RESUMO
Hyaluronic acid (HA) is a glycosaminoglycan with diverse biomedical applications including viscosupplementation of synovial fluid for the treatment osteoarthritis. Current HA viscosupplements such as Synvisc, Orthovisc, and Hyalgan have shown positive effects of reducing pain and improving joint function. The therapeutic efficacy, however, is highly transient, and these viscous fluids suffer from poor injectability. HA nanoparticles were found to modify the rheological properties of a model of the HA viscosupplement Orthovisc. Nanoparticles were successfully synthesized from 17 and 1500 kDa HA. Nanoparticle suspensions of HA were studied at different concentrations and in blends with the model viscosupplement. Nanoparticles made from 1500 kDa HA reduced the viscosupplement viscosity and elasticity to a much greater degree than nanoparticles made from 17 kDa HA. The difference in the nanoparticle effect on viscoelasticity suggested that nanoparticles made from 17 kDa HA may have dangling surface polymers that facilitated interactions with HA in solution. This hypothesis was supported by the greater compressibility of 17 kDa nanoparticles as determined by ultrasonic vibrational spectroscopy. Rheological investigations showed that the viscoelasticity of viscosupplements could be discretely titrated by modulating the concentration and type of HA nanoparticle additive (hard sphere or hairy). Thus, the injectability of viscosupplements may be enhanced while maintaining high elasticity.
Assuntos
Materiais Biocompatíveis/química , Ácido Hialurônico/química , Nanopartículas/química , Viscossuplementos/química , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
The rational design and selection of formulation composition to meet molecule-specific and product-specific needs are critical for biotherapeutics development to ensure physical and chemical stability. This work, based on three antibody-based (mAb) proteins (mAbA, mAbB, and mAbC), evaluates residue-specific impact of EDTA and methionine on protein oxidation, using an integrated biotherapeutics drug product development workflow. This workflow includes statistical experimental design, high-throughput experimental automation and execution, structure-based in silico modeling, inferential statistical analysis, and enhanced interactive data visualization of large datasets. This oxidation study evaluates the impact of formulation parameters including pH, protein concentration, and the presence of polysorbate 80 on the oxidation of specific conserved and variable residues of mAbs A, B, and C in the presence of stressors (iron, peroxide) and/or protectants (EDTA, L-methionine). Residue-specific analysis by automated high-throughput peptide mapping demonstrates differential residue-specific effects of EDTA and methionine in protecting against oxidation, highlighting the need for molecule-specific and product-specific selection of these excipients during formulation development. Computational modeling based on a homology model and the two-shell water coordination method (WCN) was employed to gain mechanistic understanding of residue-specific oxidation susceptibility of methionine residues. The computational determinants of local solvent exposure of methionine residues showed good correlation of WCN with experimentally determined oxidation for corresponding residues. The rapid generation of high-resolution data, statistical data analysis and interactive visualization of the high-throughput residue-level data containing â¼200 unique formulations facilitate residue-specific, molecule-specific and product-specific oxidation (global and local) assessment for oxidation protectants during early development for mAbs and related mAb-based modalities.
Assuntos
Metionina , Racemetionina , Metionina/química , Ácido Edético , Fluxo de Trabalho , Racemetionina/metabolismo , OxirreduçãoRESUMO
Formulation of protein-based therapeutics employ advanced formulation and analytical technologies for screening various parameters such as buffer, pH, and excipients. At a molecular level, physico-chemical properties of a protein formulation depend on self-interaction between protein molecules, protein-solvent and protein-excipient interactions. This work describes a novel in silico approach, SILCS-Biologics, for structure-based modeling of protein formulations. SILCS Biologics is based on the Site-Identification by Ligand Competitive Saturation (SILCS) technology and enables modeling of interactions among different components of a formulation at an atomistic level while accounting for protein flexibility. It predicts potential hotspot regions on the protein surface for protein-protein and protein-excipient interactions. Here we apply SILCS-Biologics on a Fab domain of a monoclonal antibody (mAbN) to model Fab-Fab interactions and interactions with three amino acid excipients, namely, arginine HCl, proline and lysine HCl. Experiments on 100 mg/ml formulations of mAbN showed that arginine increased, lysine reduced, and proline did not impact viscosity. We use SILCS-Biologics modeling to explore a structure-based hypothesis for the viscosity modulating effect of these excipients. Current efforts are aimed at further validation of this novel computational framework and expanding the scope to model full mAb and other protein therapeutics.
