RESUMO
PURPOSE: Reversible self-association (RSA) has long been a concern in therapeutic monoclonal antibody (mAb) development. Because RSA typically occurs at high mAb concentrations, accurate assessment of the underlying interaction parameters requires explicitly addressing hydrodynamic and thermodynamic nonideality. We previously examined the thermodynamics of RSA for two mAbs, C and E, in phosphate buffered saline (PBS). Here we continue to explore the mechanistic aspects of RSA by examining the thermodynamics of both mAbs under reduced pH and salt conditions. METHODS: Dynamic light scattering and sedimentation velocity (SV) studies were conducted for both mAbs at multiple protein concentrations and temperatures, with the SV data analyzed via global fitting to determine best-fit models, interaction energetics, and nonideality contributions. RESULTS: We find that mAb C self-associates isodesmically irrespective of temperature, and that association is enthalpically driven but entropically penalized. Conversely, mAb E self-associates cooperatively and via a monomer-dimer-tetramer-hexamer reaction pathway. Moreover, all mAb E reactions are entropically driven and enthalpically modest or minimal. CONCLUSIONS: The thermodynamics for mAb C self-association are classically seen as originating from van der Waals interactions and hydrogen bonding. However, relative to the energetics we determined in PBS, self-association must also be linked to proton release and/or ion uptake events. For mAb E, the thermodynamics implicate electrostatic interactions. Furthermore, self-association is instead linked to proton uptake and/or ion release, and primarily by tetramers and hexamers. Finally, although the origins of mAb E cooperativity remain unclear, ring formation remains a possibility whereas linear polymerization reactions can be eliminated.
Assuntos
Anticorpos Monoclonais , Prótons , Anticorpos Monoclonais/metabolismo , Cloreto de Sódio , Difusão Dinâmica da Luz , TermodinâmicaRESUMO
PURPOSE: Reversible self-association (RSA) remains a challenge in the development of therapeutic monoclonal antibodies (mAbs). We recently analyzed the energetics of RSA for five IgG mAbs (designated as A-E) under matched conditions and using orthogonal methods. Here we examine the thermodynamics of RSA for two of the mAbs that showed the strongest evidence of RSA (mAbs C and E) to identify underlying mechanisms. METHODS: Concentration-dependent dynamic light scattering and sedimentation velocity (SV) studies were carried out for each mAb over a range of temperatures. Because self-association was weak, the SV data were globally analyzed via direct boundary fitting to identify best-fit models, accurately determine interaction energetics, and account for the confounding effects of thermodynamic and hydrodynamic nonideality. RESULTS: mAb C undergoes isodesmic self-association at all temperatures examined, with the energetics indicative of an enthalpically-driven reaction offset by a significant entropic penalty. By contrast, mAb E undergoes monomer-dimer self-association, with the reaction being entropically-driven and comprised of only a small enthalpic contribution. CONCLUSIONS: Classical interpretations implicate van der Waals interactions and H-bond formation for mAb C RSA, and electrostatic interactions for mAb E. However, noting that RSA is likely coupled to additional equilibria, we also discuss the limitations of such interpretations.
Assuntos
Anticorpos Monoclonais/química , Termodinâmica , Anticorpos Monoclonais/uso terapêutico , Química Farmacêutica , Difusão Dinâmica da Luz , Multimerização Proteica , Eletricidade EstáticaRESUMO
To combat the COVID-19 pandemic, potential therapies have been developed and moved into clinical trials at an unprecedented pace. Some of the most promising therapies are neutralizing antibodies against SARS-CoV-2. In order to maximize the therapeutic effectiveness of such neutralizing antibodies, Fc engineering to modulate effector functions and to extend half-life is desirable. However, it is critical that Fc engineering does not negatively impact the developability properties of the antibodies, as these properties play a key role in ensuring rapid development, successful manufacturing, and improved overall chances of clinical success. In this study, we describe the biophysical characterization of a panel of Fc engineered ("TM-YTE") SARS-CoV-2 neutralizing antibodies, the same Fc modifications as those found in AstraZeneca's Evusheld (AZD7442; tixagevimab and cilgavimab), in which the TM modification (L234F/L235E/P331S) reduce binding to FcγR and C1q and the YTE modification (M252Y/S254T/T256E) extends serum half-life. We have previously shown that combining both the TM and YTE Fc modifications can reduce the thermal stability of the CH2 domain and possibly lead to developability challenges. Here we show, using a diverse panel of TM-YTE SARS-CoV-2 neutralizing antibodies, that despite lowering the thermal stability of the Fc CH2 domain, the TM-YTE platform does not have any inherent developability liabilities and shows an in vivo pharmacokinetic profile in human FcRn transgenic mice similar to the well-characterized YTE platform. The TM-YTE is therefore a developable, effector function reduced, half-life extended antibody platform.
