RESUMO
There have been significant advances in the formulation and stabilization of proteins in the liquid state over the past years since our previous review. Our mechanistic understanding of protein-excipient interactions has increased, allowing one to develop formulations in a more rational fashion. The field has moved towards more complex and challenging formulations, such as high concentration formulations to allow for subcutaneous administration and co-formulation. While much of the published work has focused on mAbs, the principles appear to apply to any therapeutic protein, although mAbs clearly have some distinctive features. In this review, we first discuss chemical degradation reactions. This is followed by a section on physical instability issues. Then, more specific topics are addressed: instability induced by interactions with interfaces, predictive methods for physical stability and interplay between chemical and physical instability. The final parts are devoted to discussions how all the above impacts (co-)formulation strategies, in particular for high protein concentration solutions.'
Assuntos
Estabilidade de Medicamentos , Estabilidade Proteica , Proteínas , Humanos , Proteínas/química , Excipientes/química , Composição de Medicamentos/métodos , Química Farmacêutica/métodos , Animais , Anticorpos Monoclonais/químicaRESUMO
While asymmetrical flow field-flow fractionation (AF4) has been widely used for separation of high molecular weight species and even particles, its ability to resolve lower molecular weight species has rarely been explored. Over the course of many projects, we have discovered that AF4 can be an effective analytical method for separating peptides from oligomers and higher molecular weight aggregates. The methodology can be used even for peptides as small as 2 kD in molecular weight. Using multi-angle laser light scattering (MALLS) detection, accurate masses of the parent peptide can be obtained, provided accurate extinction coefficients are provided. It was shown that AF4 can be stability-indicating, suggesting that AF4-MALLS may be a suitable alternative to the use of SEC to monitor the aggregation of peptides.
Assuntos
Fracionamento por Campo e Fluxo , Peso Molecular , PeptídeosRESUMO
Insecticide resistance in Aedes aegypti mosquitoes poses a major threat to public health worldwide. There are two primary biological mechanisms that can lead to insecticide resistance, target site and metabolic resistance, both of which confer resistance to specific classes of insecticides. Due to the limited number of chemical compounds available for mosquito control, it is important to determine current enzymatic profiles among mosquito populations. This study assessed resistance profiles for three metabolic pathways, α-esterases, ß-esterases, and mixed-function oxidases (MFOs), as well as insensitivity of the acetylcholinesterase (iAChE) enzyme in the presence of propoxur, among Ae. aegypti from the Central Valley and southern California. All field-collected Ae. aegypti demonstrated elevated MFOs and iAChE activity, indicating potential development of pyrethroid and organophosphate resistance, respectively. Although regional variations were found among α-esterase and ß-esterase activity, levels were generally elevated, further suggesting additional mechanisms for developing organophosphate resistance. Furthermore, mosquito samples from southern California exhibited a higher expression level to all three metabolic enzymes and iAChE activity in comparison to mosquitoes from the central region. These results could help guide future mosquito control efforts, directing the effective use of insecticides while limiting the spread of resistance.
Assuntos
Aedes/efeitos dos fármacos , Resistência a Inseticidas/genética , Mosquitos Vetores/efeitos dos fármacos , Aedes/enzimologia , Aedes/genética , Animais , California , Feminino , Proteínas de Insetos/análise , Inseticidas/farmacologia , Mosquitos Vetores/enzimologia , Mosquitos Vetores/genéticaRESUMO
We describe Yersinia pestis minimum infection prevalence in fleas collected from Tamias spp. chipmunks in the Sierra Nevadas (California, USA) during 2013-2015. Y. pestis-positive fleas were detected only in 2015 (year of plague epizootic), mostly in T. speciosus chipmunks at high-elevation sites. Plague surveillance should include testing vectors for Y. pestis.
