Mabpac sec 1 column

Size exclusion chromatography was performed using a MAbPac SEC-1 column with pore size of 300 Å and particle size of 5 µm (Thermo Fisher), pre equilibrated with 50 mM Tris pH 7.5, 80 mM NaCl, to analyse the association of DBD with ΔC1010, ΔN 133-ΔC1010 and Δ57–88-ΔC1010. For each run, Chd1 DBD was mixed with respective fragments at 3:1 molar ratio, incubated for 30 min and injected onto the column. The column was runn at the flow rate of 0.150 mL/min and 100 μL fractions were collected. The fractions from the size exclusion experiment were further analysed using SDS-PAGE gel. The runs were repeated three times for each experiment.

The mass spectrometry (MS) analysis was performed using a Q Exactive™ HF Orbitrap with BioPharma option (Thermo Fisher Scientific, Waltham, MA, USA) operating in the high mass range. The mass spectrometry spectra were deconvoluted using the Protein Deconvolution Software (Thermo Fisher Scientific, Waltham, MA, USA). UPLC was also performed on the samples. The separation was done using the MAbPAC SEC-1 column, 5 μm, 300 Å, 4 × 150 mm (Thermo Fisher Scientific, Waltham, MA, USA), and ammonium acetate 50 mM pH 7.0 at 0.3 mL/min as mobile phase. By knowing the average mass of the antibody and the center of the conjugated antibody average mass distribution (broader peak in MS spectrum than the unconjugated form), an average number of chelators linked to the antibody was calculated.

The molecular size of purified rMePUL was analysed with an MAbPac™ SEC-1 column (4 x 300 mm, Thermo Fisher Scientific™). The system was run in 25 mM phosphate buffer pH 6.8 containing 300 mM NaCl with a flowrate of 0.2 mL/min at 30°C. The signal was monitored by measuring A₂₈₀. Gel Filtration Standards (Bio-Rad) were used for molecular weight calibration. To investigate the multimeric state of rMePUL/rMeISA3 (Panpetch et al., 2018a (link)), ~ 0.26 nmol of each enzyme was mixed and incubated in 25 mM phosphate buffer pH 7.2 in 0.1 mL total volume at 4°C overnight prior to analysis.

The purified anti-CD40 fusion proteins were further analyzed regarding potential protein aggregation and degradation by size exclusion chromatography (SEC) by the UltiMate 3000 HPLC System (Thermo Fisher) with a MabPac SEC-1 column (#088460, Thermo Fisher). Calibration of the column was carried out with the aqueous SEC-1 column performance check standard (#AL0–3042, Phenomenex, Torrance, CA, USA).

To preserve non-covalent interactions, intact mass measurements were performed under native-like conditions by injecting the samples into MAbPac SEC-1 column (300 Å, 5 µm, 4 × 150 mm, Thermo Fisher Scientific, Sunnyvale, CA, USA) using a Dionex Ultimate 3000 analytical RSLC system (Dionex, Germering, Germany) coupled to a HESI source (Thermo Fisher Scientific, Bremen, Germany). The isocratic separation was performed within 7 min at flow rate of 300 µl/min and 50 mM ammonium acetate, pH 7.5 as mobile phase. Eluting fractions were analysed on high resolution QExactive HF-HT-Orbitrap-FT-MS benchtop instrument (Thermo Fisher Scientific, Bremen, Germany). High-mass-range (HMR) mode was activated with resolution of 15,000, in-source CID of 50 eV and AGC (automatic gain control) target of 5e6. The scan range was set to 1900–8000 m/z. Data analysis was performed with Protein Deconvolution 4.0 (Thermo Fischer Scientific, Sunnyvale, CA, USA) using Respect algorithm.

For the insertion of anti-EGFR antibody-lipid conjugates and ⁶⁴Cu-NOTA-lipid conjugates into the outer membrane of liposomes, the micelles containing antibody-PEG2000-DSPE or ⁶⁴Cu-NOTA-PEG2000-DSPE lipid were incubated with preformed liposomes encapsulating doxorubicin for 4 h at 37 °C with continuous stirring. Uninserted free antibody-PEG2000-DSPE or ⁶⁴Cu-NOTA-PEG2000-DSPE were removed from the ⁶⁴Cu-immunoliposomes encapsulating doxorubicin by gel filtration chromatography through sepharose CL-4B columns in HEPES buffer (25 mM HEPES, 140 mM NaCl, pH 7.4). The radioactivity of each fraction was counted in a gamma counter (WIZARD 1480, Perkin-Elmer, Waltham, MA, USA). Radiochemical purity was also analyzed by size-exclusion high-performance liquid chromatography (SE-HPLC) using a MAbPac SEC-1 column (Thermo Scientific). The mobile phase consisted of 0.3 M NaCl in 50 mM sodium phosphate buffer pH 6.8 and the column was eluted at a flow rate of 0.5 mL/min. The retention time of ⁶⁴Cu-immunoliposomes was determined at 280 nm of UV absorbance. The radioactivity of ⁶⁴Cu-immunoliposomes was determined by a radioactivity detector (Raytest, Straubenhardt, Germany).

Mass spectrometry (MS) analysis was performed using a Q Exactive HF Orbitrap (Thermo Fisher Scientific, Waltham, MA, USA) and separation was done using a MAbPAC SEC-1 column, (Thermo Fisher Scientific, Waltham, MA, USA) with a mobile phase of ammonium acetate 50 mM pH 7.0 at 0.3 mL/min as previously described [19 (link)]. After deconvolution of the mass spectrometry spectra, the drug-to-antibody ratio (DAR) is calculated using the formula:
where n = number of attached molecules for this peak and Int = intensity of the peak.