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1.
Mol Pharm ; 18(8): 3116-3124, 2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34232660

RESUMO

N-terminal glutamate can cyclize to form pyroglutamate (pGlu) in pharmaceutically relevant peptides and proteins. The reaction occurs nonenzymatically during storage for monoclonal antibodies and shows a strong 'pH' dependence in solution, but the solid-state reaction has not been studied in detail. This work investigates the effect of 'pH' and buffer species on pGlu formation for a model peptide (EVQLVESGGGLVQPGGSLR) in lyophilized solids and in solution. The model peptide was formulated from 'pH' 4 to 'pH' 9 in citrate, citrate-phosphate, phosphate, and carbonate buffers and stored at 50 °C for at least 10 weeks. pGlu formation and loss of the parent peptide were monitored by reversed-phase high-performance liquid chromatography. The apparent 'pH' dependence of the reaction rate in the solid state differed markedly from that in solution. Interestingly, in the 'pH' range often used to formulate mAbs ('pH' 5.5-6), the rate of pGlu formation in the solid state was greater than that in solution. The results have implications for the rational design of stable formulations of peptides and proteins, and for the transition from solid to solution formulations during development.

2.
Mol Pharm ; 18(7): 2657-2668, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34096731

RESUMO

Mannitol, leucine, and trehalose have been widely used in spray-dried formulations, especially for inhalation formulations. The individual contribution of these excipients on protein physical stability in spray-dried solids was studied here using bovine serum albumin (BSA) as a model protein. The spray-dried solids were characterized with scanning electron microscopy, powder X-ray diffraction, and solid-state Fourier-transform infrared spectroscopy to analyze particle morphology, crystallinity, and secondary structure change, respectively. Advanced solid-state characterizations were conducted with solid-state hydrogen-deuterium exchange (ssHDX) and solid-state nuclear magnetic resonance (ssNMR) to explore protein conformation and molecular interactions in the context of the system physical stability. Trehalose remained amorphous after spray-drying and was miscible with BSA, forming hydrogen bonds to maintain protein conformation, whereby this system showed the least monomer loss in the stability study. As indicated by ssNMR, both crystalline and amorphous forms of mannitol existed in the spray-dried BSA-mannitol solids, which explained its partial stabilizing effect on BSA. Leucine showed the strongest crystallization tendency after spray-drying and did not provide a stabilizing effect due to substantial immiscibility and phase separation with BSA as a result of crystal formation. This work showed novel applications of ssNMR in examining protein conformation and protein-excipient interaction in dry formulations. Overall, our results demonstrate the pivotal role of advanced solid-state characterization techniques in understanding the physical stability of spray-dried protein solids.

3.
J Pharm Sci ; 110(6): 2379-2385, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33711346

RESUMO

Antibody drug conjugates (ADCs) have been at the forefront in cancer therapy due to their target specificity. All the FDA approved ADCs are developed in lyophilized form to minimize instability associated with the linker that connects the cytotoxic drug and the antibody during shipping and storage. We present here solid-state hydrogen-deuterium exchange with mass spectrometric analysis (ssHDX-MS) as a tool to analyze protein structure and matrix interactions for formulations of an ADC with and without commonly used excipients. We compared results of the ssHDX-MS with accelerated stability results using size-exclusion chromatography and determined that the former technique was able to successfully identify the destabilizing effects of mannitol and polysorbate 80. In comparison, Fourier-transform infrared spectroscopy results were inconclusive. The agreement between ssHDX-MS and stressed stability studies supports the potential of ssHDX-MS as a method of predicting relative stability of different formulations.


