Oligonucleotide trapping method for purification of transcription factors

Initial purification of two serotypic variants of recombinant botulinum neurotoxin toxin heavy chain fragment [rBoNT(Hc)], produced intracellularly in the yeast Pichia pastoris, using hydrophobic charge induction chromatography (HCIC) is reported. HCIC employs a matrix containing a weakly ionizable ligand that binds proteins through hydrophobic interactions at neutral pH and elutes the proteins by charge repulsion at acidic pH. HCIC optimization led to different purification conditions for each of the proteins even though they have 58% sequence similarity. The HCIC resin has a higher affinity for the fragment of serotype A than that of serotype B. The 10% dynamic breakthrough capacity for the serotype A fragment is >12.5 mg per ml of resin and is approximately 3.5 mg or the serotype B fragment per ml of resin. Stable elution conditions are also different for the two serotypes. The serotype A fragment is unstable when citrate is used to elute the product. However the serotype B fragment is stable when eluted with citrate buffer, and it is further purified by a overnight precipitation caused by the citrate buffer. This paper reports the development strategy, dynamic capacity breakthrough curves, resin and separation reproducibility, and preliminary scale-up data. The summation of the data demonstrates that HCIC is a scaleable process step for biopharmaceutical production of rBoNT(Hc) proteins.