No services found
No Products found
Monoclonal antibodies can be produced in mammalian systems using either transient or stable expression approaches. These two approaches have well-known advantages and limitations and each remains useful for different purposes and applications. In this article, we discuss the main differences between transient and stable monoclonal antibody production and explain how each strategy can be used to its fullest potential. Check out other frequently asked questions (FAQs) about stable cell lines for monoclonal antibody production on our dedicated page.
The terms transient and stable production are used to define two different approaches to recombinant expression in mammalian systems. The main difference between the two methods lies in the duration and scale of the production process.
Transient expression implies the temporary and small-scale production of antibodies in a mammalian host; while stable expression implies the stable, virtually everlasting, and large-scale production of recombinant proteins. The former implies a temporary change, while the latter requires the stable integration of transgenes into the genome.
In transient systems, foreign DNA, unable to replicate independently from the host’s DNA, persists only for a few days. In contrast, with stable transfection, foreign DNA is integrated into the genome, replicated alongside it, and, more importantly, passed down to the progeny. In this way, vectors used for transient expression are simpler than the ones used for stable integration.
For transient production, expression vectors typically contain:
Reporter genes such as luciferase are often included in transient expression vectors because they allow the quick detection of positive cells in a heterogeneous population. Given that the numbers of copies of DNA vectors decrease with each cell cycle, no selection is carried out in transient production. Instead, the efficiency of the transfection protocol can be estimated by measuring signal intensity.
On occasion, selection markers may be included in transient expression vectors. However, given the ephemeral nature of this type of transfection, it is often most useful to optimize transfection efficiency by fine-tuning transfection conditions or optimizing vector design than to attempt enriching positively transfected clones by applying a selective pressure.
In contrast, vectors used for stable monoclonal antibody production are more complex. Unlike transient expression, the process of stable cell line generation requires the laborious selection of positive clones. Moreover, these clones need to be amplified to ensure all cells are genetically identical to each other before proceeding to the scale-up stage. For this reason, stable cell line vectors typically carry:
DNA vectors used for stable cell line generation are typically linearized, which significantly increases the chances of stable integration in the host’s genome in comparison to circular vectors.
Interestingly, even stably integrated transgenes tend to be excised from the genome if no selective pressure is applied. In this way, the growth medium of stable cell lines should still contain either the antibiotic or metabolite used during the selection step.
Transgene integration is a rare event and thus comprises one of the most important bottlenecks of this process. It requires several rounds of dilution, enrichment, and amplification in selective media. Considering the low frequency of successful transgene integration, selection of positive clones and ensuring clonality is one of the most laborious steps of the process.
Moreover, the position where the transgenes are integrated also matters. Because the genome is a complex tridimensional structure, some regions of the molecule are known to be less accessible to transcriptases and consequently having lower levels of expression.
For this reason, site-directed integration, despite being more expensive than random integration strategies, is known to have higher success rates. Several strategies can be used to direct transgene integration:
CRISPR/Cas9 technology is quickly gaining ground over other technologies for the site-directed integration of antibody-encoding genes.
Transient expression of monoclonal antibodies is an invaluable tool for producing small quantities of antibodies for research applications and lead candidate screening. As the name implies, this system allows the temporary transformation of a mammalian host into an antibody-secreting cell. But production persists only for a few days since foreign non-integrated DNA cannot replicate independently from the host’s genome or being transmitted to the progeny.
In contrast, stable expression of monoclonal antibodies allows indefinite production in synthetic medium. However, stable integration of transgenes is a rare event and the process of positive clones is laborious. For this reason, stable expression is reserved for antibodies with commercial value such as biotherapeutics or diagnostic antibodies.
Got a question or need a quote? Message us and we'll get back to you 48 hours or less.
Your cart is currently empty.