27 research outputs found
Temperature-induced unfolding, aggregation, and interaction of therapeutic monoclonal antibodies
Temperature-induced unfolding, aggregation, and interaction of therapeutic monoclonal antibodies
Exploring Chemical Space for new Substances to stabilize a therapeutic Monoclonal Antibody
The physical stability of therapeutic proteins is a major concern in the development of liquid protein formulations. The number of degrees of freedom to tweak a given protein’s stability is limited to pH, ionic strength and type and concentration of excipient. There are only very few, mostly similar excipients currently in use, limited to the short list of substances generally recognized as safe for human use by the FDA. Opposed to the limited number of molecules the formulation scientist has at hand to stabilize a protein, there is the vastness of chemical space which is hypothesized to consist of 1060 compounds. Its potential to stabilize proteins has never been explored systematically in the context of stabilization of therapeutic proteins. Here we present a screening strategy to discover new excipients to further stabilize an already stable formulation of a therapeutic antibody. We use our data to build a predictive model to evaluate the stabilizing potential of small molecules. We argue that prior to worrying about the hurdles of toxicity and approval of novel excipient candidates, it is mandatory to assess the actual potential hidden in the chemical space
β-Catenin Directs Nuclear Factor-κB p65 Output via CREB-Binding Protein/p300 in Human Airway Smooth Muscle
β-Catenin is a multifunctional protein that apart from its role in proliferative and differentiation events, also acts upon inflammatory processes, mainly via interaction with nuclear factor-κB (NF-κB). However, there is still controversy as to whether β-catenin facilitates or represses NF-κB output. Insights into the molecular mechanisms underlying the interaction between β-catenin and NF-κB have highlighted the cofactors CREB-binding protein (CBP) and p300 as important candidates. Here, we hypothesized that the interaction of β-catenin with CBP/p300 directs NF-κB output. Using human airway smooth muscle (ASM) cells, we found that β-catenin is essential in interleukin -1β (IL-1β)-mediated expression of interleukin-6 (IL-6) by promoting nuclear translocation of the p65 subunit of NF-κB. These effects were independent from WNT pathway activation or other factors that promote β-catenin signaling. In the nucleus, inhibition of either the CBP- or p300-β-catenin interaction could regulate NF-κB output, by enhancing (CBP inhibition) or inhibiting (p300 inhibition) IL-1β-induced expression of IL-6, respectively. Acetylation of p65 by p300 likely underlies these events, as inhibition of the p300-β-catenin interaction diminished levels of acetylated p65 at lysine 310, thereby reducing p65 transcriptional activity. In conclusion, β-catenin is a critical component of NF-κB-mediated inflammation in human ASM, affecting transcriptional output by interacting with the nuclear cofactors CBP and p300. Targeting β-catenin may be an alternative strategy to treat airway inflammation in patients with airway disease, such as asthma
Ongoing Challenges to Develop High Concentration Monoclonal Antibody-based Formulations for Subcutaneous Administration: Quo Vadis?
