Investigation of a failed DNA assay leads to identification of an unexpected contaminant in a commonly used chromatography buffer.

For mammalian cell-derived recombinant biotherapeutics, controlling host cell DNA levels below a threshold is a regulatory requirement to ensure patient safety. DNA removal during drug substance manufacture is accomplished by a series of chromatography-based purification steps and a qPCR-based analytical method is most used to measure DNA content in the purified drug substance to enable material disposition. While the qPCR approach is mature and its application to DNA measurement is widespread in the industry, it is susceptible to trace levels of process-related contaminants that are carried forward. In this study, we observed failures in spike recovery studies that are an integral component of the qPCR-based DNA testing, suggesting the presence of an inhibitory compound in the sample matrix. We generated hypotheses around the origin of the inhibitory compound and generated multiple sample matrices and deployed a suite of analytical techniques including Raman and NMR spectroscopy to determine the origin and identity of the inhibitory compound. The caustic wash step and depth filter extractables were ruled out as root causes after extensive experimentation and DNA testing. Subsequently, 2-(N-morpholino)ethanesulfonic acid (MES), a buffer used in the chromatography unit operations, was identified as the source of the contaminant. A 500-fold concentration followed by Raman and NMR spectroscopy analysis revealed the identity of the inhibitory compound as polyvinyl sulfone (PVS), an impurity that originates in the MES manufacturing process. We have implemented PVS concentration controls for incoming MES raw material, and our work highlights the need for rigor in raw material qualification and control.

Molecular packing of high-mobility diketo pyrrolo-pyrrole polymer semiconductors with branched alkyl side chains.

We describe a series of highly soluble diketo pyrrolo-pyrrole (DPP)-bithiophene copolymers exhibiting field effect hole mobilities up to 0.74 cm(2) V(-1) s(-1), with a common synthetic motif of bulky 2-octyldodecyl side groups on the conjugated backbone. Spectroscopy, diffraction, and microscopy measurements reveal a transition in molecular packing behavior from a preferentially edge-on orientation of the conjugated plane to a preferentially face-on orientation as the attachment density of the side chains increases. Thermal annealing generally reduces both the face-on population and the misoriented edge-on domains. The highest hole mobilities of this series were obtained from edge-on molecular packing and in-plane liquid-crystalline texture, but films with a bimodal orientation distribution and no discernible in-plane texture exhibited surprisingly comparable mobilities. The high hole mobility may therefore arise from the molecular packing feature common to the entire polymer series: backbones that are strictly oriented parallel to the substrate plane and coplanar with other backbones in the same layer.

Laser mass spectrometric detection of extraterrestrial aromatic molecules: mini-review and examination of pulsed heating effects.

Laser mass spectrometry is a powerful tool for the sensitive, selective, and spatially resolved analysis of organic compounds in extraterrestrial materials. Using microprobe two-step laser mass spectrometry (muL(2)MS), we have explored the organic composition of many different exogenous materials, including meteorites, interplanetary dust particles, and interstellar ice analogs, gaining significant insight into the nature of extraterrestrial materials. Recently, we applied muL(2)MS to analyze the effect of heating caused by hypervelocity particle capture in aerogel, which was used on the NASA Stardust Mission to capture comet particles. We show that this material exhibits complex organic molecules upon sudden heating. Similar pulsed heating of carbonaceous materials is shown to produce an artifactual fullerene signal. We review the use of muL(2)MS to investigate extraterrestrial materials, and we discuss its recent application to characterize the effect of pulsed heating on samples of interest.

Two-step laser mass spectrometry of asphaltenes.

