RESUMEN
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.
Asunto(s)
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
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
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.
Asunto(s)
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
Mpox (formerly known as monkeypox) is a zoonotic disease caused by monkeypox virus (MPXV), a DNA virus belonging to the Orthopoxvirus genus, in the Poxviridae family. The disease constitutes a moderate risk to public health at the global level. The MPXV A29L protein plays a crucial role in coordinating virion assembly and facilitating important virus-host interactions. This study focused on the expression, purification, and recombinant protein synthesis of the A29L protein of MPXV using prokaryotic systems. Using hybridoma technology, we successfully generated the monoclonal antibodies (mAbs) 1E12 and 4B2, which specifically recognize the A29L protein. These mAbs were found to be suitable for use in indirect immunofluorescence assays (IFA), Western blotting, and immunoprecipitation (IP). Our investigation also revealed that mAbs 1E12 and 4B2 could detect the A27L protein, a homologous protein found in the vaccinia virus Western Reserve (VACV WR) strain, using IFA, Western blotting, and immunoprecipitation (IP). Using mAbs 1E12 and 4B2 as primary immunological probes, A27L protein expression was detected as early as 6 h postinfection with VACV WR, with increasing protein levels being observed throughout the infection. This study enhances our understanding of the protein structure and function of MPXV and contributes to the development of specific MPXV detection methods.
Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Antivirales , Monkeypox virus , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/aislamiento & purificación , Animales , Ratones , Anticuerpos Antivirales/inmunología , Monkeypox virus/inmunología , Monkeypox virus/genética , Ratones Endogámicos BALB C , Proteínas Virales/inmunología , Proteínas Virales/genética , Humanos , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/genética , Femenino , Virus Vaccinia/inmunología , Virus Vaccinia/genética , HibridomasRESUMEN
Despite advancements in therapeutic monoclonal antibodies (mAbs) and cell line engineering, separating host cell proteins (HCPs) from mAbs during downstream purification remains challenging. Therefore, in this study, we developed a novel multimodal chromatography (MMC) resin to enhance HCP removal during mAb polishing processes. We evaluated the impact of both ligand structure and pore size of the MMC resin by purifying a post-protein A chromatography solution in flow-through mode. We observed that the efficiency of HCP clearance depended on the hydrophobic moiety structure of the ligand and predicted the mAb purification capability of MMC through linear salt-gradient elution experiments involving a mixture of transferrin, bovine serum albumin (BSA), and pepsin. Our findings revealed that the prototype immobilized 1,12-dodecanediamine via the formyl group exhibited the best performance attributed to its long alkyl chain. Furthermore, an investigation of effects of base bead pore size on HCP capacity using cellulose base beads of five different pore sizes showed that larger pore resin base beads had the highest HCP removal capacity. Specifically, MMC resins with a pore diameter exceeding 440 nm reduced the HCP level by three orders of magnitude under high mAb loading conditions (> 1000 mg/mL-resin). The MMC resin developed in this study, along with the insights gained into ligand structure and pore size, not only enhances mAb polishing efficiency but also contributes to improving downstream processes in mAb biopharmaceutical production.
