Substantial reduction in cycle time required to identify and isolate targets
High-speed sorting using FACS enables a reduction in the amount of time to sort from 3 or 4 months to 24 to 26 days—an approximate 75% improvement.
Significant increase in yield of positives identified and cloned in each cycle
DiSH methodology depositing target hybridomas in single cell wells avoids the loss of high quality prospects due to overgrowth from competing cells, resulting in nearly a two-fold increase in positives versus traditional methods.
Dramatic increase in the number of stable positives isolated in each cycle
Using traditional methods, many positive clones fail to remain viable due to issues with chromosome structure. Comparatively, 91% of positive hybridomas remained stable through the cloning process using DiSH versus 23% of positives when using conventional hybridoma technology.
Cleanly aligns with current hybridoma screening and production methods
Unlike selected competitive technologies that require specialized laboratory training, workflow and infrastructure, the DiSH protocol closely parallels the industry’s dominant hybridoma development methodology, making adoption simple and straightforward.
Monoclonal antibodies are typically created using hybridoma technology. Hybridomas are hybrid cells resulting from the fusion of antibody-producing B-cells and a non-antibody-producing cancer cell (usually a myeloma tumor cell). Once fused, the hybridoma possesses the B-cell’s ability to generate specific antibodies and the cancer cell’s potential to reproduce indefinitely. The technology was originally invented in 1975 by Drs. Kohler and Milstein and earned them a Nobel Prize for their innovation. The technique is known as limited dilution screening (LDS) and has been in practice since that time. While the method certainly works, it is clearly inefficient and frequently results in unstable clones that are lost for future development.
Limitations of current technology
The key to using hybridomas for mAb production is based on the ability to find the proverbial “needle in a haystack”—that is, identifying and isolating the small number of hybridomas that produce a desired antibody from among a large population of heterogeneous cells. Once these cells are isolated, they can then be cloned to create a homogenous population of cells producing a unique antibody.
The traditional process of identifying, isolating and cloning targetbridomas is time-consuming, inefficient and laborious. The primary bottleneck is the requirement for limiting dilution screening (LDS) to isolate target hybridomas. During this step, the relatively few hybridoma cells that are producing antibodies of interest (typically 1 to 200 cells) must be manually identified and isolated from among a very large heterogeneous population of cells (typically 10 to 100 million cells). This process is labor intensive, requiring 75 to 90 days to complete and can often take longer. Valuable antibodies are routinely lost using traditional LDS. Cells of interest are frequently slow growing, and hence, are overgrown by uninteresting cells and, therefore undetectable, before the cloning process is complete. More importantly, the LDS process results in a high number of unstable clones which greatly reduces the number of mAbs available. Conversely, the Abeome technology has consistently resulted in greater than 90% stable positives.
Direct Selection of Hybridomas (DiSH)
Abeome’s proprietary methodology is focused on breaking down the two major bottlenecks of in conventional hybridoma screening and cloning: speed and quality of results. The Company’s DiSH (Direct Selection of Hybridomas) technology offers an improved process for rapidly selecting and cloning desired hybridomas that produce antibodies with optimal binding characteristics. Details and data on the DiSH technology were published in the Journal of Immunological Methods 343 (2009) 28-41.
Abeome’s DiSH technology, offers a much-improved method for selecting desired hybridomas. Under normal circumstances, B-cells, when stimulated by an antigen, secrete antibodies but do not surface-present them. However, by using a genetically-modified myeloma partner, Abeome creates hybridomas that both secrete antigen-specific antibody and also present antibody on the cell surface of the hybridoma.
By using a fluorescent-labeled antigen, the Company can “illuminate” the hybridomas of interest and isolate them from the population of uninteresting cells. Once the cells are bound with fluorescent-labeled antigen, researchers rapidly select them directly using fluorescence- activated cell sorting (FACS) instrumentation. Once selected, the instrumentation can rapidly deposit individual cells into micro-titer plates for culturing, ensuring the purity and stability of specific colonies and eliminating the need for repetitive rounds of limiting dilution screening (LDS). Figure 2 summarizes Abeome’s approach to optimizing identification of hybridomas producing targeted mAbs.
These efficiency improvements result in a 6-fold improvement in the number mAbs Abeome is capable of generating with its proprietary technology. Thus the benefits of DiSH technology provide Abeome’s drug discovery and development programs with an important competitive edge. Successful products are likely to be those with the best combination of development speed and optimal characteristics (e.g., affinity, specificity, immunoglobulin sub-type, etc.). Abeome’s methodology can speed up hybridoma production, while increasing the number of high-affinity antibodies that might otherwise be lost using conventional methods.