Improved Stability and Manufacturability of Nucleocapsid Antigens for SARS-CoV2 Diagnostics through Protein Engineering.

The COVID-19 pandemic has had a significant impact on human health management. A rapid diagnosis of SARS-CoV2 at the point-of-care (POC) is critical to prevent disease spread. As a POC device for remote settings, a LFIA should not require cold-chain maintenance and should be kept at normal temperatures. Antigen stability can be enhanced by addressing instability issues when dealing with fragile components, such as proteinaceous capture antigens. This study used immunologically guided protein engineering to enhance the capture nucleocapsid (NP) antigen stability of SARS-CoV2. A search of the IEDB database revealed that antibodies detecting epitopes are almost uniformly distributed over NP1-419. In contrast, N-terminal stretches of NP1-419 are theoretically more unstable than C-terminal stretches. We identified NP250-365 as a NP stretch with a low instability index and B-cell epitopes. Apart from NP1-419, two other variants (NP121-419 and NP250-365) were cloned, expressed, and purified. The degradation pattern of the proteins was observed on SDS-PAGE after three days of stability studies at -20 °C, 4 °C, and 37 °C. NP1-419 was the most degraded while NP250-365 exhibited the least degradation. Also, NP1-419, NP250-365, and NP121-419 reacted with purified antibodies from COVID-19 patient serum. Our results suggest that NP250-365 may be used as a stable capture antigen in LFIA devices to detect COVID-19.

Profiling Enzyme Activity of l-Asparaginase II by NMR-Based Methyl Fingerprinting at Natural Abundance.

l-asparaginase II (MW 135 kDa) from E. coli is an FDA-approved protein drug used for the treatment of childhood leukemia. Despite its long history as a chemotherapeutic, the structural basis of enzyme action, in solution, remains widely contested. In this work, methyl-based 2D [1H-13C]-heteronuclear single-quantum correlation (HSQC) NMR, at natural abundance, has been used to profile the enzymatic activity of the commercially available enzyme drug. The [1H-13C]-HSQC NMR spectra of the protein reveal the role of a flexible loop segment in the activity of the enzyme, in solution. Addition of asparagine to the protein results in distinct conformational changes of the loop that could be signatures of intermediates formed in the catalytic reaction. To this end, an isothermal titration calorimetry (ITC)-based assay has been developed to measure the enzymatic reaction enthalpy, as a marker for its activity. Combining both ITC and NMR, it was shown that the disruption of the protein conformation can result in the loss of function. The scope, robustness, and validity of the loop fingerprints in relation to enzyme activity have been tested under different solution conditions. Overall, our results indicate that 2D NMR can be used reliably to gauge the structure-function of this enzyme, bypassing the need to label the protein. Such natural abundant NMR methods can be potentially extended to probe the structure-function aspects of high-molecular-weight protein therapeutics (glycosylated protein drugs, enzymes, therapeutic monoclonal antibodies, antibody-drug conjugates, and Fc-fusion proteins), where (a) flexible loops are required for their function and (b) isotope labeling may not be straightforward.

Multi-Level High-Throughput Screening for Discovery of Ligands That Inhibit Insulin Aggregation.

We have developed a multi-level virtual screening protocol to identify lead molecules from the FDA inactives database that can inhibit insulin aggregation. The method is based on the presence of structural and interaction specificity in non-native aggregation pathway protein-protein interactions. Some key challenges specific to the present problem, when compared with native protein association, include structural heterogeneity of the protein species involved, multiple association pathways, and relatively higher probability of conformational rearrangement of the association complex. In this multi-step method, the inactives database was first screened using the dominant pharmacophore features of previously identified molecules shown to significantly inhibit insulin aggregation nucleation by binding to its aggregation-prone conformers. We then performed ensemble docking of several low-energy ligand conformations on these aggregation-prone conformers followed by molecular dynamics simulations and binding affinity calculations on a subset of docked complexes to identify a final set of five potential lead molecules to inhibit insulin aggregation nucleation. Their effect on aggregation inhibition was extensively investigated by incubating insulin under aggregation-prone aqueous buffer conditions (low pH, high temperature). Aggregation kinetics were characterized using size exclusion chromatography and Thioflavin T fluorescence assay, and the secondary structure was determined using circular dichroism spectroscopy. Riboflavin provided the best aggregation inhibition, with 85% native monomer retention after 48 h incubation under aggregation-prone conditions, whereas the no-ligand formulation showed complete monomer loss after 36 h. Further, insulin incubated with two of the screened inactives (aspartame, riboflavin) had the characteristic α-helical dip in CD spectra, while the no-ligand formulation showed a change to ß-sheet rich conformations.

