As biologics-based therapies have expanded, LC-MS is increasingly applied to support quantitative bioanalysis to detect diverse peptides and proteins. LC-MS can offer a few notable advantages compared to the traditional approach for quantification with ligand-binding assays (LBAs), but is typically thought of as a complementary approach.
Aaron Ledvina, PhD, Senior Manager for Method Development and Brendan Powers, PhD, Staff Scientist, recently shared their thoughts on LC-MS and LBA strategies for biologic quantification.
Understanding regulatory acceptance
“Over the last five to ten years, the LC-MS platform has become increasing routine and accepted, both scientifically and from a regulatory standpoint,” said Ledvina.
While the FDA encourages the use of alternative technologies, there are a number of factors to consider when determining whether to use LC-MS or LBAs.
“You start with the biological information that you need to access and determine the specific measurements needed; this, in combination with understanding the nature of the measurement associated with LC-MS or LBA, often make the choice of platform more obvious,” he explained. “At a high level, sample cost is usually lower for LBAs but the development time tends to be faster for LC-MS, especially for certain classes of compounds such as monoclonal antibodies in preclinical studies.”
Evaluating complementary approaches
“LC-MS and LBA can be thought of as complimentary to one another, but the choice of platform depends on a number of different variables.” said Powers. “One of the most important factors is the availability of critical reagents.”
One drawback of LBA is the requirement for high-quality critical reagents, which requires careful documentation and labeling to minimize variability from batch to batch during the lengthy production process. In early discovery bioanalysis, LC-MS does not face the same concerns as generic reagents are usually readily available.
The importance of sensitivity
Sensitivity represents another important consideration. “For toxicology studies, the lower limit of quantitation (LLOQ) is usually much higher, making LC-MS more practical,” explained Powers. “But for pharmacokinetic (PK) studies, higher sensitivity is typically required due to lower doses. For example, the LLOQ is 20 ng/mL for our LBA PK assay, but our direct digest LC-MS assay can only get down to 500 ng/mL.”
In one case study with antidrug antibodies (ADA), LBA offered superior LLOQ, throughput and extraction time; whereas LC-MS (Direct Digest) offered superior precision and accuracy. When antidrug antibodies (ADA) were included, increasing ADA proportionally reduced the LBA signal, while LC-MS was not affected.
LC-MS provides an advantage over LBA for multiplexed antibody quantitation. “In conventional dosing, you evaluate one antibody or drug at a time, and then perform analysis,” explained Powers. “But because of the enhanced selectivity LC-MS provides, you can dose multiple antibodies at the same time and perform the analysis on the same run without the need for high quality critical reagents.”
Differing enrichment approaches
Other considerations include different surrogate peptide approaches. “In a recent case study, we used Protein G, which enriches antibodies,” said Powers. “Protein G-based enrichment provides a generic sample clean-up prior to digestion for antibody drugs. More targeted enrichment isn’t necessary since the LC-MS on the backend gives us the selectivity that we need. Even though the sample is still fairly complex, we’re able to mitigate interfering peaks and selectively quantitate the target antibody via surrogate (signature) peptides.”
Increasing sensitivity and looking ahead
In the past, there was a long held trend of only using LBA, but now the team increasingly asked to support pharmaceutical companies with LC-MS to evaluate their complex molecules with custom approaches.
“Our team is working to meet the needs of our clients by developing high resolution MS methods for intact protein analysis and optimizing custom immunoaffinity and digestion workflows,” said Ledvina.
“For LC-MS, we are also in the process of adding microflow capabilities, which can provide a nice bump in overall sensitivity. It’s just one area where we’re looking to improve the approach to make it more competitive to LBA and give our clients more options.”
Whether using LBA or LC-MS or both, learn more about your choices for biologic quantification.