Defined by their solubility in toluene and insolubility in n-heptane, asphaltenes are a highly aromatic, polydisperse mixture consisting of the heaviest and most polar fraction of crude oil. Although asphaltenes are critically important to the exploitation of conventional oil and are poised to rise in significance along with the exploitation of heavy oil, even as fundamental a quantity as their molecular weight distribution is unknown to within an order of magnitude. Laser desorption/ionization (LDI) mass spectra vary greatly with experimental parameters so are difficult to interpret: some groups favor high laser pulse energy measurements (yielding heavy molecular weights), arguing that high pulse energy is required to detect the heaviest components of this mixture; other groups favor low pulse energy measurements (yielding light molecular weights), arguing that low pulse energy is required to avoid aggregation in the plasma plume. Here we report asphaltene mass spectra recorded with two-step laser mass spectrometry (L2MS), in which desorption and ionization are decoupled and no plasma is produced. L2MS mass spectra of asphaltenes are insensitive to laser pulse energy and other parameters, demonstrating that the asphaltene molecular weight distribution can be measured without limitation from insufficient laser pulse energy or plasma-phase aggregation. These data resolve the controversy from LDI, showing that the asphaltene molecular weight distribution peaks near 600 Da and previous measurements reporting much heavier species suffered from aggregation effects.

Supramolecular routes towards liquid crystalline side-chain polymers.

Supramolecular attachment of mesogenic or non-mesogenic side chains to polymer backbones can result in the formation of liquid crystalline morphologies. The various parameters that can be tuned in order to achieve these morphologies, such as the type of non-covalent bonding chemistry (hydrogen bonding, ionic bonding, metal coordination, π-π interactions), the polymeric template architecture, and the side chain structure and properties, are reviewed in what follows, with emphasis placed on the role these parameters play in the determination of the final material morphologies and properties.

Gold nanoparticles as a colorimetric sensor for protein conformational changes.

Spherical gold nanoparticles and flat gold films are prepared in which yeast iso-1-cytochrome c (Cyt c) is covalently bound to the gold surface by a thiol group in the cystein 102 residue. Upon exposure to solutions of different pH, bound Cyt c unfolds at low pH and refolds at high pH. This conformational change causes measurable shifts in the color of the coated nanoparticle solutions detected by UV-VIS absorption spectroscopy and in the refractive index (RI) of the flat gold films detected by surface plasmon resonance (SPR) spectroscopy. Both experiments demonstrate the same trend with pH, suggesting the use of protein-covered gold nanoparticles as a simple colorimetric sensor for conformational change.

Extractables analysis of single-use flexible plastic biocontainers.

UNLABELLED: Studies of the extractable profiles of bioprocessing components have become an integral part of drug development efforts to minimize possible compromise in process performance, decrease in drug product quality, and potential safety risk to patients due to the possibility of small molecules leaching out from the components. In this study, an effective extraction solvent system was developed to evaluate the organic extractable profiles of single-use bioprocess equipment, which has been gaining increasing popularity in the biopharmaceutical industry because of the many advantages over the traditional stainless steel-based bioreactors and other fluid mixing and storage vessels. The chosen extraction conditions were intended to represent aggressive conditions relative to the application of single-use bags in biopharmaceutical manufacture, in which aqueous based systems are largely utilized. Those extraction conditions, along with a non-targeted analytical strategy, allowed for the generation and identification of an array of extractable compounds; a total of 53 organic compounds were identified from four types of commercially available single-use bags, the majority of which are degradation products of polymer additives. The success of this overall extractables analysis strategy was reflected partially by the effectiveness in the extraction and identification of a compound that was later found to be highly detrimental to mammalian cell growth. LAY ABSTRACT: The usage of single-use bioreactors has been increasing in biopharmaceutical industry because of the appealing advantages that it promises regarding to the cleaning, sterilization, operational flexibility, and so on, during manufacturing of biologics. However, compared to its conventional counterparts based mainly on stainless steel, single-use bioreactors are more susceptible to potential problems associated with compound leaching into the bioprocessing fluid. As a result, extractable profiling of the single-use system has become essential in the qualification of such systems for its use in drug manufacturing. The aim of this study is to evaluate the effectiveness of an extraction solvent system developed to study the extraction profile of single-use bioreactors in which aqueous-based systems are largely used. The results showed that with a non-targeted analytical approach, the extraction solvent allowed the generation and identification of an array of extractable compounds from four commercially available single-use bioreactors. Most of extractables are degradation products of polymer additives, among which was a compound that was later found to be highly detrimental to mammalian cell growth.

