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1.
Nat Med ; 25(9): 1402-1407, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31501610

RESUMO

Natalizumab (NZM), a humanized monoclonal IgG4 antibody to α4 integrins, is used to treat patients with relapsing-remitting multiple sclerosis (MS)1,2, but in about 6% of the cases persistent neutralizing anti-drug antibodies (ADAs) are induced leading to therapy discontinuation3,4. To understand the basis of the ADA response and the mechanism of ADA-mediated neutralization, we performed an in-depth analysis of the B and T cell responses in two patients. By characterizing a large panel of NZM-specific monoclonal antibodies, we found that, in both patients, the response was polyclonal and targeted different epitopes of the NZM idiotype. The neutralizing activity was acquired through somatic mutations and correlated with a slow dissociation rate, a finding that was supported by structural data. Interestingly, in both patients, the analysis of the CD4+ T cell response, combined with mass spectrometry-based peptidomics, revealed a single immunodominant T cell epitope spanning the FR2-CDR2 region of the NZM light chain. Moreover, a CDR2-modified version of NZM was not recognized by T cells, while retaining binding to α4 integrins. Collectively, our integrated analysis identifies the basis of T-B collaboration that leads to ADA-mediated therapeutic resistance and delineates an approach to design novel deimmunized antibodies for autoimmune disease and cancer treatment.


Assuntos
Anticorpos Neutralizantes/administração & dosagem , Epitopos de Linfócito T/imunologia , Esclerose Múltipla/tratamento farmacológico , Natalizumab/administração & dosagem , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Neutralizantes/química , Formação de Anticorpos/efeitos dos fármacos , Formação de Anticorpos/imunologia , Linfócitos B/efeitos dos fármacos , Humanos , Imunoglobulina G/química , Imunoglobulina G/imunologia , Integrina alfa4/antagonistas & inibidores , Integrina alfa4/imunologia , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Conformação Proteica/efeitos dos fármacos , Linfócitos T/química , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia
2.
Pharm Res ; 36(9): 129, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31254106

RESUMO

PURPOSE: Immunogenicity against biotherapeutics can lead to the formation of drug/anti-drug-antibody (ADA) immune complexes (ICs) with potential impact on safety and drug pharmacokinetics (PK). This work aimed to generate defined drug/ADA ICs, characterized by quantitative (bio) analytical methods for dedicated determination of IC sizes and IC profile changes in serum to facilitate future in vivo studies. METHODS: Defined ICs were generated and extensively characterized with chromatographic, biophysical and imaging methods. Quantification of drug fully complexed with ADAs (drug in ICs) was performed with an acid dissociation ELISA. Sequential coupling of SEC and ELISA enabled the reconstruction of IC patterns and thus analysis of IC species in serum. RESULTS: Characterization of generated ICs identified cyclic dimers, tetramers, hexamers, and larger ICs of drug and ADA as main IC species. The developed acid dissociation ELISA enabled a total quantification of drug fully complexed with ADAs. Multiplexing of SEC and ELISA allowed unbiased reconstruction of IC oligomeric states in serum. CONCLUSIONS: The developed in vitro IC model system has been properly characterized by biophysical and bioanalytical methods. The specificity of the developed methods enable discrimination between different oligomeric states of ICs and can be bench marking for future in vivo studies with ICs.


Assuntos
Anticorpos Monoclonais/química , Complexo Antígeno-Anticorpo/análise , Animais , Anticorpos Monoclonais/sangue , Complexo Antígeno-Anticorpo/sangue , Complexo Antígeno-Anticorpo/química , Cromatografia Líquida , Dimerização , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/química , Conformação Proteica , Ratos Wistar , Soroalbumina Bovina/química
3.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1122-1123: 64-72, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31154130

