RESUMEN
High-affinity and specific agents are widely applied in various areas, including diagnostics, scientific research, and disease therapy (as drugs and drug delivery systems). It takes significant time to develop them. For this reason, development of high-affinity agents extensively utilizes computer methods at various stages for the analysis and modeling of these molecules. The review describes the main affinity and specific agents, such as monoclonal antibodies and their fragments, antibody mimetics, aptamers, and molecularly imprinted polymers. The methods of their obtaining as well as their main advantages and disadvantages are briefly described, with special attention focused on the molecular modeling methods used for their analysis and development.
Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Monoclonales/química , Aptámeros de Nucleótidos/química , Modelos Moleculares , Humanos , Unión Proteica , Polímeros Impresos Molecularmente/químicaRESUMEN
Pre-existing anti-AAV antibodies can be detected using ligand binding-based assay formats. One such format is the MSD-based bridging assay, which uses sulfo-tag-labeled AAV vectors as detection reagents. However, no method has been developed to accurately measure the degree of sulfo-tag labeling on AAV vectors. To fill this gap, we developed a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method to assess the degree of labeling (DoL) of sulfo-tag on AAV5 vectors, enabling the measurement of the DoL on AAV5 at six increasing levels of sulfo-tag challenge ratio. In addition, a Biacore-based assay was used to evaluate the binding affinity between an anti-AAV5 monoclonal antibody and various sulfo-tag labeled AAV5 vectors. The results indicated that increased DoL of sulfo-tag labeling on AAV5 did not compromise the binding affinity.Our study further employed the MSD-bridging assay to detect the binding Signal/Noise (S/N) ratios of four anti-AAV5 monoclonal antibodies (mAbs) to various sulfo-tag-labeled AAV5 vectors. The findings revealed a strong correlation between the degree of sulfo-tag labeling and both the S/N ratios and the sensitivity of MSD bridging assays. This result underscores the importance of optimizing the labeling of detection reagents to enhance assay sensitivity for detecting anti-AAV5 antibodies.
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Anticuerpos Monoclonales , Dependovirus , Vectores Genéticos , Dependovirus/genética , Dependovirus/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/química , Humanos , Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Afinidad de Anticuerpos/inmunología , AnimalesRESUMEN
The formulation of biopharmaceutical drugs is designed to eliminate chemical instabilities, increase conformational and colloidal stability of proteins, and optimize interfacial stability. Among the various excipients involved, buffer composition plays a pivotal role. However, conventional buffers like histidine and phosphate buffers may not always be the optimal choice for all monoclonal antibodies (mAbs). In this study, we investigated the effects of several alternative buffer systems on seven different mAbs, exploring various combinations of ionic strengths, concentrations of the main buffer component, mAb concentrations, and stress conditions. Protein stability was assessed by analyzing soluble aggregate formation through size exclusion chromatography. At low protein concentrations, protein instability after temperature stress was exclusively observed in the bis-TRIS/ glucuronate buffer. Conversely, freeze-thaw stress led to a significant increase in aggregate formation in tested formulations, highlighting the efficacy of several alternative buffers, particularly arginine/ citrate, in preserving protein stability. Under temperature stress, the introduction of arginine to histidine buffer systems provided additional stabilization, while the addition of lysine resulted in protein destabilization. Similarly, the incorporation of arginine into histi-dine/HCl buffer further enhanced protein stability during freeze--thaw cycles. At high protein concentrations, the histidine/citrate buffer emerged as one of the most optimal choices for addressing temperature and light-induced stress. The efficacy of histidine buffers in combating light stress might be attributed to the light-absorbing properties of histidine molecules. Our findings demonstrate that the development of biopharmaceutical formulations should not be confined to conventional buffer systems, as numerous alternative options exhibit comparable or even superior performance.
