Changing the definition of productivity and performance

Over the past 30 years, hHigh pPerformance lLiquid cChromatography (HPLC) has become an indispensable technology for the modern analytical laboratory, with over 20,000 instruments sold each year. NGP talked to Dr Robert Plumb about the latest innovations in the field.

HPLC is ubiquitous in the pharmaceutical industry and is employed throughout the whole drug analysis process, including drug discovery screening, raw material analysis, impurity profiling, stability studies, pharmacokinetic studies and final product testing.

However, despite HPLC’s popularity, by the end of the 1990s it had reached its technical performance limitations with existing technology. In an increasingly competitive environment, pharmaceutical scientists were looking for a next-generation solution that would not only increase productivity and efficiency, but also improve the quality of the information output.

Waters Ultra Performance LC Technologies, first introduced with the ACQUITY UPLC System in 2004, created a new category of chromatography that uniquely addressed these needs. By utilizing high pressures and novel materials, this technological leap allowed organizations to combat their increasing bottom-line pressures with a higher productivity solution that utilizes familiar chromatographic principles, but with drastically reduced operating costs and improved decision-making from discovery to manufacturing.

Whether employed in a standalone manner in high volumes to address the complicated task of method development, in conjunction with capital-intense mass spectrometers to improve assay sensitivity and maximize performance, or in process analytical technology (PAT) analysis to facilitate real-time quality testing, UPLC has become the technique – and investment – of choice.

Streamlining development
The process of developing a reversed-phase HPLC method can take anywhere from weeks to months, incurring significant expense. Scientists have found that by utilizing UPLC technology for methods development, a six-fold improvement in throughput can be realized. This, in turn, reduces cost per sample and time of analysis considerably, while maintaining or improving separation integrity.

By developing rapid, high-resolution analytical methods, products can be brought to market faster, therefore improving the overall profitability of the assay. In one case, a leading pharmaceutical company was able to reduce its regulatory filing time by 50 percent by adopting this technology.

New method development calls for a well thought-out experimental design. A systematic screening protocol that explores selectivity factors such as pH, organic modifier and column chemistry allows chromatographers to quickly determine which experimental parameters are most effective in manipulating the selectivity of a separation. By employing this type of strategy, the total number of steps necessary to develop a method is reduced, providing an efficient and cost-effective approach.

Productivity improvements associated with employing UPLC technology for methods development are shown in the table. When we compare the UPLC methods development strategy to one directly scaled to conventional HPLC, we see a six-fold improvement in time. This significantly reduces the overall instrument time required to develop chromatographic methods to one work day, as opposed to one work week with conventional HPLC.

Extending performance
UPLC has been instrumental in facilitating unprecedented improvements in all fields of analytical chemistry; none more so than in the area of liquid chromatography/mass spectrometry (LC/MS). In countless examples, novel particle technology, low carryover and reduced cycle times have enabled extremely high-efficiency separations for improved resolution and sensitivity and increased throughput.

Waters’ partnership with MS instrument manufacturers provides seamless and direct integration, for vast improvements in sensitivity and better use of existing MS investments. In their 2006 paper, Hayes et al [1] combined ACQUITY UPLC with ABI/MDS SCIEX API 3000 and 4000 mass spectrometers to achieve improved sensitivity, and were able to successfully validate an assay for a tricyclic antihistamine and its major metabolite in human plasma.

Wang et al [2] also coupled ACQUITY UPLC with an ABI/MDS SCIEX API 4000 to produce a five-fold increase in sensitivity for the analysis of testosterone and its metabolites, while increasing throughput by a factor of two, improving analyte resolution.

In the field of human protein analysis, Pleasance et al [3] replaced a complicated, time-consuming nanoflow LC/MS/MS analysis with UPLC. The improved efficiency and resolution of UPLC when coupled to an ABI/MDS SCIEX 4000 QTRAP instrument for the quantitative measurement of a depleted serum digest allowed the analysis time to be performed “with a dramatic (~20-fold) reduction in run time.” The author concluded that “the results obtained by UPLC/MS/MS and the clinical analyser have shown that the two methods give comparable values.”

Sub-2 µm particle technology in longer LC columns has been of particular benefit for scientists performing structural elucidation experiments. Dear, James, and Sarda [4] showed how the enhanced efficiency of UPLC, coupled to a Thermo Fisher Scientific LTQ Linear Ion Trap mass spectrometer “improved chromatographic efficiency and sensitivity and at the same time providing diagnostic MSn data.” The authors observed a 45-fold increase in signal-to-noise compared to HPLC, with a 75 percent decrease in analysis time.

The overall performance of UPLC technology was evaluated by Goodwin, White and Spooner [5]. Here, the authors concluded that the technique was easily interfaced with MS instrumentation, demonstrated excellent column robustness after 1250 injections of protein-precipitated plasma, and produced an over three-fold reduction in run time.

Real-time process analysis
Manufacturers across all industries, including the pharmaceutical industry, have become highly focused on increasing quality and operational efficiencies. PAT is one component of a quality system that enables manufacturers to achieve efficiencies while increasing profitability and revenue. Quality and consistency (QC) are the two key parameters that can’t be tested into a product after it has been manufactured – a final QC test can only tell if you have met your final product specs. PAT is designed to enable manufacturers to consistently meet or exceed those final product specifications.

The benefits of PAT are significant and multifaceted. Companies that have PAT in place can increase the amount of product manufactured without adding more process equipment, increase their product portfolio without adding production lines, increase yields and make more material that will consistently meet product specifications. Conversely, companies that don’t implement PAT will feel the impact on the bottom line in scrapped material, higher processing costs and lower yields.

Most in-process QC today is done in traditional laboratories, and delays between sampling at line and receiving results are unacceptably long. A study by the Massachusetts Institute of Technology showed that producers spend more time analyzing their products than manufacturing them.

Traditional techniques like near-infrared and Raman spectroscopy have limitations: they rely heavily on chemometrics, they can’t differentiate between similar or like compounds, their sensitivity and dynamic range are poor and quantification is challenging.

On the other hand, LC has incredible resolving capability. It is very sensitive and can detect impurities down to levels of 0.01 percent in the presence of main components, and is used to validate and check the other methods like near-infrared and Raman spectroscopy.

The PATROL UPLC Process Analyzer advances LC performance capabilities by bringing UPLC technology to the production floor for real-time analysis. It draws a sample from the slipstream of a production line, fermentor, reaction tank, or process LC system; performs an automated analysis; and generates a result in just three to four minutes. Intermediates that may have very similar chemical properties to the active pharmaceutical ingredient can be easily resolved. Thus, the power of LC is combined with the speed of a real-time sensor with UPLC.

References:
[1] J Pharm Biomed Anal, 2006 Feb 24, 40(3): 689-706
[2] Rapid Commun Mass Spectrom, 2006 20(14): 2215-21
[3] Rapid Commun Mass Spectrom, 2007 21(16): 2585-93
[4] Rapid Commun Mass Spectrom, 2006 20(8): 1351-60
[5] J Chromatogr Sci, 2007 Jul 45(6): 298-304

Dr. Robert Plumb is responsible for the laboratory function within the Pharmaceutical Business Operations of Waters Corporation. He is also currently an Honorary Reader in the Faculty of Medicine at Imperial College London.