RESUMO

Glycosylation plays an important role in the maintenance of the structure and function of glycoproteins, while aberrant protein glycosylation is correlated with various diseases. Human immunoglobulin G (IgG), which is composed of four subclasses (IgG1, IgG2, IgG3 and IgG4), is one of the most dominant and significant glycoprotein in human serum. The glycosylation on IgG-Fc moiety is known to be alternated with various physiological and pathological states. We herein report an integrated approach for comprehensive profiling and quantitation of IgG-Fc glycopeptides. Firstly, IgG N-glycans were profiled by using mAb-Glyco chip quadrupole time-of-flight mass spectrometry (Q-TOF-MS), resulting characterization of 87 N­glycans originating from 29 different oligosaccharide compositions. Secondly, subclass-specific glycopeptides were identified on the basis of high-resolution MS and MS/MS data by using ultra high performance liquid chromatography (UHPLC) coupled to Q-TOF-MS. As a result, 83 IgG-Fc glycopeptides from human serum, including 17 sialylated glycopeptides, were identified. In addition, a quantitation method with high sensitivity and repeatability was established by using UHPLC triple quadrupole (QQQ) MS. We applied this approach to carry out quantitative analysis of IgG glycosylation in RA patients. Finally, 36 potential glycopeptide biomarkers, including 13 species from IgG1, 12 species from IgG2/3 and 11 species from IgG4 were identified.


Assuntos
Artrite Reumatoide/sangue , Cromatografia Líquida de Alta Pressão/métodos , Glicopeptídeos/sangue , Imunoglobulina G/química , Espectrometria de Massas em Tandem/métodos , Idoso , Artrite Reumatoide/metabolismo , Estudos de Casos e Controles , Feminino , Glicopeptídeos/química , Glicosilação , Humanos , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes
4.
Adv Mater ; 31(32): e1902542, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31183900

RESUMO

Current cancer immunotherapies including chimeric antigen receptor (CAR)-based therapies and checkpoint immune inhibitors have demonstrated significant clinical success, but always suffer from immunotoxicity and autoimmune disease. Recently, nanomaterial-based immunotherapies are developed to precisely control in vivo immune activation in tumor tissues for reducing immune-related adverse events. However, little consideration has been put on the spatial modulation of interactions between immune cells and cancer cells to optimize the efficacy of cancer immunotherapies. Herein, a rational design of immunomodulating nanoparticles is demonstrated that can in situ modify the tumor cell surface with natural killer cell (NK cell)-activating signals to achieve in situ activation of tumor-infiltrating NK cells, as well as direction of their antitumor immunity toward tumor cells. Using these immunomodulating nanoparticles, the remarkable inhibition of tumor growth is observed in mice without noticeable side effects. This study provides an accurate immunomodulation strategy that achieves safe and effective antitumor immunity through in situ NK cell activation in tumors. Further development by constructing interactions with various immune cells can potentially make this nanotechnology become a general platform for the design of advanced immunotherapies for cancer treatments.


Assuntos
Membrana Celular/imunologia , Fatores Imunológicos/química , Células Matadoras Naturais/metabolismo , Nanopartículas/química , Resinas Acrílicas/química , Animais , Ácidos Borônicos/química , Linhagem Celular Tumoral , Reagentes para Ligações Cruzadas/química , Portadores de Fármacos/química , Imunoglobulina G/química , Imunoterapia , Células Matadoras Naturais/imunologia , Camundongos , Transplante de Neoplasias , Polímeros/química , Soroalbumina Bovina/química , Propriedades de Superfície
5.
Anal Bioanal Chem ; 411(21): 5617-5629, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31214752

RESUMO

Positive identification of capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) electropherogram peaks provides information to understand protein molecular characteristics at the structural level. It is critical in the design of a robust assay that can accurately resolve, differentiate, and quantify all therapeutic protein components including fragmented species, which are considered as product related impurities. However, direct identification of the impurity peaks observed in CE-SDS is a challenging and oftentimes an ambiguous task. This paper proposed a systematic workflow for characterizing CE-SDS fragmentation peaks. Forced degradation of monoclonal antibody (mAb) by multiple stress methods was utilized to induce fragmentation and species enrichment. The characteristics, such as size and the clipped region of sequence, were then evaluated based on multiple enzymatic treatment and particle reduction. The identified fragments were further confirmed using tryptic digestion and liquid chromatography coupled with mass spectrometry (LC-MS) analysis. Common fragment sizes and clipping locations are identified after evaluating multiple IgG molecules. The methodology and procedure described in this article are readily deployable and will provide necessary information for method, process, and product characterizations. Graphical abstract.


