RESUMEN
Self-association of therapeutic monoclonal antibodies (mabs) are thought to modulate the undesirably high viscosity observed in their concentrated solutions. Computational prediction of such a self-association behavior is advantageous early during mab drug candidate selection when material availability is limited. Here, we present a coarse-grained (CG) simulation method that enables microsecond molecular dynamics simulations of full-length antibodies at high concentrations. The proposed approach differs from others in two ways: first, charges are assigned to CG beads in an effort to reproduce molecular multipole moments and charge asymmetry of full-length antibodies instead of only localized charges. This leads to great improvements in the agreement between CG and all-atom electrostatic fields. Second, the distinctive hydrophobic character of each antibody is incorporated through empirical adjustments to the short-range van der Waals terms dictated by cosolvent all-atom molecular dynamics simulations of antibody variable regions. CG simulations performed on a set of 15 different mabs reveal that diffusion coefficients in crowded environments are markedly impacted by intermolecular interactions. Diffusion coefficients computed from the simulations are in correlation with experimentally measured observables, including viscosities at a high concentration. Further, we show that the evaluation of electrostatic and hydrophobic characters of the mabs is useful in predicting the nonuniform effect of salt on the viscosity of mab solutions. This CG modeling approach is particularly applicable as a material-free screening tool for selecting antibody candidates with desirable viscosity properties.
Asunto(s)
Anticuerpos Monoclonales , Simulación de Dinámica Molecular , Interacciones Hidrofóbicas e Hidrofílicas , Electricidad Estática , ViscosidadRESUMEN
"Elegant" lyophilized pharmaceutical product cakes are expected to appear as uniform foamy plugs with little shrinkage and minimal cracking. While studying internal cake structures, we have on occasion observed some cakes that were very sharply split horizontally, roughly in halves, with foamy top and lamellar bottom regions. After many years and numerous experiments, we can finally propose a mechanism for the formation of these cakes with unusual internal structures. This phenomenon involves a complex interplay of momentum, heat, mass transfer, and phase equilibria.LAY ABSTRACT: Freeze drying (lyophilization) is a common unit operation in the manufacturing of pharmaceutical drugs. The typical final lyophilized product is expected to look like a uniform porous plug, or cake, that has foamy (sponge-like) morphology. However, we have occasionally observed cakes that were split horizontally, with the top and bottom layers exhibiting very distinctive and totally different structures. This intriguing phenomenon has not been discussed in the literature. In this report, we present experimental results that lead us to a mechanism by which split-cakes form.
Asunto(s)
Química Farmacéutica/métodos , Liofilización/métodos , Preparaciones Farmacéuticas/química , Industria Farmacéutica/métodos , Calor , Tecnología Farmacéutica/métodosRESUMEN
Monoclonal antibodies (mAbs), which are now more frequently administered by subcutaneous (SC) injection rather than intravenously, have become a tremendously successful drug format across a wide range of therapeutic areas. Preclinical evaluations of mAbs to be administered by SC injection are typically performed in species such as mice, rats, minipigs, and cynomolgus monkeys to obtain critical information regarding formulation performance and prediction of PK/PD outcomes needed to select clinical doses for first-in-human studies. Despite extensive efforts, no preclinical model has been identified to date that accurately predicts clinical outcomes for these SC injections. We have addressed this deficiency with a novel in vitro instrument, termed Scissor, to model events occurring at the SC injection site and now further validated this approach using a set of eight mAbs for which clinical PK/PD outcomes have been obtained. Diffusion of these mAbs from the Scissor system injection cartridge into a large volume physiological buffer, used to emulate mAb movement from the SC injection site into the systemic circulation, provided distinct profiles when monitored over a 6h period. Curve-fitting analysis of these profiles using the Hill equation identified parameters that were used, along with physicochemical properties for each mAb, in a partial least squares analysis to define a relationship between molecule and formulation properties with clinical PK outcomes. The results demonstrate that parameters of protein charge at neutral pH and isoelectric point (pI) along with combined formulation properties such as viscosity and mAb concentration can dictate the movement of the mAb from the injection cartridge to infinite sink compartment. Examination of profile characteristics of this movement provided a strong predictive correlation for these eight mAbs. Together, this approach demonstrates the feasibility of this in vitro modelling strategy as a tool to identify drug and formulation properties that can define the performance of SC injected medicines and provide the potential for predicting clinical outcomes that could be useful for formulation selection and a first-in-human clinical dosing strategy.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/farmacocinética , Sistemas de Liberación de Medicamentos , Modelos Biológicos , Animales , Disponibilidad Biológica , Humanos , Inyecciones Subcutáneas , Macaca fascicularis , Ratones , Ratas , Porcinos , Porcinos EnanosRESUMEN
