A  Quick Look at Human Plasma Fractionation
               The fractionation of human blood plasma came into its own as a manufacturing process with the leadership of Professor E.J Cohn during the World War II (1) and over the past sixty years evolved into an international business (2).   The current plasma fractionation processes uses the Cohn fractionation process (3,4) which uses the combined effect of pH, ionic strength, and organic solvent (solvent polarity) on the solubility of proteins (5,6) to obtain several fractions (Table 1; see also Figure 14 in reference 6).   The effect of alcohol on protein solubility had been known for some time as has the effect of ionic strength on the effect of alcohol (7).  Cohn was trained as a physical chemist at the University of Chicago and was a member of the Department of Physical Chemistry at Harvard Medical School for many years.  During his long tenure at Harvard, he worked with other outstanding scientists such as John Edsall (6) and George Scatchard (8).   It is fair to say that John Edsall, in addition to serving as the editor of The Journal of Biological Chemistry for several decades, provided a solid platform for the study of the physical biochemistry of proteins and anyone who has ever done a binding experiment is familiar with the Scatchard plot.   Cohn's early work with E.J Henderson concerned the physical chemistry of seawater (9) and later on the physical chemistry of bread (10). This latter work was done in collaboration with the Department of Defense and Professor Cohn was Lieutenant Cohn.  While Cohn was interested in other problems from 1920 into the middle 1930's, he never lost his primary interest in proteins (11-15) and was  well positioned to work with plasma fractionation during World War II.1  The original Cohn process has gone through modification over the years and alternative methods of fractionation have been developed (16).   Kistler and Friedl (17)  have summarized the technical and economic advantages of ethanol fractionation.   The original Cohn  process has been modified by the various manufacturers such that a cryoprecipitate fraction (18,19)  is removed and a Fraction I may or may not be obtained as a separate fraction.  Fraction II and Fraction III may be obtained in a single step; Fraction I,II, and III may be combined as a single step to yield a Fraction I-II-III.   Fraction IV may be obtained as a Fraction IV-1 and Fraction IV-4.  Fraction V is mostly albumin.    Fraction VI derived from the supernatant fraction from the Fraction V step.   Fraction VI has been poorly characterized and there are only a few papers (20-23).  Plasma protein fraction is a plasma fraction similar to albumin (24-26).
                The processing of plasma into various products is not dissimilar to the fractionation of crude oil into various products.  This analogy was nicely developed by Burnouf in a discussion of the economics of plasma fractionation (27).  As with the refining of oil, fractions are removed from plasma and subjected to further processing.   Cryoprecipitate is the source of fibrinogen, fibronectin,  factor VIII, and von Willebrand factor while  Fraction IV provides antithrombin, the vitamin K-dependent factors, and a1-antitrypsin.  This is similar to the refining process of oil where various derivative fractions such as subfractions such  propane and butane are used to make fuel gas, light naptha for gasoline, gas oils for lubricants, and residue for asphalt (28,29) .   Curran (29) separates the petroleum industry into four separate techology and business areas; extraction of crude oil, transportation of oil to refinery, the refinery process, and marketing.  Similar segmentation occurs in the plasma industry with donor centers, refrigerated transport to a manufacturing process, the fractionation process, and distribution and sales.  
               The Cohn fractionation process was developed for the production of albumin although the value of other proteins, most notably immune serum globulins and fibrin foam/fibrin film together with thrombin, were considered to be of value in 1945 (30).  The plasma fractionation industry slowed for a period of time after World War II with albumin being the dominant product (31,32).   Plasma protein fraction (PPF) was developed in the 1950's as a supplement to albumin (33).  There was limited work on the development of factor VIII concentrates (34) as well as immunoglobulin for intramuscular injection (35,36).    Albumin continued to be the economic driver for the fractionation of plasma until the 1960's when the development of intermediate-purity factor VIII concentrates (37) established hemophilia treatment as the economic driver for plasma fractionation.  The subsequent development of recombinant factor VIII preparations in the 1980's combined with the recognition of the value of intravenous immunoglobulin in immune modulation resulted this product replaced factor VIII as the economic driver for the plasma fractionation business (2). Other products derived from plasmas which were developed in the 1970's included the prothrombin complex concentrates which have been largely replaced by single-factor IX concentrates either from plasma or recombinant sources.  Activated prothrombin complex concentrates while still in use have been replaced by recombinant factor VIIa products which are also being used for factor VII replacement in vitamin K-antagonist intoxication and liver disease.  As an aside, it is the author's sense that there are a large number of commercial entities seeking to capitalize on the use of factor VIIa as a general hemostatic agent quite separate from the above uses.
