Recombinant AAV Purification.

Purification of rAAV is a crucial unit operation of the AAV production process. It enables the capture of AAV and removal of contaminants such as host cell proteins, host cell DNA, and other cell culture-related impurities. Here we describe the purification of rAAV produced in insect cells Sf9/rBEV by immuno-affinity capture chromatography. The method is fully scale-amenable unlike other traditional purification methods based on ultracentrifugation. The method reported herein has two main steps: (1) the clarification of cell lysate by depth filtration and (2) the selective capture and single-step purification of AAV via immune-affinity chromatography. This purification method has been successfully implemented to purify the majority of wild-type AAV serotypes.

A protocol to isolate, identify, and verify glucose- or carbohydrate-binding receptors.

Sensing, transport, and utilization of glucose is pivotal to the maintenance of energy homeostasis in animals. Although transporters involved in mobilizing glucose across different cellular compartments are fairly well known, the receptors that bind glucose to mediate its effects independently of glucose metabolism remain largely unrecognized. Establishing precise and reproducible methods to identify glucose receptors in the brain or other peripheral organs will pave the way for comprehending the role of glucose signaling pathways in maintaining, regulating, and reprogramming cellular metabolic needs. Identification of such potential glucose receptors will also likely lead to development of effective therapeutics for treatment of diabetes and related metabolic disorders. Commercially available biotin or radiolabeled glucose conjugates have low molecular weight; therefore, they do not provide enough sensitivity and density to isolate glucose receptors. Here, we describe a protocol to isolate, identify, and verify glucose-binding receptor/s using high molecular weight glucose (or other carbohydrate) conjugates. We have produced 30 kDa glucose- (or other carbohydrate-) biotin-polyacrylamide (PAA) conjugates with mole fractions of 80:5:15% respectively. These conjugates are used with biotin-streptavidin biochemistry, In-cell ELISA, and surface plasmon resonance (SPR) methods to isolate, identify, and verify glucose- or carbohydrate-binding receptors. We first demonstrate how streptavidin-coated magnetic beads are employed to immobilize glucose-biotin-PAA conjugates. Then, these beads are used to enrich and isolate glucose-binding proteins from tissue homogenates or from single-cell suspensions. The enriched or isolated proteins are subjected to mass spectrometry/proteomics to reveal the identity of top candidate proteins as potential glucose receptors. We then describe how the In-cell ELISA method is used to verify the interaction of glucose with its potential receptor through stable expression of the receptor in-vitro. We further demonstrate how a highly sensitive SPR method can be used to measure the binding kinetics of glucose with its receptor. In summary, we describe a protocol to isolate, identify, and verify glucose- or carbohydrate-binding receptors using magnetic beads, In-cell ELISA, and SPR. This protocol will form the future basis of studying glucose or carbohydrate receptor signaling pathways in health and in disease.

Binding studies of promethazine and its metabolites with human serum albumin by high-performance affinity chromatography and molecular docking in the presence of codeine.

"Purple Drank", a soft drink containing promethazine (PMZ) and codeine (COD), has gained global popularity for its hallucinogenic effects. Consuming large amounts of this combination can lead to potentially fatal events. The binding of these drugs to plasma proteins can exacerbate the issue by increasing the risk of drug interactions, side effects, and/or toxicity. Herein, the binding affinity to human serum albumin (HSA) of PMZ and its primary metabolites [N-desmethyl promethazine (DMPMZ) and promethazine sulphoxide (PMZSO)], along with COD, was investigated by high-performance affinity chromatography (HPAC) though zonal approach. PMZ and its metabolites exhibited a notable binding affinity for HSA (%b values higher than 80%), while COD exhibited a %b value of 65%. To discern the specific sites of HSA to which these compounds were bound, displacement experiments were performed using warfarin and (S)-ibuprofen as probes for sites I and II, respectively, which revealed that all analytes were bound to both sites. Molecular docking studies corroborated the experimental results, reinforcing the insights gained from the empirical data. The in silico data also suggested that competition between PMZ and its metabolites with COD can occur in both sites of HSA, but mainly in site II. As the target compounds are chiral, the enantioselectivity for HSA binding was also explored, showing that the binding for these compounds was not enantioselective.

Serodiagnosis of nasal myasis in camels (Camelus dromedaries) in Egypt using third larval instar affinity-purified glycoprotein.

