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%)…
Tag: <span>cation exchange chromatography</span>
The outstanding success and safety record of first generation monoclonal products has created an immense increase in the number of product candidates that need to be evaluated clinically. The concept of platform purification has emerged in response to this need. A platform is a semigeneric, multistep purification procedure that can be applied to a wide range of monoclonal antibodies without extensive method-scouting and optimization. This approach can substantially accelerate process development and hasten inception of clinical trials…
Cation exchange chromatography (CEX) is a versatile method for separation of proteins based on exploiting differences in positive electrostatic charges. In CEX, proteins are bound to the negatively charged stationary phase (cation exchangers) and then eluted using a salt gradient. Typically, the liquid-phase pH in CEX is lower than the isoelectric points (pI) of the proteins. CEX has been used to monitor various post-translational modifications such as glycosylation, deamidation, phosphorylation, truncation, oxidation, C-terminal and N-terminal clipping, and N-terminal cyclization. Some of these variants may exhibit different bioactivity. Therefore, it is important to characterize protein variants and monitor the stability of these variants throughout the process of drug discovery, development, and manufacture. Characterization of complex proteins such as antibodies, has traditionally been performed using slab gel-based techniques such as isoelectric focusing (IEF). This technique is qualitative and time consuming. It also generates large quantities of chemical waste from the staining process…