Recombinant Monoclonal Antibodies for Therapeutic Use
Recombinant monoclonal antibodies are one of the most promising drugs (immunotherapy) for human diseases, including infectious diseases, cancer, and various other conditions. Mammalian cells (e.g., CHO, HEK293, PER.C6) are primarily used to produce therapeutic antibodies, except single-chain antibodies without post-translational modifications (e.g., glycosylation), which are expressed in non-mammalian cells or cell-free expression systems. The generation of antibody-producing stable cell lines is the most critical factor for large-scale manufacturing of therapeutic antibodies. Several key factors are associated with generating stable cell lines for expressing therapeutically active recombinant antibodies, including the engineering of antibodies, vectors, and mammalian cells.
Engineering of Antibody Genes for Improved or Novel Therapeutic Functions
Recombinant full-length antibodies are made based on gene sequences derived from monoclonal antibody (mAb) producing B-cells (mouse), followed by humanization of mouse mAbs. Other kinds of recombinant antibodies include Fab fragments, single-chain antibodies (scFvs), bispecific antibodies, and other types of genetically engineered or modified antibodies.
Genetic Optimization of Expression Vectors for the Expression of Therapeutic Antibodies in Mammalian Cells
This process involves fine-tuning elements like promoters and coding sequences to maximize expression levels and protein quality. Strong promoters drive high transcription rates, while codon optimization tailors sequences for efficient translation. Regulatory elements like insulators stabilize expression, ensuring consistent antibody yield and quality. These optimizations are vital for developing biopharmaceuticals with potent therapeutic efficacy against various diseases. Various genetic and epigenetic elements or factors used for stable expression of therapeutically active antibodies in mammalian cells are listed in Fig. 1.
Mammalian Cell Engineering for Producing Therapeutically Active Antibodies
Mammalian cell engineering is pivotal for generating therapeutically active antibodies. By modifying cell lines, scientists optimize antibody production. Genetic modifications enhance protein yield and quality, crucial for effective therapies. Techniques like gene editing precisely alter cellular machinery, boosting antibody expression. Moreover, engineering facilitates glycosylation patterns mimicking human biology, vital for antibody function and safety. This process ensures consistency and efficacy in antibody production for treating diverse diseases. Refer to Fig. 1 for more details.
Single-Cell Cloning of Antibody-Producing Stable Cell Lines
Monoclonality of antibody-producing stable cell lines is a critical factor associated with selective genetic and physiological modifications of cells, cell viability, proliferation, and cell stability (Fig. 1). Single-cell cloning, using limited dilution cloning or disk cloning techniques, is used for generating stable cell lines. Cell-based assays (for cell viability and proliferation), genetic purity analysis (genotyping via PCR, qPCR, next-generation sequencing (NGS), or Western blotting), and structure-function analysis (ELISA), mass spectrometry, and cell-based therapeutic activity assays are used for confirming the therapeutic functions of antibodies.
Fig. 1: Genetic and physiological engineering of expression vectors and stable mammalian cell lines for producing recombinant therapeutic antibodies.
Importance of Flow Cytometry in Single-Cell Cloning
Flow cytometry plays a vital role in single-cell cloning of antibody-producing stable cell lines by enabling the isolation and characterization of individual cells based on their specific properties. In this context, flow cytometry allows for the identification and selection of cells that exhibit desirable traits, such as high antibody productivity or stable expression levels. By analyzing parameters like antibody secretion or surface marker expression at the single-cell level, flow cytometry helps researchers isolate and clone cells with the highest therapeutic potential. This process enhances the efficiency and reliability of generating stable cell lines for antibody production, ensuring the development of biopharmaceuticals with consistent quality and efficacy.
Further Reading
- The CAG promoter maintains high-level transgene expression in HEK293 cells. FEBS Open Bio (2021), 11: 95–104. Read more
- Recent advances in CHO cell line development for recombinant protein production. Drug Discovery Today: Technologies (2021). Read more
- Choice of selectable marker affects recombinant protein expression in cells and exosomes. J. Biol. Chem. (2021), 297:100838. Read more
- Advances in recombinant antibody manufacturing. Appl Microbiol Biotechnol. (2016), 100: 3451–3461. Readmore
- Characterization and Monitoring of a Novel Light-Heavy-Light Chain Mispair in a Therapeutic Bispecific Antibody. J. Pharma. Sci. (2021), 110:2904-2915. Read more
- A systematic approach for analysis and characterization of mispairing in bispecific antibodies with asymmetric architecture. MABS (2018), 10:1226–1235. Read more
- Current trends and challenges in the downstream purification of bispecific antibodies. Antibody Therapeutics (2021), 4: 73–88. Read more
- Crystal Structure and Characterization of Human Heavy-Chain Only Antibodies Reveals a Novel, Stable Dimeric Structure Similar to Monoclonal Antibodies. Antibodies (Basel) (2020), 9:66. Read more
- CHO cell engineering to prevent polypeptide aggregation and improve therapeutic protein secretion. Metab. Eng. (2014), 21:91–102. Read more
- Rapid and cost-effective development of stable clones for the production of anti-Ebola monoclonal antibodies in HEK293T cells. bioRxiv (2020) 04.21.054429. Read more
