The number and size of particles in aggregated and unaggregated IVIG, Avastin and HSA were measured using the MFI 5200 system from Proteinsimple (San Jose, California, USA) equipped with a 100-micron flow cell and MFI view system software, (version 2-R2.6.1.20.1915). The system was cleaned with purified distilled water at maximum flow rate with the plush mode. Flow cell cleanliness was visibly checked confirmed visually before running the samples. The samples were analyzed at a flow rate of 0.17 mL/mL and fixed camera. Prior to use, the MFI performance was calibrated using 5 μm particles/mL (Duke Standards, ThermoFisher Scientific, Fremont, CA) and NIST traceable size standard 3000 particles, (ThermoFisher Scientific, Fremont, CA). The samples were diluted 1000X in sodium citrate buffer. For each read 0.9 mL of product was prepared. The purge volume was 0.2 mL and analyzed sample volume was 0.6 mL. The MFI was set to capture 20,000 images/sample. Samples were measured in triplicate. The data is shown after subtraction of particles from sample buffer as background.
Duke standards
Manufactured by Thermo Fisher Scientific
Duke Standards are a line of certified reference materials designed for use in analytical laboratories. They provide precise and accurate standards for calibrating and verifying the performance of analytical instruments and methods. Duke Standards are manufactured to strict quality control standards and are accompanied by detailed certificates of analysis.
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Elisa kit for tumor necrosis factor α, by BD (1 mentions)
Pre cast 15 gels, by Bio-Rad (1 mentions)
Immbilon pdvf membranes, by Merck Group (1 mentions)
Diff quik staining kit, by Siemens (1 mentions)
Halt protease inhibitor cocktail mix, by Thermo Fisher Scientific (1 mentions)
Annexin 5 alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
8 protocols using duke standards
1
Particle Size Analysis of Protein Therapeutics
2
Polystyrene Microparticle Characterization
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Polystyrene microparticles (Duke Standards, Thermo Fisher Scientific Inc.) with narrow size dispersions (mean diameter ± standard deviation) of 1.00 ± 0.01 µm, 3.00 ± 0.03 µm, 5.0 ± 0.3 µm, 7.0 ± 0.5 µm and 10.0 ± 0.5 µm were purchased from Distrilab BV. Aqueous suspensions with 1% particle concentration were pipetted into a 0.36 mL microwell plate and promptly imaged from the top by BRAD-OCT. Datasets covering 3.5 mm × 3.5 mm × 5.0 mm were acquired. The morphology of polystyrene beads was determined by scanning electron microscopy (FEI Quanta 200). Droplets of the aqueous microsphere suspensions were placed on a sample holder equipped with double-sided sticky carbon tape and the liquid medium was evaporated at 55°C. Upon drying, the samples were sputter-coated with gold before SEM micrographs were recorded (acceleration voltage 15 kV).
3
Rapid Micro-Morphometry of Buoyant Particles
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An Archimedes system, equipped with a Hi-Q Micro Sensor (both Malvern Instruments, Malvern, UK) and the Archimedes software v1.20 was used for RMM analysis of particles up to 4 µm in size. The system was calibrated with polystyrene size standards of 0.994 µm specified diameter (Duke Standards; Thermo Fisher Scientific, Waltham, MA) and system cleanliness was verified.
Particle densities were set to 0.97 g/mL for positively buoyant particles (considered as silicone oil particles), and to 1.32 g/mL for negatively buoyant particles (considered as proteinaceous). Each sample was loaded for 40 seconds and the limit of detection was automatically determined by the instrument software. Minimum detectable particle sizes were approx. 485 nm for silicone oil particles and approx. 274 nm for protein particles.
Samples were analyzed in triplicates with a measurement time of 600 seconds as stop criterion, corresponding to an analyzed volume of ∼150 nL per replicate.
Data evaluation was performed with the LINK software platform v2.3.22.200619 (Lumetics, Nepean, ON, Canada).
Particle densities were set to 0.97 g/mL for positively buoyant particles (considered as silicone oil particles), and to 1.32 g/mL for negatively buoyant particles (considered as proteinaceous). Each sample was loaded for 40 seconds and the limit of detection was automatically determined by the instrument software. Minimum detectable particle sizes were approx. 485 nm for silicone oil particles and approx. 274 nm for protein particles.
Samples were analyzed in triplicates with a measurement time of 600 seconds as stop criterion, corresponding to an analyzed volume of ∼150 nL per replicate.
Data evaluation was performed with the LINK software platform v2.3.22.200619 (Lumetics, Nepean, ON, Canada).
4
Nanoparticle Size Analysis via Zetasizer
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A Zetasizer APS (Malvern, United Kingdom) supported by the Zetasizer Software DTS software was used to measure the size and distribution of nanometer and submicron particles. The device automatically measures liquid samples in microtiter plates. After thawing each sample (100 μl) was analysed five times at ambient temperatures and afterwards discarded. In addition to protein samples the standard of latex spheres size 60 nm and 200 nm (Duke standards™, Thermo Scientific) was also measured before and after analysis of samples to ensure correct operation. The plate filled with samples was protected with aluminium foil during the analysis.
5
Imaging Polystyrene Microspheres with QPM
6
Comprehensive Molecular Techniques Protocol
7
Characterizing Plastic and Organic Particles
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The following standard plastic beads were used during in-flow measurements; poly(methyl methacrylate) (PMMA) beads with the size of 40±18 µm (Colacryl D150, Songhan Plastic Technology Co.,Ltd.), polystyrene (PS) beads with the size of 39.5±1.0 µm (Duke Standards, ThermoFisher Scientific). Irregular-shaped LDPE particles with the maximum size of 300 µm were also used for in-flow tests (ET316031, GoodFellow Cambridge Ltd.). For static measurements of plastics, since all three plastic particles were measured in the same field of view, large PS beads with the size of 200±1.0 µm (16435-5ML, Sigma-Aldrich) were instead used to recognise particle types visually. The diameter range of PMMA beads was obtained by processing binary images created from bright field images of 224 beads using ImageJ-Fiji (Version 1.52p 36 ). The diameter ranges of PS beads and maximum size of LDPE particles were obtained from supplier information. The density of PMMA, PS and LDPE is 1.19, 1.05, and 0.94 g/cm 3 , respectively. These plastics are typically found in aquatic environments. 12 Scenedesmus, a freshwater algae (Scenedesmus Algae -Live, Breckland Scientific) was used as an organic particle.
8
Adhesive Properties of Latex Particles
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The wet mechanical properties of the latex particles were investigated with the AFM colloidal probe technique using a MultiMode IIIa AFM (Veeco Instruments Inc. Santa Barbara, CA) with a PicoForce extension. Borosilicate microspheres with the radius 4.7-5.2 μm (Duke Standards, Thermo Scientific) were attached to tipless rectangular cantilevers (CLFC-NOCAL, Bruker) using a melting glue (Epikote 1001, Shell Chemical Co.) and a manual micromanipulator (HS 6 Manuell, Marzhauser Wetzlar GmbH & Co. KG). Each AFM probe had two cantilevers with spring constants of <1.4 N m -1 and >9.5 N m -1 , respectively which were determined using AFM Tune IT version 2.7 (Force IT, Sweden). The different cantilevers were used to investigate how the applied load and the deflection sensitivity affected the adhesive properties. The latex particles (1 wt% in 10 mM NaCl solution) were adsorbed onto oxidised silicon wafers and the silica probe in situ for 1 h. The forcecurves were measured in Milli-Q with or without 120 s surface delay and were further analysed using the software AFM force IT version 2.6 (Force IT, Sweden). Typical force measurements are shown in Fig. 9.
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