The larvae of Cephalopina titillator cause nasopharyngeal myiasis in camels, which parasitize the living tissues of the nasal and paranasal sinuses, pharynx, and larynx. C. titillator infestation adversely affects camel health, meat, and milk production, and can even cause death. In our study, to improve the immunodiagnosis of camel nasal myiasis, a sensitive and specific enzyme-linked immunosorbent assay (ELISA) was developed and evaluated using the Concanavalin-A (Con-A) affinity purification for the C. titillator-N-acetylglucosamine (Ct-GlucNAc) glycoprotein fraction from third larval instars as an antigen for detecting C. titillator antibodies. Crude antigens were prepared from larval instars of C. titillator and evaluated by indirect ELISA. The third C. titillator larval antigen (L3Ct) had the highest protein content (P < 0.001) and the best diagnostic value; chi-square = 235 (P < 0.001). Four glycoprotein fractions were purified separately from the L3Ct antigen by Con-A purification and evaluated. The Ct-GlucNAc glycoprotein fraction was the fraction of choice with the highest diagnostic accuracy (P < 0.05). Using Ct-GlucNAc as a coating antigen, indirect ELISA showed a 99.3% sensitivity for positive results in camel myiasis samples and 100% specificity for negative results in healthy camel samples. The diagnostic accuracy was 99.7%, and no cross reactivity was detected for other parasitic diseases. The indirect ELISA results were confirmed by the western immunoblotting which was characterized by comparing sera from naturally infested dromedary camels with C. titillator, sera from healthy camels and sera from camels with other parasitic infections (Echinococcus granulosus, Fasciola gigantica, Hard ticks; Hyalomma dromedarii, Trichostronglid sp., Eimeria spp., and Cryptosporidium sp.). Immunoreactive antigenic bands of 63, 50, 30 and 18 kDa were predominantly detected in sera from camels with nasopharyngeal myiasis and didn't react with healthy and camel's sera from other parasitic infections. However, seven immunoreactive bands appeared at 120, 70, 63, 48, 35, 29, and 19 kDa in the crude L3Ct antigen. In addition, a positive rate of C. titillator immunodiagnosis was detected by indirect ELISA (48.6%, chi-square = 483, P < 0.001), which was significantly greater than that of postmortem diagnosis (31%). In conclusion, the current study introduces a new diagnostic immunoaffinity glycoprotein fraction of C. titillator 3rd larval instar-based ELISA as a highly accurate, simple and fast method to detect specific antibodies of nasal myiasis in camels.

Oriented immobilization of nanobodies using SpyCatcher/SpyTag significantly enhances the capacity of affinity chromatography.

The use of nanobodies (Nbs) in affinity chromatography for biomacromolecule purification is gaining popularity. However, high-performance Nb-based affinity resins are not readily available, mainly due to the lack of suitable immobilization methods. In this study, we explored an autocatalytic coupling strategy based on the SpyCatcher/SpyTag chemistry to achieve oriented immobilization of Nb ligands. To facilitate this approach, a variant cSpyCatcher003 (cSC003) was coupled onto agarose microspheres, providing a specific attachment site for SpyTagged nanobody ligands. The cSC003 easily purified from Escherichia coli through a two-step procedure, exhibits exceptional alkali resistance and structural recovery capability, highlighting its robustness as a linker in the coupling strategy. To validate the effectiveness of cSC003-derivatized support, we employed VHSA, a nanobody against human serum albumin (HSA), as the model ligand. Notably, the immobilization of SpyTagged VHSA onto the cSC003-derivatized support was achieved with a coupling efficiency of 90 %, significantly higher than that of traditional thiol-based coupling method. This improvement directly correlated to the preservation of the native conformation of nanobodies during the coupling process. In addition, the Spy-immobilized resin demonstrated better performance in the binding capacity, with a 3-fold improvement in capture efficiency, underscoring the advantages of the Spy immobilization strategy for oriented immobilization of VHSA ligands. Moreover, online purification and immobilization of SpyTagged VHSA from crude bacterial lysate was achieved using the cSC003-derivatized support. The resulting resin exhibited high binding specificity towards HSA, yielding a purity above 95 % directly from human serum, and maintained good stability throughout multiple purification cycles. These findings highlight the potential of the Spy immobilization strategy for developing Nb-based affinity chromatographic materials, with significant implications for biopharmaceutical downstream processes.

