Comprehensive characterization and optimization of Caco-2 cells enabled the development of a miniaturized 96-well permeability assay.

Assessment of compound permeability through a Caco-2 cell monolayer is a well-accepted model to evaluate its in-vivo permeability potential and transporter interaction. While this assay has commonly been conducted using a 24-well assay plate format, a miniaturised 96-well assay format is highly desirable to achieve greater capacity and higher efficiency.Previous attempts to convert this assay from 24-well to 96-well format at our lab, however, had met with varied efflux capacities and unacceptable efflux ratios for digoxin, a substrate of P-glycoprotein (Pgp), which indicated inadequate Pgp transporter expression in the 96-well format.These challenges in converting the assays were attributed to the heterogeneous and unstable nature of the Caco-2 cells. To overcome the challenges, single-cell sorting of Caco-2 cells was conducted by flow cytometry to obtain a more homogeneous and stable cell population. The sorted cells were then seeded to 96-well transwell plates and the Pgp expression under various cell culture conditions was monitored by a LC-MS/MS-based targeted proteomics method.Through cell sorting and direct Pgp expression measurement, Caco-2 cells with adequate and sustained Pgp expression in a 96-well format were obtained, which led to the successful development and implementation of a 96-well Caco-2 assay with significant efficiency gain and faster turnaround time than the historical 24-well assay.

Systematic Interpolation Method Predicts Antibody Monomer-Dimer Separation by Gradient Elution Chromatography at High Protein Loads.

A previously developed empirical interpolation (EI) method is extended to predict highly overloaded multicomponent elution behavior on a cation exchange (CEX) column based on batch isotherm data. Instead of a fully mechanistic model, the EI method employs an empirically modified multicomponent Langmuir equation to correlate two-component adsorption isotherm data at different salt concentrations. Piecewise cubic interpolating polynomials are then used to predict competitive binding at intermediate salt concentrations. The approach is tested for the separation of monoclonal antibody monomer and dimer mixtures by gradient elution on the cation exchange resin Nuvia HR-S. Adsorption isotherms are obtained over a range of salt concentrations with varying monomer and dimer concentrations. Coupled with a lumped kinetic model, the interpolated isotherms predict the column behavior for highly overloaded conditions. Predictions based on the EI method shows good agreement with experimental elution curves for protein loads up to 40 mg mL-1 column or about 50% of the column binding capacity. The approach can be extended to other chromatographic modalities and to more than two components.

Buffer exchange path influences the stability and viscosity upon storage of a high concentration protein.

High concentration protein solutions are generally produced by spin column concentration (SCC) during early development and by tangential flow filtration (TFF) during later stages, when greater quantities of protein become available. This is based on the assumption that the protein generated by the SCC process would be fairly similar to the TFF process material. In this study, we report the case of high concentration solutions of an Fc fusion protein produced by the two processes using the same upstream drug substance (DS) with very different storage stability. The TFF and SCC batches were characterized for aggregation, viscosity, and hydrodynamic radius before and after storage at different temperatures (5°C, 25 °C, and 40 °C). Aggregation and viscosity of the solutions processed by TFF were higher than those processed by SCC upon storage at 25 °C and 40 °C for three months. Differential scanning fluorimetry (DSF) revealed differences in initial protein conformation. Upon exposure to shear stress, protein solutions showed conformational instability and increased aggregation upon storage at 35 °C. In addition, protein solution showed higher aggregation upon shearing under mixed (downstream purification process and final formulation) buffer conditions - which are more likely to be encountered during the TFF, but not SCC, process. These results were further confirmed in an independent experiment by Fourier transform-infrared (FT-IR) spectroscopy and aggregation analysis. Taken together, these data indicate that shearing the protein in intermediate, unstable buffer conditions can lead to conformational perturbation during TFF processing, which led to higher rate of aggregation and viscosity upon storage. This study highlights the importance of testing shear stress sensitivity in the transitional buffer states of the TFF process early in development to de-risk process related product instability.

Gradient elution behavior of proteins in hydrophobic interaction chromatography with U-shaped retention factor curves.