               The end of World War II resulted in the dismantling of the government infrastructure established for plasma fractionation during  the conflict (31).   The private sector companies which had been involved in the fractionation of plasma during this time period continued to function and over the past 50 years, a number of private companies have been involved with commercial plasma fractionation including CSL in Australia, Grifols (Probitas), Immuno, Octapharm, Sanguin, and Behring in Europe, and Hyland, Cutter, Armour, and others in the United States.   There are also national fractionation efforts aimed at assuring self-sufficiency for blood and blood products (38-40). 
               Manufacture of biological products from human blood plasma has always been a challenging proposition,  First, there is the supply of raw material.  Blood plasma for fractionation is usually obtained from commercial sources using paid donors.  This occurs at donor centers which are either owned by the fractionator or a another company dedicated to plasma collection.    Plasma, as raw material, contributes about 50% of the cost of the manufacturing process (2).  The HIV tragedy of the 1980's and other viral issues have presented issues with blood-derived therapeutics resulting in increased emphasis on product derived by use of recombinant DNA technology (41,42).   A substantial portion is analytical costs including nucleic-based assays for virus testing as part of an overall strategy to assure viral safety.   It is noted that the methodology for viral testing continues to see development (43)  and it is possible that with multiplexed methods will reduce  the cost of testing and increase viral safety.   The current (2009) risk of infection from HCV or HIV is estimated at 1 in 1,000,000 for a blood unit and approximately 1 in 500,000 for HBV (44).  Now these are single unit odds so such cannot be directly applied to the risk with chronic use of a biological such as with factor VIII for hemophilia A but might be reasonable for an acute use product such as antithrombin.   For a better sense of risk, I commend the reader to a book (45)  by James Walsh which discusses how risk affects everyday life.  This book was published in 1998 and, as such, odds will have changed and the following are presented to put the above odds in perspective.  First, the lifetime odds of being struck by lighting is 1 in 30,000, death by excessive alcohol consumption, 1 in 100, while death by motor accident is 1 in 60.   I will grant you that these numbers are not directly comparable to the blood safety numbers but they are useful in driving analysis of risk rather than coping with the fog of uncertainty by adding some perspective.   The increased appreciation of zoonotic disease emphasizes the importance of the unknown pathogen (46-49).  Risk from the transfusion of blood is known (see above) and risk from purified protein fractions from blood is less than that for whole blood (50-56)  with removal by various processing steps demonstrated by various investigators (57-62).  It is not unreasonable to suggest, at least for the sake of argument, risk should be balanced with cost of product.  The question then becomes how much risk will society accept at what cost.  As an example, recombinant factor VIII products are available at a substantial premium to plasma-derived factor VIII with essentially equivalent therapeutic equivalence .  Mantovani and colleagues (63) presented an excellent study on the complex nature of treatment choice in hemophilia showing the combined  importance of viral safety, inhibitor development, and infusion frequency.  These investigators noted that product choice when there is cost-discrepancy between therapeutically-equivalent products is increasingly importance when resources are limited.  Other investigators have also provided insight to this issue with specific reference to the treatment of hemophilia A (64).  Outcome analysis is used for other therapeutic approaches in determining value (65) and it is clear that regardless of geography and reimbursement processes, resource allocation will be an increasing problems in health care (66).   I would be remiss if I did not mention the current issue between recombinant human thrombin and bovine-plasma-derived thrombin when there is strong effort by supporters of bovine thrombin to preserve market share for the bovine product (67).   Here the issue is the development of antibodies previously observed with the bovine product  with little thought given to potential pathogens derived from animal plasma.   The author is on record (68)  as recommending the use of plasma-derived human thrombin.