Asunto(s)
Anticuerpos Monoclonales , Cricetulus , Albúmina Sérica Bovina , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Animales , Células CHO , Ligandos , Albúmina Sérica Bovina/química , Porosidad , Cromatografía de Afinidad/métodos , Proteína Estafilocócica A/química , Transferrina/química , Transferrina/aislamiento & purificación , Pepsina A/química , Pepsina A/metabolismo , Proteínas/aislamiento & purificación , Proteínas/química , Resinas Sintéticas/química , Interacciones Hidrofóbicas e HidrofílicasRESUMEN
Protein A affinity membrane adsorbers are a promising alternative to resins to intensify the manufacturing of monoclonal antibodies. This study examined the process performance of convective diffusive membrane adsorbers operated in batch and continuous multi-column mode. Therefore, three different processes were compared regarding membrane utilization, productivity, and buffer consumption: the batch process, the rapid cycling parallel multi-column chromatography process, and the rapid cycling simulated moving bed process. The influence of the monoclonal antibody loading concentration (between 0.5 g L-1 and 5.2 g L-1) and the loading flow rate (between 1.25 MV min-1 and 10 MV min-1) on the monoclonal antibody binding behavior of the membrane adsorber were studied with breakthrough curve experiments. The determined breakthrough curves were used to calculate the monoclonal antibody dynamic binding capacity, the duration of the loading steps for each process, and the number of required membrane adsorbers for the continuous processes rapid cycling parallel multi-column chromatography and rapid cycling simulated moving bed. The highest productivity for the batch (176 g L-1 h-1) and rapid cycling parallel multi-column chromatography process (176 g L-1 h-1) was calculated for high monoclonal antibody loading concentrations and low loading flow rates. In contrast, the rapid cycling simulated moving bed process achieved the highest productivity (217 g L-1 h-1) for high monoclonal antibody loading concentrations and loading flow rates. Furthermore, due to the higher membrane utilization, the buffer consumption of the rapid cycling simulated moving bed process (1.1 L g-1) was up to 1.9 times lower than that of the batch or rapid cycling parallel multi-column chromatography operation (2.1 L g-1).
Asunto(s)
Anticuerpos Monoclonales , Cromatografía de Afinidad , Membranas Artificiales , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/química , Adsorción , Cromatografía de Afinidad/métodos , Cromatografía de Afinidad/instrumentación , Proteína Estafilocócica A/química , DifusiónRESUMEN
Secretory immunoglobulin A [sIgA] is a promising candidate for enteric therapeutics applications, and several sIgA-based constructs are currently being developed by groups utilizing clarified Chinese hamster ovary [CHO] cell culture supernatants. To the monoclonal antibody downstream processing typically entails chromatography-based purification processes beginning with Protein A chromatography. In this paper, aqueous two-phase systems [ATPS] were employed for the preliminary purification of secretory immunoglobulin A [sIgA] monoclonal antibody [mAb] from clarified CHO-cell culture supernatants. A 24 full factorial design was utilized. The influence of various process parameters such as pH, PEG molecular weight [MPEG], PEG concentration [CPEG], and phosphate salt concentration [CPHO], on the sIgA partition coefficient [K sIgA] and the recovery index [Y] in the PEG phase were evaluated. The Elisa assay revealed that, in the ATPS conditions tested, sIgA mAb was mostly detected in PEG upper phase. Run 14 with the highest sIgA activity exhibited the following conditions: MPEG 8.000 g/mol, CPEG 12,5 %, pH 7,0 and CPHO 10 %, and a sIgA K of 94.50 and a recovery index [Y] of 33.52 %. The proposed platform provides straightforward implementation, yields comparable results, and offers significantly improved economics for manufacturing sIgA mAb biotherapeutics.
Asunto(s)
Anticuerpos Monoclonales , Cricetulus , Inmunoglobulina A Secretora , Polietilenglicoles , Animales , Células CHO , Inmunoglobulina A Secretora/química , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Polietilenglicoles/química , Medios de Cultivo/química , Concentración de Iones de Hidrógeno , Cricetinae , Agua/químicaRESUMEN
Therapeutic monoclonal antibodies (mAbs) are critical for treatment of a wide range of diseases. Immunoglobulin G (IgG) is the most predominant form of mAb but is prone to aggregation during production. Detection and removal of IgG aggregates are time-consuming and laborious. Chromatography is central for purification of biopharmaceuticals in general and essential in the production of mAbs. Protein purification systems are usually equipped with detectors for monitoring pH, UV absorbance, and conductivity, to facilitate optimization and control of the purification process. However, specific in-line detection of the target products and contaminating species, such as aggregates, is currently not possible using convectional techniques. Here we show a novel fiber optical in-line sensor, based on localized surface plasmon resonance (LSPR), for specific detection of IgG and IgG aggregates during affinity chromatography. A flow cell with a Protein A sensor chip was connected to the outlet of the affinity column connected to three different chromatography systems operating at lab scale to pilot scale. Samples containing various IgG concentrations and aggregate contents were analyzed in-line during purification on a Protein A column using both pH gradient and isocratic elution. Because of avidity effects, IgG aggregates showed slower dissociation kinetics than monomers after binding to the sensor chips. Possibilities to detect aggregate concentrations below 1 % and difference in aggregate content smaller than 0.3 % between samples were demonstrated. In-line detection of aggregates can circumvent time-consuming off-line analysis and facilitate automation and process intensification.
