RESUMEN
Fc-fusion proteins are an emerging class of protein therapeutics that combine the properties of biological ligands with the unique properties of the fragment crystallizable (Fc) domain of an immunoglobulin G (IgG). Due to their diverse higher-order structures (HOSs), Fc-fusion proteins remain challenging characterization targets within biopharmaceutical pipelines. While high-resolution biophysical tools are available for HOS characterization, they frequently demand extended time frames and substantial quantities of purified samples, rendering them impractical for swiftly screening candidate molecules. Herein, we describe the development of ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU) workflows that aim to fill this technology gap, where we focus on probing the HOS of a model Fc-Interleukin-10 (Fc-IL-10) fusion protein engineered using flexible glycine-serine linkers. We evaluate the ability of these techniques to probe the flexibility of Fc-IL-10 in the absence of bulk solvent relative to other proteins of similar size, as well as localize structural changes of low charge state Fc-IL-10 ions to specific Fc and IL-10 unfolding events during CIU. We subsequently apply these tools to probe the local effects of glycine-serine linkers on the HOS and stability of IL-10 homodimer, which is the biologically active form of IL-10. Our data reveals that Fc-IL-10 produces significantly more structural transitions during CIU and broader IM profiles when compared to a wide range of model proteins, indicative of its exceptional structural dynamism. Furthermore, we use a combination of enzymatic approaches to annotate these intricate CIU data and localize specific transitions to the unfolding of domains within Fc-IL-10. Finally, we detect a strong positive, quadratic relationship between average linker mass and fusion protein stability, suggesting a cooperative influence between glycine-serine linkers and overall fusion protein stability. This is the first reported study on the use of IM-MS and CIU to characterize HOS of Fc-fusion proteins, illustrating the practical applicability of this approach.
Asunto(s)
Fragmentos Fc de Inmunoglobulinas , Espectrometría de Masas , Desplegamiento Proteico , Proteínas Recombinantes de Fusión , Fragmentos Fc de Inmunoglobulinas/química , Proteínas Recombinantes de Fusión/química , Espectrometría de Masas/métodos , Interleucina-10/química , Interleucina-10/metabolismo , Espectrometría de Movilidad Iónica/métodos , Estabilidad Proteica , Humanos , Inmunoglobulina G/químicaRESUMEN
As one of the most critical steps in process development for protein therapeutics, clone selection and cell culture optimization require a large number of samples to be screened for high titer and desirable molecular profiles. Typical analytical techniques, such as chromatographic approaches, often take minutes per sample which are inefficient for large-scale screenings. Droplet microfluidics coupled to mass spectrometry (MS) represents an attractive approach due to its low volume requirements, high-throughput capabilities, label-free nature, and ability to handle complex mixtures. In this work, we coupled a modified protein cleanup protocol with a droplet-MS workflow for mAb titer screening to guide clone selection. With this droplet approach we achieved a throughput of 0.04 samples/s with an LoD of 0.15 mg/mL and an LoQ of 0.45 mg/mL. To test its performance in a real-world setting, this workflow was applied to a 35-clone screen, where the top 20% producing clones were identified. In addition, we coupled our sample cleanup protocol to a high-resolution MS and compared the glycan profiles of the high titer clones. This work demonstrates that droplet-MS provides a rapid way of clone screening and cell culture optimization based on titer and molecular structure of the expressed proteins. Future work is aimed at increasing the throughput and automation of this droplet-MS technique.
