Osteoarthritis (OA) is characterized by degeneration of the joint cartilage, inflammation, and a change in the chondrocyte phenotype. Inflammation also promotes cell hypertrophy in human articular chondrocytes (HC-a) by activating the NF-κB pathway. Chondrocyte hypertrophy and inflammation promote extracellular matrix degradation (ECM). Chondrocytes depend on Smad signaling to control and regulate cell hypertrophy as well as to maintain the ECM. The involvement of these two pathways is crucial for preserving the homeostasis of articular cartilage. In recent years, Polynucleotides Highly Purified Technology (PN-HPT) has emerged as a promising area of research for the treatment of OA. PN-HPT involves the use of polynucleotide-based agents with controlled natural origins and high purification levels. In this study, we focused on evaluating the efficacy of a specific polynucleotide sodium agent, known as CONJURAN, which is derived from fish sperm. Polynucleotides (PN), which are physiologically present in the matrix and function as water-soluble nucleic acids with a gel-like property, have been used to treat patients with OA. However, the specific mechanisms underlying the effect remain unclear. Therefore, we investigated the effect of PN in an OA cell model in which HC-a cells were stimulated with interleukin−1β (IL−1β) with or without PN treatment. The CCK-8 assay was used to assess the cytotoxic effects of PN. Furthermore, the enzyme-linked immunosorbent assay was utilized to detect MMP13 levels, and the nitric oxide assay was utilized to determine the effect of PN on inflammation. The anti-inflammatory effects of PN and related mechanisms were investigated using quantitative PCR, Western blot analysis, and immunofluorescence to examine and analyze relative markers. PN inhibited IL−1β induced destruction of genes and proteins by downregulating the expression of MMP3, MMP13, iNOS, and COX-2 while increasing the expression of aggrecan (ACAN) and collagen II (COL2A1). This study demonstrates, for the first time, that PN exerted anti-inflammatory effects by partially inhibiting the NF-κB pathway and increasing the Smad2/3 pathway. Based on our findings, PN can potentially serve as a treatment for OA. Osteoarthritis (OA), for which there is no known cure, is a degenerative joint disease that significantly impacts people’s lives and the economy. Age, obesity, knee malalignment, biomechanical stress on joints, low-grade systemic inflammation, etc., all contribute to the onset of OA . From 19Interleukin−1β (IL−1β), a pro-inflammatory cytokine, has been implicated in the degradation of articular cartilage through the induction of chondrocyte hypertrophy . ChondroIn OA patients, elevated levels of IL−1β were detected in the synovial fluid, synovial membrane, cartilage, and subchondral bone . In addiCurrently, the most prevalent treatments for OA are surgical and nonpharmaceutical procedures. However, the lifespan of prostheses is still fairly limited, and the only effective treatments for OA are lifestyle changes and joint replacement surgery . If surgFurthermore, the effects of PN can last even longer than those of PDRN because PN creates a favorable environment for injured areas by synthesizing ECM . PolynucIL−1β is one of the most important proinflammatory cytokines involved in the pathophysiology of OA. Consequently, we evaluated the viability of HC-a treated with varying concentrations of IL−1β and discovered that IL−1β decreased cell viability in a concentration and time-dependent manner A,B. For The cytotoxic effects of PN on HC-a were determined at various concentrations  for 24 h using the cell counting kit-8 (CCK8) assay. The viability of HC-a cells was significantly enhanced at doses of PN ranging from 1 to 100 µg/mL. Therefore, based on previous research, for subsequent experiments, concentrations of 1, 10, and 100 μg/mL PN were used and HC-a chondrocytes were exposed to PN 24 h after IL−1β stimulation , demonstIn order to investigate the protective mechanism of PN in regulating IL−1β-activated inflammatory mediators in HC-a, the levels of MMP13, a matrix-degrading protein associated with OA, were measured in the presence of IL−1β alone and IL−1β combined with different concentrations of PN. The results showed that IL−1β treatment significantly increased MMP13 activity compared to controls. However, the addition of PN at 1, 10, or 100 µg/mL significantly decreased MMP13 activity, with the highest concentration of PN reducing MMP13 activity by over 70% A. In addIL−1β stimulation revealed that PN could reduce MMP13 and nitric oxide levels via ELISA and Griess reagent assays; therefore, we investigated whether PN could also reduce the gene and protein levels of MMP13, iNOS, and other hypertrophic and inflammatory mediators released in response to IL−1β stimulation. As a result, the gene expression of matrix-degrading enzymes, such as MMP13 and MMP3, and inflammatory mediators, such as iNOS and COX-2, were evaluated. Real-time PCR was utilized to assess gene expression. Treatment with IL−1β significantly increased the expression of all genes, while treatment with PN significantly inhibited the expression of MMP13, MMP3, iNOS, and COX-2 A. There We investigated whether the protective effects of PN on HC-a are related to its modulation of the NF-κB signaling pathway in cells treated with IL−1β. We examined the phosphorylated and total protein levels of p65 and IκBα in whole-cell lysates using Western blotting and 100 µg/mL of PN, which has been shown to have the greatest effect in decreasing hypertrophic and inflammatory mediators. IL−1β treatment resulted in a significant increase in pp65 protein expression relative to the control group, indicating activation of the pp65 signaling pathway. However, PN treatment effectively suppressed pp65 expression, indicating that it inhibits the NF-κB signaling pathway A. In ordSince we had previously demonstrated that PN can inhibit MMP3 and MMP13, we sought to determine whether PN could also increase the production of TGF-β, ACAN, and COL2A1 production. mRNA A and proWe also investigated whether or whether not the chondrocyte Smad protein is modified by inflammation caused by IL−1β. IL−1β has been observed to disrupt Smad signaling in chondrocytes. Therefore, we investigated whether it was possible to use PN to reestablish Smad signaling in HC-a A using aAs the population ages and more people struggle with obesity, OA has become a more severe health problem. Symptomatic OA affects approximately 240 million people worldwide, including 10% of men and 18% of women aged 60 and older . AlthougPrevious research has shown that an increase in the production of inflammatory cytokines  is a sigOur research demonstrated that PN inhibited the expression of MMP3 and MMP13. IL−1β stimulation is also believed to be characterized by chondrocyte modifications that resemble hypertrophy . The NF-In the treatment of OA, PN has been proven to be an effective alternative to HA. PN has exceptional anti-inflammatory actions that promote cartilage recovery . PN fillThe primary motivation for employing a 2D in vitro cell-based model in this study was to evaluate the mechanism of a viscosupplement (PN) for which clinical studies have been completed; however, the mechanism