Continuous Monitoring of IgG using Immobilized Fluorescent Reporters

In the manufacture of therapeutic monoclonal antibodies (mAbs), the clarified cell culture fluid 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 IgG loading to avoid wastage. Therefore, continuous real-time monitoring of IgG flowthrough is of great interest. We previously developed a fluorescence-based monitoring technology that allows mix-and-read mAb detection in cell culture fluid. Here we report the use of reporters immobilized on CNBr-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 to produce an immediately detectable change in fluorescence intensity. The technology allows rapid and reliable monitoring of IgG in a flowing stream of clarified cell culture fluid 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 2 minutes at a flow rate of 0.5 mL/min.

The cellular prion protein interacts with and promotes the activity of Na,K-ATPases

The prion protein (PrP) is best known for its ability to cause fatal neurodegenerative diseases in humans and animals. Here, we revisited its molecular environment in the brain using a well-developed affinity-capture mass spectrometry workflow that offers robust relative quantitation. The analysis confirmed many previously reported interactions. It also pointed toward a profound enrichment of Na,K-ATPases (NKAs) in proximity to cellular PrP (PrPC). Follow-on work validated the interaction, demonstrated partial co-localization of the ATP1A1 and PrPC, and revealed that cells exposed to cardiac glycoside (CG) inhibitors of NKAs exhibit correlated changes to the steady-state levels of both proteins. Moreover, the presence of PrPC was observed to promote the ion uptake activity of NKAs in a human co-culture paradigm of differentiated neurons and glia cells, and in mouse neuroblastoma cells. Consistent with this finding, changes in the expression of 5’-nucleotidase that manifest in wild-type cells in response to CG exposure can also be observed in untreated PrPC-deficient cells. Finally, the endoproteolytic cleavage of the glial fibrillary acidic protein, a hallmark of late-stage prion disease, can also be induced by CGs, raising the prospect that a loss of NKA activity may contribute to the pathobiology of prion diseases.

Affinity sedimentation and magnetic separation with plant-made immunosorbent nanoparticles for therapeutic protein purification

The virus-based immunosorbent nanoparticle is a nascent technology being developed to serve as a simple and efficacious agent in biosensing and therapeutic antibody purification. There has been particular emphasis on the use of plant virions as immunosorbent nanoparticle chassis for their diverse morphologies and accessible, high yield manufacturing via crop cultivation. To date, studies in this area have focused on proof-of-concept immunosorbent functionality in biosensing and purification contexts. Here we consolidate a previously reported pro-vector system into a single Agrobacterium tumefaciens vector to investigate and expand the utility of virus-based immunosorbent nanoparticle technology for therapeutic protein purification. We demonstrate the use of this technology for Fc-fusion protein purification, characterize key nanomaterial properties including binding capacity, stability, reusability, and particle integrity, and present an optimized processing scheme with reduced complexity and increased purity. Furthermore, we present a coupling of virus-based immunosorbent nanoparticles with magnetic particles as a strategy to overcome limitations of the immunosorbent nanoparticle sedimentation-based affinity capture methodology. We report magnetic separation results which exceed the binding capacity of current industry standards by an order of magnitude.

Quantification for Antibody-Conjugated Drug in Trastuzumab Emtansine and Application to In Vitro Linker Stability and In Vivo Pharmacokinetic Study in Rat Using an Immuno-Affinity Capture Liquid Chromatography-Mass Spectrometric Method

Trastuzumab emtansine (T-DM1, brand name: Kadcyla®) is the first FDA-approved antibody-drug conjugate (ADC) for metastatic human epidermal growth factor receptor 2 positive (HER2+) breast cancer. It consists of three components: trastuzumab, an anti-HER2 monoclonal antibody, maytansinoid (DM1) as a cytotoxic drug, and maleimidomethyl cyclohexane-1-carboxylate (MCC) as a linker. In particular, the MCC linker is known as a non-cleavable linker and has a feature of being conjugated to DM1 by a covalent thioether bond. In this study, we developed an immuno-affinity capture liquid chromatography-mass spectrometric (LC-MS/MS) assay for quantifying the antibody-conjugated drug (acDrug) component of T-DM1. To quantify acDrug, desulfurated DM1 was prepared using a chemical desulfuration pretreatment and quantified as an acDrug. A quadratic regression (weighted 1/concentration), with equation y = ax2 + bx + c, was used to fit the calibration curves over the concentration range of 17.09~1709.44 ng/mL for the acDrug of T-DM1. The quantification run met the in-house acceptance criteria of ±25% accuracy and precision values for the quality control (QC) samples. In conclusion, an immuno-affinity capture LC-MS/MS assay was successfully developed to quantify acDrug of T-DM1 and applied to evaluate in vitro plasma linker stability and preclinical pharmacokinetic (PK) study in rats. This assay could be helpful when applied to other ADCs with the same linker-cytotoxic drug platform.

