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Currently licensed vaccines require a cold-chain to maintain efficacy. This cold-chain requirement reduces the availability of vaccines in resource-poor areas of the world. Commercially available human papillomavirus (HPV) vaccines protect against the most common HPV types related to cervical cancer; however, their impact is limited in many regions due to cold-chain requirements. The goal of this study was to test the thermostability of an adjuvanted, trivalent HPV L1 capsomere-based vaccine (containing HPV types 16, 18, and 31) that was formulated by using lyophilization to embed the antigens within a solid, glassy matrix. Thermal stabilities were determined by storing the vaccine formulations for 3 months at 50 °C, followed by immunization of BALB/c mice and measurement of antibody responses. Antibody responses to capsomere vaccines formulated with alum were unchanged after storage for 3 months at 50 °C. Neutralizing responses to these vaccines were unchanged by high-temperature storage, and were equivalent to those generated after administration of the commercially available liquid HPV vaccine Gardasil®9.

This work reports the ability of hydrogel coatings to protect therapeutic proteins from cavitation-induced aggregation caused by mechanical stress. Here, we show that polyacrylamide hydrogel coatings on container surfaces suppress mechanical shock-induced cavitation and the associated aggregation of intravenous immunoglobulin (IVIg). First, crosslinked polyacrylamide hydrogels were grown on the surfaces of borosilicate glass vials. Treatment with ultrasound showed that these hydrogel surfaces suppressed cavitation events to levels below those found for unfunctionalized borosilicate glass. Next, IVIg solutions were loaded into these vials and subjected to tumbling, horizontal shaking, and drop testing. Aggregation was quantified by bisANS fluorescence staining and particle counting by flow imaging microscopy (FIM). In all cases, the presence of polyacrylamide hydrogels on the vial surfaces reduced the amount of IVIg aggregation and the number of particulates. In addition, the polyacrylamide appeared to have a protective effect that prevented additional aggregates from forming at extended tumbling times. Finally, drop test studies showed that the polyacrylamide coatings suppressed detectable cavitation. This work reveals how even a simple hydrogel vial coating can have a profound effect on stabilizing protein therapeutics.

Background: Non-steroidal anti-inflammatory drugs, e.g., celecoxib, are commonly used for inflammatory conditions, but can be associated with adverse effects. Combined glucosamine hydrochloride plus chondroitin sulfate (GH+CS) are commonly used for joint pain and have no known adverse effects. Evidence from in vitro, animal and human studies suggest that GH+CS have anti-inflammatory activity, among other mechanisms of action. Objective: We evaluated the effects of GH+CS versus celecoxib on a panel of 20 serum proteins involved in inflammation and other metabolic pathways. Methods: Samples were from a randomized, parallel, double-blind trial of pharmaceutical grade 1500 mg GH + 1200 mg CS (n=96) versus 200 mg celecoxib daily (n=93) for 6- months in knee osteoarthritis (OA) patients. Linear mixed models adjusted for age, sex, body mass index, baseline serum protein values, and rescue medicine use assessed the intervention effects of each treatment arm adjusting for multiple testing. Results: All serum proteins except WNT16 were lower after treatment with GH+CS, while about half increased after celecoxib. Serum IL-6 was significantly reduced (by 9%, P=0.001) after GH+CS, and satisfied the FDR Conclusion: The results of this study using samples from a previously conducted trial in OA patients, demonstrate that GH+CS reduces circulating IL-6, an inflammatory cytokine, but is otherwise comparable to celecoxib with regard to effects on other circulating protein biomarkers.

Concerns regarding the impact of subvisible particulate impurities on the safety and efficacy of therapeutic protein products have led manufacturers to implement strategies to minimize protein aggregation and particle formation during manufacturing, storage, and shipping. However, once these products are released, manufacturers have limited control over product handling. In this work, we investigated the effect of di(2-ethylhexyl) phthalate (DEHP) nanodroplets generated in polyvinyl chloride (PVC) bags of intravenous (IV) saline on the stability and immunogenicity of IV immunoglobulin (IVIG) formulations. We showed that PVC IV bags containing saline can release DEHP droplets into the solution when agitated or transported using a pneumatic tube transportation system in a clinical setting. We next investigated the effects of emulsified DEHP nanodroplets on IVIG stability and immunogenicity. IVIG adsorbed strongly to DEHP nanodroplets, forming a monolayer. In addition, DEHP nanodroplets accelerated IVIG aggregation in agitated samples. The immunogenicity of DEHP nanodroplets and IVIG aggregates generated in these formulations were evaluated using an in vitro assay of complement activation in human serum. The results suggested DEHP nanodroplets shed from PVC IV bags could reduce protein stability and induce activation of the complement system, potentially contributing to adverse immune responses during the administration of therapeutic proteins.

Reconstitution of lyophilized disaccharide formulations results in the formation of nano-sized air bubbles that persist in suspension for weeks. If proteins are present, interactions with nanobubbles may cause loss of monomeric protein and formation of sub-visible particles. The goals of this work are to determine the mechanism(s) by which nanobubbles form in reconstituted lyophilized formulations, and to develop strategies for reducing nanobubble generation. We hypothesize that nanobubbles are created from nano-sized gas pockets within lyophilized solids, which become bubbles when the surrounding matrix is dissolved away during reconstitution. Nano-sized voids may originate from small ice crystals formed within the concentrated liquid during freezing that subsequently sublime during drying. Nanobubble concentrations are correlated with the extent of mannitol crystallization during freezing. Nano-sized ice crystals, induced by the release of water during mannitol crystallization, were responsible for nanobubble formation. The presence of trehalose or sucrose, in formulations with low mannitol concentrations, inhibited excipient crystallization during lyophilization and reduced nanobubble levels following reconstitution. Our results show a correlation between nanobubble formation and concentrations of insoluble IL-1ra aggregates, suggesting that minimizing nanobubble generation may be an effective strategy for reducing protein aggregation following reconstitution.

When administered intravenously, various particles and nanomedicines activate complement, potentially leading to infusion reactions and other adverse drug reactions. Particles form within formulations of therapeutic proteins due to stresses incurred during shipping, handling and administration to patients. In this study, IVIg solutions were stored in multiple types of vials and prefilled syringes and exposed to agitation and freeze thawing stresses to generate particles. The stressed samples were added to human serum to determine whether these particles activated complement. Sub-visible IVIg particles ranging in size between 2 and 10 microns activated complement in a fashion that was linearly dependent on the number of particles dosed, whereas little correlation was found between doses of larger particles (>10 microns) and complement activation. Activation of complement by sub-visible particles of IVIg followed the alternative pathway, as shown by the release of complement cascade factor Bb and the production of the anaphylatoxins C3a and C5a without generation of C4a. The number and the morphology of sub-visible particles formed depended on the applied stress, formulation and on the container material. But the capacity of the 2-10 micron-sized particles to activate complement in human serum appeared to depend only on particle concentration.

