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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. In this work, a novel high-throughput host cell DNA quantitation assay was developed for rapid screening of complex vaccine drug substance samples. The developed assay utilizes the commercially available, fluorescent-sensitive Picogreen dye within a 96-well plate configuration to allow for a cost effective and rapid analysis. The assay was applied to in-process biopharmaceutical samples with known interferences to the dye, including RNA and protein. An enzymatic digestion pre-treatment was found to overcome these interferences and thus allow this method to be applied to wide-ranging, diverse analyses. In addition, the use of deoxycholate in the digestion treatment allowed for disruption of interactions in a given sample matrix in order to more accurately and selectively quantitate DNA. Critical analytical figures of merit for assay performance, such as precision and spike recovery, were evaluated and successfully demonstrated. This new analytical method can thus be successfully applied to both upstream and downstream process analysis for biologics and vaccines using an innovative and automated high-throughput approach., (© 2022. The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists.)

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. PAT involves the collection of in situ data, thereby providing real time information about the monitored process as well as increasing understanding of that process. Here, three separate PAT tools - optical particle imaging, in situ particle analysis, and Raman spectroscopy - were used to monitor two key steps involved in the formation of a stable emulsion product, emulsification and homogenization, as well as perform a stability assessment. The obtained results provided new insights-particle size decreases during emulsification and homogenization, and molecular changes do not occur during either the emulsification or homogenization steps. Further, the stability assessment indicated that the coarse emulsion product obtained from the emulsification step is stable over the course of 24 hours when mixed. To the best of our knowledge, this is the first report of an analytical methodology for in situ , real time analysis of emulsification and homogenization processes for vaccine adjuvants. Using our proposed analytical methodology, an improved understanding of emulsion-based vaccine adjuvants can now be achieved, ultimately impacting the ability to develop and deliver successful pharmaceuticals.

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. For the first time, total residual protein content in a small molecule API was quantitated using image analysis applied to SDS-PAGE. Herein, a proposed methodology for residual protein detection, quantitation, and size-based speciation is presented, in which an orthogonal technique is offered to traditional analysis methods, such as ELISA. Results indicate that our application of the analytical methodology is able to reliably quantitate both protein standards and the total residual protein present within a final API, with good agreement as compared to traditional ELISA results. Further, speciation of the residual protein within the API provides key information concerning the individual residual proteins present, including their molecular weight, which can lead to improved process development efforts for residual protein rejection and control. This analytical methodology thus offers an alternative tool for easily identifying, quantitating, and speciating residual protein content in the presence of small molecule APIs, with potential for wide applicability across industry for biocatalytic or directed enzyme evolution efforts within process development., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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. New analytical tools to investigate enzyme immobilization are therefore needed. In this work, development, application, and evaluation of an analytical methodology to study enzyme immobilization is presented. Specifically, Raman hyperspectral imaging with principal component analysis, a multivariate method, is demonstrated for the first time to investigate evolved enzymes immobilized onto microporous supports for biocatalysis. Herein we demonstrate the ability to spatially and spectrally resolve evolved pantothenate kinase (PanK) immobilized onto two commercially-available, chemically-diverse porous resins. This analytical methodology is able to chemically distinguish evolved enzyme, resin, and chemical species pertinent to immobilization. As such, a new analytical approach to study immobilized biocatalysts is demonstrated, offering potential wide application for analysis of protein or biomolecule immobilization.

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). NLRP1 and NOD2 are cytosolic innate immune proteins known to generate an immune response to PG. Both possess conserved leucine rich repeat domains (LRR) as proposed sites of molecular recognition, though limited biochemical evidence exists regarding the mechanisms of PG recognition. Here direct biochemical evidence for the association of PG fragments to NOD2 and NLRP1 with nanomolar affinity is shown. The orientations in which the fragments were presented on the SPR surface influenced the strength of PG recognition by both NLRs. This assay displays fundamental differences in binding preferences for PG by innate immune receptors and reveals unique recognition mechanisms between the LRRs. Each receptor uses specific ligand structural features to achieve optimal binding, which will be critical information to manipulate these responses and combat diseases.

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 Bacillus thermoproteolyticus rokko , 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. The non-Arrhenius behavior observed resembled results from previous studies of a thermophilic alcohol dehydrogenase enzyme, which also indicated a biphasic transition at ambient temperatures. To explore the non-Arrhenius behavior of thermolysin, room temperature crystallography was applied to characterize structural changes in a temperature range across the biphasic transition temperature. The alternate conformation of side chain fitting to electron density of a group of residues showed a higher variability in the temperature range from 26 to 29 °C, which indicated a change in conformational sampling that correlated with the non-Arrhenius break point., (© 2020 Author(s).)

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. Previous work had demonstrated that Nod2 interacts with and is stabilized by the molecular chaperone Hsp70. In this work, it is shown using purified protein and in vitro biochemical assays that the critical Nod2 CD mutations (G908R, R702W, and 1007fs) preserve the ability to bind bacterial ligands. A limited proteolysis assay and luciferase reporter assay reveal regions of Hsp70 that are capable of stabilizing Nod2 and rescuing CD mutant activity. A minimal 71-amino acid subset of Hsp70 that stabilizes the CD-associated variants of Nod2 and restores a proper immune response upon activation with MDP was identified. This work suggests that CD-associated Nod2 variants could be stabilized in vivo with a molecular chaperone.

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. Surface plasmon resonance and molecular modeling were used to investigate the interaction of the LRR domain with MDP. A functional and pure LRR domain was obtained from Escherichia coli expression in high yield. The LRR domain binds to MDP with high affinity, with a K D of 212 ± 24 nM. Critical portions of the receptor were determined by mutagenesis of putative binding residues. Fragment analysis of MDP revealed that both the peptide and carbohydrate portion contribute to the binding interaction.

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.

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). NOD2 responds to the presence of bacteria and stimulates the immune response. Mutations to NOD2 promote low diversity and dysbiosis in the microbiome, leading to impaired mucosal barrier function. Current treatments suppress the immune response rather than enhancing the function of this critical protein. New progress toward stabilizing NOD2 signaling through its interactions with chaperone proteins holds potential in the development of novel CD therapeutics., (Copyright © 2016 Elsevier Ltd. All rights reserved.)