Quartz crystal microbalance as an assay to detect anti-drug antibodies for the immunogenicity assessment of therapeutic biologics.

Because of their biological origins, therapeutic biologics can trigger an unwanted deleterious immune response with some patients. The immunogenicity of therapeutic biologics can affect drug efficacy and patient safety by the production of circulating anti-drug antibodies (ADA). In this study, quartz crystal microbalance (QCM) was developed as an assay to detect ADA. Etanercept (Enbrel®) was covalently grafted to dextran-modified QCM surfaces. Rabbits were immunized with etanercept to generate ADA. Results showed the QCM assay could detect purified ADA from rabbits at concentrations as low as 50 ng/mL, within the sensitivity range of ELISA. The QCM assay could also assess the ADA isotype. It was shown that the ADA were composed of the IgG isotype, but not IgM, as expected. Furthermore, it was shown that QCM surfaces that had been used to detect ADA could be regenerated in glycine-HCl solution and reused. The QCM assay was also demonstrated to detect ADA in crude serum samples. Serum was collected from the rabbits and analyzed before and after etanercept immunization. ADA were clearly detected in serum from rabbits after immunization, but not in serum before immunization. Serum from patients administered with etanercept for rheumatoid arthritis (RA) treatment was also analyzed and compared to serum from healthy donors. Sera from 10 RA patients were analyzed. Results showed one of the RA patient serum samples may have ADA present. In conclusion, QCM appears to be a viable assay to detect ADA for the immunogenicity assessment of therapeutic biologics.

Real-time label-free detection and kinetic analysis of Etanercept-Protein A interactions using quartz crystal microbalance.

A quartz crystal microbalance (QCM) was constructed to assess if such a biosensor has value as a complementary real-time label-free analysis platform for the biopharmaceutical industry. This was achieved through modifying QCM crystals with a low-fouling carboxymethyl-dextran layer bearing Protein A, and then injecting solutions containing Etanercept (i.e., Enbrel®) into the QCM chambers. The kinetics of Enbrel® - Protein A interactions was modeled using the Langmuir binding model and Enbrel® concentrations between 0.75-300ngmL-1. The resulting equilibrium dissociation and association constants (KD and KA) were 5.06×10-8M and 1.98×107M-1, respectively. The association and dissociation rate constants (kon and koff) decreased substantially as Enbrel® concentration, [C], increased, despite that the net binding rate, (kon[C]+koff), increased. The decrease in kon and koff was hypothesized to be a consequence of mass transport limitations. To verify this, QCM dissipation measurements were analyzed to provide insight on solution viscosity. As Enbrel® concentration increased, the net change in dissipation, ΔD, became larger. An augmentation of ΔD is associated with a higher solution viscosity, which would result in an increase in mass transport limitations. Therefore, the decrease in kon and koff for increasing Enbrel® concentration can be attributed to mass transport limitations. In conclusion, QCM is a valuable complementary real-time label-free biosensor analysis platform for the biopharmaceutical industry. Unlike the surface plasmon resonance (SPR) platform, QCM allows measuring dissipation, which can provide insight on how mass transport limitations impact interaction kinetics.

3D compartmented model to study the neurite-related toxicity of Aß aggregates included in collagen gels of adaptable porosity.

UNLABELLED: Insoluble deposits of ß-amyloid (Aß) are associated to neurodegenerative pathologies, in particular Alzheimer's Disease (AD). The toxicity of synthetic amyloid-like peptides has been largely demonstrated and shown to depend upon their aggregation state. However, standard 2D cell culture conditions are not well suited to study the role of the close vicinity of Aß aggregates and growing neurites in the degenerative process. Here, we have designed a compartmented set-up where model neural cells are differentiated on the surface of Aß-containing collagen matrices. The average pore size can be modulated, from below 0.2µm to more than 0.5µm by simple treatment with collagenase, to respectively hamper or permit neurite outgrowth towards the depth of the matrix. Dense Aß aggregates (Congo red and ThT-positive) were obtained inside the collagen matrix with a homogeneous distribution and dimensions similar to those observed in post-mortem brain slices from Alzheimer's patients. The aggregates are not toxic to cells when the pore size is small, in spite of relatively high concentrations of 0.05-0.62mg of peptide per gram of collagen (equivalent to 11.3-113µM). In contrast, on Aß-containing matrices with large pores, massive neural death is observed when the cells are seeded in the same conditions. It is the first time to our knowledge that Aß aggregates with a typical morphology of dense plaques are obtained within a porous biomimetic matrix, and are shown to be toxic only when accessible to differentiating cells. STATEMENT OF SIGNIFICANCE: Insoluble deposits of ß-amyloid (Aß) are associated to neurodegenerative pathologies, in particular Alzheimer's Disease (AD). In this study, we have formed Aß aggregates directly inside a biomimetic collagen matrix loaded with growth factors to induce the differentiation of PC12 or SH-SY6Y cells. For the first time, we show that when the contact between cells and Aß aggregates is allowed by opening up the matrix porosity, the close vicinity with aggregates induces neurite dystrophy. The compartmented 3D culture model developed and used in this study is a valuable tool to study the cytotoxicity of preformed dense Aß aggregates and proves that contact between the aggregates and neurons is required to induce neurodegenerative processes.

