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Therapeutic proteins including antibodies and Fc-fusion proteins undergo a large number of chemical modifications during cell culture, purification, storage and in human circulation. They are also exposed to harsh conditions during stress studies, including elevated temperature, extremes of pH, forced oxidation, physiological pH, UV light to assess the possible degradation pathways and suitability of methods for detecting them. Some of these modifications are located on residues in binding regions, leading to loss of binding and potency and classified as critical quality attributes. Currently, criticality of modifications is assessed by a laborious process of collecting antibody fractions from the soft chromatography techniques ion exchange and hydrophobic interaction chromatography and characterizing the fractions one-by-one for potency and chemical modifications. Here, we describe a method for large-scale, parallel identification of all critical chemical modifications in one experiment. In the first step, the antibody is stressed by one or several stress methods. It is then mixed with target protein and separated by size-exclusion chromatography (SEC) on bound antibody-target complex and unbound antibody. Peptide mapping of fractions and statistical analysis are performed to identify modifications on amino acid residues that affect binding. To identify the modifications leading to slight decreases in binding, competitive SEC of antibody and antigen mixtures was developed and described in a companion study by Shi et al, where target protein is provided at lower level, below the stoichiometry. The newly described method was successfully correlated to crystallography for assessing criticality of chemical modifications and paratope mapping. It is more sensitive to low-level modifications, better streamlined and platform ready.

Recently, cation exchange chromatography (CEX) using aqueous volatile buffers was directly coupled with mass spectrometry (MS) and applied for intact analysis of therapeutic proteins and antibodies. In our study, chemical modifications responsible for charge variants were identified by CEX-UV-MS for a monoclonal antibody (mAb), a bispecific antibody, and an Fc-fusion protein. We also report post-CEX column addition of organic solvent and acid followed by mixing at elevated temperatures, which unfolded proteins, increased ion intensity (sensitivity) and facilitated top-down analysis. mAb stressed by hydrogen peroxide oxidation was used as a model system, which produced additional CEX peaks. The on-line CEX-UV-MS top-down analysis produced gas-phase fragments containing one or two methionine residues. Oxidation of some methionine residues contributed to earlier (acidic), some to later (basic) eluting peaks, while oxidation of other residues did not change CEX elution. The abundance of the oxidized and non-oxidized fragment ions also allowed estimation of the oxidation percentage of different methionine residues in stressed mAb. CEX-UV-MS measurement revealed a new intact antibody proteoform at 5% that eluted as a basic peak and included paired modifications: high-mannose glycosylation and remaining C-terminal lysine residue (M5/M5 + K). This finding was confirmed by peptide mapping and on-column disulfide reduction coupled with reversed-phase liquid chromatography – top-down MS analysis of the collected basic peak. Overall, our results demonstrate the utility of the on-line method in providing site-specific structural information of charge modifications without fraction collection and laborious peptide mapping.

The study goal was to develop a sediment polychlorinated biphenyl (PCB) dose-response model based on benthic invertebrate effects to PCBs. The authors used an equilibrium partitioning (EqP) approach to generate predicted PCB sediment effect concentrations (largely Aroclor 1254) associated with a gradient of toxic effects in benthic organisms from effects observed in aquatic toxicity studies. The present study differs from all other EqP collective sediment investigations in that the authors examined a common dose-response gradient of effects for PCBs rather than a single, protective value. The authors reviewed the chronic aquatic toxicity literature to identify measured aqueous PCB concentrations and associated benthic invertebrate effects. The authors control-normalized the aquatic toxic effect data and expressed results from various studies as a common metric, percent injury. Then, they calculated organic carbon-normalized sediment PCB concentrations (mg/kg organic carbon) from the aqueous PCB toxicity data set using EqP theory based on the US Environmental Protection Agency's (EPIWEB 4.1) derivation of the water-organic carbon partition coefficient (KOC ). Lastly, the authors constructed a nonlinear dose-response numerical model for these synoptic sediment PCB concentrations and biological effects: Y = 100/1 + 10([logEC50-logX] × [Hill slope]) (EC50 = median effective concentration). These models were used to generate "look-up" tables reporting percent injury in benthic biota for a range of Aroclor-specific sediment concentrations. For example, the model using the EPIWEB KOC estimate predicts mean benthic injury of 23.3%, 46.0%, 70.6%, 87.1%, and 95% for hypothetical sediment concentrations of 1 mg/kg, 2 mg/kg, 4 mg/kg, 8 mg/kg, and 16 mg/kg dry weight of Aroclor 1254, respectively (at 1% organic carbon). The authors recommend the model presented for screening but suggest, when possible, determining a site-specific KOC that, along with the tables and equations, allows users to create their own protective dose-response sediment concentration. Environ Toxicol Chem 2017;36:1311-1329. © 2016 SETAC.

Agonism of cell surface receptors by monoclonal antibodies is dependent not only on its ability to bind the target, but also to deliver a biological signal through receptors to the cell. Immunoglobulin G2 antibodies (IgG2s) are made up of a mixture of distinct isoforms (IgG2-A, -B and A/B), which differ by the disulfide connectivity at the hinge region. When evaluating panels of agonistic antibodies against CD200 receptor (CD200R) or βklotho receptor (βklotho), we noticed striking activity differences of IgG1 or IgG2 antibodies with the same variable domains. For the CD200R antibody, the IgG2 antibody demonstrated higher activity than the IgG1 or IgG4 antibody. More significantly, for βklotho, agonist antibodies with higher biological activity as either IgG2 or IgG1 were identified. In both cases, ion exchange chromatography was able to isolate the bioactivity to the IgG2-B isoform from the IgG2 parental mixture. The subclass-related increase in agonist activity was not correlated with antibody aggregation or binding affinity, but was driven by enhanced avidity for the CD200R antibody. These results add to the growing body of evidence that show that conformational differences in the antibody hinge region can have a dramatic impact on the antibody activity and must be considered when screening and engineering therapeutic antibody candidates. The results also demonstrate that the IgG1 (IgG2-A like) or the IgG2-B form may provide the most active form of agonist antibodies for different antibodies and targets.

