Bringing Mass Spec to the Masses - From niche tool to necessity: the history of MS in the pharmaceutical industry

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  • Published: Mar 1, 2014
  • Channels: Proteomics & Genomics / HPLC / Proteomics / Base Peak
thumbnail image: Bringing Mass Spec to the Masses - From niche tool to necessity: the history of MS in the pharmaceutical industry

This article has been taken in its entirety from Pharmaceutical Formulation & Quality - the premier source of news and information for pharmaceutical and biopharmaceutical industry professionals.

By Gina Shaw
 

Darwin Asa, PhD, now the manager of marketing for mass spectrometry vendor Bruker Daltonics Inc., recalls all too vividly the first time he encountered a mass spectrometer. He was working on carbohydrates at a startup biotech firm in the 1980s. The firm's chief scientific officer, enamored of mass spectrometry for analyzing carbs, plowed about half the company's startup money into two mass spectrometers and an NMR [nuclear magnetic resonance] spectrometer.

"We had some of the first instruments that were MS-MS. It was supposed to be really special, but all I knew was that they cost $2 million apiece and we had to hire a guy from Switzerland just to run these instruments," said Dr. Asa. "You'd give him a sample of stuff you'd spent six months trying to purify, and he'd come back and say 'It's keratin,' or 'It's junk,' or 'I don't see any signal.' It wouldn't deliver what you wanted unless you could meet some very specific criteria."

The history of mass spectrometry in the pharmaceutical industry can, in some ways, be separated into two eras: BLC-MS (before liquid chromatography mass spec) - or, as Dr. Asa puts it, the "expert from Switzerland" mode - and ALC-MS (after LC-MS).

In the early decades of the 20th century, the newly developed mass spectrometer - the first fully functional one having been built by Francis William Aston of Britain's Trinity College in 1919 - was mostly the purview of physicists and academics. But soon after commercial production of mass spectrometers began in the 1940s, pharmaceutical companies began to recognize that the rapidly evolving instruments held great potential for the identification of organic compounds.

"Mass spectrometry has long been a very important and useful tool for the structural elucidation and structural confirmation of molecules, drugs, and drug metabolites," said Ian Jardine, PhD, vice president for global research and development with the analytical technologies group at Thermo Fisher Scientific. "For 40 or 50 years, that's been the core application of mass spectrometry in the pharmaceutical industry. Analyzing the pure drug, or the drug in mixture, or an isolated metabolite, by directly inserting it into the mass spectrometer was a common method."


GC Marries MS

PHOTO COURTESY OF BRIAN MURPHY, WATERS CORPORATION

PHOTO COURTESY OF BRIAN MURPHY, WATERS CORPORATION
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A VG Analytical Inc. Ltd Micromass Model 7070 Magnetic Sector Mass Spectrometer on display at the Manchester, England, Museum of Science and Industry. This instrument was made around 1974, weighs 1,600 pounds, and cost about $400,000 brand new. Manchester has a long and storied history of invention and is where John Dalton, the British chemist and physicist whose theory of atomic weights forms the basis for mass spectrometry, did most of his fundamental work in chemistry.

Mass spectrometry in drug development took a great leap forward in the 1950s with the coupling of gas chromatography (GC) with quadrupole mass spectrometers, which use a quadrupolar electrical field (comprising radiofrequency and direct current components) to separate ions. Wolfgang Paul's development of the quadrupole and quadrupole ion trap later garnered the Nobel Prize in Physics.

"When the GC was attached, it then allowed you to look at mixtures and move from the qualitative to the quantitative arena, which was a very useful technique," said Dr. Jardine. "But it did come with the clear disadvantage that you had to chemically derivatize the molecules before they would volatilize into the gas chromatograph. Still, GC-MS for both identification of drug/metabolite structures and also quantitation was a very powerful and useful technique through the 1970s and 1980s."

But in that era, mass spectrometry was definitely not for the "masses." "These things for a long time were temperamental toys that you couldn't really use seriously," said Ian Wilson, PhD, a senior principal scientist at AstraZeneca. "They were fun for research and fun for getting papers out of, but you couldn't use them for a very long period, because they kept falling to pieces. It used to require very specialized people to operate them - becoming a mass spectrometry expert meant years of training."

