Thierry Burnouf

Modern Plasma Fractionation

Protein products fractionated from human plasma are an essential class of therapeutics used, often as the only available option, in the prevention, management, and treatment of life-threatening conditions resulting from trauma, congenital deficiencies, immunologic disorders, or infections. Modern plasma product production technology remains largely based on the ethanol fractionation process, but much has evolved in the last few years to improve product purity, to enhance the recovery of immunoglobulin G, and to isolate new plasma proteins, such as α1-protease inhibitor, von Willebrand factor, and protein C. Because of the human origin of the starting material and the pooling of 10000 to 50000 donations required for industrial processing, the major risk associated to plasma products is the transmission of blood-borne infectious agents. A complete set of measures—and, most particularly, the use of dedicated viral inactivation and removal treatments—has been implemented throughout the production chain of fractionated plasma products over the last 20 years to ensure optimal safety, in particular, and not exclusively, against HIV, hepatitis B virus, and hepatitis C virus. In this review, we summarize the practices of the modern plasma fractionation industry from the collection of the raw plasma material to the industrial manufacture of fractionated products. We describe the quality requirements of plasma for fractionation and the various treatments applied for the inactivation and removal of blood-borne infectious agents and provide examples of methods used for the purification of the various classes of plasma protein therapies. We also highlight aspects of the good manufacturing practices and the regulatory environment that govern the whole chain of production. In a regulated and professional environment, fractionated plasma products manufactured by modern processes are certainly among the lowest-risk therapeutic biological products in use today.

Affinity chromatography in the industrial purification of plasma proteins for therapeutic use

Affinity chromatography is a powerful technique for the purification of many proteins in human plasma. Applications cover the isolation of proteins for research purposes but also, to a large extent, for the production of therapeutic products. In industrial plasma fractionation, affinity chromatography has been found to be particularly advantageous for fine and rapid capture of plasma proteins from industrial plasma fractions pre-purified by ethanol fractionation or by ion-exchange chromatography. To date, affinity chromatography is being used in the production of various licensed therapeutic plasma products, such as the concentrates of Factor VIII, Factor IX, von Willebrand Factor, Protein C, Antithrombin III, and Factor XI. Most commonly used ligands are heparin, gelatin, murine antibodies, and, to a lesser extent, Cu(2+). Possible development of the use of affinity chromatography in industrial plasma fractionation should be associated to the current development of phage display and combinatorial chemistry. Both approaches may lead to the development of tailor-made synthetic ligands that would allow implementation of protein capture technology, providing improved productivity and yield for plasma products.

Chromatography in plasma fractionation: benefits and future trends

Industrial-scale chromatographic fractionation and purification methods have been used increasingly in the last few years for plasma fractionation. This has resulted in the development of a new generation of therapeutic plasma derivatives, especially coagulation factors, protease inhibitors and anticoagulants. Implementation and combination of ion-exchange, affinity and size-exclusion chromatography have allowed the development of new therapeutic products with improved purity and safety for treating congenital or acquired plasma protein deficiencies in patients. More recently, the benefit of chromatographic purification of plasma proteins in the removal of plasma-borne viruses has been revealed. Development of packing materials with improved characteristics for industrial applications, including higher capacity and rigidity, should further promote the use of chromatography as an essential plasma fractionation tool and confine more and more the traditional ethanol precipitation methods to the final processing stages used to recover albumin.

Biochemical and physical properties of a solvent-detergent-treated fibrin glue

Abstract. A fibrin glue preparation has been obtained from pooled human plasma using a procedure which includes a solvent‐detergent (SD) treatment to inactivate lipid‐enveloped viruses. The SD treatment inactivated ≥ 5.5 log10 of HIV in less than 45 min, and ≥5 log10 and ≥6.5 log10 of VSV and Sindbis virus, respectively, in less than 2 h. The product was found to contain high quantities of fibrinogen (116±2.49 g/l; n = 12), factor XIII (35±2.88 U/ml) and von Willebrand factor (23±1.9 U/ml ristocetin cofactor activity), and relatively low levels of fibronectin (5.9±0.51 g/l). Plasminogen, the precursor of plasmin, which may play a negative role by decreasing the resistance of the fibrin clot, was at only 0.03 g/l. Cellulose acetate electrophoresis showed 95% gamma‐proteins and 5% alpha‐2‐beta proteins. Sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions detected three main protein bands with apparent molecular weights of 65, 56 and 47 kilodaltons, probably corresponding to the alpha, beta, and gamma fibrinogen subunits. Other characteristics of the product included (1) high clottability of fibrinogen (over 85%); (2) absence of low molecular weight fibrin degradation products; (3) rapid solubilization at room temperature (less than 10 min); (4) high tensile strength (202±27 g/cm2 after 2 h of application), and (5) high elasticity of the fibrin clot. In addition, scanning electron microscopy revealed a highly organized structure showing tridimensional arrangement of the fibrin fibers. SD treated fibrin glue should efficiently replace autologous fibrinogen or cryoprecipitate preparations for surgical application.