of action has not been adequately discussed. In addition, very little is known about the mechanism by which PN exerts these properties, despite the fact that it is said to have high regenerative properties and an efficient repair mechanism. Since the 1990s, PN has been utilized in skin regeneration therapies and OA treatment. When applied for skin regeneration, PN has been shown to stimulate the production of a stronger ECM, as well as collagen, and elastin, which restores youthful midface convexity in a consistent and durable manner. In addition, since PNs are efficient water binders that reorient their spatial structure and orientation after binding water molecules in dermal tissues, they are also effective water binders. The resultant three-dimensional viscoelastic PN gel demonstrated prolonged hydration of the ECM. Based on clinical studies demonstrating the role of PN in rotator cuff healing, skin rejuvenation, and management of OA, we investigated two main signaling pathways. The involvement of PN in other signaling pathways is unknown and necessitates further investigation. When evaluating IA viscosupplements, it is critical to understand the advantages and disadvantages of the IA in in vitro models. Therefore, 2D models of cells, such as synoviocytes or chondrocytes, that respond to cytokine stimulation  are the optimal choice for screening anti-inflammatory or chondroprotective molecules for the evaluation of IA delivery systems . In vitrPolynucleotide (PN) from this study  was supplied in prefilled sterile syringes containing 2 mL of a clear, colorless solution. At 37 °C, PN was dissolved directly in a standard cell culture medium. When cells were in a standard cell culture medium, the optimal concentrations of this solution were determined. Cell culture media 1X Dulbecco’s Modified Eagle’s Medium (DMEM), Penicillin streptomycin (pen strep), and fetal bovine serum (FBS) were supplied from Gibco . R&D systems  provided human recombinant IL−1β, which was dissolved in PBS containing 0.5% bovine serum albumin. Antibodies against type II collagen (COL2A1) , aggrecan (ACAN) , COX-2 , iNOS , MMP3 , MMP13 , Total-Smad , p-Smad2/3 , p65 , pp65 , IκBα , and p-IκBα  were purchased. Additionally, we purchased conjugated goat anti-rabbit secondary antibody (H+L)  and goat antimouse IgG .The human articular chondrocyte (HC-a) cell line was purchased from ScienCell  (#4650) ,71. CellWhen cells reached 80% confluence, we adopted a pretreatment approach by exposing HC-a to IL−1β for 24 h, followed by the administration of PN for the subsequent 24 h. The IL−1β concentration was 10 ng/mL, while the concentrations of PN used were 1, 10, and 100 µg/mL. We chose to examine post-treatment of PN after exposure to proinflammatory cytokines rather than pretreatment before stimulation with inflammatory cytokines because pretreatment may not reflect clinical reality. Due to the prolonged exposure to an inflammatory environment, patients with Kellgren–Lawrence grade II or grade III knee OA frequently experience pain and limited mobility. Therefore, we investigated whether administering PN after exposure could aid in reducing inflammation in HC-a.4 cells/well, stimulated with IL−1β for 24 h, and then treated with or without PN at concentrations of 1, 10, and 100 µg/mL for 24 h. After appropriate treatment, according to the experimental grouping, 10 µL of CCK-8 solution was added to each well and incubated for 2 h at room temperature. The level of the dye formed was measured with a BioTek Synergy HTX multimode plate reader  with the absorbance at 450 nm in each well.CCK-8 test kit  was used to determine cell viability after IL−1β and PN treatment. In summary, cells were seeded in 96-well plates at a density of 1 × 10Next, using the biorbyt MMP13 ELISA Kit, we analyzed the ability of PN to inhibit MMP13 activity in vitro . Cells were plated in 12-well plates and treated with IL−1β (10 ng/mL) alone or with PN  at various concentrations for 24 h. Following the manufacturer’s instructions, MMP13 activity was determined using the biorbyt MMP13 Assay Kit  on the culture medium.The nitric oxide colorimetric assay was conducted following a previously established protocol . To asse® GreenStar PCR premix  using the MyGenieTM 96/384 thermal cycler system .To assess the effects of IL−1β and PN on the transcription of MMP3, MMP13, ACAN, COL2A1, TGF-β, COX-2, iNOS, and the housekeeping enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH)  and lysis solution containing protease and phosphatase inhibitor cocktails that had been cooled. The samples were incubated on ice for 30 min before being centrifuged at 12,000 rpm and 4 °C for 10 min. The protein concentration of the whole cell lysate was determined using a bicinchoninic acid (BCA) protein assay kit  per the manufacturer’s instructions. The protein was then combined with a sodium dodecyl sulfate sampling buffer, followed by a 5-min incubation at 95 °C. A total of 20 μg protein was loaded onto sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and separated by electrophoresis for each sample. The proteins were then transferred to PVDF membranes ; the membranes were blocked with 5% skim milk  for 2 h and overnight incubated with primary antibodies directed for iNOS (1:1000), COX-2 (1:1000), MMP13 (1:1000), MMP3 (1:1000), ACAN (1:1000), COL2A1 (1:1000), p65 (1:1000), pp65 (1:1000), IκBα (1:1000), TGF-β (1:1000), total Smad2/3 (1:1000), p-Smad2/3 (1:1000), and β-actin (1:5000) at 4 °C. The membranes were incubated for 2 h at room temperature with the appropriate enzyme-linked secondary antibodies. Blots were visualized using enhanced chemiluminescence (ECL) solution as per the manufacturer’s instructions. The band intensity was quantified using ImageJ software .6/mL) were allowed to grow for 2 to 3 days in 10% DMEM and FBS supplemented with penicillin (100 U/mL) before being used in the experiments. HC-a were serum starved for 6 h to equilibrate all cells, pretreated with 10 μM of the NF-kB signaling inhibitor 5HPP-33 . Lastly, nuclei were stained with DAPI solution.t-test was employed for comparisons between two groups, while, for multivariable analyses involving multiple groups, analysis of variance (ANOVA) with Tukey’s multiple comparison test was implemented. Statistical significance was defined as p < 0.05 for one asterisk , p < 0.01 for two asterisks , p < 0.001 for three asterisks , and p < 0.0001 for four asterisks .All experiments in this study were conducted in triplicate to ensure reproducibility. The data are presented as the mean ± standard error. GraphPad Prism 9.0 software  was utilized for generating the graphical representations of the data. Statistical analysis was performed to determine the significance of differences between two or more groups. The student’s In conclusion, we found that PN decreased the expression of MMP3, MMP13, iNOS, and COX-2 in HC-a while increasing TGF-β, COL2A1, and ACAN expression. In this preliminary study, we demonstrate that PN protects chondrocytes by inhibiting key molecular events involved in inflammation through NF-κB activation and matrix synthesis through the Smad pathway. In addition, our research reveals novel information regarding the potential of PN as an anti-inflammatory and chondroprotective agent.


