Introduction
Virus clearance studies constitute an important part of drug product validation in biopharma manufacturing. For virus filtration steps, these studies are typically carried out with manually operated pressure vessel equipment. However, this method is labor intensive and error prone and requires specialized equipment. Here we present a way to automate virus filtration testing using an ÄKTA™ avant chromatography system and UNICORN™ software.
We also show that the same volumetric throughput can be achieved with the ÄKTA™ avant system as with a pressure vessel, and we demonstrate use of the ÄKTA™ avant autosampler for facile collection of samples during virus clearance experiments.
In addition to eliminating hands-on sample processing, this approach enables automatic data logging and analysis, continuous real-time mAb throughput measurements via the inline UV sensor, and automation of stop/start studies via pre-coded script.
Fig 1. ÄKTA™ avant chromatography system
Materials and methods
Materials
These experiments were done with an ÄKTA™ avant 25 chromatography system operated by UNICORN™ control software through a feedback loop at constant pressure. The filters used are Pegasus™ Prime virus filters, which are highly virus retentive and available in a variety of formats and sizes, and Cygnus Technologies MVM-MVP mock virus.
PI tuning on the ÄKTA™ avant chromatography system
Whereas pressure vessels commonly use a controlled air source to administer constant pressure, the ÄKTA™ avant systems use a closed-loop feedback system for control of constant pressure. To utilize pressure control, this mode must be enabled in the UNICORN™ software method settings for each method step. This is done by inserting a line using Text Instructions for the equilibration step (Fig 2).
- Click Advanced
- Specify constant pressure amount (delta column pressure should be used as pressure sensor)
- Click Insert
Fig 2. How to use the Text Instructions function to enable pressure control in UNICORN™ software for the ÄKTA™ avant system.
The PI (proportional integral) tuning may be adjusted depending on a series of parameters, including the size rating of the virus filter and the viscosity of the solution to be filtered. This tuning is performed through the same Advanced menu of each method step (Fig 3). Proportional (P) reacts proportionally to the current error (difference between setpoint and actual value). Integral (I) reacts based on the accumulated error over time and helps eliminate steady-state error. The P factor regulates how fast the response of the flow regulation and adjusts how fast the re-adaption to the original flow rate will take. Our recommendation is to increase P gradually to improve responsiveness without overshooting when using more viscous solutions. In this case, it will also be beneficial to reduce I slightly to avoid integral windup. Setting the factors to default values of P = 8.0 and I = 40.0 works well for low-viscosity fluids such as commonly used Tris or acetate buffers.
Fig 3. Adjustment of PI tuning parameters is necessary for solutions with varying viscosity.
Comparison of flow rates in ÄKTA™ avant system vs pressure vessels
To demonstrate equivalent performance of the ÄKTA™ avant system and pressure vessels for virus filtration, we measured the throughput of two different mAb solutions (one with low and one with high aggregate content) under constant pressure (30 PSI). Both approaches resulted in comparable throughput values and flow profiles (Table 1 and Fig 4).
Table 1.The throughput values obtained using ÄKTA™ avant system and pressure vessels with low and high aggregate mAb. n = 3
| Throughput (L/m2) | ||
| 1% aggregate | 3% aggregate | |
| ÄKTA™ avant | 430 ± 34 | 940 ± 40 |
| Pressure vessel | 490 ± 3 | 890 ± 81 |
Fig 4. Flow rate and throughput data from Pegasus Prime virus filter experiments with mAb containing 3% aggregate. (A) ÄKTA™ avant 25 chromatography system flow rate vs time. (B) pressure vessel flow rate vs time. (C) ÄKTA™ avant 25 chromatography system throughput. (D) Pressure vessel throughput.
Hold experiments
One advantage of the ÄKTA™ avant system for use in virus filtration experiments is its ability to automate holds and sampling, thus reducing the amount of hands-on time needed compared to pressure vessel operation, which often requires full manual oversight. To demonstrate equivalent performance between operating Pegasus™ Prime virus filters with the ÄKTA™ avant chromatography system and the pressure vessel, we programmed a 2 h hold during the viral clearance experiment (using 1% MVM-MVP mock virus from Cygnus Technologies) and collected samples at various time points and intervals (Table 2). We observed equivalent log reduction values (LRV) with both methods, with the automated method requiring fewer steps and less manual processing.| Sample | LRV automated | LRV manual |
| A First 5 mL | ≥ 4.0 | ≥ 3.9 |
| B Pre-hold pool | ≥ 4.0 | ≥ 3.9 |
| C Post-hold 5 mL | ≥ 4.0 | ≥ 3.9 |
| D Post-hold pool | ≥ 4.0 | ≥ 3.9 |
| E Last 5 mL | ≥ 4.0 | ≥ 3.9 |
Table 2. Comparison of LRVs obtained with the automated method (using an ÄKTA™ avant system) and manual collection (using pressure vessels).
Summary
Here we describe the use of an ÄKTA™ avant chromatography system and advanced control features in UNICORN™ software to automate virus clearance testing. The performance of the ÄKTA™ avant system was comparable to that of pressure vessels typically used for this type of experiment, as demonstrated by throughput and LRV measurements. Moreover, this approach reduces the time and manual input required for sample collection and data logging and enables automation data analysis, holds, and stop/start studies—without the need for additional equipment.