Plantibody purification is not as efficient as antibody purification from serum, ascites, or mammalian cell cultures. It is characterized by the application of inefficient plantibody solid-liquid extraction systems, low plantibody recovery, and short lifetimes of expensive chromatography matrices. To overcome it, several protocols of liquid-liquid aqueous two-phase extraction (ATPE) combined with affinity chromatography were previously studied to purify the CB.Hep-1 monoclonal antibody, which showed an unexpectedly high recovery. However, a study of ATPE combined with several affinity chromatography matrices to purify plantibodies has not been reported so far. Therefore, a combination of the best ATPE protocol with five specific affinity chromatography matrices to purify a plantibody for vaccine manufacturing is described in this study. Positive outcomes from plantibody recovery (%), specific activity (%), yield (mg purified IgG/L of leaf extract), and productivity (mg purified IgG/L of leaf extract/h) were achieved. Plantibody purity did not show statistical differences among all samples (> 97%, p < 0.05), and protein A leakage was thousands of times smaller than toxic protein A for non-human primates. In summary, the combination of ATPE (10% PEG 4000/15% K2PO4, pH 5.5) with two specific affinity resins were well-suited for large-scale plantibody purification from tobacco plant leaves...
Tag: <span>affinity chromatography</span>
Cancer is one of the leading causes of death worldwide, and the second leading cause of death in Cuba. To address this serious health problem, some research has involved suppressing tumor growth by inhibiting the angiogenesis process using several molecules including antibodies. A divalent version of antibody fragments, the CIGB-598a, with a molecular weight between 100 and 110 kDa, has been expressed in CHO cells specific for a novel epitope of the human vascular endothelium growth factor (VEGF). This material has been generated at the Center for Genetic Engineering and Biotechnology to support cancer research efforts. As in other studies involving the purification of recombinant molecules, CIGB-598a exhibited a high degree of aggregation in the CHO cell culture supernatant. This required the design of a downstream process capable of removing high levels of aggregates to obtain a highly pure target molecule for use in preclinical studies and human applications further down the road. We have developed a suitable downstream method based on the combination of three chromatography processes: affinity, cation-exchange, and anion-exchange that recover a relatively low level of CIGB-598a, but at a level of high purity (greater than 95 %) with fewer aggregates (below 1%)…
A variety of affinity chromatography mechanisms exist that exploit either immobilized proteins or small molecule ligands. Among them, protein-A chromatography is widely used as a platform technology for a commercial-scale manufacturing of therapeutic monoclonal antibodies. Antigen, dye and specific ligand-columns are used to purify target proteins for research uses and diagnostic applications. It uses specific interactions between affinity columns and target proteins. In general, the samples containing a protein of interest, such as cell culture media, serum, and tissue or cell lysates, are loaded on to the affinity columns; the proteins will normally bind to the column due to high affinity…
Current expression technologies have enabled the production of thousands of recombinant proteins in diverse production hosts. Therapeutic recombinant proteins have been engineered for a variety of purposes including reduced antigenicity, longer half-life, simplified process development, and increased affinity. Protein engineering has relied on various high throughput methods (e.g., directed evolution, phage display) to identify candidate proteins with the desired therapeutic properties. The physiological and biochemical diversity of native and engineered proteins reflects on the abundance of production hosts, expression tools, and different approaches for protein purification. Notably, a key step in high-throughput protein production is purification, which is a bottleneck where large numbers of samples are involved. Universal purification methods that can be applied to virtually any protein, and that are amenable to automation, can be used to address this problem…