C-terminal Poly-histidine Tags Alter Escherichia coli Polyphosphate Kinase Activity and Susceptibility to Inhibition.

In Escherichia coli, many environmental stressors trigger polyphosphate (polyP) synthesis by polyphosphate kinase (PPK1), including heat, nutrient restriction, toxic compounds, and osmotic imbalances. PPK1 is essential for virulence in many pathogens and has been the target of multiple screens for small molecule inhibitors that might serve as new anti-virulence drugs. However, the mechanisms by which PPK1 activity and polyP synthesis are regulated are poorly understood. Our previous attempts to uncover PPK1 regulatory elements resulted in the discovery of PPK1* mutants, which accumulate more polyP in vivo, but do not produce more in vitro. In attempting to further characterize these mutant enzymes, we discovered that the most commonly-used PPK1 purification method - Ni-affinity chromatography using a C-terminal poly-histidine tag - altered intrinsic aspects of the PPK1 enzyme, including specific activity, oligomeric state, and kinetic values. We developed an alternative purification strategy using a C-terminal C-tag which did not have these effects. Using this strategy, we were able to demonstrate major differences in the in vitro response of PPK1 to 5-aminosalicylic acid, a known PPK1 inhibitor, and observed several key differences between the wild-type and PPK1* enzymes, including changes in oligomeric distribution, increased enzymatic activity, and increased resistance to both product (ADP) and substrate (ATP) inhibition, that help to explain their in vivo effects. Importantly, our results indicate that the C-terminal poly-histidine tag is inappropriate for purification of PPK1, and that any in vitro studies or inhibitor screens performed with such tags need to be reconsidered in that light.

Production and Purification of Adeno-Associated Viral Vectors (AAVs) Using Orbitally Shaken HEK293 Cells.

Here, we describe methods for the production of adeno-associated viral (AAV) vectors by transient transfection of HEK293 cells grown in serum-free medium using orbital shaken bioreactors and the subsequent purification of vector particles. The protocol for expression of AAV components is based on polyethyleneimine (PEI)-mediated transfection of a three-plasmid system and is specified for production in milliliter-to-liter scales. After PEI and plasmid DNA (pDNA) complex formation, the diluted cell culture is transfected without a prior concentration step or medium exchange. Following a 7-day batch process, cell cultures are further processed using a set of methods for cell lysis and vector recovery. Methods for the purification of viral particles are described, including immunoaffinity and anion-exchange chromatography, ultrafiltration, as well as digital PCR to quantify the concentration of vector particles.

A strategy for inhibitors screening of xanthine oxidase based on colorimetric sensor combined with affinity chromatography technology.

The discovery of enzyme inhibitors from natural products is a crucial aspect in the development of therapeutic drugs. However, the complexity of natural products presents a challenge in developing simple and efficient methods for inhibitor screening. Herein, we have developed an integrated analytical model for screening xanthine oxidase (XOD) inhibitors that combines simplicity, accuracy, and efficiency. This model utilizes a colorimetric sensor and affinity chromatography technology with immobilized XOD. The colorimetric sensor procedure can quickly identify whether there are active components in complex samples. Subsequently, the active components in the samples identified by the colorimetric sensor procedure were further captured, separated, and identified through affinity chromatography. The integrated analytical model can significantly enhance the efficiency and accuracy of inhibitor screening. The proposed method was applied to screen for an activity inhibitor of XOD in five natural medicines. As a result, a potential active ingredient for XOD, polydatin, was successfully identified from Polygoni Cuspidati Rhizoma et Radix. This work is anticipated to offer new insights for the screening of enzyme inhibitors from natural medicines.

Purification of Recombinant N-terminal Histidine-Tagged Arabidopsis thaliana Phosphoglycolate Phosphatase 1, Glycolate Oxidase 1 and 2, and Hydroxypyruvate Reductase 1.

To measure the kinetic properties of photorespiratory enzymes, it is necessary to work with purified proteins. Protocols to purify photorespiratory enzymes from leaves of various plant species require several time-consuming steps. It is now possible to produce large quantities of recombinant proteins in bacterial cells. They can be rapidly purified as histidine-tagged recombinant proteins by immobilized metal affinity chromatography using Ni2+-NTA-agarose. This chapter describes protocols to purify several Arabidopsis thaliana His-tagged recombinant photorespiratory enzymes (phosphoglycolate phosphatase, glycolate oxidase, and hydroxypyruvate reductase) from Escherichia coli cell cultures using two bacterial strain-plasmid systems: BL21(DE3)-pET and LMG194-pBAD.

