The efficient synthesis and purification of 2'3'- cGAMP from Escherichia coli.

Agonists of the stimulator of interferon genes (STING) pathway are being explored as potential immunotherapeutics for the treatment of cancer and as vaccine adjuvants for infectious diseases. Although chemical synthesis of 2'3' - cyclic Guanosine Monophosphate-Adenosine Monophosphate (cGAMP) is commercially feasible, the process results in low yields and utilizes organic solvents. To pursue an efficient and environmentally friendly process for the production of cGAMP, we focused on the recombinant production of cGAMP via a whole-cell biocatalysis platform utilizing the murine cyclic Guanosine monophosphate-Adenosine monophosphate synthase (mcGAS). In E. coli BL21(DE3) cells, recombinant expression of mcGAS, a DNA-dependent enzyme, led to the secretion of cGAMP to the supernatants. By evaluating the: (1) media composition, (2) supplementation of divalent cations, (3) temperature of protein expression, and (4) amino acid substitutions pertaining to DNA binding; we showed that the maximum yield of cGAMP in the supernatants was improved by 30% from 146 mg/L to 186 ± 7 mg/mL under optimized conditions. To simplify the downstream processing, we developed and validated a single-step purification process for cGAMP using anion exchange chromatography. The method does not require protein affinity chromatography and it achieved a yield of 60 ± 2 mg/L cGAMP, with <20 EU/mL (<0.3 EU/µg) of endotoxin. Unlike chemical synthesis, our method provides a route for the recombinant production of cGAMP without the need for organic solvents and supports the goal of moving toward shorter, more sustainable, and more environmentally friendly processes.

Expression and Characterization of Intein-Cyclized Trimer of Staphylococcus aureus Protein A Domain Z.

Staphylococcus aureus protein A (SpA) is an IgG Fc-binding virulence factor that is widely used in antibody purification and as a scaffold to develop affinity molecules. A cyclized SpA Z domain could offer exopeptidase resistance, reduced chromatographic ligand leaching after single-site endopeptidase cleavage, and enhanced IgG binding properties by preorganization, potentially reducing conformational entropy loss upon binding. In this work, a Z domain trimer (Z3) was cyclized using protein intein splicing. Interactions of cyclic and linear Z3 with human IgG1 were characterized by differential scanning fluorimetry (DSF), surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC). DSF showed a 5 ℃ increase in IgG1 melting temperature when bound by each Z3 variant. SPR showed the dissociation constants of linear and cyclized Z3 with IgG1 to be 2.9 nM and 3.3 nM, respectively. ITC gave association enthalpies for linear and cyclic Z3 with IgG1 of -33.0 kcal/mol and -32.7 kcal/mol, and -T∆S of association 21.2 kcal/mol and 21.6 kcal/mol, respectively. The compact cyclic Z3 protein contains 2 functional binding sites and exhibits carboxypeptidase Y-resistance. The results suggest cyclization as a potential approach toward more stable SpA-based affinity ligands, and this analysis may advance our understanding of protein engineering for ligand and drug development.

Continuous monitoring of IgG using immobilized fluorescent reporters.

In the manufacture of therapeutic monoclonal antibodies, the clarified cell culture fluid (CCF) is typically loaded onto an initial protein A affinity capture column. Imperfect mass transfer and loading to maximum capacity can risk antibody breakthrough and loss of valuable product, but conservative underloading wastes expensive protein A resin. In addition, the effects of column fouling and ligand degradation require the frequent optimization of immunoglobulin G (IgG) loading to avoid wastage. Continuous real-time monitoring of IgG flowthrough is of great interest, therefore. We previously developed a fluorescence-based monitoring technology that allows batch mix-and-read mAb detection in the CCF. Here, we report the use of reporters immobilized on cyanogenbromide-activated Sepharose 4B resin for continuous detection of IgG in column breakthrough. The column effluent is continuously contacted with immobilized fluorescein-labeled Fc-binding ligands in a small monitoring column to produce an immediately-detectable change in fluorescence intensity. The technology allows rapid and reliable monitoring of IgG in a flowing stream of clarified CCF emerging from a protein A column, without prior sample preparation. We observed a significant change in fluorescence intensity at 0.5 g/L human IgG, sufficient to detect a 5% breakthrough of a 10 g/L load, within 18 s at a flow rate of 0.5 ml/min. The current small-scale technology is suitable for use in process development, but the chemistry should be readily adaptable to larger scale applications using fiber-optic sensors, and continuous IgG monitoring could be applicable in a variety of upstream and downstream process settings.

Protein A-Nanoluciferase fusion protein for generalized, sensitive detection of immunoglobulin G.

