Modulation of half antibody and aggregate formation in a CHO cell line expressing a bispecific antibody

Therapeutic bispecific antibodies are formed by assembly of multichain polypeptides. In general, a bispecific antibody has two different light chains and two heavy chains that fold and correctly pair via a diversity of engineered interchain interactions (e.g., orthogonal interface, domain crossover, charged mutations, sterically complementary mutations [1-3]). As a consequence of these complex mechanisms that mediate chain assembly, product-related impurities (e.g., half antibodies, homodimers, mispaired light chain species) can be prevalent when expressing bispecific antibodies in cell culture, requiring its removal during subsequent purification. In this study, we investigated the modulation of impurity levels in a stable CHO cell line X expressing a bispecific antibody formed by light chains LC1 and LC2 and heavy chains HC1 and HC2. In particular, this cell line responded to cell culture temperature by decreasing half antibody formation from ~14% to less than 3% when temperature changed from 36°C to 32.5°C. However, the decrease in half antibody also correlated with increased protein aggregates from ~4% to ~10%. We established that half antibody and aggregate formation correlated to intracellular events and not to extracellular degradation mechanism (studies included Western blots of cell lysates and extended supernatant incubation). Analytical characterization showed that protein A-purified pools from cell line X cultured at lower temperatures were enriched in LC1-contaning species, whereas pools from cultures at 36°C were enriched in LC2-containing species. When comparing the LC1 to LC2 ratio in antibodies secreted by cell line X to the ratio in another different 30 cell lines expressing the same bispecific antibody, it revealed a pattern with half antibody formation only present in ratios lower than one, and with enhanced aggregation in ratios larger than one. These results suggested the imbalance of expressed light chains led to one of the two main impurities being preferentially formed. Further studies for cell line X showed that cell culture temperature also modulated mRNA levels of the four expressed chains, which possibly led to misassembled species that contributed to either increased half antibody levels at 36°C (enriched with LC2-containing species), or increased aggregate formation at 32.5°C (enriched with LC1-containing species). Overall, we identified culture conditions that could alter the overall process yield by adjusting impurity amounts and types and consequently, the impurity separation in subsequent purification steps such as cation exchange chromatography. References Lewis SM et al. Nature Biotechnology (2014), 32:191 Gunasekaran K et al. The Journal of Biological Chemistry (2010), 25:19637 Merchant AM et al. Nature Biotechnology (1998), 16:67

Excitation of spin waves by a current-driven magnetic nanocontact in a perpendicularly magnetized waveguide

It is demonstrated both analytically and numerically that the properties of spin wave modes excited by a current-driven nanocontact of length $L$ in a quasi-one-dimensional magnetic waveguide magnetized by a perpendicular bias magnetic field ${H}_{e}$ are qualitatively different from the properties of spin waves excited by a similar nanocontact in a two-dimensional unrestricted magnetic film (``free layer''). In particular, there is an optimum nanocontact length ${L}_{\mathrm{opt}}$ corresponding to the minimum critical current of the spin wave excitation. This optimum length is determined by the magnitude of ${H}_{e}$, the exchange length, and the Gilbert dissipation constant of the waveguide material. Also, for $Ll{L}_{\mathrm{opt}}$ the wavelength \ensuremath{\lambda} (and the wave number $k$) of the excited spin wave can be controlled by the variation of ${H}_{e}$ (\ensuremath{\lambda} decreases with the increase of ${H}_{e}$), while for $Lg{L}_{\mathrm{opt}}$ the wave number $k$ is fully determined by the contact length $L$ (k\ensuremath{\sim}1/L), similar to the case of an unrestricted two-dimensional free layer

Metabolic control and determinants among HIV- infected Type 2 diabetes mellitus patients attending a tertiary clinic in Botswana

Purpose: We primarily aimed at determining the prevalence of metabolic syndrome and abnormal individual metabolic control variables in HIV-infected participants as compared to HIV-uninfected participants given current concerns. Our secondary objective was to determine the predictors of metabolic syndrome and individual metabolic control variables among the study participants to guide future management. Patients and methods: A descriptive, case-matched cross sectional study for four months from 15th June 2019 to 15th October 2019 at Block 6 Diabetes Reference clinic in Gaborone, Botswana. We compared the proportions of metabolic syndrome and individual metabolic control variables based on gender and HIV status by means of bivariate analysis (Chi-squared test or Fisher’s exact test) to determine factors associated with metabolic control. A p-value of less than 0.05 was considered statistically significant. Results: Overall, 86% of the study participants were found to have metabolic syndrome by International Diabetes Federation (IDF) criteria with 79.8% among HIV-infected and 89.1% among HIV-negative participants (p-value = 0.018). Older age was significantly associated with metabolic syndrome (p-value = 0.008). Female gender was significantly associated with metabolic syndrome as compared to male gender (P-value < 0.001), and with a statistically significant higher proportion of low HDL-C compared to males (P-value < 0.001). Female participants were significantly more likely to be obese as compared to males (P-value < 0.001). High trigerylcerides were more common in HIV-infected compared to HIV-negative participants (P-value = 0.004). HIV-negative participants were more likely to be obese as compared to HIV-infected participants (P-value = 0.003). Conclusion: Metabolic syndrome is an appreciable problem in this tertiary clinic in Botswana for both HIV-infected and HIV-negative participants. Future prospective studies are warranted in our setting and similar sub-Saharan settings to enhance understanding of the role played by HAART in causing the metabolic syndrome, and the implications for future patient management

