Product and contaminant measurement in bioprocess development by SELDI‐MS

Bioprocesses for therapeutic protein production typically require significant resources to be invested in their development. Underlying these efforts are analytical methods, which must be fit for the purpose of monitoring product and contaminants in the process. It is highly desirable, especially in early‐phase development when material and established analytical methods are limiting, to be able to determine what happens to the product and impurities at each process step with small sample volumes in a rapid and readily performed manner. This study evaluates the utility of surface‐enhanced laser desorption ionization mass spectroscopy (SELDI‐MS), known for its rapid analysis and minimal sample volumes, as an analytical process development tool. In‐process samples from an E. coli process for apolipoprotein A‐IM (ApoA‐IM) manufacture were used along with traditional analytical methods such as HPLC to check the SELDI‐MS results. ApoA‐IM is a naturally occurring variant of ApoA‐I that appears to confer protection against cardiovascular disease to those that carry the mutated gene. The results show that, unlike many other analytical methods, SELDI‐MS can handle early process samples that contain complex mixtures of biological molecules with limited sample pretreatment and thereby provide meaningful process‐relevant information. At present, this technique seems most suited to early‐phase development particularly when methods for traditional analytical approaches are still being established. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Background and overview of current sediment toxicity identification evaluation procedures

Laboratory bioassays can provide an integrated assessment of the potential toxicity of contaminated sediments to aquatic organisms; however, toxicity as a sole endpoint is not particularly useful in terms of identifying remedial options. To focus possible remediation (e.g., source control), it is essential to know which contaminants are responsible for toxicity. Unfortunately, contaminated sediments can contain literally thousands of potentially toxic compounds. Methods which rely solely on correlation to identify contaminants responsible for toxicity are limited in several aspects: (a) actual compounds causing toxicity might not be measured, (b) concentrations of potentially toxic compounds may covary, (c) it may be difficult to assess the bioavailability of contaminants measured in a sediment, and (d) interactions may not be accounted for among potential toxicants (e.g., additivity). Toxicity identification evaluation (TIE) procedures attempt to circumvent these problems by using toxicity-based fractionation procedures to implicate specific contaminants as causative toxicants. Phase I of TIE characterizes the general physio-chemical nature of sample toxicants. Phase II employs methods to measure toxicants via different analytical methods, and Phase III consists of techniques to confirm that the suspect toxicants identified in Phases I and II of the TIE actually are responsible for toxicity. These TIE procedures have been used to investigate the toxicity of a variety of samples, including sediments. Herein we present a brief conceptual overview of the TIE process, and discuss specific considerations associated with sediment TIE research. Points addressed include: (a) selection and preparation of appropriate test fractions, (b) use of benthic organisms for sediment TIE work, and (c) methods for the identification of common sediment contaminants.     

Specific blocking to improve biopanning in biological samples such as serum and hybridoma supernatants

Screening phage-displayed peptide libraries (biopanning) is an important technique for acquiring peptide ligands and for mapping peptide epitopes recognized by antibodies (Ab). In biological samples, other materials, not only contaminants but also natural constituents, often interfere with biopanning. Capture methods use anchoring Abs that reduce the need for purification of the intended panning target from a crude sample. This capture method is analogous to sandwich ELISA. However, when the target molecule concentration is low in the initial mixture, the panning of a captured target often yields epitopes that bind to the capture antibody rather than the target of interest. We have developed a methodology that utilizes specific blocking reagents of the capture Ab to extend the sensitivity and applicability of the capture approach to phage panning. A flowchart is presented to enable the worker to begin panning with the simplest approach and then to employ sandwich capture and specific blocking reagents as necessary.     

Sorptive extraction techniques in sample preparation for organophosphorus pesticides in complex matrices

Organophosphorus pesticides (OPPs), widely known as persistent organic pollutants, are the most popular contaminants in agriculture products in developing countries. The determination of OPPs in complex matrices, such as food, environmental and biological samples, usually requires extensive sample pretreatment. This review focuses on the sorptive extraction techniques applied as sample pretreatment for OPPs in complex matrices, including solid-phase extraction (SPE) and solid-phase microextraction (SPME). These methods are evaluated and the applications of each technique are demonstrated extensively with many practical examples.     