Consumption of dietary lignans has been associated with reduced risk of chronic diseases, although the underlying mechanisms are unclear. We sought to determine if urinary excretion of ENL, the predominant microbial metabolite of dietary lignans, was associated with plasma protein abundance using a cross-sectional design based on data and plasma collected from 80 healthy participants in a randomized crossover, controlled feeding study. Proteomic analysis was performed on plasma samples collected at the end of each of two diet periods (160 samples total) using a customized antibody array corresponding to 2072 unique proteins. GC-MS was used to measure ENL excretion in 24-h urine samples collected in tandem with plasma. Linear mixed models tested the association between urinary enterolignan excretion and plasma protein abundance. Subsequently, over-representation analysis was conducted considering 17 a priori pathways with putative associations with enterolignan bioactivity in an exploratory approach. Controlling the false discovery rate at 10%, ENL excretion was inversely associated with seven proteins (FCRL5, PSCA, GAB1, LAPTM5, CCS, REG4, CEACAM1), and positively associated with two proteins (WAS and FBLN5). Over-representation analysis revealed associations of ENL excretion with estrogen and TNF signaling pathways, which were not significant after adjustment for false discovery. These ENL-associated proteins and pathways have potential implications in cancer prevention.

Zaire ebolavirus (EBOV), Sudan ebolavirus (SUDV), and Marburg marburgvirus (MARV) are the most prevalent and pathogenic species of filovirus. Previously, we showed that glycoprotein antigens from each virus could be lyophilized to create thermostable monovalent subunit vaccines. However, cross-protection is not expected from the monovalent vaccines and therefore developing a trivalent filovirus vaccine would be desirable. Subunit protein vaccines often require the addition of an adjuvant to sufficiently boost the immunogenicity. Typically, liquid suspensions or emulsions of adjuvants and lyophilized antigens are stored in separate vials to avoid destabilizing interactions and are only mixed immediately before administration. Herein, we describe the development and characterization of monovalent and trivalent filovirus vaccines that are co-lyophilized with a squalane-in-water emulsion adjuvant. We found that the single-vial presentation retained adjuvant particle diameter and zeta potential after lyophilization and reconstitution. Furthermore, the trivalent vaccines elicited high antibody levels against all three antigens in mice and non-human primates. These results advance the prospect of developing a single-vial trivalent filovirus vaccine, which would enable easier distribution and administration of the vaccine to resource-poor areas.

Cavitation can occur when liquids are exposed to pressure waves of sufficient amplitude, producing rapidly expanding and collapsing gas bubbles that generate localized regions of high energy dissipation. When vials containing insulin were subjected to mechanical shock or when ultrasound was applied to the vials, the resulting cavitation events induced formation of insulin amyloid fibril nuclei that were detected by transmission electron microscopy and quantified by fluorescence spectroscopy following staining with the amyloid-sensitive dye thioflavin-T. Dropping insulin solutions in glass vials produced only minute amounts of insulin fibril nuclei, which could be detected by allowing the nuclei to grow. Cavitation-induced formation of amyloid aggregates may be relevant for iatrogenic insulin deposition disease, where insulin fibrils formed in vitro prior to administration to patients could serve as nuclei for growing fibril deposits in vivo.

BACKGROUND: Low–glycemic load dietary patterns, characterized by consumption of whole grains, legumes, fruits, and vegetables, are associated with reduced risk of several chronic diseases. METHODS: Using samples from a randomized, controlled, crossover feeding trial, we evaluated the effects on metabolic profiles of a low-glycemic whole-grain dietary pattern (WG) compared with a dietary pattern high in refined grains and added sugars (RG) for 28 d. LC-MS-based targeted metabolomics analysis was performed on fasting plasma samples from 80 healthy participants (n = 40 men, n = 40 women) aged 18–45 y. Linear mixed models were used to evaluate differences in response between diets for individual metabolites. Kyoto Encyclopedia of Genes and Genomes (KEGG)–defined pathways and 2 novel data-driven analyses were conducted to consider differences at the pathway level. RESULTS: There were 121 metabolites with detectable signal in >98% of all plasma samples. Eighteen metabolites were significantly different between diets at day 28 [false discovery rate (FDR)

Silicone oil, used as a lubricating coating in pharmaceutical containers, has been implicated as a cause of therapeutic protein aggregation. After adsorbing to silicone oil-water interfaces, proteins may form interfacial gels, which can be transported into solution as insoluble aggregates if the interfaces are perturbed. Mechanical interfacial perturbation of both monomeric recombinant human interleukin-1 receptor antagonist (rhIL-1ra) and PEGylated rhIL-1ra (PEG rhIL-1ra) in siliconized syringes resulted in losses of soluble monomeric protein. However, the loss of rhIL-1ra was twice that for PEG rhIL-1ra, even though in solution, PEG rhIL-1ra had a lower ΔG(unf) and exhibited a more perturbed tertiary structure at the interface. Net proteinprotein interactions in solution for rhIL-1ra were attractive, but increased steric repulsion due to PEGylation led to net repulsive interactions for PEG rhIL-1ra. Attractive interactions for rhIL-1ra were associated with increases in intermolecular β-sheet content at the interface, whereas no intermolecular β-sheet structures were observed for adsorbed PEG rhIL-1ra. rhIL-1ra formed interfacial gels that were five times stronger than those formed by PEG rhIL-1ra. Thus, the steric repulsion contributed by the PEGylation resulted in decreased interfacial gelation and in the reduction of aggregation, in spite of the destabilizing effects of PEGylation on the protein’s conformational stability.

Therapeutic proteins are among the most widely prescribed medications, with wide distribution and complex supply chains. Shipping exposes protein formulations to stresses that can trigger aggregation, although the exact mechanism(s) responsible for aggregation are unknown. To better understand how shipping causes aggregation, we compared populations of aggregates that were formed in a polyclonal antibody formulation during live shipping studies to populations observed in accelerated stability studies designed to mimic both the sporadic high g-force and continuous low g-force stresses encountered during shipping. Additionally, we compared the effects on aggregation levels generated in two types of secondary packaging, one of which was designed to mitigate the effects of large g-force stresses. Aggregation was quantified using fluorescence intensity of 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) dye, size exclusion high performance liquid chromatography (SECHPLC), and flow imaging microscopy (FIM). FIM was also combined with machine learning methods to analyze particle morphology distributions. These comparisons revealed that the morphology distributions of aggregates formed during live shipping resemble distributions that result from low g-force events, but not those observed following high g-force events, suggesting that low g-force stresses play a predominant role in shipping-induced aggregation.

BACKGROUND: Results from observational studies suggest high diet quality favorably influences the human gut microbiome. Fruit and vegetable consumption is often a key contributor to high diet quality. OBJECTIVE: To evaluate measures of gut bacterial diversity and abundance in relation to serum biomarkers of fruit and vegetable intake. DESIGN: Secondary analysis of cross-sectional data. PARTICIPANTS/SETTING: Men and women from Los Angeles, CA and Hawai’i who participated in the Multiethnic Cohort - Adiposity Phenotype Study from 2013–2016 (n=1709). MAIN OUTCOME MEASURES: Gut microbiome diversity and composition in relation to dietary biomarkers. STATISTICAL ANALYSES: Carotenoid (beta-carotene, alpha-carotene, cryptoxanthins, lutein, lycopene, and zeaxanthin), tocopherol (alpha, beta+gamma, and delta), and retinol concentrations were assessed in serum. Alpha and beta diversity and composition of the gut microbiome were classified based on 16S rRNA gene sequencing of bacterial DNA from self-collected fecal samples. Global differences in microbial community profiles in relation dietary biomarkers were evaluated using multivariable permutational analysis of variance (PERMANOVA). Associations of alpha diversity (Shannon index), beta diversity (weighted and unweighted UniFrac) with center log-ratio-transformed phyla and genera abundances were evaluated using linear regression, adjusted for covariates. RESULTS: Increasing total carotenoid, beta-carotene, alpha-carotene, cryptoxanthin, and lycopene concentrations were associated with higher gut bacterial diversity (Shannon Index) (p

