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State-of-the-art characterization of in vivo biotransformations of biotherapeutics on the new timsOmni platform

Structural Proteomics Top-down & Middle-down

Abstract #313 Posted on28 March 20252 June 2025 2 June 2025 23h30

Author(s)

Lucile KOGEY-FUCHS (Presenting Author) | Mass Spectrometry for Biology Unit, Institut Pasteur & DMPK, Sanofi R&D | 28 rue du Docteur Roux, 75015, Paris, France
Athanasios Smyrnakis | Fasmatech Science & Technology | Andrea Kalvou 5, 15232, Chalandri, Greece
Anastasios Grigoriadis | Fasmatech Science & Technology | Andrea Kalvou 5, 15232, Chalandri, Greece
Maria-Aggeliki Kosmopoulou | Fasmatech Science & Technology | Andrea Kalvou 5, 15232, Chalandri, France
Megan Gant | Mass Spectrometry for Biology Unit, Institut Pasteur | 28 rue du Docteur Roux, 75015, Paris, France
Florian Busch | Bruker Switzerland AG | Industriestrasse 26, 8117, Fällanden, Switzerland
Jonathan Dhenin | Novartis Pharma AG | Klybeckstrasse 141, 4057, Basel, Switzerland
Alain Krick | DMPK, Sanofi R&D | 13 Quai Jules Guesdes, 94403, Vitry-sur-Seine, France
Christine Mauriac | DMPK, Sanofi R&D | 13 Quai Jules Guesdes, 94403, Vitry-sur-Seine, France
Dimitris Papanastasiou | Fasmatech Science & Technology | Andrea Kalvou 5, 15232, Chalandri, Greece
Julia Chamot-Rooke | Mass Spectrometry for Biology Unit, Institut Pasteur | 28 rue du Docteur Roux, 75015, Paris, France

Abstract

An important step in the development of new biotherapeutics (BTx) is assessing their in vivo stability and identifying biotransformations that may impact their efficacy and safety. The standard peptide-level LC-MS/MS approach fails to analyze BTx at the proteoform level. We introduce here a novel middle-down workflow using the timsOmni multimodal platform to characterize low-abundance in vivo biotransformation products.
Adalimumab subunits (25-100 kDa) were used for the optimization of the timsOmni instrument (using ECD, CID, EID) either in direct infusion or LC-MS/MS. The best sequence coverage was achieved using ECD followed by collisional activation of charge-reduced species, ranging from 68% for Fc/2 (25 kDa) to 35% for F(ab')2 (100 kDa), with 100% coverage of CDR3 regions. By combining subunit coverage, we achieved 46% sequence coverage of intact mAb without reducing disulfide bonds.
These optimized parameters were further used for analyzing the plasma of mice administered with a hybrid mAb (150 kDa) across five timepoints after immunoenrichment. Previous analysis of these sample (Orbitrap Exploris 480) identified 16 distinct proteoforms (100-189 kDa), revealing extensive biotransformation. However, only the most abundant proteoform could be characterized by MS/MS, the others being too low in abundance. We thus applied our optimized timsOmni middle-down workflow to these in vivo samples, to enable deeper characterization of all biotransformation products.

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An important step in the development of new biotherapeutics (BTx) is assessing their in vivo stability and identifying biotransformations that may impact their efficacy and safety. The standard peptide-level LC-MS/MS approach fails to analyze BTx at the proteoform level. We introduce here a novel middle-down workflow using the timsOmni multimodal platform to characterize low-abundance in vivo biotransformation products. 
Adalimumab subunits (25-100 kDa) were used for the optimization of the timsOmni instrument (using ECD, CID, EID) either in direct infusion or LC-MS/MS. The best sequence coverage was achieved using ECD followed by collisional activation of charge-reduced species, ranging from 68% for Fc/2 (25 kDa) to 35% for F(ab')2 (100 kDa), with 100% coverage of CDR3 regions. By combining subunit coverage, we achieved 46% sequence coverage of intact mAb without reducing disulfide bonds.
These optimized parameters were further used for analyzing the plasma of mice administered with a hybrid mAb (150 kDa) across five timepoints after immunoenrichment. Previous analysis of these sample (Orbitrap Exploris 480) identified 16 distinct proteoforms (100-189 kDa), revealing extensive biotransformation. However, only the most abundant proteoform could be characterized by MS/MS, the others being too low in abundance. We thus applied our optimized timsOmni middle-down workflow to these in vivo samples, to enable deeper characterization of all biotransformation products.

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