RESUMO
The computational prediction of the viscosity of dense protein solutions is highly desirable, for example, in the early development phase of high-concentration biopharmaceutical formulations where the material needed for experimental determination is typically limited. Here, we use large-scale atomistic molecular dynamics (MD) simulations with explicit solvation to de novo predict the dynamic viscosities of solutions of a monoclonal IgG1 antibody (mAb) from the pressure fluctuations using a Green-Kubo approach. The viscosities at simulated mAb concentrations of 200 and 250 mg/mL are compared to the experimental values, which we measured with rotational rheometry. The computational viscosity of 24 mPa·s at the mAb concentration of 250 mg/mL matches the experimental value of 23 mPa·s obtained at a concentration of 213 mg/mL, indicating slightly different effective concentrations (or activities) in the MD simulations and in the experiments. This difference is assigned to a slight underestimation of the effective mAb-mAb interactions in the simulations, leading to a too loose dynamic mAb network that governs the viscosity. Taken together, this study demonstrates the feasibility of all-atom MD simulations for predicting the properties of dense mAb solutions and provides detailed microscopic insights into the underlying molecular interactions. At the same time, it also shows that there is room for further improvements and highlights challenges, such as the massive sampling required for computing collective properties of dense biomolecular solutions in the high-viscosity regime with reasonable statistical precision.
RESUMO
Molecular interaction mechanisms in high-concentrated protein systems are of fundamental importance for the rational development of biopharmaceuticals such as monoclonal antibody (mAb) formulations. In such high-concentrated protein systems, the intermolecular distances between mAb molecules are reduced to the size of the protein diameter (approx. 10 nm). Thus, protein-protein interactions are more pronounced at high concentrations; so a direct extrapolation of physicochemical properties obtained from measurements at a low protein concentration of the corresponding properties at a high protein concentration is highly questionable. Besides the charge-charge interaction, the effects of molecular crowding, dipolar interaction, changes in protein hydration, and self-assembling tendency become more relevant. Here, protein hydration, protein dipole moment, and protein-protein interactions were studied in protein concentrations up to 200 mg/mL (= 1.3 mM) in different formulations for selected mAbs using dielectric relaxation spectroscopy (DRS). These data are correlated with the second virial coefficient, A2, the diffusion interaction parameter, kD, the elastic shear modulus, G', and the dynamic viscosity, η. When large contributions of dipolar protein-protein interactions were observed, the tendency of self-assembling and an increase in solution viscosity were detected. These effects were examined using specific buffer conditions. Furthermore, different types of protein-water interactions were identified via DRS, whereby the effect of high protein concentration on protein hydration was investigated for different high-concentrated liquid formulations (HCLFs).
Assuntos
Anticorpos Monoclonais , Anticorpos Monoclonais/química , Difusão , ViscosidadeRESUMO
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.
Assuntos
Substituição de Aminoácidos , Anticorpos Monoclonais , Imunoglobulina G , Modelos Moleculares , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/isolamento & purificação , Cromatografia por Troca Iônica , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Imunoglobulina G/química , Imunoglobulina G/genética , Imunoglobulina G/isolamento & purificação , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/genéticaRESUMO
PURPOSE: Introduction of the activation energy (Ea) as a kinetic parameter to describe and discriminate monoclonal antibody (mAb) stability. METHODS: Ea is derived from intrinsic fluorescence (IF) unfolding thermograms. An apparent irreversible three-state fit model based on the Arrhenius integral is developed to determine Ea of respective unfolding transitions. These activation energies are compared to the thermodynamic parameter of van´t Hoff enthalpies (∆Hvh). Using a set of 34 mAbs formulated in four different formulations, both the apparent thermodynamic and kinetic parameters together with apparent melting temperatures are correlated collectively with each other to storage stabilities to evaluate its predictive power with respect to long-term effects potentially reflected in shelf-life. RESULTS: Ea allows for the discrimination of (i) different parent mAbs, (ii) different variants that originate from parent mAbs, and (iii) different formulations. Interestingly, we observed that the Ea of the CH2 unfolding transition shows strongest correlations with monomer and aggregate content after storage at accelerated and stress conditions when collectively compared to ∆Hvh and Tm of the CH2 transition. Moreover, the predictive parameters determined for the CH2 domain show generally stronger correlations with monomer and aggregate content than those derived for the Fab. Qualitative assessment by ranking Ea of the Fab domain showed good agreement with monomer content in storage stabilities of individual mAb sub-sets. CONCLUSION: Ea from IF unfolding transitions can be used in addition to other commonly used thermodynamic predictive parameters to discriminate and characterize thermal stability of different mAbs in different formulations. Hence, it shows great potential for antibody engineering and formulation scientists.