The Hui people are the second-largest ethnic minority in China, and they are distributed throughout the country. A previous study explored the paternal genetic structure of the Hui population in nine different regions of China, but it overlooked the Liaoning province. In this study, we examined the paternal genetic makeup and forensic traits of the Hui population in Liaoning province by analyzing 157 Y-chromosome single nucleotide polymorphisms (Y-SNPs) and 26 short tandem repeats (Y-STRs). We successfully genotyped 282 unrelated male individuals from the Hui population of Liaoning province using the SNaPshot® single base extension assay and Goldeneye™ Y26 system kit . The results revealed high haplotypic diversity (0.9998) and identified 46 terminal haplogroups for the Hui population. Additional analyses, such as heat maps, principal component analysis (PCA), genetic distance (FST), Multidimensional scaling (MDS) analysis, and median-joining network (MJ) analysis, showed that the Hui population could be classified into three groups: Northwest Hui populations (NWH), including Liaoning, Xinjiang, Qinghai, Gansu, Ningxia, Shaanxi, and Henan; Hui populations from Sichuan and Shandong (SSH); and Yunnan Hui populations (YNH). Pairwise genetic distance (Rst) comparisons with other Chinese populations revealed that the Hui population displayed genetic affinity with the Han population. The comprehensive understanding of the Hui population in Liaoning province, explored by Y-SNPs and Y-STRs, can be utilized to interpret their genetic structure and enhance the accuracy of forensic databases.The online version contains supplementary material available at 10.1186/s12864-023-09774-8. The ancestors of the Hui came to China from Central Asia and Islamic Persia as handicraftsmen, merchants, scholars, and soldiers. This human movement started in the seventh century and continued until the thirteenth century. After settling in China, they intermingled with Han Chinese, Mongols, and Uyghurs, ultimately assimilating their phenotype, cultural characteristics, and language into Chinese culture. In Liaoning province, the Hui population represents only 0.632% of the population and is the fourth largest ethnic group after Han (83.94%), Manchu (12.88%), and Mongol (1.60%) [Short tandem repeats (Y-STRs) and single nucleotide polymorphisms (Y-SNPs) on the male-specific Y chromosome are valuable genetic markers used in population genetics and forensic investigations. These markers have distinctive inheritance patterns that enable the identification of paternal lineages and the reconstruction of male ancestry , 2. Y-ST (1.60%) . Previou (1.60%) . Another (1.60%) . Our pre (1.60%)  showed a (1.60%) , 18. How (1.60%) , 20. In This study was approved by the Ethical Review Board of China Medical University in Shenyang, Liaoning Province, People's Republic of China, in accordance with the standards of the Declaration of Helsinki. Blood samples on FTA cards were randomly collected from 282 unrelated healthy male individuals of the Hui population living in Liaoning province for at least three generations. The aims and procedures of the study were explained to all volunteers, and they signed informed consent forms before participating.DNA was extracted from bloodstains collected on FTA cards  using the Chelex-100 method . DNA quaA total of 157 Y-SNPs were selected from elsewhere , represeNext, the PCR products were purified by adding 1 μl of exonuclease I and 2 μl of shrimp alkaline phosphatase (SAP)  Co., Ltd) to a 9 μl multiplex PCR product. The mixture was then incubated at 37°C for 60 min, followed by enzyme denaturation at 80°C for 10 min . This reFollowing the SNaPshot reaction, the reaction products were purified by adding 1.25 μl of SAP and incubating at 37°C for 60 min, followed by enzyme denaturation at 80°C for 10 min. Further analysis was performed by mixing 1 μl of the purified SNaPshot reaction product with 9.5 μl of Hi-Di formamide and 0.50 μl of LIZ-120 internal sizing standard . The mixture was then subjected to denaturation at 95°C for 3 min, followed by cooling to 4°C. The samples were separated on an ABI Prism 3100 Genetic Analyzer  using a 36 cm capillary array and the default instrument settings recommended by the manufacturer. The Goldeneye™ 26Y system kit , which contains PowerPlex Y23 loci and three additional Y-STRs , were co-amplified in a GeneAmp® PCR 9700  thermal cycler according to the manufacturer's recommendations . The 280th haplogroup, and n is the sample size. Heat map matrices were performed based on haplogroup frequency in the R program, and the ggplot2 module was used. Arlequin 3.5.2 [Haplogroup frequencies were calculated by direct counting and presented in pie charts using Microsoft Excel. Haplotype diversity was calculated according to the formula:in 3.5.2  was usedAllelic frequencies and haplotypes were calculated by the direct counting method, while haplotype diversity (HD) was calculated using the formula:S385a/b) , 11 lociS385a/b) , PowerplS385a/b) , Yfiler™S385a/b) , PowerplS385a/b) , GoldeneS385a/b) . The disS385a/b) . ReducedThe median-joining networks was constructed using NETWORK 5.0.0.3 `. We setThe genetic diversity of the study population, consisting of 282 self-identified Hui individuals from Liaoning, China, and their genotypic profiles are summarized in Supplementary Table The Hui population from Liaoning showed high haplogroup diversity with 46 terminal groups. The predominant haplogroups were O (57%), J (8.86%), C 8.51%), Q (8.15%) and D (5.31%) in Hui population from Liaoning. When we have combined our data with previously published data of Hui population , 99 dist%, Q  using Y SNPs and Y STRs between Hui population from Liaoning PR China and nine other Hui populations across China.Additional file 6: Supplementary Table S6. Pairwise Rst values  and P values  for Hui population from Liaoning and other relevant population across China.Additional file 7: Supplementary Figure 1. Phylogenetic tree showing Y-haplogroups found in the ten Hui populations.Additional file 8: Supplementary Figure 2. Interactivity test between Hui population from Liaoning PR China and nine other Hui populations across China.Additional file 9: Supplementary Figure 3. Heatmap analysis between Hui population from Liaoning and other relevant population across China.Additional file 10: Supplementary Figure 4. Principal component analysis between Hui population from Liaoning and other relevant population across China.