Human IgG2 consists of disulfide-mediated structural isoforms, classified by the number of Fab arms disulfide-linked to the heavy chain hinge. In the IgG2-B isoform, both Fab arms are linked to the hinge region, and in IgG2-A, neither Fab arm are linked to the hinge. IgG2-A/B is a hybrid between these two forms, with only one Fab arm disulfide-linked to the hinge. Within each of these isoform types are subtypes, with subtle disulfide-linkage differences. Here we explored the structural basis for the A1 and A2 isoform subtypes. Whereas A1 isoform converts into the A/B and B isoforms under mild redox conditions, A2 does not. Characterization of the disulfide connectivities of A2 isoform revealed a similar structure to A1 isoform, with parallel inter heavy chain disulfide linkages in the hinge region. However, the hinge disulfides in A2 isoform were resistant to reduction under conditions where A1 isoform hinge disulfides became reduced and they required thermal treatment (>55°C) to obtain thiol-dependent disulfide reduction. Structural analysis of the hinge region indicated that the protected disulfides were restricted to cysteines 219 and 220 of the upper hinge. Disruption of the upper hinge through insertion mutagenesis eliminated A2 isoform behavior. 1H NMR studies showed that the A1 isoform Fc glycan was more dynamic than that on A2 isoform and showed some other conformational differences. Results point to an IgG2-A2 upper hinge region that is more akin to the interior of a globular protein than the flexible hinge region expected on an IgG.

Human IgG2 antibodies contain three types of disulfide isoforms, classified by the number of Fab arms having disulfide links to the heavy chain hinge region. In the IgG2-B form, both Fab arms have interchain disulfide bonds to the hinge region, and in IgG2-A, neither Fab arm are disulfide linked to the hinge. The IgG2-A/B is a hybrid between these two forms, with only one Fab arm disulfide linked to the hinge. Changes in the relative levels of these forms over time are observed while IgG2 circulates in humans, suggesting IgG2-A→IgG2-A/B→IgG2-B conversion. Using a flow-through dialysis system, we studied the conversion kinetics of these forms in vitro under physiological conditions. For two IgG2κ antibodies, in vivo results closely matched the kinetics observed in vitro, indicating that the changes observed in vivo were solely conversions between isoforms, not differential clearance of specific forms. Moreover, the combined results validate the accuracy of the physiological model for the study of blood redox reactions. Further exploration of the conversion kinetics using material enriched in the IgG2-A forms revealed that the IgG2-A→IgG2-A/B rate was similar between IgG2κ and IgG2λ antibodies. In IgG2κ antibodies, conversion of IgG2-A/B→IgG2-B was slower than the IgG2-A→IgG2-A/B reaction. However, in IgG2λ antibodies, little IgG2-A/B→IgG2-B conversion was detected under physiological conditions. Thus, small differences in the C-terminus of the light chain sequences affect the disulfide conversion kinetics and impact the IgG2 disulfide isoforms produced in vivo.

Analysis of the strength and stoichiometry of immunoglobulin G (IgG) binding to neonatal Fc receptor (FcRn) and Fcγ receptor (FcγR) is important for evaluating the pharmacokinetics and effector functions of therapeutic monoclonal antibody (mAb) products, respectively. The current standard for assessing FcγR and FcRn binding is composed of cell-based and surface plasmon resonance (SPR) assays. In this work, asymmetrical flow field flow fractionation (AF4) was evaluated to establish the true stoichiometry of IgG binding in solution. AF4 and liquid chromatography-mass spectrometry (LC-MS) were applied to directly observe IgG/FcγR and IgG/FcRn complexes, which were not observed using nonequilibrium size exclusion chromatography (SEC) analysis. Human serum albumin (HSA), an abundant component of human blood and capable of binding FcRn, was studied in combination with FcRn and IgG. AF4 demonstrated that the majority of large complexes of IgG/FcRn/HSA were at an approximate 1:2:1 molar ratio. In addition, affinity measurements of the complex were performed in the sub-micromolar affinity range. A significant decrease in binding was detected for IgG molecules with increased oxidation in the Fc region. AF4 was useful in detecting weak binding between full-length IgG/Fc fragments and Fc receptors and the effect of chemical modifications on binding. AF4 is a useful technique in the assessment of mAb product quality attributes.

Human IgG2 exists as a mixture of disulfide-linked structural isoforms that can show different activities. To probe the contribution of specific cysteine residues to the formation of structural isoforms, we characterized a series of Cys-->Ser mutant IgG2 recombinant monoclonal antibodies, focused on the first C(H)1 cysteine and the first two hinge cysteines. These residues participate in the formation of structural isoforms that have been noted by nonreduced capillary sodium dodecyl sulfate polyacrylamide gel electrophoresis, reversed-phase high-performance liquid chromatography, and cation exchange chromatography. We show that single Cys-->Ser mutants can greatly reduce heterogeneous disulfide bonding in human IgG2 and maintain in vitro activity. The data demonstrate the feasibility of applying site-directed mutagenesis to reduce disulfide bond heterogeneity in human IgG2 while preserving the activity of this therapeutically important class of human antibodies.

In the accompanying report ( Wypych, J., Li, M., Guo, A., Zhang, Z., Martinez, T., Allen, M. J., Fodor, S., Kelner, D. N., Flynn, G. C., Liu, Y. D., Bondarenko, P. V., Ricci, M. S., Dillon, T. M., and Balland, A. (2008) J. Biol. Chem. 283, 16194-16205 ), we have identified that the human IgG2 subclass exists as an ensemble of distinct isoforms, designated IgG2-A, -B, and -A/B, which differ by the disulfide connectivity at the hinge region. In this report, we studied the structural and functional properties of the IgG2 disulfide isoforms and compared them to IgG1. Human monoclonal IgG1 and IgG2 antibodies were designed with identical antigen binding regions, specific to interleukin-1 cell surface receptor type 1. In vitro biological activity measurements showed an increased activity of the IgG1 relative to the IgG2 in blocking interleukin-1beta ligand from binding to the receptor, suggesting that some of the IgG2 isoforms had lower activity. Under reduction-oxidation conditions, the IgG2 disulfide isoforms converted to IgG2-A when 1 m guanidine was used, whereas IgG2-B was enriched in the absence of guanidine. The relative potency of the antibodies in cell-based assays was: IgG1 > IgG2-A > IgG2 >> IgG2-B. This difference correlated with an increased hydrodynamic radius of IgG2-A relative to IgG2-B, as shown by biophysical characterization. The enrichment of disulfide isoforms and activity studies were extended to additional IgG2 monoclonal antibodies with various antigen targets. All IgG2 antibodies displayed the same disulfide conversion, but only a subset showed activity differences between IgG2-A and IgG2-B. Additionally, the distribution of isoforms was influenced by the light chain type, with IgG2lambda composed mostly of IgG2-A. Based on crystal structure analysis, we propose that IgG2 disulfide exchange is caused by the close proximity of several cysteine residues at the hinge and the reactivity of tandem cysteines within the hinge. Furthermore, the IgG2 isoforms were shown to interconvert in whole blood or a "blood-like" environment, thereby suggesting that the in vivo activity of human IgG2 may be dependent on the distribution of isoforms.