Assuntos
Medição da Troca de Deutério , Imunoconjugados , Deutério , Estabilidade de Medicamentos , Liofilização , Hidrogênio , Espectrometria de Massas
4.
Int J Pharm ; 596: 120263, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33486024

RESUMO

Lyophilized powders containing myoglobin and various excipients were subjected to ssHDX-MS at different temperatures and D2O vapor activity (RH). Deuterium incorporation was fitted to a bi-exponential association model for each formulation and the dependence of regression parameters on temperature and RH was evaluated. Data fitted best to a simplified model in which the slow exponential term was considered invariant with temperature and RH while the fast exponential term retained its temperature and RH dependence. This suggests that rapid rate processes such as water vapor sorption and initial deuterium labeling may be more dependent on temperature and RH than slower processes such as sequential exchange and transport in the solid matrix.


Assuntos
Medição da Troca de Deutério , Espectrometria de Massa com Troca Hidrogênio-Deutério , Liofilização , Umidade , Hidrogênio , Temperatura
5.
Int J Pharm ; 594: 120169, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33333176

RESUMO

This study aims to determine the impacts of drying method and excipient on changes in protein structure and physical stability of model protein solids. Protein solids containing one of two model proteins (lysozyme or myoglobin) were produced with or without excipients (sucrose or mannitol) using freeze drying or spray freeze drying (SFD). The protein powders were then characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), differential scanning calorimetry (DSC), circular dichroism spectrometry (CD), size exclusion chromatography (SEC), BET surface area measurements and solid-state hydrogen deuterium exchange with mass spectrometry (ssHDX-MS). ssFTIR and CD could identify little to no difference in structure of the proteins in the formulation. ssHDX-MS was able to identify the population heterogeneity, which was undetectable by conventional characterization techniques of ssFTIR and CD. ssHDX-MS metrics such as Dmax and peak area showed a good correlation with the protein physical instability (loss of the monomeric peak area by size exclusion chromatography) in 90-day stability studies conducted at 40 °C for lysozyme. Higher specific surface area was associated with greater loss in monomer content for myoglobin-mannitol formulations as compared to myoglobin-only formulations. Spray freeze drying seems a viable manufacturing technique for protein solids with appropriate optimization of formulations. The differences observed within the formulations and between the processes using ssHDX-MS, BET surface area measurements and SEC in this study provide an insight into the influence of drying methods and excipients on protein physical stability.


Assuntos
Química Farmacêutica , Excipientes , Composição de Medicamentos , Estabilidade de Medicamentos , Liofilização , Espectrometria de Massas
6.
Biophys J ; 120(1): 86-100, 2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33220304

RESUMO

Some therapeutic peptides self-assemble in solution to form ordered, insoluble, ß-sheet-rich amyloid fibrils. This physical instability can result in reduced potency, cause immunogenic side effects, and limit options for formulation. Understanding the mechanisms of fibrillation is key to developing rational mitigation strategies. Here, amide hydrogen-deuterium exchange with mass spectrometric analysis (HDX-MS) coupled with proteolytic digestion was used to identify the early stage interactions leading to fibrillation of human calcitonin (hCT), a peptide hormone important in calcium metabolism. hCT fibrillation kinetics was sigmoidal, with lag, growth, and plateau phases as shown by thioflavin T and turbidity measurements. HDX-MS of fibrillating hCT (pH 7.4; 25°C) suggested early involvement of the N-terminal (1-11) and central (12-19) fragments in interactions during the lag phase, whereas C-terminal fragments (20-32 and 26-32) showed limited involvement during this period. The residue-level information was used to develop phosphorylated hCT analogs that showed modified fibrillation that depended on phosphorylation site. Phosphorylation in the central region resulted in complete inhibition of fibrillation for the phospho-Thr-13 hCT analog, whereas phosphorylation in the N-terminal and C-terminal regions inhibited but did not prevent fibrillation. Reduction of the Cys1-Cys7 disulfide bond resulted in faster fibrillation with involvement of different hCT residues as indicated by pulsed HDX-MS. Together, the results demonstrate that small structural changes have significant effects on hCT fibrillation and that understanding these effects can inform the rational development of fibrillation-resistant hCT analogs.


