Minidawn treos multi angle light scattering detector

Example 3

Purification

The polypeptides were purified by means of a two-step protocol. First, the harvested and clarified culture supernatant was loaded on a HiScale 16/20 column (GE Healthcare) packed with an affinity resin (Capture Select) that consists of a HA specific single domain antibody, immobilized on Poros beads (obtained from ThermoFisher Scientific). This resin is highly specific for H1 strain derived hemagglutinin proteins. The column was intentionally overloaded by ˜15% to improve isolation of the trimer. Following binding and equilibration in 50 mM Tris, 0.5 M NaCl, pH 7.4 the polypeptides were eluted by applying a step gradient to 0.1 M Tris, 2 M MgCl₂, 40% propylene glycol, pH 7.4. Based on the UV signal (A280) the elution fractions were pooled and filtrated through a Millex-GV 0.22 μm filter membrane (Merck Millipore). Subsequently, the collected elution peak was applied to a Superdex 200 pg 26/60 column (GE Healthcare) equilibrated in running buffer (20 mM Tris, 150 mM NaCl, pH 7.8) for polishing purpose, i.e. remove the minimal amount of multimeric and monomeric protein. The trimer fractions were pooled and purity was assessed by analytical SEC-MALS in a High Performance Liquid Chromatography (HPLC) Infinity 1260 series setup (Agilent). Of each purified polypeptide 40 μg was run (1 mL/min.) over a TSK gel G3000SWxl column (Sigma-Aldrich) and the molar mass of the eluted material was measured by a miniDAWN Treos Multi Angle Light Scattering detector and Optilab T-rex differential refractometer (Wyatt Technology). The data were analyzed by the Astra 6 software package and molecular weight calculations were derived from the refractive index signal.

Results and Conclusion

The elution profile of the second purification step (Size Exclusion Chromatography) indicated that a large amount of non-trimeric (aggregates and monomer) polypeptide 5367 was present in the pooled elution fractions of the affinity column. In contrast, the chromatogram of UFV160656 showed only a small amount of aggregates whereas the major species present was the trimeric polypeptide (FIGS. 6A and B).

The yield of the parental constructs UFV5367 and UFV5369 was 5 and 70 mg/L, respectively, whereas the yields for the polypeptides of the invention varied from 120-240 mg/L. The recovery percentage, calculated by comparison of the amount of protein present in the culture supernatant and the trimeric end product, was roughly 40-60%. Analysis of the pooled trimer fractions by analytical SEC-MALS showed that the purified material is pure; no other peaks in the UV signal were observed (FIG. 6B). Furthermore, the calculated molecular weight, ˜96-106 kDa, corresponded with the expected molecular weight of the glycosylated trimeric polypeptide (FIG. 6C).

The above data indicated that purification of the polypeptides was achieved by a straightforward 2 step protocol resulting in very pure trimeric protein with high efficiency and yields well above 100 mg/L culture supernatant.