Column-free optical deconvolution of intrinsic fluorescence for a monoclonal antibody and its product-related impurities.

The quantification of monoclonal antibody (mAb) aggregates and fragments using high pressure liquid chromatography-size exclusion chromatography (HPLC-SEC) typically requires off-line measurements that are time-consuming and therefore not compatible with real-time monitoring. However, it has been crucial to manufacturing and process development, and remains the industrial standard in the assessment of product-related impurities. Here we demonstrate that our previously established intrinsic time-resolved fluorescence (TRF) approach can be used to quantify the bioprocess critical quality attribute (CQA) of antibody product purity at various stages of a typical downstream process, with the potential to be developed for in-line bioprocess monitoring. This was directly benchmarked against industry-standard HPLC-SEC. Strong linear correlations were observed between outputs from TRF spectroscopy and HPLC-SEC, for the monomer and aggregate-fragment content, with R2 coefficients of 0.99 and 0.69, respectively. At total protein concentrations above 1.41 mg/mL, HPLC-SEC UV-Vis chromatograms displayed signs of detector saturation which reduced the accuracy of protein quantification, thus requiring additional sample dilution steps. By contrast, TRF spectroscopy increased in accuracy at these concentrations due to higher signal-to-noise ratios. Our approach opens the potential for reducing the time and labour required for validating aggregate content in mAb bioprocess stages from the several hours required for HPLC-SEC to a few minutes per sample.

Enhanced Thermal Stability and Reduced Aggregation in an Antibody Fab Fragment at Elevated Concentrations.

The aggregation of protein therapeutics such as antibodies remains a major challenge in the biopharmaceutical industry. The present study aimed to characterize the impact of the protein concentration on the mechanisms and potential pathways for aggregation, using the antibody Fab fragment A33 as the model protein. Aggregation kinetics were determined for 0.05 to 100 mg/mL Fab A33, at 65 °C. A surprising trend was observed whereby increasing the concentration decreased the relative aggregation rate, ln(v) (% day-1), from 8.5 at 0.05 mg/mL to 4.4 at 100 mg/mL. The absolute aggregation rate (mol L-1 h-1) increased with the concentration following a rate order of approximately 1 up to a concentration of 25 mg/mL. Above this concentration, there was a transition to an apparently negative rate order of -1.1 up to 100 mg/mL. Several potential mechanisms were examined as possible explanations. A greater apparent conformational stability at 100 mg/mL was observed from an increase in the thermal transition midpoint (Tm) by 7-9 °C, relative to those at 1-4 mg/mL. The associated change in unfolding entropy (△Svh) also increased by 14-18% at 25-100 mg/mL, relative to those at 1-4 mg/mL, indicating reduced conformational flexibility in the native ensemble. Addition of Tween or the crowding agents Ficoll and dextran, showed that neither surface adsorption, diffusion limitations nor simple volume crowding affected the aggregation rate. Fitting of kinetic data to a wide range of mechanistic models implied a reversible two-state conformational switch mechanism from aggregation-prone monomers (N*) into non-aggregating native forms (N) at higher concentrations. kD measurements from DLS data also suggested a weak self-attraction while remaining colloidally stable, consistent with macromolecular self-crowding within weakly associated reversible oligomers. Such a model is also consistent with compaction of the native ensemble observed through changes in Tm and △Svh.

Proof-of-concept analytical instrument for label-free optical deconvolution of protein species in a mixture.

The adoption of process analytical technologies by the biopharmaceutical industry can reduce the cost of therapeutic drugs and facilitate investigation of new bioprocesses. Control of critical process parameters to retain critical product quality attributes within strict bounds is important for ensuring a consistently high product quality, but developing the sophisticated analytical technologies required has proven to be a major challenge. Here, we demonstrate a new optical technique for continuous monitoring of protein species as they are eluted from a chromatographic column, even when they fully co-elute with other protein species, without making any assumption about or peak-fitting to the elution profile. To achieve this, we designed and constructed a time-resolved intrinsic fluorescence lifetime chromatograph, and established an analytical framework for deconvolving and quantifying distinct but co-eluting protein species in real time. This proof-of-concept technology has potentially useful applications as a process analytical technology and more generally as an analytical technique for label-free quantification of proteins in mixtures.

Virus lasers for biological detection.

The selective amplification of DNA in the polymerase chain reaction is used to exponentially increase the signal in molecular diagnostics for nucleic acids, but there are no analogous techniques for signal enhancement in clinical tests for proteins or cells. Instead, the signal from affinity-based measurements of these biomolecules depends linearly on the probe concentration. Substituting antibody-based probes tagged for fluorescent quantification with lasing detection probes would create a new platform for biomarker quantification based on optical rather than enzymatic amplification. Here, we construct a virus laser which bridges synthetic biology and laser physics, and demonstrate virus-lasing probes for biosensing. Our virus-lasing probes display an unprecedented > 10,000 times increase in signal from only a 50% increase in probe concentration, using fluorimeter-compatible optics, and can detect biomolecules at sub-100 fmol mL-1 concentrations.

