Superdex 200 prep grade resin

Example 10

Treatment of Purified Plasma-Derived A1PI with 10 mM DTT

A plasma-derived A1PI product that has a yellow appearance was tested to determine if treatment with reducing agent would also result in a similar reduction in the yellow color. A 10 mL aliquot of plasma-derived A1PI at approximately 50 mg/mL was treated with DTT at a final concentration of 10 mM. Gel filtration chromatography on Superdex™ 200 prep grade resin was performed as described herein (GE Healthcare Life Sciences). The chromatogram from the run with the DTT-treated plasma-derived A1PI is shown in FIG. 14.

The fractions corresponding to recA1PI under the main peak were pooled and concentrated to 50 mg/mL. The color of the sample was no different from that of the untreated sample. This indicated that the source of yellow color of cell culture-derived recA1PI was different from that of the plasma-derived A1PI.

Example 7

DTT Concentration Analysis

The effects of lower concentrations of DTT were analyzed by incubating recA1PI with DTT, purifying the sample using gel filtration chromatography, and then obtaining the UV-Vis spectra of the concentrated purified samples. A 1.6 cm inner diameter×90 cm bed height (181 mL column volume) column of Superdex™ 200 prep grade resin was used (GE Healthcare Life Sciences). This column was pre-equilibrated with PBS containing 5 mM DTT. The recA1PI in a volume of 9 mL was treated with DTT at the appropriate concentration at ambient temperature, overnight. The load constituted 5% of the column volume and the column was run at 2 mL/minute (60 cm/h). Constant volume fractions of 14.5 mL were collected. Appropriate fractions were pooled and concentrated back to approximately 50 mg/mL using 30 kDa MWCO spin concentrators.

The UV-Vis spectra of the samples treated with 10 mM DTT and 50 mM DTT shows that the 10 mM DTT concentration is as effective as 50 mM DTT for diminishing the characteristic spectral signature of the yellow colored recA1PI (FIG. 11).

Example 4

Gel Filtration Chromatography with Superdex™ 200 Resin

A gel filtration experiment (aka size exclusion chromatography; SEC) was also attempted to remove the yellow species from recA1PI. A 1.6 cm inner diameter×90 cm column (bed volume of 181 mL) of Superdex™ 200 prep grade resin was packed (GE Healthcare Life Sciences). The column was equilibrated with phosphate buffered saline (PBS; 12 mM phosphates, pH 7.4, 137 mM NaCl, 2.7 mM KCl). recA1PI was loaded at 5% of the column volume (9 mL) and run at 2 mL/minute (60 cm/h). Fractions of 12 mL volume were collected for analysis.

The chromatogram for this column is shown in FIG. 5. Although, the load is 99% recA1PI, a split peak was observed, possibly owing to saturation of the UV detector. The fractions under this peak were analyzed by UV-Vis spectrophotometry (FIG. 6). The recA1P fractions were visibly yellow and the UV-Vis spectra of these fractions were similar to that of the loading material. Accordingly, the gel filtration column did not remove the yellow color from recA1PI.