Transparency and reproducibility in the Adolescent Brain Cognitive Development (ABCD) study.

BACKGROUND: Transparency can build trust in the scientific process, but scientific findings can be undermined by poor and obscure data use and reporting practices. The purpose of this work is to report how data from the Adolescent Brain Cognitive Development (ABCD) Study has been used to date, and to provide practical recommendations on how to improve the transparency and reproducibility of findings. METHODS: Articles published from 2017 to 2023 that used ABCD Study data were reviewed using more than 30 data extraction items to gather information on data use practices. Total frequencies were reported for each extraction item, along with computation of a Level of Completeness (LOC) score that represented overall endorsement of extraction items. Univariate linear regression models were used to examine the correlation between LOC scores and individual extraction items. Post hoc analysis included examination of whether LOC scores were correlated with the logged 2-year journal impact factor. RESULTS: There were 549 full-length articles included in the main analysis. Analytic scripts were shared in 30 % of full-length articles. The number of participants excluded due to missing data was reported in 60 % of articles, and information on missing data for individual variables (e.g., household income) was provided in 38 % of articles. A table describing the analytic sample was included in 83 % of articles. A race and/or ethnicity variable was included in 78 % of reviewed articles, while its inclusion was justified in only 41 % of these articles. LOC scores were highly correlated with extraction items related to examination of missing data. A bottom 10 % of LOC score was significantly correlated with a lower logged journal impact factor when compared to the top 10 % of LOC scores (ß=-0.77, 95 % -1.02, -0.51; p-value < 0.0001). CONCLUSION: These findings highlight opportunities for improvement in future papers using ABCD Study data to readily adapt analytic practices for better transparency and reproducibility efforts. A list of recommendations is provided to facilitate adherence in future research.

Transparency and Reproducibility in the Adolescent Brain Cognitive Development (ABCD) Study.

Background: Transparency can build trust in the scientific process, but scientific findings can be undermined by poor and obscure data use and reporting practices. The purpose of this work is to report how data from the Adolescent Brain Cognitive Development (ABCD) Study has been used to date, and to provide practical recommendations on how to improve the transparency and reproducibility of findings. Methods: Articles published from 2017 to 2023 that used ABCD Study data were reviewed using more than 30 data extraction items to gather information on data use practices. Total frequencies were reported for each extraction item, along with computation of a Level of Completeness (LOC) score that represented overall endorsement of extraction items. Univariate linear regression models were used to examine the correlation between LOC scores and individual extraction items. Post hoc analysis included examination of whether LOC scores were correlated with the logged 2-year journal impact factor. Results: There were 549 full-length articles included in the main analysis. Analytic scripts were shared in 30% of full-length articles. The number of participants excluded due to missing data was reported in 60% of articles, and information on missing data for individual variables (e.g., household income) was provided in 38% of articles. A table describing the analytic sample was included in 83% of articles. A race and/or ethnicity variable was included in 78% of reviewed articles, while its inclusion was justified in only 41% of these articles. LOC scores were highly correlated with extraction items related to examination of missing data. A bottom 10% of LOC score was significantly correlated with a lower logged journal impact factor when compared to the top 10% of LOC scores (ß=-0.77, 95% -1.02, -0.51; p-value < 0.0001). Conclusion: These findings highlight opportunities for improvement in future papers using ABCD Study data to readily adapt analytic practices for better transparency and reproducibility efforts. A list of recommendations is provided to facilitate adherence in future research.

Effect of residual platelets in frozen-thawed plasma on results of dilute Russell's viper venom time assay for lupus anticoagulant testing.

OBJECTIVES: To determine the impact of residual platelets on dilute Russell's viper venom time (DRVVT) assay in frozen-thawed plasma submitted for lupus anticoagulant (LAC) testing. METHODS: We measured platelet counts in frozen-thawed samples submitted for LAC testing and evaluated the association between platelet count and the DRVVT screening time and ratios. We also spiked platelets into a LAC-positive sample to observe the effect on the DRVVT. RESULTS: Progressive increase in platelet count resulted in a statistically significant shortening of the DRVVT assay results on plasma after 1 freeze-thaw cycle. A similar effect was noted on the LAC-positive sample. CONCLUSIONS: Residual platelets in plasma samples result in shortening of DRVVT assay after 1 freeze-thaw cycle. This may result in a false-negative LAC test result.

