Applications of Multiple-heartcut 2D-LC/MS at Quality Assistance:

• Characterisation of monoclonal antibodies

• Characterisation of ADCs : 
  Hydrophobic Interaction Chromatography (HIC) is a method of choice for the characterisation of ADCs. Due to high salt content in mobile phase, it is impossible to hyphenate the chromatography to MS for peak identification. Fraction collection and desalting is time-consuming and is not easily amenable to low concentration species (due to dilution effects)

• Quantification of free drugs in ADCs:
  Quantification of free drug in ADCs is critical for batch release and during stability studies. Cytotoxic payloads are extremely toxic and low limit of quantifications should be achieved. MS is often required to reach low LOQ, and the protein has to be removed (offline or online) before the analysis. 2D-LC/MS is a way to meet all those criteria

• Analysis of in-process samples (work in progress)

Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) is an innovative tool for the characterisation of biotherapeutics. Besides the study of higher-order structures, HDX-MS can also be used for epitope mapping studies in order to determine to which region of the target a monoclonal antibody binds. HDX-MS is a good alternative to techniques such as X-ray diffraction or NMR.

Glycosylation is critical for the safety and efficacy of biotherapeutics.

Orthogonal methods have to be applied for characterisation studies and QC testing. LC/MS is a valuable tool for glycosylation analyses. All these methods can be applied in a regulated environment.

• Limitations of AAA in terms of precision, accuracy and throughput 

• Development of an innovative method based on sulfur quantification by ICP-MS/MS 

• Presentation of validation data that confirm the unparalleled performance of the ICP-MS/MS method 

• Application to ADCs

La spectrométrie de masse est un outil indispensable pour la caractérisation des protéines thérapeutiques à différents niveaux :

• Intact
• Sous-unités
• Peptides
• Glycosylations

Phosphodiester to phosphorothioate ratio (P=O/P=S) is a critical quality attribute of phosphorothioate oligonucleotides. The determination of this ratio is commonly performed by P-NMR, which requires specific equipment and cannot be easily applied in a regulated (GxP) environment. 

At Quality Assistance, we have developed an ICP-MS/MS method based on the quantification of P and S after microwave digestion to determine the P=O/P=S ratio in oligonucleotide samples. Preliminary results are very encouraging, with excellent precision and accuracy on reference standards and therapeutic oligonucleotide samples.

The method could be used in routine testing of oligonucleotides in a GxP environment, as an orthogonal method to P-NMR. In addition, the method is currently evaluated for the potential simultaneous absolute quantification of the tested oligonucleotide in place of the classical UV determination.