Dense fibrillar collagen matrices sustain osteoblast phenotype in vitro and promote bone formation in rat calvaria defect.

Two pure collagen materials were prepared from acidic collagen solutions at 5 and 40 mg/mL. Benefits of collagen concentration on bone repair were evaluated in vitro with human calvaria cells and in vivo in a rat cranial defect. Both materials exhibited specific structures, 5 mg/mL was soft with an open porous network of fibrils; 40 mg/mL was stiffer with a plugged surface and bundles of collagen fibrils. Osteoblasts seeded on 5 mg/mL formed an epithelioid layer with ultrastructural characteristics of mature osteoblasts and induced mineralization. Numerous osteoblasts migrated inside 5 mg/mL, triggering reorganization of their actin cytoskeleton, whereas on 40 mg/mL osteoblasts remained in a resting state. In rat calvaria defects, both materials induced active bone formation. Dual-energy X-ray absorption bone area measures after 4 weeks averaged 84.0% with 5 mg/mL, 88.4% with 40 mg/mL, and 36.7% in the controls (p < 0.05). Tartrate-resistant acid phosphatase-positive giant cells releasing amounts of metalloproteinase-2 progressively degraded the implants at 76.5% with 5 mg/mL and 38.2% with 40 mg/mL (p < 0.05), whereas alkaline phosphatase-positive osteoprogenitors invaded collagen remnant. Hence, the dense structure of collagen materials allowed cell invasion and raise their mechanical behavior without addition of chemical cross-linkers. Collagen concentration can be tuned to form 3D matrices for in vitro investigations or to fit degradation rate to different bone repair purposes.

Collagen supramolecular and suprafibrillar organizations on osteoblasts long-term behavior: benefits for bone healing materials.

This study compares the behavior of osteoblastic cells seeded on three structurally distinct collagen-based materials. Adhesion and long-term behavior were evaluated in vitro in regard to collagen scaffolds forming loose or dense fibrillar networks or exempt of fibrils. In this purpose collagen solutions at concentrations of 5 and 40 mg/mL were processed by freeze-drying or by sol/gel fibrillogenesis to form either sponges or hydrogels. Macroscopic and microscopic images of sponges showed a light material exhibiting large pores surrounded by dense collagen walls made of thin unstriated microfibrils of 20 nm in diameter. In comparison collagen hydrogels are more homogeneous materials, at 5 mg/mL the material consists of a regular network of cross-striated collagen fibrils of 100 nm in diameter. At 40 mg/mL the material appears stiffer, the ultrastructure exhibits cross-striated collagen fibrils packed in large bundles of 300-800 nm of width. Human osteoblastic cells seeded on top of the 5 mg/mL matrices exhibit a squared shaped osteoblast-like morphology over 28 days of culture and express both alkaline phosphatase and osteocalcin. Osteoblastic cells seeded on top of sponges or of 40 mg/mL matrices exhibit both flat and elongated resting-osteoblast morphology. Osteoblastic cells have mineralized the three collagen-based materials after 28 days of culture but collagen sponges spontaneously mineralized in absence of cells. These results highlight, in an in vitro cell culture approach, the benefit of fibrils and of dense fibrillar networks close to in vivo-like tissues, as positive criteria for new bone tissue repair materials.

Dense fibrillar collagen matrices for tissue repair.

The preparation of dense fibrillar collagen matrices, through a sol/gel transition at variable concentrations, offers routes to produce a range of simple, non toxic materials. Concentrated hydrogels entrapping cells show enhanced properties in terms of reduced contraction and enhanced cell proliferation . Dense fibrillar matrices attain tissue like mechanical properties and show ultrastructures described in connective tissues, namely liquid crystalline cholesteric geometries. Their colonization by cells and possible association with a mineral phase in a tissue like manner validate their use as biomimetic materials for regenerative medicine.

[Molecular aspects of cadherin-mediated cell adhesion: the first moments of the interaction]. / Aspects moléculaires de l'adherence cellulaire cadhérine-dépendante: les premiers moments de l'interaction.

Cadherins play a major role in the development and maintenance of all solid tissues. These transmembrane glycoproteins are responsible for calcium-dependent homophilic cell interactions. Recently, many different experimental approaches have been used to untangle the molecular basis of cadherin-mediated adherence. Various models have been suggested, particularly from high-resolution structures. Whilst the adherence mechanism is still under controversy, it is widely accepted that the specificity of the adherent interaction is localized to the N-terminal domain. New biophysical techniques together with biological approaches will allow a better understanding of how cadherins regulate cell-cell adherence. Integrating kinetics properties of cadherin interaction at the single molecule level has led to a greater understanding of cadherin molecular regulations.