MHC-II-associated peptide proteomics (MAPPs) is a mass spectrometry-based (MS) method to identify naturally presented MHC-II-associated peptides that could elicit CD4+T cell activation. MAPPs assay is considered one of the assays that better characterize the safety of biotherapeutics by driving the selection of the best candidates concerning their immunogenicity risk. However, there is little knowledge about the impact of bead material on the recovery of MHC-II MS-eluted ligands in MAPPs assays. Here, we firstly describe a robust MAPPs protocol by implementing streptavidin magnetic beads for the isolation of these peptides instead of commonly used NHS-activated beads. Moreover, we assessed the impact of the cell medium used for cell cultures on the morphology and recovery of the in vitro-generated APCs, and its potential implications in the amount of MHC-II isolated peptides. We also described an example of a MAPPs assay application to investigate drug-induced immunogenicity of two bispecific antibodies and compared them with monospecific trastuzumab IgG1 control. This work highlighted the importance of MAPPs in the preclinical in vitro strategy to mitigate the immunogenicity risk of biotherapeutics.

The complexity of therapeutic proteins like antibody–drug conjugates (ADCs) holds a tremendous analytical challenge. Complementary mass spectrometry approaches such as peptide mapping and intact mass analysis are required for the in-depth characterization of these bioconjugates. Cysteine-linked ADCs have shown a unique challenge for characterization, mainly when the conjugation is carried out on interchain cysteines, because their intact analysis requires native mass spectrometry conditions to preserve non-covalent binding between antibody chains. In this work, two different approaches were proposed. Specifically, a full scan data-independent all ion fragmentation (FS-AIF) and a full scan data-dependent targeted MS2 (FS-ddtMS2) were applied to generate complementary datasets for a cysteine-linked ADC characterization with a highly reactive payload. These two methods were applied to in vitro plasma stability and in vivo PK samples to calculate and refine mean drug-to-antibody ratio over time. Using this approach, we successfully characterized an ADC containing a hydrolysis-sensitive payload and refined the “active” drug-to-antibody ratio on in vitro stability and in vivo samples. These two methods allowed the confirmation of the different ADC species and potential metabolites of conjugated payload attached to the antibody backbone in a single analysis without needing a dedicated method for the conjugated payload metabolite identification.

The development of biotherapeutics, particularly multi-domain biotherapeutics (MDBs) and antibody drug conjugates (ADCs), presents unique challenges due to their complexity and increased immunogenicity risks. Bioanalytical approaches play a pivotal role in addressing these challenges. Robust bioanalytical methods are essential for effectively assessing the pharmacokinetics (PK), pharmacodynamics (PD), and immunogenic responses of novel biologics. Advanced techniques, such as multiplexed assays using ligand binding assays (LBAs) and Liquid Chromatography-Mass Spectrometry (LC-MS) methods, are employed to handle the complexity of MDBs and ADCs effectively. Additionally, high-resolution mass spectrometry can investigate the potential biotransformation of biologics directly in in vivo studies. Furthermore, the Context of Use (CoU) is critical for defining assay purposes across nonclinical and clinical settings, ensuring their relevance and efficiency. Regulatory compliance is paramount to ensure assays meet standards for drug approval. Practical considerations include assay sensitivity, specificity, and the method's ability to investigate the structural integrity of MDBs and ADCs in in vivo studies. This paper aims to review recent publications on the application of bioanalytical techniques, specifically LBA and LC-MS, in supporting the development of pioneering biotherapeutics. Additionally, it will discuss optimal strategies for bioanalytical methods and immunogenicity assessment.