Asunto(s)
Anticuerpos Monoclonales , Excipientes , Estabilidad Proteica , Tampones (Química) , Anticuerpos Monoclonales/química , Excipientes/química , Concentración Osmolar , Composición de Medicamentos/métodos , Temperatura , Estabilidad de Medicamentos , Histidina/química , Congelación , Química Farmacéutica/métodos , Arginina/química , Agregado de ProteínasRESUMEN
Metamizole (MET) is an antipyretic and analgesic drug, the illegal use of which can result in residues of MET metabolites in edible tissues of animals. In this study, a computational chemistry-assisted hapten screening strategy was used to screen for the optimal immunogenic hapten-A and the optimal coating antigen hapten-G-OVA. A monoclonal antibody capable of recognizing two pharmacologically active metabolites of MET, 4-methylamidinoantipyrine (MAA) and 4-aminoantipyrine (AA), was prepared from the hapten-A. The antibody showed excellent specificity for MAA and AA and almost no cross-reactivity with the pharmacologically inactive metabolites 4-formamidinoantipyrine (FAA) and 4-acetamidinoantipyrine (AAA). An ic-ELISA was developed for the simultaneous detection of MAA and AA in animal-derived food, the limits of detection for MAA ranged from 0.93 to 1.18 µg/kg, while those for AA ranged from 1.74 to 4.61 µg/kg. The recovery rate fell within the range of 82 %-110 %, with a coefficient of variation less than 16.39 %.
Asunto(s)
Anticuerpos Monoclonales , Dipirona , Haptenos , Haptenos/química , Haptenos/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/química , Animales , Dipirona/inmunología , Dipirona/análisis , Dipirona/química , Ratones , Contaminación de Alimentos/análisis , Ensayo de Inmunoadsorción Enzimática/métodos , Ratones Endogámicos BALB C , Análisis de los Alimentos/métodosRESUMEN
BACKGROUND: Charge heterogeneity is a critical quality attribute for therapeutic biologics including antibody-drug conjugates (ADCs). Developing an ion exchange chromatography (IEX) or an imaged capillary isoelectric focusing (icIEF) method for ADCs with high drug-to-antibody ratio (DAR) is challenging because of the increased hydrophobicity from the payload-linker, DAR heterogeneity, and payload-linker instability. A sub-optimal method can be poorly stability-indicating due to the inability to discern contributions from charge and size variants conjugated with different number of drugs/payloads. Systematic strategy and guidance on charge variant method development is highly desired for high DAR ADCs with various complex structures. RESULTS: This work encompasses the development and optimization of icIEF methods for high DAR ADCs of various DAR values (4-8) and payload linker chemistry. Method optimization focuses on improving resolution and stability indicating capabilities and differentiating contributions from the protein and payload-linker. Types, proportion, and combination of solubilizers and carrier ampholytes, as well as focusing parameters were interrogated. Our findings show that the structural units of the linker, the DAR, and the payload chemistry prescribe the selection of buffer, solubilizer, and ampholyte. We demonstrate that a stronger denaturant or solubilizer is needed for high DAR ADCs with polyethylene glycol (PEG)-containing linker structure compared to peptide linker. For unstable payload-linker, buffer system enhances sample stability which is vital to method robustness. In addition, a longer isoelectric focusing time is necessary for an ADC than its corresponding antibody to reach optimal focusing. SIGNIFICANCE: To the best of our knowledge, this is the first comprehensive study on icIEF method development for charge variant determination of high DAR ADCs with unique physicochemical properties.