A combinatory approach for the characterization of post-translational and chemical modifications in high molecular weight therapeutic proteins like antibodies and peptide-Fc fusion proteins (MWor = 50 000 Da) is presented. In this approach, well-established techniques such as limited proteolysis, reversed-phase (RP) high-performance liquid chromatography (HPLC), and in-line mass spectrometry (MS) were combined for the characterization of a monoclonal IgG1 antibody and three different peptide-Fc fusion proteins. The one commonality of these molecules is the presence of a similarly accessible lysine residue either located in the flexible hinge region of the antibody or in the flexible linker of the peptide-Fc fusion proteins. Applying limited proteolysis using endoproteinase Lys-C resulted in the predominant cleavage C-terminal of this lysine residue. The created fragments, two identical Fab domain fragments and one Fc domain fragment derived from the IgG1 antibody and one Fc domain fragment and each of the three individual peptide moieties generated from the peptide-Fc fusion proteins, were readily accessible for complete separation by RP-HPLC and detailed characterization by in-line MS analysis. This approach facilitated rapid detection of a variety of chemical modifications such as methionine oxidation, disulfide bond scrambling, and reduction as well as the characterization of various carbohydrate chains. We found limited proteolysis followed by RP-HPLC-MS to be less time-consuming for sample preparation, analysis, and data interpretation than traditional peptide mapping procedures. At the same time, the reduced sample complexity provided superior chromatographic and mass spectral resolution than the analysis of the corresponding intact molecules or a large number of enzymatically generated fragments.

The antibody MAB007 was recently shown to be cysteinylated on an unpaired cysteine residue in the CDR3 variable region. Cysteinylation at this position was not complete and resulted in heterogeneous lots of MAB007 with respect to this posttranslational modification. In this report, a mild redox step was used that effectively removed cysteinylation while keeping native inter and intra-molecular disulfide bonds intact. Biophysical methods were employed to determine what consequences cysteinylation of the variable region had by directly comparing cysteinylated and de-cysteinylated MAB007 antibodies. No differences were detected in secondary structure; however, several pieces of evidence indicated that cysteinylation may result in tertiary or quaternary structural perturbations. These included differences in the cation-exchange chromatography and fluorescence-emission spectra of the cysteinylated and de-cysteinylated antibodies as well as differences in the solvent accessibility of the unpaired cysteine residue determined by labeling experiments. Such structural changes induced by cysteinylation were shown to increase the rate of MAB007 aggregation and to decrease the melting temperature of the Fab region by as much as 6 degrees C. The bioactivity of MAB007 was also shown to be adversely affected by cysteinylation and a direct correlation was made between the percent cysteinylation and biological activity.

A new isoform of the light chain of a fully human monoclonal immunoglobulin gamma2 (IgG2) antibody panitumumab against human epidermal growth factor receptor (EGFR) was generated by in vitro aging. The isoform was attributed to the isomerization of aspartate 92 located between phenylalanine 91 and histidine 93 residues in the antigen-binding region. The isomerization rate increased with increased temperature and decreased pH. A size-exclusion chromatography binding assay was used to show that one antibody molecule was able to bind two soluble extracellular EGFR molecules in solution, and isomerization of one or both Asp-92 residues deactivated one or both antigen-binding regions, respectively. In addition, isomerization of Asp-92 showed a decrease in in vitro potency as measured by a cell proliferation assay with a 32D cell line that expressed the full-length human EGFR. The data indicate that antibodies containing either one or two isomerized residues were not effective in inhibiting EGFR-mediated cell proliferation, and that two unmodified antigen binding regions were needed to achieve full efficacy. For comparison, the potency of an intact IgG1 antibody cetuximab against the same receptor was correlated with the bioactivity of its individual antigen-binding fragments. The intact IgG1 antibody with two antigen-binding fragments was also much more active in suppressing cell proliferation than the individual fragments, similar to the IgG2 results. These results indicated that avidity played a key role in the inhibition of cell proliferation by these antibodies against the human EGFR, suggesting that their mechanisms of action are similar.

The glycosylation profile of intact antibody due to the galactose and fucose heterogeneity in the N-linked sugars was determined with instrument resolution of 5000 and 10,000. After deconvolution of electrospray ionization mass spectra to complete convergence, several extra peaks appeared in addition to the peaks observed in the original mass spectra. The artificial peaks were avoided if deconvolution was stopped after a smaller number of iterations. A standard antibody was used as an external calibrant to minimize mass measurement errors during long-period experiments. Precision of four consecutive LC/MS measurements of the same antibody was 10 ppm (+/-1.5 Da). By using this approach, the masses of 11 intact antibodies were measured. All antibodies containing N-terminal glutamines had a negative mass shift due to the formation of pyroglutamate (-17 Da). Although the pyroglutamate variant of intact antibody was not resolved from the unmodified variant, this modification led to a mass shift proportional to the percentage of N-terminal pyroglutamate. By accurately measuring the mass shift we were able to quantify the abundance of pyroglutamic acid on intact antibodies. Mass accuracy in measuring different antibodies was below 30 ppm (+/-4 Da). The accurate mass measurement can be an effective tool for monitoring chemical degradations in therapeutic antibodies.

The status of the N-terminus of proteins is important for amino acid sequencing by Edman degradation, protein identification by shotgun and top-down techniques, and to uncover biological functions, which may be associated with modifications. In this study, we investigated the pyroglutamic acid formation from N-terminal glutamic acid residues in recombinant monoclonal antibodies. Almost half the antibodies reported in the literature contain a glutamic acid residue at the N-terminus of the light or the heavy chain. Our reversed-phase high-performance liquid chromatography-mass spectrometry method could separate the pyroglutamic acid-containing light chains from the native light chains of reduced and alkylated recombinant monoclonal antibodies. Tryptic peptide mapping and tandem mass spectrometry of the reduced and alkylated proteins was used for the identification of the pyroglutamic acid. We identified the formation of pyroglutamic acid from N-terminal glutamic acid in the heavy chains and light chains of several antibodies, indicating that this nonenzymatic reaction does occur very commonly and can be detected after a few weeks of incubation at 37 and 45 degrees C. The rate of this reaction was measured in several aqueous buffers with different pH values, showing minimal formation of pyroglutamic acid at pH 6.2 and increased formation of pyroglutamic acid at pH 4 and pH 8. The half-life of the N-terminal glutamic acid was approximately 9 months in a pH 4.1 buffer at 45 degrees C. To our knowledge, we showed for the first time that glutamic acid residues located at the N-terminus of proteins undergo pyroglutamic acid formation in vitro.