Asunto(s)
Anticuerpos Monoclonales , Cromatografía de Afinidad , Inmunoglobulina G , Proteína Estafilocócica A , Resonancia por Plasmón de Superficie , Inmunoglobulina G/aislamiento & purificación , Cromatografía de Afinidad/métodos , Cromatografía de Afinidad/instrumentación , Resonancia por Plasmón de Superficie/métodos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Proteína Estafilocócica A/química , Agregado de Proteínas , Concentración de Iones de HidrógenoRESUMEN
Appended bispecific antibody (aBsAb) with two single chain variable fragments (scFv) linked at the c-terminus of its heavy chains is one of the promising formats in bispecific therapeutics. The presence of hydrophobic and flexible scFv fragments render aBsAb molecules higher molecule hydrophobicity and structural flexibility compared to monoclonal antibody (mAb), thus making its purification more challenging. We set out to investigate how the unique molecular properties of aBsAb affect its performance on Protein A chromatography. We showed that aBsAb has a high propensity for chromatography-induced aggregation due to its high molecule hydrophobicity, and this couldn't be improved by the addition of common chaotropic salts. Moreover, the presence of chaotropic salts, such as arginine hydrochloride (Arg-HCl), retarded aBsAb elution during Protein A chromatography rather than facilitating which was widely observed in mAb Protein A elution. Nevertheless, we were able to overcome the aggregation issue by optimizing elution condition and improved aBsAb purity from 29 % to 93 % in Protein A eluate with a high molecular weight (HMW) species of less than 5 %. We also showed that the high molecular flexibility of aBsAb leads to different hydrodynamic sizes of the aBsAb molecule post Protein A elution, neutralization, and re-acidification, which are pH dependent. This is different from mAbs where their sizes do not change post neutralization even with re-exposure to acid. The above unique observations of aBsAb in Protein A chromatography were clearly explained from the perspectives of its high molecular hydrophobicity and structural flexibility.
Asunto(s)
Anticuerpos Biespecíficos , Interacciones Hidrofóbicas e Hidrofílicas , Proteína Estafilocócica A , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/aislamiento & purificación , Proteína Estafilocócica A/química , Cromatografía de Afinidad/métodos , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/aislamiento & purificación , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Concentración de Iones de Hidrógeno , HumanosRESUMEN
Antibody drugs are becoming increasingly popular in disease diagnosis, targeted therapy, and immunoprevention owing to their characteristics of high targeting ability, strong specificity, low toxicity, and mild side effects. The demand for antibody drugs is steadily increasing, and their production scale is expanding. Upstream cell culture technology has been greatly improved by the high-capacity production of monoclonal antibodies. However, the downstream purification of antibodies presents a bottleneck in the production process. Moreover, the purification cost of antibodies is extremely high, accounting for approximately 50%-80% of the total cost of antibody production. Chromatographic technology, given its selectivity and high separation efficiency, is the main method for antibody purification. This process usually involves three stages: antibody capture, intermediate purification, and polishing. Different chromatographic techniques, such as affinity chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, mixed-mode chromatography, and temperature-responsive chromatography, are used in each stage. Affinity chromatography, mainly protein A affinity chromatography, is applied for the selective capture and purification of antibodies from raw biofluids or harvested cell culture supernatants. Other chromatographic techniques, such as ion-exchange chromatography, hydrophobic interaction chromatography, and mixed-mode chromatography, are used for intermediate purification and antibody polishing. Affinity biomimetic chromatography and hydrophobic charge-induction chromatography can produce antibodies with purities comparable with those obtained through protein A chromatography, by employing artificial chemical/short peptide ligands with good selectivity, high stability, and low cost. Temperature-responsive chromatography is a promising technique for the separation and purification of antibodies. In this technique, antibody capture and elution is controlled by simply adjusting the column temperature, which greatly eliminates the risk of antibody aggregation and inactivation under acidic elution conditions. The combination of different chromatographic methods to improve separation selectivity and achieve effective elution under mild conditions is another useful strategy to enhance the yield and quality of antibodies. This review provides an overview of recent advances in the field of antibody purification using chromatography and discusses future developments in this technology.