Asunto(s)
Microfluídica , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Ionización de Electrospray/métodos , Microfluídica/métodos , Formación de Anticuerpos , Anticuerpos Monoclonales , Células ClonalesRESUMEN
Bispecific antibodies (bsAbs) represent a critically important class of emerging therapeutics capable of targeting two different antigens simultaneously. As such, bsAbs have been developed as effective treatment agents for diseases that remain challenging for conventional monoclonal antibody (mAb) therapeutics to access. Despite these advantages, bsAbs are intricate molecules, requiring both the appropriate engineering and pairing of heavy and light chains derived from separate parent mAbs. Current analytical tools for tracking the bsAb construction process have demonstrated a limited ability to robustly probe the higher-order structure (HOS) of bsAbs. Native ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU) have proven to be useful tools in probing the HOS of mAb therapeutics. In this report, we describe a series of detailed and quantitative IM-MS and CIU data sets that reveal HOS details associated with a knob-into-hole (KiH) bsAb model system and its corresponding parent mAbs. We find that quantitative analysis of CIU data indicates that global KiH bsAb stability occupies an intermediate space between the stabilities recorded for its parent mAbs. Furthermore, our CIU data identify the hole-containing half of the KiH bsAb construct to be the least stable, thus driving much of the overall stability of the KiH bsAb. An analysis of both intact bsAb and enzymatic fragments allows us to associate the first and second CIU transitions observed for the intact KiH bsAb to the unfolding Fab and Fc domains, respectively. This result is likely general for CIU data collected for low charge state mAb ions and is supported by data acquired for deglycosylated KiH bsAb and mAb constructs, each of which indicates greater destabilization of the second CIU transition observed in our data. When integrated, our CIU analysis allows us to link changes in the first CIU transition primarily to the Fab region of the hole-containing halfmer, while the second CIU transition is likely strongly connected to the Fc region of the knob-containing halfmer. Taken together, our results provide an unprecedented road map for evaluating the domain-level stabilities and HOS of both KiH bsAb and mAb constructs using CIU.
Asunto(s)
Anticuerpos Biespecíficos , Anticuerpos Biespecíficos/química , Anticuerpos Monoclonales , Espectrometría de MasasRESUMEN
Antibody-based therapeutic proteins have highly complex molecular structures. The final therapeutic protein product may contain a wide range of charge variants. Accurate analysis of this charge variant composition is critical to determine manufacturing process consistency and protein stability and ultimately helps to ensure that patients receive a safe and efficacious product. Here, a highly sialylated bispecific antibody (bsAb-1) challenged the ability to monitor stability by imaged capillary isoelectric focusing (iCIEF). This challenge was overcome by optimization of the iCIEF master mix buffer (adjustment of urea concentration, addition of l-arginine) and enzymatic removal of sialic acid. The method was qualified by assessing linearity, precision, LOD, LOQ, accuracy, and robustness in accordance with ICH guidance. Main species loss detectability increased up to approximately fivefold compared to the iCIEF method without desialylation when monitoring changes in stressed samples. Importantly, the results of the iCIEF method with desialylation correlated with results obtained through LC-MS tryptic peptide mapping and enabled analysis of formulation development stability samples. Finally, this analytical method shows the potential to assess low-concentration formulation development samples down to a sample concentration of 0.1 mg/ml.
Asunto(s)
Electroforesis Capilar , Ácido N-Acetilneuramínico , Cromatografía Liquida , Electroforesis Capilar/métodos , Humanos , Focalización Isoeléctrica/métodos , Espectrometría de MasasRESUMEN
Liquid chromatography-mass spectrometry (LC-MS)-based proteomics approaches have been widely used to identify residual host-cell proteins (HCPs) in support of process and product characterization for protein therapeutics. Particularly, these methods can provide a general and unbiased approach for the detection of HCPs and may generate critical information on HCPs that are outside the coverage provided by a conventional immunoassay. A significant technical hurdle for HCP analysis is the overwhelmingly large background of biotherapeutic that obscures HCP detection and quantification. In this work, we developed a method that relies on hydrophilic interaction chromatography (HILIC) for HCP enrichment followed by in situ concentration and digestion prior to LC-MS analysis. This approach has enabled detection of HCPs in a drug substance that were not observed in other conventional flow rate LC-MS strategies. For example, 28% of HCPs identified in NISTmAb (20 out of 71) were not previously published or identified by established methods such as the native digestion technique. For an IgG1 protein spiked with 1000 ppm HCP standards, we detected 83 HCPs, 61 out of which were not identified by the native digestion method. Similar improvement in performance was demonstrated for an Fc-fusion protein therapeutic. Our method can be readily implemented in most protein mass spectrometry laboratories to support process development for protein therapeutics.
