RESUMEN
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
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
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
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
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
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
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
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
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
Background: Allergen extracts and recombinant allergens are used in allergy diagnostics and immunotherapy. Since allergen extracts from different manufacturers lack proper standardization regarding their composition, monoclonal antibodies (MAbs) against specific allergen components can be used for their identification and quantification in allergen extracts. This study aimed to generate MAbs against allergen Der p 21 of Dermatophagoides pteronyssinus for the analysis of allergen extracts. Methods: Recombinant Der p 21 was expressed in E. coli and purified using affinity chromatography. MAbs against Der p 21 were generated using hybridoma technology. House dust mite (HDM) allergen extracts were analyzed using the newly developed sandwich enzyme-linked immunosorbent assay, Western blotting and microarray immunoassay. Results: MAbs raised against recombinant Der p 21 were characterized in detail and proven to be reactive with natural Der p 21. Highly specific sandwich enzyme-linked immunosorbent assay for the quantification of Der p 21 was developed and optimized. The allergen was detected and its concentration was determined in only three of six analyzed HDM allergen extracts from different manufacturers. Conclusion: HDM analysis by MAb-based immunoassays shows their differences in allergen composition. The results demonstrate the importance of allergen-specific MAbs as a tool for the characterization of allergen extracts and the need for their appropriate standardization before their use for allergy diagnostics or immunotherapy.
Asunto(s)
Anticuerpos Monoclonales , Antígenos Dermatofagoides , Ensayo de Inmunoadsorción Enzimática , Proteínas Recombinantes , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Animales , Antígenos Dermatofagoides/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Proteínas Recombinantes/inmunología , Proteínas de Artrópodos/inmunología , Ratones , Alérgenos/inmunología , Alérgenos/análisis , Western Blotting , Pyroglyphidae/inmunología , Ratones Endogámicos BALB CRESUMEN
The 21st century has been particularly productive for the biopharmaceutical industry, with the introduction of several classes of innovative therapeutics, such as monoclonal antibodies and related compounds, gene therapy products, and RNA-based modalities. All these new molecules are susceptible to aggregation and fragmentation, which necessitates a size variant analysis for their comprehensive characterization. Size exclusion chromatography (SEC) is one of the reference techniques that can be applied. The analytical techniques for mAbs are now well established and some of them are now emerging for the newer modalities. In this context, the objective of this review article is: i) to provide a short historical background on SEC, ii) to suggest some clear guidelines on the selection of packing material and mobile phase for successful method development in modern SEC; and iii) to highlight recent advances in SEC, such as the use of narrow-bore and micro-bore columns, ultra-wide pore columns, and low-adsorption column hardware. Some important innovations, such as recycling SEC, the coupling of SEC with mass spectrometry, and the use of alternative detectors such as charge detection mass spectrometry and mass photometry are also described. In addition, this review discusses the use of SEC in multidimensional setups and shows some of the most recent advances at the preparative scale. In the third part of the article, the possibility of SEC for the characterization of new modalities is also reviewed. The final objective of this review is to provide a clear summary of opportunities and limitations of SEC for the analysis of different biopharmaceutical products.
Asunto(s)
Cromatografía en Gel , Liposomas , Nanopartículas , Cromatografía en Gel/métodos , Nanopartículas/química , Productos Biológicos/análisis , Productos Biológicos/química , Ácidos Nucleicos/análisis , Vectores Genéticos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/aislamiento & purificación , Proteínas/análisis , Proteínas/química , Humanos , Lípidos/química , Lípidos/análisis , Espectrometría de Masas/métodosRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales , Bordetella pertussis , Cromatografía de Afinidad , Factores de Virulencia de Bordetella , Cromatografía de Afinidad/métodos , Animales , Bordetella pertussis/inmunología , Bordetella pertussis/química , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/inmunología , Ratones , Factores de Virulencia de Bordetella/inmunología , Factores de Virulencia de Bordetella/química , Adhesinas Bacterianas/inmunología , Adhesinas Bacterianas/química , Adhesinas Bacterianas/aislamiento & purificación , Ratones Endogámicos BALB C , Ovinos , Anticuerpos Antibacterianos/inmunología , Anticuerpos Antibacterianos/química , Ensayo de Inmunoadsorción Enzimática/métodosRESUMEN
When purifying mAb from serum-containing hybridoma culture supernatant, it is essential that mouse IgG remains free from contaminations of bovine IgG. However, the broadly used Protein A resin cannot achieve this goal due to binding between both mouse and bovine IgG. Here, a novel nanobody-based affinity purification magnetic beads that discriminates mouse IgG from bovine IgG was developed. To bind all subtypes of mouse IgG (IgG1, IgG2a, IgG2b and IgG3) that contain the kappa light chain, mCK (mouse kappa constant region)-specific nanobody binders were selected from an immune phage display VHH library; this library was constructed with peripheral blood mononuclear cells (PBMCs), which were collected from Bactrian camels immunized with a mix of intact mouse IgGs (IgG1, IgG2a, IgG2b and IgG3). A novel clone that exhibited a higher expression level and a higher binding affinity was selected (4E6). Then, the 4E6 nanobody in the format of VHH-hFC (human Fc) was conjugated on magnetic beads with a maximal binding capacity of 15.41±0.69 mg mouse IgG/mL beads. Furthermore, no bovine IgG could be copurified from hybridoma culture supernatant with immunomagnetic beads. This approach is valuable for the large-scale in vitro production of highly pure antibodies by hybridoma cells.
