Selexis CHO Cells in Suspension-1

Technology FAQs

Selexis enables biopharmaceutical companies to produce virtually any recombinant therapeutic protein, because we have the ability to understand and modify our cells to address productivity and expression challenges.

Selexis tools and technologies
  • Do you have a proprietary CHO cell line that is used for transfections?

    Yes, our proprietary CHO-M is cell line used for our Selexis-KBI Integrated programs.

    CHO-M is a CHO-K1 derived cell line deposited as ATCC CCL-61. It has been adapted to suspension and cultivated in animal component free medium/feeds. This cell line is genetically unmodified, and the full genome sequence is available.

  • How do you address cells and best clone selection? Are you using FACS, MACSQuant® Tyto® or Berkeley Lights?

    First, we must distinguish between clone isolation and clone screening and selection.

    For the isolation of clones, while ensuring high productivity, selectivity and monoclonality of the cell line, we use two technologies at Selexis:

    - ClonePix, from Molecular Devices,
    - Beacon, from Berkely Lights.


    ClonePix monoclonality assessment

    The ClonePix platform is based on the incorporation of a heterogeneous population of cells (or pool) into semi-solid medium containing selective agent. Individual cells are growing on this solid medium and forming colonies. Those colonies are then imaged by ClonePix. Thanks to a fluorescent detection reagent targeting the Fc part, we can sort the most productive colonies and pick the most promising clones.

    Monoclonality is ensured by statistics considering:

    - Cell density,
    - Distance between colonies,
    - Size of colonies.

    Two runs of isolation are performed to ensure monoclonality.


    Beacon monoclonality assessment

    The Beacon platform is slightly different. The liquid pools are loaded onto chips that contain thousands of nano pens (or cell growth chambers). The cells are then isolated using a technology called optoelectro positioning - light patterns that activate photoconductors to gently repel cells -. It enables the distribution of one cell per pen.

    Cell productivity can be assessed using in-pen fluorescent assays and allows for ranking and export of the most promising candidates. With Beacon platform, monoclonality is based on imaging, providing proof of single cell progenitor per pen. Only one round of isolation is required for ensuring monoclonality.


    Selection of the best expressing clones

    In both approaches, ClonePix and Beacon, hundreds of selected clones are further narrowed down by screening selection. This ensures high producing cell lines with correct quality attributes are finally selected as lead clones.

  • Do Selexis vectors contain an amplification cassette, such as DHFR or the GS system?

    No, the Selexis SUREtechnology Platform™ does not require gene amplification methods such as MTX/DHFR or GS/MSX.

  • Can Selexis conduct genomic characterization to support RCB generation?

    Yes, Selexis has one of the most comprehensive genomic characterization platforms in the industry. Selexis SUREscan™ technology is based on Selexis’ proprietary bioinformatics tools, which were co-developed with the Swiss Institute of Bioinformatics.

    At Selexis, we use NGS (next generation sequencing) Illumina technology for top-of-the-line transgenes quality control as well as genomic characterization. On top of transgenes integrity quality control, Selexis SUREscan® technology platform enables rapid assembly of DNA sequences on reference genomes. Therefore, SUREscan® allows for both mapping of transgene(s) integration site(s) and transgene(s) copy number(s) determination.

    Selexis can perform such assays in any mammalian cell, including CHO-K1, SP2/0, HEK-293, and PER.C6 cells.

  • Does Selexis have CHO host cell lines that have been adapted or modified to address secretion issues or to perform directed post-translational modifications available?

    Yes, the SURE CHO-Mplus Libraries™ were designed to address such complex issues. These libraries, based on the SURE CHO-M® transcriptome and genome analysis, can help generate products with various post-translational characteristics and attributes, including glycan modifications. Furthermore, the SURE CHO-Mplus Libraries™ enable complex proteins (e.g., multimeric proteins and bispecific mAbs) and difficult-to-express proteins (e.g., fusion proteins) to be folded and secreted in the SURE CHO-M Cell Line™ (Selexis’ CHO K1 derivative cell line).

  • Does the SUREtechnology Platform™ use serum-free or chemically defined media and feed from commercial suppliers in large quantities?

    The Selexis CHO-M Cell Line™ (CHO K1 derived) has been optimized for chemically defined (CD), animal-component-free, off-the-shelf media, as well as CD feeds that are commercially available from multiple suppliers.

  • How is the Selexis SUREtechnology Platform™ different from other available technologies for generating high-expressing and stable mammalian research cell banks (RCBs)?

    There are several patented and proprietary technologies that are unique to the Selexis SUREtechnology Platform™. Embedded in our transfection vectors, Selexis SGE® (Selexis Genetic Elements) are patented chromatin modifiers that unwind the DNA at the site of transgene integration, boost transcription, and thus increase recombinant protein expression. Selexis has optimized the DNA sequence of its SGEs that are integrated into the DNA expression vector to obtain the highest and most reproducible gene expression levels across all recombinant protein types.

