Increasing Productivity with UHPLC

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Ultra high-pressure liquid chromatography (UHPLC) is enabling faster product development and production batch approvals with increased sensitivity.

Pharmaceutical laboratories are under pressure to increase productivity in order to speed up product development. The growing interest in process analytical technology has increased demand for more rapid analytical techniques that are appropriate for use in the manufacturing environment. More rapid analysis of production batches is needed to get material to market faster. Ultra high-pressure liquid chromatography (UHPLC), introduced in 2004, has become an important analytical tool for meeting these varied needs for higher throughput. Analytical instrument makers believe that further advances in the technology will enable UHPLC to have an even bigger impact on the pharmaceutical industry.

UHPLC meets many needs
“The field of separation science was revolutionized with the introduction of the first commercially available UHPLC system,” asserts Eric Grumbach, senior product marketing manager for separations technologies with Waters Corporation. “Today, there are tens of thousands of these systems in use, clearly indicating that the inherent business and scientific benefits of UHPLC technology are being realized,” he continues.Many factors are contributing to the demand for UHPLC. First, according to Grumbach, is the fact that the pharmaceutical and other industries, are under increasing pressure to improve overall profitability, from research and development through manufacturing and distribution, all the while facing the challenge of diversifying their product portfolio into new and unfamiliar territory. “One avenue towards improving profitability is to increase the throughput of their laboratory and manufacturing operations in an effort to decrease product development timelines and bring their products to market faster,” he notes.

The need for quicker results, but with moderate sample sizes, is another driving force behind the implementation of high throughput UHPLC, according to Michael Frank, marketing manager for HPLC at Agilent Technologies. “For the release of a production batch, not only the one sample has to be analyzed, but easily up to 20 if counting blanks, standards, repeated analyses, etc. The approval process using conventional HPLC, with typical run times of 20 minutes, can take nearly 7 hours. UHPLC, which is often 5 to 10 times faster, allows a significant reduction in the analysis time, and thus, production batches can be released in much less time. As a result, produced goods can be packaged and shipped sooner, streamlining the complete process and saving money.”

The online monitoring of products directly in the manufacturing facility is also much more feasible when using UHPLC compared to HPLC, according to Rainer Bauder, HPLC solutions manager for Thermo Fisher Scientific. “With UHPLC, the results are available within a few minutes rather than 30 or 45 minutes, which enables an immediate response to any undesired condition within the production cycle. Similar benefits can also be applied to process development and cleaning validation.”

Multiresidue methods, including UHPLC, are also becoming the preferred way to monitor for drug residue contamination as the number of contaminants that are monitored increases, according to April DeAtley, LC product planner from PerkinElmer. “The combination of more complex analyses and a growing number of samples means that longer runs cannot be tolerated in high throughput labs, where the emphasis is put on achieving the maximum chromatographic resolution in dramatically reduced times.”

The chemistry of UHPLC
UHPLC is based on stationary phases using smaller particles. While conventional HPLC assays use 2.5–5 µm separation media, UHPLC assays use smaller 1.7–1.8 µm column chemistries that offer up to a three-fold higher separation efficiency. Thus, the same exact separation can be achieved in a column that is threefold shorter, which directly translates into higher throughput due to shorter analysis times. Alternatively, better chromatographic resolution can be achieved with UHPLC compared to conventional HPLC if the same method conditions are used. Bauder adds that in addition to faster results, the power of UHPLC as a technique lies in its extreme versatility, the opportunity to reduce operation costs and solvent consumption, and the ability to develop higher resolving methods for new products, which mitigates the risk of missing problematic impurities during drug development or production. Nearly all instrument suppliers offer online calculators for establishing equivalent conditions for UHPLC and HPLC. Thus, the analytical chemist now has the flexibility to decide between the same resolution and higher speed or the same speed and higher resolution, according to Frank.

Advancing the technology
While it may seem contradictory, one recent advance in UHPLC technology has been the introduction of the ability to run conventional HPLC methods on UHPLC systems. “Pharmaceutical customers need to be able to run legacy HPLC methods because these methods have been validated for approved drugs, and any change in the QC method requires revalidation, a halt in production, and an interruption of the revenue stream,” explains Grumbach.

There have also been a number of developments with respect to separation column technologies, according to Bauder. “New sub-2 micron particle and core-enhanced column technologies boost separation efficiencies and are triggering the development of new generations of column chemistries that offer improved selectivity for previously problematic target molecules, such as small, highly charged ions and mixtures of acidic, neutral and basic analytes. Grumbach points to the development of alternatives to U(H)PLC systems based on supercritical fluid chromatography (SFC), which he believes has enabled the development of a truly exceptional selectivity tool that is orthogonal to traditional reversed-phase methodologies. “This mode of chromatography has proven to be a clear replacement for normal-phase chromatography for most applications, and one that is ideally suited for chiral analysis. As an added benefit, the reduced solvent usage has significantly reduced assay cost and allows organizations to meet sustainability initiatives.”

The ability of the instrumentation to withstand increasingly higher pressures is also critical to the advancement of UHPLC technology. “Entire systems are now able to tolerate greater pressures, including pump backpressures and detector flowcells. As a result, UHPLC systems are increasing in their overall performance,” notes DeAtley. Bauder adds that the development of novel detection techniques fully supporting UHPLC-type separation deliver additional leverage for faster and more in-depth sample assaying. Thermo Fisher Scientific has also focused on improving workflows around and within UHPLC instruments, including the introduction of automation tools, such as its x2 dual gradient UHPLC pumps and autosamplers that perform sample injection and fraction collection.

Challenges to overcome
Workflow, in fact, is one of the factors limiting the ability of pharmaceutical companies to fully leverage UHPLC technology. “A consistent theme we have heard from many of our customers is that the bottleneck of their workflow has moved from analyzing their samples, to preparing those samples prior to analysis. There is a need to make sample preparation workflows more efficient such that they can keep pace with the higher throughput analysis that UHPLC technology inherently provides,” Grumbach observes. Frank believes, however, that automated liquid-handling systems and the ability to make automated changes with different ultra high-pressure valve solutions for rapid switching to different solvents and columns is increasing the flexibility of UHPLC systems.

Other limitations of UHPLC are being addressed with column technology, according to DeAtley. “Solid core column technology is allowing for very fast runs, incredible resolution, and lower pressures, which takes much of the stress off of the system,” he comments. Bauder believes that, in addition to needed advances in system control and sample management, there remains significant improvement potential with respect to data processing and detection technologies, and that new and exciting column technologies are also showing great promise.

Anticipating the future
Clearly, much more can be expected from UHPLC in the future. DeAtley believes that column technology coupled with UHPLC capabilities will work together for lower pressure UHPLC applications with the benefit of fast run times and overall cost of ownership benefits. “We are definitely continuing to explore the boundaries of UHPLC technology. Further advances, however, may not come in the form of smaller particles (e.g. 1-µm particles) and higher pressures, but rather through the use of miniaturization, microfluidics, and different particle technologies,” says Grumbach. Concludes Bauder, “The ultimate goal for UHPLC is to reduce the variety of separation methods required by end users while optimizing run time and sample throughput. Near-universal separation methods, as well as near-universal or (alternatively), highly selective detection technologies, have the potential to transform the chromatography lab and make UHPLC the methodology of choice.”