Although many subcutaneously (s.c.) delivered, high-concentration antibody formulations (HCAF) have received regulatory approval and are widely used commercially, formulation scientists are still presented with many ongoing challenges during HCAF development with new mAb and mAb-based candidates. Depending on the specific physicochemical and biological properties of a particular mAb-based molecule, such challenges vary from pharmaceutical attributes e.g., stability, viscosity, manufacturability, to clinical performance e.g., bioavailability, immunogenicity, and finally to patient experience e.g., preference for s.c. vs. intravenous delivery and/or preferred interactions with health-care professionals. This commentary focuses on one key formulation obstacle encountered during HCAF development: how to maximize the dose of the drug? We examine methodologies for increasing the protein concentration, increasing the volume delivered, or combining both approaches together. We discuss commonly encountered hurdles, i.e., physical protein instability and solution volume limitations, and we provide recommendations to formulation scientists to facilitate their development of s.c. administered HCAF with new mAb-based product candidates
Smooth-muscle-derived WNT5A augments allergen-induced airway remodelling and Th2 type inflammation
Asthma is a heterogeneous disease characterized by chronic inflammation and structural changes in the airways. The airway smooth muscle (ASM) is responsible for airway narrowing and an important source of inflammatory mediators. We and others have previously shown that WNT5A mRNA and protein expression is higher in the ASM of asthmatics compared to healthy controls. Here, we aimed to characterize the functional role of (smooth muscle-derived) WNT5A in asthma. We generated a tet-ON smooth-muscle-specific WNT5A transgenic mouse model, enabling in vivo characterization of smooth-muscle-derived WNT5A in response to ovalbumin. Smooth muscle specific WNT5A overexpression showed a clear trend towards enhanced actin (α-SMA) expression in the ASM in ovalbumin challenged animals, but had no effect on collagen content. WNT5A overexpression in ASM also significantly enhanced the production of the Th2-cytokines IL4 and IL5 in lung tissue after ovalbumin exposure. In line with this, WNT5A increased mucus production, and enhanced eosinophilic infiltration and serum IgE production in ovalbumin-treated animals. In addition, CD4+ T cells of asthma patients and healthy controls were stimulated with WNT5A and changes in gene transcription assessed by RNA-seq. WNT5A promoted expression of 234 genes in human CD4+ T cells, among which the Th2 cytokine IL31 was among the top 5 upregulated genes. IL31 was also upregulated in response to smooth muscle-specific WNT5A overexpression in the mouse. In conclusion, smooth-muscle derived WNT5A augments Th2 type inflammation and remodelling. Our findings imply a pro-inflammatory role for smooth muscle-derived WNT5A in asthma, resulting in increased airway wall inflammation and remodelling
Smooth-muscle-derived WNT5A augments allergen-induced airway remodelling and Th2 type inflammation
Asthma is a heterogeneous disease characterized by chronic inflammation and structural changes in the airways. The airway smooth muscle (ASM) is responsible for airway narrowing and an important source of inflammatory mediators. We and others have previously shown that WNT5A mRNA and protein expression is higher in the ASM of asthmatics compared to healthy controls. Here, we aimed to characterize the functional role of (smooth muscle-derived) WNT5A in asthma. We generated a tet-ON smooth-muscle-specific WNT5A transgenic mouse model, enabling in vivo characterization of smooth-muscle-derived WNT5A in response to ovalbumin. Smooth muscle specific WNT5A overexpression showed a clear trend towards enhanced actin (α-SMA) expression in the ASM in ovalbumin challenged animals, but had no effect on collagen content. WNT5A overexpression in ASM also significantly enhanced the production of the Th2-cytokines IL4 and IL5 in lung tissue after ovalbumin exposure. In line with this, WNT5A increased mucus production, and enhanced eosinophilic infiltration and serum IgE production in ovalbumin-treated animals. In addition, CD4+ T cells of asthma patients and healthy controls were stimulated with WNT5A and changes in gene transcription assessed by RNA-seq. WNT5A promoted expression of 234 genes in human CD4+ T cells, among which the Th2 cytokine IL31 was among the top 5 upregulated genes. IL31 was also upregulated in response to smooth muscle-specific WNT5A overexpression in the mouse. In conclusion, smooth-muscle derived WNT5A augments Th2 type inflammation and remodelling. Our findings imply a pro-inflammatory role for smooth muscle-derived WNT5A in asthma, resulting in increased airway wall inflammation and remodelling
Prediction of Unwanted Crystallization of Freeze-Dried Protein Formulations Using α-Relaxation Measurements
