RESUMEN
Subvisible particles (SbVPs) are a critical quality attribute for biotherapeutics. Particle content in prefilled syringes (PFSs) of a biotherapeutic can include protein particles and silicone oil particles (SiOP). Here, a real-world protein therapeutic PFS shows that although polysorbate is effective in preventing protein particle formation, it also leads to the formation of SiOP. PFSs of protein and buffer formulations in the presence and absence of polysorbate are subjected to a drop shock to generate SbVP and the effect of polysorbate and protein in generating SbVP is investigated. Particle characterization by light obscuration and flow imaging shows that polysorbate prevents protein particle formation as intended, but the presence of polysorbate substantially increases the formation of SiOP. The protein itself also acts as a surfactant and leads to increased SiOP, but to a lesser degree compared to polysorbate. In a separate companion study by Joh et al., the risk of immunogenicity was assessed using in vivo and in vitro models. Flow imaging distinguishes between SiOP and protein particles and enables risk assessment of the natures of different SbVP in PFSs.
Asunto(s)
Anticuerpos Monoclonales/química , Inmunoglobulina G/química , Polisorbatos/química , Aceites de Silicona/química , Tensoactivos/química , Tampones (Química) , Composición de Medicamentos , Embalaje de Medicamentos , Estabilidad de Medicamentos , Concentración de Iones de Hidrógeno , Tamaño de la Partícula , Agregado de Proteínas , Estabilidad Proteica , Proteolisis , Estrés Mecánico , JeringasRESUMEN
Monoclonal antibodies are attractive therapeutic agents because of their impressive biological activities and favorable biophysical properties. Nevertheless, antibodies are susceptible to various types of chemical modifications, and the impact of such modifications on antibody physical stability and aggregation remains understudied. Here, we report a systematic analysis of the impact of methionine oxidation, tryptophan oxidation, and asparagine deamidation on antibody conformational and colloidal stability, hydrophobicity, solubility, and aggregation. Interestingly, we find little correlation between the impact of these chemical modifications on antibody conformational stability and aggregation. Methionine oxidation leads to significant reductions in antibody conformational stability while having little impact on antibody aggregation except at extreme conditions (low pH and elevated temperature). Conversely, tryptophan oxidation and asparagine deamidation have little impact on antibody conformational stability while promoting aggregation at a wide range of solution conditions, and the aggregation mechanisms appear linked to unique types of reducible and nonreducible covalent crosslinks and, in some cases, to increased levels of attractive colloidal interactions. These findings highlight that even related types of chemical modifications can lead to dissimilar antibody aggregation mechanisms, and evaluating these findings for additional antibodies will be important for improving the systematic generation of antibodies with high chemical and physical stability.
Asunto(s)
Anticuerpos Monoclonales/química , Asparagina/química , Metionina/química , Triptófano/química , Coloides , Composición de Medicamentos , Estabilidad de Medicamentos , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Oxidación-Reducción , Agregado de Proteínas , Estabilidad Proteica , Solubilidad , TemperaturaRESUMEN
Monoclonal antibodies must be both chemically and physically stable to be developed into safe and effective drugs. Although there has been considerable progress in separately understanding the molecular determinants of antibody chemical and physical stability, it remains poorly understood how defects in one property (e.g., chemical stability) impact the other property (e.g., physical stability). Here, we have investigated the impact of a common chemical modification (deamidation) on the physical stability of two monoclonal antibodies as a function of pH (from pH 3.8 to 7.4). Interestingly, we find that deamidation has significant, antibody-specific impacts on physical stability at low pH values that are common during antibody purification. Deamidation causes increases in self-association and/or aggregation at low pH (3.8), and a key contributor to this behavior appears to be deamidation-dependent increases in antibody hydrophobicity at low pH. Our findings highlight pH-dependent impacts of deamidation on antibody colloidal stability and aggregation, which are important to understand in order to improve the development and production of potent antibody therapeutics with high chemical and physical stabilities.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Composición de Medicamentos/métodos , Diseño de Fármacos , Estabilidad de Medicamentos , Concentración de Iones de Hidrógeno , Agregado de Proteínas , Asparagina/química , Cromatografía/métodos , Dispersión Dinámica de Luz/métodos , Oro/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Inmunoglobulina G/química , Nanopartículas del Metal/química , Solubilidad , Temperatura de TransiciónRESUMEN