Recent years have witnessed impressive growth in applying natural medicine in tumor treatment. Saffron is reported to elicit an inhibitory property against BC. Herein, we sought to explore the specific components and mechanistic basis of saffron’s anti-breast carcinoma (BC) function.Bioinformatics analysis was employed to analyze saffron components' anti-BC activity and screen the corresponding target genes involved in BC. Then, the roles of the main saffron ingredient quercetin in the activity of BC cells were examined using CCK-8, MTS, flow cytometry, colony formation, Transwell, and Gelatin zymogram assays. Additionally, the interactions among Quercetin, EET, and Stat3 were assessed by immunofluorescence and Western blot, and LC–MS/MS determined the levels of AA, EETs, and CYP3A. Finally, BC xenograft mouse models were established to verify the anti-BC function of Quercetin in vivo.Quercetin, the main active component of saffron, inhibited BC progression. Quercetin suppressed BC cell growth, migration, and invasion and inhibited CYP3A4 expression and activity in BC. Mechanistically, Quercetin down-regulated CYP3A4 to block the nuclear translocation of Stat3 by decreasing the metabolization of AA to EETs, thereby alleviating BC. Moreover, exogenously added EETs counteracted the anti-tumor effect of Quercetin on BC. Quercetin also inhibited the tumor growth of tumor-bearing nude mice.Quercetin could inhibit the activity of CYP3A to down-regulate AA metabolites EETs, consequently hampering p-Stat3 and nuclear translocation, thus impeding BC development.The online version contains supplementary material available at 10.1186/s10020-023-00720-8. Crocus sativus flower, is proposed to induce cell apoptosis in multiple types of cancers, such as colorectal, bladder, and pancreatic cancer, thus eliciting its anti-tumor effect in cancers  remains one of the most frequently diagnosed cancers worldwide. It is the primary cause of cancer-associated mortality among women in over 100 countries  is a metabolite of arachidonic acid (AA) . Adequate measures were taken to minimize the number of mice and their suffering.http://ctdbase.org/detail.go? acc = C452899 & type = chem) was used to retrieve the target genes of the main components in saffron. Next, the potential target genes were determined by the intersection of the DisGeNET, GeneCards, and CTD databases concerning the BC-related target genes. Finally, the STRING database was employed to plot the protein–protein interaction network diagram  database was adopted to retrieve the main components of saffron for extensive conditional screening . Next, the Comparative Toxicogenomics Database (CTD) data , CC ≥ Avg (CC), and De ≥ Avg (De)]. Additionally, the Molecular Complex Detection (MCODE) plugin in the Cytoscape software was adopted for cluster analysis of the network diagram  online were employed for the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and functional enrichment analysis. In addition, Cytoscape was employed to plot a network diagram of the KEGG analysis results , serum-free cell freezing medium (RPMI)-1640, and L-15 culture medium, respectively. The medium contained 10% (v/v) FBS , 100 µg/mL penicillin, and 100 µg/mL streptomycin  and two normal human breast epithelial cell lines (MCF10A and MDA-kb2), all of which were purchased from Biobw . Cells were cultured at 37 °C with 5% CO3 cells/well density. After incubation at 37 °C for 24 h, the cells were supplemented in a medium containing different concentrations of Quercetin . Following incubation for another 48 h, 20 μL Aqueous One Solution was added for 2 h culture, after which the absorbance value at the wavelength of 490 nm was measured using a microplate reader. According to the value of absorbance, the survival of cells was converted into survival rate /(mean OD of control group-mean OD of the blank group) × 100%)  were seeded in 96-well plates at 5 × 103 cells/well density. After incubation at 37 °C for 24 h, the cells were added to the culture medium in the presence of different concentrations of Quercetin . After continuing the culture for 48 h, each well was added with 100 μL of CCK8 solution, incubated for about 1 h in the culture, and then the absorbance was measured at 450 nm with a microplate reader. The absorbance value OD450 was used to evaluate the ability of cell proliferation -8 assay detected cell proliferation. ZR-75-1 and MCF-7 cells were seeded in 96-well plates at a 5 × 10Quercetin was purchased from Sigma  in the experiment. Quercetin was insoluble in water . DMSO was added as the solvent to dissolve Quercetin according to the instructions. ZR-75-1 and MCF-7 cells were treated with designated concentrations of Quercetin  for 24 h and trypsinized. Following centrifugation at 800 rpm for 5 min, the collected cells were resuspended using 0.3 mL phosphate buffer saline (PBS) to prepare a single-cell suspension, which was then added into 0.7 mL of pre-cooled anhydrous ethanol and fixed at 4 °C overnight. After centrifugation and two PBS rinses, the fixed cells were resuspended in 500 μL PBS supplemented with RNase A (100 μg/mL) and propidium iodide  solution and incubated at 37 °C for 30 min in conditions devoid of light. The distribution of the cell cycle was analyzed using a flow cytometer  for 24 h and trypsinized. Following centrifugation at 800 rpm for 5 min, the collected cells were resuspended using 0.3 mL PBS to make a single-cell suspension. Next, the cells were detached and resuspended using 0.1 mL 1 × binding buffer  and incubated with PI and Annexin V for 30 min in conditions devoid of light. Cell apoptosis was also assessed using a flow cytometer  were inoculated in 6-well plates. After 24 h of culture, a fresh medium with different concentrations of Quercetin  was added to the wells. After incubation for another 8 days, 4% paraformaldehyde was added to fix cells and stained with 0.1% crystal violet  were inoculated in 96-well plates pre-plated with fibronectin and supplemented with a quercetin of different concentrations . After incubation at 37 °C for 25 min, the adhered cells were stained with 0.1% crystal violet. Following PBS rinse, the crystal violet in stained cells was dissolved using 30% ice acetic acid, and the absorbance value at 590 nm was read using a microplate reader  for 12 h, followed by trypsinization and counting. Then, the cells  kit (Thermo) was used to determine the concentration of the extracted protein. Next, the protein was separated by 8–15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then electrotransferred onto a polyvinylidene fluoride membrane. Then, a membrane blockade was conducted using 5% bovine serum albumin (BSA) and incubated with corresponding primary antibodies to CYP3A4 , PCNA , p21 , caspase3 , cleaved caspase3 , PARP , cleaved-PARP , β-actin , Stat3 , p-Stat3 , matrix metalloproteinase 2 (MMP2) , MMP9 , TIMP1 , and TIMP2 . Following three rinses with Tris Buffered Saline Tween (TBST), the membrane was incubated with the horseradish peroxidase (HRP)-labeled anti-rabbit or anti-mouse secondary antibody (Sigma). After another three TBST rinses, the LI-COROdyssey System was employed for membrane scanning and image analysis  were employed, which can be placed in a 24-well plate.For detecting cell migration, the cells were detached, counted, and prepared into cell suspensions containing different concentrations of Quercetin  , 100 μL of which were added to the apical chamber, whereas 600 μL of Quercetin with the corresponding concentration was added to the basolateral chamber. After about 10 h of incubation at 37 °C, the chamber was fixed using paraformaldehyde for 15 min and stained with 0.1% crystal violet. Then the cells were photographed and counted. Finally, the chamber was pre-coated with Matrigel in the cell invasion assay, and the remaining steps were the same as mentioned above , ac-tubulin (ab179484), and p-Stat3 (ab76315)] at 4 °C followed by three rinses with phosphate-buffered saline with tween detergent (PBST). Next, the cells were incubated with the corresponding fluorescent secondary antibody for at least 1 h (depending on the microscopy results) and rinsed 3 times with PBST. Following a 3-min staining regimen with 4′,6-diamidine-2-phenylindole (DAPI) for nuclear labeling and three rinses with PBST, the coverslip was placed upside down on a glass slide supplemented with the mounting solution and dried with filter paper. Finally, the coverslip was sealed with nail polish and photographed under a confocal laser microscope   containing 1% type I bovine skin gelatin. Pre-stained SDS-PAGE standards  were used as low molecular weight markers. The gel was run at 120V/25A, then incubated in buffer I , pH 7.5) at 