Assessment of the role of afucosylated glycoforms on the in vitro antibody-dependent phagocytosis activity of an antibody to Aß aggregates.

The terminal sugars of Fc glycans can influence the Fc-dependent biological activities of monoclonal antibody therapeutics. Afucosylated N-glycans have been shown to significantly alter binding to FcγRIIIa and affect antibody-dependent cell-mediated cytotoxicity (ADCC). Therefore, in order to maintain and ensure safety and efficacy for antibodies whose predominant mechanism of action (MOA) is ADCC, afucosylation is routinely monitored and controlled within appropriate limits. However, it is unclear how the composition and levels of afucosylated N-glycans can modulate the biological activities for a recombinant antibody whose target is not a cell surface receptor, as is the case with ADCC. The impact of different types and varying levels of enriched afucosylated N-glycan species on the in vitro bioactivities is assessed for an antibody whose target is aggregated amyloid beta (Aß). While either the presence of complex biantennary or high mannose afucosylated glycoforms significantly increased FcγRIIIa binding activity compared to fucosylated glycoforms, they did not similarly increase aggregated Aß uptake activity mediated by different effector cells. These experiments suggest that afucosylated N-glycans are not critical for the in vitro phagocytic activity of a recombinant antibody whose target is aggregated Aß and uses Fc effector function as part of its MOA.

Biolayer Interferometry-based FcγRIIa binding assay for a therapeutic antibody with strong effector function.

FcγRIIa receptor binding is part of the mechanism of action for many therapeutic antibodies. AlphaScreen® technology and Biolayer Interferometry (BLI) are often used to assess protein-protein interactions. Recently we demonstrated that the presence of aggregates in samples significantly increased binding potency values in AlphaScreen®-based FcRn binding assays, sometimes masking the loss of potency. Even bigger effect of aggregates was observed in an AlphaScreen®-based FcγRIIa binding assay for a monoclonal antibody with strong effector function. To resolve this issue a novel BLI-based FcγRIIa binding assay was developed and qualified. The assay measures association binding responses and calculates the binding potency of the samples relative to the standard using Parallel Line Analysis. The method overcomes interference of aggregates present in the samples, distinguishes different Fc glycosylation patterns, and is stability-indicating. It can be used for sample characterization, drug product release and stability testing.

Integration of Fluorescence Detection and Image-Based Automated Counting Increases Speed, Sensitivity, and Robustness of Plaque Assays.

Plaque assays are used to measure the infectious titer of viral samples. These assays are multi-day and low-throughput and may be subject to analyst variability from biased or subjective manual plaque counting. Typically, on day 1, cells are adhered to plates overnight. On day 2, cells are infected with virus. After 3 additional days, plaques are fixed, stained with a horseradish peroxidase (HRP)-conjugated antibody and a HRP substrate, and counted by eye. Manual-based visual counting of plaques is time-consuming and laborious and may be subject to variability between analysts. Also, the assay must proceed for several days to allow the plaques to increase to sufficiently large sizes for manual identification. Here, we integrate fluorescent detection and automated plaque counting to increase the sensitivity and speed of the assay. First, we stain plaques with a fluorescent-labeled antibody. Second, we implement a plate-based cell imager to perform non-biased, non-subjective plaque counting. The integration of these two technologies decreases the assay length by 40%, from 5 days to 3 days, because plaque size, plaque signal to noise, and manual visualization are no longer limiting. This optimized plaque assay is sensitive, fast, and robust and expands the throughput and usage of this method for measuring plaque formation.

Challenging the workhorse: Comparative analysis of eukaryotic micro-organisms for expressing monoclonal antibodies.

