Mark S. Lesney and Lynn Willis

Whether for natural or engineered products, chromatography is key.

In medicine and mythology, blood has always possessed a special significance as the font of human life. Using blood as a drug has a long history, from external application in bizarre poultices to physician-authorized blood drinking. The purported curative power of nonlethal bloodletting was the basis of much of medicine since the time of the ancient Greeks, finding willing proponents even at the dawn of so-called scientific medicine in the 19th century. Even transfusions have a long history of failure. Only when the knowledge of blood groups was achieved at the turn of the century would routine transfusions become less than a gamble with fate—as Prof. Van Helsing must have known when he took the chance and gave Miss Lucy his own blood in an ultimately vain attempt to save her from the more lethal exsanguinations of Count Dracula in Bram Stoker’s 1897 novel.

And only in the 20th century, with the foundations of sterile technique, chemical diagnostics, and centrifugation equipment, were the true medicinal possibilities of blood made apparent. From the first life-giving whole blood and serum transfusions to the use of isolated platelets and other clotting factors, blood showed its promise. This realization that blood components could be exploited for their medical benefits created an increasing demand for better methods to isolate and purify the compounds of interest. Chromatographic techniques for separating the more subtle of the natural drugs available in blood became paramount—especially the use of various forms of affinity and ion-exchange chromatography.

The Bounty of Blood
Natural blood is a complex mixture of living cells and a host of biochemicals such as sugars, lipids, vitamins, and amino acids. Also included are the various proteins whose functions are critical to the cardiovascular system and to the body as a whole. Red blood cells get their color from hemoglobin (this iron-containing protein carries oxygen throughout the body). White blood cells fight disease invaders by devouring them; platelets are subcellular bodies that induce blood coagulation when appropriately triggered. Antibodies are protein molecules that specifically bind to foreign proteins and to microbial and viral invaders; at their best, antibodies deactivate and hold invaders in place, providing a lure to the white blood cells. (The mix of antibody-precipitated invading material and living and dead white blood cells is what makes up pus in a wound.) Serum is the straw-colored liquid component of the blood in which the rest flows.

Each of these various blood components has a potential therapeutic use. The most obvious use of whole blood and serum is in transfusions to replace traumatic blood loss. The uses for various other protein components are manifold, such as stimulating clotting in serious wounds, preventing clotting in strokes and heart attacks, improving sports performance, and providing antibodies for diagnostics or therapeutics for numerous diseases.

Engineered Blood Components
With the advent of biotechnology, blood-based genetically engineered products obtained from microorganisms or cellular fermentation have been in greater demand from considerations of purity as well as cost. The discovery of ever newer bloodborne diseases, such as AIDS and hepatitis C and G, and even fears of the transmission of prion-associated illnesses, such as Creuzfeldt–Jacob disease and mad cow disease, have greatly accelerated this push for engineered rather than naturally derived blood-based medicines. To engineer a blood protein in a transgenic plant, animal, microbe, or cell culture, one must first identify and sequence the gene coding for the protein, clone it, and then transfer it into the production vehicle best suited to it. Many blood proteins are complex and require posttranslational processing typical of eukaryotic cells, making bacterial fermentation impossible or at best impractical (with the requirement of expensive, postharvest modifications that drastically affect cost and purity. According to the American Red Cross, “transgenic technology is expected to lessen the cost of factor VIII, factor IX, and fibrinogen, making improved therapy more economical for more patients.”

Milking Another Vein One of the most exciting modern developments in bulk production of therapeutic blood products is the use of transgenic animals. Sheep, cows, pigs, and goats are genetically engineered to contain the gene sequence of specific human blood proteins. Attached to each gene is the regulatory sequence for specific production of these proteins in the milk of the transgenic females. Milk is a well-defined, easily purifiable biological liquid that already contains many natural-blood proteins, especially in the first days after giving birth (this early milk is known as colostrum). Pharming Holding NV (Leiden, The Netherlands), in conjunction with The Netherlands Red Cross, is developing recombinant human esterase inhibitor as a therapeutic plasma product, as well as recombinant lactoferrin as a heparin neutralization factor. A subsidiary, Pharming Healthcare, Inc. (Rockville, MD), has entered a partnership with the American Red Cross Biomedical Services to develop transgenically produced human factor VIII for treatment of hemophilia A; human factor IX for treatment of hemophilia B; and human fibrinogen to act as a component of sealants for surgical procedures and wound repair. Recombinant cattle and pigs will generate these products in their milk. Clinical trials are expected to begin within two years. As with purification from blood or cell culture medium, the various chromatographic techniques described in this article are typically also used to isolate specific compounds from the engineered milk for use in drug therapy. Milk is not, of course, the only source of transgenic blood proteins, as reported in a previous article (TCAW, August 1999); antibodies are also being expressed in transgenic plants.

