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Discovery and optimization of a biotherapeutic monoclonal antibody requires a careful balance of target engagement and physicochemical developability properties. To take full advantage of the sequence diversity provided by different antibody discovery platforms, a rapid and reliable process for humanization of antibodies from nonhuman sources is required. Canonically, maximizing homology of the human variable region (V-region) to the original germline was believed to result in preservation of binding, often without much consideration for inherent molecular properties. We expand on this approach by grafting the complementary determining regions (CDRs) of a mouse anti-LAG3 antibody into an extensive matrix of human variable heavy chain (VH) and variable light chain (VL) framework regions with substantially broader sequence homology to assess the impact on complementary determining region-framework compatibility through progressive evaluation of expression, affinity, biophysical developability, and function. Specific VH and VL framework sequences were associated with major expression and purification phenotypes. Greater VL sequence conservation was correlated with retained or improved affinity. Analysis of grafts that bound the target demonstrated that initial developability criteria were significantly impacted by VH, but not VL. In contrast, cell binding and functional characteristics were significantly impacted by VL, but not VH. Principal component analysis of all factors identified multiple grafts that exhibited more favorable antibody properties, notably with nonoptimal sequence conservation. Overall, this study demonstrates that modern throughput systems enable a more thorough, customizable, and systematic analysis of graft-framework combinations, resulting in humanized antibodies with improved global properties that may progress through development more quickly and with a greater probability of success.
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Anticuerpos Monoclonales Humanizados , Anticuerpos Monoclonales , Animales , Humanos , Ratones , Anticuerpos Monoclonales Humanizados/química , Afinidad de Anticuerpos , Regiones Determinantes de Complementariedad/químicaRESUMEN
Solubility is a property of central importance for the use of proteins in research in molecular and cell biology and in applications in biotechnology and medicine. Since experimental methods for measuring protein solubility are material intensive and time consuming, computational methods have recently emerged to enable the rapid and inexpensive screening of solubility for large libraries of proteins, as it is routinely required in development pipelines. Here, we describe the development of one such method to include in the predictions the effect of the pH on solubility. We illustrate the resulting pH-dependent predictions on a variety of antibodies and other proteins to demonstrate that these predictions achieve an accuracy comparable with that of experimental methods. We make this method publicly available at https://www-cohsoftware.ch.cam.ac.uk/index.php/camsolph, as the version 3.0 of CamSol.
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Proteínas , Programas Informáticos , Bovinos , Humanos , Albúminas/química , Secuencia de Aminoácidos , Anticuerpos/química , Pollos , Concentración de Iones de Hidrógeno , Internet , Proteínas/química , Solubilidad , AnimalesRESUMEN
Antibody drugs have become a key part of biotherapeutics. Patients suffering from various diseases have benefited from antibody therapies. However, its development process is rather long, expensive and risky. To speed up the process, reduce cost and improve success rate, artificial intelligence, especially deep learning methods, have been widely used in all aspects of preclinical antibody drug development, from library generation to hit identification, developability screening, lead selection and optimization. In this review, we systematically summarize antibody encodings, deep learning architectures and models used in preclinical antibody drug discovery and development. We also critically discuss challenges and opportunities, problems and possible solutions, current applications and future directions of deep learning in antibody drug development.
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Inteligencia Artificial , Aprendizaje Profundo , Humanos , Descubrimiento de DrogasRESUMEN
Feeding biopharma pipelines with biotherapeutic candidates that possess desirable developability profiles can help improve the productivity of biologic drug discovery and development. Here, we have derived an in silico profile by analyzing computed physicochemical descriptors for the variable regions (Fv) found in 77 marketed antibody-based biotherapeutics. Fv regions of these biotherapeutics demonstrate significant diversities in their germlines, complementarity determining region loop lengths, hydrophobicity, and charge distributions. Furthermore, an analysis of 24 physicochemical descriptors, calculated using homology-based molecular models, has yielded five nonredundant descriptors whose distributions represent stability, isoelectric point, and molecular surface characteristics of their Fv regions. Fv regions of candidates from our internal discovery campaigns, human next-generation sequencing repertoires, and those in clinical-stages (CST) were assessed for similarity with the physicochemical profile derived here. The Fv regions in 33% of CST antibodies show physicochemical properties that are dissimilar to currently marketed biotherapeutics. In comparison, physicochemical characteristics of â¼29% of the Fv regions in human antibodies and â¼27% of our internal hits deviated significantly from those of marketed biotherapeutics. The early availability of this information can help guide hit selection, lead identification, and optimization of biotherapeutic candidates. Insights from this work can also help support portfolio risk assessment, in-licensing, and biopharma collaborations.