Assuntos
Peste , Sifonápteros , Yersinia pestis , Animais , California/epidemiologia , Peste/epidemiologia , Peste/veterinária , Sciuridae , Yersinia pestis/genéticaRESUMO
The first breeding populations of Aedes aegypti (Linnaeus) were identified in California in 2013, and have since been detected in 13 counties. Recent studies suggest two introductions likely occurred, with genetically distinct populations in the central and southern regions of the state. Given the threat of dengue, chikungunya, and Zika virus transmission, it is imperative to understand if these populations harbor genes that could confer resistance to pyrethrin-based insecticides, known as pyrethroids, the most commonly used class of adulticides in the state. In 2017, the California Department of Public Health initiated a pesticide resistance screening program for Ae. aegypti to assess the presence of specific mutations on the sodium channel gene (V1016I and F1534C) associated with knockdown resistance to pyrethroids. Mosquitoes collected between 2015 and 2017 from 11 counties were screened for mutations using real-time polymerase chain reaction assays. Results revealed distinctly different resistance profiles between the central and southern regions. The central population displayed nearly fixed resistant mutations at both loci, whereas the southern population was more variable. The relative proportion of resistant alleles observed in sampled mosquitoes collected in southern California increased each year from 2015 through 2017, indicating potential increases in resistance across this region. The presence of these mutations indicates that these mosquitoes may be predisposed to surviving pyrethroid treatments. Additional biological and biochemical assays will help better elucidate the mechanisms underlying insecticide resistance in California Ae. aegypti and prompt the use of pesticides that are most effective at controlling these mosquitoes.
Assuntos
Aedes/genética , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Mosquitos Vetores/genética , Piretrinas/farmacologia , Aedes/efeitos dos fármacos , Animais , California , Genótipo , Mosquitos Vetores/efeitos dos fármacosRESUMO
PURPOSE: To evaluate the different degrees of residual structure in the unfolded state of interferon-τ using chemical denaturation as a function of temperature by both urea and guanidinium hydrochloride. METHODS: Asymmetrical flow field-flow fractionation (AF4) using both UV and multi-angle laser light scattering (MALLS). Flow Microscopy. All subvisible particle imaging measurements were made using a FlowCAM flow imaging system. RESULTS: The two different denaturants provided different estimates of the conformational stability of the protein when extrapolated back to zero denaturant concentration. This suggests that urea and guanidinium hydrochloride (GnHCl) produce different degrees of residual structure in the unfolded state of interferon-τ. The differences were most pronounced at low temperature, suggesting that the residual structure in the denatured state is progressively lost when samples are heated above 25°C. The extent of expansion in the unfolded states was estimated from the m-values and was also measured using AF4. In contrast, the overall size of interferon-τ was determined by AF4 to decrease in the presence of histidine, which is known to bind to the native state, thereby providing conformational stabilization. Addition of histidine as the buffer resulted in formation of fewer subvisible particles over time at 50°C. Finally, the thermal aggregation was monitored using AF4 and the rate constants were found to be comparable to those determined previously by SEC and DLS. The thermal aggregation appears to be consistent with a nucleation-dependent mechanism with a critical nucleus size of 4 ± 1. CONCLUSION: Chemical denaturation of interferon-τ by urea or GnHCl produces differing amounts of residual structure in the denatured state, leading to differing estimates of conformational stability. AF4 was used to determine changes in size, both upon ligand binding as well as upon denaturation with GnHCl. Histidine appears to be the preferred buffer for interferon-τ, as shown by slower formation of soluble aggregates and reduced levels of subvisible particles when heated at 50°C.
Assuntos
Interferon Tipo I/química , Proteínas da Gravidez/química , Agregados Proteicos , Desnaturação Proteica , Desdobramento de Proteína , Água/química , Interferon Tipo I/análise , Interferon Tipo I/metabolismo , Soluções Farmacêuticas/química , Soluções Farmacêuticas/metabolismo , Espectroscopia Fotoeletrônica/métodos , Proteínas da Gravidez/análise , Proteínas da Gravidez/metabolismo , Agregados Proteicos/fisiologia , Água/metabolismoRESUMO
Buffers comprise an integral component of protein formulations. Not only do they function to regulate shifts in pH, they also can stabilize proteins by a variety of mechanisms. The ability of buffers to stabilize therapeutic proteins whether in liquid formulations, frozen solutions, or the solid state is highlighted in this review. Addition of buffers can result in increased conformational stability of proteins, whether by ligand binding or by an excluded solute mechanism. In addition, they can alter the colloidal stability of proteins and modulate interfacial damage. Buffers can also lead to destabilization of proteins, and the stability of buffers themselves is presented. Furthermore, the potential safety and toxicity issues of buffers are discussed, with a special emphasis on the influence of buffers on the perceived pain upon injection. Finally, the interaction of buffers with other excipients is examined.