Cell-Penetrating D-Peptides Retain Antisense Morpholino Oligomer Delivery Activity

Cell-penetrating peptides (CPPs) can cross the cell membrane to enter the cytosol and deliver otherwise non-penetrant macromolecules such as proteins and oligonucleotides. For example, recent clinical trials have shown that a CPP attached to phosphorodiamidate morpholino oligomers (PMO) resulted in higher muscle concentration, increased exon-skipping and dystrophin production relative to another study of the PMO alone in patients of Duchenne muscular dystrophy. Therefore, effective design and study of CPPs could help enhance therapies for difficult-to-treat diseases. So far, the study of CPPs for PMO delivery has been restricted to predominantly canonical L-peptides. We hypothesized that mirror-image D-peptides could have similar PMO delivery activity as well as enhanced proteolytic stability, facilitating their characterization and quantification from biological milieu. We found that several enantiomeric peptide sequences could deliver a PMO-biotin cargo with similar activities, while remaining stable against serum proteolysis. The biotin label allowed for affinity capture of fully intact PMO-peptide conjugates from whole cell and cytosolic lysates. By profiling a mixture of these constructs in cells, we determined their relative intracellular concentrations. When combined with PMO activity, these concentrations provide a new metric for delivery efficiency which may be useful for determining which peptide sequence to pursue in further pre-clinical studies.Abstract Figure

Immunofluorescence of RAB5 and FLAG-EEA1 puncta after Dynamin-1 and -2 inhibition with Dyngo4a v1

Selective purification of early endosomes can be achieved through affinity capture of the early endosome-associated protein EEA1 (termed Endo-IP) (Park et al. in submission). These purified endosomes can be used for proteomic and lipidomic studies to obtain snapshots of early endosomes. Here we present an immunofluorescence protocol to assess the extent of colocalization between FLAG-EEA1 and RAB5 with and without the Dynamin-1 and -2 (DNM1/2) inhibitor Dyngo4a.

Plant Stress Granules: Trends and Beyond

Stress granules (SGs) are dynamic membrane-less condensates transiently assembled through liquid–liquid phase separation (LLPS) in response to stress. SGs display a biphasic architecture constituted of core and shell phases. The core is a conserved SG fraction fundamental for its assembly and consists primarily of proteins with intrinsically disordered regions and RNA-binding domains, along with translational-related proteins. The shell fraction contains specific SG components that differ among species, cell type, and developmental stage and might include metabolic enzymes, receptors, transcription factors, untranslated mRNAs, and small molecules. SGs assembly positively correlates with stalled translation associated with stress responses playing a pivotal role during the adaptive cellular response, post-stress recovery, signaling, and metabolic rewire. After stress, SG disassembly releases mRNA and proteins to the cytoplasm to reactivate translation and reassume cell growth and development. However, under severe stress conditions or aberrant cellular behavior, SG dynamics are severely disturbed, affecting cellular homeostasis and leading to cell death in the most critical cases. The majority of research on SGs has focused on yeast and mammals as model organism. Nevertheless, the study of plant SGs has attracted attention in the last few years. Genetics studies and adapted techniques from other non-plant models, such as affinity capture coupled with multi-omics analyses, have enriched our understanding of SG composition in plants. Despite these efforts, the investigation of plant SGs is still an emerging field in plant biology research. In this review, we compile and discuss the accumulated progress of plant SGs regarding their composition, organization, dynamics, regulation, and their relation to other cytoplasmic foci. Lastly, we will explore the possible connections among the most exciting findings of SGs from mammalian, yeast, and plants, which might help provide a complete view of the biology of plant SGs in the future.