Assuntos
COVID-19 , SARS-CoV-2 , Animais , Camundongos , Humanos , SARS-CoV-2/genética , Pandemias , Anticorpos NeutralizantesRESUMO
Electrostatically driven attractions between proteins can result in issues for therapeutic protein formulations such as solubility limits, aggregation, and high solution viscosity. Previous work showed that a model monoclonal antibody displayed large and potentially problematic electrostatically driven attractions at typical pH (5-8) and ionic strength conditions (â¼10-100 mM). Molecular simulations of a hybrid coarse-grained model (1bC/D, one bead per charged site and per domain) were used to predict potential point mutations to identify key charge changes (charge-to-neutral or charge-swap) that could greatly reduce the net attractive protein-protein self-interactions. A series of variants were tested experimentally with static and dynamic light scattering to quantify interactions and compared to model predictions at low and intermediate ionic strength. Differential scanning calorimetry and circular dichroism confirmed minimal impact on structural or thermal stability of the variants. The model provided quantitative/semiquantitative predictions of protein self-interactions compared to experimental results as well as showed which amino acid pairings or groups had the most impact.
Assuntos
Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Células HEK293 , Humanos , Modelos Moleculares , Mutação Puntual , Ligação Proteica , Eletricidade EstáticaRESUMO
Weak macromolecular interactions assume a dominant role in the behavior of highly concentrated solutions, and are at the center of a variety of fields ranging from colloidal chemistry to cell biology, neurodegenerative diseases, and manufacturing of protein drugs. They are frequently measured in different biophysical techniques in the form of second virial coefficients, and nonideality coefficients of sedimentation and diffusion, which may be related mechanistically to macromolecular distance distributions in solution and interparticle potentials. A problem arises for proteins where reversible self-association often complicates the concentration-dependent behavior, such that grossly inconsistent coefficients are measured in experiments based on different techniques, confounding quantitative conclusions. Here we present a global multi-method analysis that synergistically bridges gaps in resolution and sensitivity of orthogonal techniques. We demonstrate the method with a panel of monoclonal antibodies exhibiting different degrees of self-association. We show how their concentration-dependent behavior, examined by static and dynamic light scattering and sedimentation velocity, can be jointly described in a self-consistent framework that separates nonideality coefficients from self-association properties, and thereby extends the quantitative interpretation of nonideality coefficients to probe dynamics in highly concentrated protein solutions.
Assuntos
Substâncias Macromoleculares/química , Algoritmos , Anticorpos Monoclonais/química , Difusão Dinâmica da Luz , Hidrodinâmica , Temperatura , UltracentrifugaçãoRESUMO
Asparagine (Asn) deamidation is a common posttranslational modification in which Asn is converted to aspartic acid or isoaspartic acid. By introducing a negative charge, deamidation could potentially impact the binding interface and biological activities of protein therapeutics. We identified a deamidation variant in moxetumomab pasudotox, an immunotoxin Fv fusion protein drug derived from a 38-kDa truncated Pseudomonas exotoxin A (PE38) for the treatment of hairy-cell leukemia. Although the deamidation site, Asn-358, was outside of the binding interface, the modification had a significant impact on the biological activity of moxetumomab pasudotox. Surprisingly, the variant eluted earlier than its unmodified form on anion exchange chromatography, which often leads to the conclusion that it has a higher positive charge. Here we describe the characterization of the deamidation variant with differential scanning calorimetry and hydrogen-deuterium exchange mass spectrometry, which revealed that the Asn-358 deamidation caused the conformational changes in the catalytic domain of the PE38 region. These results provide an explanation for why the deamidation affected the biological activity of moxetumomab pasudotox and suggest the approach that can be used for process control to ensure product quality and process consistency.
