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1.
J Endocr Soc ; 3(11): 2051-2063, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31637346

RESUMO

Background: Nonobese nonalcoholic fatty liver disease is reported in several populations. However, because persons of African origin display unique fat accumulation, insulin resistance, and lipid profiles, we investigated fatty liver in nonobese persons of African origin. Method: We recruited 78 urban Jamaican volunteers. CT was used to estimate liver and abdominal fat and dual-energy X-ray absorptiometry to measure body composition. Fasting blood was collected for lipids, alanine aminotransferase (ALT), adiponectin, and fetuin-A. Homeostatic model assessment of insulin resistance (HOMA-IR), whole-body insulin sensitivity index (WBISI), insulinogenic index (IGI), and oral disposition index (oDI) were calculated after a 75-g oral glucose tolerance test. Results: Fifty-two percent of participants were male; mean (±SD) age was 28.5 ± 7.8 years, and body mass index was 22.4 ± 3.0 kg/m2. Mean liver attenuation (MLA) and liver/spleen (LS) ratio, both inversely correlated to liver fat, were 62.8 ± 4.3 HU and 1.2 ± 0.1, respectively; 3.8% of participants had liver fat >30% (LS ratio < 1). In age, sex, and BMI-adjusted correlations, MLA was negatively associated with weight (r = -0.30; P = 0.009) and height (r = -0.28; P = 0.017) and was associated with fasting glucose (r = 0.23; P = 0.05), fasting insulin (r = 0.42; P ≤ 0.001) and HOMA-IR (r = 0.35; P = 0.004). Serum lipids, ALT, adiponectin, fetuin-A, WBISI, IGI, and oDI were not associated with liver fat. Conclusions: In nonobese Afro-Caribbean participants, greater liver fat was associated with weight and height and lower fasting insulin and hyperinsulinemia appears to be influential in the reduction of NAFLD. These findings may be influenced by ethnicity, body size, and method of estimating liver fat.

3.
PLoS One ; 13(6): e0198626, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29879181

RESUMO

AIMS/HYPOTHESES: We hypothesized that there is decreased synthesis of glutathione (GSH) in type 2 diabetes (T2DM) especially in the presence of microvascular complications, and this is dependent on the degree of hyperglycemia. METHODS: In this case-control study, we recruited 16 patients with T2DM (7 without and 9 with microvascular complications), and 8 age- and sex-matched non-diabetic controls. We measured GSH synthesis rate using an infusion of [2H2]-glycine as isotopic tracer and collection of blood samples for liquid chromatography mass spectrometric analysis. RESULTS: Compared to the controls, T2DM patients had lower erythrocyte GSH concentrations (0.90 ± 0.42 vs. 0.35 ± 0.30 mmol/L; P = 0.001) and absolute synthesis rates (1.03 ± 0.55 vs. 0.50 ± 0.69 mmol/L/day; P = 0.01), but not fractional synthesis rates (114 ± 45 vs. 143 ± 82%/day; P = 0.07). The magnitudes of changes in patients with complications were greater for both GSH concentrations and absolute synthesis rates (P-values ≤ 0.01) compared to controls. There were no differences in GSH concentrations and synthesis rates between T2DM patients with and without complications (P-values > 0.1). Fasting glucose and HbA1c did not correlate with GSH concentration or synthesis rates (P-values > 0.17). CONCLUSIONS: Compared to non-diabetic controls, patients with T2DM have glutathione deficiency, especially if they have microvascular complications. This is probably due to reduced synthesis and increased irreversible utilization by non-glycemic mechanisms.