Asunto(s)
Inmunoconjugados , Focalización Isoeléctrica , Focalización Isoeléctrica/métodos , Inmunoconjugados/química , Inmunoconjugados/análisis , Electroforesis Capilar/métodos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/análisis , Focalización Isoeléctrica CapilarRESUMEN
Host cell proteins (HCPs) are one of the process-related impurities that need to be well characterized and controlled throughout biomanufacturing processes to assure the quality, safety, and efficacy of monoclonal antibodies (mAbs) and other protein-based biopharmaceuticals. Although ELISA remains the gold standard method for quantification of total HCPs, it lacks the specificity and coverage to identify and quantify individual HCPs. As a complementary method to ELISA, the LC-MS/MS method has emerged as a powerful tool to identify and profile individual HCPs during the downstream purification process. In this study, we developed a sensitive, robust, and reproducible analytical flow ultra-high-pressure LC (UHPLC)-high-resolution accurate mass (HRAM) data-dependent MS/MS method for HCP identification and monitoring using an Orbitrap Ascend BioPharma Tribrid mass spectrometer. As a case study, the developed method was applied to an in-house trastuzumab product to assess HCP clearance efficiency of the newly introduced POROS™ Caprylate Mixed-Mode Cation Exchange Chromatography resin (POROS Caprylate mixed-mode resin) by monitoring individual HCP changes between the trastuzumab sample collected from the Protein A pool (purified by Protein A chromatography) and polish pool (purified by Protein A first and then further purified by POROS Caprylate mixed-mode resin). The new method successfully identified the total number of individual HCPs in both samples and quantified the abundance changes in the remaining HCPs in the polish purification sample.
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Anticuerpos Monoclonales , Cricetulus , Espectrometría de Masas en Tándem , Espectrometría de Masas en Tándem/métodos , Cromatografía Líquida de Alta Presión/métodos , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/química , Células CHO , Animales , Trastuzumab/química , Trastuzumab/análisis , HumanosRESUMEN
We develop a multiscale coarse-grain model of the NIST Monoclonal Antibody Reference Material 8671 (NISTmAb) to enable systematic computational investigations of high-concentration physical instabilities such as phase separation, clustering, and aggregation. Our multiscale coarse-graining strategy captures atomic-resolution interactions with a computational approach that is orders of magnitude more efficient than atomistic models, assuming the biomolecule can be decomposed into one or more rigid bodies with known, fixed structures. This method reduces interactions between tens of thousands of atoms to a single anisotropic interaction site. The anisotropic interaction between unique pairs of rigid bodies is precomputed over a discrete set of relative orientations and stored, allowing interactions between arbitrarily oriented rigid bodies to be interpolated from the precomputed table during coarse-grained Monte Carlo simulations. We present this approach for lysozyme and lactoferrin as a single rigid body and for the NISTmAb as three rigid bodies bound by a flexible hinge with an implicit solvent model. This coarse-graining strategy predicts experimentally measured radius of gyration and second osmotic virial coefficient data, enabling routine Monte Carlo simulation of medically relevant concentrations of interacting proteins while retaining atomistic detail. All methodologies used in this work are available in the open-source software Free Energy and Advanced Sampling Simulation Toolkit.
Asunto(s)
Lactoferrina , Método de Montecarlo , Muramidasa , Lactoferrina/química , Muramidasa/química , Anisotropía , Anticuerpos Monoclonales/químicaRESUMEN
During the development process of therapeutic monoclonal antibodies (mAbs), it is crucial to control (critical) quality attributes such as N-glycosylation influencing pharmacokinetics (PK) and Fc effector functions. Previous reports have shown that mAbs containing high-mannose N-glycans are cleared faster from blood circulation, leading to reduced half-lives. The high-mannose N-glycan content of mAbs can be influenced during the cell culture process by factors such as cell lines, process conditions, and media. Furthermore, mAbs have either one high mannose N-glycan (asymmetrical high-mannose glyco-pair) or two high mannose N-glycans (symmetrical high-mannose glyco-pair). The hypothesis that the mannose receptor (MR, CD206) accelerates clearance by facilitating their internalization and subsequent lysosomal degradation is widespread. However, the interaction between MR and mAbs has not been explicitly demonstrated. This study aimed to investigate this interaction, providing the first systematic demonstration of MR binding to the Fc region of mAbs with high-mannose N-glycans. Two novel analytical methods, MR surface plasmon resonance and MR affinity chromatography, were developed and applied to investigate the MR-mAb interaction. The interaction is found to be dependent on high-mannose content, but is independent of the mAb format or sequence. However, different glyco-pairs exhibited varying binding affinities to the MR, with the symmetrical high-mannose glyco-pair showing the strongest binding properties. These findings strengthen the hypothesis for the MR-mediated mAb interaction and contribute to a deeper understanding of the MR-mAb interaction, which could affect the criticality of high-mannose containing mAbs development strategies of IgG-based molecules and improve their PK profiles.
