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ECVAM report: monoclonal antibody production
Monoclonal antibodies (mAbs) have many important roles in fundamental research, biotechnology research and development, clinical diagnosis and some therapies. In many ways they are a good example of the importance of animal research, since the initial step in making a mAb involves the immunisation of a rodent.
However, once the hybridoma secreting the desired mAb has been produced, there is a choice of methods for the actual production of the monoclonal. Either the hybridoma can be cultured in vitro and the mAb purified from the culture supernatant, or the hybridoma can be injected into a rodent where it will form an ascitic tumour, containing a fluid with a high concentration of the mAB. Obviously, animal welfare considerations would argue against using the animal method of production because the process undoubtedly will involve some animal suffering. However, there are some disadvantages with the in vitro method of production which have made it unsuitable for use in some cases.
A recent report from an workshop of the European Centre for the Validation of Alternative Methods (ECVAM) has examined the arguments in favour of these two approaches to producing monoclonal antibodies (published in ATLA 25, 121-137) and has concluded that "for all levels of mAb production, one or more in vitro methods are scientifically acceptable and reasonably or practically available." Accordingly, they recommend that the use of the animal method of production should be banned although they accept that there will still be a small number of cases where the animal method of production will still need to be used and that, where appropriate justification is shown, exceptions to the ban should be granted.
Apart from the animal welfare considerations, there are three main factors which have influenced the choice between the animal and the in vitro methods of mAb production: mAb yield, technical difficulty and cost.
The ascites method yields monoclonal antibody concentrations of between 1 and 20mg/ml with up to 5ml fluid obtained per mouse (and up to 20ml in a rat). By comparison, standard flask culture of hybridomas will yield between 20 and 100µg/ml in a 500ml flask. However, there are several high-density cell culture methods which have been developed to harvest products such as mAbs, which produce significantly higher yields. Modern membrane-based or matrix based culture systems have been shown to produce up to 500µg/ml of mAb in the culture medium. High-density cell bioreactors have prod-ucedbetween 500µg/ml and 5mg/ml mAb.
The technical difficulty of using these modern high-density cell culture techniques has been a barrier to their greater use, but the technology has improved and become much easier in recent years. One complaint which was commonly voiced was that a particular hybridoma would not grow very well (or at all) in cell culture. With improved cell culture technology, this appears to be less of a problem these days. There will still be the rare hybridoma which will only yield high concentrations of mAb in an ascites and cannot be adapted to a cell culture system and these should be permitted to continue using the animal system of production.
The costs of using animals for mAb production have risen over the years with increasing health and welfare standards. At the same time, the cost of cell culture apparatus has decreased. Currently, the costs of the two systems of production are similar. The main cost for the cell culture system is fetal calf serum. The development of several serum-free, or low serum, culture mediums for mAb production have made it possible to reduce this cost and result in considerable economic savings.
The ECVAM workshop report also contains a very useful summary of the regulatory position regarding the use of animals for mAb production in each of five main European countries. This showed a variable regulatory position, from the Netherlands and Switzerland,which have a formal ban on the use of the ascites method of production but will allow exceptions to the ban where scientifically justified: to Germany, where the production of a mAb by the ascites method, for diagnostic or therapeutic use, is not actually considered to be part of an experimental procedure and is not covered by the German Animal Protection Act.
This ECVAM report is worthy of serious study. The authors have brought together a lot of relevant detail and have made their case effectively. The only problem with the report is that it makes a case. It is clearly arguing from the belief that the animal method of mAb production should be banned. The authors may well be correct, but they would have made their case much stronger if they had presented an impartial and objective assessment.