Assuntos
Glicemia , Diabetes Mellitus Tipo 2/metabolismo , Angiopatias Diabéticas/metabolismo , Glutationa/metabolismo , Microvasos/patologia , Adulto , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/complicações , Angiopatias Diabéticas/sangue , Angiopatias Diabéticas/etiologia , Angiopatias Diabéticas/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
4.
Expert Rev Proteomics ; 15(5): 431-449, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29694790

RESUMO

INTRODUCTION: Mass spectrometry (MS) is widely used in the characterization of biomolecules including peptide and protein therapeutics. These biotechnology products have seen rapid growth over the past few decades and continue to dominate the global pharmaceutical market. Advances in MS instrumentation and techniques have enhanced protein characterization capabilities and supported an increased development of biopharmaceutical products. Areas covered: This review describes recent developments in MS-based biotherapeutic analysis including sequence determination, post-translational modifications (PTMs) and higher order structure (HOS) analysis along with improvements in ionization and dissociation methods. An outlook of emerging applications of MS in the lifecycle of product development such as comparability, biosimilarity and quality control practices is also presented. Expert commentary: MS-based methods have established their utility in the analysis of new biotechnology products and their lifecycle appropriate implementation. In the future, MS will likely continue to grow as one of the leading protein identification and characterization techniques in the biopharmaceutical industry landscape.


Assuntos
Produtos Biológicos/farmacologia , Espectrometria de Massas/métodos , Animais , Biotecnologia , Fatores Celulares Derivados do Hospedeiro/metabolismo , Humanos , Mapeamento de Peptídeos , Polissacarídeos/análise
5.
J Nutr ; 148(2): 170-171, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29490106
6.
Arch Med Sci Atheroscler Dis ; 2: e61-e67, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29242846

RESUMO

Introduction: The aim of the study was to determine the prevalence of dyslipidemia among primary care patients with hypertension and diabetes in Jamaica and the proportion of patients who achieve recommended targets. Material and methods: An audit of 500 dockets of adult patients with chronic disease attending public primary care clinics in Jamaica was conducted between October and December 2013. Data were collected on patient characteristics including medical history, medications, anthropometry, and lipid profiles (since January 1, 2011). Lipid targets were based on the Ministry of Health 2007 management guidelines. Stepwise multivariable logistic regression analysis was performed to determine the predictors of achieving lipid targets. Results: Four hundred and thirty-seven patient records had a lipid profile done and 90% of these had at least one abnormal lipid value. 15.3% of the patients achieved the low density lipoprotein cholesterol (LDL-C) target, 63.2% high density lipoprotein cholesterol (HDL-C), 85.1% triglycerides and 57.4% the total cholesterol target. Statins were prescribed for 49% and these patients were less likely to achieve LDL-C (OR = 0.57; 95% CI: 0.33-0.97; p = 0.04) or total cholesterol (OR = 0.21; 95% CI: 0.13-0.33; p < 0.001) targets. Patients over 80 years were more likely to achieve the LDL-C target (OR = 3.21; 95% CI: 1.64-6.28; p = 0.002) than those less than 50 years old. More men than women achieved total cholesterol targets (OR = 2.2; 95% CI: 1.4-3.6; p = 0.001). Conclusions: Dyslipidemia is widespread among primary care patients with hypertension and diabetes. The proportion of patients who achieve the respective lipid targets must be documented and routinely monitored and appropriate medication and lifestyle changes implemented to improve this.