Assuntos
Anticorpos Monoclonais/química , Eletroforese Capilar/métodos , Dodecilsulfato de Sódio/química , Cromatografia Líquida/métodos , Imunoglobulina G/química , Espectrometria de Massas em Tandem/métodos
6.
Int J Pharm ; 565: 162-173, 2019 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-31054877

RESUMO

Determining the stability of downstream process (DSP) intermediates is an extremely important parameter used to maintain product quality attributes within their acceptance ranges. The IgG4 monoclonal antibody studied (mAb1) showed aggregation under acidic conditions, inhibiting the use of low pH treatment to inactivate endogenous retroviruses, and poor virus filtration performance. Both manufacturing steps are included in mAb DSP for viral clearance. The impact of several new compounds on the aggregation and stabilization of mAb1 in process intermediate pools encountered during these critical DSP steps was investigated. Results showed that, in the presence of a protein stabilizer at pH 3.2, 27% less aggregation was observed compared to controls, during the low pH treatment for viral inactivation. The impact of a novel protein stabilizer on virus filter throughput during mAb1 filtration was compared to L-arginine using an innovative high-throughput automation technique. Compared to control experiments without additives, conditions were found where a 70% increase in filter volumetric throughput was achieved in the presence of the novel stabilizer, and a 56% decrease in volumetric throughput observed with L-arginine. These findings present the possibility of using these novel compounds to stabilize proteins during DSP and permitting the use of platform DSP elements such as low pH treatment and high-throughput virus filtration to challenging and unstable proteins.


Assuntos
Anticorpos Monoclonais/química , Imunoglobulina G/química , Química Farmacêutica , Estabilidade de Medicamentos , Filtração , Concentração de Íons de Hidrogênio , Vírus
7.
Pharm Res ; 36(8): 109, 2019 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-31127417

RESUMO

PURPOSE: To investigate differences in the preferential exclusion of trehalose, sucrose, sorbitol and mannitol from the surface of three IgG1 monoclonal antibodies (mAbs) and understand its effect on the aggregation and reversible self-association of mAbs at high-concentrations. METHODS: Preferential exclusion was measured using vapor pressure osmometry. Effect of excipient addition on accelerated aggregation kinetics was quantified using size exclusion chromatography and on reversible self-association was quantified using dynamic light scattering. RESULTS: The doubling of excipient concentration in the 0 to 0.5 m range resulted in a doubling of the mAb transfer free energy for all excipients and antibodies tested in this study. Solution pH and choice of buffering agent did not significantly affect the magnitude of preferential exclusion. We find that aggregation suppression for trehalose, sucrose and sorbitol (but not mannitol) correlates with the magnitude of their preferential exclusion from the native state of the three IgG1 mAbs. We also find that addition of sugars and polyols reduced the tendency for reversible self-association in two mAbs that had weakly repulsive or neutral self-interactions in the presence of buffer alone. CONCLUSIONS: The magnitude of preferential exclusion for trehalose, sucrose and sorbitol correlates well with their partial molar volumes in solution. Mannitol is excluded to a greater extent than that expected from its partial molar volume, suggesting specific interactions of mannitol that might be different than the other sugars and polyols tested in this study. Local interactions play a role in the effect of excipient addition on the reversible self-association of mAbs. These results provide further insights into the stabilization of high-concentration mAb formulations by sugars and polyols.


