Over the past century, small chemical molecules have dominated the pharmaceutical market. The manufacturing of small-molecule drugs is supported by highly skilled organic and physical chemists, along with deep supply chains, a thorough theoretical understanding of processes, and rigorous standardization. This allows for the efficient large-scale manufacturing of even complicated small-molecule drugs with multiple chiral centers.
Then came Orthoclone OKT3, marking the first monoclonal antibody approved as a human drug in the US in 1986. Notably, 2010 was the last year when all the top 10 drugs in the US were small molecules. The introduction of Humira in 2002 and many other antibody-based drugs shifted the trillion-dollar pharmaceutical business from a chemical industry towards a biological industry almost overnight. In the most recent 2023 top 10 list, only two drugs were small molecules (Eliquis and Biktarvy). Instead, biologics now dominate the top 10 list, with six of the top 10 drugs being antibodies, one being RNA, and one a peptide. The upcoming waves of future drugs are also predominantly biologics, with acronyms like BsAbs, CAR-T, AAV, TCR, ADC, and many more on the horizon.
Biologics development
Biologics manufacturing has become a cornerstone of the biopharma industry, playing a vital role in developing innovative treatments for a wide range of diseases. As the demand for biologics continues to grow, understanding the complexities of production systems is critical. Contrary to chemically synthesized small molecule drugs of ~100 atoms with a dozen chiral centers, the typical antibody contains ~30,000 atoms and thousands of chiral centers. The antibody often requires post-translational modifications, and it may aggregate, be heterogenic, or have assembly issues, to mention a few of the concerns that must be addressed during production. In addition, biological production systems are inherently unstable because of evolutionary pressure and an abundance of epistatic variables complicating predictability. The quality, consistency, and yield of an antibody is dependent on cell line, media, gene configuration, and much more, making for interesting days for the CMC scientist in a biotech company.
Complexities of antibodies
Despite antibodies' massive health and economic value, the research and development stage of antibody development and the manufacturing stage are typically performed in different host cell lines, potentially resulting in inconsistencies in Critical Quality Attributes (CQA) between the R&D version of the antibody and the manufacturing version. During drug discovery and early development stages, antibodies are produced using transient platforms where episomal vectors encode the antibody genes. Cell lines such as HEK293 derived from Human Embryonic Kidney cells grow well in suspension and are favored due to the ease and speed of production of workable quantities of antibodies for preliminary studies. HEK293 cells are easy to transfect at high titer. In addition, growth media, vectors, and other components have been developed and standardized to support this cell line over several decades. However, transient expression yield from genes encoded on the episomal vector is genetically unstable, and over time, the episomal copy is diluted, resulting in diminishing yield.
Once the lead molecules have been identified, the antibody encoding genes are integrated into the chromosome of CHO-cells to ensure stable protein output over many generations. Historically, this chromosomal insertion was done using random integration, resulting in duplications, truncations, concatemerizations, and other genetic rearrangements and instabilities of the antibody encoding genes. Finding the “best” clone required screening of many thousands of isolated clones. Over the last few years, most commercial-scale cell line development has instead been performed using Leap-In or PiggyBac transposon-mediated insertions resulting in the need to screen just ~100 clones while still generating CHO clones with higher expression yield and exceptional genetic stability. CHO cells are derived from Chinese Hamster Ovaries and are preferred hosts for stable cell lines due to their high yield, long history of regulatory approval, well-characterized glycosylation pattern, and other post-translational modifications similar to those produced in human cells. CHO cells are exceptionally robust, with high viability and high tolerance to variations of pH, oxygen levels, and cell density, making them ideal for large-scale GMP production.
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Since the isolation of the original CHO cell line, many variants of the CHO lineage have been developed using extensive mutagenesis and clonal selection. The most commonly used variants include CHO-K1, CHO-S, and CHO-DG44. Studies suggest that CHO-K1 metabolism favors mAb expression, whereas CHO-S metabolism prefers biomass formation. CHO-DG44 ranks in between the two.
Tools to address these challenges are now being developed and implemented across the biotech industry. Cell lines and platforms that minimize the host cell differences are now available when transferring from high-throughput transient screening to subsequent scale-up in a stable cell line format. One early example is the ExpiCHO platform built on a CHO-S lineage developed with its proprietary media for high expression and CQA consistency from early transient to late stable CHO. A more recent development is the discovery CHO-K1 derived cell line lineage discoCHO that streamlines the process from early transient discovery to late-stage stable production in the CHO-K1 derivative miCHO cell line.
Emerging development workflows and strategies are increasingly pushed to the forefront as part of the focus on antibody developability to increase the likelihood of success in moving the right candidate into the clinic instead of realizing too late that the CQA from the transient screen didn’t conform with the clinical goal, the constructs are not genetically stable, or the productivity of the cell line is not sufficient.
For early-stage research, transient expression systems like HEK293 can expedite initial screening and characterization due to their rapid and flexible production capabilities. However, as projects progress toward large-scale manufacturing, transitioning to stable expression systems like CHO cells becomes critical to ensure consistency, genetic stability, and regulatory compliance.
To optimize this transition, lab managers should:
- Leverage advanced cell line platforms: Utilize platforms that minimize differences between transient and stable expression, to streamline workflows and reduce discrepancies in productivity, stability and Critical Quality Attributes (CQA).
- Invest in screening efficiency: Adopt newer technologies like transposon-mediated integration to reduce the number of clones needed for screening, thereby saving time and resources.
- Focus on consistent CQA monitoring: Implement robust monitoring protocols to track CQA from the early stages of development through to large-scale production, ensuring alignment with clinical requirements.