7.
EBioMedicine ; 18: 274-280, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28330812

RESUMO

BACKGROUND: Severe acute malnutrition (SAM) in infants may present as one of two distinct syndromic forms: non-edematous (marasmus), with severe wasting and no nutritional edema; or edematous (kwashiorkor) with moderately severe wasting. These differences may be related to developmental changes prior to the exposure to SAM and phenotypic changes appear to persist into adulthood with differences between the two groups. We examined whether the different response to SAM and subsequent trajectories may be explained by developmentally-induced epigenetic differences. METHODS: We extracted genomic DNA from muscle biopsies obtained from adult survivors of kwashiorkor (n=21) or marasmus (n=23) and compared epigenetic profiles (CpG methylation) between the two groups using the Infinium® 450K BeadChip array. FINDINGS: We found significant differences in methylation of CpG sites from 63 genes in skeletal muscle DNA. Gene ontology studies showed significant differential methylation of genes in immune, body composition, metabolic, musculoskeletal growth, neuronal function and cardiovascular pathways, pathways compatible with the differences in the pathophysiology of adult survivors of SAM. INTERPRETATION: These findings suggest persistent developmental influences on adult physiology in survivors of SAM. Since children who develop marasmus have lower birth weights and after rehabilitation have different intermediary metabolism, these studies provide further support for persistent developmentally-induced phenomena mediated by epigenetic processes affecting both the infant response to acute malnutrition and later life consequences. FUNDING: Supported by a Grant from the Bill and Melinda Gates Foundation (Global Health OPP1066846), Grand Challenge "Discover New Ways to Achieve Healthy Growth." EVIDENCE BEFORE THIS STUDY: Previous research has shown that infants who develop either kwashiorkor or marasmus in response to SAM differ in birth weight and subsequently have different metabolic patterns in both infancy and adulthood. ADDED VALUE OF THIS STUDY: This study demonstrates epigenetic differences in the skeletal muscle of adult survivors of marasmus versus kwashiorkor and these differences are in genes that may underlie the longer-term consequences. IMPLICATIONS OF ALL THE AVAILABLE EVIDENCE: These data are compatible with the different clinical responses to SAM arising from developmentally-induced epigenetic changes laid down largely before birth and provide evidence for the predictive adaptive response model operating in human development.


Assuntos
DNA/metabolismo , Desnutrição Aguda Grave/patologia , Adulto , Proteína C-Reativa/genética , Ilhas de CpG , DNA/química , DNA/isolamento & purificação , Metilação de DNA , Epigenômica , Feminino , Genoma Humano , Hexoquinase/genética , Proteínas de Homeodomínio/genética , Humanos , Kwashiorkor , Masculino , Músculo Esquelético/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Desnutrição Proteico-Calórica , Proteínas Serina-Treonina Quinases/genética , Análise de Regressão , Desnutrição Aguda Grave/genética , Desnutrição Aguda Grave/metabolismo , Fatores de Transcrição/genética , Adulto Jovem
8.
PLoS One ; 12(3): e0173101, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28291805

RESUMO

OBJECTIVES: Severe acute malnutrition (SAM) is an important risk factor for illness and death globally, contributing to more than half of deaths in children worldwide. We hypothesized that SAM is positively correlated to poverty, low educational attainment, major crime and higher mean soil concentrations of lead, cadmium and arsenic. METHODS: We reviewed admission records of infants admitted with a diagnosis of SAM over 14 years (2000-2013) in Jamaica. Poverty index, educational attainment, major crime and environmental heavy metal exposure were represented in a Geographic Information System (GIS). Cases of SAM were grouped by community and the number of cases per community/year correlated to socioeconomic variables and geochemistry data for the relevant year. RESULTS: 375 cases of SAM were mapped across 204 urban and rural communities in Jamaica. The mean age at admission was 9 months (range 1-45 months) and 57% were male. SAM had a positive correlation with major crime (r = 0.53; P < 0.001), but not with educational attainment or the poverty index. For every one unit increase in the number of crimes reported, the rate of occurrence of SAM cases increased by 1.01% [Incidence rate ratio (IRR) = 1.01 (95% CI = 1.006-1.014); P P<0.001]. The geochemistry data yielded no correlation between levels of heavy metals and the prevalence of malnutrition. CONCLUSION: Major crime has an independent positive association with severe acute malnutrition in Jamaican infants. This could suggest that SAM and major crime might have similar sociological origins or that criminality at the community level may be indicative of reduced income opportunities with the attendant increase in poor nutrition in the home.