Asunto(s)
Cromatografía Liquida/métodos , Proteínas/química , Proteínas/uso terapéutico , Animales , Anticuerpos Monoclonales , Anticuerpos Monoclonales Humanizados , Células CHO , Cricetulus , Inmunoglobulina G/química , Espectrometría de Masas en Tándem/métodos , Tripsina/metabolismoRESUMEN
Monoclonal antibodies are attractive therapeutic agents because of their impressive biological activities and favorable biophysical properties. Nevertheless, antibodies are susceptible to various types of chemical modifications, and the impact of such modifications on antibody physical stability and aggregation remains understudied. Here, we report a systematic analysis of the impact of methionine oxidation, tryptophan oxidation, and asparagine deamidation on antibody conformational and colloidal stability, hydrophobicity, solubility, and aggregation. Interestingly, we find little correlation between the impact of these chemical modifications on antibody conformational stability and aggregation. Methionine oxidation leads to significant reductions in antibody conformational stability while having little impact on antibody aggregation except at extreme conditions (low pH and elevated temperature). Conversely, tryptophan oxidation and asparagine deamidation have little impact on antibody conformational stability while promoting aggregation at a wide range of solution conditions, and the aggregation mechanisms appear linked to unique types of reducible and nonreducible covalent crosslinks and, in some cases, to increased levels of attractive colloidal interactions. These findings highlight that even related types of chemical modifications can lead to dissimilar antibody aggregation mechanisms, and evaluating these findings for additional antibodies will be important for improving the systematic generation of antibodies with high chemical and physical stability.
Asunto(s)
Anticuerpos Monoclonales/química , Asparagina/química , Metionina/química , Triptófano/química , Coloides , Composición de Medicamentos , Estabilidad de Medicamentos , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Oxidación-Reducción , Agregado de Proteínas , Estabilidad Proteica , Solubilidad , TemperaturaRESUMEN
Monoclonal antibodies must be both chemically and physically stable to be developed into safe and effective drugs. Although there has been considerable progress in separately understanding the molecular determinants of antibody chemical and physical stability, it remains poorly understood how defects in one property (e.g., chemical stability) impact the other property (e.g., physical stability). Here, we have investigated the impact of a common chemical modification (deamidation) on the physical stability of two monoclonal antibodies as a function of pH (from pH 3.8 to 7.4). Interestingly, we find that deamidation has significant, antibody-specific impacts on physical stability at low pH values that are common during antibody purification. Deamidation causes increases in self-association and/or aggregation at low pH (3.8), and a key contributor to this behavior appears to be deamidation-dependent increases in antibody hydrophobicity at low pH. Our findings highlight pH-dependent impacts of deamidation on antibody colloidal stability and aggregation, which are important to understand in order to improve the development and production of potent antibody therapeutics with high chemical and physical stabilities.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Composición de Medicamentos/métodos , Diseño de Fármacos , Estabilidad de Medicamentos , Concentración de Iones de Hidrógeno , Agregado de Proteínas , Asparagina/química , Cromatografía/métodos , Dispersión Dinámica de Luz/métodos , Oro/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Inmunoglobulina G/química , Nanopartículas del Metal/química , Solubilidad , Temperatura de TransiciónRESUMEN
Recombinant monoclonal antibodies (mAbs) manufactured from immortalized mammalian cell lines are becoming increasingly important as therapies. Ensuring the quality of expressed proteins is critical when developing manufacturing processes. Protein sequence variants (PSVs) are a type of product-related variant in which errors in the protein sequence are present. Detecting PSVs and determining their origins, either by DNA mutation or mRNA mistranslation, is critical. Mutations cannot be remediated without developing new clones, which can be costly and time-consuming. In contrast, mistranslation can usually be mitigated by optimizing cell culture conditions. In this work, we first developed a new method to detect low-abundance PSVs with improved sensitivity. Then, a statistical metric was proposed to determine whether the observed PSVs originate from mutation or mistranslation by characterizing the distribution of PSVs. This method was applied to the evaluation of 50 clones from five mAbs programs, allowing for identification of five mutation and 139 mistranslation PSVs. The presence of even a few mutations demonstrates the necessity of clone screening during process development.