Background The gut microbiome may play a role in inflammation associated with type 2 diabetes (T2D) development. This cross-sectional study examined its relation with glycemic status within a subset of the Multiethnic Cohort (MEC) and estimated the association of circulating bacterial endotoxin (measured as plasma lipopolysaccharide-binding protein (LBP)) with T2D, which may be mediated by C-reactive protein (CRP). Methods In 2013–16, cohort members from five ethnic groups completed clinic visits, questionnaires, and stool and blood collections. Participants with self-reported T2D and/or taking medication were considered T2D cases. Those with fasting glucose >125 and 100–125 mg/dL were classified as undiagnosed (UT2D) and pre-diabetes (PT2D) cases, respectively. We characterized the gut microbiome through 16S rRNA gene sequencing and measured plasma LBP and CRP by standard assays. Linear regression was applied to estimate associations of the gut microbiome community structure and LBP with T2D status adjusting for relevant confounders. Results Among 1,702 participants (59.9–77.4 years), 735 (43%) were normoglycemic (NG), 506 (30%) PT2D, 154 (9%) UT2D, and 307 (18%) T2D. The Shannon diversity index decreased (ptrend = 0.05), while endotoxin, measured as LBP, increased (ptrend = 0.0003) from NG to T2D. Of 10 phyla, Actinobacteria (ptrend = 0.007), Firmicutes (ptrend = 0.003), and Synergistetes (ptrend = 0.02) were inversely associated and Lentisphaerae (ptrend = 0.01) was positively associated with T2D status. Clostridium sensu stricto 1, Lachnospira, and Peptostreptococcaceae were less, while Escherichia-Shigella and Lachnospiraceae were more abundant among T2D patients, but the associations with Actinobacteria, Clostridium sensu stricto 1, and Escherichia-Shigella may be due metformin use. PT2D/UT2D values were closer to NG than T2D. No indication was detected that CRP mediated the association of LBP with T2D. Conclusions T2D but not PT2D/UT2D status was associated with lower abundance of SCFA-producing genera and a higher abundance of gram-negative endotoxin-producing bacteria suggesting that the gut microbiome may contribute to chronic systemic inflammation and T2D through bacterial translocation.

Busulfan-based conditioning is the most commonly used high-dose conditioning regimen for allogeneic hematopoietic cell transplant (HCT). The alkylating agent busulfan has a narrow therapeutic index, with busulfan doses personalized to a target plasma exposure (targeted busulfan). Using a global pharmacometabonomics approach, we sought to identify novel biomarkers of relapse or acute graft versus host disease (GVHD) in a cohort of 84 patients receiving targeted busulfan before allogeneic HCT. A total of 763 endogenous metabolomic compounds (EMCs) were quantitated in 230 longitudinal blood samples before, during, and shortly after intravenous busulfan administration. We performed both univariate linear regression and pathway enrichment analyses using global testing. The cysteine/methionine pathway and the glycine, serine, and threonine metabolism pathway were most associated with relapse. The latter be explained by the fact that glutathione S-transferases conjugate both busulfan and glutathione, which contains glycine as a component. The d-arginine and d-ornithine metabolism pathway and arginine and proline metabolism pathway were most associated with acute GVHD. None of these associations were significant after correcting for false discovery rate (FDR) with a strict cutoff of FDR-adjusted p < 0.1. Although larger studies are needed to substantiate these findings, the results show that EMCs may be used as predictive biomarkers in HCT patients.

Aggregation of therapeutic proteins can result from a number of stress conditions encountered during their manufacture, transportation, and storage. This work shows the effects of two interrelated sources of protein aggregation: the chemistry and structure of the surface of the container in which the protein is stored, and mechanical shocks that may result from handling of the formulation. We investigated how different mechanical stress conditions (dropping, tumbling and agitation) and container surface passivation affected the stability of solutions of intravenous immunoglobulin (IVIG). Application of mechanical shock caused cavitation to occur in the protein solution, followed by bubble collapse and the formation of high-velocity fluid microjets that impinged on container surfaces, leading to particle formation. We also observed cavitation following dropping of vials from heights as low as 5 cm and that polyethylene glycol (PEG) grafting could provide temporary protection against drop-induced cavitation. PEG treatment of the vial surface reduced the formation of protein aggregates observed after repeated dropping events, most likely by reducing protein adsorption to container surfaces. These studies enable the development of new coatings and surface chemistries that can reduce the particulate formation induced by surface adsorption or/and mechanical shock.

Cold-chain requirements affect worldwide distribution of many vaccines. In addition, vaccines requiring multiple doses impose logistical and financial burdens, as well as patient compliance barriers. To address such limitations, we have developed new technologies to prepare thermostable, single-shot, prime-boost microparticle vaccines. Antigen/adjuvant formulations containing glass-forming polymers and trehalose first are spray-dried to form glassy microparticles that confer thermostability. Atomic layer deposition (ALD) reactions conducted in fluidized beds are then used to coat the microparticles with defined numbers of molecular layers of alumina that modulate the timed release of the internalized antigen and act as adjuvants. We have used a model HPV16 L1 capsomere antigen to evaluate the properties of these technologies. Thermostabilized powders containing HPV16 L1 capsomeres were prepared by spray-drying, coated by ALD with up to 500 molecular layers of alumina, and injected into mice. Antigen distribution was assessed by live-animal IR dye tracking of injected labeled antigen. Antibody responses were measured weekly by ELISA, and neutralizing antibodies were measured by pseudovirus neutralization assays at selected time points. Thermostability was evaluated by measuring antibody responses after incubating ALD-coated antigen powders for one month at 50 °C. Single doses of the ALD-coated vaccine formulations elicited a prime-boost immune response, and produced neutralizing responses and antibody titers that were equivalent or superior to conventional prime-boost doses of liquid formulations. Antibody titers were unaffected by month-long incubation of the formulations at 50 °C. Single-dose, thermostable antigen preparations may overcome current limitations in HPV vaccine delivery as well as being widely applicable to other antigens.

Plant lignans and their microbial metabolites, e.g., enterolactone (ENL), may affect bile acid (BA) metabolism through interaction with hepatic receptors. We evaluated the effects of a flaxseed lignan extract (50 mg/day secoisolariciresinol diglucoside) compared to a placebo for 60 days each on plasma BA concentrations in 46 healthy men and women (20&ndash, 45 years) using samples from a completed randomized, crossover intervention. Twenty BA species were measured in fasting plasma using LC-MS. ENL was measured in 24-h urines by GC-MS. We tested for a) effects of the intervention on BA concentrations overall and stratified by ENL excretion, and b) cross-sectional associations between plasma BA and ENL. We also explored the overlap in bacterial metabolism at the genus level and conducted in vitro anaerobic incubations of stool with lignan substrate to identify genes that are enriched in response to lignan metabolism. There were no intervention effects, overall or stratified by ENL at FDR &lt, 0.05. In the cross-sectional analysis, irrespective of treatment, five secondary BAs were associated with ENL excretion (FDR &lt, 0.05). In vitro analyses showed positive associations between ENL production and bacterial gene expression of the bile acid-inducible gene cluster and hydroxysteroid dehydrogenases. These data suggest overlap in community bacterial metabolism of secondary BA and ENL.