Assuntos
Anticorpos Monoclonais/química , Modelos Químicos , Química Farmacêutica , Cinética , Desnaturação Proteica , Estabilidade Proteica , TermodinâmicaRESUMO
The thermal unfolding of a recombinant monoclonal antibody IgG1 (mAb) was measured with differential scanning calorimetry (DSC). The DSC thermograms reveal a pretransition at 72°C with an unfolding enthalpy of ΔHcal â¼200-300 kcal/mol and a main transition at 85°C with an enthalpy of â¼900-1000 kcal/mol. In contrast to small single-domain proteins, mAb unfolding is a complex reaction that is analyzed with the multistate Zimm-Bragg theory. For the investigated mAb, unfolding is characterized by a cooperativity parameter σ â¼6 × 10-5 and a Gibbs free energy of unfolding of gnu â¼100 cal/mol per amino acid. The enthalpy of unfolding provides the number of amino acid residues ν participating in the unfolding reaction. On average, νâ¼220 ± 50 amino acids are involved in the pretransition and νâ¼850 ± 30 in the main transition, accounting for â¼90% of all amino acids. Thermal unfolding was further studied in the presence of guanidineHCl. The chemical denaturant reduces the unfolding enthalpy ΔHcal and lowers the midpoint temperature Tm. Both parameters depend linearly on the concentration of denaturant. The guanidineHCl concentrations needed to unfold mAb at 25°C are predicted to be 2-3 M for the pretransition and 5-7 M for the main transition, varying with pH. GuanidineHCl binds to mAb with an exothermic binding enthalpy, which partially compensates the endothermic mAb unfolding enthalpy. The number of guanidineHCl molecules bound upon unfolding is deduced from the DSC thermograms. The bound guanidineHCl-to-unfolded amino acid ratio is 0.79 for the pretransition and 0.55 for the main transition. The pretransition binds more denaturant molecules and is more sensitive to unfolding than the main transition. The current study shows the strength of the Zimm-Bragg theory for the quantitative description of unfolding events of large, therapeutic proteins, such as a monoclonal antibody.
Assuntos
Anticorpos Monoclonais , Imunoglobulina G , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Desnaturação Proteica , Dobramento de Proteína , TermodinâmicaRESUMO
PURPOSE: Predicting thermal protein stability is of major interest in the development of protein-based biopharmaceuticals. Therefore, this study provides a predictive tool for determining transition enthalpies, which can be used for ranking different proteins according to their thermal stability. METHODS: Unfolding and aggregation profiles of eight different therapeutic monoclonal antibodies (mAbs) of type G, isotype 1 were investigated. The unfolding profiles were determined by intrinsic fluorescence (IF) spectroscopy and differential scanning calorimetry (DSC). A three-state unfolding fitting model was used to determine thermodynamic parameters for macromolecular multi-domain mAbs in IF experiments, like the van't Hoff enthalpy change (∆Hvh) and the entropy change (∆S) of the unfolding event. The derived values were compared to thermodynamic parameters obtained directly by calorimetry. Moreover, differences in the Fab enthalpies were used to predict aggregation behavior and protein thermal stabilities. To do so, the liquid-formulated mAbs were investigated exemplarily by size exclusion chromatography (SEC) after accelerated thermal-induced stress conditions. RESULTS: Comparing the thermodynamic parameters derived from IF spectroscopy and DSC resulted in similar values. Data generated by thermal-induced stress at 40°C show similar stability ranking as postulated through the Fab enthalpies for mAbs in two different formulations, while at 25°C a meaningful ranking is not possible, because distinct differences in the thermal stability cannot be observed. The additional consideration of Fab enthalpies to predict the 40 °C SEC ranking seems to be more reliable compared to the use of exclusively the melting temperatures or aggregation onset temperatures and times. CONCLUSION: We show that thermodynamic profiling can help predicting unfolding and aggregation properties of therapeutic mAbs at 40°C. Therefore, analyzing thermodynamic unfolding parameters is a useful and supportive tool discriminating thermal stability profiles of mAbs for further pharmaceutical development and clinical studies.
