Pharmaceutical Manufacturing Handbook: Production and

ANALYTICAL CHARACTERIZATION 405
immunization with a 38 - kDa protein antigen against tuberculosis [85] . Similarly,
Kazzaz et al. [86] created anionic PLGA microparticles by substituting the standard
nonionic emulsifi er PVA with anionic SDS during microsphere preparation. In addition
to eliciting elevated antibody responses in mice relative to the soluble antigen,
the adsorbed antigen elicited a potent cytotoxic T - cell (CTL) response, similar to
that observed after infection from virus expressing the p55 gag and polymerase
proteins. Moreover, the CTLs were formed from the more challenging intramuscular
route but not signifi cantly by the soluble antigen, even at elevated doses. The
SDS - PLGA particles could also be gamma irradiated before adsorption and were
shown to effectively boost antigen in nonhuman primates [87] .
5.2.3 ANALYTICAL CHARACTERIZATION OF PEPTIDE/
PROTEIN - LOADED MICROSPHERES
An area requiring additional efforts is analytical characterization of peptides and
proteins encapsulated in PLGA microspheres. The high complexity of the therapeutic
peptides and proteins requires not only physicochemical methodologies but
also immunochemical and biological techniques for the characterization and quality
control of these substances. In general, the analytical methods can be broadly
viewed from the following study perspectives: methods meant for microsphere
product quality checking, methods used for peptide/protein stability identifi cation
inside the microspheres, and methods called for peptide integrity detection following
liberation from the microspheres immediately upon placement in release
medium either in vitro or in vivo. Therefore, in most cases, a combination of several
analytical methods is necessary for a comprehensive characterization of the
peptide/protein under investigation and for appropriate quality control of the
product concerning identity, purity, and potency. However, some of the analytical
methods have potentially appealing applications to interplay among the mentioned
perspectives. In Table 4 , a selection of widely used analytical methods is given,
showing which technology is applicable for the testing of identity, purity, and
potency of peptides and proteins. In addition, peptide/protein integrity evaluation
is indeed likely to be affected by artefacts during the sample preparation before
analysis and during the analysis itself. Therefore, artefacts might prevent the scientist
from critically ascribing detected protein denaturation to manufacturing
conditions [88] .
In order to measure the extent of peptide/protein degradation within the carriers
and during release, the encapsulated molecule has to be removed from the polymeric
matrix. Moreover, for avoiding artefacts such as underestimation of drug
content, recovery methods need to be tried by an empirical trial - and - error approach
as each peptide/protein is different one from the other. Recovery methods so far
reported include extraction - based method with the help of potentially deleterious
organic solvents, hydrolysis of the polymer matrix with alkaline medium, dissolution
of polymer matrix in an organic solvent, recovery of suspended insoluble protein
by fi ltration [89] , total protein quantifi cation after complete digestion of carriers
followed by amino acid analysis [90, 91] , electrophoretic extraction of the protein
using sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS – PAGE) [92,
93] , and direct dissolution of both the polymer and the protein drug in a single liquid