Somatic mutations within the antibody variable domains are critical to the immense capacity of the immune repertoire. Here, via a deep mutational scan, we dissect how mutations at all positions of the variable domains of a high-affinity anti-VEGF antibody G6.31 impact its antigen-binding function. The resulting mutational landscape demonstrates that large portions of antibody variable domain positions are open to mutation, and that beneficial mutations can be found throughout the variable domains. We determine the role of one antigen-distal light chain position 83, demonstrating that mutation at this site optimizes both antigen affinity and thermostability by modulating the interdomain conformational dynamics of the antigen-binding fragment. Furthermore, by analyzing a large number of human antibody sequences and structures, we demonstrate that somatic mutations occur frequently at position 83, with corresponding domain conformations observed for G6.31. Therefore, the modulation of interdomain dynamics represents an important mechanism during antibody maturation in vivo.
Asunto(s)
Anticuerpos/genética , Fragmentos Fab de Inmunoglobulinas/genética , Anticuerpos/química , Anticuerpos/inmunología , Afinidad de Anticuerpos , Antígenos/inmunología , Sitios de Unión de Anticuerpos , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Mutación , Conformación ProteicaRESUMEN
Concentration determination is an important method of protein characterization required in the development of protein therapeutics. There are many known methods for determining the concentration of a protein solution, but the easiest to implement in a manufacturing setting is absorption spectroscopy in the ultraviolet region. For typical proteins composed of the standard amino acids, absorption at wavelengths near 280 nm is due to the three amino acid chromophores tryptophan, tyrosine, and phenylalanine in addition to a contribution from disulfide bonds. According to the Beer-Lambert law, absorbance is proportional to concentration and path length, with the proportionality constant being the extinction coefficient. Typically the extinction coefficient of proteins is experimentally determined by measuring a solution absorbance then experimentally determining the concentration, a measurement with some inherent variability depending on the method used. In this study, extinction coefficients were calculated based on the measured absorbance of model compounds of the four amino acid chromophores. These calculated values for an unfolded protein were then compared with an experimental concentration determination based on enzymatic digestion of proteins. The experimentally determined extinction coefficient for the native proteins was consistently found to be 1.05 times the calculated value for the unfolded proteins for a wide range of proteins with good accuracy and precision under well-controlled experimental conditions. The value of 1.05 times the calculated value was termed the predicted extinction coefficient. Statistical analysis shows that the differences between predicted and experimentally determined coefficients are scattered randomly, indicating no systematic bias between the values among the proteins measured. The predicted extinction coefficient was found to be accurate and not subject to the inherent variability of experimental methods. We propose the use of a predicted extinction coefficient for determining the protein concentration of therapeutic proteins starting from early development through the lifecycle of the product.LAY ABSTRACT: Knowing the concentration of a protein in a pharmaceutical solution is important to the drug's development and posology. There are many ways to determine the concentration, but the easiest one to use in a testing lab employs absorption spectroscopy. Absorbance of ultraviolet light by a protein solution is proportional to its concentration and path length; the proportionality constant is the extinction coefficient. The extinction coefficient of a protein therapeutic is usually determined experimentally during early product development and has some inherent method variability. In this study, extinction coefficients of several proteins were calculated based on the measured absorbance of model compounds. These calculated values for an unfolded protein were then compared with experimental concentration determinations based on enzymatic digestion of the proteins. The experimentally determined extinction coefficient for the native protein was 1.05 times the calculated value for the unfolded protein with good accuracy and precision under controlled experimental conditions, so the value of 1.05 times the calculated coefficient was called the predicted extinction coefficient. Comparison of predicted and measured extinction coefficients indicated that the predicted value was very close to the experimentally determined values for the proteins. The predicted extinction coefficient was accurate and removed the variability inherent in experimental methods.