Assuntos
Anticorpos Monoclonais/química , Modelos Químicos , Agregados Proteicos , Desdobramento de Proteína , Varredura Diferencial de Calorimetria , Conformação Proteica , Dobramento de Proteína , Estabilidade Proteica , Espectrometria de Fluorescência , TermodinâmicaRESUMO
We report the x-ray crystal structure of intact, full-length human immunoglobulin (IgG4) at 1.8 Å resolution. The data for IgG4 (S228P), an antibody targeting the natriuretic peptide receptor A, show a previously unrecognized type of Fab-Fc orientation with a distorted λ-shape in which one Fab-arm is oriented toward the Fc portion. Detailed structural analysis by x-ray crystallography and molecular simulations suggest that this is one of several conformations coexisting in a dynamic equilibrium state. These results were confirmed by small angle x-ray scattering in solution. Furthermore, electron microscopy supported these findings by preserving molecule classes of different conformations. This study fosters our understanding of IgG4 in particular and our appreciation of antibody flexibility in general. Moreover, we give insights into potential biological implications, specifically for the interaction of human anti-natriuretic peptide receptor A IgG4 with the neonatal Fc receptor, Fcγ receptors, and complement-activating C1q by considering conformational flexibility.
Assuntos
Anticorpos/química , Imunoglobulina G/química , Receptores do Fator Natriurético Atrial/imunologia , Animais , Sítios de Ligação , Células CHO , Cricetulus , Cristalização , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Receptores de IgG/químicaRESUMO
Monoclonal antibodies (mAbs) are often needed and applied in high concentration solutions, >100 mg/mL. Due to close intermolecular distances between mAbs at high concentrations (~10-20 nm at 200 mg/mL), intermolecular interactions between mAbs and mAbs and solvent/co-solute molecules become non-negligible. Here, EPR spectroscopy is used to study the high-concentration solutions of mAbs and their effect on co-solvated small molecules, using EPR "spin probing" assay in aqueous and buffered solutions. Such, information regarding the surrounding environments of mAbs at high concentrations were obtained and comparisons between EPR-obtained micro-viscosities (rotational correlation times) and macroscopic viscosities measured by rheology were possible. In comparison with highly viscous systems like glycerol-water mixtures, it was found that up to concentrations of 50 mg/mL, the mAb-spin probe systems have similar trends in their macro- (rheology) and micro-viscosities (EPR), whereas at very high concentrations they deviate strongly. The charged spin probes sense an almost unchanged aqueous solution even at very high concentrations, which in turn indicates the existence of large solvent regions that despite their proximity to large mAbs essentially offer pure water reservoirs for co-solvated charged molecules. In contrast, in buffered solutions, amphiphilic spin probes like TEMPO interact with the mAb network, due to slight charge screening. The application of EPR spectroscopy in the present work has enabled us to observe and discriminate between electrostatic and hydrophobic kinds of interactions and depict the potential underlying mechanisms of network formation at high concentrations of mAbs. These findings could be of importance as well for the development of liquid-liquid phase separations often observed in highly concentrated protein solutions.
