Small-volume Experiments Research Articles

Efficient and scalable clarification of Orf virus from HEK suspension for vaccine development

The Orf virus (ORFV) is a promising vector platform for the generation of vaccines against infectious diseases and cancer, highlighted by its progression to clinical testing phases. One of the critical steps during GMP manufacturing is the clarification of crude harvest because of the enveloped nature and large size of ORFV. This study presents the first description of ORFV clarification process from a HEK suspension batch process. We examined various filter materials, membrane pore sizes, harvest timings, and nuclease treatments. Employing the Ambr® crossflow system for high-throughput, small-volume experiments, we identified polypropylene-based Sartopure® PP3 filters as ideal. These filters, used in two consecutive stages with reducing pore sizes, significantly enhanced ORFV recovery and addressed scalability challenges. Moreover, we demonstrated that the time of harvest and the use of a nuclease play a decisive role to increase ORFV yields. With these findings, we were able to establish an efficient and scalable clarification process of ORFV derived from a suspension production process, essential for advancing ORFV vaccine manufacturing.

Screening Fuels for Autoignition with Small-Volume Experiments and Gaussian Process Classification

Partially reacting candidate fuels under highly dilute conditions across a range of temperatures provides a means to classify the candidates based on traditional ignition characteristics using much lower quantities (sub-mL) than the full octane tests. Using a classifier based on a Gaussian Process model, synthetic species profiles obtained by plug flow reactor simulations at seven temperatures are used to demonstrate that the configuration can be used to classify 95% of the samples correctly for autoignition sensitivity exceeding a threshold (S ≥ 8) and 100%of the samples correctly for research octane number exceeding a threshold (RON ≥ 90). Molecular beam mass spectrometry (MBMS) experimental data at four temperatures is then used as the model input in a real-world test. Despite the nontrivial relationship between the MBMS measurements and speciation as well as experimental noise it is still possible to classify 95% of the samples correctly for RON and 85% of the samples correctly for S in a “leave-one-o...

The promoting effect of natural sand on methane hydrate formation: Grain sizes and mineral composition

Investigations regarding the effects of particle size on gas hydrate growth are commonly carried out using glass beads, silica powder, and various clay minerals. Comparisons of different grain sizes of sands are less common as are natural samples. Since the highest gas hydrate saturations and possible energy resource targets are associated with natural sandy sediments, the presented experiments address the possible effects of different grain sizes from a natural sand sample on methane hydrate formation. Influences on the kinetics of hydrate formation in natural deposits are of particular interest in hydrate production or carbon dioxide sequestration in methane hydrates. In both cases an undesired secondary hydrate formation may occur on short time scales, usually under non-equilibrium conditions.The static small-volume experiments are carried out far within the methane hydrate stability field (7MPa/274K) using five different grain size classes of a natural quartz sand: <125μm, < 250μm, 250–500μm, 500–1000μm, and 1000–2000μm. A constant volume of water or salt water is added to saturate the sediment sample and pressure is built up using methane gas. Pressure and temperature are continuously recorded and a glass window allows for microscopic observation and Raman spectroscopy to verify methane hydrate formation.For the chosen experimental set-up there is a strong particle size effect on the kinetics of methane hydrate formation. A high concentration of fine sands with a grain size <125μm led to explicitly faster gas hydrate formation compared to coarser sand or a small fraction of fine particles diluted in a sample of coarser sand grains. Our article discusses possible causes in conjunction with changes of the mineral surface. These include accelerated hydrate nucleation or growth due to the surface area (1) or mineral composition (2) and changes in dissolution kinetics (3) caused by enhanced dissolution rates or changes in the ratio of bound to bulk water volumes. The addition of salt significantly increased induction times but did not supplant the particle size effect.

Inherent variability in the kinetics of autocatalytic protein self-assembly.

In small volumes, the kinetics of filamentous protein self-assembly is expected to show significant variability, arising from intrinsic molecular noise. This is not accounted for in existing deterministic models. We introduce a simple stochastic model including nucleation and autocatalytic growth via elongation and fragmentation, which allows us to predict the effects of molecular noise on the kinetics of autocatalytic self-assembly. We derive an analytic expression for the lag-time distribution, which agrees well with experimental results for the fibrillation of bovine insulin. Our expression decomposes the lag-time variability into contributions from primary nucleation and autocatalytic growth and reveals how each of these scales with the key kinetic parameters. Our analysis shows that significant lag-time variability can arise from both primary nucleation and from autocatalytic growth and should provide a way to extract mechanistic information on early-stage aggregation from small-volume experiments.