Assuntos
Anticorpos Monoclonais/química , Imunoglobulina G/química , Polímeros/química , Agregados Proteicos , Sacarose/química , Álcoois Açúcares/química , Trealose/química , Excipientes/química , Cinética , Simulação de Dinâmica Molecular , Conformação Proteica , Propriedades de Superfície
8.
Nanoscale ; 11(22): 10819-10827, 2019 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-31135010

RESUMO

Myxovirus protein A (MxA) is a biomarker that can be used to distinguish between viral and bacterial infections. While MxA lateral flow assays (LFAs) have been successfully used for viral vs. bacterial differential diagnosis for children, the clinically relevant level of MxA for adults has been reported to be 100 times lower, which is too low for traditional LFAs. We present results applying the use of surface enhanced Raman spectroscopy (SERS) to detect MxA. AuAg nanoshells (AuAg NSs) were used to enhance the Raman signal of mercaptobenzoic acid (4-MBA), enabling readout by SERS. The AuAg NSs were conjugated to antibodies for the biomarker of interest, resulting in a "nanotag", that could be used in a dipstick immunoassay for detection. We first optimized the nanotag parameters using anti-human IgG/human IgG as a model antibody/antigen system, and then demonstrated detection of MxA using anti-MxA antibodies. We show that SERS readout of immunoassays for MxA can quantify MxA levels at clinically relevant levels for adult viral infection.


Assuntos
Anticorpos Antivirais/química , Ouro/química , Imunoglobulina G/química , Nanopartículas Metálicas/química , Proteínas de Resistência a Myxovirus/imunologia , Nanoconchas/química , Infecções por Orthomyxoviridae , Infecções Bacterianas/diagnóstico , Infecções Bacterianas/imunologia , Criança , Humanos , Imunoensaio , Orthomyxoviridae , Infecções por Orthomyxoviridae/diagnóstico , Infecções por Orthomyxoviridae/imunologia , Papel
9.
J Chromatogr A ; 1601: 133-144, 2019 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-31054833

RESUMO

In Part I of this work we determined the experimental cation exchange behavior of bivalent bispecific antibodies (BiSAb) comprising a pair of single chain variable fragment (scFv) domains flexibly linked to a framework immunoglobulin G (IgG), which exhibit a complex, three-peak elution pattern dependent on the residence time. A phenomenological model was developed assuming that the BiSAb molecules exist in multiple configurations that interact differently with the resin surface and interconvert at finite rates. In Part II of this work we provide relevant biomolecular perspectives that shed light on the underlying mechanisms. Firstly, we show that crosslinking the BiSAb molecules with a bifunctional reagent, which limits conformational flexibility, suppresses multiple peak elution. Secondly, we show that of the fragments obtained by enzymatic digestion of the BiSAb molecules only those that exhibit a pair of scFv domains show three-peak elution, while only two peaks are observed if a single scFv is present. Thirdly, we analyze the roles of electrostatic and hydrophobic surface properties of the BiSAb domains, identifying regions that are likely responsible for inter-domain and protein-surface interactions. The results demonstrate that the complex elution behavior catalyzed by the combination of surface charge and hydrophobicity of the stationary phase is associated with outstretched and collapsed configurations of the scFv domains relative to the framework IgG.


Assuntos
Anticorpos Biespecíficos/química , Resinas de Troca de Cátion/química , Cromatografia por Troca Iônica , Interações Hidrofóbicas e Hidrofílicas , Imunoglobulina G/química , Anticorpos de Cadeia Única/química , Eletricidade Estática
10.
J Chromatogr A ; 1598: 101-112, 2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-30954243

RESUMO

When developing purification processes for monoclonal antibodies (mAbs), ensuring the effective removal of high molecular weight (HMW) species is often challenging and labor intensive. In this work, we present a bottom-up characterization approach to achieve streamlined polishing step development as well as a more fundamental understanding of the protein of interest. Prior to physicochemical characterization, in-process HMW species of two IgG4 mAbs (mAb A and mAb B) were isolated via semi-preparative size exclusion chromatography (SEC). Key differences in approximate molecular weight, net charge, and native surface hydrophobicity were then identified using multi-angle light scattering (SEC-MALS), analytical-scale chromatographic screening, isoelectric focusing, and structural aggregation propensity modeling. SEC-MALS revealed two main HMW isoforms for each mAb: dimers and 1.7-mers for mAb A, and tetramers and dimers for mAb B. Analytical-scale chromatographic screening showed promising trends in charge-based separation for mAb A, and hydrophobic-based separation for mAb B. Isoelectric focusing data detected a 30% increase in acidic variants for mAb A HMW species relative to monomer, and a 20% increase in basic variants for mAb B HMW species. Lastly, analytical-scale characterization data was successfully applied to preparative scale purification conditions, producing results highly similar to those observed during analytical characterization of the isolated species. By using this high-throughput approach as a template for preparative-scale process development, key physicochemical differences between aggregate and monomer species were utilized to determine optimal polishing steps for HMW removal.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Química Farmacêutica/métodos , Cromatografia em Gel , Interações Hidrofóbicas e Hidrofílicas , Imunoglobulina G/química , Imunoglobulina G/isolamento & purificação , Peso Molecular
11.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1114-1115: 93-99, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30939413