OBJECTIVES: Health benefits of high-fiber foods may be attributed, in part, to microbial metabolites of plant compounds. Lignans and their microbial metabolites, the enterolignans [enterolactone (ENL) and enterodiol (END)], reduce serum lipids through a variety of mechanisms, including regulation of bile acid (BA) synthesis. BA, released into the gut lumen in response to dietary fat, undergo microbial metabolism to secondary (2°) BA, which have been positively associated with chronic disease, e.g., liver disease and colorectal cancer. Our aims were to evaluate the effects of a flaxseed lignan supplement on circulating BA and examine associations between enterolignans and 2° BA. METHODS: We conducted a randomized, crossover trial of a flaxseed lignan supplement (50 mg/d secoisolariciresinol diglucoside) compared to placebo in 46 healthy men and women (20-45 y). Each period lasted 60 days, separated by a 60-day washout period. Six primary and fourteen 2° BA species were measured in fasting plasma using LC-MS. ENL and END were measured in 24-h urines by GC-MS. Low- and high-ENL excreters were defined as below and above the median 24-h ENL excretion at the end of the flaxseed lignan intervention (23.4 µmol/24 h). Linear mixed models were used to a) test the effects of the intervention on individual BA concentrations, overall and stratified by low and high ENL excreters; and b) to cross-sectionally determine the association between plasma 2(o) BA and ENL and END. RESULTS: There was no significant effect of the flaxseed lignan intervention compared to placebo on BA concentrations overall, or by ENL-excreter status, after FDR adjustment. In the cross-sectional analysis, irrespective of treatment, six 2° BA were statistically significantly associated with ENL (FDR

The filoviruses Zaire ebolavirus (EBOV), Marburg marburgvirus (MARV), and Sudan ebolavirus (SUDV) are some of the most lethal infectious agents known. To date, the Zaire ebolavirus vaccine (ERVEBO®) is the only United States Food and Drug Administration (FDA) approved vaccine available for any species of filovirus. However, the ERVEBO® vaccine requires cold-chain storage not to exceed −60 °C. Such cold-chain requirements are difficult to maintain in low- and middle-income countries where filovirus outbreaks originate. To improve the thermostability of filovirus vaccines in order to potentially relax or eliminate these cold-chain requirements, monovalent subunit vaccines consisting of glycoproteins from EBOV, MARV, and SUDV were stabilized within amorphous disaccharide glasses through lyophilization. Lyophilized formulations and liquid controls were incubated for up to 12 weeks at 50 °C to accelerate degradation. To identify a stability-indicating assay appropriate for monitoring protein degradation and immunogenicity loss during these accelerated stability studies, filovirus glycoprotein secondary, tertiary, and quaternary structures and vaccine immunogenicity were measured. Size-exclusion chromatography was the most sensitive indicator of glycoprotein stability in the various formulations for all three filovirus immunogens. Degradation of the test vaccines during accelerated stability studies was reflected in changes in quaternary structure, which were discernible with size-exclusion chromatography. Filovirus glycoproteins in glassy lyophilized formulations retained secondary, tertiary, and quaternary protein structure over the incubation period, whereas the proteins within liquid controls both aggregated to form higher molecular weight species and dissociated from their native quaternary structure to form a variety of structurally-perturbed lower molecular weight species.

BACKGROUND: Trimethylamine N-oxide (TMAO), a compound derived from diet and metabolism by the gut microbiome, has been associated with several chronic diseases, although the mechanisms of action are not well understood and few human studies have investigated microbes involved in its production. OBJECTIVES: Our study aims were 1) to investigate associations of TMAO and its precursors (choline, carnitine, and betaine) with inflammatory and cardiometabolic risk biomarkers; and 2) to identify fecal microbiome profiles associated with TMAO. METHODS: We conducted a cross-sectional analysis using data collected from 1653 participants (826 men and 827 women, aged 60–77 y) in the Multiethnic Cohort Study. Plasma concentrations of TMAO and its precursors were measured by LC-tandem MS. We also analyzed fasting blood for markers of inflammation, glucose and insulin, cholesterol, and triglycerides (TGs), and further measured blood pressure. Fecal microbiome composition was evaluated by sequencing the 16S ribosomal RNA gene V1–V3 region. Associations of TMAO and its precursors with disease risk biomarkers were assessed by multivariable linear regression, whereas associations between TMAO and the fecal microbiome were assessed by permutational multivariate ANOVA and hurdle regression models using the negative binomial distribution. RESULTS: Median (IQR) concentration of plasma TMAO was 3.05 μmol/L (2.10–4.60 μmol/L). Higher concentrations of TMAO and carnitine, and lower concentrations of betaine, were associated with greater insulin resistance (all P

Stable formulations of inactivated polio vaccine (IPV) could reduce cold-chain requirements and increase distribution of the vaccine to developing countries. Recently, significant improvement in thermal stability of IPV vaccines has been achieved by including urea in lyophilized formulations. In the present study, we investigated the effects of urea on recovery of potency of IPV after lyophilization and storage at 37°C and the correlation of potency recovery with key biophysical properties of IPV. By dynamic light scattering and transmission light microscopy, we found that loss of potency appeared to be due to agglomeration of virus particles during lyophilization and that moderate concentrations (e.g., 0.4 M) of urea reduced agglomeration and improved potency recovery. In addition, the relative thermal stability of the viron proteins was assessed after rehydration with temperature-dependent intrinsic fluorescence. Lyophilization of formulations without urea and postdrying storage resulted in reduced apparent melting temperatures in rehydrated samples. In formulations with urea, the rehydrated samples had thermal transitions and melting temperatures that were similar to those observed in aqueous control samples. Overall, the results indicated that in IPV formulations, urea improved potency recovery by inhibiting viron particle agglomeration and reducing denaturation of viron proteins.

The presence of subvisible particles in formulations of therapeutic proteins is a risk factor for adverse immune responses. Although the immunogenic potential of particulate contaminants likely depends on particle structural characteristics (e.g., composition, size, and shape), exact structure-immunogenicity relationships are unknown. Images recorded by flow imaging microscopy reflect information about particle morphology, but flow microscopy is typically used to determine only particle size distributions, neglecting information on particle morphological features that may be immunologically relevant. We recently developed computational techniques that utilize the Kullback-Leibler divergence and multidimensional scaling to compare the morphological properties of particles in sets of flow microscopy images. In the current work, we combined these techniques with expectation maximization cluster analyses and used them to compare flow imaging microscopy data sets that had been collected by the U.S. Food and Drug Administration after severe adverse drug reactions (including 7 fatalities) were observed in patients who had been administered some lots of peginesatide formulations. Flow microscopy images of particle populations found in the peginesatide lots associated with severe adverse reactions in patients were readily distinguishable from images of particles in lots where severe adverse reactions did not occur.

