Ever since hybridoma technology was discovered in the 1970’s, scientists have been improving the technology. They have worked to enhance the antibodies themselves by improving the way they are accepted by the human body and reducing side effects. Through genetic engineering scientists have been able to move away from a final product that is full mouse antibody, to a chimeric antibody (about 33% mouse and 67% human) and now some companies are manufacturing humanized antibodies (generally 5-10% mouse and 90-95% human). In addition to improving the genetic make-up of the antibody, work has continually been done to improve manufacturing techniques and antibody yield. In this blog we will look at three areas in the process where antibody production can be improved. This will be the first in a series of blogs on improving hybridoma antibody production. In blogs to follow over the next few weeks, we will delve into each of the technologies presented here in further detail.
At the beginning of the hybridoma culture process, a clone must be generated and selected. The best clones are stable and good producers with high antibody production. One technology that allows an alternative to traditional cloning and selection is to utilize an automated process. These automated systems offer a quicker way to develop a stable cell line by utilizing the machine to screen several more clones than would be possible using traditional means. By screening hundreds of clones at a time, it increases the likelihood that a very high expressing, stable clone will be found. This not only increases the yield and stability of hybridoma lines but also reduces opportunity for human error by turning more processes over to robotics. There are different methods utilized in automated systems and several companies have automated systems designed for hybridoma cloning including: Stemcell Technologies’ ClonaCell Easy Pick System and Molecular Devices (formerly Genetix) ClonePix System. Despite their differences, each system offers the benefit of cutting development time, the ability to screen more clones and elimination of the need for a person to conduct time consuming ELISA assays. These are all very attractive benefits, but these systems can be costly and may not be an option for all labs.
Another evolution for hybridoma technology has been the media in which the cells are grown. In many instances hybridoma media consists of base media plus serum. However, serum and other animal derived ingredients are undesirable because they are undefined and as such have high batch-to-batch variation that leads to unpredictable results. Serum also contains contaminating IgG, which makes purification of the antibody difficult. These issues have caused a migration toward serum-free hybridoma culture. To assist in the endeavor to become animal-free, animal components can now be replaced by utilizing innovative animal-free supplements such as recombinant albumin and recombinant transferrin that are completely IgG free. Today companies including Sigma, Fisher Scientific, InVitria, Sheffield Bioscience and Mediatech sell recombinant albumin and recombinant transferrin that can be added to base media to provide an alternative to using serum in hybridoma culture. InVitria has a supplement that is specifically designed for this purpose called Zap-Hybridoma. According to InVitria, Zap-Hybridoma can provide an animal-free replacement for fetal bovine serum (FBS) when used with DMEM-F12.
In production there are also opportunities for improvement and many believe that perfusion bioreactors can improve hybridoma antibody production. In particular with hybridoma culture, perfusion bioreactors can offer a great benefit because they can produce gram quantities of antibody very quickly in a small amount of space. This is important because often early in the drug discovery process, antibodies need to be generated quickly for proof of concept, toxicology and animal studies. The amount does not require larger scale antibody production and frequently the antibody itself has not been approved for further development, thus it would not be feasible to manufacture in CHO. High yield, quick hybridoma culture can produce the necessary antibodies to complete these initial examinations. The sooner an antibody can be generated for initial studies, the earlier a go/no go decision can be made, resulting in a reduced overall drug discovery timeline. Using perfusion bioreactors in hybridoma culture typically results in higher cell densities, faster cell growth times, higher antibody concentration and ultimately fewer runs to manufacture the same amount that could be generated using a T-flask or shake flask. Companies that manufacture perfusion systems include Wave Biotech, FiberCell, Integra, Xcellerex and Biovest.
As part of the hybridoma series, I would like to hear from readers who have used any of these techniques or have others to recommend.