Blood protein purification and simultaneous removal of nonenveloped viruses using tangential-flow preparative electrophoresis

Abstract

Gradiflow is new technology allowing purification of important blood proteins from viral contaminated plasma. Protein purification is based on unique scalable tangential-flow preparative electrophoresis, and is distinct from current technology because protein purification and virus removal are performed in the same step. This one-step removal and purification exploits both the size and charge of target proteins. The medically important blood proteins, immunoglobulin G (IgG) and alpha-1-antitrypsin, were chosen to demonstrate the ability of this process to purify proteins from contaminated plasma. Clearance factors achieved by infectivity assays and polymerase chain reaction (PCR) that meet regulatory requirements demonstrated removal of canine parvovirus (CPV). CPV is a model virus for pathogenic nonenveloped viruses, including parvovirus B19, not adequately removed or inactivated by most processes currently in practice. The recovery of proteins from plasma with high purity, recovery, and function, while simultaneously removing viruses, provides blood products with a level of purity compatible with clinical use more quickly and cheaply than available techniques.

Purification of intravenous immunoglobulin G from human plasma – aspects of yield and virus safety

“…The Gradiflow technology is a membrane-based preparative electrophoresis technique, which isolates proteins by exploiting the dual intrinsic characteristics of molecular weight and charge. IgG from human plasma was purified in 2 steps with a recovery of 93.5% [59] or even in 1 step [60].…”

“…Temperature (¡C) 60 70 80 90 Cp (cal/mole/¡C) Process step 1 from preparation A (A) showed a very wide intensity distribution, which suggested the presence of aggregates with many different molecular masses. By the following process step (B) only a light improvement of the sample composition concerning the aggregate pattern could be achieved.…”

Membrane Processes and Devices for Separation of Bioactive Peptides

“…Indeed, the large-scale prototype of Gradiflow was designed to process only some liters of solution. Furthermore, low flowrates (ranging from 20 to 600 mL/h) [64] as well as high voltage electric field are generally applied (ranging between 150 and 380 V/cm) [64,68,71] for carrying-out the gradiflow technology ; the flowrates limit the volume to be treated and the high electric field applied generates a heating of the solution by joule effect demanding a cooling of the separation chamber.…”

“Go no Go” in plasma fractionation in the world's emerging economies: Still a question asked 70 years after the COHN process was developed!

“…New technologies, however, are being evaluated in both developed and emerging economies that intend to provide either higher yields for current market drivers, higher flexibility to adjust to countries' needs, or ease of implementation [17]. They include fractionation processes based on multi-sequential purification processes using mimetic ligands [18], mixed mode, high-density, expanding bed adsorbents [19], large-scale preparative electrophoresis technology across membranes of controlled porosity [20], viral inactivation and mini-pool protein fractionation in closed-bag production systems [21,22], and aqueous twophase systems (ATPSs) using a mixture of polymer(s), salt, and water [23]. These novel technologies should be proven to meet current quality, consistency, and safety standards.…”