Pharmaceutical Analysis

Pharmaceutical analysis continues to be a major focus of the analytical chemistry industry. Participants in this discussion are Richard Larsen of Agilent Technologies, Gary Dowthwaite of Biotage, Richard M. LeLacheur and Rohan Thakur of Taylor Technology, and Pat Bennett of Thermo Fisher Scientific.

What are the current major challenges in pharmaceutical analysis?

Larsen: Instruments that enable faster sample turn-around in the lab thereby reducing time to market and a prolonged life cycle of the drug will continue to be attractive to Pharma. Reinforced cost containment required a higher cost/performance ratio for new investments in all price segments. Automated sample preparation to increase through-put and quality of results is always a challenge. The need for faster measurement requires more effective data processing and content management. Regulatory requirements need to be satisfied with less overhead.

Dowthwaite:Sample size reduction and the need to simplify the analytical workflow are of paramount importance. In order to minimize sample preparation, less selective methods are often employed to remove phospholipids, salts, and proteins. However, supported liquid extraction plates can be used with acidic, neutral, or basic analytes, and provide a simple load-wait-elute methodology. The analytes are eluted in an organic solvent compatible with analysis. The increased use of supported liquid extraction has dramatically simplified sample preparation and offers a cost effective alternative to liquid–liquid extraction and solid-phase extraction.

LeLacheur and Thakur:Challenges continue to exist in pharmaceutical analysis, particularly at the intersection of scientific and regulatory concerns. The recent addition of incurred sample reproducibility testing has raised awareness of unstable metabolites that can affect sample results, but that are absent from quality control (QC) sample testing for short and long term stability. The conditions of sample preparation such as pH and temperature exposures are of particular concern, but it may be difficult or impossible to assess their impact when neither reference standards nor incurred samples are available during method development and validation.

The differences in global regulatory requirements are also challenging when a new molecular entity is in simultaneous trials around the world. Harmonization of requirements, method validation, and sample analysis will significantly streamline our ability to meet both regulatory requirements and critical timelines efficiently.

Bennett: Over the last three years, the main challenges facing pharmaceutical scientists performing sample analysis have been wide-ranging and more extreme than in previous years and are not all analytical in nature. These differences result from multiple sources including dramatic organizational changes, analytical instrumentation and separation technology performance and availability, more complex projects, and maintaining GLP compliance concurrent with the implementation of new technology and requirements.

The recent recession accelerated a consolidation process within the pharmaceutical industry resulting in the loss of thousands of scientific positions. Also, because investors moved to a capital conservation mode, the recession resulted in changes to the way small, innovative pharmaceutical companies managed their portfolios and restricted the entry of new companies into the R&D arena. The resulting changes to the focus and needs of pharma companies create organizational challenges irrespective of the organization’s size or geographical location. These challenges affect not only the internal operations, but also affect their suppliers, contract research organizations (CROs), and instrument vendors. The primary consequences of these changes are limitations for personnel, instrumentation, laboratory space, training and other resource availability, the need for improved information flow, and challenges communicating analytical requirements.

Emphasis will be less on high throughput and more on the effectiveness of throughput. For the scientists, this means learning how to maintain productivity and quality with fewer resources concurrent with a reduced ability to invest in new or unproven technology. Fewer scientists will be able to work on the cutting edge of technology and those that do will do so while continuing with their expected role and output.

There are specific challenges that result from the recent recession. Travel restrictions reduce the ability of scientists to share and exchange ideas and scientific findings, as well as continued education. This can have a significant impact on less experienced scientists. Because scientists have less time and resources to explore and evaluate new technology, techniques, and software, the vendors that research, develop, and sell these products and services cannot thrive. This can result in prevention or delays in new product introductions and exacerbate the challenges analytical scientists face.

Historically, regulatory pressures have been challenging. While the core aspects of GLP and GMP have not changed and there has been significant industry agreement on topics including method validations, security, electronic audits, and system validations, there have been recent additions that create new challenges.

The most significant addition was the implementation of incurred sample repeats (ISR) for bioanalytical sample analysis. The industry adapted to this reasonably quickly, but it is becoming clear that a primary reason for ISR failures is the instability of analytes or their metabolites in the biological matrix. This creates a new challenge on how and when to test for incurred sample stability.

Other regulatory challenges include the Metabolites in Safety Testing (MIST guidance) and how regulatory guidelines are applied to both large molecule and biomarker studies. The expectation to apply CLIA or GLP to certain biomarker studies has resulted in confusion and unclear application of these guidelines. The rapid change in technology available for large molecule quantitation creates significant issues when they are used to analyze samples that fall under GLP or GMP guidelines. As more biologics are approved by regulatory agencies, the issues that are present in GLP laboratories will become prevalent in GMP laboratories.

