Conjugation Site Research Articles

Enhanced Payload Localization in Antibody-Drug Conjugates Using a Middle-Down Mass Spectrometry Approach with Proton Transfer Charge Reduction.

Antibody-drug conjugates (ADCs) represent a novel class of immunoconjugates with growing therapeutic relevance, since they combine the efficacy of cytotoxic drugs with the specificity of antibodies. However, by design, ADCs introduce structural features into the monoclonal antibody scaffold that complicate their analysis. Payload attachment to cysteine or lysine residues can often result in product heterogeneity, regarding both the number of attached drug molecules and their conjugation site, necessitating the use of state-of-the-art MS instrumentation to elucidate their complexity. In middle-down mass spectrometry (MD MS), the gas-phase sequencing of ∼25 kDa ADC subunits with different ion activation techniques generally produces rich fragmentation mass spectra; however, spectral congestion can cause some fragment ions to go undetected, including those that can pinpoint the exact location of payload conjugation sites. Proton transfer charge reduction (PTCR) can substantially simplify fragment ion spectra, thereby unveiling the presence of product ions whose signals were previously suppressed. Herein, we present an MD MS strategy relying on the use of PTCR to investigate a cysteine-based ADC mimic with a variable drug-to-antibody ratio, targeting the unambiguous localization of payload conjugation sites. Unlike traditional tandem MS experiments (MS2), which could not provide a complete map of conjugation sites, a single PTCR-based experiment (MS3) proved to be sufficient to achieve this goal across all variably modified ADC subunits, including isomeric ones. Combining the results obtained from orthogonal ion activation techniques followed by PTCR further strengthened the confidence in the assignments.

Generation of binder-format-payload conjugate-matrices by antibody chain-exchange

The generation of antibody-drug conjugates with optimal functionality depends on many parameters. These include binder epitope, antibody format, linker composition, conjugation site(s), drug-to-antibody ratio, and conjugation method. The production of matrices that cover all possible parameters is a major challenge in identifying optimal antibody-drug conjugates. To address this bottleneck, we adapted our Format Chain Exchange technology (FORCE), originally established for bispecific antibodies, toward the generation of binder-format-payload matrices (pair-FORCE). Antibody derivatives with exchange-enabled Fc-heterodimers are combined with payload-conjugated Fc donors, and subsequent chain-exchange transfers payloads to antibody derivatives in different formats. The resulting binder-format-conjugate matrices can be generated with cytotoxic payloads, dyes, haptens, and large molecules, resulting in versatile tools for ADC screening campaigns. We show the relevance of pair-FORCE for identifying optimal HER2-targeting antibody-drug conjugates. Analysis of this matrix reveals that the notion of format-defines-function applies not only to bispecific antibodies, but also to antibody-drug conjugates.

Design of a Genetically Programmable and Customizable Protein Scaffolding System for the Hierarchical Assembly of Robust, Functional Macroscale Materials.

Inspired by the properties of natural protein-based biomaterials, protein nanomaterials are increasingly designed with natural or engineered peptides or with protein building blocks. Few examples describe the design of functional protein-based materials for biotechnological applications that can be readily manufactured, are amenable to functionalization, and exhibit robust assembly properties for macroscale material formation. Here, we designed a protein-scaffolding system that self-assembles into robust, macroscale materials suitable for in vitro cell-free applications. By controlling the coexpression in Escherichia coli of self-assembling scaffold building blocks with and without modifications for covalent attachment of cross-linking cargo proteins, hybrid scaffolds with spatially organized conjugation sites are overproduced that can be readily isolated. Cargo proteins, including enzymes, are rapidly cross-linked onto scaffolds for the formation of functional materials. We show that these materials can be used for the in vitro operation of a coimmobilized two-enzyme reaction and that the protein material can be recovered and reused. We believe that this work will provide a versatile platform for the design and scalable production of functional materials with customizable properties and the robustness required for biotechnological applications.