                Robert (69) argues that plasma fractionation will increase at a  greater rate if intravenous immunoglobulin is approved for use in Alzheimer's Disease (70).  Regardless of potential use in Alzheimer's Disease, there will be an increase in use of intravenous immunoglobulin for immunomodulation as rationale is provided for the various indications (71).   There will also be an increase in use for infectious disease.  As an example, it is clear that donor plasma could  be selected for action against specific pathogens (72,73)  suggesting that donor plasmas could be preselected for the presence of antibodies of value in specific infectious disease  as it is likely that a maximum therapeutic effect  will be obtained from a polyclonal antibody preparation.    In principle, the repertoire of immunoglobulin within a population should mirror pathogen challenges in the local environment and, therefore, represent an immediate therapeutic for an emerging pathogen.   As an aside, it is of some interest that, considering the studies on the presence of antibodies to measles virus in intravenous immunoglobulin cited above (72),  that one of the first application of human immune serum globulin was in the treatment of measles (74).
               Judged by today's biotechnology industry, the methods used for plasma fractionation seem quite primitive.   However, it must be recognized that the Cohn fractionation procedure was developed well in advance of the various separation technologies currently available for commercial biotechnology in the 21st century.  Perspective may be obtained by considering reviews by Taylor in 1953 (75)  and a bit later by Pennell in 1960 (76).  Taylor reviews the state-of-the-art of protein purification at midpoint of the 20th century.   Techniques available for protein fractionation prior to approximately 1955 were based on differential solubility, physical methods such as ultracentrifugation, preparative electrophoresis (free boundary), and adsorption/elution from insoluble salts as well as the use of partition chromatography. Partition chromatography was developed by Gordon and coworkers (77)in 1943 to separate amino acid and found application for proteins in work by Martin and Porter in 1951 (78).  Adsorption chromatography for proteins on silica gel was reported by Shepard and Tiselius in 1949 (79).  The latter paper (79)  is prescient of hydrophobic interaction chromatography.   Thus, the use of chromatography was in the earliest stage of development when Cohn and colleagues developed the alcohol fractionation scheme and it would be some 40 years before Michael Griffith and colleagues in the Hyland Division of Baxter Healthcare applied immunoaffinity chromatography to the purification of plasma factor VIII (80)  and by other groups for albumin and IgG (81-84).   To the best of my knowledge, although various chromatographic "trains" were discussed for plasma fractionation, Factor VIII was the only plasma-derived product which utilized chromatography in a GMP manufacturing process  until more recent application to intravenous immunoglobulin (85, 86)  and albumin (87).   Lihme and colleagues (88)  have recently advanced a fractionation for plasma using expanded bed adsorption chromatography.
               Four issues impinge on the future of plasma-derived biopharmaceutical products.   First, the emergence of another "HIV-like" pathogen would be devastating for the industry limiting the market to only absolutely unique biopharmaceuticals such as intravenous immunoglobulin.  Experience over the past two decades would suggest that a combination of donor screening and a rigorous manufacturing process can provide adequate risk reduction (50).     Second, new indications for existing products such as the use of intravenous immunoglobulin in Alzheimer disease (70,89).   Thirdly, the is the potential for the development of new products (2.90).   This could be done with change to the basic Cohn fractionation process thus eliminating any influence on the licensure of existing products.   A recent example is the development of a1-antitrypsin from Cohn fraction IV (91,92).   Where there is change in process that might influence a downstream product, for example a change in processing of Cohn Fraction I-III to maximize intravenous immunoglobulin yields might influence the Cohn IV process,  advanced characterization technologies (93)  combined with insight gained through considerations of biosimilars (94,95)  and Quality-by-Design (QbD)(96)  should facilitate the approval of changes.   Fourth is the potential for the establishment of new markets for existing products.   Curling and Bryant, in their review of the plasma fractionation industry in 2005 (2), observed that developing economies represent an underserved market for all biopharmaceutical including plasma-derived biological products.    The use of plasma-derived biologicals in developing economies raises the perennial issue of self-sufficiency of resources (97-100).    Blood could be collected in the local geography and either processed locally or processed in another geography using contract manufacturing.  The reader is referred to an article by Farrugia (39) which discusses the international movement of plasma and contract manufacturing. 
               It can be argued that "local" plasma is invaluable in reflecting local immune experience (101,102)  which would then be critical in guarding against local pathogens (103) such as H1N1 virus (104,105).  Not all geographies have the same quality of plasma collection (106)  and there may be different standards for the processing and storage of human plasma (38).  The are significant economic issues in considering local versus imported materials (39).   For example, in Brazil, the cost of imported plasma protein products for 2006 was approximately $(US) 300,000 of which half was for intravenous immunoglobulin.  It may well be useful for a country such as Brazil consider developing a local fractionation industry.  The primary products could intravenous immunoglobulin and albumin.  In that case, a manufacturing process could consist a a cryoprecipitate step followed by a column chromatography scheme such as introduced by Limhe and coworkers (88).   The cryoprecipitate could be exported for contract manufacture of factor VIII and fibrinogen.   Since the immunoglobulin and albumin would be manufactured for internal or regional consumption, it is possible that an expedited regulatory path could developed.   It noted that Brazil has a local company (Hemobrás) (107)  developing a fractionation facility (39).