Obesity in Sexual and Gender Minority Populations: Prevalence and Correlates.

PURPOSE OF REVIEW: The purpose of this review is to explore the prevalence and correlates of obesity among members of sexual and gender minority (SGM) populations. RECENT FINDINGS: Research overall finds higher rates of obesity among lesbian and bisexual women than heterosexual women, lower rates of obesity among gay and bisexual men than heterosexual men, and inconsistent findings on obesity among transgender individuals. Rates of mental health disorders and disordered eating are high among all SGM groups. Rates of comorbid medical conditions differ among groups. More research needs to be conducted within all SGM groups but particularly among transgender populations. All members of SGM encounter stigma, including when seeking healthcare, and this may lead to individuals avoiding healthcare. Therefore, it is important to educate providers about population-specific factors. This article serves as an overview of important considerations for providers treating individuals within SGM populations.

Internalization of the Thin-Ideal and Eating Pathology in Kuwaiti Adult Women.

Rapid socioeconomic growth, Western acculturation, and thin-ideal internalization are theorized to be linked to dieting, weight concerns, and disordered eating in Arab countries. The purpose of this study was to examine eating pathology and the internalization of Western messages regarding the importance of thinness in a community sample of Kuwaiti women. Participants (N = 83) aged 18-35 years with BMIs between 17.58 and 24.88 (M = 21.30; SD = 1.83) completed measures of thin-ideal internalization and eating pathology. Results demonstrated that approximately 28% of the sample was at risk for eating pathology. Thin-ideal internalization was also found to significantly predict eating pathology. Study findings extend prior research on eating pathology in Arabic nations. This study is the first to examine the internalization of the thin-ideal in a community sample of Kuwait adult women and lends further support to the importance of continued research in this population.

Expression of MAPK and PI3K/AKT/mTOR Proteins according to the Chronic Liver Disease Etiology in Hepatocellular Carcinoma.

AIMS: Chronic liver disease (CLD) of different etiologies leads to hepatocellular carcinoma (HCC) by multiple mechanisms that may be translated into clinicopathological differences. We evaluated the tissue expression of the MAPK and PI3K/Akt/mTOR pathway proteins and their association with long-term outcome and other parameters, according to the etiology of the CLD, in HCC patients. METHODS: Clinicopathological data from 80 patients who underwent orthotopic liver transplantation for HCC treatment in a Brazilian referral center were compared according to CLD etiology. Event (tumor recurrence or death from any cause) occurrence and event-free survival (EFS) were analyzed. Pathway protein expression was assessed by immunohistochemistry (IHQ) in both tumor and underlying cirrhosis and by RT-PCR in tumor tissue. RESULTS: Strong expression (SE) of KRAS was more frequent in tumors arising from viral (26.8%) than the nonviral group of liver disease (7.7%, p=0.024) and also than cirrhotic parenchyma (0%, p=0.004). SE of PI3K was more frequent in tumor than in cirrhosis (p=0.048, p < 0.01), without differences in its tumor expression among etiologic groups (p=0.111). mRNA of ERK, PI3K, and BRAF was expressed in the tumor, without differences between CLD etiologies, and there was no association with IHQ findings. Older age and microvascular invasion (MIV) were the only parameters independently associated with the event. MIV was also associated with shorter EFS. CONCLUSIONS: Hepatitis B and C virus can lead to HCC by different mechanisms compared with nonviral hepatopathy. KRAS and PI3K may have a role in carcinogenesis. The prognostic and therapeutic implications need to be investigated.

Optimization Rules for SARS-CoV-2 Mpro Antivirals: Ensemble Docking and Exploration of the Coronavirus Protease Active Site.