There is a lack of methods to predict the isothermal crystallization behavior of amorphous freeze-dried formulations stored below the glass transition temperature. This study applies isothermal microcalorimetry to predict long-term crystallization during product storage time. The relaxation curve of a fresh sample recorded within 12 h after lyophilization is correlated with the long-term crystallization time at the same temperature. Storage conditions of 25 °C and 40 °C are examined and five model formulations containing either sucrose or trehalose with different concentrations of an IgG1 antibody are investigated. The amorphous formulations were created by different freeze-drying processes only differing in their freezing step (random nucleation; additional annealing step of 1.5 h and 3 h, controlled nucleation; quench cooling). Samples that crystallized during the study time of 12 months showed a promising correlation between their relaxation time and crystallization behavior upon storage. Furthermore, the study shows that polysorbate 20 strongly accelerates crystallization of sucrose and that the freezing step itself has a strong impact on the relaxation phenomena that is not levelled out by primary and secondary drying
Modulated scanning fluorimetry can quickly assess thermal protein unfolding reversibility in microvolume samples
Determining the temperature at which the thermal unfolding of a protein starts becoming irreversible is relevant for many areas of protein research. Until now, published methods cannot determine, within a reasonable time frame and with moderate sample consumption, the exposure temperature that starts causing irreversible protein unfolding. We present modulated scanning fluorimetry (MSF) and share a software (MSF Analyzer), which can be used to derive nonreversibility curves of thermal protein unfolding from a series of incremental temperature cycles performed on only 10 μL samples, consuming as low as a few micrograms of protein. Further processing of the data can yield the onset temperature that starts causing nonreversible protein unfolding. The MSF method is based on the hardware of the already existing nanoDSF technology and can be applied to dozens of samples simultaneously. Here, we use MSF to study how solution pH affects the reversibility of thermal protein unfolding of several model proteins to show that the nonreversibility onset temperature (Tnr) is a unique biophysical parameter, providing orthogonal information from thermal protein denaturation data and insights into the validity of thermal unfolding analysis in the context of equilibrium thermodynamics. We also show that MSF can be used to study enzyme stability after exposure to high temperatures. Besides, we demonstrate that protein thermal unfolding and nonreversibility can be affected in different ways upon modifications like PEG-ylation or labeling with fluorescent dyes. Finally, we show that MSF can be used to study the effect of various protein interactions on thermal protein unfolding reversibility. With the diverse examples in this work, we reveal how MSF can provide orthogonal information from thermal denaturation experiments that can bring benefits to various areas of protein research. The MSF Analyzer software is available at https://github.com/CoriolisPharmaResearch/MSFAnalyser
Combining unfolding reversibility studies and molecular dynamics simulations to select aggregation-resistant antibodies
The efficient development of new therapeutic antibodies relies on developability assessment with biophysical and computational methods to find molecules with drug-like properties such as resistance to aggregation. Despite the many novel approaches to select well-behaved proteins, antibody aggregation during storage is still challenging to predict. For this reason, there is a high demand for methods that can identify aggregation-resistant antibodies. Here, we show that three straightforward techniques can select the aggregation-resistant antibodies from a dataset with 13 molecules. The ReFOLD assay provided information about the ability of the antibodies to refold to monomers after unfolding with chemical denaturants. Modulated scanning fluorimetry (MSF) yielded the temperatures that start causing irreversible unfolding of the proteins. Aggregation was the main reason for poor unfolding reversibility in both ReFOLD and MSF experiments. We therefore performed temperature ramps in molecular dynamics (MD) simulations to obtain partially unfolded antibody domains in silico and used CamSol to assess their aggregation potential. We compared the information from ReFOLD, MSF, and MD to size-exclusion chromatography (SEC) data that shows whether the antibodies aggregated during storage at 4, 25, and 40 °C. Contrary to the aggregation-prone molecules, the antibodies that were resistant to aggregation during storage at 40 °C shared three common features: (i) higher tendency to refold to monomers after unfolding with chemical denaturants, (ii) higher onset temperature of nonreversible unfolding, and (iii) unfolding of regions containing aggregation-prone sequences at higher temperatures in MD simulations