1.  Blood Program in World War II, ed. G.B. Kendrick, Superintendent of Documents, U.S. Government  Printing Office, Washington, DC, 1964.
2. Curling, J. and Bryant, C., The plasma fractionation industry. New opportunities to move forward?, Bioprocess International, March, pps. 18-27, 2005.
3. Cohn, E.J., Strong, L.E., Hughes, W.L., Jr., et al., Preparation and properties of serum and plasma proteins. IV. A system for the separation into fractions of the protein and lipoprotein components of biological tissues and fluids, J.Am.Chem.Soc. 68, 459-475, 1946.
4. Cohn, E.J., The separation of blood into fractions of therapeutic value, Ann.Int.Med. 26, 341-352, 1947.
5.  Edsall, J. T., The development of the physical chemistry of proteins, 1898-1940, Ann.N.Y.Acad.Sci. 325, 52-74, 1979
6. Hughes, W.L., Interstitual proteins: The proteins of blood plasma and lymph, in The Proteins, ed. H.Neurath and K. Bailey, Volume II, Part B., Chapter 21, pps. 663-754, Academic Press, New York, New York, USA, 1953.
7. Loeb, J., Proteins and The Theory of Colloidal Behavior,  Chapter XX, McGraw-Hill, New York, New York, USA, 1924.
8.  Scatchard, G., Edwin J. Cohn and protein chemistry, Vox Sang. 17, 37-44, 1969.
9.  Henderson, L.J. and Cohn, L.J., The equilibrium between acids and bases in sea water, Proc.Natl.Acad.Sci.USA 2, 618-622, 1916.
10. Cohn, E.J. and Henderson, L.J., The physical chemistry of bread making, Science  48, 501-505, 1918. 
11. Cohn, E.J., Study on the physical chemistry of proteins I. The solubility of certain proteins at their isoelectric point, J.Gen.Physiol. 4, 697-722, 1922.
12. Cohn, E.J., McMeekin, T.L., Edsall, J.T., and Weare, J.H., Studies on the physical chemistry of amino acids, peptides and related substances II. The solubility of a-amino acids in water and alcohol-water mixtures, J.Am.Chem.Soc. 56, 2270-2282, 1934
13. Cohn, E.J., McMeekin, T.L., Ferry, J.D., and Blanchard, M.H., The physical chemistry of amino acids, peptides, and related substances. XII. Interaction between dipolar ions in aqueous solutions, J.Phys.Chem. 43, 169-188, 1939.
14. Cohn, E.J., McMeekin, T.L., Oncley, J.L., et al., Preparation and properties of serum and plasma proteins. I. Size and charge of proteins separating  upon equilibrium across membranes with ammonium sulfate solutions of controlled pH, ionic strength, and temperature,  J.Am.Chem.Soc. 62, 3386-3398, 1940.
15. Cohn, E.J., Luetscher, J.A., Jr., Oncley, J.L., et al., Preparation and properties of serum and plasma proteins. III. Size and charge of proteins separating on equilibrium across membranes with ethanol-water mixtures of controlled pH, ionic strength and temperature, J.Am.Chem.Soc. 62, 3396-3400, 1960.
16. Methods of Plasma Fractionation, ed J.M. Curling, Academic Press, London, United Kingdom, 1980.
17. Kistler, P. and Friedl, H., Ethanol precipitation,  in Methods of Plasma Fractionation, ed. J.M. Curling, Chapter 1, pps. 1-15, Academic Press, London, United Kingdom, 1980.
18. Tanaka, K., Shigueoka, E.M., Sawatani, E., et al., Purification of human albumin by the combination of the method of Cohn with liquid chromatography, Brazilian J.Med.Biomed.Res. 31,1383-1388, 1998.
19. Brown, P., Rohwer, R.G., Dunstan, B.C., The distribution of infectivity in blood components  and plasma derivatives in experimental models of transmissable spongiform encephalopathy, Transfusion 38, 810-818, 1998.