Coronaviruses are viral infections that have a significant ability to impact human health. Coronaviruses have produced two pandemics and one epidemic in the last two decades. The current pandemic has created a worldwide catastrophe threatening the lives of over 15 million as of July 2020. Current research efforts have been focused on producing a vaccine or repurposing current drug compounds to develop a therapeutic. There is, however, a need to study the active site preferences of relevant targets, such as the SARS-CoV-2 main protease (SARS-CoV-2 Mpro), to determine ways to optimize these drug compounds. The ensemble docking and characterization work described in this article demonstrates the multifaceted features of the SARS-CoV-2 Mpro active site, molecular guidelines to improving binding affinity, and ultimately the optimization of drug candidates. A total of 220 compounds were docked into both the 5R7Z and 6LU7 SARS-CoV-2 Mpro crystal structures. Several key preferences for strong binding to the four subsites (S1, S1', S2, and S4) were identified, such as accessing hydrogen binding hotspots, hydrophobic patches, and utilization of primarily aliphatic instead of aromatic substituents. After optimization efforts using the design guidelines developed from the molecular docking studies, the average docking score of the parent compounds was improved by 6.59 -log10(Kd) in binding affinity which represents an increase of greater than six orders of magnitude. Using the optimization guidelines, the SARS-CoV-2 Mpro inhibitor cinanserin was optimized resulting in an increase in binding affinity of 4.59 -log10(Kd) and increased protease inhibitor bioactivity. The results of molecular dynamic (MD) simulation of cinanserin-optimized compounds CM02, CM06, and CM07 revealed that CM02 and CM06 fit well into the active site of SARS-CoV-2 Mpro [Protein Data Bank (PDB) accession number 6LU7] and formed strong and stable interactions with the key residues, Ser-144, His-163, and Glu-166. The enhanced binding affinity produced demonstrates the utility of the design guidelines described. The work described herein will assist scientists in developing potent COVID-19 antivirals.

Prevalence and Correlates of Body Image Dissatisfaction in Patients with Multiple Sclerosis.

BACKGROUND: Body image dissatisfaction (BID) strongly predicts undesirable outcomes, including disordered eating, depression, and low self-esteem. People with multiple sclerosis (MS) may have higher BID due to changes in mobility and functioning and high rates of depression; however, little research has explored BID in people with MS. Identifying factors predicting BID in people with MS would help providers become more aware of BID and its possible negative outcomes. METHODS: The sample included 151 adult patients with MS receiving care at the Cleveland Clinic Mellen Center for MS. The Body Shape Questionnaire was administered, and demographic information was collected from medical records. Data on MS-specific variables were collected via computerized testing. A one-sample t test, an independent-samples t test, and a hierarchical linear regression were conducted. RESULTS: Average scores on BID were not significantly different from the population mean. Patients with moderate/marked concern were more likely to be female and had higher body mass index values, Patient Health Questionnaire-9 scores, and Quality of Life in Neurological Disorders Stigma T-scores. There were no age differences. No MS-specific variables significantly predicted BID. CONCLUSIONS: People with MS show approximately the same levels of BID as the general population. Higher BID was associated with being female and with higher body mass index, depression, and stigma. No MS-specific variables predicted higher BID after controlling for significant variables. Given the evidence in the literature of the negative effects of BID on health behaviors and mood, it is important to explore whether other factors affect BID in people with MS.

Modified Dose Efficacy Trial of a Canine Distemper-Measles Vaccine for Use in Rhesus Macaques (Macaca mulatta).

