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1.
J Pharm Sci ; 113(2): 314-331, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-37944666

RESUMO

Therapeutic biology encompasses different modalities, and their manufacturing processes may be vastly different. However, there are many similarities that run across the different modalities during the drug product (DP) development process and manufacturing. Similarities include the need for Quality Target Product Profile (QTTP), analytical development, formulation development, container/closure studies, drug product process development, manufacturing and technical requirements set out by numerous regulatory documents such as the FDA, EMA, and ICH for pharmaceuticals for human use and other country specific requirements. While there is a plethora of knowledge on studies needed for development of a drug product, there is no specific guidance set out in a phase dependent manner delineating what studies should be completed in alignment with the different phases of clinical development from pre-clinical through commercialization. Because of this reason, we assembled a high-level drug product development and manufacturing roadmap. The roadmap is applicable across the different modalities with the intention of providing a unified framework from early phase development to commercialization of biologic drug products.


Assuntos
Produtos Biológicos , Humanos , Desenvolvimento de Medicamentos , Indústria Farmacêutica
2.
J Pharm Sci ; 113(2): 366-376, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-38042344

RESUMO

Aflibercept is a recombinant fusion protein that is commercially available for several ocular diseases impacting millions of people worldwide. Here, we use a case study approach to examine alternative liquid formulations for aflibercept for ocular delivery, utilizing different stabilizers, buffering agents, and surfactants with the goal of improving the thermostability to allow for limited storage outside the cold chain. The formulations were developed by studying the effects of pH changes, substituting amino acids for sucrose and salt, and using polysorbate 80 or poloxamer 188 instead of polysorbate 20. A formulation containing acetate, proline, and poloxamer 188 had lower rates of aggregate formation at 4, 30, and 40°C when compared to the marketed commercial formulation containing phosphate, sucrose, sodium chloride, and polysorbate 20. Further studies examining subvisible particles after exposure to a transport stress and long-term stability at 4°C, post-translational modifications by multi-attribute method, purity by reduced and non-reduced capillary electrophoresis, and potency by cell proliferation also demonstrated a comparable or improved stability for the enhanced formulation of acetate, proline, and poloxamer 188. This enhanced stability could enable limited storage outside of the cold chain, allowing for easier distribution in low to middle income countries.


Assuntos
Poloxâmero , Polissorbatos , Receptores de Fatores de Crescimento do Endotélio Vascular , Humanos , Polissorbatos/química , Proteínas Recombinantes de Fusão , Cloreto de Sódio , Acetatos , Sacarose , Prolina , Estabilidade de Medicamentos
3.
Antibodies (Basel) ; 11(2)2022 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-35466277

RESUMO

In this study, we used sodium chloride (NaCl) to extensively modulate non-specific protein-protein interactions (PPI) of a humanized anti-streptavidin monoclonal antibody class 2 molecule (ASA-IgG2). The changes in PPI with varying NaCl (CNaCl) and monoclonal antibody (mAb) concentration (CmAb) were assessed using the diffusion interaction parameter kD and second virial coefficient B22 measured from solutions with low to moderate CmAb. The effective structure factor S(q)eff measured from concentrated mAb solutions using small-angle X-ray and neutron scattering (SAXS/SANS) was also used to characterize the PPI. Our results found that the nature of net PPI changed not only with CNaCl, but also with increasing CmAb. As a result, parameters measured from dilute and concentrated mAb samples could lead to different predictions on the stability of mAb formulations. We also compared experimentally determined viscosity results with those predicted from interaction parameters, including kD and S(q)eff. The lack of a clear correlation between interaction parameters and measured viscosity values indicates that the relationship between viscosity and PPI is concentration-dependent. Collectively, the behavior of flexible mAb molecules in concentrated solutions may not be correctly predicted using models where proteins are considered to be uniform colloid particles defined by parameters derived from low CmAb.

