Developing high-producing, stable cell lines is fundamental to the production of biotherapeutics such as monoclonal antibodies, bispecific monoclonal antibodies, fusion proteins, and vaccines. The expense of the drug discovery process is well recognized. To reduce development costs, there is increasing pressure on researchers to quickly and efficiently maneuver through the early stages of biotherapeutic discovery and biotherapeutic candidate selection. Key to driving efficiency is to quickly generate and select clonal cell lines that deliver high concentrations of quality recombinant protein, while screening out clone candidates that may fail in-process scale-up as early as possible.
Filtration applications can play a critical role throughout the cell line development process. Cytiva supplies filtration, separation, and sample preparation products that can help streamline workflows and ensure sterility from gene editing, through to cell culture and omics analysis.
Gene editing and filtration
Gene editing has advanced in recent years through molecular tools such as zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs), and most recently, the CRISPR/Cas9 system. These techniques have driven considerable improvements in accuracy and ease-of-use when engineering cell lines for optimal biotherapeutic production.
Despite technical advancements, multi-step gene editing workflows remain resource intensive involving: gene selection; PCR amplification; DNA extraction; DNA clean up; restriction enzyme digest; ligation; and insert verification. Constant vigilance is required to avoid contamination and downstream complications. Standard techniques often involve multiple purification steps, each one associated with significant product loss. Large amounts of quality DNA are therefore necessary to guarantee a successful cellular transformation or transfection.
Our simple-to-use, nucleic acid binding centrifugal devices incorporate double layer glass fiber membranes, providing high yields and the flexibility to purify genomic DNA and mRNA in a single device to support multiple downstream applications. These nucleic acid purification solutions save time by removing unnecessary steps. The automation-friendly and high-throughput 96-well format eliminates the need for sequential sample preparations further increasing the processing speed of workflows.
Nucleic acid binding Nanosep™ centrifugal devices (Pall™ Life Sciences products).
AcroPrep™ multi-well filter plates (Pall™ Life Sciences products).
Strain construction and filtration
Effective strain construction through the transformation of bacterial cells (such as E. coli), or transfection of yeast (such as S. cerevisiae) or mammalian cells (such as CHO, HEK) requires efficient recovery, concentration, and delivery of the target nucleic acid fragments.
Preparation of small volumes of media for transformation or transfection requires specific strain constructs with variations in amino acids or antibiotics. Sterile syringe filters help you to maintain sterility when handling small media volumes that cannot be autoclaved. Our 0.2 µm sterile filters and capsules ensure that small or large batches of liquid media are efficiently filtered to remove the risk of contamination. Where further concentration of the construct is required prior to introduction into the cells, our wide range of low molecular weight cut-off (3 to 1000 kDa) ultrafiltration centrifugation devices and multi-well filter plates can help streamline sample handling and preparation processes.
AcroPak™ capsules (Pall™ Life Sciences products).
Cell culture and filtration
Cloning and expression of proteins through bacterial, yeast, insect, or mammalian cell culture presents specific challenges. Aseptic techniques and sterility in cell culture are paramount to avoid contamination and downstream complications. Contamination of cell cultures can lead to poor cell health and cell death, reduced cell line performance, low product yield, and loss of confidence in the integrity of the sample extracted.
In addition to ensuring aseptic or sterile culture conditions, media and nutrient feeds need to be filter sterilized to avoid introducing contaminants to the cell culture environment. Cross-contamination when handling cell lines also needs to be avoided to ensure reproducible results.
We supply low protein binding 0.2 µm filter media, encapsulated in pre-sterilized devices and capsules, which offer fast and efficient filtration to maintain sterility in cell culture applications. From small feed volumes of a few milliliters to over 100 liters, proprietary dual-layer membranes enable sterile filtration of nutrient-rich media and supplements while ensuring valuable nutrients are not lost.
Our 0.1 µm Supor™ membrane (a Pall™ Life Sciences product) is an inherently hydrophilic polyethersulfone (PES) membrane that is retentive of mycoplasma and can help protect against mycoplasma contamination in serum or serum-containing media for cell culture.
Our range of in-line vent air filters can help ensure sterility of air coming into the cell growing vessels, and gasses coming out of the vessel while protecting liquids from entering devices such as a vacuum pump.
Clone screening and filtration
Screening clones to identify those carrying the desired genomic changes and expressing the target protein, can be carried out through traditional approaches such as: Western blotting; enzyme-linked immunosorbent assay (ELISA); fluorescence-activated cell sorting (FACS); flow cytometry; or utilizing high content imaging systems. Maintaining sterility and clonality is important for purity and homogeneity of the target protein. Screening large numbers of clones as quickly as possible to select those of value for further analysis is essential.
In addition to sterile filtration systems to ensure media and environmental sterility, our centrifugal devices and filter plates simplify protein sample preparation, genomic DNA (gDNA), and mRNA purification. The AcroPrep™ Advance filter plate (a Pall™ Life Sciences product) is designed to reduce the risk of cross-contamination and create a smooth liquid flow for sample processing and high-throughput screening. Cytiva also supplies high-sensitivity protein binding membranes designed for protein detection analysis applications, such as Western blotting, which exhibit low background noise for clarity and sensitivity of results. Immobilized proteins can be used directly for sequencing, providing flexibility in the workflow.
Ensure clear results for flow cytometry and cell labeling by preparing samples with AcroPrep Advance filter plates equipped with 30-40 µm PP/PE media. These plates are designed to capture clumps of cellular aggregates that could otherwise clog the flow chamber, disrupt the workflow, and impact clarity of results. All cell labeling and detection processes can be carried out in one plate (detection of cell surface antigens or intracellular proteins) without the need for aspiration after centrifugation reducing the potential for product loss. Our filter plates allow you to remove, wash, or label cells by vacuum or centrifugation simply, quickly, and easily.
Sample preparation and filtration
Following identification of clones expressing the protein of interest, clonal amplification, cell harvest, media collection, and sampling is required. Streamlining these processes through efficient sample preparation and purification of the biomolecules for analysis can save significant time and resources. Comprehensive characterization of the expressed target protein in terms of yield, purity, stability, structure, and function is essential to select suitable clones for further optimization and scale-up. To aid in this determination several techniques can be used, including next generation sequencing, immunohistochemistry, and mass spectroscopy.
Cytiva offers a range of devices that can be used to perform sample preparation filtration prior to functional analysis steps such as HPLC. Sample preparation helps remove particulate contamination that can damage analysis equipment and chromatography columns, causing system downtime, the premature replacement of parts, and even potentially impacting the quality of results obtained.
Acrodisc™ PSF syringe filters with PTFE, Nylon, PVDF or wwPTFE membrane (Pall™ Life Sciences products) are certified to be low in UV absorbing extractables. They have been tested for compatibility with common HPLC solvents (water, acetonitrile, and methanol) using established HPLC procedures.
Acrodisc™ PSF syringe filters.