Assuntos
Amiloide , Calcitonina , Amiloide/metabolismo , Calcitonina/metabolismo , Dissulfetos , Humanos , Cinética , Fosforilação
7.
Mol Pharm ; 17(9): 3541-3552, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32786954

RESUMO

The reversibility of solid-state hydrogen-deuterium exchange (ssHDX) and the effects of prehydration on the rate and extent of deuterium incorporation were evaluated using poly-d,l-alanine (PDLA) peptides colyophilized with various excipients. In prehydration studies, samples were equilibrated at a controlled relative humidity (6% or 11% RH) for 12 h and then transferred to corresponding D2O humidity conditions (6% or 11% RD) for deuterium labeling. In amorphous samples, the rate and extent of deuterium incorporation were similar in prehydrated samples and controls not subjected to prehydration. In reversibility studies, PDLA samples were maximally deuterated in controlled D2O humidity conditions (6% or 11% RD) and then transferred to corresponding H2O relative humidity (0%, 6%, 11%, or 43% RH). Hysteresis in deuterium removal was observed when compared with the deuterium incorporation kinetics for all formulations and conditions, confirming that the reaction is reversible in the solid state and that the forward and reverse processes differ. The extent of deuterium loss reached a plateau that depended on the delabeling relative humidity. Reverse reaction rate constants were quantified using a first-order kinetic model, a limiting case of the reversible first-order model applicable under sink conditions. For other conditions, plateau (steady-state) deuteration levels were related to forward and reverse rate constants in a reversible first-order kinetic model. The results support a mechanistic interpretation of ssHDX kinetics as a reversible first-order process, in which the forward (deuteration) rate depends on the activity of the deuterium donor.


Assuntos
Deutério/química , Hidrogênio/química , Química Farmacêutica/métodos , Medição da Troca de Deutério/métodos , Excipientes/química , Umidade , Cinética , Peptídeos/química
8.
Mol Pharm ; 17(9): 3501-3512, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32672982

RESUMO

The effects of peptide secondary structure on the rate and extent of deuterium incorporation in solid-state hydrogen deuterium exchange mass spectrometry (ssHDX-MS) were assessed. Unstructured poly-d,l-alanine (PDLA) peptides, an α-helical model peptide (peptide A) and a ß-sheet model peptide (peptide B), were co-lyophilized with various excipients. Peptide structures were confirmed in solution using circular dichroism (CD) spectroscopy and in the solid state with Fourier transform infrared (FTIR) spectroscopy. ssHDX-MS was conducted at two relative humidities (11 and 23% RH D2O) and deuterium uptake kinetics were monitored over 10 days. The relative contributions of peptide secondary structure and matrix interactions to deuteration incorporation were evaluated by comparing the ssHDX-MS kinetic data of peptide A and peptide B with PDLA of similar molecular weight. The results demonstrate that both peptide secondary structure and interactions with the solid matrix contribute to the protection from exchange in ssHDX-MS. A quantitative data analysis and interpretation method is presented, in which the number of protected amide bonds is calculated as the difference between the maximum deuterium incorporation in solution and in solid samples.


Assuntos
Deutério/química , Hidrogênio/química , Peptídeos/química , Medição da Troca de Deutério/métodos , Excipientes/química , Cinética , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta
9.
J Pharm Sci ; 109(1): 709-718, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31034909

RESUMO

Excipients used in lyophilized protein drug products are often selected by a trial-and-error method, in part, because the analytical methods used to detect protein-excipient interactions in lyophilized solids are limited. In this study, photolytic labeling was used to probe interactions between salmon calcitonin (sCT) and excipients in lyophilized solids. Two diazirine-derived photo-excipients, photo-leucine (pLeu) and photo-glucosamine (pGlcN), were incorporated into lyophilized solids containing sCT, together with an unlabeled excipient (sucrose or histidine) at prelyophilization pH values from 6 to 9.9. Commercially available pLeu was selected as an ionizable photo-excipient and amino acid analog, while pGlcN was synthesized as an analog of sugar-based excipients. Photolytic labeling was induced by exposing the solids to UV light (365 nm, 30-60 min), and the resulting products were identified and quantified with liquid-chromatography mass spectrometry. The distribution of photo-reaction products was affected by the photoreactive reagent used, the type of unlabeled excipient, and the solution pH before lyophilization. When other components of the solid were identical, the extent and sites of labeling on sCT were different for pGlcN and pLeu. The results suggest that ionizable and nonionizable excipients interact differently with sCT in lyophilized solids and that photo-excipients can be used to map these interactions.


