Fred E. Regnier: The Road One Chromatographer Took to Reach the Lifetime Achievement Award
LCGC recently spoke with the 2014 LCGC Lifetime Achievement Award winner, Fred E. Regnier, the John H. Law Distinguished Professor in the Department of Chemistry at Purdue University in West Lafayette, Indiana, about his early work, his numerous patents and start-up companies, as well as his vast accomplishments in separation science and his role as a teacher.
Were you always interested in a scientific career? What were the focus of your B.S., PhD, and postdoctoral studies and why?
Regnier: Getting into science as a career didn’t occur to me until I was out of college. Actually, I only went to college to get out of working outside in the cold taking care of cattle all winter. I came into science through the back door. As a teenager I built cars and fixed farm machinery. Automobile and tractor engines are based on a lot of physics, so that subject was really easy for me. When I entered college I asked if I could get out of taking humanities classes by majoring in physics, chemistry, and math. They let me have a triple major, but still made me take all of the humanities; for which I am grateful.
My undergraduate thesis was on paper electrophoresis of amino acids, my PhD was on the biosynthesis of terpenes and pheromones, and my postdocs with Ed Wilson and John Law were on the identification of insect hormones and pheromones. (Wilson and Law were collaborators.) I loved those projects because I got to do tons of gas chromatography (GC), liquid chromatography (LC), and mass spectrometry (MS). I built a high performance liquid chromatography (HPLC) system to purify trail pheromones from 100-lb bags of fire ants before HPLC instruments were commercially available. All of these projects were about analytical chemistry. Actually, it took a while for it to dawn on me that I liked analytical chemistry more than biology.
How did you get started working with chromatography?
Regnier: I shelved books in the library as an undergraduate and came across books on chromatography and electrophoresis. I was fascinated by separations, so much so that it became a dominant theme in my life; as I mentioned earlier, paper electrophoresis of amino acids was my undergraduate thesis project. It wasn’t until I was 30 or so that I realized I could make a living in separation science.
Who was the biggest influence on your career when you were just getting started?
Regnier: Ed Wilson and John Law; they were both members of the National Academy and scientists that I considered to have great wisdom. They told me I should focus on method development, their rationale being that developing new, innovative, original methods always keeps one at the cutting edge.“You will always be relevant,” Ed said. They also told me that as a young assistant professor I should go out of my way not to offend a full professor. Full professors run promotion committees. That too was enormously valuable advice.
What led you to develop the first high-performance chromatography columns for size-exclusion, anion-exchange, cation-exchange, hydrophobic interaction, and macroporous reversed-phase chromatography separations of proteins?
Regnier: As a young assistant professor in the biochemistry department I was asked to develop and introduce an analytical biochemistry course that involved protein separations. At the same time, a graduate student named Dale Deutch asked me to interpret papers about the immobilization of proteins on controlled porosity glass (CPG). The juxtaposition of the two caused me to realize that by immobilizing oligosaccharides and ion-exchanging polymers on the surface of CPG I could construct chromatographic media that would be pressure stable to hundreds of atmospheres. It worked. We were able to accelerate protein separations 100-fold within a few months. In 1975, we completely resolved the five isoenzymes of lactate dehydrogenase in 5 min by anion-exchange chromatography. Size-exclusion chromatography (SEC) separations were achieved in the same time frame.
Reversed-phase chromatography of proteins on macroporous materials came about in another way, 6–8 years after the SEC and ion-exchange chromatography (IEC) materials. There are two parts to the story. Because of the CPG work, we were looking at macroporous silicas. We noticed that when the diameter of proteins became more than roughly 10% of the pore diameter of the sorbent you started to have mass transfer and recovery problems. It got really bad when the protein approached the size of the pores. That led us to silica sorbents with pore diameters greater than 300 Å. The second part of the story is the mobile phase. The protein chemists at Purdue pointed out to us that tricholoracetic acid precipitates proteins whereas trifluoroacetic acid solubilizes them. Jim Pearson in our group found that trifluoroacetic acid in fact was a great mobile-phase additive. The use of 0.1% trifluoroacetic acid came about because Jim decided that higher concentrations of trifluoroacetic acid caused too much baseline elevation at the end of gradient elution. The trifluoroacetic acid–acetonitrile mobile phase from that work has been used unchanged for more than 30 years.
Of your 50+ patents, which do you think have had the greatest impact on the field?
Regnier: Our coating patents have been the most widely licensed, particularly those in which stationary phases are attached to support matrices by adsorption. The most successful sorbent type produced with these patents would be the Poros packing materials. Poros is widely used in the purification of biopharmaceuticals. This family of packings uses a poly(styrene–divinylbenzene) matrix with pore diameters ranging from 800 Å to greater than 5000 Å. The coating is a fimbriated polymer that is sort of like shag carpeting. The fimbria greatly increase surface area and loading capacity. Together these two features allow very large molecules to reach the particle interior while still allowing loading capacities of 100 mg of protein per 1 mL of sorbent.
What has been the most challenging research project you have undertaken? What has been the most rewarding?
Regnier: The most challenging was our attempt to make protein-imprinted sorbents. We were able to make hemoglobin imprints in agarose that had the selectivity of antibodies, but there were a series of major problems. One was that imprinting efficiency relative to the number of hemoglobins used was low. It took a lot of protein to make a small number of imprints. Second, this meant you had to do a lot of separation runs to recover the cost of the imprinting protein. Third, you got the equivalent of polyclonal antibodies — that is, there were a lot of different imprints of very different binding affinity. And finally, all of this made columns very expensive. Protein imprinting was not economically viable.