20.  Covaci, A., Laub, R., Di Giambattista, M., et al., Polychlorinated biphenyls and organochlorine pesticides are eliminated from therapeutic factor VIII and immunoglobulin concentrates and reduced in albumin by plasma fractionation,  Organohalogen Compounds 52, 172-175, 2001.
21.    Burgi, W. and Schmid, K., Preparation and properties of zinc-a2-glycoprotein of normal human plasma, J.Biol.Chem. 236, 1066-1074, 1961.
22.  Polet, H. and Spieker-Polet, H., Mechanism of the growth-promoting effect of serum albumin on concanavalin A- activated lymphocytes: protective effect of the plasma proteins, J.Immunol. 117, 1275-1281, 1976.
23.  Papp, A.C., Hai, E.R., and Wu, K.K., Binding of prostacyclin by plasma glycoproteins, Prostaglandins 30, 1057-1068, 1985.
24.  Hink, J.H., Jr., Pappenhagen, A.R., Lundblad, J., and Johnson, F.F., Plasma protein fraction (human). Physical and clinical properties after storage for 7-8 years, Vox. Sang. 18, 527-541, 1970.
25. Vogelaar, E.F., Brummelhuis, H.G., Beentjes, S.P., and Krijnen, H.W., Contributions to the optimal use of human blood.  I. Analysis and optimalization of the production of plasma protein fraction (PPF),  Vox. Sang. 23, 481-492, 1972.
26. Lane, R.S. and Vallet, L., Human albumin and plasma protein fraction, Lancet  323 (8388), 1245-1246, 1984.
27.  Burnouf, T., Plasma proteins: Unique biopharmaceuticals - Unique economics, Pharmaceutical Policy & Law 7, 209-218, 2005-2006.
28. Lynk, E.L., On the economics of the oil refining industry in the United Kingdom, Appld.Economics 18, 113-126, 1986.
29. Curran, L.M., Waste minimization procedures in the petroleum industry, J.Hazardous Mat. 29, 189-197, 1992.
30. Anon, Byproducts of plasma fractionation. General consideration, in Blood Programs in World War II, ed. G.B. Kendrick, Chapter 13, pps. 350-369,  Superintendent of Documents, U.S. Government  Printing Office, Washington, DC, 1964.
31. Palmer, J.W., The evolution of large-scale human plasma fractionation in the United States, in Proceedings of the Workshop on Albumin, February 12-13, 1975, DHEW Publication No. (NIH) 76-925. pps. 255-268, U.S.Government Printing Office, Washington, DC, USA, 1975.
32. Finlayson, J.S., Therapeutic plasma fractions and plasma fractionation, Sem.Thromb.Hemost. 6, 1-11, 1979
33.  Hink,J.H., Jr., Hidalgo, J., Seeberg, V.P., and Johnson, F.F., Preparation and properties of heat-treated plasma protein fraction, Vox.Sang. 2, 174-178, 1957.
34.  Soulier, J.-P., Gobbi, F., and  Larrieu, M.J., Séparation du fibrinogène et du facteur antihemophilique A, Rev.Hémat. 2, 481-496, 1957.
35. Bodian, D., Experimental studies on passive immunization against poliomyelitis. I. Protection with human gamma globulin against intramuscular inoculation, and combined passive and active immunization, Am.J.Hyg. 54, 132-143, 1951.
36.Janaway, C.A., Rosen, E.S., Merler, E., and Alper, C.A., The Gamma Globulins, Little, Brown, Boston, Massachusetts, 1966.
37. Sharrer, I. and Becker, T., Products used to treat hemophilia: evolution of treatment for hemophilia A and B, in Textbook of Hemophilia, ed. C.A. Lee, E.E. Berntorp, and W.K. Hoots, Blackwell, Malden, Massachusetts, USA, 2005.
38.  Farrugia, A., International movement of plasma and plasma contracting, Dev.Biol.(Basal), 120, 85-96, 2005.
39. Farrugia, A., Evers, T., Falcon, P.-F., et al., Plasma fractionation issues, Biologicals 37, 88-93, 2009.
40.  Cheraghali, A.M. and Abolghasemi, H., Improving availability and affordability of plasma-derived medicines, Biologicals  38, 81-86, 2010.
