A simple scheme for large scale purification of urine - Derived Bence Jones Kappa protein.

We have investigated and optimized purification process, suitable for industrial scale, to obtain high purity grade Bence Jones Kappa Protein ('BJK-Protein'), while preserving its physiological properties and functions. BJK-Protein was obtained from a biological waste product i.e. human urine of renal failure patients. Isolated 'BJK-Protein' was analyzed by electrophoresis, western blotting, double immunodiffusion, SEC-HPLC assay and Mass Spectrometry (MS). The relative molecular mass of 'BJK-Protein' is 23054.2 Da. Moreover, dimer forms of 'BJK-Protein' were also detected in SDS-PAGE and mass spectrum corresponding to 46054.4 Da. The results of western blotting, immunoelectrophoresis, SEC-HPLC assay, and mass spectrometry analysis indicate a high purity (>99 %) of 'BJK-Protein'. Peptide mass fingerprint analysis of 'BJK-Protein' yielded peptides that partially matches the known database sequences of kappa variable region (KV139_HUMAN) of immunoglobulin. This protein was found to be stable up to 20 months at 2-8 °C temperature and also found negative for major undesirable viral markers as well as bacterial endotoxin. With this purification approach, the cost of purified 'BJK-Protein' is significantly reduced as compared to the current market price of Kappa light chain available in international market.

An improved protocol for large scale production of high purity 'Fc' fragment of human immunoglobulin G (IgG-Fc).

We describe a simplified approach for purification and characterization of human 'IgG-Fc' fragment used widely as immunochemical tool and for therapeutic purposes. The 'Fc' fragment was purified from human IgG in a 3-stage column chromatography. The purified 'Fc' fragment appeared as a dimer glycoprotein with an apparent molecular mass of 52,981 Dalton (Ultraflex MALDI TOF/TOF). The Size-exclusion HPLC profile of the purified 'Fc' fragment of human IgG matched that of a commercially procured reference 'Fc' fragment material. The purity of the 'Fc' fragments was >99% by SDS-PAGE and size-exclusion HPLC. The results of Western blotting, immunoelectrophoresis, and mass spectrometry analysis indicate a high purity of the 'Fc' fragment. Peptide mass fingerprint analysis of the purified 'Fc' protein yielded peptides that partially match the known database sequences of FCG3B_HUMAN (Uniprot ID: O75015). This method of purification of the 'Fc' fragment is suitable for achieving high purity level of 'Fc' fragment protein. With this purification approach, the cost of the purified 'Fc' fragment of human IgG is significantly reduced as compared with the current market price of IgG-Fc fragment protein in international market. The purified 'IgG-Fc' fragment protein was found to be negative for major viral markers.