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Anticuerpos Monoclonales/química , Regiones Determinantes de Complementariedad/química , Diseño de Fármacos , Descubrimiento de Drogas , Ingeniería de Proteínas/normas , Simulación por Computador , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Estabilidad ProteicaRESUMEN
Proteins require high developability-quantified by expression, solubility, and stability-for robust utility as therapeutics, diagnostics, and in other biotechnological applications. Measuring traditional developability metrics is low throughput in nature, often slowing the developmental pipeline. We evaluated the ability of 10 variations of three high-throughput developability assays to predict the bacterial recombinant expression of paratope variants of the protein scaffold Gp2. Enabled by a phenotype/genotype linkage, assay performance for 105 variants was calculated via deep sequencing of populations sorted by proxied developability. We identified the most informative assay combination via cross-validation accuracy and correlation feature selection and demonstrated the ability of machine learning models to exploit nonlinear mutual information to increase the assays' predictive utility. We trained a random forest model that predicts expression from assay performance that is 35% closer to the experimental variance and trains 80% more efficiently than a model predicting from sequence information alone. Utilizing the predicted expression, we performed a site-wise analysis and predicted mutations consistent with enhanced developability. The validated assays offer the ability to identify developable proteins at unprecedented scales, reducing the bottleneck of protein commercialization.
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Bases de Datos de Ácidos Nucleicos , Biblioteca de Genes , Ensayos Analíticos de Alto Rendimiento , Aprendizaje Automático , Proteínas/genéticaRESUMEN
The androgen receptor (AR) has been shown to be a key determinant in the pathogenesis of castration-resistant prostate cancer (CRPC). The current standard of care therapies targets the ligand-binding domain of the receptor and can afford improvements to life expectancy often only in the order of months before resistance occurs. Emerging preclinical and clinical compounds that inhibit receptor activity via differentiated mechanisms of action which are orthogonal to current antiandrogens show promise for overcoming treatment resistance. In this review, we present an authoritative summary of molecules that noncompetitively target the AR. Emerging small molecule strategies for targeting alternative domains of the AR represent a promising area of research that shows significant potential for future therapies. The overall quality of lead candidates in the area of noncompetitive AR inhibition is discussed, and it identifies the key chemotypes and associated properties which are likely to be, or are currently, positioned to be first in human applications.
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Neoplasias de la Próstata Resistentes a la Castración , Receptores Androgénicos , Masculino , Humanos , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/patología , Antagonistas de Andrógenos/uso terapéutico , Línea Celular TumoralRESUMEN
Advancement in all disciplines (art, science, education, and engineering) requires a careful balance of disruption and advancement of classical techniques. Often technologies are created with a limited understanding of fundamental principles and are prematurely abandoned. Over time, knowledge improves, new opportunities are identified, and technology is reassessed in a different light leading to a renaissance. Recovery of biological products is currently experiencing such a renaissance. Crystallization is one example of an elegant and ancient technology that has been applied in many fields and was employed to purify insulins from naturally occurring sources. Crystallization can also be utilized to determine protein structures. However, a multitude of parameters can impact protein crystallization and the "hit rate" for identifying protein crystals is relatively low, so much so that the development of a crystallization process is often viewed as a combination of art and science even today. Supplying the worldwide requirement for insulin (and associated variants) requires significant advances in process intensification to support scale of production and to minimize the overall cost to enable broader access. Expanding beyond insulin, the increasing complexity and diversity of biologics agents challenge the current purification methodologies. To harness the full potential of biologics, there is a need to fully explore a broader range of purification technologies, including nonchromatographic approaches. This impetus requires one to challenge and revisit the classical techniques including crystallization, chromatography, and filtration from a different vantage point and with a new set of tools, including molecular modeling. Fortunately, computational biophysics tools now exist to provide insights into mechanisms of protein/ligand interactions and molecular assembly processes (including crystallization) that can be used to support de novo process development. For example, specific regions or motifs of insulins and ligands can be identified and used as targets to support crystallization or purification development. Although the modeling tools have been developed and validated for insulin systems, the same tools can be applied to more complex modalities and to other areas including formulation, where the issue of aggregation and concentration-dependent oligomerization could be mechanistically modeled. This paper will illustrate a case study juxtaposing historical approaches to insulin downstream processes to a recent production process highlighting the application and evolution of technologies. Insulin production from Escherichia coli via inclusion bodies is an elegant example since it incorporates virtually all the unit operations associated with protein production-recovery of cells, lysis, solubilization, refolding, purification, and crystallization. The case study will include an example of an innovative application of existing membrane technology to combine three-unit operations into one, significantly reducing solids handling and buffer consumption. Ironically, a new separations technology was developed over the course of the case study that could further simplify and intensify the downstream process, emphasizing and highlighting the ever-accelerating pace of innovation in downstream processing. Molecular biophysics modeling was also employed to enhance the mechanistic understanding of the crystallization and purification processes.