Protein retention in hydrophobic interaction chromatography is described by the solvophobic theory as a function of the kosmostropic salt concentration. In general, an increase in salt concentration drives protein partitioning to the hydrophobic surface while a decrease reduces it. In some cases, however, protein retention also increases at low salt concentrations resulting in a U-shaped retention factor curve. During gradient elution the salt concentration is gradually decreased from a high value thereby reducing the retention factor and increasing the protein chromatographic velocity. For these conditions, a steep gradient can overtake the protein in the column, causing it to rebind. Two dynamic models, one based on the local equilibrium theory and the other based on the linear driving force approximation, are presented. We show that the normalized gradient slope determines whether the protein elutes in the gradient, partially elutes, or is trapped in the column. Experimental results are presented for two different monoclonal antibodies and for lysozyme on Capto Phenyl (High Sub) resin. One of the mAbs and lysozyme exhibit U-shaped retention factor curves and for each, we determine the critical gradient slope beyond which 100% recovery is no longer possible. Elution with a reverse gradient is also demonstrated at low salt concentrations for these proteins. Understanding this behavior has implications in the design of gradient elution since the gradient slope impacts protein recovery.

Systematic interpolation method predicts protein chromatographic elution with salt gradients, pH gradients and combined salt/pH gradients.

A methodology is presented to predict protein elution behavior from an ion exchange column using both individual or combined pH and salt gradients based on high-throughput batch isotherm data. The buffer compositions are first optimized to generate linear pH gradients from pH 5.5 to 7 with defined concentrations of sodium chloride. Next, high-throughput batch isotherm data are collected for a monoclonal antibody on the cation exchange resin POROS XS over a range of protein concentrations, salt concentrations, and solution pH. Finally, a previously developed empirical interpolation (EI) method is extended to describe protein binding as a function of the protein and salt concentration and solution pH without using an explicit isotherm model. The interpolated isotherm data are then used with a lumped kinetic model to predict the protein elution behavior. Experimental results obtained for laboratory scale columns show excellent agreement with the predicted elution curves for both individual or combined pH and salt gradients at protein loads up to 45 mg/mL of column. Numerical studies show that the model predictions are robust as long as the isotherm data cover the range of mobile phase compositions where the protein actually elutes from the column.

Surface induced three-peak elution behavior of a monoclonal antibody during cation exchange chromatography.

A monoclonal antibody exhibits a two- or three-peak elution behavior when loaded on the CEX resin POROS XS and eluted with a salt gradient. Two peaks are observed without a hold step while a third more strongly retained peak becomes noticeable with a hold time as low as 10min. As the hold time is increased further, the first peak gradually disappears, the second peak initially increases and then decreases, and the third peak continuously increases. Dynamic light scattering shows that the third peak contains significant levels of aggregates formed in the column. Circular dichroism, HX-MS analyses of the eluted fraction, in-line fluorescence detection, and bound-state HX-MS analysis indicate that the aggregates derive from an unfolded intermediate that is slowly formed while the protein is bound to the resin. Aggregate formation does not occur on a different CEX resin, Nuvia HR-S, with similar particle size but with a more homogenous structure or when the sodium acetate load buffer is replaced with arginine acetate. The two early eluting peaks observed for POROS XS regardless of hold time are shown to comprise exclusively monomeric species. A set of biophysical measurements as well as mechanistic modeling support the hypothesis that these two peaks form as a result of the presence of weak and strong binding sites on the resin having, respectively, fast and slow binding kinetics.

Systematic interpolation method predicts protein chromatographic elution from batch isotherm data without a detailed mechanistic isotherm model.

Predicting protein elution for overloaded ion exchange columns requires models capable of describing protein binding over broad ranges of protein and salt concentrations. Although approximate mechanistic models are available, they do not always have the accuracy needed for precise predictions. The aim of this work is to develop a method to predict protein chromatographic behavior from batch isotherm data without relying on a mechanistic model. The method uses a systematic empirical interpolation (EI) scheme coupled with a lumped kinetic model with rate parameters determined from HETP measurements for non-binding conditions, to numerically predict the column behavior. For two experimental systems considered in this work, predictions based on the EI scheme are in excellent agreement with experimental elution profiles under highly overloaded conditions without using any adjustable parameters. A qualitative study of the sensitivity of predicting protein elution profiles to the precision, granularity, and extent of the batch adsorption data shows that the EI scheme is relatively insensitive to the properties of the dataset used, requiring only that the experimental ranges of protein and salt concentrations overlap those under which the protein actually elutes from the column and possess a ± 10% measurement precision.

