Sepharose phenyl high performance hic resin

Example 39

1.75 L of NPBA-PEG_5k-Tf crude mixture (20 g/L) was added to a reaction vessel. 7 L of mobile phase A (1.5 M ammonium sulfate, 20 mM sodium phosphate, pH 7.4) was added to crude mixture to achieve a 1.2 M ammonium sulfate, 16 mM sodium phosphate, pH 7.4 concentration. After mixing, sample mixture was loaded onto an AxioChrom 140 column (1.7 L) packed with GE Sepharose Phenyl High Performance HIC resin with a pump flow rate of 200 mL/min. Approximately 10 minutes of residence time was allowed for loading the sample mixture onto the HIC column. IPA/10 mM sodium phosphate, pH 7.4 (1:4 v/v, mobile phase B) was used as the eluent. Prior to the elution step, column was washed with 20% mobile phase B for 2 column volumes (CV) to remove unbound species. Purified NPBA-PEGk-Tf was eluted with gradient 20-100% mobile phase B over 15 CV. Fractions of mono-PEGylated Tf (mono-NPBA-PEGk-Tf) were collected and combined for ultrafiltration and diafiltration (UF/DF). Sartocon Slice Hydrosart Cassette 30 kDa membrane was used for UF/DF of mono-NPBA-PEG_5k-Tf. Mono-NPBA-PEG_5k-Tf conjugate was concentrated to 50 g/L by diafiltration using 7 volumes of PBS pH 7.4. After that, mono-NPBA-PEGk-Tf was sterile filtered through a 0.22 μm polyethersulfone (PES) membrane.

Example 42

0.55 L of NPBA-PEG_5k-Tras crude mixture (10 g/L) was added to a reaction vessel. 1.1 L of mobile phase A (1.5 M ammonium sulfate, 20 mM sodium phosphate, pH 7.4) was added to crude mixture to achieve a 1 M ammonium sulfate, 13.3 mM sodium phosphate, pH 7.4 concentration. After mixing, sample mixture was loaded onto an AxioChrom 140 column (1.7 L) packed with GE Sepharose Phenyl High Performance HIC resin at a flow rate of 200 mL/min. Approximately 10 minutes of residence time was allowed for loading the sample mixture onto the HIC column. IPA/10 mM sodium phosphate, pH 7.4 (1:4 v/v, mobile phase B) was used as the eluent. Prior to the elution step, the column was washed with two CV of 40% mobile phase B to remove unbound species. Purified NPBA-PEG_5k-Tras was eluted with a gradient of 40-100% mobile phase B over 15 CV. Fractions of mono-PEGylated Tras (mono-NPBA-PEGk-Tras) were collected and combined for UF/DF. Sartocon Slice Hydrosart Cassette 30 kDa membrane was used for UF/DF of mono-NPBA-PEGk-Tras. Mono-NPBA-PEGk-Tras conjugate was concentrated to 50 g/L by diafiltration using 7 volumes of PBS pH 7.4. Following diafiltration, mono-NPBA-PEGk-Tras was sterile filtered through a 0.22 μm PES membrane.