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Analytical characterization of proteins is a critical task for developing therapeutics and subunit vaccine candidates. Assessing candidates with a battery of biophysical assays can inform the selection of one that exhibits properties consistent with a given target product profile (TPP). Such assessments, however, require several milligrams of purified protein, and ideal assessments of the physicochemical attributes of the proteins should not include unnatural modifications like peptide tags for purification. Here, we describe a fast two-stage minimal purification process for recombinant proteins secreted by the yeast host Komagataella phaffii from a 20 mL culture supernatant. This method comprises a buffer exchange and filtration with a Q-membrane filter and we demonstrate sufficient removal of key supernatant impurities including host-cell proteins (HCPs) and DNA with yields of 1-2 mg and >60% purity. This degree of purity enables characterizing the resulting proteins using affinity binding, mass spectrometry, and differential scanning calorimetry. We first evaluated this method to purify an engineered SARS-CoV-2 subunit protein antigen and compared the purified protein to a conventional two-step chromatographic process. We then applied this method to compare several SARS-CoV-2 RBD sequences. Finally, we show this simple process can be applied to a range of other proteins, including a single-domain antibody, a rotavirus protein subunit, and a human growth hormone. This simple and fast developability methodology obviates the need for genetic tagging or full chromatographic development when assessing and comparing early-stage protein therapeutics and vaccine candidates produced in K. phaffii.
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Monoclonal antibodies (mAbs) are often formulated as high-protein-concentration solutions, which in some cases can exhibit physical stability issues such as high viscosity and opalescence. To ensure that mAb-based drugs can meet their manufacturing, stability, and delivery requirements, it is advantageous to screen for and select mAbs during discovery that are not prone to such behaviors. It has been recently shown that both these macroscopic properties can be predicted to a certain extent from the diffusion interaction parameter (kD), which is a measure of self-association under dilute conditions.1 However, kD can be challenging to measure at the early stage of discovery, where a relatively large amount of a high-purity material, which is required by traditional methods, is often not available. In this study, we demonstrate asymmetric field-flow fractionation (AF4) as a tool to measure self-association and therefore identify antibodies with problematic issues at high concentrations. The principle lies on the ability to concentrate the sample close to the membrane during the injection mode, which can reach formulation-relevant concentrations (>100 mg/mL).2 By analyzing a well-characterized library of commercial antibodies, we show that the measured retention time of the antibodies allows us to pinpoint molecules that exhibit issues at high concentrations. Remarkably, our AF4 assay requires very little (30 µg) sample under dilute conditions and does not need extensive sample purification. Furthermore, we show that a polyethylene glycol (PEG) precipitation assay provides results consistent with AF4 and moreover can further differentiate molecules with issues of opalescence or high viscosity. Overall, our results delineate a two-step strategy for the identification of problematic variants at high concentrations, with AF4 for early developability screening, followed by a PEG assay to validate the problematic molecules and further discriminate between opalescence or high-viscosity issues. This two-step antibody selection strategy enables us to select antibodies early in the discovery process, which are compatible with high-concentration formulations.
