Improving Chromatography for Analysis of Biological and Pharmaceutical Molecules: Barry L. Karger, the 2022 winner of the Lifetime Achievement in Chromatography Award

Barry L Karger is Director Emeritus of the Barnett Institute, and is the James L. Waters Emeritus Chair and Distinguished Professor Emeritus at Northeastern University in Boston. Professor Karger has spent a lifetime career in the field of gas chromatography (GC), liquid chromatography (LC), capillary electrophoresis (CE), and mass spectrometry (MS) making major advances to their understanding and application. He is the winner of the 2022 Lifetime Achievement in Chromatography Award, which is presented by LCGC magazine. This annual award honors an outstanding and seasoned professional for a lifetime of contributions to the advancement of chromatographic techniques and applications. Recipients are selected by an independent scientific committee. We recently interviewed him about his research work and career. This interview script includes multiple references describing the career of Barry L. Karger (1–7).

What was it like for you to be in the forefront of separation science 60 years ago? There were no lab-based computers, look-up devices, or automated software—how much work did it take to measure, record, and verify the accuracy of data in those times?

The early 1960s was a time of the beginning days of modern chromatography research—the first instruments for gas chromatography (GC) were commercialized in the late 1950s. At that time many people conducting separations were using paper chromatography, and thin layer chromatography was just being introduced. These techniques were not automated, they had limited quantitation abilities, and they were more of an art than a science.

It was true that we did not have computers or the internet. When I think back to the types of analyses we were able to do, we did separations by GC and the detector results were traced out on a strip chart recorder. The question was “how do you quantitate that?” You could quantitate the peak height, but peak area was more challenging. For peak area, I remember using an instrument such as a planimeter, which is simply a device to trace a peak on the chart recorder printout. Another popular approach we used was to cut out the individual chart printout peaks and carefully weigh them. I remember in meetings we used to discuss the humidity in the room and how it affected the cutout weights, as a humid environment would increase the paper weight. Those were the early days, but it was exciting that we were able to do separation science at all. Furthermore, if you wanted to research information you had to physically go to a library. We obviously couldn’t use Wikipedia or the internet (since they did not exist)—so you would often have to wait until the book you were interested in was returned to the library in order to read it. But the good news in those days was we did not get so many emails or Twitter, so we had time to really think about our research. It was a time when one had to speak directly with people in order to get information, by seeking them in person, or by phone or snail mail.

Would you tell us about the analytical history of the technology challenges when going from GC, in which you were involved, to HPLC—and what prompted you to become more active in HPLC?

Chromatography advanced quickly in the 1960s, particularly with the work of Marcel Golay on capillary GC columns. This type of GC led to high resolution separations. One of the early and challenging issues with GC involved analysis of polar compounds where one had to be able to vaporize the solutes. Increasing solute volatility involved derivatizing and blocking the polar groups of the molecule so you could get vaporization to occur. Very clever ways of doing derivatization were developed. However, it became obvious there were still many compounds that could not be analyzed using GC. We became interested in high performance liquid chromatography (HPLC) when we first met Csaba Horváth in the mid–1960s, at a time before he went to Yale; he was a post-doc at Harvard Medical School. I had talked to him several times, and he encouraged me to move into HPLC research. So we started exploring HPLC and quickly realized that the separation fundamentals for GC and HPLC are very similar. Our initial interest was to begin to separate polar compounds such as polar metabolites and drugs, and amino acids and peptides.

You have been active in research for 60 years with over 370 publications; which one or two of your research areas would you highlight?

The first research area that I would highlight is the work we did on the Human Genome Project (HGP). We began our work in capillary electrophoresis (CE) in the late 1980s, and we were immediately interested in separating DNA samples. At that time, DNA sequencing was being conducted using slab gel electrophoresis where radioactive labeling was applied in order to detect the separated peaks. This was a slow process—not automated—and obviously was not going to be successful for separating 3.2 billion base pairs needed for the HGP. So in the late 1980s, we first showed that CE had the resolving power to begin to separate closely sized DNA substances. We developed a very high resolving separating polymer matrix—linear polyacrylamide. Probably the highest impact advance we made was to show that you could make a high-resolution DNA sequencing separation, blow out the linear polymer, and then reload fresh polymer in the capillary and perform a second separation which was just as good. The reason this was so important is it provided a means for automating DNA sequencing. We then studied the fundamentals of the separation and optimized the conditions and the matrix, to be able to first show the separation and sequencing of 1000 bases, ending up at 1300 bases in a single run. We generated 10 million theoretical plates in the CE column; such ultrahigh efficiency was needed to separate the species that differed only slightly from each other. In the end, our linear polyacrylamide polymer was used as part of the HGP—for DNA sequencing as much as 40% of the total project.

