Pharmaceutical Manufacturing Handbook: Production and

INJECTABLE PEPTIDE/PROTEIN-LOADED MICROSPHERES 417
model monoclonal antibody, mannitol alone at 60 m M provided less protection
during storage than sucrose or trehalose alone. A specifi c sugar/protein molar ratio
was suffi cient to provide storage stability for this particular monoclonal antibody
[196, 197] .
Low - molecular - weight additives such as osmolytes ( N,N - dimethylglycine, trehalose,
and sucrose) or salts (sodium chloride, sodium phosphate, ammonium sulfate,
and sodium citrate) were found to be highly effective in stabilizing keratinocyte
growth factor, both against thermal denaturation and enhancing long - term storage
stability [198] . Nevertheless, the stabilizing properties of osmolytes appear to be
balanced between their binding to (deteriorating effect) and exclusion from (stabilizing
effect) the peptide/protein surface. As binding or exclusion predominantly
results from hydrophobic interactions, hydrogen bonding, and electrostatic interactions,
the sum of the various interaction parameters are dissimilar for different
proteins. Therefore, it becomes crucial to examine the individual nature of the additive
toward each individual protein and to assess whether it will offer a stabilizing
or destabilizing effect [199, 200] .
Polyvinyl pyrrolidone (PVP) and glycine were found to stabilize lyophilized
sodium prasterone sulfate whereas dextran 40 or mannitol did not [201] . PVP and
glycine stabilized the pH of the reconstituted solution by neutralizing the acidic
degradation product, sodium bisulfate, formed by the hydrolysis of prasterone
sulfate. Dextran 40 or mannitol was ineffective because of no buffer capacity. Buffering
agents, such as phosphate – citrate buffer and some neutral and basic amino
acids ( l - arginine, l - lysine, and l - histidine), also stabilized prasterone sulfate. l - Cysteine
is an example of an amino acid that did not stabilize the drug, presumably
because of its weak buffer capacity. Although the effi ciency of proteinic additives
for protein stabilization has been clearly demonstrated in several occasions even
during encapsulation processes [31, 72, 98, 144] , their use in pharmaceuticals is at
present not desirable from a strictly regulatory point of view. Additionally, such
agents might contribute to complicate all subsequent protein characterization within
the formulation. Among these additives, albumins and gelatins are those mainly
used for protection purposes. The protective effect of albumins against protein
unfolding and aggregation has been extensively documented and is likely due to
their surface - active properties (see Bilati et al. [130] for details).
Prevention/Minimization of Microclimate pH -Induced Instability Evidence for
acidifi cation within degrading microspheres is investigated and local pH values
between 1.5 and 4.7 are being reported [202 – 204] . Methods to measure microclimate
pH in PLGA microspheres include (i) ensemble average measurements using electron
paramagnetic resonance (EPR) [203, 204] , nuclear magnetic resonance (NMR)
[205] , and potentiometry and (ii) direct visualization techniques such as confocal
imaging of pH - sensitive dyes [206, 207] . In the EPR method, the constant of hyperfi
ne splitting, 2 aN , was used to determine an average pH inside PLGA microspheres.
Because the experiments relied on the mobility of spin - labeled protein, with an
increase of the microviscosity in the later hours of the experiments, the spectra of
EPR was changed and the signal - to - noise ratio decreased to prevent the measurement
of pH throughout the release period [203] . The potentiometric measurements
can give rapid values of pH for thin polymer fi lms, and the pH of the thin water fi lm
between the electrode and polymer mimics the microclimate pH of aqueous pores
inside the polymer - based drug delivery system. However, it is diffi cult to mimic