In this application note, we tested the volume reduction capacity of Cadence™ inline concentrator (ILC) modules during continuous feed processing of human immunoglobulin G (IgG) for purification. We used Cadence™ ILC modules with Omega™ and Delta membranes and performed 24 h tests to concentrate human IgG. The flow rate and feed pressure of the modules were used to determine the volumetric concentration factor (VCF).
Introduction
The Cadence™ inline concentrator (ILC) allows direct flow-through in-process volume reduction and can be implemented into a process system or operated as a stand-alone unit. When using Cadence™ ILC modules, the potential concentration factor and throughput capacity are key determinants to be evaluated. In this study, we benchmarked the concentration performance and determined the capacity of Cadence™ ILC modules with both Omega™ and Delta membranes with human immunoglobulin G (IgG) in downstream processing applications.
Materials and methods
For this study, we selected Cadence™ ILC modules with T01 Centramate™ cassettes with Delta and Omega™ 30 kDa membranes. A schematic of the laboratory equipment we used for this study is shown in Figure 1. We performed two types of tests with a human IgG feed solution, a feed flux excursion and a 24 h concentration. The Cadence™ ILC modules were cleaned, pre-conditioned, and characterized for normalized water permeability (NWP) before re-use by following the recommended procedures in the Cadence™ ILC user guide.
Fig 1. Laboratory equipment setup.
Equipment
The equipment we used in this study was:
- Cadence™ ILC module with T01 Centramate™ cassettes with Delta 30 kDa membrane
- Cadence™ ILC module with T01 Centramate™ cassettes with Omega™ 30 kDa membrane
- QuattroFlow 150 pump
- Pump tubing (PharmaPureu, Sta-Pureu)
- PendoTECH PMAT-STND benchtop monitor
- PendoTECH PMAT pressure sensors
- Laptop with PendoTECH DAQ System software
- 2 LEVIFLOW LFS-Series flow meters
- Laptop with LEVIFLOW data recording software
- Magnetic stir plate with stir bar
- 100 mL measuring cylinders
- Supor™ ECV sterilizing grade filter
- 2 L vessel with stir bar
Buffers and solutions
The feed solutions and buffers we used in this study were:
- Feed solution 1: 10 g/L human IgG solution, 10 mM histidine, 100 mM sodium chloride, pH 6; (human gamma globulin powder was purchased from Nova Biologics, Inc.)
- Feed solution 2: 50 g/L human IgG solution, 10 mM histidine, 100 mM sodium chloride, pH 6
- Conditioning buffer: 10 mM histidine, 100 mM sodium chloride, pH 6
- Deionized water
- Cleaning buffer: 0.1 N sodium hydroxide
- Cleaning solutions:
- Modules with Delta membrane: 0.1 N sodium hydroxide
- Modules with Omega™ membrane: 0.5 N sodium hydroxide with 400 ppm sodium hypochlorite
- Storage buffer: 0.1 N sodium hydroxide
Test method summary
In the feed flux excursion tests, we tested 2 L of human IgG feed solution at concentrations of 10 and 50 g/L. We used the flow rates and feed pressure data to calculate the volumetric concentration factor (VCF [VCF = the feed flow rate divided by the retentate flow rate]). We started the tests at the highest feed flux possible while maintaining an operating feed pressure below the recommended maximum of ≤ 4.1 barg (≤ 60 psig, ≤ 0.41 MPag). Multiple VCF conditions were stabilized and recorded at reduced feed fluxes.
In the 24 h concentration tests, we challenged the Cadence™ ILC modules with a 10 g/L human IgG feed solution in recirculation mode (with the retentate and permeate lines directed back to the stirred feed vessel). The process parameters for the human IgG concentration test were approximately 74 L/m2/h (LMH) feed flux, for 24 h. During the tests, we monitored the feed pressure, feed flow rate, and retentate flow rate.
Results and discussion
The feed flux excursion results for the Cadence™ ILC modules using the 10 g/L human IgG feed solution are displayed in Figure 2. The Cadence™ ILC module with Omega™ membrane showed higher VCF (5× to 7×) and higher feed pressure than the Cadence™ ILC module with Delta membrane (4× to 5×) when operated in a feed flux range of 40 to 120 LMH. At lower feed fluxes (40 to 80 LMH), the VCF increased as the feed flux was decreased for the Cadence™ ILC modules with both Omega™ and Delta membranes. At higher feed fluxes (80 to 120 LMH), the VCF values for both Cadence™ ILC modules were relatively stable.
Fig 2. Feed flux excursion test results for 10 g/L human IgG solution using Cadence™ ILC modules with Delta and Omega™ membranes.
The feed flux excursion results for the Cadence™ ILC modules using the 50 g/L human IgG feed solution are displayed in Figure 3. The Cadence™ ILC module with Omega™ membrane showed slightly higher VCF than the module with Delta membrane in the feed flux range of 40 to 65 LMH. However, the feed pressure for Cadence™ ILC module with Omega™ membrane was higher than for the module with Delta membrane, and it reached the pressure limit of 4.1 barg (60 psig, 0.41 MPag) at a feed flux of 65 LMH. Therefore, these feed flux excursion results also indicate that the Cadence™ ILC module with Delta membrane can be operated at a higher feed flux compared to the Cadence™ ILC module with Omega™ membrane.
Fig 3. Feed flux excursion test results for 50 g/L human IgG solution using Cadence™ ILC modules with Delta and Omega™ membrane.
In summary, the feed flux excursion data shows that the 10 g/L human IgG solution could be concentrated by approximately 4× to 7×, and the 50 g/L human IgG solution could be concentrated by approximately 2× to 3× by the Cadence™ ILC modules.
Figure 4 illustrates the VCF and feed pressure curves for the Cadence™ ILC modules that were operated in recirculation mode with a feed solution of 10 g/L human IgG at a constant feed flux of approximately 74 LMH for 24 h. The results show that the Cadence™ ILC modules with Delta and Omega™ 30 kDa membranes could be operated for 24 h with acceptable human IgG concentration performance.
The Cadence™ ILC module with Omega™ membrane showed higher VCF and feed pressure than the Cadence™ ILC module with Delta membrane. The VCF of the Cadence™ ILC module with Omega™ membrane started at 5.7-fold and gradually decreased to 4.7-fold over the 24 h operation, indicating a small amount of membrane fouling. The feed pressure increased slightly from approximately 1.9 barg (28 psig, 0.19 MPag) to approximately 2.1 barg (31 psig, 0.21 MPag) over the 24 h operation. The VCF of the Cadence™ ILC module with Delta membranes started at 3.9-fold and remained constant over the 24 h operation, indicating minimal membrane fouling. The feed pressure was stable at approximately 1.7 to 1.8 barg (25 to 26 psig, 0.17 to 0.18 MPag) for the 24 h operation.
Fig 4. 24 h concentration test results. VCF and feed pressure for the Cadence™ ILC modules with Delta and Omega™ 30 kDa membranes processed with a 10 g/L human IgG solution.
Conclusion
We evaluated the ability of Cadence™ ILC modules with Delta and Omega™ 30 kDa membranes to concentrate human IgG under multiple conditions for downstream bioprocessing applications. These results show that Cadence™ ILC modules provide an effective solution for inline volume reduction in continuous downstream purification processes.
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