4.
J Pharm Sci ; 109(1): 233-246, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31348937

RESUMO

The broadly neutralizing anti-HIV antibody, 10-1074, is a highly somatically hypermutated IgG1 being developed for prophylaxis in sub-Saharan Africa. A series of algorithms were applied to identify potentially destabilizing residues in the framework of the Fv region. Of 17 residues defined, a variant was identified encompassing 1 light and 3 heavy chain residues, with significantly increased conformational stability while maintaining full neutralization activity. Central to the stabilization was the replacement of the heavy chain residue T108 with R108 at the base of the CDR3 loop which allowed for the formation of a nascent salt bridge with heavy chain residue D137. Three additional mutations were necessary to confer increased conformational stability as evidenced by differential scanning fluorimetry and isothermal chemical unfolding. In addition, we observed increased stability during low pH incubation in which 40% of the parental monomer aggregated while the combinatorial variant showed no increase in aggregation. Incubation of the variant at 100 mg/mL for 6 weeks at 40°C showed a 9-fold decrease in subvisible particles ≥2 µm relative to the parental molecule. Stability-based designs have also translated to improved pharmacokinetics. Together, these data show that increasing conformational stability of the Fab can have profound effects on the manufacturability and long-term stability of a monoclonal antibody.


Assuntos
Anticorpos Amplamente Neutralizantes/química , Anticorpos Amplamente Neutralizantes/genética , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/genética , Mutação/fisiologia , Animais , Anticorpos Amplamente Neutralizantes/metabolismo , Células HEK293 , Anticorpos Anti-HIV/metabolismo , Humanos , Camundongos , Conformação Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína
5.
J Pharm Sci ; 109(1): 690-695, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31689428

RESUMO

An early-phase development shipping study was designed to interrogate the stability of liquid formulations under normal shipping conditions. Parcel shipments were made between Seattle, WA, and Indianapolis, IN, during 2018-2019. Each parcel contained a data recorder that tracked the shipment by GPS and measured shock and temperature. During the transport process, the parcels received up to 40 shock events with strengths ranging from 8 to 36G. After shipment, the formulations without polysorbate showed considerable increases in submicron and visible particles while little to no change occurred when polysorbate was present. Samples dropped repeatedly from a height of 18 inches to produce a shock of ∼25G caused visible particle formation with little increase in the subvisible particles, suggesting that other factors, such as vibration, in addition to the shock, were necessary to produce particle formation. These results provide a basis for further studies in the relationships between physical stability of mAbs and the challenges introduced by the shipment network, specifically shock and vibration. The findings indicate that the shock events as measured are repeatable and attributable to the layout of the sorting facility.


Assuntos
Embalagem de Medicamentos , Meios de Transporte , Ustekinumab/química , Composição de Medicamentos , Estabilidade de Medicamentos , Arquitetura de Instituições de Saúde , Agregados Proteicos , Conformação Proteica , Dobramento de Proteína , Estabilidade Proteica , Estresse Mecânico , Temperatura , Instalações de Transporte
6.
J Pharm Sci ; 109(1): 532-542, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31669607

RESUMO

Formulation screening for biotherapeutics can cover a vast array of excipients and stress conditions. These studies consume quantities of limited material and, with higher concentrated therapeutics, more material is needed. Here, we evaluate the use of crystal zenith (CZ) microtiter plates in conjunction with FluoroTec-coated butyl rubber mats as a small-volume, high-throughput system for formulation stability studies. The system was studied for evaporation, edge effects, and stability with comparisons to type 1 glass and CZ vials for multiple antibodies and formulations. Evaporation was minimal at 4°C and could be reduced at elevated temperatures using sealed, mylar bags. Edge effects were not observed until 12 weeks at 40°C. The overall stability ranking as measured by the rate of change in high molecular weight and percent main peak species was comparable across both vials and plates at 4°C and 40°C out to 12 weeks. Product quality attributes as measured by the multi-attribute method were also comparable across all containers for each molecule formulation. A potential difference was measured for subvisible particle analysis, with the plates measuring lower particle counts than the vials. Overall, CZ plates are a viable alternative to traditional vials for small-volume, high-throughput formulation stability screening studies.


Assuntos
Anticorpos Monoclonais/química , Cicloparafinas/química , Ensaios de Triagem em Larga Escala/instrumentação , Fragmentos Fc das Imunoglobulinas/química , Espectrometria de Massas/instrumentação , Cromatografia em Gel , Cromatografia de Fase Reversa , Composição de Medicamentos , Estabilidade de Medicamentos , Eletroforese Capilar , Desenho de Equipamento , Miniaturização , Desnaturação Proteica , Estabilidade Proteica , Proteínas Recombinantes de Fusão/química , Temperatura , Fatores de Tempo
7.
J Pharm Sci ; 107(12): 3032-3046, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30176252

