Analytical SEC was performed to assess the purity and aggregation behavior of the antibody derivatives. 7.5 µg protein were injected on TSKgel UP-SW3000 column (4.6 × 300 mm, Tosoh Bioscience LLC) in an Agilent HPLC system under a flow rate of 0.35 ml/min using 50 mM sodium phosphate, 0.4 M NaClO4 pH 6.3 as mobile phase. Molecular mass of the proteins was determined using a molecular standard (Bio-Rad).
Tskgel up sw3000 column
Manufactured by Tosoh
Sourced in United States
The TSKgel UP-SW3000 column is a size exclusion chromatography (SEC) column designed for the analysis of macromolecules such as proteins, polymers, and other large biomolecules. The column features a porous silica-based stationary phase that separates analytes based on their hydrodynamic volume. It is suitable for the analysis of a wide range of molecular weights.
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5 protocols using tskgel up sw3000 column
1
Antibody Purity and Aggregation Analysis
2
SEC Analysis of Monoclonal Antibody
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SEC analysis was performed using a Waters H-Class HPLC system. Aliquots of mAb1 fractions were diluted to 1 mg/mL with mobile phase (0.2 M potassium, 0.25 M potassium chloride, pH 6.2). Samples were separated on a TSKgel UP-SW3000 column (Tosoh Bioscience, LLC, South San Francisco, CA, USA) with isocratic elution. The flow rate was at 0.3 mL/min for 18 min, and the column temperature was at ambient. The elution profile was monitored at 280 nm.
3
Recombinant Antibody Expression and Purification
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For antibody expression, the pTT5 vector system was employed (Durocher 2002) . Chimeric bovine × human IgGs as well as adalimumab and engineered derivatives were expressed using a Fc effector silenced backbone (Pekar et al. 2021a ). The B.01-derived Knobbody and Fc knob engraftments as well as adaFc knob derivatives were constructed as described elsewhere (Pekar et al. 2021b; Yanakieva et al. 2023) . Sequences are given in Supplementary Figure 2. All proteins were expressed in a volume of 5 ml using the ExpiCHO™ expression system (Thermo Fisher Scientific) or at 25 ml scale harnessing Expi293™ cells (Thermo Fisher Scientific) according to the manufacturers manual, employing a 2:1 ratio for the heavy chain and light chain, respectively. After 7 days the protein containing supernatants were purified exploiting MabSelect™ antibody purification chromatography resin (Cytiva). After sterile filtration, protein concentrations were determined by A 280 absorption measurement. For the assessment of protein sample quality regarding monomer content [%], analytical size exclusion chromatography (SEC) was applied using 7.5 µg protein per sample on a TSKgel UP-SW3000 column (Tosoh Bioscience).
4
SEC Quantification of Protein Aggregates
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SEC was carried out in triplicates on an Ultra High-Performance Liquid Chromatography (UHPLC) (Agilent 1290 system, Santa Clara, CA, USA) with a TSK gel® UP-SW3000 column (4.6 × 300 mm, 2 μm; Tosoh Bioscience, Japan). The elution was conducted by 0.1 M sodium phosphate pH 6.8, containing 0.2 M Arg-HCl, and 0.05% sodium azide, at a flow rate of 0.35 mL/min. The detection was obtained at 280 nm. Sample aliquots (10 µL) were loaded at a concentration of 1 mg/mL, after centrifugation (15000 rpm per 15 min at 4 • C) to remove eventual insoluble particles. The calibration of the column was obtained by loading a mixture of proteins with known molecular weight (Thyroglobulin, γ-globulin, Ovalbumin, Ribonuclease A, p-aminobenzoic acid) (Fig. S1). Quantification was done by OriginPro 2018b software, integrating the area under the peaks after the baseline subtraction. The areas (Au) of the monomer and HMW species were calculated from SEC profiles and expressed as percentage of aggregated species (Au HMW /Au TOT × 100).
5
Size-Exclusion Chromatography of Bispecific Antibody
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SEC was carried out using
a TSKgel UP-SW3000 column (4.6 × 300 mm, 2 μm particle
size; Tosoh Bioscience, Cat. No. 0023448). An isocratic elution using
200 mM KH2PO4, 250 mM KCl, pH 6.2 at 0.3 mL/min
as the solvent was used for chromatographic separation on a Dionex
UltiMate3000 BioRS HPLC-system (Thermo Scientific) equipped with UV
detection at 280 nm. Then, 50 μg of bsAb1 was injected for the
chromatographic analysis. Data acquisition and relative quantification
by manual integration and comparison of peak areas was performed by
Chromeleon software (Thermo Scientific).
a TSKgel UP-SW3000 column (4.6 × 300 mm, 2 μm particle
size; Tosoh Bioscience, Cat. No. 0023448). An isocratic elution using
200 mM KH2PO4, 250 mM KCl, pH 6.2 at 0.3 mL/min
as the solvent was used for chromatographic separation on a Dionex
UltiMate3000 BioRS HPLC-system (Thermo Scientific) equipped with UV
detection at 280 nm. Then, 50 μg of bsAb1 was injected for the
chromatographic analysis. Data acquisition and relative quantification
by manual integration and comparison of peak areas was performed by
Chromeleon software (Thermo Scientific).
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