41.   Hoots, W.K., History of plasma-product safety, Transfus.Med.Rev. 15 (2 suppl 1), 3-10, 2001.
42. Key, N.S. and Negrier, C., Coagulation factor concentrates: past, present, and future, Lancer 370, 439-448, 2007
43.  Fournier-Wirth, C., Jaffrezic-Renault, N., and Coste, J., Detection of blood-transmissible agents: can screening be miniaturized?, Transfusion 50, 2032-2045, 2010.
44.  Dodd, R.,Managing the microbiological safety of blood for transfusion: a US perspective, Future Microbiol. 4, 807-818, 2009.
45.  Walsh, J., The Odds, Silver Lake Publishing, Los Angeles, California, USA, 1998.
46.  Tang, J.W., Shetty, N., Lam, T.T., et al., Emerging, novel, and known influenza virus infections in humans, Infect.Dis.Clin.North Am. 24, 603-617, 2010.
47.  Roess, A.A., Galan, A., Kitces, E., et al., Novel deer-associated parapoxvirus infection in deer hunters, New Engl. 363, 2621-2627, 2010.
48.  Leiby,D.A., Transfusion-transmitted Babesia spp.: Bull's-eye on Babesia microti, Clin.Microbiol.Rev. 24, 14-28, 2011.
49. Altizer, S.,Bartel, R., and Han,B .A., Animal migration and infectious disease risk, Science 331, 296-302, 2011.
50. Burnouf, T. and Radosevich, M., Reducing the risk of infection from plasma products: specific preventative strategies, Blood Rev. 14, 94-110, 2000.
51. Cai, K., Gierman, T.M., Hotta, J., et al., Ensuring the biologic safety of plasma-derived therapeutic proteins - Detection, inactivation, and removal of pathogens, BioDrugs 19, 79-96, 2005.
52. Burdick, M.D., Pifat, D.Y., Petteway, S.R., and Cai, K., Clearance of prions during plasma protein manufacture, Transfusion Med. 20, 57062, 2006.
53. Poelsler, G., Berting, A., Kindermann, J., et al., A new liquid intravenous immunoglobulin with three dedicated virus reduction steps: virus and prion reduction capacity, Vox Sang. 94, 184-192, 2008.
54. Agjaie, A., Pourfathollah, A.A., Bathaie, S.Z., et al., Structural study on immunoglobulin G solution after pasteurization with and without stabilizer, Transfus.Med. 18, 62-70, 2008.
55. Roberts, P.L., Lloyd, D., and Marshall, P.,  Virus inactivation in a factor VIII/VWF concentrate treated using solvent/detergent procedure based on polysorbate 20, Biologicals 3, 26-31, 2009.
56. Jeong, E.K., Sung, H.M., and Kim, I.S., Inactivation and removal of influenza  A virus H1N1 during the manufacture of plasma derivatives, Biologicals 38, 652-657, 2010.
57. Prince, A.M., Piët, M.P.,  and Horowitz, B., Effect of Cohn fractionation conditions on infectivity of the AIDS virus, N.Engl.J.Med. 314, 386-387, 1986.
58. Mitra, G., Wong, M.F., Mozen, M.M., et al., Elimination of infectious retroviruses during preparation of immunoglobulins, Transfusion 26, 394-397, 1986.
59. Yei, S., Yu, M.W., and Tankersley,  D.L.,Partitioning of hepatitis C virus during Cohn-Oncley fractionation of plasma, Transfusion 32, 824-828, 1992.
60. Bos, O.J., Sunyé, D.G., Nieuweboer, C.E., et al., Virus validation of pH 4-treated human immunglobulin products producted by the Cohn fractionation process, Biologicals 26, 267-276, 1998.
61. Horwith, G. and Revie, D.R., Efficacy of viral clearance methods used in the manufacture of activated protehrombin complex concentrate: focus on AUTOPLEX T, Haemophilia 5 (suppl 3), 19-23, 1999.
62.  Gregori, L., Maring, J.-A., MacAuley, C., et al., Partitioning of TSE infectivity during ethanol fractionation of human plasma, Biologicals 32, 1-10, 2004.
63.  Mantovani, L.G., Monzini, M.S., Mannucci, P.M., et al., Differences between patients', physicians' and pharmacists' preferences for treatment products in haemophilia: a discrete choice experiment, Haemophilia 11, 589-597, 2005.