Asunto(s)
Anticuerpos Monoclonales/análisis , Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , Animales , Anticuerpos Monoclonales/genética , Células CHO , Codón/genética , Cricetulus , Mutación , Biosíntesis de Proteínas/genéticaRESUMEN
Microfabricated fluidic systems have emerged as a powerful approach for chemical analysis. Relatively unexplored is the use of microfabrication to create sampling probes. We have developed a sampling probe microfabricated in Si by bulk micromachining and lithography. The probe is 70 µm wide by 85 µm thick by 11 mm long and incorporates two buried channels that are 20 µm in diameter. The tip of the probe has two 20 µm holes where fluid is ejected or collected for sampling. Utility of the probe was demonstrated by sampling from the brain of live rats. For sampling, artificial cerebral spinal fluid was infused in through one channel at 50 nL/min while sample was withdrawn at the same flow rate from the other channel. Analysis of resulting fractions collected every 20 min from the striatum of rats by liquid chromatography with mass spectrometry demonstrated reliable detection of 17 neurotransmitters and metabolites. The small probe dimensions suggest it is less perturbing to tissue and can be used to sample smaller brain nuclei than larger sampling devices, such as microdialysis probes. This sampling probe may have other applications such as sampling from cells in culture. The use of microfabrication may also enable incorporation of electrodes for electrochemical or electrophysiological recording and other channels that enable more complex sample preparation on the device.
Asunto(s)
Cuerpo Estriado/química , Microelectrodos/veterinaria , Microtecnología/instrumentación , Neurotransmisores/análisis , Animales , Cromatografía Liquida , Espectrometría de Masas , Microelectrodos/normas , Microtecnología/métodos , Ratas , Técnicas EstereotáxicasRESUMEN
Although populations of neurons are known to vary on the micrometer scale, little is known about whether basal concentrations of neurotransmitters also vary on this scale. We used low-flow push-pull perfusion to test if such chemical gradients exist between several small brain nuclei. A miniaturized polyimide-encased push-pull probe was developed and used to measure basal neurotransmitter spatial gradients within brain of live animals with 0.004 mm(3) resolution. We simultaneously measured dopamine (DA), norepinephrine, serotonin (5-HT), glutamate, γ-aminobutyric acid (GABA), aspartate (Asp), glycine (Gly), acetylcholine (ACh), and several neurotransmitter metabolites. Significant differences in basal concentrations between midbrain regions as little as 200 µm apart were observed. For example, dopamine in the ventral tegmental area (VTA) was 4.8 ± 1.5 nM but in the red nucleus was 0.5 ± 0.2 nM. Regions of high glutamate concentration and variability were found within the VTA of some individuals, suggesting hot spots of glutamatergic activity. Measurements were also made within the nucleus accumbens core and shell. Differences were not observed in dopamine and 5-HT in the core and shell; but their metabolites homovanillic acid (460 ± 60 nM and 130 ± 60 nM respectively) and 5-hydroxyindoleacetic acid (720 ± 200 nM and 220 ± 50 nM respectively) did differ significantly, suggesting differences in dopamine and 5-HT activity in these brain regions. Maintenance of these gradients depends upon a variety of mechanisms. Such gradients likely underlie highly localized effects of drugs and control of behavior that have been found using other techniques.
Asunto(s)
Encéfalo/metabolismo , Espacio Extracelular/metabolismo , Microdiálisis/métodos , Neurotransmisores/metabolismo , Perfusión/métodos , Acetilcolina/metabolismo , Animales , Ácido Aspártico/metabolismo , Dopamina/metabolismo , Ácido Glutámico/metabolismo , Glicina/metabolismo , Masculino , Microdiálisis/instrumentación , Norepinefrina/metabolismo , Núcleo Accumbens/metabolismo , Perfusión/instrumentación , Ratas , Núcleo Rojo/metabolismo , Serotonina/metabolismo , Área Tegmental Ventral/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Droplet-based microfluidics is an attractive platform for screening and optimizing chemical reactions. Using this approach, it is possible to reliably manipulate nanoliter volume samples and perform operations such as reagent addition with high precision, automation, and throughput. Most studies using droplet microfluidics have relied on optical techniques to detect the reaction; however, this requires engineering color or fluorescence change into the reaction being studied. In this work, we couple electrospray ionization mass spectrometry (ESI-MS) to nanoliter scale segmented flow reactions to enable direct (label-free) analysis of reaction products. The system is applied to a screen of inhibitors for cathepsin B. In this approach, solutions of test compounds (including three known inhibitors) are arranged as an array of nanoliter droplets in a tube segmented by perfluorodecalin. The samples are pumped through a series of tees to add enzyme, substrate (peptides), and quenchant. The resulting reaction mixtures are then infused into a metal-coated, fused silica ESI emitter for MS analysis. The system has potential for high-throughput as reagent addition steps are performed at 0.7 s per sample and ESI-MS at up to 1.2 s per sample. Carryover is inconsequential in the ESI emitter and between 2 and 9% per reagent addition depending on the tee utilized. The assay was reliable with a Z-factor of ~0.8. The method required 0.8 pmol of test compound, 1.6 pmol of substrate, and 5 fmol of enzyme per reaction. Segmented flow ESI-MS allows direct, label free screening of reactions at good throughput and ultralow sample consumption.