Adaptation to high throughput batch chromatography enhances multivariate screening.

High throughput process development offers unique approaches to explore complex process design spaces with relatively low material consumption. Batch chromatography is one technique that can be used to screen chromatographic conditions in a 96-well plate. Typical batch chromatography workflows examine variations in buffer conditions or comparison of multiple resins in a given process, as opposed to the assessment of protein loading conditions in combination with other factors. A modification to the batch chromatography paradigm is described here where experimental planning, programming, and a staggered loading approach increase the multivariate space that can be explored with a liquid handling system. The iterative batch chromatography (IBC) approach is described, which treats every well in a 96-well plate as an individual experiment, wherein protein loading conditions can be varied alongside other factors such as wash and elution buffer conditions. As all of these factors are explored in the same experiment, the interactions between them are characterized and the number of follow-up confirmatory experiments is reduced. This in turn improves statistical power and throughput. Two examples of the IBC method are shown and the impact of the load conditions are assessed in combination with the other factors explored.

Development and scale-up of the recovery and purification of a domain antibody Fc fusion protein-comparison of a two and three-step approach.

A robust, economical process should leverage proven technology, yet be flexible enough to adopt emerging technologies which show significant benefit. Antibody and Fc-fusion processes may capitalize on the high selectivity of an affinity capture step by reducing the total number of chromatographic steps to 2. Risk associated with this approach stems from the potentially increased time frame needed for process development as well as unforeseen changes in impurity profile during first scale-up of drug substance (DS) for animal toxicology and clinical phase I trials (FIH) production, which could challenge a two-step process to the point of failure. Two different purification strategies were pursued during process development for an FIH process of a dAB-Fc fusion protein. A two-step process was compared to a three-step process. The two-step process leveraged additives to maximize impurity reduction during affinity capture. While wash additives in combination with a mixed mode chromatography met all impurity reduction requirements, HCP levels were still higher as compared to the three-step process. The three-step process was implemented for manufacture of clinical material to mitigate risk.

Optimization of preoperative status in hypoplastic left heart syndrome with intact atrial septum by left atrial decompression and bilateral pulmonary artery bands.

Hypoplastic left heart syndrome (HLHS) with intact (IAS) or highly restrictive atrial septum (RAS) has extremely poor outcomes largely related to pulmonary pathology. At birth, immediate left atrial (LA) decompression is required to remain viable, but there is a tradeoff between residual increase in LA pressure and pulmonary overcirculation, either of which exacerbates the pulmonary status. From August 2010 to April 2013, a retrospective chart review was performed on consecutive patients with a prenatal diagnosis of HLHS with IAS/RAS presenting to a single center. The management strategy was immediate LA decompression followed by placement of bilateral pulmonary artery bands (bPAB) and subsequent conventional Norwood procedure. Six patients were born with HLHS with IAS/RAS during this time period with this planned management strategy. Four patients underwent LA decompression and subsequently developed low cardiac output with pulmonary overcirculation. bPAB were used with improvement in cardiac output and pronounced diuresis. These patients all survived the Norwood and subsequent Glenn procedures and remain alive [median follow-up 2.2 years (range 11 months-2.7 years)]. Two patients did not survive with therapy being withdrawn before the Norwood procedure. It is hypothesized that a strategy of total LA decompression followed by bPAB maximizes preoperative systemic perfusion and minimizes ongoing injury to the pulmonary system. This may enhance patient candidacy for the Norwood procedure and long-term survival.

Temporary atrial pacing for cardiac output after pediatric cardiac surgery.

