RESUMO
This study presents a comprehensive investigation of the mechanistic understanding of retention and selectivity in hydrophobic interaction chromatography. It provides valuable insights into crucial method-development parameters involved in achieving chromatographic resolution for profiling molecular variants of trastuzumab. Retention characteristics have been assessed for three column chemistries, i.e., butyl, alkylamide, and long-stranded multialkylamide ligands, while distinguishing column hydrophobicity and surface area. Salt type and specifically chloride ions proved to be the key driver for improving chromatographic selectivity, and this was attributed to the spatial distribution of ions at the protein surface, which is ion-specific. The effect was notably more pronounced on the multialkylamide column, as proteins intercalated between the multiamide polymer strands, enabling steric effects. Column coupling proved to be an effective approach for maximizing resolution between molecular variants present in the trastuzumab reference sample and trastuzumab variants induced by forced oxidation. Liquid chromatography-mass spectrometry (LC-MS)/MS peptide mapping experiments after fraction collection indicate that the presence of chloride in the mobile phase enables the selectivity of site-specific deamidation (N30) situated at the heavy chain. Moreover, site-specific oxidation of peptides (M255, W420, and M431) was observed for peptides situated at the Fc region close to the CH2-CH3 interface, previously reported to activate unfolding of trastuzumab, increasing the accessible surface area and hence resulting in an increase in chromatographic retention.
Assuntos
Anticorpos Monoclonais , Cloretos , Anticorpos Monoclonais/química , Cromatografia , Trastuzumab , Peptídeos , Interações Hidrofóbicas e HidrofílicasRESUMO
The Caspase-based fusion protein technology (CASPON) allows for universal cleavage of fusion tags from proteins of interest to reconstitute the native N-terminus. While the CASPON enzyme has been optimized to be promiscuous against a diversity of N-terminal peptides, the cleavage efficacy for larger proteins can be surprisingly low. We develop an efficient means to rationalize and predict the cleavage efficiency based on a structural representation of the intrinsically disordered N-terminal peptides and their putative interactions with the CASPON enzyme. The number of favorably interacting N-terminal conformations shows a very good agreement with the experimentally observed cleavage efficiency, in agreement with a conformational selection model. The method relies on computationally cheap molecular dynamics simulations to efficiently generate a diverse collection of N-terminal conformations, followed by a simple fitting procedure into the CASPON enzyme. It can be readily used to assess the CASPON cleavability a priori.
Assuntos
Simulação de Dinâmica Molecular , Conformação Proteica , Caspases/metabolismo , Caspases/química , Especificidade por Substrato , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Peptídeos/química , Peptídeos/metabolismoRESUMO
Advances in affinity chromatography now make it possible to analyze immunoglobulin G from plasma and its fractions with a simple chromatographic method. Ligands derived from camelid antibodies have been developed which have affinity to all 4 subclasses of human IgG without a cross reactivity to other immunoglobulins. The commercially available Capture Select FcXL is the basis for a simple method for direct quantification of immunoglobulin G from plasma or from fractions from cold ethanol precipitation. After direct injection of the sample into the column the unbound proteins are washed out with equilibration buffer and eluted with a pH-step. The elution the peak is integrated, and quantity is derived form a standard curve. The limit of detection with 40 µg/mL, and a linearity up to 250 µg/mL allows an analysis of samples ranging from 0.04 to 50 mg/mL using varying injection volume without further dilution and the two-wavelength detection. A full cycle is completed within five minutes. This method can serve as orthogonal method for in-process control but also for process development.
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Pertuzumab is a monoclonal antibody used for the treatment of HER2-positive breast cancer in combination with trastuzumab. Charge variants of trastuzumab have been extensively described in the literature; however, little is known about the charge heterogeneity of pertuzumab. Here, changes in the ion-exchange profile of pertuzumab were evaluated by pH gradient cation-exchange chromatography after stressing it for up to 3 weeks at physiological and elevated pH and 37 °C. Isolated charge variants arising under stress conditions were characterized by peptide mapping. The results of peptide mapping showed that deamidation in the Fc domain and N-terminal pyroglutamate formation in the heavy chain are the main contributors to charge heterogeneity. The heavy chain CDR2, which is the only CDR containing asparagine residues, was quite resistant to deamidation under stress conditions according to peptide mapping results. Using surface plasmon resonance, it was shown that the affinity of pertuzumab for the HER2 target receptor does not change under stress conditions. Peptide mapping analysis of clinical samples showed an average of 2-3% deamidation in the heavy chain CDR2, 20-25% deamidation in the Fc domain, and 10-15% N-terminal pyroglutamate formation in the heavy chain. These findings suggest that in vitro stress studies are able to predict in vivo modifications.