Asunto(s)
Catepsina B/antagonistas & inhibidores , Pruebas de Enzimas/instrumentación , Inhibidores Enzimáticos/farmacología , Técnicas Analíticas Microfluídicas/instrumentación , Espectrometría de Masa por Ionización de Electrospray , Dimetilpolisiloxanos/química , Evaluación Preclínica de Medicamentos/economía , Evaluación Preclínica de Medicamentos/instrumentación , Pruebas de Enzimas/economía , Diseño de Equipo , Humanos , Técnicas Analíticas Microfluídicas/economíaRESUMEN
Developing sensors for in vivo chemical monitoring is a daunting challenge. An alternative approach is to couple sampling methods with online analytical techniques; however, such approaches are generally hampered by lower temporal resolution and slow analysis. In this work, microdialysis sampling was coupled with segmented flow electrospray ionization mass spectrometry (ESI-MS) to perform in vivo chemical monitoring. The use of segmented flow to prevent Taylor dispersion of collected zones and rapid analysis with direct ESI-MS allowed 5 s temporal resolution to be achieved. The MS "sensor" was applied to monitor acetylcholine in the brain of live rats. The detection limit of 5 nM was sufficient to monitor basal acetylcholine as well as dynamic changes elicited by microinjection of neostigmine, an inhibitor of acetylcholinesterase, that evoked rapid increases in acetycholine and tetrodotoxin, a blocker of Na(+) channels, that lowered the acetylcholine concentration. The versatility of the sensor was demonstrated by simultaneously monitoring metabolites and infused drugs.
Asunto(s)
Acetilcolina/análisis , Acetilcolinesterasa/metabolismo , Técnicas Biosensibles/métodos , Encéfalo/efectos de los fármacos , Microdiálisis/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Acetilcolina/metabolismo , Animales , Encéfalo/metabolismo , Inhibidores de la Colinesterasa/farmacología , Inyecciones Intraventriculares , Masculino , Microinyecciones , Neostigmina/farmacología , Ratas , Ratas Sprague-Dawley , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/metabolismo , Tetrodotoxina/farmacología , Factores de TiempoRESUMEN
Low-flow push-pull perfusion is a sampling method that yields better spatial resolution than competitive methods like microdialysis. Because of the low flow rates used (50 nL/min), it is challenging to use this technique at high temporal resolution which requires methods of collecting, manipulating, and analyzing nanoliter samples. High temporal resolution also requires control of Taylor dispersion during sampling. To meet these challenges, push-pull perfusion was coupled with segmented flow to achieve in vivo sampling at 7 s temporal resolution at 50 nL/min flow rates. By further miniaturizing the probe inlet, sampling with 200 ms resolution at 30 nL/min (pull only) was demonstrated in vitro. Using this method, L-glutamate was monitored in the striatum of anesthetized rats. Up to 500 samples of 6 nL each were collected at 7 s intervals, segmented by an immiscible oil and stored in a capillary tube. The samples were assayed offline for L-glutamate at a rate of 15 samples/min by pumping them into a reagent addition tee fabricated from Teflon where reagents were added for a fluorescent enzyme assay. Fluorescence of the resulting plugs was monitored downstream. Microinjection of 70 mM potassium in physiological buffered saline evoked l-glutamate concentration transients that had an average maxima of 4.5 ± 1.1 µM (n = 6 animals, 3-4 injections each) and rise times of 22 ± 2 s. These results demonstrate that low-flow push-pull perfusion with segmented flow can be used for high temporal resolution chemical monitoring and in complex biological environments.