Tryptophan (Trp) oxidation in proteins leads to a number of events, including changes in color, higher order structure (HOS), and biological activity. We describe here a number of new findings through a comprehensive characterization of 6 monoclonal antibodies (mAbs) following selective oxidation of Trp residues by 2,2'-azobis(2-amidinopropane) dihydrochloride. Fluorescence spectroscopy, in combination with second derivative analysis, demonstrates that the loss of Trp fluorescence intensity is a sensitive indicator of Trp oxidation in mAbs. Size-exclusion chromatography with UV and intrinsic Trp fluorescence detection was demonstrated to be a useful method to monitor Trp oxidation levels in mAbs. Furthermore, the Trp oxidation levels measured by size-exclusion chromatography with UV and intrinsic Trp fluorescence detection were found to be in agreement with the values obtained from tryptic peptide mapping by liquid chromatography with mass spectrometric detection and correlate with the total solvent accessible surface area of the exposed Trp residues from in silico modeling. Finally, near-UV circular dichroism and Raman spectroscopy were used to evaluate the impact of Trp oxidation on HOS and identify specific oxidation products, respectively. This work demonstrates that protein HOS is altered on Trp oxidation in mAbs and multiple spectroscopic markers can be used to monitor the molecule-dependent Trp oxidation behavior.
Asunto(s)
Anticuerpos Monoclonales/química , Triptófano/química , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/uso terapéutico , Células CHO , Dicroismo Circular , Cricetulus , Espectrometría de Masas , Simulación de Dinámica Molecular , Oxidación-Reducción , Mapeo Peptídico , Estructura Terciaria de Proteína , Espectrometría de FluorescenciaRESUMEN
Protein higher order structure (HOS) is an essential quality attribute to ensure protein stability and proper biological function. Protein HOS characterization is performed during comparability assessments for product consistency as well as during forced degradation studies for structural alteration upon stress. Circular dichroism (CD) spectroscopy is a widely used technique for measuring protein HOS, but it remains difficult to assess HOS with a high degree of accuracy and precision. Moreover, once spectral changes are detected, interpreting the differences in terms of specific structural attributes is challenging. Spectral normalization by the protein concentration remains one of the largest sources of error and reduces the ability to confidently detect differences in CD spectra. This work develops a simple method to enhance the precision of the CD spectral measurements through normalization of the CD spectra by the protein concentration determined directly from the CD measurement. This method is implemented to successfully detect small CD spectral changes in multiple forced degradation studies as well as comparability assessments during biologics drug development. Furthermore, the interpretation of CD spectral changes in terms of HOS differences are provided based on orthogonal data in conjunction with structural insights gained through in silico homology modeling of the protein structure.
Asunto(s)
Productos Biológicos/química , Proteínas/química , Dicroismo Circular/métodos , Conformación ProteicaRESUMEN
Methionine oxidation is a major degradation pathway in therapeutic proteins which can impact the structure and function of proteins as well as risk to drug product quality. Detecting Met oxidation in proteins by peptide mapping followed by liquid chromatography with mass spectrometry (LC-MS) is the industry standard but is also labor intensive and susceptible to artifacts. In this work, vibrational difference spectroscopy in combination with 18O isotopic shift enabled us to demonstrate the application of Raman and FTIR techniques for the detection and quantification of Met oxidation in various therapeutic proteins, including mAbs, fusion proteins, and antibody drug conjugate. Vibrational markers of Met oxidation products, such as sulfoxide and sulfone, corresponding to SâO and C-SâO stretching frequencies were unequivocally identified based 18O isotoptic shifts. The intensity of the isolated νC-S Raman band at 702 cm-1 was successfully applied to quantify the average Met oxidation level in multiple proteins. These results are further corroborated by oxidation levels measured by tryptic peptide mapping, and thus the confirmed Met oxidation levels derived from Raman and mass spectrometry are indeed consistent with each other. Thus, we demonstrate the broader application of vibrational spectroscopy to detect the subtle spectral changes associated with various chemical or physical degradation of proteins, including Met oxidation as well as higher order structural changes.