Assuntos
Neoplasias da Mama , Regiões Determinantes de Complementaridade , Humanos , Feminino , Ácido Pirrolidonocarboxílico , Anticorpos Monoclonais Humanizados , Trastuzumab , Neoplasias da Mama/tratamento farmacológico , Receptor ErbB-2RESUMO
The CASPON enzyme became an interesting enzyme for fusion protein processing because it generates an authentic N-terminus. However, the high cysteine content of the CASPON enzyme may induce aggregation via disulfide-bond formation, which can reduce enzymatic activity and be considered a critical quality attribute. Different multimerization states of the CASPON enzyme were isolated by preparative size exclusion chromatography and analyzed with respect to multimerization propensity and enzymatic activity. The impact of co-solutes on multimerization was studied in solution and in adsorbed state. Furthermore, protein-protein interactions in the presence of different co-solutes were measured by self-interaction chromatography and were then correlated to the multimerization propensity. The dimer was the most stable and active species with 50% higher enzymatic activity than the tetramer. Multimerization was mainly governed by a cysteine-mediated pathway, as indicated by DTT-induced reduction of most caspase multimers. In the presence of ammonium sulfate, attractive protein-protein interactions were consistent with those observed for higher multimerization when the cysteine-mediated pathway was followed. Multimerization was also observed under attractive conditions on a chromatographic stationary phase. These findings corroborate common rules to perform protein purification with low residence time to avoid disulfide bond formation and conformational change of the protein upon adsorption.
Assuntos
Cisteína , Dissulfetos , Cisteína/química , Cromatografia em Gel , Dissulfetos/química , Multimerização ProteicaRESUMO
Proteases serve as important tools in biotechnology and as valuable drugs or drug targets. Efficient protein engineering methods to study and modulate protease properties are thus of great interest for a plethora of applications. We established PROFICS (PRotease Optimization via Fusion-Inhibited Carbamoyltransferase-based Selection), a bacterial selection system, which enables the optimization of proteases for biotechnology, therapeutics or diagnosis in a simple overnight process. During the PROFICS process, proteases are selected for their ability to specifically cut a tag from a reporter enzyme and leave a native N-terminus. Precise and efficient cleavage after the recognition sequence reverses the phenotype of an Escherichia coli knockout strain deficient in an essential enzyme of pyrimidine synthesis. A toolbox was generated to select for proteases with different preferences for P1' residues (the residue immediately following the cleavage site). The functionality of PROFICS is demonstrated with viral proteases and human caspase-2. PROFICS improved caspase-2 activity up to 25-fold after only one round of mutation and selection. Additionally, we found a significantly improved tolerance for all P1' residues caused by a mutation in a substrate interaction site. We showed that this improved activity enables cells containing the new variant to outgrow cells containing all other mutants, facilitating its straightforward selection. Apart from optimizing enzymatic activity and P1' tolerance, PROFICS can be used to reprogram specificities, erase off-target activity, optimize expression via tags/codon usage, or even to screen for potential drug-resistance-conferring mutations in therapeutic targets such as viral proteases in an unbiased manner.
Assuntos
Caspase 2 , Cisteína Endopeptidases , Evolução Molecular Direcionada , Escherichia coli , Engenharia de Proteínas , Caspase 2/biossíntese , Caspase 2/química , Caspase 2/genética , Cisteína Endopeptidases/biossíntese , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Escherichia coli/enzimologia , Escherichia coli/genética , HumanosRESUMO
Fusion protein technologies to facilitate soluble expression, detection, or subsequent affinity purification in Escherichia coli are widely used but may also be associated with negative consequences. Although commonly employed solubility tags have a positive influence on titers, their large molecular mass inherently results in stochiometric losses of product yield. Furthermore, the introduction of affinity tags, especially the polyhistidine tag, has been associated with undesirable changes in expression levels. Fusion tags are also known to influence the functionality of the protein of interest due to conformational changes. Therefore, particularly for biopharmaceutical applications, the removal of the fusion tag is a requirement to ensure the safety and efficacy of the therapeutic protein. The design of suitable fusion tags enabling the efficient manufacturing of the recombinant protein remains a challenge. Here, we evaluated several N-terminal fusion tag combinations and their influence on product titer and cell growth to find an ideal design for a generic fusion tag. For enhancing soluble expression, a negatively charged peptide tag derived from the T7 bacteriophage was combined with affinity tags and a caspase-2 cleavage site applicable for CASPase-based fusiON (CASPON) platform technology. The effects of each combinatorial tag element were investigated in an integrated manner using human fibroblast growth factor 2 as a model protein in fed-batch lab-scale bioreactor cultivations. To confirm the generic applicability for manufacturing, seven additional pharmaceutically relevant proteins were produced using the best performing tag of this study, named CASPON-tag, and tag removal was demonstrated.
