SUREtechnology Platform™

Yes, our proprietary SURE CHO-M Cell Line is the main cell line used for our cell line development 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 media/feeds. This cell line is genetically unmodified, and the full genome sequence is available. We use a broad panel of proprietary patented technologies, tools, and processes to develop a cell line, all grouped as the SUREtechnology Platform. This Platform is fully optimized for our SURE CHO-M Cell Line to offer you the best of its capacities.

Our extensive knowledge of mammalian cells also enables us to remain flexible enough to adapt to other CHO cells and even other mammalian cells if requested – HEK-293, for example.

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:

• ClonePix, from Molecular Devices
• Beacon, from Berkeley Lights

ClonePix monoclonality assessment

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

Monoclonality is ensured by statistical analysis 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 move the cells individually. This enables the distribution of one cell per pen.

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

Selection of the best-expressing clones

With both approaches, hundreds of selected clones are further narrowed down by screening selection. This ensures that high-producing cell lines with the correct quality attributes are finally selected as lead clones.

No, our vectors do not require gene amplification methods such as MTX/DHFR or GS/MSX. This is the result of SGE^®, which are Selexis proprietary, patented, unique epigenetic DNA-based elements that control the dynamic organization of chromatin in all mammalian cells and enable higher and more stable expression of recombinant proteins. SGE are included in our SUREtechnology Platform™ vectors, designed and optimized for our proprietary SURE CHO-M Cell Line. 

Yes, we have one of the most comprehensive genomic characterization platforms in the industry. Our monoclonality assessment and genomic characterization technology are based on our proprietary bioinformatics tools, which were co-developed with the Swiss Institute of Bioinformatics.

We use NGS (next-generation sequencing) Illumina technology for top-of-the-line transgene quality control as well as genomic characterization. On top of transgenes integrity quality control, our technology platform enables rapid assembly of DNA sequences on reference genomes. From there, we can both deduce mapping of transgene(s) integration site(s) and determine transgene(s) copy number(s).

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

Yes, KBI SURE CHO-Mplus Libraries™ were designed to address such complex issues. These libraries, based on the SURE CHO-M Cell Line 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 antibodies) and difficult-to-express proteins (e.g., fusion proteins) to be correctly folded and secreted.

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

In additional, further media optimization have been performed recently, resulting in medium and feed that are optimized for the SURE CHO-M Cell Line.
CELLiST™ BASAL CHO MX medium, proprietary to Ajinomoto, was co-developped with our parent company JSR Life Sciences in-house at KBI Biopharma. More about this media can be found on JSR Life Sciences website.

There are several patented and proprietary technologies that are unique to the SUREtechnology Platform. Embedded in our transfection vectors, there are patented chromatin modifiers that unwind the DNA at the site of transgene integration, boost transcription, and thus increase recombinant protein expression. We have optimized the DNA sequence of the genetic elements 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 these types of issues, we have sequenced the transcriptome of the SURE CHO-M Cell Line to measure the expression levels of its host CHO proteins. We then developed the SURE CHO-Mplus Libraries™ to repair and improve the pathways that are significantly challenged with difficult-to-express proteins.

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, we have helped clients rescue promising candidates that have now entered clinical trials.

To enable comprehensive genomic characterization of our RCBs, we use unique bioinformatics combined with next-generation sequencing (NGS). By analyzing whole genomes, we can provide the complete map of any genome in a mammalian cell, mapping transgene integration sites and determining transgene(s) copy number(s). By sequencing whole genomes, we get rid of bias in characterizing and documenting RCBs, strengthening IND applications with thorough cell line history and traceability.

Among the ten marketed products and 170+ 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.  When there are expression problems, the most common ones are 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 as a whole: interleukins tend to aggregate, for example.

To overcome these challenges, we have developed our custom-engineered SURE CHO-Mplus Libraries, stably transfected SURE CHO-M cell lines with helper proteins. These chaperones target specific expression pathways and help to de-bottleneck secretion, folding, or aggregation problems.

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

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 the SUREtechnology Platform.

In October 2024, leveraging CRISPR gene editing technologies, we have created and  launched a brand new FUT8 KO SURE CHO-M Cell line, that produces completely afucosylated mAbs and has proven to enhancing ADCC 13-fold over our standard SURE CHO-M Cell Line.

We can also play on process parameters (media and additives) for afucosylated mAbs.

We use patented epigenetic elements optimized four the SURE CHO-M Cell Line in all of our vectors. These epigenetic elements 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 many transgene copies into the cell’s chromosomal DNA. As the necessary transgene copy number is reduced, it is easier for the cell to keep them, resulting in increased stability over generations at the locus of integration.

Hence, when we incorporate auxiliary proteins and gene of interest into the cell’s chromosomal DNA, the epigenetic elements 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.

To further increase the consistency of our pools to RCBs, we have developed and launched in October 2024 our transposase-based gene of interest insertion. Transposase are known for decades in the industry as promoting stability, so this new mode of transgene insertion will not be an issue from a stability standpoint.

In addition, alongside our epigenetic elements, we offer our 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. We have successfully offered this alternative to rescue promising programs that could not be expressed at commercially viable levels.

With the use of our 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.

For standard IgGs, as part of our SUREmAb offering, we have launched in October 2024 new guarantees on titer. At the RCB stage, you can be guaranteed a titer of minimum 4 g/L.

In addition, and for information, as of 2022, our typical cell line productivity per product type at CLD stage, before process development, is:

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.

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 phenotype 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.

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, we can fully characterize any research cell bank (RCB) by combining unique bioinformatics with next-generation sequencing (NGS) to demonstrate the location of any given gene against available reference genome.

As the SUREtechnology Platform is a premium service, once contractual negotiations are completed, there is no delay - the CLD project starts immediately.

Usually, we do not exceed three different selections for cell line development. When we apply this strategy, it is mainly when we are using SURE CHO-Mplus 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. The rationale behind this is that the light chain may play the role of chaperone. Therefore, it is essential to ensure the proper integration and selection of a clone expressing this chain at high levels.

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

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, radioactive and toxic payloads would have to undergo a distinct conjugation step after CLD and we cannot perform these steps within the same labs.

This depends on your choice of program type. We offer an accelerated program for mAbs specifically. However, would you wish to develop a mAb with our classical program to keep full control and make all the decisions yourself,  the timelines would be similar in general. From the cell line development perspective, as far as best producing cell line isolation, there is no difference in timelines between classical IgG formats and bsAbs.

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 are primarily molecule-specific.