Determination of the internal morphology of nanostructures patterned by directed self assembly.

The directed self-assembly (DSA) of block copolymers (BCP) is an emerging resolution enhancement tool that can multiply or subdivide the pitch of a lithographically defined chemical or topological pattern and is a resolution enhancement candidate to augment conventional lithography for patterning sub-20 nm features. Continuing the development of this technology will require an improved understanding of the polymer physics involved as well as experimental confirmation of the simulations used to guide the design process. Both of these endeavors would be greatly facilitated by a metrology, which is capable of probing the internal morphology of a DSA film. We have developed a new measurement technique, resonant critical-dimension small-angle X-ray scattering (res-CDSAXS), to evaluate the 3D buried features inside the film. This is an X-ray scattering measurement where the sample angle is varied to probe the 3D structure of the film, while resonant soft X-rays are used to enhance the scattering contrast. By measuring the same sample with both res-CDSAXS and traditional CDSAXS (with hard X-rays), we are able to demonstrate the dramatic improvement in scattering obtained through the use of resonant soft X-rays. Analysis of the reciprocal space map constructed from the res-CDSAXS measurements allowed us to reconstruct the complex buried features in DSA BCP films. We studied a series of DSA BCP films with varying template widths, and the internal morphologies for these samples were compared to the results of single chain in mean-field simulations. The measurements revealed a range of morphologies that occur with changing template width, including results that suggest the presence of mixed morphologies composed of both whole and necking lamella. The development of res-CDSAXS will enable a better understanding of the fundamental physics behind the formation of buried features in DSA BCP films.

Molecular order in high-efficiency polymer/fullerene bulk heterojunction solar cells.

We report quantitative measurements of ordering, molecular orientation, and nanoscale morphology in the active layer of bulk heterojunction (BHJ) organic photovoltaic cells based on a thieno[3,4-b]thiophene-alt-benzodithiophene copolymer (PTB7), which has been shown to yield very high power conversion efficiency when blended with [6,6]-phenyl-C71-butyric acid methyl ester (PC(71)BM). A surprisingly low degree of order was found in the polymer-far lower in the bulk heterojunction than in pure PTB7. X-ray diffraction data yielded a nearly full orientation distribution for the polymer π-stacking direction within well-ordered regions, revealing a moderate preference for π-stacking in the vertical direction ("face-on"). By combining molecular orientation information from polarizing absorption spectroscopies with the orientation distribution of ordered material from diffraction, we propose a model describing the PTB7 molecular orientation distribution (ordered and disordered), with the fraction of ordered polymer as a model parameter. This model shows that only a small fraction (≈20%) of the polymer in the PTB7/PC(71)BM blend is ordered. Energy-filtered transmission electron microscopy shows that the morphology of PTB7/PC(71)BM is composed of nanoscale fullerene-rich aggregates separated by polymer-rich regions. The addition of diiodooctane (DIO) to the casting solvent, as a processing additive, results in smaller domains and a more finely interpenetrating BHJ morphology, relative to blend films cast without DIO.

Denaturation and renaturation of self-assembled yeast iso-1-cytochrome c on Au.

We have made surface plasmon resonance (SPR) measurements of yeast iso-1-cytochrome c (Cyt c) on a gold surface. Angle-resolved SPR curves are recorded as a function of urea concentration before and after self-assembly of the Cyt c. Exposure to a urea solution causes denaturation of Cyt c, which shifts the minimum in the SPR curve to a larger angle and decreases the signal amplitude. The Gibbs free energy change for denaturation of the protein on Au is calculated from the change of the SPR signal amplitude with urea concentration. We find that (1) Cyt c can be reversibly denatured and renatured, depending on the urea concentration, and (2) the Gibbs free energy change for denaturation of Cyt c on Au surface in water, DeltaG degrees (water), is 1.5 kcal/mol, which is approximately 4 times less than that in bulk solution.