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1.
Mol Pharm ; 21(5): 2223-2237, 2024 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-38552144

RESUMO

The fibrillation of therapeutic peptides can present significant quality concerns and poses challenges for manufacturing and storage. A fundamental understanding of the mechanisms of fibrillation is critical for the rational design of fibrillation-resistant peptide drugs and can accelerate product development by guiding the selection of solution-stable candidates and formulations. The studies reported here investigated the effects of structural modifications on the fibrillation of a 29-residue peptide (PepA) and two sequence modified variants (PepB, PepC). The C-terminus of PepA was amidated, whereas both PepB and PepC retained the carboxylate, and Ser16 in PepA and PepB was substituted with a helix-stabilizing residue, α-aminoisobutyric acid (Aib), in PepC. In thermal denaturation studies by far-UV CD spectroscopy and fibrillation kinetic studies by fluorescence and turbidity measurements, PepA and PepB showed heat-induced conformational changes and were found to form fibrils, whereas PepC did not fibrillate and showed only minor changes in the CD signal. Pulsed hydrogen-deuterium exchange mass spectrometry (HDX-MS) showed a high degree of protection from HD exchange in mature PepA fibrils and its proteolytic fragments, indicating that most of the sequence had been incorporated into the fibril structure and occurred nearly simultaneously throughout the sequence. The effects of the net peptide charge and formulation pH on fibrillation kinetics were investigated. In real-time stability studies of two formulations of PepA at pH's 7.4 and 8.0, analytical methods detected significant changes in the stability of the formulations at different time points during the study, which were not observed during accelerated studies. Additionally, PepA samples were withdrawn from real-time stability and subjected to additional stress (40 °C, continuous shaking) to induce fibrillation; an approach that successfully amplified oligomers or prefibrillar species previously undetected in a thioflavin T assay. Taken together, these studies present an approach to differentiate and characterize fibrillation risk in structurally related peptides under accelerated and real-time conditions, providing a model for rapid, iterative structural design to optimize the stability of therapeutic peptides.


Assuntos
Desenho de Fármacos , Peptídeos , Peptídeos/química , Dicroísmo Circular/métodos , Estabilidade de Medicamentos , Sequência de Aminoácidos , Cinética , Ácidos Aminoisobutíricos/química , Estabilidade Proteica , Espectrometria de Massas/métodos
2.
Mol Pharm ; 21(4): 1965-1976, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38516985

RESUMO

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) previously elucidated the interactions between excipients and proteins for liquid granulocyte colony stimulating factor (G-CSF) formulations, confirming predictions made using computational structure docking. More recently, solid-state HDX mass spectrometry (ssHDX-MS) was developed for proteins in the lyophilized state. Deuterium uptake in ssHDX-MS has been shown for various proteins, including monoclonal antibodies, to be highly correlated with storage stability, as measured by protein aggregation and chemical degradation. As G-CSF is known to lose activity through aggregation upon lyophilization, we applied the ssHDX-MS method with peptide mapping to four different lyophilized formulations of G-CSF to compare the impact of three excipients on local structure and exchange dynamics. HDX at 22 °C was confirmed to correlate well with the monomer content remaining after lyophilization and storage at -20 °C, with sucrose providing the greatest protection, and then phenylalanine, mannitol, and no excipient leading to progressively less protection. Storage at 45 °C led to little difference in final monomer content among the formulations, and so there was no discernible relationship with total deuterium uptake on ssHDX. Incubation at 45 °C may have led to a structural conformation and/or aggregation mechanism no longer probed by HDX at 22 °C. Such a conformational change was observed previously at 37 °C for liquid-formulated G-CSF using NMR. Peptide mapping revealed that tolerance to lyophilization and -20 °C storage was linked to increased stability in the small helix, loop AB, helix C, and loop CD. LC-MS HDX and NMR had previously linked loop AB and loop CD to the formation of a native-like state (N*) prior to aggregation in liquid formulations, suggesting a similar structural basis for G-CSF aggregation in the liquid and solid states.