Assuntos
Imunotoxinas , Leucemia de Células Pilosas , Asparagina , Toxinas Bacterianas , Exotoxinas , HumanosRESUMO
Monoclonal antibodies are a class of biotherapeutics used for an increasing variety of disorders, including cancer, autoimmune, neurodegenerative, and viral diseases. Besides their antigen specificity, therapeutic use also mandates control of their solution interactions and colloidal properties in order to achieve a stable, efficacious, non-immunogenic, and low viscosity antibody solution at concentrations in the range of 50-150 mg/mL. This requires characterization of their reversible self-association, aggregation, and weak attractive and repulsive interactions governing macromolecular distance distributions in solution. Simultaneous measurement of these properties, however, has been hampered by solution nonideality. Based on a recently introduced sedimentation velocity method for measuring macromolecular size distributions in a mean-field approximation for hydrodynamic interactions, we demonstrate simultaneous measurement of polydispersity and weak and strong solution interactions in a panel of antibodies with concentrations up to 45 mg/mL. By allowing approximately an order of magnitude higher concentrations than previously possible in sedimentation velocity size distribution analysis, this approach can substantially improve efficiency and sensitivity for characterizing polydispersity and interactions of therapeutic antibodies at or close to formulation conditions.
Assuntos
Anticorpos Monoclonais/química , Agregados Proteicos , Hidrodinâmica , Concentração de Íons de Hidrogênio , Ultracentrifugação , ViscosidadeRESUMO
The post-fusion form of Respiratory Syncytial Virus (RSV) fusion (F) protein has been used recently in clinical trials as a potential vaccine antigen with the objective of eliciting protective immune response against RSV. In this paper, in vitro antigenicity and in vivo immunogenicity of recombinant, soluble F protein of RSV (RSVsF) were evaluated by several assays. In Vitro Relative Potency (IVRP) of RSVsF was measured in a sandwich ELISA using two antibodies, each specific for epitope site A or C. Therefore, IVRP reflected the integrity of the antigen in terms of changes in antibody binding affinity of either or both of these sites. RSVsF samples with a wide range of IVRP values were generated by applying UV irradiation (photo) and high temperature (heat) induced stress for varying lengths of time. These samples were characterized in terms of stress induced modifications in primary and secondary structures as well as aggregation of RSVsF. Immunogenicity, also referred to as In vivo potency, was measured by induction of total F-protein specific IgG and RSV-neutralizing antibodies in mice dosed with these RSVsF samples. Comparison of results between IVRP and these immunogenicity assays revealed that IVRP provided a sensitive read-out of the integrity of epitope sites A and C, and a conservative and reliable evaluation of the potency of RSVsF as a vaccine antigen. This high throughput and fast turn-around assay allowed us to efficiently screen many different RSVsF antigen lots, thereby acting as an effective filter for ensuring high quality antigen that delivered in vivo potency. In vitro and in vivo potencies were further probed at the level of individual epitope sites, A and C. Results of these experiments indicated that site A was relatively resistant to stress induced loss of potency, in vitro or in vivo, compared to site C.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Imunogenicidade da Vacina , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vírus Sincicial Respiratório Humano/imunologia , Proteínas Virais de Fusão/imunologia , Animais , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Humanos , Imunoglobulina G/imunologia , Camundongos , Testes de Neutralização , Estresse Fisiológico , Vacinas/imunologiaRESUMO
Monoclonal antibodies (mAbs) represent a major class of biotherapeutics and are the fastest growing category of biologic drugs on the market. However, mAb development and formulation are often impeded by reversible self-association (RSA), defined as the dynamic exchange of monomers with native-state oligomers. Here, we present a comparative analysis of the self-association properties for 5 IgG mAbs, under matched conditions and using orthogonal methods. Concentration-dependent dynamic light scattering and sedimentation velocity studies revealed that the majority of mAbs examined exhibited weak to moderate RSA. However, because these studies were carried out at mAb concentrations in the mg/mL range, we also observed significant nonideality. Noting that nonideality frequently masks RSA and vice versa, we conducted direct boundary fitting of the sedimentation velocity data to determine stoichiometric binding models, interaction affinities, and nonideality terms for each mAb. These analyses revealed equilibrium constants from micromolar to millimolar and stoichiometric models from monomer-dimer to isodesmic. Moreover, even for those mAbs described by identical models, we observed distinct kinetics of self-association. The accuracy of the models and their corresponding equilibrium constants were addressed using sedimentation equilibrium and simulations. Overall, these results serve as the starting point for the comparative dissection of RSA mechanisms in therapeutic mAbs.