RESUMO

Characterization of free thiol variants in antibody therapeutics is important for biopharmaceutical development, as the presence of free thiols may have an impact on aggregate formation, structural and thermal stability, as well as antigen-binding potency of monoclonal antibodies. Most current methods for free thiol quantification involve labeling of free thiol groups by different tagging molecules followed by UV, fluorescence or mass spectrometry (MS) detection. Here, we optimized a label-free liquid chromatography (LC)-UV/MS method for free thiol quantification at a subunit level and compared this method with two orthogonal and conventional approaches, Ellman's assay and peptide mapping with differential alkylation. This subunit unit approach was demonstrated to be able to provide domain-specific free thiol quantification and comparable results with labeling approaches, using a relatively simple and efficient workflow.


Assuntos
Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Cromatografia Líquida/métodos , Compostos de Sulfidrila/análise , Compostos de Sulfidrila/química , Animais , Ácido Ditionitrobenzoico , Imunoglobulina G/análise , Imunoglobulina G/química , Espectrometria de Massas/métodos , Mapeamento de Peptídeos , Reprodutibilidade dos Testes
12.
J Chem Phys ; 150(15): 155102, 2019 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-31005084

RESUMO

As revealed by previous experiments, protein mechanical stability can be effectively regulated by ligand binding with the binding site distant from the force-bearing region. However, the mechanism for such long-range allosteric control of protein mechanics is still largely unknown. In this work, we use protein topology-based elastic network model (ENM) and all-atomic steered molecular dynamics (SMD) simulations to study the impact of ligand binding on protein mechanical stability in two systems, i.e., GB1 and CheY-binding P2-domain of CheA (CBDCheA). Both ENM and SMD results show that the ligand binding has considerable and negligible effects on the mechanical stability of these two proteins, respectively. These results are consistent with the experimental observations. A physical mechanism for the enhancement of protein mechanical stability was then proposed: the correlated deformations of the force-bearing region and the binding site are handcuffed by the binding of ligand. The handcuff effect suppresses the propagation of internal force in the force-bearing region, thus improving the resistance to the loading force. Our study indicates that ENM method can effectively identify the structure motifs allosterically related to the deformation in the force bearing region, as well as the force propagation pathway within the structure of the studied proteins. Hence, it should be helpful to understand the molecular origin of the different mechanical properties in response to ligand binding for GB1 and CBDCheA.


Assuntos
Estabilidade Proteica , Fenômenos Biomecânicos , Elasticidade , Ligações de Hidrogênio , Imunoglobulina G/química , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios Proteicos , Receptores de GABA-B
13.
Int J Mol Sci ; 20(8)2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30991723

RESUMO

Antibodies leverage on their unique architecture to bind with an array of antigens. The strength of interaction has a direct relation to the affinity of the antibodies towards the antigen. In vivo affinity maturation is performed through multiple rounds of somatic hypermutation and selection in the germinal centre. This unique process involves intricate sequence rearrangements at the gene level via molecular mechanisms. The emergence of in vitro display technologies, mainly phage display and recombinant DNA technology, has helped revolutionize the way antibody improvements are being carried out in the laboratory. The adaptation of molecular approaches in vitro to replicate the in vivo processes has allowed for improvements in the way recombinant antibodies are designed and tuned. Combinatorial libraries, consisting of a myriad of possible antibodies, are capable of replicating the diversity of the natural human antibody repertoire. The isolation of target-specific antibodies with specific affinity characteristics can also be accomplished through modification of stringent protocols. Despite the ability to screen and select for high-affinity binders, some 'fine tuning' may be required to enhance antibody binding in terms of its affinity. This review will provide a brief account of phage display technology used for antibody generation followed by a summary of different combinatorial library characteristics. The review will focus on available strategies, which include molecular approaches, next generation sequencing, and in silico approaches used for antibody affinity maturation in both therapeutic and diagnostic applications.


