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Purification of recombinant brain derived neurotrophic factor using reversed phase displacement chromatography.

Biotechnology progress (2001-10-06)
K M Sunasara, R G Rupp, S M Cramer
ABSTRACT

This work investigates the utility of RPLC displacement chromatography for the purification of recombinant brain derived neurotrophic factor (rHu-BDNF) from its variants and E. coli. protein (ECP) impurities. The closely associated variants (six in total) differ by one amino acid from the native BDNF and thus pose a challenging separation problem. Several operational parameters were investigated to study their effects on the yield of the displacement process. The results indicated that the concentration of trifluoroacetic acid (TFA) in the buffer was a key factor in achieving the desired purification. Displacement chromatography on an analytical scale column resulted in extremely high purity and yield in a single chromatographic step. The process was successfully scaled-up with respect to particle and column diameter. The production rate of a pilot scale RPLC displacement process was shown to be 23 times higher than the combined production rates of the current preparative ion exchange and hydrophobic interaction gradient elution steps that are used to remove variant and ECP impurities, respectively.

MATERIALS
Product Number
Brand
Product Description

Supelco
Tetrahexylammonium hydrogensulfate, suitable for ion pair chromatography, LiChropur, ≥99.0% (T)
Sigma-Aldrich
Tetrahexylammonium hydrogensulfate, ≥98.0% (T)
Sigma-Aldrich
Tetrahexylammonium bromide, 99%
Sigma-Aldrich
Tetrahexylammonium chloride, 96%
Supelco
Tetrahexylammonium bromide, suitable for ion pair chromatography, LiChropur, ≥99.0% (AT)
Sigma-Aldrich
Tetrahexylammonium hydroxide solution, ~40% in H2O (T)
Sigma-Aldrich
Tetrahexylammonium iodide, ≥99.0% (AT)
Sigma-Aldrich
Tetrahexylammonium hydroxide solution, ~10% in methanol (T)

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