We investigated the effects of protein-protein interaction strength on interfacial viscoelastic properties and aggregation of recombinant human interleukin-1 receptor antagonist (rhIL-1ra) at silicone oil-water interfaces. Osmotic second virial coefficients determined by static light scattering were used to quantify protein-protein interactions in bulk solution. Attractive protein-protein interactions dominated at low ionic strengths and their magnitude decreased with increasing ionic strength, in contrast to repulsive interactions that would be expected based on uniformly charged sphere models. Interfacial shear rheometry was used to characterize rhIL-1ra interfacial layers. More attractive protein-protein interactions in bulk solution correlated with stronger interfacial gels. Thioflavin-T fluorescence measurements indicated that the intermolecular β-sheet content of rhIL-1ra incubated in the presence of silicone oil-water interfaces correlated with gel strength. Siliconized syringes were used to probe the effects of mechanical perturbation of the interfacial gel layers. When rhIL-1ra solutions in siliconized glass syringes were subjected to end-over-end rotation, monomeric rhIL-1ra was lost from solution, and particles containing aggregated protein were released into the bulk aqueous phase. The loss of monomeric rhIL-1ra in response to mechanical perturbation was highest under the conditions where the strongest gels were observed. Aggregation of rhIL-1ra was strictly interface-induced and growth of aggregates in the bulk solution was not observed, even in the presence of particles released from silicone oil-water interfaces.

Objectives We previously reported that compared with night sleep, day sleep among shift workers was associated with reduced urinary excretion of 8-hydroxydeoxyguanosine (8-OH-dG), potentially reflecting a reduced ability to repair 8-OH-dG lesions in DNA. We identified the absence of melatonin during day sleep as the likely causative factor. We now investigate whether night work is also associated with reduced urinary excretion of 8-OH-dG. Methods For this cross-sectional study, 50 shift workers with the largest negative differences in night work versus night sleep circulating melatonin levels (measured as 6-sulfatoxymelatonin in urine) were selected from among the 223 shift workers included in our previous study. 8-OH-dG concentrations were measured in stored urine samples using high performance liquid chromatography with electrochemical detection. Mixed effects models were used to compare night work versus night sleep 8-OH-dG levels. Results Circulating melatonin levels during night work (mean=17.1 ng/mg creatinine/mg creatinine) were much lower than during night sleep (mean=51.7 ng/mg creatinine). In adjusted analyses, average urinary 8-OH-dG levels during the night work period were only 20% of those observed during the night sleep period (95% CI 10% to 30%; p Conclusions This study suggests that night work, relative to night sleep, is associated with reduced repair of 8-OH-dG lesions in DNA and that the effect is likely driven by melatonin suppression occurring during night work relative to night sleep. If confirmed, future studies should evaluate melatonin supplementation as a means to restore oxidative DNA damage repair capacity among shift workers.

The effect of an aqueous/organic interface on the folding and aggregation of amphipathic peptides is examined by applying discontinuous molecular dynamics (DMD) simulations combined with an intermediate resolution protein model, PRIME20, to a peptide/interface system. The systems contain 48 (KLLK)4 peptides in random coil or α-helical conformations interacting with both strong and weak interfaces. In the absence of an interface, most of the oligomers form helical bundles, a small fraction of which convert to β-sheets when the temperature is above the folding transition. Adding a weak interface decreases oligomer formation above the folding temperature and increases it below. Little monolayer formation is observed at the weak interface; instead reversible adsorption increases the local peptide concentration near the interface, promoting helical bundle formation in the aqueous phase below the folding temperature and β-sheet formation above the folding temperature. Introducing a strong interface lead...

Aggregates and particles may be generated by positive displacement piston pumps during fill-finishing operations for protein formulations. We investigated potential factors that might contribute to aggregation in intravenous IgG (IVIG) formulations during pumping, including electrostatic interactions between protein molecules and pump surfaces, cavitation, and aggregate nucleation from particles shed from pumps. Electrostatic interactions were investigated by modifying pump surface chemistry. Cavitation as a potential cause of particle formation was investigated by changing pumping speeds, and the possibility that particles shed from pump surfaces act to nucleate protein aggregation was explored by spiking prepumped buffer solutions into IVIG formulations. Neither cavitation nor particles shed from pump surfaces played dominant roles in generating particles. Per pump cycle, production of particles and protein aggregates was constant, and corresponded with the amount of protein expected to adsorb on pump surfaces at monolayer coverage. More subvisible particles and protein aggregates were generated in formulations containing higher concentrations of IVIG, but they reached a plateau at protein concentrations above 2 mg/mL, where adsorption isotherms saturated. Negatively charged pump surfaces interacted with the positively charged IVIG to produce more particles and aggregates than positively charged surfaces, an effect ascribed to electrostatic interactions that moderated rates of protein adsorption.

Mechanical shock may cause cavitation in vials containing liquid formulations of therapeutic proteins and generate protein aggregates and other particulates. To test whether common formulation components such as protein molecules, air bubbles, or polysorbate 20 (PS20) micelles might nucleate cavitation, a high-speed video camera was used to detect cavitation in vials containing antibody formulations after application of controlled mechanical shock using a shock test. Higher concentrations of subvisible particles were found in formulations where cavitation had occurred. Bubbles trapped on vial surfaces were a primary site for cavitation nucleation; other potential cavitation nuclei were ineffective. The incidence of cavitation events observed after application of mechanical shock was lower in type I glass vials than in cyclic olefin polymer vials or in SiOPlas™ cyclic olefin polymer vials and correlated with the surface roughness of the different vials. To reduce the incidence of cavitation and the adsorption of mAb on glass-water and silicone oil-water interfaces and thus minimize protein damage due to cavitation, PS20, a common nonionic surfactant, was added to formulations. Addition of PS20 to formulations in glass and silicone oil-coated glass vials significantly reduced both incidence of mechanical shock-induced cavitation and the particle formation that resulted from cavitation events.

BACKGROUND: Microbial metabolism of lignans from high-fiber plant foods produces bioactive enterolignans, such as enterolactone (ENL) and enterodiol (END). Enterolignan exposure influences cellular pathways important to cancer risk and is associated with reduced colon tumorigenesis in animal models and lower colorectal cancer risk in humans. OBJECTIVES: The aim of this study was to test the effects of a flaxseed lignan supplement (50 mg secoisolariciresinol diglucoside/d) compared with placebo on host gene expression in colon biopsies and exfoliated colonocyte RNA in feces and fecal microbial community composition, and to compare responses in relation to ENL excretion. METHODS: We conducted a 2-period randomized, crossover intervention in 42 healthy men and women (20–45 y). We used RNA-seq to measure differentially expressed (DE) genes in colonic mucosa and fecal exfoliated cells through the use of edgeR and functional analysis with Ingenuity Pathway Analysis. We used 16S ribosomal RNA gene (V1–V3) analysis to characterize the fecal microbiome, and measured END and ENL in 24-h urine samples by gas chromatography–mass spectrometry. RESULTS: We detected 32 DE genes (false discovery rate

No United States Food and Drug Administration-licensed vaccines protective against Ebola virus (EBOV) infections are currently available. EBOV vaccine candidates currently in development, as well as most currently licensed vaccines in general, require transport and storage under a continuous cold chain in order to prevent potential decreases in product efficacy. Cold chain requirements are particularly difficult to maintain in developing countries. To improve thermostability and reduce costly cold chain requirements, a subunit protein vaccine against EBOV was formulated as a glassy solid using lyophilization. Formulations of the key antigen, Ebola glycoprotein (EBOV-GP), adjuvanted with microparticulate aluminum hydroxide were prepared in liquid and lyophilized forms, and the vaccines were incubated at 40 °C for 12 weeks. Aggregation and degradation of EBOV-GP were observed in liquid formulations during the 12-week incubation period, whereas changes were minimal in lyophilized formulations. Antibody responses against EBOV-GP following three intramuscular immunizations in BALB/c mice were used to determine vaccine immunogenicity. EBOV-GP formulations were equally immunogenic in liquid and lyophilized forms. After lyophilization and reconstitution, adjuvanted vaccine formulations produced anti-EBOV-GP IgG antibody responses in mice similar to those generated against corresponding adjuvanted liquid vaccine formulations. More importantly, antibody responses in mice injected with reconstituted lyophilized vaccine formulations that had been incubated at 40 °C for 12 weeks prior to injection indicated that vaccine immunogenicity was fully retained after high-temperature storage, showing promise for future vaccine development efforts.

