QUALITY CONTROL - Analytical Methods | LC/MS/MS Laboratory Workhorse for Generics

By Johnny Cardenas Drug companies leverage new opportunities
The window of opportunity opens wide for generic pharmaceutical companies when blockbuster drugs come off patent. As we approach the next few years, when a number of notable blockbusters such as Lipitor, Plavix, Advair, and Singulair come to the end of their patent protection, generic pharmaceutical companies are noticeably ramping up their drug development capabilities and are poised to reap big profits. These companies have evolved into giants in recent years, and the contract research organizations (CROs) they depend on are proliferating in many parts of the world. Because the window of opportunity is relatively short and time-sensitive, generic drug development must start early and move quickly so that products are available the minute they can be put on the market legally.

Whether increasing their own laboratory bandwidth or outsourcing more work to CROs, generic pharmaceutical companies are implementing more studies with shorter and shorter timelines. By far the most common research activity in generic drug development is the bioequivalency study, which identifies and quantitates metabolized molecules to document the pharmaceutical/therapeutic equivalence between a generic drug and its brandname counterpart.
Many generic companies have dozens of studies going on simultaneously, requiring the processing of hundreds of thousands of samples. High performance liquid chromatography (HPLC) with triple quadrupole mass spectrometry (LC/MS/MS) is the gold standard for this pharmaceutical bioanalytical quantitation, and instrument manufacturers have been working at breakneck speed to deliver system improvements that will help drug developers move faster and more efficiently.
LC/MS/MS replaced LC-UV (liquid chromatography with UV detection) analysis for these bioequivalent studies in the 1990s, providing vast improvements in sensitivity, selectivity, and throughput. Emerging powerhouses such as India and China are also adopting this analytical technology as they join the global competition.

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Figure 1: This figure illustrates 1,300 plasma injections over three days on the AB SCIEX QTRAP 5500 System. It demonstrates excellent reproducibility for the target compound and internal standard responses with a 3.3% coefficient of variation (CV) on the peak areas and a 2.3% CV on the peak area rations.

LC/MS/MS has revolutionized the speed at which clinical and preclinical studies can be done, but throughput is only one advantage required by generic pharmaceutical companies. They also need ruggedness and robustness, systems that can deliver reproducible results over time and run the highest number of sample injections without cleanup. A rugged LC/MS/MS system will produce the same data day to day, and a robust one will deliver maximum time between failures (see Figure 1, above).

Canadian Case Study

Pharmascience is one of two large generic pharmaceutical companies based in Canada and produces an impressive portfolio of single-source prescription drugs. In Canada alone, the company fills more than 21 million prescriptions a year. According to Tristan Booth, PhD, the company’s vice president of innovative development, “we’ve improved our high-throughput bioanalytical lab with multiple high performance LC/MS/MS systems, to the point where we can do 50 studies in one year, processing 80,000 to 100,000 samples.” With seven AB SCIEX LC/MS/MS systems powering this bioanalytical lab, the company has now been able to devote more resources to discovery, including Phase 1 clinical trials.
Increased detection sensitivity has also improved the efficiency of generic pharmaceutical research. Biological samples contain compounds that can interfere with an assay. Preparation steps such as liquid- or solid-phase extraction can clean up the sample before processing, but they add time and cost to the study. With increased LC/MS/MS instrument sensitivity, these samples can be diluted rather than prepared with extraction techniques, allowing the injection of five to ten times less sample to obtain results.
Even highly polar analytes that are difficult to separate from a matrix can be analyzed more clearly with the latest highly sensitive instruments. The limit of quantitation (LOQ), which is the lowest concentration at which quantitative results can be accurately reproduced, can be lowered with newer, more sensitive systems such as the AB SCIEX QTRAP 5500 System.

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Figure 2a: The figure to the right shows a liquid chromatography chromatogram displaying the excellent signal-to-noise (S/N) ratio in an analysis of budesonide from a dried blood spot sample using the new LC/MS/MS method. The ratio is 15 with a 3.2% coefficient of variation (CV). The figure below shows a calibration curve for budesonide verifying that the accuracy and precision for this new LC/MS/MS method on dried blood spot samples are well within the limits of acceptance.