For commercial protein therapeutics, Chinese hamster ovary (CHO) cells have an established history of safety, proven capability to express a wide range of therapeutic proteins and high volumetric productivities. Expanding global markets for therapeutic proteins and increasing concerns for broadened access of these medicines has catalyzed consideration of alternative approaches to this platform. Reaching these objectives likely will require an order of magnitude increase in volumetric productivity and a corresponding reduction in the costs of manufacture. For CHO-based manufacturing, achieving this combination of targeted improvements presents challenges. Based on a holistic analysis, the choice of host cells was identified as the single most influential factor for both increasing productivity and decreasing costs. Here we evaluated eight wild-type eukaryotic micro-organisms with prior histories of recombinant protein expression. The evaluation focused on assessing the potential of each host, and their corresponding phyla, with respect to key attributes relevant for manufacturing, namely (a) growth rates in industry-relevant media, (b) adaptability to modern techniques for genome editing, and (c) initial characterization of product quality. These characterizations showed that multiple organisms may be suitable for production with appropriate engineering and development and highlighted that yeast in general present advantages for rapid genome engineering and development cycles.

Isolation and Characterization of Monoclonal Antibody Charge Variants by Free Flow Isoelectric Focusing.

Capillary isoelectric focusing (cIEF) is widely used in the biopharmaceutical industry to measure the charge distribution of therapeutic proteins. The implementation of this technology has created a new challenge. Capillary volumes are on the order of hundreds of nanoliters and cannot be scaled up for the preparative collection of charge variants. This makes it difficult to identify the charge variants in a cIEF electropherogram. Therefore, preparative IEF methods are needed to fractionate charge variants for characterization. We used free-flow electrophoresis (FFE) to isolate monoclonal antibody charge variants observed in a cIEF electropherogram. The same antibody was also fractionated using the Rotofor and Offgel instruments for comparison. A strategy for purifying the fractionated charge variants and downstream characterization is described. Acidic and basic variants were identified and related back to the analytical cIEF charge profile. This study establishes free-flow isoelectric focusing as a valuable tool for characterizing therapeutic proteins.

A simple enzyme-substrate localized conjugation method to generate immobilized, functional glutathione S-transferase fusion protein columns for affinity enrichment.

Immobilized protein receptors and enzymes are tools for isolating or enriching ligands and substrates based on affinity. For example, glutathione S-transferase (GST) is fused to proteins as a tag for binding to its substrate glutathione (GSH) linked to solid supports. One issue with this approach is that high-affinity interactions between receptors and ligands require harsh elution conditions such as low pH, which can result in leached receptor. Another issue is the inherent nonspecific chemical conjugation of reactive groups such as N-hydroxysuccinimide (NHS) that couple lysines to solid supports; the nonspecificity of NHS may result in residue modifications near the binding site(s) of the receptor that can affect ligand specificity. In this study, a simple conjugation procedure is presented that overcomes these limitations and results in immobilized GST fusion proteins that are functional and specific. Here, the affinity of GST for GSH was used to generate an enzyme-substrate site-specific cross-linking reaction; GSH-Sepharose was preactivated with 1-ethyl-3-(dimethylaminopropyl)carbodiimide (EDC) and then incubated Fc gamma receptor IIIa (FcγRIIIa)-GST. The immobilized FcγRIIIa-GST more specifically bound glycosylated immunoglobulin G1s (IgG1s) and was used to enrich nonfucosylated IgG1s from weaker binding species. This technique can be used when modifications of amino acids lead to changes in activity.

A mass conserved reaction-diffusion system captures properties of cell polarity.

