Comparison of Tumor Non-specific and PD-L1 Specific Imaging by Near-Infrared Fluorescence/Cerenkov Luminescence Dual-Modality In-situ Imaging.

Background: Labeled antibodies are excellent imaging agents in oncology to non-invasively visualize cancer-related antigens expression levels. However, tumor tracer uptake (TTU) of specific antibodies in-vivo may be inferior to non-specific IgG in some cases. Objectives: To explore factors affecting labeled antibody visualization by PD-L1 specific and non-specific imaging of nude mouse tumors. Methods: TTU was observed in RKO model on Cerenkov luminescence (CL) and near-infrared fluorescence (NIRF) imaging of radionuclide 131I or NIRF dyes labeled Atezolizumab and IgG. A mixture of NIRF dyes labeled Atezolizumab and 131I-labeled IgG was injected, and TTU was observed in the RKO and HCT8 model by NIRF/CL dual-modality in-situ imaging. TTU were observed by 131I-labeled Atezolizumab and IgG in-vitro distribution. Results: Labeled IgG concentrated more in tumors than Atezolizumab. NIRF/CL imaging in 24 to 168 h showed that TTU gradually decreased over time, which decreased more slowly on CL imaging compared to NIRF imaging. The distribution data in-vitro showed that TTU of 131I-labeled IgG was higher than that of 131I-labeled Atezolizumab at any time point. Conclusion: Non-specific IgG may not be suitable as a control for Atezolizumab in comparing tumor PD-L1 expression in nude mice via labeled antibody optical imaging under certain circumstances.

Co-crosslinking strategy for dual functionalization of small magnetic nanoparticles with redox probes and biological probes.

Surface functionalization strategy is becoming a crucial bridge from magnetic nanoparticles (MNPs) to their broad bio-application. To realize the multiple functions of MNPs such as magnetic manipulation, target capture, and signal amplification in their use of electrochemical biosensing, co-crosslinking strategy was proposed here to construct dual-functionalized MNPs by combining ultra-sensitive redox moieties and specific biological probes. In this work, MNPs with a TEM size of 10 nm were synthesized by co-precipitation for amination and PEGylation to maintain colloid stability once dispersed in high-ionic-strength buffer (such as phosphate-buffered saline). Then, MNPs@IgG were prepared via the bis(sulfosuccinimidyl) suberate (BS3) cross-linker to conjugate these IgG onto the MNP surface, with a binding efficiency of 73%. To construct dual-functionalized MNPs, these redox probes of ferrocene-NHS (Fc) were co-crosslinked onto the MNP surface, together with IgG, by using BS3. The developed MNPs@Redox@IgG were characterized by SDS‒PAGE to identify IgG binding and by square wave voltammetry (SWV) to validate the redox signal. Additionally, the anti-CD63 antibodies were selected for the development of MNPs@anti-CD63 for use in the bio-testing of exosome sample capture. Therefore, co-crosslinking strategy paved a way to develop dual-functionalized MNPs that can be an aid of their potential utilization in diagnostic assay or electrochemical methods.

Exploiting the Fc base of IgG antibodies to create functional nanoparticle conjugates.

The structures of the Fc base of various IgG antibodies have been examined with a view to understanding how this region can be used to conjugate IgG to nanoparticles. The base structure is found to be largely consistent across a range of species and subtypes, comprising a hydrophobic region surrounded by hydrophilic residues, some of which are charged at physiological conditions. In addition, atomistic Molecular Dynamics simulations were performed to explore how model nanoparticles interact with the base using neutral and negatively charged gold nanoparticles. Both types of nanoparticle interacted readily with the base, leading to an adaptation of the antibody base surface to enhance the interactions. Furthermore, these interactions left the rest of the domain at the base of the Fc region structurally intact. This implies that coupling nanoparticles to the base of an IgG molecule is both feasible and desirable, since it leaves the antibody free to interact with its surroundings so that antigen-binding functionality can be retained. These results will therefore help guide future attempts to develop new nanotechnologies that exploit the unique properties of both antibodies and nanoparticles.

Insight into the avidity-affinity relationship of the bivalent, pH-dependent interaction between IgG and FcRn.