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Anticuerpos Monoclonales , Polietilenglicoles/químicaRESUMEN
OBJECTIVE: This work introduces a material-sparing process that rapidly screens the solid form landscape for ophthalmic compound candidates. SIGNIFICANCE: Crystalline form of compound candidates generated by a Form Risk Assessment (FRA) can be used to reduce their downstream development risk. METHODS: This workflow evaluated nine model compounds with various molecular and polymorphic profiles by using less than 350 mg of drug substances. Kinetic solubility of the model compounds in a variety of solvents was screened to support the experimental design. The FRA workflow integrated several crystallization methods such as temperature-cycled slurrying (thermocycling), cooling, and evaporation. The FRA was also applied on ten ophthalmic compound candidates for verification. X-ray powder diffractometry (XRPD) was used for form identification. RESULTS: For the nine model compounds studied, multiple crystalline forms were generated. This demonstrates the potential of the FRA workflow to reveal polymorphic tendency. In addition, thermocycling process was found to be the most effective technique to capture the thermodynamically most stable form. Satisfactory results were observed with the discovery compounds intended for ophthalmic formulations. CONCLUSIONS: This work introduces a form risk assessment workflow by using sub-gram level of drug substances. The capability of this material-sparing workflow to discover polymorphs and capture the thermodynamically most stable forms within 2-3 weeks makes it suitable for discovery stage compounds, especially for ophthalmic candidates.
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Flujo de Trabajo , Cristalización/métodos , Composición de Medicamentos/métodos , Temperatura , Solubilidad , Medición de Riesgo , Rastreo Diferencial de Calorimetría , Difracción de Rayos XRESUMEN
Soft tissue defects resulting from head and neck tumor resection seriously impact the physical appearance and psychological well-being of patients. The complex curvature of the human head and neck poses a formidable challenge for maxillofacial surgeons to achieve precise aesthetic and functional restoration after surgery. To this end, a normal head and neck volunteer was selected as the subject of investigation. Employing Gaussian curvature analysis, combined with mechanical constraints and principal curvature analysis methods of soft tissue clinical treatment, a precise developable/non-developable area partition map of the head and neck surface was obtained, and a non-developable surface was constructed. Subsequently, a digital design method was proposed for the repair of head and neck soft tissue defects, and an in vitro simulated surgery experiment was conducted. Clinical verification was performed on a patient with tonsil tumor, and the results demonstrated that digital technology-designed flaps improved the accuracy and aesthetic outcome of head and neck soft tissue defect repair surgery. This study validates the feasibility of digital precision repair technology for soft tissue defects after head and neck tumor resection, which effectively assists surgeons in achieving precise flap transplantation reconstruction and improves patients' postoperative satisfaction.
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Neoplasias de Cabeza y Cuello , Procedimientos de Cirugía Plástica , Humanos , Colgajos Quirúrgicos/cirugía , Neoplasias de Cabeza y Cuello/cirugía , Cabeza/cirugía , Cuello/cirugíaRESUMEN
Understanding of peptide aggregation propensity is an important aspect in pharmaceutical development of peptide drugs. In this work, methodologies based on all-atom molecular dynamics (AA-MD) simulations and 1H NMR (in neat H2O) were evaluated as tools for identification and investigation of peptide aggregation. A series of structurally similar, pharmaceutically relevant peptides with known differences in aggregation behavior (D-Phe6-GnRH, ozarelix, cetrorelix, and degarelix) were investigated. The 1H NMR methodology was used to systematically investigate variations in aggregation with peptide concentration and time. Results show that 1H NMR can be used to detect the presence of coexisting classes of aggregates and the inclusion or exclusion of counterions in peptide aggregates. Interestingly, results suggest that the acetate counterions are included in aggregates of ozarelix and cetrorelix but not in aggregates of degarelix. The peptides investigated in AA-MD simulations (D-Phe6-GnRH, ozarelix, and cetrorelix) showed the same rank order of aggregation propensity as in the NMR experiments. The AA-MD simulations also provided molecular-level insights into aggregation dynamics, aggregation pathways, and the influence of different structural elements on peptide aggregation propensity and intermolecular interactions within the aggregates. Taken together, the findings from this study illustrate that 1H NMR and AA-MD simulations can be useful, complementary tools in early evaluation of aggregation propensity and formulation development for peptide drugs.
