We are musicians in the same band: how LBA and LC-MS assays complement each other

How do you put together a band? Do you get 4 guitar players? Or do you try to diversify? The same applies to bioanalytical groups/organizations. The “Hot Topics in Large Molecule Bioanalysis” survey and recent discussions at the 2014 AAPS National Biotechnology Conference (NBC) in San Diego show a divide between labs utilizing LBA platforms and those labs using LC-MS assay platforms. I believe that better integration and collaboration between both technologies is the key to addressing today’s bioanalytical challenges. It is difficult to determine from this small survey if integration is on-going but I believe that this collaboration is where our focus should be, rather than worrying if one platform is going to replace the other.

The survey touched on challenges and assay acceptance criteria, the latter particularly relevant as the FDA reviews comments from the industry to the new draft on Bioanalytical Method Validation. The use of LC-MS to support bioanalysis of large molecules is still relatively new, as pointed out by representatives of regulatory agencies. That is why there is no clear guidance in that space. From my perspective, it is scientifically sound to let the pre-study validation data drive the acceptance criteria for sample analysis while using the same target acceptance criteria as that set for LBAs. But it is not surprising that people do not want to set tighter acceptance criteria as it may lead to higher failure rates in sample analysis.

Matrix interference is one of the main challenges in quantification of large molecules by LBAs, since they rely on protein-protein interactions. As the most typical sample pretreatment step consists of a simple dilution, matrix components may hamper or enhance the signal affecting accuracy, particularly at the LLOQ of the assay. However this phenomenon (matrix interference) is not unique to LBAs; we also observe this in small molecule LC-MS platform assays, where the ionization efficiency and the extraction efficiency can be affected by matrix components.

Some of the survey results were hard to interpret. For example, despite the answers, most bioanalytical labs continue to utilize ligand binding assays to support large molecule bioanalysis, especially in the regulated space. LC-MS assays have proven to be very useful; especially in cases where no affinity capture pre-treatment step is required as it helps avoid generation of capture and detection antibodies, which may be expensive and time consuming. With the right extraction, peptide selection and IS, LC-MS can offer great specificity with good sensitivity and dynamic range, and contrary to some comments there are applications in development for isotyping of anti-drug antibodies (ADAs). But, as it was nicely stated by one of the attendees at NBC this year, if we use affinity capture in an LC-MS assay, that is the same as a LBA which happens to have MS detection. In this respect, it is shocking to see that 53 responders (out of 145) think that LC-MS will replace LBAs. What if you ever need to answer the question of free drug vs. total? However, at least 83 people (out of 145) see them as complementary platforms and view the value of LBAs also as a more economical platform which in some cases can still deliver superior sensitivity over the LC-MS platform (reagent dependent). Hopefully, the leaders of bioanalytical labs are among those 83 responders, and will work to forge more collaboration between LBA and LC-MS platform groups to create a harmonious “music band”.