Clarifying mammalian cell cultures in 24-well formats can be time-consuming and costly, especially when using traditional methods like centrifugation, filtration, or flocculation. This application note evaluates AcroPrep™ 24-well clarification and sterilization filter plates as an efficient alternative for processing Chinese hamster ovary (CHO) and HEK293T cell cultures at densities ranging from 2 to 26 × 106 cells/mL. We assessed performance under both vacuum and centrifugation workflows, measuring parameters such as pH, conductivity, turbidity, and protein recovery. The results show consistent filtration performance with minimal media breakthrough and > 95% total protein recovery, supporting the suitability of these plates for small-scale upstream workflow.
Introduction
Biologics like monoclonal antibodies (mAbs) are increasingly being used to treat diseases like cancer and autoimmune disorders. Recently, evolving biopharmaceutical manufacturing processes have led to major improvements in the quality and recovery of mAbs, partly due to culturing cell lines at high densities. This situation generated challenges in clarification and further downstream processing, as it requires the removal of substantial quantities of biomass and elevated levels of cell debris generated during cell culture and harvesting (1).
To reduce costs and improve efficiency, many labs are optimizing individual steps, such as increasing antibody yield per volume of culture. Centrifugation and a combination of filtration techniques, such as cross-flow or tangential flow filtration (TFF), and depth filtration (DF), are widely accepted techniques for clarifying complex suspension cell cultures. Single-use bioreactors have gained widespread acceptance for accelerating biomolecule production, but harvest and clarification operations remain largely dependent on centrifugation, TFF, and DF—all techniques that have yet to be widely adapted to single-use formats.
Flocculation combined with depth filtration (2) has been slow to gain acceptance in mAb manufacturing, particularly in mammalian cell culture processes such as CHO cell cultures. Flocculants are evaluated for their mechanism of action, impact on downstream processing and product quality, and potential disadvantages. Diatomaceous earth (DE) is used to clarify mAb samples for analytical purposes by separating whole cells and coarse debris; while suspended particles are removed by microfiltration at the same time. This method (flocculation) reduces the processing time by more than half with the wetted-DE remaining porous, preventing blockage throughout filtration (3).
Centrifugation is suitable for cultures with a high solid content, but product recovery can be low due to high levels of solid particulates leading to large pellet volumes, and shear induced cell disruption from shear forces, which can cause product damage and/or entrapment. Depth filters offer single-use convenience and reduce cleaning requirements, but struggle handling high-solids cultures and are therefore often used in series with centrifugation. TFF can handle high solids loading, but can cause poor yield due to product dilution and shear induced cell lysis.
Traditionally, mammalian cell line development involves screening the cell lines in shake flasks prior to scale-up—an often time-consuming process due to the need to balance the complex interactions involved in mammalian cell culture parameters. Commercial small scale systems with controllable microbioreactors can help accelerate the development process by mimicking lab-scale bioreactors, and allow several cell cultivations in parallel under different conditions (4).
Regardless of the development path taken, 24-well culture plates for growth are often a pivotal part of these processes. However, clarification and sterilization using 24-well plates present unique challenges. To help streamline upstream process using 24-well plates, we offer an AcroPrep™ 24-well filter plate which combines cell clarification and sterile filtration functions.
This application note details the integration of our AcroPrep™ 24-well clarification and sterilization filter plate and AcroPrep™ 24-well sterilization filter plate for clarification CHO and HEK293T cell cultures (2 to 26 × 106 cells/mL) and recovery of proteins present in the supernatant.