Asunto(s)
Anticuerpos Monoclonales , Animales , Bovinos , Humanos , Ratones , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Camelus , Cromatografía de Afinidad/métodos , Hibridomas , Regiones Constantes de Inmunoglobulina/química , Inmunoglobulina G/aislamiento & purificación , Inmunoglobulina G/inmunología , Cadenas kappa de Inmunoglobulina/inmunología , Cadenas kappa de Inmunoglobulina/química , Biblioteca de Péptidos , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/aislamiento & purificaciónRESUMEN
Biopharmaceutical manufacturing processes may include a low pH treatment step as a means of inactivating enveloped viruses. Small scale virus clearance studies are routinely performed using model enveloped viruses such as murine leukemia virus to assess inactivation at the pH range used in the downstream manufacturing process. Further, as a means of bioburden reduction, chromatography resins may be cleaned and stored using sodium hydroxide and this can also inactivate viruses. The susceptibility of SARS-CoV-2 and SARS-CoV to low pH conditions using protein A eluate derived material from a monoclonal antibody production process as well as high pH cleaning conditions was addressed. SARS-CoV-2 was effectively inactivated at pH 3.0, moderately inactivated at pH 3.4, but not inactivated at pH 3.8. Low pH was less effective at inactivating SARS-CoV. Both viruses were inactivated at a high pH of ca.13.4. These studies provide important information regarding the effectiveness of viral clearance and inactivation steps of novel coronaviruses when compared to other enveloped viruses.
Asunto(s)
Anticuerpos Monoclonales , SARS-CoV-2 , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo , Inactivación de Virus , Concentración de Iones de Hidrógeno , SARS-CoV-2/efectos de los fármacos , Inactivación de Virus/efectos de los fármacos , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Humanos , Proteína Estafilocócica A/química , Animales , COVID-19/virología , Chlorocebus aethiops , Células VeroRESUMEN
Downstream processing is the bottleneck in the continuous manufacturing of monoclonal antibodies (mAbs). To overcome throughput limitations, two different continuous processes with a novel convective diffusive protein A membrane adsorber (MA) were investigated: the rapid cycling parallel multi-column chromatography (RC-PMCC) process and the rapid cycling simulated moving bed (RC-BioSMB) process. First, breakthrough curve experiments were performed to investigate the influence of the flow rate on the mAb dynamic binding capacity and to calculate the duration of the loading steps. In addition, customized control software was developed for an automated MA exchange in case of pressure increase due to membrane fouling to enable robust, uninterrupted, and continuous processing. Both processes were performed for 4 days with 0.61 g L-1 mAb-containing filtrate and process performance, product purity, productivity, and buffer consumption were compared. The mAb was recovered with a yield of approximately 90% and productivities of 1010 g L-1 d-1 (RC-PMCC) and 574 g L-1 d-1 (RC-BioSMB). At the same time, high removal of process-related impurities was achieved with both processes, whereas the buffer consumption was lower for the RC-BioSMB process. Finally, the attainable productivity for perfusion bioreactors of different sizes with suitable MA sizes was calculated to demonstrate the potential to operate both processes on a manufacturing scale with bioreactor volumes of up to 2000 L.