One of the papers that was most rewarding for Bing He and me was the one on collicated monolith support structures (COMOSS), now referred to as pillar columns (1). It was rewarding because it was so hard to do, but in the end we fabricated every support structure (equivalent to a particle) in a whole chromatography column. All the pillars were exactly spaced and we knew how many pillars (particles) there were in the column. Unfortunately, this idea was far ahead of our ability to fabricate the system. As fabrication technology catches up with the concept, this type of column may become a viable option for producing chromatography columns.
Of the multiple polymeric bonded phases you synthesized for protein separations, which have had the most lasting effect on the field?
Regnier: It would be the bonded phase used on the Poros packing material. Actually, I have to qualify that. Poros had the largest impact on protein separations. It is harder to know which chromatographic media per se had the largest impact. What we do know is that the paper we published titled “High-performance liquid chromatography of proteins” (2) was the first ever to describe the use of polymeric bonded phases on the surface of a support matrix that would cover the surface features of the support, provide a high density coating, and be fimbriated to increase loading capacity. We always described this technique as being like paving a parking lot to cover up the bad things underneath. At the time we did this work, polymeric coatings were thought to cause very serious mass transfer problems. This paper showed that wasn’t true with very large pore diameter supports. Today, most chromatography matrices used in protein separations have a polymeric coating.
Why did you choose an academic career path?
Regnier: Early in my career I was told by a pharmaceutical company I had “too many ideas and would be too hard to control” to work in their environment. That was the exact comment. That scared me, and it was easy to get a job in academia when I entered. It is interesting how small things that are probably not true can have a big impact on you when you are young and inexperienced.
While academic scientists occasionally form start-up companies, you have started five. How have you been able to create so many?
Regnier: It is my hobby. Some people play golf; I start companies. I was never confused about who I was — I am an academic who has a corporate hobby. That means I never get involved in starting a company without a really good team of people who know far more about business than I do and who will work at the company every day. If you lift up the hood and look at the engine in all the companies I have been involved with it was a team who did the heavy lifting and made them work.
Do you have any advice for other academics who are thinking about forming a company related to their research?
Regnier: Actually I don’t. My reason for starting companies was that I was really passionate about what we were doing and believed we could impact the world. Publishing papers doesn’t always bring something to the world. A paper is a little like the birth of a child. It is beautiful, but requires an enormous amount of parenting and work to reach adulthood. Parenting and development of an idea is what happens in corporate development. That determines how the child will impact the world.
Over the years you have mentored more than 80 graduate students and more than 25 postdoctoral associates and visiting scientists. How have your students impacted your career?
Regnier: The great value of students is that they bring a fresh, new way of looking at the world and science. I won’t be specific on who impacted me most because I might leave someone out, but in probably half of the things we did in our lab students either got us started in the field or made major intellectual contributions toward the outcome. Most of the people who went through the lab will tell you they pretty much did whatever they wanted, and I encouraged that. Many successes in our lab came from things students suggested that I flat out told them wouldn’t work but they should try it if they wanted. They did and it worked. I was wrong. I was very aware of the great value young students brought to our lab.
Your work has been published quite a number of times (about 300 journal articles, two books, more than 30 book chapters, and more than 35 review papers). How important is it to you to share your work with the scientific community? Has anything you published led to unexpected collaborations?
Regnier: We in academia have a contract with society; they provide us with money to examine scientific problems and in turn we have an obligation to share what we find with society. Hopefully that in turn will lead to the solution of societal problems and will create products and jobs, and the ensuing income and taxes will repay society. I have done that with products we helped create. Investment in academic research has benefited the American taxpayer by orders of magnitude. I view our relationship with the American taxpayer as a collaboration for which I am enormously grateful.
How has being a member on more than 13 editorial advisory boards affected your career?
Regnier: I came to view my job as a peer reviewer of manuscripts as that of trying to help people improve the presentation of their work to society in the form of a publication, in part fulfilling the obligation noted above. That is the case with 80% of the manuscripts I read. Unfortunately, in a small number of cases the research was either so poorly planned or described in a manuscript that it could not be easily fixed with editing and, in such cases, I had to recommend that those manuscripts not be published. The rationale was that those manuscripts would mislead readers for all kinds of reasons. These experiences helped us write better papers describing our work.
In a guest editorial article you wrote for LCGC in 2012 (3), you suggested that a new “selectivity era” was emerging in liquid chromatography. Do you still think that? Has the change in selectivity already begun?
Regnier: Indeed it has! Targeted proteomics, based on high selectivity, structure-specific separations of proteins was the method of the year in Nature Methods (2013). Moreover, at the recent Hyphenated Techniques for Chromatography (HTC) meeting in Bruges, Belgium, and the Mass Spectrometry: Applications to the Clinical Lab (MSACL) conference in San Diego, California, multiple papers were present on structure-specific selection of analytes.
What advice would you offer a scientist just starting out?
Regnier: Pursue whatever you do with enormous vigor and passion, don’t let others discourage you, believe you can change the way to world does things, and visualize yourself doing it, over and over.
Do you have any closing thoughts?
Regnier: I just saw the movie Nebraska (4) and as a gray-haired Nebraskan it caused me to think of something. It is often the case that car keys are taken away from an elderly person because they might hurt someone. Science is a little like that as well. Administrators, granting agencies, and even youngsters tell those of us who have been around a long time we don’t really understand how to drive very well anymore. That might be true, but, like the pride of the old man driving the new truck down Main Street at the end of the movie, there is nothing like having the keys to a lab. I absolutely, totally love analytical chemistry and everything that goes on in labs, and will forever. I won’t give up the keys easily.