[Recent progress of chromatographic techniques for antibody purification].

Antibody drugs are becoming increasingly popular in disease diagnosis, targeted therapy, and immunoprevention owing to their characteristics of high targeting ability, strong specificity, low toxicity, and mild side effects. The demand for antibody drugs is steadily increasing, and their production scale is expanding. Upstream cell culture technology has been greatly improved by the high-capacity production of monoclonal antibodies. However, the downstream purification of antibodies presents a bottleneck in the production process. Moreover, the purification cost of antibodies is extremely high, accounting for approximately 50%-80% of the total cost of antibody production. Chromatographic technology, given its selectivity and high separation efficiency, is the main method for antibody purification. This process usually involves three stages: antibody capture, intermediate purification, and polishing. Different chromatographic techniques, such as affinity chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, mixed-mode chromatography, and temperature-responsive chromatography, are used in each stage. Affinity chromatography, mainly protein A affinity chromatography, is applied for the selective capture and purification of antibodies from raw biofluids or harvested cell culture supernatants. Other chromatographic techniques, such as ion-exchange chromatography, hydrophobic interaction chromatography, and mixed-mode chromatography, are used for intermediate purification and antibody polishing. Affinity biomimetic chromatography and hydrophobic charge-induction chromatography can produce antibodies with purities comparable with those obtained through protein A chromatography, by employing artificial chemical/short peptide ligands with good selectivity, high stability, and low cost. Temperature-responsive chromatography is a promising technique for the separation and purification of antibodies. In this technique, antibody capture and elution is controlled by simply adjusting the column temperature, which greatly eliminates the risk of antibody aggregation and inactivation under acidic elution conditions. The combination of different chromatographic methods to improve separation selectivity and achieve effective elution under mild conditions is another useful strategy to enhance the yield and quality of antibodies. This review provides an overview of recent advances in the field of antibody purification using chromatography and discusses future developments in this technology.

Antibody-ligand interactions on a high-capacity staphylococcal protein A resin.

Staphylococcal protein-A affinity chromatography has been optimized for antibody purification, achieving a current capacity of up to 90 mg/ml in packed bed. The morphology of the particles, the number of antibodies bound per ligand and the spatial arrangement of the ligands were assessed by in-situ Small-angle X-ray scattering (SAXS) and scanning electron microscopy (SEM) combined with measurement of adsorption isotherms. We employed SAXS measurements to probe the nanoscale structure of the chromatographic resin. From scanning electron microcopy, the morphology and area of the beads were obtained. The adsorption isotherm revealed a bi-Langmuirian behavior where the association constant varied with the critical bulk concentration, indicating multilayer adsorption. Determining the antibody-ligand stoichiometry was crucial for understanding the adsorption mechanism, which was estimated to be 4 at lower concentrations and 4.5 at higher concentrations, suggestive of reversible protein-protein interactions. The same results were reached from the in-situ small angle X-ray scattering measurements. A stoichiometry of 6 cannot be achieved since the two protein A monomers are anchored to the stationary phase and thus sterically hindered. Normalization through ellipsoids facilitated SAXS analysis, enabling the determination of distances between ligands and antibody-ligand complexes. Density fluctuations were examined by subtracting the elliptical fit, providing insights into ligand density distribution. The dense ligand packing of TOYOPEARL® AF-rProtein A HC was confirmed, making further increases in ligand density impractical. Additionally, SAXS analysis revealed structural rearrangements of the antibody-ligand complex with increasing antibody surface load, suggesting reversible association of antibodies.

Ligand Screening of Membrane Proteins Embedded in Nanodiscs: How to Manage Non-Specific Interactions in Weak Affinity Chromatography?

