Increased and detailed knowledge about the precise carbohydrate
structures has revealed the importance of post-translational
modifications for functionality of therapeutic proteins. However, these
findings also exposed that the list of modifications is frighteningly
long and many of them might affect the immunogenicity, stability,
pharmacokinetics and thus efficacy of the protein. The most important
capability that distinguishes mammalian cells from other expression
systems is N- and O-linked glycosylation and it is assumed that around
2% of the human genome encodes proteins that contribute to
glycosylation [28].
Although almost any mammalian cell line possesses the machinery to
produce and secrete proteins, only a limited number meet the
fermentation requirements and thus can be used for industrial
manufacturing: Chinese hamster ovary (CHO), baby hamster kidney (BHK)
and mouse myeloma cells NS0 and Sp2/0.
Glycan structures differ significantly among different cell types
and species. Only human cells lines promise to produce proteins in a
way that species-specific and thus immunogenic differences in
glycosylation are absent. For safety and regulatory reasons human cells
used for production should not be of tumor origin. In addition, only a
limited number of human cell lines are available to date. The
development of permanent cell lines using primary human cells and
recombinant DNA techniques has been hampered by the fact that human
cells are highly resistant to transformation by viral functions. In
fact, from human tissue sources only human embryonic kidney (HEK) cells
[17], human embryonic lung (HEL) cells [29], human embryonic retinoblasts (HER cells) [30,19] and primary human amniocytes [20]
have been successfully transformed with adenoviral functions. For
practical and ethical reasons it is very difficult to obtain primary
cells from fetal origin. Primary human amniocytes are the only cell
type that is readily available without ethical concerns: they can be
collected by routine amniocentesis. Human amniocytes can be cultivated
as adherent cultures for several passages under standard conditions and
consist of three main cell types, fibroblast-like, epithelial-like and
amniotic fluid (AF) cells [31]. Recently it has been shown, that about 1% of cells found in amniotic fluid are amniotic-fluid derived stem (AFS) cells [32].
Considering the low transfection efficiency (< 1% in the present
analyses) of human amniocytes, the transformation efficiency using an
E1-expressing plasmid is surprisingly high. In earlier studies we have
calculated to obtain at least one or two transformants in 1 × 105 cells transfected [20].
Even though in the present analyses we have transfected two plasmids,
one containing the E1 and pIX genes and a second expressing hAAT with a
plasmid ratio of 1:1, the transformation efficiency was comparable to
transfection with the E1-plasmid alone (data not shown).
Cotransfection of two plasmids expressing the transforming
E1-functions and hAAT, respectively, resulted in stably transformed
cell pools, with 6 out of 10 pools expressing hAAT. Moreover, 4 out of
6 cell pools show long-lasting expression for more than 35 passages,
with expression levels up to 6 μg/ml or 8
pg/cell/day. Cell cloning by limited dilution from two cell pools
resulted in genetically identical cell lines which show stable
expression of hAAT for more than 65 passages (the time course of the
present study) with expression levels up to 30 pg/cell/day. Since
cloning of cells was started in passage 26, this indicates stable
expression of hAAT for more than 90 passages. Again it has to be
emphasized that this stable protein expression was achieved without any
antibiotic selection.
The majority of the mammalian genome is transcriptionally silent.
Since integration of transfected plasmids occurs randomly, the position
effects generally manifest partial or complete loss of expression. In
order to overcome the position effect numerous regulatory elements
introduced into the plasmid expressing the gene of interest have been
tested including insulators, MARs, strong promoters and enhancers. Even
when using such elements a time consuming testing of numerous cell
clones over multiple passages cannot be avoided. Other crucial
parameters like the choice of promoters for expressing the gene of
interest and the marker gene, the ratio of gene of interest to marker
gene expressing plasmid, concentration of selection marker in the
medium or using linear versus circular plasmids considerably influence
cell line development and expression levels.