Asunto(s)
Anticuerpos Monoclonales/química , Metionina/análisis , Proteínas Recombinantes de Fusión/química , Sulfonas/análisis , Sulfóxidos/análisis , Anticuerpos Monoclonales/metabolismo , Biomarcadores/análisis , Cromatografía Liquida , Espectrometría de Masas , Metionina/metabolismo , Oxidación-Reducción , Proteínas Recombinantes de Fusión/metabolismo , VibraciónRESUMEN
PURPOSE: Characterizing submicron protein particles (approximately 0.1-1µm) is challenging due to a limited number of suitable instruments capable of monitoring a relatively large continuum of particle size and concentration. In this work, we report for the first time the characterization of submicron protein particles using the high size resolution technique of resistive pulse sensing (RPS). METHODS: Resistive pulse sensing, dynamic light scattering and size-exclusion chromatography with in-line multi-angle light scattering (SEC-MALS) are performed on protein and placebo formulations, polystyrene size standards, placebo formulations spiked with silicone oil, and protein formulations stressed via freeze-thaw cycling, thermal incubation, and acid treatment. RESULTS: A method is developed for monitoring submicron protein particles using RPS. The suitable particle concentration range for RPS is found to be approximately 4 × 107-1 × 1011 particles/mL using polystyrene size standards. Particle size distributions by RPS are consistent with hydrodynamic diameter distributions from batch DLS and to radius of gyration profiles from SEC-MALS. RPS particle size distributions provide an estimate of particle counts and better size resolution compared to light scattering. CONCLUSION: RPS is applicable for characterizing submicron particles in protein formulations with a high degree of size polydispersity. Data on submicron particle distributions provide insights into particles formation under different stresses encountered during biologics drug development.
Asunto(s)
Anticuerpos Monoclonales/química , Productos Biológicos/química , Desarrollo de Medicamentos/métodos , Tamaño de la Partícula , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Productos Biológicos/aislamiento & purificación , Células CHO , Química Farmacéutica , Cromatografía en Gel/instrumentación , Cromatografía en Gel/métodos , Cricetulus , Desarrollo de Medicamentos/instrumentación , Dispersión Dinámica de Luz/instrumentación , Dispersión Dinámica de Luz/métodos , Estudios de Factibilidad , Ensayos Analíticos de Alto Rendimiento/instrumentación , Ensayos Analíticos de Alto Rendimiento/métodos , Microfluídica/instrumentación , Microfluídica/métodosRESUMEN
Inverse Kirkwood-Buff (KB) solution theory can be used to relate macroscopic quantities with molecular scale interactions and correlation functions, in the form of KB integrals. Protein partial specific volumes ([Formula: see text]) from high-precision density measurements can be used to quantify solvent-solute and solute-solute KB integrals. Currently, general expressions for [Formula: see text] as a function of cosolute concentration (c3) have been provided for only binary and ternary solutions. We derive a general multicomponent expression for [Formula: see text] in terms of the relevant KB integrals for the case of low (infinite dilution) protein concentration but arbitrary cosolute concentrations. To test the utility of treating a quaternary system with a pseudoternary approximation, α-Chymotrypsinogen (aCgn) solutions with a series of solutes (NaCl, sucrose, and trehalose) were compared as a function of solute concentration with and without buffer present. Comparison between those ternary and quaternary solutions shows equivalent results within experimental uncertainty and suggests the pseudoternary approximation may be reasonable. In the case of aCgn, doing so also revealed that the preferential interactions can depend on pH. Analysis of steric contributions also provides an example that illustrates how KB integrals allow one to interpret [Formula: see text] in terms of contributions from molecular volume, excluded volume, and hydration/solvation effects.