If I were to pick a second research area to highlight, it would be the chromatographic separation of proteins. In the 1980s, as reversed-phase LC was being rapidly developed and applied, we began looking at protein separations. We injected native state proteins into the chromatographic column and all of a sudden we started seeing multiple peaks. We noticed if we first let the proteins sit on the reversed-phase during analysis, the amount of one peak grew at the expense of the other. To make a long story short, what was happening is that the native species were being denatured when it was in contact with the reversed-phase matrix. This denaturation process was clearly demonstrated when the denatured protein species separated at a different retention time from the native state protein. Understanding this denaturation process was important because people didn’t want to think they had an impurity in their protein-based products, when in fact the difference in separation was due to the denaturing effect of the reversed-phase matrix. The other main topic, we worked on in the 1980s was hydrophobic interaction chromatography (HIC). Using HIC we were able to separate the native state proteins and collect purified samples, which was important for biotherapeutic product development because one has to purify products such as enzymes in their native state. Now HIC is one of the important tools available in biotechnology for downstream processing where one is tasked with purifying biotherapeutic products, for example, as in the production of specific therapeutic protein molecules. In the 2000’s, we have been interested in LC– and CE–MS for protein analysis, including intact analysis, especially for protein therapeutics.

You have written and edited a ground-breaking and comprehensive book on separation science in 1973, entitled An Introduction to Separation Science (1). Why did you publish this book, and what was it like to collaborate with Lloyd Snyder and Csaba Horváth on this project?

As I noted, modern chromatographic separations were rapidly being developed in the 1960s, and a better understanding of the fundamental science by the community was needed to move the field forward. To address this need, Lloyd, Csaba, and I felt it would be really important from an educational point of view that there be a book available on the principles of separation along with a discussion of specific separation methods available at that time. So we wrote the first part of the book, the three of us, on the fundamentals of separation, and we invited experts in the various separation methods to write chapters describing the specific methods. It is interesting that as this book was written in 1973, there is no mention of the field of mass spectrometry. In writing the book, Lloyd Snyder was always ahead of us, and he would finish his writing before we did— Csaba and I would struggle to keep up with him. The book turned out to have a high impact, and it was used for 25 years or more as the textbook at senior level advanced undergraduate and graduate level courses in separations, along with being used by practitioners in the field.

One might ask “how do you make an index for a book without a computer?” There is one funny and interesting story that relates to our lack of computers at that time. To compose the book index, we first started by writing the words we wanted in the index on little pieces of paper. We would then read each page and write the page numbers for each topic on the pieces of paper. To alphabetize, we spread the papers on the floor. We had to read through the manuscript multiple times to make sure we found all the page numbers. Today, one does a simple word search, copies and pastes all of the page numbers for each topic, and then automatically alphabetizes the indexed words. We were in the dark ages in the 1970’s!

How did you begin your research using high performance capillary electrophoresis (HPCE), and how did this technique provide new opportunities for advancing separation science?

We were aware of the topic of electrophoresis from the book we wrote, and also I had been teaching a course on separations for many years. In 1981, at the Avignon HPLC meeting, I heard a talk by Jim Jorgenson on CE that was very exciting. Then I became really enthused by listening to a panel discussion at another scientific meeting comparing LC and electrophoresis. At that meeting, Fred Regnier was the panel member assigned to defend electrophoresis; several other people were on the panel to tout the benefits of chromatography. As I was listening to the panel discussion, I heard of the many problems associated with electrophoresis—to me these problems indicated research opportunities, which I later shared with Fred. So we became more interested in this technique. I went to Japan to another meeting and met Shigeru Terabe, who was already active in the field of CE. I invited Shigeru to come to my laboratory, which he did, and that helped push us along in our CE research. The reason we were quite interested in electrophoresis is because it fit well with our concurrent interest in biological molecules, specifically proteins and DNA. We realized that the analytical approaches being used at that time were not optimal, such as two-dimensional slab gel electrophoresis—which was slow, not very quantitative, and difficult to reproduce. So we believed that a method using a separation column operating electrophoretically would be very powerful—and that is how we got started in HPCE. In 1989 we started the international meeting series of HPCE to advance the field; the series is now called MSB. Also in 1989, we wrote a review article for the Journal of Chromatography on the subject of CE, which included the principles of CE and a review of CE applications—that review article was widely cited. The review and our early research led to our deeper involvement in the HGP.

You completed work in the Human Genome Project (HGP) and then began work in peptide mapping using electrospray ionization and mass spectrometry. What were some of your early advances and challenges using mass spectrometry (MS)?

In the late 1990s and moving into the 2000s, work for the HGP was coming to an end, and there was a fork in the road for me. The question was “should we now continue our research into the next generation of the HGP, where there would surely be a high degree of instrument development and advancement of new approaches beyond CE, or should we do something else, like biotechnology product testing?” At that time, mass spectrometry was being more widely applied, and we had been working with biotechnology companies such as Genentech. What was important in biotechnology was the capability of fully analyzing biotherapeutic products, most of which were protein-based. At that time, it was not possible to analyze an intact protein using MS, but we were able to analyze peptides. The standard peptide analysis method was to perform peptide mapping separation on digested protein fragments, collecting each one of the peak fractions, and then completing an Edman sequencing for each peptide fraction. This technique was extremely cumbersome, and we, as others, started to perform the analysis by coupling chromatography with MS—this technique has, of course, now become very powerful. We turned to explore the use of MS in many biotechnology applications. At the beginning, we focused on protein analysis and became interested in increasing the analytical sensitivity of the technique. We began looking at narrow bore columns where the flow rates were in the nL/min range. At the lower flow rates, the electrospray-MS becomes more sensitive. So we began work using a low flow rate MS while developing narrow bore columns of 15–20 µm I.D. for high sensitivity protein analysis. We are still interested in protein analysis using LC–MS and CE–MS to this day.

