Biology Reference
In-Depth Information
stability to rHuEPO. The carbohydrate content was increased from 40% to
51%, the size from approximately 30 Kd to approximately 37 Kd, and the
maximum number of sialic acids was increased from 14 to 22.
Studies in mice administered darbepoetin alfa revealed that more rHuEPO
was required to obtain a response similar to that of darbepoetin alfa [18, 19].
In pre-clinical studies, animals were administered rHuEPO or darbepoetin alfa
at various dose intervals. The relative
in vivo
activity difference of rHuEPO
and darbepoetin alfa increased as the dosing interval increased. Three-fold
more rHuEPO than darbepoetin alfa was required to elicit a similar response
when the drugs were administered three times per week. This difference
increased to 13-fold when the molecules were administered at weekly intervals
[19]. The increased
in vivo
activity and the differing potencies with changes in
dose interval could be explained by an observed three-fold increase in serum
half-life of darbepoetin alfa over rHuEPO [19, 31]. The observation that the
serum half-life increased in proportion to the number of added carbohydrate
chains indicated that the carbohydrate directly affected clearance. Testing in
humans mirrored the results found in animals. The serum half-life of darbepo-
etin administered intravenously was increased approximately three-fold [31].
In clinical trials, patients were converted from rHuEPO administered two to
three times per week to darbepoetin alfa administered once per week or from
weekly rHuEPO to once-every-other-week darbepoetin alfa. The hemoglobin
concentrations were successfully maintained with the less frequent dosing
schedule [32, 33]. More recently, clinical results suggest that hemoglobin con-
centrations can be successfully maintained when darbepoetin alfa is adminis-
tered at once every three to four weeks dosing intervals [34].
One theoretical concern with any alteration in a protein's amino acid sequence
or structure is immunogenicity. Several characteristics of darbepoetin alfa and its
methods of manufacture, minimize the potential for antibody formation. The
particular amino acid substitutions in darbepoetin alfa had a minimal effect on
structure and stability. The carbohydrate and sialic acid content of the material
selected during the purification process was maximized for several reasons: first,
the higher carbohydrate content enhances the
in vivo
activity. Secondly, carbo-
hydrate can increase solubility and stability of proteins thereby inhibiting for-
mation of aggregates and other byproducts [35-38]. Finally,
N
-linked carbohy-
N
drate is large relative to the peptide backbone giving the carbohydrate a “shield-
ing” effect potentially inhibiting antibody formation. Antibody formation was
monitored during clinical trials with darbepoetin alfa and in all patient samples
examined, no neutralizing antibody was detected [33, 39].
Pegylation
Pegylation of proteins has been used successfully to increase serum half-life
of proteins [40]. Pegylation involves chemical attachment of the polymer,
polyethylene glycol (PEG), to reactive regions of proteins or carbohydrates.