Asunto(s)
Anticuerpos Monoclonales , Lectinas Tipo C , Receptor de Manosa , Lectinas de Unión a Manosa , Manosa , Polisacáridos , Receptores de Superficie Celular , Polisacáridos/metabolismo , Polisacáridos/química , Lectinas de Unión a Manosa/metabolismo , Receptores de Superficie Celular/metabolismo , Lectinas Tipo C/metabolismo , Manosa/metabolismo , Manosa/química , Humanos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/inmunología , Animales , Glicosilación , Cricetulus , Células CHO , Resonancia por Plasmón de Superficie , Unión ProteicaRESUMEN
Therapeutic formats derived from the monoclonal antibody structure have been gaining significant traction in the biopharmaceutical market. Being structurally similar to mAbs, most Fc-containing therapeutics exhibit product-related impurities in the form of aggregates, charge variants, fragments, and glycoforms, which are inherently challenging to remove. In this work, we developed a workflow that employed rapid resin screening in conjunction with an in silico tool to identify and rank orthogonally selective processes for the removal of product-related impurities from a Fc-containing therapeutic product. Linear salt gradient screens were performed at various pH conditions on a set of ion-exchange, multimodal ion-exchange, and hydrophobic interaction resins. Select fractions from the screening experiments were analyzed by three different analytical techniques to characterize aggregates, charge variants, fragments, and glycoforms. The retention database generated by the resin screens and subsequent impurity characterization were then processed by an in silico tool that generated and ranked all possible two-step resin sequences for the removal of product-related impurities. A highly-ranked process was then evaluated and refined at the bench-scale to develop a completely flowthrough two-step polishing process which resulted in complete removal of the Man5 glycoform and aggregate impurities with a 73% overall yield. The successful implementation of the in silico mediated workflow suggests the possibility of a platformable workflow that could facilitate polishing process development for a wide variety of mAb-based therapeutics.
Asunto(s)
Anticuerpos Monoclonales , Simulación por Computador , Contaminación de Medicamentos , Fragmentos Fc de Inmunoglobulinas , Flujo de Trabajo , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/aislamiento & purificación , Cromatografía por Intercambio Iónico/métodos , Cricetulus , Interacciones Hidrofóbicas e Hidrofílicas , Células CHO , AnimalesRESUMEN
Aflatoxin B1 (AFB1) accumulates in crops, where it poses a threat to human health. To detect AFB1, anti-AFB1 monoclonal antibodies have been developed and are widely used. While the sensitivity and specificity of these antibodies have been extensively studied, information regarding the atomic-level docking of AFB1 (and its derivatives) with these antibodies is limited. Such information is crucial for understanding the key interactions that are required for high affinity and specificity in aflatoxin binding. First, a 3D comparative model of anti-AFB1 antibody (Ab-4B5G6) was predicted from the sequence using RosettaAntibody. We then utilized RosettaLigand to dock AFB1 onto ten homology models, producing a total of 10,000 binding modes. Interestingly, the best-scoring mode predicted strong interactions involving four sites within the heavy chain: ALA33, ASN52, HIS95, and TRP99. Importantly, these strong binding interactions exclusively involve the variable domain of the heavy chain. The best-scoring mode with AFB1 was also obtained through AF multimer combined with RosettaLigand, and two interactions at TRP and HIS were consistent with those found by Rosetta antibody-ligand computational simulation. The role of tryptophan in π interactions in antibodies was confirmed through mutation experiments, and the resulting mutant (W99A) exhibited a >1000-fold reduction in binding affinity for AFB1 and analogs, indicating the effect of tryptophan on the stability of CDR-H3 region. Additionally, we evaluated the binding of two glycolic acid-derived molecular derivatives (with impaired