Assuntos
Desnutrição/etiologia , Fatores Socioeconômicos , Doença Aguda , Pré-Escolar , Humanos , Lactente , Recém-Nascido , Jamaica , Metais Pesados/análise , Pobreza , Fatores de Risco , Poluentes do Solo/análise
9.
Xenobiotica ; 47(5): 431-438, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-27321253

RESUMO

1. Topical anesthesia with benzocaine or lidocaine occasionally causes methemoglobinemia, an uncommon but potentially fatal disorder where the blood has a reduced ability to transport oxygen. Previous in vitro studies using human whole blood have shown that benzocaine causes more methemoglobin (MetHb) formation than lidocaine, and that both compounds require metabolic transformation to form the MetHb producing species. In the current investigation, the active species of benzocaine forming the MetHb was investigated. 2. HPLC analysis of benzocaine samples incubated with human hepatic S9 showed the formation of a peak with the same UV spectrum and retention time as benzocaine hydroxylamine (BenzNOH). To confirm the activity of BenzNOH, MetHb production following exposure to the compound was determined in whole human blood using an Avoximeter 4000 CO-oximeter. 3. BenzNOH produced MetHb in a concentration dependent manner without the need for metabolic activation. Benzocaine in the presence of metabolic activation required a concentration of 500 µM to produce a similar degree of MetHb formation as 20 µM BenzNOH without activation. Previous work suggested that two metabolites of lidocaine may also form MetHb; N-hydroxyxylidine and 4-hydroxyxylidine. Of these two metabolites 4-hydroxyxylidine produced the most MetHb in whole blood in vitro in the absence of metabolic activation, however BenzNOH produced up to 14.2 times more MetHb than 4-hydroxyxylidine at a similar concentration. 4. These results suggest that the ability of benzocaine to form MetHb is likely to be mediated through its hydroxylamine metabolite and that this metabolite is inherently more active than the potentially MetHb-forming metabolites of lidocaine.


Assuntos
Benzocaína/metabolismo , Lidocaína/metabolismo , Metemoglobina/metabolismo , Acetaminofen/análogos & derivados , Anestésicos Locais/metabolismo , Humanos , Metemoglobinemia
10.
Biotechnol Prog ; 33(1): 163-170, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27813291

RESUMO

Linkage of upstream cell culture with downstream processing and purification is an aspect of Quality by Design crucial for efficient and consistent production of high quality biopharmaceutical proteins. In a previous Plackett-Burman screening study of parallel bioreactor cultures we evaluated main effects of 11 process variables, such as agitation, sparge rate, feeding regimens, dissolved oxygen set point, inoculation density, supplement addition, temperature, and pH shifts. In this follow-up study, we observed linkages between cell culture process parameters and downstream capture chromatography performance and subsequent antibody attributes. In depth analysis of the capture chromatography purification of harvested cell culture fluid yielded significant effects of upstream process parameters on host cell protein abundance and behavior. A variety of methods were used to characterize the antibody both after purification and buffer formulation. This analysis provided insight in to the significant impacts of upstream process parameters on aggregate formation, impurities, and protein structure. This report highlights the utility of linkage studies in identifying how changes in upstream parameters can impact downstream critical quality attributes. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:163-170, 2017.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Reatores Biológicos , Técnicas de Cultura de Células/métodos , Cromatografia/métodos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/química , Biotecnologia/métodos , Células CHO , Cricetulus , Concentração de Íons de Hidrogênio , Temperatura
11.
J Am Soc Mass Spectrom ; 28(5): 786-794, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-27873217

RESUMO

The characterization sections of biologics license applications (BLAs) approved by the United States Food and Drug Administration (FDA) between 2000 and 2015 were investigated to examine the extent of the use of mass spectrometry. Mass spectrometry was found to be integral to the characterization of these biotherapeutics. Of the 80 electronically submitted monoclonal antibody and protein biotherapeutic BLAs included in this study, 79 were found to use mass spectrometric workflows for protein or impurity characterization. To further examine how MS is being used in successful BLAs, the applications were filtered based on the type and number of quality attributes characterized, the mass spectrometric workflows used (peptide mapping, intact mass analysis, and cleaved glycan analysis), the methods used to introduce the proteins into the gas phase (ESI, MALDI, or LC-ESI), and the specific types of instrumentation used. Analyses were conducted over a time course based on the FDA BLA approval to determine if any trends in utilization could be observed over time. Additionally, the different classes of protein-based biotherapeutics among the approved BLAs were clustered to determine if any trends could be attributed to the specific type of biotherapeutic. Graphical Abstract ᅟ.


