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1.
Artigo em Inglês | MEDLINE | ID: mdl-30594826

RESUMO

Intravenous (IV) infusion of therapeutic proteins typically involves dilution of the formulated product into infusion media such as normal saline or dextrose, 5% m/v in water. We report results from a rigorous evaluation of imaged capillary isoelectric focusing (iCIEF) for monitoring dextrose-mediated glycation of proteins in IV infusion solutions. In addition to detecting stable Amadori glycation products, iCIEF was able to detect the labile Schiff base (SB) glycation adducts since the equilibrium with free dextrose is maintained on capillary. Method parameters such as sample dilution factor and ampholyte composition (but not urea) were found to influence the observed level of SB glycation adducts. The impacts of dextrose and urea on the apparent pI values are also reported. iCIEF results were compared with results from cation exchange chromatography, which was found to preferentially detect the more stable Amadori glycation products due to the on-column decomposition of the SB adducts resulting from the separation of the protein from free dextrose which in turn altered the SB adduct- free dextrose equilibrium. These results demonstrate the need for careful consideration when selecting the analytical methodology to investigate protein sensitivity to dextrose and to monitor protein stability in dextrose-containing infusion solutions.


Assuntos
Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Eletroforese Capilar/métodos , Glucose/química , Focalização Isoelétrica/métodos , Administração Intravenosa , Anticorpos Monoclonais/administração & dosagem , Cromatografia por Troca Iônica/métodos , Glicosilação , Soluções/administração & dosagem , Soluções/química
2.
J Pharm Sci ; 108(2): 842-850, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30257193

RESUMO

Protein formulation stability is difficult to predict a priori and generally involves long-term stability studies. It is of interest to develop an analytical method that can predict stability trends reliably. Here, pulse proteolysis was evaluated as an analytical tool to predict solution-state stability in different formulations. Four proteins formulated in different buffer and excipient compositions were subjected to urea-induced unfolding and brief enzymatic digestion ("pulse" proteolysis), and relative resistance to proteolysis was measured by microfluidics-based capillary electrophoresis-sodium dodecyl sulfate. Biophysical properties of each formulation were measured using orthogonal biophysical techniques such as differential scanning fluorimetry, differential scanning calorimetry, dynamic light scattering, circular dichroism, and fluorescence spectroscopy. Protein stability in all formulations was monitored by size exclusion chromatography on storage at 5°C and 40°C. For all 4 proteins, formulations with greater proteolytic resistance also showed higher monomer content on thermal stability. In contrast, standard biophysical techniques showed reasonable-to-no correlation with size exclusion chromatography data. The data support the use of pulse proteolysis as an orthogonal, quantitative, and predictive tool to measure protein conformational stability and rank-order formulations.


Assuntos
Anticorpos Monoclonais/química , Varredura Diferencial de Calorimetria , Composição de Medicamentos , Excipientes/química , Agregados Proteicos , Conformação Proteica , Estabilidade Proteica , Desdobramento de Proteína , Proteólise , Proteínas Recombinantes de Fusão/química
3.
J Pharm Sci ; 105(5): 1684-1692, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27044943

RESUMO

Myoglobin (Mb) was lyophilized in the absence (Mb-A) and presence (Mb-B) of sucrose in a pilot-scale lyophilizer with or without controlled ice nucleation. Cake morphology was characterized using scanning electron microscopy, and changes in protein structure were monitored using solid-state Fourier-transform infrared spectroscopy, solid-state hydrogen-deuterium exchange-mass spectrometry, and solid-state photolytic labeling-mass spectrometry (ssPL-MS). The results showed greater variability in nucleation temperature and irregular cake structure for formulations lyophilized without controlled nucleation. Controlled nucleation resulted in nucleation at ∼(-5°C) and uniform cake structure. Formulations containing sucrose showed better retention of protein structure by all measures than formulations without sucrose. Samples lyophilized with and without controlled nucleation were similar by most measures of protein structure. However, ssPL-MS showed the greatest photoleucine incorporation and more labeled regions for Mb-B lyophilized with controlled nucleation. The data support the use of solid-state hydrogen-deuterium exchange-mass spectrometry and ssPL-MS to study formulation and process-induced conformational changes in lyophilized proteins.