RESUMO

In this study, we investigated analytical challenges associated with the formulation of 2 anti-HIV broadly neutralizing antibodies (bnAbs), 3BNC117 and PGT121, both separately at 100 mg/mL and together at 50 mg/mL each. The bnAb formulations were characterized for relative solubility and conformational stability followed by accelerated and real-time stability studies. Although the bnAbs were stable during 4°C storage, incubation at 40°C differentiated their stability profiles. Specific concentration-dependent aggregation rates at 30°C and 40°C were measured by size exclusion chromatography for the individual bnAbs with the mixture showing intermediate behavior. Interestingly, although the relative ratio of the 2 bnAbs remained constant at 4°C, the ratio of 3BNC117 to PGT121 increased in the dimer that formed during storage at 40°C. A mass spectrometry-based multiattribute method, identified and quantified differences in modifications of the Fab regions for each bnAb within the mixture including clipping, oxidation, deamidation, and isomerization sites. Each bnAb showed slight differences in the levels and sites of lysine residue glycations. Together, these data demonstrate the ability to differentiate degradation products from individual antibodies within the bnAb mixture, and that degradation rates are influenced not only by the individual bnAb concentrations but also by the mixture concentration.


Assuntos
Anticorpos Neutralizantes/química , Anticorpos Anti-HIV/química , Anticorpos Monoclonais Humanizados , Anticorpos Neutralizantes/imunologia , Anticorpos Amplamente Neutralizantes , Composição de Medicamentos , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Humanos , Oxirredução , Agregados Proteicos , Conformação Proteica , Estabilidade Proteica , Solubilidade
8.
Anal Biochem ; 563: 1-8, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30236889

RESUMO

The chemical unfolding (denaturation) assay can be used to calculate the change in the Gibbs free energy of unfolding, ΔG, and inflection point of unfolding, to collectively inform on molecule stability. Here, we evaluated methods for calculating the ΔG across 23 monoclonal antibody sequence variants. These methods are based on how the measured output (intrinsic fluorescence intensity) is treated, including utilizing (a) a single wavelength, (b) a ratio of two wavelengths, (c) a ratio of a single wavelength to an area, and (d) a scatter correction plus a ratio of a single wavelength to an area. When applied to the variants, the three ratio methods showed comparable results, with a similar pooled standard deviation for the ΔG calculation, while the single-wavelength method is shown as inadequate for the data in this study. However, when light scattering is introduced to simulated data, only the scatter-correction area normalization method proves robust. Using this method, common plate-based spectrophotometers found in many laboratories can be used for high-throughput screening of mAb variants and formulation stability studies.


Assuntos
Proteínas/química , Varredura Diferencial de Calorimetria , Luz , Modelos Químicos , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Desdobramento de Proteína , Termodinâmica
10.
J Phys Chem B ; 120(13): 3318-30, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-27007711

RESUMO

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.


Assuntos
Anticorpos Monoclonais/química , Imunoglobulina G/química , Concentração Osmolar , Polietilenoglicóis/química , Estabilidade Proteica , Sorbitol/química , Sacarose/química , Termodinâmica , Trealose/química , Água/química
11.
J Pharm Sci ; 105(3): 1066-73, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26886346

RESUMO

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.


Assuntos
Acetatos/química , Anticorpos Monoclonais/química , Ácido Cítrico/química , Imunoglobulina G/química , Íons/química , Agregados Proteicos , Estreptavidina/imunologia , Soluções Tampão , Química Farmacêutica/métodos , Excipientes/química , Concentração de Íons de Hidrogênio , Desdobramento de Proteína , Temperatura
12.
J Phys Chem B ; 119(49): 15150-63, 2015 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-26563591

RESUMO

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 (∼5 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.


Assuntos
Imunoglobulina G/química , Estreptavidina/imunologia , Dicroísmo Circular , Imunoglobulina G/imunologia , Cinética , Conformação Proteica , Espectrofotometria Ultravioleta , Análise Espectral Raman
13.
J Phys Chem B ; 119(18): 5793-804, 2015 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-25885209

RESUMO

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.