In common strategies to select for highly expressing cell lines, the
marker gene is linked to the gene of interest and employing a selection
strategy can circumvent the problem of silencing. However, even under
selective pressure only a very minor number of cell clones yield in
high and stable expression of the gene of interest. In addition, the
selective pressure has to be maintained throughout cell line
development. Currently, aminoglycoside antibiotics such as hygyromycin
B and Geneticin are frequently used. These substances interfere with
protein translation and exhibit highly toxic effects in mammalian cells
not containing the corresponding bacterial genes. However, these
antibiotics are also described to have undesired side effects like
increasing frequency of sister chromatid exchange [33] and altering expression of glucose-regulated genes [34].
A different very popular strategy for high-level protein production is based on the use of dhfr-deficient CHO cells in combination with expression vectors carrying a functional dhfr gene
and the gene of interest. Cultivation of these transfected cells in
methotrexate (MTX) containing medium results in amplification of the dhfr gene
and gene of interest sequences and thus high-level expression of the
gene of interest. The disadvantage of this production system however
is, that cells grown in the presence of MTX often show substantial
heterogenicity in chromosomal location and copy number of amplified
sequences, they exhibit rearrangement and highly variable amplification
of transfected sequences, and they contain chromosomes with highly
extended regions, chromosomes joined at amplified regions or even
circular chromosomes consisting entirely of exogenous DNA. Thus the use
of the dhfr-amplification system very often results in a high degree of genetic instability in the production cell line [35].
As described earlier, the permanent expression of E1-functions is
crucial for maintaining the transformed character of stable cell lines [36].
Thus, the E1-functions are replacing the selection marker and prevent
the repressive effect of the surrounding heterochromatin. Earlier
analyses have shown, that transfection of two plasmids into CHO cells
results in co-integration at a common site in all clones examined [37].
Since in the present analyses 4 out of 6 cell pools show long-lasting
expression of hAAT, we assume that the hAAT-expressing plasmid has
co-integrated in a transcriptionally active site. Single cell cloning
of the cell pools resulted in stable cell lines exhibiting very high
protein expression of up to 30 pg/cell/day. The E1-functions have been
shown to increase expression from several promoters including the CMV
promoter [38-40] used in the present analyses, which might contribute to the high expression levels.
Primary human amniocytes are efficiently transformed by adenoviral
E1-functions. Based on this observation, we exhibit an improved method
for developing high protein expressing stable human cell lines. These
cell lines can be easily adapted to serum-free suspension culture by
gradually exchanging the medium to a serum-free, chemically defined
medium for suspension cells (data not shown). Moreover, hAAT expressed
as reference protein in human amniocyte cell lines is fully
glycosylated and sialylated. In future experiments we plan to perform a
more detailed analyses of the glycan structure of hAAT expressed in
human amniocyte cell lines in comparison to the protein expressed in
CHO cells.
Industrial protein expression demand short time lines for cell line
development, use of chemically derived serum free medium, growth in
suspension and the possibility to scale up production process. Only
during the very early passages the primary amniocytes depend on fetal
serum but are soon transferred to chemically derived serum-free medium.
For technical reasons transformation and isolation of permanent cell
clones occurs in adherent culture, and thus an additional step for
adaptation to growth in suspension has to be admitted. We have started
additional experiments in order to simplify and speed up this
adaptation step and thus shorten the time frame for cell line
development.
The potential of the present novel method of cell line development
however is not restricted to production of biopharmaceuticals. For
example, cotransfection of primary amniocytes with the E1-expressing
plasmid and a second plasmid expressing SV40 T-antigen or Epstein-Barr
virus EBNA-1 protein would result in cell lines that would be optimized
for transient protein expression. Moreover, overexpression of
glycosylation enzymes like the α2–6
sialyltransferase would result in cells optimized for production of
glycosylated proteins with high sialic acid content. Additional
examples would be the expression of certain viral proteins in cells
lines for improved production of viruses for vaccination or gene
therapy. The current method would also allow the insertion of certain
DNA-sequences like FLP recombinase targets (FRT) sites by simply
introducing FRT sites in the E1-expressing plasmid. By cotransfecting
such new cells with a plasmid expressing the gene of interest flanked
by FRT sites and a FLP expressing plasmid, a recombinase mediated
cassette exchange would occur. The insertion of the gene of interest
would then occur at a predicted site and thus would drastically
simplify cell line development.
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