Biotherapeutic immunogenicity remains a great challenge for researchers because multiple factors trigger immune responses. Predicting and assessing the potential human immune response against biological drugs could represent an impressive breakthrough toward generating potentially safer and more efficacious therapeutic proteins. This article describes an in vitro assay that can contribute to evaluating the potential immunogenicity of biotherapeutics by focusing on lysosomal proteolysis. We selected human liver lysosomes (hLLs) from four different donors as a surrogate in vitro model instead of APC lysosomes because they are a ready-to-use lysosomal source. To assess the biological comparability of this surrogate to APC lysosomal extract, we compared the proteome content of hLLs with literature data of lysosomal fractions extracted from murine bone marrow and human blood-derived dendritic cells. Then we tested infliximab (IFX; Remicade) under different proteolytic conditions using liquid chromatography and high-resolution and -accuracy mass spectrometry to better define the degradation kinetics inside the lysosomes. hLLs revealed similar enzymatic content compared with human and murine dendritic cell lysosomes. Degradation assays demonstrated that our liquid chromatography and high-resolution and -accuracy mass spectrometry method could identify both the intact protein and the peptides resulting from proteolysis with high specificity and resolution. The rapid and easy assay described in this article can be extremely useful for evaluating the immunogenic risk associated with therapeutic proteins. In addition, this method can complement information from MHC class II–associated peptide proteomics assays and other in vitro and in silico techniques.

Cross-linking mass spectrometry (XL-MS) has emerged as a powerful approach for probing protein structure and conformational dynamics. Conventional cross-linkers typically contain two N-hydroxysuccinimide (NHS) ester groups that primarily target lysine residues. Here, we report the optimization of the in-solution reactivity of disuccinimidyl sulfoxide carbamate (DSSO-carbamate), an analogue of DSSO in which the two NHS ester groups are replaced by NHS carbamates. The enhanced stability of the carbamate functionality reduces the degradation of DSSO through retro-ene sulfoxide elimination under standard XL-MS buffer conditions, thereby improving cross-linking efficiency. We further characterized the gas-phase dissociation behavior of DSSO-carbamate and optimized the collision energy (CE) parameters for automated data analysis with XL-MS search engines. Mapping of cross-linking sites for bovine serum albumin revealed an unexpectedly high frequency of cross-links involving the protein N-terminus, suggesting increased N-terminal reactivity of NHS carbamates relative to NHS esters. This hypothesis was corroborated by comparative cross-linking of nonacetylated and N-terminally acetylated α-synuclein using DSSO-carbamate and the NHS ester-based disuccinimidyl dibutyric urea (DSBU). We observed the same reactivity trend for the NHS carbamate-based cross-linker NNP9. Proteome-wide XL-MS analysis confirmed a higher propensity of NHS carbamate-based reagents to form cross-links with protein N-termini compared to NHS ester-based cross-linkers. Together, these results show that NHS carbamate-based reagents provide complementary XL-MS restraints to NHS ester-based cross-linkers and are particularly useful for investigating systems where N-terminal interactions are functionally relevant. We anticipate that this unique N-terminal selectivity of NHS carbamates will find broader applications in bioconjugation and chemical proteomics.

Bioanalytical methods to quantitate conjugated payloads are essential for assessing antibody-drug conjugate (ADC) stability and pharmacokinetics (PK). Dual-payload ADCs present analytical challenges; different linker chemistries can require complex digestion conditions and to perform the cleavage. Developing separate methods for each linker combination can be time and resource demanding. Rat tritosomes—purified lysosomal fractions from Triton-treated rat liver—provide a comprehensive enzymatic mixture that mimics the lysosomal environment. The presented bioanalytical method combines immunoaffinity purification with tritosome-mediated digestion for simultaneous quantitation of dual conjugated payloads. The method was applied to a model dual-payload ADC containing two different cytotoxic payloads, conjugated using different enzymatically cleavable linkers, with not related Dar (drug-to-antibody-ratio). Method validation in mouse plasma demonstrated excellent accuracy (bias ± 20%, LLOQ and ULOQ ± 25%) and precision (coefficient of variation CV% ≤ 20%, LLOQ and ULOQ ± 25%) across all concentration levels (lower to upper limit of quantitation ,LLOQ to ULOQ) for both payloads, with 100% of quality control samples (QCs) meeting acceptance criteria for hybrid LC-MS/MS quantitation methods. This tritosome-based approach provides a unified, efficient platform for multi-payload ADC bioanalysis, eliminates linker-specific method optimization and enables robust support for preclinical studies. The method has been tested for accuracy and precision on 4 different model ADCs and employed to quantify the conjugated payloads in in-vivo samples from a homozygous hFcRn transgenic mouse model (Tg32) PK study, resulting in reliable data in accordance with total antibody measurements.