Toward complete continuity in antibody biomanufacture: Multi-column continuous chromatography for Protein A capture and mixed mode hydroxyapatite polishing

Monoclonal antibodies (mAbs) are a predominant modality for a broad range of clinical indications including oncology and inflammatory diseases. Increasing manufacturing capacity and decreasing cost per purification campaign are critical factors for making antibody therapies more affordable. Cell culture mAb expression levels have steadily increased over the past ten years with titers of five grams per liter frequently achieved. Higher titers reduce production costs and allow processing of kilogram quantities for clinical trials from single use cell culture vessels. Drawbacks of increased titers include higher levels of aggregates, fragments, variants, and process impurities. These combined titer and impurity burdens further stress downstream processing that already accounts for up to 70% of mAb production cost. Protein A and other batch chromatography methods are the primary contributors to this cost. Multi-column continuous chromatography (MCC), a form of simulated moving bed chromatography (SMBC), is a scalable technology previously demonstrated to improve productivity and lower the cost of Protein A affinity chromatography versus the standard single column batch process. This study incorporates another MCC process using hydroxyapatite for aggregate removal and concurrent depletion of impurities following Protein A purification of mAb. Results indicate that a completely continuous downstream process, including only two chromatographic steps, may be possible to increase efficiency and reduce cost in mAb biomanufacture

Toward complete continuity in antibody biomanufacture: Multi-column continuous chromatography for Protein A capture and mixed mode hydroxyapatite polishing

Monoclonal antibodies (mAbs) are a predominant modality for a broad range of clinical indications including oncology and inflammatory diseases. Increasing manufacturing capacity and decreasing cost per purification campaign are critical factors for making antibody therapies more affordable. Cell culture mAb expression levels have steadily increased over the past ten years with titers of five grams per liter frequently achieved. Higher titers reduce production costs and allow processing of kilogram quantities for clinical trials from single use cell culture vessels. Drawbacks of increased titers include higher levels of aggregates, fragments, variants, and process impurities. These combined titer and impurity burdens further stress downstream processing that already accounts for up to 70% of mAb production cost. Protein A and other batch chromatography methods are the primary contributors to this cost. Multi-column continuous chromatography (MCC), a form of simulated moving bed chromatography (SMBC), is a scalable technology previously demonstrated to improve productivity and lower the cost of Protein A affinity chromatography versus the standard single column batch process. This study incorporates another MCC process using hydroxyapatite for aggregate removal and concurrent depletion of impurities following Protein A purification of mAb. Results indicate that a completely continuous downstream process, including only two chromatographic steps, may be possible to increase efficiency and reduce cost in mAb biomanufacture

Analysis of Behavior and Trafficking of Dendritic Cells within the Brain during Toxoplasmic Encephalitis

Under normal conditions the immune system has limited access to the brain; however, during toxoplasmic encephalitis (TE), large numbers of T cells and APCs accumulate within this site. A combination of real time imaging, transgenic reporter mice, and recombinant parasites allowed a comprehensive analysis of CD11c+ cells during TE. These studies reveal that the CNS CD11c+ cells consist of a mixture of microglia and dendritic cells (DCs) with distinct behavior associated with their ability to interact with parasites or effector T cells. The CNS DCs upregulated several chemokine receptors during TE, but none of these individual receptors tested was required for migration of DCs into the brain. However, this process was pertussis toxin sensitive and dependent on the integrin LFA-1, suggesting that the synergistic effect of signaling through multiple chemokine receptors, possibly leading to changes in the affinity of LFA-1, is involved in the recruitment/retention of DCs to the CNS and thus provides new insights into how the immune system accesses this unique site

The Dual Origin of Stellar Halos

We investigate the formation of the stellar halos of four simulated disk galaxies using high resolution, cosmological SPH + N-Body simulations. These simulations include a self-consistent treatment of all the major physical processes involved in galaxy formation. The simulated galaxies presented here each have a total mass of ~10^12 M_sun, but span a range of merger histories. These simulations allow us to study the competing importance of in-situ star formation (stars formed in the primary galaxy) and accretion of stars from subhalos in the building of stellar halos in a LambdaCDM universe. All four simulated galaxies are surrounded by a stellar halo, whose inner regions (r < 20 kpc) contain both accreted stars, and an in-situ stellar population. The outer regions of the galaxies' halos were assembled through pure accretion and disruption of satellites. Most of the in-situ halo stars formed at high redshift out of smoothly accreted cold gas in the inner 1 kpc of the galaxies' potential wells, possibly as part of their primordial disks. These stars were displaced from their central locations into the halos through a succession of major mergers. We find that the two galaxies with recently quiescent merger histories have a higher fraction of in-situ stars (~20-50%) in their inner halos than the two galaxies with many recent mergers (~5-10% in-situ fraction). Observational studies concentrating on stellar populations in the inner halo of the Milky Way will be the most affected by the presence of in-situ stars with halo kinematics, as we find that their existence in the inner few tens of kpc is a generic feature of galaxy formation.Comment: Version accepted to ApJ. Content is unchanged from previous version, but paper has been restructured for clarit