Assuntos
Anticorpos Monoclonais/química , Imunoglobulina G/química , Algoritmos , Difusão Dinâmica da Luz , Humanos , Multimerização Proteica , Termodinâmica , UltracentrifugaçãoRESUMO
Antigenic drift of the influenza A virus requires that vaccine production is targeted to the strains circulating each year. Live-attenuated influenza A vaccine manufacturing is used to produce intact virions with the surface antigens of the circulating strains. Influenza A typically contains a large percentage (>90%) of non-infective virions. The ribonucleoprotein (RNP) content, virion structure, and aggregation are factors that are thought to have an impact on infectivity. However, these factors are difficult to study because of the intrinsic variability in virion size, shape and overall structural integrity. Negative stain TEM for total particle counts and cryoTEM for detailed size/structural analysis are established benchmark techniques for virus characterization. Other methods may be valuable for certain sample types or circumstances. The aim of this work is to establish a benchmark comparison between orthogonal biophysical techniques for particle counts, population size distribution, structural integrity, and aggregate levels. NTA and FFF-MALS rapidly provided total counts, size distribution, and aggregate/elongated virion content. CryoTEM with size analysis and fraction counting yielded detailed information about the pleomorphism of the sample. The structural integrity of virions was inferred from multi-signal AUC-SV and CryoTEM. The current work provides a comparative assessment and a baseline for the selection of biophysical tools for the determination of particle counts, aggregation and pleomorphic characteristics of influenza A virus.
Assuntos
Fenômenos Biofísicos , Vírus da Influenza A/fisiologia , Vírus da Influenza A/ultraestrutura , Carga Viral/métodos , Vírion/fisiologia , Vírion/ultraestrutura , Virologia/métodos , Microscopia Crioeletrônica/métodos , Difusão Dinâmica da Luz/métodos , Ultracentrifugação/métodosRESUMO
This study explores the structural and functional changes associated with a low-temperature thermal transition of 2 engineered bacterial uricase mutants. Uricase has a noncovalent homotetrameric structure, with 4 active sites located at the interface of subunits. Using differential scanning calorimetry, a low-temperature transition was identified at 42°C for mutant A and at 33°C for mutant B. This transition was stabilized by the uricase inhibitor, oxonic acid, suggesting a strong structural relationship to the active site. For mutant B, there was a reversible loss of enzymatic activity above the low-temperature transition. Spectroscopic measurements demonstrated that there was also a reversible loss of secondary and tertiary structures and an increase in surface hydrophobicity. However, the hydrophobic core environment and the tetrameric structure were not altered over the low-temperature transition suggesting that the changes occurred primarily at the surface of the enzyme. The protein became aggregation-prone at temperatures approaching the cluster of higher-temperature melting transitions at 84°C, indicating these transitions represent a global unfolding of the protein. Our findings shed light on the structural changes that affect the uricase mechanism of action and provide new insights into how enzyme therapeutic development may be approached.
Assuntos
Arthrobacter/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Mutação/genética , Urato Oxidase/química , Urato Oxidase/genética , Arthrobacter/enzimologia , Proteínas de Bactérias/metabolismo , Ativação Enzimática/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Urato Oxidase/metabolismoRESUMO
The role of antibody structure (conformation) in solution rheology is probed. It is demonstrated here that pH-dependent changes in the tertiary structure of 2 mAb solutions lead to viscoelasticity and not merely a shear viscosity (η) increase. Steady shear flow curves on mAb solutions are reported over broad pH (3.0 ≤ pH ≤ 8.7) and concentration (2 mg/mL ≤ c ≤ 120 mg/mL) ranges to comprehensively characterize their rheology. Results are interpreted using size exclusion chromatography, differential scanning calorimetry, analytical ultracentrifugation, near-UV circular dichroism, and dynamic light scattering. Changes in tertiary structure with concentration lead to elastic yield stress and increased solution viscosity in solution of "mAb1." These findings are supported by dynamic light scattering and differential scanning calorimetry, which show increased hydrodynamic radius of mAb1 at low pH and a reduced melting temperature Tm, respectively. Conversely, another molecule at 120 mg/mL solution concentration is a strong viscoelastic gel due to perturbed tertiary structure (seen in circular dichroism) at pH 3.0, but the same molecule responds as a viscous liquid due to reversible self-association at pH 7.4 (verified by analytical ultracentrifugation). Both protein-protein interactions and structural perturbations govern pH-dependent viscoelasticity of mAb solutions.