A reversed-phase LC/MS method was developed for reduced antibodies that provides efficient separation of light chain and two variants of heavy chain containing N-terminal glutamine and pyroglutamic acid. The best separation was achieved on Zorbax CN and Varian Pursuit DiPhenyl columns eluted with increasing percentage of n-propanol and acetonitrile in 0.1% trifluoroacetic acid. Although glutamine was genetically coded for the N-terminal residue of heavy chain of a monoclonal antibody used in this study, we found that most of it (70%) was converted to pyroglutamate during production. The conversion process continued in vitro and was monitored by the method. Deconvoluted electrospray ionization mass spectrum of the heavy chain revealed the glycosylation profile of a single N-linked sugar including a-, mono-, and di-galactosylated biantennary glycans and a 5-mannose sugar form.

Spectroscopic studies have identified a number of proteins that appear to retain significant residual structure under even strongly denaturing conditions. Intrinsic viscosity, hydrodynamic radii, and small-angle x-ray scattering studies, in contrast, indicate that the dimensions of most chemically denatured proteins scale with polypeptide length by means of the power-law relationship expected for random-coil behavior. Here we further explore this discrepancy by expanding the length range of characterized denatured-state radii of gyration ( R G ) and by reexamining proteins that reportedly do not fit the expected dimensional scaling. We find that only 2 of 28 crosslink-free, prosthetic-group-free, chemically denatured polypeptides deviate significantly from a power-law relationship with polymer length. The R G of the remaining 26 polypeptides, which range from 16 to 549 residues, are well fitted ( r 2 = 0.988) by a power-law relationship with a best-fit exponent, 0.598 ± 0.028, coinciding closely with the 0.588 predicted for an excluded volume random coil. Therefore, it appears that the mean dimensions of the large majority of chemically denatured proteins are effectively indistinguishable from the mean dimensions of a random-coil ensemble.

A new high-frequency turbulence measuring instrument, MicroSoar, has been developed, tested, and used to make scalar variance dissipation rate measurements. MicroSoar was mounted on the undercarriage of SeaSoar, a depth-programmable winged platform, and towed by a ship, at speeds up to 7 kt, in a depth range of the sea surface to 120 m. Sensors carried by MicroSoar were a fast thermistor, a pressure sensor, a microscale capillary conductivity sensor, and a three-axis accelerometer. With appropriate assumptions about the local T–S relation, measurements of microscale conductivity fluctuations can often be used to directly determine temperature variance dissipation rate (χT), the Cox number (Cx), and the scalar diathermal turbulent diffusivity (KT). Compared to conventional quasi-free-fall tethered vertically profiling instruments, MicroSoar's major advantage lies in its ability to sample large fluid volumes and large geographic areas in a short time, and to provide, rapidly and simply, two-dimensi...

Microstructure measurements taken prior and after some strong atmospheric events on the shallow Black Sea shelf allowed to track the formation and evolution of the thermohaline structure caused first by wind-induced mixing and local convection, later by a storm surge, and finally by intense heating during a short period of the ‘Indian summer’. It was found that even a day long mild surge can decrease the temperature by 2.5°C and increase salinity by 2 psu over the whole 20–25 m quasi-homogeneous upper layer, which was formed by previous intense vertical storm-induced mixing. During the following period of upwelling-favorable winds, the near-bottom temperature decreased by 8–9°C. The upwelling was accompanied by a series of thermohaline intrusions overlaying the inclined boundary of the sharp near-bottom thermocline. Restratification in the upper 10 m layer in the form of a series of quasi-homogeneous steps was successfully reproduced by a numerical model of wind-induced daytime mixing, followed by nighttime convection, using the measured sea-surface fluxes as background conditions. The vertical turbulence structure was depicted by the logarithm of the averaged kinetic energy dissipation, which showed a parabolic decrease from the boundary layers to the midpoint of internal weakly-stratified part of the water column. Intermittent turbulent patches were superimposed at this background profile, which closely coincided with mean structure of the vertical shear. A correlation between the averaged vertical profiles of the turbulent buoyancy Reynolds number Re b , mixing activity  G , and the gradient Richardson number Ri was found. The  G cannot be solely determined by Re b , it also strongly depends on Ri at all depths. Analytical relations betweeen these variables that may be used for mixing parameterization on the shelf are proposed.

A critical component of the ecological risk assessment process, identification of contaminants and receptors of concern, is currently lacking an objective, data-driven, broadly applicable methodology. The paired comparison technique (PCT) can be used to meet this need by providing a standardized technique for making these decisions. PCT condenses the decision-making process down to individual components and ultimately generates an objective ranking for each alternative based on pertinent attributes. Factors considered in the PCT may be selected and weighted via a formal consensus building process which can incorporate stakeholder concerns. Once factors have been selected and weighted, each alternative is ranked relative to each other alter native for each factor. Each alternative ranking for each factor is multiplied by the factor's weight and the weighted rankings are summed. These sums are then used to rank alternatives relative to one another. This ranking provides decision-makers with a semi quantitat...

