Bioanalysis Zone

What challenges do you experience when utilizing hybrid LBA/LC–MS assays in research and development? How do you avoid/overcome these?


Fabrizia Fusetti (QPS)

FF (2)

“The use of LC–MS or LC–MS/MS for large molecules is generally more complex compared with applying the technique for the quantification of small synthetic compounds. One key aspect in the development of a fit-for-purpose assay is the availability of a representative internal standard. Stable labeled internal standards for various therapeutic antibodies are commercially available and specialized companies offer customized production of labeled recombinant proteins. Extended synthetic peptides represent a valuable alternative, although they do not allow monitoring of the full sample preparation procedure.

In addition, the need to extract or enrich while maintaining compound stability and integrity, can represent a challenge for achieving maximum recovery and minimize matrix interferences and experimental molecular stress. Hybrid LBA/LC–MS assays are often made up of several steps and therefore, sample preparation procedure is time-consuming and work-intensive. The possibility to partially automate the sample preparation helps to prevent technical variability and somewhat increases the throughput.

The efficacy of enzymatic digestion in bottom-up approaches, as well as optimal chromatographic conditions and instrumental set-up to fine tune ionization and fragmentation in the mass spectrometer, are crucial for obtaining robust assays.”

Eric Ma (PPD)


“Because surrogate peptides are used for protein quantitation, as in LBA methods, only part of the molecule is used for determination. As a result, conclusions based on measurements could be misleading.  Hopefully, in the near future, this limitation can be overcome by analyzing proteins at intact level.

Bioanalysis of ADCs remains challenging due to their heterogeneous nature. The heterogenicity arises from two aspects: Manufacturing processes, such as conjugation through lysine side-chain amines on an antibody, which typically result in a complex mixture of conjugate species that differ in the number of drugs attached as well as in the sites of drug linkage; and in vivo changes, such as catabolism/de-conjugation which result in dynamically changing the mixture of ADC species creating a different drug-antibody ratio in circulation.

LC–MS is a reliable, unique and complementary technique to LBA in protein bioanalysis. Results from published data have demonstrated that in many cases there was a good correlation between the results obtained via LBA and LC–MS.  Choosing LBA or LC–MS for protein bioanalysis can be determined based on scientific considerations and the specific questions that need to be answered and where in the development process a particular drug product is.”

Olivier Heudi (Novartis)

OH (2)“The sample preparation step is a critical step during the development of hybrid LBA–LC–MS as the enrichment phase with the desired compound/protein should be reproducible and enough to produce a sensitive and robust assay. Following the enrichment step, the sample clean-up appears to be important since the sample pretreatment with different chemicals and enzymes generates high amounts of compounds, leading to poor method sensitivity and selectivity. The analysis by mass spectrometry is also challenging due to the environment of the sample. In addition, the mass range of the mass spectrometer is not large enough to cope with the mass of all the detected molecules in the case of large molecules.

In order to improve the sample preparation, an internal standard is used during the sample work up procedure. Different approaches are considered for the use of internal standards Furthermore, there are multiple different types of internal standards. Ideally, the internal standard should be as close as possible to the analyzed molecules – for the analysis of therapeutic proteins it is recommended to use stable isotope labeled proteins. The sample clean-up is also tackled using various cleaning procedures that tend to reduce the sample complexity. Regarding the detection, different mass spectrometric acquisition methods are used and in some instances a high-resolution mass spectrometer has helped to analyze very complex samples and intact proteins.”

Steven Piccoli (GSK)

Steve_0135 (3) (002)“The first challenge is always determination of the exact molecular species to be analyzed to correctly inform on the clinical decision under consideration. Vigorous discussions between the /biomarker scientists and the developmental/clinical team will ensure alignment on the context of use and success of the experimental outcome. The second challenge is ensuring that the binding characteristics of the capture reagent have been appropriately interrogated and are suitable for the end purpose. This is easily remediated by assessing serum interactions and determination of individual binding rate constants (kon and koff, not simply KA) to allow the best performance. The third challenge is generally the time allowed for development. I wish I had a better answer for this but, planning, communication, more planning and more communication seem to be the tried and true path. Automation helps as well.”


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