Development and biophysical characterization of a humanized FSH-blocking monoclonal antibody therapeutic formulated at an ultra-high concentration.

Highly concentrated antibody formulations are oftentimes required for subcutaneous, self-administered biologics. Here, we report the development of a unique formulation for our first-in-class FSH-blocking humanized antibody, MS-Hu6, which we propose to move to the clinic for osteoporosis, obesity, and Alzheimer's disease. The studies were carried out using our Good Laboratory Practice (GLP) platform, compliant with the Code of Federal Regulations (Title 21, Part 58). We first used protein thermal shift, size exclusion chromatography, and dynamic light scattering to examine MS-Hu6 concentrations between 1 and 100 mg/mL. We found that thermal, monomeric, and colloidal stability of formulated MS-Hu6 was maintained at a concentration of 100 mg/mL. The addition of the antioxidant L-methionine and chelating agent disodium EDTA improved the formulation's long-term colloidal and thermal stability. Thermal stability was further confirmed by Nano differential scanning calorimetry (DSC). Physiochemical properties of formulated MS-Hu6, including viscosity, turbidity, and clarity, confirmed with acceptable industry standards. That the structural integrity of MS-Hu6 in formulation was maintained was proven through Circular Dichroism (CD) and Fourier Transform Infrared (FTIR) Spectroscopy. Three rapid freeze-thaw cycles at -80 °C/25 °C or -80 °C/37 °C further revealed excellent thermal and colloidal stability. Furthermore, formulated MS-Hu6, particularly its Fab domain, displayed thermal and monomeric storage stability for more than 90 days at 4°C and 25°C. Finally, the unfolding temperature (Tm) for formulated MS-Hu6 increased by >4.80 °C upon binding to recombinant FSH, indicating highly specific ligand binding. Overall, we document the feasibility of developing a stable, manufacturable and transportable MS-Hu6 formulation at a ultra-high concentration at industry standards. The study should become a resource for developing biologic formulations in academic medical centers.

Development and Biophysical Characterization of a Humanized FSH-Blocking Monoclonal Antibody Therapeutic Formulated at an Ultra-High Concentration.

Highly concentrated antibody formulations are oftentimes required for subcutaneous, self-administered biologics. Here, we report the creation of a unique formulation for our first-in- class FSH-blocking humanized antibody, MS-Hu6, which we propose to move to the clinic for osteoporosis, obesity, and Alzheimer's disease. The studies were carried out using our Good Laboratory Practice (GLP) platform, compliant with the Code of Federal Regulations (Title 21, Part 58). We first used protein thermal shift, size exclusion chromatography, and dynamic light scattering to examine MS-Hu6 concentrations between 1 and 100 mg/mL. We found that thermal, monomeric, and colloidal stability of formulated MS-Hu6 was maintained at a concentration of 100 mg/mL. The addition of the antioxidant L-methionine and chelating agent disodium EDTA improved the formulation's long-term colloidal and thermal stability. Thermal stability was further confirmed by Nano differential scanning calorimetry (DSC). Physiochemical properties of formulated MS-Hu6, including viscosity, turbidity, and clarity, conformed with acceptable industry standards. That the structural integrity of MS-Hu6 in formulation was maintained was proven through Circular Dichroism (CD) and Fourier Transform Infrared (FTIR) spectroscopy. Three rapid freeze-thaw cycles at -80°C/25°C or -80°C/37°C further revealed excellent thermal and colloidal stability. Furthermore, formulated MS-Hu6, particularly its Fab domain, displayed thermal and monomeric storage stability for more than 90 days at 4°C and 25°C. Finally, the unfolding temperature (T m ) for formulated MS-Hu6 increased by >4.80°C upon binding to recombinant FSH, indicating highly specific ligand binding. Overall, we document the feasibility of developing a stable, manufacturable and transportable MS-Hu6 formulation at a ultra-high concentration at industry standards. The study should become a resource for developing biologic formulations in academic medical centers.

Optimizing a therapeutic humanized follicle-stimulating hormone-blocking antibody formulation by protein thermal shift assay.