Assuntos
Aminoácidos , Proteínas , Simulação por Computador , LigantesRESUMO
Developing ultra-high concentration biotherapeutics drug products can be challenging due to increased viscosity, processing, and stability issues. Excipients used to alleviate these concerns are traditionally evaluated at lower protein concentrations. This study investigates whether classically known modulators of stability and viscosity at low (<50 mg/mL) to high (>50 - 150 mg/mL) protein concentrations are beneficial in ultra-high (>150 mg/mL) concentration protein formulations and drug products. This study evaluates the effect of arginine monohydrochloride, proline, and lysine monohydrochloride on viscosity and concentratability at different high and ultra-high protein concentrations using a monoclonal antibody, mAbN, formulation as a candidate protein system. The effect of excipients on the viscosity and concentratability (rate and extent) was different at high versus ultra-high protein concentrations. These results highlight that classical excipients in literature known to modulate protein interactions at low protein concentrations and reduce viscosity at high protein concentrations may need to be evaluated at target protein concentrations in a product-specific manner while developing ultra-high concentration biologics drug products.
Assuntos
Produtos Biológicos , Excipientes , Anticorpos Monoclonais , Desenvolvimento de Medicamentos , Estabilidade de Medicamentos , ViscosidadeRESUMO
Production of recombinant proteins generates a variety of process-related impurities. The multistep manufacturing processes may introduce many potential contaminants into the final pharmaceutical products. These residual impurities and contaminants can potentially impact the protein stability significantly. In this short review, the authors intend to discuss major sources and types of residual process-related impurities and potential product contaminants, their impact on protein quality/stability, and possible mitigations during product development and manufacturing processes.
Assuntos
Contaminação de Medicamentos , Estabilidade Proteica , Proteínas Recombinantes/química , Biotecnologia , HumanosRESUMO
Aggregation of monoclonal antibodies is often a multi-step process involving structural alterations in monomeric proteins and subsequent formation of soluble or insoluble oligomers. The role of local conformational stability and dynamics of native and/or partially altered structures in determining the aggregation propensity of monoclonal antibodies, however, is not well understood. Here, we investigate the role of conformational stability and dynamics of regions with distinct solvent exposure in determining the aggregation propensity of an IgG1 and IgG2 monoclonal antibody. The temperatures employed span the pre-unfolding range (10-40°C) and the onset temperatures (T onset ) for exposure of apolar residues (≈ 50°C), alterations in secondary structures (≈ 60°C) and initiation of visible aggregate formation (≈ 60°C). Solvent-exposed regions were found to precede solvent-shielded regions in an initiation of aggregation for both proteins. Such a process was observed upon alterations in overall tertiary structure while retaining the secondary structures in both the proteins. In addition, a greater dynamic nature of solvent-shielded regions in potential intermediates of IgG1 and the improved conformational stability increased its resistance to aggregation when compared to IgG2. These results suggest that local conformational stability and fluctuations of partially altered structures can influence the aggregation propensity of immunoglobulins.
Assuntos
Anticorpos Monoclonais/química , Imunoglobulina G/química , Acrilamida/farmacologia , Dicroísmo Circular , Fluorescência , Cinética , Complexos Multiproteicos/química , Conformação Proteica , Dobramento de Proteína , Multimerização Proteica , Estabilidade Proteica , Temperatura , Triptofano/químicaRESUMO
The effects of sucrose and arginine on the conformational and storage stability of an IgG1 monoclonal antibody (mAb) were monitored by differential scanning calorimetry (DSC) and size-exclusion chromatography (SEC), respectively. Excipient effects on protein physical stability were then compared with their effects on the local flexibility of the mAb in solution at pH 6, 25°C using hydrogen/deuterium-exchange mass spectrometry (H/D-MS). Compared with a 0.1 M NaCl control, sucrose (0.5 M) increased conformational stability (T(m) values), slowed the rate of monomer loss, reduced the formation of insoluble aggregates, and resulted in a global trend of small decreases in local flexibility across most regions of the mAb. In contrast, the addition of arginine (0.5 M) decreased the mAb's conformational stability, increased the rate of loss of monomer with elevated levels of soluble and insoluble aggregates, and led to significant increases in the local flexibility in specific regions of the mAb, most notably within the constant domain 2 of the heavy chain (C(H)2). These results provide new insights into the effect of sucrose and arginine on the local dynamics of IgG1 domains as well as preliminary correlations between local flexibility within specific segments of the C(H)2 domain (notably heavy chain 241-251) and the mAb's overall physical stability.