Measles virus causes a highly infectious disease in NHP. Clinical signs range from asymptomatic to fatal, although measles virus is most well-known for its characteristic generalized maculopapular rash. Along with appropriate quarantine practices, restricted human access, and appropriate personal protective equipment, vaccines are used to combat the risk of infection. The canine distemper-measles vaccine (CDMV), administered at the manufacturer's standard dose (1.0 mL IM), has been shown to be effective against clinical measles disease in rhesus macaques (Macaca mulatta). The goal of the current study was to test whether doses smaller than the manufacturer's recommended dose stimulated adequate antibody production to protect against infection. We hypothesized that either 0.25 or 0.5 mL IM of CDMV would stimulate antibody production comparable to the manufacturer's recommended dose. We found that the 0.25-mL dose was less effective at inducing antibodies than either the standard (1.0 mL) or 0.5-mL dose, which both yielded similar titers. The primary implication of this study informs balancing resource allocation and providing efficacious immunity. By using half the manufacturer-recommended dose, the 50% cost reduction may provide sufficient monetary incentive to implement, maintain, or modify measles vaccination programs at NHP facilities.

Structure and function insights garnered from in silico modeling of the thrombospondin type-1 domain-containing 7A antigen.

The thrombospondin type-1 domain containing 7A (THSD7A) protein is known to be one of the antigens responsible for the autoimmune disorder idiopathic membranous nephropathy. The structure of this antigen is currently unsolved experimentally. Here we present a homology model of the extracellular portion of the THSD7A antigen. The structure was evaluated for folding patterns, epitope site prediction, and function was predicted. Results show that this protein contains 21 extracellular domains and with the exception of the first two domains, has a regular repeating pattern of TSP-1-like followed by F-spondin-like domains. Our results indicate the presence of a novel Trp-ladder sequence of WxxxxW in the TSP-1-like domains. Of the 21 domains, 18 were shown to have epitope binding sites as predicted by epitopia. Several of the F-spondin-like domains have insertions in the canonical TSP fold, most notably the coiled coil region in domain 4, which may be utilized in protein-protein binding interactions, suggesting that this protein functions as a heparan sulfate binding site.

The mediating role of appearance comparisons on the relationship between negative appearance commentary and binge eating symptoms.

While the frequency of weight-based teasing during childhood/adolescence has been linked to eating disturbance, limited research has examined the impact of negative appearance commentary experienced in young adulthood. Appearance comparisons have been shown to mediate the relationship between weight-based teasing frequency in childhood/adolescence and eating disturbance; however, less is known about the impact of other forms of negative appearance commentary and binge eating symptoms. The current study examined appearance comparisons to others as a mediator of the relationship between the impact of negative appearance commentary from interpersonal sources and binge eating symptoms in college women. Three hundred and seventeen young adult women from universities in the southwest and midsouth U.S. completed measures of appearance-related commentary, physical appearance comparisons, and binge eating symptoms. Controlling for body mass index, the mediation effect of appearance comparisons was tested using the PROCESS macro for SPSS. Appearance comparisons was a significant mediator of the relationship between the impact of negative appearance comments and binge eating symptoms, F(3,312)=40.256, p<0.001, R2=0.279. There was no direct effect of the impact of negative appearance comments on binge eating symptoms. Study findings indicate that appearance comparisons play a role in the extent to which the impact of negative appearance commentary influences binge eating symptoms. Results also suggest the need to address the impact of negative appearance commentary and appearance comparisons simultaneously in interventions for eating disorder psychopathology among college women.

Positive comments, negative outcomes? The potential downsides of appearance-related commentary in ethnically diverse women.

Although research has shown that appearance-related commentary influences body dissatisfaction and disordered eating, limited research has studied such commentary among ethnically diverse women. The current study examined ethnic group differences in the frequency and impact of appearance-related commentary and associations with body dissatisfaction and eating disorder psychopathology. Participants included 280 undergraduate women aged 18-25 (56.1% European American, 28.6% African American, and 15.3% Latina American). Results indicated no ethnic group differences in frequencies of positive weight/shape, positive general appearance, or negative weight/shape commentary while controlling for BMI. However, African American and Latina American women reported stronger negative responses to positive weight/shape commentary than European American women. Negative responses to positive weight/shape commentary were correlated with more body dissatisfaction in African American women, after controlling for frequency of commentary. Findings suggest that positive weight/shape commentary may be associated with poor outcomes in a subgroup of ethnic minority college women.

Adult and childhood weight influence body image and depression through weight stigmatization.

