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Distribution of Protein Content and Number of Aggregates in Monoclonal Antibody Formulation After Large-Scale Freezing
No full textCryoconcentration of an in-house IgG1 and number of aggregates in a formulation containing trehalose were determined in dependence on freezing protocol and volume. Morphology changes of ice crystals depending on cooling rates were captured by optical cryomicroscopy (OCM) images. UV-Vis and affinity chromatography (ALC) was used to determine protein content and size-exclusion chromatography (SEC) for detection of aggregates. Cooling to − 80°C rather than − 20°C is beneficial in avoiding hot spots of high protein concentration. An upscaling of 250 ml to 2 L bottles results in an up to fourfold increase of macroscopic cryoconcentration. There is no direct correlation between number of aggregates and macroscopic cryoconcentration. Aggregate formation of that specific mAb is not caused by macroscopic cryoconcentration but can be directly linked to microscopic cryoconcentration in between the ice dendrites. Slower cooling with set-point and storage temperatures below Tg’ has proven to be advantageous for the prevention of aggregate formation. We reveal that the subcooling prior to freezing plays a key role in avoiding aggregates. The lower the solution is supercooled the more likely aggregates form. As a consequence, we suggest controlled initiation of the freezing process to avoid large supercooling
Benchmarking network algorithms for contextualizing genes of interest
No full textComputational approaches have shown promise in contextualizing genes of interest with known molecular interactions. In this work, we evaluate seventeen previously published algorithms based on characteristics of their output and their performance in three tasks: cross validation, prediction of drug targets, and behavior with random input. Our work highlights strengths and weaknesses of each algorithm and results in a recommendation of algorithms best suited for performing different tasks
A Genome-wide CRISPR Screen Identifies ZCCHC14 as a Host Factor Required for Hepatitis B Surface Antigen Production
No full textA hallmark of chronic hepatitis B (CHB) virus infection is the presence of high circulating levels of non-infectious small lipid HBV surface antigen (HBsAg) vesicles. Although rare, sustained HBsAg loss is the idealized endpoint of any CHB therapy. A small molecule, RG7834, has been previously reported to inhibit HBsAg expression by targeting terminal nucleotidyltransferase proteins 4A and 4B (TENT4A and TENT4B). In this study, we describe a genome-wide CRISPR screen to identify other potential host factors required for HBsAg expression and to gain further insights into the mechanism of RG7834. We report more than 60 genes involved in regulating HBsAg and identify additional factors involved in RG7834 activity, including a zinc finger CCHC-type containing 14 (ZCCHC14) protein. We show that ZCCHC14, together with TENT4A/B, stabilizes HBsAg expression through HBV RNA tailing, providing a potential new therapeutic target to achieve functional cure in CHB patients.
Hyrina et al. employ a non-biased functional CRISPR screening approach to identify host factors regulating HBsAg expression as well as those targeted by RG7834, a HBsAg inhibitor. The screen highlighted over 60 genes and identified a mechanism by which ZCCHC14, together with TENT4A/B, stabilizes HBsAg expression through HBV RNA tailing
Team Coaching through CDAI and the GLP
No full textThis chapter illustrates how Collaborative Developmental Action Inquiry (CDAI) theory, method, and practice can be used to simultaneously assess and transform leaders, teams, and organizations, through a non-formulaic coaching and consulting process that becomes increasingly self-transforming and collaborative as it evolves. The chapter provides two mini-cases illustrating how CDAI and Global Leadership Profile (GLP) can be used in team coaching contexts to shape coaching interventions on the ground and support the growth of individuals and teams toward more transforming/collaborative ways of operating. The first case was written by Peter Hill during his period of supervised debriefings en route to GLP certification. The second case was written by Nancy Wallis based on her experience coaching a technically brilliant but irascible senior executive toward becoming a more effective team leader and coach of his manufacturing quality team in a global biopharmaceutical firm
Innovations in addressing pediatric diarrhea in low resource settings
No full textDiarrhea has long been recognized as an important cause of mortality during childhood. In parallel with ensuring access to proven care practices is the imperative to apply modern advances in medicine, science, and technology to accelerate progress against diarrheal disease, particularly in developing countries where the burden of avoidable harm is the greatest. In order to highlight achievements and identify outstanding areas of need, we reviewed the landscape of recent innovations that have significance for the study and clinical management of pediatric diarrhea in low resource settings
Reactivity of Carbenes in the Aqueous Micelles Containing Palladium Nanoparticles
No full textArguably, water, if appropriately used, is the
most sustainable and green solvent for conducting organic synthesis.