Pediatric cardiothoracic surgery is often associated with low cardiac output in the postoperative period. This study sought to determine whether increasing heart rate via temporary atrial pacing is beneficial in augmenting cardiac output. Patients younger than 18 years who underwent cardiothoracic surgery and had no perioperative arrhythmias were eligible for the study. Patients not paced postoperatively were atrial paced at a rate of 15 % above the intrinsic sinus rate (not to exceed 170 beats per minute, less for older patients) for 15 min. Patients paced for cardiac output postoperatively had their pacemakers paused for 15 min. Markers of cardiac output were measured before and after the intervention. Of the 60 patients who consented to participate, 30 completed the study. Failure to complete the study was due to tachycardia (n = 13), lack of pacing wires (n = 7), junctional rhythm (n = 4), advanced atrioventricular block (n = 3), and other cause (n = 3). Three patients were paced at baseline. There was no change in arteriovenous oxygen saturation difference, mean arterial blood pressure, central venous pressure, toe temperature, or lactate with atrial pacing. Atrial pacing was associated with a decrease in head and flank near-infrared spectroscopy (p = 0.01 and <0.01 respectively). Secondary analysis found an inverse relationship between mean arterial pressure response to pacing and bypass time. Temporary atrial pacing does not improve cardiac output after pediatric cardiac surgery and may be deleterious. Future research may identify subsets of patients who benefit from this strategy. Practitioners considering this strategy should carefully evaluate each patient's response to atrial pacing before its implementation.

Bilateral pulmonary artery banding for resuscitation in high-risk, single-ventricle neonates and infants: a single-center experience.

OBJECTIVES: Bilateral pulmonary artery banding with or without ductal stenting has been performed as a resuscitative intervention for patients considered at too high risk for conventional single ventricle palliation. The purpose of the present study was to determine the outcomes using this strategy. METHODS: We performed a retrospective review of 24 patients with single ventricle anatomy who were younger than 3 months who had undergone bilateral pulmonary artery banding and ductal stenting or maintenance of prostaglandin E(1) from January 2007 to October 2011 at our institution. The echocardiographic, angiographic, operative, and clinical data were reviewed. Follow-up data were available for 100% of the patients. RESULTS: All 24 patients (13 male patients) underwent bilateral pulmonary artery banding at a median age of 8 days (range, 2-44 days). Their gestational age was 38 weeks (range, 27-41 weeks), and their weight was 3.01 kg (range, 1.5-4.4 kg). The cardiac diagnoses included hypoplastic left heart syndrome/variant hypoplastic left heart syndrome in 18, unbalanced atrioventricular canal in 4, and tricuspid atresia in 2. In the hypoplastic left heart syndrome group, 9 (50%) had an intact or a highly restrictive atrial septum requiring open (n = 1) or transcatheter (n = 8) atrial septostomy with or without atrial stent placement (n = 4). Ductal stenting was performed in 14 patients, and 10 patients were continued with prostaglandin E(1). Fifteen patients (62.5%) survived to undergo a Norwood procedure (n = 7), comprehensive stage 2 (n = 1), or primary cardiac transplantation (n = 7). Of the 9 who died, support was withdrawn in 5 because of a contraindication to transplantation, 1 because of sepsis and/or multiorgan system failure, and 1 for whom palliative care was desired. Two died awaiting transplantation. All 7 patients who underwent a conventional Norwood operation survived to discharge, and 6 of the 7 (85.7%) underwent bidirectional Glenn shunt placement. Of the 7 patients who underwent transplantation, 6 (85.7%) were alive at a median follow-up of 33.6 months. CONCLUSIONS: Bilateral pulmonary artery banding with or without ductal stenting is an effective method of resuscitation for high-risk neonates and infants with a single ventricle, allowing for reasonable survival to conventional first-stage palliation or primary transplantation.

Enhancers of adeno-associated virus AAV2 transduction via high throughput siRNA screening.

Intracellular barriers to adeno-associated virus (AAV) transduction may limit gene delivery. We screened a short interfering RNA (siRNA) library targeting 5,520 genes to help identify pathways that modulate AAV transduction of human endothelium. In replicate screening, 50 pools (three siRNAs per gene) resulted in greater than eightfold reporter gene expression enhancement. Single siRNA confirmation tests demonstrated that at least one siRNA from each of the top 10 pools provided greater than twofold enhancement. Several siRNAs when used together resulted in additive effects and two of the most potent siRNA sequences were enhancers in cultured airway epithelium. However, enhanced transduction was not correlated with mRNA knockdown by quantitative real time PCR, indicating an off-target mechanism. In fact, four of the five most potent siRNAs contained a consensus hexamer region 5'-UGUUUC-3' at positions 2-7 of the antisense strand. The point mutation U4A within this region (but not mutations at positions 1 or 14) disrupted transduction enhancement, indicating a microRNA (miRNA)-like mechanism. Transcription profiling indicated that the hexamer also resulted in perturbation of the interferon pathway via reduced interferon-induced protein 44-like (IFI44L), interferon-inducible myxovirus resistance 1 (MX1), and interferon-induced protein with tetratricopeptide repeats (IFIT5) mRNAs. Direct interferon (α, ß, and ω) receptor 2 (IFNAR2) knockdown resulted in greater than twofold transduction enhancement. In addition to providing insight into AAV biology and enhanced transduction, the results demonstrate certain beneficial siRNA off-target effects.