22 °C for 30 min. Next, the gel was further incubated in buffer II , 1μM ZnCl2 and 5 mmol/L CaCl2 at 22 °C) for 30 min. After that, the gel was incubated in buffer III  at 37 °C) for 12 h. Next, the gel was stained with 0.1% Coomassie Brilliant Blue R-250 at 22 °C for 60 min and then for 30 min in a solution containing 10% acetic acid and 10% methanol . After attaining 80–90% confluence, the cells were treated with a complete medium containing different concentrations of Quercetin  for 2 h. Afterward, the cells were cultured with a complete medium containing the CYP3A probe substrate midazolam (10 μM) for another 2 h, detached using trypsin, and resuspended in deionized water. The cells were disrupted after treatment with repetitive freezing and thawing (three times) and ultrasound . The AA and its metabolites EETs in the cell sap were extracted with ethyl acetate, the organic extracts were saponified, the obtained fatty acids were extracted into the acidified ether and evaporated under NFor the detection of AA and its metabolite EETs in the tissue, 100 mg of the sample was taken out, added with 10 times the volume of pre-cooled 90% methanol (addition of 1 mL of 90% methanol to 100 mg sample), frozen with liquid nitrogen, and homogenate crushing was performed using MP homogenizer , followed by low-temperature ultrasound 30 min/time for 2 times. Then, the samples were placed at – 20 °C for 60 min, centrifuged at 13,000 g at 4 °C for 15 min, and the supernatant (divide) was taken and aliquoted as 900 μL/tube), vacuum dried, freeze-dried powder, and stored at – 80 °C for later use. Next, 100 μL acetonitrile aqueous solution  was added to reconstitute during mass spectrometry, vortexed, centrifuged at 14,000 g at 4 °C for 15 min. Finally, the supernatant was taken for sample analysis, and for measurement of AA and EETs, the AA and its metabolites EETs in the cell fluid extracted with ethyl acetate were analyzed by LC–MS/MS, which was described previously , and 100 μL of the suspension was randomly injected into the right dorsal of each nude mouse. When the average tumor volume reached 50 mm3, the nude mice with no significant difference in tumor volume were randomly divided into a control group and an administration group . The control group was subjected to intraperitoneal injection with 0.5% CMC-Na . In contrast, the administration group was subjected to intraperitoneal injection with Quercetin . The tumor volume (TV) was measured daily and calculated using the formula: TV = A × B2 × 0.52, where A is the longest diameter of solid tumors (mm). B is the shortest diameter of solid tumors (mm). Finally, tumors excised from the sacrificed mice were photographed, weighed, and fixed. All experiments were conducted blindingly  were raised in a specific pathogen-free animal house one week before acclimatization to the breeding environment. Meanwhile, ZR-75-1 cells were resuspended  or repeated measures ANOVA was adopted for comparison between multiple groups, followed by Tukey’s post hoc test  . The first 20 interaction targets and network diagrams of Quercetin were obtained -705) phosphorylation could induce dimerization and nuclear translocation, thus ultimately regulating the expression patterns of several growth-related genes  in ZR-75-1 and MCF-7 cells  Flow cytometry was used to detect the apoptosis of ZR-751 breast cancer cells treated with saffron extract at different concentrations; (B) MTS assay was used to determine the viability of breast cancer cells treated with different concentrations of saffron extract. * represents P < 0.05 compared to 0 μg/mL group, *** represents P < 0.001, **** represents P < 0.0001.Additional file 2: Figure S2. Crocetin, isorhamnetin, kaempferol, and Quercetin affect the cytotoxicity of BC MCF-7 and MDA-MB-231 cells at different concentrations. The effect of Crocetin (A), isorhamnetin (B), kaempferol (C), Quercetin (D), and n–n-heptanal (E) on the cytotoxicity and viability of BC MCF-7 and MDA-MB-231 cells was detected by MTS assay. (F) The cytotoxic effect of Quercetin on human normal breast cells MCF10A and MDA-kb2 cells was determined with an MTS assay.Additional file 3: Figure S3. Analysis of the main signaling pathway of five major components of saffron in breast cancer. (A) Network relationship diagram of the target point; (B) Network relationship diagram of the target point based on BC, CC, De screening; (C) Network relationship diagram of the signaling pathway and target genes; (D) Key target gene interaction diagram of Quercetin's anti-cancer effect on breast cancer.Additional file 4: Figure S4. Analysis of the main signaling pathways of the 5 major components of saffron in breast cancer. (A-F) Network relationship diagram of potential target genes for cluster analysis.


Solanum tuberosum) chips are the most consumed snacks worldwide today. Colored potato chips prepared from potato cultivars with red and purple flesh are a novel alternative to traditional potato chips because of their higher phenolic compound content, such as anthocyanins and hydroxycinnamic acid derivatives (HCADs), which might make these chips healthier compared with traditional chips. There is little information on the stability of these compounds. In this study, the nutritional value of these chips was evaluated by determining phenolic profiles, antioxidant activity and color parameters with liquid chromatography diode array and mass spectrometry detection (HPLC-DAD-ESI-MS/MS) and spectrophotometric methods during storage for four months. Five anthocyanins and three HCADs were detected, with the latter compounds being the most abundant, with concentrations on average between the first (97.82 mg kg−1) and the last (31.44 mg kg−1) week of storage. Similar trends were observed in antioxidant activity and stability, with the CUPRAC method showing the highest response among all the methods employed. The color indices were stable throughout the storage time. Based on these results, colored-flesh potato chips are an optimal alternative for consumption because of their high retention of phenolic compounds and antioxidant activity during storage, providing potential benefits to human health.Potato ( Solanum tuberosum sp. tuberosum) is the fourth most important crop in the world in terms of human food after maize, wheat and rice [Potato  in the pulp relative to light-yellow potato varieties. Furthermore, it was observed that the pre-drying and frying stages resulted in the highest losses of the determined hydroxycinnamic acids, at 91% and 97%, respectively. Stability studies of colored potato extracts carried out by Ercoli et al. [A recent study by Tajner-Czopek et al.  revealedi et al.  showed aAccording to these preceding studies, the general aim of this study is to evaluate the nutritional value of colored potato chips by determining their phenolic profiles and antioxidant activity.A total of five different anthocyanins were detected in the samples, whereas, for HCADs, two compounds were detected up to week 12, but from this point onward, a third HCAD was detected .−1, and it was not detected (nd) in the last week of storage. In contrast, peonidin-3-p-coumaroylrutinoside-5-glucoside and an unidentified compound (peak 8) showed the lowest values, and peonidin-3-p-coumaroylrutinoside-5-glucoside reached the highest concentration of 1.21 ± 0.24 mg kg−1 at week 18 of analysis, but with no significant differences between the initial and final conditions. The quantification of HCADs was carried out using an external calibration curve using chlorogenic acid (CQA) as a standard. The compound with the highest values corresponded to 5-caffeoylquinic acid with an initial concentration of 97.82 ± 34.47 mg kg−1 and a concentration of 26.08 ± 8.20 mg kg−1 in the last week of analysis; in contrast, the compound with the lowest values corresponded to the caffeoylquinic acid isomer, with concentrations ranging from 1.79 to 1.33 mg kg−1 between the initial and final determinations  as a standard . Among tinations .