64. Brown, S.A., Aledort, L.M., and others, Economic Challenges in haemophilia, Haemophilia 11, 64-72, 2005.
65.  Plosker, G.L. and Lyseng-Williamson, K.A., Atorvastatin: a pharmacoeconomic review of its use in the primary and secondary prevention of cardiovascular events, Pharmacoeconomics  25, 1031-1053, 2007.
66. Bodrogi, J. and Kaló, Z., Priniciples of pharmacoeconomics and their impact on strategic imperatives of pharmaceutical research and development, Br.J.Pharmacol. 159, 1367-1373, 2010.
67.  Bhandari, M., Ofosu, F.A., Mackman, N., et al., Safety and efficacy of Thrombin-JMI: A multidisciplinary expert group consensus, Clin.Appl.Thromb.Hemost. 17, 39-45, 2011.
68.  Lundblad, R.L., Bradshaw, R.A., Gabriel, D., et al., A review of the therapeutic uses of thrombin, Thromb.Haemost. 91, 851-860, 2004.
69. Robert, P., Global plasma demand in 2015, Pharmaceutical Policy & Law 11, 359-367, 2009.
70. Stangel,M. and Gold, R., Einsatz intravenöser immunglobuline in der neurologie, Nerveartz, in press, 2011.
71.  Sapan, C.V., Reisner, H.M., and Lundblad, R.L., Antibody therapy (IVIG): evaluation of the use genomics and proteomics for the study of immunomodulation therapeutics, Vox Sang. 92, 197-205, 2007.
72.  Audet, S., Virata-Theimer, M.L., Beeler, J.A., et al., Measles-virus-neutralizing antibodies in intravenous immunoglobulin, J.Infect.Dis. 194, 781-789, 2006.
73. Sullivan, J.S., Selleck, P.W., Downton, T., et al., Heterosubtypic anti-avian H5N1 influenza antibodies in intravenous immunoglobulins from globally separate populations protect against H5N1 infection in cell culture, J.Mol.Genet.Med. 3, 217-224, 2009.
74. Stokes, J., Jr., Maris, E.P., and Gellis, S.S., Chemical, clinical, and immunological studies on the products of human plasma fractionation. XI. The use of concentrated normal human serum gamma globulin (human immune serum globulin) in the prophylaxis and treatment of measles, J.Clin.Invest. 23, 531-549, 1944.
75.  Taylor, J.F., The isolation of proteins, in The Proteins, ed. H. Neurath and K. Bailey, Volume 1, Pt. B., Chapter 1, pps. 1-85, Academic Press, New York, New York, USA, 1953.
76.  Pennell, R.B., Fractionation and isolation of purified components by precipitation methods, in The Plasma Proteins, Chapter 2, pps. 9-50, Academic Press, New York, New York, USA, 1960.
77. Gordon, A.H., Martin, A.J.P., and Synge, R.L.M., Partition chromatography in the study of protein constituents, Biochem.J.37, 79-86, 1943.
78.  Martin, A.J.P. and Porter, R.B., The chromatographic fractionation of ribonuclease, Biochem.J. 49, 215-218, 1951.
79. Shepard, C.C. and Tiselius, A., The chromatography of proteins. The effect of salt concentration on the adsorption of proteins to silica gel, Discussions of the Faraday Society, No. 7, 275-283, 1949.
80.  Addiego, J.E., Jr., Gomperts, E., Liu, S.L., et al., Treatment of hemophilia A with a highly purified factor VIII concentrate prepared by anti-FVIIIc immunoaffinity chromatography, Thromb.Haemost. 67, 19-27, 1992.
81.  Fourcart, J., Saint-Blancard, J., Girot, P., and Boschetti, E., Préparation de l'albumine et des immunoglobulines G par fractionnment chromatographique direct du plasma humain sur DEAE et CM - Trisacryl M, Rev.Franc.Trans.Immunohematol. 25, 7-17, 1982.
82.  Berglöf, J.H., Eriksson, S., and Curling, J.M., Chromatographic preparation and in vitro properties of albumin from human plasma, J.Appl.Biochem. 5, 282-292, 1983
83. Tayot, J.L., Tardy, M., Gattel, P., et al., Large scale use of Spherosil ion exchangers in plasma fractionation, Dev.Biol.Stand. 67, 15-24, 1987.
84.  Farrugia, A., Spiers, D., Young, I., et al., Effects of plasma collection systems and processing parameters on the quality of factor IX concentrates, Vox Sang. 57, 4-9, 1989.