hydrogen bonding potential), and these derivatives (AFB2-GA and AFG2-GA) demonstrated a very weak binding affinity for Ab-4B5G6. The heavy chain was successfully isolated, and its sensitivity and specificity were consistent with those of the intact antibody. The homology models of variable heavy (VH) single-domain antibodies were established by RosettaAntibody, and the docking analysis revealed the same residues, including Ala, His, and Trp. Compared to the potential binding mode of fragment variable (FV) region, the results from a model of VH indicated that there are seven models involved in hydrophobic interaction with TYR32, which is usually referred to as polar amino acid and has both hydrophobic and hydrophilic features depending on the circumstances. Our work encompasses the entire process of Rosetta antibody-ligand computational simulation, highlighting the significance of variable heavy domain structural design in enhancing molecular interactions.
Asunto(s)
Aflatoxina B1 , Anticuerpos Monoclonales , Simulación del Acoplamiento Molecular , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Ligandos , Aflatoxina B1/química , Aflatoxina B1/inmunología , Especificidad de Anticuerpos , Aflatoxinas/química , Afinidad de Anticuerpos , Conformación Proteica , Secuencia de Aminoácidos , Simulación por Computador , Humanos , Simulación de Dinámica MolecularRESUMEN
Stability of conjugated critical reagents supporting ligand binding assays to enable biotherapeutic drug development is a universal concern. Formulation buffer employed for long-term cold storage may be key to mitigate protein aggregation issues. We investigated biophysical and functional attributes of murine mAb and human multispecific drug labeled with biotin, ruthenium, and Alexa fluor 647 frozen at -80 °C in PBS or a protein storage buffer for 3-15 months. Aggregation was observed at 4 months in mAb A-Ru (11.2%) and -Alexa (10%) in PBS followed by precipitation and reduced biological binding at 15 months. Increased aggregation in drug Ru (11.7%, 6 months) and Alexa (6.9%, 15 months) were noted but without impact on performance. There were no observations with biotin labeled reagents.
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Anticuerpos Monoclonales , Tampones (Química) , Humanos , Animales , Anticuerpos Monoclonales/química , Ratones , Biotina/química , Estabilidad de Medicamentos , Agregado de Proteínas , Rutenio/química , Estabilidad ProteicaRESUMEN
Methods for the reliable and effective detection and identification of impurities are crucial to ensure the quality and safety of biopharmaceutical products. Technical limitations constrain the accurate identification of individual impurity peaks by size-based electrophoresis separations followed by mass spectrometry. This study presents a size-based electrophoretic method for detecting and identifying impurity peaks in antibody production. A hydrogen sulfide-accelerated degradation method was employed to generate known degradation products observed in bioreactors that forms the basis for size calibration. LabChip GXII channel electrophoresis enabled the rapid (< 1 min) detection of impurity peaks based on size, while capillary zone electrophoresis-mass spectrometry (CZE-MS) facilitated their accurate identification. We combine these techniques to examine impurities resulting from cell culture harvest conditions and forced degradation to assess antibody stability. To mimic cell culture harvest conditions and the impact of forced degradation, we subjected samples to cathepsin at different pH buffers or exposed them to high pH and temperature. Our method demonstrated the feasibility and broad applicability of using a CZE-MS generated spectral library to unambiguously assign peaks in high throughput size-based electrophoresis (i.e., LabChip GXII) with identifications or likely mass of the antibody impurity. Overall, this strategy combines the utility of CZE-MS as a high-resolution separation and detection method for impurities with size-based electrophoresis methods that are typically used to detect (not identify) impurities during the discovery and development of antibody therapeutics.