Asunto(s)
Aminoácidos/análisis , Química Farmacéutica/métodos , Proteínas/análisis , Absorción Fisicoquímica , Aminoácidos/química , Química Farmacéutica/instrumentación , Valor Predictivo de las Pruebas , Conformación Proteica , Proteínas/química , Sensibilidad y Especificidad , Espectrofotometría UltravioletaRESUMEN
Micellar aggregation behavior of polysorbate 20 (PS20) has generated significant interest because of the wide use of PS20 as a surfactant to minimize protein surface adsorption and mitigate protein aggregation. Thus, there is a need for better molecular understanding of what drives the biophysical behavior of PS20 in solution. We observe that a complex amphipathic PS20 molecule, which contains both hydrophobic tail and relatively large hydrophilic head, self-associates strongly within the course of a molecular dynamics simulation performed with a fully atomistic representation of the molecule and an explicit water solvent model. The in silico behavior is consistent with micellar models of PS20 in solution. The dynamics of this self-association is rather complex involving both internal reorganization of the molecule and diffusion to form stable micelle-like aggregates. The micellar aggregates of PS20 are long-lived and are formed by the balance between the large hydrophobic interactions associated with the aliphatic tail of PS20, and the steric repulsion of the hydrophilic sorbitan head structure. In the present work, molecular models of PS20 that represent naturally occurring PS20 fractions were produced and characterized in silico. The study investigated the monoester and diester fractions: PS20M, and PS20D. These fractions present differences in the strength of their hydrophobic effect, which influences the aggregation behavior. Adaptive biasing force (ABF) simulations were carried out with the PS20M and PS20D molecular constructs to calculate the free energy of their pairwise interaction. The free energy barrier for the dissociation is higher for PS20D compared with PS20M. The results show that hydrogen bonds can form when head groups are in close proximity, such as in the PS20 aggregate assembly, and the free energy of interaction can be used to predict the morphology of the micellar aggregate for the different PS20 fractions. We were also able to simulate PS20 in the presence of N-phenyl-1-naphthylamine (NPN) to study the solution behavior of the hydrophobic molecule and of the mechanism in which it is sequestered in the hydrophobic core of the PS20 micellar aggregate.
Asunto(s)
Micelas , Simulación de Dinámica Molecular , Polisorbatos/química , Tensoactivos/química , 1-Naftilamina/análogos & derivados , 1-Naftilamina/química , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Químicos , TermodinámicaRESUMEN
Color is an important quality attribute for biotherapeutics. In the biotechnology industry, a visual method is most commonly utilized for color characterization of liquid drug protein solutions. The color testing method is used for both batch release and on stability testing for quality control. Using that method, an analyst visually determines the color of the sample by choosing the closest matching European Pharmacopeia reference color solution. The requirement to judge the best match makes it a subjective method. Furthermore, the visual method does not capture data on hue or chroma that would allow for improved product characterization and the ability to detect subtle differences between samples. To overcome these challenges, we describe a quantitative method for color determination that greatly reduces the variability in measuring color and allows for a more precise understanding of color differences. Following color industry standards established by International Commission on Illumination, this method converts a protein solution's visible absorption spectra to L*a*b* color space. Color matching is achieved within the L*a*b* color space, a practice that is already widely used in other industries. The work performed here is to facilitate the adoption and transition for the traditional visual assessment method to a quantitative spectral method. We describe here the algorithm used such that the quantitative spectral method correlates with the currently used visual method. In addition, we provide the L*a*b* values for the European Pharmacopeia reference color solutions required for the quantitative method. We have determined these L*a*b* values by gravimetrically preparing and measuring multiple lots of the reference color solutions. We demonstrate that the visual assessment and the quantitative spectral method are comparable using both low- and high-concentration antibody solutions and solutions with varying turbidity. LAY ABSTRACT: In the biotechnology industry, a visual assessment is the most commonly used method for color characterization, batch release, and stability testing of liquid protein drug solutions. Using this method, an analyst visually determines the color of the sample by choosing the closest match to a standard color series. This visual method can be subjective because it requires an analyst to make a judgment of the best match of color of the sample to the standard color series, and it does not capture data on hue and chroma that would allow for improved product characterization and the ability to detect subtle differences between samples. To overcome these challenges, we developed a quantitative spectral method for color determination that greatly reduces the variability in measuring color and allows for a more precise understanding of color differences. The details of the spectral quantitative method are described. A comparison between the visual assessment method and spectral quantitative method is presented. This study supports the transition to a quantitative spectral method from the visual assessment method for quality testing of protein solutions.