Assuntos
Anticorpos Monoclonais/química , Produtos Biológicos/química , Aprovação de Drogas/métodos , Espectrometria de Massas/métodos , Humanos , Mapeamento de Peptídeos/métodos , Estados Unidos , United States Food and Drug Administration , Fluxo de Trabalho
12.
AAPS J ; 19(1): 4-14, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27709452

RESUMO

Protein therapeutics have unique critical quality attributes (CQAs) that define their purity, potency, and safety. The analytical methods used to assess CQAs must be able to distinguish clinically meaningful differences in comparator products, and the most important CQAs should be evaluated with the most statistical rigor. High-risk CQA measurements assess the most important attributes that directly impact the clinical mechanism of action or have known implications for safety, while the moderate- to low-risk characteristics may have a lower direct impact and thereby may have a broader range to establish similarity. Statistical equivalence testing is applied for high-risk CQA measurements to establish the degree of similarity (e.g., highly similar fingerprint, highly similar, or similar) of selected attributes. Notably, some high-risk CQAs (e.g., primary sequence or disulfide bonding) are qualitative (e.g., the same as the originator or not the same) and therefore not amenable to equivalence testing. For biosimilars, an important step is the acquisition of a sufficient number of unique originator drug product lots to measure the variability in the originator drug manufacturing process and provide sufficient statistical power for the analytical data comparisons. Together, these analytical evaluations, along with PK/PD and safety data (immunogenicity), provide the data necessary to determine if the totality of the evidence warrants a designation of biosimilarity and subsequent licensure for marketing in the USA. In this paper, a case study approach is used to provide examples of analytical similarity exercises and the appropriateness of statistical approaches for the example data.


Assuntos
Medicamentos Biossimilares/normas , Avaliação de Medicamentos/estatística & dados numéricos , Indústria Farmacêutica/normas , Aprovação de Drogas , Indústria Farmacêutica/tendências , Controle de Qualidade , Estados Unidos , United States Food and Drug Administration
13.
Dis Markers ; 2016: 9296457, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27110056

RESUMO

Lethal influenza A virus infection leads to acute lung injury and possibly lethal complications. There has been a continuous effort to identify the possible predictors of disease severity. Unlike earlier studies, where biomarkers were analyzed on certain time points or days after infection, in this study biomarkers were evaluated over the entire course of infection. Circulating proinflammatory cytokines and/or miRNAs that track with the onset and progression of lethal A/Puerto Rico/8/34 (PR8) influenza A virus infection and their response to oseltamivir treatment were investigated up to 10 days after infection. Changes in plasma cytokines (IL-1ß, IL-10, IL-12p70, IL-6, KC, TNF-α, and IFN-γ) and several candidate miRNAs were profiled. Among the cytokines analyzed, IL-6 and KC/GRO cytokines appeared to correlate with peak viral titer. Over the selected 48 miRNAs profiled, certain miRNAs were up- or downregulated in a manner that was dependent on the oseltamivir treatment and disease severity. Our findings suggest that IL-6 and KC/GRO cytokines can be a potential disease severity biomarker and/or marker for the progression/remission of infection. Further studies to explore other cytokines, miRNAs, and lung injury proteins in serum with different subtypes of influenza A viruses with varying disease severity may provide new insight into other unique biomarkers.


