February 15, 2018 (Vol. 38, No. 4)

Polishing and Concentration Connected in One Automated Method

For biomanufacturing operations, production processes are typically performed in a series of separate unit operations. Included equipment is often controlled at an instrument level. Such a setup demands intermediate handling of the bioprocess material, with manual product transfer between steps.

Integration of the different unit operations streamlines the manufacturing process and reduces manual interaction between process steps. Here, we describe integration of a polishing step with the subsequent concentration step-by-direct connection of the ÄKTA™ ready chromatography system to the ÄKTA readyflux filtration system.

ÄKTA ready is a liquid chromatography system built for process scale-up and manufacturing. ÄKTA readyflux is a tangential flow filtration (TFF) system intended for both microfiltration and ultrafiltration applications in both upstream and downstream processes, ranging from pilot to small manufacturing scales.

For flexibility and ease-of-use, ÄKTA ready and ÄKTA readyflux operate with single-use flow paths, and both systems are controlled through the UNICORN™ software, eliminating the need for a separate automation software.

For systems controlled by UNICORN, connected processing can be facilitated by the Method Queue function and Ready instruction. Here, the polishing and concentration process was based on individual methods executed using Method Queue, which defines which systems are included as well as the order of individual methods and their initiator: at a set time point immediately after the preceding method or at the Ready instruction.

Once a set stage in an individual method has been executed on one system, the Ready instruction initiates start of another individual method on another system.

Materials and Methods

A process for polishing and concentration of bovine serum albumin (BSA) was used as model. For the polishing step, a ReadyToProcess™ DEAE Sepharose™ Fast Flow 1-L column was connected to the ÄKTA ready system. The column was operated in flow through mode, where the flowthrough and wash fractions were directly transferred to ÄKTA readyflux.

For the concentration step, ÄKTA readyflux was equipped with a hollow fiber filter size 6 with an Mr 10,000 nominal molecular weight cut-off (NMWC). In this step, BSA was five times concentrated and sevenfold diafiltered before final concentration and recovery.

ÄKTA ready and ÄKTA readyflux were connected to one computer each, both installed with UNICORN 7.1 software with instrument configurations for both systems. In a setup with two integrated systems in a method queue, one of the system computers needs to be set as the “controlling” computer (via Connect to System), and the other should be set as “view only” to enable execution of the methods.

Here, the ÄKTA ready system computer was set as the “controlling” computer, whereas the system computer of ÄKTA readyflux was set as “view only.”

A database must be installed on one of the system computers or on an external server. Here, the database was installed on the ÄKTA ready computer, and the two systems were interlinked over a local area network. The two computers were equipped with two network cards each, one for communication between the system and its computer, and one for communication between the system and the database. A schematic description over the setup is given in Figure 1.

For transfer of the process material, process steps were interconnected via tubing from the ÄKTA ready outlet to the ÄKTA readyflux recirculation bag.

Figure 1. Setup of hardware and software for ÄKTA ready and ÄKTA readyflux. EQ: equilibration; FT: flow through; DF: diafiltration; IC: instrument configuration; LAN: local area network; NA: not applicable.

Method Queue Setup

For polishing using ÄKTA ready, individual methods were created for column cleaning-in-place (CIP), equilibration, load, strip, and storage operations. For concentration using ÄKTA readyflux, individual methods were created for CIP, equilibration, batch concentration, diafiltration, final concentration, recovery, and storage operations using predefined methods in the Phase Editor. The methods were executed using the Method Queue function of the Unicorn software (Figure 2).

The Ready instruction was used as initiator and included in two individual methods on ÄKTA ready: in the beginning of the CIP method to initiate CIP of ÄKTA readyflux, and in the beginning of the strip method to initiate batch concentration on ÄKTA readyflux. Immediately after the Preceding Method was used as start initiator for all other individual methods.

To avoid interruption in the execution of the individual methods, it is essential to define in the start protocol for each individual method where and how the created data is saved, and the start questions should be disabled.

Figure 2. Screenshot showing the Method Queue function of the UNICORN software, listing methods to control the two systems: ÄKTA ready and ÄKTA readyflux. Subsequent methods are initiated immediately after the preceding method or by the Ready instructions.

Secure Handover of Instructions

It is also of importance that the system receiving the Ready instruction is in a passive mode to assure a secure handover of the instructions. The instruction is sent once to the “receiver,” and there is no receipt to the “sender” if the instruction is received. The Ready instruction can be used in both directions: “controlling” to or from “view only.”

Here, the Ready instruction was programmed to be sent from the “controlling” computer (ÄKTA ready) to be received by the “view only” computer (ÄKTA readyflux).

For programming of the systems, parameter time was used as base, as it is recommended to use the same base for all methods included in the method queue. For ÄKTA ready, all methods were initially designed for a flow rate of 8 L/h. CIP was designed to have a contact time of 60 min, and the equilibration step was designed to run for 30 min. The load and wash steps were merged into one method, denoted Load. The strip and storage methods were designed to both run for 22.5 min.

For ÄKTA readyflux, all methods were designed for a transmembrane pressure of 1.5 bar and a feed flow rate of 2 L/min. CIP and rinse were merged into one method (CIP) designed with an intermediate pause for 30 min between CIP and rinse to reach a 60 min total contact time for cleaning.

Equilibration was designed to work with 6 L of equilibration buffer, which was added to the recirculation bag and flushed through the filter. Batch concentration was designed to read the start volume of the bag and concentrate this volume five times.

The diafiltration step was designed to read the bag volume at start and retain this volume by adding diafiltration buffer until the permeate conductivity exceeded 17 mS/cm or until more than seven diafiltration volumes were achieved. Final concentration was designed to concentrate the recirculation bag volume to less than 2 L.

Recovery was designed to recirculate the concentrate for 1 min and then to empty the system via the recovery valve until the recirculation bag weight was less than 0.2 L, after which 2 L of diafiltration buffer was added to the system and recirculated for 3 min before the system was emptied again via the recovery valve until the recirculation bag weight was again less than 0.2 L. Recovery by recirculation was followed by recovery by air blowdown, which lasted 2 min. The storage method was designed to add storage solution until 2 L permeate was collected.

When predefined method phases for programming are used, it should be noted that the End Block instruction after the Watch functions are by default set to 9999 min. To avoid overconcentration, the default setting should be changed from 9999 min to an applicable time interval, where 0 min is directly after occurred watch condition.


Here, we demonstrate integration of the single-use ÄKTA ready chromatography system and ÄKTA readyflux filtration system (Figure 3). Method creation was straightforward, using method phases predefined in the software. The polishing step and the subsequent concentration step were successfully executed in a consecutive manner using the Method Queue function and the Ready instruction. Alarm functions alerted if anything needed operator attention.

When unit operations are integrated, resources can be released and overall process times reduced. Direct connection of unit operations minimizes intermediate hold steps, which would otherwise require manual intervention, and enables the operator to control several instruments from one computer.

As both systems used in this work are controlled through the same software, the integrated process steps can be executed without the requirement of any additional automation software.

The single-use flow paths omit the need for costly and time-consuming cleaning and cleaning validation operations. Bringing flexibility and speed to operations, the described solution is especially beneficial for manufacturers operating in smaller scales, with many parallel productions.

Figure 3. Execution of individual methods on ÄKTA ready and ÄKTA readyflux using the Method Queue function and the Ready instruction of the Unicorn software.

Markus Pitkanen ([email protected]) is senior research engineer and Fredrik Lundström ([email protected]) serves as global product manager at GE Healthcare Life Sciences.
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