Pharmaceutical Manufacturing Handbook: Production and

PEPTIDE/PROTEIN-LOADED MICROSPHERE PRODUCTION METHODS 403
why these methods were not credited as encapsulation techniques for protein within
PLGA may be the tendency of several polymers to rapidly precipitate and agglomerate
during the process [70] .
ASES has been compared with conventional spray drying in terms of effects on
the stability of the peptide tetracosactide [71] . Almost no intact peptide was recovered
from spray - dried PLA particles, whereas the tetracosactide was well protected
against oxidation during ASES ( ∼ 94% unmodifi ed peptide). In general, the particle
formation step seems to be less detrimental to proteins than the loading step. For
example, emulsifi cation in an aqueous phase or spray drying of rhEPO/PLGA
emulsions was mild compared to the fi rst emulsifi cation step [72] . Also, variation of
the particle formation step (spray drying or coacervation) had a minor impact on
diphtheria toxoid (DTd) antigenicity when compared to other process variables
[73] .
A serious limitation of GAS, ASES, and RESS for producing microspheres is the
need of polymer types that form discrete crystalline domains upon solidifi cation,
such as l - PLA [74, 75] . The advantages of these methods offer (e.g., over spray
drying) are the low critical temperatures for processing (34 ° C) and the avoidance
of oxygen exposure during atomization, with both parameters being potentially
important to peptide/protein stability.
Ultrasonic Atomization Ultrasonic atomization of w/o dispersions is presently
under investigation for preparing especially protein antigen containing microspheres.
In one setup, the atomized antigen/polymer dispersion was sprayed into a
nonsolvent where the polymer solvent was extracted, resulting in microspheres [76] .
A comparable technique was proposed where the antigen or polymer dispersion
was atomized into a reduced pressure atmosphere and the preformed microspheres
hardened in a collection liquid [77] . Similarly, PLGA solutions were also atomized
by acoustical excitation and the atomized droplets transported by an annular stream
of a nonsolvent phase [aqueous polyvinyl alcohol (PVA)] into a vessel containing
aqueous PVA [78] . Solvent evaporation and microsphere hardening occurred in the
vessel over several hours. The main advantages of these atomization techniques
encompass the possibility of easy particle size control and scale - up, processing at
ambient or reduced temperature, and the suitability for aseptic manufacturing in
a small containment chamber such as an isolator.
In Situ Formed Injectable Microspheres All the encapsulation techniques discussed
so far rely on the preparation of solid microspheres. However, a method for
preparing a stable dispersion of protein containing semisolid PLGA microglobules
has been reported [79] . Here, a protein dissolved in PEG 400 was added to a solution
of PLGA in triacetin or triethyl citrate. This mixture, stabilized by Tween 80,
was added dropwise and under stirring to a solution of Miglyol 812 or soybean oil,
containing Span 80, resulting in a stable dispersion of protein inside semisolid
PLGA microglobules. The microglobules remained in an embryonic state until
mixed with an aqueous medium, so that the water - miscible components were
extracted and protein containing matrix - type microspheres formed. Myoglobin was
encapsulated and found to remain physically unchanged (circular dichroism
analysis) after the process and during storage of the microglobular dispersion (15
days, 4 ° C).