Materials and methods
Table 1. Materials used in the study
| Category | Item description | Notes |
|---|---|---|
| Consumables | Adhesive plate foil (-80°C to 130°C) | |
| Consumables | AcroPrep™ 24-well cell clarification and sterile filtration plate-depth filter plus 0.65/0.2 µm Supor™ EKV membrane | |
| Consumables | AcroPrep™ 24-well sterilization filter plates with 0.2 µm Supor™ EKV membrane | |
| Consumables | AcroPrep™ 24-well filter plates, collection plates and lids | ɤ-irradiation at 17.4 to 30 kGy |
| Consumables | Reverse osmosis water | |
| Organisms |
CHO cells, S-line (suspension culture) | Internal cell culture collection |
| Organisms | HEK293T | Internal cell culture collection |
| Equipment | WPA Biowave II UV-Vis spectrophotometer (Biochrom) | Adherent cells for suspension culture |
| Equipment | 5810 centrifuge with deep well-plate rotors (Eppendorf) | |
| Equipment | Multi-well plate vacuum manifold | |
| Equipment | NucleoCounter NC-200 automated cell counter and disposables (Chemotech) | Cell density and viability determination |
AcroPrep™ 24-well filter plates processed under vacuum or centrifugation centrifuge processing
- Centrifuge processing: The AcroPrep™ 24 well filter and collection plates were centrifuged at 1000 × g in an Eppendorf 5810 centrifuge with rotor A-2-DWP or A-2-DWP-AT.
- Vacuum processing: The AcroPrep™ 24 well plates were processed using a multi-well plate vacuum manifold, with vacuum at 15 inHg.
CHO cell culture
We grew the CHO cell cultures under sterile conditions after reviving from a cell bank stock (passage 8) in Hyclone™ ActiSM™ cell culture medium supplemented with 0.87 g/L of UltraGlutamine I (Lonza Bioscience). Cultures were maintained at 37°C, 135 rpm, and 8% CO2. Cells were passaged every 2 to 3 days in the ActiSM™ adaptation medium for 1 week, before continuing culture under the same conditions in Gibco Dynamis medium (Thermo Fisher Scientific), supplemented with 0.87 g/L of UltraGlutamine I and puromycin (1.25 mL/L medium, 12.5 μg/mL).
At passages 19 to 22, we harvested the cells in Dynamis medium supplemented with only UltraGlutamine I. Cell density was ~ 2 to 3 × 106 cells/mL with viability > 98%.
High CHO cell density culture (artificial)
To reach a high cell density of ≥ 26 × 106 cells/mL, we centrifuged 2 to 3 × 106 cells/mL CHO cells at 1000 × g for 10 min. We removed the supernatant to resuspend the cells to a final density of 2.6 × 106 cells/mL. CHO cells were used and concentrated on the day of harvesting.
Due to the number of tests to be performed, different CHO cell populations at different passage levels (19 to 22) were used to perform the testing with fresh concentrated CHO cells culture. The high CHO cell density culture was used with the AcroPrep™ 24-well clarification and sterilization filter plates to remove the cells from media.
HEK293T cell culture
We grew the HEK293T cell cultures under sterile conditions, from a cell bank stock (stored in liquid nitrogen at ‘passage 8’) of adherent cells, thawed in FreeStyle 293 expression medium supplemented with 0.1% of Pluronic F-68 non-ionic surfactant (both Thermo Fisher Scientific) at the following growth conditions: 37°C, 135 rpm, and 5% CO2. Cells were passaged every 2 to 3 days for 2 weeks under the same growth conditions, until very little or no cell aggregation was observed under a microscope. Cells were harvested at passages 13 and 14, with a cell density in suspension culture of ~ 2 to 4 × 106 cells/mL and a viability of > 98%.
HEK293T cell cultures were processed using the clarification and sterilization plate. The supernatant of the HEK293T culture was filtered using the sterilization plate.
Clarification of HEK293T cell culture by centrifugation
We removed cells from an aliquot of the HEK293T culture at a viability > 98% and a cell density of 4 × 106 cells/mL, pelleted by centrifugation for 5 min at 700 × g. The supernatant was recovered and filtered using the AcroPrep™ 24-well sterilization filter plate. We measured the turbidity, pH, and protein recovery before and after filtration of the cell-free extract.
Hold-up volume / Time-through / pH / Conductivity / Turbidity / Optical density
To assess hold-up, we compared the volume of filtrate per well (downstream) to the initial volume, 5 mL, of CHO cells (upstream). The difference represents the unrecoverable sample (or hold-up) volume.