Chromatographic Techniques
Whole blood is normally purified by various centrifugation and filtration techniques. Centrifugation can easily separate cellular components from serum and red blood cells from platelets; this procedure is commonly used at blood banks to produce materials used in normal hospital practice. But for stringent isolation of unique blood proteins, whether from natural donor blood or from culture medium into which genetically engineered blood components have been secreted by living, cultured cells, chromatographic techniques are generally required.

In purifying blood proteins, the cellular components are first separated from the serum by centrifugation and/or filtration. If the protein of interest is a component of the cells (such as hemoglobin from the red blood cells or various clotting proteins from the platelets), then the cells must first be broken up to release the proteins and centrifuged free of debris. Then chromatographic techniques can be applied to quickly separate the various proteins in the blood from other serum components.

Chief among the separation methods used are ion-exchange chromatography (IEC), size-exclusion chromatography (SEC), and affinity chromatography. IEC separates the components on the basis of charge. Cationic beads bind negatively charged proteins; anionic beads bind positively charged ones. Binding strength varies from weak to strong depending on the type of ligand ion bound to the particular beads. IEC media are available in differing charges, pore sizes, and support strengths (from low-pressure to high-pressure tolerant) and from a wide variety of companies, including Amersham-Pharmacia, Bio-Rad, Dionex, Hewlett Packard, Merck, Perseptive Biosystems, and TosoHaas, among others.

SEC separates on the basis of molecular weight. Gels with defined pore sizes retard molecules that can slide within their openings and allow larger molecules to pass around the beads relatively unimpeded. Typical low-pressure gel beads are capable of separating molecules from a molecular weight of a few hundred to multimeric proteins weighing in the millions range. These gels are made from polyacrylamide, dextran, or agarose and include the familiar Bio-Gel (Bio-Rad) and Sephadex/Sepharose (Amersham Pharmacia Biotech) brand names. Once these first purification steps are completed, affinity chromatography generally follows.

Affinity chromatography is probably one of the most efficient and sensitive methods of protein purification available. Chromatographic beads are cross-linked with a protein or carbohydrate moiety that binds in a noncovalent lock-and-key fashion (similar to the way an enzyme binds to its substrate) to the particular molecule to be purified.

Protein A, for example, is a bacterially derived protein that binds preferentially to gamma globulin (IgG antibody molecules) and is ideal for use in column chromatographic purification. The unpurified serum or the cell culture medium that contains secreted antibodies is put through the affinity column, and the antibodies bind to the protein A attached to the column gel. All other proteins flow through in the weak buffer wash. The very highly purified antibodies are easily released without damage to either the column or the protein by a subsequent high-salt elution. Other ligands useful for purifying blood products include heparin, which when linked to an affinity gel, binds specifically to several known clotting-factor proteins (see the discussion of hemophilia below). Different lectins can be used to bind to specific glycoproteins. Antibodies can be used to bind and purify their unique antigens; enzyme inhibitors or cofactors can be used to bind their particular enzymes. The same vendors that produce IEC and SEC media generally also supply various lines of affinity chromatography supports.

Cleaning Up
Typically, all stages of protein isolation for therapeutic purposes—especially fine fractionation techniques—are monitored for purity. HPLC and gel electrophoresis are commonly used to demonstrate degree of purification. Presence of a single peak or band under stringent conditions (or a defined, identifiable spectrum of peaks in more complex mixtures) is often considered the necessary signature of final purity.

The final and critical step, antiseptic treatment, is often performed because of the increasing virulence and prevalence of bloodborne diseases. This step is especially important if the product is obtained as a natural product fraction or from a fermentation process that uses serum-derived components to grow its cells or to stabilize the purified product. (Some blood proteins require the presence of serum albumin for stability—at least before defined stabilizing media are developed on a case-by-case basis.) Whole blood or serum products are potential carriers of numerous viral and prion diseases. Detergent washing and UV treatments are two of the techniques used to destroy such contaminants.

The Case of Hemophilia
A perfect example of the many considerations and techniques involved in using blood as a drug is the case of hemophilia, which has two main forms: A and B. The American Red Cross estimates there are more than 55,000 hemophilia patients in Europe, Japan, and North America. Hemophilia A is the “classic” form the so-called royal blood disease that afflicted several of Queen Victoria’s male descendants, including the ill-fated Czarevitch, Alexei Romanov. It is currently treated with blood factor VIII, a natural clotting protein; its gene was first sequenced in 1984. Within 10 years, recombinant factor VIII was in clinical use. Bayer produces the Kogenate brand name from transgenic baby hamster kidney cells, and Baxter produces its Recombinate brand from transgenic Chinese hamster ovary (CHO) cells.

Hemophilia B is a similar condition that results from insufficient or abnormal production factor IX, a blood-clotting protein. The disorder is also caused by an inherited sex-linked recessive trait; the defective gene is located on the X chromosome. It is treated by administering clotting factors during bleeding episodes to prevent the loss of large amounts of blood. Hemophilia B is also known as Christmas disease, named after the family in which the disease was first examined in detail in 1952.

