Introduction
Monoclonal antibodies are antibodies which
have a single, selected specificity and which
are continuously secreted by “immortalised”
hybridoma cells. A hybridoma is a biologically
constructed hybrid of a mortal, antibody-
producing, lymphoid cell, and a
malignant, or “immortal”, myeloma cell. Following
the discovery of hybridoma technology
in 1975 (2), developments in mAb
production and in their application have had
profound implications not only on medical
research, diagnosis and therapy, but also on
biology in general. Hybridoma technology
represents a significant advance because, in
principle, it provides a means for obtaining
unlimited supplies of highly specific antibodies.
In the production of mAbs, animals (generally
rats or mice) first have to be immunised
with the target antigen to obtain
mortal antibody-producing cells. The biological
construction of hybrids, and the selection
of hybridomas which produce antibodies
with the desired specificities, are carried out
in vitro. In the early days of hybridoma technology
(the late 1970s), the hybridomas
developed in vitro were injected into the peritoneal
cavity of an animal so that useful
amounts of the desired mAb could be harvested
from the ascitic fluid. This procedure
was considered necessary at the time, since
no efficient large-scale in vitro methods were
available. By the mid-1980s, there were
already serious doubts regarding the necessity
of such a painful animal procedure. Nevertheless,
as a result of its early introduction
as part of the hybridoma technology, ascites
production of mAbs is now employed worldwide,
in spite of the ongoing development
of in vitro technologies and the growing public
pressure to replace or reduce animal
experiments. The urgent need for experts to
disseminate information and make recommendations
about antibody production, taking
animal welfare issues into consideration,
was recognised by ECVAM in holding a
workshop on avian antibodies in March 1996
(3) and, subsequently, in organising this
workshop on mAb production.
Hybridoma Technology
There are essentially two stages in the production
of mAbs: a) the induction of antibodyproducing
lymphoid cells in vivo and the
selection of antibody-producing hybridoma
cells in vitro; and b) the in vitro/in vivo propagation
of selected hybridoma clones. The
first stage, the formation and selection of the
hybridoma clone, involves the use of one or
more animals (except in rare cases when a
human mAb is being developed), and is carried
out in the following way:
1. The antigen is injected into mice (or
rats). The antigen is often injected in
combination with an adjuvant, to
enhance the immune response, even
though the use of adjuvant generally
leads to severe side-effects.
2. After an appropriate interval (5–21 days),
the immunised animals are killed and
User group B: 0.1–0.5g
This group accounts for approximately 30%
of mAb users and encompasses a significant
number of people still using the in vivo
method. Antibodies in these amounts are
required for the development and production
of a wide range of in vitro diagnostic kits and
reagents, as well as for evaluating the usefulness
of novel therapeutic mAbs in animal
experiments.
User group C: 0.5–10g
In this group, which accounts for approximately
10% of mAb users, adoption of the in
vivo method is comparatively rare. The
mAbs produced are used in routine diagnostic
procedures and in preclinical evaluation
studies. They are usually produced by large
biotechnology companies but, during the last
few years, the production of these mAbs has
increasingly been contracted out to smaller
facilities.
User group D: > 10g
Users in this group, who require mAbs for
prophylactic and therapeutic purposes in
vivo, make up less than 1% of all mAb users
in Europe. The mAb production processes
they use are first developed and validated by
the pharmaceutical industry, and are then
submitted to a regulatory body for approval.
The extensive use of the ascites method by
groups A and B can be attributed to its supposed
economic advantage as well as to a
lack of inclination to adopt the new techniques.
Most of the mAbs produced by
these groups are not used in clinical studies
and therefore do not have to comply with
the standard requirements for pharmaceutical
products. This has led to a lack of
awareness in these user groups of the disadvantages
of ascites production, such as
the potential for infection by animal
viruses, and the reduced immunoreactivity
of the mAb due to contamination with nonspecific
animal immunoglobulins.
Monoclonal Antibody Production In
Vivo
The in vivo procedures entail the use of mice
or rats. Initially, the immune systems of the
experimental animals are suppressed (1–2
weeks before the intraperitoneal [i.p.] injection
of hybridoma cells) by injection (i.p.) of
a primer, such as pristane (2,6,10,14-tetramethylpentadecane)
or Freund’s incomplete
adjuvant. The hybridoma cells then multiply
in the peritoneal cavity, and the ascitic fluid
which forms is a very rich source of the
secreted antibody.
When an adequate amount of ascites has
formed, the animal is killed and the ascitic
fluid is collected. Sometimes, the ascitic fluid
is first “tapped” or drained from the peritoneal
cavity while the animal is under
anaesthetic, with a second and final harvest
being taken once the ascites has reformed.
The mAb product can be harvested 5–21 days
after the injection of hybridoma cells.
Approximately 5ml of ascites can be obtained
from a mouse, and 10–40ml from a rat. Thus,
for the production of a mAb with a given
specificity, it may be necessary to use one or
more mice, depending on the amount of antibody
required.
The main advantage of the ascites method
is the extremely high yield of antibody,
which generally lies in the range 1–20mg/ml.
In addition, the method is not excessively
labour-intensive.
However, these advantages are outweighed
by a number of disadvantages. The
main disadvantage of the ascites method is
that it is extremely painful for the animals
used, due to the following: a) the injection of
primer; b) the resulting peritonitis caused by
the primer; c) abdominal tension; and d) the
invasive tumours which result (4–6). Proper
animal husbandry facilities are mandatory.
The mAbs produced generally show a
reduced immunoreactivity of 60–70%, as
opposed to an immunoreactivity of 90–95%
for antibodies produced in vitro, due to contamination
by biochemically identical
immunoglobulins. There is also a potential
risk of product contamination by viruses
which are pathogenic to humans. A further
disadvantage is that the individual batches
of harvested ascitic mAb are of variable quality,
and they are contaminated with bioreactive
cytokines.