The effects of five di-ortho polychlorinated biphenyl (PCB) congeners on survival, growth, and reproduction in the fathead minnow. Pimephales promelas, were determined. Fish were exposed to one of two nominal concentrations of each congener for a total of 13 weeks (7-week prespawning period plus 6-week spawning period) under flow-through conditions using methanol as a carrier solvent. At termination, P. promelas survival was high in all PCB congener treatments. PCB congeners had no significant sublethal effects on reproductive success within the concentration range examined. The total number of eggs, clutch size, number of clutches, percent hatchability, and presence of terata in offspring were not significantly affected. Reproductive success was slightly but not significantly enhanced in the solvent control and high treatment concentrations for congeners 52, 101, 138, and 153. Growth expressed as wet weight was not affected for both adult males or females. Male lengths were not significantly affected but female lengths were significantly reduced when exposed to congeners 101, 138, 153, and 180. Pimephales promelas accumulated substantial amounts of all PCBs, with tissue concentrations ranging from 13 to 183 mg/kg wet weight at termination in PCB-exposed fish. These tissue residues are one to several orders of magnitude greater than thosemore » reported for these congeners in aquatic biota previously collected in industrial waterways of the Great Lakes. Several di-ortho PCB congeners frequently found in aquatic biota showed minimal adverse effects on fathead minnow survival, growth, and reproduction even though bioaccumulation of these congeners was substantial.« less

The Shannon entropy is a measure of the degree of intricacy contained in any graphable n-dimensional realization of an observable quantity. We use the Shannon entropy to measure the intricacy of density overturns in the oceanic thermocline and find that the Shannon entropy is related to the Thorpe scale LT [Thorpe, 1977] and the Ozmidov scale LO [Ozmidov, 1965]. We find that (1) small Shannon entropy corresponds to small values of ROT (≡LO/LT), while large Shannon entropy is associated with large values of ROT; (2) density spectra are typically more steep than inertial subrange spectra when both Shannon entropy and ROT are small, whereas the spectral slope tends to be flatter than inertial subrange spectra when both Shannon entropy and ROT are large; (3) the Grashof number is very large (O(1010)) when Shannon entropy is small, indicating that these patches are extremely density unstable; (4) spectral bandwidth is much larger for patches with small Shannon entropy than for those with large entropy, indicating that large-scale, or “bulk” Reynolds number is large when entropy is small. We discuss the hypothesis that the degree of intricacy, and hence the Shannon entropy, increases with increasing time in a turbulent overturn and is observed to decrease only when the resolution limits of the measuring system are exceeded. On the basis of these arguments we suggest that some classes of overturns are created with Thorpe scale larger than the Ozmidov scale. In these overturns the kinetic energy dissipation rate (e) is small during the initial growth of the overturn. Later, a small-scale structure develops, and a more complex, higher-order flow evolves. This behavior is discussed and compared with gridgenerated laboratory turbulence, in which initially small, energetic, rapidly growing boundary layers detached from the grid and advect downstream, forcing ROT to be largest adjacent to the grid and thereafter decrease as a result of entrainment.

Juvenile Nereis (Neanthes) arenaceodentata survival and growth were used to evaluate the effect of storage time on the toxicity of sediments with moderate PAH and metal contamination. Seven San Francisco Bay area sediments and a clean control sediment were stored (4° C) and then periodically evaluated (up to two years after collection). During each test, juvenile worms (2–3 weeks post emergence) were exposed for 21 days. Test endpoints were survival and growth rate (mg dry weight/day). In general, survival was high (>75%) and long-term cold storage (740 days) did not significantly alter growth or survival. In half of the sediments a cyclical phenomenon was observed associated with the appearance of ammonia in the overlying water of bioassay beakers. The periodicity of this phenomenon was approximately one year. It was not associated with any geophysical characteristic of the test sediments (i.e. grain size, % TOC, % TKN). Significant mortality (0% survival on day 427) was associated with the largest of these peaks in overlying water ammonia concentration. Results of this study suggest that ammonia in stored sediments is an important, potentially confounding factor in sediment toxicity tests.

Ammonia has been found to be toxic to many species of marine organisms, but little data address the toxicity of ammonia to benthic infaunal species such as amphipods. Organisms closely associated with marine or estuarine sediments may be exposed to higher ammonia concentrations than are found in the water column, especially when sediments are disturbed. In this study, four species of marine or estuarine amphipods ( Rhepoxynius abronius, Eohaustorius estuarius, Ampelisca abdita and Grandidierella japonica ) were exposed to ammonia in seawater, in the absence of sediment, under controlled laboratory conditions, to determine the concentrations of ammonia that would be acutely toxic to those species. A. abdita was found to be the most sensitive to ammonia, with a median lethal concentration (LC 50 ) of 49·8 mg/liter total ammonia (0·83 mg/liter as un-ionized ammonia). R. abronius was also relatively sensitive: LC 50 = 78·7 mg/liter total ammonia (1·59 mg/liter un-ionized ammonia). E. estuarius and G. japonica were less sensitive, with estimated LC 50 values of 125·5 mg/liter and 148·3 mg/liter total ammonia, respectively (2·49 mg/liter and 3·35 mg/liter un-ionized ammonia). The absence of sediment did not appear to influence the response of the amphipods over the 96-h exposure period.

Many biological systems are primarily governed by protein-protein interactions. It is important to develop sensitive analytical techniques to identify and characterize these bimolecular interactions in order to understand their fundamental roles in biological processes and in disease. In this book chapter, we summarize three case studies that applied asymmetrical flow field-flow fractionation (AF4) to access the protein-protein interactions of therapeutic proteins with their counterparts. These new applications of AF4 provide a unique and innovative tool that extends the bioanalytical capability to study protein complexes beyond micro-molar affinity.

Development of a chronic sublethal sediment bioassay with the polychaete Nereis (Neanthes) arenaceodentata is described. The sublethal test end point was estimated individual somatic growth rate (milligrams dry weight per day). The test was initiated with two- to three-week-old post-emergent juvenile worms and continued for 28 d. The potential bias due to selected nontreatment factors on polychaete survival and growth was evaluated. For example, grain size had no significant effect, whereas the number of worms placed in each exposure vessel was critical. Direct transfer from 30%0 seawater to salinites ≤15%0 had a highly significant and adverse effect on survival and growth. Both survival and growth of juvenile worms may be adversely affected if test conditions involve exposures to ≥ 0.7 mg/L un-ionized ammonia or ≥ 5 mg/L hydrogen sulfide. Survival of juvenile worms to concentrations of the reference toxicant, cadmium chloride, approximating the 96-h LC50 (5 mg/L) was used as a quality control measure. Results are expressed in control chart format analogous to methods used in analytical chemistry.

Several models now exist for predicting the dissipation rate of turbulent kinetic energy, ϵ, in the oceanic thermocline as a function of the large-scale properties of the internal gravity wave field. These models are based on the transfer of energy toward smaller vertical scales by wave-wave interactions, and their predictions are typically evaluated for a canonical internal wave field as described by Garrett and Munk. Much of the total oceanic dissipation may occur, however, in regions where the wave field deviates in some way from the canonical form. In this paper simultaneous measurements of the internal wave field and ϵ from a drifting ice camp in the eastern Arctic Ocean are used to evaluate the efficacy of existing models in a region with an anomalous wave field and energetic mixing. By explicitly retaining the vertical wavenumber bandwidth parameter, β*, models can still provide reasonable estimates of the dissipation rate. The amount of data required to estimate β*, is, however, substanti...