Thermally Activated Delayed Fluorescence Sensitizer with Through‐Space Charge Transfer Manipulated by Atom Distribution for Solution‐Processed Deep‐Blue Multi‐Resonance OLEDs

AbstractOrganic light‐emitting diodes (OLEDs) employing multi‐resonance (MR) emitters have attracted much attention due to their high luminescent efficiency and color purity, however, the development of deep‐blue multi‐resonance OLEDs is hindered by the lack of suitable sensitizers. Here a novel design strategy is reported for thermally activated delayed fluorescence sensitizer with high reverse intersystem crossing (kRISC) and radiative transition (kR) rate by regulating atom distribution of a spatially‐aligned carbazole donor/diphenylpyrimidine acceptor structure to manipulate through‐space charge transfer (CT) process. It is found that diphenylpyrimidine acceptor with 1,3‐position nitrogen atoms is un‐conjugated to bridging phenyl moiety by the conjugation node of pyrimidine, while the acceptor with 1,5‐position nitrogen atoms is conjugated to bridging phenyl through conjugation site, opening through‐bond CT pathway via bridging phenyl besides through‐space CT from spatial donor‐acceptor interactions. As a result, the sensitizer with 1,5‐position nitrogen atoms exhibits enhanced oscillator strength by 3.5 folds and accelerated kR of 3.57 × 107 s−1, while keeping high kRISC of 4.03 × 106 s−1 and high singlet (S1) state (2.88 eV). Solution‐processed OLEDs using the sensitizer and deep‐blue multi‐resonance emitter exhibit efficient narrowband electroluminescence with CIE coordinates of (0.14, 0.13) and EQEmax of 15.6%, representing the state‐of‐the‐art device performance for deep‐blue multi‐resonance OLEDs by solution process.

Fluorescent Antimicrobial Peptides Based on Nile Red: Effect of Conjugation Site and Chemistry on Wash-Free Staining of Bacteria.

Fluorescent probes for bacterial detection can be obtained by conjugating antimicrobial peptides with fluorescent dyes. However, little is known about the effect of the conjugation site and linker chemistry on staining efficiency. We synthesized three conjugates of the antimicrobial peptide ubiquicidin with the environmentally sensitive fluorophore Nile Red that differed by the attachment site and the chemical composition of the linker. We showed that incorporating fluorophore as a minimalistic non-natural amino acid resulted in a superior probe compared with the typically used bioconjugation approaches. The new peptide-based probe named UNR-1 displayed red fluorescence and enabled robust wash-free staining of Gram-positive and Gram-negative bacteria. The probe exhibited selectivity over mammalian cells and enabled rapid fluorescence detection of bacteria by fluorescence microscopy and flow cytometry in an add-and-read format. Our results may foster the development of next-generation fluorescent AMPs for clinical laboratory diagnostics and medical imaging.

Extensive Biotransformation Profiling of AZD8205, an Anti-B7-H4 Antibody-Drug Conjugate, Elucidates Pathways Underlying Its Stability In Vivo.

What happens to macromolecules in vivo? What drives the structure-activity relationship and in vivo stability for antibody-drug conjugates (ADCs)? These interrelated questions are increasingly relevant due to the re-emerging importance of ADCs as an impactful therapeutic modality and the gaps that exist in our understanding of ADC structural determinants that underlie ADC in vivo stability. Complex macromolecules, such as ADCs, may undergo changes in vivo due to their intricate structure as biotransformations may occur on the linker, the payload, and/or at the modified conjugation site. Furthermore, the dissection of ADC metabolism presents a substantial analytical challenge due to the difficulty in the identification or quantification of minor changes on a large macromolecule. We employed immunocapture-LCMS methods to evaluate in vivo changes in the drug-antibody ratio (DAR) profile in four different lead ADCs. This comprehensive characterization revealed that a critical structural determinant contributing to the ADC design was the linker, and competition of the thio-succinimide hydrolysis reaction over retro-Michael deconjugation can result in superb conjugation stability in vivo. These data, in conjunction with additional factors, informed the selection of AZD8205, puxitatug samrotecan, a B7-H4-directed cysteine-conjugated ADC bearing a novel topoisomerase I inhibitor payload, with durable DAR, currently being studied in the clinic for the potential treatment of solid malignancies (NCT05123482). These results highlight the relevance of studying macromolecule biotransformation and elucidating the ADC structure-in vivo stability relationship. The comprehensive nature of this work increases our confidence in the understanding of these processes. We hope this analytical approach can inform future development of bioconjugate drug candidates.

Computer-Aided Site-Specific PEGylation of PET Hydrolases for Enhanced PET Degradation.

