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50 Years of Sephadex Gel Learning from a Scandinavian Saga

by Eric Grund and Jan-Christer Janson

t has been fifty years since the separate biomolecules. He was first Sephadex paper was awarded the Nobel Prize in 1948, “for published (1). Readers of BioProcess his research on electrophoresis and I International work in a field that adsorption analysis, especially for his was fundamentally affected by what discoveries concerning the complex happened after that paper was nature of the serum proteins.” He published in 1959. So this anniversary coined the names alpha-, beta- and is certainly worthy of a party and a gamma-globulins (3). Six decades few speeches. But there are lessons to later, following enormous technical be learned, too. Let’s look at threads refinements and combining connecting events before and after the knowledge of the structure of DNA discovery of gel filtration with the discovery of specialized chromatography and introduction of bacterial enzymes, electrophoresis was the Sephadex product. used to perform decisive steps in the sequencing of the human genome (4). In t e r d i s c i p l i n a r y Re s e a r c h When someone with expertise in one Wh e n Pu r e Sc i e n c e field joins a team from another field, Collaborates wi t h In d u s t r y great leaps of mind can occur, and In Uppsala, Svedberg was a keen longstanding problems can be solved ge Healthcare (www.gehealthcare.com) advocate of applying scientific with ease. Science has seen numerous discoveries to make useful products. examples of this phenomenon, There, Svedberg’s eyes were opened Indeed, he thought it was almost particularly the field of bioseparations. to biological colloids, particularly sinful to not do so, and he argued One such example involves Theodor protein molecules, which were not strongly against the artificial division Svedberg, a physical chemist, who very well characterized at that time. of “pure” and “applied” science: helped define what we call Because of his background in physical We must become actively aware biochemistry today. In 1908, as a chemistry, he decided to construct an that research and industry are young and gifted student of physical ultracentrifuge and went on to show equally necessary. And that both of chemistry, Svedberg was studying that certain proteins had distinct these fields of endeavor must be metal colloids at Uppsala University in molecular weights rather than being adequately addressed. . . . Our Sweden and using sophisticated polydisperse (2). In those days, protein scientists cannot be occupied optical methods to characterize them. structure was largely a mystery, so the exclusively with rationing out Having been appointed a professor of ultracentrifuge was a breakthrough knowledge; they must be given physical chemistry in 1912, he that allowed their separation and opportunity for productive action. continued to make groundbreaking further study as never before. In fact, One must extinguish the notion advances in colloid science and Svedberg was awarded the Nobel that research is a disconnected acquired a worldwide reputation in the Prize in chemistry for 1926 as a result. pastime for periods when you are field. That led to an assignment as Back in Uppsala, another physical away from work. (5) guest professor at the University of chemist and a research fellow of Wisconsin, Madison, in the United Svedberg, Arne Tiselius developed For many years two Swedish States. methods using electric potential to companies with close ties to academia,

2 BioProcess International Oc t o b e r 2009 Pharmacia and LKB (after having Q&A wi t h Pr o f e s s o r Je r k e r Po r a t h merged in 1986, the resulting company How did you get involved in research became Amersham Pharmacia Biotech that lead to the Sephadex discovery? in 1997, then Amersham Biosciences I started my career as an organic chemist in 2001, and was finally acquired by during World War II. I studied terpenes for GE Healthcare in 2004), reaped the about seven years in the department of rewards of close collaboration between organic chemistry next door to two world- renowned experts in the field of university research and industry. separation: Theodor Svedberg and Arne Examples of such achievements Tiselius, who were my teachers in physical include chemistry and biochemistry, respectively. • Svensson-Rilbe and Vesterberg’s To cut a long story short, I joined their work in the field of isoelectric team and began working on proteins. focusing that gave rise to Ampholine What was the key turning point in your carrier ampholytes (6, 7) research? In 1955, Herbert Sober and • Porath’s work with affinity Elbert Peterson at the National Institutes of chromatography for the introduction Health in Bethesda, MD, introduced of CNBr-activated Sepharose gel (8) cellulose ion exchangers as adsorbents for proteins. I realized