Platelet-cancer interactions.

Platelets play a crucial role in the pathophysiological processes of hemostasis and thrombosis. Increasing evidence indicates that they fulfill much broader roles in balancing health and disease. The presence of tumor cells affects platelets both numerically, through a wide variety of mediators and cytokines, or functionally through tumor cell-induced platelet activation, the first step toward cancer-induced thrombosis. This induction results from signaling events through the different platelet receptors, or may be cytokine-mediated. Reciprocally, upon activation, the platelets will release a myriad of growth factors from their dense and α-granules and peroxisomes; these will directly impact tumor growth, tethering, and spread. A similar cross-talk is initiated between tumor microvesicles stimulating the platelets and platelet microparticles, promoting both thrombosis and tumor growth. A vicious loop of activation thereafter takes place. Platelets directly and indirectly promote tumor growth, and enable a molecular mimicry coating the malignant growth and allowing metastasizing cells to escape T-cell-mediated immunity and natural killer cell surveillance. Breaking this vicious activation loop with nonspecific platelet inhibitors, such as aspirin, or by targeting specific sites on the activation cascade may offer a mean to reduce both the risks of development and progression of cancer and the risk of thrombosis.

The role of microparticles in inflammation and transfusion: A concise review

Microparticles are small membrane-bound vesicles found in body fluids including peripheral blood. Microparticles are an intrinsic part of blood labile products delivered to transfused patients and have active roles in inflammation. They are delimited by a lipid bilayer composed mainly of phospholipids, cholesterol, membrane-associated proteins, intracellular components such as metabolic enzymes, proteins-involved in adhesion and fusion, cytoskeletal-associated proteins, surface glycoproteins and/or chemokines. Microparticles can trigger a pro-inflammatory message to neighbouring or target cells. Microparticles originating from platelets, leukocytes, erythrocytes, and endothelial cells are associated with a variety of pathophysiological conditions. This review summarises the role of Microparticles in modulating inflammation.

Plasma fractionation issues

Procurement and processing of human plasma for fractionation of therapeutic proteins or biological medicines used in clinical practice is a multi-billion dollar international trade. Together the private sector and public sector (non-profit) provide large amounts of safe and effective therapeutic plasma proteins needed worldwide. The principal therapeutic proteins produced by the dichotomous industry include gamma globulins or immunoglobulins (including pathogen-specific hyperimmune globulins, such as hepatitis B immune globulins) albumin, factor VIII and Factor IX concentrates. Viral inactivation, principally by solvent detergent and other processes, has proven highly effective in preventing transmission of enveloped viruses, viz. HBV, HIV, and HCV.

Recombinant plasma proteins

For almost 50 years, the fractionation of human plasma has been the sole possible source of a wide range of therapeutic proteins–such as coagulation factors, anticoagulants, immunoglobulins, and albumin – essential to the treatment of serious congenital or acquired bleeding or immunological diseases. In the last 20 years, the application of recombinant technologies to mammalian cell cultures has made possible – although with some limitations in productivity, costs, and immunogenic risks – to produce and commercialize complex plasma glycoproteins for human therapeutic applications and has opened the way to the development of new molecular entities. Today, the advanced exploration of alternative cell lines and enhanced cell culture systems, as well as the development of alternative expression technologies, such as transgenic animals, is opening a new era in the production of the full range of recombinant plasma protein therapeutics. In this review, we examine the achievements and ongoing challenges of the recombinant DNA technology as a platform for the production of plasma proteins and the advantages and limitations of such products compared to fractionated plasma proteins.

Nanofiltration of single plasma donations: Feasibility study

Background and Objectives Major technical developments have been made in recent years to improve the quality and safety of human plasma for transfusion and fractionation. The present study was performed to assess, for the first time, the feasibility of applying a nanofiltration process, using 75‐nm and 35‐nm mean pore size membranes (Planova® 75N and Planova® 35N), to human plasma.

Application of bioaffinity technology in therapeutic extracorporeal plasmapheresis and large-scale fractionation of human plasma

This paper describes the increasingly unique and powerful role that affinity chromatography is occupying both as a tool for the treatment of extracorporeal plasma exchange (to discard biological compounds with noxious metabolic or immunologic effects in patients) and as a purification tool in the production of therapeutic plasma protein derivatives. Management of both applications requires careful monitoring of the parameters applied to the plasma material, to avoid immunological stimulation or activation of the coagulation cascade. Examples of direct current applications of affinity ligands in therapeutic removal and industrial production of plasma compounds are presented.