The enormous accumulation of poly(ethylene terephthalate) (PET) waste has posed a serious threat to the environment and human health, and biodegradation with PET hydrolase (PETase) can be a possible solution. Herein, we propose site-specifically modifying PETase with amphiphilic polymers to improve the enzyme performance at ambient temperature. For this purpose, we devise a computer-aided strategy to prioritize the conjugation site, and polyethylene glycol (PEG) preparations of 0.55 to 10 kDa are site-specifically conjugated to PETase. The most active conjugate PETase-PEG 5k (PETase-5K) shows an increase of melting temperature (3.88 °C) and significantly improves PET degradation performance (3.5- and 3.1-fold increases at 30 and 40 °C, respectively). Experimental investigation and molecular dynamics simulations reveal that the site-specific PEGylation increases the hydrophobic solvent-accessible surface area and the binding capability to the PET surface, thickens the hydration layer, increases the intramolecular hydrogen bonding, reduces the interactions between water and the conjugated enzyme surface, and rigidifies the enzyme structure via hydrogen bonding and hydrophobic interactions between the polymer and the enzyme, thus leading to improved enzymatic performance of PETase-5K. We further validate the versatility of the site-specific PEGylation in one of the most evolved variants of PETase, FAST-PETase, by 1.8-fold improvement in PET degradation at 30 °C. The presented computer-aided site-specific conjugation strategy has opened a new avenue to enhancing PETase performance at ambient temperature, and the contribution of PEGylation to PETase unraveled in this work laid a foundation for the rational engineering of PET hydrolases.

Site-Specific Stability Evaluation of Antibody-Drug Conjugate in Serum Using a Validated Liquid Chromatography-Mass Spectrometry Method.

Antibody-drug conjugate (ADC) consists of engineered antibodies and cytotoxic drugs linked via a chemical linker, and the stability of ADC plays a crucial role in ensuring its safety and efficacy. The stability of ADC is closely related to the conjugation site; however, no method has been developed to assess the stability of different conjugation sites due to the low response of conjugated peptides. In this study, an integrated strategy was developed and validated to assess the stability of different conjugation sites on ADC in serum. Initial identification of the conjugated peptides of the model drug ado-trastuzumab emtansine (T-DM1) was achieved by the proteomic method. Subsequently, a semiquantitative method for conjugated peptides was established in liquid chromatography-hybrid linear ion trap triple quadrupole mass spectrometry (LC-QTRAP-MS/MS) based on the qualitative information. The pretreatment method of the serum sample was optimized to reduce matrix interference. The method was then validated and applied to evaluate the stability of the conjugation sites on T-DM1. The results highlighted differences in stability among the different conjugation sites on T-DM1. This is the first study to assess the stability of different conjugation sites on the ADC in serum, which will be helpful for the design and screening of ADCs in the early stages of development.

Investigation and Development of the BODIPY-Embedded Isotopic Signature for Chemoproteomics Labeling and Targeted Profiling.

A common goal in mass spectrometry-based chemoproteomics is to directly measure the site of conjugation between the target protein and the small molecule ligand. However, these experiments are inherently challenging due to the low abundance of labeled proteins and the difficulty in identifying modification sites using standard proteomics software. Reporter tags that either generate signature fragment ions or isotopically encode target peptides can be used for the preemptive discovery of labeled peptides even in the absence of identification. We investigated the potential of BODIPY FL azide as a click chemistry enabled chemoproteomics reagent due to the presence of boron and the unique 1:4 natural abundance ratio of 10B:11B. The isotopes of boron encode BODIPY-labeled peptides with a predictable pattern between the monoisotopic (M) and M+1 peaks. BODIPY-labeled peptides were identified in MS1 spectra using an R script that filters for the signature 10B:11B intensity ratio and mass defect. Application of the boron detection script resulted in three times the labeled peptide coverage achieved for a BODIPY-conjugated BSA sample compared with untargeted data-dependent acquisition sequencing. Furthermore, we used the inherent HF neutral loss signature from BODIPY to assist with BODIPY-modified peptide identification. Finally, we demonstrate the application of this approach using the BODIPY-conjugated BSA sample spiked into a complex E. coli. digest. In summary, our results show that the commercially available BODIPY FL azide clicked to alkyne-labeled peptides provides a unique isotopic signature for pinpointing the site(s) of modification with the added potential for on- or off-line UV or fluorescence detection.