Processing time was measured using 5 mL/well of high-density CHO cell culture, using the AcroPrep™ 24-well filter plate, and 6 and 7 mL/well of HEK293T cell suspension, processed under centrifugation or vacuum, respectively.
We used the AcroPrep™ 24-well filter plate (upstream) and the collection plate (downstream) with our multi-well plate vacuum manifold at 15 inHg, or with the Eppendorf 5810 centrifuge at 1000 × g. Time taken for the mammalian cultures to filter through the AcroPrep™ 24-well filter plate was recorded.
pH and conductivity of the mammalian cell cultures were measured before and after filtration was carried out for several wells in various plates. The variation could reflect the release of molecules from the filters to the downstream samples. Consistent conductivity readings indicate minimal interference.
We determined the turbidity/optical density (OD600) of the mammalian cell culture (upstream), before filtration, by dilution in the growth media to record measurements at a wavelength of 600 nm. Filtered samples (in collection plate) were not diluted and several wells were pooled to have 10 mL solution for measurements at 600 nm. Turbidity and optical density indicate the removal of cells and cell debris by the filtration.
Protein recovery
An aliquot of the initial mammalian cell culture (upstream) was spiked with IgG as a control.
Initial total protein (Pi)
Pi (upstream) was measured in a cell-free culture using fluorometry and interferometry. Cells were removed by:
- Centrifugation at 1000 × g for 5 min, with or without filtration through a 0.2 μm nylon filter.
- Addition of DE to a small volume of concentrated CHO cell culture, followed by centrifugation at 1000 × g for 5 min, and/or filtration with a 0.2 μm filter.
Total protein (Pt) recovery
The total protein (Pt) concentration was measured using a Qubit 4 Fluorometer and Qubit Protein Assay Kit (Thermo Fisher Scientific). Recovery was determined by comparing the quantity of proteins in the samples filtered using the AcroPrep™ 24-well filter plates (downstream, in the collection plate) and the Pi in the cell-free sample.
Immunoglobulin G (IgG) recovery
IgG concentration was assessed by bio-layer interferometry (BLI) on a FortéBio Octet instrument (Sartorius) with protein A Biosensors, which allowed specific quantification of IgG.
The recovery of IgG in the filtrated samples (downstream, in the collection plate) was assessed and compared to the initial IgG concentration in the free-cell samples.
Results and discussion
24-well clarification and sterile filtration plates
Table 2 presents the mean values obtained from high CHO cell density cultures. Table 3 provides a summary of data gathered from HEK293T cell cultures and their corresponding supernatant.
Table 2. Parameters recorded for the CHO cell culture with a concentration of 26 × 106 CHO cells/mL and processing with the AcroPrep™ clarification and sterile filtration plate
| 5 mL concentrated CHO cell culture at 26 million cells/mL upstream | Upstream culture | Downstream filtrate (vacuum: 15 inHg) | Downstream filtrate (centrifugation: 1000 × g) |
|---|---|---|---|
| Processing time (min) | - | 20.2 ± 6.3 | 15 |
| Hold-up volume (µL) | - | 300 to 450 | 400 to 450 |
| pH | 7.2 | 7.3 | 6.8 |
| Conductivity (µS/cm) | ≈ 10 100 | ≈ 9200 | ≈ 9800 |
| Turbidity (NTU) | ≈ 1900 to 2600 | ≈ 1.8 | ≈ 2.4 |
| OD at 600 nm |
≈ 18 to 19 | 0 | 0 |
| Total protein recovery (%) | - | 98.3 ± 8.2 | 95.4 ± 11.4 |
| IgG recovery (%) | - | 91.3 ± 11 | 85.0 ± 6.9 |
Table 3. Parameters recorded for the HEK293T cell cultures at 2 to 4 × 106 cells/mL and processing with the AcroPrep™ 24-well clarification and sterile filtration plate or the AcroPrep™ 24-well sterile filtration plate
| Step | Initial culture | Clarification | Filtration supernatant (0.2 µm filter) |
|
|---|---|---|---|---|
