Automated Concentration and Diafiltration of Multiple siRNA Samples - A team of scientists from GE Healthcare collaborated with Merck scientists on a project to transfer a conventional manual concentr

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Automated Concentration and Diafiltration of Multiple siRNA Samples
A team of scientists from GE Healthcare collaborated with Merck scientists on a project to transfer a conventional manual concentration/diafiltration process for siRNA production.


BioPharm International
Volume 26, Issue 2, pp. 22-26

EXPERIMENTAL METHOD

To implement the automation strategy for concentration and diafiltration, the following equipment reconfigurations were performed:

1) the air sensor was moved from transfer inlet 1 to the common transfer valve block outlet

2) a fraction collector was added to retentate outlet 3 to allow for multiple sample processing

3) the inlet ports on the ÄKTAcrossflow system were reassigned in the method.


Figure 2: ÄKTAcrossflow modified transfer inlet configuration enabling automated multiple sample processing.
Transfer inlet configuration. The system air sensor controlled sample volumes processed across all inlets. Water from a hydro purification system was used for rinsing, water flux test, flush, and diafiltration steps. Water was used to condition the filter. The modified inlet configuration is shown in Figure 2.

Fraction collector configuration. The Frac-920 fraction collector was programmed to collect five fractions for each sample processed. The collection sequence was programmed as shown below:

Tube 1: Reservoir volume at product end

Tube 2: Product chase

Tube 3: Reservoir volume at buffer flush end

Tube 4: Flush chase

Tube 5: Fraction collector tubing clean via 25 mL buffer flush.

Merck reported that there were no cross contamination issues with their samples using the 25 mL buffer flush as determined by HPLC analysis.


Table I: The product step endpoints.
Method configuration. The ÄKTAcross-flow method wizard in the UNICORN software was used to create the initial method framework, including the pre- and postproduct steps (see Figure 3). Scouting was enabled to vary the sample inlet used for each run. The ÄKTAcrossflow method was modified to prevent the loading of the subsequent sample onto a fouled filter. This was achieved with inclusion of the flux drop calculation mode, combined with conditional programming on the percent flux drop. Pre- and postwater flux test results were compared within the same run. If the percent flux drop, as set by the user, was outside the threshold, a message would appear on screen for the user to take action while the system entered into a hold state with the permeate outlet in recycle mode. The goal of the method was two-fold—firstly, to concentrate the product to the minimum working volume, and secondly, to remove as much salt as possible.


Figure 3: Flow scheme of ÄKTAcrossflow system. The air sensor is moved from transfer inlet 1 to the common transfer outlet. The fraction collector is connected at the retentate valve block 3 outlet for product recovery.
Starting volumes were typically 0.6–2 L [5–20 OD/mL]. The system holdup volume (recirculation loop including filter) was 45 mL. The molecular weight cut-off for the membrane filters was 1 KD and the membrane surface area was 1 ft2. The product step endpoints were as shown in Table I.


Figure 4: UNICORN scouting method setup. Sample names have been removed to protect proprietary information.
An example of the method scouting setup in the UNICORN software is shown in Figure 4. User configured options are available each time a set of samples is processed with the same method. Parameters such as sample inlet and sample volume are updated as needed for each sample set. Parameters that are identical for each sample, such as diafiltration endpoint and flux drop percent, can also be shown for verification of status.


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