Superose 6 prep grade sec columns

Example 4

Example 4 represents an optimized method for commercial manufacturing of rVWF by separating rVWF and rVWF propeptide (rVWF-PP) via size exclusion chromatography. Sodium citrate is added to the SEC running buffer to provide an efficient split of rVWF and rVWF-PP.

A rVWF containing ultrafiltrated UNOsphere™ S-eluate was loaded directly onto an array of two Superose 6 prep grade SEC columns in series (GE Healthcare, Cat. No.: 28-9913-16), both with an inner diameter of 16 mm each, a bed height 82.0 cm (2×41 cm), and the volume of both columns was approximately 165 ml. The load was applied at a rate of 7 cm/h. The running buffer was 20 mM HEPES free acid, 150 mM NaCl, 15 mM Na citrate dihydrate pH 7.5±0.2. The size exclusion chromatography was carried out with isocratic conditions at a linear flow rate of 12 cm/h.

FIG. 10 shows a chromatogram, a chromatography scheme, and buffer compositions for Example 4.

FIG. 11 provides a table of the results for Example 4.

FIG. 12 shows a silver stained protein gel and a western blot illustrating the separation of rVWF and rVWF propeptide by the method of Example 4.

Example 5

Example 5 represents a method for separating rVWF and rVWF-PP by size exclusion chromatography by applying a pH conditioned rVWF containing start material onto size exclusion chromatography.

A rVWF containing ultrafiltrated UNOsphere™ S-eluate was conditioned to a pH of 7.5±0.2 with 1 M glycine pH 9.0 prior loading onto the column. This solution was loaded onto an array of two Superose 6 prep grade SEC columns in series (GE Healthcare, Cat. No.: 28-9913-16), both with an inner diameter of 16 mm each, a bed height 82.0 cm (2×41 cm), and the volume of both columns was approximately 165 ml. The load was applied at a flow rate of 7 cm/h. The SEC running buffer comprised 20 mM HEPES free acid and 150 mM NaCl, pH 7.5±0.2. The size exclusion chromatography was carried out with isocratic conditions at a linear flow rate of 12 cm/h.

FIG. 13 shows a chromatogram, a chromatography scheme, and buffer compositions for Example 5.

FIG. 14 provides a table of the results for Example 5.