Asunto(s)
Anticuerpos Monoclonales , Cricetulus , Membranas Artificiales , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/química , Adsorción , Células CHO , Reactores Biológicos , Proteína Estafilocócica A/química , Animales , Cromatografía de Afinidad/métodos , Cromatografía de Afinidad/instrumentaciónRESUMEN
Chromatography resins used for purifying biopharmaceuticals are generally dedicated to a single product. For clinical manufacturing, this can result in resin being used only for a fraction of its potential lifetime. Extending the use of resins to multiple products can significantly reduce resin waste and cost. It can also improve manufacturing flexibility in case of raw material shortage during times such as the COVID-19 pandemic. The work presented herein describes an overarching multiproduct resin reuse (MRR) strategy, which includes a risk assessment, strategic planning, small-scale feasibility runs, and the successful execution of the MRR strategy to support Good manufacturing practice (GMP) clinical manufacturing of an antibody-based therapeutic. Specifically, an anion exchange (AEX) and cation exchange (CEX) MRR strategy is described. Clearance of carryover biological product is demonstrated by first cleaning the AEX and CEX manufacturing columns with sodium hydroxide to ensure inactivation and degradation of the carryover protein and followed by a blank buffer elution that is tested using various analytical methodologies to ensure reduction of the carryover protein to an acceptable level. To our knowledge, this is the first time an MRR approach has been successfully implemented and submitted to health authorities to support biologic GMP clinical manufacture.
Asunto(s)
COVID-19 , Humanos , Cromatografía por Intercambio Iónico/métodos , SARS-CoV-2 , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/biosíntesis , Resinas de Intercambio de Catión/química , Productos Biológicos/química , Productos Biológicos/aislamiento & purificaciónRESUMEN
Neuraminidase (NA) is one of the two influenza virus surface glycoproteins, and antibodies that target it are an independent correlate of protection. However, our current understanding of NA antigenicity is incomplete. Here, we describe human monoclonal antibodies (mAbs) from a patient with a pandemic H1N1 virus infection in 2009. Two mAbs exhibited broad reactivity and inhibited NA enzyme activity of seasonal H1N1 viruses circulating before and after 2009, as well as viruses with avian or swine N1s. The mAbs provided robust protection from lethal challenge with human H1N1 and avian H5N1 viruses in mice, and both target an epitope on the lateral face of NA. In summary, we identified two broadly protective NA antibodies that share a novel epitope, inhibited NA activity, and provide protection against virus challenge in mice. Our work reaffirms that NA should be included as a target in future broadly protective or universal influenza virus vaccines.
Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Antivirales , Subtipo H1N1 del Virus de la Influenza A , Gripe Humana , Neuraminidasa , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/metabolismo , Anticuerpos Antivirales/aislamiento & purificación , Anticuerpos Antivirales/metabolismo , Neuraminidasa/química , Neuraminidasa/metabolismo , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Microscopía por Crioelectrón , Epítopos , Ratones Endogámicos BALB C , Animales , Ratones , Gripe Humana/tratamiento farmacológico , Modelos Animales de EnfermedadRESUMEN
The monoclonal antibodies (MAbs) NCI05 and NCI09, isolated from a vaccinated macaque that was protected from multiple simian immunodeficiency virus (SIV) challenges, both target an overlapping, conformationally dynamic epitope in SIV envelope variable region 2 (V2). Here, we show that NCI05 recognizes a CH59-like coil/helical epitope, whereas NCI09 recognizes a ß-hairpin linear epitope. In vitro, NCI05 and, to a lesser extent, NCI09 mediate the killing of SIV-infected cells in a CD4-dependent manner. Compared to NCI05, NCI09 mediates higher titers of antibody-dependent cellular cytotoxicity (ADCC) to gp120-coated cells, as well as higher levels of trogocytosis, a monocyte function that contributes to immune evasion. We also found that passive administration of NCI05 or NCI09 to macaques did not affect the risk of SIVmac251 acquisition compared to controls, demonstrating that these anti-V2 antibodies alone are not protective. However, NCI05 but not NCI09 mucosal levels strongly correlated with delayed SIVmac251 acquisition, and functional and structural data suggest that NCI05 targets a transient state of the viral spike apex that is partially opened, compared to its prefusion-closed conformation. IMPORTANCE Studies suggest that the protection against SIV/simian-human immunodeficiency virus (SHIV) acquisition afforded by the SIV/HIV V1 deletion-containing envelope immunogens, delivered by the DNA/ALVAC vaccine platform, requires multiple innate and adaptive host responses. Anti-inflammatory macrophages and tolerogenic dendritic cells (DC-10), together with CD14+ efferocytes, are consistently found to correlate with a vaccine-induced decrease in the risk of SIV/SHIV acquisition. Similarly, V2-specific antibody responses mediating ADCC, Th1 and Th2 cells expressing no or low levels of CCR5, and envelope-specific NKp44+ cells producing interleukin 17 (IL-17) also are reproducible correlates of decreased risk of virus acquisition. We focused on the function and the antiviral potential of two monoclonal antibodies (NCI05 and NCI09) isolated from vaccinated animals that differ in antiviral function in vitro and recognize V2 in a linear (NCI09) or coil/helical (NCI05) conformation. We demonstrate that NCI05, but not NCI09, delays SIVmac251 acquisition, highlighting the complexity of antibody responses to V2.