Asunto(s)
Percepción de Color , Color , Soluciones Farmacéuticas/análisis , Farmacopeas como Asunto , Proteínas/análisis , Color/normas , Humanos , Soluciones Farmacéuticas/normas , Farmacopeas como Asunto/normas , Estándares de Referencia , Espectrofotometría/métodosRESUMEN
A quantitative spectral method has been developed to precisely measure the color of protein solutions. In this method, a spectrophotometer is utilized for capturing the visible absorption spectrum of a protein solution, which can then be converted to color values (L*a*b*) that represent human perception of color in a quantitative three-dimensional space. These quantitative values (L*a*b*) allow for calculating the best match of a sample's color to a European Pharmacopoeia reference color solution. In order to qualify this instrument and assay for use in clinical quality control, a technical assessment was conducted to evaluate the assay suitability and precision. Setting acceptance criteria for this study required development and implementation of a unique statistical method for assessing precision in 3-dimensional space. Different instruments, cuvettes, protein solutions, and analysts were compared in this study. The instrument accuracy, repeatability, and assay precision were determined. The instrument and assay are found suitable for use in assessing color of drug substances and drug products and is comparable to the current European Pharmacopoeia visual assessment method. LAY ABSTRACT: In the biotechnology industry, a visual assessment is the most commonly used method for color characterization, batch release, and stability testing of liquid protein drug solutions. Using this method, an analyst visually determines the color of the sample by choosing the closest match to a standard color series. This visual method can be subjective because it requires an analyst to make a judgment of the best match of color of the sample to the standard color series, and it does not capture data on hue and chroma that would allow for improved product characterization and the ability to detect subtle differences between samples. To overcome these challenges, we developed a quantitative spectral method for color determination that greatly reduces the variability in measuring color and allows for a more precise understanding of color differences. In this study, we established a statistical method for assessing precision in 3-dimensional space and demonstrated that the quantitative spectral method is comparable with respect to precision and accuracy to the current European Pharmacopoeia visual assessment method.
Asunto(s)
Química Farmacéutica/normas , Color/normas , Soluciones Farmacéuticas/análisis , Proteínas/análisis , Química Farmacéutica/métodos , Humanos , Reproducibilidad de los Resultados , Espectrofotometría/métodos , Espectrofotometría/normasRESUMEN
Antibody-drug conjugates (ADCs) require thorough characterization and understanding of product quality attributes. The framework of many ADCs comprises one molecule of antibody that is usually conjugated with multiple drug molecules at various locations. It is unknown whether the drug release rate from the ADC is dependent on drug location, and/or local environment, dictated by the sequence and structure of the antibody carrier. This study addresses these issues with valine-citrulline-monomethylauristatin E (vc-MMAE)-based ADC molecules conjugated at reduced disulfide bonds, by evaluating the cathepsin B catalyzed drug release rate of ADC molecules with different drug distributions or antibody carriers. MMAE drug release rates at different locations on ADC I were compared to evaluate the impact of drug location. No difference in rates was observed for drug released from the V(H), V(L), or C(H)2 domains of ADC I. Furthermore, four vc-MMAE ADC molecules were chosen as substrates for cathepsin B for evaluation of Michaelis-Menten parameters. There was no significant difference in K(M) or k(cat) values, suggesting that different sequences of the antibody carrier do not result in different drug