Miniaturized weak affinity chromatography is emerging as an interesting alternative to conventional biophysical tools for performing fragment-screening studies in the context of fragment-based drug discovery. In order to push back the analytical limits, it is necessary not only to control non-specific interactions with chromatographic support, but also to adapt this methodology by comparing the results obtained on an affinity column to a control column. The work presented in this study focused on fragment screening that targets a model membrane protein, the adenosine A2A receptor, embedded in nanodiscs (NDs) as biomimetic membranes. By studying the retention behavior of test fragment mixtures on supports modified with different types of NDs, we were able to determine the contribution of ND-related non-specific interactions, in particular the electrostatic effect of anionic phospholipids and the hydrophobic effect of neutral phospholipids. Different strategies for the preparation of control columns (empty NDs, orthosteric site blocking) were investigated and are presented for the first time. With these two types of control columns, the screening enabled the identification of two new fragments of AA2AR, which were confirmed by competition experiments and whose Kd values, estimated directly during the screening or after the competition experiments in frontal mode, were in good agreement.

Expression and Purification of Mammalian NMDA Receptor Protein for Functional Characterization.

N-methyl-D-aspartate (NMDA) receptors are critical for brain function and serve as drug targets for the treatment of neurological and psychiatric disorders. They typically form the tetrameric assembly of GluN1-GluN2 (2A to 2D) subtypes, with their diverse three-dimensional conformations linked with the physiologically relevant function in vivo. Purified proteins of tetrameric assembled NMDA receptors have broad applications in the structural elucidation, hybridoma technology for antibody production, and high-throughput drug screening. However, obtaining sufficient quantity and monodisperse NMDA receptor protein is still technically challenging. Here, we summarize a paradigm for the expression and purification of diverse NMDA receptor subtypes, with detailed descriptions on screening constructs by fluorescence size-exclusion chromatography (FSEC), generation of recombinant baculovirus, expression in the eukaryotic expression system, protein purification by affinity chromatography and size-exclusion chromatography (SEC), biochemical and functional validation assays.

Soluble expression of recombinant human interleukin-2 in Escherichia coli and its facile production.

Recombinant human interleukin-2 (rhIL-2) represents one of the most difficult-to-produce cytokines in E. coli due to its extreme hydrophobicity and high tendency to formation of inclusion bodies. Refolding of rhIL-2 inclusion bodies always represents cumbersome downstream processes and low production efficiency. Herein, we disclosed a fusion strategy for efficiently soluble expression and facile production of rhIL-2 in E. coli Origami B (DE3) host. A two-tandem SUMO fusion partner (His-2SUMO) with a unique SUMO protease cleavage site at C-terminus was devised to fuse with the N-terminus of rhIL-2 and the fusion protein (His-2SUMO-rhIL-2) was almost completely expressed in a soluble from. The fusion partner could be efficiently removed by Ulp1 cleavage and the rhIL-2 was simply produced by a two-step Ni-NTA affinity chromatography with a considerable purity and whole recovery. The eventually obtained rhIL-2 was well-characterized and the results showed that the purified rhIL-2 exhibits a compact and ordered structure. Although the finally obtained rhIL-2 exists in a soluble aggregates form and the aggregation probably has been occurred during expression stage, the soluble rhIL-2 aggregates remain exhibit comparable bioactivity with the commercially available rhIL-2 drug formulation.

[Preparation technology comparison and performance evaluation of different protein A affinity chromatographic materials].