Assuntos
Anticorpos Monoclonais/química , Espectroscopia de Ressonância de Spin Eletrônica , Algoritmos , Glicerol/química , Cinética , Modelos Químicos , Solubilidade , Solventes/química , ViscosidadeRESUMO
In cases of subcutaneous injection of therapeutic monoclonal antibodies, high protein concentrations (>50â¯mg/ml) are often required. During the development of these high concentration liquid formulations (HCLF), challenges such as aggregation, gelation, opalescence, phase separation, and high solution viscosities are more prone compared to low concentrated protein formulations. These properties can impair manufacturing processes, as well as protein stability and shelf life. To avoid such unfavourable solution properties, a detailed understanding about the nature of these properties and their driving forces are required. However, the fundamental mechanisms that lead to macroscopic solution properties, as above mentioned, are complex and not fully understood, yet. Established analytical methods for assessing the colloidal stability, i.e. the ability of a native protein to remain dispersed in solution, are restricted to dilute conditions and provide parameters such as the second osmotic virial coefficient, B22, and the diffusion interaction coefficient, kD. These parameters are routinely applied for qualitative estimations and identifications of proteins with challenging solution behaviours, such as high viscosities and aggregation, although the assays are prepared for low protein concentration conditions, typically between 0.1 and 20â¯mg/ml ("ideal" solution conditions). Quantitative analysis of samples of high protein concentration is difficult and it is hard to obtain information about the driving forces of such solution properties and corresponding protein-protein self-interactions. An advantage of using specific spectroscopic methods is the potential of directly analysing highly concentrated protein solutions at different solution conditions. This allows for collecting/gaining valuable information about the fundamental mechanisms of solution properties of the high protein concentration regime. In addition, the derived parameters might be more predictive as compared to the parameters originating from assays which are optimized for the low protein concentration range. The provided information includes structural data, molecular dynamics at various timescales and protein-solvent interactions, which can be obtained at molecular resolution. Herein, we provide an overview about spectroscopic techniques for analysing the origins of macroscopic solution behaviours in general, with a specific focus on pharmaceutically relevant high protein concentration and formulation conditions.
Assuntos
Proteínas/análise , Proteínas/química , Análise Espectral/métodos , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Difusão , SoluçõesRESUMO
N-glycosylation of the Fc part is a (critical) quality attribute of therapeutic antibodies and Fc-containing biotherapeutics, that impacts their stability, immunogenicity, pharmacokinetics, and effector functions. Current glycosylation analysis methods focus on the absolute amounts of glycans, neglecting the apparent glycan distribution over the entirety of proteins. The combination of the two Fc N-glycans, herein referred to as glyco-pair, therefore remains unknown, which is a major drawback for N-glycan impact assessment. This study presents a comprehensive workflow for the analysis and characterization of Fc N-glycan pairing in biotherapeutics, addressing the limitations of current glycosylation analysis methods. The applicability of the method across various biotherapeutic proteins including antibodies, bispecific antibody formats, and a Fc-Fusion protein is demonstrated, and the impact of method conditions on glycan pairing analysis is highlighted. Moreover, the influence of the molecular format, Fc backbone, production process, and cell line on glycan pairing pattern was investigated. The results underscore the significance of comprehensive glycan pairing analysis to accurately assess the impact of N-glycans on important product quality attributes of therapeutic antibodies and Fc-containing biotherapeutics.