Cell polarity is a general cellular process that can be seen in various cell types such as migrating neutrophils and Dictyostelium cells. The Rho small GTP(guanosine 5'-tri phosphate)ases have been shown to regulate cell polarity; however, its mechanism of emergence has yet to be clarified. We first developed a reaction-diffusion model of the Rho GTPases, which exhibits switch-like reversible response to a gradient of extracellular signals, exclusive accumulation of Cdc42 and Rac, or RhoA at the maximal or minimal intensity of the signal, respectively, and tracking of changes of a signal gradient by the polarized peak. The previous cell polarity models proposed by Subramanian and Narang show similar behaviors to our Rho GTPase model, despite the difference in molecular networks. This led us to compare these models, and we found that these models commonly share instability and a mass conservation of components. Based on these common properties, we developed conceptual models of a mass conserved reaction-diffusion system with diffusion-driven instability. These conceptual models retained similar behaviors of cell polarity in the Rho GTPase model. Using these models, we numerically and analytically found that multiple polarized peaks are unstable, resulting in a single stable peak (uniqueness of axis), and that sensitivity toward changes of a signal gradient is specifically restricted at the polarized peak (localized sensitivity). Although molecular networks may differ from one cell type to another, the behaviors of cell polarity in migrating cells seem similar, suggesting that there should be a fundamental principle. Thus, we propose that a mass conserved reaction-diffusion system with diffusion-driven instability is one of such principles of cell polarity.

Mechanism of actin network attachment to moving membranes: barbed end capture by N-WASP WH2 domains.

Actin filament networks exert protrusive and attachment forces on membranes and thereby drive membrane deformation and movement. Here, we show that N-WASP WH2 domains play a previously unanticipated role in vesicle movement by transiently attaching actin filament barbed ends to the membrane. To dissect the attachment mechanism, we reconstituted the propulsive motility of lipid-coated glass beads, using purified soluble proteins. N-WASP WH2 mutants assembled actin comet tails and initiated movement, but the comet tails catastrophically detached from the membrane. When presented on the surface of a lipid-coated bead, WH2 domains were sufficient to maintain comet tail attachment. In v-Src-transformed fibroblasts, N-WASP WH2 mutants were severely defective in the formation of circular podosome arrays. In addition to creating an attachment force, interactions between WH2 domains and barbed ends may locally amplify signals for dendritic actin nucleation.

A polybasic motif allows N-WASP to act as a sensor of PIP(2) density.

Phosphatidylinositol 4,5-bisphosphate (PIP(2)) activates the actin regulatory protein N-WASP by binding to a short polybasic region involved in N-WASP autoinhibition. Here, we show that unlike canonical lipid binding modules, such as PH domains, this polybasic motif binds PIP(2) in a multivalent, cooperative manner. As a result, PIP(2) activation of N-WASP-mediated actin polymerization in vitro and in extracts is ultrasensitive: above a certain threshold, N-WASP responds in a switch-like manner to a small increase in the density of PIP(2) (Hill coefficient n(H) = approximately 20). We show that the sharpness of the PIP(2) activation threshold can be tuned by varying the length of the polybasic motif. This sharp activation threshold may help suppress N-WASP activation by quiescent PIP(2) levels yet leave it poised for activation upon subtle, signaling-induced perturbations in PIP(2) distribution.

Cdc42-dependent actin polymerization during compensatory endocytosis in Xenopus eggs.

The actin filament (F-actin) cytoskeleton associates dynamically with the plasma membrane and is thus ideally positioned to participate in endocytosis. Indeed, a wealth of genetic and biochemical evidence has confirmed that actin interacts with components of the endocytic machinery, although its precise function in endocytosis remains unclear. Here, we use 4D microscopy to visualize the contribution of actin during compensatory endocytosis in Xenopus laevis eggs. We show that the actin cytoskeleton maintains exocytosing cortical granules as discrete invaginated compartments, such that when actin is disrupted, they collapse into the plasma membrane. Invaginated, exocytosing cortical granule compartments are directly retrieved from the plasma membrane by F-actin coats that assemble on their surface. These dynamic F-actin coats seem to drive closure of the exocytic fusion pores and ultimately compress the cortical granule compartments. Active Cdc42 and N-WASP are recruited to exocytosing cortical granule membranes before F-actin coat assembly and coats assemble by Cdc42-dependent, de novo actin polymerization. Thus, F-actin may power fusion pore resealing and function in two novel endocytic capacities: the maintenance of invaginated compartments and the processing of endosomes.