Development and evaluation of aptamer magnetic capture assay in conjunction with real-time PCR for detection of Campylobacter jejuni

A prototype method for the concentration and detection of Campylobacter jejuni was developed using a previously reported biotinylated DNA aptamer in conjunction with qPCR. The so-called aptamer-based magnetic capture-qPCR (AMC-qPCR) assay was compared to a similar immunomagnetic separation (IMS)-qPCR assay. In small volume experiments (300 μl) applied to serially diluted C. jejuni suspended in buffer containing a mixed culture of other common food borne pathogens, the lower detection limit of the AMC-qPCR method was 1.1 log10/300 μl C. jejuni cells, one log10 better (lower) than that of IMS-qPCR (2.1 log10 CFU/300 μl). AMC-qPCR capture efficiency was 10–13% at assay detection limit. In 10 ml scale-up experiments, the lower detection limit of AMC-qPCR was 2.0 log10 CFU/10 ml with corresponding capture efficiency of 4–7%. Nucleic acid aptamers are promising alternatives to antibodies for magnetic bead-based capture followed by qPCR detection.

On the relevance of iron adsorption to container materials in small-volume experiments on iron marine chemistry: 55Fe-aided assessment of capacity, affinity and kinetics

Iron chemistry in seawater has been extensively studied in the laboratory, mostly in small-volume sample bottles. However, little has been reported about iron wall sorption in these bottles. In this paper, radio-iron 55Fe was used to assess iron wall adsorption, both in terms of capacity, affinity and kinetics. Various bottle materials were tested. Iron sorption increased from polyethylene/polycarbonate to polymethylmetacrylate (PMMA)/high-density polyethylene/polytetrafluoroethylene to glass/quartz, reaching equilibrium in a 25–70 h period. PMMA was studied in more detail: ferric iron (Fe(III)) adsorbed on the walls of the bottles, whereas ferrous iron (Fe(II)) did not. Considering that in seawater the inorganic iron pool mostly consists of ferric iron, the wall will be a factor that needs to be considered in bottle experiments. The present data indicate that for PMMA with specific surface ( S)-to-volume ( V) ratio S/ V, both iron capacity (42 ± 16 × 10 − 9 mol/m 2 or 1.7 × 10 − 9 mol/L recalculated for the S/ V-specific PMMA bottles used) and affinity (log K Fe'W = 11.0 ± 0.3 m 2/mol or 12.4 ± 0.3 L/mol, recalculated for the S/ V-specific PMMA bottles used) are of similar magnitude as the iron capacity and -affinity of the natural ligands in the presently used seawater and thus cannot be ignored. Calculation of rate constants for association and dissociation of both Fe'L (iron bound to natural occurring organic ligands) and Fe'W (iron adsorbed on the wall of vessels) suggests that the two iron complexes are also of rather similar kinetics, with rate constants for dissociation in the order of 10 −4–10 − 5 L/s and rate constants for association in the order of 10 8 L/(mol s). This makes that iron wall sorption should be seriously considered in small-volume experiments, both in assessments of shorter-term dynamics and in end-point observations in equilibrium conditions. Therefore, the present data strongly advocate making use of iron mass balances throughout in experiments in smaller volume set-ups on marine iron (bio) chemistry.

Heterogeneous fouling in dead-end ultrafiltration

In the literature membrane fouling is defined as a homogenous fouling of the membrane surface. Pore blocking as well as cake formation increase the resistance of a membrane at each spot on the membrane to the same extent. This homogenous fouling is probably true for the first filtration cycle. But if the hydraulic cleaning of the membrane (backwash and/or forward flush) is not capable of removing all the fouling in the membrane module, we may expect a heterogeneous membrane surface and have to face different mechanisms in the next filtration cycles. In our research we found evidence for the existence of such heterogeneous fouling in dead-end ultrafiltration. Experiments are performed with small scale constant flux equipment with the possibility to perform an automated backwash and forward flush. Because of the small membrane surface only 8 liters per hour is filtered. With such a small volume experiments can be done with one batch of water or with synthetic water. Our results indicate that there are two different types of heterogeneous fouling: (1) Patches of fouling remaining on the membrane surface after a backwash causes a decrease of the available membrane surface. This fouling mechanism can be recognized by a disproportional increase of the build-up of the resistance during filtration. (2) Blocking of capillaries especially in the potting of the modules. This type of fouling is not necessarily affecting the transmembrane pressure. This type of fouling can be recognized by an increase of the forward flush pressure.