Assuntos
Antivirais/administração & dosagem , Citocinas/sangue , MicroRNAs/genética , Infecções por Orthomyxoviridae/tratamento farmacológico , Oseltamivir/administração & dosagem , Animais , Antivirais/farmacologia , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Modelos Animais de Doenças , Progressão da Doença , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Vírus da Influenza A/efeitos dos fármacos , Camundongos , MicroRNAs/sangue , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/virologia , Oseltamivir/farmacologia , Resultado do Tratamento , Carga Viral/efeitos dos fármacos
14.
Biomed Res Int ; 2016: 2074149, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27042659

RESUMO

Formulating appropriate storage conditions for biopharmaceutical proteins is essential for ensuring their stability and thereby their purity, potency, and safety over their shelf-life. Using a model murine IgG3 produced in a bioreactor system, multiple formulation compositions were systematically explored in a DoE design to optimize the stability of a challenging antibody formulation worst case. The stability of the antibody in each buffer formulation was assessed by UV/VIS absorbance at 280 nm and 410 nm and size exclusion high performance liquid chromatography (SEC) to determine overall solubility, opalescence, and aggregate formation, respectively. Upon preliminary testing, acetate was eliminated as a potential storage buffer due to significant visible precipitate formation. An additional 2(4) full factorial DoE was performed that combined the stabilizing effect of arginine with the buffering capacity of histidine. From this final DoE, an optimized formulation of 200 mM arginine, 50 mM histidine, and 100 mM NaCl at a pH of 6.5 was identified to substantially improve stability under long-term storage conditions and after multiple freeze/thaw cycles. Thus, our data highlights the power of DoE based formulation screening approaches even for challenging monoclonal antibody molecules.


Assuntos
Anticorpos Monoclonais/química , Formação de Anticorpos , Imunoglobulina G/química , Animais , Anticorpos Monoclonais/imunologia , Tampões (Química) , Cromatografia Líquida de Alta Pressão , Congelamento , Concentração de Íons de Hidrogênio , Imunoglobulina G/biossíntese , Imunoglobulina G/imunologia , Camundongos , Estabilidade Proteica
15.
Evol Med Public Health ; 2016(1): 158-69, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26817484

RESUMO

BACKGROUND AND OBJECTIVES: Birthweight differences between kwashiorkor and marasmus suggest that intrauterine factors influence the development of these syndromes of malnutrition and may modulate risk of obesity through dietary intake. We tested the hypotheses that the target protein intake in adulthood is associated with birthweight, and that protein leveraging to maintain this target protein intake would influence energy intake (EI) and body weight in adult survivors of malnutrition. METHODOLOGY: Sixty-three adult survivors of marasmus and kwashiorkor could freely compose a diet from foods containing 10, 15 and 25 percentage energy from protein (percentage of energy derived from protein (PEP); Phase 1) for 3 days. Participants were then randomized in Phase 2 (5 days) to diets with PEP fixed at 10%, 15% or 25%. RESULTS: Self-selected PEP was similar in both groups. In the groups combined, selected PEP was 14.7, which differed significantly (P < 0.0001) from the null expectation (16.7%) of no selection. Self-selected PEP was inversely related to birthweight, the effect disappearing after adjusting for sex and current body weight. In Phase 2, PEP correlated inversely with EI (P = 0.002) and weight change from Phase 1 to 2 (P = 0.002). Protein intake increased with increasing PEP, but to a lesser extent than energy increased with decreasing PEP. CONCLUSIONS AND IMPLICATIONS: Macronutrient intakes were not independently related to birthweight or diagnosis. In a free-choice situation (Phase 1), subjects selected a dietary PEP significantly lower than random. Lower PEP diets induce increased energy and decreased protein intake, and are associated with weight gain.