Monoclonal antibodies (mAbs) as therapeutics necessitate favorable pharmacokinetic properties, including extended serum half-life, achieved through pH-dependent binding to the neonatal Fc receptor (FcRn). While prior research has mainly investigated IgG-FcRn binding kinetics with a focus on single affinity values, it has been shown that each IgG molecule can engage two FcRn molecules throughout an endosomal pH gradient. As such, we present here a more comprehensive analysis of these interactions with an emphasis on both affinity and avidity by taking advantage of switchSENSE technology, a surface-based biosensor where recombinant FcRn was immobilized via short DNA nanolevers, mimicking the membranous orientation of the receptor. The results revealed insight into the avidity-to-affinity relationship, where assessing binding through a pH gradient ranging from pH 5.8 to 7.4 showed that the half-life extended IgG1-YTE has an affinity inflection point at pH 7.2, reflecting its engineering for improved FcRn binding compared with the wild-type counterpart. Furthermore, IgG1-YTE displayed a pH switch for the avidity enhancement factor at pH 6.2, reflecting strong receptor binding to both sides of the YTE-containing Fc, while avidity was abolished at pH 7.4. When compared with classical surface plasmon resonance (SPR) technology and complementary methods, the use of switchSENSE demonstrated superior capabilities in differentiating affinity from avidity within a single measurement. Thus, the methodology provides reliable kinetic rate parameters for both binding modes and their direct relationship as a function of pH. Also, it deciphers the potential effect of the variable Fab arms on FcRn binding, in which SPR has limitations. Our study offers guidance for how FcRn binding properties can be studied for IgG engineering strategies.

Use of phase plate cryo-EM reveals conformation diversity of therapeutic IgG with 50 kDa Fab fragment resolved below 6 Å.

While cryogenic electron microscopy (cryo-EM) is fruitfully used for harvesting high-resolution structures of sizable macromolecules, its application to small or flexible proteins composed of small domains like immunoglobulin (IgG) remain challenging. Here, we applied single particle cryo-EM to Rituximab, a therapeutic IgG mediating anti-tumor toxicity, to explore its solution conformations. We found Rituximab molecules exhibited aggregates in cryo-EM specimens contrary to its solution behavior, and utilized a non-ionic detergent to successfully disperse them as isolated particles amenable to single particle analysis. As the detergent adversely reduced the protein-to-solvent contrast, we employed phase plate contrast to mitigate the impaired protein visibility. Assisted by phase plate imaging, we obtained a canonical three-arm IgG structure with other structures displaying variable arm densities co-existing in solution, affirming high flexibility of arm-connecting linkers. Furthermore, we showed phase plate imaging enables reliable structure determination of Fab to sub-nanometer resolution from ab initio, yielding a characteristic two-lobe structure that could be unambiguously docked with crystal structure. Our findings revealed conformation diversity of IgG and demonstrated phase plate was viable for cryo-EM analysis of small proteins without symmetry. This work helps extend cryo-EM boundaries, providing a valuable imaging and structural analysis framework for macromolecules with similar challenging features.

Ion Mobility-Mass Spectrometry and Collision-Induced Unfolding Rapidly Characterize the Structural Polydispersity and Stability of an Fc-Fusion Protein.

Fc-fusion proteins are an emerging class of protein therapeutics that combine the properties of biological ligands with the unique properties of the fragment crystallizable (Fc) domain of an immunoglobulin G (IgG). Due to their diverse higher-order structures (HOSs), Fc-fusion proteins remain challenging characterization targets within biopharmaceutical pipelines. While high-resolution biophysical tools are available for HOS characterization, they frequently demand extended time frames and substantial quantities of purified samples, rendering them impractical for swiftly screening candidate molecules. Herein, we describe the development of ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU) workflows that aim to fill this technology gap, where we focus on probing the HOS of a model Fc-Interleukin-10 (Fc-IL-10) fusion protein engineered using flexible glycine-serine linkers. We evaluate the ability of these techniques to probe the flexibility of Fc-IL-10 in the absence of bulk solvent relative to other proteins of similar size, as well as localize structural changes of low charge state Fc-IL-10 ions to specific Fc and IL-10 unfolding events during CIU. We subsequently apply these tools to probe the local effects of glycine-serine linkers on the HOS and stability of IL-10 homodimer, which is the biologically active form of IL-10. Our data reveals that Fc-IL-10 produces significantly more structural transitions during CIU and broader IM profiles when compared to a wide range of model proteins, indicative of its exceptional structural dynamism. Furthermore, we use a combination of enzymatic approaches to annotate these intricate CIU data and localize specific transitions to the unfolding of domains within Fc-IL-10. Finally, we detect a strong positive, quadratic relationship between average linker mass and fusion protein stability, suggesting a cooperative influence between glycine-serine linkers and overall fusion protein stability. This is the first reported study on the use of IM-MS and CIU to characterize HOS of Fc-fusion proteins, illustrating the practical applicability of this approach.

