In this application note, we highlight the capabilities of the Allegro™ Connect depth filtration system to accurately and consistently conduct fully automated, in situ flow kit leak testing.
In addition, we demonstrate how the depth filtration system can be used for effective depth filter processing, through strategic process control in terms of flow and pressure. We also highlight effective product recovery (100%) of IgG solution, including recovery of excess product that is held up within the filters during processing.
Introduction
The Allegro™ Connect depth filtration system is a single-use, automated filtration system designed to deliver robust process control during the harvest and clarification steps, which are the primary recovery steps of purification in downstream processing.
The compact design of the system minimizes risk through a fully automated process with recipe-controlled steps. The steps include a pre-use flow kit leak test, system priming, product filtration, and buffer chase with all data stored in a batch reporting system, significantly reducing nonconformities and manual labor.
Design features allow configurability of each flow kit to meet various process needs, supporting different process trending sensors and a wide range of liquid filter capsules.
Materials and methods
All flow kits were installed and operated on the system according to the Allegro™ Connect depth filtration system Operating Instructions.
System and flow kits
Fig 1. Allegro™ Connect depth filtration system set up for this study.
Equipment and single-use flow kit
Some of the main equipment used in this study were:
Allegro™ Connect depth filtration advanced system: PLC 400 VAC, software automation
762 mm (30 in.) Supor™ EAV membrane in Kleenpak™ Nova capsules
Leak test
We installed flow kits according to the instructions in the standard installation phase on the system. Once installation was completed, the recipe continued to an automated flow kit leak test phase.
Before water or product commitment, the Allegro™ Connect depth filtration system raised the flow kit pressure to 0.5 bar (7.25 psi, 0.05 MPa) and measured the pressure decay over 10 min.
Constant flow control
To demonstrate the process control of the system under a typical depth filter clarification/harvest challenge, we filtered a cell culture simulant (chemically lysed brewer’s yeast, Saccharomyces cerevisiae) through a first stage of 6 m2 single-layer media grade K250P, and a second stage of 2 m2 single-layer media grade EKSP using fixed flow control. Before operation, we used the standard phases for filter priming, flushing, and buffer conditioning.
Proportional-integral-derivative (PID) control parameters were set for optimal flow control at 10 L/min.
Constant flow to constant pressure transition
High initial flows and high final pressures cause poor filter performance. An alternative control strategy to protect against this is to switch from constant flow to constant pressure control during the same run at a predetermined critical pressure. To show this transition, we simulated fouling of a filter using a single pinch valve in place of the filters.
We pumped a dilute salt solution through the pinch valve and operated the processing phase at a constant flow of 20 L/min until a pressure of 1.0 bar (14.5 psi, 0.1 MPa) was reached at approximately 20 min. The processing phase then automatically switches to control the pressure at 1.0 bar for a further 20 min by reducing the flow. PID control parameters were set for optimal control of both flow and pressure with separate PID settings for each parameter.
Product recovery
We used a 400 L human immunoglobulin G (IgG) solution in phosphate buffered saline (PBS) to demonstrate product recovery performance without the interference of cellular material. We used the standard phases for processing and buffer flush with the buffer chase material collected in 31 L aliquots, equivalent to the system and filter hold-up volumes. We measured concentrations of the IgG in the 400 L initial feed and in the aliquots to demonstrate the capability to rinse out and recover the product. IgG concentrations were determined using ultraviolet-visible (UV-vis) spectroscopy at a wavelength of 280 nm.
Results and discussion
Leak test
We performed the flow kit leak test dry following the installation process to assure a leak-free process before any product is committed to the process or any of the flow path is wetted with water. An example data set is displayed in Figure 2, demonstrating the highly accurate pressure control and a typical drop in pressure (note: some tests may not exhibit any pressure drop). The pass result confirms the leak-free status of the installed flow kit. Table1 details multiple leak test results for a variety of system configurations.
Fig 2. Example of an automated flow kit leak test performed post-installation by the Allegro™ Connect depth filtration system. The minimum pressure threshold is determined by the maximum acceptable pressure decay from the test pressure.