Asunto(s)
Cromatografía de Afinidad , Anticuerpos/aislamiento & purificación , Anticuerpos/química , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/química , Cromatografía/métodos , Cromatografía de Afinidad/métodos , Cromatografía por Intercambio Iónico/métodos , Interacciones Hidrofóbicas e HidrofílicasRESUMEN
Maximizing product quality attributes by optimizing process parameters and performance attributes is a crucial aspect of bioprocess chromatography process design. Process parameters include but are not limited to bed height, eluate cut points, and elution pH. An under-characterized chromatography process parameter for protein A chromatography is process temperature. Here, we present a mechanistic understanding of the effects of temperature on the protein A purification of a monoclonal antibody (mAb) using a commercial chromatography resin for batch and continuous counter-current systems. A self-designed 3D-printed heating jacket controlled the 1 mL chromatography process temperature during the loading, wash, elution, and cleaning-in-place (CIP) steps. Batch loading experiments at 10, 20, and 30 °C demonstrated increased dynamic binding capacity (DBC) with temperature. The experimental data were fit to mechanistic and correlation-based models that predicted the optimal operating conditions over a range of temperatures. These model-based predictions optimized the development of a 3-column temperature-controlled periodic counter-current chromatography (TCPCC) and were validated experimentally. Operating a 3-column TCPCC at 30 °C led to a 47% increase in DBC relative to 20 °C batch chromatography. The DBC increase resulted in a two-fold increase in productivity relative to 20 °C batch. Increasing the number of columns to the TCPCC to optimize for increasing feed concentration resulted in further improvements to productivity. The feed-optimized TCPCC showed a respective two, three, and four-fold increase in productivity at feed concentrations of 1, 5, and 15 mg/mL mAb, respectively. The derived and experimentally validated temperature-dependent models offer a valuable tool for optimizing both batch and continuous chromatography systems under various operating conditions.
Asunto(s)
Anticuerpos Monoclonales , Distribución en Contracorriente , Proteína Estafilocócica A , Temperatura , Proteína Estafilocócica A/química , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Distribución en Contracorriente/métodos , Cromatografía de Afinidad/métodos , Cricetulus , Células CHO , AnimalesRESUMEN
In previous publications we have described the pISep dual simultaneous, independent gradients (DSIGs) liquid chromatography (LC) for uncoupling gradients of non-buffering solute (NaCl, urea or acetonitrile) from externally generated pH gradients. In DSIGs the shape and slope of the [salute] gradient does not depend on the shape and slope of the pH gradient. The technique allows in a single run true simultaneous two dimensional LC separation of complex protein mixtures on various stationary phases including anion, cation exchangers (AEX, CEX), reversed phase (RP), mixed mode and mixed bed. Using a humanized IgG1 (HIgG1) monoclonal antibody (MAb) and a variety of pH & [NaCl] DSIGs, we show that most of MAb isoforms can be successfully separated from each other. These experimental observations are supported by an initial theoretical argument presented here predicting an overall improvement of all MAb isoforms separation by DSIGs of pH & [NaCl]. Theoretical calculations predict that, in general, there exists an optimal non-zero isocratic salt concentration in a pH gradient separation that will resolve isoforms close in binding energy, but a wide range of salt concentrations will be required for acceptable resolution of all isoforms. Theory also predicts better separation of weaker rather than stronger binding isoforms. Experimentally, we have found that no one set of DSIGs LC conditions could optimally baseline resolve all identifiable MAb isoforms in a single run of reasonable duration. The versatility and simplicity of the pH & [NaCl] pISep DSIGs LC allows fast, automated scouting of protein separations over any range of pH from 2.4 to 10.8 and [NaCl] from 0 to 1 M without changing the chemistry of the buffering system. Due to the universal applicability of the pISep buffering system in IEX LC, the researcher is given a powerful tool to easily develop pH & [NaCl] DSIGs protocols that vary mobile phase compositions to achieve high resolution separations of targeted proteins.