Use of Raman spectroscopy and PCA for quality evaluation and out-of-specification identification in biopharmaceutical products.

Over the past three decades, there was a remarkable growth in the approval of antibody-based biopharmaceutical products. These molecules are notably susceptible to the stresses occurring during drug manufacturing, often leading to structural alterations. A key concern is thus the ability to detect and comprehend these alterations caused by processes, such as aggregation, fragmentation, oxidation levels, as well as the change in protein concentration throughout the process steps, potentially resulting in out-of-spec products. In the present study, Raman spectroscopy, coupled with Principal Component Analysis (PCA), has proven to be an excellent tool for characterizing protein-based products. Notably, it offers the advantages of being minimally invasive, rapid and relatively insensitive to water. Therefore, it was successfully employed to discriminate between various stresses impacting a monoclonal antibody (mAb). The molecule used in this study is a fully human IgG1 fusion protein. Thermal stress was induced by incubating the samples at 50 °C for one month, while oxidative stress was induced by introducing hydrogen peroxide. Additionally, dilutions were performed to explore a broader range of protein concentrations. Specific key bands were identified in the Raman spectra, which facilitated the PCA classification and allowed for their association with distinct changes in the secondary and tertiary structures of the protein. Notably, it was observed that signals corresponding to amino acids exhibited a decrease in intensity with increasing levels of thermal stress, while other alterations were noted in the amide bands. It was shown that changes in the range 2800-3000 cm-1 pertains to the dilution process, while specific peaks of C-H stretching were essential for the discrimination between the oxidative-stressed samples and the thermal and diluted counterparts. Furthermore, the model calibrated on the mAb demonstrated remarkable performance when used to evaluate a different product, e.g. a hormone.

An inert tracer: The binding site of a fluorescent dye on the antibody and its effects on Protein A chromatography.

Fluorescently labeled antibodies are widely used to visualize the adsorption process in protein chromatography using confocal laser scanning microscopy (CLSM), but also as a tracer for determination of residence time distribution (RTD) in continuous chromatography. It is assumed that the labeled protein is inert and representative of the unlabeled antibody, ignoring the fact that labeling with a fluorescent dye can change the characteristics of the original molecule. It became evident that the fluorescently labeled antibody has a higher affinity toward protein A resins such as MabSelect Sure. This can be due to slight differences in hydrophobicity and net charge, which are caused by the addition of the fluorescent dye. However, this difference is eliminated when using high salt concentrations in the adsorption studies. In this work, the site occupancy of two labeled antibodies, MAb1 (IgG1 subclass) and MAb2 (IgG2 subclass) conjugated with the fluorescent dye Alexa Fluor™ 488 was elucidated by intact mass spectrometry (MS) and peptide mapping LC-MS/MS, employing a sequential cleavage with Endoproteinase Lys-C and trypsin and in parallel with chymotrypsin alone. It was shown that the main binding site for the dye was a specific lysine in the heavy chains of the MAb1 and MAb2 molecules, in positions 188 and 189 respectively. Other lysine residues distributed throughout the protein sequence were labeled to a lot lesser extent. The labeled antibody had a slightly different affinity to MabSelect Sure although its primary binding site (to Protein A) was not affected by labeling, despite the secondary region responsible for binding to the protein A was partly labeled. Overall, the fluorescent-labeled antibodies are a good compromise as an inert tracer in residence time distribution and chromatography studies because they are much cheaper than isotope-labeled antibodies; However, the differences between the labeled and unlabeled antibodies should be considered.