Assuntos
Medição da Troca de Deutério , Fator Estimulador de Colônias de Granulócitos , Humanos , Deutério/química , Medição da Troca de Deutério/métodos , Excipientes/química , Fator Estimulador de Colônias de Granulócitos/química , Espectrometria de Massas/métodos , Proteínas/química
3.
Mol Pharm ; 20(8): 4086-4099, 2023 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-37466053

RESUMO

The effects of atomic layer (ALC) coating on physical properties and storage stability were examined in solid powders containing myoglobin, a model protein. Powders containing myoglobin and mannitol (1:1 w/w) were prepared by lyophilization or spray drying and subjected to aluminum oxide or silicon oxide ALC coating. Uncoated samples of these powders as well as coated and uncoated samples of myoglobin as received served as controls. After preparation (t0), samples were analyzed for moisture content, reconstitution time, myoglobin secondary structure, crystallinity, and protein aggregate content. Samples were stored for 3 months (t3) under controlled conditions (53% RH, 40 °C) in both open and closed vials and then analyzed as above. At t3, the recovery of soluble native (i.e., monomeric) protein depended on formulation, coating type, and drying method and was up to 2-fold greater in coated samples than in uncoated controls. Promisingly, some samples with high recovery also showed low soluble aggregate content (<10%) at t3 and low total monomer loss; the latter was correlated to sample moisture content. Overall, the results demonstrate that ALC coatings can stabilize solid protein formulations during storage, providing benefits over uncoated controls.


Assuntos
Mioglobina , Mioglobina/química , Pós/química , Liofilização , Estrutura Secundária de Proteína , Estabilidade de Medicamentos
4.
Biophys J ; 121(23): 4505-4516, 2022 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-36325616

RESUMO

Insulin forms amyloid fibrils under slightly destabilizing conditions, and B-chain residues are thought to play an important role in insulin fibrillation. Here, pulsed hydrogen-deuterium exchange mass spectrometry (HDX-MS), far-UV circular dichroism spectroscopy, thioflavin T (ThioT) fluorescence, turbidity, and soluble fraction measurements were used to monitor the kinetics and mechanisms of fibrillation of human insulin B-chain (INSB) in acidic solution (1 mg/mL, pH 4.5) under stressed conditions (40°C, continuous shaking). Initially, INSB rapidly formed ß-sheet-rich oligomers that were protected from HD exchange and showed weak ThioT binding. Subsequent fibril growth and maturation was accompanied by even greater protection from HD exchange and stronger ThioT binding. With peptic digestion of deuterated INSB, HDX-MS suggested early involvement of the N-terminal (1-11, 1-15) and central (12-15, 16-25) fragments in fibril-forming interactions, whereas the C-terminal fragment (25-30) showed limited involvement. The results provide mechanistic understanding of the intermolecular interactions and structural changes during INSB fibrillation under stressed conditions and demonstrate the application of pulsed HDX-MS to probe peptide fibrillation.


Assuntos
Espectrometria de Massa com Troca Hidrogênio-Deutério , Insulina , Humanos , Medição da Troca de Deutério , Hidrogênio
5.
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
6.
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.


Assuntos
Concentração de Íons de Hidrogênio , Peptídeos/química , Ácido Pirrolidonocarboxílico/química , Anticorpos Monoclonais/química , Soluções Tampão , Catálise , Química Farmacêutica/métodos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Ciclização , Estabilidade de Medicamentos , Liofilização , Cinética , Estabilidade Proteica , Soluções
7.
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.