Asunto(s)
Anticuerpos Monoclonales , Cloruro de Sodio , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Concentración de Iones de Hidrógeno , Cromatografía por Intercambio Iónico/métodos , Cloruro de Sodio/química , Inmunoglobulina G/aislamiento & purificación , Inmunoglobulina G/química , Humanos , Cromatografía Liquida/métodos , Fuerza Protón-Motriz , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/químicaRESUMEN
Bi- and multispecific antibody formats allow the development of new therapeutic strategies to address previously unmet medical needs. However, due to the increased complexity (e.g., the interface design and the presence of multiple binders), such molecules are generally more challenging to express and purify compared to standard monoclonal antibodies (mAbs). We describe here an optimized methodology to express and purify basic bispecific antibodies using the BEAT® interface. This interface allows to generate antibodies with very high levels of heterodimer product (reported titers exceed 10 g/L) and comes with a built-in purification strategy allowing removal of residual levels of undesired product-related impurities (e.g., homodimers and half molecules).
Asunto(s)
Anticuerpos Biespecíficos , Anticuerpos Biespecíficos/aislamiento & purificación , Humanos , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/biosíntesis , Expresión Génica , Ingeniería de Proteínas/métodos , AnimalesRESUMEN
Current postexposure prophylaxis of rabies includes vaccines, human rabies immunoglobulin (RIG), equine RIG, and recombinant monoclonal antibodies (mAb). In the manufacturing of rabies recombinant mAb, charge variants are the most common source of heterogeneity. Charge variants of rabies mAb were isolated by salt gradient cation exchange chromatography (CEX) to separate acidic and basic and main charge variants. Separated variants were further extensively characterized using orthogonal analytical techniques, which include secondary and tertiary structure determination by far and near ultraviolet circular dichroism spectroscopy. Charge and size heterogeneity were evaluated using CEX, isoelectric focusing (IEF), capillary-IEF, size exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and western blotting. Antigen binding affinity was assessed by enzyme linked immuno-sorbent assay and rapid florescence foci inhibition test. Results from structural and physicochemical characterizations concluded that charge variants are formed due to posttranslational modification demonstrating that the charge heterogeneity, these charge variants did neither show any considerable physicochemical change nor affect its biological function. This study shows that charge variants are effective components of mAb and there is no need of deliberate removal, until biological functions of rabies mAb will get affected.
Asunto(s)
Anticuerpos Monoclonales , Focalización Isoeléctrica , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Animales , Focalización Isoeléctrica/métodos , Cromatografía por Intercambio Iónico/métodos , Humanos , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/aislamiento & purificación , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Virus de la Rabia/inmunología , Cromatografía en Gel/métodos , Rabia , Western BlottingRESUMEN
Magnetic separation is a promising alternative to chromatography for enhancing the downstream processing (DSP) of monoclonal antibodies (mAbs). However, there is a lack of efficient magnetic particles for successful application. Aiming to fill this gap, we demonstrate the suitability of bare iron oxide nanoparticles (BION) with physical site-directed immobilization of an engineered Protein A affinity ligand (rSpA) as an innovative magnetic material. The rSpA ligand contains a short peptide tag that enables the direct and stable immobilization onto the uncoated BION surface without commonly required laborious particle activation. The resulting BION@rSpA have beneficial characteristics outperforming conventional Protein A-functionalized magnetic particles: a simple, fast, low-cost synthesis, a particle size in the nanometer range with a large effective specific surface area enabling large immunoglobulin G (IgG) binding capacity, and a high magnetophoretic velocity advantageous for fast processing. We further show rapid interactions of IgG with the easily accessible rSpA ligands. The binding of IgG to BION@rSpA is thereby highly selective and not impeded by impurity molecules in perfusion cell culture supernatant. Regarding the subsequent acidic IgG elution from BION@rSpA@IgG, we observed a hampering pH increase caused by the protonation of large iron oxide surfaces after concentrating the particles in 100 mM sodium acetate buffer. However, the pH can be stabilized by adding 50 mM glycine to the elution buffer, resulting in recoveries above 85% even at high particle concentrations. Our work shows that BION@rSpA enable efficient magnetic mAb separation and could help to overcome emerging bottlenecks in DSP.