Assuntos
Anticorpos , Terapia Biológica , Polissacarídeos , Polissacarídeos/química , Polissacarídeos/metabolismo , Anticorpos/química , Anticorpos/uso terapêutico , Glicosilação , Terapia Biológica/métodos , Fluxo de Trabalho , Glicosídeo Hidrolases/metabolismo , Fucose/químicaRESUMO
Bispecific antibodies (BsAbs) capable of recognizing two distinct epitopes or antigens offer promising therapeutic options for various diseases by targeting multiple pathways. The favorable pharmacokinetic (PK) properties of monoclonal antibodies (mAbs) are crucial, as they directly influence patient safety and therapeutic efficacy. For numerous mAb therapeutics, optimization of neonatal Fc receptor (FcRn) interactions and elimination of unfavorable molecular properties have led to improved PK properties. However, many BsAbs exhibit unfavorable PK, which has precluded their development as drugs. In this report, we present studies on the molecular determinants underlying the distinct PK profiles of three IgG1-scFv BsAbs. Our study indicated that high levels of nonspecific interactions, elevated isoelectric point (pI), and increased number of positively charged patches contributed to the fast clearance of IgG1-scFv. FcRn chromatography results revealed specific scFv-FcRn interactions that are unique to the IgG1-scFv, which was further supported by molecular dynamics (MD) simulation. These interactions likely stabilize the BsAb FcRn interaction at physiological pH, which in turn could disrupt FcRn-mediated BsAb recycling. In addition to the empirical observations, we also evaluated the impact of in silico properties, including pI differential between the Fab and scFv and the ratio of dipole moment to hydrophobic moment (RM) and their correlation with the observed clearance. These findings highlight that the PK properties of BsAbs may be governed by novel determinants, owing to their increased structural complexity compared to immunoglobulin G (IgG) 1 antibodies.
Assuntos
Anticorpos Biespecíficos , Recém-Nascido , Humanos , Anticorpos Monoclonais , Epitopos , Imunoglobulina G , Ponto IsoelétricoRESUMO
Polyclonal autoantibodies against human GM-CSF (granulocyte/macrophage colony-stimulating factor) are a hallmark of PAP (pulmonary alveolar proteinosis) and several other reported autoimmune diseases. MB007 is a high-affinity anti-(human GM-CSF) autoantibody isolated from a patient suffering from PAP which shows only modest neutralization of GM-CSF bioactivity. We describe the first crystal structure of a cytokine-directed human IgG1λ autoantibody-binding fragment (Fab) at 1.9 Å (1 Å=0.1 nm) resolution. Its CDR3-H substantially differs from all VH7 germline IgG1 structures reported previously. We derive a reliable model of the antigen-autoantibody complex by using NMR chemical shift perturbation data in combination with computational methods. Superposition of the modelled complex structure with the human GM-CSF-GM-CSF ternary receptor complex reveals only little overlap between receptor and Fab when bound to GM-CSF. Our model provides a structural basis for understanding the mode of action of the MB007 autoantibody.
Assuntos
Autoanticorpos/química , Autoanticorpos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Imunoglobulina G/química , Proteinose Alveolar Pulmonar/imunologia , Complexo Antígeno-Anticorpo/química , Autoanticorpos/uso terapêutico , Sítios de Ligação de Anticorpos/efeitos dos fármacos , Cristalização , Mapeamento de Epitopos , Humanos , Fragmentos Fab das Imunoglobulinas/química , Modelos MolecularesRESUMO
Sub-visible particles (SVPs) in pharmaceutical products are a critical quality attribute, and therefore should be monitored during development. Although light obscuration (LO) and microscopic particle count tests are the primary pharmacopeial methods used to quantify SVPs, flow imaging methods like Micro-Flow Imaging (MFI™) appear to overcome shortcomings of LO such as limited sensitivity concerning smaller translucent SVPs in the size range < 10 µm. Nowadays, MFI™ is routinely utilized during development of biologicals. Oftentimes multiple devices are distributed across several laboratories and departments. This poses challenges in data interpretation and consistency as well as in the use of multiple devices for one purpose. In this study, we systematically evaluated seven MFI™ instruments concerning their counting and size precision and accuracy, using an inter-comparable approach to mimic daily working routine. Therefore, we investigated three different types of particles (i) NIST certified counting standards, (ii) protein-coated particles, and (iii) stress-induced particles from a monoclonal antibody. We compared the results to alternative particle detection methods: LO and Backgrounded Membrane Imaging (BMI). Our results showed that the precision and accuracy of particle count and size, as well as the comparability of instruments, depended on the particle source and its material properties. The various MFI™ instruments investigated showed high precision (<15 %) and data generated on different instruments were of the same order of magnitude within pharmacopeial relevant size ranges for NIST certified counting standards. However, we found limitations in the upper and lower detection limits, contrary to the limits claimed by the manufacturer. In addition, proteinaceous and protein-containing particles showed statistically significant differences in particle counts, while the measured particle diameters of all sizes were quite consistent.