From an instrumentation and separations technology perspective, matrix effects and analyte carryover in autosampler and high performance liquid chromatography (HPLC) columns persist as significant issues. Because the volume of matrix sample available continues to be reduced, sensitivity can still be challenging even as we see gains in the sensitivity of the mass spectrometers. ISR and the use of dried blood spots are important drivers in the need for sensitivity and selectivity in situations where there is limited sample volume.

From a chemistry perspective, endogenous materials (for example, phospholipids), increasingly complex formulations (for example, PEGs, lipids, protein linked molecules), degradation products or metabolites of these formulations, unstable molecules, and pro-drugs all present scientists with an array of challenges that continue to require ingenuity and skill to overcome.

Which techniques and instrumentation have shown the greatest change over the past few years in response to changing requirements in the pharmaceutical industry?

Larsen:The greatest change is the move from HPLC to ultrahigh-pressure liquid chromatography (UHPLC) and higher sensitivity for both UV and LC–mass spectrometry (MS) detection. The entire market had to adjust to solvent reduction and green chemistry is leading to a revival of supercritical fluid chromatography (SFC). We are seeing a higher degree of automation for method development and multi-method execution as well.

Dowthwaite:The uptake of supported liquid extraction in the pharmaceutical and CRO industry has highlighted the need for quick, simple, generic clean-up techniques. The methodology is much simpler than many SPE protocols and mimics liquid–liquid extraction. Because there are no offline steps, supported liquid extraction is automatable with industry standard liquid handlers. The overall extraction process is quick, providing very low prep cost per sample. Also, because the samples are free from compounds that cause ion suppression–enhancement in mass spectrometry, sensitivity can be greatly increased.

LeLacheur and Thakur:For quantitative bioanalysis, the triple-quadrupole mass spectrometer has seen little change beyond sensitivity improvement for many years. However, there have been significant improvements in liquid chromatography, which feeds into these systems with the advent of UHPLC systems. These systems offer the potential to improve separations quality and reduce analysis time — a pairing that was often seen as mutually exclusive.

The routine use of high-resolution mass spectrometry (HRMS) has revolutionized qualitative bioanalysis over the last decade and is now poised to enter quantitative bioanalysis. HRMS systems offer the discovery lab improved throughput by eliminating the need for compound-specific tuning, yet simultaneously offer enhanced data content by allowing the analyst to screen and estimate metabolites in the same analysis. During development, HRMS offers a complementary tool to the triple quadrupole, and will yield improved specificity and sensitivity for certain compounds.

The drive to use LC–MS-MS or LC–HRMS for quantitation of peptides and proteins is particularly exciting. These analytes offer unique challenges in all phases of sample preparation, chromatographic separation, and MS detection. In the latter for example, determination of the optimal charge state and possible role of HRMS in quantitative analysis are new questions for the chemist with small molecule experience. Likewise, standard sample preparation techniques need to be reexamined to control protein digestion, adsorption, and stability issues.

Bennett:Mass spectrometers and the instruments used to detect and quantitate large molecules such as proteins have shown the greatest change. The improvement of high resolution, for example accurate mass (HRAM) instruments, has been significant. These instruments include the orbital trap and time-of-flight–based mass spectrometers. The improvements have resulted in instruments that are significantly simpler to use, more robust, and more useful to a broader group of scientists. Current HRAM mass spectrometers are finally allowing a merger of two distinct workflows — that is, quantitative and qualitative analysis. They also allow greater throughput and more accurate information for proteomic, metabolomic, and metabolic sample analysis.

The increased implementation of alternative mass spectrometer interfaces including laser diode thermal desorption (LDTD), direct analysis in real time (DART), and desorption electrospray ionization (DESI) are indicators that scientists need to analyze samples faster and with little or no sample preparation. This is particularly important in discovery and product release laboratories where speed to results reporting is paramount.

Because of the increased research and development of large molecule therapeutics, new technologies that make the quantitative analysis of these compounds simpler, faster, and, in some cases, more specific have been introduced over the last five years. These include multiplexed electrochemiluminesence– and flow cytometry–based instruments.

The technique that has shown the greatest change is not a change in a technique itself, but in the application of an older technology to a new area. The use of dried blood spot (DBS) to acquire, ship, store, and test samples is not new. However, it has gained tremendous visibility and is rapidly being adopted in all aspects of bioanalysis. In the near term, it is actually more challenging for bioanalytical scientists. The development of automation for the DBS format and newer technology similar to dried blood spotting will be developed to overcome that challenge.