Assuntos
Química Farmacêutica/métodos , Proteínas/química , Proteínas/metabolismo , Soluções Tampão , Fenômenos Químicos , Composição de Medicamentos/métodos , Excipientes/química , Excipientes/metabolismo , Humanos , Ligação Proteica/fisiologiaRESUMO
The peptide teriparatide, also known as parathyroid hormone (1-34), PTH(1-34), was developed for intranasal delivery, requiring extended stability of the reconstituted product for up to four weeks at room temperature. Lyophilized formulations of PTH(1-34), containing glycine and trehalose and using lactate as the buffer, are stable for months upon storage. However, the physical stability of the peptide after reconstitution unexpectedly varied considerably, depending on peptide concentration and storage temperature, with precipitation seen within two to four weeks in some samples. By comparison, equivalent samples that did not undergo lyophilization did not display any precipitation upon storage in the liquid state for as long as twelve weeks. PTH(1-34) appears to adopt a higher order structure that is perturbed by the combined stresses of freezing and drying, leading to greater propensity to aggregate, which is accentuated at higher peptide concentrations and at higher temperatures. The precipitation seems to be correlated with increased amounts of subvisible particles. This study shows the importance of peptide conformation in long-term stability and illustrates the ability of lyophilization to cause increased propensity to aggregate, even in a peptide.
Assuntos
Hormônio Paratireóideo/química , Hormônio Paratireóideo/normas , Teriparatida/química , Teriparatida/normas , Estabilidade de Medicamentos , Liofilização , Espectroscopia de Infravermelho com Transformada de Fourier/métodosRESUMO
BACKGROUND: Remote ischemic preconditioning (RIPC) is the process of inducing brief ischemia in a tissue to prevent ischemic damage in another. This preconditioning can be induced simply by inflating a blood pressure cuff on a limb. Previous randomized controlled trials (RCT) have suggested that RIPC may infer myocardial protection during open cardiac surgery. One method of assessing the degree of myocardial damage incurred in these studies is to assay troponin concentration. Troponin is a cardiac enzyme released by damaged myocardiocytes. With the recent publication of several large RCTs in this area, a meta-analysis of the evidence was undertaken. METHODS: A systematic search of PubMed, EMBASE, and clinicaltrials.gov.uk was conducted using MeSH terms "ischaemic preconditioning" and "cardiac surgery." RCTs that examined post-surgery troponin concentrations were included in this review. The primary outcome investigated was troponin levels at six hours post-cardiac surgery. Secondary outcomes included six to eight hour and twenty-four hour troponin release. RESULTS: Thirteen RCTs, comprising 1398 participants, were identified for inclusion in this meta-analysis. Twelve hour postoperative troponin was significantly reduced by RIPC, standardized mean difference 1.29 (95% CI 0.34-2.24). Six to eight and twenty-four hour troponin were also significantly reduced, standardized mean differences 1.23 (95% CI 0.62-1.84) and 1.25 (95% CI 0.31-2.19) respectively. CONCLUSIONS: The reduction in troponin concentration suggests that RIPC reduces myocardial damage during open cardiac surgery, however, the degree of bias in the studies assessed may have had a significant impact on this result.