Assuntos
Mioglobina/análise , Mioglobina/química , Espectrometria de Massas em Tandem/métodos , Animais , Composição de Medicamentos , Liofilização/métodos , Cavalos , Espectrometria de Massas/métodos , Estrutura Secundária de Proteína , Difração de Raios X/métodos
4.
Curr Pharm Des ; 21(40): 5845-53, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26446463

RESUMO

The long-term stability of protein therapeutics in the solid-state depends on the preservation of native structure during lyophilization and in the lyophilized powder. Proteins can reversibly or irreversibly unfold upon lyophilization, acquiring conformations susceptible to degradation during storage. Therefore, characterizing proteins in the dried state is crucial for the design of safe and efficacious formulations. This review summarizes the basic principles and applications of the analytical techniques that are commonly used to characterize protein structure, dynamics and conformation in lyophilized solids. The review also discusses the applications of recently developed mass spectrometry based methods (solid-state hydrogen deuterium exchange mass spectrometry (ssHDX-MS) and solid-state photolytic labeling mass spectrometry (ssPL-MS)) and their ability to study proteins in the solid-state at high resolution.


Assuntos
Química Farmacêutica , Liofilização/métodos , Espectrometria de Massas/métodos , Conformação Proteica , Proteínas/química , Proteínas/metabolismo , Humanos
5.
Mol Pharm ; 12(9): 3237-49, 2015 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-26204425

RESUMO

Protein structure and local environment in lyophilized formulations were probed using high-resolution solid-state photolytic cross-linking with mass spectrometric analysis (ssPC-MS). In order to characterize structure and microenvironment, protein-protein, protein-excipient, and protein-water interactions in lyophilized powders were identified. Myoglobin (Mb) was derivatized in solution with the heterobifunctional probe succinimidyl 4,4'-azipentanoate (SDA) and the structural integrity of the labeled protein (Mb-SDA) confirmed using CD spectroscopy and liquid chromatography/mass spectrometry (LC-MS). Mb-SDA was then formulated with and without excipients (raffinose, guanidine hydrochloride (Gdn HCl)) and lyophilized. The freeze-dried powder was irradiated with ultraviolet light at 365 nm for 30 min to produce cross-linked adducts that were analyzed at the intact protein level and after trypsin digestion. SDA-labeling produced Mb carrying up to five labels, as detected by LC-MS. Following lyophilization and irradiation, cross-linked peptide-peptide, peptide-water, and peptide-raffinose adducts were detected. The exposure of Mb side chains to the matrix was quantified based on the number of different peptide-peptide, peptide-water, and peptide-excipient adducts detected. In the absence of excipients, peptide-peptide adducts involving the CD, DE, and EF loops and helix H were common. In the raffinose formulation, peptide-peptide adducts were more distributed throughout the molecule. The Gdn HCl formulation showed more protein-protein and protein-water adducts than the other formulations, consistent with protein unfolding and increased matrix interactions. The results demonstrate that ssPC-MS can be used to distinguish excipient effects and characterize the local protein environment in lyophilized formulations with high resolution.


Assuntos
Reagentes para Ligações Cruzadas/metabolismo , Excipientes/química , Liofilização/métodos , Mioglobina/metabolismo , Fragmentos de Peptídeos/metabolismo , Fotólise , Pós/química , Animais , Química Farmacêutica , Cromatografia Líquida/métodos , Guanidina/química , Cavalos , Espectrometria de Massas/métodos , Mioglobina/química , Fragmentos de Peptídeos/química , Água/química
6.
J Vis Exp ; (98): 52503, 2015 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-25938927

RESUMO

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for characterizing lyophilized formulations of protein therapeutics. Labeling followed by suitable proteolytic digestion allows the protein structure and interactions to be mapped with peptide-level resolution. Since the protein structural elements are stabilized by a network of chemical bonds from the main-chains and side-chains of amino acids, specific labeling of atoms in the amino acid residues provides insight into the structure and conformation of the protein. In contrast to routine methods used to study proteins in lyophilized solids (e.g., FTIR), ssHDX-MS and ssPL-MS provide quantitative and site-specific information. The extent of deuterium incorporation and kinetic parameters can be related to rapidly and slowly exchanging amide pools (N fast, N slow) and directly reflects the degree of protein folding and structure in lyophilized formulations. Stable photolytic labeling does not undergo back-exchange, an advantage over ssHDX-MS. Here, we provide detailed protocols for both ssHDX-MS and ssPL-MS, using myoglobin (Mb) as a model protein in lyophilized formulations containing either trehalose or sorbitol.