Assuntos
Anticorpos Monoclonais , Produtos Biológicos , Tamanho da PartículaRESUMO
Visible light (400-800 nm) can lead to photooxidation of protein formulations, which might impair protein integrity. However, the relevant mechanism of photooxidation upon visible light exposure is still unclear for therapeutic proteins, since proteinogenic structures do not absorb light in the visible range. Here, we show that exposure of monoclonal antibody formulations to visible light, lead to the formation of reactive oxygen species (ROS), which subsequently induce specific protein degradations. The formation of ROS and singlet oxygen upon visible light exposure is investigated using electron paramagnetic resonance (EPR) spectroscopy. We describe the initial formation of ROS, most likely after direct reaction of molecular oxygen with a triplet state photosensitizer, generated from intersystem crossing of the excited singlet state. Since these radicals affect the oxygen content in the headspace of the vial, we monitored photooxidation of these mAb formulations. With increasing protein concentrations, we found (i) a decreasing headspace oxygen content in the sample, (ii) a higher relative number of radicals in solution and (iii) a higher protein degradation. Thus, the protein concentration dependence indicates the presence of higher concentration of a currently unknown photosensitizer.
RESUMO
Biological drugs intended for multi-dose application require the presence of antimicrobial preservatives to avoid microbial growth. As the presence of certain preservatives has been reported to increase protein and peptide particle formation, it is essential to choose a preservative compatible with the active pharmaceutical ingredient in addition to its preservation function. Thus, this review describes the current status of the use of antimicrobial preservatives in biologic formulations considering (i) appropriate preservatives for protein and peptide formulations, (ii) their physico-chemical properties, (iii) their in-/compatibilities with other excipients or packaging material, and (iv) their interactions with the biological compound. Further, (v) we present an overview of licensed protein and peptide formulations.
RESUMO
UV and ambient light-induced modifications and related degradation of therapeutic proteins are observed during manufacturing and storage. Therefore, to ensure product quality, protein formulations need to be analyzed with respect to photo-degradation processes and eventually protected from light exposure. This task usually demands the application and combination of various analytical methods. This review addresses analytical aspects of investigating photo-oxidation products and related mediators such as reactive oxygen species generated via UV and ambient light with well-established and novel techniques.
RESUMO
The present study investigated the photodegradation of three different monoclonal antibodies (mAb) by visible light. Several chromatographic techniques, such as size-exclusion and hydrophobic interaction chromatography as well as mass spectrometry were used to measure relative changes of various oxidation related monoclonal antibody species. The results show that visible light is indeed capable of inducing the formation of protein photo-oxidation products, such as acidic, basic, hydrophilic, and several other protein species with altered physicochemical properties. Although, the formation rate of degradants of these three protein species was dependent on the light source's intensity (I), their yield is clearly correlated to the applied light dosage (ld), which is defined as the product of light intensity I and irradiation time t (light dosage = I·t). Hence, our findings indicate that the degradation of monoclonal antibodies can be described according to the Bunsen-Roscoe reciprocity law. This correlation can be useful to assess the impact of photodegradation of biologics with regards to changes in light intensity and/or duration of light exposure of the protein, e.g. during the manufacturing of biologics.