Protein A affinity chromatographic materials are widely used in clinical medicine and biomedicine because of their specific interactions with immunoglobulin G (IgG). Both the characteristics of the matrix, such as its structure and morphology, and the surface modification method contribute to the affinity properties of the packing materials. The specific, orderly, and oriented immobilization of protein A can reduce its steric hindrance with the matrix and preserve its bioactive sites. In this study, four types of affinity chromatographic materials were obtained using agarose and polyglycidyl methacrylate (PGMA) spheres as substrates, and multifunctional epoxy and maleimide groups were used to fix protein A. The effects of the ethylenediamine concentration, reaction pH, buffer concentration, and other conditions on the coupling efficiency of protein A and adsorption performance of IgG were evaluated. Multifunctional epoxy materials were prepared by converting part of the epoxy groups of the agarose and PGMA matrices into amino groups using 0.2 and 1.6 mol/L ethylenediamine, respectively. Protein A was coupled to the multifunctional epoxy materials using 5 mmol/L borate buffer (pH 8) as the reaction solution. When protein A was immobilized on the substrates by maleimide groups, the agarose and PGMA substrates were activated with 25% (v/v) ethylenediamine for 16 h to convert all epoxy groups into amino groups. The maleimide materials were then converted into amino-modified materials by adding 3 mg/mL 3-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) dissolved in dimethyl sulfoxide (DMSO) and then suspended in 5 mmol/L borate buffer (pH 8). The maleimide groups reacted specifically with the C-terminal of the sulfhydryl group of recombinant protein A to achieve highly selective fixation on both the agarose and PGMA substrates. The adsorption performance of the affinity materials for IgG was improved by optimizing the bonding conditions of protein A, such as the matrix type, matrix particle size, and protein A content, and the adsorption properties of each affinity material for IgG were determined. The column pressure of the protein A affinity materials prepared using agarose or PGMA as the matrix via the maleimide method was subsequently evaluated at different flow rates. The affinity materials prepared with PGMA as the matrix exhibited superior mechanical strength compared with the materials prepared with agarose. Moreover, an excellent linear relationship between the flow rate and column pressure of 80 mL/min was observed for this affinity material. Subsequently, the effect of the particle size of the PGMA matrix on the binding capacity of IgG was investigated. Under the same protein A content, the dynamic binding capacity of the affinity materials on the PGMA matrix was higher when the particle size was 44-88 µm than when other particle sizes were used. The properties of the affinity materials prepared using the multifunctional epoxy and maleimide-modified materials were compared by synthesizing affinity materials with different protein A coupling amounts of 1, 2, 4, 6, 8, and 10 mg/mL. The dynamic and static binding capacities of each material for bovine IgG were then determined. The prepared affinity material was packed into a chromatographic column to purify IgG from bovine colostrum. Although all materials showed specific adsorption selectivity for IgG, the affinity material prepared by immobilizing protein A on the PGMA matrix with maleimide showed significantly better performance and achieved a higher dynamic binding capacity at a lower protein grafting amount. When the protein grafting amount was 15.71 mg/mL, the dynamic binding capacity of bovine IgG was 32.23 mg/mL, and the dynamic binding capacity of human IgG reached 54.41 mg/mL. After 160 cycles of alkali treatment, the dynamic binding capacity of the material reached 94.6% of the initial value, indicating its good stability. The developed method is appropriate for the production of protein A affinity chromatographic materials and shows great potential in the fields of protein immobilization and immunoadsorption material synthesis.

Quality by Digital Design for Developing Platform RNA Vaccine and Therapeutic Manufacturing Processes.

Quality by digital design (QbDD) utilizes data-driven, mechanistic, or hybrid models to define and optimize a manufacturing design space. It improves upon the QbD approach used extensively in the pharmaceutical industry. The computational models developed in this approach identify and quantify the relationship between the product's critical quality attributes (CQAs) and the critical process parameters (CPPs) of unit operations within the manufacturing process. This chapter discusses the QbDD approach in developing and optimizing unit operations such as in vitro transcription, tangential flow filtration, affinity chromatography, and lipid nanoparticle (LNP) formulation in mRNA vaccine manufacturing. QbDD can be an efficient framework for developing a production process for a disease-agnostic product that requires extensive experimental and model-based process-product interaction characterization during the early process development phase.

Exploring affinity chromatography in proteomics: A comprehensive review.

Over the past decades, the proteomics field has undergone rapid growth. Progress in mass spectrometry and bioinformatics, together with separation methods, has brought many innovative approaches to the study of the molecular biology of the cell. The potential of affinity chromatography was recognized immediately after its first application in proteomics, and since that time, it has become one of the cornerstones of many proteomic protocols. Indeed, this chromatographic technique exploiting the specific binding between two molecules has been employed for numerous purposes, from selective removal of interfering (over)abundant proteins or enrichment of scarce biomarkers in complex biological samples to mapping the post-translational modifications and protein interactions with other proteins, nucleic acids or biologically active small molecules. This review presents a comprehensive survey of this versatile analytical tool in current proteomics. To navigate the reader, the haphazard space of affinity separations is classified according to the experiment's aims and the separated molecule's nature. Different types of available ligands and experimental strategies are discussed in further detail for each of the mentioned procedures.

Pore-blocking steric mass-action model for adsorption of bioparticles.