Until recently, the products used to treat people with hemophilia and related clotting disorders only could be obtained from concentrates of human blood provided by donors. Much has been done to ensure the safety of these mixtures, but viruses (particularly parvovirus B19 and hepatitis A) and prions are still of great concern. A great tragedy of the AIDS era has been the many hemophiliacs who became infected with HIV and/or hepatitis C before the introduction in 1985 of physical and chemical methods of viral inactivation for coagulant concentrates obtained from bulk human blood. Today, many of the products still used to treat hemophilia B are actually mixtures of clotting factors (prothrombin in complex concentrates). These products use various chromatography methods to fractionate clotting factors: DEAE-Sephadex, affinity chromatography (usually heparin ligand), ion exchange, and metal chelate. One product with a high percentage of factor IX, Mononine from Armour Pharmaceuticals, uses immunoaffinity chromatography (monoclonal antibodies) to concentrate factor IX. Sodium thiocyanate treatment and ultrafiltration deactivate and remove any remaining viruses.

The gene sequence for factor IX was determined almost simultaneously in two separate labs, those of G. G. Brownlee of the University of Oxford and Earl W. Davie at the Washington Research Foundation. The company BTG acquired the rights to the sequence from both groups and then divided the licenses to use the information among four companies. Genetics Institute has the viral and retroviral license and made the first commercial version of the engineered fraction IX product: BeneFIX (U.S. patent no. 4,770,999, 1988). Genetic Therapy and Transkaryotic Therapies have the rights to develop gene therapies using factor IX. PPL Therapeutics plc has the license to create transgenic sheep that express factor IX in their milk.

BeneFIX was brought to market in slightly more than three years. The speedy approval was based on its bioequivalence to blood fraction product. Currently, it has about 70% of the market (about 3000 patients in the United States), almost 100% of the HIV and pediatric markets, due to safety issues. BeneFIX is also cheaper to produce than natural blood products.

The protein is synthesized in CHO cells, which provide the necessary posttranslational modifications and yet are suitable for large-scale suspension culture. Factor IX cDNA is coexpressed with the enzymes necessary for proper cleavage and secretion. The mammalian cells are grown in serum-free medium—no bovine serum or animal proteins are present, which avoids potential contamination by serum-associated viruses and prions.

Factor IX is secreted into the growth medium, which is then removed from the cells and purified through a series of chromatographic steps: Q-Sepharose-FF (a form of IEC and pseudo-affinity chromatography) and Cellufine sulfate (heparin-like) affinity chromatography, followed by ceramic hydroxyapatite, and Chelate EMD copper chromatography to remove trace contaminants. Affinity chromatography with monoclonal antibody is deliberately avoided to prevent the potential for contamination with extraneous animal proteins (e.g., mouse protein). After chromatography, the purified product is formulated in a buffer of polysorbate 80, histidine, glycine, and sucrose. (Unlike most previous factor IX products, no human albumin or other proteins are used to provide stability.)

Other Drugs from Blood
New genetic engineering/fermentation and chromatographic methods of purification offer exciting prospects for blood pharmacology in bulk production. These methods use specific blood-derived proteins for nongenetic diseases and traumas, such as during heart attacks and surgery, and treatment following massive injury in accidents. Naturally purified or genetically engineered human hemoglobin can be modified specifically for use in blood substitutes. These processes ultimately may provide the greatest use for trauma in which massive and continual transfusions are required, eliminating the need for donors and fear of disease transmission. The most promising such compound to date in clinical trials is a pyridoxalated polymerized hemoglobin solution called PolyHeme, developed by Northfield Laboratories (IL). Toxicity issues and the risks of bacterial sepsis caused by increased bacterial growth with higher available free iron remain potential problems.

A (Non)Bloody End
Blood’s modern medical history is only just beginning. We are a far cry from the days of the ancient Romans when gladiators would drink the blood of their slain opponents as a rejuvenating liquid (the original sports drink?). As a living biochemical soup, blood not only keeps us all alive, but is also beginning to provide researchers and doctors with some of the most useful modern therapeutics and diagnostic agents. Dracula’s dependence on the vital fluid, although different in style, is no less fundamental than our own. Luckily, with modern chemical techniques, we have no need to feed off each other like vampires for relief.

The Bloody Web Numerous Internet sites deal with blood, blood-related products, purification, and diseases. For a start, check the following sites. The American Red Cross (www.crossnet.org). The site directory can be searched by topics of interest. Enzyme Research Ltd. (www.enzymeresearch.co.uk). A corporate Web site whose open catalog provides a comprehensive list of human and bovine blood proteins with information on properties and uses. Pharming Group NV (www.pharming.com). This company is working with the International Red Cross to develop transgenic blood products in milk. World Federation of Hemophilia (www.wfh.org). This international organization provides information and links. An Online Library of Literature (www.literature.org/authors/stoker-bram/dracula/). This site provides the text of Bram Stoker's Dracula (among other literature).

Mark S. Lesney is an assistant editor and Lynn Willis is a senior editor of Today’s Chemist at Work.

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Copyright © 1999 American Chemical Society.