that protein adsorbents • Sjöquist’s use of protein A to must be hydrophilic in nature. This conviction became a turning point in my research isolate immunoglubulins that resulted and has guided my work ever since. in protein A Sepharose gel (9) What else has influenced your work? I was approached by a Swedish paper pulp • Hjerten’s development of agarose company with a patent application for sulphomethyl cellulose, which was to be used media for chromatography that led to as an adsorbent as well as for other purposes such as wallpaper adhesive. I learned Sepharose products (10) from this experience that there is a large step from the laboratory bench to the • Pertoft’s density gradient factory. separation of cells that gave rise to How would you describe the Sephadex discovery? Was it luck, or did your work Percoll media (11) lead you to plan for commercial opportunities in gel filtration? Molecular size is • Porath and coworkers’ studies on an important physical parameter for fractionation and characterization of macromolecular substances. Svedberg’s ultracentrifuge instrument was excellent for immobilized metal-affinity the purpose, but it was expensive and cumbersome to use. A chromatographic chromatography, which brought about technique would be much simpler and cheaper, so I looked forward to such a chelating Sepharose media. method. Then Noris Siliprandi, an absent-minded Italian professor in medicine, forgot Recently, in collaboration with to connect the electric power in an electrophoresis experiment. We were surprised to Uhlen’s team, the advanced, base- find a separation but ready for it too, I suppose. Going from starch to dextran gels was stable MabSelect SuRe affinity the result of a systematic search for a better medium. adsorbent for purification of Which scientists have had the greatest influences on your career? A. Fredga and monoclonal antibodies was developed A. Tiselius; my teachers H. Sober and E. Peterson (cellulose ion exchangers); L.C. Craig (13). Without the enthusiasm of and S. Moore (chromatography); R. Mullican, R Pearson, L. Pauling, and others academic scientists to collaborate with (biological affinity); G. Manneke and E. Katzir (immobilized enzymes) industry so their discoveries could be How do you see separation technologies developing in the next decade? What developed further by other scientists developments could drive this? I expect more efficient separation technology for and technologists, many of the environmental research and for applications such as the elimination of contaminating analytical and purification techniques hormones and drugs in water for human consumption. For these large-scale we use today to produce biomolecules applications in water treatment, the cost of media and adsorbents would need to be or manipulate cells would be stuck in reduced to a fraction of a percent of their present price. This should be possible. The a few dedicated research laboratories future prospects seem very promising. rather than being available to the industry as a whole. dextran in Uppsala. When sugar beet a “better” anticonvective agent for his Allowing Serendipity: The final extracts were accidentally infected electrophoretic protein experiments, lesson that can be learned from the with the bacterium Leuconostoc Per Flodin suggested Ingelman’s cross- Sephadex story is not to destroy the mesenteroides, he found they produced linked dextran. At the time, starch environment in which discoveries can a long a-1,6-linked d-glucose gels were widely used in occur by chance. Louis Pasteur said polysaccharide with interesting electrophoresis. Porath and Flodin had that “in the fields of observation, properties. Ingelman speculated that observed macromolecule separation in fortune favors only the prepared by chemical cross-linking he might a starch-gel column electrophoresis mind.” Back in the 1940s, the Second produce a useful gelling substance that experiment when someone forgot to World War led to many pressures and could be used instead of pectin. switch on the current! Together with needs that inventive people tried to About 10 years later, when organic Tiselius, they speculated that the effect fill. One example involves Björn chemist Jerker Porath was looking for was caused by size-exclusion of Ingelman, who was working on