release rates. Comparison between ADCs and small molecules containing vc-MMAE moieties as substrates for cathepsin B suggests that the presence of IgG1 antibody carrier, regardless of its bulkiness, does not impact drug release rate. Finally, a molecular dynamics simulation on ADC II revealed that the val-cit moiety at each of the eight possible conjugation sites was, on average, solvent accessible over 50% of its maximum solvent accessible surface area (SASA) during a 500 ns trajectory. Combined, these results suggest that the cathepsin cleavage sites for conjugated drugs are exposed enough for the enzyme to access and that the drug release rate is rather independent of drug location or monoclonal antibody carrier. Therefore, the distribution of drug conjugation at different sites is not a critical parameter to control in manufacturing of the vc-MMAE-based ADC conjugated at reduced disulfide bonds.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Catepsina B/metabolismo , Inmunoconjugados/metabolismo , Oligopéptidos/metabolismo , Cinética , Mapeo Peptídico , ProteolisisRESUMEN
This study was designed to assess the effects of cooling rate, storage temperature, and formulation composition on trehalose phase distribution and protein stability in frozen solutions. The data demonstrate that faster cooling rates (>100°C/min) result in trehalose crystallization and protein aggregation as determined by Fourier Transform Near-Infrared (FT-NIR) spectroscopy and size-exclusion chromatography, respectively. Conversely, at slower cooling rates (≤1°C/min), trehalose remains predominantly amorphous and there is no effect on protein stability. Evaluation of storage temperatures demonstrates that aggregation increases more rapidly at -14°C compared with higher (-8°C) and lower (-20°C) storage temperatures; however, a relatively higher amount of cumulative aggregation was observed at lower (-20°C) temperature compared with higher storage temperatures (-14°C and -8°C). Further evaluation of the effects of formulation composition suggests that the phase distribution of amorphous and crystallized trehalose dihydrate in frozen solutions depends on the ratio of trehalose to mAb. The results identify an optimal range of trehalose-mAb (w/w) ratio, 0.2-2.4, capable of physically stabilizing mAb formulations during long-term frozen storage-even for fast cooled (>100°C/min) formulations.
Asunto(s)
Proteínas/química , Trehalosa/química , Química Farmacéutica/métodos , Cromatografía en Gel/métodos , Cristalización/métodos , Estabilidad de Medicamentos , Almacenaje de Medicamentos/métodos , Liofilización/métodos , Congelación , Estabilidad Proteica , Soluciones/química , Espectroscopía Infrarroja por Transformada de Fourier/métodos , TemperaturaRESUMEN
Modeling ion exchange chromatography (IEC) behavior has generated significant interest because of the wide use of IEC as an analytical technique as well as a preparative protein purification process; indeed there is a need for better understanding of what drives the unique behavior of protein charge variants. We hypothesize that a complex protein molecule, which contains both hydrophobic and charged moieties, would interact strongly with an in silico designed resin through charged electrostatic patches on the surface of the protein. In the present work, variants of recombinant human growth hormone that mimic naturally-occurring deamidation products were produced and characterized in silico. The study included these four variants: rhGH, N149D, N152D, and N149D/N152D. Poisson-Boltzmann calculations were used to determine surface electrostatic potential. Metropolis Monte Carlo simulations were carried out with the resulting variants to simulate IEC systems, examining the free energy of the interaction of the protein with an in silico anion exchange column represented by polylysine polypeptide. The results show that the charge variants have different average binding energies and the free energy of interaction can be used to predict the retention time for the different variants.
