Ktaprime plus liquid chromatography system

The production of recombinant gp120 that was encoded by the Env from NL(AD8) [50 (link)] (herein referred to as AD8 strain) utilized a stable cleavable gp140-expressing HeLa cell line. The cell line was generated and the Env-containing supernatant was produced, as described previously [37 (link)]. The gp120 was purified from culture supernatant by lentil lectin affinity chromatography, as described previously [51 (link)]. The purified gp120 was concentrated and further purified by size exclusion chromatography while using a Superdex™ 200 16/600 pg with an ÄKTAprime plus liquid chromatography system (GE Healthcare Life Sciences, Little Chalfont, UK). The protein was injected into the column that was equilibrated with PBS and the flow rate was 1 mL/min. Fractions containing monomeric gp120 were pooled and concentrated. Recombinant p24 that was encoded by the Gag from isolate BH10 was purchased from Aalto Bio Reagents.

Secreted twin StrepTagged gp16-V1V2 proteins in the harvested and filtered supernatant were supplemented with BioLock biotin blocking solution (IBA Lifesciences) at 5 µl/ml to mask the biotin present in the supernatant. After 30 min of incubation, the supernatant was loaded onto a 1 ml StrepTactin column (QIAGEN) at a flow rate of 0.7 ml/min in the ÄKTA prime-plus liquid chromatography system (GE Healthcare). Non-specifically bound proteins were washed off by passing at least 20 column volumes of the wash buffer (300 mM NaCl, 50 mM Tris-HCl, pH 8) or until the absorbance reached the baseline level. Bound gp16-V1V2 proteins were eluted with StrepTactin elution buffer (5 mM d-Desthiobiotin, 300 mM NaCl, 50 mM Tris-HCl, pH 8) at a flow rate of 1 ml/min. Eluted peak fractions were buffer exchanged into 100 mM NaCl, 50 mM Tris-HCl, pH 8 buffer. Protein fractions were stored with 10% glycerol at –80 °C until use for antigenicity and immunogenicity studies. GnTi expressed His-tagged gp140s and gp120s were purified from the harvested and clarified supernatant using Ni-NTA agarose beads (Qiagen) following manufacturer’s instructions.

Five hundred microlitres of plasma was loaded onto a HiLoad 16/60 Superdex 75 (120 ml) size‐exclusion column in combination with a 25 mmol/l Tris/0·52 mol/l NaCl buffer mobile phase at pH 7·4, with a flow rate of 1 ml/min. Optimization studies demonstrated that the addition of bovine serum albumin (BSA) to fraction collector tubes before GFC improved insulin recovery from the column, achieving >70%. Six millilitre elution volume fractions with 1 ml bovine serum albumin (BSA) (final volume 7 ml, calculated BSA concentration 40 g/l) were collected in Cellstar^® polypropylene tubes (Greiner Bio‐One, Stonehouse, Gloucestershire, UK). A total of 36–114 ml eluted volume was collected.
GFC was performed using the ÄKTAprime plus^™ liquid chromatography system (GE Healthcare, Uppsala, Sweden), and ultraviolet (UV) absorbance at 280 nm was recorded. The chromatography method demonstrated good precision with elution volume coefficient of variation of 6% for immunoglobulin (n = 30; mean elution volume 49 ml).
Samples were analysed for insulin using the LIAISON^® XL immunoassay. This assay was chosen because in‐house data supported high analytical sensitivity (1·2 pmol/l) and acceptable coefficient of variation at lower insulin concentrations (8·6% at 34 pmol/l; n = 244).