Preparation Of SP Ion Exchange Media And Its Application In The Purification Of Recombinant Human Lactoferrin

Lactoferrin (LF) is a valuable pharmaceutical protein abundant in human milk. Although the limited source of human milk hampered its commercial application, the advancements in recombinant technology have made it possible to express human lactoferrin (HLF) efficiently in cattle mammary bioreactor. Therefore, an efficient purification process of recombinant HLF (rHLF) from transgenic milk must be established. In this study, scale-up chromatographic purification of rHLF from transgenic milk was achieved using a commercial SP cation-exchange media. The overall process scheme includes milk pretreatment, Ion Exchange Chromatography (IEC), ultrafiltration desalting and freeze drying. The treatment capacity can reach 200 L transgenic milk per batch with a production of 600 g rHLF. The results of electrophoresis and HPSEC (High-Performance Size-Exclusion Chromatography) indicated that the purity of rHLF was over 95%. However, high price of the commercial ion exchange adsorbent used in the purification process may limit more extensive application of the protein.To reduce the cost of production, novel ion exchange adsorbents were synthesized by immobilizing Sulfopropyl derivative onto homemade highly cross-linked agarose beads, which were successfully applied in the purification of rHLF from transgenic milk. In the allyl activating step, the effects of various factors, including amount of Allylglycidyl ether (AGE), NaBH4 and Na2SO4, reaction time, temperature, initial concentration of NaOH on allyl density were investigated. In the coupling step, the effects of Na2S2O5 amount, pH value of the buffer, reaction temperature on ligand density were evaluated using orthogonal experiment. The results indicated that the factor order was pH> Na2S2O5 amount> reaction temperature. By varying reaction conditions, SP ion exchange adsorbents with controllable ligand densities could be produced and a high density of 0.24 mmol/mL was achieved.Finally, reproducibility and adsorption capacity of SP ion exchange adsorbents were also investigated. The results indicated that the adsorbents had good reproducibility from batch to batch of purification. The adsorbents also had good static and dynamic adsorption capacity. rHLF could be purified from transgenic milk using the adsorbents with different ligand densities.1 mL high density (0.24 mmol/mL) adsorbent could handle 50 mL rHLF-containing milk. The mass recovery of rHLF was 86.5% and the purity was 98.5%. CD spectra demonstrated that the native structure of rHLF was not affected in the purification process. The biological functions of the purified rHLF, including iron binding, releasing and antimicrobial activities were then investigated. The results showed that rHLF had comparable iron binding and releasing activity to that of native HLF.5 g/L concentration of rHLF significantly inhibited the growth of Escherichia coli. These studies lay a solid foundation for the wide application of our self-prepared ion exchange adsorbents in protein purification.