connected and integrated processing has emerged as a promising approach to address these challenges. By physically linking multiple unit operations and controlling them through a single automation system, connected processing enables streamlined workflows that reduce production costs while maintaining product quality and throughput. Integrating unit operations can also minimize equipment footprint, reduce manual interventions, and improve overall operational efficiency, supporting better process economics and consistent performance.
In this article, we demonstrate the implementation of connected processing using the communication interface of the ÄKTA ready™ chromatography platform. A chromatography unit operation was integrated with a subsequent virus inactivation step into a coordinated process flow. The study was performed using the ÄKTA ready™ chromatography system XL SCS (standard configuration system) in combination with two Xcellerex™ XDUO mixers, highlighting the practical application and benefits of connected downstream processing.
Process setup for practical demonstration of connected capture and virus inactivation
The chromatography unit operation comprised of one SCS system, two mixer units—one designated for eluate collection and low pH titration, and one for incubation and subsequent neutralization—as well as an external pump facilitating controlled transfer between the two XDUO units (Fig 1).
Fig 1. Process set up.
The Xcellerex XDUO mixers are highly configurable to meet a wide range of application requirements, supporting upstream and downstream applications for automated mixing of buffer, cell culture media, product and intermediates, and other process fluids. Control can be performed locally or remotely for Xcellerex XDUO mixers used in FlexFactory™ and other biomanufacturing platforms.
With this setup, we show how mixers can be integrated with the ÄKTA platform using UNICORN™ control software, with the mixer data stored in the UNICORN™ result file and batch report giving a fully traceable process step. This plug-and-play functionality removes expensive and time-consuming automation work.
Process setup for virus inactivation
Virus inactivation is an example of a downstream unit operation that can be performed in Xcellerex™ XDUO mixers used in conjunction with chromatography. A commonly applied approach involves lowering the pH of the chromatography eluate (typically to below pH 3.6), maintaining the eluate at this pH for a defined incubation period (typically 30 to 60 min), and subsequently neutralizing the pH to preserve the integrity of the target molecule.
From a regulatory perspective, it is critical to ensure that the entire eluate pool is exposed to the virus inactivation conditions (low pH environment). Residual droplets adhering to the walls or roof of the mixer bag represent a potential risk, as they may not be sufficiently subjected to the low pH treatment and could contaminate the inactivated processed eluate. To mitigate this risk, the eluate is commonly titrated to low pH in one mixer and subsequently transferred to a second mixer dedicated to incubation and neutralization.
Our ÄKTA ready™ chromatography system XL unit has an updated design that allows control of up to five XDUO mixers. This enables a closed and connected process where functions like virus inactivation or feed conditioning can be integrated into the automation of the chromatography step with process data from the mixers stored in the UNICORN™ software. The UNICORN™ batch report includes all data without additional software needed for generating reports.
Materials and methods
In this proof-of-concept study (without feed material), ÄKTA ready™ chromatography system was used with an AxiChrom™ 600/300 column, two Xcellerex™ XDUO 200 mixers, and a peristaltic pump to form an integrated capture and virus inactivation process. This was followed by a cleaning-in-place (CIP) step on the column.
Buffers:
Elution buffer (Inlet A2): 25 mM acetate, pH 4.2
For 200 L of buffer, the following recipe was used:
- 222 mL acetic acid glacial
- 153 g Na-acetate (× 3 H2O)
- Distilled water to final volume of 200 L
Solution simulating feed and equilibration buffer (Inlet A1): Distilled water, NaCl added to a conductivity of > 0.5 mS/cm to enable using flow meter reading.
Solution simulating wash buffer and CIP solution (Inlet A3): Distilled water, NaCl added to a conductivity of > 10 mS/cm
Acid for pH adjustment in XD1: 1 M HCl
Base for pH adjustment in XD2: 2 M Tris base
The method phases are shown in Figure 2.
Fig 2. High level phase description of the connected operation. CIP = cleaning-in-place; CV = column volume; EQ = equilibration; VI = virus inactivation.
Notes:
- The mixer agitators will not start before level exceeds the low limit required by the hardware.
