50 Years of Sephadex Gel Learning from a Scandinavian Saga

FOCUS ON... TECHNOLOGY 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). INTERD I SC I PL I NARY RESEARCH When someone with expertise in one WHEN PURE SC I ENCE field joins a team from another field, COLLABORATES wi TH INDUSTRY 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 OCT O BER 2009 Pharmacia and LKB (after having Q&A wi TH PROFESSOR JERKER PORATH 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 OCT O BER 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

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