FDS at a protein concentration of 40 mg/mL was diluted 1.6-fold with WFI, filtered as above, and 8.8 mL was filled in 20-mL
vial, stoppered, and weighed. Shelf temperature ramp rate was constant at 0.5 °C/min in all steps below.
There were five vials per group. Each group was separately cooled at 5 °C for 1 h and frozen at –40 °C for 2 h prior to initiating
the annealing described below. The control group without annealing was transferred to a –40 °C freezer at this point.
To determine the effects of annealing temperature on the primary drying rate, each of the four sample groups was annealed
for 3 h at either –3 °C, –8 °C, –13 °C, or –18 °C. Samples were resolidified at –40 °C for 1 h and transferred to the –40
At the completion of the annealing for all the groups, the vials including the controls were transferred from the –40 °C freezer
to the –40 °C shelf in the lyophilizer, surrounded by two layers of placebo-containing vials, and held at –40 °C for 1 h.
Primary drying was initiated by first evacuating to 100 mTorr and then raising the shelf temperature to –5 °C. Vials were
stoppered after 3.5–4.5 h when 20–40% of the crystalline water had sublimed. Samples were reweighed, and the primary drying
rate was calculated using the weight loss during the partial drying.
To determine the effects of annealing time on the primary drying rate, each of the three sample groups was annealed at –8
°C for 1 h, 2.5 h, or 4 h. Samples were resolidified at –40 °C for 1 h and transferred to the –40 °C freezer, then the lyophization
procedure above was followed.
Size exclusion-high performance liquid chromatography (SE-HPLC)
SE-HPLC was performed using an Agilent 1100 system and detection was at 215 nm. A TSK-Gel G4000SWXL column (TOSOH Bioscience)
was used with mobile phase consisting of 0.5 N NaCl, 10 mM sodium phosphate, pH 7.4, at a flow rate of 0.4 mL/min. The mass
load of the protein was 25 µg. In preliminary experiments with multiangle light scattering and refractive index detectors
connected in series, the molecular weight (MW) of eluted species was determined. The main peak eluted at ~21 min was assigned
to native monomeric protein. Higher MW species eluted at ~19 min were defined as soluble aggregates, and lower MW species
eluted at ~23 min as degradation products. Empower 2 software (Waters) quantifies each as percent of the total protein.
Fourier-transform infrared (FTIR) spectroscopy
Samples were analyzed on an ABB FTLA 2000 spectrometer equipped with a DuraSampleIR II ATR. Prota (Grams/32) software was
used for data acquisition, subtraction of buffer and vapor signals, protein spectra normalization, and secondary derivative
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
All supplies were from Invitrogen. Samples were diluted with NuPAGE LDS nonreducing 4x sampled buffer to a protein concentration
of 0.05 mg/mL and heated at 70 °C for 10 min. Samples (1 µg per lane) and SeeBlue Plus 2 molecular weight marker were applied
onto 4–12% Bis-Tris precast 1.0 mm x 10 well gel with 1x MOPS running buffer in a Mini-Cell. Electrophoresis was performed
at 125 volts for 1.75 h. The gel was stained with Simply Blue Safestain, scanned via Amersham Biosciences Image scanner, and
quantified using ImageQuant software.