−1 and 99.60 mg kg−1, respectively. At the end of week 2, decreases of 69.02% and 46.08% for each group were observed. The anthocyanin content varied throughout the experiment, reaching the lowest point after 23 weeks of storage, with contents below the quantification limit of our method  as the standard C. The in−1, followed by a decrease of 35.02% at the end of week 2, and then, stable values were observed until week 9. Then, an increase in activity was observed between weeks 9 and 12, with a subsequent decrease to values close to 2 µmol Trolox g−1. In contrast, the TEAC assay showed stable antioxidant activity throughout the storage period, with a 52.94% decrease in the last week compared with the initial measurements (1.14 µmol g−1) , 2,2-diphenyl-1-picrylhydrazyl (DPPH), cupric reducing antioxidant capacity (CUPRAC) and oxygen radical absorbance capacity (ORAC) methods. The highest level of antioxidant activity was found with the CUPRAC method A, reachimol g−1) B. In themol g−1) C, resultC method D, the inYellow, red and blue colorations were evaluated in the potato chips. The initial conditions of the potato chips represented, on average, 79.82% yellow, 15.98% red and 5.08% blue coloration. The proportion of each color remained stable during the entire storage period A. For thPrincipal component analysis (PCA) was carried out considering all the evaluated parameters . This anIn other studies on colored-flesh potatoes, after the cooking and frying processes, a variety of compounds were identified ,4. In thIn the present study, total HCAD concentrations were higher than the total anthocyanin concentrations. A similar finding was reported by Ruiz et al. , who sho−1, anthocyanin degradation rates in sour cherry and pomegranate juices increased significantly [Comparing the stability of anthocyanins from colored potato chips with other sources of anthocyanins could be important in the food industry. For example, it has been reported that the stability of anthocyanins from strawberry nectars is quite low after forty-two days of storage . One facficantly ,26. Simificantly . The difficantly , potatoeficantly ,4, whichficantly  observedficantly  conducteficantly  on bluebficantly  conducteficantly  on milk ficantly . These rficantly ,33,34.−1. The results obtained with the TEAC method were low compared with studies performed by Ruiz et al. [−1. However, in the same study by Ruiz et al. [−1 dry weight (DW) to 33.2 μmol g−1 DW [−1 DW and 24 μmol 100 g−1 DW, respectively. These results were lower relative to the values obtained in our study on colored-flesh potato chips.The high response detected with the CUPRAC method may be attributed to the high concentration of HCADs in the samples. Ruiz et al.  demonstrz et al.  on colorz et al. , higher l g−1 DW . ORAC isl g−1 DW . There il g−1 DW  performeFor antioxidant activity, our results, based on the CUPRAC method, revealed a 45.23% decrease relative to the initial measurement and showed higher stabilities compared with those achieved in studies on colored-flesh potato extracts, in which researchers observed an 81% decrease, on average, in antioxidant activity under acidic conditions  . The actColor is one of the key factors that affect the quality of food products because some products attract consumer attention because of their appearance . RegardiInterestingly, although a relationship between anthocyanin degradation and loss of color quality was mentioned, in our study, no change in the color of the potato chips could be seen despite the decrease in anthocyanin concentrations. Studies on color stability in different food products, e.g., jam or yogurt made from berries, have shown that the rate of color loss is slower than the rate of anthocyanin degradation, and the presence of other compounds can have a considerable effect on color ,42. The In general, the phenolic compounds identified in this study decreased drastically during the storage period, which directly correlated with a decrease in the antioxidant activity of these compounds. Tereucán et al.  presente2PO4), dibasic potassium phosphate (K2HPO4), potassium persulfate (K2O8S2), ammonium acetate and copper (II) chloride (CuCl2) were obtained from Sigma-Aldrich . Water (HPLC grade), ethanol (HPLC grade), methanol (HPLC grade), acetonitrile (HPLC grade), formic acid (p.a. grade) and sodium carbonate (K2O8S2) were obtained from Merck .Standard petunidin-3-glucoside (98.95%) was obtained from PhytoLab . Trolox  (97%), ABTS ) (>98%), gallic acid (97.5–102.5% titration), chlorogenic acid (≥95%), neocuproin (≥98%), DPPH , Folin–Ciocalteu reagent, AAPH  (97%), fluorescein (95%), monobasic potassium phosphate (KHPotato chips (“PatPot Chips”) were provided by C-M SpA  . Coloredv/v) in a test tube protected from light. Subsequently, the samples were sonicated with an ultrasonic processor at 130 Watt  for 60 s at 40% amplitude, shaken for 10 min at 200× g and centrifuged  for 10 min at 3000× g. Finally, the supernatant was recovered and stored at −20 °C until analysis.Five grams of potato chips was smashed and mixed with 10 mL of extraction solvent  protected with a C18 precolumn  at 40 °C. The mobile phases were A) water:acetonitrile:formic acid 87:3:10 (v:v:v) and B) water:acetonitrile:formic acid 40:50:10 (v:v:v). The mobile phase B gradient was from 6% to 30% over 15 min; from 30% to 50% over 15 min; from 50% to 60% over 5 min; and from 60% to 6% over 6 min, followed by stabilization for 10 min at a flow rate of 0.8 mL min−1. The injection volume was 20 μL. Anthocyanins and HCADs were quantified at 520 nm and 320 nm, respectively, using petunidin-3-glucoside and chlorogenic acid as external calibration standards . The tests were performed using gallic acid as a standard , and finally, the second reading was performed. The measurements were made using a UV-Vis Epoch microplate spectrophotometer , and the results were expressed as µmol Trolox equivalents g−1.For the determination of Trolox equivalent antioxidant capacity (TEAC), solutions must be adjusted to an absorbance of 0.70 ± 0.05 (734 nm) via dilution with ethanol or water before use . Briefly2, 50 µL of 7.5 mM neocuproine and 50 µL of 1 M ammonium acetate buffer (pH 7), and the mixture was incubated in the dark at 27 °C for 15 min. Next, 100 µL of Trolox standard or sample was added, and the solution was incubated for 30 min at 27 °C. Quantification was carried out at 450 nm using a UV-Vis Epoch microplate spectrophotometer , and the results were expressed as µmol Trolox equivalents g−1 [For cupric reducing antioxidant capacity (CUPRAC), the following reagents were added to a 96-well microplate: 50 µL of 10 mM CuCl−1.The determination of antioxidant activity using DPPH was based on a methodology described by Maldonado et al. , with so−2 µM), and the solution was incubated for 30 min at 37 °C before adding 25 µL of AAPH solution. Fluorescence was measured every 5 min for 90 min in a Synergy HTX Multi-Mode reader , and the values were expressed as µmol Trolox equivalents g−1.Determination of oxygen radical absorbance capacity (ORAC) was performed following a methodology described by Cao et al. . In brie420 + A520 + A620), color tonality (To = A420/A520), percentage of yellow (%Ye = A420 × 100/CI), percentage of red (%Red = A520 × 100/CI), percentage of blue (%Bl = A620 × 100/CI), redness a = 3.282 −  + (74.509 × A520) − (81.731 × A620), yellowness b = −0.716 + (91.261 × A420) − (41.675 × A520) − (54.352 × A620), lightness L = 98.172 − (17.497 × A420) − (35.817 × A520) − (10.416 × A620), saturation C = 3.035 − (14.582 × A420) + (59.488 × A520) − (91.112 × A620) and hue angle h = 9.569 + (299.84 × A420) − (186.894 × A520) − (93.708 × A620). Determinations were carried out in a 1 mm quartz cuvette using a Genesys 10 s UV-Vis spectrometer .Color determinations were carried out using the CIELab method , in whicAll the statistical analyses and calculations were conducted using R version 4.2.1. One-way ANOVA was employed to examine significant variances between the measurements of each experimental variable. For the variables that exhibited significant differences, the means were compared using Tukey’s HSD test along with the appropriate post hoc analysis with “agricolae” v. 1.3.5 package. Furthermore, the datasets corresponding to weeks 0, 4, 8, 13, 17, 21 and 23 were subjected to principal component analysis (PCA). Confidence ellipses representing the group means for each week were generated using the “FactoMineR” v. 2.7 and “factoextra” v. 1.0.7 packages.The results obtained in this study indicate that chips made from colored-flesh potatoes are a good source of phenolic compounds, mainly HCADs, which remained stable during a storage period of twenty-four weeks. Although colored potatoes are rich in anthocyanins, they do not constitute a stable food matrix for anthocyanins when cooked as chips since the anthocyanins were almost completely degraded after 23 weeks of storage. Similar trends were observed in the total phenolic compound content and antioxidant activity using all methodologies, with a slight decrease over time. The color index did not show large differences in relation to stability. Potato chips made from colored-flesh potatoes might be an optimal alternative snack for global consumption given their high phenolic compound content, including anthocyanins and HCADs, which possess high antioxidant activity and other potential benefits for human health.