Identification of the parathyroid glands during thyroid surgery may prevent their inadvertent surgical removal and thus provide a better postoperative quality of life. Nevertheless, the most common “technique” for intraoperative evaluation of perfusion of parathyroid gland tissues during thyroid surgery is visual inspection of the physical condition of tissues, e.g., their color and bleeding edges. Another technique is measurement of intact parathyroid hormone. Recently, indocyanine green-enhanced fluorescence has been used in various surgical techniques, particularly laparoscopic surgery, to improve visualization and to provide detailed anatomical information. Fluorescent optical guidance helps surgeons to avoid inadvertent tissue injury while enhancing procedural efficiency. This technique has potential use for evaluating perfusion of the parathyroid gland in real-time intraoperative angiography.

The generation of nanobubbles following reconstitution of lyophilized trehalose formulations has recently been reported. Here, we characterize particle formation and aggregation of recombinant human interleukin-1 receptor antagonist (rhIL-1ra) in reconstituted formulations of lyophilized trehalose. Particle characterization methods including resonant mass measurement and nanoparticle tracking analysis were used to count and size particles generated upon reconstitution of lyophilized trehalose formulations. In addition, accelerated degradation studies were conducted to monitor rhIL-1ra aggregation in solutions containing various concentrations of suspended nanobubbles. Reconstitution of lyophilized trehalose formulations with solutions containing rhIL-1ra reduced nanobubble concentrations and generated negatively buoyant particles attributed to aggregated rhIL-1ra. Furthermore, levels of rhIL-1ra aggregation following incubation in aqueous solution correlated with concentrations of suspended nanobubbles. The results of this study suggest that nanobubbles may be a contributor to protein aggregation and particle formation in reconstituted, lyophilized therapeutic protein formulations.

In this study, we investigated the effect of ionic strength (1.25-231 mM) on viscoelastic interfacial gels formed by a monoclonal antibody at silicone oil-water interfaces, and the formation of subvisible particles due to rupture of these gels. Rates of gel formation and their elastic moduli did not vary significantly with ionic strength. Likewise, during gel rupture no significant effects of ionic strength were observed on particle formation and aggregation as detected by microflow imaging, resonance mass measurement, and size exclusion chromatography. Subvisible particles formed by mechanical rupturing of the gels agglomerated over time, even during quiescent incubation, due to the colloidal instability of the particles.

Intravenous (IV) busulfan doses are often personalized to a concentration at steady state (Css) using the patient’s clearance, which is estimated with therapeutic drug monitoring. We sought to identify biomarkers of IV busulfan clearance using a targeted pharmacometabonomics approach. A total of 200 metabolites were quantitated in 106 plasma samples, each obtained before IV busulfan administration in hematopoietic cell transplant (HCT) recipients. Both univariate linear regression with false discovery rate (FDR), and pathway enrichment analyses using the Global test were performed. In the univariate analysis, glycine, N-acetylglycine, 2-hydroxyisovaleric acid, creatine, serine, and tyrosine and were statistically significantly associated with IV busulfan clearance at P0.1. Glycine is a component of glutathione, which is conjugated with busulfan via glutathione transferase enzymes. These results demonstrate the potential utility of pharmacometabonomics to inform IV busulfan dosing. Future studies are required to validate these findings.

The impact of drug conjugation on intra- and intermolecular interactions of trastuzumab (TmAb) was determined by comparing the conformational and colloidal stabilities of TmAb and trastuzumab emtansine (T-DM1). In low ionic strength formulations, drug conjugation to native lysine residues of TmAb significantly reduced the repulsive electrostatic interactions between T-DM1 molecules. When these electrostatic interactions were screened in solutions with high ionic strength, intermolecular interactions between T-DM1 molecules were found to be more attractive than those between TmAb molecules. Drug conjugation lowered the colloidal stability of T-DM1 compared to TmAb, making T-DM1 more susceptible to agitation-induced aggregation. The presence of polysorbate-20 in the formulations inhibited aggregation of TmAb and T-DM1 induced by the hydrophobic air-water interface. Furthermore, the effect of increased hydrophobic interactions between T-DM1 molecules was studied by monitoring aggregation in TmAb and T-DM1 solutions that were incubated at 4°C, 25°C, and 50°C. Conjugating DM1 to TmAb increased the hydrophobicity of the molecule, and faster aggregation of T-DM1 at 50°C could be attributed to a temperature-dependent increase in hydrophobic interactions between T-DM1 molecules.

Current technologies for monitoring the subvisible particles that may be generated during fill-finish operations for protein formulations are cumbersome. Measurement times are generally too long for real-time analysis, and the high protein concentrations that are characteristic of many antibody products interfere with common optical techniques for particle analysis. To rapidly monitor protein particle levels in high-concentration protein solutions, we developed a fluorescence-based method that uses extrinsic fluorescent dyes such as 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid that are sensitive to the presence of aggregated protein. To test the method, antibody formulations containing various concentrations of protein particles were generated by application of various mechanical and freeze-thaw stresses. After addition of fluorescent dyes, fluorescence intensities were measured and compared to fluorescence intensities in particle-free formulations. The differences in fluorescence intensities were linearly proportional to protein particle levels, which for calibration purposes were measured offline by fluid imaging microscopy and protein assays. Protein particle levels could be measured without requiring sample dilution, even in high-concentration (e.g., 40 mg/mL) antibody formulations.

Human serum albumin (HSA) is an excipient present in formulations of several recombinant protein products that are approved for clinical use. We investigated the relative contributions of HSA and HSA particles to the generation of antibody responses against recombinant human erythropoietin (rhEPO) and the excipient HSA itself. Protein samples were characterized before injection for quantities of monomeric proteins, soluble protein aggregates, and nano- and micron-sized particles. rhEPO, containing various concentrations of HSA particles, were injected three times a week for 8 weeks into mice. Hematocrits and the production of anti-rhEPO and anti-HSA antibodies were determined at various time points. Levels of antibodies against rhEPO in mice injected with HSA-containing rhEPO were higher than those in mice treated with HSA-free rhEPO. Mice injected with formulations that contained particles of HSA produced strong anti-HSA antibody responses; whereas these responses were greatly reduced when particle-free formulations were administered. In contrast, anti-rhEPO antibody responses were not affected by the presence of particles.