Bioavailability of Soil-Borne Chemicals: Abiotic Assessment Tools

The abiotic tools that are available, or under development, for evaluating the oral and dermal bioavailability of contaminants from soils are described in this article. These tools generally rely on one of two approaches: (1) characterizing the form of the contaminant and the chemical binding of the contaminant to the soil matrix, and (2) chemical extractions intended to evaluate the fraction of the chemical that would be liberated in biological fluids (gastrointestinal fluid or sweat). For the purpose of human health risk assessment, abiotic methods to estimate the bioavailability of inorganic contaminants in soil are considered generally to be “screening” level tools at this time. Development work for physiologically based extraction tests (PBETs) is ongoing for many inorganic contaminants, and these methods hold great promise for eventual use in making quantitative bioavailability adjustments in risk assessment. The availability of abiotic tools to evaluate the bioavailability of organic contaminants from soils lags behind that for metals, due to the difficulty in conducting in vivo bioavailability studies with organic compounds and their complex interactions with soil. However, considerable research is being conducted in this field, and new assessment tools are being validated for use in human health risk assessment.     

PCR inhibitor levels in concentrates of biosolid samples predicted by a new method based on excitation-emission matrix spectroscopy

Biosolids contain a wide variety of organic contaminants that are known for their ability to inhibit PCR. During sample processing, these contaminants are coconcentrated with microorganisms. Elevated concentrations of these compounds in concentrates render samples unsuitable for molecular applications. Glycine-based elution and recovery methods have been shown to generate samples with fewer PCR inhibitory compounds than the current U.S. EPA-recommended method for pathogen recovery from biosolids. Even with glycine-based methods, PCR inhibitors still persist in concentrations that may interfere with nucleic acid amplification. This results in considerable loss of time and resources and increases the probability of false negatives. A method to estimate the degree of inhibition prior to application of molecular methods is desirable. Here we report fluorescence excitation-emission matrix (EEM) profiling as a tool for predicting levels of molecular inhibition in sample concentrates of biosolids.     

Historical Assessment of the Impacts of Chemical Contaminants in Sediments on Benthic Invertebrates in the Tidal Passaic River,New Jersey

A historical impact assessment was conducted for chemical contaminants in sediments of the tidal Passaic River in northern New Jersey. The assessment was based on sediment cores collected from 1990 to 1995. Each sediment core was segregated into fractions and the fractions dated using radioisotope analysis. Chemical concentrations, including a variety of metals and organic compounds, were estimated by decade for most of the 20th century in five reaches of the River. The chemical data for each decade were compared to available benchmark sediment quality values that represent levels of chemicals that may be toxic to benthic invertebrates in estuarine systems. Benchmark exceedances in the River were then calculated and characterized spatially and temporally using quantitative Geographic Information System (GIS) analyses, and the area of “impacted” sediments was calculated for each chemical and decade. Results of this assessment suggest that the ability of Passaic River sediments to support “normal” benthic invertebrate populations was limited by sediment contaminants throughout the 20^th century. Conditions have improved somewhat since the 1950s, although impacts to benthic populations remain from several metals and organic compounds despite some overall improvements in sediment quality in recent years.     

Mass spectrometry to describe product and contaminant adsorption properties for bioprocess development

Process development for biologics is expensive and lengthy, tools are needed to rapidly understand where the difficulties will lie, and, hence, rationally deploy resources. In this work we introduce and evaluate a methodology to determine the manufacturability of a protein candidate. The methodology determines protein impurities by mass spectrometry and separation difficulty from the product based on adsorption properties deduced from a single set of experiments. This information can aid early process strategy decisions to target hard to remove protein impurities (nearest neighbors) and allow the re-evaluation of conventional process synthesis. The methodology chosen gives consideration to the fact that at this point in early phase development, material, and established analytical methods are limiting. This study uses surface enhanced laser desorption ionization mass spectroscopy (SELDI-MS), for its rapid analysis and minimal sample requirement to measure product and contaminant adsorption properties. The technique is used to provide an array of hydrophobic and electrostatic conditions for protein adsorption. The adsorption pattern produced for each protein is analyzed and visualized via a star plot. Dendrograms then define nearest neighbor protein contaminants by quantifying differences in the adsorption pattern between the product and contaminants. By comparison to an existing process to manufacture a 28 kDa recombinant protein expressed in Escherichia coli, we confirm the method is capable of determining where the greatest separation difficulty lies and what separation methods should be considered. The technique identified that the nearest neighbor contaminants were product-related proteins (28.6 and 29.1 kDa/e). Thus demonstrating a capability to measure the relative difficulty of purifying early stage protein candidates where little is known about the separation properties of products and contaminants, or the process sequence for their production.     