Assuntos
Escherichia coli , Fusão Gênica , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SolubilidadeRESUMO
The N-terminal cleavage of fusion tags to restore the native N-terminus of recombinant proteins is a challenging task and up to today, protocols need to be optimized for different proteins individually. Within this work, we present a novel protease that was designed in-silico to yield enhanced promiscuity toward different N-terminal amino acids. Two mutations in the active-site amino acids of human Caspase-2 were determined to increase the recognition of branched amino-acids, which show only poor binding capabilities in the unmutated protease. These mutations were determined by sequential and structural comparisons of Caspase-2 and Caspase-3 and their effect was additionally predicted using free-energy calculations. The two mutants proposed in the in-silico studies were expressed and in-vitro experiments confirmed the simulation results. Both mutants showed not only enhanced activities toward branched amino acids, but also smaller, unbranched amino acids. We believe that the created mutants constitute an important step toward generalized procedures to restore original N-termini of recombinant fusion proteins.
Assuntos
Aminoácidos de Cadeia Ramificada/química , Caspase 2/química , Caspase 3/química , Cisteína Endopeptidases/química , Mutação , Proteínas Recombinantes de Fusão/química , Sequência de Aminoácidos , Aminoácidos de Cadeia Ramificada/metabolismo , Caspase 2/genética , Caspase 2/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Domínio Catalítico , Clonagem Molecular , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Engenharia de Proteínas/métodos , Domínios e Motivos de Interação entre Proteínas , Proteólise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , TermodinâmicaRESUMO
Recombinant monoclonal antibodies are predominantly produced in mammalian cell culture bioprocesses. Post-translational modifications affect the micro-heterogeneity of the product and thereby influence important quality attributes, such as stability, solubility, pharmacodynamics and pharmacokinetics. The analysis of the surface charge distribution of monoclonal antibodies provides aggregated information about these modifications. In this work, we established a direct injection pH gradient cation exchange chromatography method, which determines charge heterogeneity from cell culture supernatant without any purification steps. This tool was further applied to monitor processes that were performed under certain process conditions. Concretely, we were able to provide insights into charge variant formation during a fed-batch process of a Chinese hamster ovary cell culture, in turn producing a monoclonal antibody under varying temperatures and glucose feed strategies. Glucose concentration impacted the total emergence of acidic variants, whereas the variation of basic species was mainly dependent on process temperature. The formation rates of acidic species were described with a second-order reaction, where a temperature increase favored the conversion. This platform method will aid as a sophisticated optimization tool for mammalian cell culture processes. It provides a quality fingerprint for the produced mAb, which can be tested, compared to the desired target and confirmed early in the process chain.
Assuntos
Anticorpos Monoclonais/metabolismo , Animais , Anticorpos Monoclonais/genética , Células CHO , Técnicas de Cultura de Células/métodos , Cricetinae , Cricetulus , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TemperaturaRESUMO
The aim of this study was the development of a scalable production process for high titer (108 pfu/mL and above) recombinant baculovirus stocks with low cell line-derived impurities for the production of virus-like particles (VLP). To achieve this, we developed a high cell density (HCD) culture for low footprint cell proliferation, compared different infection strategies at multiplicity of infection (MOI) 0.05 and 0.005, different infection strategies and validated generally applicable harvest criteria of cell viability ≤ 80%. We also investigated online measurable parameters to observe the baculovirus production. The infection strategy employing a very low virus inoculum of MOI 0.005 and a 1:2 dilution with fresh medium one day after infection proved to be the most resource efficient. There, we achieved higher cell-specific titers and lower host cell protein concentrations at harvest than other tested infection strategies with the same MOI, while saving half of the virus stock for infecting the culture compared to other tested infection strategies. HCD culture by daily medium exchange was confirmed as suitable for seed train propagation, infection, and baculovirus production, equally efficient as the conventionally propagated seed train. Online measurable parameters for cell concentration and average cell diameter were found to be effective in monitoring the production process. The study concluded that a more efficient VLP production process in large scale can be achieved using this virus stock production strategy, which could also be extended to produce other proteins or extracellular vesicles with the baculovirus expression system.
