Production of erythropoietin-specific polyclonal antibodies

Background: Erythropoietin, as a principal hormone promotes red blood cell production in bone marrow. Varieties of erythropoietin biosimilar are being produced by recombinant DNA technology in cell cultures. The detection or quantification of these molecules are being performed by different methods which some of theme such as Western blot and enzymelinked immunosorbent assay [ELISA] require specific antibodies. High cost, inappropriate shipping [cold chain failures], reduced sensitivity and thus poor detection performance are common pitfalls of using commercial kits for performing immunological tests

Objectives: To produce in-house polyclonal antibody against active pharmaceutical ingredient [API] of recombinant human erythropoietin [rh-EPO] was the aim of this study

Materials and Methods: Two healthy female albino rabbits were injected four times in 14 days interval using rh-EPO API as antigen. The produced antibody was separated from plasma via either caprylic acid or saturated ammonium sulfate precipitation and the results were compared from each purification methodologies. The antibody was further purified by ion exchange chromatography. Acceptable purity and good immunogenicity were detected respectively by SDS-PAGE and western blot analysis. The purified antibody was compared with a commercial kit to determine rh-EPO concentration in different steps of production batches via ELISA

Results: The purity of antibodies after ion exchange chromatography, obtained from caprylic acid and ammonium sulfate precipitation were 97 and 80%, respectively

Conclusions: As producing in house kits is one of the important challenges of bio- pharmaceutical manufacturers, a simple, cost- and time-effective, and easy to scale up strategy for making in-house polyclonal antibody was set up. Caprylic acid precipitation resulted higher purity than ammonium sulfate and finally purified antibody [97% purity] used as a capture antibody in sandwich ELISA test was able to detect erythropoietin antigen as sensitive [100%] and specific [100%] as commercial kits

Optimization of dynamic binding capacity of anion exchange chromatography media for recombinant erythropoietin purification

The dynamic binding capacity [DBC] of a chromatography matrix in protein purification is the amount of the total protein absorbed into the matrix, before occurrence of a significant break in the breakthrough curve. Optimization of the process criteria for maximum DBC avoids extra process scale-up and reduces the processing time, costs and protein loss. Taguchi method is a simple useful tool in experimental design to estimate the optimal condition with minimum experiments. In this research, linear flow rate, pH and protein concentration of the feed were checked according to an L9 orthogonal Taguchi array, to estimate the best conditions for maximum DBC of Q-sepharose fast flow [QSFF] resin in recombinant human erythropoietin purification process. A crud sample containing human recombinant erythropoietin was harvested from a cell culture of Chinese hamster ovary [CHO] cell line. Desalted harvests with different total protein concentrations [30, 40 and 50 microg.mL[-1]] and pH values [5, 6 and 7] were loaded into a packed column of QSFFwith different linear flow rates [60, 120 and 280 cm.h[-1]] up to 10% of the breakthrough curve. The total protein loading to the column was checked by UV absorbance and Lowry method, and erythropoietin concentration was measured by ELISA. Analysis of variance [ANOVA] was applied to determine the optimum condition. Finally, total protein concentration of 50 microg.mL[-1], pH of 5 and flow rate of 120 cm.h[-1], were anticipated as the optimal process conditions with 5.85 mg.mL[-1]of resin as the dynamic binding capacity. Experiments with anticipated optimal criteria were performed three times and no significant difference was observed [p = 0.136, and 6.06 mg/mL as the average dynamic binding capacity]

Increased the specificity and sensitivity of monospecific antibody against host cell protein [HCP] in quality control of hepatitis B recombinant vaccine

One of the most important aspects in recombinant biologic production, based on GMP rules, is the accuracy of final product quality control, especially assessment of host cell macromolecules contamination rate in final product. The purification requirement can be eliminated when the yeast cell containing the recombinant protein is used as a host cell. It is possibile that the final product contaminated to the host cell protein during purification stages of HBsAg [HBV vaccine]. The protein purification costs depend on the purification procedures required. Nowadays several companies produce commercial kits for identification and assessment of host cell protein contamination based on ELISA and Western blotting methods. But high prices, difference in sensitivity and lack of easy access to these kits sometimes create problems. So, in this study, two methods of Ammonium sulphate and caprilic acid precipitation technique were used separately for IgG purification. The results showed that IgG purification increased by 97% in caprylic acid method, compared with only a 77% increase in ammonium sulphate method. There were also significant differences in specificity and sensitivity between our standardized ELISA technique and using commercial kit [Cygnus CHO HCP]