Impact of lipidation site on the activity of α-helical antimicrobial peptides

Antimicrobial peptides (AMPs) display advantages over traditional antibiotics due to their broad spectrum of activity against various pathogens, and may even overcome bacterial drug resistance. However, despite their potential therapeutic benefits, widespread application of AMPs is limited by their instability, sensitivity to high salt concentrations, toxicity, and immunogenicity. Lipidation is a promising strategy in overcoming these drawbacks and potential problems for drug candidates. While N-terminal lipidation is a well-studied form of acylation of biologically active peptides, fatty acylation of the lysine side chain has still been poorly explored. In this study, we examined systematic introduction of octanoic (C8) or decanoic (C10) acid into the sequences of three antimicrobial α-helical peptides, namely LL-I, LK6, and ATRA-1, by acylation of subsequent lysine residues, resulting in 17 lipopeptides. Fatty acid lengths optimal for antimicrobial activity were selected based on a previous study on the N-terminal lipidated counterparts of these peptides. Shuffling the position of the fatty acid tails in the sequences of the peptides preserved high activity against Gram-positive bacteria, increased activity against Gram-negative strains and reduced cytotoxicity, compared to the N-terminal acylated counterparts. In the case of the LL-I and LK6 conjugates, the interactions with artificial negatively charged membranes induced formation of an α-helical structure but without a direct correlation between helicity and amphipathicity. Unexpectedly, the ATRA-1 derivatives showed only a small tendency, if any, to adopt a helical structure upon binding to POPG vesicles, which may indicate a non-helical active conformation. A more detailed study of the selected analogues, namely LL-I-4C8, LK6-7C8, and ATRA-1-11C10, provided evidence of a tendency to self-assemble into clumped and/or isolated fibrils, micelles or clusters of micelles, and proved that the lipid bilayer is the main target of action of the tested lipopeptides. In summary, the results of the present study highlight that alternative conjugation sites for lipid modification of AMPs, rather than the commonly applied N-terminal conjugation site, may improve the selectivity of action and be feasible in testing for the development of new lipid-peptide conjugates.

A donor-acceptor π-stacked multifunctional Eu-MOF based on a novel viologen-like photosensitive ligand: Degradation of tetracycline, photochromism, and photoluminescence

The photosensitive building blocks of photochromic MOFs are mostly 4,4′-bipyridine. However, the linear structure and kind of 4,4′-bipyridine derivatives limit the development of its properties and applications. Therefore, a “Y"-shaped 2,4,6-tri (4-pyridyl)-1,3,5-triazine derivative ([H3p-Batpt]Cl3, TPT) with high conjugation, bare nitrogen and oxygen sites that was selected as a novel photosensitive ligand to replace the traditional 4,4′-bipyridine ligand for photochromic MOFs. Then, [H3p-Batpt]Cl3 was combined with EuIII and terephthalic acid (H2TPA) to construct a multifunctional Eu-MOF [Eu2 (p-Batpt) (TPA)3(H2O)]n (1). 1 possess degradation of tetracycline (TC), photochromic and photoluminescence properties. The colored 1 (1P) after irradiation with UV–vis light (300 W, Xe-lamp) is difficult to fade due to donor-acceptor (D-A) π-π and radical-π interactions. In addition, 1 is used in photocatalysis because it is an excellent N-type semiconductor. The TC degradation rate of 1 can reach 67.42 % under visible light and be recycled six times. Interestingly, the TC degradation rate of 1P was 6.78 % higher than that of 1. Electrochemical tests show that 1P has higher charge separation efficiency and a smaller band gap. The free radicals capture and ESR experiments of 1 revealed that holes (h+) played a leading role in the photocatalytic degradation of TC. This research expands the application of photochromic MOFs and provides a reference for removing organic pollutants.

Structure-Activity Relationship of Antibody-Oligonucleotide Conjugates: Evaluating Bioconjugation Strategies for Antibody-siRNA Conjugates for Drug Development.

Antibody-oligonucleotide conjugates are a promising class of therapeutics for extrahepatic delivery of small interfering ribonucleic acids (siRNAs). These conjugates can be optimized for improved delivery and mRNA knockdown (KD) through understanding of structure-activity relationships. In this study, we systematically examined factors including antibody isotype, siRNA chemistry, linkers, conjugation chemistry, PEGylation, and drug-to-antibody ratios (DARs) for their impact on bioconjugation, pharmacokinetics (PK), siRNA delivery, and bioactivity. Conjugation site (cysteine, lysine, and Asn297 glycan) and DAR proved critical for optimal conjugate PK and siRNA delivery. SiRNA chemistry including 2' sugar modifications and positioning of phosphorothioates were found to be critical for delivery and duration of action. By utilizing cleavable and noncleavable linkers, we demonstrated the impact of linkers on PK and mRNA KD. To achieve optimal properties of antibody-siRNA conjugates, a careful selection of siRNA chemistry, DAR, conjugation sites, linkers, and antibody isotype is necessary.

