Peptide ligands for the universal purification of exosomes by affinity chromatography.

Exosomes are gaining prominence as vectors for drug delivery, vaccination, and regenerative medicine. Owing to their surface biochemistry, which reflects the parent cell membrane, these nanoscale biologics feature low immunogenicity, tunable tissue tropism, and the ability to carry a variety of payloads across biological barriers. The heterogeneity of exosomes' size and composition, however, makes their purification challenging. Traditional techniques, like ultracentrifugation and filtration, afford low product yield and purity, and jeopardizes particle integrity. Affinity chromatography represents an excellent avenue for exosome purification. Yet, current affinity media rely on antibody ligands whose selectivity grants high product purity, but mandates the customization of adsorbents for exosomes with different surface biochemistry while their binding strength imposes elution conditions that may harm product's activity. Addressing these issues, this study introduces the first peptide affinity ligands for the universal purification of exosomes from recombinant feedstocks. The peptides were designed to (1) possess promiscuous biorecognition of exosome markers, without binding process-related contaminants and (2) elute the product under conditions that safeguard product stability. Selected ligands SNGFKKHI and TAHFKKKH demonstrated the ability to capture of exosomes secreted by 14 cell sources and purified exosomes derived from HEK293, PC3, MM1, U87, and COLO1 cells with yields of up to 80% and up-to 50-fold reduction of host cell proteins (HCPs) upon eluting with pH gradient from 7.4 to 10.5, recommended for exosome stability. SNGFKKHI-Toyopearl resin was finally employed in a two-step purification process to isolate exosomes from HEK293 cell fluids, affording a yield of 68% and reducing the titer of HCPs to 68 ng/mL. The biomolecular and morphological features of the isolated exosomes were confirmed by analytical chromatography, Western blot analysis, transmission electron microscopy, nanoparticle tracking analysis.

Advances in high-throughput, high-capacity nonwoven membranes for chromatography in downstream processing: A review.

Nonwoven membranes are highly engineered fibrous materials that can be manufactured on a large scale from a wide range of different polymers, and their surfaces can be modified using a large variety of different chemistries and ligands. The fiber diameters, surface areas, pore sizes, total porosities, and thicknesses of the nonwoven mats can be carefully controlled, providing many opportunities for creative approaches for the development of novel membranes with unique properties to meet the needs of the future of downstream processing. Fibrous membranes are already finding use in ultrafiltration, microfiltration, depth filtration, and, more recently, in membrane chromatography for product capture and impurity removal. This article summarizes the various methods of manufacturing nonwoven fabrics, and the many methods available for the modification of the fiber surfaces. It also reviews recent studies focused on the use of nonwoven fabric devices in membrane chromatography and provides some perspectives on the challenges that need to be overcome to increase binding capacities, decrease residence times, and reduce pressure drops so that eventually they can replace resin column chromatography in downstream process operations.

Novel multimodal cation-exchange membrane for the purification of a single-chain variable fragment from Pichia pastoris supernatant.

A novel salt-tolerant cation-exchange membrane, prepared with a multimodal ligand, 2-mercaptopyridine-3-carboxylic acid (MMC-MPCA), was examined for its purification properties in a bind-and-elute mode from the high conductivity supernatant of a Pichia pastoris fermentation producing and secreting a single-chain variable fragment (scFv). If successful, this approach would eliminate the need for a buffer exchange prior to product capture by ion-exchange. Two fed-batch fermentations of Pichia pastoris resulted in fermentation supernatants reaching an scFv titer of 395.0 mg/L and 555.7 mg/L, both with a purity of approximately 83 %. The MMC-MPCA membrane performance was characterized in terms of pH, residence time (RT), scFv load, and scFv concentration to identify the resulting dynamic binding capacity (DBC), yield, and purity achieved under optimal conditions. The MMC-MPCA membrane exhibited the highest DBC of 39.06 mg/mL at pH 5.5, with a residence time of 1 min, while reducing the pH below 5.0 resulted in a significant decrease of the DBC to around 2.5 mg/mL. With almost no diffusional limitations, reducing the RT from 2 to 0.2 min did not negatively impact the DBC of the MMC-MPCA membrane, resulting in a significant improvement in productivity of up to 180 mg/mL/min at 0.2 min RT. Membrane fouling was observed when reusing the membranes at 0.2 and 0.5 min RT, likely due to the enhanced adsorption of impurities on the membrane. Changing the amount of scFv loaded onto the membrane column did not show any changes in yield, instead a 10-20 % loss of scFv was observed, which suggested that some of the produced scFv were fragmented or had aggregated. When performing the purification under the optimized conditions, the resulting purity of the product improved from 83 % to approximately 92-95 %.

Situational, interpersonal, and intrapersonal characteristic associations with adolescent conflict forgiveness.

ABSTRACT. Given the voluntary nature of adolescent friendships, forgiveness of interpersonal transgressions has been identified as a critical aspect of maintaining these relationships. However, transgression forgiveness is related to a range of situational (e.g., transgression severity), interpersonal (e.g., friendship commitment), and intrapersonal (e.g., victim's empathy) factors. Data from 161 adolescents were used to examine the nature of the relationships between these factors and forgiveness and to examine the differential association patterns for adolescent boys and girls. Results for the overall adolescent sample indicated both situational and interpersonal factor associations with forgiveness (R2 = .52, p < .001). Examination of separate female and male forgiveness reports indicated similar interpersonal factor associations and differential situational factor associations with female (R2 = .46, p < .001), and male (R2 = .60, p < .001) forgiveness. Findings suggest the likelihood of forgiving may be contextually dependent, and that researchers should consider transgression, relationship, and intrapersonal characteristics when examining forgiveness. Further, the present study suggests the contextual factors associated with forgiveness may be further differentiated by gender.