Equilibrium Ensembles for Insulin Folding from Bias-Exchange Metadynamics.

Earliest events in the aggregation process, such as single molecule reconfiguration, are extremely important and the most difficult to characterize in experiments. To this end, we have used well-tempered bias exchange metadynamics simulations to determine the equilibrium ensembles of an insulin molecule under amyloidogenic conditions of low pH and high temperature. A bin-based clustering method that uses statistics accumulated in bias exchange metadynamics trajectories was employed to construct a detailed thermodynamic and kinetic model of insulin folding. The highest lifetime, lowest free-energy ensemble identified consisted of native conformations adopted by a folded insulin monomer in solution, namely, the R-, the Rf-, and the T-states of insulin. The lowest free-energy structure had a root mean square deviation of only 0.15 nm from native x-ray structure. The second longest-lived metastable state was an unfolded, compact monomer with little similarity to the native structure. We have identified three additional long-lived, metastable states from the bin-based model. We then carried out an exhaustive structural characterization of metastable states on the basis of tertiary contact maps and per-residue accessible surface areas. We have also determined the lowest free-energy path between two longest-lived metastable states and confirm earlier findings of non-two-state folding for insulin through a folding intermediate. The ensemble containing the monomeric intermediate retained 58% of native hydrophobic contacts, however, accompanied by a complete loss of native secondary structure. We have discussed the relative importance of nativelike versus nonnative tertiary contacts for the folding transition. We also provide a simple measure to determine the importance of an individual residue for folding transition. Finally, we have compared and contrasted this intermediate with experimental data obtained in spectroscopic, crystallographic, and calorimetric measurements during early stages of insulin aggregation. We have also determined stability of monomeric insulin by incubation at a very low concentration to isolate protein-protein interaction effects.

Analysis for residual host cell proteins and DNA in process streams of a recombinant protein product expressed in Escherichia coli cells.

Analyses of crude samples from biotechnology processes are often required in order to demonstrate that residual host cell impurities are reduced or eliminated during purification. In later stages of development, as the processes are further developed and finalized, there is a tremendous volume of testing required to confirm the absence of residual host cell proteins (HCP) and DNA. Analytical tests for these components are very challenging since (1). they may be present at levels that span a million-fold range, requiring substantial dilutions; (2). are not a single component, often existing as fragments and a variety of structures; (3). require high sensitivity for final steps in process; and (4). are present in very complex matrices including other impurities, the product, buffers, salts and solvents. Due to the complex matrices and the variety of potential analytes, the methods of analysis are not truly quantitative for all species. Although these limitations are well known, the assays are still very much in demand since they are required for approval of new products. Methods for final products, described elsewhere, focus on approaches to achieve regulatory requirements. The study described herein will describe the technical rationale for measuring the clearance of HCP and DNA in the entire bioprocessing to purification from an Escherichia coli-derived expression system. Three analytical assays, namely, reversed-phase high-performance liquid chromatography (RP-HPLC), enzyme-linked immunosorbent assay (ELISA), and Threshold Total DNA Assay, were utilized to quantify the protein product, HCP and DNA, respectively. Product quantification is often required for yield estimation and is useful since DNA and HCP results are best expressed as a ratio to product for calculation of relative purification factors. The recombinant E. coli were grown to express the protein of interest as insoluble inclusion bodies (IB) within the cells. The IB were isolated by repeated homogenization and centrifugation and the inclusion body slurry (IBS) was solubilized with urea. After refolding the product, the solution was loaded on several commonly used ion exchangers (CM, SP, DEAE, and Q). Product was eluted in a salt gradient mode and fractions were collected and analyzed for product, HCP and DNA. The IBS used for this study contained about 15 mg/ml product, 38 mg/ml HCP and 1.1 mg/ml DNA. Thus, the relative amounts of HCP and DNA in the IBS was excessive, and about 10(3) times greater than typical (because the cells and IB were not processed with the normal number of washing steps during isolation). This was of interest since similar samples may be encountered when working with non-inclusion body systems, such as periplasmic expressions, or in cases where the upstream unit operations under-perform in IB cleaning. The study described herein describes the development of three robust methods that provide the essential process data needed. These findings are of general interest to other projects since applications of similar analytical technology may be used as a tool to develop processes, evaluate clearance of impurities, and produce a suitable product.