Assuntos
Anticorpos Monoclonais/genética , Técnicas de Visualização da Superfície Celular/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Humanos , Imunoglobulina G/química , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Mutagênese , Biblioteca de Peptídeos
14.
Appl Biochem Biotechnol ; 189(2): 374-383, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31020512

RESUMO

A capacitive sensor was developed to analyze the presence and enzymatic activity of a model protease from standard solutions by following the degradation of the substrate in real time. The enzyme was chosen based on its specific digestion of the hinge region of immunoglobulin G (IgG). Real-time enzyme activity was monitored by measuring the change in capacitance (∆C) based on the release of IgG fragments after enzymatic digestion by the enzyme. The results indicated that the developed capacitive system might be used successfully for label-free and real-time monitoring of enzymatic activity of different enzymes in a sensitive, rapid, and inexpensive manner in biotechnological, environmental, and clinical applications.


Assuntos
Capacitância Elétrica , Técnicas Eletroquímicas/métodos , Imunoglobulina G/química , Peptídeo Hidrolases/química
15.
Int J Pharm ; 564: 106-116, 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-30999044

RESUMO

Crystalline bulking agent in lyophilized biopharmaceutical formulations provides an elegant lyophilized cake structure and allows aggressive primary drying conditions. The interplay between amorphous and crystalline state of excipients heavily influence the stability of lyophilized biological products and should be carefully evaluated in the formulation and process development phase. This study focuses on: (1) elucidating the influence of formulation and lyophilization process variables on the formation of different states of mannitol and (2) its impact on model monoclonal antibody stability when compared to sucrose. The main aim of the present research work was to study the influence of different mannitol to sucrose ratios and monoclonal antibody concentrations on mannitol physical form established during lyophilization. In addition, also the effect of process variables on mannitol hemihydrate (MHH) formation was under investigation. Thermal analysis and powder X-ray diffraction results revealed that the ratio between sucrose and mannitol and mAb concentration have a decisive impact on mannitol crystallization. Namely, increasing amount of mannitol and monoclonal antibody resulted in decreasing formation of MHH. From the process parameters investigated, a higher secondary drying temperature has the biggest impact on the complete dehydration of MHH. Specifically, higher secondary drying temperature reflected in complete dehydration of MHH. Annealing temperature was shown to affect the MHH content in the final product, wherein the higher annealing temperature was preferential for formation of anhydrous mannitol. Temperature stress stability study revealed that the most important parameter influencing monoclonal antibody stability is the ratio of protein to sucrose. Contrary to widespread assumption, we did not detect any impact of MHH on the stability of the investigated monoclonal antibody.


Assuntos
Anticorpos Monoclonais/química , Imunoglobulina G/química , Manitol/química , Sacarose/química , Estabilidade de Medicamentos , Liofilização , Estabilidade Proteica , Temperatura Ambiente
16.
J Chem Theory Comput ; 15(4): 2548-2560, 2019 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-30822382

RESUMO

In protein adsorption, the surrounding solvent has an important role in mediating protein-surface interactions. Therefore, it is of paramount importance that the solvent methods employed to model these kinds of processes are able to correctly capture the complex mechanisms occurring in the protein-water-surface interface. Here, we test the suitability of the two most popular implicit solvent methods based on the Generalized Born formalism to describe the adsorption process of the immunoglobulin G (IgG) on a hydrophobic graphene surface. Our results show that in both cases, IgG experiences an extreme and early (in less than 40 ns) unfolding as a result of the adsorption to the surface in contrast with previous experimental findings. A detailed energy decomposition analysis of explicit and implicit solvent simulations reveals that this discrepancy arises from the ill-characterization of two energy components in implicit solvent methods. These findings help to elucidate how implicit solvent models may be improved to accurately characterize the protein adsorption process.