Aptasensor for Detection of Influenza-A in Human Saliva.

Access to low-cost, rapid, individualized diagnostics at point-of-care and point-of-need is vital to minimize the impact of highly infectious viruses, such as influenza. Herein, a biosensor for detecting hemagglutinin (HA), an abundant capsid protein in H1N1 viruses, is demonstrated. A gold working electrode was functionalized with a thiol-modified, HA-binding aptamer derivatized with a methylene blue modification for redox reporting. The aptamer was characterized by surface plasmon resonance to confirm its biorecognition activity for HA. The aptasensor was characterized by square wave voltammetry to quantify the sensor's response to varying concentrations of HA. The sensor exhibited a lower limit of detection of 1.5 pM with linear detection of up to 1.2 nM in both Tris buffer and simulated human saliva, thus encompassing the clinically relevant HA range in saliva. Average sensitivity was measured at 21.083 nA·nM-1in Tris and 14.5 nA·nM-1in artificial saliva across clinically relevant HA titers. Sensor stability across time was also investigated, providing a preliminary understanding of the translational viability of the aptasensors for mobile and remote diagnostic applications.

Iminodiacetic Acid (IDA) Cation-Exchange Nonwoven Membranes for Efficient Capture of Antibodies and Antibody Fragments.

There is strong need to reduce the manufacturing costs and increase the downstream purification efficiency of high-value therapeutic monoclonal antibodies (mAbs). This paper explores the performance of a weak cation-exchange membrane based on the coupling of IDA to poly(butylene terephthalate) (PBT) nonwoven fabrics. Uniform and conformal layers of poly(glycidyl methacrylate) (GMA) were first grafted to the surface of the nonwovens. Then IDA was coupled to the polyGMA layers under optimized conditions, resulting in membranes with very high permeability and binding capacity. This resulted in IgG dynamic binding capacities at very short residence times (0.1-2.0 min) that are much higher than those achieved by the best cation-exchange resins. Similar results were obtained in the purification of a single-chain (scFv) antibody fragment. As is customary with membrane systems, the dynamic binding capacities did not change significantly over a wide range of residence times. Finally, the excellent separation efficiency and potential reusability of the membrane were confirmed by five consecutive cycles of mAb capture from its cell culture harvest. The present work provides significant evidence that this weak cation-exchange nonwoven fabric platform might be a suitable alternative to packed resin chromatography for low-cost, higher productivity manufacturing of therapeutic mAbs and antibody fragments.

Migration Effects of Fluorochemical Melt Additives for Alcohol Repellency in Polypropylene Nonwoven Materials.

The use of bulk polymer melt additives provides a facile, industrially relevant approach to tailor properties of polymer surfaces for many different applications. These melt additives, when blended with polymers prior to melt spinning, migrate to the fiber surface and influence surface functionality. While the use of bulk polymer melt additives to impart hydrophilicity or oleophobicity is well studied, the impact of the fiber formation process on additive migration and resultant repellency of nonwoven media products remains largely unexplored. In this study, we produce fluorochemical melt additive containing meltblown nonwovens, and establish methods for characterization of fiber mat surface composition and repellency. Repellency of low surface tension fluids is a significant challenge and is of particular importance in the creation of medical garments such as surgical gowns and masks which must perform as liquid barriers even when exposed to alcohol based solutions. Similarly, melt additives are also used in the production of electret air filtration devices. Electret filters are imbued with charges to enhance particle capture performance, but this charge can be negated through wetting by low surface tension fluids. To address this challenge, the changing composition of fiber surfaces due to the migration of additives is monitored via X-ray Photoelectron Spectroscopy, then related to repellency of alcohol solutions by contact angle analysis. We demonstrate that for the samples tested a fluorine to carbon (F/C) ratio of 0.35 is sufficient to prevent wicking of isopropanol droplets, and higher surface tension fluids could be repelled by fiber mats with lower fluorine content. Through the use of cross-sectional ToF-SIMS analysis, we find that migration of additives is key to the performance of samples with low additive loadings, and that these phenomena are heavily influenced by many nonwoven manufacturing parameters including fiber size, die-to-collector distance, and polymer resin melt flow rates.

Individuation and parents as people: measurement concerns regarding two aspects of autonomy.

The focus of this study was on examining two assumed components of adolescent autonomy-individuation and perceiving parents as people and the validity of their measurement. Participating in the study were 67 students in grades 9 and 11 (ages 14-17 years) and 118 college students (ages 18-27 years). Each student completed two measures of individuation and two measures of parents as people. The sample was divided into four data sets for analyses based on gender and age grouping. Neither the two measures of individuation nor the two measures of parents as people correlated with each other indicating a problem with concurrent validity. The problems with adequate grounding in theory and validity for the measures are discussed.

Physical and Interpersonal Attractiveness of the Model and Imitation in Adults.

The influence of physical attractiveness and warmth of a model on imitation and social perception in adults was assessed in this study with the use of 108 male and female college students. The Ss were randomly paired with a model who, depending on experimental conditions, was either a male or female, of high or low facial attractiveness, and who interacted with the S in a warm or cold manner. The Ss were then placed in a task situation which provided an opportunity to imitate the model, after which the Ss were asked to rate their experience and the model's personality. Warmth of the model was a more influential determinant of imitation, personality rating, and experience than physical attractiveness. However, the warmth affect was influenced by sex of the model and sex of the S. Possible explanations for the warmth effect were explored.