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Simulación de Dinámica Molecular , Espectroscopía de Resonancia Magnética , Espectroscopía de Protones por Resonancia MagnéticaRESUMEN
The widespread interest in antibody therapeutics has led to much focus on identifying antibody candidates with favorable developability properties. In particular, there is broad interest in identifying antibody candidates with highly repulsive self-interactions in standard formulations (e.g., low ionic strength buffers at pH 5-6) for high solubility and low viscosity. Likewise, there is also broad interest in identifying antibody candidates with low levels of non-specific interactions in physiological solution conditions (PBS, pH 7.4) to promote favorable pharmacokinetic properties. To what extent antibodies that possess both highly repulsive self-interactions in standard formulations and weak non-specific interactions in physiological solution conditions can be systematically identified remains unclear and is a potential impediment to successful therapeutic drug development. Here, we evaluate these two properties for 42 IgG1 variants based on the variable fragments (Fvs) from four clinical-stage antibodies and complementarity-determining regions from 10 clinical-stage antibodies. Interestingly, we find that antibodies with the strongest repulsive self-interactions in a standard formulation (pH 6 and 10 mM histidine) display the strongest non-specific interactions in physiological solution conditions. Conversely, antibodies with the weakest non-specific interactions under physiological conditions display the least repulsive self-interactions in standard formulations. This behavior can be largely explained by the antibody isoelectric point, as highly basic antibodies that are highly positively charged under standard formulation conditions (pH 5-6) promote repulsive self-interactions that mediate high colloidal stability but also mediate strong non-specific interactions with negatively charged biomolecules at physiological pH and vice versa for antibodies with negatively charged Fv regions. Therefore, IgG1s with weakly basic isoelectric points between 8 and 8.5 and Fv isoelectric points between 7.5 and 9 typically display the best combinations of strong repulsive self-interactions and weak non-specific interactions. We expect that these findings will improve the identification and engineering of antibody candidates with drug-like biophysical properties.
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Anticuerpos Monoclonales , Regiones Determinantes de Complementariedad , Anticuerpos Monoclonales/química , Regiones Determinantes de Complementariedad/química , Inmunoglobulina G/química , Punto IsoeléctricoRESUMEN
Therapeutic mAbs must not only bind to their target but must also be free from "developability issues" such as poor stability or high levels of aggregation. While small-molecule drug discovery benefits from Lipinski's rule of five to guide the selection of molecules with appropriate biophysical properties, there is currently no in silico analog for antibody design. Here, we model the variable domain structures of a large set of post-phase-I clinical-stage antibody therapeutics (CSTs) and calculate in silico metrics to estimate their typical properties. In each case, we contextualize the CST distribution against a snapshot of the human antibody gene repertoire. We describe guideline values for five metrics thought to be implicated in poor developability: the total length of the complementarity-determining regions (CDRs), the extent and magnitude of surface hydrophobicity, positive charge and negative charge in the CDRs, and asymmetry in the net heavy- and light-chain surface charges. The guideline cutoffs for each property were derived from the values seen in CSTs, and a flagging system is proposed to identify nonconforming candidates. On two mAb drug discovery sets, we were able to selectively highlight sequences with developability issues. We make available the Therapeutic Antibody Profiler (TAP), a computational tool that builds downloadable homology models of variable domain sequences, tests them against our five developability guidelines, and reports potential sequence liabilities and canonical forms. TAP is freely available at opig.stats.ox.ac.uk/webapps/sabdab-sabpred/TAP.php.