DNA-directed immobilization fluorescent immunoarray for multiplexed antibiotic residue determination in milk.

The presence of antibiotic residues in cow's milk entails high risk for consumers, the dairy industry, and the environment. Therefore, the development of highly specific and sensitive screening tools for the rapid and cost-effective identification of traces of these compounds is urgently needed. A multiplexed screening platform utilizing DNA-directed immobilization (DDI) was developed aiming to detect three classes of antibiotic residues (fluoroquinolones, sulfonamides, and tylosin) prevalently found in milk. Throughout this work, each oligonucleotide sequence was conjugated to a different hapten molecule, while the three complementary strands were immobilized in 24 independent microarray chips on a single glass slide. First, the array was incubated with the pool of hapten-oligonucleotide conjugate site encoded the signal through DNA hybridization. Next, commercial milk samples were incubated with the cocktail of monoclonal antibodies following a secondary fluorophore-labeled antibody which was required for fluorescent readout. Direct sample detection was achieved in milk diluting 20 times in assay buffer. The limits of detection (LODs) reached were 1.43µgkg-1, 1.67µgkg-1, and 0.89µgkg-1 for TYLA, STZ, and CIP, respectively, which represented in raw milk 7.15µgkg-1, 8.35µgkg-1, and 4.45µgkg-1 for TYLA, STZ, and CIP, respectively, that are below the EU regulatory limits. Cross-reactivity profiles were evaluated against the family of structurally related antibiotics in order to demonstrate the capability to detect antibiotics from the same family of compounds. A pre-validation study was performed by spiking 20 blind samples above and below the maximum residue limits established by the EU guidelines. The system was successfully implemented towards randomized sample classification as compliant or non-compliant. The proposed DDI-based immunoarray provides a fast and cost-effective alternative to obtain semi-quantitative information about the presence of three veterinary residues simultaneously in milk samples.

Developing analytical ion exchange chromatography methods for antibody drug conjugates containing the hydrolysis-prone succinimide-thioether conjugation chemistry

Charge variants are one of the most important quality attributes for protein therapeutics, including antibody drug conjugates (ADCs). ADCs are conjugation products between monoclonal antibodies (mAbs) and highly potent payloads. After attaching a payload, the charge profile of a mAb can be modified due to the change in net charge or surface charge. In this study, we present a unique challenge of charge assay development that arises from a desirable engineering of ADCs that incorporates the hydrolysis-prone succinimide-thioether conjugation chemistry. This engineered hydrolysis at conjugation sites is usually not complete during conjugation process and continuously progressing during mild stress. This hydrolysis also creates a carboxylic functional group, which manifests as acidic peaks in the ADC charge profiles. As a result, ion exchange chromatograms become sensitive measurements of this hydrolysis, which often masks the charge profile change due to other important post-translational modifications. In this study, two approaches were explored to address this unique challenge: to remove the hydrolysis heterogeneity by incubating ADCs under high pH conditions to drive complete hydrolysis; and to analyze charge variants at the subunit level after IdeS digestion. Acceptable charge profiles and quantitative integration results were successfully obtained by both approaches.

Advancing structural elucidation of conjugation drug metabolites in metabolite profiling with novel electron-activated dissociation.