Natriuresis and “dilutional” hyponatremia after infusion of glycine 1.5%

Study Objective: To challenge the view that the hyponatremia resulting from absorption of glycine 1.5% is attributed to the expansion of the extracellular fluid (ECF) volume, and that the change in serum sodium is therefore widely used to calculate the amount of irrigant absorbed. Design: Retrospective analysis of six studies. Setting: University-affiliated hospital. Measurements: Approximately 1.2 L of glycine 1.5% was infused in 23 volunteers (two studies), 1 L in 10 prostatectomy patients, 2.4 L in 6 sheep, 4 L in 6 other sheep, and 3 L in 9 piglets. The distribution of the irrigant water was estimated from the difference between the measured and the expected serum sodium levels, taking into account the urinary losses of sodium and water. Main Results: Between 30% and 50% of the irrigant volume had diffused into the cells 30 minutes after the infusion, and only between 0% and 50% of the fluid remained in the ECF. One hour later, natriuresis accounted for 100% of the residual hyponatremia in the small-volume experiments, and it accounted for approximately 50% in the large-volume experiments. The change in serum sodium could not be used to quantify the small infusion volumes, but the large ones could be quantified fairly accurately if assessed at the very end of the infusion. Conclusions: Diffusion of water into the cells and natriuresis reduced and prolonged the hyponatremia associated with infusions of glycine 1.5%. This finding makes IV fluid with sodium added a more rational alternative than furosemide in the treatment of fluid overload with this irrigating solution.

The rate of change of pH of lactic acid exposed to glass-ionomer dental cements

The rate of change of pH of aqueous lactic acid at pH 4.2–4.5 (i.e. a little below that of active caries in vivo) in contact with disks of various commercial glass-ionomer cements has been determined in two configurations. In the first of them, a thin film set-up, 20 μl of solution was spread across the surface of a cement disk (diameter: 13 mm), and its pH determined by pressing a flat-ended electrode against the film at varying time intervals. In the second, a similar disk was immersed in 1.5 ml of solution, removed after varying time intervals, after which the pH of the solution was measured using a round-ended electrode. The latter measurement was more reliable, in that the pH electrode had time to equilibrate, whereas the former was more realistic because the film was approximately the same thickness as that of saliva on a tooth surface. Both series of experiments showed measurable differences in pH after only 30 s, with the thin-film configuration showing a range of pH changes of 0.5–1.2 units depending on the cement and the small volume configuration showing a range of 0.1–0.5 units, also depending on the cement. After 10 min, in the small volume experiments, the pH had generally increased further. The extent and speed of the change in pH led to the conclusion that ability of glass-ionomers to increase pH is likely to be an important mechanism of caries protection under clinical conditions.

Characterization of Electrochemical Responses in Picoliter Volumes

Cyclic voltammetry is conducted in picoliter microvials to investigate the influence of small volumes on the electrochemical response. Electrochemical experiments using a standard reduction-oxidation couple, ferrocene-carboxylic acid, have been performed in volumes as small as 1 pL. Peak-shaped voltammetry and an increase in the current on the reverse wave of the cyclic voltammogram are observed in the voltammetric response when ultrasmall volumes (16 pL or less) are used. This deviation from bulk microelectrode behavior is observed only at slower scan rates in the smaller microvials. The origins of the voltammetric behavior in the small-volume experiments have been probed by varying experimental conditions such as scan rate, vial size, and concentration. The voltammetric properties have been shown to be dependent on all three parameters. The peak-shaped voltammetry and increases in the current on the reverse wave are attributed to depletion of oxidizable species on the forward scan and reduction of the analyte restricted at the electrode surface, respectively. A physical model based on restriction of analyte in these well-defined microenvironments is proposed to explain the differences in current compared to that predicted by microelectrode theory in bulk solutions.