Wrongly perceived arguments on the difficulties in water
recycling and generation of more waste, when it is used as a
solvent in synthesis so far posed many hinderances toward its
rampant usage in organic synthesis. Although according to the
Pfizer’s solvent selection guide, water is the most preferred solvent,
still it did not gain the popularity that it deserves. It is an
ideal solvent for chemist safety, process safety, and environmental
safety. Contributions from Kobayashi, Lipshutz, Uozumi,
and our group set the foundation to explore chemistry in
water for highly selective reaction pathways, especially for industrial
applications. Very recently, our group developed an environmentally
benign proline-based amphiphile PS-750-M that
when dissolved in water, instantaneously form nanomicelles
with less-polar interior, unlike other well established amphiphiles.
The rationale behind the design of PS-750-M is the
inclusion of tert-amide functionality inside the micelle to mimic
DMF, DMAc, NMP, and other toxic polar-aprotic solvents
(Figure 1). These toxic solvents have a common tert-amide
group which imparts polarity to these solvent as well as acts as
a residue to participate in secondary interactions in some catalytic
pathways. PS-750-M has been explored on variety of
transformations including those which traditionally require
toxic polar-aprotic solvents. The transformation such as monofluorination
of indoles, which can not be cleanly achieved in
any organic solvent is achieved in PS-750-M. To date, very less
knowledge is available on the behavior of reaction intermediates
in the micellar media. For example, does carbene or carbenoid
exists in the micelle; whether it reacts with micelle or not;
does it impact micellization; can reactions involving carbene intermediates
be successfully achieved in water while leveraging
the positive features of micellar catalysis
Quantitative Systems Pharmacology models as a key to translational medicine
No full textRecent advances in experimentation and computation have allowed us to build multiscale models of unprecedented scale. Quantitative Systems Pharmacology (QSP) models are multiscale models that link protein-interaction or drug-interaction kinetics to cellular response in the context of animal or human (disease) physiology. One of the areas where QSP models hold the most promise is in translating preclinical science into the clinic. In the following, I describe recent examples of multiscale models such as bacterial whole-cell models, multiscale tumor growth inhibition models and human disease models. Challenges of multiscale models and emerging solutions will be discussed
Modular synthesis and post-modification of novel bifunctional dendrons
No full textHerein, we report the design and synthesis of two novel bifunctional dendrons bearing multiple amine termini at the periphery and an azide at the focal point. Copper-catalyzed alkyne-azide cycloaddition enabled modular dendritic scaffold assembly resulting in a first generation dendron carrying six amines and a second generation dendron carrying eighteen amines. Peripheral amines were labeled with multiple copies of a metal isotope, whereas the azide functionality at the focal point was employed in conjugation to a single anti-human CD4 antibody. We demonstrated that the highly monomeric first generation dendron-antibody conjugate selectively detected CD4+ T cells in PMBCs culture
Ligand-Based Fluorine NMR Screening: Principles and Applications in Drug Discovery Projects
No full textLigand-based fluorine NMR screening has gained popularity in drug discovery projects during the past decade and has become a powerful methodology to produce high quality hits. Its high sensitivity to protein binding makes it particularly suitable for fragment screening, allowing detection and binding strength measurement of very weak affinity ligands. The screening can be performed in direct or competition format, and its versatility allows application to complex biological and chemical systems. As the potential of the methodology has now been recognized and successfully demonstrated in several relevant medicinal chemistry projects, it is now an appropriate time to report the learned lessons and point the way to the future. In this Perspective the principles of the methodology along with several applications to pharmaceutical projects are
presented