This study focuses on the advantage of using the novel electron-activated dissociation (EAD) technology on the QTOF system for structural elucidation of conjugation metabolites. In drug metabolite identification, conceptual "boxes" are generally used to represent potential sites of modifications, which are proposed based on MS/MS data. Electron-activated dissociation (EAD) provides unique fragmentation patterns, potentially allowing for more precise localization of the metabolic modification sites compared to CID, particularly for conjugations. Known compounds were incubated with rat liver microsomes in the presence of nicotinamide adenine dinucleotide phosphate (NADPH), uridine dihosphate-glucuronic acid (UDPGA), and glutathione. Conjugation metabolites were analyzed using the QTOF system. High-resolution MS/MS spectra were collected using EAD and CID fragmentations along with TOF MS full scan for tested drugs and metabolites. Fragmentation patterns were compared to evaluate their efficiency in structural elucidation. Metabolite profiling identified conjugation metabolites (glucuronides and GSH adducts), using characteristic mass shifts. A comparison of EAD and CID fragmentation revealed EAD-specific fragments for most conjugates. EAD was able to break the relatively stable bonds on parent drug motifs while keeping relatively weak conjugation bonds intact, despite the generally low intensity of EAD. EAD effectively narrowed the conceptual "box" representing modification sites, providing more definitive information on conjugation sites and facilitating the structural elucidation of conjugated metabolites. EAD is a powerful tool for metabolite profiling in drug development, particularly for identifying conjugation sites. EAD-enabled MS/MS spectra offer a greater variety of signature fragments compared to CID, resulting in more comprehensive and unique structural information for metabolic modification analysis. Overall, EAD, complementary to CID, has the potential to narrow down potential modification sites, significantly enhancing the precision of conjugation metabolite structure elucidation.

Effective Immobilization of Novel Antimicrobial Peptides via Conjugation onto Activated Silicon Catheter Surfaces.

Antibiotic-resistant microorganisms have become a serious threat to public health, resulting in hospital infections, the majority of which are caused by commonly used urinary tract catheters. Strategies for preventing bacterial adhesion to the catheters' surfaces have been potentially shown as effective methods, such as coating thesurface with antimicrobial biomolecules. Here, novel antimicrobial peptides (AMPs) were designed as potential biomolecules to prevent antibiotic-resistant bacteria from binding to catheter surfaces. Thiolated AMPs were synthesized using solid-phase peptide synthesis (SPPS), and prep-HPLC was used to obtain AMPs with purity greater than 90%. On the other side, the silicone catheter surface was activated by UV/ozone treatment, followed by functionalization with allyl moieties for conjugation to the free thiol group of cystein in AMPs using thiol-ene click chemistry. Peptide-immobilized surfaces were found to become more resistant to bacterial adhesion while remaining biocompatible with mammalian cells. The presence and site of conjugation of peptide molecules were investigated by immobilizing them to catheter surfaces from both ends (C-Pep and Pep-C). It was clearly demonstrated that AMPs conjugated to the surface via theirN terminus have a higher antimicrobial activity. This strategy stands out for its effective conjugation of AMPs to silicone-based implant surfaces for the elimination of bacterial infections.

A hydrolyzed N-propionylthiosuccinimide linker is cleaved by metastable fragmentation, increasing reliability of conjugation site identification in conjugate vaccines.

Conjugation sites are a quality attribute of conjugate vaccines. Proteolysis of bioconjugates synthesized by maleimide-thiol chemistry generates type 2 peptides with a hydrolyzed thiosuccinimide linker containing information on the conjugation sites. A mass spectrometry (MS)-cleavable linker could make the identification of conjugation sites by MS more reliable. Four synthetic type 2 peptides with a hydrolyzed thiosuccinimide linker were analyzed by matrix-assisted laser desorption ionization (MALDI) MS/MS with and without collision gas. These peptides were also partially labeled with 18O in the linker to confirm the proposed fragmentation mechanism. A conjugate vaccine with the hydrolyzed thiosuccinimide linker was reduced and S-alkylated, digested with trypsin and analyzed by liquid chromatography-MS/MS using collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) fragmentation methods at a normalized collision energy of 30. A metastable fragmentation preferentially cleaves the newly formed pseudopeptide bond within the hydrolyzed thiosuccinimide linker of type 2 peptides to yield P + 71 and C + 98 ions. These ions make the assignment of conjugation sites more reliable. Partial 18O-labeling and MS/MS analysis confirmed the proposed structures. CID produces these ions as the two most intense signals more favorably than HCD. The latter also yields these ions, guarantees better sequence coverage and promotes other fragmentations in the linker. Hydrolyzed thiosuccinimide linker is cleavable in MALDI and electrospray ionization MS/MS analysis by a gas-phase metastable fragmentation. The resulting fragment ions (P + 71 and C + 98) make the identification of conjugation sites more reliable. These results could be extended to self-hydrolyzing maleimides, which efficiently stabilize the thiosuccinimide linker upon hydrolysis, in antibody-drug conjugates.