Assuntos
Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , Proteínas/química , Amidas/química , Liofilização/métodos , Cinética , Peptídeos/química , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína
7.
Mol Pharm ; 10(12): 4629-39, 2013 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-24125175

RESUMO

Local side-chain interactions in lyophilized protein formulations were mapped using solid-state photolytic labeling-mass spectrometry (ssPL-MS). Photoactive amino acid analogues (PAAs) were used as probes and either added to the lyophilized matrix or incorporated within the amino acid sequence of a peptide. In the first approach, apomyoglobin was lyophilized with sucrose and varying concentrations of photoleucine (L-2-amino-4,4'-azipentanoic acid; pLeu). The lyophilized solid was irradiated at 365 nm to initiate photolabeling. The rate and extent of labeling were measured using electrospray ionization/high-performance liquid chromatography/mass spectrometry (ESI-HPLC-MS), with labeling reaching a plateau at ~30 min, forming up to six labeled populations. Bottom-up MS/MS analysis was able to provide peptide-level resolution of the location of pLeu. ssPL-MS was also able to detect differences in side-chain environment between sucrose and guanidine hydrochloride formulations. In the second approach, peptide GCG (1-8)* containing p-benzoyl-L-phenylalanine (pBpA) in the amino acid sequence was lyophilized with various excipients and irradiated. Peptide-peptide and peptide-excipient adducts were detected using MS. Top-down MS/MS on the peptide dimer provided amino acid-level resolution regarding interactions and the cross-linking partner for pBpA in the solid state. The results show that ssPL-MS can provide high-resolution information about protein interactions in the lyophilized environment.


Assuntos
Pós/química , Sequência de Aminoácidos , Aminoácidos/química , Apoproteínas/química , Benzofenonas/química , Cromatografia Líquida de Alta Pressão/métodos , Liofilização/métodos , Guanidina/química , Espectrometria de Massas/métodos , Mioglobina/química , Peptídeos/química , Fenilalanina/análogos & derivados , Fenilalanina/química , Fotólise , Sacarose/química
8.
Expert Opin Drug Deliv ; 10(8): 1155-70, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23662940

RESUMO

INTRODUCTION: Peptide and protein microarray and microneedle array technology provides direct information on protein function and potential drug targets in drug discovery and delivery. Because of this unique ability, these arrays are well suited for protein profiling, drug target identification/validation and studies of protein interaction, biochemical activity, immune responses, clinical prognosis and diagnosis and for gene, protein and drug delivery. AREAS COVERED: The aim of this review is to describe and summarize past and recent developments of microarrays in their construction, characterization and production and applications of microneedles in drug delivery. The scope and limitations of various technologies in this respect are discussed. EXPERT OPINION: This article offers a review of microarray/microneedle technologies and possible future directions in targeting and in the delivery of pharmacologically active compounds for unmet needs in biopharmaceutical research. A better understanding of the production and use of microarrays and microneedles for delivery of peptides, proteins and vaccines is needed.


Assuntos
Sistemas de Liberação de Medicamentos , Peptídeos/administração & dosagem , Preparações Farmacêuticas/administração & dosagem , Análise Serial de Proteínas , Proteínas/administração & dosagem , Vacinas/administração & dosagem , Animais , Humanos , Análise em Microsséries/instrumentação
9.
Comput Chem Eng ; 58(2013): 369-377, 2013 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-24516290

RESUMO

Lyophilization can induce aggregation in therapeutic proteins, but the relative importance of protein structure, formulation and processing conditions are poorly understood. To evaluate the contribution of protein structure to lyophilization-induced aggregation, fifteen proteins were co-lyophilized with each of five excipients. Extent of aggregation following lyophilization, measured using size-exclusion chromatography, was correlated with computational and biophysical protein structural descriptors via multiple linear regression. Descriptor selection was performed using exhaustive search and forward selection. The results demonstrate that, for a given excipient, extent of aggregation is highly correlated by eight to twelve structural descriptors. Leave-one-out cross validation showed that the correlations were able to successfully predict the aggregation for a protein "left out" of the data set. Selected descriptors varied with excipient, indicating both protein structure and excipient type contribute to lyophilization-induced aggregation. The results show some descriptors used to predict protein aggregation in solution are useful in predicting lyophilized protein aggregation.

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