RESUMO
This work investigated the use of hydrophobic interaction membrane chromatography for intermediate purification of recombinant human Factor IX (rFIX) produced by CHO cells. The first purification step was based on a strong anion exchange monolith, thus forming a purification process fully based on convective media, which allow operation at high flow rates and low pressure drops, as well as modular scale-up. Although the starting material was challenging (CHO cell culture supernatant harvested at 70% cell viability), the two-step purification process showed promising results, with a global purification factor of 298, a global recovery of 69%, and DNA and endotoxin levels close to regulatory limits. Final host cell DNA (68.8 ng per dose of 500 IU), endotoxins (60 EU per dose of 500 IU) and activated FIX (FIXa/FIX = 2.33%) were in levels close to those recommended by regulatory authorities. HCP removal was of 99.98%, decreasing from 9 424 358 ppm in the supernatant to a final HCP value of 2071 ppm. The use of a supernatant harvested at higher viability and/or the addition of a third polishing step focusing on HCP removal could allow meeting the desired HCP range of 50-100 ppm, as well as the regulatory requirements for the other critical contaminants.
Assuntos
Cromatografia de Afinidade/métodos , Fator IX/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Interações Hidrofóbicas e Hidrofílicas , Sulfatos/químicaRESUMO
Both recombinant and plasma-derived factor IX concentrates are used in replacement therapies for the treatment of haemophilia B. In the present work, the capture step for a recombinant FIX (rFIX) purification process was investigated. Different strong anion-exchange chromatography media (the resins Q Sepharose(®) FF and Fractogel(®) TMAE, the monolith CIM(®) QA and the membrane adsorber Sartobind(®) Q) were tested for their rFIX binding capacity under dynamic conditions. In these experiments, crude supernatant from CHO cells was used, thus in the presence of supernatant contaminants and mimicking process conditions. The highest dynamic binding capacity was obtained for the monolith, which was then further investigated. To study pseudoaffinity elution of functional rFIX with Ca(2+) ions, a design of experiments to evaluate the effects of pH, NaCl and CaCl2 on yield and purification factor was carried out. The effect of pH was not statistically significant, and a combination of no NaCl and 45mM CaCl2 yielded a good purification factor combined with a high yield of active rFIX. Under these conditions, activity yield of rFIX was higher than the mass yield, confirming selective elution of functional, γ-carboxylated rFIX. Scaling-up of this process 8 fold resulted in very similar process performance. Monitoring of the undesired activated FIX (FIXa) revealed that the FIXa/FIX ratio (1.94%) was higher in the eluate than in the loaded sample, but was still within an acceptable range. HCP and DNA clearances were high (1256 and 7182 fold, respectively), indicating that the proposed process is adequate for the intended rFIX capture step.