Assuntos
Procedimentos Cirúrgicos Cardíacos/estatística & dados numéricos , Precondicionamento Isquêmico Miocárdico/estatística & dados numéricos , Isquemia Miocárdica/epidemiologia , Isquemia Miocárdica/prevenção & controle , Complicações Pós-Operatórias/epidemiologia , Complicações Pós-Operatórias/prevenção & controle , Biomarcadores/sangue , Medicina Baseada em Evidências , Humanos , Isquemia Miocárdica/sangue , Complicações Pós-Operatórias/sangue , Prevalência , Fatores de Risco , Resultado do Tratamento , Troponina C/sangueRESUMO
BACKGROUND: Limited availability of fish meal and whey protein concentrate increases overall feed costs. Availability of increased number of supplemental amino acids including Lys, Met, Thr, Trp, Val, and Ile allows replacing expensive protein supplements to reduce feed costs. This study was to evaluate the effect of replacing fish meal and/or whey protein concentrate in nursery diets with 6 supplemental amino acids on growth performance and gut health of post-weaning pigs. Treatments were 1) FM-WPC: diet with fish meal (FM) and whey protein concentrate (WPC); 2) FM-AA: diet with FM and crystalline amino acids (L-Lys, L-Thr, L-Trp, DL-Met, L-Val, and L-Ile); 3) WPC-AA: diet with WPC and crystalline amino acid; and 4) AA: diet with crystalline amino acid. RESULTS: Pigs in FM-AA, WPC-AA, and AA had greater (P < 0.05) ADG and gain:feed than pigs in FM-WPC during wk 1 (phase 1). Plasma insulin concentration of pigs in AA tended to be greater (P = 0.064) than that of FM-WPC at the end of wk 1(phase 1). Plasma concentrations of IgG in AA was lower (P < 0.05) compared with WPC-AA and FW, and FM-AA had lower (P < 0.05) IgG concentration than WPC-AA at the end of wk 1 (phase 1). Concentration of acetate in cecum digesta in FM-AA tended to be greater (P = 0.054) than that of FM-WPC and WPC-AA. Concentration of isovalerate in cecum digesta of pigs in FM-AA was greater (P < 0.05) than that of FW and WPC-AA. CONCLUSIONS: This study indicates that use of 6 supplemental amino acids can replace fish meal and/or whey protein concentrate without adverse effects on growth performance, immune status, and gut health of pigs at d 21 to 49 of age. Positive response with the use of 6 supplemental amino acids in growth during the first week of post-weaning may due to increased plasma insulin potentially improving uptake of nutrients for protein synthesis and energy utilization. The replacement of fish meal and/or whey protein concentrate with 6 supplemental amino acids could decrease the crude protein level in nursery diets, and potentially lead to substantial cost savings in expensive nursery diets.
RESUMO
Short peptides are important biopharmaceuticals as agonistic or antagonistic ligands, aggregation inhibitors, and vaccines, as well as in many other applications. They behave differently from globular proteins in solution. Many short peptides are unstructured and tend to aggregate and undergo structural transition in response to changes in solvent environment, including pH, temperature, ionic strength, presence of organic solvents or surfactants, and exposure to lipid membranes. Such structural transitions are often associated with fibril or ß-amyloid formation. These structural characteristics of short peptides have drastic impact on their function, immunogenicity, and storage stability.
Assuntos
Peptídeos/química , Peptídeos/metabolismo , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Amiloide/química , Amiloide/metabolismo , Dados de Sequência Molecular , Maleabilidade , Conformação ProteicaRESUMO
Eight lyophilized formulations of a IgG1 monoclonal antibody (MAb) were prepared containing increasing levels of sucrose. In addition, three of the formulations had sorbitol added at a level of 5% w/w relative to sucrose. The samples were stored for up to 4 weeks at 40°C, which is well below the Tg. Upon reconstitution, the levels of subvisible particles were measured using microflow imaging (MFI). The formulation containing no sucrose contained exceedingly high levels of subvisible particles, accounting for as much as 25% of the weight of the protein. Addition of sucrose markedly decreased the number of subvisible particles, with the maximal sucrose:protein weight ratio being 2:1 (the highest level tested). Addition of sorbitol further decreased subvisible particle levels, even for formulations where the sucrose:protein ratio was relatively high. This suggests that even small amounts of a plasticizer like sorbitol can improve the storage stability of a lyophilized antibody formulation, probably by dampening ß-relaxations within the amorphous glass.