85.  Li, G., Stewart, R., Conlan, B., et al., Purification of human immunoglobulin G: A new approach to plasma fractionation,  Vox Sang. 83, 332-338, 2002.
86. Buchacher, A. and Iberer, G., Purification of intravenous immunoglobulin G from human plasma - - aspects of yield and virus safety, Biotechnol. J. 1, 148-163, 2006.
87.  Wolf, M., Kronenberg, H., Dodds, A., et al., A safety study of Albumex 5, a human albumin solution produced by ion exchange chromatography, Vox Sang. 70, 198-202, 1996.
88. Lihme, A., Hansen, M.B., Anderson, I.V., and Burnouf, T., A novel core fractionation process of human plasma by expanded bed adsorption chromatography, Anal.Biochem. 399, 102-109, 2010.
89.  Dodel, R., Neff, F., Noelker, C., et al., Intravenous immunoglobulins as a treatment for Alzheimer's disease: rationale and current evidence, Drugs 70, 513-528, 2010.
90. Farrugia, A., Trialing plasma protein therapies for rare disorders: Thinking outside the box, Pharmaceutical Policy & Law 11, 345-352, 2009.
91.  Chen, S.X., Hammond, D.J., Lang, J.M., and Lebing, W.R., Purificaton of a1 proteinase inhibitor from human plasma fraction IV-1 by ion exchange chromatography, Vox Sang. 74, 232-241, 1998.
92. Zimmerman, T.P., Yield improvement for manufacture of a1-proteinase inhibitor, Vox.Sang.91, 309-315, 2006.
93.  Lundblad, R.L., Approaches to the Conformational Analysis of Biopharmaceuticals, CRC Press, Boca Raton, Florida, USA, 2010
94.  Jelkmann, W., Biosimilar epoetins and other "follow-on" biologics: update on the European experiences, Am.J.Hematol. 85, 771-780, 2010.
95. Wang, Y.M. and Chow, A.T., Development of biosimilars - - pharmacokinetic and pharmacodynamic considerations,  J.Biopharm.Stat. 20, 46-61, 2010.
96. de Val, I.J., Kontorvavdi, C., and Nagy, J.M., Towards the implementation of quality by design to the production of thereapeutic monoclonal antibodies with desired glycosylation patterns, Biotechnol.Prog. 26, 1505-1527, 2010.
97. Bocciardo, L., Martinengo, M., Ardenghi, D., et al., Plasma derivatives and strategies for reaching self-sufficiency in Liguria: the role of the Transfusion Medicine Service of the Gaslini Institute, Blood Transfus. 5, 85-92, 2007.
98. Dyer, C., "Human tragedy" was due to delay in achieving national self sufficiency in blood products, BMJ  333:b808, 2009.
99.   Park, Q., Kim, M.J., Lee, J., et al., Plasma fractionation in Korea: working towards self-sufficiency, Korean J.Hematol. 45, 3-5, 2010.
100.  Rautonen, J.,Self-sufficiency, free trade and safety, Biologicals 38, 97-99, 2010.
101. Matejtschuk, P., Chitwick, Y., Prince, A., et al., A direct comparison of the antigen-specifity antibody profile of intravenous immunoglobulins derived by US and UK donor plasma, Vox.Sang. 83, 17-22, 2002.
102.  Farcet, M.R., Planitzer, C.B., Stein, O., et al., Hepatitis A virus antibodies in immunoglobulin preparations, J.Allergy Clin.Immunol. 125, 198-202, 2010.
103. Luke, T.C., Casadevall, A., Watowich, S.J., et al., Hark back: passive immunotherapy for influenza and other serious infections, Crit.Care Med. 38 (4 Suppl):e66-e73, 2010
104.   Hung, I.F., To, K.K., Lee, C.K., et al., Effect of clinical and virological parameters on the level of neutralizing  antibody against pandemic influenza A virus H1N1 2009, Clin.Infect.Dis. 51, 274-279, 2010.
105.  Wong,H.K., Lee, C.K., Hung, I.F., et al., Practical limitations of convalescent plasma collection: a case scenario in pandemic preparation for influenza A (H1N1) infection, Transfusion 50, 1967-1971, 2010.
106. Emmanuel, J.C., Material & equipment, procurement & maintenance: impact on blood safety, Biologicals 38, 78-80, 2010.
107. Hemobrás (http://www.hemobras.gov.br/site/conteudo/index.asp )