Assuntos
Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Elasticidade , Animais , Células CHO , Varredura Diferencial de Calorimetria/métodos , Cromatografia em Gel/métodos , Cricetinae , Cricetulus , Soluções Farmacêuticas/análise , Soluções Farmacêuticas/química , Domínios e Motivos de Interação entre Proteínas , Reologia/métodos , ViscosidadeRESUMO
UNLABELLED: Nanoparticle Tracking Analysis (NTA) is an emerging analytical technique developed for detection, sizing, and counting of sub-micron particles in liquid media. Its feasibility for use in biopharmaceutical development was evaluated with particle standards and recombinant protein solutions. Measurements of aqueous suspensions of NIST-traceable polystyrene particle standards showed accurate particle concentration detection between 2 × 10(7) and 5 × 10(9) particles/mL. Sizing was accurate for particle standards up to 200 nm. Smaller than nominal value sizes were detected by NTA for the 300-900 nm particles. Measurements of protein solutions showed that NTA performance is solution-specific. Reduced sensitivity, especially in opalescent solutions, was observed. Measurements in such solutions may require sample dilution; however, common sample manipulations, such as dilution and filtration, may result in particle formation. Dilution and filtration case studies are presented to further illustrate such behavior. To benchmark general performance, NTA was compared against asymmetric flow field flow fractionation coupled with multi-angle light scattering (aF4-MALS) and dynamic light scattering, which are other techniques for sub-micron particles. Data shows that all three methods have limitations and may not work equally well under certain conditions. Nevertheless, the ability of NTA to directly detect and count sub-micron particles is a feature not matched by aF4-MALS or dynamic light scattering. LAY ABSTRACT: Thorough characterization of particulate matter present in protein therapeutics is limited by the lack of analytical methods for particles in the sub-micron size range. Emerging techniques are being developed to bridge this analytical gap. In this study, Nanoparticle Tracking Analysis is evaluated as a potential tool for biologics development. Our results indicate that method performance is molecule-specific and may not work as well under all solution conditions, especially when testing opalescent solutions. Advantages and disadvantages of Nanoparticle Tracking Analysis are discussed in comparison to other analytical techniques for particles in the sub-micron size range.
Assuntos
Produtos Biológicos/química , Nanopartículas , Proteínas Recombinantes/química , Tecnologia Farmacêutica/métodos , Desenho de Fármacos , Difusão Dinâmica da Luz , Fracionamento por Campo e Fluxo , Tamanho da PartículaRESUMO
Reversible self-association of protein therapeutics, the phenomenon of formation of native reversible oligomeric species as a result of noncovalent intermolecular interactions, can add additional manufacturing, stability, delivery, and safety complexities in biopharmaceutical development. Its early detection, characterization, and mitigation can therefore contribute to the success of drug development. A variety of structural and environmental factors can contribute to the modulation of self-association with mechanisms still elusive in some cases due to the inherent structural complexity of proteins. By combining the capabilities of dynamic and static light scattering techniques, the modulatory effects of a variety of solution conditions on a model IgG1's (mAbA) intermolecular interactions have been utilized to derive mechanism of its self-association at relatively low-protein concentration. The analysis of the effect of pH, buffer type, Hofmeister salts, and aromatic amino acids utilizing light scattering supported a combined role of hydrophobic and electrostatic interactions in mAbA self-association. Fitting of the data into the equilibrium models obtained from the multiangle static light scattering provided the enthalpic and entropic contributions of self-association, highlighting the more dominant effect of electrostatic interactions. In addition, studies of the Fab and Fc fragments of mAbA suggested the key role of the former in observed self-association.
Assuntos
Anticorpos Monoclonais/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Espalhamento de Radiação , Eletricidade Estática , Citratos/metabolismo , Glucose/metabolismo , Histidina/metabolismo , Imunoglobulina G/metabolismo , Ligação Proteica/fisiologiaRESUMO
Some monoclonal antibodies (mAbs) are reported to display concentration-dependent reversible self-association (RSA). There are multiple studies that investigate the effect of RSA on product characteristics such as viscosity, opalescence, phase separation and aggregation. This work investigates the effects of RSA on a bind-and-elute mode cation exchange chromatography (CEX) unit operation. We report a case study in which the RSA of an IgG2 (mAb X) resulted in significant peak splitting during salt gradient elution in CEX. Multiple factors including resin type, load challenge, residence time and gradient slope were evaluated and demonstrated little effect on the peak splitting of mAb X. It was determined that high NaCl concentrations in combination with high protein concentrations induced mAb X to form one RSA species that binds more strongly to the column, resulting in a large second elution peak. The finding of NaCl-induced RSA suggested that lower NaCl elution concentrations and different types of salts could mitigate RSA and thus eliminate peak splitting. Different salts were tested, showing that chaotropic salts such as CaCl2 reduced the second elution peak by inducing less RSA. The addition of a positively charged amino acid (such as 50mM histidine) into the CEX elution buffer resulted in elution at lower NaCl concentrations and also effectively reduced peak splitting. However, experiments that were intended to reduce salt concentration by increasing the elution buffer pH did not significantly mitigate peak splitting. This is because higher pH conditions also increase RSA. This work identifies salt-induced RSA as the cause of peak splitting of a mAb in CEX and also provides solutions to reduce the phenomenon.