New method to produce equine antirabies immunoglobulin F(ab′)2 fragments from crude plasma in high quality and yield

Rabies is still a major cause of human deaths in several developing countries. According to the World Health Organization, administration of antirabies serum or antirabies immunoglobulin is recommended for patients who have experienced a category-III exposure to rabies. Improvement of antirabies immunoglobulin production is required to enhance safety and efficacy of the products. In this paper, a new method to produce equine antirabies immunoglobulin F(ab′)2 fragments from crude plasma is proposed. First, protein G affinity chromatography was used to purify IgG from equine plasma. Moreover, purification of IgG was shown to facilitate its digestion by pepsin. Compared to the direct digestion of crude plasma, a lower amount of pepsin and a shorter digestion time were required to completely digest the purified IgG to F(ab′)2. Complete digestion of purified IgG to F(ab′)2 was achieved at a pepsin/IgG (w/w) ratio of 5:45 with preservation of structure and potency. Finally, purification of F(ab′)2 was accomplished by a combination of protein A affinity chromatography and ultrafiltration with a 50-kDa nominal molecular weight cut-off membrane. The new process resulted in 68.9 ± 0.6 (%) total recovery of F(ab′)2 and a F(ab′)2 product of high potency.

Graphical abstractA new method consisting of protein G affinity chromatography, pepsin digestion, followed by a combination of protein A affinity chromatography and ultrafiltration, was proposed to produce equine antirabies immunoglobulin F(ab′)2 fragments in higher yield and bioactivity than the current WHO method.Figure optionsDownload full-size imageDownload as PowerPoint slide