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Regiones Determinantes de Complementariedad , Simulación por Computador , Modelos Moleculares , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Descubrimiento de Drogas , HumanosRESUMEN
As the number of antibody drugs being approved and marketed increases, our knowledge of what makes potential drug candidates a successful product has increased tremendously. One of the critical parameters that have become clear in the field is the importance of mAb "developability." Efforts are being increasingly focused on simultaneously selecting molecules that exhibit both desirable biological potencies and manufacturability attributes. In the current study mutations to improve the developability profile of a problematic antibody that inconsistently precipitates in a batch scale-dependent fashion using a standard platform purification process are described. Initial bioinformatic analysis showed the molecule has no obvious sequence or structural liabilities that might lead it to precipitate. Subsequent analysis of the molecule revealed the presence of two unusual positively charged mutations on the light chain at the interface of VH and VL domains, which were hypothesized to be the primary contributor to molecule precipitation during process development. To investigate this hypothesis, straightforward reversion to the germline of these residues was carried out. The resulting mutants have improved expression titers and recovered stability within a forced precipitation assay, without any change to biological activity. Given the time pressures of drug development in industry, process optimization of the lead molecule was carried out in parallel to the "retrospective" mutagenesis approach. Bespoke process optimization for large-scale manufacturing was successful. However, we propose that such context-dependent sequence liabilities should be included in the arsenal of in silico developability screening early in development; particularly since this specific issue can be efficiently mitigated without the requirement for extensive screening of lead molecule variants.
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Anticuerpos Monoclonales , Ingeniería de Proteínas , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/genética , Línea Celular , Humanos , SolubilidadRESUMEN
A vital part of biopharmaceutical research is decision making around which lead candidate should be progressed in early-phase development. When multiple antibody candidates show similar biological activity, developability aspects are taken into account to ease the challenges of manufacturing the potential drug candidate. While current strategies for developability assessment mainly focus on drug product stability, only limited information is available on how antibody candidates with minimal differences in their primary structure behave during downstream processing. With increasing time-to-market pressure and an abundance of monoclonal antibodies (mAbs) in development pipelines, developability assessments should also consider the ability of mAbs to integrate into the downstream platform. This study investigates the influence of amino acid substitutions in the complementarity-determining region (CDR) of a full-length IgG1 mAb on the elution behavior in preparative cation exchange chromatography. Single amino acid substitutions within the investigated mAb resulted in an additional positive charge in the light chain (L) and heavy chain (H) CDR, respectively. The mAb variants showed an increased retention volume in linear gradient elution compared with the wild-type antibody. Furthermore, the substitution of tryptophan with lysine in the H-CDR3 increased charge heterogeneity of the product. A multiscale in silico analysis, consisting of homology modeling, protein surface analysis, and mechanistic chromatography modeling increased understanding of the adsorption mechanism. The results reveal the potential effects of lead optimization during antibody drug discovery on downstream processing.
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Sustitución de Aminoácidos , Anticuerpos Monoclonales , Inmunoglobulina G , Modelos Moleculares , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/aislamiento & purificación , Cromatografía por Intercambio Iónico , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Inmunoglobulina G/química , Inmunoglobulina G/genética , Inmunoglobulina G/aislamiento & purificación , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/genéticaRESUMEN
The efficient development of new therapeutic antibodies relies on developability assessment with biophysical and computational methods to find molecules with drug-like properties such as resistance to aggregation. Despite the many novel approaches to select well-behaved proteins, antibody aggregation during storage is still challenging to predict. For this reason, there is a high demand for methods that can identify aggregation-resistant antibodies. Here, we show that three straightforward techniques can select the aggregation-resistant antibodies from a dataset with 13 molecules. The ReFOLD assay provided information about the ability of the antibodies to refold to monomers after unfolding with chemical denaturants. Modulated scanning fluorimetry (MSF) yielded the temperatures that start causing irreversible unfolding of the proteins. Aggregation was the main reason for poor unfolding reversibility in both ReFOLD and MSF experiments. We therefore performed temperature ramps in molecular dynamics (MD) simulations to obtain partially unfolded antibody domains in silico and used CamSol to assess their aggregation potential. We compared the information from ReFOLD, MSF, and MD to size-exclusion chromatography (SEC) data that shows whether the antibodies aggregated during storage at 4, 25, and 40 °C. Contrary to the aggregation-prone molecules, the antibodies that were resistant to aggregation during storage at 40 °C shared three common features: (i) higher tendency to refold to monomers after unfolding with chemical denaturants, (ii) higher onset temperature of nonreversible unfolding, and (iii) unfolding of regions containing aggregation-prone sequences at higher temperatures in MD simulations.