Global timber investments, 2005 to 2017

We estimated timber investment returns for 22 countries and 54 species/management regimes in 2017, for a range of global timber plantation species and countries at the stand level, using capital budgeting criteria, without land costs, at a real discount rate of 8%. Returns were estimated for the principal plantation countries in the Americas-Brazil, Argentina, Uruguay, Chile, Colombia, Venezuela, Paraguay, Mexico, and the United States-as well as New Zealand, Australia, South Africa, China, Vietnam, Laos, Spain, Finland, Poland, Scotland, and France. South American plantation growth rates and their concomitant returns were generally greater, at more than 12% Internal Rates of Return (IRRs), as were those in China, Vietnam, and Laos. These IRRs were followed by those for plantations in southern hemisphere countries of Australia and New Zealand and in Mexico, with IRRs around 8%. Temperate forest plantations in the U.S. and Europe returned less, from 4% to 8%, but those countries have less financial risk, better timber markets, and more infrastructure. Returns to most planted species in all countries except Asia have decreased from 2005 to 2017. If land costs were included in calculating the overall timberland investment returns, the IRRs would decrease from 3 percentage points less for loblolly pine in the U.S. South to 8 percentage points less for eucalypts in Brazil.Peer reviewe

Gradual polyploid genome evolution revealed by pan-genomic analysis of Brachypodium hybridum and its diploid progenitors

Our understanding of polyploid genome evolution is constrained because we cannot know the exact founders of a&nbsp;particular polyploid. To differentiate between founder effects and post polyploidization evolution, we use a pan-genomic approach to study the allotetraploid Brachypodium hybridum and its diploid progenitors. Comparative analysis suggests that most B. hybridum whole gene presence/absence variation is part of the standing variation in its diploid progenitors. Analysis of nuclear single nucleotide variants, plastomes and k-mers associated with retrotransposons reveals two independent origins for B. hybridum, ~1.4 and ~0.14 million years ago. Examination of gene expression in the younger B. hybridum lineage reveals no bias in overall subgenome expression. Our results are consistent with a gradual accumulation of genomic changes after polyploidization and a lack of subgenome expression dominance. Significantly, if we did not use a pan-genomic approach, we would grossly overestimate the number of genomic changes attributable to post polyploidization evolution

Energy Estimation of Cosmic Rays with the Engineering Radio Array of the Pierre Auger Observatory

The Auger Engineering Radio Array (AERA) is part of the Pierre Auger Observatory and is used to detect the radio emission of cosmic-ray air showers. These observations are compared to the data of the surface detector stations of the Observatory, which provide well-calibrated information on the cosmic-ray energies and arrival directions. The response of the radio stations in the 30 to 80 MHz regime has been thoroughly calibrated to enable the reconstruction of the incoming electric field. For the latter, the energy deposit per area is determined from the radio pulses at each observer position and is interpolated using a two-dimensional function that takes into account signal asymmetries due to interference between the geomagnetic and charge-excess emission components. The spatial integral over the signal distribution gives a direct measurement of the energy transferred from the primary cosmic ray into radio emission in the AERA frequency range. We measure 15.8 MeV of radiation energy for a 1 EeV air shower arriving perpendicularly to the geomagnetic field. This radiation energy -- corrected for geometrical effects -- is used as a cosmic-ray energy estimator. Performing an absolute energy calibration against the surface-detector information, we observe that this radio-energy estimator scales quadratically with the cosmic-ray energy as expected for coherent emission. We find an energy resolution of the radio reconstruction of 22% for the data set and 17% for a high-quality subset containing only events with at least five radio stations with signal.Comment: Replaced with published version. Added journal reference and DO

Measurement of the Radiation Energy in the Radio Signal of Extensive Air Showers as a Universal Estimator of Cosmic-Ray Energy

We measure the energy emitted by extensive air showers in the form of radio emission in the frequency range from 30 to 80 MHz. Exploiting the accurate energy scale of the Pierre Auger Observatory, we obtain a radiation energy of 15.8 \pm 0.7 (stat) \pm 6.7 (sys) MeV for cosmic rays with an energy of 1 EeV arriving perpendicularly to a geomagnetic field of 0.24 G, scaling quadratically with the cosmic-ray energy. A comparison with predictions from state-of-the-art first-principle calculations shows agreement with our measurement. The radiation energy provides direct access to the calorimetric energy in the electromagnetic cascade of extensive air showers. Comparison with our result thus allows the direct calibration of any cosmic-ray radio detector against the well-established energy scale of the Pierre Auger Observatory.Comment: Replaced with published version. Added journal reference and DOI. Supplemental material in the ancillary file

Measurement of the cosmic ray spectrum above 4×10184{\times}10^{18} eV using inclined events detected with the Pierre Auger Observatory

A measurement of the cosmic-ray spectrum for energies exceeding $4{\times}10^{18}$ eV is presented, which is based on the analysis of showers with zenith angles greater than $60^{\circ}$ detected with the Pierre Auger Observatory between 1 January 2004 and 31 December 2013. The measured spectrum confirms a flux suppression at the highest energies. Above $5.3{\times}10^{18}$ eV, the "ankle", the flux can be described by a power law $E^{-\gamma}$ with index $\gamma=2.70 \pm 0.02 \,\text{(stat)} \pm 0.1\,\text{(sys)}$ followed by a smooth suppression region. For the energy ($E_\text{s}$) at which the spectral flux has fallen to one-half of its extrapolated value in the absence of suppression, we find $E_\text{s}=(5.12\pm0.25\,\text{(stat)}^{+1.0}_{-1.2}\,\text{(sys)}){\times}10^{19}$ eV.Comment: Replaced with published version. Added journal reference and DO