Direct Affinity Ligand Immobilization onto Bare Iron Oxide Nanoparticles Enables Efficient Magnetic Separation of Antibodies.

Magnetic separation is a promising alternative to chromatography for enhancing the downstream processing (DSP) of monoclonal antibodies (mAbs). However, there is a lack of efficient magnetic particles for successful application. Aiming to fill this gap, we demonstrate the suitability of bare iron oxide nanoparticles (BION) with physical site-directed immobilization of an engineered Protein A affinity ligand (rSpA) as an innovative magnetic material. The rSpA ligand contains a short peptide tag that enables the direct and stable immobilization onto the uncoated BION surface without commonly required laborious particle activation. The resulting BION@rSpA have beneficial characteristics outperforming conventional Protein A-functionalized magnetic particles: a simple, fast, low-cost synthesis, a particle size in the nanometer range with a large effective specific surface area enabling large immunoglobulin G (IgG) binding capacity, and a high magnetophoretic velocity advantageous for fast processing. We further show rapid interactions of IgG with the easily accessible rSpA ligands. The binding of IgG to BION@rSpA is thereby highly selective and not impeded by impurity molecules in perfusion cell culture supernatant. Regarding the subsequent acidic IgG elution from BION@rSpA@IgG, we observed a hampering pH increase caused by the protonation of large iron oxide surfaces after concentrating the particles in 100 mM sodium acetate buffer. However, the pH can be stabilized by adding 50 mM glycine to the elution buffer, resulting in recoveries above 85% even at high particle concentrations. Our work shows that BION@rSpA enable efficient magnetic mAb separation and could help to overcome emerging bottlenecks in DSP.

DNA Nanostructure-Templated Antibody Complexes Provide Insights into the Geometric Requirements of Human Complement Cascade Activation.

The classical complement pathway is activated by antigen-bound IgG antibodies. Monomeric IgG must oligomerize to activate complement via the hexameric C1q complex, and hexamerizing mutants of IgG appear as promising therapeutic candidates. However, structural data have shown that it is not necessary to bind all six C1q arms to initiate complement, revealing a symmetry mismatch between C1 and the hexameric IgG complex that has not been adequately explained. Here, we use DNA nanotechnology to produce specific nanostructures to template antigens and thereby spatially control IgG valency. These DNA-nanotemplated IgG complexes can activate complement on cell-mimetic lipid membranes, which enabled us to determine the effect of IgG valency on complement activation without the requirement to mutate antibodies. We investigated this using biophysical assays together with 3D cryo-electron tomography. Our data revealed the importance of interantigen distance on antibody-mediated complement activation, and that the cleavage of complement component C4 by the C1 complex is proportional to the number of ideally spaced antigens. Increased IgG valency also translated to better terminal pathway activation and membrane attack complex formation. Together, these data provide insights into how nanopatterning antigen-antibody complexes influence the activation of the C1 complex and suggest routes to modulate complement activation by antibody engineering. Furthermore, to our knowledge, this is the first time DNA nanotechnology has been used to study the activation of the complement system.

Protein loss during membrane processes in biopharmaceutical manufacturing.

Maximizing product yield in biopharmaceutical manufacturing processes is a critical factor in determining the overall cost of goods, especially given the high value of these biological products. However, there has been relatively limited research on the quantitative analysis of protein losses due to adsorption and fouling during the different membrane filtration processes employed in typical downstream operations. This study aims to provide a comprehensive analysis of protein loss in the range of membrane systems used in downstream processing including clarification, virus removal filtration, ultrafiltration/diafiltration for formulation, and final sterile filtration, all using commercially available membranes with three model proteins (bovine serum albumin, human serum albumin, and immunoglobulin G). The correlation between protein loss and various parameters (i.e., protein type, protein concentration, throughput, membrane morphology, and protein removal mechanism) was also investigated. This study provides important insights into the nature of protein loss during membrane processes as well as a methodology for quantifying protein yield loss in bioprocesses.

[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.

Transforming waste into valuables: Preparation and evaluation of dual-ligand hydrophobic charge-induction chromatography using two poor performing ligands.