Ray Farmen, PhD, president of Eurofins AvTech Laboratories, recalls how difficult MS was to use during his early days at Bristol-Myers in 1980. "At the time, 85% of our assays used HPLC [high performance liquid chromatography], and only about 7% GC-MS, because although it was a really nice tool, it was a pain in the neck. It took a lot more skill and a lot more time than HPLC," he said. "If it took a day to prepare 80 samples for HPLC, it would take two or three days to prepare the sample for GC-MS."

Still, mass spectrometry offered advantages. "It was more sensitive - if there was a small amount you had to look at, GC-MS was the only tool you could use," said Dr. Farmen. "You always had this nagging concern with HPLC - is this assay really specific? I just see a peak in HPLC, and I'm thinking and praying that the peak I'm saying is my drug doesn't have a metabolite or another interfering substance under it."

But although GC-MS offered more sensitivity, the analytes had to be volatile. "That's not very useful for drug companies," said Dr. Wilson. "You can, with a lot of hard work, make involatile compounds volatile, but it requires specialized knowledge of chemistry, and it's certainly not quick."

Dr. Wilson compares the use of MS "before we could hyphenate" to the production process of making Jack Daniels. "You certainly didn't do things in a hurry. You had to isolate your material from the bulk drug or the urine or fecal sample you had, get it into pure form using preparative liquid chromatography, and insert it into the mass spectrometer, hoping that it was volatile enough to give you molecular ions before it fried on the probe."

And then, in the late 1980s and early 1990s, came a series of developments that would take mass spectrometry out of the "expert from Switzerland" mode into an era that put mass spectrometers on virtually every drug development benchtop.

In 1989, John Bennett Fenn published his groundbreaking paper, "Electrospray ionization for mass spectrometry of large biomolecules," which eventually earned him the 2002 Nobel Prize in Chemistry. Electrospray ionization produces ions from macromolecules without fragmentation, and it allowed LC to be directly linked with MC.


The Age of Electrospray

PHOTO COURTESY OF GARY SIUZDAK, PHD

PHOTO COURTESY OF GARY SIUZDAK, PHD
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Gary Siuzdak, senior director of the Scripps Center for Mass Spectrometry, pictured with a magnetic sector instrument that came on the market in 1990. This bulky mass spectrometer was state-of-the-art prior to the modern electrospray triple quadrupoles, time-of-flight, and Fourier transform mass spectrometry technology that have revolutionized pharmaceutical mass spectrometry.

"The part that's great about the mass spec is the detector; the part that's great about the HPLC is the pump and column. A lot of people tried to play around and combine the two, but it didn't work," said Dr. Farmen. "The problem was that in HPLC, what's coming out of the column is liquid, not ionized. How the heck are you going to get the liquid into an ion phase and into a vacuum?"

Electrospray solved that problem, and when used in combination with atmospheric pressure chemical ionization (APCI) - which was first reported in 1975 but came of age in the 1980s - it flung open the door for LC-MS.

"With APCI you took the end of your HPLC column, then applied a high voltage to it that ionized the heck out of everything," said Dr. Farmen. "Now they could spray tiny little droplets that were filled with ions and put them in a very drying environment where they would dry quickly and ions would shed from the droplet as it was drying. Your vacuum pump could then suck in the drying ions."

"Some other attempts for structural elucidation and quantitation with mass spectrometry worked, but nothing was terribly satisfactory until the development of electrospray and APCI," said Dr. Jardine. "These two techniques really allowed, for the first time, the easy coupling of LC to mass spectrometry without having to chemically derivatize the compound."

When that happened, mass spectrometers rapidly became ubiquitous in the pharmaceutical industry. Waters introduced its first benchtop LC-MS instrument in 1993, and others rapidly followed. "Once the manufacturers saw the gold mine ahead, within five years they made tremendous strides," said Michael Balogh, PhD, principal scientist in MS Technology Development at Waters Corporation.

