Improving protein therapeutic development through cloud-based data integration.

Pharma 4.0 is a digital evolution of the pharmaceutical industry that automates scientists' traditional workflows with the implementation of modern technologies like cloud pipelines, artificial intelligence, robotic platforms, and augmented reality. Lab data capture (LDC) is an essential strategy for initiating Pharma 4.0 that aggregates and harmonizes siloed lab data from analytical instruments, reporting systems, and operational platforms. This publication describes the execution of LDC within a quantitative PCR (qPCR) workflow using the Tetra Data Platform (TDP). We selected this workflow because the qPCR instrument, the ViiA7, generates discrete file-based data that documents execution of individual assays for quantifying residual DNA throughout biologics process development and product profiling. TDP executes LDC through the deployment of file scanning software agents, scanning and ingestion processes, and a cloud-based parsing pipeline that harmonizes source data. Web applications were developed to query, visualize, and interpret harmonized qPCR data for automated experiment data processing and process control charting from the TDP platform. Our implementation of LDC enables analytical researchers to harness FAIR (Findable, Accessible, Interoperable, Reproducible) data practices across the organization and establishes a "compliance-by-code" culture in development labs.

Dissociation between Cervical Mucus and Urinary Hormones during the Postpartum Return of Fertility in Breastfeeding Women.

Identifying the return of fertility with cervical mucus observations is challenging during the postpartum period. Use of urinary measurements of estrogen and progesterone can assist in understanding the return to fertility during this period. The purposes of this study were to describe the postpartum return of fertility by an analysis of total estrogen (TE) and pregnanediol glucuronide (PDG) profiles and to correlate these profiles with cervical mucus observations. Twenty-six participants collected urine samples during the postpartum period and recorded mucus scores. TE and PDG hormones were analyzed and compared with mucus scores. During amenorrhea, mucus reflected TE changes in only 35 percent of women; after amenorrhea, typical mucus patterns were seen in 33 percent of cycles. We concluded that postpartum mucus and hormone profiles are significantly dissociated but that monitoring urinary hormones may assist in identifying the return of fertility. We also identified different hormonal patterns in the return to fertility. The postpartum period is a challenging time for identifying the return of fertility. The purposes of this study were to describe the hormonal patterns during the return of fertility and to correlate these patterns with cervical mucus observations. Twenty-six postpartum women collected urine samples and recorded mucus scores. Urinary estrogen and progesterone hormones were analyzed and compared with mucus scores. Before the return of menses, mucus reflected hormonal changes in only 35 percent women and after first menses in 33 percent of cycles. We found that hormone profiles do not correlate well with mucus observations during the postpartum return of fertility.

Ff-nano, short functionalized nanorods derived from Ff (f1, fd, or M13) filamentous bacteriophage.

F-specific filamentous phage of Escherichia coli (Ff: f1, M13, or fd) are long thin filaments (860 nm × 6 nm). They have been a major workhorse in display technologies and bionanotechnology; however, some applications are limited by the high length-to-diameter ratio of Ff. Furthermore, use of functionalized Ff outside of laboratory containment is in part hampered by the fact that they are genetically modified viruses. We have now developed a system for production and purification of very short functionalized Ff-phage-derived nanorods, named Ff-nano, that are only 50 nm in length. In contrast to standard Ff-derived vectors that replicate in E. coli and contain antibiotic-resistance genes, Ff-nano are protein-DNA complexes that cannot replicate on their own and do not contain any coding sequences. These nanorods show an increased resistance to heating at 70(∘)C in 1% SDS in comparison to the full-length Ff phage of the same coat composition. We demonstrate that functionalized Ff-nano particles are suitable for application as detection particles in sensitive and quantitative "dipstick" lateral flow diagnostic assay for human plasma fibronectin.

In vivo self-assembly of fluorescent protein microparticles displaying specific binding domains.

In this study, fluorescent proteins (FPs) were engineered to self-assemble into protein particles inside recombinant Escherichia coli while mediating the display of various protein functionalities such as maltose binding protein or IgG binding domains of Protein A or G, respectively. Escherichia coli produced functional FP particles of up to 30% of cellular dry weight. The use of respective FP particles displaying certain binding domains in diagnostics and as bioseparation resins was demonstrated by direct comparison to commercial offerings. It was demonstrated that variable extensions (AVTS, FHKP, LAVG, or TS) of the N-terminus of FPs (GFP, YFP, CFP, HcRed) in combination with large C-terminal extensions such as translational fusion of the polyester synthase from Ralstonia eutropha or an aldolase from Escherichia coli led to extensive intracellular self-assembly of strongly fluorescent fusion protein particles of oval shape (0.5×1 µm). The strong fluorescent label of these bioparticles in combination with covalent display of protein functions provides a molecular toolbox for the design of self-assembled microparticles suitable for antibody-capture or ligand binding based diagnostic assays as well as the high affinity purification of target compounds such as antibodies.

Sensitive determination of estriol-16-glucuronide using surface plasmon resonance sensing.

For the quantitative evaluation of low levels of an estriol metabolite of estriol (estriol-16-glucuronide (E3-16G)) in liquid media, we developed a simple and highly sensitive immunoassay using a surface plasmon resonance (SPR) biosensor which did not require any time-consuming sample pretreatment steps. E3-16G was conjugated to ovalbumin (OVA) through an oligoethylene glycol (OEG) linker to form protein conjugates (E3-16G-OEG-OVA), which were then immobilized on a carboxymethyl dextran-coated sensor chip via amine coupling to develop inhibition immunoassays. A limit of detection (LOD) of 76 pg/mL was achieved using a rabbit anti-sheep primary antibody as a binding agent. The detection limit was further improved by using synthesized gold colloids (15 nm) as high mass labels conjugated to the primary antibody. In this Au nanoparticle-enhanced assay, the concentration of E3-16G in aqueous samples could be determined in 7.5 min at a level as low as 14 pg/mL. In addition, the high stability of the E3-16G-OEG-OVA surface gave no obvious drop in antibody-binding capability after more than 1000 binding/regeneration cycles which significantly lowered the research cost.