Assuntos
Excipientes/metabolismo , Manitol/química , Pós/metabolismo , Soroalbumina Bovina/metabolismo , Animais , Bovinos , Cristalização , Excipientes/química , Liofilização , Pós/química , Conformação Proteica , Estabilidade Proteica , Soroalbumina Bovina/química
8.
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
9.
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
10.
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
11.
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
13.
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
14.
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
15.
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 de Ligações Cruzadas/química , Estabilidade de Medicamentos , Liofilização , Espectrometria de Massas/métodos , Peptídeos/metabolismo , Peptídeos/uso terapêutico , Fotólise , Estabilidade Proteica
16.
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
17.
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
18.
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
19.
Biochemistry ; 56(50): 6555-6564, 2017 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-29166011

RESUMO

Previously, we demonstrated that binding of a ligand to Escherichia coli cofactor-dependent phosphoglycerate mutase (dPGM), a homodimeric protein, is energetically coupled with dimerization. The equilibrium unfolding of dPGM occurs with a stable, monomeric intermediate. Binding of several nonsubstrate metabolites stabilizes the dimeric native form over the monomeric intermediate, reducing the population of the intermediate. Both the active site and the dimer interface appear to be unfolded in the intermediate. We hypothesized that a loop containing residues 118-152 was responsible for the energetic coupling between the dimer interface and the distal active site and was unfolded in the intermediate. Here, we investigated the structure of the dPGM intermediate by probing side-chain interactions and solvent accessibility of the peptide backbone. By comparing the effect of a mutation on the global stability and the stability of the intermediate, we determine an equilibrium φ value (φeq value), which provides information about whether side-chain interactions are retained or lost in the intermediate. Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) was used to investigate differences in the solvent accessibility of the peptide backbone in the intermediate and native forms of dPGM. The results of φeq value analysis and HDX-MS reveal the least stable folding unit of dPGM, which is unfolded in the intermediate and links the active site to the dimer interface. The structure of the intermediate reveals how the cooperative network of residues in dPGM gives rise to the observed energetic coupling between dimerization and ligand binding.


Assuntos
Fosfoglicerato Mutase/química , Fosfoglicerato Mutase/metabolismo , Sequência de Aminoácidos , Deutério , Dimerização , Escherichia coli/química , Proteínas de Escherichia coli/química , Hidrogênio , Ligantes , Espectrometria de Massas/métodos , Modelos Moleculares , Fosfoglicerato Mutase/genética , Ligação Proteica/fisiologia , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína
20.
Pharm Res ; 33(6): 1370-82, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26887678

RESUMO

PURPOSE: Thiol-disulfide exchange was monitored in recombinant human growth hormone (hGH) and in model tryptic peptides derived from hGH to investigate the effects of higher-order structure on the reaction. METHODS: Different free thiol-containing peptides, varying in length and amino acid sequence, were used to initiate the reaction at pH 7.0 and 37°C in hGH. Protein samples were digested with trypsin and analyzed for native disulfides, scrambled disulfides and free thiols using LC/MS. The loss of native disulfide and disulfide exchange was compared with model peptides derived from hGH. RESULTS: Loss of native disulfide in cyclic (cT20-T21) and linear peptides (T20-T21pep) derived from the C-terminal hGH disulfide during the first 60 min of reaction was greater than loss of the C-terminal disulfide in hGH itself. Of the thiols tested, glutathione (GSH) was the most reactive, forming the highest percentage of mixed disulfides in intact hGH and in the model peptides. At longer reaction times (>240 min), native disulfides in both hGH and cT20-T21 were regenerated. The fastest rates of regeneration were observed for Cys and the di- or tripeptide containing an Arg residue adjacent to Cys, suggesting that they may be useful in refolding. CONCLUSIONS: Thiol-disulfide exchange reactions in hGH and related model peptides were influenced by higher order structure, by the size of the thiol reactant and by an Arg residue adjacent to Cys in the thiol reactant. Reduction of disulfide bonds in hGH did not affect higher order structure as measured by CD and HDX-MS.


Assuntos
Dissulfetos/química , Hormônio do Crescimento Humano/química , Compostos de Sulfidrila/química , Arginina , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Cisteína , Estabilidade de Medicamentos , Glutationa/química , Humanos , Cinética , Espectrometria de Massas , Modelos Químicos , Fragmentos de Peptídeos/química , Conformação Proteica , Desnaturação Proteica , Redobramento de Proteína , Estabilidade Proteica , Proteínas Recombinantes/química , Relação Estrutura-Atividade , Tripsina/química
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