Myosin-I nomenclature

We suggest that the vertebrate myosin-I field adopt a common nomenclature system based on the names adopted by the Human Genome Organization (HUGO). At present, the myosin-I nomenclature is very confusing; not only are several systems in use, but several different genes have been given the same name. Despite their faults, we believe that the names adopted by the HUGO nomenclature group for genome annotation are the best compromise, and we recommend universal adoption

Experiences of patients with chronic gastrointestinal conditions: in their own words

<p>Abstract</p> <p>Background</p> <p>Irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) are chronic conditions affecting millions of individuals in the United States. The symptoms are well-documented and can be debilitating. How these chronic gastrointestinal (GI) conditions impact the daily lives of those afflicted is not well documented, especially from a patient's perspective.</p> <p>Methods</p> <p>Here we describe data from a series of 22 focus groups held at three different academic medical centers with individuals suffering from chronic GI conditions. All focus groups were audio recorded and transcribed. Two research team members independently analyzed transcripts from each focus group following an agreed upon coding scheme.</p> <p>Results</p> <p>One-hundred-thirty-six individuals participated in our study, all with a chronic GI related condition. They candidly discussed three broad themes that characterize their daily lives: identification of disease and personal identity, medications and therapeutics, and daily adaptations. These all tie to our participants trying to deal with symptoms on a daily basis. We find that a recurrent topic underlying these themes is the dichotomy of experiencing uncertainty and striving for control.</p> <p>Conclusions</p> <p>Study participants' open dialogue and exchange of experiences living with a chronic GI condition provide insight into how these conditions shape day-to-day activities. Our findings provide fertile ground for discussions about how clinicians might best facilitate, acknowledge, and elicit patients' stories in routine care to better address their experience of illness.</p

Community-developed checklists for publishing images and image analysis

Images document scientific discoveries and are prevalent in modern biomedical research. Microscopy imaging in particular is currently undergoing rapid technological advancements. However for scientists wishing to publish the obtained images and image analyses results, there are to date no unified guidelines. Consequently, microscopy images and image data in publications may be unclear or difficult to interpret. Here we present community-developed checklists for preparing light microscopy images and image analysis for publications. These checklists offer authors, readers, and publishers key recommendations for image formatting and annotation, color selection, data availability, and for reporting image analysis workflows. The goal of our guidelines is to increase the clarity and reproducibility of image figures and thereby heighten the quality of microscopy data is in publications.Comment: 28 pages, 8 Figures, 3 Supplmentary Figures, Manuscript, Essential recommendations for publication of microscopy image dat

Evolution of the mammalian lysozyme gene family

<p>Abstract</p> <p>Background</p> <p>Lysozyme <it>c </it>(chicken-type lysozyme) has an important role in host defense, and has been extensively studied as a model in molecular biology, enzymology, protein chemistry, and crystallography. Traditionally, lysozyme <it>c </it>has been considered to be part of a small family that includes genes for two other proteins, lactalbumin, which is found only in mammals, and calcium-binding lysozyme, which is found in only a few species of birds and mammals. More recently, additional testes-expressed members of this family have been identified in human and mouse, suggesting that the mammalian lysozyme gene family is larger than previously known.</p> <p>Results</p> <p>Here we characterize the extent and diversity of the lysozyme gene family in the genomes of phylogenetically diverse mammals, and show that this family contains at least eight different genes that likely duplicated prior to the diversification of extant mammals. These duplicated genes have largely been maintained, both in intron-exon structure and in genomic context, throughout mammalian evolution.</p> <p>Conclusions</p> <p>The mammalian lysozyme gene family is much larger than previously appreciated and consists of at least eight distinct genes scattered around the genome. Since the lysozyme <it>c </it>and lactalbumin proteins have acquired very different functions during evolution, it is likely that many of the other members of the lysozyme-like family will also have diverse and unexpected biological properties.</p