Assuntos
Baculoviridae , Baculoviridae/metabolismo , Linhagem Celular , Proliferação de Células , Contagem de CélulasRESUMO
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.
Assuntos
Anticorpos Monoclonais , Cricetulus , Imunoglobulina A Secretora , Polietilenoglicóis , Animais , Células CHO , Imunoglobulina A Secretora/química , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Polietilenoglicóis/química , Meios de Cultura/química , Concentração de Íons de Hidrogênio , Cricetinae , Água/químicaRESUMO
Immobilized metal affinity chromatography (IMAC) is a powerful technique for capture and purification of relevant biopharmaceuticals in complex biological matrices. However, protein recovery can be drastically compromised due to surface induced spreading and unfolding of the analyte, leading to fouling of the stationary phase. Here, we report on the kinetics of irreversible adsorption of a protease on an IMAC resin in a time span ranging from minutes to several hours. This trend correlated with the thermal data measured by nano differential scanning calorimetry, and showed a time-dependent change in protein unfolding temperature. Our results highlight that 'soft' proteins show a strong time dependent increase in irreversible adsorption. Furthermore, commonly used co-solvents for preservation of the native protein conformation are tested for their ability to reduce fouling. Thermal data suggests that the amino acid l-arginine is beneficial in preventing unfolding, which was confirmed in batch adsorption experiments. The choice of counter-ions has to be considered when using this amino acid. These results show that l-arginine sulfate decelerates the irreversible adsorption kinetics of proteins on the IMAC stationary phase to a greater extent than l-arginine chloride.
Assuntos
Cromatografia de Afinidade , Arginina/química , Sulfatos/química , Ligação Proteica , Cromatografia de Afinidade/métodos , Caspase 2/química , Proteínas de Fluorescência Verde/química , Fator de Necrose Tumoral alfa/química , Níquel/químicaRESUMO
Hydrophobic interaction chromatography (HIC) is a chromatographic technique that mainly targets the separation of biomolecules (intact proteins, monoclonal antibodies, etc.) based on the difference in surface hydrophobicity while applying non-denaturing conditions. This protocol paper provides guidelines for setting-up robust HIC analysis and considers the instrument configuration, mobile-phase and sample preparation, as well as chromatographic conditions and settings. The separation of a mixture of intact proteins and monoclonal antibodies is demonstrated by applying conventional HIC conditions, that is, using a mildly hydrophobic (C4) stationary phase in combination with an inverse ammonium sulphate gradient dissolved in aqueous phosphate buffer. The effect of sample-preparation conditions on sample breakthroughs is presented. Finally, good run-to-run repeatability (relative standard deviation < 2%) is demonstrated for five different columns obtained from three different column lots, considering chromatographic retention, peak width, peak area and column pressure.
RESUMO
In preparative and industrial chromatography, the current viewpoint is that the dynamic binding capacity governs the process economy, and increased dynamic binding capacity and column utilization are achieved at the expense of productivity. The dynamic binding capacity in chromatography increases with residence time until it reaches a plateau, whereas productivity has an optimum. Therefore, the loading step of a chromatographic process is a balancing act between productivity, column utilization, and buffer consumption. This work presents an online optimization approach for capture chromatography that employs a residence time gradient during the loading step to improve the traditional trade-off between productivity and resin utilization. The approach uses the extended Kalman filter as a soft sensor for product concentration in the system and a model predictive controller to accomplish online optimization using the pore diffusion model as a simple mechanistic model. When a soft sensor for the product is placed before and after the column, the model predictive controller can forecast the optimal condition to maximize productivity and resin utilization. The controller can also account for varying feed concentrations. This study examined the robustness as the feed concentration varied within a range of 50%. The online optimization was demonstrated with two model systems: purification of a monoclonal antibody by protein A affinity and lysozyme by cation-exchange chromatography. Using the presented optimization strategy with a controller saves up to 43% of the buffer and increases the productivity together with resin utilization in a similar range as a multi-column continuous counter-current loading process.