Proteins destined to circulate in the blood are first folded and assembled in the endoplasmic reticulum of secretory cells. For antibodies, like many other serum proteins, the folding and assembly steps involve the formation of disulfide bonds. Such bonds have been thought to be static features of proteins, stabilizing domains, and linking polypeptide chains, although some cases of extracellular disulfide bond cleavage have been noted. Recently, the human IgG2 antibody subclass was found to possess multiple structures differing in specific disulfide linkages. These structures are naturally occurring and can, in some cases, affect the activity of the antibody. Here we show that these IgG2 disulfide linkages interconvert while circulating in humans. Secretory cells initially produce primarily one form (IgG2-A), which is rapidly converted to a second form (IgG2-A/B) while circulating in the blood, followed by a slower conversion to a third form (IgG2-B). This work demonstrates that the disulfide structure of the IgG2 antibody is dynamic in vivo, on a time scale similar to that of the protein's lifetime. Thus, changes to the IgG2 disulfide structure provide a marker of the protein's age and may alter its activity over its lifetime.

Antibody formulation development relies on accelerated stability data at elevated temperatures to optimize formulation parameters. However, the pH- and temperature-dependence of aggregation is complicated for antibody formulations. In this study, a human monoclonal IgG2 antibody exhibited typical pH-dependent dimer formation under normal storage conditions (4 and/or 29°C). However, an inversed pH-dependence was discovered for high molecular weight aggregate formation at elevated temperatures (37°C). The different stability profiles exhibited at the various storage conditions resulted in nonlinearity of the Arrhenius kinetics. Thermal unfolding at or below 37°C was not evident by differential scanning calorimetry. Enriched populations of the structural isoforms of the IgG2 subclass were tested for their unique temperature and pH-dependence of aggregation. The Arrhenius kinetics of aggregation for each of the individual IgG2 isoforms was also nonlinear. However, the temperature-dependence of clipping suggested that clip-mediated aggregation was responsible for the increased higher order aggregates at low pH and elevated temperatures. Unique clip species resulting from the conformational differences between the IgG2 isoforms lead to increased aggregation. These results have implications on the mechanisms of antibody aggregation and on the validity of accelerated data to predict shelf-life accurately. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3031–3042, 2009

In this work, we present studies of the covalent structure of human IgG2 molecules. Detailed analysis showed that recombinant human IgG2 monoclonal antibody could be partially resolved into structurally distinct forms caused by multiple disulfide bond structures. In addition to the presently accepted structure for the human IgG2 subclass, we also found major structures that differ from those documented in the current literature. These novel structural isoforms are defined by the light chain constant domain (CL) and the heavy chain CH1 domain covalently linked via disulfide bonds to the hinge region of the molecule. Our results demonstrate the presence of three main types of structures within the human IgG2 subclass, and we have named these structures IgG2-A, -B, and -A/B. IgG2-A is the known classic structure for the IgG2 subclass defined by structurally independent Fab domains and hinge region. IgG2-B is a structure defined by a symmetrical arrangement of a (CH1-CL-hinge)2 complex with both Fab regions covalently linked to the hinge. IgG2-A/B represents an intermediate form, defined by an asymmetrical arrangement involving one Fab arm covalently linked to the hinge through disulfide bonds. The newly discovered structural isoforms are present in native human IgG2 antibodies isolated from myeloma plasma and from normal serum. Furthermore, the isoforms are present in native human IgG2 with either κ or λ light chains, although the ratios differ between the light chain classes. These findings indicate that disulfide structural heterogeneity is a naturally occurring feature of antibodies belonging to the human IgG2 subclass.

The rapid growth of antibody drugs and drug candidates in the biopharmaceutical industry has created a demand for automated proteolytic digestion to assist in pharmaceutical stability studies, identity assays and quality control of these therapeutic proteins. Here, we describe the development of a fully automated proteolytic digestion procedure for monoclonal antibodies in solution, which requires a high concentration of denaturants for unfolding. The antibody samples were placed in a 96-well plate or in 0.5-mL Eppendorf tubes. The proteins were then reduced and alkylated in a denaturing solution of 6M guanidine HCl. The denaturing solution was replaced with a digestion buffer using a custom-designed 96-well size-exclusion plate for desalting. The sample was digested for 5 h with two additions of trypsin. The completeness and reproducibility of digestion were verified by reversed-phase high-performance liquid chromatography tandem mass spectrometry (HPLC/MS) analysis of the digestion products. The performance of the automatic digestion was comparable to the currently used manual digestion procedure, but saved time, reduced manual labor, and increased the reproducibility of the tryptic digests. Our method should be useful not only for high-throughput analysis of antibodies, but for other therapeutic protein samples as well. Other applications like gel-free proteomics, where the analysis of a large number of samples is often needed and the completeness of the liquid digestion is critical for the identification of a large number of different proteins, should also benefit from this fully automated liquid proteolytic digestion procedure.

Glycation of a recombinant monoclonal IgG2 molecule, in sucrose containing liquid formulations, was studied using reversed-phase LC/MS analysis of the intact IgG, the F(ab')2 fragments and after complete tryptic digestion. The extent of glycation in sucrose containing formulations was monitored at different temperatures over a period of 21 months using the Hexose index (Hex(I)). Hex(I) represents the average number of hexose molecules per molecule of IgG and was calculated by using the intensity values of peaks corresponding to hexose isoforms in the deconvoluted mass spectra. The rate of glycation in mildly acidic sucrose containing formulations was proportional to the incubation temperature. No glycation was observed in sucrose containing formulations incubated at 4 degrees C even after 18 months. However, when the same formulations were incubated at 37 degrees C glycation was observed after just 1 month. The glycation sites were mapped to 10 lysine residues distributed throughout the molecule. The amino terminal end of the light chain was also shown to contain glycation. The surface accessibility of the lysine side chain could influence its susceptibility to glycation.