Protein aggregation and particle formation have been observed when protein solutions contact hydrophobic interfaces, and it has been suggested that this undesirable phenomenon may be initiated by interfacial adsorption and subsequent gelation of the protein. The addition of surfactants, such as polysorbate 20, to protein formulations has been proposed as a way to reduce protein adsorption at silicone oil-water interfaces and mitigate the production of aggregates and particles. In an accelerated stability study, monoclonal antibody formulations containing varying concentrations of polysorbate 20 were incubated and agitated in pre-filled glass syringes (PFS), exposing the protein to silicone oil-water interfaces at the siliconized syringe walls, air-water interfaces, and agitation stress. Following agitation in siliconized syringes that contained an air bubble, lower particle concentrations were measured in the surfactant-containing antibody formulations than in surfactant-free formulations. Polysorbate 20 reduced particle formation when added at concentrations above or below the critical micelle concentration (CMC). The ability of polysorbate 20 to decrease particle generation in PFS corresponded with its ability to inhibit gelation of the adsorbed protein layer, which was assessed by measuring the interfacial diffusion of individual antibody molecules at the silicone oil-water interface using total internal reflectance fluorescence (TIRF) microscopy with single-molecule tracking.

High-throughput single-molecule total internal reflection fluorescence microscopy was used to investigate the effects of pH and ionic strength on bovine serum albumin (BSA) adsorption, desorption, and interfacial diffusion at the aqueous-fused silica interface. At high pH and low ionic strength, negatively charged BSA adsorbed slowly to the negatively charged fused silica surface. At low pH and low ionic strength, where BSA was positively charged, or in solutions at higher ionic strength, adsorption was approximately 1000 times faster. Interestingly, neither surface residence times nor the interfacial diffusion coefficients of BSA were influenced by pH or ionic strength. These findings suggested that adsorption kinetics were dominated by energy barriers associated with electrostatic interactions, but once adsorbed, protein-surface interactions were dominated by short-range nonelectrostatic interactions. These results highlight the ability of single-molecule techniques to isolate elementary processes (e.g., adsorption and desorption) under steady-state conditions, which would be impossible to measure using ensemble-averaging methods.

Using high throughput single-molecule total internal reflection fluorescence microscopy (TIRFM), we have acquired molecular trajectories of bovine serum albumin (BSA) and hen egg white lysozyme during protein layer formation at the silicone oil-water interface. These trajectories were analyzed to determine the distribution of molecular diffusion coefficients, and for signatures of molecular crowding/caging, including subdiffusive motion and temporal anticorrelation of the instantaneous velocity vector. The evolution of these properties with aging time of the interface was compared with dynamic interfacial tension measurements. For both lysozyme and BSA, we observed an overall slowing of protein objects, the onset of both subdiffusive and anticorrelated motion (associated with crowding), and a decrease in the interfacial tension with aging time. For lysozyme, all of these phenomena occurred virtually simultaneously, consistent with a homogeneous model of layer formation that involves gradual crowding of weakly interacting proteins. For BSA, however, the slowing occurred first, followed by the signatures of crowding/caging, followed by a decrease in interfacial tension, consistent with a heterogeneous model of layer formation involving the formation of protein clusters. The application of microrheological methods to single molecule trajectories described here provides an unprecedented level of mechanistic interpretation of interfacial events that occurred over a wide range of interfacial protein coverage.

Neuroendocrine tumors (NETs) are a collection of complex tumors that arise from the diffuse endocrine system, primarily from the digestive tract. Carcinoid tumors most commonly originate from the small intestine. These tumors are either referred to as small intestinal neuroendocrine tumors or midgut carcinoids (MGCs). The purpose of this review article is to survey the diagnostic and therapeutic pathways for patients with MGC and provide an overview of the complex multidisciplinary care involved in improving their quality of life, treatment outcomes, and survival.The current literature regarding the diagnosis and management of MGCs was reviewed.Dry flushing and secretory diarrhea are the hallmarks of the clinical syndrome of MGC. Managing MGC requires attention to the overall symptom complex, including the physical effects of the tumor and biomarker levels. The somatostatin analogs (SAs) octreotide and lanreotide are highly efficacious for symptomatic improvement. MGCs require resection to encompass the primary tumor and mesenteric lymph node metastases and should include cholecystectomy if the patient is likely to receive SA therapy. Debulking of liver metastasis by resection in combination with ablative therapies and other liver-directed modalities may help palliate symptoms and hormonal overproduction in carefully selected patients. Quality of life is an important measure of patients' perception of the burden of their disease and impact of treatment modalities and may be a useful guide in deciding changes in therapy to alter apparent health status.MGC is a challenging malignancy that requires the input of a multidisciplinary team to develop the best treatment plan. Consultation with expert centers that specialize in NETs may also be indicated for complex cases. With expert care, patients can be cured or live with the disease and enjoy good quality of life.

Silicone oil is used as a lubricant in glass pre-filled syringes (PFS) but can contribute to the generation of particles within protein formulations in PFS. To mitigate the production of such particles, various silicone oil coating processes have been proposed. In this study, three siliconization methods (the "covalent" method, the "baked silicone oil" method, and the "liquid silicone oil" method) were used to coat glass syringes with silicone oil. Glide forces were determined for syringes coated by each method. Then, a monoclonal antibody formulation or a buffer solution were incubated in the coated syringes in either the presence or absence of an air bubble, and the syringes were rotated end-over-end to induce air bubble movement within the syringe. The particle concentrations were measured throughout the incubation period using flow microscopy. The coating method did not affect particle concentrations measured in buffer alone, nor did the coating method affect particle concentrations measured in antibody formulations in the absence of an air bubble. Particle concentrations were influenced by the syringe coating method in protein formulations agitated in the presence of an air bubble, with the most particles formed in syringes lubricated with liquid silicone oil. Fewer particles were produced in syringes lubricated with baked silicone oil, and the fewest particles were produced in syringes with covalently-attached silicone oil. However, the glide forces measured in syringes coated with silicone oil by each method are inversely correlated with the measured particle concentrations.

Background: Lignans in plant foods are metabolized by gut bacteria to the enterolignans, enterodiol (END) and enterolactone (ENL). Enterolignans have biologic activities important to the prevention of cancer and chronic diseases. We examined the composition of the gut microbial community (GMC) as a contributor to human enterolignan exposure. Methods: We evaluated the association between the GMC in stool, urinary enterolignan excretion, and diet from a 3-day food record in 115 premenopausal (ages 40–45 years) women in the United States. Urinary enterolignans were measured using gas chromatography–mass spectroscopy. The GMC was evaluated using 454 pyrosequencing of the 16S rRNA gene. Sequences were aligned in SILVA (www.arb-silva.de). Operational taxonomic units were identified at 97% sequence similarity. Taxonomic classification was performed and alpha and beta diversity in relationship to ENL production were assessed. Multivariate analysis and regression were used to model the association between enterolignan excretion and the GMC. Bacteria associated with ENL production were identified using univariate analysis and ridge regression. Results: After adjusting for dietary fiber intake and adiposity, we found a significant positive association between ENL excretion and either the GMC (P = 0.0007), or the diversity of the GMC (P = 0.01). The GMC associated with high ENL production was distinct (UNIFRAC, P < 0.003, MRPP) and enriched in Moryella spp., Acetanaerobacterium spp., Fastidiosipila spp., and Streptobacillus spp. Conclusion: Diversity and composition of the GMC are associated with increased human exposure to enterolignans. Impact: Differences in gut microbial diversity and composition explain variation in gut metabolic processes that affect environmental exposures and influence human health. Cancer Epidemiol Biomarkers Prev; 24(3); 546–54. ©2014 AACR.