16.
Anal Bioanal Chem ; 407(29): 8647-59, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26458562

RESUMO

Glatiramer acetate (GA) is a mixture of synthetic copolymers consisting of four amino acids (glutamic acid, lysine, alanine, and tyrosine) with a labeled molecular weight range of 5000 to 9000 Da. GA is marketed as Copaxone™ by Teva for the treatment of multiple sclerosis. Here, the agency has evaluated the structure and composition of GA and a commercially available comparator, Copolymer-1. Modern analytical technologies which can characterize these complex mixtures are desirable for analysis of their comparability and structural "sameness." In the studies herein, a molecular fingerprinting approach is taken using mass-accurate mass spectrometry (MS) analysis, nuclear magnetic resonance (NMR) (1D-(1)H-NMR, 1D-(13)C-NMR, and 2D NMR), and asymmetric field flow fractionation (AFFF) coupled with multi-angle light scattering (MALS) for an in-depth characterization of three lots of the marketplace drug and a formulated sample of the comparator. Statistical analyses were applied to the MS and AFFF-MALS data to assess these methods' ability to detect analytical differences in the mixtures. The combination of multiple orthogonal measurements by liquid chromatography coupled with MS (LC-MS), AFFF-MALS, and NMR on the same sample set was found to be fit for the intended purpose of distinguishing analytical differences between these complex mixtures of peptide chains.


Assuntos
Acetato de Glatiramer/química , Imunossupressores/química , Fracionamento por Campo e Fluxo , Espectroscopia de Ressonância Magnética , Espectrometria de Massas
17.
AAPS J ; 17(6): 1438-45, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26242210

RESUMO

Conjugated estrogens purified from pregnant mares urine has been used as estrogen hormone replacement therapy since 1942. Previously, methods were proposed to identify and quantify the components of this complex mixture but ultimately were withdrawn due to incomplete characterization of the product and difficulties in transferring the method between laboratories. The aim of the current study is to develop a LC method that can reliably detect multiple steroidal components in conjugated estrogen tablets and measure their relative amount. The method developed was optimized for UHPLC columns, and the elution profile was analyzed using high-resolution mass spectrometry. A total of 60 steroidal components were identified using their exact m/z, product ion spectra of known, and predicted conjugated estrogen structures. These components were consistently present in 23 lots of Premarin tablets spanning two production years. The ten conjugated estrogens identified in the USP monograph and other additional estrogens reported elsewhere are among the 60 steroidal components reported here. The LC-MS method was tested in different laboratories using multiple samples, and the obtained results were reproducible among laboratories.


Assuntos
Contaminação de Medicamentos , Estrogênios Conjugados (USP)/análise , Estrogênios Conjugados (USP)/química , Espectrometria de Massas/métodos , Animais , Cromatografia Líquida/métodos , Feminino , Cavalos , Gravidez
18.
MAbs ; 7(6): 1104-17, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26218711

RESUMO

The use of liquid chromatography--mass spectrometry (LC-MS) for the characterization of proteins can provide a plethora of information related to their structure, including amino acid sequence determination and analysis of posttranslational modifications. The variety of LC-MS based applications has led to the use of LC-MS characterization of therapeutic proteins and monoclonal antibodies as an integral part of the regulatory approval process. However, the improper use of an LC-MS system, related to intrinsic instrument limitations, improper tuning parameters, or poorly optimized methods may result in the production of low quality data. Improper system performance may arise from subtle changes in operating conditions that limit the ability to detect low abundance species. To address this issue, we systematically evaluated LC-MS/MS operating parameters to identify a set of metrics that can be used in a workflow to determine if a system is suitable for its intended purpose. Development of this workflow utilized a bovine serum albumin (BSA) digest standard spiked with synthetic peptides present at 0.1% to 100% of the BSA digest peptide concentration to simulate the detection of low abundance species using a traditional bottom-up workflow and data-dependent MS(2) acquisition. BSA sequence coverage, a commonly used indicator for instrument performance did not effectively identify settings that led to limited dynamic range or poorer absolute mass accuracy on 2 separate LC-MS systems. Additional metrics focusing on the detection limit and sensitivity for peptide identification were determined to be necessary to establish system suitability for protein therapeutic characterization by LC-MS.