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Cromatografia por Troca Iônica/métodos , Imunoglobulina G/isolamento & purificação , Cloreto de Sódio/química , Anticorpos Monoclonais/química , Cátions/química , Humanos , Imunoglobulina G/química , Multimerização Proteica , Soluções/químicaRESUMO
In addition to controlling typical instabilities such as physical and chemical degradations, understanding monoclonal antibodies' (mAbs) solution behavior is a key step in designing and developing process and formulation controls during their development. Reversible self-association (RSA), a unique solution property in which native, reversible oligomeric species are formed as a result of the noncovalent intermolecular interactions has been recognized as a developability risk with the potential to negatively impact manufacturing, storage stability, and delivery of mAbs. Therefore, its identification, characterization, and mitigation are key requirements during formulation development. Considering the large number of available analytical methods, choice of the employed technique is an important contributing factor for successful investigation of RSA. Herein, a multitechnique (dynamic light scattering, multiangle static light scattering, and analytical ultracentrifugation) approach is employed to comprehensively characterize the self-association of a model immunoglobulin G1 molecule. Studies herein discuss an effective approach for detection and characterization of RSA during biopharmaceutical development based on the capabilities of each technique, their complementarity, and more importantly their suitability for the stage of development in which RSA is investigated.
Assuntos
Anticorpos Monoclonais/química , Imunoglobulina G/química , Química Farmacêutica/métodos , Humanos , Luz , Espalhamento de Radiação , Soluções , Ultracentrifugação/métodosRESUMO
In addition to controlling typical instabilities such as physical and chemical degradations, understanding monoclonal antibodies' (mAbs) solution behavior is a key step in designing and developing process and formulation controls during their development. Reversible self-association (RSA), a unique solution property in which native, reversible oligomeric species are formed as a result of the noncovalent intermolecular interactions has been recognized as a developability risk with the potential to negatively impact manufacturing, storage stability, and delivery of mAbs. Therefore, its identification, characterization, and mitigation are key requirements during formulation development. Considering the large number of available analytical methods, choice of the employed technique is an important contributing factor for successful investigation of RSA. Herein, a multitechnique (dynamic light scattering, multiangle static light scattering, and analytical ultracentrifugation) approach is employed to comprehensively characterize the self-association of a model immunoglobulin G1 molecule. Studies herein discuss an effective approach for detection and characterization of RSA during biopharmaceutical development based on the capabilities of each technique, their complementarity, and more importantly their suitability for the stage of development in which RSA is investigated.
Assuntos
Anticorpos Monoclonais/química , Imunoglobulina G/química , Tecnologia Farmacêutica , Anticorpos Monoclonais/uso terapêutico , Química Farmacêutica , Estabilidade de Medicamentos , Ensaios de Triagem em Larga Escala , Imunoglobulina G/uso terapêutico , Luz , Conformação Proteica , Desnaturação Proteica , Estabilidade Proteica , Espalhamento de Radiação , Tecnologia Farmacêutica/métodos , UltracentrifugaçãoRESUMO
PURPOSE: Demonstrate the ability of an artificial neural network (ANN), trained on a formulation screen of measured second virial coefficients to predict protein self-interactions for untested formulation conditions. MATERIALS AND METHODS: Protein self-interactions, quantified by the second virial coefficient, B22, were measured by self-interaction chromatography (SIC). The B22 values of lysozyme were measured for an incomplete factorial distribution of 81 formulation conditions of the screen components. The influence of screen parameters (pH, salt and additives) on B22 value was modeled by training an ANN using B22 value measurements. After training, the ANN was asked to predict the B22 value for the complete factorial of parameters screened (12,636 conditions). Twenty of these predicted values (distributed throughout the range of predictions) were experimentally measured for comparison. RESULTS: The ANN was able to predict lysozyme B22 values with a significance of p<0.0001 and RMSE of 2.6x10(-4) mol ml/g2. CONCLUSIONS: The results indicate that an ANN trained on measured B22 values for a small set of formulation conditions can accurately predict B22 values for untested formulation conditions. As a measure of protein-protein interactions correlated with solubility, B22 value predictions based on a small screen may enable rapid determination of high solubility formulations.