Assuntos
Grafite/química , Imunoglobulina G/química , Adsorção , Interações Hidrofóbicas e Hidrofílicas , Modelos Químicos , Simulação de Dinâmica Molecular , Eletricidade Estática , Propriedades de Superfície , Termodinâmica
17.
J Chromatogr A ; 1597: 100-108, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-30922716

RESUMO

Platform manufacturing processes are widely adopted to simplify and standardize the development and manufacturing of monoclonal antibodies (mAbs). However, there are mAbs that do not conform to a platform design due to instability or other protein properties leading to a negative impact on product quality or process performance (non-platform mAb). Non-platform mAbs typically require prolonged development times and significant deviations from the platform process to address these issues due to the need to sequentially optimize individual process steps. In this study, we describe an IgG2 mAb (mAb A) that is susceptible to aggregation and reversible self-association (RSA) under platform conditions. In lieu of a sequential optimization approach, we evaluated the solution stability of mAb A across the platform operating space (solution stability screen). This screening design was used to identify interacting parameters that affected the non-platform mAb stability. A subsequent response surface design was found to predict an acceptable operating space that minimized aggregate formation and RSA across the entire process. This information guided the selection of optimal parameters best suited to avoid destabilizing conditions for each process step. Substantial time savings was achieved by focusing development around these factors including protein concentration, buffer pH, salt concentration, and excipient type. In addition, this work enabled the optimization of a cation exchange chromatography step that removed aggregate without yield losses due to the presence of reversible aggregation. The final optimized process derived from this study resulted in an increase in yield of ˜30% over the original process while maintaining the same level of aggregate clearance to match product quality. Solution stability screening is readily adapted to high throughput technologies to minimize material requirements and accelerate analytical data availability. Implementation of high throughput approaches will further expedite process development and enable enhanced selection of candidate drugs by including process development objectives.


Assuntos
Anticorpos Monoclonais/química , Biotecnologia/métodos , Cromatografia , Anticorpos Monoclonais/isolamento & purificação , Biotecnologia/instrumentação , Cátions/química , Concentração de Íons de Hidrogênio , Imunoglobulina G/química , Cloreto de Sódio
18.
Anal Chim Acta ; 1058: 97-106, 2019 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-30851859

RESUMO

Owing to their multiscale pore size regimes and unique properties, the materials with hierarchically porous structures have become an important family of functional materials in recent years. They have been applied from energy conversion and storage, catalysis, separation to drug delivery, etc. The synthesis of them is difficult by the need to employ multiple templates and take complicated steps. Herein, we successfully prepared epoxy-functionalized hierarchically porous hybrid monoliths (HPHMs) with micro/meso/macro-structures in an easy way. Firstly, a bulk monolithic material was formed via free radical polymerization between polyhedral oligomeric vinylsilsesquioxanes (vinylPOSS) and allyl glycidyl ether (AGE) in the presence of polycaprolactone (PCL). Then PCL was degraded with hydrochloric acid solution, and the epoxy-functionalized HPHM was obtained. This approach was very simple and suitable for large-scale preparation. Hybrid monoliths with different specific surface area (from 5.4 to 636.7 m2/g) were prepared by adjusting the mole ratio of vinylPOSS to AGE and the content of PCL. The results of several characterization methods, including nitrogen adsorption/desorption measurements, scanning electron microscopy (SEM) and mercury intrusion porosimetry (MIP), showed that these materials contained not only micropores and mesopores but also macropores. The materials were further modified with penicillamine to be used as hydrophilic interaction chromatography (HILIC) adsorbents for enriching N-glycopeptides in IgG and serum protein tryptic digests. Up to 23 N-glycopeptides were identified from IgG digest, and 385 N-glycopeptides and 283 N-glycosylation sites were identified from human serum digest.


