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.

Preclinical in vivo and in vitro characterization of Antibody-Drug Conjugates (ADCs) involves the development of several bioanalytical methods to address many drug exposure questions. The current pharma industry approach requires at least three different assays that must be run, i.e., total antibody (mAb), conjugated payload or conjugated mAb, and free payload assays. Herein we present analytical performances of a quantitative hybrid Ligand Binding/Liquid Chromatography High Resolution and Accuracy Mass Spectrometry (LB/LCHRAM) method that can condense much of the necessary bioanalytical information in one method. The method includes an immuno-capture step, and it detects whole ADC molecules. It was applied to plasma mouse samples and showed reliable bioanalytical performance according to full method validation standards. Quantitation using extracted ion chromatograms and deconvoluted mass peaks was evaluated. The limit of quantitation resulted in 0.5ng of protein on column with a linear dynamic range spanning from 0.5 to 10μg/mL. Moreover, lower drug-to-antibody ratio (DAR) ADC species can be simultaneously detected, also enabling qualitative characterization of in vivo ADC conjugation.

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.

Fibroblast growth factor 18 (FGF18) is involved in chondrogenesis and articular cartilage repair. We investigated tissue distribution and pharmacokinetics of radioactive [³H]sprifermin, a recombinant human FGF18, in rats after a single intravenous (i.v.) or intra-articular (i.a.) injection. In two studies (48–96-h [n = 23] and 28-day [n = 12]), 35 male albino (Sprague Dawley) rats received single i.v. or i.a. dose [³H]sprifermin (0.24 mg/kg). Radioactivity was measured in blood, serum, and (in animals receiving i.a. administration) in the knee joint by liquid scintillation counting. Radioactivity in organs, tissues, and distribution in the whole body were measured with whole-body autoradiography. After i.v. injection, radioactivity peaked in serum and whole blood after 4 and 24 h, respectively, with greater total radioactivity in serum. After i.a. injection, radioactivity peaked in serum and whole blood after 24 and 48 h, respectively; intact [³H]sprifermin was not detected in vena caval serum and systemic exposure was low, approximately 20% of that with i.v. injection. Following i.v. injection, radioactivity was mainly found in the liver, adrenal glands, kidney, and spleen; following i.a. injection, radioactivity was preferentially concentrated in articular cartilage after initial distribution in the joint capsule, and still evident in the joint after 28 days. After i.a. injection of [3H]sprifermin in rats, radioactivity was concentrated in the knee joint, particularly articular cartilage, with low levels in other investigated tissues. Systemic exposure to sprifermin was greater with i.v. than i.a. injection. Subsequent clinical investigation in patients with osteoarthritis has reported consistent results.

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.