In the end, a proof of concept was performed using two different mAbs from serum-free CHO cell cultures.
Following the main purification, purified IgG was characterized in terms of isoelectric point, size and activity. HPLC analysis of carbohydrates on POLYSPHERCH(R)OH columns using pulsed amperometric detection (PAD) with sodium hydroxide as post column detection reagent Anal Bioanal Chem. The purification outcome was also compared with protein A chromatography that revealed a recovery of 99%, 87% protein purity and 29 out of a maximum of 33 purification factor. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 1.06462). Buffers mitigate the influence of hydrogen/hydronium and hydroxide. In analytical chemistry, buffers are typically used in reversed-phase high performance liquid chromatography (RP-HPLC), when the sample contains acidic or basic functional groups. From the different conditions tested, best recoveries - 99% - and purifications - protein purity of 81% and a purification factor of 16 out of a maximum of 20 - were achieved using 100 mM D-sorbitol in 10 mM Tris-HCl as washing buffer and 0.5 M D-sorbitol with 150 mM NaCl in 10 mM Tris-HCl as elution buffer. Size exclusion chromatography (SEC) or gel filtration is a hydrodynamic technique that. Buffers are solutions of a weak acid and its conjugate base, or a weak base and its conjugate acid. In particular, the study was focused on the development of a washing step and in the optimization of the elution step using a serum containing supernatant. In this work, phenylboronic acid (PBA) was thoroughly investigated as a synthetic ligand for the purification of an immunoglobulin G (IgG) from a clarified cell supernatant from Chinese Hamster Ovary (CHO) cell cultures.
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