Process for the purification of recombinant human erythropoietin (EPO), EPO thus purified and pharmaceutical compositions comprising same
An erythropoietin, mixture technology, applied in the field of erythropoietin (EPO), can solve problems such as reducing liver clearance rate and increasing circulation time
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Embodiment 1
[0094] Example 1: Capture of EPO and reduction of possible contaminants by affinity chromatography using Blue Sepharose 6FF (Blue Sepharose 6FF)
[0095] Blue Sepharose 6FF is an agarose resin covalently attached to the dye Cibacron Blue and used to preferentially bind EPO in the presence of contaminants contained in the fermentation harvest. The product stream was first purified by affinity chromatography as described in Table 2.
[0096] Table 2: Parameters used to perform Blue Sepharose chromatography
[0097]
[0098] The column was packed with Blue Sepharose 6FF resin. After packing, the columns were qualified for theoretical plates and asymmetry factors. The column was sterilized with 0.5 M NaOH for 1 h and then washed with water for injection (WFI). Before loading, the column was equilibrated with 20 mM Tris.HCl, 1.5M NaCl, pH 7.5, then with 20 mM Tris.HCl, pH 7.5. The sample was loaded onto the column and the column was washed with 20 mM Tris.HCl, pH 7.5. Sampl...
Embodiment 2
[0101] Example 2: Concentration of EPO by Diafiltration
[0102] The eluate was concentrated by ultrafiltration and diafiltration was performed on a tangential flow filtration device using a 10 kDa cut-off membrane; see Table 3.
[0103] Table 3: Parameters used to perform diafiltration
[0104]
[0105] Perform a normal water flux assay (NWP) before using the filter unit. The filter unit was sterilized with 1M NaOH for at least 1 h, then washed with WFI. After washing, filter devices were equilibrated with 20 mM Tris-HCl, pH 7.0. The sample is then loaded and filtered at a transmembrane pressure not to exceed 1 bar. Diafiltration was terminated when the conductivity of the permeate was below 2.5 mS / cm.
Embodiment 3
[0106] Example 3: Enrichment of acidic isoforms of EPO and further removal of contaminants by anion exchange chromatography using Q-Sepharose HP
[0107] Anion exchange chromatography using Q-Sepharose HP resin was used to enrich for the acidic isoforms of EPO, further removing contaminants (e.g. DNA, HCP) and eliminating any dye ligands that might have leached from the first column. Furthermore, anion exchange chromatography is an effective step for the removal of adventitious viruses. Therefore, the leachate was processed by anion exchange chromatography as described in Table 4, followed by 0.2 μm filtration of the Q eluate fraction and fraction pool.
[0108] Table 4: Parameters used to perform anion exchange chromatography
[0109]
[0110] The column was packed with Q-Sepharose HP resin. After packing, the columns were qualified for theoretical plates and asymmetry factors. The column was rinsed with WFI and then sanitized with 1M NaOH for 1 hour. After sanitization,...
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