Assuntos
Cromatografia Líquida/métodos , Glicopeptídeos/sangue , Compostos de Organossilício/química , Polivinil/química , Adsorção , Animais , Bovinos , Compostos de Epóxi/química , Glicopeptídeos/química , Glicosilação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunoglobulina G/química , Compostos de Organossilício/síntese química , Penicilamina/química , Poliésteres/síntese química , Poliésteres/química , Polimerização , Polivinil/síntese química , Porosidade , Proteólise , Soroalbumina Bovina/química , Tripsina/química
19.
AAPS PharmSciTech ; 20(4): 154, 2019 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-30919164

RESUMO

The physical and structural stability of freeze-dried immunoglobulin G (IgG) were examined by applying trehalose and amino acids (glycine, phenylalanine, and serine). The efficacy of amino acids was statistically compared considering their side-chain characteristics. The amount of amino acids (X1) and trehalose (X2) was considered as independent variables. Size exclusion chromatography (SEC-HPLC) was utilized to calculate the soluble aggregates, as dependent variables. The amounts of excipients were optimized through the central composite design (CCD). The beta-sheet conformation of IgG was quantified by Fourier transform infrared spectroscopy (FTIR). Thermal behavior and molecular integrity of IgG were evaluated by differential scanning calorimetry (DSC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Optimized formulations of powders were as follows: 24.5 mg serine-139.5 mg trehalose, 14 mg glycine-118 mg Trehalose, and 25 mg phenylalanine-139.5 mg trehalose. The amounts of soluble aggregates after processing were 0, 4.50, and 2.20%, respectively. The corresponding induced aggregates following storage conditions were 1.02, 7.0, and 3.70%. In all preparations, there were no detectable fragments. The native conformation of IgG was well preserved in the presence of amino acids. Excluding the glycine-based sample with minor endotherm at about 45°C, serine and phenylalanine incorporating powders were fully amorphous at examination temperatures. Trehalose was more potent than the amino acids in the stabilization of IgG. Serine was the most effective amino acid; phenylalanine and glycine were the next ones, respectively. Glycine crystallization was assumed to have accounted for low stabilization capability. The statistically synergistic phenomenon was only observed in the co-application of trehalose and phenylalanine. Graphical abstract.


Assuntos
Aminoácidos/química , Imunoglobulina G/química , Trealose/química , Composição de Medicamentos , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Liofilização , Glicina/química
20.
Spectrochim Acta A Mol Biomol Spectrosc ; 215: 340-344, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30852281

RESUMO

A dual sites affinity protocol was developed for fluorescent analysis of Staphylococcus aureus (S. aureus) by employing daptomycin and immunoglobulin G (IgG) as the recognition elements. Pig IgG immobilized on microplate was employed as the first recognition element to capture S. aureus owing to the fact that the Fc segment of mammal IgG can selectively bind with protein A on the surface of the target bacteria. Meanwhile, fluorescein isothiocyanate-conjugated daptomycin was employed as the second recognition element as well as the signal tracer for the target bacteria utilizing the binding capability of daptomycin to Gram-positive bacteria. S. aureus can be analyzed within a concentration range of 5.0 × 103-5.0 × 108 CFU mL-1 with a detection limit of 3.6 × 103 CFU mL-1. The analytical process can be accomplished within 1.5 h by using a pre-coated microplate. The dual sites affinity protocol can exclude the interference led by Gram-negative bacteria and other common Gram-positive bacteria. We have successfully applied it to analyze S. aureus in spiked lake water and physiological saline injection samples, and the recovery values ranged from 88.0% to 120.0%. The results demonstrate its application potential for environmental sanitation and drug safety control.


Assuntos
Daptomicina/química , Corantes Fluorescentes/química , Imunoglobulina G/química , Staphylococcus aureus/química , Anticorpos Antibacterianos/química , Anticorpos Antibacterianos/metabolismo , Anticorpos Imobilizados/química , Anticorpos Imobilizados/metabolismo , Daptomicina/metabolismo , Corantes Fluorescentes/metabolismo , Imunoglobulina G/metabolismo , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes , Espectrometria de Fluorescência/métodos , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/metabolismo
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