The hybridoma technology allowed us to leverage potent and highly specific antibodies for therapy, research, and diagnostics. However, monoclonal antibody production by hybridoma technology has remained largely unaltered during the past decades. With the increasing demand for new antibody reagents, hybridomas can be impractical when urgent and time-sensitive solutions are needed.
Development of monoclonal antibody production by hybridoma technology
Hybridomas are hybrid cell lines created by the fusion of short-lived antibody-producing B cells and immortal myeloma cells. The process, optimized and perfected over the past decades, endows the hybrid cell line with the ability to produce antibodies indefinitely under laboratory conditions.
However, in recent years, monoclonal antibody production by hybridoma technology has raised some concerns. For instance, even though hybridomas are immortal cell lines, some studies have shown they are prone to genetic drift often resulting in the loss of antibody-encoding genes. Moreover, researchers have also recently found that hybridomas can co-produce additional unspecific antibody heavy or light chains, contaminating the production batch.
As a result, with a few exceptions, most monoclonal antibodies are now produced recombinantly, and hybridomas have ceased to be the main production platform and come to represent only an intermediary step.
But despite the problems solved by hybridoma sequencing and recombinant technology, the development of these hybrid cell lines is still time-consuming and technically challenging. Thus, it may be impractical when urgent solutions are necessary.
Typical hybridoma development projects can take up to 5 months and are restricted to only a few hosts, such as mouse, rabbit, and less frequently chicken. However, despite the technical hurdles and limitations, hybridoma is still popular due to its natural advantages:
- Possibility to adapt to fully human antibody production by using transgenic mice – thus leveraging the benefits of a robust technology and forgoing the need for further antibody humanization
- Efficient in vivo affinity maturation – reducing potential antibody engineering costs
- Preservation of natural chain pairing information (heavy and light) – thus reduced risk for immunogenicity or instability on the resulting antibody
Recent improvements in monoclonal antibody production by hybridoma technology
Antibodies can now be generated in vitro, thus considerably shortening the typical timeframes associated with monoclonal antibody production by hybridoma technology. However, in vitro methods (e.g. phage display) still present an important disadvantage – the risk for weak antibody affinity that may need to be compensated by additional antibody engineering approaches.
Thus, as some researchers attempt to improve in vitro antibody generation, others focus on improving the robust hybridoma technologies. In recent years, some approaches were reported to increase efficiency and reduce developmental times for hybridomas, namely:
- The use of type 1 interferons to boost antibody production targeting antigens delivered via Fc-fusion proteins
- The co-inoculation of anti-CD40 agonist antibodies and antigens to shorten the immunization process and increase the number of hybridomas containing unique antibodies
- Direct cloning of antibody variable genes for immediate transfection into recombinant antibody expression systems
These improvements are shown to shorten the timeframes for monoclonal antibody production by hybridoma technology. However, one of the biggest bottlenecks for antibody development by hybridoma persists – the reduced fusion efficiency between B cells and their corresponding myeloma partners which compromises the chances of discovering the best antibodies.
Many researchers agree it is time to shift from monoclonal antibody production by hybridoma technology to single-cell approaches. Antigen-specific antibodies can now be obtained by single-cell screening technologies on peripheral blood samples coupled with single-cell sequencing for immediate transfection of antibody-encoding genes. This would eliminate the need to produce hybridomas and consequently the need to develop adequate fusion partners for B cells from different host species.
Several studies showed it is possible to isolate and select high quality B cells using FACS (fluorescence-activated cell sorting) with fluorochrome-labeled antigens or antigen-coated magnetic beads. mRNA transcripts for each cell can then be obtained by RT-PCR (reverse transcription PCR). Moreover, by including restriction sites in the primer sequences these can be immediately cloned and transfected into suitable expression systems.
However, single-cell technologies still suffer from many important drawbacks, including difficulty in maintaining cell structure and viability during the screening process, difficulty in isolating low-abundance cells, and high risks of introducing mutations during mRNA transcription and amplification. Moreover, these techniques generate a high amount of complex data that needs to be correctly interpreted to generate the best possible results.
Researchers believe these limitations may soon be bypassed with the use of microfluidic technologies for single-cell-based antibody discovery. These technologies can be easily coupled with next-generation sequencing platforms to speed up monoclonal antibody production. Nevertheless, it is important to consider that these technologies are still expensive, technically demanding, and not fully mature, which may lead to prohibitive antibody production costs.
Monoclonal antibody production by hybridoma technology is still a highly valuable method. Unlike in vitro antibody generation, hybridomas leverage the fast and inexpensive in vivo maturation process as well as maintain the natural pairing information between antibody heavy and light chains.
However, the greatest bottleneck of this technology is the long time-frame necessary to generate, screen, and clone antigen-specific cell lines. For this reason, researchers are urging us to invest in alternative technologies for antibody discovery.
In recent years, several studies showed it is possible to use single-cell technologies to eliminate the need for hybridoma development. But, presently, these technologies may not be robust or reliable enough to provide a practical alternative to hybridoma development.
- Bradbury, A. R. M. et al. When monoclonal antibodies are not monospecific: Hybridomas frequently express additional functional variable regions. MAbs. 2018; 10(4): 539–546. doi: 10.1080/19420862.2018.1445456
- Fundamental technologies for antibody engineering. Therapeutic Antibody Engineering. Woodhead Publishing. 2012; chapter 4:57-595. doi: 10.1533/9781908818096.57
- Lei, L. et al. Antigen-Specific Single B Cell Sorting and Monoclonal Antibody Cloning in Guinea Pigs. Front Microbiol. 2019; 10:672. doi: 10.3389/fmicb.2019.00672
- Stark, J. and Coe, D. Limitations in Single-Cell Analysis Must Be Addressed with Innovation. GEN. March 19, 2019. Retrieved from https://www.genengnews.com/insights/limitations-in-single-cell-analysis-must-be-addressed-with-innovation/