Industry experience suggests that g-forces sustained when vials containing protein formulations are accidentally dropped can cause aggregation and particle formation. To study this phenomenon, a shock tower was used to apply controlled g-forces to glass vials containing formulations of two monoclonal antibodies and recombinant human growth hormone (rhGH). High-speed video analysis showed cavitation bubbles forming within 30 μs and subsequently collapsing in the formulations. As a result of echoing shock waves, bubbles collapsed and reappeared periodically over a millisecond time course. Fluid mechanics simulations showed low-pressure regions within the fluid where cavitation would be favored. A hydroxyphenylfluorescein assay determined that cavitation produced hydroxyl radicals. When mechanical shock was applied to vials containing protein formulations, gelatinous particles appeared on the vial walls. Size-exclusion chromatographic analysis of the formulations after shock did not detect changes in monomer or soluble aggregate concentrations. However, subvisible particle counts determined by microflow image analysis increased. The mass of protein attached to the vial walls increased with increasing drop height. Both protein in bulk solution and protein that became attached to the vial walls after shock were analyzed by mass spectrometry. rhGH recovered from the vial walls in some samples revealed oxidation of Met and/or Trp residues.

OBJECTIVE: The effects of diets high in refined grains on biliary and colonic bile acids have been investigated extensively. However, the effects of diets high in whole versus refined grains on circulating bile acids, which can influence glucose homeostasis and inflammation through activation of farnesoid X receptor (FXR) and G protein-coupled bile acid receptor 1 (TGR5), have not been studied. MATERIALS AND METHODS: We conducted a secondary analysis from a randomized controlled crossover feeding trial (NCT00622661) in 80 healthy adults (40 women/40 men, age 18–45 years) from the greater Seattle Area, half of which were normal weight (BMI 18.5–25.0 kg/m(2)) and half overweight to obese (BMI 28.0–39.9 kg/m(2)). Participants consumed two four-week controlled diets in randomized order: 1) a whole grain diet (WG diet), designed to be low in glycemic load (GL), high in whole grains, legumes, and fruits and vegetables, and 2) a refined grain diet (RG diet), designed to be high GL, high in refined grains and added sugars, separated by a four-week washout period. Quantitative targeted analysis of 55 bile acid species in fasting plasma was performed using liquid chromatography tandem mass spectrometry. Concentrations of glucose, insulin, and CRP were measured in fasting serum. Linear mixed models were used to test the effects of diet on bile acid concentrations, and determine the association between plasma bile acid concentrations and HOMA-IR and CRP. Benjamini-Hochberg false discovery rate (FDR) < 0.05 was used to control for multiple testing. RESULTS: A total of 29 plasma bile acids were reliably detected and retained for analysis. Taurolithocholic acid (TLCA), taurocholic acid (TCA) and glycocholic acid (GCA) were statistically significantly higher after the WG compared to the RG diet (FDR

In this study we investigated the potential roles of nanoparticles (< 100 nm) and submicron (100-1000 nm) particles in the formation of microparticles (> 1000 nm) in protein formulations under some pharmaceutically-relevant stress conditions. Exposure of intravenous immunoglobulin (IVIG) solutions to the interface-associated stresses of freeze-thawing or agitation resulted in relatively large increases in microparticle concentrations, which depended directly on the levels of preexisting nano- and submicron particles. Thus, agglomeration of nanoparticles and submicron particles appears to play a role in microparticle formation under these stresses. In contrast, increases in microparticle concentrations during quiescent incubation at elevated temperatures were independent of the initial nano- and submicron particle concentrations in solution.

Enterolignans, products of gut bacterial metabolism of plant lignans, have been associated with reduced risk of chronic diseases, but their association with other plasma metabolites is unknown. We examined plasma metabolite profiles according to urinary enterolignan excretion in a cross-sectional analysis using data from a randomized crossover, controlled feeding study. Eighty healthy adult males and females completed two 28-day feeding periods differing by glycemic load, refined carbohydrate, and fiber content. Lignan intake was calculated from food records using a polyphenol database. Targeted metabolomics was performed by LC-MS on plasma from fasting blood samples collected at the end of each feeding period. Enterolactone (ENL) and enterodiol, were measured in 24 h urine samples collected on the penultimate day of each study period using GC-MS. Linear mixed models were used to test the association between enterolignan excretion and metabolite abundances. Pathway analyses were conducted using the Global Test. Benjamini–Hochberg false discovery rate (FDR) was used to control for multiple testing. Of the metabolites assayed, 121 were detected in all samples. ENL excretion was associated positively with plasma hippuric acid and melatonin, and inversely with epinephrine, creatine, glycochenodeoxycholate, and glyceraldehyde (P < 0.05). Hippuric acid only satisfied the FDR of q < 0.1. END excretion was associated with myristic acid and glycine (q < 0.5). Two of 57 pathways tested were associated significantly with ENL, ubiquinone and terpenoid-quinone biosynthesis, and inositol phosphate metabolism. These results suggest a potential role for ENL or ENL-metabolizing gut bacteria in regulating plasma metabolites.

The presence of micron aggregates in protein formulations has recently attracted increased interest from regulatory authorities, industry, and academia because of the potential undesired side effects of their presence. In this study, we characterized the micron aggregate formation of hen egg-white lysozyme (Lyz) and its diPEGylated (5 kDa) analog as a result of typical handling stress conditions. Both proteins were subjected to mechanical stress in the absence and presence of silicone oil (SO), elevated temperatures, and freeze-thaw cycles. Flow imaging microscopy showed that PEGylated Lyz formed approximately half as many particles as Lyz, despite its lower apparent thermodynamic stability and more loose protein fold. Further characterization showed that the PEGylation led to a change from attractive to repulsive protein-protein interactions, which may partly explain the reduced particle formation. Surprisingly, the PEGylated Lyz adsorbed an order of magnitude faster onto SO, despite being much larger in size, as determined by small-angle X-ray scattering and dynamic light scattering measurements. Thus, PEGylation may significantly reduce, but not prevent, micron aggregate formation of a protein during typical handling stresses.

Lyophilized formulations of keratinocyte growth factor-2 (KGF-2) were prepared with a range of disaccharide (sucrose or trehalose) and hydroxyethyl starch (HES) mass ratios. Protein degradation was assessed as a function of time of storage of the dried formulations at 40, 50 and 60 °C. Lyophilized and stored samples were rehydrated, and protein degradation was quantified by measuring loss of monomeric protein with size exclusion chromatography and by determining chemical degradation in the soluble fraction with reverse-phase chromatography. The secondary structure of the protein in the lyophilized formulations was studied with infrared spectroscopy. The magnitudes of degradation were compared the key physical properties of the formulations including retention of protein native secondary structure, glass transition temperature (Tg), inverse mean square displacements 〈 u 2 〉 −1 for hydrogen atoms (fast β relaxation), and the relaxation time τ β , which correlates with relaxation due to fast Johari–Goldstein motions in the glass (Xu et al., 2013) [1]. In addition, specific surface areas of the lyophilized formulations were determined by Brunauer–Emmet–Teller analysis of krypton adsorption isotherms and used to estimate the fraction of the KGF-2 molecules residing at the solid–air interface. KGF-2 degradation rates were highest in formulations wherein the protein’s structure was most perturbed, and wherein β relaxations were fastest, but the dominant factor governing KGF-2 degradation in freeze-dried formulations was the fraction of the protein found at the glass solid–air interface.