“Advancements in LC/MS/MS technology have delivered increased sensitivity for metabolized molecule identification from small sample concentrations in the sub-picogram range,” said Dr. Booth. Increases in selectivity allow Pharmascience to analyze 1,000 samples in two to three days, a significant improvement in throughput over just a few years ago.
Technological advances in LC/MS/MS, such as the TurboV Ion Source (AB SCIEX), provide increased sensitivity that reduces sample prep requirements, delivering improvements in LOQ, throughput, and robustness. Curtain Gas technology (AB SCIEX) protects the mass spec interface region and quadrupole analyzer from contamination, reducing routine maintenance requirements.
Data processing and advanced algorithms are another dynamic factor in successful bioanalytical analysis for generic drug development. As throughput increases on smaller and smaller sample volumes and concentrations, quantitation becomes more challenging. The ability of new triple quadrupole systems to monitor ever-increasing numbers of components has led to the need to improve data processing speed and accuracy. Mass spec companies continue to develop novel algorithms and software to keep pace with throughput and data generation advances.

CROs Add Analysis Bandwidth

Generic pharmaceutical companies are also ramping up by using CROs, which have significantly more LC/MS/MS analysis bandwidth. Ideally, both organizations will use the same LC/MS/MS platform to speed up the cross-validation process and eliminate method transfer issues. “Any CRO we use must have validated our methods,” said Dr. Booth. “If they are using the same LC/MS/MS platform that we have, validation is not an issue.”
As these generic drug companies look to the future, it’s clear that growth in opportunities will slow significantly as industry reaches the impending patent cliff, when the last of the big blockbusters go off patent. What looks like gloom and doom for big pharma will be a major catalyst for the growth of generic drug companies, which are embarking on new areas, including researching novel drug formulas, exploring new dosage forms, and even looking at drug repurposing. “We are diversifying our pipeline in anticipation of this market change,” said Dr. Booth. “Our LC/MS/MS capability has freed up resources so we can do more work in Phase 1 clinical trials, safety testing, dose escalations, etc.”

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Figure 2b.

As generic companies move beyond synthesizing blockbuster drugs to new and innovative endeavors, their analysis challenges expand to include qualitative pharmacokinetic studies, such as absorption, distribution, metabolism, and excretion. Complementary LC/MS/MS technology can meet these needs and streamline the transfer of methods from discovery to development. “It’s important to us to have backup and downstream LC/MS/MS systems that are all on one platform so we don’t waste time changing methods as we move down the pipeline,” said Dr. Booth.
The latest development for the analysis of small molecules in drug development is dried blood spot (DBS) sampling. DBS sample collection is easier and less invasive, and shipping and storage costs are significantly reduced. However, these small spots, between 20 and 100 times smaller than plasma samples, present new analysis challenges and require very high sensitivity detection. Many pharmaceutical companies and instrument manufacturers are working on ways to improve DBS sampling and analysis.
To demonstrate the feasibility of LC/MS/MS analysis on these samples, AB SCIEX recently developed a method for the quantitation of inhaled corticosteroids used in the treatment of asthma using DBS samples. These drugs, which are absorbed through the nasal passage and lungs before systemic metabolism, are found in very low concentrations in the blood. The use of DBS sampling for the pharmacokinetic study of these bronchodilators presents even lower sample concentrations.
Using the latest LC/MC/MS technology in the AB SCIEX QTRAP 5500 System, we were able to develop an accurate, verifiable method that enables pharmacokinetic and toxicokinetic studies of bronchodilators such as budesonide and fluticasone propionate. Figures 2a and 2b (see above) shows that quantification of inhalation drugs with low systemic circulation is feasible with DBS using ultra HPLC and high sensitivity triple quadrupole instruments. Sufficient sensitivity for 5-10 pg/mL LOQs can be achieved.
At virtually every point in the generic drug discovery pipeline, LC/MS/MS is an essential analytical tool, enabling research to advance and keep pace with ever-changing market requirements.