Asunto(s)
Resinas de Intercambio Aniónico/química , Hormona de Crecimiento Humana/química , Simulación de Dinámica Molecular , Hormona de Crecimiento Humana/genética , Hormona de Crecimiento Humana/metabolismo , Humanos , Método de Montecarlo , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Electricidad EstáticaRESUMEN
Subcutaneous (SC) injection is becoming a more common route for the administration of biopharmaceuticals. Currently, there is no reliable in vitro method that can be used to anticipate the in vivo performance of a biopharmaceutical formulation intended for SC injection. Nor is there an animal model that can predict in vivo outcomes such as bioavailability in humans. We address this unmet need by the development of a novel in vitro system, termed Scissor (Subcutaneous Injection Site Simulator). The system models environmental changes that a biopharmaceutical could experience as it transitions from conditions of a drug product formulation to the homeostatic state of the hypodermis following SC injection. Scissor uses a dialysis-based injection chamber, which can incorporate various concentrations and combinations of acellular extracellular matrix (ECM) components that may affect the release of a biopharmaceutical from the SC injection site. This chamber is immersed in a container of a bicarbonate-based physiological buffer that mimics the SC injection site and the infinite sink of the body. Such an arrangement allows for real-time monitoring of the biopharmaceutical within the injection chamber, and can be used to characterize physicochemical changes of the drug and its interactions with ECM components. Movement of a biopharmaceutical from the injection chamber to the infinite sink compartment simulates the drug migration from the injection site and uptake by the blood and/or lymph capillaries. Here, we present an initial evaluation of the Scissor system using the ECM element hyaluronic acid and test formulations of insulin and four different monoclonal antibodies. Our findings suggest that Scissor can provide a tractable method to examine the potential fate of a biopharmaceutical formulation after its SC injection in humans and that this approach may provide a reliable and representative alternative to animal testing for the initial screening of SC formulations.
Asunto(s)
Productos Biológicos/administración & dosificación , Productos Biológicos/farmacocinética , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacocinética , Bicarbonatos/química , Productos Biológicos/química , Tampones (Química) , Química Farmacéutica , Diálisis , Diseño de Fármacos , Matriz Extracelular/metabolismo , Humanos , Ácido Hialurónico/metabolismo , Inyecciones Subcutáneas , Linfa/metabolismo , Tejido Subcutáneo/metabolismoRESUMEN
For mAbs to be viable therapeutics, they must be formulated to have low viscosity, be chemically stable, and have normal in vivo clearance rates. We explored these properties by observing correlations of up to 60 different antibodies of the IgG1 isotype. Unexpectedly, we observe significant correlations with simple physical properties obtainable from antibody sequences and by molecular dynamics simulations of individual antibody molecules. mAbs viscosities increase strongly with hydrophobicity and charge dipole distribution and decrease with net charge. Fast clearance correlates with high hydrophobicities of certain complementarity determining regions and with high positive or high negative net charge. Chemical degradation from tryptophan oxidation correlates with the average solvent exposure time of tryptophan residues. Aspartic acid isomerization rates can be predicted from solvent exposure and flexibility as determined by molecular dynamics simulations. These studies should aid in more rapid screening and selection of mAb candidates during early discovery.
Asunto(s)
Anticuerpos Monoclonales/química , Inmunoglobulina G/química , Animales , Anticuerpos Monoclonales/uso terapéutico , Células CHO , Cricetinae , Cricetulus , Humanos , Inmunoglobulina G/uso terapéutico , Estabilidad Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/uso terapéutico , ViscosidadRESUMEN
Molecular simulations are employed to compute the free energy of pairwise monoclonal antibodies (mAbs) association using a conformational sampling algorithm with a scoring function. The work reported here is aimed at investigating the mAb-mAb association driven by weak interactions with a computational method capable of predicting experimental observations of low binding affinity. The simulations are able to explore the free energy landscape. A steric interaction component, electrostatic interactions, and a nonpolar component of the free energy form the energy scoring function. Electrostatic interactions are calculated by solving the Poisson-Boltzmann equation. The nonpolar component is derived from the van der Waals interactions upon close contact of the protein surfaces. Two mAbs with similar IgG1 framework but with small sequence differences, mAb1 and mAb2, are considered for their different viscosity and propensity to form a weak interacting dimer. mAb1 