244
Mohamed A1-Rubeai
diseases, viral diseases and autoimmunity, but the implications of this activity
outside the medical field have also been recently realised. In particular, the rapid
advances of the last few years have heightened interest in the mechanisms of cell
death in the bioreactors used for the production of biopharmaceuticals. The
question as to why there should be any interest in the mechanism of cell death in
animal cell culture technology can be answered by illustrations from experience
in the culture of hybridoma and myeloma, two cell lines which are used
extensively for the large scale production of antibodies. Apoptosis in hybridoma
cells was first reported by A1-Rubeai et al. [29] in a study of the cellular
ultrastructre during batch culture and by the Franek group [90, 91] in proteinfree media. A recent kinetics study demonstrated an association between DNA
fragmentation and cell density during batch cultivation of 24 hybridoma cell
lines with the presence of short fragments of 100 bp at the mid exponential phase
and unresolved long fragments at the late death phase [92]. In the physical and
chemical environment of a bioreactor, it has been recently shown that apoptosis
plays a critical role in the determination of growth and survival of both
hybridoma and myeloma cell lines [21]. It has also been demonstrated that
apoptotic responses can be caused by serum, glutamine and glucose depletion,
cytotoxic metabolite accumulation and by the stressful hydrodynamic conditions often found in bioreactors [-93-95]. Significant levels of cell death occur in
high cell density cultures, specifically when process intensification by means of
cell retention or immobilisation is employed. For example, in fixed bed systems
the cells are exposed to oxygen limitation within the porous carriers [96].
Depreviation of hybridoma cells of oxygen in bioreactor culture for 45 h
resulted in 70% dead cells, of which 90% were apoptotic [97]. The induction of
apoptosis under anoxic conditions was attributed to a critical reduction in the
intracellular energetic balance and protein synthesis and probably also to
the production of stress proteins similar to those synthesised following heat
shock.
In seeking to implement higher performance bioproduction strategies it is
necessary to identify ways to reduce apoptotic cell death of cultured cells. Prior
research strongly suggests that such apoptotic cell death might be reduced by
specific genetic modification of the cells. Changes in bioreactor configuration
and/or operating protocols may further reduce apoptosis.
The growing appreciation of the central role of apoptosis in controlling cell
number and viability in the cultures of cell lines that are used for the production
of biopharmaceuticals has stimulated interest in metabolic engineering of cell
survival. The most obvious application is the transfection of cell lines with
anti-apoptotic genes, such as bcl-2, bcr-abl, v-abl and E1B to inhibit cell death,
thereby prolonging culture duration. The successful transfection and expression
of these genes in cell lines producing products of commercial importance should
lead to the development of more robust cell lines which are able to survive in
sub-optimal conditions, including nutrient limitation, and in stressful physical
environments. The feasibility of such objectives was demonstrated in my laboratory by showing that, unlike unmodified cells, a lymphoblastoid cell line
Mohamed A1-Rubeai
diseases, viral diseases and autoimmunity, but the implications of this activity
outside the medical field have also been recently realised. In particular, the rapid
advances of the last few years have heightened interest in the mechanisms of cell
death in the bioreactors used for the production of biopharmaceuticals. The
question as to why there should be any interest in the mechanism of cell death in
animal cell culture technology can be answered by illustrations from experience
in the culture of hybridoma and myeloma, two cell lines which are used
extensively for the large scale production of antibodies. Apoptosis in hybridoma
cells was first reported by A1-Rubeai et al. [29] in a study of the cellular
ultrastructre during batch culture and by the Franek group [90, 91] in proteinfree media. A recent kinetics study demonstrated an association between DNA
fragmentation and cell density during batch cultivation of 24 hybridoma cell
lines with the presence of short fragments of 100 bp at the mid exponential phase
and unresolved long fragments at the late death phase [92]. In the physical and
chemical environment of a bioreactor, it has been recently shown that apoptosis
plays a critical role in the determination of growth and survival of both
hybridoma and myeloma cell lines [21]. It has also been demonstrated that
apoptotic responses can be caused by serum, glutamine and glucose depletion,
cytotoxic metabolite accumulation and by the stressful hydrodynamic conditions often found in bioreactors [-93-95]. Significant levels of cell death occur in
high cell density cultures, specifically when process intensification by means of
cell retention or immobilisation is employed. For example, in fixed bed systems
the cells are exposed to oxygen limitation within the porous carriers [96].
Depreviation of hybridoma cells of oxygen in bioreactor culture for 45 h
resulted in 70% dead cells, of which 90% were apoptotic [97]. The induction of
apoptosis under anoxic conditions was attributed to a critical reduction in the
intracellular energetic balance and protein synthesis and probably also to
the production of stress proteins similar to those synthesised following heat
shock.
In seeking to implement higher performance bioproduction strategies it is
necessary to identify ways to reduce apoptotic cell death of cultured cells. Prior
research strongly suggests that such apoptotic cell death might be reduced by
specific genetic modification of the cells. Changes in bioreactor configuration
and/or operating protocols may further reduce apoptosis.
The growing appreciation of the central role of apoptosis in controlling cell
number and viability in the cultures of cell lines that are used for the production
of biopharmaceuticals has stimulated interest in metabolic engineering of cell
survival. The most obvious application is the transfection of cell lines with
anti-apoptotic genes, such as bcl-2, bcr-abl, v-abl and E1B to inhibit cell death,
thereby prolonging culture duration. The successful transfection and expression
of these genes in cell lines producing products of commercial importance should
lead to the development of more robust cell lines which are able to survive in
sub-optimal conditions, including nutrient limitation, and in stressful physical
environments. The feasibility of such objectives was demonstrated in my laboratory by showing that, unlike unmodified cells, a lymphoblastoid cell line