Major infection after pediatric cardiac surgery: a risk estimation model.

BACKGROUND: In pediatric cardiac surgery, infection is a leading cause of morbidity and mortality. We created a model to predict risk of major infection in this population. METHODS: Using the Society of Thoracic Surgeons Congenital Heart Surgery Database, we created a multivariable model in which the primary outcome was major infection (septicemia, mediastinitis, or endocarditis). Candidate-independent variables included demographic characteristics, comorbid conditions, preoperative factors, and cardiac surgical procedures. We created a reduced model by backward selection and then created an integer scoring system using a scaling factor with scores corresponding to percent risk of infection. RESULTS: Of 30,078 children from 48 centers, 2.8% had major infection (2.6% septicemia, 0.3% mediastinitis, and 0.09% endocarditis). Mortality and postoperative length of stay were greater in those with major infection (mortality, 22.2% versus 3.0%; length of stay >21 days, 69.9% versus 10.7%). Young age, high complexity, previous cardiothoracic operation, preoperative length of stay more than 1 day, preoperative ventilator support, and presence of a genetic abnormality were associated with major infection after backward selection (p < 0.001). Estimated infection risk ranged from less than 0.1% to 13.3%; the model discrimination was good (c index, 0.79). CONCLUSIONS: We created a simple bedside tool to identify children at high risk for major infection after cardiac surgery. These patients may be targeted for interventions to reduce the risk of infection and for inclusion in future clinical trials.

RNA interference screen to identify pathways that enhance or reduce nonviral gene transfer during lipofection.

Some barriers to DNA lipofection are well characterized; however, there is as yet no method of finding unknown pathways that impact the process. A druggable genome small-interfering RNA (siRNA) screen against 5,520 genes was tested for its effect on lipofection of human aortic endothelial cells (HAECs). We found 130 gene targets which, when silenced by pooled siRNAs (three siRNAs per gene), resulted in enhanced luminescence after lipofection (86 gene targets showed reduced expression). In confirmation tests with single siRNAs, 18 of the 130 hits showed enhanced lipofection with two or more individual siRNAs in the absence of cytotoxicity. Of these confirmed gene targets, we identified five leading candidates, two of which are isoforms of the regulatory subunit of protein phosphatase 2A (PP2A). The best candidate siRNA targeted the PPP2R2C gene and produced a 65% increase in luminescence from lipofection, with a quantitative PCR-validated knockdown of approximately 76%. Flow cytometric analysis confirmed that the silencing of the PPP2R2C gene resulted in an improvement of 10% in transfection efficiency, thereby demonstrating an increase in the number of transfected cells. These results show that an RNA interference (RNAi) high-throughput screen (HTS) can be applied to nonviral gene transfer. We have also demonstrated that siRNAs can be co-delivered with lipofected DNA to increase the transfection efficiency in vitro.

The personal protective behaviors of college student drinkers: evidence of indigenous protective norms.

Given the prevalence of alcohol consumption and the relative infrequency of harm among college students, the authors sought to determine how most college students protect themselves from alcohol-related harm. An analysis of the aggregate National College Health Assessment data identified a cluster of personal protective behaviors that correlated with reduced risk when drinking. Further analysis revealed that nearly three-quarters of student drinkers regularly employ at least 1 protective behavior, and well over half of the students who use protective behaviors routinely employ 2 or more. In addition, the data reveal that student drinkers employ situational abstinence, with nearly 7 out of 10 students reporting that they sometimes or usually refrain from drinking alcohol when they socialize. The use of these protective behaviors is a strong predictor of safety and harm for college-student drinkers.