Phosphonic acids represent one of the most important categories of organophosphorus compounds, with myriad examples found in chemical biology, medicine, materials, and other domains. Phosphonic acids are rapidly and conveniently prepared from their simple dialkyl esters by silyldealkylation with bromotrimethylsilane (BTMS), followed by desilylation upon contact with water or methanol. Introduced originally by McKenna, the BTMS route to phosphonic acids has long been a favored method due to its convenience, high yields, very mild conditions, and chemoselectivity. We systematically investigated microwave irradiation as a means to accelerate the BTMS silyldealkylations (MW-BTMS) of a series of dialkyl methylphosphonates with respect to solvent polarity , alkyl group , electron-withdrawing P-substitution, and phosphonate–carboxylate triester chemoselectivity. Control reactions were performed using conventional heating. We also applied MW-BTMS to the preparation of three acyclic nucleoside phosphonates , which were reported to undergo partial nucleoside degradation under MW hydrolysis with HCl at 130–140 °C . In all cases, MW-BTMS dramatically accelerated quantitative silyldealkylation compared to BTMS with conventional heating and was highly chemoselective, confirming it to be an important enhancement of the conventional BTMS method with significant advantages over the MW-HCl method. Phosphonic acids constitute one of the most important categories of organophosphorus compounds. The diverse utility of phosphonates encompasses applications as antibiotic and antiviral agents, chelating agents in imaging, catalysts, ion exchange materials, and components of polymers used in paints and adhesives, among many others . As a reWhen present as part of a complex molecule, phosphonates are normally maintained as simple alkyl esters until final conversion to the final phosphonic acid product. Since its introduction by the senior author ,6,7, silUnder typical reaction conditions, BTMS silyldealkylations of phosphonate dialkyl esters proceed at room temperature or with moderate thermal heating. A growing trend in organic synthesis during recent years has been the substitution of microwave (MW) irradiation for conventional heating, with organophosphorus chemistry being no exception . Some yeIn 2012, Jansa et al. studied the acid-catalyzed hydrolysis of acyclic nucleoside phosphonate (ANP) esters under microwave irradiation . ANPs ar2, (S)-PMPDAP (OEt)2, and PMEA (OiPr)2 [Here, we present a systematic investigation of the scope of the microwave-assisted BTMS (MW-BTMS) dealkylation of phosphonate alkyl esters, comparing the influence of the alkyl  ester groups on the 100% conversion time and examining the role of the solvent. The effect of an electron-withdrawing P-substituent is examined, as is the chemoselectivity for trialkyl phosphonoacetates. Finally, we assess its compatibility with three ANP phosphonate esters possessing acid-sensitive groups: PMEDAP (OiPr)31P NMR, and after treatment with MeOH, the yield of isolated methylphosphonic acid was obtained.Reaction completion times were compared for four aprotic solvents with markedly different structures, with sulfolane being the most polar (ε = 43), followed by ACN (ε = 37), DMF (ε = 38), and 1,4-dioxane, which is the least polar (ε = 2.2) . As a reFor the methyl ester, shorter reaction times were observed in DMF and sulfolane compared to ACN when the temperature and other conditions were held constant, although the dielectric constant (ε) of DMF is marginally lower than that of ACN. Significantly, in 1,4-dioxane, the least polar of the solvents tested, this reaction proceeded to completion at least as rapidly as in ACN. Rapid conversions were also observed for the reactions in neat excess BTMS. For the ethyl ester, the reaction in all four solvents was complete at 40 °C within 6–15 min. The reaction of the isopropyl ester required only 2 min in sulfolane at 80 °C and 20–30 min in ACN, DMF, or 1,4-dioxane at 60–80 °C.In all cases, MW-BTMS reactions were up to >100 times faster (often complete within a few min) than their conventionally heated counterparts , depending on the ester alkyl group, temperature, and BTMS molar excess. The reaction of all three esters was complete in neat BTMS within 2–15 min at 40 °C.− creates a positive charge at phosphorus, decreasing reactivity when R’ is an electron-withdrawing substituent such as a carboxyl or α-halo alkyl group [The marked P-O vs. C-O chemoselectivity of BTMS for silyldealkylation in carboxylate-phosphonate mixed esters under standard conditions is well established ,19,20. Tyl group ,21. ThisIn contrast to the ethyl triester , Entry 2Slight modifications to the workup procedures for the carboxylphosphonate products were necessary. For the silyl ester intermediate of 2-phosphonoacetic acid , Entry 3ANPs, especially those containing DAP or PMEA groups, may undergo the partial degradation of the nucleoside moiety during phosphonate ester dealkylation under the vigorous HCl hydrolysis conditions described by Jansa et al. . In contThe initial report on the acceleration of BTMS silyldealkylations of certain diethyl phosphonates in ACN  was follUsing BTMS reacting with a uniform dialkyl methylphosphonate substrate, MW irradiation under the experimental conditions  was found to accelerate reactions dramatically for all three types of alkyl ester in all four aprotic solvents and neat excess BTMS, with quantitative conversion by NMR obtained for the dimethyl ester in as little as 2 min at 40–60 °C in solvents with different polarities and neat BTMS . NotablyIn ACN at 40 °C, the methyl ester reaction time was longer than for the other solvents (10 min vs. 2–5 min), but this was more than halved by raising the temperature to 60 °C. Results for the diethyl esters showed only a small decrease in reactivity, depending on the solvent. For the diethyl ester in 1,4-dioxane, the reaction time increased slightly to 15 min at 40 °C , but it was reduced to 6 min at 60 °C and 4 min at 80 °C. The reaction was virtually immediate in DMF, sulfolane, and ACN at 60 °C (2–5 min) and greatly accelerated compared to thermal heating alone. MW-BTMS silyldealkylation times of the sterically more hindered isopropyl diesters, which are typically less reactive with BTMS under conventional heating, did show a dependence on solvent polarity; sulfolane was the most effective (10 min at 60 °C and 2 min at 80 °C), compared to DMF , 1,4-dioxane (30 min at 80 °C), and ACN (90 min at 40 °C and 30 min at 60 °C). The ability of MW-BTMS to achieve the rapid quantitative silylation of the isopropyl esters under these mild conditions is a signal improvement over the traditional BTMS method, where limitations to methyl or ethyl ester precursors of targeted phosphonic acids to avoid higher temperatures and longer reaction times may be encountered.Neat BTMS reactions were used in the initially reported phosphonate diester silyldealkylations ,6. With 2P(O)(OEt)2 moiety, which has an exceptionally electronegative phosphonate carbon. This compound required 1 h in ACN at 60 °C to complete silyldealkylation, suggesting that MW irradiation does not change the mechanism of the reaction in terms of it proceeding via an electron-deficient transition state.A