Separation of truncated basic fibroblast growth factor from the full-length protein by hydrophobic interaction chromatography

Basic fibroblast growth factor (FGF-2) is a labile protein which degrades over time or forms aggregates. A purification process combining a cation ion exchange chromatography (CIEX) capture step with a polishing step by hydrophobic interaction chromatography (HIC) efficiently depleted process-related impurities such as host cell proteins (HCPs), dsDNA and endotoxins from the clarified E. coli homogenate containing the overexpressed FGF-2. Fresh homogenates did not contain verifiable amounts of process-related impurities such as degraded product variants. However, the storage of the capture eluates for longer than two weeks at 4 degrees C led to product degradation up to 49%. Mass spectrometry showed that the truncated FGF-2 was devoid of the nine N-terminal amino acids. Removal of such product-related impurities is included in state of the art purifications because these byproducts bear the risk of having different biological activities. Therefore, the established purification process has been evaluated for its potential to deplete such truncated FGF-2 variants. The HIC polishing step using Toyopearl (R) Hexyl-650C with a linear salt gradient over 4 column volumes from 1.65 M trisodium citrate to 0 M separated this variant from the full-length FGF-2. The truncated form eluted before the full length product due to the loss of 9 hydrophobic amino acids. The developed ion exchange HPLC method enabled us to quantify the truncated variant and full-length FGF-2. A purity of > 98% full-length FGF-2 with a step yield of up 86% is achieved. As long as the degree of truncation does not exceed 25% of the stored eluates, the purification process delivers the product in the desired quality of > 98% full-length FGF-2, HCP content below 10 ppm/dose, dsDNA content below 10 ng/mL/dose and endotoxin level below 400 EU/dose. This purification sequence allowed the production of FGF-2 which meets distinctive quality standards defined by regulatory bodies with respect to the depletion of both - process- and product-related impurities.