MAB007, an IgG1 monoclonal antibody, is unique because of the presence of a free cysteine residue in the Fab region at position 104 on the heavy chain in the CDR3 region. Mass spectrometric analysis of intact MAB007 showed multiple peaks varying in mass by 120-140 Da that cannot be fully attributed to glycosylation isoforms typically present in IgG molecules. Limited proteolysis of MAB007 with Lys-C led to a single cleavage at the C-terminus of a lysine residue in the hinge region of the heavy chain at position 222, generating free Fab and Fc fragments. Reversed-phase liquid chromatography/mass spectrometry analysis of the Fab and Fc fragments revealed several modifications. The Fab fraction showed cysteinylation of a free cysteine in the CDR3 region resulting in a mass shift of 119 Da. Using limited proteolysis, we also identified modifications resulting in a mass increase of 127 Da in the Fc region, corresponding to C-terminal lysine variants in the heavy chain. Other modifications, such as oxidation (+16 Da) and succinimide formation (-17 Da), were also detected in the Fab fragment. The cysteinylation observed after limited proteolysis was confirmed by peptide mapping coupled with tandem mass spectrometry analysis.

An enhanced analytical RP-HPLC/MS method was developed for monitoring the stability and production of intact and fragmented monoclonal antibodies (MAbs). The use of high column temperatures (70-80 degrees C), organic solvents with high eluotropic strength coefficients (isopropyl and n-propyl alcohols), and Zorbax StableBond columns, were critical for good recovery and resolution of immunoglobulin G1 (IgG1) and IgG2 monoclonal antibodies. Using this method, cleavage products of a degraded IgG1 antibody were clearly separated and identified by in-line electrospray ionization time-of-flight (ESI-TOF) mass spectrometry generating exact masses and unique terminal ladder sequences. The glycosylation profile, including mapping of the terminal galactose and fucose heterogeneity of the N-linked sugars, was determined by mass spectrometry of intact MAbs. In addition, we discovered that several IgG2 MAbs exhibited greater structural heterogeneity compared to IgG1s. Mass spectral characterization data and reduction data suggested that the heterogeneity is disulfide related. This reversed-phase LC/MS method represents a key advancement in monitoring intact MAb production and stability.

Analytical characterization of monoclonal antibodies has been hindered by the lack of appropriate chromatographic methods to be used in conjunction with high-resolution MS. Current methodologies for standard RP-HPLC are incompatible with antibodies due to irreproducibility, low recovery, short column lifetimes, and poor resolution of degradation products. An analytical RP-HPLC-MS method was developed for monitoring and characterizing intact IgG1antibodies. Key parameters required for improved chromatographic resolution included long alkyl chains of the stationary phase (Zorbax SB300 C8), column temperatures elevated to 65-70 degrees C and combination of trifluoroacetic acid and heptafluorobutyric acid ion-pairing agents. RP chromatographic separation of degradation species and C-terminal lysine variants along with the characterization of glycosylation profile by mass spectrometry demonstrates the capability of this method for whole antibody analysis.

This review summarizes information obtained from published literature to determine to what degree biomagnification of organic compounds and metals occurs in freshwater and marine food webs. This review was conducted by: (1) examining data from studies conducted in laboratory experiments to establish body burden ratios between trophic levels (trophic transfer coefficients; TTCs); (2) comparing laboratory-derived TTCs with data obtained from field studies; and (3) comparing biomagnification predictions described by published aquatic food-web models with data obtained in this review. It was determined that: (1) the majority of chemicals evaluated (both organic and metals) do not biomagnify in aquatic food webs; (2) for many of the compounds examined, considerable trophic transfer does occur in aquatic food webs; (3) DDT, DDE, PCBs, toxaphene, methyl mercury, total mercury, and arsenic have the potential to biomagnify in aquatic systems; (4) the lipid fraction of receptors directly influences biomagnification of lipophilic compounds; (5) the food web model reviewed provided similar estimates for most of the organic compounds examined (log Kow values between 5 and 7), with model predictions falling within the range of values of all compounds except dieldrin; (6) for many organic compounds, lack of information precludes assessing the relative importance of biomagnification for these contaminants; and (7) even those compounds for which evidence for biomagnification is strongest show considerable variability and uncertainty regarding the magnitude and existence of food-web biomagnification in aquatic systems.

Growth in the polychaete worm Nereis (Neanthes) arenaceodentata has been suggested as a good sublethal end point for marine sediment bioassays. While growth is biologically important, eventual population success is more directly dependent on survival and reproduction. Growth may affect population success indirectly via survival and reproduction. To provide interpretative guidance for bioassays measuring worm growth, the quantitative relationship between growth and reproduction must be established. Juvenile N. arenaceodentata were exposed to five geometrically decreasing food rations. Growth, as individual worm wet weight, was measured after 3, 6, and 9 weeks. A significant gradient in worm growth was observed at each time interval. Eggs were first observed in the coelom of female worms at the 6-week interval. Subsequent reproduction, measured as fecundity and number of juveniles produced, closely mirrored the reduced growth rates. Based on results reported herein, a somatic growth rate greater than or equal 0.65 mg/day measured between 0 to 6 weeks resulted in no significant effect on either survival or reproduction. Growth rates less than or equal 0.45 mg/day resulted in significant reductions in reproduction. Low growth rates (0.05 mg/day) were associated with a nearly complete cessation of reproduction and very poor survival (5 to 11 percent). The importance of distinguishing somatic growth from gametic growth in chronic sublethal sediment bioassays with N. arenaceodentata is discussed.... Bioassay, Reproduction, Growth, Sublethal, Neanthes

This technical note describes a risk-based framework for testing and evaluating dredged material scheduled for open-water disposal.

This technical note describes a prototype expert system being developed to assist managers and scientists in the interpretation of bioaccumulation test results and their potential effect on the disposal of dredged material. This is a microcomputer (MS-DOS(TM)) based system, operating in the Microsoft Windows(TM) environment.

Initial development of a chronic sublethal sediment bioassay with the polychaete Nereis (Neanthes) arenaceodentata is described. The test was initiated with 2to 3-week-old postemergent juvenile worms and terminated after 21 days. The sublethal test end point was estimated individual somatic growth rate (milligrams dry weight/day). The potential bias due to selected nontreatment factors on polychaete survival and growth was evaluated. For example, grain size had no effect, while the number of worms added to each exposure vessel was critical. Direct transfer from 30 ppt to salinities or = mg/L ammonia or > or = mg/L hydrogen sulfide.... Chronic, Growth, Neanthes, Sediment, Sublethal.

