Biology Reference
In-Depth Information
branched with two to four arms. The end of each arm is usually capped by a
sialic acid; sialic acid content exhibits microheterogeneity in the different gly-
coforms. The sialic acid is of importance because it is the only negatively-
charged sugar on the carbohydrate. Variations in the amount of sialic acid can
affect the electrostatic properties of the molecules to which it is attached.
The carbohydrate is essential for
in vivo
but not for
in vitro
biological activ-
ity [13, 24]. The sialic acid component of carbohydrate, in particular, plays a
critical role in the
in vivo
biological activity of rHuEPO. Removal of sialic acid
from the carbohydrate of EPO results in almost complete loss of
in vivo
activ-
ity [14, 25]. Studies on glycoforms of rHuEPO containing different sialic acid
contents demonstrated a direct relationship between increased sialic acid con-
tent and increased
in vivo
activity [19]. The increased
in vivo
activity was due
to an increased serum half-life of the molecule and not increased receptor
affinity. The theoretical maximum number of sialic acids on rHuEPO is 14 (up
to four sialic acids for each of the three
N
-linked carbohydrates and up to two
N
sialic acids for the
O
-linked carbohydrate) [23]. It was hypothesized that
in
vivo
activity may be increased beyond that observed with rHuEPO by adding
new sialic acid containing
N
-linked carbohydrates. Each new
N
N
-linked chain
N
could add up to four additional sialic acids.
To add more
N
-linked carbohydrate, new
N N
-linked glycosylation sites were
N
introduced into the amino acid sequence of EPO.
N
-linked carbohydrate is
N
attached to an Asn present in the consensus sequence Asn-Xxx-Ser/Thr (where
Xxx can be any amino acid except proline) [26]. This sequence is necessary
but not sufficient for
N
-linked carbohydrate addition [27]. During synthesis of
N
a glycoprotein, appropriate consensus sequences are recognized by oligotrans-
ferases in the cell, resulting in attachment of carbohydrate. This carbohydrate
is subsequently modified by the action of additional intracellular enzymes. The
protein is then secreted from the cell into the circulation [28].
For the purpose of potentially developing a new drug with properties supe-
rior to then available products, it became apparent that simply adding an
N
-linked consensus sequence to rHuEPO would not be sufficient. The changes
N
needed to be introduced in such a way that the resultant molecule was effi-
ciently glycosylated and retained activity, conformation, and stability. To
increase the likelihood of success, the amino acid changes were introduced
into a region of the molecule distal to the receptor-binding site to ensure that
the molecule would efficiently activate EPOR. This effort was aided by struc-
ture-function studies that defined the active sites of rHuEPO and determination
of amino acids important for maintenance of structure [17, 29, 30].
EPO glycosylation analogs with introduced
N
-linked glycosylation consen-
N
sus sequences were constructed and tested [18]. Several candidates containing
additional carbohydrate had acceptable activity and conformation characteris-
tics. Two of these consensus sequences (Asn30-Thr32 and Val87-Asn88-
Thr90) were combined to generate a new molecule with two additional
N
-linked carbohydrates. This molecule (darbepoetin alfa) had near-normal
N in
vitro
activity, was glycosylated efficiently, and had a similar conformation and
