Application Note USD 3412 Dilution of Multiple Buffer Concentrates with One Single-Use Flow Path

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www.pall.com/biotech

Application Note

USD 3412

Dilution of Multiple Buffer Concentrates with

One Single-Use Flow Path

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1 Introduction

Buffers are used in a variety of bioprocessing steps, ranging across multiple product recovery, purification and formulation processes such as diafiltration and chromatography. The use of buffers within downstream processing is particularly important as they are used to protect proteins from variations in pH and to ensure that proteins interact correctly with various media to maximize performance of the specified purification process. Thousands of liters of buffers are produced each year for use in the manufacturing of

biopharmaceutical products in clinical or commercial-scale processes, which requires a large resource input from the operator’s time producing the buffer solutions, to the facility space for storing the vast amount of buffers created, and the logistics of using them across the unit operations.

Within traditional facilities, multiple buffers of varying specifications are made for bioprocessing operations. Buffers are generated in vast quantities (a typical monoclonal antibody (mAb) process could require

approximately 2,000 L of a buffer, or greater than 100,000 L of buffer per batch depending on the mAb titer, and the bioreactor volume) and are stored in large containers with significant footprint within the bioprocess facility until use. More buffer is subsequently created where needed, keeping buffer equipment at high utilization levels. This applies certain pressures on the buffer preparation operators, as well as the facility, to ensure buffers are readily made and can be stored efficiently. If multiple batches of buffer are required for a bioprocessing step, there is a risk of batch-to-batch variability which adds resource and time pressures as extra quality control (QC) procedures are required for each additional batch. In addition, there is an increasing consumables and cleaning costs associated with current buffer preparation systems, in order to ensure there is minimal risk of buffer contamination.

To optimize the buffer process, the Allegro Connect buffer management system (BMS) can be used to provide users with an efficient and optimized design for buffer management. The Allegro Connect BMS is an automated in-line buffer dilution system designed to manage the production of diluted process buffers on-demand and just-in-time from 5x to 20x stock buffer concentrates to feed various unit operations. The system consequently reduces the operator labor time, the facility space requirements and logistical bottlenecks when compared to traditional buffer management approaches, improving overall flexibility and ease-of-use.

In this study, the capacity for the Allegro Connect BMS to produce multiple diluted buffer solutions from stock buffer concentrates using one single-use flow path for bioprocessing was evaluated. This required the dilution of multiple pre-made stock buffers to desired specifications using the Allegro Connect BMS.

Results from the study demonstrate the system functionality to switch between multiple different buffer concentrates with minimal downtime on the system using the same single-use flow path. The findings also establish how applying a fast, effective flush between each buffer dilution can ensure the flow path was suitably cleaned and maintained minimal contamination risk.

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Figure 1.

The Allegro Connect buffer management system connected to one stock buffer work station (pictured left) and one process buffer work station (pictured right). The system pictured contains a Siemens♦

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2 Materials and Methods

2.1 Materials

The equipment and materials used for this study are shown in the Tables 1 to 3 below.

Table 1.

Equipment used for study investigating the dilution of multiple stock buffers

Part Number Description Manufacturer

ACBMSEUPLC Allegro Connect buffer management system Pall

ACBMSWS Buffer management workstation assembly Pall

ACBMSFT Buffer management filter trolley Pall

ACBMSFSK Buffer management flow sensor kit Pall

Table 2.

Single-use consumables used for study investigating the dilution of multiple stock buffers

6431-1420S Allegro Connect buffer management ½ in. feed including flow sensor Pall

6431-1420U Allegro Connect buffer management ½ in. outlet Pall

6431-1418E Allegro Connect buffer management ½ in. transfer line, 0.7 m tubing Pall 6431-1418F Allegro Connect buffer management ½ in. transfer line, 1.6 m tubing Pall 6431-1417W Allegro Connect buffer management ½ in. 100 L biocontainer Pall

6431-1420V Allegro Connect buffer management ½ in. drain control Pall

Table 3.

Materials used for study investigating the dilution of multiple stock buffers

10119CU Glycine VWR

30018.32 5 M Hydrochloric acid VWR

103157P Tris VWR

27808.468 Sodium chloride VWR

APPC183508.1211 10 N Sodium hydroxide PanReac Applichem

11591 Sodium monobasic phosphate VWR

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2.2 Method

This testing incorporated the use of the Allegro Connect BMS and two workstations, one to contain the stock buffers in 100 L single-use (SU) biocontainers, and the other to contain the diluted process buffers in 100 L SU biocontainers. The system was set-up in accordance with the instructions for use (Pall document reference USD 3416), please see Figure 2 for reference (the optional filter trolley, although not used for this study, can hold up to six 254 mm (10 in.) filters, which can be incorporated in to the process flow for additional sterility).

Figure 2.

Allegro Connect buffer management system set-up

The Allegro Connect BMS houses a single-use manifold made of three key parts, these are:

 Inlet manifold

 Outlet manifold

 Drain manifold

One overall manifold made from the three parts was used in this study to demonstrate the application of creating multiple process buffers using the same single-use flow path.

The cycle fill functionality (used for downstream process (DSP) operations which have multiple, simultaneous buffer draws or low flow singular draws) of the Allegro Connect BMS was used to continually fill process buffers into the process biocontainers on the workstation. This was performed by the system mixing the desired stock buffers with the water feed pumped into the system. Each stock and process buffer used has a known pH and conductivity determined via benchtop studies prior to application on the Allegro Connect BMS system.