Asunto(s)
Inmunoglobulina G , Nanopartículas Magnéticas de Óxido de Hierro , Ensayo de Materiales , Tamaño de la Partícula , Nanopartículas Magnéticas de Óxido de Hierro/química , Ligandos , Inmunoglobulina G/química , Inmunoglobulina G/aislamiento & purificación , Materiales Biocompatibles/química , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Proteína Estafilocócica A/química , Proteína Estafilocócica A/metabolismo , Propiedades de Superficie , Compuestos Férricos/químicaRESUMEN
The importance of humoral immunity to fungal infections remains to be elucidated. In cryptococcosis, patients that fail to generate antibodies against antigens of the fungus Cryptococcus neoformans are more susceptible to the disease, demonstrating the importance of these molecules to the antifungal immune response. Historically, antibodies against C. neoformans have been applied in diagnosis, therapeutics, and as important research tools to elucidate fungal biology. Throughout the process of generating monoclonal antibodies (mAbs) from a single B-cell clone and targeting a single epitope, several immunization steps might be required for the detection of responsive antibodies to the antigen of interest in the serum. This complex mixture of antibodies comprises the polyclonal antibodies. To obtain mAbs, B-lymphocytes are harvested (from spleen or peripheral blood) and fused with tumor myeloma cells, to generate hybridomas that are individually cloned and specifically screened for mAb production. In this chapter, we describe all the necessary steps, from the immunization to polyclonal antibody harvesting, hybridoma generation, and mAb production and purification. Additionally, we discuss new cutting-edge approaches for generating interspecies mAbs, such as humanized mAbs, or for similar species in distinct host backgrounds.
Asunto(s)
Anticuerpos Antifúngicos , Anticuerpos Monoclonales , Cryptococcus neoformans , Hibridomas , Cryptococcus neoformans/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Animales , Humanos , Hibridomas/inmunología , Anticuerpos Antifúngicos/inmunología , Anticuerpos Antifúngicos/aislamiento & purificación , Ratones , Linfocitos B/inmunología , Criptococosis/inmunología , Criptococosis/diagnóstico , Antígenos Fúngicos/inmunología , InmunizaciónRESUMEN
Objective To prepare a monoclonal antibody (mAb) against mouse NOD-like receptor family pyrin domain-containing 3 (NLRP3) and assess its specificity. Methods A gene fragment encoding mouse NLRP3 exon3 (Ms-N3) was inserted into the vector p36-G3-throhFc to construct a recombinant plasmid named Ms-N3-throhFc. This plasmid was then transfected into HEK293F cells for eukaryotic expression. NLRP3-/- mice were immunized with Ms-N3 protein purified using a protein A chromatography column, and splenocytes from the immunized mice were fused with SP2/0 myeloma cells to generate hybridoma cells. Specific mAbs against murine NLRP3 from hybridoma cells were screened using ELISA and immunofluorescence assay(IFA). Results The Ms-N3-throhFc recombinant plasmid was successfully constructed and exhibited stable expression in HEK293F cells. Twelve hybridoma cell lines were initially screened using ELISA. IFA revealed that the mAb secreted by the 9-B8-3-2-C5 cell line specifically recognized the native form of mouse NLRP3 protein. The heavy and light chain subtypes of this mAb were identified as IgM and κ, respectively. Conclusion A monoclonal antibody against mouse NLRP3 has been successfully prepared.