Assuntos
Anticorpos Monoclonais/química , Arginina/química , Excipientes/química , Imunoglobulina G/química , Sacarose/química , Armazenamento de Medicamentos , Espectrometria de Massas , Simulação de Dinâmica Molecular , Conformação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacosRESUMO
Since immunoglobulins are conformationally dynamic molecules in solution, we studied the effect of stabilizing and destabilizing excipients on the conformational stability and dynamics of two IgG1 monoclonal antibodies (mAbs; mAb-A and mAb-B) using a variety of biophysical approaches. Even though the two mAbs are of the same IgG1 subtype, the unfolding patterns, aggregation behavior, and pretransition dynamics of these two antibodies were strikingly different in response to external perturbations such as pH, temperature, and presence of excipients. Sucrose and arginine were identified as stabilizers and destabilizers, respectively, on the basis of their influence on conformational stability for both the IgG1 mAbs. The two excipients, however, had distinct effective concentrations and different effects on the conformational stability and pretransition dynamics of the two mAbs as measured by a combination of differential scanning calorimetry, high-resolution ultrasonic spectroscopy, and red-edge excitation shift fluorescence studies. Stabilizing concentrations of sucrose were found to decrease the internal motions of mAb-B, whereas arginine marginally increased its adiabatic compressibility in the pretransition region. Both sucrose and arginine did not influence the pretransition dynamics of mAb-A. The potential reasons for such differences in excipient effects between two IgG1 mAbs are discussed.
Assuntos
Anticorpos Monoclonais/química , Arginina/química , Excipientes/química , Imunoglobulina G/química , Sacarose/química , Varredura Diferencial de Calorimetria , Química Farmacêutica , Estabilidade de Medicamentos , Concentração de Íons de Hidrogênio , Conformação Proteica , Desnaturação Proteica , Espectrometria de Fluorescência , Tecnologia Farmacêutica/métodos , Temperatura , UltrassomRESUMO
Studies of protein-protein interactions (PPIs), especially in high-concentration solutions, have become increasingly important from a pharmaceutical perspective. Analytical methods used to study protein interactions, however, rely primarily on the detection of nonideality in relatively dilute (<50 mg/mL) solutions. We present here an application of variable-pathlength ultraviolet (UV)-visible absorption spectroscopy to examine and better understand such interactions over a wide concentration range (5-240 mg/mL) using several representative proteins. In this study, the change in UV absorption (or extinction coefficient) was monitored by determining delta absorbance (ΔAbs), the difference between the measured absorbance and the corresponding theoretical absorbance (calculated from gravimetric dilution), over a wide range of protein concentrations. The ΔAbs, corrected for light scattering, was found to increase with protein concentration for three model proteins (bovine serum albumin, lysozyme, and monoclonal antibody). Because PPIs influence solution viscosity, we studied the correlation between ΔAbs measurements and viscosity as a function of protein concentration. The magnitude of ΔAbs and solution viscosity followed similar trends with increasing protein concentration, albeit to different extents for different proteins. These data support the use of such ΔAbs measurements as an alternative approach to monitor and evaluate interactions in protein solutions at high concentration.
