Purification of Recombinant Brain Derived Neurotrophic Factor Using Reversed Phase Displacement Chromatography

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.

Chromatographic and electrophoretic characterization of protein variants

“…They presented the separation of two closely related insulin variants in a process-scale and outlined that the applied system is suitable for theoretical modeling for optimization and scale-up. Sunasara et al [77] used reversed phase displacement chromatography for the purification of recombinant brain-derived neurotrophic factor from its variants, where tetrahexylammonium chloride (THAC) was used as a displacer. Operationally, displacement chromatography is similar to step gradient chromatography, with the difference lying in the fact that the displacer has the highest affinity for the stationary phase compared to all other solutes.…”

“…Therefore, the displacer stays behind the protein zones and back mixing is avoided. Sunasara et al [77] were also able to scale-up this separation step from an analytical column (Phenomenex Jupiter C 4 column, Vydac C 4 column) to a pilot-scale system (Phenomenex Jupiter 10 m C 4 , 250 mm × 50 mm). The system was successful in removing an oxidized protein variant and a protein variant containing norleucine instead of leucine from the native form of recombinant brain derived neurotrophic factor.…”

Comparison of displacement versus gradient mode for separation of a complex protein mixture by anion-exchange chromatography

“…Different examples have already demonstrated that displacement chromatography is well suited for protein purification. Cramer and colleagues were able to show that the displacement elution mode can be used in combination with hydrophobic interaction, reversed phase and ion-exchange chromatography [8][9][10].…”

Application of hydrophobic interaction displacement chromatography for an industrial protein purification

“…While linear and stepgradient are the most widely used elution modes, displacement chromatography can offer several advantages over these traditional modes. These advantages include (i) high effective separation factors, leading to enhanced selectivi-ties; (ii) control over the concentration of the purified product; and (iii) high loadings, leading to enhanced production rates (Frenz and Horvath, 1988;Kundu and Cramer, 1997;Sunasara et al, 2001).…”

“…While low molecular weight displacers have been successfully employed for the purification of complex industrial mixtures in ion exchange (Barnthouse et al, 1998; and RPLC systems (Sunasara et al, 2001), there are no reports in the literature, to date, on using these molecules in HIC systems for the purification of recombinant protein therapeutics. Proof of concept is demonstrated for the purification of an industrial protein mixture from its aggregate impurities.…”