In 1975 George Kohler and Cesar Milstein (13) published a short paper
describing their new method for producing monoclonal antibodies (MAbs).
Their work included the observations that: (1) "the manufacture of
predefined specific antibodies by means of permanent tissue culture cell
lines is of general interest," (2) "such cells can be grown in vitro in
massive cultures," and (3) "such cultures could be valuable for medical
and industrial use." These comments were prophetic, since their new
alternative technique was subsequently adopted in virtually every field
of biomedical research and biotechnology and in many areas of clinical
diagnosis and therapy. So important was this approach to antibody
production that Kohler and Milstein received the 1984 Nobel Prize for
their discovery.
Although the original research was principally an in vitro technique,
it was also apparent that monoclonal antibodies could be produced by
injecting the hybridoma cells into the abdominal cavities of different
species of rodents. This was the initial use of the ascites method.
Since these MAbs were easily made in any laboratory and the ascites
process, widely viewed as both simple and inexpensive, was introduced
early, its use rapidly expanded. Unfortunately, the original possibility
of MAb production replacing uses of laboratory animals was and often
continues to be overlooked or ignored. In the decades that followed the
original discovery, tens of millions of animals suffered and died
despite the availability of more humane alternatives.
During this same period of time the appropriateness of using the
ascites method was increasingly being questioned in Europe. Milstein
noted that "in later years, both on practical and humane grounds, I
became concerned with the use of ascitic fluids" (personal
communication, 1997). As new in vitro alternative techniques were
developed and validated, it became more difficult to justify the
suffering associated with the use of ascites. It simply was not possible
to humanely produce MAbs using animal-based procedures.
In 1989, The Netherlands government introduced a Code of Practice for
the Production of Monoclonal Antibodies (1), which provided detailed
descriptions of the veterinary problems and pathophysiology associated
with the ascites process and placed restrictions on its use. The
resulting increased humane awareness among Dutch researchers provided
further encouragement for adoption of in vitro approaches to MAb
production. In 1995, a symposium held in Bilthoven, The Netherlands,
concluded that progress in development of such alternatives (both in
efficacy and cost) was sufficient that the use of ascites could no
longer be justified. The resulting prohibition of animal-based MAb
production caused no serious difficulties within the Dutch biomedical
research community. Despite initial academic resistance, bans on ascites
in Germany and Switzerland experienced similar results, as did the
restrictions placed on ascites use in Sweden and the United Kingdom.
By 1996 in vitro production of MAbs was the method of choice in
Europe for commercial concerns and others needing large quantities and
on the increase for the smaller-scale needs of individual researchers.
This latter group made up about 60 percent of European MAb users,
primarily for basic research and some diagnostic procedures. Their MAb
needs were often met using the ascites technique. A similar situation
exists in the United States.
Scientist representatives from several member states of the European
Union met in October 1996 at the European Center for the Validation of
Alternative Methods to discuss the current status of in vitro and in
vivo methods of monoclonal antibody production. After careful
consideration of the different types of research and commercial needs
for MAbs and the available production options, they concluded that "for
all levels of MAb production; there are one or more in vitro methods
which are not only scientifically acceptable, but are also reasonably
and practically available; and as a consequence, in vivo production can
no longer be justified and should cease." (15) The group further called
for a Europe-wide prohibition on the routine use of ascites methods of
MAb production. In Europe the trend is toward adoption of in vitro
alternatives at all stages of the MAb process.
There are three principal steps in production of monoclonal
antibodies: 1) immunization, 2) hybridoma formation and 3) MAb
production. Each has its own potential for adoption of alternative
methods. Although currently done primarily as an in vivo procedure using
a few animals, it is possible, especially with human MAbs, to conduct
the immunization process entirely in cell cultures. Some technical
difficulties remain to be solved before this becomes a routine
non-animal-based procedure.
Formation of the hybridoma cells has always been an in vitro
technique. However, final production of the monoclonal antibodies
involves use of either the ascites or in vitro alternative approaches.
The present review briefly focuses on this last step, with an emphasis
on the availability of multiple alternative MAb production techniques,
suitable for the small-to-medium-scale research and commercial
laboratories.