Affinity Tag Coating Enables Reliable Detection of Antigen-Specific B Cells in Immunospot Assays

Assessment of humoral immunity to SARS-CoV-2 and other infectious agents is typically restricted to detecting antigen-specific antibodies in the serum. Rarely does immune monitoring entail assessment of the memory B-cell compartment itself, although it is these cells that engage in secondary antibody responses capable of mediating immune protection when pre-existing antibodies fail to prevent re-infection. There are few techniques that are capable of detecting rare antigen-specific B cells while also providing information regarding their relative abundance, class/subclass usage and functional affinity. In theory, the ELISPOT/FluoroSpot (collectively ImmunoSpot) assay platform is ideally suited for antigen-specific B-cell assessments since it provides this information at single-cell resolution for individual antibody-secreting cells (ASC). Here, we tested the hypothesis that antigen-coating efficiency could be universally improved across a diverse set of viral antigens if the standard direct (non-specific, low affinity) antigen absorption to the membrane was substituted by high-affinity capture. Specifically, we report an enhancement in assay sensitivity and a reduction in required protein concentrations through the capture of recombinant proteins via their encoded hexahistidine (6XHis) affinity tag. Affinity tag antigen coating enabled detection of SARS-CoV-2 Spike receptor binding domain (RBD)-reactive ASC, and also significantly improved assay performance using additional control antigens. Collectively, establishment of a universal antigen-coating approach streamlines characterization of the memory B-cell compartment after SARS-CoV-2 infection or COVID-19 vaccinations, and facilitates high-throughput immune-monitoring efforts of large donor cohorts in general.

Identification of Fucosylated SERPINA1 as a Novel Plasma Marker for Pancreatic Cancer Using Lectin Affinity Capture Coupled with iTRAQ-Based Quantitative Glycoproteomics

Pancreatic cancer (PC) is an aggressive cancer with a high mortality rate, necessitating the development of effective diagnostic, prognostic and predictive biomarkers for disease management. Aberrantly fucosylated proteins in PC are considered a valuable resource of clinically useful biomarkers. The main objective of the present study was to identify novel plasma glycobiomarkers of PC using the iTRAQ quantitative proteomics approach coupled with Aleuria aurantia lectin (AAL)-based glycopeptide enrichment and isotope-coded glycosylation site-specific tagging, with a view to analyzing the glycoproteome profiles of plasma samples from patients with non-metastatic and metastatic PC and gallstones (GS). As a result, 22 glycopeptides with significantly elevated levels in plasma samples of PC were identified. Fucosylated SERPINA1 (fuco-SERPINA1) was selected for further validation in 121 plasma samples (50 GS and 71 PC) using an AAL-based reverse lectin ELISA technique developed in-house. Our analyses revealed significantly higher plasma levels of fuco-SERPINA1 in PC than GS subjects (310.7 ng/mL v.s. 153.6 ng/mL, p = 0.0114). Elevated fuco-SERPINA1 levels were associated with higher TNM stage (p = 0.024) and poorer prognosis for overall survival (log-rank test, p = 0.0083). The increased plasma fuco-SERPINA1 levels support the utility of this protein as a novel prognosticator for PC.

Affinity tag coating enables reliable detection of antigen-specific B cells in ImmunoSpot assays

Assessment of humoral immunity to SARS-CoV-2 and other infectious agents is typically restricted to detecting antigen-specific antibody in the serum. Rarely does immune monitoring entail assessment of the memory B cell compartment itself, although it is these cells that engage in secondary antibody responses capable of mediating immune protection when pre-existing antibodies fail to prevent re-infection. There are few techniques that are capable of detecting rare antigen-specific B cells while also providing information regarding their precursory frequency, class/subclass usage and functional affinity. In theory, the ELISPOT/FluoroSpot (collectively ImmunoSpot) assay platform is ideally-suited for antigen-specific B cell assessments since it provides this information at single-cell resolution for individual antibody-secreting cells (ASC). Here, we tested the hypothesis that antigen coating efficiency could be universally improved across a diverse set of viral antigens if the standard direct (non-specific, low affinity) antigen absorption to the membrane was substituted by high affinity capture. Specifically, we report an enhancement in assay sensitivity and a reduction in required protein concentrations through the capture of recombinant proteins via their encoded hexahistidine (6XHis) affinity tag. Affinity tag antigen coating enabled detection of SARS-CoV-2 Spike receptor binding domain (RBD)-reactive ASC, and also significantly improved assay performance using additional control antigens. Collectively, establishment of a universal antigen coating approach streamlines characterization of the memory B cell compartment after SARS-CoV-2 infection or COVID-19 vaccinations, and facilitates high-throughput immune monitoring efforts of large donor cohorts in general.