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Contaminación de Medicamentos , Electroforesis Capilar , Espectrometría de Masas , Electroforesis Capilar/métodos , Espectrometría de Masas/métodos , Contaminación de Medicamentos/prevención & control , Animales , Células CHO , Cricetulus , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/análisis , Concentración de Iones de Hidrógeno , Catepsinas/análisis , Reactores BiológicosRESUMEN
Co-formulated antibodies can bring clinical benefits to patients by combining two or more antibodies in a single dosage form. However, the quality analysis of co-formulated antibodies raises additional challenges, compared to individual antibodies, due to the need for accurate analysis of multiple antibodies in one solution. It is extremely difficult to effectively separate the charge variants of the two co-formulated antibodies using one ion exchange chromatography (IEC) method because of their similar characteristics. In this study, a novel method was developed for the charge variants characterization of co-formulated antibodies using three-dimensional liquid chromatography-mass spectrometry (3D-LC-MS). Hydrophobic interaction chromatography (HIC) was used as the first dimension to separate and collect the two co-formulated antibodies. The two collections were then injected into the second-dimension IEC separately for charge variants separation and analysis. Subsequently, the separated charge variants underwent on-line desalting in the third-dimension reverse-phase chromatography (RPC) and subsequent mass spectroscopy analysis. The novel method could simultaneously provide a charge variants ratio and post-translational modification (PTM) data for the two co-formulated antibodies. Therefore, it could be used for release testing and stability studies of co-formulated antibodies, making up for the shortcomings of the existing approaches. It was the first time that charge variants of co-formulated antibodies were characterized by the 3D-LC-MS method, to the best of our knowledge.
Asunto(s)
Espectrometría de Masas , Espectrometría de Masas/métodos , Cromatografía Liquida/métodos , Cromatografía por Intercambio Iónico/métodos , Interacciones Hidrofóbicas e Hidrofílicas , Humanos , Cromatografía de Fase Inversa/métodos , Animales , Anticuerpos Monoclonales/química , Procesamiento Proteico-Postraduccional , Cromatografía Líquida con Espectrometría de MasasRESUMEN
The central immunological role of HLA class I (HLA-I) in presenting peptide Ags to cellular components of the immune system has been the focus of intense study for >60 y. A confounding factor in the study of HLA-I has been the extreme polymorphism of these molecules. The mAb W6/32 has been a fundamental reagent bypassing the issue of polymorphism by recognizing an epitope that is conserved across diverse HLA-I allotypes. However, despite the widespread use of W6/32, the epitope of this Ab has not been definitively mapped. In this study, we present the crystal structure of the Fab fragment of W6/32 in complex with peptide-HLA-B*27:05. W6/32 bound to HLA-B*27:05 beneath the Ag-binding groove, recognizing a discontinuous epitope comprised of the α1, α2, and α3 domains of HLA-I and ß2-microglobulin. The epitope comprises a region of low polymorphism reflecting the pan-HLA-I nature of the binding. Notably, the W6/32 epitope neither overlaps the HLA-I binding sites of either T cell Ag receptors or killer cell Ig-like receptors. However, it does coincide with the binding sites for leukocyte Ig-like receptors and CD8 coreceptors. Consistent with this, the use of W6/32 to block the interaction of NK cells with HLA-I only weakly impaired inhibition mediated by KIR3DL1, but impacted HLA-LILR recognition.