Asunto(s)
Proteínas/química , Cloruro de Sodio/química , Sacarosa/química , Termodinámica , Trehalosa/química , Modelos Moleculares , SolucionesRESUMEN
PURPOSE: Characterization of submicron protein particles continues to be challenging despite active developments in the field. NTA is a submicron particle enumeration technique, which optically tracks the light scattering signal from suspended particles undergoing Brownian motion. The submicron particle size range NTA can monitor in common protein formulations is not well established. We conducted a comprehensive investigation with several protein formulations along with corresponding placebos using NTA to determine submicron particle size distributions and shed light on potential non-particle origin of size distribution in the range of approximately 50-300 nm. METHODS: NTA and DLS are performed on polystyrene size standards as well as protein and placebo formulations. RESULTS: Protein formulations filtered through a 20 nm filter, with and without polysorbate-80, show NTA particle counts. As such, particle counts above 20 nm are not expected in these solutions. Several other systems including positive and negative controls were studied using NTA and DLS. CONCLUSIONS: These apparent particles measured by NTA are not observed in DLS measurements and may not correspond to real particles. The intent of this article is to raise awareness about the need to interpret particle counts and size distribution from NTA with caution.
Asunto(s)
Nanopartículas/química , Proteínas/química , Tensoactivos/química , Química Farmacéutica , Humanos , Tamaño de la Partícula , Agregado de Proteínas , Unión Proteica , Conformación ProteicaRESUMEN
Parallel temperature initial rates (PTIR) from chromatographic separation of aggregating protein solutions are combined with continuous simultaneous multiple sample light scattering (SMSLS) to make quantitative deductions about protein aggregation kinetics and mechanisms. PTIR determines the rates at which initially monomeric proteins are converted to aggregates over a range of temperatures, under initial-rate conditions. Using SMSLS for the same set of conditions provides time courses of the absolute Rayleigh scattering ratio, IR(t), from which a potentially different measure of aggregation rates can be quantified. The present report compares these measures of aggregation rates across a range of solution conditions that result in different aggregation mechanisms for anti-streptavidin (AS) immunoglobulin gamma-1 (IgG1). The results illustrate how the two methods provide complementary information when deducing aggregation mechanisms, as well as cases where they provide new mechanistic details that were not possible to deduce in previous work. Criteria are presented for when the two techniques are expected to give equivalent results for quantitative rates, the potential limitations when solution non-idealities are large, as well as a comparison of the temperature dependence of AS-IgG1 aggregation rates with published data for other antibodies.
Asunto(s)
Inmunoglobulina G/química , Luz , Modelos Químicos , Agregado de Proteínas , Dispersión de Radiación , Animales , Ratones , Estreptavidina/antagonistas & inhibidores , Estreptavidina/químicaRESUMEN
Preferential interactions of proteins with water and osmolytes play a major role in controlling the thermodynamics of protein solutions. While changes in protein stability and shifts in phase behavior are often reported with the addition of osmolytes, the underlying protein interactions with water and/or osmolytes are typically inferred rather than measured directly. In this work, Kirkwood-Buff integrals for protein-water interactions (G12) and protein-osmolyte interactions (G23) were determined as a function of osmolyte concentration from density measurements of antistreptavidin immunoglobulin gamma-1 (AS-IgG1) in ternary aqueous solutions for a set of common neutral osmolytes: sucrose, trehalose, sorbitol, and poly(ethylene glycol) (PEG). For sucrose and PEG solutions, both protein-water and protein-osmolyte interactions depend strongly on osmolyte concentrations (c3). Strikingly, both osmolytes change from being preferentially excluded to preferentially accumulated with increasing c3. In contrast, sorbitol and trehalose solutions do not show large enough preferential interactions to be detected by densimetry. G12 and G23 values are used to estimate the transfer free energy for native AS-IgG1 (Δµ2N) and compared with existing models. AS-IgG1 unfolding via calorimetry shows a linear increase in midpoint temperatures as a function of trehalose, sucrose, and sorbitol concentrations, but the opposite behavior for PEG. Together, the results highlight limitations of existing models and common assumptions regarding the mechanisms of protein stabilization by osmolytes. Finally, PEG preferential interactions destabilize the Fab regions of AS-IgG1 more so than the CH2 or CH3 domains, illustrating preferential interactions can be specific to different protein domains.
