A two-step process for capture and purification of human basic fibroblast growth factor from E. coli homogenate: Yield versus endotoxin clearance

Abstract

A two-step purification process for human basic fibroblast growth factor (FGF-2) from clarified E. coli homogenate has been developed in which the impurity level after the second step is below the limit of quantification. Endotoxin content is cleared to 0.02 EU/μg FGF-2 and the overall yield is 67%. The performance of the cation exchanger Carboxymethyl-Sepharose Fast Flow (CM-SFF) was compared to the affinity resin Heparin-SFF regarding the impurity profile and product quality in the elution peak. The CM-SFF eluate was further purified using hydrophobic interaction resin Toyopearl-Hexyl-650C. The relative amounts of target product, host cell proteins (HCPs), dsDNA, endotoxin, monomer content, and high molecular weight impurities differed along the elution peak depending on the applied method. The bioactive monomer (>99%) was obtained with a yield of 48% for CM-SFF and 68% for Heparin-SFF. A half-load reduction in CM-SFF increased the yield up to 67% without deterioration of the impurity content. Assuming a dose of 400 μg FGF-2, endotoxin was reduced to 188 EU/dose, dsDNA <10 ng/dose, and HCP <2 ppm/dose using the cation exchanger. In the pooled eluate fractions, dsDNA was removed 4-fold (291 ng/mL) and endotoxin 14-fold (0.47 EU/μg FGF-2) more efficiently by CM-SFF than by affinity chromatography. In contrast, HCP clearance was 3-fold (13 ppm) more efficient with Heparin-SFF than CM-SFF. In contrast to process monitoring by UV or SDS-PAGE, this characterization is the basis for a Process Analytical Technology attempt when correlated with online monitored signals, as it enables knowledge-based pooling according to defined quality criteria.

Semi-automation of process analytics reduces operator effect

“…IgG1 concentrations were determined by high-performance monolith affinity chromatography as described in [16]. Human fibroblast growth factor 2 (FGF-2) was expressed in E. coli, captured by cation exchange or affinity chromatography, polished by hydrophobic interaction chromatography, and quantified by reversed phase HPLC, all as described in [17].…”

“…Binding affinities of anti-TNFα-IgG against TNFα (10,602-HNAE-100, Sino Biological, China) and of FGF-2 to FGFreceptor 2 were determined by a SPR assay using a Biacore 2000 system (GE Healthcare, USA) as described in [17].…”

Real‐time monitoring and model‐based prediction of purity and quantity during a chromatographic capture of fibroblast growth factor 2

“…Fibroblast growth factor 2 (FGF‐2) was overexpressed in Escherichia coli ( E. coli ) BL21 cells (Sauer et al, ). FGF‐2 has a molecular weight of 17 kDa and an isoelectric point pI of 9.6 (Gasparian et al, ).…”

“…10 CV of clarified E. coli homogenate (1.7 ± 0.2 mg/ml) were loaded and elution was performed with a linear gradient from 0 to 1 M NaCl in 100 mM Na‐phosphate (pH 7.0). For each performed chromatographic run, 15 fractions (UV 280 nm signal >50 mAU) of 1 ml were collected and used for all offline assays (Sauer et al, ). Clarified homogenate compositions of fermentation batches are provided in Table SI.…”

“…Based on the FGF‐2 concentration, the relative dsDNA content (ppm) was quantified by PicoGreen ® assay and the HCP content (ppm) by enzyme‐linked immunosorbent assay (ELISA). The intra‐assay variabilities were 20% for HCP ELISA, 15% for the PicoGreen ® assay, and 5% for the RP‐HPLC quantification (Sauer et al, ).…”