This report is designed to address concerns regarding the potential toxicity of dredged material from San Francisco Bay and to provide input into the San Francisco District's Long-Term Management Strategy for dredged material disposal. To this end, a review of the regulatory history of dredged material management within San Francisco Bay and the development of sediment toxicity tests to assess dredged material is provided. Included in this discussion is a national overview of sediment toxicity, as well as the toxicity of San Francisco Bay sediments. Information gaps are identified and prioritized. Finally, specific research hypotheses are posed that will allow selected technical questions to be addressed by direct experimental examination. Keywords: Chronic sublethal, Noncontaminant effects, San Francisco Bay, Sediment toxicity.

During the ice camp component of the Coordinated Eastern Arctic Experiment (CEAREX) in March–April 1989, 1500 profiles of temperature, conductivity, and velocity shear microstructure were obtained near the Yermak plateau. The dominant signal in isopycnal displacements was the diurnal variability near the plateau, consistent with the enhanced diurnal currents found in this region previously from the Fram III and Fram IV ice camps. The dissipation rate of turbulent kinetic energy e reached maximum values of about 10−6 W kg−1 in two distinct regions, the surface mixing layer and the pycnocline between 120 and 220 m. There was a significant component of diurnal variability in e in each region. In the surface layer, e was proportional to the cube of the ice-relative current speed at 30 m below the ice, and it decayed approximately exponentially with increasing depth. Major mixing events in the pycnocline were correlated with large-amplitude, short-duration, isopycnal displacements. The lowest dissipation rates were recorded over the deep Nansen basin, north of the plateau, and in the subsurface core of a submesoscale baroclinic eddy in northern Fram Strait. The time-averaged vertical eddy diffusivity near the slope of the plateau was about 2.5×10−4 m2 s−1 in the pycnocline above the Atlantic layer, implying an upward heat flux of 25 W m−2, although only a small fraction of this heat reached the base of the mixed layer. The results confirm that current interactions with steep topography are critical to the modification of Atlantic Water in the eastern Arctic Ocean.

Microstructure measurements in the equatorial Pacific at 140°W in late 1984 show a pronounced diurnal variation in both high-frequency internal wave energy and kinetic energy dissipation rate. Observations indicated that after sunset, internal waves (presumably generated by convective overturns in the mixed layer) propagate downward and increase turbulence levels in the pycnocline. It is proposed that large mixed layer eddies in the South Equatorial Current interact with the large shear caused by the Equatorial Undercurrent to generate a westward going anisotropic wave field. The momentum transport in the radiated wave field results in a drag force on the equatorial mean flow field. The observed mean wind stress at 140°W during Tropic Heat I (which is twice as large as the annual mean wind) is closer to the estimated radiation stress (∼−10−4 m2 s−2) at the base of the mixed layer (≈30 m) than to the estimated turbulent stress (∼−10−5 m2 s−2). A wave dissipation model based on the observed turbulent kinetic energy dissipation rate is introduced in order to estimate the wave momentum flux divergence in the stratified region above the undercurrent core. The model predicts that most of the downward wave momentum flux penetrates through the undercurrent core. It is hypothesized that when the wind stress is strong, the equatorial Pacific Ocean responds by generating a westward traveling internal wave field which transports much of the surface wind stress below the actively mixing surface layer.

Hydrographic and velocity profiles were made through a small baroclinic cyclonic eddy during the Arctic Internal Wave Experiment in the Canada Basin in April 1985. The maximum measured azimuthal velocity was 0.38 m s -1 at a depth of 115 m, with velocities decaying to near zero at 30 and 270 m. Maximum isopycnal displacements at the closest approach to the eddy's axis were about 40 m. The deduced radius of maximum velocity is r0 = 7 -+ 2 km, and the total radius is about 13 km. Kinetic energy dissipation rate, e, was enhanced within 70 m of the surface throughout the transect, and for radius r < r0 near 60 m and 180 m. The subsurface maxima in e do not correspond to regions of low Richardson number, but are colocated with locally reduced shear, consistent with the observed dissipation rates being merely the remnants of recent, more energetic mixing events. The time scale for the decay of the eddy, based on its total energy and the measured dissipation rates, is O(10) years. Given this large dissipation time scale, critical layer absorption of vertically propagating internal wave momentum may be dynamically significant to the eddy's evolution. The Arctic Internal Wave Experiment (AIWEX) was conducted from an ice camp drifting in the Canada Basin of the Arctic Ocean during March-May 1985. Velocity, temperature, and conductivity were sampled at fixed depths, and profiles of these variables from the surface to below 300 m were made. Toward the end of the experiment the camp drifted across a small eddy with a total inferred radius (see section 3.1) of about 13 km. From both the hydrographic data and current meter records, the rotation sense (parity) of the eddy was cyclonic, with an azimuthal velocity maximum near 115 m, decreasing to approximately zero near 30 m and 270 m. Small eddies are a common feature of the Canada Basin, being present in about 25% of the hydrographic and current meter profiles during the extensive Arctic Ice Dynamics Joint Experiment (AIDJEX) program in 1975/1976 [Manley and Hunkins, 1985]. They contain much of the kinetic energy in the depth range 0-250 m, and transport significant amounts of anomalous heat, salt, and nutrients within the Canada Basin. Understanding these eddies is therefore essential to modeling the upper ocean in the Canada Basin. Manley and Hunkins [ 1985] found that approximately 95% of eddies identified during AIDJEX were anticyclonic. Hunkins [1974] documented one cyclonic eddy extending from near the surface to below 200 m from the AIDJEX pilot program in 1972. Manley and Hunkins [1985] found three cyclonic eddies during the main AIDJEX program in 19751976, but two of these were below the Atlantic layer core, i.e, below 500 m, while the third was very weak. D'Asaro

A large number of oceanic and freshwater microstructure observations are analyzed to determine the energetic state of the turbulence. The total available energy and a time scale for dissipating the total energy are estimated. It is found that the time scale for dissipating the total energy in overturns is usually much shorter than the time scale for gravitational collapse found in laboratory studies. This implies that the energy being dissipated in an overturn could not be supplied fast enough to support the dissipation if gravitational collapse were the only source supplying the energy. Two criteria used in Gibson's theory of fossil turbulence for establishing the state of overturns are compared: a length wale (or available potential energy) criterion suggested by Gibson and tested in the laboratory by Stillinger and Gibson's “activity parameter” criterion. It is found that most overturns are “active” according to the available potential-energy criterion, but “fossil” according to the activity p...