Assuntos
Anticorpos Monoclonais/química , Excipientes/química , Imunoglobulina G/imunologia , Sacarose/química , Anticorpos Monoclonais/imunologia , Composição de Medicamentos , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Liofilização , Transição de Fase , Sorbitol/química , TemperaturaRESUMO
There are many aspects of stabilization of lyophilized proteins. Of these various factors, retention of native structure, having sufficient amount of stabilizer to embed the protein within an amorphous matrix, and dampening ß-relaxations have been shown to be critical in optimizing protein stability during storage. In this study, an IgG1 was lyophilized with varying amounts of sucrose. In some formulations, a small amount of sorbitol was added as a plasticizer. The structure of the protein in dried state was monitored using infrared (IR) spectroscopy. The IR spectra indicated increasing retention of the native structure, which correlated with stability as indicated by size-exclusion chromatography as well as micro-flow imaging. Maximal stability was achieved with a 2:1 mass ratio of sucrose to protein, which is more than that would be expected based on earlier studies. Analysis of both high and low frequency bands associated with intramolecular ß-sheet structure provides additional information on the structure of antibodies in the solid state. Finally, there is a correlation between the bandwidth of the ß-sheet bands and the enthalpy of relaxation, suggesting that amide I bands can provide some indication of the degree of coupling to the sugar matrix, as well as structural heterogeneity of the protein.
Assuntos
Anticorpos Monoclonais/química , Imunoglobulina G/química , Química Farmacêutica/métodos , Cromatografia em Gel/métodos , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Liofilização/métodos , Estabilidade Proteica , Estrutura Secundária de Proteína , Espectrofotometria Infravermelho/métodos , Sacarose/químicaRESUMO
Bacteria in their natural environments frequently exist as mixed surface-associated communities, protected by extracellular material, termed biofilms. Biofilms formed by the human pathogen Campylobacter jejuni may arise in the gastrointestinal tract of animals but also in water pipes and other industrial situations, leading to their possible transmission into the human food chain either directly or via farm animals. Bacteriophages are natural predators of bacteria that usually kill their prey by cell lysis and have potential application for the biocontrol and dispersal of target bacteria in biofilms. The effects of virulent Campylobacter specific-bacteriophages CP8 and CP30 on C. jejuni biofilms formed on glass by strains NCTC 11168 and PT14 at 37°C under microaerobic conditions were investigated. Independent bacteriophage treatments (n ≥ 3) led to 1 to 3 log10 CFU/cm² reductions in the viable count 24 h postinfection compared with control levels. In contrast, bacteriophages applied under these conditions effected a reduction of less than 1 log10 CFU/ml in planktonic cells. Resistance to bacteriophage in bacteria surviving bacteriophage treatment of C. jejuni NCTC 11168 biofilms was 84% and 90% for CP8 and CP30, respectively, whereas bacteriophage resistance was not found in similarly recovered C. jejuni PT14 cells. Dispersal of the biofilm matrix by bacteriophage was demonstrated by crystal violet staining and transmission electron microscopy. Bacteriophage may play an important role in the control of attachment and biofilm formation by Campylobacter in situations where biofilms occur in nature, and they have the potential for application in industrial situations leading to improvements in food safety.
Assuntos
Bacteriólise , Bacteriófagos/crescimento & desenvolvimento , Biofilmes/crescimento & desenvolvimento , Campylobacter jejuni/crescimento & desenvolvimento , Campylobacter jejuni/virologia , Viabilidade MicrobianaRESUMO
Covalent attachment of poly(ethylene) glycol (PEG) groups to proteins, a process commonly called PEGylation, is often used to improve the performance of a protein in vivo. To date, at least eight such PEGylated peptide and protein conjugates have been approved as therapeutic agents and many more have undergone clinical trials. This review examines PEGylation from the perspective of developing a commercially viable drug product. The first section focuses on obtaining a pure and well-characterized drug substance. The latter section discusses formulation and manufacturing issues, with an emphasis on analytical methodology that provides the most detailed description of the purity and stability of PEGylated proteins.
