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Overcoming Challenges in Biopharma Quantitation with LC-MS

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Biopharmaceuticals comprising peptide and protein based therapeutics are amongst the fastest growing classes of therapeutics. Typically, biopharmaceuticals offer several advantages over traditional pharmaceuticals in that they are more targeted, better tolerated by our bodies, and generally offer fewer side effects. Essentially, they are popular as they offer better efficiency, efficacy, and safety for the treatment of a wide range of diseases.

But despite their many benefits, the design and development of biotherapeutics can be challenging. For starters, compared to small molecules, proteins and peptides are much larger molecules, and hence demand a different approach for efficient characterization and quantitation. However, with the right tools and techniques, working with larger biological molecules don’t have to mean larger workflow bottlenecks.

Achieving targeted quantitation with LC-MS

Targeted quantitation plays an important role in understanding the pharmacokinetics of biotherapeutics. While liquid chromatography-mass spectrometry (LC-MS) techniques have long been employed for bioanalysis of small molecule drugs, for proteins, ligand binding assays have been the traditional approach. But owing to increased selectivity, specificity and dynamic range offered by mass spectrometers, LC-MS-based analysis has established itself as a versatile and capable alternative.

Although both sample preparation and LC play extremely important roles in optimizing protein quantitation assays, here we focus on the two main MS technologies: triple quadrupole (QqQ) mass spectrometry and high-resolution accurate mass (HRAM) analysis. Each has its strengths, and the decision to use a particular strategy often comes down to what analysts are looking to achieve.

The high levels of sensitivity, selectivity, and speed that can be routinely achieved using modern QqQ analyzers allows researchers to confidently quantify peptides and proteins at extremely low concentrations. Combined with the reliability and robustness that these instruments offer, QqQ based approaches can offer a cost-effective solution for high-throughput analyses. With the constructive knowledge of the peptide(s) that one needs to follow (this can be ascertained with several software tools), QqQs offer superior robust, sensitive, reliable, quantitation assays for biotherapeutics across a host of matrices that can be achieved by MS users with different levels of expertise.

HRAM technology also delivers exceptional performance when it comes to non-targeted and targeted protein quantitation. Its resolution and sensitivity allows analysts to extract even more information from the most complex of biological samples. And because HRAM permits retrospective analysis, scientists who are trying to monitor every analyte, retrospectively looking for unknowns, metabolites, and/or biotransformed products, can gain significantly. 

Developing effective methods for biotherapeutic quantification

Biopharmaceuticals differ to small molecule drugs, not just in size but also in their physical characteristics and behavior. For example, unlike most small organic compounds, proteins and peptides have multiple charge states.

From a method development standpoint, the choice of selected reaction monitoring mode may not depend on the most intense signal. Instead, a greater emphasis may need to be placed on the type of fragmentation. Orbitrap-based technology can select the most promising precursor ions and characterize fragments to their isotopic fine structure level.

In order to develop an effective LC-MS method to quantify biotherapeutics, technologies should be paired with reliable protein digestion and purification strategies, supported by liquid chromatography instruments that can deliver optimum levels of resolution, sensitivity and throughput.

It’s also necessary to have effective instrument control and data analysis software that can help analysts acquire and review the large amounts of information generated in LC-MS workflows. This software needs to be robust, easy to use and should be capable of processing data in the minimum amount of time.

From sample digestion through to peptide and protein quantitation, by using best-in-class technologies and the latest workflow solutions, the unique challenges associated with biopharmaceutical quantitation can be elegantly overcome.