Polyclonal Antibody Purification
While recombinant monoclonal antibodies have dominated the biotherapeutics market in recent years, there still is a place for conventional polyclonal reagents. Protherics (www.protherics.com) is exploiting this technology, addressing an old problem with a new strategy. Over the years sepsis treatment has been a black hole for biotech companies, with over 70 Phase II and III trials failing when monoclonal antibodies were targeted against putative cytokine mediators.
However, according to Richard Francis, Ph.D., of the process development group at Protherics, a polyclonal ovine anti-TNF produced a statistically significant response in sepsis patients in a trial conducted 10 years ago. The product, CytoFab, is part of the Protherics R&D portfolio and further clinical trials are planned for 2007.
These results placed demands on Dr. Francis’ team for a robust purification protocol that worked from a holistic point of view, in manner similar to that employed by Dr. Sardonini. This resulted in evaluation of a 1.8 M diameter column, whose behemoth size was dictated by the vast amounts of antibody (running to tens of kilograms) obtained from an Australian sheep herd immunized with a-TNF. Average amounts of antibody hovered around 5–6 g/L in serum. Since Australia is free of Mad Cow disease, this concern is obviated.
The purification modeling involved small initial volumes, which were gradually increased. Dr. Francis emphasizes that the scaleup process required close cooperation with the FDA, as many minor changes were required during its development. This flexibility allowed a large reduction in cost and better consistency in batches as the protocol moved forward.
The Cytofab polyclonals provide rapid neutralization of TNF, according to Dr. Francis, an outcome not achieved in years past through the use of monoclonal antibodies, which may explain their superior performance over Remicade. There is a substantial interest in recombinant polyclonals, however, Dr. Francis believes that the Cytofab technology at this stage is more cost effective.
Because of the cost of gargantuan quantities of purification reagents, such as Proteins A, L, and G, for such a project, Dr. Francis’ team elected to use MABsorbent® A2P (Prometic Biosciences; www.prometic.com) as a feasible alternative. The compound is composed of a di-substitued phenolic derivative of tri-chlorotriazine and is commercially available coupled to a 6% cross-linked agarose base matrix. The ligand is thought to mimic the structure of two critical amino acids side chains of Protein A, essential for the complexing of Protein A to the Fc region of the IgG molecule.
Dr. Francis’ team found that the material could be reused over 100 cycles with no drop in IgG purity. “It is important to consider GMP when developing a purification process and A2P provides a feasible alternative for large-scale antibody fragment purification.”
“The increasing demand for proteins such as antibodies has led to rapid expansion of global manufacturing capacity, increased reactor size (up to 20,000 L), and a drive for improved process efficiency to reduce manufacturing costs,” according to John Birch, Ph.D., of Lonza Biologics (www.lonza.com). “We are concerned with expression technology and process optimization, especially the development of fed-batch cultures.”