In conventional chromatographic ligand screening, underperforming ligands are often dismissed. However, this practice may inadvertently overlook potential opportunities. This study aims to investigate whether these underperforming ligands can be repurposed as valuable assets. Hydrophobic charge-induction chromatography (HCIC) is chosen as the validation target for its potential as an innovative chromatographic mode. A novel dual-ligand approach is employed, combining two suboptimal ligands (5-Aminobenzimidazole and Tryptamine) to explore enhanced performance and optimization prospects. Various dual-ligand HCIC resins with different ligand densities were synthesized by adjusting the ligand ratio and concentration. The resins were characterized to assess appearance, functional groups, and pore features using SEM, FTIR, and ISEC techniques. Performance assessments were conducted using single-ligand mode resins as controls, evaluating the selectivity against human immunoglobulin G and human serum albumin. Static adsorption experiments were performed to understand pH and salt influence on adsorption. Breakthrough experiments were conducted to assess dynamic adsorption capacity of the novel resin. Finally, chromatographic separation using human serum was performed to evaluate the purity and yield of the resin. Results indicated that the dual-ligand HCIC resin designed for human antibodies demonstrates exceptional selectivity, surpassing not only single ligand states but also outperforming certain high-performing ligand types, particularly under specific salt and pH conditions. Ultimately, a high yield of 83.9 % and purity of 96.7 % were achieved in the separation of hIgG from human serum with the dual-ligand HCIC, significantly superior to the single-ligand resins. In conclusion, through rational design and proper operational conditions, the dual-ligand mode can revitalize underutilized ligands, potentially introducing novel and promising chromatographic modes.

Site-selective antibody-lipid conjugates for surface functionalization of red blood cells and targeted drug delivery.

Displaying antibodies on carrier surfaces facilitates precise targeting and delivery of drugs to diseased cells. Here, we report the synthesis of antibody-lipid conjugates (ALCs) through site-selective acetylation of Lys 248 in human Immunoglobulin G (IgG) and the development of antibody-functionalized red blood cells (immunoRBC) for targeted drug delivery. ImmunoRBC with the HER2-selective antibody trastuzumab displayed on the surface (called Tras-RBC) was constructed following a three-step procedure. First, a peptide-guided, proximity-induced reaction transferred an azidoacetyl group to the ε-amino group of Lys 248 in the Fc domain. Second, the azide-modified IgG was subsequently conjugated with dibenzocyclooctyne (DBCO)-functionalized lipids via strain-promoted azide-alkyne cycloaddition (SPAAC) to result in ALCs. Third, the lipid portion of ALCs was then inserted into the cell membranes, and IgGs were displayed on red blood cells (RBCs) to construct immunoRBCs. We then loaded Tras-RBC with a photosensitizer (PS), Zinc phthalocyanine (ZnPc), to selectively target HER2-overexpressing cells, release ZnPc into cancer cells following photolysis, and induce photodynamic cytotoxicity in the cancer cells. This work showcases assembling immunoRBCs following site-selective lipid conjugation on therapeutic antibodies and the targeted introduction of PS into cancer cells. This method could apply to the surface functionalization of other membrane-bound vesicles or lipid nanoparticles for antibody-directed drug delivery.

Protein-Centric Analysis of Personalized Antibody Repertoires Using LC-MS-Based Fab-Profiling on a timsTOF.