A combination of multiple LC-MS approaches for the comprehensive characterization of cysteine-linked ADCs

Antibody-drug conjugates (ADCs) represent the forefront of the next generation of biopharmaceuticals. An ADC typically comprises an antibody covalently linked to a cytotoxic drug via a linker, resulting in a highly heterogeneous product. This study focuses on the analysis of a custom-made cysteine-linked ADC. Initially, we developed a LC-MS-based characterization workflow using brentuximab vedotin (Adcetris®), encompassing native intact MS, analysis of reduced chains and subunits under denaturing condition, peptide mapping and online strong cation exchange chromatography coupled with UV and mass spectrometry detection (SCX-UV-MS) applied for brentuximab vedotin first time reported. Subsequently, we applied this in-depth characterization workflow to a custom-made cysteine-linked ADC. The measured drug-to-antibody ratio(DAR) of this ADC is 6.9, further analysis shown that there is a small amount of unexpected over-conjugation. Over-conjugation sites were successfully identified using multiple UHPLC-MS based characterization techniques. Also, one competitively cysteine-conjugated impurity was observed in native intact MS results, by combing native intact MS, reduced chains, subunit analysis and peptide mapping results, the impurity conjugation sites were also identified. Since this molecule is at early development stage, this provides important information for conjugation process improvement and link-drug material purification. SCX-UV-MS approach can separate the custom-made cysteine-linked ADC carrying different payloads and reduce the complexity of the spectra. The integrated approach underscores the significance of combining the SCX-UV-MS online coupling technique with other characterization methods to elucidate the heterogeneity of cysteine-linked ADCs.

On the utility of the extracted ion chromatograms for assigning the conjugation sites and side reactions in bioconjugates synthesized by the maleimide-thiol chemistry

Maleimide-thiol chemistry is widely used in the synthesis of protein–protein conjugate vaccines. In this reaction, thiol and maleimide groups, either in the carrier protein or in the antigen molecules, are linked via a thiosuccinimide linker. The assignment of the conjugation sites is key and is usually done by LC-MS/MS analysis of the conjugate proteolytic digestion, resulting in a very complex mixture of linear peptides and cross-linked peptide pairs. During proteolysis, the thiosuccinimide linker is converted to its hydrolyzed and transcyclized forms. The set of MS/MS spectra assigned to cross-linked peptide pairs contains valuable information about the conjugation sites that must be validated using objective criteria for more reliable assignment. The extracted ion chromatograms (XICs) have not previously been considered for these validation purposes. In the LC-MS/MS analysis, linear peptides eluted with a single-peak XIC pattern, while the cross-linked peptide pairs with a transcyclized linker (consisting of a mixture of diastereomers) eluted with a two-peak XIC pattern in 68–100 % of cases. Cross-linked peptide pairs with the hydrolyzed linker were detected with a multi-peak XIC pattern consisting of two, three and four peaks in 77 %, 15 % and 8 % of cases, respectively. The four-peak XIC pattern corresponds to cross-linked peptide pairs with two positional isomers of the hydrolyzed linker containing a mixture of diastereomers. Tandem digestions increased the analytical value of the XICs by converting the large and hydrophobic cross-linked peptide pairs, that show a single-peak XIC pattern, to more hydrophilic species eluting in multi-peak XIC patterns. XICs are easily obtained in any LC-MS/MS analysis, contain valuable information about conjugation sites and side reaction detection, and are worthy of routine inclusion in bioconjugate characterization.

Optimization of quinolinium-carbazole theranostics for the improved photooxidation of β-amyloid aggregates with the variation of conjugation site

Alzheimer’s disease (AD), the most prevalent neurodegenerative condition, is characterized by a gradual decline in cognitive function and memory loss. Presently, it ranks as the fifth leading cause of death worldwide. Photodynamic therapy, with its operational flexibility, noninvasive approach, and high spatial and temporal resolution, has emerged as an innovative therapeutic method, enhancing therapeutic efficacy and minimizing systemic toxicity. In this work, we have designed two donor-π-acceptor (D-π-A) type photosensitizers targeting amyloid, differing in the donor and acceptor junction sites. Surprisingly we found that by altering the junction site of the donor and acceptor from the 2-position to the 3-position of carbazole, the binding affinity and singlet oxygen efficiency of compound 3-QM16 improved compared to compound 2-QM16. This modification facilitated β-amyloid imaging and photooxidative therapy. Our study might shed light on the further optimization of the photooxidation performance of photosensitizers on misfolded proteins.