Asunto(s)
Anticuerpos Monoclonales/química , Inmunoglobulina G/química , Concentración Osmolar , Polietilenglicoles/química , Estabilidad Proteica , Sorbitol/química , Sacarosa/química , Termodinámica , Trehalosa/química , Agua/químicaRESUMEN
Controlling and predicting unwanted degradation, such as non-native aggregation, is a long-standing challenge for mAbs and other protein-based products. mAb aggregation rates are typically sensitive to temperature, pH, and the addition of excipients. Quantitatively comparing temperature-dependent aggregation rates across multiple possible formulations is a challenge in product development. A parallel temperature initial rate method is used to efficiently and accurately determine initial rates for anti-streptavidin (AS) IgG1 aggregation as a function of pH, [NaCl], and in the presence of acetate versus citrate buffer. Parallel temperature initial rates are shown to agree with results from a traditional, isothermal method and permits direct comparison of the formulations across almost 3 orders of magnitude of aggregation rates. The apparent midpoint unfolding temperatures (through differential scanning calorimetry) and the effective activation energy values (Ea) are generally higher in acetate buffer compared with citrate buffer, which is consistent with preferential accumulation of citrate ions compared with acetate ions that was speculated in previous work (Barnett et al., J Phys Chem B, 2015). Static light scattering and Kirkwood-Buff analysis show that AS-IgG1 has stronger net repulsive protein-protein interactions in acetate compared with citrate buffer, also consistent with increased values of Ea. In an extreme case, aggregation of AS-IgG1 is effectively eliminated across all practical temperatures at pH 4 in 10 mM sodium acetate but proceeds readily in citrate buffer.
Asunto(s)
Acetatos/química , Anticuerpos Monoclonales/química , Ácido Cítrico/química , Inmunoglobulina G/química , Iones/química , Agregado de Proteínas , Estreptavidina/inmunología , Tampones (Química) , Química Farmacéutica/métodos , Excipientes/química , Concentración de Iones de Hidrógeno , Desplegamiento Proteico , TemperaturaRESUMEN
Non-native protein aggregation may occur during manufacturing and storage of protein therapeutics, and this may decrease drug efficacy or jeopardize patient safety. From a regulatory perspective, changes in higher order structure due to aggregation are of particular interest but can be difficult to monitor directly at elevated protein concentrations. The present report focuses on non-native aggregation of antistreptavidin (AS) IgG1 at 30 mg/mL under solution conditions that prior work at dilute concentrations (e.g., 1 mg/mL) indicated would result in different aggregation mechanisms. Time-dependent aggregation and structural changes were monitored in situ with dynamic light scattering, small-angle neutron scattering, and Raman scattering and ex situ with far-UV circular dichroism and second-derivative UV spectroscopy. The effects of adding 0.15 M (â¼5 w/w %) sucrose were also assessed. The addition of sucrose decreased monomer loss rates but did not change protein-protein interactions, aggregation mechanism(s), or aggregate structure and morphology. Consistent with prior results, altering the pD or salt concentration had the primary effect of changing the aggregation mechanism. Overall, the results provide a comparison of aggregate structure and morphology created via different growth mechanisms using orthogonal techniques and show that the techniques agree at least qualitatively. Interestingly, AS-IgG1 aggregates created at pD 5.3 with no added salt formed the smallest aggregates but had the largest structural changes compared to other solution conditions. The observation that the larger aggregates were also those with less structural perturbation compared to folded AS-IgG1 might be expected to extend to other proteins if the same strong electrostatic repulsions that mediate aggregate growth also mediate structural changes of the constituent proteins within aggregates.