Small-Sample Blood Culture Method for Identification of Bacteria in Central Arterial and Peripheral Blood

A blood culture technique that utilized small arterial blood samples or peripheral capillary blood was tested in beagle dogs and pig-tailed macaque monkeys. A bolus of 2.0 x 10(7)Escherichia coli (ATCC 25922) was injected intravenously into five animals of each species. Blood samples were taken before injection of the organisms and 10, 15, 20, 30, 60, and 120 min after injection. Arterial blood samples (2.0 and 0.2 ml) and peripheral capillary samples (0.14 ml) were taken at each sampling time. Pour plates were prepared from arterial blood for colony counts. All three blood sampling methods were equally effective in detecting sepsis when 10 or more organisms per ml of blood were present. Below this level, the 2.0-ml sample was more effective. Contamination of the peripheral sample with air or skin contaminants was a problem.     

Impact of processing method on recovery of bacteria from wipes used in biological surface sampling

Environmental sampling for microbiological contaminants is a key component of hygiene monitoring and risk characterization practices utilized across diverse fields of application. However, confidence in surface sampling results, both in the field and in controlled laboratory studies, has been undermined by large variation in sampling performance results. Sources of variation include controlled parameters, such as sampling materials and processing methods, which often differ among studies, as well as random and systematic errors; however, the relative contributions of these factors remain unclear. The objective of this study was to determine the relative impacts of sample processing methods, including extraction solution and physical dissociation method (vortexing and sonication), on recovery of Gram-positive (Bacillus cereus) and Gram-negative (Burkholderia thailandensis and Escherichia coli) bacteria from directly inoculated wipes. This work showed that target organism had the largest impact on extraction efficiency and recovery precision, as measured by traditional colony counts. The physical dissociation method (PDM) had negligible impact, while the effect of the extraction solution was organism dependent. Overall, however, extraction of organisms from wipes using phosphate-buffered saline with 0.04% Tween 80 (PBST) resulted in the highest mean recovery across all three organisms. The results from this study contribute to a better understanding of the factors that influence sampling performance, which is critical to the development of efficient and reliable sampling methodologies relevant to public health and biodefense.     

Sediment-associated contaminants — an overview of scientific bases for developing remedial options

The review article covers three major aspects of scientific research on sediment-associated contaminants during the last 20 years: (i) identification and monitoring of sources and distribution (sampling; sample preparation; analyses, mainly of non-residual fractions; estimation of pollution potential); (ii) study of processes and mechanisms of pollutant transfer (interactions between dissolved and particulate element species; particle environments; transport and diagenesis: colloids; surface microlayers; particle related processes; bioturbation; dredging operations; remobilization of toxic elements; bioaccumulation of organic chemicals: solid/dissolved distribution of contaminants); (iii) assessment of the environmental impact of particle-bound pollutations (chemical extraction sequence; sediment bioassay; combined chemical/biological test procedures). Enstead, empirical tests developed from multi-disciplinary research on biological, chemical and physical factors are applied for assessing the reactivity, mobility and bioavailability of sediment-bound pollutations and for estimating the validity of remedial measures.     