    Even with high transcription levels, high-level protein expression can be thwarted by translation and secretion bottlenecks. To address such issues, Selexis has sequenced the transcriptome of its CHO K1 cell line to obtain the expression levels of its CHO proteins. Selexis then developed the SURE CHO-Mplus Libraries™ to repair and improve the pathways that are impaired in its CHO K1 cells.

    This unique and sophisticated platform is designed to rapidly and simultaneously address several translation and secretion bottlenecks that can occur with difficult-to-express proteins, such as blood coagulation factors, bispecific monoclonal antibodies, and Fc fusion proteins. With the SURE CHO-Mplus Libraries, Selexis has helped clients rescue promising candidates that have now entered clinical trials.

    Selexis has launched the Selexis SUREscan® platform, which uses unique bioinformatics combined with next-generation sequencing (NGS) to fully characterize research cell banks (RCBs). By analyzing whole genomes, Selexis has no bias in characterizing and documenting RCBs and thus can provide the complete map of any genome in a mammalian cell. Selexis SUREscan® maps transgene integration sites and determines transgene(s) copy number(s).

Selexis experience and pipeline
  • Could you describe some of the molecules you have worked with where an additional domain/molecule was appended to the Fc domain? Any challenges associated with expressing those molecules?

    Among the 10 marketed product and 160+ clinical phase products that we have developed with our technology, we have worked with many different Fc fusions:

    - Orientations in N terminus or C terminus, on one arm or the two, leading to asymmetric or symmetric molecules which can further complexify the expression of the desired format.
    - Maintain the hinge or remove it.
    - Design with different kinds of linkers in terms of amino acid composition or length.

    Depending on those engineering choices, the expression may vary from easy- to difficult-to-express. There is no general trend.

    We regularly observe problems of expression such as Steric hindrance, toxicity and aggregation.

    Expression bottlenecks can also be linked to the nature of the protein found in the Fc portion. Receptors and enzymes are difficult to express in a whole: interleukins tend to aggregate, for example. Therefore, we have developed our engineered CHO-M cell lines, which we call libraries, that are stably transfected CHO-M cell lines with helper proteins. These chaperones target specific expression pathways and help to de-bottleneck secretion, folding, or aggregation problems.

    Note: to see a glint of the variety of different scaffolds Selexis has successfully expressed, please click here.

  • How does understanding of the process and use of the early candidate pools impact the final clone?

    Typically, process and titer improvements observed during studies using the early candidate pools translates very well to the final clones for CHO-M cell lines. While the absolute values often vary, the trends that we observe as a response to tested factors are very consistent.

  • How many different types of pairing technologies have you worked with?

    We have worked with a wide range of pairing technologies:

    - Knob-into-hole: this represents almost 90% of the usual engineering designs
    - Sophisticated combinations of electrostatic interactions
    - Charge opposite,
    - Introduction of artificial disulfide bonds.

    Success in expressing the right format is dependent on this molecular engineering. From two identical formats, we can observe big differences in expression depending on the force of the pairing system.

    However, there is still room to help our clients to favor their incomplete pairing with the transgene molar ratio approach and rescue some of their programs. That is one of the strengths of Selexis SUREtechnology Platform.

  • What is your experience using cell lines that produce afucosylated mAbs for enhanced ADCC?

    Selexis plays on process parameters (media and additives) for afucosylated mAbs. In addition, work is in progress to modify genetically the CHO-M and create such cell lines.

  • Are there cell line stability issues when all auxiliary proteins and genes of interest are incorporated into the cell’s chromosomal DNA?

    Selexis uses SGE (Selexis Genetic Elements) in all of its vectors. These are epigenetic elements that stabilize the chromatin and open it at both ends of each transgene-genome junction integration site. This results in enhanced expression without having to add lots of transgene’s copies into the cell’s chromosomal DNA. As the necessary transgene copy number is reduced, it is easier for the cell to keep them, hence increasing stability over generations at the locus of integration.

    So, when Selexis incorporates auxiliary proteins and gene of interest into the cell’s chromosomal DNA, the SGE contained in each vector greatly favors both expression and stability at the locus of integration. We have demonstrated the stability of the expression of auxiliary proteins throughout the cell line development for over 60 generations.

    In addition with the SGE, Selexis offers its proprietary SURE CHO-Mplus Libraries™. These libraries have been specifically engineered and designed to address secretion issues for difficult-to-express proteins thanks to the stable co-expression of auxiliary – or chaperone – proteins. Selexis has successfully offered this alternative to rescue promising programs that our client could not express at commercially viable levels.

    With the use of Selexis SURE CHO-Mplus Libraries™ we have not experienced any compromise in the stability of the expression of a transgene in our RCBs. We have evaluated this concern for over 60 generations.

  • What is the typical (g/L) cell line productivity for mAb, fusion protein, bsAb, and alternative scaffold proteins using the SUREtechnology Platform™?

    As of 2022, our typical cell line productivity per product type is:


    Here, the bars represent the avarage titer for different types of biomolecules expressed in CHO-M cell clones, and the dots represent the highest titer reached over the past two years at Selexis with the CHO-M and SUREtechnology PlatformTM


Selexis process
  • For cell line stability studies, how many generations do you go out to?