Asunto(s)
Anticuerpos Monoclonales , Humanos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/química , Cristalografía por Rayos X , Antígenos HLA-B/inmunología , Antígenos HLA-B/química , Antígenos HLA-B/genética , Unión Proteica , Epítopos/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/química , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/química , Antígeno HLA-B27RESUMEN
Established in recent years as an important approach to unraveling the heterogeneity of intact monoclonal antibodies, native mass spectrometry has been rarely utilized for sequencing these complex biomolecules via tandem mass spectrometry. Typically, top-down mass spectrometry has been performed starting from highly charged precursor ions obtained via electrospray ionization under denaturing conditions (i.e., in the presence of organic solvents and acidic pH). Here we systematically benchmark four distinct ion dissociation methodsânamely, higher-energy collisional dissociation, electron transfer dissociation, electron transfer dissociation/higher-energy collisional dissociation, and 213 nm ultraviolet photodissociationâin their capability to characterize a therapeutic monoclonal antibody, trastuzumab, starting from denatured and native-like precursor ions. Interestingly, native top-down mass spectrometry results in higher sequence coverage than the experiments carried out under denaturing conditions, with the exception of ultraviolet photodissociation. Globally, electron transfer dissociation followed by collision-based activation of product ions generates the largest number of backbone cleavages in disulfide protected regions, including the complementarity determining regions, regardless of electrospray ionization conditions. Overall, these findings suggest that native mass spectrometry can certainly be used for the gas-phase sequencing of whole monoclonal antibodies, although the dissociation of denatured precursor ions still returns a few backbone cleavages not identified in native experiments. Finally, a comparison of the fragmentation maps obtained under denaturing and native conditions strongly points toward disulfide bonds as the primary reason behind the largely overlapping dissociation patterns.
Asunto(s)
Anticuerpos Monoclonales , Desnaturalización Proteica , Trastuzumab , Trastuzumab/química , Trastuzumab/análisis , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Secuencia de AminoácidosRESUMEN
HER2 receptors, overexpressed in certain human cancers, have drawn significant attention in cancer research due to their correlation with poor survival rates. Researchers have developed monoclonal antibodies like Trastuzumab and Pertuzumab against HER2 receptors, which have proven highly beneficial in cancer therapy. Bispecific antibodies like Zanidatamab and antibody-drug conjugates like T-DM1 have been developed to overcome the resistance associated with monotherapy. Small molecules such as Lapatinib, Neratinib, and Pyrotinib were initially developed for treating breast cancer. However, ongoing research is investigating their potential use in other types of cancer, often in combination with other medications. EGFR/HER2 dual-targeted drugs have overcome drug resistance associated with HER2-targeted monotherapy. This comprehensive review covers the structural characteristics of HER2, the HER family signaling pathway mechanism, recent findings regarding HER2 receptor involvement in various cancers, and diverse HER2-targeted therapies. This information provides a comprehensive understanding of HER2-targeted strategies in the evolving field of cancer treatment.
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Anticuerpos Monoclonales , Antineoplásicos , Neoplasias , Receptor ErbB-2 , Humanos , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Estructura Molecular , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/metabolismoRESUMEN
Previous work has shown that binding of target proteins to a sparse, unbiased sample of all possible peptide sequences is sufficient to train a machine learning model that can then predict, with statistically high accuracy, target binding to any possible peptide sequence of similar length. Here, highly sequence-specific molecular recognition is explored by measuring binding of 8 monoclonal antibodies (mAbs) with specific linear cognate epitopes to an array containing 121,715 near-random sequences about 10 residues in length. Network models trained on resulting sequence-binding values are used to predict the binding of each mAb to its cognate sequence and to an in silico generated one million random sequences. The model always ranks the binding of the cognate sequence in the top 100 sequences, and for 6 of the 8 mAbs, the cognate sequence ranks in the top ten. Practically, this approach has potential utility in selecting highly specific mAbs for therapeutics or diagnostics. More fundamentally, this demonstrates that very sparse random sampling of a large amino acid sequence spaces is sufficient to generate comprehensive models predictive of highly specific molecular recognition.
Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/química , Secuencia de Aminoácidos , Aprendizaje Automático , Epítopos/inmunología , Epítopos/química , Humanos , Unión Proteica , Sitios de Unión de Anticuerpos , Simulación por ComputadorRESUMEN
A design procedure for the separation of charge variants of a monoclonal antibody (mAb) was developed, which was based on the coupling of cation-exchange chromatography (CEX) and anion-exchange chromatography (AEX) under high loading conditions. The design of the coupled process was supported by a dynamic model. The model was calibrated on the basis of band profiles of variants determined experimentally for the mAb materials of different variant compositions. The numerical simulations were used to select the coupling configuration and the loading conditions that allowed for efficient separation of the mAb materials into three products enriched with each individual variant: the acidic (av), main (mv) and basic (bv) one. In the CEX section, a two-step pH gradient was used to split the loaded mass of mAb into a weakly bound fraction enriched with av and mv, and a strongly bound fraction containing the bv-rich product. The weakly bound fraction was further processed in the AEX section, where the mv-rich product was eluted in flowthrough, while the av-rich product was collected by a step change in pH. The choice of flow distribution and the number of columns in the CEX and AEX sections depended on the variant composition of the mAb material. For the selected configurations, the optimized mAb loading density in the CEX columns ranged from 10 to 26 mg mL-1, while in the AEX columns it was as high as 300 or 600 mg mL-1, depending on the variant composition of the mAb material. By proper selection of the loading condition, a trade-off between yield and purity of the products could be reached.
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Anticuerpos Monoclonales , Cromatografía por Intercambio Iónico/métodos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Concentración de Iones de Hidrógeno , Cricetulus , Células CHO , AnimalesRESUMEN
This study investigates the impact of 2-methyl imidazolium dihydrogen phosphate (2-MIDHP) on monoclonal antibody (mAb) aggregation during the Protein A purification stage, at a low pH (pH 3.0), and the viral inactivation phase. Size-exclusion high-performance liquid chromatography (SE-HPLC) and dynamic light scattering (DLS) were used to assess the mAb aggregation. Additionally, the influence of 2-MIDHP on mAb recovery, host cell protein (HCP) clearance, and Protein A leaching was investigated. Thermal stability of mAb, eluted in buffers containing 5 % to 25 % 2-MIDHP was analysed, using differential scanning calorimetry (DSC). Structural insights were obtained via circular dichroism (CD) and fluorescence spectroscopy. Our findings indicated that 2-MIDHP exerted a concentration-dependent protective effect against mAb aggregation, at the pH of 3.0. As the concentration of 2-MIDHP was increased from 0 % to 25 %, the aggregation was significantly reduced from 3.8 ± 0.01 % to 0.56 ± 0.002 %, as analysed by SE-HPLC. Addition of 2-MIDHP did not significantly impact the mAb recovery, HCP clearance, or Protein A leaching. DSC data supported these results, with higher 2-MIDHP concentrations leading to increased melting temperatures of mAb. CD and fluorescence spectroscopy revealed no significant changes in the secondary structure or aromatic residue environment in 2-MIDHP-treated samples, despite the observed reduction in aggregation. The results suggested that 2-MIDHP mitigated mAb aggregation during Protein A purification, possibly by stabilizing the protein structure under acidic stress conditions. These findings offer valuable insights for improving the robustness of mAb purification processes, enhancing product quality and yield.
Asunto(s)
Anticuerpos Monoclonales , Imidazoles , Estabilidad Proteica , Proteína Estafilocócica A , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Imidazoles/química , Proteína Estafilocócica A/química , Animales , Cromatografía Líquida de Alta Presión/métodos , Concentración de Iones de Hidrógeno , Células CHO , Cricetulus , Dicroismo Circular , Cromatografía en Gel/métodos , Cromatografía de Afinidad/métodos , Rastreo Diferencial de Calorimetría , Espectrometría de Fluorescencia , Agregado de ProteínasRESUMEN
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