Nanofiltration as an alternative bioprocessing platform offers the possibility of cost reduction while achieving effective purification of plasma proteins, reported Merche Faro, Ph.D., of Grifols. Dr. Faro discussed her group’s experiences with nanofiltration-based purification of fibrinogen and intravenous immune globulin, which is used to treat immune disorders and boost the immune response.
According to Dr. Faro, therapeutic products obtained from biological sources pose a particular challenge, as they must be demonstrated to be free of pathogenic agents, both as raw materials and as final products.
Furthermore, regulatory governance bodies require viral safety steps, based on inactivation and removal platforms. Inactivation options include solvents and treatment with caprylate, dry heat, and pasteurization. The removal step detailed by Dr. Faro is achieved by nanofiltration through small (15−50 nm) pore-size membranes.
“This is a robust, reliable, and flexible clearance technology,” stated Dr. Faro, “It permits efficient removal of both enveloped and nonenveloped viruses, and we have achieved high protein recovery with no undesirable effects on the product.”
The company has included a nanotechnology process in its purification train for over 15 years, to a point where it is now a routine step. Proteins filtered through the small virus retentive filter membranes include thrombin, Factor IX, and a number of other candidates.
Dr. Faro explained that each application must be individually tailored for that protein. “One must take into account the features of that individual protein and the manufacturing process to which it will be subjected,” she pointed out.
Not only is the molecular weight of the protein critical, but stability, isoelectric point, and the concentration of the protein solution must all be factored into the protocol. These properties will affect the optimization of the filtration medium and decisions regarding additional polishing steps. In choosing and monitoring membrane performance, membrane structure and filtration conditions will critically impact recovery yield and thereby the cost of goods.
Dr. Faro further discussed her experiences with nanofiltration of the fibrinogen molecule. This 340 KD glycoprotein is a critical component of the coagulation cascade and is widely used as a supplement in surgical procedures. It is structurally complex, containing two sets of three different polypeptide chains.
For this reason it must be handled with care, and Dr. Faro’s team incorporated gentle filtration conditions into their protocol, including the addition of stabilizers to the filtration medium. Addition of a freeze/thaw step greatly improved recovery by removing aggregates that tend to block the filter. This optimized process could be expanded to industrial scale with excellent performance, she said.
The second application of the nanofiltration technology discussed by Dr. Faro was to intravenous immune globulin purification, a major protein found in high concentrations in plasma. It possesses a wide range of critical therapeutic applications, such as treatment of immune deficiencies and autoimmune diseases.
Dr. Faro and her colleagues measured a number of parameters, including different membrane pore sizes and protein concentrations of the filtration solutions. The 20 nm pore size with a 2−3% protein concentration proved to be optimal. Filter size and brand were also important variables that were considered in the optimization process.
“We found that a dedicated optimization protocol is required in the development of a nanofiltration-based platform for each protein,” said Dr. Faro. “Critical aspects that allow optimal nanofiltration performance are unique for each protein, manufacturing process and nanofilter. Once successfully achieved, we observe robust and consistent virus retention capacity under a wide range of process conditions.”