Assuntos
Calcitonina/química , Excipientes/efeitos da radiação , Glucosamina/efeitos da radiação , Leucina/efeitos da radiação , Raios Ultravioleta , Composição de Medicamentos , Excipientes/química , Liofilização , Glucosamina/química , Histidina/química , Concentração de Íons de Hidrogênio , Leucina/química , Fotólise , Estudo de Prova de Conceito , Sacarose/química , Fatores de Tempo
10.
Pharm Res ; 37(1): 14, 2019 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-31873808

RESUMO

PURPOSE: The aim of this study is to determine the effects of saccharide-containing excipients on the surface composition of spray-dried protein formulations and their matrix heterogeneity. METHODS: Spray-dried formulations of myoglobin or bovine serum albumin (BSA) were prepared without excipient or with sucrose, trehalose, or dextrans. Samples were characterized by solid-state Fourier-transform infrared spectroscopy (ssFTIR), differential scanning calorimetry (DSC), size exclusion chromatography (SEC) and scanning electron microscopy (SEM). Protein surface coverage was determined by X-ray photoelectron spectroscopy (XPS), while conformational differences were determined by solid-state hydrogen/deuterium exchange with mass spectrometry (ssHDX-MS). RESULTS: Structural differences were exhibited with the inclusion of different excipients, with dextran formulations indicating perturbation of secondary structure. XPS indicated sucrose and trehalose reduced protein surface concentration better than dextran-containing formulations. Using ssHDX-MS, the amount of deuterium incorporation and populations present were the largest in the samples processed with dextrans. Linear correlation was found between protein surface coverage and ssHDX-MS peak area (R2 = 0.853) for all formulations with saccharide-containing excipients. CONCLUSIONS: Lower molecular weight species of saccharides tend to enrich the particle surface and reduce protein concentration at the air-liquid interface, resulting in reduced population heterogeneity and improved physical stability, as identified by ssHDX-MS.


Assuntos
Excipientes/química , Mioglobina/química , Soroalbumina Bovina/química , Química Farmacêutica/métodos , Dessecação/métodos , Deutério/química , Dextranos/química , Espectrometria de Massas/métodos , Sacarose/química , Propriedades de Superfície , Trealose/química
11.
Mol Pharm ; 16(11): 4485-4495, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31568722

RESUMO

Solid-state hydrogen-deuterium exchange with mass spectrometry (ssHDX-MS) was evaluated as an analytical method to rapidly screen and select an optimal lyophilized fragment antigen binding protein (Fab) formulation and the optimal lyophilization cycle. ssHDX-MS in lyophilized Fab formulations, varying in stabilizer type and stabilizer/protein ratio, was conducted under controlled humidity and temperature. The extent of deuterium incorporation was measured using mass spectrometry and correlated with solid-state stress degradation at 50 °C as measured by size exclusion chromatography (SEC) and ion-exchange chromatography (IEC). ssHDX-MS was also used to evaluate the impact of three different types of lyophilization processing on storage stability: controlled ice nucleation (CN), uncontrolled ice nucleation (UCN), and annealing (AN). The extent of deuterium incorporation for different Fab formulations agreed with the order of solid-state stress degradation, with formulations having lower deuterium incorporation showing lower stress-induced degradation (aggregation and charge modifications). For lyophilization processing, no significant effect of ice nucleation was observed in either solid-state stress degradation or in the extent of deuterium incorporation for high concentration Fab formulations (25 mg/mL). In contrast, for low concentration Fab formulations (2.5 mg/mL), solid-state stability from different lyophilization processes correlated with the extent of deuterium incorporation. The order of solid-state degradation (AN < CN < UCN) was the same as the extent of deuterium incorporation on ssHDX-MS (AN < CN < UCN). The extent of deuterium incorporation on ssHDX-MS correlated well with the solid-state stress degradation for different Fab formulations and lyophilization processing methods. Thus, ssHDX-MS can be used to rapidly screen and optimize the formulation and lyophilization process for a lyophilized Fab, reducing the need for time-consuming stress degradation studies.


