Strategies For Generating Stable Cell Lines For Producing Therapeutic Antibodies
Posted on Aug 26, 2021
Recombinant monoclonal antibodies are one of the most promising drugs (immunotherapy) for human diseases which include infectious diseases, cancer, and various other diseases. Mammalian cells (CHO, HEK293, PER.6) are primarily being used to produce therapeutic antibodies, except single chain antibodies without PTMs (glycosylation) which are expressed in non-mammalian cells or cell-free expression systems. Generation of antibody producing stable cell lines is the single most factor for large-scale manufacturing of therapeutic antibodies. There are several critical factors that are associated with generating stable cell lines for expressing therapeutically active recombinant antibodies. These factors include 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
Genetically optimized expression vectors are required for efficient expression of functionally active antibody drugs and creation of stable cell lines producing these antibodies. The genetic optimization of expression vectors includes increased and stabilized transcription and translation of target antibody genes. 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
Multiple strategies and methods for mammalian cell engineering are adopted for the expression of therapeutically functional antibodies and generation of stable cell lines expressing these antibodies. These cell engineering methods or strategies include controlled dose dependent expression or copy number using site specific integration (e.g., CRISPR), inhibition of apoptosis and gene silencing using genomic modification of mammalian cells, glycoengineering, etc. 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 which is associated with selective genetic & physiological modifications of cells, cell viability & proliferation, and cell stability (Fig.1). Single cell cloning using limited dilution cloning or disk cloning techniques are used for generating stable cell lines. Cell based assays (for cell viability and proliferation), genetic purity analysis (genotyping PCR, qPCR, next-generation sequencing (NGS), or Western blotting), and structure-function analysis ELISA, mass spectrometry, cell based therapeutic activity assays) are used for confirming therapeutic functions of antibodies
Fig. 1: Genetic and physiological engineering of expression vectors and stable mammalian cell lines for producing recombinant therapeutic antibodies.
- The CAG promoter maintains high‐level transgene expression in HEK293 cells. FEBS Open Bio (2021), 11: 95–104. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7780116/
- Recent advances in CHO cell line development for recombinant protein production.
Drug Discovery Today: Technologies (2021).
- Choice of selectable marker affects recombinant protein expression in cells and exosomes. J. Biol. Chem. (2021), 297:100838. https://www.jbc.org/article/S0021-9258(21)00636-0/pdf
- Advances in recombinant antibody manufacturing Appl Microbiol Biotechnol. (2016), 100: 3451–3461.
- Characterization and Monitoring of a Novel Light-heavy-light Chain Mispair in a Therapeutic Bispecific Antibody. J. Pharma. Sci. (2021),110:2904-2915.
- A systematic approach for analysis and characterization of mispairing in bispecific antibodies with asymmetric architecture. MABS (2018),10:1226–1235. https://www.tandfonline.com/doi/pdf/10.1080/19420862.2018.1511198.
- Current trends and challenges in the downstream purification of bispecific antibodies. Antibody Therapeutics (2021), 4: 73–88.
- 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. https://pubmed.ncbi.nlm.nih.gov/33266498/
- CHO cell engineering to prevent polypeptide aggregation and improve therapeutic protein secretion. Metab. Eng. (2014), 21:91–102. https://pubmed.ncbi.nlm.nih.gov/23380542/
- Rapid and cost-effective development of stable clones for the production of anti-Ebola monoclonal antibodies in HEK293T cells. bioRxiv (2020) 04.21.054429. https://www.biorxiv.org/content/10.1101/2020.04.21.054429v1
Warning: sizeof(): Parameter must be an array or an object that implements Countable in /nfs/c04/h06/mnt/63860/domains/marinbio.com/html/wp-content/themes/marin-biologic-laboratories/template-parts/content-single.php on line 43
Tags : Therapeutic Antibodies