Method development for the analysis and characterization of proteins and mRNA as biotherapeutics by UHPLC-QTOF


Losada Baranera, Mar


Biotherapeutics are currently the fastest growing group of drugs amongst the pharmaceutical market [1]. Protein-based therapeutics account for almost half of recent new drugs approvals since they present advantages including minimal safety/toxicity issues and high target specificity. They have been used for a wide range of treatments. On the other hand, mRNA has recently become a promising biotherapeutic drug substance, currently used in the new generation of vaccines, composed of polymeric or lipid nanosystems. Nevertheless, a long path has precluded the tremendous success of mRNA prophylactic Covid-19 vaccines, focused mainly on the in vitro transcription method of production set up and in the modification of the mRNA structure to enhance its stability and functionality. Thus, analyzing these modifications is key to achieve functional mRNA. It must be taken into account that these biomolecules present much more complexity and heterogeneity, which requires an extensive characterization to enable their use as therapeutic treatments. Here the need to develop specific analytical and bioanalytical methods to fully characterize them.
For protein analysis, two approaches were studied. (1) Top-down approach for intact protein analysis with reversed-phase chromatography coupled to a quadrupole time-of-flight (QTOF) mass detector in positive electrospray ionization. That provided the MS spectrum with the characteristic envelope of ions at different charges. Through deconvolution, the molecular weight can be obtained. (2) Bottom-up approach for peptide mapping was studied, where the protein is submitted to an enzymatic digestion to obtain peptides that were separated with reversed-phase chromatography coupled to a QTOF with a data-independent acquisition mode to obtain the MSMS spectra with the peptide fragmentation. This provides the sequence coverage for each peptide and for the initial protein. Further studies were performed to know if the most abundant peptides were proteotypic.
For mRNA analysis, digestion was performed to obtain shorter oligonucleotides, which were further characterized to identify the modifications in the nucleic acid backbone. Both amide and ion pair reversed-phase high-performance liquid chromatography were tested, coupled to a QTOF mass detector in negative electrospray ionization that provided the exact mass from the deconvoluted MS spectra. Then, with a specific variant of data-independent acquisition mode, the MSMS spectra provided information of the fragmentation to elucidate the sequence. The UV detection was also used at 260 nm to compare integrated areas. In order to study the enzymatic digestion, two custom oligonucleotides were analyzed. Digestion with ribonuclease T1 was performed at different ratios of enzyme-substrate and different times of digestion, incubated at 37°C. Then eGFP mRNA was digested and different additions of the custom oligonucleotides were performed with different purposes, as an internal standard and as a digestion control. After all this process, oligonucleotides and their digested fragments were characterized with both intact mass analysis (TOF MS) and fragmentation analysis (MSMS spectra), thus concluding that the method set up is useful for the analysis of mRNA fragments.



Fornaguera i Puigvert, Cristina
Gotor Navarra, Gemma


IQS SE - Master’s Degree in Analytical Chemistry