Developability Assessments of Monoclonal Antibody Candidates to Minimize Aggregation During Large-Scale Ultrafiltration and Diafiltration (UF-DF) Processing.

Therapeutic proteins are subjected to a variety of stresses during manufacturing, storage or administration, that often lead to undesired protein aggregation and particle formation. Ultrafiltration-diafiltration (UF-DF) processing of monoclonal antibodies (mAbs) is one such manufacturing step that has been shown to result in such physical degradation. In this work, we explore the use of different analytical techniques and lab-scale setups as methodologies to predict and rank-order the aggregation potential of four different mAbs during large-scale UF-DF processing. In the first part of the study, a suite of biophysical techniques was applied to assess differences in their inherent bulk protein properties including conformational and colloidal stability in a PBS buffer. Additionally, the inherent interfacial properties of these mAbs in PBS were measured using a Langmuir trough technique. In the next part of the study, several different scale-down lab models were evaluated including a lab bench-scale UF-DF setup, mechanical stress (shaking/stirring) studies in vials, and application of interfacial dilatational stress using a Langmuir trough to assess protein particle formation in different UF-DF processing buffers. Taken together, our results demonstrate the ability of a Langmuir-trough methodology to accurately predict the mAb instability profile observed during large scale UF-DF processing.

Effect of substrate stiffness on human intestinal enteroids' infectivity by enteroaggregative Escherichia coli.

Human intestinal enteroids (HIE) models have contributed significantly to our understanding of diarrheal diseases and other intestinal infections, but their routine culture conditions fail to mimic the mechanical environment of the native intestinal wall. Because the mechanical characteristics of the intestine significantly alter how pathogens interact with the intestinal epithelium, we used different concentrations of polyethylene glycol (PEG) to generate soft (~2 kPa), medium (~10 kPa), and stiff (~100 kPa) hydrogel biomaterial scaffolds. The height of HIEs cultured in monolayers atop these hydrogels was 18 µm whereas HIEs grown on rigid tissue culture surfaces (with stiffness in the GPa range) were 10 µm. Substrate stiffness also influenced the amount of enteroaggregative E. coli (EAEC strain 042) adhered to the HIEs. We quantified a striking difference in adherence pattern; on the medium and soft gels, the bacteria formed clusters of > 100 and even > 1000 on both duodenal and jejunal HIEs (such as would be found in biofilms), but did not on glass slides and stiff hydrogels. All hydrogel cultured HIEs showed significant enrichment for gene and signaling pathways related to epithelial differentiation, cell junctions and adhesions, extracellular matrix, mucins, and cell signaling compared to the HIEs cultured on rigid tissue culture surfaces. Collectively, these results indicate that the HIE monolayers cultured on the hydrogels are primed for a robust engagement with their mechanical environment, and that the soft hydrogels promote the formation of larger EAEC aggregates, likely through an indirect differential effect on mucus. STATEMENT OF SIGNIFICANCE: Enteroids are a form of in vitro experimental mini-guts created from intestinal stem cells. Enteroids are usually cultured in 3D within Matrigel atop rigid glass or plastic substrates, which fail to mimic the native intestinal mechanical environment. Because intestinal mechanics significantly alter how pathogens interact with the intestinal epithelium, we grew human intestinal enteroids in 2D atop polyethylene glycol (PEG) hydrogel scaffolds that were soft, medium, or stiff. Compared with enteroids grown in 2D atop glass or plastic, the enteroids grown on hydrogels were taller and more enriched in mechanobiology-related gene signaling pathways. Additionally, enteroids on the softest hydrogels supported adhesion of large aggregates of enteroaggregative E. coli. Thus, this platform offers a more biomimetic model for studying enteric diseases.

Approaching the compressive modulus of articular cartilage with a decellularized cartilage-based hydrogel.

UNLABELLED: ECM-based materials are appealing for tissue engineering strategies because they may promote stem cell recruitment, cell infiltration, and cell differentiation without the need to supplement with additional biological factors. Cartilage ECM has recently shown potential to be chondroinductive, particularly in a hydrogel-based system, which may be revolutionary in orthopedic medicine. However, hydrogels composed of natural materials are often mechanically inferior to synthetic materials, which is a major limitation for load-bearing tissue applications. The objective was therefore to create an unprecedented hydrogel derived entirely from native cartilage ECM that was both mechanically more similar to native cartilage tissue and capable of inducing chondrogenesis. Porcine cartilage was decellularized, solubilized, and then methacrylated and UV photocrosslinked to create methacrylated solubilized decellularized cartilage (MeSDCC) gels. Methacrylated gelatin (GelMA) was employed as a control for both biomechanics and bioactivity. Rat bone marrow-derived mesenchymal stem cells were encapsulated in these networks, which were cultured in vitro for 6weeks, where chondrogenic gene expression, the compressive modulus, swelling, and histology were analyzed. One day after crosslinking, the elastic compressive modulus of the 20% MeSDCC gels was 1070±150kPa. Most notably, the stress strain profile of the 20% MeSDCC gels fell within the 95% confidence interval range of native porcine cartilage. Additionally, MeSDCC gels significantly upregulated chondrogenic genes compared to GelMA as early as day 1 and supported extensive matrix synthesis as observed histologically. Given that these gels approached the mechanics of native cartilage tissue, supported matrix synthesis, and induced chondrogenic gene expression, MeSDCC hydrogels may be promising materials for cartilage tissue engineering applications. Future efforts will focus on improving fracture mechanics as well to benefit overall biomechanical performance. STATEMENT OF SIGNIFICANCE: Extracellular matrix (ECM)-based materials are appealing for tissue engineering strategies because they may promote stem cell recruitment, cell infiltration, and cell differentiation without the need to supplement with additional biological factors. One such ECM-based material, cartilage ECM, has recently shown potential to be chondroinductive; however, hydrogels composed of natural materials are often mechanically inferior to synthetic materials, which is a major limitation for load-bearing tissue applications. Therefore, this work is significant because we were the first to create hydrogels derived entirely from cartilage ECM that had mechanical properties similar to that of native cartilage until hydrogel failure. Furthermore, these hydrogels had a compressive modulus of 1070±150kPa, they were chondroinductive, and they supported extensive matrix synthesis. In the current study, we have shown that these new hydrogels may prove to be a promising biomaterial for cartilage tissue engineering applications.

Chondroinductive Hydrogel Pastes Composed of Naturally Derived Devitalized Cartilage.

Hydrogel precursors are liquid solutions that are prone to leaking from the defect site once implanted in vivo. Therefore, the objective of the current study was to create a hydrogel precursor that exhibited a yield stress. Additionally, devitalized cartilage extracellular matrix (DVC) was mixed with DVC that had been solubilized and methacrylated (MeSDVC) to create hydrogels that were chondroinductive. Precursors composed of 10% MeSDVC or 10% MeSDVC with 10% DVC were first evaluated rheologically, where non-Newtonian behavior was observed in all hydrogel precursors. Rat bone marrow stem cells (rBMSCs) were mixed in the precursor solutions, and the solutions were then crosslinked and cultured in vitro for 6 weeks with and without exposure to human transforming growth factor ß3 (TGF-ß3). The compressive modulus, gene expression, biochemical content, swelling, and histology of the gels were analyzed. The DVC-containing gels consistently outperformed the MeSDVC-only group in chondrogenic gene expression, especially at 6 weeks, where the relative collagen II expression of the DVC-containing groups with and without TGF-ß3 exposure was 40- and 78-fold higher, respectively, than that of MeSDVC alone. Future work will test for chondrogenesis in vivo and overall, these two cartilage-derived components are promising materials for cartilage tissue engineering applications.