presents favorable free energy of association at pH 6 with 15 mM of ion concentration reproducing experimental trends of high viscosity and dimer formation at high concentration. Free energy landscape and minimum free energy configurations of the dimer, as well as the second virial coefficient (B22) values are calculated. The energy distributions for mAb1 are obtained, and the most probable configurations are seen to be consistent with experimental measurements. In contrast, mAb2 shows an unfavorable average free energy at the same buffer conditions due to poor electrostatic complementarity, and reversible dimer configurations with favorable free energy are found to be unlikely. Finally, the simulations of the mAb association dynamics provide insights on the self-association responsible for bulk solution behavior and aggregation, which are important to the processing and the quality of biopharmaceuticals.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/química , Concentración de Iones de Hidrógeno , Simulación de Dinámica Molecular , Análisis de Componente Principal , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Electricidad Estática , Termodinámica , ViscosidadRESUMEN
Glycosylation is an important post-translational modification during protein production in eukaryotic cells, and it is essential for protein structure, stability, half-life, and biological functions. In this study, we produced various monoclonal antibody (mAb) glycoforms from Chinese hamster ovary (CHO) cells, including the natively glycosylated antibody, the enriched G0 form, the deglycosylated form, the afucosylated form, and the high mannose form, and we compared their intrinsic properties, side-by-side, through biophysical and biochemical approaches. Spectroscopic analysis indicates no measureable secondary or tertiary structural changes after in vitro or in vivo modification of the glycosylation pattern. Thermal unfolding experiments show that the high mannose and deglycosylated forms have reduced thermal stability of the CH2 domain compared with the other tested glycoforms. We also observed that the individual domain's thermal stability could be pH dependent. Proteolysis analysis indicates that glycosylation plays an important role in stabilizing mAbs against proteases. The stability of antibody glycoforms at the storage condition (2-8 °C) and at accelerated conditions (30 and 40 °C) was evaluated, and the results indicate that glycosylation patterns do not substantially affect the storage stability of the antibody we studied.
Asunto(s)
Anticuerpos Monoclonales Humanizados/química , Animales , Fenómenos Biofísicos , Células CHO , Rastreo Diferencial de Calorimetría , Cricetinae , Cricetulus , Glicosilación , Calor , Humanos , Espectrometría de Masas , Modelos Moleculares , Péptido Hidrolasas , Conformación Proteica , Estabilidad Proteica , Estructura Terciaria de Proteína , Espectrometría de Fluorescencia , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
The conjugation of hydrophobic cytotoxic agents such as monomethyl auristatin E (MMAE) to the interchain sulfhydryl groups of monoclonal antibodies (Mabs) through a protease-labile linker generates a heterogeneous drug load distribution. The conjugation process can generate high-drug-load species that can affect the physical stability of antibody-drug conjugates (ADCs). In this study, the mechanism of physical instability of ADCs was investigated by formulating the ADC pool as well as isolated drug load species in high and low ionic strength buffers to understand the effect of ionic strength on the stability of drug-conjugated Mabs. The results showed that the presence of high ionic strength buffer led to time-dependent aggregate and fragment formation of ADCs, predominantly ADCs with high-drug-load species under stress conditions. In addition, differential scanning calorimetry (DSC) results confirmed that there is a direct correlation between thermal unfolding and drug payload and that specific changes in the DSC thermogram profiles can be assigned to modifications by MMAE.
Asunto(s)
Anticuerpos Monoclonales/química , Inmunoconjugados/química , Oligopéptidos/química , Animales , Células CHO , Rastreo Diferencial de Calorimetría , Cricetulus , Estabilidad de Medicamentos , Humanos , Concentración Osmolar , Factores de TiempoRESUMEN
We investigated the photo-protective effect of sugar-based surfactants--hexyl glucoside and hexyl maltoside--against light-induced oxidation of a monoclonal antibody. Reactive oxygen species are generated in solutions in the presence of light; these reactive species readily oxidize amino acids such as tryptophan. Hexyl glucosides and hexyl maltosides scavenge these reactive species and protect tryptophan residues from light-induced oxidation in a concentration-dependent manner. As a result of the scavenging process, hydrogen peroxide is formed, especially at high (millimolar) concentrations of the alkyl glycoside surfactants. These results suggest that hexyl glucoside and hexyl maltoside have the potential to protect tryptophan residues against light-induced oxidation.
