Biomedical Engineering Reference
In-Depth Information
exploitation.The use of human cell lines for expressing protein is rare.However,
the human lymphoblastoid Namalwa cell line has been used for the production
of tPA. The cell line performs O -linked and N -linked glycosylation efficiently,
and preliminary study shows that the glycosylation pattern is identical to the
normal human cell type [177].
5.1.2.1
Mouse Cells
Most mammals express the enzyme
a
1,3-GalT, which generates Gal
a
1,3-Gal
b
1,4-Gn residues on membrane and secreted glycoproteins.Humans are notable
exceptions where the gene has become inactivated [178]. Certain mouse cell
lines such as hybridomas, mouse-human heterohybridomas, and C127 cells
synthesize some glycans terminating in Gal
1,4-Gn [179]. Moreover,
the levels of N -glycolylneuraminic acid (NeuGc), a SA derivative, are more
prevalent in antibodies derived from mouse or human-mouse hybridomas
[180].In contrast,glycoprotein in adult humans do not normally contain NeuGc.
Low levels of NeuGc (1% of total sialic acid) are tolerated in recombinant
proteins such as EPO, but higher levels (around 7%) can elicit an anti-NeuGc
antibody [181]. Furthermore, high levels of terminal NeuGc are correlated with
a rapid removal of molecule from circulation, compared to the same protein
bearing terminal SA residues [182]. Other rodent cell lines, such as mouse NSO
or rat YO myeloma, producing humanized antibodies, do not add this unwanted
residue (Gal
a
1,3-Gal
b
1,4-Gn) and therefore are only mildly immunogenic
[183]. In spite of nonnative forms of glycosylation, mouse cell lines seem to be
one of the powerful expression system commercially exploitable. Mouse C127
cell line under BPV promoter has been found to produce tPA at peak titre of
55 mg l -1 [184]. The highest yield of antibody (1 g l -1 ) so far reported is in NSO
cells using glutamine synthetase amplification system [185].
a
1,3-Gal
b
5.1.2.2
Hamster Cells
Chinese hamster ovary (CHO) and baby hamster kidney (BHK) cells have a “set”
of glycosylation enzymes that are similar (but not identical) to those in human
cells [165].However,these lack a functional
a
2,6-ST required to add SA through
a
2,6 linkages.As the rat gene coding for this enzyme has been cloned,the defect
can be corrected by
2,6-linked
SAs [186]. In addition, mutants of CHO cells that display altered glycosylation
pattern have proven useful in expressing glycoproteins with minimal hetero-
geneity [187]. Thus, CHO-K1 cell produces terminally sialylated glycans, while
CHO-Lec2 cell produces carbohydrate with a >90% decrease in SA content, and
CHO-Lec1 cell, lacks GnT I activity, leading to the accumulation of high-Man
intermediates [188]. The highest expression obtained in CHO-K1 cell line was
for tPA,and the maximum product concentration obtained was 136.4 mg l -1 in a
high cell density perfusion culture [189].
a
2,6-ST transfection,generating both
a
2,3- and
a
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