Table 1. Summary of flow kit leak tests for various configurations at 0.5 bar for 600 s
| Configuration | Acceptable pressure decay (bar) | Measured pressure decay (bar) | Test result |
|---|---|---|---|
| First stage: Stax™ P250 ×1 | ≤ 0.05 | 0.03 | Pass |
| Second stage: Stax™ EKSP ×2 | |||
| Bioburden: NP8 EAV ×2 | |||
| First stage: Stax™ P250 ×1 | ≤ 0.05 | 0.04 | Pass |
| Second stage: Stax™ EKSP ×2 | |||
| Bioburden: NP8 EAV ×2 | |||
| First stage: Stax™ P250 ×1 | ≤ 0.05 | 0.00 | Pass |
| Second stage: Stax™ EKSP ×2 | |||
| Bioburden: NP8 EAV ×2 | |||
| First stage: Stax™ P250 ×1 | ≤ 0.05 | 0.00 | Pass |
| Second stage: Stax™ EKSP ×1 | |||
| Bioburden: NP8 EAV ×2 | |||
| First stage: Stax™ P250 ×1 | ≤ 0.05 | 0.00 | Pass |
| Second stage: Stax™ EKSP ×1 | |||
| Bioburden: NP8 EAV ×2 | |||
| First stage: Stax™ P250 ×2 | ≤ 0.05 | 0.03 | Pass |
| Second stage: Stax™ EKSP ×1 | |||
| Bioburden: NP8 EAV ×2 | |||
| First stage: Stax™ P250 ×1 | ≤ 0.05 | 0.01 | Pass |
| Second stage: Stax™ EKSP ×1 | |||
| Bioburden: NP8 EAV ×2 | |||
| First stage: Stax™ P250 ×2 | ≤ 0.05 | 0.03 | Pass |
| Second stage: Stax™ EKSP ×1 | |||
| Bioburden: NP8 EAV ×2 | |||
| First stage: Stax™ P250 ×2 | ≤ 0.05 | 0.00 | Pass |
| Second stage: Stax™ EKSP ×1 | |||
| Bioburden: NP8 EAV ×2 | |||
| Flow kit only, no filters | ≤ 0.05 | 0.00 | Pass |
| Flow kit only, no filters | ≤ 0.05 | 0.00 | Pass |
| First stage: Stax™ P250 ×12 (2 chassis) | ≤ 0.05 | 0.03 | Pass |
| Second stage: Stax™ EKSP ×6 | |||
| Bioburden: NP8 EAV ×2 |
Processing control
Effective depth filter processing must allow accurate control of the flow rate and pressure in the depth filtration train. Typical control strategy results are detailed in Figure 3 (fixed flow control) and Figure 4 (fixed flow control transitioning to fixed inlet pressure control).
The Allegro™ Connect depth filtration system seamlessly transitions from fixed flow control to inlet pressure control to prevent any excess fouling or general poor filter performance that might be associated with compression at elevated pressures.
Maintaining a fixed flow control in the early stages of the process maintains the adsorptive performance of the depth filters by avoiding short residence times associated with high initial flows in constant pressure operation. If required, the system could be operated directly in fixed inlet pressure control mode by setting a high initial flow and optimizing the PID settings.
Fig 3. Fixed flow rate control during yeast processing to simulate typical control during cell culture processing.
Fig 4. Fixed flow rate control progressing to inlet pressure control during simulated filter fouling. Testing carried out in triplicate.
Product recovery
After processing 400 L of IgG during fixed flow control processing, we collected the buffer flush in 31 L aliquots, equivalent to one hold-up volume of the flow kits and filters used during the test.
Table 2 summarizes the concentrations measured in each of the three hold-up volumes of buffer flush. The data demonstrates that more than 100% of the IgG present in one hold-up volume of feed is recovered, indicating that the system does not hinder product recovery and can effectively recover excess product that is held up within the filters during processing.
The concentration of IgG in the buffer flush drops with each subsequent hold-up volume flushed, as expected for depth filtration. Also typical for depth filtration, there remains a small proportion of material held up even after three flushes due to the large volumes and complex interactions within the depth filter capsules. The choice of the buffer flush volume is a product-specific and user-preference choice, but we recommend just one or two hold-up volumes of buffer flush followed by blow-down of the filters to the product biocontainer.
Table 2. IgG concentration during buffer flush
| Sample | IgG concentration (g/L) | Concentration relative to feed |
|---|---|---|
| Feed | 1.03 | - |
| Buffer flush hold-up volume 1 | 0.97 | 94% |
| Buffer flush hold-up volume 2 | 0.51 | 49% |
| Buffer flush hold-up volume 3 | 0.19 | 19% |
Conclusions
The Allegro™ Connect depth filtration system facilitates fully automated, in situ flow kit leak testing throughout the system tubing and filter assemblies before water or product commitment to assure a leak-free process.
In addition, the system facilitates fully automated processing during primary recovery (harvesting and clarification) depth filtration. This application note successfully details the following performance capabilities of the system:
- Effective pump control to a fixed flow rate or a subsequent fixed critical inlet pressure, which keeps the critical parameters of pressure and flow consistent across the whole process.
- Successful product recovery through buffer flushing without negative impact from the flow kit design, with one or two hold-up volumes of buffer sufficient to optimize yield when used in conjunction with a blow-down to the product biocontainer.
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