Asunto(s)
Inmunoglobulina G/química , Estreptavidina/inmunología , Dicroismo Circular , Inmunoglobulina G/inmunología , Cinética , Conformación Proteica , Espectrofotometría Ultravioleta , Espectrometría RamanRESUMEN
Non-native aggregation is a common issue in a number of degenerative diseases and during manufacturing of protein-based therapeutics. There is a growing interest to monitor protein stability at intermediate to high protein concentrations, which are required for therapeutic dosing of subcutaneous injections. An understanding of the impact of protein structural changes and interactions on the protein aggregation mechanisms and resulting aggregate size and morphology may lead to improved strategies to reduce aggregation and solution viscosity. This report investigates non-native aggregation of a model protein, α-chymotrypsinogen, under accelerated conditions at elevated protein concentrations. Far-UV circular dichroism and Raman scattering show structural changes during aggregation. Size exclusion chromatography and laser light scattering are used to monitor the progression of aggregate growth and monomer loss. Monomer loss is concomitant with increased ß-sheet structures as monomers are added to aggregates, which illustrate a transition from a native monomeric state to an aggregate state. Aggregates grow predominantly through monomer-addition, resulting in a semi-flexible polymer morphology. Analysis of aggregation growth kinetics shows that pH strongly affects the characteristic timescales for nucleation (τn) and growth (τg), while the initial protein concentration has only minor effects on τn or τg. Low-shear viscosity measurements follow a common scaling relationship between average aggregate molecular weight (Mw(agg)) and concentration (σ), which is consistent with semi-dilute polymer-solution theory. The results establish a link between aggregate growth mechanisms, which couple Mw(agg) and σ, to increases in solution viscosity even at these intermediate protein concentrations (less than 3w/v %).
Asunto(s)
Quimotripsinógeno/química , Quimotripsinógeno/metabolismo , Dicroismo Circular , Dispersión Dinámica de Luz , Concentración de Iones de Hidrógeno , Espectrometría Raman , ViscosidadRESUMEN
Non-native protein aggregation is common in the biopharmaceutical industry and potentially jeopardizes product shelf life, therapeutic efficacy, and patient safety. The present article focuses on the relationship(s) among protein-protein interactions, aggregate growth mechanisms, aggregate morphologies, and specific-ion effects for an anti-streptavidin (AS) immunoglobulin gamma 1 (IgG1). Aggregation mechanisms of AS-IgG1 were determined as a function of pH and NaCl concentration with sodium acetate buffer and compared to previous work with sodium citrate. Aggregate size and shape were determined using a combination of laser light scattering and small-angle neutron or X-ray scattering. Protein-protein interactions were quantified in terms of the protein-protein Kirkwood-Buff integral (G22) determined from static light scattering and in terms of the protein effective charge (Zeff) measured using electrophoretic light scattering. Changing from citrate to acetate resulted in significantly different protein-protein interactions as a function of pH for low NaCl concentrations when the protein displayed positive Zeff. Overall, the results suggest that electrostatic repulsions between proteins were lessened because of preferential accumulation of citrate anions, compared to acetate anions, at the protein surface. The predominant aggregation mechanisms correlated well with G22, indicating that ion-specific effects beyond traditional mean-field descriptions of electrostatic protein-protein interactions are important for predicting qualitative shifts in protein aggregation state diagrams. Interestingly, while solution conditions dictated which mechanisms predominated, aggregate average molecular weight and size displayed a common scaling behavior across both citrate- and acetate-based systems.
Asunto(s)
Inmunoglobulina G/química , Iones/química , Agregado de Proteínas , Estreptavidina/inmunología , Citratos/química , Coloides/química , Hidrodinámica , Concentración de Iones de Hidrógeno , Peso Molecular , Dispersión de Radiación , Acetato de Sodio/química , Cloruro de Sodio/química , Citrato de Sodio , Soluciones , Electricidad Estática , Propiedades de SuperficieRESUMEN
Zeolitic imidazolate frameworks (ZIFs) are hybrid organic-inorganic microporous materials that exhibit zeolite-like structures and can be synthesized with a wide range of pore sizes and chemical functionality. ZIFs as thin films and membranes are of interest for their applications in sensors and gas separation. Here, we report a method for ZIF film and membrane fabrication, based on support surface modification and in situ solvothermal growth, which has potential for general application to other ZIF membranes. Our simple surface modification method results in strong covalent bonds between α-Al(2)O(3) supports and imidazolate ligands, which promote the heterogeneous nucleation and growth of ZIF crystals. The microstructure of ZIF-8 films can be controlled by controlling the pH of the growth solution. ZIF-7 films were fabricated to demonstrate the potential for general applicability of our method. Finally, the separation performance of several ZIF-8 membranes was evaluated, revealing molecular sieving behavior with an ideal selectivity for H(2)/CH(4) of 13.