The goal of this study was to examine lifetime weight stigmatization as a mediator of the relationships among current body mass index, childhood overweight, depression, and body dissatisfaction. Participants were 299 female undergraduates (mean age = 20.52, standard deviation = 2.57; mean body mass index = 23.29, standard deviation = 4.51). Weight stigmatization significantly mediated the relationships between body mass index and body dissatisfaction, body mass index and depressive symptoms, and childhood overweight and depressive symptoms. The model accounted for 44.7 percent of the variance in depressive symptoms and 28.2 percent of the variance in body image dissatisfaction. Findings indicated that a decrease in weight stigmatization may predict better mental health.

Dietary Adherence, Glycemic Control, and Psychological Factors Associated with Binge Eating Among Indigenous and Non-Indigenous Chileans with Type 2 Diabetes.

BACKGROUND: Despite the strong association between obesity and binge eating, limited research has examined the implications of binge eating on dietary adherence and psychological factors in ethnically diverse type 2 diabetes patients. PURPOSE: This study investigated the prevalence of binge eating and its association with dietary adherence, glycemic control, and psychological factors among indigenous and non-indigenous type 2 diabetes patients in Chile. METHOD: Participants were 387 indigenous (Mapuche) and non-indigenous (non-Mapuche) adults with type 2 diabetes. Self-report measures of binge eating, dietary adherence, diet self-efficacy, body image dissatisfaction, and psychological well-being were administered. Participants' weight, height, and glycemic control (HbA(1c)) were also obtained. RESULTS: Approximately 8 % of the type 2 diabetes patients reported binge eating. The prevalence among Mapuche patients was 4.9 %, and among non-Mapuche patients, it was 9.9 %. Compared to non-binge eaters, binge eating diabetes patients had greater body mass index values, consumed more high-fat foods, were less likely to adhere to their eating plan, and reported poorer body image and emotional well-being. CONCLUSION: Results of this study extend previous research by examining the co-occurrence of binge eating and type 2 diabetes as well as the associated dietary behaviors, glycemic control, and psychological factors among indigenous and non-indigenous patients in Chile. These findings may increase our understanding of the health challenges faced by indigenous populations from other countries and highlight the need for additional research that may inform interventions addressing binge eating in diverse patients with type 2 diabetes.

Source-sink estimates of genetic introgression show influence of hatchery strays on wild chum salmon populations in Prince William Sound, Alaska.

The extent to which stray, hatchery-reared salmon affect wild populations is much debated. Although experiments show that artificial breeding and culture influence the genetics of hatchery salmon, little is known about the interaction between hatchery and wild salmon in a natural setting. Here, we estimated historical and contemporary genetic population structures of chum salmon (Oncorhynchus keta) in Prince William Sound (PWS), Alaska, with 135 single nucleotide polymorphism (SNP) markers. Historical population structure was inferred from the analysis of DNA from fish scales, which had been archived since the late 1960's for several populations in PWS. Parallel analyses with microsatellites and a test based on Hardy-Weinberg proportions showed that about 50% of the fish-scale DNA was cross-contaminated with DNA from other fish. These samples were removed from the analysis. We used a novel application of the classical source-sink model to compare SNP allele frequencies in these archived fish-scales (1964-1982) with frequencies in contemporary samples (2008-2010) and found a temporal shift toward hatchery allele frequencies in some wild populations. Other populations showed markedly less introgression, despite moderate amounts of hatchery straying. The extent of introgression may reflect similarities in spawning time and life-history traits between hatchery and wild fish, or the degree that hybrids return to a natal spawning area. The source-sink model is a powerful means of detecting low levels of introgression over several generations.

The disappearing northern leopard frog (Lithobates pipiens): conservation genetics and implications for remnant populations in western Nevada.

