Streamlining the production process

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

Markus Pitkanen Senior Research Engineer GE Healthcare Life Sciences
Fredrik Lundström Global Product Manager GE Healthcare Life Sciences

Polishing and concentration connected in one automated method

For biofabrication operations, production processes are usually carried out in a series of separate unit operations. The equipment included is often controlled at the instrument level. Such a configuration requires intermediate handling of the bioprocess material, with manual transfer of the product between steps.

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

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

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

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

After a step defined in an individual method has been executed on one system, the Ready statement initiates the start of another individual method on another system.


Materials and methods

A method of polishing and concentrating bovine serum albumin (BSA) was used as a 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 mode, where the flow and wash fractions were directly transferred to ÄKTA readyflux.

For the concentration step, ÄKTA readyflux was fitted with a size 6 hollow fiber filter with a nominal molecular weight cutoff of Mr 10,000 (NMWC). In this step, the BSA was concentrated five times and diafiltered seven times 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 configuration with two systems integrated in a method queue, one of the computers in the system must be defined as the “controlling” computer (via Connect to System), and the other must be defined as ” display only ”to allow the execution of the methods.

Here, the ÄKTA ready system computer has been defined as the “control” computer, while the ÄKTA readyflux system computer has been defined as “display only”.

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

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

Figure 1. Hardware and software configuration for ÄKTA ready and ÄKTA readyflux. EQ: balancing; FT: flow through; DF: diafiltration; IC: instrument configuration; LAN: local network; NA: not applicable.

Configuring the Method Queue

For polishing using ÄKTA ready, individual methods have been created for column cleaning in place (CIP), equilibration, loading, stripping and storage. For concentration using ÄKTA readyflux, individual methods have been created for CIP, equilibration, batch concentration, diafiltration, final concentration, recovery and storage using methods. predefined 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 the initiator and included in two individual methods on ÄKTA ready: at the start of the CIP method to initiate the CIP of ÄKTA readyflux and at the start of the strip method to initiate batch concentration on ÄKTA readyflux. Immediately after, the previous method was used as the boot initiator for all other individual methods.

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


Figure 2. Screen capture showing the Method Queue function of the UNICORN software, listing the methods to control the two systems: ÄKTA ready and ÄKTA readyflux. The following methods are started immediately after the previous method or by the Ready statements.

Secure delivery of instructions

It is also important that the system receiving the Ready instruction is in passive mode to ensure safe transfer of instructions. The instruction is sent once to the “receiver”, and there is no reception at the “sender” if the instruction is received. The Ready instruction can be used in either direction: “control” to or from “view only”.

Here the Ready instruction has been programmed to be sent from the “control” computer (ÄKTA ready) to be received by the “view only” computer (ÄKTA readyflux).

For system programming, the time parameter was used as the basis, as it is recommended to use the same basis for all methods included in the method queue. For ÄKTA ready, all methods were initially designed for a flow rate of 8 L / h. The CIP was designed to have a contact time of 60 min, and the balancing step was designed to last 30 min. The loading and washing steps have been merged into a single method, denoted Load. The tape and storage methods were designed to both run for 22.5 min.

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

The balancing was designed to work with 6 L of balancing buffer, which was added to the recirculation bag and rinsed through the filter. The batch concentration was designed to read the starting volume of the bag and concentrate that volume five times.

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

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

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


Conclusion

Here we demonstrate the integration of the ÄKTA ready single-use chromatography system and the ÄKTA readyflux filtration system (figure 3). Creating the method was straightforward, using method phases predefined in the software. The polishing step and the subsequent concentration step were successfully executed consecutively using the Method Queue function and the Ready statement. Alarm functions alerted if something requires operator attention.

When unit operations are integrated, resources can be freed up and overall processing times reduced. Direct connection of unit operations minimizes intermediate holding steps, which would otherwise require manual intervention, and allows the operator to control multiple instruments from a single computer.

As the two systems used in this job are controlled by the same software, integrated process steps can be performed without the need for additional automation software.

Single-use flow paths omit the need for costly and time-consuming cleaning and cleaning validation operations. Bringing flexibility and speed to operations, the solution described is particularly beneficial for manufacturers operating on a smaller scale, with many parallel productions.


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



























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