Stock Buffer

Workstation Management Buffer

System

Filter Trolley

(Optional) Process Buffer Workstation

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www.pall.com/biotech 7 Each process buffer was created using the cycle fill phase on the Allegro Connect BMS at the maximum pump flow rate for both stock buffer and water feed to achieve a desired dilution factor, and to reach the conductivity and pH values pre-determined from the benchtop studies. As the buffer mixing on the system enters and remains within the desired conductivity band, the flow of the diluted buffer switches to direct filling of the specified process biocontainers on the workstation.

Between each process buffer created in a cycle fill phase, the single-use manifold on the system was flushed with water to drain until the conductivity within the system manifold decreased below 0.022 mS/cm. Once this set-point has been reached the system recognizes that the manifold is suitably cleaned, and begins to stabilize the next process buffer for re-fill of the biocontainers (this flushing process happens after every buffer dilution in every dilution cycle).

The study used four stock buffer solutions for cycle filling which are shown in Table 4.

Table 4.

Stock buffers and process buffers used for study

Stock Buffer Dilution Factor

Process Buffer Conductivity Set-Point (mS/cm) pH Set-Point 1 M Glycine-HCl, pH 3.0 10x 0.1 M Glycine-HCl 2.36 3.0 0.1 M Tris-HCl, 0.5 M NaCl, pH 8.0 10x 0.01 M Tris-HCl, 0.05 M NaCl 5.83 8.0 0.2 M Tris-HCl, 1 M NaCl, pH 8.1 20x 0.01M Tris-HCl, 0.05 M NaCl 5.83 8.0 0.2 M Sodium phosphate, 1 M NaCl, pH 6.4 20x 0.01 M Sodium phosphate, 0.05 M NaCl 6.49 7.0

The process control parameters to maintain optimum buffer development on the system are shown in Table 5.

Table 5.

In-line process parameters

Parameter Value

pH tolerance Target set-point ± 0.15

Conductivity tolerance Target set-point ± 5%

Flow rate range (LPH) 1360 – 1450

Flushing flow rate (LPH) 1530

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3 Results

The Allegro Connect BMS demonstrated throughout the buffer cycle fill study that flushing with reverse osmosis water between buffers to reach conductivity measurements below 0.022 mS/cm, can be achieved within approximately 30 seconds. The average conductivity readings for the outlet conductivity probe, post-flush, was approximately 0.015 mS/cm. Once flushing was completed, the stabilization for the next buffer as part of the cycle filling can be achieved within approximately 40 seconds before beginning the next process buffer biocontainer filling phase, this is illustrated in Figure 3. This was repeatable throughout the study using all four buffer concentrates over a range of conductivity and pH specifications. The study exemplifies the minimal downtime experienced when switching between buffers on the Allegro Connect BMS. Figure 4 illustrates how within a cycle fill phase the buffer remains within specification for both conductivity and pH. Figures 5 and 6 also highlight the consistency across multiple fill cycles (6 total) for conductivity and pH, respectively, for a single buffer to be flushed, stabilized, and begin filling.

For both conductivity and pH measurements across all testing, once the in-line dilution was stable, measurements remained within specification with only slight deviations by no more than approximately 2 seconds for all data sets, showing good repeatability of buffer stabilization.

Figure 3.

Graph showing multi-buffer filling, stabilization and flushing cycles for the 20x dilution of the 0.2 M Sodium phosphate, 1 M NaCl, pH 6.4 stock buffer, and the 10x dilution of the 1 M Glycine-HCl, pH 3.0 stock buffer

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Figure 4.

Graph showing the accuracy of buffer management system to remain within specification for pH and conductivity during the 20x dilution of the 0.2 M Sodium phosphate, 1 M NaCl, pH 6.4 stock buffer

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Figure 5.

Graph showing the accuracy of the buffer management system to remain within specification for conductivity during the 10x dilution of 0.1 M Tris-HCl, 0.5 M NaCl, stock buffer, following flushing and stabilization cycles

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Figure 6.

Graph showing the accuracy of the buffer management system to remain within specification for pH during the 10x dilution of 0.1 M Tris-HCl, 0.5 M NaCl, stock buffer, following flushing and stabilization cycles

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4 Conclusion

This study demonstrated that the buffer management system provides multiple buffers in-specification and ready to use by running the cycle filling operational phase. Each buffer stayed within defined tolerance bands for both pH and conductivity during dilution while filling the process biocontainers. This indicated the

consistency of the system to produce continuous in-specification process buffer with short stabilization times (<60 seconds) irrespective of dilution factor.

The results showed that the system reproducibly flushed the flow-path sufficiently between buffer cycles to clear any residual buffers (<30 seconds), before moving on to the subsequent buffer as part of the next cycle fill phase. This data demonstrated the consistency of the system to reduce carry-over with fast effective flushes (<30 seconds) and the capacity to create multiple buffers using one single-use flow path. The Allegro Connect buffer management system is suitable for application with capture, purification and polishing chromatographic operations. Furthermore, the system can provide a continuous supply of multiple in-specification diluted buffers with minimal downtime and is suitable to support continuous operations such as multi-column chromatography and multi-buffer filtration operations.

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Application Note USD 3412 Dilution of Multiple Buffer Concentrates with One Single-Use Flow Path