Assuntos
Anticorpos Monoclonais , Anticorpos Monoclonais/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas , Oxirredução , FotóliseRESUMO
The paralogous endoribonucleases, RNase E and RNase G, play major roles in intracellular RNA metabolism in Escherichia coli and related organisms. To assay the relative importance of the principal RNA binding sites identified by crystallographic analysis, we introduced mutations into the 5'-sensor, the S1 domain, and the Mg(+2)/Mn(+2) binding sites. The effect of such mutations has been measured by assays of activity on several substrates as well as by an assay of RNA binding. RNase E R169Q and the equivalent mutation in RNase G (R171Q) exhibit the strongest reductions in both activity (the k(cat) decrease approximately 40- to 100-fold) and RNA binding consistent with a key role for the 5'-sensor. Our analysis also supports a model in which the binding of substrate results in an increase in catalytic efficiency. Although the phosphate sensor plays a key role in vitro, it is unexpectedly dispensable in vivo. A strain expressing only RNase E R169Q as the sole source of RNase E activity is viable, exhibits a modest reduction in doubling time and colony size, and accumulates immature 5 S rRNA. Our results point to the importance of alternative RNA binding sites in RNase E and to alternative pathways of RNA recognition.
Assuntos
Endorribonucleases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , RNA Bacteriano/genética , RNA Ribossômico 5S/genética , Sítios de Ligação , Northern Blotting , Domínio Catalítico , Endorribonucleases/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Modelos Moleculares , Mutação/genética , Fosfatos/metabolismo , Conformação Proteica , RNA Bacteriano/metabolismo , RNA Ribossômico 5S/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Polysorbate is one of the most commonly employed non-ionic surfactant in protein containing biological formulations, whereby, it can stabilize these biomolecules under different stress conditions. Despite the fact that polysorbates are present in almost 70% of currently marketed parenteral biological drugs, polysorbate degradation in biopharmaceutical formulations has emerged as a specific quality concern. Different degradation pathways have been explored in the recent years with the aim of understanding the root cause for polysorbate degradation in biopharmaceutical formulations. In an attempt to explore hydrolytic degradation of polysorbates in accelerated degradation conditions, we studied extreme pH conditions. We investigated specific polysorbate degradation profiles depending on acidic or alkaline solution conditions. The acidic and alkaline hydrolysis of polysorbate is monitored for the total content using a fluorescence micelle assay (FMA). Additionally, the compositional changes in polysorbates were detected using reversed phase high performance liquid chromatography coupled to a charged aerosol detector (RP-HPLC-CAD). We show that the stability of polysorbate against chemical hydrolysis is dependent upon selected pH condition and differ for polysorbate 20 and polysorbate 80. Additionally, we were able to show that a degradation pathway dependent fingerprint may support the identification of the degradation root cause.
Assuntos
Química Farmacêutica/métodos , Polissorbatos/química , Aerossóis , Produtos Biológicos/química , Cromatografia Líquida de Alta Pressão , Composição de Medicamentos/métodos , Concentração de Íons de Hidrogênio , Hidrólise , Espectrometria de Massas , Micelas , Tensoativos , ÁguaRESUMO
Detection and characterization of particles in the visible and subvisible size range is critical in many fields of industrial research. Commercial particle analysis systems have proliferated over the last decade. Despite that growth, most systems continue to be based on well-established principles, and only a handful of new approaches have emerged. Identifying the right particle-analysis approach remains a challenge in research and development. The choice depends on each individual application, the sample, and the information the operator needs to obtain. In biopharmaceutical applications, particle analysis decisions must take product safety, product quality, and regulatory requirements into account. Biopharmaceutical process samples and formulations are dynamic, polydisperse, and very susceptible to chemical and physical degradation: improperly handled product can degrade, becoming inactive or in specific cases immunogenic. This article reviews current methods for detecting, analyzing, and characterizing particles in the biopharmaceutical context. The first part of our article represents an overview about current particle detection and characterization principles, which are in part the base of the emerging techniques. It is very important to understand the measuring principle, in order to be adequately able to judge the outcome of the used assay. Typical principles used in all application fields, including particle-light interactions, the Coulter principle, suspended microchannel resonators, sedimentation processes, and further separation principles, are summarized to illustrate their potentials and limitations considering the investigated samples. In the second part, we describe potential technical approaches for biopharmaceutical particle analysis as some promising techniques, such as nanoparticle tracking analysis (NTA), micro flow imaging (MFI), tunable resistive pulse sensing (TRPS), flow cytometry, and the space- and time-resolved extinction profile (STEP®) technology.