Comparison of Methods for the Recovery of Virus Inoculated into Ground Beef

Various methods for the recovery of virus inoculated into ground beef were investigated in an attempt to develop a sensitive system that could be used to detect viral contaminants in market foods. A 100-g sample, inoculated with poliovirus 1, was suspended in 150 to 900 ml of Eagle minimum essential medium, pH 8.5, and mixed in either plastic bags or plastic cups on a mechanical shaker. The particulate materials were removed by means of cheese cloth, glass wool, woven fiber glass, or low-speed centrifugation. Large volumes of fluid were concentrated by ultrafiltration. Microbiological contamination was controlled by high antibiotic concentrations or by filtration. Quantitative plaque-forming-unit recovery of the virus was determined by utilizing an agar overlay technique on Vero cell cultures. The data indicated that from 20 to 50% of the seeded virus could be recovered from a 100-g sample of ground beef. The glass wool and woven fiber glass methods were the most effective, with recovery of approximately 50% of the inoculated virus.     

A comparison of microbial community characteristics among petroleum-contaminated and uncontaminated subsurface soil samples

Measurements of microbial community size, including total cell counts and specific degrader enumerations, were conducted on subsurface soil samples from both petroleum-contaminated and pristine aquifers. Samples were collected from both uncontaminated and contaminated areas of the petroleum-contaminated sites. In pristine and uncontaminated samples, total cell counts (acridine orange direct counts) were related to depth. The deeper samples contained smaller total microbial populations. However, indices of microbial activity varied considerably from sample to sample and probably reflect soil and site heterogeneity. Exposure to petroleum contamination apparently altered the microbial community structure. In samples exposed to low levels of contaminants as vapors and/or dissolved phases (ppb concentrations), and not free product, the toluene-specific degrader populations were larger at greater depths, and the numbers of amino acid-specific degraders were highly correlated to the numbers of decane-specific degraders, indicating that petroleum-adapted microbial communities were present in the contaminated samples. In highly contaminated samples, total microbial population densities decreased with increasing depth; however, microbial activity tended to increase with depth. These results indicate that petroleum contaminants exert toxic effects on the active microbial community at high exposures and enrich specific degraders at ppb levels of dissolved contaminants. Correspondence to: S.C. Long     

Lithospermic acid B isolated from Salvia miltiorrhiza ameliorates ischemia/reperfusion-induced renal injury in rats

The present study was designed to examine whether lithospermic acid B (LSB) isolated from Salvia miltiorrhiza has an ameliorative effect on renal functional parameters in association with the expression of aquaporin 2 (AQP 2) and Na,K-ATPase in the ischemia-reperfusion induced acute renal failure (ARF) rats. LSB showed strong antioxidant activity against production of reactive oxygen species (ROS), ROS-induced hemolysis, and production of lipid peroxide in a dose-dependent manner. Polyuria caused by down-regulation of renal AQP 2 in the ischemia-reperfusion induced ARF rats was partially restored by administration of LSB (40 mg/kg, i.p.), restoring expression of AQP 2, in renal inner and outer medulla. The expression of Na,K-ATPase alpha1 subunit in outer medulla of the ARF rats was also restored in the ARF rats by administration of LSB, while beta1 subunit level was not altered. The renal functional parameters including creatinine clearance, urinary sodium excretion, urinary osmolality, and solute-free reabsorption were also partially restored in ischemia-ARF rats by administration of LSB. Histological study also showed that renal damages in the ARF rats were abrogated by administration of LSB. Taken together, these data indicate that LSB ameliorates renal defects in rats with ischemia-reperfusion induced ARF, most likely via scavenging of ROS.     

Protein purification and crystallization artifacts: The tale usually not told

The misidentification of a protein sample, or contamination of a sample with the wrong protein, may be a potential reason for the non‐reproducibility of experiments. This problem may occur in the process of heterologous overexpression and purification of recombinant proteins, as well as purification of proteins from natural sources. If the contaminated or misidentified sample is used for crystallization, in many cases the problem may not be detected until structures are determined. In the case of functional studies, the problem may not be detected for years. Here several procedures that can be successfully used for the identification of crystallized protein contaminants, including: (i) a lattice parameter search against known structures, (ii) sequence or fold identification from partially built models, and (iii) molecular replacement with common contaminants as search templates have been presented. A list of common contaminant structures to be used as alternative search models was provided. These methods were used to identify four cases of purification and crystallization artifacts. This report provides troubleshooting pointers for researchers facing difficulties in phasing or model building.     