The steric mass-action (SMA) model has been widely reported to describe the adsorption of proteins in different types of chromatographic adsorbents. Here in the present work, a pore-blocking steric mass-action model (PB-SMA) was developed for the adsorption of large-size bioparticles, which usually exhibit the unique pore-blocking characteristic on the adsorbent and thus lead to a fraction of ligands in the deep channels physically inaccessible to bioparticles adsorption, instead of being shielded due to steric hindrance by adsorbed bioparticles. This unique phenomenon was taken into account by introducing an additional parameter, Lin, which is defined as the inaccessible ligand densities in the physically blocked pore area, into the PB-SMA model. This fraction of ligand densities (Lin) will be deducted from the total ligand (Lt) for model development, thus the steric factor (σ) in the proposed PB-SMA will reflect the steric shielding effect on binding sites by adsorbed bioparticles more accurately than the conventional SMA model, which assumes that all ligands on the adsorbent have the same accessibility to the bioparticles. Based on a series of model assumptions, a PB-SMA model was firstly developed for inactivated foot-and-mouth disease virus (iFMDV) adsorption on immobilized metal affinity chromatography (IMAC) adsorbents. Model parameters for static adsorption including equilibrium constant (K), characteristic number of binding sites (n), and steric factor (σ) were determined. Compared with those derived from the conventional SMA model, the σ values derived from the PB-SMA model were dozens of times smaller and much closer to the theoretical maximum number of ligands shielded by a single adsorbed iFMDV, indicating the modified model was more accurate for bioparticles adsorption. The applicability of the PB-SMA model was further validated by the adsorption of hepatitis B surface antigen virus-like particles (HBsAg VLPs) on an ion exchange adsorbent with reasonably improved accuracy. Thus, it is considered that the PB-SMA model would be more accurate in describing the adsorption of bioparticles on different types of chromatographic adsorbents.

Multidimensional LC-MS with 1D multi-method option and parallel middle-up and bottom-up MS acquisition for in-depth characterization of antibodies.

Monoclonal antibodies (mAbs) are large and highly heterogeneous species typically characterized using a plethora of analytical methodologies. There is a trend within the biopharmaceutical industry to combine several of these methods in one analytical platform to simultaneously assess multiple structural attributes. Here, a protein analyzer for the fully automated middle-up and bottom-up liquid chromatography-mass spectrometry (LC-MS) analysis of charge, size and hydrophobic variants is described. The multidimensional set-up combines a multi-method option in the first dimension (1D) (choice between size exclusion - SEC, cation exchange - CEX or hydrophobic interaction chromatography - HIC) with second dimension (2D) on-column reversed-phase (RPLC) based desalting, denaturation and reduction prior to middle-up LC-MS analysis of collected 1D peaks and parallel on-column trypsin digestion of denatured and reduced peaks in the third dimension (3D) followed by bottom-up LC-MS analysis in the fourth dimension (4D). The versatile and comprehensive workflow is applied to the characterization of charge, hydrophobic and size heterogeneities associated with an engineered Fc fragment and is complemented with hydrogen-deuterium exchange (HDX) MS and FcRn affinity chromatography - native MS to explain observations in a structural/functional context.

Expression and Purification of His-Tagged Variants of Human Hepatitis A Virus 3C Protease.

BACKGROUND: Protease 3C (3Cpro) is the only protease encoded in the human hepatitis A virus genome and is considered a potential target for antiviral drugs due to its critical role in the viral life cycle. Additionally, 3Cpro has been identified as a potent inducer of ferroptosis, a newly described type of cell death. Therefore, studying the molecular mechanism of 3Cpro functioning can provide new insights into viral-host interaction and the biological role of ferroptosis. However, such studies require a reliable technique for producing the functionally active recombinant enzyme. OBJECTIVE: Here, we expressed different modified forms of 3Cpro with a hexahistidine tag on the N- or C-terminus to investigate the applicability of Immobilized Metal Ion Affinity Chromatography (IMAC) for producing 3Cpro. METHODS: We expressed the proteins in Escherichia coli and purified them using IMAC, followed by gel permeation chromatography. The enzymatic activity of the produced proteins was assayed using a specific chromogenic substrate. RESULTS: Our findings showed that the introduction and position of the hexahistidine tag did not affect the activity of the enzyme. However, the yield of the target protein was highest for the variant with seven C-terminal residues replaced by a hexahistidine sequence. CONCLUSION: We demonstrated the applicability of our approach for producing recombinant, enzymatically active 3Cpro.