- The external pump used in this setup starts with Digital out 1 is set to Off.
- The manual pH control for the mixer works as follows:
Manual controlled value = 50%: no pump running
Manual controlled valve < 50%: acid pump running
Manual controlled valve > 50%: base pump running
- Trigger 2 was not strictly necessary for this sequence but was introduced to separate XD1 and XD2 sequences into independent Watch blocks.
Digital connectivity
The ÄKTA ready™ chromatography system XL provides built-in digital connectivity that enables integration with surrounding process equipment. Using PROFIBUS communication, the system supports digital connection for up to five XDUO mixers, allowing coordinated control and data exchange across chromatography and adjacent unit operations (Fig 3).
In addition, the system offers plug-and-play digital connectivity to the automatic bypass valve kit to connect to AxiChrom™ columns. This allows the bypass valve to be directly controlled from the chromatography skid without additional configuration, simplifying setup and ensuring reliable, automated operation.
Together, these digital connectivity features enable easier process integration, reduced manual intervention, and a more robust, scalable, and GMP-aligned manufacturing workflow.
Fig 3. The Xcellerex™ XDUO mixers controlled directly within UNICORN™ software.
Results
Using the integrated setup, the chromatography eluate was successfully collected in mixer 1 via Outlet 3 and titrated to low pH through controlled acid addition. The pH was reduced below 3.4 and maintained until a stable pH signal was confirmed, ensuring uniform exposure of the eluate to virus inactivation conditions. Following acidification, the eluate was transferred from mixer 1 to mixer 2 using an external pump.
The virus inactivation step was continued in mixer 2, by incubating the eluate at low pH for the defined hold time. Subsequent neutralization was achieved by controlled base addition until the pH exceeded 5.7, followed by verification of pH stability. After completion of the virus inactivation sequence, we performed a cleaning-in-place of the chromatography column and then equilibration to prepare the system for subsequent operations.
Fig 4. The chromatogram in UNICORN™ software displays the chromatography step combined with the virus inactivation steps performed in the two mixers. The chromatography system can be cleaned and re-equilibrated while the mixer steps are performed, saving time.
A: The eluate is collected in mixer 1 via Outlet 3.
B: Acid is added to mixer 1 until the pH in XD1 is reduced below 3.4, followed by a hold period to confirm pH stability.
C: The contents of mixer 1 are transferred to mixer 2 using an external pump.
D: The eluate is incubated at low pH in XD2.
E: Base is added to mixer 2 until the pH in XD2 rises above 5.7, followed by a hold period to confirm pH stability.
F: A CIP procedure is performed on the system.
G: The system is equilibrated in preparation for the next operation.
Example of production set-up
A proposed capture chromatography and virus inactivation solution that integrates into a coordinated and automated process flow could look as below. See Figure 5.
Fig 5. Automated process flow for capture chromatography (A) and virus inactivation (B).
The capture chromatography is performed using the SCS model of ÄKTA ready™ chromatography system XL controlled by UNICORN™ software, with an AxiChrom™ 600/300 column equipped with an automatic bypass valve kit connected directly to and controlled from the chromatography skid.
Following capture, the product stream is transferred to Xcellerex™ XDUO 100 L single-use mixers for low pH virus inactivation, enabling controlled titration, incubation, and neutralization within a closed, GMP-aligned setup. All process buffers are supplied from Allegro™ plastic totes, supporting flexible buffer management and single-use operation (Fig 6).
Fig 6. Aerial view of a cleanroom suite layout of an integrated solution with a capture chromatography step and automatic virus inactivation.
Conclusion
This application note demonstrates how digital and physical downstream connectivity can enable true process intensification in biopharmaceutical manufacturing. By integrating capture chromatography and virus inactivation into a single, coordinated workflow, the solution shows how multiple downstream unit operations can be automated and controlled through one platform. Using UNICORN software to integrate Xcellerex™ XDUO mixers with an ÄKTA ready™ chromatography system XL delivers a closed, traceable, and GMP‑aligned process. The approach reduces manual interventions, improves process control, and supports scalable, connected manufacturing—addressing one of today’s key downstream bottlenecks.