br Cy maleimide and Cy
Cy5-maleimide and Cy5-NHS ester were acquired from GE Healthcare (Uppsala, Sweden). N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) was from Proteochem (Loves Park, IL, USA). BCA Protein Assay Kit for protein concentration quantification,
trifluoroacetic acid, D2O and all other reagents including salts and solvents as well as McCoy’s 5A cell medium and FACS buﬀer were ac-quired from Sigma-Aldrich s.r.l. (Milan, Italy). PEGs were from NOF Corporation (Tokyo, Japan). Precast gels for SDS-PAGE 8–16% and 4–15% were purchased from Thermo Fisher Scientific (Waltham, MA, USA) and Bio-Rad (Milan, Italy). Pierce™ Dye Removal Columns for the elimination of unconjugated Cy5 were from Thermo Fisher Scientific (Waltham, MA, USA). Tubulysin A was obtained from Tube Pharma (Wien, Austria). RPMI 1640 and DMEM cell media were from EuroClone (Milan, Italy); L-glutamine, HEPES, penicillin, streptomycin, and sodium pyruvate were obtained from Lonza (Basel, Switzerland). Heat-inactivated fetal bovine serum was from Gibco BRL (Paisley, UK). R-phycoerythrin (PE)-conjugated mouse anti-human CD20 mAb (clone 2H7) was acquired from BD Biosciences (San Diego, USA), and PE-conjugated mouse anti-human IgG1 mAb from Miltenyi Biotec (Bergisch Gladbach, Germany).
The HPLC/FPLC systems used were the following: (i) HPLC Shimadzu composed of two LC-10AD and Dalbavancin UV–Vis SPD-10AV detector (Kyoto, Japan); (ii) HPLC Agilent Technologies, 1260 Infinity model (Santa Clara, CA, USA); (iii) AKTA purifier (GE Healthcare, Uppsala, Sweden).
2.2. Analytical methods
NMR. NMR spectra were obtained using a Brüker Avance 400 spectrometer (Rheinstetten, Germany). operating at 400.132 MHz, and the spectra were processed with MestReNova software.
UV–Vis. Protein concentrations were determined spectro-photometrically on a Thermo Scientific Evolution 201 spectro-photometer (Waltham, MA, USA) or via BCA assay, as described else-
SDS-PAGE. Electrophoresis (SDS-PAGE) was performed in ac-cordance with the Laemmli-SDS-PAGE protocol [54,55]; the gel was stained with Blue Coomassie for protein detection and with iodine for PEG detection. Electrophoretic runs were made using an Electrophor-esis Power Supply 300 (Pharmacia, New Jersey, USA).
Far-UV circular dichroism (FUV-CD). FUV-CD spectra were measured on a Jasco J-810 spectropolarimeter equipped with a Peltier temperature control unit at 25 °C. The samples were dissolved in PBS pH 7.4 at a protein concentration of 0.1–0.2 mg/ml. The spectra were collected between 200 and 250 nm with an average of 3 scans and the data at each wavelength were averaged for 8 s. The sample cell path length was 1 mm. The CD data were converted to mean residue ellip-ticity, expressed in deg cm2 dmol−1 by applying the following formula:
where Θobs is the observed ellipticity in degrees, the MRW is the mean residue weight of the protein (molecular weight divided by the number of amino acids), [C] is the protein concentration in mg/ml, and L is the optical path length in centimeters.
MALDI-TOF MS. Mass spectra were obtained with a REFLEX time-of-flight instrument (4800 Plus MALDI TOF/TOF, AB Sciex, Framingham, Massachusetts, USA) equipped with a SCOUT ion source, operating in positive linear mode. A pulsed UV laser beam (nitrogen laser, λ 337 nm) generated ions that were accelerated to 25 kV. Matrix (a saturated solution of sinapic acid in water/ACN (1:1, v/v) + 0.1% TFA (v/v)) was mixed with an equal volume of sample, and 1 μl was loaded on the plate.
Dynamic light scattering (DLS). Hydrodynamic diameters were measured using a Zetasizer Nano ZS apparatus (Malvern Instruments Ltd., Worcestershire, United Kingdom) at 25 °C the results of three
measurements were averaged. A series of solutions (PBS pH 7.4) with diﬀerent mAb/protein molar ratios (1:0.25, 1:0.5, 1:1 and 1:2) were prepared to maintain a fixed mAb concentration and varying protein concentrations. The solutions were left to equilibrate for 1 h at room temperature and filtered with a 0.22 µm cellulose acetate centrifuge tube filter before being analyzed. The size values are reported based on volume.