Sephadex g 25 pd 10 desalting column

Human haemoglobin (Sigma Aldrich) was dissolved in water (20 mg/ml) and reduced by a 10-fold molar excess of sodium dithionite (Na₂S₂O₄; Sigma Aldrich). Excess reductant was removed by gel filtration over Sephadex G-25 (PD10 desalting column; GE Healthcare) according to the manufacturer’s instructions. Oxyhaemoglobin (OxyHb) was eluted with 3.5 ml of water, and only the middle run was collected. The concentration of OxyHb was determined spectrophotometrically, as described in.²⁶ Aliquots of the OxyHb stock solution were kept at −80°C, thawed on the day of experimentation and discarded after use.

To produce adenovirus containing each of our KRIT1 constructs, HEK293A cells were transfected with parent plasmid pBHGloxΔE1,3Cre and shuttle vector pDC315 containing the gene of interest using TurboFect transfection reagent (Thermo Scientific). Harvested viral particles were desalted using a Sephadex G-25 PD-10 Desalting Column (GE Life Sciences, Marlborough, MA) and collected in 10 mM Tris, pH 8.0, containing 10% glycerol. Viral titers were determined by immunoreactivity ‘spot’ assay, as previously described (Bewig and Schmidt, 2000 (link)), and KRIT1 constructs were transduced into HPAEC at appropriate multiplicities of infection (MOI) to achieve equal expression levels between constructs.
Lentiviral particles were produced by transfecting HEK293T cells with packaging plasmid pMDLg/pRRE, envelope plasmid pMD2.G, transcription factor plasmid pRSV-Rev, and transfer vector pLKO.1 containing either scramble shRNA or KRIT1 shRNA TRCN0000072879, using Lipofectamine 2000 (Invitrogen). Lentiviral particles were precipitated, and then collected in serum-free DMEM/F-12 (1:1). HPAEC were transduced with lentivirus in complete medium containing 8 µg/ml hexadimethrine bromide (Polybrene; Sigma-Aldrich, St Louis, MO).

¹⁷⁷Lu-chloride (370 MBq) was added to a solution containing DOTA-9E7.4 mAb, prepared in 0.25M NH₄OAc pH 7 and 0.05M ascorbic acid buffer pH 5.5, and was adjusted to pH 5.5 with 0.5M NH₄OAc. The reaction mixture was incubated at 42 ± 1°C for 3 h. Complexation was then stopped by adding a 10M excess of EDTA and mixing for 15 min at RT. Labeling of anti-CD138 9E7.4 mAb with ²¹³Bi was performed as described in Chérel et al. (23 (link)). Radioiodination of anti-CD138 antibodies was performed as described in Fraker and Speck (33 (link)). All radiolabeled immunoconjugates were purified by gel filtration on a Sephadex G-25 PD-10 desalting column (GE Healthcare Life Science, France) and eluted in 0.9% NaCl. The radiochemical purity was verified by instant thin layer chromatography (ITLC-SG) as described in Koppe et al. (34 (link)), using citrate buffer (0.1M pH 4.5) as the mobile phase. For all experiments, purities of the ¹⁷⁷Lu and ²¹³Bi immunoconjugates were >98%, and purity of the ¹²⁵I immunoconjugate was >95%. The immunoreactive fraction of the radiolabeled conjugates was determined using CD138-peptide-coated magnetic beads. In all cases, the immunoreactive fraction was estimated at ≥80%.

Antibodies were dialyzed with BupH™ PBS Packs (Thermo Fisher Scientific) and conjugated with smcc-DM1 (MedChemExpress, Monmouth Junction, NJ, USA) at RT at a molar ratio of 1:20 for 1.5 h. The mixture was centrifuged at 21,000× g for 20 min at 20 °C, to remove aggregates. ADCs were purified using Sephadex G-25 PD-10 desalting column (GE Healthcare, Chicago, IL, USA) and stored in 10 mM sodium succinate, 6% (v/v) sucrose, and 0.05% (v/v) Tween-20 pH 5.0. The ADCs were further analyzed using SDS-PAGE and ultraviolet spectrometry. Analysis of the DAR ratio was performed as previously described [32 (link)].

HuMab-5B1 was produced and provided by MabVax Therapeutics (San Diego, CA) Preparation of Zr-89-labeled HuMab-5B1 ([⁸⁹Zr]DFO-HuMab-5B1 or MVT-2163) was achieved in accordance with previously described methods, including conjugation of p-SCN-Bn-DFO, purification, and subsequent radiolabeling [13 (link)]. Briefly, Zr-89 oxalate in oxalic acid (1 M) was neutralized to pH 7.0–7.2, using Na₂CO₃ (1 M) followed by addition of the appropriate construct in PBS (pH 7.4). The mixture was incubated at room temperature (RT) for 30–60 min and monitored using radio-iTLC with silica-gel impregnated glass-microfiber paper strips (iTLC-SG, Varian, Lake Forest, CA, analyzed using an AR-2000, Bioscan Inc., Washington, DC), eluted with a mobile phase of aqueous solution of EDTA (50 mM, pH 5.5). The reaction was quenched by addition of EDTA solution (10–20 μl). Gel-filtration chromatography (Sephadex G-25, PD10 desalting column; GE Healthcare, Chicago, IL) with 0.9 % saline was used to purify radiolabeled construct, and radiochemical purity was determined by radio-iTLC as described above. Zr-89 was obtained from and produced by MSKCC via the ⁸⁹Y(p,n)⁸⁹Zr transmutation reaction using a TR19/9 variable-beam energy cyclotron (Ebco Industries, Richmond, British Columbia, Canada) [14 (link)].