Assuntos
Deutério/química , Hidrogênio/química , Fragmentos Fab das Imunoglobulinas/química , Química Farmacêutica/métodos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia por Troca Iônica/métodos , Medição da Troca de Deutério/métodos , Liofilização/métodos , Espectrometria de Massa com Troca Hidrogênio-Deutério/métodos , Cinética , Ligação Proteica
12.
Int J Pharm ; 568: 118512, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31301464

RESUMO

Deuterium incorporation in solid-state hydrogen deuterium exchange with mass spectrometry (ssHDX-MS) has been correlated with protein aggregation on storage in sugar-based solid matrices. Here, the effects of sucrose, arginine and histidine buffer on the rate of aggregation of a lyophilized monoclonal antibody (mAb) were assessed using design of experiments (DoE) and response surface methodology. Lyophilized formulations were characterized using ssHDX-MS and Fourier transform infrared spectroscopy (ssFTIR) to assess potential correlation with stability in solid state. The samples were subjected to storage stability at 5 °C and stressed stability at 40 °C/75% RH for 6 months, and the aggregation rate was measured using size exclusion chromatography (SEC). Different levels of arginine had no significant effect on deuterium uptake in ssHDX-MS, although stability studies showed that aggregation rate decreased with increasing arginine concentration. Similarly, when histidine buffer was replaced with phosphate buffer at the same pH and molarity, ssHDX-MS showed no differences in deuterium uptake, but storage stability studies showed a significant increase in aggregation rate. The results suggest that proteins can be stabilized in amorphous solids by ionic interactions which ssHDX-MS does not detect, an important indication of the limitations of the method.


Assuntos
Anticorpos Monoclonais/química , Arginina/química , Imunoglobulina G/química , Sacarose/química , Deutério/química , Medição da Troca de Deutério , Histidina/química , Espectrometria de Massas , Fosfatos/química , Conformação Proteica , Estabilidade Proteica
13.
Int J Pharm ; 567: 118470, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31252148

RESUMO

Powders containing one of four model proteins (myoglobin, bovine serum albumin, lysozyme, ß-lactoglobulin) were formulated with either sucrose, trehalose, or mannitol and dried using lyophilization or spray-drying. The powders were characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), solid-state fluorescence spectroscopy, differential scanning calorimetry (DSC) and solid-state hydrogen/deuterium exchange mass spectrometry (ssHDX-MS). ssFTIR and fluorescence spectroscopy identified minor structural differences among powders with different excipients and drying methods for some proteins. Using ssHDX-MS, differences in protein structure were observed among protein formulations containing sucrose or trehalose and mannitol, and/or with varying processing conditions, including proteins like ß-lactoglobulin, for which standard characterization techniques showed no differences. Proteins processed by spray-drying typically showed greater heterogeneity by ssHDX-MS than those lyophilized; these differences were not detected by ssFTIR or solid-state fluorescence spectroscopy. The ssHDX-MS metrics were better correlated with protein physical instability measured by size-exclusion chromatography in 90-day stability studies (40 °C, 33% RH) than with the results of DSC, ssFTIR, or fluorescence spectroscopy. Thus, ssHDX-MS detected subtle changes in conformation and/or matrix interactions for these proteins that were correlated with storage stability, suggesting that the method can be used to design robust solid-state pharmaceutical protein products more rapidly.