How the doughnut hole affects prescription fulfillment decisions involving cardiovascular medications for Medicare Part D enrollees.

PURPOSE: The unsupplemented Medicare Part D prescription drug benefit does not provide coverage for stand-alone prescription drug plan (PDP) beneficiaries within the coverage gap (often called a doughnut hole) in Medicare Part D. DESIGN: We evaluated whether the doughnut hole was a factor in altering prescription fulfillment decisions regarding cardiovascular medications in Part D beneficiaries. METHODOLOGY: We investigated 500 Medicare Part D members' pharmacy adjudication records (all prescription transactions) for one full year from a blinded, national Part D HMO pharmacy database. We selected 250 stand-alone PDP beneficiaries without coverage in the doughnut hole and 250 Medicare Advantage-Prescription Drug (MA-PD) plan beneficiaries whose prescription coverage did not lapse in the doughnut hole by way of random stratified sampling and analyzed the records using multinomial logistic regression for their prescription fulfillment decision endpoints of filling, delaying, switching, or stopping their medications. FINDINGS: Of stand-alone PDP beneficiaries, 16.8% delayed medication, 12.4% switched medication, 10.4% both delayed and stopped medication, and 9.6% stopped at least one medication. Part D enrollees who entered the doughnut hole are 1.5 times as likely to delay their cardiovascular prescriptions (OR = 1.54, 95% CI 0.924, 2.562), 1.5 times as likely to switch and delay their cardiovascular prescriptions (OR = 1.52, 95% CI 0.532, 4.332), and 2.3 times as likely to delay and stop their cardiovascular prescriptions than beneficiaries with coverage through the doughnut hole period (OR = 2.30, 95% CI 1.134, 4.673). CONCLUSIONS: For cardiovascular medications, the presence of a doughnut hole affects the prescription fulfillment decisions made by Part D beneficiaries.

Thermodynamic stability and electronic structure of small carbon nitride nanotubes.

In order to tune the electronic properties of carbon-based nanotubes, attention is now turning to new avenues based on chemical manipulation. The introduction of nitrogen at either doping or alloying concentrations has been shown to give rise to new tubular structures and desirable electronic properties, but a detailed understanding of the strain and thermodynamic properties is still lacking. In this paper a systematic computational study of the structure and thermodynamics of small C(x)N nanotubes is presented (x = 1, 2, 3, 5, and 7). The aim of this work is to investigate which stoichiometries and atomic distributions are likely to be stable under ambient and operating conditions, thereby offering viable candidates for future synthesis efforts. In addition to this, the electronic properties of stable structures are briefly examined, to establish whether small carbon nitride nanotubes may be tailored for emerging technological applications.

Variations in excitability of single human motor axons, related to stochastic properties of nodal sodium channels.

Threshold variability of single human motor axons was studied by delivering 0.1 ms constant current stimuli of randomly varied intensity over the ulnar nerve at the elbow, and recording all-or-none potentials from flexor carpi ulnaris. In nine normal subjects, a single unit was tested with 8-11,000 stimuli at intervals of 0.5 s. After allowing for slow changes in excitability, the probability of excitation was in all cases well fitted by a cumulative Gaussian function. The relative spread (RS) of thresholds (S.D./mean) averaged 1.65 +/- 0.26% (mean +/- S.D., n = 9). When threshold was tested 20 ms after the start of a polarizing current, RS increased on depolarization and decreased on hyperpolarization. The product RS x mean threshold also increased on depolarization, especially when threshold was reduced by more than 50%. When the mean was reduced by 90%, RS increased above 50% and the axon sometimes fired spontaneously. Threshold variability was simulated by a computer model of a single node of Ranvier, in which the variability arose because of the stochastic behaviour of nodal sodium channels. The observed values of RS, and potential dependence of RS, were well modelled by a node with 60,000 sodium channels, of which about 1% were modelled as persistent sodium channels. Threshold variations in the model at resting potential were not primarily due to fluctuations in the state of the node before the stimulus was delivered, but rather to the variable activation of channels by the stimulus pulse. On depolarization, however, current through (mainly persistent) sodium channels caused appreciable fluctuations in membrane potential, which increased RS and the probability of spontaneous firing.

Business-office challenges of small and rural hospitals.

All hospitals face tremendous challenges in optimizing their business-office operations these days. Tighter and more complex reimbursement requirements, ongoing human resources shortages, rising HR costs, and changes in information technology all affect operations and the bottom line. But small and rural hospitals, with their more limited resources and greater geographic and corporate isolation, are particularly challenged to optimize their business-office operations to improve revenue cycle management and reimbursement.