Assuntos
Imunoglobulina G/química , Íons/química , Agregados Proteicos , Estreptavidina/imunologia , Citratos/química , Coloides/química , Hidrodinâmica , Concentração de Íons de Hidrogênio , Peso Molecular , Espalhamento de Radiação , Acetato de Sódio/química , Cloreto de Sódio/química , Citrato de Sódio , Soluções , Eletricidade Estática , Propriedades de Superfície
14.
J Pharm Sci ; 104(2): 485-94, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25362835

RESUMO

The structural integrity and conformational stability of a genetically modified live, oncolytic herpes simplex virus (o-HSV) were investigated across a wide pH (5.5-8.0) and temperature (10°C-87.5°C) range. A combination of circular dichroism, intrinsic and extrinsic fluorescence, and static light scattering results was visualized using an empirical phase diagram approach to provide a global assessment of physical stability. Distinct phases were identified including the native state of the virus, an intermediate phase that could represent gradual swelling and/or shedding of the viral envelope, and a highly disrupted, aggregated phase. The nature of these altered forms of the virus was further evaluated by transmission electron microscopy and viral plaque assays. The effect of freeze-thaw (F/T) stress on o-HSV was also examined. After one F/T cycle, a loss of infectious virus titers was observed. In addition, the monomeric virus particle concentration decreased during F/T stress, whereas there was a concurrent increase in larger particles (2-10 µm). The comprehensive biophysical characterization of viral stability conducted in this study identified major degradation events leading to loss of infectivity of o-HSV and represents an important step toward stabilization of the virus against thermal and F/T stresses.


Assuntos
Neoplasias/terapia , Terapia Viral Oncolítica , Simplexvirus , Temperatura , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Espalhamento de Radiação , Simplexvirus/química , Simplexvirus/fisiologia , Simplexvirus/ultraestrutura
15.
J Phys Chem B ; 118(19): 5044-9, 2014 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-24758234

RESUMO

Antibody solutions are typically much more viscous than solutions of globular proteins at equivalent volume fraction. Here we propose that this is due to molecular entanglements that are caused by the elongated shape and intrinsic flexibility of antibody molecules. We present a simple theory in which the antibodies are modeled as linear polymers that can grow via reversible bonds between the antigen binding domains. This mechanism explains the observation that relatively subtle changes to the interparticle interaction can lead to large changes in the viscosity. The theory explains the presence of distinct power law regimes in the concentration dependence of the viscosity as well as the correlation between the viscosity and the charge on the variable domain in our antistreptavidin IgG1 model system.


Assuntos
Anticorpos Monoclonais/química , Complexo Antígeno-Anticorpo/química , Imunoglobulina G/química , Modelos Químicos , Estreptavidina/química , Módulo de Elasticidade , Cinética , Agregados Proteicos , Ligação Proteica , Soluções , Termodinâmica , Viscosidade
16.
Mol Pharm ; 11(5): 1391-402, 2014 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-24679215

RESUMO

Colloidal stability of antibody solutions, i.e., the propensity of the folded protein to precipitate, is an important consideration in formulation development of therapeutic monoclonal antibodies. In a protein solution, different pathways including crystallization, colloidal aggregation, and liquid-liquid phase separation (LLPS) can lead to the formation of precipitates. The kinetics of crystallization and aggregation are often slow and vary from protein to protein. Due to the diverse mechanisms of these protein condensation processes, it is a challenge to develop a standardized test for an early evaluation of the colloidal stability of antibody solutions. LLPS would normally occur in antibody solutions at sufficiently low temperature, provided that it is not preempted by freezing of the solution. Poly(ethylene glycol) (PEG) can be used to induce LLPS at temperatures above the freezing point. Here, we propose a colloidal stability test based on inducing LLPS in antibody solutions and measuring the antibody concentration of the dilute phase. We demonstrate experimentally that such a PEG-induced LLPS test can be used to compare colloidal stability of different antibodies in different solution conditions and can be readily applied to high-throughput screening. We have derived an equation for the effects of PEG concentration and molecular weight on the results of the LLPS test. Finally, this equation defines a binding energy in the condensed phase, which can be determined in the PEG-induced LLPS test. This binding energy is a measure of attractive interactions between antibody molecules and can be used for quantitative characterization of the colloidal stability of antibody solutions.