Assuntos
Dessecação/métodos , Lactoglobulinas/química , Muramidase/química , Mioglobina/química , Soroalbumina Bovina/química , Excipientes/química , Liofilização , Espectrometria de Massa com Troca Hidrogênio-Deutério , Conformação Proteica , Estabilidade Proteica
14.
Mol Pharm ; 16(7): 2935-2946, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31244225

RESUMO

Solid-state hydrogen-deuterium exchange mass spectrometry (ssHDX-MS) has been developed to study proteins in amorphous solids, but the relative contributions of protein structure and protein-matrix interactions to exchange are not known. In this work, short unstructured poly-d,l-alanine (PDLA) peptides were colyophilized with sucrose, trehalose, mannitol, sodium chloride, or guanidine hydrochloride to quantify the contributions of protein-matrix interactions to deuterium uptake in ssHDX-MS in the absence of a higher order structure. Deuterium incorporation differed with the excipient type and relative humidity (RH) in D2O(g), effects that were not observed in solution controls and are not described by the Linderstrom-Lang model for solution HDX. A reversible pseudo first-order kinetic model for deuterium uptake in ssHDX-MS is proposed. The model agrees with the experimentally observed dependences of the apparent deuteration rate and plateau value on RH in ssHDX-MS of PDLA and reduces to the Linderstrom-Lang limit when the forward rate of exchange is much greater than the reverse rate.


Assuntos
Química Farmacêutica/métodos , Composição de Medicamentos/métodos , Excipientes/química , Espectrometria de Massa com Troca Hidrogênio-Deutério/métodos , Peptídeos/química , Deutério/química , Liofilização/métodos , Guanidina/química , Umidade , Cinética , Manitol/química , Modelos Químicos , Estrutura Secundária de Proteína , Cloreto de Sódio/química , Sacarose/química , Trealose/química
15.
Mol Pharm ; 16(3): 1053-1064, 2019 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-30721080

RESUMO

Interactions of a lyophilized peptide with water and excipients in a solid matrix were explored using photolytic labeling. A model peptide "KLQ" (Ac-QELHKLQ-NHCH3) was covalently labeled with NHS-diazirine (succinimidyl 4,4'-azipentanoate), and the labeled peptide (KLQ-SDA) was formulated and exposed to UV light in both solution and lyophilized solids. Solid samples contained the following excipients at a 1:400 molar ratio: sucrose, trehalose, mannitol, histidine, or arginine. Prior to UV exposure, the lyophilized solids were exposed to various relative humidity (RH) environments (8, 13, 33, 45, and 78%), and the resulting solid moisture content (Karl Fischer titration) and glass transition temperature ( Tg; differential scanning calorimetry, DSC) were measured. To initiate photolytic labeling, solution and solid samples were exposed to UV light at 365 nm for 30 min. Photolytic-labeling products were quantified using reversed-phase high-performance liquid chromatography (rp-HPLC) and mass spectrometry (MS). In lyophilized solids, studies excluding oxygen and using H218O confirmed that the source of oxygen in KLQ adducts with a mass increase of 18 amu are attributable to reaction with water, while those with a mass increase of 16 amu are not attributable to reaction with either water or molecular oxygen. In solids containing sucrose or trehalose, peptide-excipient adducts decreased with increasing solid moisture content, while peptide-water adducts increased only at lower RH exposure and then plateaued, in partial agreement with the water replacement hypothesis.