    Typically, cell line stability studies go out to 60 generations.

    Depending on the project timeline and needs, we have conducted shorter stability studies down to 45 generations, and longer stability studies up to 90 generations.

  • How do you determine the copy number of your plasmid in the cells and how do you control this parameter? As it for sure has an influence on the performance of your "winner clone".

    Copy number assessment is done by ddPCR by using specific probes directed against each transgene sequence.

    We usually monitor copy number evolution during the stability study at different timepoints: generations 0, 20, 40 and 60.

    Nevertheless, it may happen that genetic instability is observed while phenotypic remains constant (no change in productivity). This would then be a case-by-case study of the different lead candidates in terms of absolute number range of copy numbers (units versus tens…) and titer.

  • How do Selexis and KBI coordinate project activities with one another?

    For Selexis-KBI integrated programs, we effectively function as one company.

    We have successfully completed more than 65 integrated programs, including projects for:

    - mAbs
    - bsAbs
    - tri-specifics
    - and Fc-fusion

    We hold bi-weekly meetings for all integrated programs to:

    - Track timelines and shipments
    - Share and exchange data and test results
    - Discuss program progress and status on both sides
    - Plan future activities accordingly

    This includes planning and tracking of international shipment of cell bank vials when necessary. Our Selexis-KBI integrated programs typically have very aggressive and expedited timelines, and our coordination and close collaboration has proven to be essential for the success of these programs.

    Recently, our collocated facility in Geneva for Selexis and KBI will improve collaboration and reduce the long-distance constraints, which will result in a benefit for clients.

  • After the contract is signed, what is the typical delay to get a project into the company’s production queue?

    Once contractual negotiations are completed, there is no delay—the project starts immediately.

  • What is the estimated time that is necessary to achieve a high-producing and stable research cell bank (RCB) to support Phase I clinical development?

    Selexis reduces average cell line development timelines thanks to robust proprietary technologies and efficient project management. Our typical timelines are:

    − Technology transfer and shipping arrangements are conducted in parallel to the cell line development activities (no extra time necessary).

    − 2-3 weeks for vector preparation

    − From transfection to RCB generation: 10 weeks including tech transfer to KBI (this can vary as it is product dependent).

    − Stability studies: 2 months (8 weeks) for 60 generations. This can be run in parallel with the CMO transfer.

  • Are some cell lines better adapted for bispecific formats?

    Usually, bispecific production choice relies mostly on the technology rather than the cell line. From time to time, some particular attribute is required from the cell line. Still, even in such cases, the cell line choice is independent from the format of the bispecific and is related to other considerations (e.g., glycosylation patterns).

    When we use the Selexis platform, we are independently cloning each transgene chain in different vectors. This allows us to play on molar ratio for chain representativity and complexity, leading to a better assembly of the bispecific molecule.

    In addition, each of our vectors encoding the different polypeptide chains bear Selexis Genetic elements (SGEs) that may stabilize the integration of each chain into the CHO-M genome. This removes restrictions around the number of chains constituting a bispecific construct without impacting expression level potential.

  • For the transfection of 2 heavy chains and 1 light chain, do you use 3 different selections for the 3 vectors?

    Usually, we do not exceed three different selections for cell line development. When we apply this strategy, it is mainly when we are using CHO-M libraries to bring the chaperone with the third selection.

    For this particular bispecific case, we would rather maintain the same selection on both heavy chains and a different one for the light chain. Rationale behind is that the light chain may play a role of chaperone. So, it is essential to ensure its proper integration and selecting clone expressing this chain at high levels.

    For the heavy chains, we do not want to complexify too much the selection procedure and preferentially play on ratio testing.

  • Are you developing any bsAbs with a payload? If so, what types of payloads (radioactive, toxic, or other)?

    KBI has worked with ADC and other conjugated molecules in the past, such as PEGylation and non-toxic peptide and oligopeptide conjugates. However, due to GMP suite and lab safety restrictions, we have not worked directly with radioactive and toxic payloads.

  • Are development timelines of bispecific projects similar to mAb projects?

    In general, the timelines are similar. From the cell line development perspective, as far as best producing cell line isolation, there is no difference in timelines with classical IgG formats.

    For example, certain mAb programs may not include an additional optimization study and/or confirmation run. For heterodimeric molecules in size, miniaturization of qualitative assays with µCE- enable us to discriminate formats in parallel to titer determination.

    However, bispecific programs typically require additional upstream, downstream, and analytical development. This is to optimize heterodimer levels and purification of product-related impurities, such as homodimer species, which is primarily molecule-specific.

Regulatory agencies approval
  • Does the expression of a chaperone or other protein create a problem for FDA or EMEA approval?

    No, it is believed that the expression of chaperones and other proteins from different pathways does not create a problem for FDA or EMEA approval. These proteins are CHO proteins—naturally expressed by CHO cells but at very low levels, as shown by transcriptomic analysis. In any case, Selexis can fully characterize any research cell bank (RCB) using the Selexis SUREscan® platform, which combines unique bioinformatics with next-generation sequencing (NGS) and demonstrates the location of any given gene.