Assuntos
Técnicas de Química Analítica/métodos , Química Farmacêutica/métodos , Polietilenoglicóis/química , Proteínas/química , Humanos , Peptídeos/química , Peptídeos/uso terapêutico , Polietilenoglicóis/farmacocinética , Estabilidade Proteica , Proteínas/farmacocinéticaRESUMO
In 1989, Manning, Patel, and Borchardt wrote a review of protein stability (Manning et al., Pharm. Res. 6:903-918, 1989), which has been widely referenced ever since. At the time, recombinant protein therapy was still in its infancy. This review summarizes the advances that have been made since then regarding protein stabilization and formulation. In addition to a discussion of the current understanding of chemical and physical instability, sections are included on stabilization in aqueous solution and the dried state, the use of chemical modification and mutagenesis to improve stability, and the interrelationship between chemical and physical instability.
Assuntos
Preparações Farmacêuticas/química , Preparações Farmacêuticas/metabolismo , Proteínas/química , Proteínas/metabolismo , Animais , Estabilidade de Medicamentos , Humanos , Estabilidade ProteicaRESUMO
BACKGROUND: Local activation of Rho GTPases is important for many functions including cell polarity, morphology, movement, and growth. Although a number of molecules affecting Rho-of-Plants small GTPase (ROP) signalling are known, it remains unclear how ROP activity becomes spatially organised. Arabidopsis root hair cells produce patches of ROP at consistent and predictable subcellular locations, where root hair growth subsequently occurs. METHODOLOGY/PRINCIPAL FINDINGS: We present a mathematical model to show how interaction of the plant hormone auxin with ROPs could spontaneously lead to localised patches of active ROP via a Turing or Turing-like mechanism. Our results suggest that correct positioning of the ROP patch depends on the cell length, low diffusion of active ROP, a gradient in auxin concentration, and ROP levels. Our theory provides a unique explanation linking the molecular biology to the root hair phenotypes of multiple mutants and transgenic lines, including OX-ROP, CA-rop, aux1, axr3, tip1, eto1, etr1, and the triple mutant aux1 ein2 gnom(eb). CONCLUSIONS/SIGNIFICANCE: We show how interactions between Rho GTPases (in this case ROPs) and regulatory molecules (in this case auxin) could produce characteristic subcellular patterning that subsequently affects cell shape. This has important implications for research on the morphogenesis of plants and other eukaryotes. Our results also illustrate how gradient-regulated Turing systems provide a particularly robust and flexible mechanism for pattern formation.
Assuntos
Arabidopsis/citologia , Arabidopsis/enzimologia , Ácidos Indolacéticos/metabolismo , Modelos Biológicos , Raízes de Plantas/citologia , Raízes de Plantas/enzimologia , Proteínas rho de Ligação ao GTP/metabolismo , Genótipo , Mutação/genética , Fenótipo , Transporte Proteico , Fatores de TempoRESUMO
The dominant metric for setting public health priorities, the disability-adjusted life year (DALY), is unsuited to parasitic infections. In particular, the current DALY framework fails to acknowledge the non-linear pathologies of infection, the community level dynamics of epidemiology and the co-morbidities of polyparasitism. Parasitologists must urgently provide a better way of accounting for the true costs of parasitic disease.
Assuntos
Avaliação da Deficiência , Doenças Parasitárias/epidemiologia , Saúde Pública/normas , Anos de Vida Ajustados por Qualidade de Vida , Adulto , Fatores Etários , Animais , Comorbidade , Humanos , Doenças Parasitárias/parasitologia , Doenças Parasitárias/patologia , Adulto JovemRESUMO
The monthly incidence of listeriosis infections in England and Wales had 2 sudden increases during April 2001 (41%) and March 2003 (48%). Although no causative association is demonstrated, these increases correspond to key dates relating to the onset and aftermath of the 2001 foot and mouth disease outbreak in the United Kingdom.