Just a couple of years ago, in 2017, the original Chinese Hamster Ovary (CHO) cell line hit the big “six O.” Originally created in 1957, the Chinese Hamster Ovary (CHO) cell line was one of the first stable mammalian cell lines developed and continues to deliver immense value to the biotherapeutics industry. While other mammalian cell lines have been developed, the regulatory pathway for biologics produced using CHO stable cell lines is well established with 70% of today’s protein therapeutics are manufactured using CHO stable cell lines.1

CHO Cells May be the Way to Go, But What’s Next?

Generation of stable cell lines is a resource-intensive process that can add as much as 12-18 months to therapeutic development timelines. While manufacturing therapeutics via stable CHO cells is unlikely to be displaced any time soon, there is immense pressure to compress development timelines and of course, develop more efficacious therapeutics. Enabling technologies have once again led the way, this time taking the form of automation & gene editing.

Enabling Instrumentation

When using stable cell lines for manufacturing the FDA requires proof of clonality or if not available requires additional manufacturing and control steps that can add cost and time to the development pathway. Conventional cell line generation processes rely on limiting dilution and/or fluorescence-activated cell sorting (FACS). Thousands of wells must be screened to identify high-producing clones with good growth properties. This is a resource-intensive process which is liable to variability.

Two advances in cell line development instrumentation have come together to greatly reduce time, improve clone quality and provide documentation of clonality – automated colony selection and image-based cell sorting and growth monitoring.

For example, the ClonePix™ 2 Mammalian Colony Picker from Molecular Devices images and selects cells based on user-defined parameters. Plate handling, barcode reading, and picking are all integrated and data, including images, are saved for downstream analysis. This picking technology increases the probability of finding optimally produced cell lines and significantly reduces time and labor.

A variety of other vendors, including Solentim, Kbiosystems, Cytena, Nexcelom Biosciences and Hudson Robotics, offer systems that range in ability from automated colony picking to full evaluation and documentation of transfection efficiency, clonality, growth kinetics, media optimization and more. Systems such as these provide for reliable selection of quality clones in substantially reduced timeframes while enhancing FDA compliance, thereby overcoming many of the challenges faced during cell line development — but the innovation doesn’t stop there.

Don’t Settle for Just Your Average CHO – Applying Gene Editing Technology

Since the CHO genome was sequenced several years ago (www.chogenome.org), gene editing technologies like zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas systems are proving to have significant impacts not only on biotherapeutic manufacturing and purification, but also on therapeutic efficacy through precision genetic engineering.

Computational models of metabolism, glycosylation, signaling, DNA damage response and transcriptional regulation are aiding in the design of novel expression hosts.2 These models, when combined with “omics” data are helping to identify pathways that when engineered will yield desired traits – traits that deliver desired glycosylation profiles, other bioavailability factors impacting efficacy, and engineering for manufacturability, for example. As the pathways to the desired traits are better understood and off-target genome modifications are minimized, the creation of CHO cell lines with the desired performance characteristics will become increasingly efficient.

Arguably equally important to understanding the genetics of desired cell characteristics, is the ability to deliver the DNA into host cells. Robust electroporation technologies, such as MaxCyte’s ExPERT platform, allows CHO cells to be efficiently transfected with gene-editing machinery while maintaining the high levels of cell viability needed for successful cell line development.

What’s Next for Protein Production?

The question of what’s next for cell line production and protein production might be best answered by looking at work currently underway. Dr. Phillip Berman’s group of the Department of Molecular Biology at the University of California (UC) Santa Cruz gives us a fascinating glimpse into how recent advancements — ranging from new automated methods of colony selection to creation of CRISPR-engineered CHO cell lines — can be applied not only to improve protein manufacturing and shorten development timelines by as much as a year, but also positively impact efficacy.3-7

Dr. Berman’s work looked to improve the recombinant HIV antigen used in the RV144 trial, the first HIV trial to demonstrate that vaccination could deliver HIV protection, although only modest efficacy of 31% was reported. gp120, a major component of the RV144 trial, was produced in CHO cells; however, the particular CHO cell line produced gp120 with a high level of glycosylation heterogeneity that generally lacked N-linked glycosylation sites critical for binding of anti-HIV broadly neutralizing antibodies (bN-mAbs). Researchers believe, at least in part, that the lack of N-linked glycosylation may play a role in the poor efficacy.

His group sought to generate a CHO MGAT1-cell line via CRISPR gene knockout that produced rgp120 enriched for mannose-5 terminal glycans yet maintained growth properties suitable for use in protein manufacturing.5

Using a combination of MaxCyte’s ExPERT cell engineering platform, CRISPR/Cas9 gene editing and automated colony selection, the UC Santa Cruz team developed an approach to engineer a tailored CHO cell line while decreasing the time required for stable cell line production to two to three months. Since then, the group has gone on to show the HIV rgp120 produced from the CHO-MGAT- stable cell line has the desired improved binding to anti-HIV bN-mAbs.6,7

So, what’s next for protein production? Facilitated by enabling technologies and gene editing expertise, protein therapeutics can deliver efficacy that was previously not possible, enjoy greatly shortened cell line development timelines, and make promising improvements to manufacturing productivity. In short, there is no reason to settle for just an average CHO.

References:

  1. Cell line techniques and gene editing tools for antibody production: A Review. (2018) Front. Pharmacol., 9: 630.
  2. Advances in mammalian cell line development technologies for recombinant protein production. (2013), Pharmaceuticals, 6(5): 579-603.
  3. Development and characterization of an automated imaging workflow to generate clonally-derived cell lines for therapeutic proteins. (2018) Biotechnol. Prog., 34(3): 584-592.
  4. Glycan modification to the gp120 immunogens used in the RV144 vaccine trial improve binding to broadly neutralizing antibodies. (2018) PLoS ONE, 13(4):e0196370.
  5. CRISPR/Cas9 Gene Editing for the Creation of an MGAT1-deficienct CHO Cell Line to Control HIV-1 Vaccine Glycosylation. (2018) PLoS Biol, 16(8): e2005817.
  6. Robotic selection for the rapid development of stable CHO cell lines for HIV vaccine production. (2018) PLoS ONE, 13(8):e0197656.
  7. Development of a stable MGAT1- CHO cell line to produce clade C gp 120 with improved binding to broadly neutralizing antibodies. (2018) Front. Immunol., 9: 2313.