In the early 1990s, as the drug industry became more adept at making more potent drugs, dose levels began decreasing by orders of magnitude - which meant that blood levels also went down by orders of magnitude. This made the newly hybridized LC-MS all the more important to pharmaceuticals. "It had become more and more difficult to develop assays to quantitate drugs in plasma with liquid chromatography and the UV detector," said Dr. Jardine. "The investigator would have to turn to fairly tedious techniques like derivatization of the drug through a fluorescent molecule, then using LC fluorescence. It could take anywhere from three to six months, and sometimes even longer with particularly difficult compounds, to set up a validated assay."

Gary Siuzdak, PhD, senior director of the Scripps Center for Mass Spectrometry in La Jolla, Calif., also points to a seminal paper in 1989 - the first reported application of electrospray with a triple-quadrupole MS for the direct analysis of enzyme kinetics. "I don't think Jack Henion gets enough credit for this work. He was the first to show that you could get great quantitative information on enzyme kinetics with this technology. Essentially, this work was a precursor to how MS has been used in the pharmaceutical arena," said Dr. Siuzdak.

New technologies have brought mass spectrometry to the masses. Pretty much any intelligent person in a lab can learn to operate today's LC-MS.

- Ian Wilson, PhD, Astra Zeneca


The Transformative Power of LC-MS

From the late 1980s to the early years of the 20th century, the success of LC-MS transformed drug development - and mass spectrometry itself. "Once an annual event for thought leaders and innovators in MS research, the American Society for Mass Spectrometry (ASMS) annual conference grew from 2,200 members in 1997 to twice that number in 2007," Dr. Balogh said. ASMS now has some 6,000 members.

If liquid chromatography hadn't been linked to mass spectrometry, the field would still be a niche structural identification technology, said Dr. Wilson. "New technologies have brought mass spectrometry to the masses. Pretty much any intelligent person in a lab can learn to operate today's LC-MS. They may not understand the physics of what's going on, but they can get an answer. It's the difference between Henry Ford building a car and you and me driving it."

The best instruments for quantitative analysis - those with the greatest sensitivity, specificity, and precision - came when electrospray, LC, and APCI were combined with triple-quadrupole mass spectrometry. "The triple quad dramatically improves selectively by analyzing not just the precursor ion, but for the precursor and its fragments simultaneously," said Dr. Siuzdak. "It adds a high level of specificity as well as sensitivity. Combine that with the LC separation, and you have a really wonderful technology that allows you to get great specificity and sensitivity."

More recent improvements in triple-quadrupole systems in the early part of the 21st century have made these systems almost like a GC-MS technology, said Dr. Siuzdak. "The down side for mass spec companies is that the investigators don't need to replace the instruments that much - in that sense, the companies ultimately have been their own worst enemy, by creating such great instruments."

Dr. Wilson also credits the advent of computerized data collection and analysis for the widespread adoption of MS in the pharmaceutical industry. "When I first started doing structural identification with mass spectrometry, what you got was a piece of pink photosensitive paper with your mass spectrum on it," he recalls. "You stuck two weights on each end of your meter-long piece of paper, and if something had a molecular mass of 500, you had to count, literally, one, two, three... Then somebody would phone you up, and you'd lose your place and start all over again. Today's metabolite profiling, comparing 40 or 50 chromatograms each, we simply could not do 20 years ago without an army of people all prepared to sit there doing excruciatingly boring stuff."

Within less than 20 years, MS has evolved from a niche tool for specialists to an absolute necessity for almost every pharmaceutical lab bench. Most experts today could not imagine the modern drug development process without MS. "Drugs to attack certain therapeutic targets have, of necessity, become more toxic. They have to be administered in smaller and smaller doses, and different pathways will lead to different metabolites, which can be toxic," said Dr. Balogh. "A lot of work has to be done at low levels, with high sensitivity and good specificity. Without mass spec, I think we'd be back to grinding plants."


Shaw is a freelance writer based in Montclair, NJ. Reach her at ginashaw@vagabondmedia.com.

This article has been taken in its entirety from Pharmaceutical Formulation & Quality - the premier source of news and information for pharmaceutical and biopharmaceutical industry professionals.

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