Global amphibian declines suggest a major shift in the amount and quality of habitat for these sensitive taxa. Many species that were once widespread are now experiencing declines either in part of or across their historic range. The northern leopard frog (Rana [Lithobates] pipiens] has undergone significant declines particularly in the western United States and Canada. Leopard frog population losses in Nevada are largely due to habitat fragmentation and the introduction of nonnative fish, amphibian, and plant species. Only two populations remain in the Truckee and Carson River watersheds of western Nevada which represents the western boundary of this species range. We used sequence data for an 812 base pair fragment of the mitochondrial NADH dehydrogenase 1 (ND1) gene to support a native origin for western Nevada populations. All frogs had a single haplotype (W07) from the distinct western North America ND1 haplotype clade. Data from seven polymorphic microsatellite loci show that Truckee and Carson River populations are highly differentiated from each other and from leopard frogs collected from eastern Nevada sites. Lack of gene flow among and distinct color morphs among the western Nevada populations likely predates the current geographical isolation. Comparisons with other peripheral L. pipiens populations show western Nevada populations have similar levels of gene diversity despite their contemporary isolation (H(E) 0.411, 0.482). Restoration of leopard frog populations in these watersheds will be challenging given well-entrenched nonnative bullfrog populations and major changes to the riparian zone over the past century. Declines of once common amphibian species has become a major conservation concern. Contemporary isolation of populations on a species range periphery such as the leopard frog populations in the Truckee and Carson rivers further exacerbate extirpation risk as these populations are likely to have fewer genetic resources to adaptively respond to rapidly changing biotic and abiotic environments.

Childhood obesity and dental caries among paediatric dental clinic attenders.

BACKGROUND: More than one-quarter of New Zealand children are overweight or obese. Research on the causes of obesity has found associations with high consumption of sweetened foods and beverages, which have also been shown to be risk factors for dental caries, but studies investigating a possible association between dental caries and obesity have had conflicting findings. AIM: The aim of this study was to determine whether deciduous dental caries experience was associated with BMI among paediatric dental clinic attenders. DESIGN: This was a cross-sectional study of clinical records of 200 children aged eight and under (70% European) treated in the University of Otago undergraduate paediatric dentistry clinic between 2004 and 2006. Height and weight were measured and used to calculate BMI. Deciduous dental caries experience was recorded. RESULTS: The overall mean BMI was 16.0 (SD = 2.0). Pacific Island children had a higher mean BMI (at 17.0) than NZ European, Maori, and Asian/Other children (15.7, 16.8, and 15.9 respectively; P < 0.05). The dmft ranged from 0 to 15, with a mean of 6.1 (SD = 3.8); 24% had dmft <3, and 38% had dmft >8. No significant association was found between the BMI and caries experience (P-value = 0.932). CONCLUSIONS: There was no association between BMI and dental caries experience in this convenient sample.

Effects of annealing lyophilized and spray-lyophilized formulations of recombinant human interferon-gamma.

The purpose of this study was to examine the effects of adsorption of recombinant human interferon-gamma (rhIFN-gamma) on ice surfaces and subsequent drying during processing by spray-lyophilization and lyophilization. Ice/liquid interfacial areas were manipulated by the freezing method as well as by the addition of an annealing step during lyophilization; that is, rhIFN-gamma adsorption was modified by the addition of nonionic surfactants. rhIFN-gamma was lyophilized or spray-lyophilized at a concentration of 1 mg/mL in 5% sucrose, 5% hydroxyethyl starch (HES) +/- 0.03% polysorbate 20 in 140 mM KCl, and 10 mM potassium phosphate, pH 7.5. After the samples were frozen, half were annealed on the lyophilizer shelf. Recovery of soluble protein was measured at intermediate points during processing. On drying, the secondary structure of rhIFN-gamma was determined by second-derivative infrared (IR) spectroscopy, specific surface areas (SSAs) were measured, scanning electron micrographs (SEM) were taken, and dissolution times were recorded. Adsorption of rhIFN-gamma to ice/liquid interfaces alone was not responsible for aggregation. Rather, drying was necessary to cause aggregation in lyophilized sucrose formulations. Addition of an annealing step to the lyophilization cycle resulted in more native-like secondary protein structure in the dried solid, eliminated cracking of the dried cakes, and suppressed both the formation of air/liquid interfaces and rhIFN-gamma aggregation on reconstitution.