Assuntos
Anticorpos Monoclonais/química , Cromatografia Líquida/métodos , Mapeamento de Peptídeos/métodos , Soroalbumina Bovina/química , Espectrometria de Massas em Tandem/métodos , Animais , Anticorpos Monoclonais/uso terapêutico , Bovinos , Humanos , Reprodutibilidade dos Testes , Soroalbumina Bovina/uso terapêutico , Fatores de Tempo
19.
J Pharm Sci ; 104(8): 2464-72, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26053232

RESUMO

Monoclonal antibody therapeutics are a heterogeneous mixture of glycoforms. Multiple methods exist for defining the glycan composition and relative abundance of species present. In the current report, two MS-based methods were compared for their ability to both identify glycans and monitor differences in the glycoprofile. Gross changes in the glycoprofile can be identified either by visual inspection of fluorescence profiles and correlated to glycan identities when coupled with online MS/MS (LC-F-MS/MS) or through extracted ion chromatograms using LC-MS. In the present study, both an LC-F-MS/MS method and an automated LC-MS label free approach were able to identify minor differences in low abundance glycoforms, and data indicate a disparity in glycosylation between the analyzed batches of US and foreign-sourced mAb X. Thus, either method may be useful in characterizing monoclonal antibody therapeutics products and could serve as a potential screening test for understanding process, comparability, similarity, and possibly detecting counterfeit agents.


Assuntos
Anticorpos Monoclonais/química , Corantes Fluorescentes/química , Glicoproteínas/química , Preparações Farmacêuticas/química , Polissacarídeos/análise , ortoaminobenzoatos/química , Métodos Analíticos de Preparação de Amostras , Animais , Anticorpos Monoclonais/metabolismo , Automação Laboratorial , Química Farmacêutica , Cromatografia Líquida de Alta Pressão , Medicamentos Falsificados/química , Medicamentos Falsificados/metabolismo , Enzimas Imobilizadas/metabolismo , Glicoproteínas/metabolismo , Glicosilação , Humanos , Hidrólise , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Preparações Farmacêuticas/metabolismo , Polissacarídeos/metabolismo , Processamento de Proteína Pós-Traducional , Controle de Qualidade , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
20.
Toxicol Appl Pharmacol ; 287(3): 246-52, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26079829

RESUMO

The erythropoietin analog peginesatide was withdrawn from marketing due to unexpected severe anaphylactic reactions associated with administration of the multi-use formulation. The adverse events occurred rapidly following the first ever administration of the drug with most affected patients becoming symptomatic in less than 30min. This is most consistent with an anaphylactoid reaction due to direct activation of mast cells. Laboratory evaluation was undertaken using rat peritoneal mast cells as the model system. Initial studies showed that high concentrations of the formulated drug as well as formulated vehicle alone could cause mast cell degranulation as measured by histamine release. The purified active drug was not able to cause histamine release whereas the vehicle filtrate and lab created drug vehicle were equally potent at causing histamine release. Individual formulations of vehicle leaving one component out showed that histamine release was due to phenol. Dose response studies with phenol showed a very sharp dose response curve that was similar in three buffer systems. Cellular analysis by flow cytometry showed that the histamine release was not due to cell death, and that changes in light scatter parameters consistent with degranulation were rapidly observed. Limited testing with primary human mast cells showed a similar dose response of histamine release with exposure to phenol. To provide in vivo confirmation, rats were injected with vehicle formulated with various concentrations of phenol via a jugular vein cannula. Significant release of histamine was detected in blood samples taken 2min after dosing at the highest concentrations tested.


Assuntos
Degranulação Celular/efeitos dos fármacos , Excipientes/toxicidade , Hematínicos/toxicidade , Histamina/metabolismo , Mastócitos/efeitos dos fármacos , Peptídeos/toxicidade , Fenol/toxicidade , Animais , Células Cultivadas , Química Farmacêutica , Relação Dose-Resposta a Droga , Excipientes/administração & dosagem , Excipientes/química , Feminino , Hematínicos/química , Histamina/sangue , Humanos , Injeções Intravenosas , Mastócitos/metabolismo , Camundongos Endogâmicos NOD , Peptídeos/química , Fenol/administração & dosagem , Fenol/química , Cultura Primária de Células , Ratos Sprague-Dawley , Medição de Risco , Fatores de Tempo
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