| Plate | 24-well depth + Supor EKV membrane |
24-well Supor EKV membrane |
||
| Material (HEK293T cell culture/supernatant) | HEK293T at 2 to 4 million cells/mL (upstream) | HEK293T cell culture at 2 million cells/mL (upstream) | Supernatant of HEK293T cell culture at 4 million cells/mL (upstream) |
|
| Sample | Upstream culture | Downstream filtrate (vacuum: 7 mL/15 inHg) | Downstream filtrate (vacuum: 7 mL/15 inHg) |
Downstream filtrate (centrifugation: 6 mL, 1000 × g) |
| Processing time (min) | 4.3 ± 0.4 | 2.7 ± 0.7 |
5.0 |
|
| pH | 7.1 to 7.6 | 7.2 |
||
| Conductivity (µS/cm) |
≈ 10 836 | ≈ 10 906 |
||
| Turbidity (NTU) | ≈ 81 to 226 | ≈ 0.91 |
||
| OD at 600 nm | ≈ 1.3 to 2.0 | 0.001 |
||
| Total protein recovery* (%) | 101.2 ± 0.4 |
101.2 ± 1.0 |
99.4 ± 1.0 |
|
*Max of 5.4 mg total protein
The data show that the AcroPrep™ 24-well filter plates perform consistently under both vacuum and centrifugation, with respect to parameters such as media pH, conductivity, optical density, and protein recovery.
pH and conductivity
Measurements taken before and after filtration of the CHO (Table 2) and the HEK293T cell cultures (Table 3), indicating that none or negligible amounts of filtering media material are released downstream to the filtered samples. While cell removal can influence these parameters, no significant changes were observed in this study.
Turbidity and optical density
Filtration of the mammalian cell cultures and HEK293T supernatants resulted in clarified media with minimal breakthrough. Turbidity and OD600 readings confirmed effective removal of cells and debris by both AcroPrep™ 24-well filter plate formats.
Protein recovery
Total protein recovery from the CHO or HEK293T cell cultures was greater than 95% after filtration when both AcroPrep™ 24-well filter plates were used. This indicates high efficiency in retaining extracellular proteins during processing.
IgG recovery
IgG recovery was lower than total protein recovery in CHO cultures. This is likely due to differences in detection methods, and challenges in removing the cells in the initial concentrated CHO cell cultures without causing cell damage—which could release intracellular molecules and cellular debris, creating inconsistency in the data.
Conclusion
In this study, we aimed to assess the efficiency of the AcroPrep™ 24-well clarification and sterilization filter plates, including the 0.2 µm sterilization filter plates, for processing mammalian cell suspensions.
We tested high-density CHO cell suspensions (artificially concentrated) and HEK293T cell cultures, using both vacuum and centrifugation methods.
Results show that both processes perform equivalently. The filter plates effectively clarified:
- CHO cells at densities up to 26 × 106 cells/mL
- HEK293T cells up to 4 × 106 cells/mL
- Total protein recovery ranged from ≈ 5 to 10 mg and was consistently > 95% regardless of which plate type was used. These findings support the suitability of AcroPrep™ 24-well filter plates for efficient clarification and protein recovery in small-scale mammalian cell culture workflows.
References
- Identification and tracking of problematic host cell proteins removed by a synthetic, highly functionalized nonwoven media in downstream bioprocessing of monoclonal antibodies. 2019. Journal of chromatography A, 1595, pp28-38.
- Burgstaller D, Krepper W, Haas J, et al. Continuous cell flocculation for recombinant antibody harvesting. J Chem Technol Biotechnol. 2018;93(7):1881–1890. doi:10.1002/jctb.5500
- Overcoming the clarification challenge of high cell density culture. 2015. L. Gimenez, E. E. Kawkabani, [...], and L. Malphettes, BMC Proc., 9 (Suppl 9) p35.
- Miniature bioreactors: current practices and future opportunities. 2006. J. I. Betts & F. Baganz, Microb Cell Fact, 2006, 5: 21.
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