Assuntos
Proteínas/química , Espectrofotometria Ultravioleta , Tecnologia Farmacêutica/métodos , Absorção , Anticorpos Monoclonais/química , Luz , Modelos Químicos , Muramidase/química , Ligação Proteica , Espalhamento de Radiação , Soroalbumina Bovina/química , Solubilidade , ViscosidadeRESUMO
Protective antigen (PA) is the primary protein antigenic component of both the currently used anthrax vaccine and related recombinant vaccines under development. An analogue of recombinant PA (2-FHis rPA) has been recently shown to block the key steps of pore formation in the process of inducing cytotoxicity in cells, and thus can potentially be used as an antitoxin or a vaccine. This rPA analogue was produced by fermentation to incorporate the unnatural amino acid 2-fluorohistidine (2-FHis). In this study, the effects of 2-FHis labeling on rPA antigen's conformational stability and dynamic properties were investigated by various biophysical techniques. Temperature/pH stability profiles of rPA and 2-FHis rPA were analyzed by the empirical phase diagram (EPD) approach, and physical stability differences between them were identified. Results showed that rPA and 2-FHis rPA had similar stability at pH 7-8. With decreasing solution pH, however, 2-FHis rPA was found to be more stable. Dynamic sensitive measurements of the two proteins at pH 5 found that 2-FHis rPA was more dynamic and/or differentially hydrated under acidic pH conditions. The biophysical characterization and stability data provide information useful for the potential development of 2-FHis rPA as a more stable rPA vaccine candidate.
Assuntos
Antígenos de Bactérias/química , Toxinas Bacterianas/química , Histidina/análogos & derivados , Dicroísmo Circular , Histidina/química , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/química , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , TemperaturaRESUMO
A molecular understanding of excipient effects on the interrelationship(s) between dynamics and conformational stability of proteins, such as monoclonal antibodies (mAbs), can be important for their pharmaceutical development. The current study examines stabilizing and destabilizing effects of excipients on the conformational stability and local dynamics of distinct solvent-exposed regions within an IgG1 monoclonal antibody (mAb-B). The principles of site-selective photoselection upon red-edge excitation, accompanied by acrylamide quenching of tryptophan fluorescence were employed in this study. The initiation of mAb-B thermal unfolding occurs by structural alterations in the more solvent-exposed regions of the antibody, which subsequently leads to a cascade of structural alterations in its relatively more solvent-shielded regions. In addition, an increase in internal dynamics of solvent-shielded regions made mAb-B more susceptible to thermally induced structural perturbations resulting in its global destabilization. Sucrose and arginine exert their stabilizing and destabilizing effects by predominantly influencing the conformational stability of solvent-exposed regions in mAb-B. The complex molecular effects of sucrose and arginine on local dynamics of different regions in mAb-B and their correlation with the protein's conformational stability are described within the pretransition range, at the onset temperature (T(onset)) and at the thermal melting temperature (T(M)).
Assuntos
Anticorpos Monoclonais/química , Excipientes/química , Imunoglobulina G/química , Arginina/química , Conformação Proteica , Estabilidade Proteica , Desdobramento de Proteína , Sacarose/química , TemperaturaRESUMO
We recently identified a novel opioid peptide transport system in the retinal pigment epithelium that transports opioid peptides by a Na+/Cl--dependent process. Here we describe a similar transport system expressed in SK-N-SH cells (a human neuronal cell line) and show for the first time that the activity of the transport system is modulated differentially by lysine and small nonopioid peptides. The transport process in SK-N-SH cells, monitored with deltorphin II as the substrate, is Na+/Cl--dependent and interacts with several opioid peptides, consisting of 5 to 13 amino acids. The activity of this transport system is markedly stimulated by specific dipeptides and tripeptides, with significant stimulation observable at low micromolar concentrations. The ion dependence, Na+/Cl--activation kinetics, and opioid peptide selectivity of the transport system, however, remain unchanged. The stimulation by the modulatory peptides is associated with an increase in maximal velocity with no change in substrate affinity of the system. Amino acids have no or little effect on the transport system, with the exception of lysine. This cationic amino acid inhibits the transport system, with significant inhibition occurring at physiologic concentrations of the amino acid. The inhibitory effect is primarily associated with a decrease in the maximal velocity of the transport system with little change in substrate affinity. Methyl and ethyl esters of lysine retain the inhibitory potency, but most other structural analogs have no effect. The differential modulation of the transport system by lysine and specific small peptides has important implications in the biology and pharmacology of opioid peptides.