Traditional downstream processing for the purification of monoclonal antibodies (mAbs) can lead to a diluted product solution, which results in longer loading times in subsequent chromatography purification steps.
Here, we used a Cadence™ inline concentrator (ILC) unit to reduce the volume of a mAb solution.
- Inline concentration of dilute mAb intermediates can significantly reduce process volume before chromatography.
- Using a Cadence ILC with an ÄKTA pilot™ 600 system achieved ~ 3-fold concentration under the tested conditions.
- Volume reduction helps shorten chromatography loading time, improving downstream process efficiency.
- The ILC approach demonstrated high product recovery (~ 97%), maintaining process performance.
- Inline concentration can be integrated into existing workflows without increasing overall process time when operated in parallel with capture steps.
Introduction
Downstream purification of a mAb can lead to diluted intermediates in the process, as a consequence of a larger elution volume from chromatography. If these intermediates are loaded onto another chromatography column, the loading time will be disproportionately long with no added value to the process.
An easy way to reduce the volume of the intermediate product is to concentrate it using an ILC unit. In this study, we connected a T02 ILC unit of 0.13 m2 to an ÄKTA pilot 600 system and ran an ILC concentration of a dilute chromatography eluate pool. The ILC unit we selected was small, but sufficient to concentrate the eluate over several hours. A larger unit can be used to reduce the time for concentration. A few ILC units are shown in Figure 1 and the principle behind the flow of product solution through an ILC unit is shown in Figure 2.
The principle behind ILC is the same as for single-pass tangential flow filtration (SPTFF). The difference is that ILC units are preassembled and have a preconfigured restriction on the retentate port. Therefore, ILC units do not require an external valve or pump on the retentate port for flow control. Moreover, an ILC unit as preconfigured can concentrate between two to four times, whereas an SPTFF can reach higher concentration factors.
Fig 1. ILC units, four stages in series consisting of seven membranes.
Fig 2. The principle behind the direction of product flow through an ILC unit.
Materials and methods
Intermediate mAb product
A dilute chromatography eluate with a titer of 8 mg/mL was used for this ILC concentration. The total weight of the eluate pool was 146.7 kg.
ILC concentration
Before the ILC concentration, the ILC unit was prepared. We performed cleaning in place (CIP) using 0.25 M NaOH for 1 h with a recirculation flow of 220 mL/min. After the CIP, the unit was rinsed with water until a neutral pH was achieved. The unit was then drained and integrity tested at 4.1 bar (59.5 psi, 0.41 MPa). The retentate was closed and the air diffusion measured through the permeate.
A 0.13 m2 Cadence ILC unit (30 kDa MWCO) stored in 0.1 M NaOH was connected to an ÄKTA pilot 600 system, with column inlet to the ILC feed port and column outlet to the ILC retentate port. The permeate port tubing was placed to drain waste. The ILC unit was filled with 0.25 M NaOH and the top and bottom permeate ports, as well as the retentate port, were opened until fully saturating the unit. The ILC cleaning was then performed for 1 h recirculating the 0.25 M NaOH at a flow of 220 mL/min and the bottom permeate port was opened 1 min every 5 min during the cleaning.
After the cleaning, the ILC unit was flushed with water at 300 mL/min, staying above 1.4 bar (20.3 psi, 0.14 MPa) in feed pressure (measured as pre-column pressure on the ÄKTA pilot 600 system). The ILC unit was flushed until the pH in the retentate and permeate lines was neutral. A normalized water permeability (NWP) test was performed on the ILC unit by connecting Pendotech pressure sensors on the feed, retentate, and top permeate tubing. The test was performed at 170, 220, and 270 mL/min, resulting in an NWP of 133.4 LMH/bar. The unit was drained from water and filled with air at 4.1 bar (59.5 psi, 0.41 MPa), closing the retentate and performing an integrity test which was passed with an air diffusion value of 3 mL/min through the permeate. The hold-up volume of the unit was 42 mL.
Before the concentration of the mAb intermediate, the unit was equilibrated with elution buffer (100 mM sodium acetate, pH 3.5) through inlet A1 at 220 mL/min. The mAb intermediate was also filtered with a 0.2 µm filter before the ILC concentration.
Inlet A1 and A2, and outlet 2 on the ÄKTA pilot 600 system were primed with elution buffer, and the UV was autozeroed.
