Overcoming Biopharmaceutical Interferents for Quantitation of Host Cell DNA Using an Automated, High-Throughput Methodology.

The rapid development of biologics and vaccines in response to the current pandemic has highlighted the need for robust platform assays to characterize diverse biopharmaceuticals. A critical aspect of biopharmaceutical development is achieving a highly pure product, especially with respect to residual host cell material. Specifically, two important host cell impurities of focus within biopharmaceuticals are residual DNA and protein.

real time monitoring of emulsification and homogenization processes for vaccine adjuvants.

Adjuvants are commonly employed to enhance the efficacy of a vaccine and thereby increase the resulting immune response in a patient. The activity and effectiveness of emulsion-based adjuvants has been heavily studied throughout pharmaceuticals; however, there exists a lack in research which monitors the formation of a stable emulsion in real time. Process analytical technology (PAT) provides a solution to meet this need.

Quantitation and speciation of residual protein within active pharmaceutical ingredients using image analysis with SDS-PAGE.

Recent advances in biocatalysis and directed enzyme evolution has led to a variety of enzymatically-driven, elegant processes for active pharmaceutical ingredient (API) production. For biocatalytic processes, quantitation of any residual protein within a given API is of great importance to ensure process robustness and quality, pure pharmaceutical products. Typical analytical methods for analyzing residual enzymes within an API, such as enzyme-linked immunosorbent assays (ELISA), colorimetric assays, and liquid chromatographic techniques, are limited for determining only the concentration of known proteins and require harsh solvents with high API levels for analysis.

Raman hyperspectral imaging with multivariate analysis for investigating enzyme immobilization.

Directed enzyme evolution has led to significant application of biocatalysis for improved chemical transformations throughout the scientific and industrial communities. Biocatalytic reactions utilizing evolved enzymes immobilized within microporous supports have realized unique advantages, including notably higher enzyme stability, higher enzyme load, enzyme reusability, and efficient product-enzyme separation. To date, limited analytical methodology is available to discern the spatial and chemical distribution of immobilized enzymes, in which techniques for surface visualization, enzyme stability, or activity are instead employed.

Differential Peptidoglycan Recognition Assay Using Varied Surface Presentations.

Bacterial peptidoglycan (PG) is recognized by the human innate immune system to generate an appropriate response. To gain an appreciation of how this essential polymer is sensed, a surface plasmon resonance (SPR) assay using varied PG surface presentation was developed. PG derivatives were synthesized and immobilized on the surface at different positions on the molecule to assess effects of ligand orientation on the binding affinities of NOD-like receptors (NLRs).

Conformational sampling and kinetics changes across a non-Arrhenius break point in the enzyme thermolysin.

Numerous studies have suggested a significant role that protein dynamics play in optimizing enzyme catalysis, and changes in conformational sampling offer a window to explore this role. Thermolysin from , which is a heat-stable zinc metalloproteinase, serves here as a model system to study changes of protein function and conformational sampling across a temperature range of 16-36 °C. The temperature dependence of kinetics of thermolysin showed a biphasic transition at 26 °C that points to potential conformational and dynamic differences across this temperature.

Crohn's Disease Variants of Nod2 Are Stabilized by the Critical Contact Region of Hsp70.

Nod2 is a cytosolic, innate immune receptor responsible for binding to bacterial cell wall fragments such as muramyl dipeptide (MDP). Upon binding, subsequent downstream activation of the NF-κB pathway leads to an immune response. Nod2 mutations are correlated with an increased susceptibility to Crohn's disease (CD) and ultimately result in a misregulated immune response.

Molecular Recognition of Muramyl Dipeptide Occurs in the Leucine-rich Repeat Domain of Nod2.

Genetic mutations in the innate immune receptor nucleotide-binding oligomerization domain-containing 2 (Nod2) have demonstrated increased susceptibility to Crohn's disease, an inflammatory bowel disease that is hypothesized to be accompanied by changes in the gut microbiota. Nod2 responds to the presence of bacteria, specifically a fragment of the bacterial cell wall, muramyl dipeptide (MDP). The proposed site of this interaction is the leucine-rich repeat (LRR) domain.

Redefining the Defensive Line: Critical Components of the Innate Immune System.

The human body harbors over a trillion microorganisms; the innate immune system is charged with a tremendous task: to recognize "the needle in the haystack" or, in other words, to sense the pathogen in this milieu. In this viewpoint, three recent discoveries in the field of innate immunity are discussed, highlighting how in each case multiple disciplines worked together to expand the elements of the innate immune system.

The effect of NOD2 on the microbiota in Crohn's disease.

Recent advancements toward the treatment of Crohn's disease (CD) indicate great promise for long-term remission. CD patients suffer from a complex host of dysregulated interactions between their innate immune system and microbiome. The most predominant link to the onset of CD is a genetic mutation in the innate immune receptor nucleotide-binding oligomerization domain-containing 2 (NOD2).