Ultrafree mc centrifugal filter devices 0.22μm

To remove antibodies from NHS, κ/λ-light chain specific affinity chromatography was employed. The matrix was prepared by mixing three parts of κ-light chain specific beads with one part of λ-light chain specific beads (083310 and 084910, respectively, Life Technology). To avoid clogging of the resin, NHS was spin-filtered before incubating with double volume of resin at 4°C for 10min. The supernatant was separated from the matrix by centrifugation (Ig depleted serum, Ig-dep). As control, equivalent volume of serum was treated identically but without the chromatography matrix (mock). Both Ig-depleted and mock sera were concentrated with Vivaspin 500 (10,000 MWCO PES, Sartorius). The dilution factor was determined based on absorbance measurement at 405, 490, and 650 nm, compared to that of untreated serum. To remove beads, fibers and precipitates, the samples were spin-filtered. Bound antibodies were recovered by washing the resin with DPBS with Ca/Mg, eluting with 0.1M glycine pH 2.0 buffer and neutralizing with 1M Tris pH 8.0. This immunoglobulin fraction was concentrated with Vivaspin 20 (10,000 MWCO PES, Sartorius) followed by Vivaspin 500 and finally spin-filtered. In all cases Ultrafree™-MC Centrifugal Filter Devices 0.22μm (EMD Millipore) was employed for spin-filtration. Samples were stored at −80°C.

C1-INH was concentrated to about 2 mg/mL (Vivaspin 500, Sartorius), mixed with about 0.2–0.4 mg/mL C1 and 25 mg/mL HSA, and incubated for 10min on ice. This mixture or C1 alone was dialyzed twice against DPBS with Ca/Mg at 4°C for 3 h followed by concentration to ~1.3 mg/mL of C1 (Vivaspin 500, Sartorius). In the C1/C1-INH mixture, the C1-INH to C1 ratio was 1.8 times the physiological ratio. The samples were spin filtered to ensure sterility (Ultrafree™-MC Centrifugal Filter Devices 0.22μm; EMD Millipore) and then aliquoted and stored at −80°C until use. At least of three different batches of C1/C1-INH were prepared and used.

Fab fragments of human IVIg (ClairYg®) were prepared by digestion with agarose-immobilized papain (20341, ThermoFisher) according to the manufacturer's instructions. DPBS with 10mM EDTA pH 7.0 was used as buffer. The uncleaved Igs and Fc fragments were removed by using MabSelect Sure® (17543801, GE Healthcare) according to the manufacturer's instructions. The flow-through, containing the Fabs, was buffer exchanged in DPBS, concentrated (Vivaspin 500, Sartorius) and spin filtered (Ultrafree™-MC Centrifugal Filter Devices 0.22μm; EMD Millipore) to ensure sterility. An untreated aliquot of ClairYg® was buffer exchanged and treated the same way to be used as control. The concertation of both Fab and full IgG in DPBS was determined by bicinchoninic acid (BCA) assay (23225, ThermoFisher).