Software is also a significant area showing change in response to changing timeline and regulatory requirements. Shorter timelines are a part of every aspect of drug discovery and development. Because software is critical to simplifying and shortening many workflows, it is not surprising to see an increase in providers and products. These include electronic laboratory notebooks (ELN) that can be used for basic R&D, discovery, and routine regulated sample analysis; specialized software used for metabolism and structure elucidation, automated mass spectrometer tuning for compound libraries, proteomic and metabolomic applications and databases; and software that can link data from a variety of sources into a central application for simplified reporting.

Have pharmaceutical analysts successfully adopted UHPLC as a routine technique?

Larsen:Customers require flexibility to choose between HPLC and UHPLC. Still a large number of validated methods do require standard HPLC methods. For new developments, UHPLC is the way to go. Still, customers look for solutions to achieve the same or better analytical results with better column technology, such as superficially porous particles.

Dowthwaite:Those scientists who have migrated to UHPLC request sample prep methods that are free from particulates. Supported liquid extraction products provide extracts compatible with this technology.

LeLacheur and Thakur:UHPLC has absolutely been a success, yet as a routine technique it is still not universal. We have seen rapid adoption by some companies, but reluctance from others. Early issues included the typical challenges of reliable connectivity, particularly when the UHPLC–MS-MS system worked across vendor brands, validation concerns for regulated bioanalysis, and of course reluctance to change. At present, the performance of these systems is unchallenged, and we continue to increase steadily our UHPLC capacity.

Bennett:Yes, the pharmaceutical industry has successfully adopted UHPLC as a routine technique. However, its implementation will not replace traditional HPLC, and UHPLC has not been implemented as broadly as expected. The initial reason for this was not unexpected and resulted from the narrow selection of chemistries and dimensions available commercially. However, the reduction in capital equipment purchases caused by the recession is likely a significant reason for the slower implementation. The reduced funds for capital equipment forced scientists to be creative in the way they increased throughput without buying UHPLC systems. The careful use of HPLC columns with 2- or 3-µm particle size allowed scientists to perform very fast chromatography with acceptably high resolution on traditional HPLC pumps. Scientists also recognized that they could obtain adequate resolution at higher flow rates with shorter columns (for example, 10–50 mm length) with 5-µm particle size and take advantage of modern atmospheric pressure ionization (API) MS sources that can handle flow rates up to and above 1 mL/min. Column switching and multiplexed sample introduction systems also allowed scientists to increase throughput without purchasing UHPLC systems. There are still advantages to UHPLC and these systems will be implemented increasingly in research, GLP and GMP applications for proteomics, metabolomics, lipidomics, formulations, and traditional small molecule analysis.

What challenges are on the horizon for analytical chemists in the pharmaceutical industry?

Larsen:We see method compatibility and method transfer as a significant challenge. Fewer chemists in the lab require flexible use of systems, including ease of operation and maintenance. It takes a long time to revalidate a method once it is changed. With the advent of fast separation techniques, routine labs would like to take advantage of them and transfer them as efficiently as possible. Another challenge is the cooperation between the drug development department and QC. Methods, which are developed, need to be validated and there must be good cooperation to ensure that knowledge is transferred into labs. The adoption to emerging regulations such as process analytical technology (PAT) and quality by design (QbD) will play a role as well. Recently, a focus has been put on genotoxic impurities.

Dowthwaite:The challenge in sample prep is to provide methods or instrumentation for emerging molecules of pharmaceutical and clinical interest from novel matrices.

LeLacheur and Thakur:On an individual level, the landscape has shifted in two major ways. First, the era of secure employment at the major pharmaceutical companies has ended, and jobs continue to shift to both CROs and to smaller pharmaceutical companies. Second, and simultaneously, the geography of employment has changed from a heavy focus on Western Europe and North America to a more balanced global presence.

In addition, chemists must grow their individual skill sets. Often overlooked are the skills necessary to thrive in the fluid global industry — communication skills across languages, cultures, and large distances, for example, are not a focus of education or training, yet are critical in mastering the modern environment. Similarly, our technical skill sets must evolve. We need to embrace and drive technological change by continually reeducating and training ourselves in areas such as HRMS.

Bennett:Workflow will be a keyword within organizations. The “top down” pressure to reduce costs and “do more with less” will drive scientists to use simpler and integrated workflows — that is, from designing experiments to performing and reporting results. But also, materials, reagents, equipment, and software that are designed to work together will increasingly be important. This includes how they can be ordered, stocked, used and reordered, maintained, and so forth. The ability of chemists to integrate themselves and their workflow into a rapidly changing organization is the primary challenge. This will be true for analytical chemists around the globe. A challenge that will continue to increase is the integration and harmonization of analytical chemistry in a true global industry. Other industries have faced this and are working their way through their issues. It is important for analytical scientists to observe these other industries to identify what might work and might not work to overcome these challenges.

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