Comparative evaluation of purification strategies for anti-rabies immunoglobulins: Impact on yield, functional activity, and stability

The study aimed to evaluate the effectiveness of different purification methods for anti-rabies immunoglobulins obtained from the plasma of hyperimmunised cattle. The study included a total of 30 animals, randomly divided into three groups based on the purification method: ammonium sulphate precipitation, affinity chromatography on a Protein G column, and a combined method with the addition of ultrafiltration. The purification methods differed in their ability to preserve protein yield, specific antibody activity, and virus-neutralising capacity. The combined protocol produced the highest final protein concentration; however, total protein was interpreted only as an indicator of recovery and concentration efficiency, not as evidence of IgG purity. The superiority of the combined method was supported by the parallel increase in anti-rabies IgG reactivity and virus-neutralising activity. In contrast, the method of precipitation with ammonium sulphate revealed the lowest values for each of these parameters, indicating the lower selectivity of this method and the loss of the functionally active immunoglobulins. However, the results obtained with the precipitation method, Protein G affinity chromatography and ultrafiltration suggest that these methods increase both the amount of anti-rabies immunoglobulin that is obtained and the quality of that immunoglobulin. The method of precipitation with ammonium sulphate and the storage of the obtained immunoglobulin without the step of freezing revealed the greatest decrease in each of the indicators over the 60-day period. However, the method that employed the use of all three steps revealed the best results in the preservation of the protein, specific antibodies, and virus- neutralising activity, with only a 4% loss of each of these initial values after storage at -20°C. Statistical analysis using repeated measures ANOVA confirmed the significant influence of the purification method and storage conditions on all the parameters studied (p < 0.05). In conclusion, the integration of precipitation, affinity chromatography, and ultrafiltration resulted in a better yield, functional activity, and stability of anti-rabies immunoglobulins. The practical significance of the present findings lies in the possibility of applying them in the development, standardisation, and production of highly effective immunoglobulin preparations for the prevention and treatment of rabies in veterinary and medical practice.