Asunto(s)
Glucósidos/química , Maltosa/química , Tensoactivos/química , Triptófano/química , Triptófano/efectos de la radiación , Aminoácidos/química , Cromatografía en Gel , Estabilidad de Medicamentos , Humanos , Peróxido de Hidrógeno/análisis , Hidrólisis , Inmunoglobulina G/química , Luz , Oxidación-Reducción , Oxígeno/química , Espectrometría de Fluorescencia , TripsinaRESUMEN
Polymer implants are promising systems for sustained release applications but their utility for protein delivery has been hindered because of concerns over drug stability at elevated temperatures required for processing. Using bovine serum albumin (BSA) as a model, we have assessed whether proteins can be formulated for processing at elevated temperatures. Specifically, the effect of trehalose and histidine-HCl buffer on BSA stability in a spray-dried formulation has been investigated at temperatures ranging from 80°C to 110°C. When both the sugar and buffer are present, aggregation is suppressed even when exposed to 100°C, the extrusion temperature of poly(lactide-co-glycolide) (PLGA), a bioresorbable polymer. Estimation of aggregation rate constants (k) indicate that though both trehalose and histidine-HCl buffer contribute to BSA stability, the effect because of trehalose alone is more pronounced. BSA-loaded PLGA implants were prepared using hot-melt extrusion process and in vitro release was conducted in phosphate buffered saline at 37°C. Comparison of drug released from implants prepared using four different formulations confirmed that maximal release was achieved from the formulation in which BSA was least aggregated. These studies demonstrate that when trehalose and histidine-HCl buffer are included in spray-dried formulations, BSA stability is maintained both during processing at 100°C and long-term residence within implants.
Asunto(s)
Preparaciones de Acción Retardada/química , Ácido Láctico/química , Ácido Poliglicólico/química , Albúmina Sérica Bovina/administración & dosificación , Trehalosa/química , Implantes Absorbibles , Animales , Bovinos , Desecación , Calor , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Estabilidad Proteica , Albúmina Sérica Bovina/químicaRESUMEN
In this study, the nature of interactions between monoclonal antibodies and polyols was studied using static light scattering. Solutions of mAb-U and mAb-P (4-12 mg/mL) were analyzed using static light scattering in buffer, 10% w/v trehalose and ethylene glycol solutions at pH 5.0, 7.0 and 9.0. Mechanical stress studies were conducted by shaking the mAb-U solutions (5mg/mL, pH 5.0, 7.0 and 9.0) and mAb-P solutions (5mg/mL, pH 7.0) at 200 rpm for 5 days at 25°C. Addition of trehalose and ethylene glycol resulted in a decrease in the attractive interactions between mAb-U molecules at pH 7.0 and 9.0, and at pH 9.0 between mAb-P molecules. At a higher ionic strength (300 mM, pH 5.0) trehalose and ethylene glycol decreased attractive interactions for both mAbs. Mechanical stress studies showed higher aggregation of mAb-U in trehalose solutions than ethylene glycol and buffer solutions at pH 7.0 and 9.0. A converse trend was seen for mAb-P at pH 7.0. This study showed that polyols, conformational stabilizers or destabilizers, decrease attractive interactions between protein molecules. The decrease is a result of masking of the hydrophobic sites on a protein as polyols can have favorable hydrophobic interactions with the surface exposed hydrophobic groups.
Asunto(s)
Anticuerpos Monoclonales/química , Glicol de Etileno/química , Excipientes/química , Trehalosa/química , Glicerol/química , Interacciones Hidrofóbicas e Hidrofílicas , Luz , Concentración Osmolar , Estabilidad Proteica , Dispersión de Radiación , Solubilidad , Estrés Mecánico , Sacarosa/química , Factores de TiempoRESUMEN
The solution dynamics of antibodies are critical to antibody function. We explore the internal solution dynamics of antibody molecules through the combination of time-resolved fluorescence anisotropy experiments on IgG1 with more than two microseconds of all-atom molecular dynamics (MD) simulations in explicit water, an order of magnitude more than in previous simulations. We analyze the correlated motions with a mutual information entropy quantity, and examine state transition rates in a Markov-state model, to give coarse-grained descriptors of the motions. Our MD simulations show that while there are many strongly correlated motions, antibodies are highly flexible, with F(ab) and F(c) domains constantly forming and breaking contacts, both polar and non-polar. We find that salt bridges break and reform, and not always with the same partners. While the MD simulations in explicit water give the right time scales for the motions, the simulated motions are about 3-fold faster than the experiments. Overall, the picture that emerges is that antibodies do not simply fluctuate around a single state of atomic contacts. Rather, in these large molecules, different atoms come in contact during different motions.