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Anticuerpos Monoclonales/química , Desnaturalización Proteica , Anticuerpos Monoclonales/uso terapéutico , Rastreo Diferencial de Calorimetría , Química Farmacéutica/métodos , Cromatografía en Gel , Almacenaje de Medicamentos , Dispersión Dinámica de Luz , Calor/efectos adversos , Concentración de Iones de Hidrógeno , Simulación de Dinámica Molecular , Conformación Proteica , Pliegue de Proteína , Desplegamiento ProteicoRESUMEN
There is significant interest in formulating antibody therapeutics as concentrated liquid solutions, but early identification of developable antibodies with optimal manufacturability, stability, and delivery attributes remains challenging. Traditional methods of identifying developable mAbs with low self-association in common antibody formulations require relatively concentrated protein solutions (>1 mg/mL), and this single challenge has frustrated early-stage and large-scale identification of antibody candidates with drug-like colloidal properties. Here, we describe charge-stabilized self-interaction nanoparticle spectroscopy (CS-SINS), an affinity-capture nanoparticle assay that measures colloidal self-interactions at ultradilute antibody concentrations (0.01 mg/mL), and is predictive of antibody developability issues of high viscosity and opalescence that manifest at four orders of magnitude higher concentrations (>100 mg/mL). CS-SINS enables large-scale, high-throughput selection of developable antibodies during early discovery.
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Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Oro/química , Nanopartículas del Metal/química , Ensayos Analíticos de Alto Rendimiento , Humanos , Multimerización de Proteína , Solubilidad , ViscosidadRESUMEN
In addition to activity, successful biological drugs must exhibit a series of suitable developability properties, which depend on both protein sequence and buffer composition. In the context of this high-dimensional optimization problem, advanced algorithms from the domain of machine learning are highly beneficial in complementing analytical screening and rational design. Here, we propose a Bayesian optimization algorithm to accelerate the design of biopharmaceutical formulations. We demonstrate the power of this approach by identifying the formulation that optimizes the thermal stability of three tandem single-chain Fv variants within 25 experiments, a number which is less than one-third of the experiments that would be required by a classical DoE method and several orders of magnitude smaller compared to detailed experimental analysis of full combinatorial space. We further show the advantage of this method over conventional approaches to efficiently transfer historical information as prior knowledge for the development of new biologics or when new buffer agents are available. Moreover, we highlight the benefit of our technique in engineering multiple biophysical properties by simultaneously optimizing both thermal and interface stabilities. This optimization minimizes the amount of surfactant in the formulation, which is important to decrease the risks associated with corresponding degradation processes. Overall, this method can provide high speed of converging to optimal conditions, the ability to transfer prior knowledge, and the identification of new nonlinear combinations of excipients. We envision that these features can lead to a considerable acceleration in formulation design and to parallelization of operations during drug development.
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Productos Biológicos/administración & dosificación , Composición de Medicamentos/métodos , Aprendizaje Automático , Teorema de Bayes , Productos Biológicos/uso terapéutico , Evaluación Preclínica de Medicamentos/métodos , Humanos , Sistema de Administración de Fármacos con Nanopartículas/administración & dosificaciónRESUMEN
Various screening approaches are used by industry to evaluate development risks associated with discovery candidates. This process has become more complicated with biological therapeutics, a class dominated by monoclonal antibodies (mAb), and, increasingly, their derivative constructs. Effective early assessment for drug-like properties (DLP) can save time and costs by allowing a more complete consideration of issues that could impact the desired end result of a stable drug product. Here we report a case study of four IgG1 mAbs, with sequence variations in the variable domain region, screened as a set of possible drug candidates. Our comprehensive, tiered approach used a battery of analytical tools to assess molecular characteristics, conformational stability, colloidal stability, and short-term storage stability. While most DLP for the four candidates were developmentally acceptable and comparable, mAb-2 was associated with adverse colloidal properties. Further investigation of mAb-2 in an expanded pH range revealed a propensity for phase separation, indicating a need for the additional product development effort. Our results support that comprehensive DLP assessments in an expanded pH range are beneficial in identifying development options for promising molecules that show challenging stability trends. This adaptable approach may be especially useful in the development of increasingly complex antibody constructs.