eBook: Formulation of Dry Powder Inhaler - 25

2.9. Residual Solvent Quantitation by
Gas Chromatography (GC) Headspace
The concentration of residual solvent from spray dying
in the powder was quantified by GC headspace.
A known mass of sample was dissolved in 4 mL of
dimethylacetamide in a 20 mL headspace vial. GC
was performed on the headspace using an Agilent
G7890 (Agilent Technologies, Santa Clara, CA, USA)
equipped with a flame ionization detector and split
injection capability as well as an Agilent 7697 automated
headspace sampler. An Agilent DB-624
column was used with 30 m 0.32 mm 1.8 microns.
The headspace sampler and instrument parameters
used for the experiments are summarized in the
Supplementary Information.
2.10. VEGF Binding ELISA Assay
To assess the in vitro binding of spray-dried BEV
simul-sprays to VEGF, a Human VEGF Quantikine
ELISA kit (Part DVE00, R&D Systems, Minneapolis,
MN, USA) was repurposed for a competitive ELISA
assay. As a control, the as-received BEV solution was
diluted to a 4 mg/mL active in 0.01 M pH 7.4 phosphate
buffered saline. Spray-dried powders or as-received
API were reconstituted to a 4 mg/mL active
in the buffered saline and allowed to dissolve for 60
min. All samples were then centrifuged at 10,000 g
for 1 min to remove undissolved solids (as would
be expected for simul-sprays containing low-solubility
actives such as PTX and ERL). Samples were
then prepared containing 2.5 nM human recombinant
VEGF (R&D Systems, Minneapolis, MN, USA)
and 0.75 mg/mL BEV (from spray-dried powder or
stock) in the ELISA kit's calibrator diluent RD5K. For
controls which did not contain bevacizumab, identical
volumes were transferred. Samples were incubated
for 60 min at 37 °C to allow BEV to bind VEGF
to equilibrium. Standards were prepared according
to the kit protocol.
After incubation, 50 µL of the assay diluent was
added to each of the wells of the supplied plate.
A total of 200 µL each of the sample (in triplicate)
and standards were added to respective wells
and incubated for 2 h at ambient temperature.
The plate was washed three times with 400 µL
of the kit's wash buffer, then 200 µL of the VEGFconjugate
was added. The plate was incubated for
another 2 h, then washed again three times, as
previously. A 1:1 mixture of the kit's color reagent
A and B were mixed, then 200 µL was added to
each well. After a 30 min incubation at ambient
temperature, 50 µL of stop solution was added to
each well. The absorbances were read on an M5e
plate reader (Molecular Devices, San Jose, CA,
USA) using a 450 nm detection wavelength and
540 nm as the blank wavelength for baseline correction.
The quantified concentration of unbound
VEGF in pg/mL was reported. All samples were analyzed
in triplicate.
2.11. Solubility
To measure the equilibrium solubility of the API
and excipients, a saturated solution of each substance
was prepared by stirring the excess solids
in the solvent for ~2 h. The solutions were centrifuged
to remove excess solids. A total of 50 µL
of the supernatant was pipetted into an aluminum
pan and placed into a Thermogravimetric
Analyzer (Discovery TGA, TA Instruments, New
Castle, DE, USA). The sample was heated to 130
°C at a ramp rate of 50 °C/min and then held isothermal
for 10 min to allow all of the solvent to
evaporate. The final mass of the sample (of the
known 50 µL volume) was recorded and used to
determine the solubility in mg/mL. Samples were
measured in triplicate.
25

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