Detection and quantification of antibodies, especially immunoglobulin G (IgG), is a cornerstone of ELISAs, many diagnostics, and the development of antibody-based drugs. Current state-of-the-art immunoassay techniques for antibody detection require species-specific secondary antibodies and carefully-controlled bioconjugations. Poor conjugation efficiency degrades assay performance and increases the risk of clinical false positives due to non-specific binding. We developed a generic, highly-sensitive platform for IgG quantification by fusing the IgG-Fc binding Z domain of Staphylococcal Protein A with the ultrabright bioluminescence reporter Nanoluc-luciferase (Nluc). We demonstrated the application of this fusion protein in a sandwich IgG detection immunoassay using surface-bound antigens to capture target IgG and protein A-Nanoluc fusion as the detector. We optimized the platform's sensitivity by incorporating multiple repeats of the Z domain into the fusion protein constructs. Using rabbit and mouse anti-SARS-CoV-2 Nucleoprotein IgGs as model analytes, we performed ELISAs in two different formats, either with SARS-CoV-2 Nucleoprotein as the capture antigen or with polyclonal chicken IgY as the capture antibody. Using standard laboratory equipment, the platform enabled the quantitation of antibody analytes at concentrations as low as 10 pg/mL (67 fM).

Multi-wavelength UV-based PAT tool for measuring protein concentration.

Process chromatography is commonly used for purification of therapeutic proteins. Most chromatography skids that are used in such operations utilize single ultraviolet (UV) absorbance for monitoring and quantification of protein content. While the signal from such UV measurement is linear with respect to protein concentration at low values of protein concentrations, as the concentration increases across an eluting product peak, it goes manifold over the linear range, resulting in saturation of the UV signal and as a result incomplete quantification of the protein concentration. This can hamper our ability to decide on where to pool the process chromatography peak. It is evident that a simple, fast, and cost-effective methodology for on-line estimation of protein concentration is the need of the hour. In this paper, a multi-wavelength UV-based approach has been proposed for dilution-free on-line concentration estimation in the range of 0.8-100 g/L. Stable absorbance regions are picked up in the proposed approach from the multi-wavelength UV spectra, thereby offering a solution to the problem of saturation and non-linearity of the UV signal that is otherwise observed at higher concentrations. Further, using chemometrics tools such as principal component analysis (PCA) and partial least squares (PLS), the model has been validated for rapid quantification of protein concentration from the spectra. The predictions from the model were comparable to values measured using an existing UV-based offline method with an R2 of>98%. The proposed process analytical technology (PAT) tool was successfully tested online and exhibited<8% variability and could effectively be used from capture to formulation to enable dilution-free online concentration measurement of IgG. The proposed tool is a simple, low-cost alternative to other methods and could enable integrated/continuous operations throughout the downstream train.

Antibody mix-and-read assays based on fluorescence intensity probes.

Antibodies and Fc fusion proteins are a rapidly growing class of pharmaceuticals. Cell culture and purification process development and operation require frequent measurement of product concentrations, commonly by complex enzyme-linked immunosorbent assay and high-performance liquid chromatography methods. Here we report a fast (<30 s), and simple antibody Fc assay based on mix-and-read reporting by fluorescence emission. A soluble fluorescein-labeled Fc-affinity reporter produced by standard peptide synthesis is mixed with an Fc-containing sample to produce an immediate shift in both fluorescence polarization and intensity, compatible with on- and at-line measurements and microbioreactor monitoring. We observed significant shifts in fluorescence intensity in Chinese hamster ovary cell culture fluid spiked with IgG and detected an adalimumab biosimilar down to 100 ng/mL (10-4 g/L), despite the interferents in the complex sample matrix. Neither the fluorescence polarization nor the fluorescence intensity assay is significantly affected by the addition of clarified lysate of 2 million CHO-k1 cells/mL, suggesting applicability even to cultures of low viability. Biochemical and molecular docking approaches suggest that the fluorescence intensity enhancement is caused by changes in the fluorophore's local microenvironment upon binding to IgG Fc, especially by interactions with Fc His433.Abbreviations: CCF: Cell Culture Fluid; CHO: Chinese Hamster Ovary cells; ELISA: Enzyme Linked Immunosorbent Assay; Fc: Fragment Crystallizable of antibody; HPLC: High-Performance Liquid Chromatography; HPßCD: hydroxypropyl-ß-cyclodextrin; IgG: ImmunoglobulinG; mAb: Monoclonal Antibody; PBS: Phosphate-Buffered Saline; PDB: Protein Data Bank; SpA: Staphylococcal protein A; SpG: Staphylococcal protein G.