Biopharmaceutical products are formulated using several Food and Drug Administration (FDA) approved excipients within the inactive ingredient limit to maintain their storage stability and shelf life. Here, we have screened and optimized different sets of excipient combinations to yield a thermally stable formulation for the humanized follicle-stimulating hormone (FSH)-blocking antibody, MS-Hu6. We used a protein thermal shift assay in which rising temperatures resulted in the maximal unfolding of the protein at the melting temperature (Tm ). To determine the buffer and pH for a stable solution, four different buffers with a pH range from 3 to 8 were screened. This resulted in maximal Tm s at pH 5.62 for Fab in phosphate buffer and at pH 6.85 for Fc in histidine buffer. Upon testing a range of salt concentrations, MS-Hu6 was found to be more stable at lower concentrations, likely due to reduced hydrophobic effects. Molecular dynamics simulations revealed a higher root-mean-square deviation with 1 mM than with 100 mM salt, indicating enhanced stability, as noted experimentally. Among the stabilizers tested, Tween 20 was found to yield the highest Tm and reversed the salt effect. Among several polyols/sugars, trehalose and sucrose were found to produce higher thermal stabilities. Finally, binding of recombinant human FSH to MS-Hu6 in a final formulation (20 mM phosphate buffer, 1 mM NaCl, 0.001% w/v Tween 20, and 260 mM trehalose) resulted in a thermal shift (increase in Tm ) for the Fab, but expectedly not in the Fc domain. Given that we used a low dose of MS-Hu6 (1 µM), the next challenge would be to determine whether 100-fold higher, industry-standard concentrations are equally stable.

Lysine245 plays a crucial role in stability and function of glycerol 3-phosphate dehydrogenase (Gpd1) in Saccharomyces cerevisiae.

Glycerol 3-phosphate dehydrogenase (Gpd1 isoform) catalyzes the rate limiting step of glycerol synthesis and is a critical component of the osmo-responsive machinery in yeast. The three-dimensional structure of the enzyme is similar to the enzyme from many other organisms, including humans. A recent study with the human enzyme has proposed K120 (K152 in yeast) to be in the correct orientation for catalysis; K204 (K245 in yeast) is out of plane and is not a participant in the catalytic cycle. The current work was carried out to establish the role of K245 in the catalytic cycle of yeast Gpd1. K245A mutant was found to possess lower catalytic activity. Osmotically stressed cells expressing Gpd1 (K245A) showed no change in intracellular glycerol as compared with wild-type cells which showed ~60% increase. Fluorescence microscopy, native polyacrylamide gel electrophoresis (PAGE) analysis, fluorescence spectroscopy, and Thioflavin T spectrofluorimetry showed a relatively unstable, aggregation- and degradation-prone conformation for the mutant. In silico studies showed an aggregation "hotspot" around K245. This study establishes the requirement of K245 for conformational stability and functional adaptation of Gpd1 in Saccharomyces cerevisiae.

Does N-terminal huntingtin function as a 'holdase' for inhibiting cellular protein aggregation?

Proteolytic cleavage of huntingtin gives rise to N-terminal fragments. While the role of truncated mutant huntingtin is described in Huntington's disease (HD) pathogenesis, the function of N-terminal wild-type protein is less studied. The yeast model of HD is generated by the presence of FLAG tag and absence of polyproline tract as flanking sequences of the elongated polyglutamine stretch. We show that the same sequence derived from wild-type huntingtin exon1 is able to inhibit the aggregation of proteins in vitro and in yeast cells. It is able to stabilize client proteins as varied as luciferase, α-synuclein, and p53 in a soluble but non-native state. This is somewhat similar to the 'holdase' function of small heat shock proteins and 'nonchaperone proteins' which are able to stabilize partially unfolded client proteins in a nonspecific manner, slowing down their aggregation. Mutagenesis studies show this property to be localized at the N17 domain preceding the polyglutamine tract. Distortion of this ordered segment, either by deletion of this segment or mutation of a single residue (L4A), leads to decreased stability and increased aggregation of client proteins. It is interesting to note that the helical conformation of the N17 domain is also essential for aggregation of the N-terminal mutant protein. Our results provide evidence for a novel function for the amphipathic helix derived from exon1 of wild-type huntingtin.