Oc t o b e r 2009 BioProcess International 3 molecules inside the gel (14). Visiting were other early industrial environment in which such scientists took that knowledge away applications. breakthroughs and advances can with them and succeeded in separating Today’s World: So here we are today occur. The principle of the cross- milk proteins (15). with monoclonal antibodies (MAbs), disciplinary approach is alive and well Now Flodin and Porath decided hormones, novel vaccines, and safe in biotechnology, with molecular they would test the chromatographic plasma proteins on the market and in biologists, cell biologists, clinicians, separating power of cross-linked development. These are often biochemists, and chemical engineers dextran gel. The Sephadex produced using genetically engineered all working together in successful (SEparation, PHArmacia, DEXtran) organisms followed by downstream R&D teams. The application of G-25 (a designation indicating that 1 g processing, which inevitably involves scientific understanding to creating of crosslinked dextran absorbed 1.5 g products that are the successors of useful products is fundamentally what of water) product was born. Several Sephadex gel or even the original we all do. To advance our industry, we papers showing its application were itself. Single-use Sephadex ion must stay in touch with the cutting published during 1959, about the time exchangers are still used in the edge of academic research. it became available commercially (16– manufacture of many commercially Unfortunately, this kind of industrial– 18). Tiselius didn’t like the term available plasma products (19). academic collaboration is not always chromatography, which he considered easy to achieve when companies are a misnomer, so the name gel filtration driven by metrics, dashboards, and was coined for this new technique. monthly sales statistics. Today it’s often called size-exclusion Finally, and perhaps most chromatography (SEC) which later important, we must recognize the scientists have considered a more significance of accidental observations accurate description. for technological advances. Although Industry Boom: Pharmacia at the we strive to achieve hyperefficient time was a small Swedish drug industrial processes through lean and company that manufactured and six-sigma approaches — and for very commercialized the Sephadex product good reason — we must also provide — and suddenly found itself at the sufficient room for serendipity and center of a global “gold-rush” as creativity to make broader scientific scientists from Tokyo to Boston and discoveries. Dextran would not have Vancouver to Moscow rapidly exploited been developed if a sugar production gel filtration. The Sephadex product “Stack” columns packed with Sepahdex gel process had been under better control. proved reliable, was manufactured and were used for insulin purification in the Gel filtration’s discovery depended on controlled to pharmaceutical quality 1970’s. It’s made from the hydrophilic someone forgetting to turn on the dextran gel, which is today a component standards, and was marketed using a of many products such as Capto S power in an electrophoresis high standard of scientific expertise. It chromatography media from GE Healthcare. experiment. How do we control the had arrived at just the right time: Sephadex technology kick-started what is discovery process while increasing its now hyperefficient biomanufacturing, e.g., Enzymes, hormones, and other through lean and six sigma programs. output rather than reducing it? We biomolecules were the subject of ge Healthcare (www.gehealthcare.com) must learn from past successes, as intense research, and many of the described here, while we improve our fundamental chemical processes of life The hydrophilic dextran, from development and manufacturing were being elucidated. which Sephadex gel is made today processes through application of Pharmacia carefully documented serves as a component of many quality by design (QbD), design of the use of its products and by the late chromatography media in combination experiments (DoE), multivariate 1970s had ~10,000 scientific with other materials. It gives analysis, and operational excellence publications in its archives. The list of molecular sieving properties to rigid through six-sigma and lean discoveries, seminal papers, and Nobel SEC media and acts as a flexible approaches. prizes that depended on Sephadex gel ligand suspension system for modern, is a long one. The product enabled a high-capacity ion exchangers (20, 21). Re f e r e n c e s generation of biochemists to study the Dextran itself is used clinically as a 1 Porath J, Flodin P. Gel Filtration: A macromolecules that are the plasma volume expander because of its Method for Desalting and Group Separation. Nature 183, 1959: 1657–1659. foundation of the biopharmaceutical general biocompatibility, and it is a 2 Svedberg T, Fåhraeus R. A New industry. Sephadex gel and ion molecule that continues to have a Method for the Determination of the exchangers enabled both Eli Lilly and bright future. Molecular Weight of the Proteins. J. Am. Chem. Novo Nordisk to produce highly Soc. 48(2), 1926: 430–438. purified, single-component insulin. Le a r n i n g f r o m t h e Pa s t 3 Tiselius A, Kabat EA. Electrophoresis Milk proteins, plasma albumin, For us, the main message in this of Immune Serum. Science 87(2262), 1938: immunoglobulins, and clotting factors Scandinavian saga relates to the 416–417.