Assuntos
Química Farmacêutica/métodos , Composição de Medicamentos/métodos , Liofilização/métodos , Peptídeos/química , Fotólise/efeitos da radiação , Água/química , Varredura Diferencial de Calorimetria , Cromatografia de Fase Reversa , Diazometano/análogos & derivados , Diazometano/química , Excipientes/química , Umidade , Ligação de Hidrogênio , Espectrometria de Massas , Oxigênio/química , Sacarose/química , Temperatura de Transição , Trealose/química , Raios Ultravioleta , Vitrificação
16.
J Pharm Sci ; 108(2): 791-797, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30339867

RESUMO

In this mini-review, the major types of photolytic labeling reagents are presented together with their reaction mechanisms. The applications of photolytic labeling in protein drug discovery and development are then discussed; these have expanded from studies of protein-protein interactions in vivo to protein-matrix interactions in lyophilized solids. The mini-review concludes with recommendations for further development of the approach, which include the need for new and more chemically diverse photo-reactive reagents and better understanding of the mechanisms of photolytic labeling reactions in various media.


Assuntos
Desenvolvimento de Medicamentos/métodos , Descoberta de Drogas/métodos , Proteínas/metabolismo , Animais , Humanos , Indicadores e Reagentes/química , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas/análise
17.
Mol Pharm ; 15(7): 2797-2806, 2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29792715

RESUMO

Peptide-matrix interactions in lyophilized solids were explored using photolytic labeling with reversed phase high performance liquid chromatography (rp-HPLC) and mass spectrometric (MS) analysis. A model peptide (Ac-QELHKLQ-NHCH3) derived from salmon calcitonin was first labeled with a heterobifunctional cross-linker NHS-diazirine (succinimidyl 4,4'-azipentanoate; SDA) at Lys5 in solution, with ∼100% conversion. The SDA labeled peptide was then formulated with the following excipients at a 1:400 molar ratio and lyophilized: sucrose, trehalose, mannitol, histidine, arginine, urea, and NaCl. The lyophilized samples and corresponding solution controls were exposed to UV at 365 nm to induce photolytic labeling, and the products were identified by MS and quantified with rp-HPLC or MS. Peptide-excipient adducts were detected in the lyophilized solids except the NaCl formulation. With the exception of the histidine formulation, peptide-excipient adducts were not detected in solution and the fractional conversion to peptide-water adducts in solution was significantly greater than in lyophilized solids, as expected. In lyophilized solids, the fractional conversion to peptide-water adducts was poorly correlated with bulk moisture content, suggesting that the local water content near the labeled lysine residue differs from the measured bulk average. In lyophilized solids, the fractional conversion to peptide-excipient adducts was assessed using MS extracted ion chromatograms (EIC); subject to the assumption of equal ionization efficiencies, the fractional conversion to excipient adducts varied with excipient type. The results demonstrate that the local environment near the lysine residue of the peptide in the lyophilized solids can be quantitatively probed with a photolytic labeling method.


Assuntos
Composição de Medicamentos/métodos , Excipientes/química , Peptídeos/química , Coloração e Rotulagem/métodos , Química Farmacêutica , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Reagentes para Ligações Cruzadas/química , Estabilidade de Medicamentos , Liofilização , Espectrometria de Massas/métodos , Peptídeos/metabolismo , Peptídeos/uso terapêutico , Fotólise , Estabilidade Proteica
18.
Mol Pharm ; 15(2): 356-368, 2018 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-29355022

RESUMO

Therapeutic proteins are often formulated as lyophilized products to improve their stability and prolong shelf life. The stability of proteins in the solid-state has been correlated with preservation of native higher order structure and/or molecular mobility in the solid matrix, with varying success. In the studies reported here, we used solid-state hydrogen-deuterium exchange with mass spectrometric analysis (ssHDX-MS) to study the conformation of an IgG1 monoclonal antibody (mAb) in lyophilized solids and related the extent of ssHDX to aggregation during storage in the solid phase. The results demonstrate that the extent of ssHDX correlated better with aggregation rate during storage than did solid-state Fourier-transform infrared (ssFTIR) spectroscopic measurements. Interestingly, adding histidine to sucrose at different formulation pH conditions decreased aggregation of the mAb, an effect that did not correlate with structural or conformational changes as measured by ssFTIR or ssHDX-MS. Moreover, peptide-level ssHDX-MS analysis in four selected formulations demonstrated global changes across the structure of the mAb when lyophilized with sucrose, trehalose, or mannitol, whereas site-specific changes were observed when lyophilized with histidine as the sole excipient.


