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).
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