Asunto(s)
Anticuerpos Monoclonales , Proteína con Dominio Pirina 3 de la Familia NLR , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/aislamiento & purificación , Animales , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Humanos , Células HEK293 , Hibridomas , Transfección , Exones , Clonación Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Ensayo de Inmunoadsorción EnzimáticaRESUMEN
3D printing offers the unprecedented ability to fabricate chromatography stationary phases with bespoke 3D morphology as opposed to traditional packed beds of spherical beads. The restricted range of printable materials compatible with chromatography is considered a setback for its industrial implementation. Recently, we proposed a novel ink that exhibits favourable printing performance (printing time â¼100 mL/h, resolution â¼200 µm) and broadens the possibilities for a range of chromatography applications thanks to its customisable surface chemistry. In this work, this ink was used to fabricate 3D printed ordered columns with 300 µm channels for the capture and polishing of therapeutic monoclonal antibodies. The columns were initially assessed for leachables and extractables, revealing no material propensity for leaching. Columns were then functionalised with protein A and SO3 ligands to obtain affinity and strong cation exchangers, respectively. 3D printed protein A columns showed >85 % IgG recovery from harvested cell culture fluid with purities above 98 %. Column reusability was evaluated over 20 cycles showing unaffected performance. Eluate samples were analysed for co-eluted protein A fragments, host cell protein and aggregates. Results demonstrate excellent HCP clearance (logarithmic reduction value of > 2.5) and protein A leakage in the range of commercial affinity resins (<100 ng/mg). SO3 functionalised columns employed for polishing achieved removal of leaked Protein A (down to 10 ng/mg) to meet regulatory expectations of product purity. This work is the first implementation of 3D printed columns for mAb purification and provides strong evidence for their potential in industrial bioseparations.
Asunto(s)
Anticuerpos Monoclonales , Cricetulus , Inmunoglobulina G , Impresión Tridimensional , Proteína Estafilocócica A , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/química , Proteína Estafilocócica A/química , Inmunoglobulina G/aislamiento & purificación , Inmunoglobulina G/química , Células CHO , Cromatografía de Afinidad/métodos , Animales , Cromatografía por Intercambio Iónico/métodos , TintaRESUMEN
Filamentous hemagglutinin (FHA) is a critical adhesion molecule produced by Bordetella pertussis (BP), the causative agent of highly contagious respiratory infection known as whooping cough. FHA plays a pivotal role in the pathogenesis of whooping cough and is a key component of acellular pertussis vaccines (aPV). However, conventional purification methods for FHA often involve labor-intensive processes and result in low purity and recovery rates. Therefore, this study explores the use of monoclonal and polyclonal antibodies as specific tools to achieve highly pure and efficient FHA purification. To generate FHA-specific antibodies, polyclonal antibodies were produced by immunizing sheep and monoclonal antibodies (MAbs) were generated by immunizing mice with recombinant and native FHA. The MAbs were selected based on affinity, isotypes, and specificity, which were assessed through ELISA and Western blot assays. Two immunoaffinity columns, one monoclonal and one polyclonal, were prepared for FHA antigen purification. The purity and recovery rates of these purifications were determined using ELISA, SDS-PAGE, and immunoblotting. Furthermore, the MAbs were employed to develop an ELISA assay for FHA antigen concentration determination. The study's findings revealed that immunoaffinity column-based purification of FHA resulted in a highly pure antigen with recovery rates of approximately 57% ± 6.5% and 59% ± 7.9% for monoclonal and polyclonal columns, respectively. Additionally, the developed ELISA exhibited appropriate reactivity for determining FHA antigen concentration. This research demonstrates that affinity chromatography is a viable and advantageous method for purifying FHA, offering superior purity and recovery rates compared to traditional techniques. This approach provides a practical alternative for FHA purification in the context of aPV development.