Assuntos
Anticorpos/química , Coloides/química , Polietilenoglicóis/química , Soluções/química , Soluções Farmacêuticas/química
17.
J Phys Chem B ; 117(45): 14029-38, 2013 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-24171386

RESUMO

Monoclonal antibodies (mAbs) contain hinge-like regions that enable structural flexibility of globular domains that have a direct effect on biological function. A subclass of mAbs, IgG2, have several interchain disulfide bonds in the hinge region that could potentially limit structural flexibility of the globular domains and affect the overall configuration space available to the mAb. We have characterized human IgG2 mAb in solution via small-angle neutron scattering (SANS) and interpreted the scattering data using atomistic models. Molecular Monte Carlo combined with molecular dynamics simulations of a model mAb indicate that a wide range of structural configurations are plausible, spanning radius of gyration values from ∼39 to ∼55 Å. Structural ensembles and representative single structure solutions were derived by comparison of theoretical SANS profiles of mAb models to experimental SANS data. Additionally, molecular mechanical and solvation free-energy calculations were carried out on the ensemble of best-fitting mAb structures. The results of this study indicate that low-resolution techniques like small-angle scattering combined with atomistic molecular simulations with free-energy analysis may be helpful to determine the types of intramolecular interactions that influence function and could lead to deleterious changes to mAb structure. This methodology will be useful to analyze small-angle scattering data of many macromolecular systems.


Assuntos
Anticorpos Monoclonais/química , Difração de Nêutrons , Espalhamento a Baixo Ângulo , Anticorpos Monoclonais/metabolismo , Humanos , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Simulação de Dinâmica Molecular , Método de Monte Carlo , Estreptavidina/imunologia , Termodinâmica
18.
J Pharm Sci ; 102(12): 4291-304, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24142544

RESUMO

Although the viscosity of concentrated antibody solutions has been the focus of many recent studies, less attention has been concentrated on how changes in protein structure impact viscosity. This study examines viscosity profiles of an immunoglobulin G (IgG) 2 monoclonal antibody at 150 mg/mL as a function of temperature and pH. Although the structure of the antibody at pH 4.0-7.0 was comparable at lower temperatures as measured by second derivative UV absorbance and Fourier transform infrared spectroscopy, differences in 8-anilino-1-naphthalene sulfonate (ANS) fluorescence intensity indicated small structural alterations as a function of pH. Below the structural transition onset temperature, the viscosity profiles were pH dependent and linearly correlated with fluorescence intensity, and followed semilogarithmic behavior as a function of temperature. The transitions of the viscosity profiles correlated well with the major structure transitions at a protein concentration of 150 mg/mL. The viscosity correlated particularly well with ANS fluorescence intensity at 0.2 mg/mL below and above the structural transition temperatures. These results suggest: (1) ANS can be an important measure of the overall structure and (2) hydrophobic interactions and charge-charge interactions are the two major physical factors that contribute collectively to the high viscosity of concentrated IgG solutions.


Assuntos
Imunoglobulina G/química , Concentração de Íons de Hidrogênio , Conformação Proteica , Estabilidade Proteica , Espectrometria de Fluorescência , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura , Viscosidade
19.
J Pharm Sci ; 102(10): 3545-55, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23925953

RESUMO

We describe a novel human immunoglobulin G2 (IgG2 )-tolerant and immune-competent heterozygous mouse model (Xeno-het) developed by crossbreeding a human Ig-tolerized XenoMouse® with a C57BL/6J wild-type mouse. The Xeno-het mouse expresses both mouse and human immunoglobulin G (IgG) genes, resulting in B-cells expressing human and mouse IgG, and secretion of human and mouse Ig into serum. This model was utilized to evaluate the immunogenicity risk of aggregated and chemically modified human antibodies. The mice were tested for their ability to break tolerance to self-tolerant monomeric antibodies. Aggregates made by mechanical stirring elicited an anti-drug antibody (ADA) response, but did not induce a robust and long-term memory B and T-cell response. Chemically modified antibodies made by oxidation were only weak and transient inducers of an immune response, as measured by a lack of both an ADA response and a B-cell antigen-specific response. Aggregate size was an important characteristic, as specific-sized protein-coated beads were able to elicit an immune response. We propose the use of this model to identify risk factors such as aggregation during manufacturing at early development for an increased potential immunogenicity risk.


Assuntos
Anticorpos/imunologia , Formação de Anticorpos/imunologia , Fatores Biológicos/imunologia , Tolerância Imunológica/imunologia , Animais , Linfócitos B/imunologia , Humanos , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia
20.
Protein Sci ; 22(10): 1295-305, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23893936

RESUMO

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/química
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