What is your opinion about the quality of science and science publications in the 2020’s vs. earlier years?

The current era is so different from earlier times; clearly the tools available today are much more powerful. Not only do we have better and more sensitive means of detection, but we also now have digitization tools enabling us to process data faster and in a more powerful way. We also see that artificial intelligence (AI) technology is beginning to have a significant impact on analytical science, as it is on biology and medicine as a whole. AI is being directly applied in the field of chromatography. For example, deep learning techniques are being used to look at the sequence of peptides and predict their retention times. We were always interested in predicting retention in LC but the tools we had available were primitive compared to those available today. I see real advances in AI such that all scientists will require at least a basic understanding of this field as we move forward with technological improvements.

In your estimation, what are the greatest challenges scientists face today?

One challenge results from the power of modern technology tools to generate data and the increased knowledge base that has been built up over so many years. Scientific progress is moving at an ever-increasing pace and that presents a big challenge for scientists to stay current. One needs to be intellectually nimble and avoid getting stuck in one limited field—because knowledge will continue to change. Even today, I am still involved and continue to learn new things, and that is very exciting.

Do you have any words of advice for young scientists just beginning their careers?

My first advice to young scientists is to not be afraid to move into new fields, because you will have to. Be prepared to build up your ability to learn new areas of science rapidly in order to stay relevant. My second point of advice for young analytical scientists is to understand that not only the measurements themselves are important—to understand how correct the measurements are quantitatively and qualitatively—but to also comprehend why you are performing the particular analysis. I think too often some analysts get so involved in just getting the analysis finished that they don’t consider what the analytical results mean. So it is very important to understand the big picture of why you are doing the analysis. The third important point of advice is to develop the ability to collaborate with others, which is becoming ever more important and easier to do than in my time. My fourth point is to realize that this is an unbelievable time of excitement and opportunity, so stay encouraged and enthusiastic about your work.

I read the Walter Isaacson book about Jennifer Doudna, who received the 2020 Nobel Prize in chemistry. In the book, the author relays a particular thought I would like to leave the readers with. Looking back 50 years from now, it is likely that the biological revolution will have been shown to have had a bigger impact on the world than even the digital revolution. We are moving into an era where unbelievable discoveries that impact health and longevity will occur. As one final note, the analytical chromatography part of the biological revolution will play an important role as will the preparative chromatography part. The analytical community should realize that without the preparative chromatography contributions of researchers to achieve purification of the cellular produced biopharmaceutical (downstream processing), there would be no biotechnology field. The future is bright for all the young analytical scientists.

References

(1) B.L. Karger, L.R. Snyder, and C. Horvath, An Introduction to Separation Science (Wiley-Interscience, New York, 1973), 608 pages.

(2) Bill Hancock, “A Celebration for Professor Barry L. Karger,” Chromatographia 69, 607 (2009). https://doi.org/10.1365/s10337-009-1092-1

(3) Andras Guttman, “Professor Barry L. Karger turns seventy,”

Electrophoresis 30(7):1096-1097, 2009. https://pubmed.ncbi.nlm.nih.gov/19373803/

https://doi.org/10.1002/elps.200990037

(4) Howard G. Barth, “Professor Barry L. Karger: Scientist, Mentor, and Innovator,”LCGC North America 32 (7), 494–502, 2014. https://www.chromatographyonline.com/view/professor-barry-l-karger-scientist-mentor-and-innovator-0

(5) Rich Whitworth, “Bioanalytical Living Legend,” The Analytical Scientist, 07/17/2015. https://theanalyticalscientist.com/techniques-tools/bioanalytical-living-legend

(6) Robert L. Stevenson, “Interview With Professor Emeritus Barry L. Karger,” American Laboratory, September 28, 2018. https://www.americanlaboratory.com/353159-Interview-With-Professor-Emeritus-Barry-L-Karger/

(7) Jerome Workman, Jr., “The 2022 Lifetime Achievement and Emerging Leader in Chromatography Awards,” LCGC North America, 40 (2), 82–92 (2022). https://www.chromatographyonline.com/view/the-2022-winners-of-the-lifetime-achievement-and-emerging-leader-in-chromatography-awards

Barry L. Karger, the 2022 winner of the Lifetime Achievement in Chromatography Award, is Director Emeritus of the Barnett Institute, and is the James L. Waters Emeritus Chair and Distinguished Professor Emeritus at Northeastern University in Boston. He received his PhD from Cornell University in 1963, and his BS degree in 1960 from the Massachusetts Institute of Technology. Professor Karger has spent a lifetime career of over 60 years in the field of separation science, and contributed major advances to the understanding of the fundamentals and applications of GC, LC, CE, and MS.