Optimized sensitivity of allele-specific PCR for prenatal typing of human platelet alloantigen single nucleotide polymorphisms

PCR using sequence-specific primers (PCR-SSP) is widely employed for the genotyping of single nucleotide polymorphisms (SNPs) in both routine diagnosis and medical research. The human platelet alloantigens (HPAs) represent SNPs in platelet-specific glycoproteins, and HPA-1, -2, -3, and -5 are the most relevant in immunohematology. In most protocols, the respective HPA-SNPs are analyzed in allele-specific reactions, each with at least 100 ng DNA. In many cases, prenatal HPA typing in the diagnosis of neonatal alloimmune thrombocytopenia is often limited by the restricted amounts of fetal DNA that are obtainable. We developed a novel PCR-SSP technique to achieve accurate HPA genotypes using only 1 ng DNA per reaction. The concentration of HPA-specific primers was increased to 1 microM each and exhibited a higher sensitivity compared to a commercial PCR-SSP kit. The modified PCR-SSP technique enabled the identification of fetal HPA genotypes using only 0.5 mL amniotic fluid (from week 16 of gestation) and from a maternal plasma sample (from week 38 of gestation). The principle of the modified PCR-SSP technique may also be applied for the genotyping of other SNPs from limited amounts of DNA.     

Monitoring river water quality with transplanted bryophytes: A methodological review

The aim of this literature review, which considers 47 articles published between 1989 and 2015, is to ascertain the current status of the active biomonitoring technique for assessing water quality and to evaluate the degree to which different aspects of the method have been standardized. Use of the tool is largely limited to Europe (83% of the articles reviewed). The technique has been used to biomonitor inorganic contaminants (in 96% of the studies) and, to a lesser extent, organic contaminants (4% of the studies). Only 25% of the articles concern methodological aspects of the technique. Moreover, most authors (78%) have only published one article on the topic, and many different protocols have been used in the various studies. As a result, the technique is not standardized, which hampers comparison of the results of different studies. We propose a protocol that would facilitate use of the technique for routine monitoring of the quality of river waters.     

Analysis of Volatile and Oxidation Sensitive Compounds Using a Cold Inlet System and Electron Impact Mass Spectrometry

This video presents a protocol for the mass spectrometrical analysis of volatile and oxidation sensitive compounds using electron impact ionization. The analysis of volatile and oxidation sensitive compounds by mass spectrometry is not easily achieved, as all state-of-the-art mass spectrometric methods require at least one sample preparation step, e.g., dissolution and dilution of the analyte (electrospray ionization), co-crystallization of the analyte with a matrix compound (matrix-assisted laser desorption/ionization), or transfer of the prepared samples into the ionization source of the mass spectrometer, to be conducted under atmospheric conditions. Here, the use of a sample inlet system is described which enables the analysis of volatile metal organyls, silanes, and phosphanes using a sector field mass spectrometer equipped with an electron impact ionization source. All sample preparation steps and the sample introduction into the ion source of the mass spectrometer take place either under air-free conditions or under vacuum, enabling the analysis of compounds highly susceptible to oxidation. The presented technique is especially of interest for inorganic chemists, working with metal organyls, silanes, or phosphanes, which have to be handled using inert conditions, such as the Schlenk technique. The principle of operation is presented in this video.     

使用电子断层技术对IgG1抗体逐个分子的三维重构与结构分析

抗体(antibody)又称免疫球蛋白(immunoglobulin,Ig),是人体免疫反应的重要参与者.了解抗体的结构和结构动态特征,是理解人体免疫作用机理、修复或提高免疫能力、定向设计抗体以治疗各种疾病的基础.本文以人体IgG1抗体为对象,综述了使用透射电子显微学方法研究IgG1抗体结构方向的最新进展.详细介绍了使用逐个分子的电子断层三维重构技术(individual-particle electron tomography,IPET)对抗体进行结构研究的方法,包括样品制备、图像处理和数据分析等.并描述了利用该技术,在研究抗体结合肽分子后的结构形变和通过收集不同构象来研究抗体动态结构特征方面所取得的阶段性成果.最后,对尚待解决的关键问题与该技术未来的发展方向进行了讨论与展望.