The ILC concentration started at a flow rate of 150 mL/min, gradually increased to 220 mL/min with a dCP alarm at 3 bar (43.5 psi, 0.3 MPa). The retentate with the concentrated mAb was collected through outlet 2. The permeate was sent directly to drain waste.
At the end of the ILC concentration, about 0.5 L of elution buffer were used to wash out the remaining mAb from the ILC unit. When the UV280 absorbance was < 100 mAU, collection of the concentrated mAb through outlet 2 was ended. The ILC unit was further rinsed with 2 L elution buffer.
The retentate with the concentrated mAb weighed 46.8 kg, had a conductivity of 2.63 mS/cm, the pH was 4.44, the titer 27.4 mg/mL, and the turbidity was 12.8 FNU. The pH of the concentrated mAb was adjusted to 5.0 using a 2 M Tris base before the final 0.2 µm filtration.
The ILC unit was cleaned with 0.25 M NaOH for 1 h running at 150 mL/min. The unit was rinsed with water, and an NWP test was performed at 170, 220, and 270 mL/min. The NWP after the ILC run was 128.1 LMH/bar, displaying a recovery NWP of 96.1%.
Results and discussion
The ILC concentration ran for about 11 h at constant flow through the ÄKTA pilot 600 system (Fig 3). The pressure gradually increased during the concentration, which was expected since the mAb product becomes concentrated in the unit and processing over several hours generates mAb build-up over the membrane, increasing pressure over time.
Fig 3. ILC chromatogram displaying the feed pressure and pressure over the ILC unit (delta pressure) at constant flow.
The results from the Cadence ILC concentration are displayed in the table below showing the weight, turbidity, mAb titer and amount, host cell protein (HCP) content, and protein A ligand leakage. The 0.2 µm filtered capture eluate pool had a starting titer of 9.0 mg/mL, the ILC retentate had a titer of 27.4 mg/mL resulting in a concentration factor of 3.1. The yield was 97.4%.
Table 1. Parameters of the mAb solution before and after ILC concentration
|
Sample |
Weight (kg) |
Turbidity (FNU) |
Concentration (mg/mL) |
Amount mAb8 (g) |
HCP (ppm) |
Protein A ligand (ppm) |
|
Pool Protein A eluates, 0.2 µm filtered |
146.7 |
6.29 |
9.0 |
1316.3 |
720 |
4 |
|
ILC retentate eluates |
46.80 |
12.80 |
27.4 |
1281.9 |
708 |
4 |
Conclusion
In this application note we have shown the simplicity of using an inline concentrator to concentrate a mAb product as an intermediate process step with a 97.4% mAb recovery. Inline concentration adds value by reducing the volume of dilute mAb intermediates generated during chromatography steps, which can shorten loading times in subsequent purification steps.
Using our Cadence ILC with an ÄKTA pilot 600 system achieved ~ 3-fold mAb concentration under the tested conditions. This small ILC unit operates at a lower flow and has shown that it can be run in a continuous mode in parallel with the chromatography capture step (if cycling is performed over the capture step). Therefore, an ILC step can be included within the same total process time.
Frequently asked questions
What is an inline concentrator (ILC)?
An ILC is a preassembled filtration unit used to reduce the volume of a liquid stream during processing. It enables concentration of dilute intermediates by removing solvent while retaining the product.
Why use inline concentration in mAb downstream processing?
Inline concentration helps reduce the volume of dilute mAb intermediates generated during chromatography steps, which can shorten loading times in subsequent purification steps.
How does inline concentration compare with tangential flow filtration (TFF)?
Inline concentration operates on a similar principle to filtration-based methods such as TFF but uses preconfigured units that simplify setup and operation. The achievable concentration factor and flexibility may vary depending on the system and application.
What level of concentration can be achieved?
In this study, approximately a 3-fold increase in mAb concentration was achieved under the tested conditions.
Does inline concentration affect product yield or quality?
The results demonstrated high product recovery (~ 97%) under the tested conditions, with no significant changes observed in key process parameters.
Can inline concentration be integrated into existing processes?
Inline concentrators can be connected directly to chromatography systems, enabling integration into existing workflows. In this study, the system was operated alongside chromatography without extending overall process time.
When should inline concentration be considered?
Inline concentration is particularly useful when chromatography steps generate dilute product pools that increase loading time or reduce process efficiency.
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