Endogenous antibodies, or immunoglobulins (Igs), abundantly present in body fluids, represent some of the most challenging samples to analyze, largely due to the immense variability in their sequences and concentrations. It has been estimated that our body can produce billions of different Ig proteins with different isotypes, making their individual analysis seemingly impossible. However, recent advances in protein-centric proteomics using LC-MS coupled to Orbitrap mass analyzers to profile intact Fab fragments formed by selective cleavage at the IgG-hinge revealed that IgG repertoires may be less diverse, albeit unique for each donor. Serum repertoires seem to be dominated by a few hundred clones that cumulatively make up 50-95% of the total IgG content. Enabling such analyses required careful optimization of the chromatography and mass analysis, as all Fab analytes are highly alike in mass (46-51 kDa) and sequence. To extend the opportunities of this mass-spectrometry-based profiling of antibody repertoires, we here report the optimization and evaluation of an alternative MS platform, namely, the timsTOF, for antibody repertoire profiling. The timsTOF mass analyzer has gained traction in recent years for peptide-centric proteomics and found wide applicability in plasma proteomics, affinity proteomics, and HLA peptidomics, to name a few. However, for protein-centric analysis, this platform has been less explored. Here, we demonstrate that the timsTOF platform can be adapted to perform protein-centric LC-MS-based profiling of antibody repertoires. In a side-by-side comparison of the timsTOF and the Orbitrap we demonstrate that the extracted serum antibody repertoires are alike qualitatively and quantitatively, whereby in particular the sensitivity of the timsTOF platform excels. Future incorporation of advanced top-down capabilities on the timsTOF may make this platform a very valuable alternative for protein-centric proteomics and top-down proteomics and thus also for personalized antibody repertoire profiling.

AptaShield: A Universal Signal-Transduction System for Fast and High-Throughput Optical Molecular Biosensing.

Biosensing technologies are often described to provide facile, sensitive, and minimally to noninvasive detection of molecular analytes across diverse scientific, environmental, and clinical diagnostic disciplines. However, commercialization has been very limited mostly due to the difficulty of biosensor reconfiguration for different analyte(s) and limited high-throughput capabilities. The immobilization of different biomolecular probes (e.g., antibodies, peptides, and aptamers) requires the sensor surface chemistry to be tailored to provide optimal probe coupling, orientation, and passivation and prevent nonspecific interactions. To overcome these challenges, here we report the development of a solution-phase biosensor consisting of an engineered aptamer, the AptaShield, capable of universally binding to any antigen recognition site (Fab') of fluorescently labeled immunoglobulins (IgG) produced in rabbits. The resulting AptaShield biosensor relies on a low affinity dynamic equilibrium between the fluorescently tagged aptamer and IgG to generate a specific Förster resonance energy transfer (FRET) signal. As the analyte binds to the IgG, the AptaShield DNA aptamer-IgG complex dissociates, leading to an analyte concentration-dependent decrease of the FRET signal. The biosensor demonstrates high selectivity, specificity, and reproducibility for analyte quantification in different biological fluids (e.g., urine and blood serum) in a one-step and low sample volume (0.5-6.25 µL) format. The AptaShield provides a universal signal transduction mechanism as it can be coupled to different rabbit antibodies without the need for aptamer modification, therefore representing a robust high-throughput solution-phase technology suitable for point-of-care applications, overcoming the current limitations of gold standard enzyme-linked immunosorbent assays (ELISA) for molecular profiling.

The IgG-specific endoglycosidases EndoS and EndoS2 are distinguished by conformation and antibody recognition.

The IgG-specific endoglycosidases EndoS and EndoS2 from Streptococcus pyogenes can remove conserved N-linked glycans present on the Fc region of host antibodies to inhibit Fc-mediated effector functions. These enzymes are therefore being investigated as therapeutics for suppressing unwanted immune activation, and have additional application as tools for antibody glycan remodeling. EndoS and EndoS2 differ in Fc glycan substrate specificity due to structural differences within their catalytic glycosyl hydrolase domains. However, a chimeric EndoS enzyme with a substituted glycosyl hydrolase from EndoS2 loses catalytic activity, despite high structural homology between the two enzymes, indicating either mechanistic divergence of EndoS and EndoS2, or improperly-formed domain interfaces in the chimeric enzyme. Here, we present the crystal structure of the EndoS2-IgG1 Fc complex determined to 3.0 Å resolution. Comparison of complexed and unliganded EndoS2 reveals relative reorientation of the glycosyl hydrolase, leucine-rich repeat and hybrid immunoglobulin domains. The conformation of the complexed EndoS2 enzyme is also different when compared to the earlier EndoS-IgG1 Fc complex, and results in distinct contact surfaces between the two enzymes and their Fc substrate. These findings indicate mechanistic divergence of EndoS2 and EndoS. It will be important to consider these differences in the design of IgG-specific enzymes, developed to enable customizable antibody glycosylation.