Surface adsorption of recombinant human interferon-gamma in lyophilized and spray-lyophilized formulations.

Recombinant human interferon-gamma (rhIFN-gamma) was lyophilized or spray-lyophilized in 9.5% trehalose, +/- 0.12% polysorbate 20 in 10 mM potassium phosphate, pH 7.5. We measured recovery of soluble protein after spraying, freeze-thawing, and drying and reconstitution. Infrared spectroscopy showed rhIFN-gamma secondary structure to be native-like in all dried powders. Powders were characterized using electron spectroscopy for chemical analysis, time-of-flight secondary ion mass spectroscopy, X-ray diffraction, and gas adsorption isotherms. rhIFN-gamma adsorbed at air/liquid interfaces during spraying, and to ice/liquid interfaces during lyophilization. The concentration of rhIFN-gamma at ice/liquid interfaces was approximately one-fourth that adsorbed at air/liquid interfaces. Addition of 0.12% polysorbate 20 reduced the concentration of rhIFN-gamma at both interfaces. Time-of-flight secondary ion mass spectroscopy detected polysorbate 20 on surfaces of lyophilized powders. Lyophilized samples dried more slowly but reconstituted more quickly than spray-lyophilized samples. rhIFN-gamma aggregated after nebulization, but aggregation decreased in 0.12% polysorbate 20. Addition of 0.12% polysorbate 20 reduced protein surface adsorption and decreased but did not completely prevent aggregation. Insignificant aggregation occurred after exposure to ice/liquid interfaces, but subsequent drying and reconstitution caused aggregation. The majority of the aggregation is due to adsorption at air-liquid and solid-air interfaces formed during spray-lyophilization or lyophilization.

A new mechanism for decreasing aggregation of recombinant human interferon-gamma by a surfactant: slowed dissolution of lyophilized formulations in a solution containing 0.03% polysorbate 20.

To study the mechanisms by which Tween 20 (polysorbate 20) used in a reconstitution solution affects the aggregation of lyophilized recombinant human interferon-gamma (rhIFN-gamma), we used four types of buffered formulations containing 0.4-5 mg/mL rhIFN-gamma in either 10 mM potassium phosphate or phosphate buffered saline: (1) without excipients, (2) with 5% sucrose, (3) with 0.03% polysorbate 20, or (4) with the combination of 5% sucrose and 0.03% polysorbate 20. After lyophilization, infrared spectroscopy was used to analyze the secondary structure of the protein in the freeze-dried solid. Each solid showed structural perturbation of the protein. Each formulation was reconstituted with water or a 0.03% polysorbate 20 solution. Aggregation of rhIFN-gamma after reconstitution was measured by optical density at A(350), and recovery of soluble protein was determined by high-performance liquid chromatography and ultraviolet spectroscopy. After reconstitution with a 0.03% polysorbate 20 solution, aggregation levels in all formulations were either reduced or similar to those found after reconstitution with water. These results revealed the potential for recovery of native protein using the appropriate reconstitution conditions, even though the protein is non-native in the lyophilized state. Urea-induced unfolding with and without polysorbate 20 as measured by second-derivative ultraviolet spectroscopy indicated that a concentration of 0.03% polysorbate 20 lowered the free energy of unfolding for rhIFN-gamma (destabilizing). Polysorbate 20 also retarded refolding from urea solutions and increased aggregation. At a level of 0.03%, polysorbate 20 did not protect the protein against surface-induced aggregation during agitation. Dissolution times in water versus a 0.03% polysorbate 20 solution were measured using a rotating disk electrode for lyophilized formulations containing an electrochemically reactive species. The presence of 0.03% polysorbate 20 in the reconstitution solution nearly doubled the time required for dissolution of the phosphate buffered saline formulation, and the sucrose formulations dissolved 33-57% more slowly. Slowing the dissolution rates of lyophilized powders allows more time for the protein to refold while it decreases the maximum concentration of the protein at the dissolution interface, thus reducing the total amount of aggregation.