Assuntos
Cromatografia , Proteína Estafilocócica A , Anticorpos Monoclonais/química , Modelos Biológicos , Proteína Estafilocócica A/químicaRESUMO
Cycle stability is important for preparative chromatography resins. Up to 200 cycles have been reported for Protein A affinity resins when used under optimized operating conditions. Through engineered ligands, alkaline resistant Protein A resins are available that can withstand repeated cleaning-in-place cycles with even 1 M NaOH. This enables an increase of purification cycles through the reduction of fouling while maintaining high binding capacities. Previously, non-intuitive changes in dynamic binding capacity after alkaline treatment have been observed for these novel Protein A resins, where sharper breakthrough curves and increased capacities were reported. In this work, we have systematically investigated resins with both low and high alkaline stability and studied the changes in static and dynamic binding capacities and elution behavior. We propose that the observed mass transfer increases of up to 40% are due to a switch in diffusion mechanism, as shown by confocal laser scanning microscopy. Based on our results, only a small window of alkaline treatment conditions exists, where dynamic binding capacity can be increased. Our findings may help to explain previous findings and observations of others.
Assuntos
Proteína Estafilocócica A , Cromatografia de Afinidade/métodos , Difusão , Ligantes , Proteína Estafilocócica A/químicaRESUMO
Fusion protein technologies improve the expression and purification of recombinant proteins, but the removal of the tags involved requires specific proteases. The circularly permuted caspase-2 (cpCasp2) with its specific cleavage site, efficiently generates the untagged protein. While cleavage with cpCasp2 is possible before all 20 proteinogenic amino acids, cleavage before valine, leucine, isoleucine, aspartate and glutamate suffers from slow, and before proline extremely slow, turnover. To make the platform fusion protein process even more general such that any protein with an authentic N-terminus can be produced with high efficiency, the bacterial selection system PROFICS (PRotease Optimization via Fusion-Inhibited Carbamoyltransferase-based Selection) was used to evolve cpCasp2 into a variant with a catalytic turnover two orders of magnitude higher and the ability to cleave before any amino acid. The high specificity and the stability of the original circularly permuted protease was fully retained in this mutant, while the high manufacturability was mostly retained, albeit with decreased soluble titer. Four point-mutations are responsible for this change in activity, two of which are located in or near the binding pocket of the active site. This variant was named CASPON enzyme and is a major component of the CASPase-based fusiON (CASPON) platform technology. Applicability for the production of recombinant proteins was demonstrated by enzymatic removal of the CASPON tag from five model proteins. The CASPON tag enables high soluble expressions, affinity purification and good accessibility for cleavage. The five industry-relevant proteins of interest were FGF2, TNF, GH, GCSF and PTH.
Assuntos
Aminoácidos , Caspase 2 , Cromatografia de Afinidade , Proteínas Recombinantes de Fusão/metabolismo , Proteínas RecombinantesRESUMO
Deploying two salts in hydrophobic interaction chromatography can significantly increase dynamic binding capacities. Nevertheless, the mechanistic understanding of this phenomenon is lacking. Here, we investigate whether surface tension or ionic strength govern dynamic binding capacities of the chromatographic resin Toyopearl Butyl-650 M in dual salt systems. Small-angle X-ray scattering was employed to analyze the model proteins and the protein-resin adduct in the respective dual salt systems. The dual salt systems incorporate sodium citrate and a secondary sodium salt (acetate, sulfate, or phosphate). As model proteins, we used lysozyme, GFP, and a monoclonal antibody (adalimumab). Moreover, for the protein-resin adduct, we determined the model parameters of a self-avoiding random walk model fitted into the pair density distribution function of the SAXS data. Ionic strength is more predictive for dynamic binding capacities in HIC dual salt systems than surface tension. However, dynamic binding capacities still differ by up to 30 % between the investigated dual salt systems. The proteins exhibit extensive protein-protein interactions in the studied dual salt HIC buffers. We found a correlation of protein-protein interactions with the well-known Hofmeister series. For systems with elevated protein-protein interactions, adsorption isotherms deviate from Langmuirian behavior. This highlights the importance of lateral protein-protein interactions in protein adsorption, where monomolecular protein layers are usually assumed. SAXS analysis of the protein-resin adduct indicates an inverse correlation of the binding capacity and the excluded volume parameter. This is indicative of the deposition of proteins in the cavities of the stationary phase. We hypothesize that increasing protein-protein interactions allow the formation of attractive clusters and multilayers in the cavities, respectively.