Controlling nanoparticle-induced endothelial leakiness with the protein corona.

Understanding nanoparticle-cell interaction is essential for advancing research in nanomedicine and nanotoxicology. Apart from the transcytotic pathway mediated by cellular recognition and energetics, nanoparticles (including nanomedicines) may harness the paracellular route for their transport by inducing endothelial leakiness at cadherin junctions. This phenomenon, termed as NanoEL, is correlated with the physicochemical properties of the nanoparticles in close association with cellular signalling, membrane mechanics, as well as cytoskeletal remodelling. However, nanoparticles in biological systems are transformed by the ubiquitous protein corona and yet the potential effect of the protein corona on NanoEL remains unclear. Using confocal fluorescence microscopy, biolayer interferometry, transwell, toxicity, and molecular inhibition assays, complemented by molecular docking, here we reveal the minimal to significant effects of the anionic human serum albumin and fibrinogen, the charge neutral immunoglobulin G as well as the cationic lysozyme on negating gold nanoparticle-induced endothelial leakiness in vitro and in vivo. This study suggests that nanoparticle-cadherin interaction and hence the extent of NanoEL may be partially controlled by pre-exposing the nanoparticles to plasma proteins of specific charge and topology to facilitate their biomedical applications.

Exploring the effects of resin particle sizes on enhancing antibody binding capacity of a hybrid biomimetic ligand.

Particle size is a critical parameter of chromatographic resins that significantly affects protein separation. In this study, effects of resin particle sizes (31.26 µm, 59.85 µm and 85.22 µm named Aga-31, Aga-60 and Aga-85, respectively) on antibody adsorption capacity and separation performance of a hybrid biomimetic ligand were evaluated. Their performance was investigated through static adsorption and breakthrough assays to quantify static and dynamic binding capacity (Qmax and DBC). The static adsorption results revealed that the Qmax for hIgG was 152 mg/g resin with Aga-31, 151 mg/g resin with Aga-60, and 125 mg/g resin with Aga-85. Moreover, the DBC at 10% breakthrough for hIgG with a residence time of 2 min was determined to be 49.4 mg/mL for Aga-31, 45.9 mg/mL for Aga-60, and 38.9 mg/mL for Aga-85. The resins with smaller particle sizes exhibited significantly higher capacity compared to typical commercial agarose resins and a Protein A resin (MabSelect SuRe). Furthermore, the Aga-31 resin with the hybrid biomimetic ligand demonstrated exceptional performance in terms of IgG purity (>98%) and recovery (>96%) after undergoing 20 separation cycles from CHO cell supernatant. These findings are helpful in further chromatographic resin design for the industrial application of antibody separation and purification.

Purification of monoclonal antibodies using novel 3D printed ordered stationary phases.

3D printing offers the unprecedented ability to fabricate chromatography stationary phases with bespoke 3D morphology as opposed to traditional packed beds of spherical beads. The restricted range of printable materials compatible with chromatography is considered a setback for its industrial implementation. Recently, we proposed a novel ink that exhibits favourable printing performance (printing time ∼100 mL/h, resolution ∼200 µm) and broadens the possibilities for a range of chromatography applications thanks to its customisable surface chemistry. In this work, this ink was used to fabricate 3D printed ordered columns with 300 µm channels for the capture and polishing of therapeutic monoclonal antibodies. The columns were initially assessed for leachables and extractables, revealing no material propensity for leaching. Columns were then functionalised with protein A and SO3 ligands to obtain affinity and strong cation exchangers, respectively. 3D printed protein A columns showed >85 % IgG recovery from harvested cell culture fluid with purities above 98 %. Column reusability was evaluated over 20 cycles showing unaffected performance. Eluate samples were analysed for co-eluted protein A fragments, host cell protein and aggregates. Results demonstrate excellent HCP clearance (logarithmic reduction value of > 2.5) and protein A leakage in the range of commercial affinity resins (<100 ng/mg). SO3 functionalised columns employed for polishing achieved removal of leaked Protein A (down to 10 ng/mg) to meet regulatory expectations of product purity. This work is the first implementation of 3D printed columns for mAb purification and provides strong evidence for their potential in industrial bioseparations.