4 BioProcess International Oc t o b e r 2009 4 Venter JC, et al. The Sequence of the 11 Pertoft H, et al. Density Gradients 19 Over J. Impact of Technology Human Genome. Science 291(5507), 2001: Prepared from Colloidal Silica Particles Transitions on the Plasma Business. Sixth 1304–51. Coated By Polyvinylpyrrolidone (Percoll). Plasma Product Biotechnology Meeting, Menorca, 5 Svedberg T. Forskning och Industri. Analyt. Biochem. 88(1), 1978: 271–282. Spain, 11–15 May 2009. Hugo Gebers Förlag: Stockholm, Sweden, 12 Porath J, et al. Metal Chelate Affinity 20 Kågedal L, et al. Chemical, Physical, 1918. Chromatography, a New Approach to Protein and Chromatographic Properties of Superdex 6 Svensson H. Isoelectric Fractionation, Fractionation. Nature 258(5536) 1975: 598– 75 Prep Grade and Superdex 200 Prep Grade Analysis, and Characterization of Ampholytes 599. Gelfiltration Media. J. Chromatogr. 537, 1991: in Natural pH Gradients: I, The Differential 13 Hober S, Nord K, Linhult M. Protein 17–32. Equation of Solute Concentrations at a Steady A Chromatography for Antibody Purification. 21 Berg H, Svensson J, Nilsson CH. State and Its Solution for Simple Cases. Acta J. Chromatogr. B 848, 2007: 40–47. Highly Rigid Agarose-Based Chromatographic Chemica Scandinavica 15, 1961: 325–341. 14 Porath J, personal communication. Matrices: Increasing Throughput in 7 Vesterberg O. Synthesis and Isoelectric 15 Lindqvist B, Storgårds T. Molecular- Downstream Processing. BioProcess Int. 13(2) Fractionation of Carrier Ampholytes. Acta Sieving Properties of Starch. Nature 175, 1955: 2005: 68–72. c Chemica Scandinavica 23, 1969: 2653–2666. 511–512. 8 Axen R, Porath J, Emback S. Chemical 16 Porath J, Flodin P. Gel Filtration: A Eric Grund is senior director of bioprocess Coupling of Peptides and Proteins to Method for Desalting and Group Separation. applications at GE Healthcare; ... Jan- Polysaccharides By Means of Cyanogen Nature 183(4676), 1959: 1657–1659. Christer Janson is a professor emeritus for Halides. Nature 214, 1967: 1302. 17 Porath J. Fractionation of Polypeptides the department of physical and analytical 9 Hjelm H, Hjelm K, Sjöquist J. Protein and Proteins on Dextran Gels. Clin. Chim. chemistry in surface biotechnology at the A from Staphylococcus aureus: Its Isolation By Acta. 4, 1959: 776–778. Affinity Chromatography and Its Use as an Uppsala Biomedical Center of Uppsala 18 Lindner EB, Elmqvist A, Porath J. Gel University; Uppsala, Sweden... Immunosorbent for Isolation of Filtration as a Method for Purification of Immunoglobulins. FEBS Lett. 28(1) 1972: Protein-Bound Peptides Exemplified By 73–76. Oxytocin and Vasopressin. Nature 184(20) 10 Hjertén S. The Preparation of Agarose 1959: S1565–S1566. Spheres for Chromatography of Molecules and Particles. Biochim. Biophys. Acta 79, 1964: 393.

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