Assuntos
Anticorpos Monoclonais/química , Química Farmacêutica/métodos , Imunoglobulina G/química , Medição da Troca de Deutério/métodos , Estabilidade de Medicamentos , Excipientes/química , Liofilização , Concentração de Íons de Hidrogênio , Espectrometria de Massas/métodos , Peptídeos/química , Conformação Proteica , Espectroscopia de Infravermelho com Transformada de Fourier/métodos
19.
Pharm Res ; 35(1): 12, 2018 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-29299701

RESUMO

PURPOSE: Lyophilization and spray drying are widely used to manufacture solid forms of therapeutic proteins. Lyophilization is used to stabilize proteins vulnerable to degradation in solution, whereas spray drying is mainly used to prepare inhalation powders or as an alternative to freezing for storing bulk drug substance. Both processes impose stresses that may adversely affect protein structure, stability and bioactivity. Here, we compared lyophilization with and without controlled ice nucleation, and spray drying for their effects on the solid-state conformation and matrix interactions of a model IgG1 monoclonal antibody (mAb). METHODS: Solid-state conformation and matrix interactions of the mAb were probed using solid-state hydrogen-deuterium exchange with mass spectrometric analysis (ssHDX-MS), and solid-state Fourier transform infrared (ssFTIR) and solid-state fluorescence spectroscopies. RESULTS: mAb conformation and/or matrix interactions were most perturbed in mannitol-containing samples and the distribution of states was more heterogeneous in sucrose and trehalose samples that were spray dried. CONCLUSIONS: The findings demonstrate the sensitivity of ssHDX-MS to changes weakly indicated by spectroscopic methods, and support the broader use of ssHDX-MS to probe formulation and process effects on proteins in solid samples.


Assuntos
Medição da Troca de Deutério/métodos , Deutério/química , Hidrogênio/química , Imunoglobulina G/química , Espectrometria de Massas/métodos , Química Farmacêutica/métodos , Cristalização , Liofilização/métodos , Humanos , Manitol/química , Microscopia Eletrônica de Varredura/métodos , Pós/química , Ligação Proteica , Conformação Proteica , Espectrometria de Fluorescência/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Sacarose/química , Trealose/química , Difração de Raios X/métodos
20.
Mol Pharm ; 15(1): 1-11, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29182876

RESUMO

Solid state hydrogen-deuterium exchange with mass spectrometric analysis (ssHDX-MS) has been used to assess protein conformation and matrix interactions in lyophilized solids. ssHDX-MS metrics have been previously correlated to the formation of aggregates of lyophilized myoglobin on storage. Here, ssHDX-MS was applied to lyophilized monoclonal antibody (mAb) formulations and correlated to their long-term stability. After exposing lyophilized samples to D2O(g), the amount of deuterium incorporated at various time points was determined by mass spectrometry for four different lyophilized mAb formulations. Hydrogen-deuterium exchange data were then correlated with mAb aggregation and chemical degradation, which was obtained in stability studies of >2.5 years. Deuterium uptake on ssHDX-MS of four lyophilized mAb formulations determined at the initial time point prior to storage in the dry state was directly and strongly correlated with the extent of aggregation and chemical degradation during storage. Other measures of physical and chemical properties of the solids were weakly or poorly correlated with stability. The data demonstrate, for the first time, that ssHDX-MS results are highly correlated with the stability of lyophilized mAb formulations. The findings thus suggest that ssHDX-MS can be used as an early read-out of differences in long-term stability between formulations helping to accelerate formulation screening and selection.


Assuntos
Anticorpos Monoclonais/química , Medição da Troca de Deutério/métodos , Liofilização/métodos , Espectrometria de Massas/métodos , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Cromatografia Líquida , Composição de Medicamentos , Microscopia Eletrônica de Varredura
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