Optimize drug development with advanced bioanalysis

We have come a long way since discovering the first therapeutic protein, insulin, in 1921¹. In fact, contemporary medicine is driven by an ever-expanding knowledge of the therapeutic utility of biological molecules. The options available to today’s patients – including simple proteins, antibodies (and antibody-drug conjugates), and oligonucleotides – is testament to this. But as our knowledge of these molecules has grown, so has the need for a diverse array of bioanalytical methods to evaluate them and provide data that supports regulatory submissions. Historically, bioanalytical laboratories developed their own standards for validating methods to align with guidelines issued by their home countries’ regulatory agencies. This led to major variations between centers, and therefore the need for global harmonization.

Alturas Analytics is a contract research organization (CRO) that offers solutions to the challenges that arise during bioanalysis, including support with method development, feasibility testing, and validation. The company utilizes validated LC-MS/MS instruments (including ultra-performance microflow LC systems) and offers GC-MS/MS bioanalytical services for small molecules, volatile or semi-volatile compounds such as prodrug biproducts. Alongside its bioanalytical services, Alturas Analytics provides PK and TK analysis and reporting to deliver good laboratory practice-compliant analytical packages to its sponsors. The result is a team of experts that can help sponsors translate their data into insights to make informed decisions during drug development.

Bioanalytical methods and regulatory standards

Bioanalytical methods employed for the quantitative determination of drugs and their metabolites in biological fluids provide essential regulatory data for pharmacokinetic, toxicokinetic, bioavailability, and bioequivalence studies. The quality of these studies is directly related to the integrity of bioanalytical data. In 2001 the US FDA finalized the first ‘Guidance for Industry on Bioanalytical Method Validation’ that was instrumental in setting standards for assays supporting drug submissions within the US.

The bioanalytical workplace has become global, necessitating universal rules for quality and compliance for bioanalysis. Data generated by a typical bioanalytical laboratory may be submitted to multiple regulatory agencies. “The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) formed a working group in 2016 to address regulatory differences within the scope of drug development. The ICH M10 ‘Harmonized Guideline’ provides recommendations to ensure reliable data is generated to support regulatory decisions globally,” says Ann Hoffman, Vice President of Strategic Development for Alturas Analytics. “This guidance has helped standardize testing within countries and around the globe. Advantages of this alignment include the generation of data that are consistent despite their geographical origins, increased predictability, and the capacity to make more informed decisions.” However, which techniques are used and how has this changed over time? And what should we expect from future analyses?

Ann Hoffman
Vice President of Strategic Development at Alturas Analytics

A shifting landscape

A myriad of techniques are applied to analyze molecules, including variations of gas or liquid chromatography coupled with mass spectrometry (tandem MS/MS and hybrid assays), ligand-binding assays (LBAs), and the polymerase chain reaction (PCR). The key factor driving methodological decisions is the nature of the target analyte. “The type of compound, its size, and stability are key considerations when choosing an analytical platform,” Hoffman explains. “For example, GC-MS can be useful for very small and volatile molecules that are not amenable to LC-MS. The sensitivity and lower limit of quantitation are also important factors to consider, as is the time taken to complete the analysis. The expectation is to produce a reliable assay that runs as quickly as possible without compromising the integrity of the results.”

“The most common method for quantitation to support pharmacokinetic (PK) analysis and toxicokinetic (TK) studies is LC-MS/MS using triple quadrupole instruments,” Hoffman affirms. “This is especially true for small molecules, but LC-MS/MS is being increasingly used for smaller oligonucleotides and proteins (these are typically digested into smaller signature peptides before analysis). The increasing availability and affordability of high-resolution MS approaches is also leading to the wider use of LC-MS in situations requiring higher specificity, such as the analysis of target analytes within a complex matrix, biomarkers, and oligonucleotides.”

Ann Hoffman
Vice President of Strategic Development at Alturas Analytics

LBAs are typically used to study larger biomolecules, and these approaches can also be coupled with LC-MS to improve analytical specificity. When it comes to cell and gene therapies or immunotherapy, PCR, flow cytometry or both often constitute the methods of choice. “Standardized methods for analyzing these new compounds are still evolving. Researchers along with instrument manufacturers are challenged to develop technologies in order to keep the pace,” Hoffman explains. “The development of highly specific and highly potent compounds has also led to a need for very selective methods with greater sensitivity. It’s all very positive, but also makes our job as bioanalytical scientists more complex.”

Jumping over hurdles

“The greatest challenge in analyzing these compounds is the speed at which the landscape has changed,” Hoffman says. “And, despite the ICH guidelines, harmonization still represents an issue for teams in this space. The global bioanalytical industry is much more aligned than it once was, but guidelines change regularly and newer recommendations tend to add more complexity to their predecessors. Teams must adapt to these changes by updating their standard operating procedures that often require additional experiments. Though these changes tend to lead to more robust, consistent data, it can be difficult to keep pace with evolving expectations.”

There are conditions when fit-for-purpose validations are acceptable. Robustness of the method must be demonstrated and suitable for its intended use. For analysis of emerging modalities, methods may not yet be standardized and specific experiments recommended in the guidances may not all be applicable to techniques used to quantitate the target analyte. In Hoffman’s words, “There are many variables with fit-for-purpose validations because the choice of experiments is context-driven. Biomarker assays are typically fit-for-purpose as we’re looking at variations in levels of endogenous compounds, but may also apply for quantitation of analytes in complex matrices like tissue homogenates.”

Ann Hoffman
Vice President of Strategic Development at Alturas Analytics

Shifting gears

Bioanalytical method validation is an essential practice that supports key experiments during the preclinical and clinical stages of drug development. Harmonized guidelines from the ICH have provided some much-needed standardization for the field, but continued progress will rely on collaboration between centers of excellence. “There has been an increase among researchers throughout the industry for collaboration especially over the last decade,” Hoffman states. “This has led to sharing information that was once highly protected between key stakeholders, including biotech companies, pharma, and CROs. For CROs, organizations like the Global CRO Council have stimulated further cooperation. This is all contributing to the rapid pace in which more effective new therapies are emerging.”

Artificial intelligence-based approaches, automation, and development of more sensitive instrumentation are also contributing factors that support the rapid progression of drug development. Automation is particularly helpful for labor-intensive stages of the workflow, like sample preparation in speeding the turnaround times of producing reliable and solid data. Looking further ahead, increased knowledge of our biology may spur on revolutionary research. “The Human Genome Project had a huge impact on drug development,” Hoffman explains. “A heightened understanding of our own biology will continue to support the development of drugs to target and treat specific conditions at the source. Acquiring this knowledge will be key to unlocking key opportunities in our field – and treating patients.”

References

Diem P., Ducluzeau P.H., Scheen A. The discovery of insulin. Diabet Epidemiol Manag (2022).