Beh sec column

To determine the release
of bevacizumab from the different hydrogels, bevacizumab-loaded hydrogels
(10, 16, or 20 wt % with molar ratio 1:2, 1:3, or 1:5 ratio of maleimide:furan)
were prepared as described in Section 2.3. The release studies were performed at
37 °C, and the in vitro release buffer (IVR
buffer) consisted of PBS (0.13 M NaCl, 2.7 mM KCl, and 11.9 mM phosphates,
pH 7.4) supplemented with 0.02% NaN₃. Bevacizumab-loaded
hydrogels (100 mg) were first immersed in 500 μL of PBS. After
incubation, the release samples of 200 μL were taken at predetermined
time points, and 200 μL of fresh IVR buffer was added. The release
samples were stored at 4 °C until analysis of protein content
by size exclusion ultra-performance liquid chromatography (SE-UPLC)
on an Acquity UPLC (Waters Corporation, Milford) with an FLR-detector,
operated at λ_ex and λ_em of 276 and
310 nm, respectively. BEH SEC column (200 Å, 1.7 μm, 4.6
mm ×150 mm; Waters) was attached to the system and used for all
measurements at room temperature. The filtered (0.2 μm) mobile
phase consisted of an aqueous solution of sodium phosphate 100 mM
and sodium sulfate 300 mM at pH 6.7 and was operated at a flow rate
of 0.3 mL/min. Sample aliquots of 7.5 μL were injected, and
the retention time of bevacizumab was 4.90 min under these conditions.
The bevacizumab calibration curve’s linear range was from 7.8
μg/mL (detection limit) to 1250 μg/mL.

All size exclusion chromatography experiments were performed on Waters Acquity Classic UPLC, equipped with a multi-wavelength detector. Protein species separation by an isocratic elution was achieved using a flow rate of 0.4 mL/min on a BEH SEC column (200 Å, 1.7 µm, 4.6 mm × 150 mm, Waters, Baden, Switzerland) maintained at 40 °C. The mobile phase was 50 mM sodium phosphate, 400 mM sodium perchlorate, and pH6.0. The detection of signals was performed at 280 nm.
The obtained chromatograms of individual protein components and complexes in PBS matrix were evaluated directly. For complexes incubated in a matrix containing mouse serum, a subtraction procedure was introduced. Hereby, chromatograms of mouse serum control samples (addition of PBS instead of protein components) were subtracted from the chromatograms of samples containing formed complexes in mouse serum matrix prior to the data interpretation. Data collection, evaluation, integration, and processing by subtraction were performed with Chromeleon 7.3 (Thermo Scientific, Reinach, Switzerland) software.