Assuntos
Cromatografia Líquida/métodos , Proteínas/química , Adsorção , Interações Hidrofóbicas e Hidrofílicas , Concentração Osmolar , Mapas de Interação de Proteínas , Espalhamento a Baixo Ângulo , Cloreto de Sódio/química , Tensão Superficial , Difração de Raios XRESUMO
The nucleocapsid protein (NP) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for several steps of the viral life cycle, and is abundantly expressed during infection, making it an ideal diagnostic target protein. This protein has a strong tendency for dimerization and interaction with nucleic acids. For the first time, high titers of NP were expressed in E. coli with a CASPON tag, using a growth-decoupled protein expression system. Purification was accomplished by nuclease treatment of the cell homogenate and a sequence of downstream processing (DSP) steps. An analytical method consisting of native hydrophobic interaction chromatography hyphenated to multi-angle light scattering detection (HIC-MALS) was established for in-process control, in particular, to monitor product fragmentation and multimerization throughout the purification process. 730 mg purified NP per liter of fermentation could be produced by the optimized process, corresponding to a yield of 77% after cell lysis. The HIC-MALS method was used to demonstrate that the NP product can be produced with a purity of 95%. The molecular mass of the main NP fraction is consistent with dimerized protein as was verified by a complementary native size-exclusion separation (SEC)-MALS analysis. Peptide mapping mass spectrometry and host cell specific enzyme-linked immunosorbent assay confirmed the high product purity, and the presence of a minor endogenous chaperone explained the residual impurities. The optimized HIC-MALS method enables monitoring of the product purity, and simultaneously access its molecular mass, providing orthogonal information complementary to established SEC-MALS methods. Enhanced resolving power can be achieved over SEC, attributed to the extended variables to tune selectivity in HIC mode.
Assuntos
COVID-19 , Proteínas do Nucleocapsídeo , Cromatografia , Escherichia coli/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas do Nucleocapsídeo/genética , SARS-CoV-2RESUMO
Cation-exchange chromatography is a widely used approach to study charge heterogeneity of monoclonal antibodies. Heterogeneity may arise both in vitro and in vivo because of the susceptibility of monoclonal antibodies to undergo chemical modifications. Modifications may adversely affect the potency of the drug, induce immunogenicity or affect pharmacokinetics. In this study, we evaluated the application of optimized pH gradient systems for the separation of charge variants of trastuzumab after forced degradation study. pH gradient-based elution resulted in high-resolution separation of some 20 charge variants after 3 weeks at 37°C under physiological conditions. The charge variants were further characterized by LC-MS-based peptide mapping. There was no significant difference in the binding properties to HER2 or a range of Fcγ receptors between non-stressed and stressed trastuzumab.
Assuntos
Anticorpos Monoclonais , Cromatografia por Troca Iônica , Concentração de Íons de Hidrogênio , Espectrometria de Massas , TrastuzumabRESUMO
BACKGROUND: Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection or vaccination. While first-generation antibody tests have primarily provided qualitative results, accurate seroprevalence studies and tracking of antibody levels over time require highly specific, sensitive and quantitative test setups. METHODS: We have developed two quantitative, easy-to-implement SARS-CoV-2 antibody tests, based on the spike receptor binding domain and the nucleocapsid protein. Comprehensive evaluation of antigens from several biotechnological platforms enabled the identification of superior antigen designs for reliable serodiagnostic. Cut-off modelling based on unprecedented large and heterogeneous multicentric validation cohorts allowed us to define optimal thresholds for the tests' broad applications in different aspects of clinical use, such as seroprevalence studies and convalescent plasma donor qualification. FINDINGS: Both developed serotests individually performed similarly-well as fully-automated CE-marked test systems. Our described sensitivity-improved orthogonal test approach assures highest specificity (99.8%); thereby enabling robust serodiagnosis in low-prevalence settings with simple test formats. The inclusion of a calibrator permits accurate quantitative monitoring of antibody concentrations in samples collected at different time points during the acute and convalescent phase of COVID-19 and disclosed antibody level thresholds that correlate well with robust neutralization of authentic SARS-CoV-2 virus. INTERPRETATION: We demonstrate that antigen source and purity strongly impact serotest performance. Comprehensive biotechnology-assisted selection of antigens and in-depth characterisation of the assays allowed us to overcome limitations of simple ELISA-based antibody test formats based on chromometric reporters, to yield comparable assay performance as fully-automated platforms. FUNDING: WWTF, Project No. COV20-016; BOKU, LBI/LBG.