hallmark of the BTMS approach to the synthesis of phosphonic acids from their diesters is its chemoselectivity against the dealkylation of a carboxylic acid ester present in the same molecule . Consequ2, (S)-PMPDAP(OEt)2, and PMEA(OiPr)2. The reaction times required for the quantitative conversion of these compounds to the bis(trimethylsilyl) esters by treatment with BTMS in ACN at 60 °C  ranged from 4 to 6 h  and distilled under nitrogen (greaseless glass joints) prior to use. Dimethyl methylphosphonate, trimethyl phosphonoacetate, and triethyl phosphonoacetate were purchased from Aldrich. Diethyl methylphosphonate was purchased from Alfa Aesar , while diisopropyl methylphosphonate was purchased from Lancaster Synthesis . Diethyl (bromodifluoromethyl)phosphonate was purchased from Oakwood Products . Acetonitrile was purchased from EMD Chemicals  and distilled prior to use; Drisolv N,N-dimethylformamide was purchased from EMD Chemicals; 1,4-dioxane was purchased from Macron Chemicals  and distilled over sodium; sulfolane was purchased from Sigma-Aldrich and distilled under reduced pressure. Diethyl phosphonoacetate  and the 1H) or 202 (31P) MHz on a Varian VNMRS-500 NMR. The microwave irradiation of reaction mixtures was carried out in a Milestone Ethos Synth Microwave Synthesis Labstation. The chemical shift references for 1H NMR spectra were the residual HDO (δ 4.79) in D2O or residual CHCl3 (δ 7.26) in CDCl3, while the 31P NMR spectra were referenced against an external 85% H3PO4 standard (δ 0.00). All chemical shift values (δ) are given in ppm, and NMR sample pH values were measured in 99.9% D2O without deuterium isotope corrections. The approximate concentration of the NMR samples was 1–3 mg/mL for isolated compounds. All glassware was oven-dried, solvents were AR reagent grade, and NMR spectra were obtained at 500  δ: 1.20–1.24 (d) ppm (31P NMR (D2O) δ: 31.05 ppm (31P NMR (D2O) δ: 31.11 and 1H NMR (D2O) δ: 1.24–1.31 [General procedure for the microwave-assisted dealkylation of methylphosphonate dialkyl esters : the app (d) ppm ; 31P NMR1.05 ppm  These va.24–1.31 . 31P NMR spectra of product mixtures in CDCl3 were obtained for the bis(trimethylsilyl) esters of (2-methoxy-2-oxoethyl)phosphonic acid 2 (0.134 mmol), (S)-PMPDAP(OEt)2, or PMEA(OiPr)2 (0.140 mmol) were dissolved in 1.5 mL of acetonitrile, and 4 or 6 molar equivalents of BTMS was quickly added. All microwave reaction times included a 1-minute ramp to 60 °C , the conventional BTMS silyldealkylation of methyl, ethyl, and isopropyl phosphonate diesters is greatly accelerated in aprotic solvents having both low and high dialectric constants, including ACN , 1,4-dio


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.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 . 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 As monoclonal antibodies, particularly blocking antibodies, are generally of low affinity, and therefore must be administered at relatively high doses to elicit therapeutic responses7. Mostly given subcutaneously3, they are utilized at a maximally allowable volume of 1.5 mL5. It has therefore become critical to develop formulations of monoclonal antibodies at ultra–high concentrations8. These formulations also allow single–dose self–administration using auto–injection devices, reduced dosing frequency, and improved patient compliance9.Biotherapeutics have transformed the treatment of chronic inflammatory diseases and cancers12. Notably, due to the higher concentrations, these formulations may be very viscous or gelatinous, leading to increased tissue back pressure and pain at the injection site13. Manufacturing challenges for highly concentrated formulations also include difficulty in pumping due to high shear stress, clogging of the membrane, high back pressure, and aggregation during the manufacturing17.However, ultra–high concentration monoclonal antibody formulations are associated with physical and chemical instabilities depending on the physicochemical properties of the specific monoclonal antibody8. The very first ultra–high concentration monoclonal antibody formulation, Palivizumab , was approved in 199818, followed by others including Alirocumab , Cosentyx , Actemra , and Belimumab , that constitute almost one third of all approved biotherapeutics, have been developed with high protein concentrations (≥100 mg/mL); 76 % of the approved formulations are administered by the subcutaneous route9 resulting from high viscosity and a crowded environment21. Such interactions are reversible or irreversible, and may cause undesirable liquid–liquid separation, protein aggregation, or opalescence22. The effects may also be caused by a variety of transient interactions in the solution, including hydrophobic, electrostatic, dipole–dipole, hydrogen bonding (inter and intra), or van der Waals interactions25via Fab-Fab20 or, in rare instances, Fab-Fc26 interactions between antibodies.The main issue with highly concentrated formulations is protein–protein interactions28. Native monomers self–associate into the oligomers through transient interactions and regain their native structure—most such associations result in reversible aggregates. However, upon stressing, and over time, monomers can associate to form multimers, resulting in irreversible (insoluble) aggregates28. While 5–10 % soluble aggregates are acceptable for biologic therapeutics, insoluble aggregates must be limited for stable formulations29. The US Pharmacopeia (USP) and the FDA have set guidelines to control aggregates in a final biologic formulation31. For example, sub–visible particles with a hydrodynamic radius ≤50–100 μm are considered optimal29. Hence, per the FDA, the acceptable limit of sub–visible particulate matter in a container of ≤100 mL should be 6000 particles ≥10 μm and 600 particles ≥25 μm30. Particle sizes ≤10 μm can cause vascular occlusion.Proteins generally undergo aggregation using native monomers, denatured proteins, and pre–existing aggregates39. MS-Hu6 was humanized from the parent murine antibody clone, Hf2, generated against a short 13–amino–acid–long epitope of the receptor–binding sequence of FSHβ40. We have shown that MS-Hu6 binds to FSHβ and blocks its interaction with the FSH receptor (FSHR) with an affinity of 7.5 nM, comparable to trastuzumab 37. We have also confirmed the stability of MS-Hu6 and its binding to FSH using a protein thermal shift assay 41.We have reported the development of a first–of–its–kind humanized FSH–blocking antibody, MS-Hu6, based on studies that have established direct actions of FSH in promoting bone loss, body fat accrual, and neurodegeneration. MS-Hu6 reduces body fat, induces thermogenic adipose tissue, improves bone density, and prevents cognitive decline in mouse modelsper Code of Federal Regulations , applied perhaps for the first time, in this context, in an academic medical center.Here, we have formulated an ultra–high concentration of MS-Hu6 at 100 mg/mL. Namely, we have fully characterized the effect of antibody concentration on formulation stability using the protein thermal shift assay, dynamic light scattering (DLS), and size exclusion chromatography. For long–term and optimum stability, we screened antioxidant and chelating agent concentrations using the protein thermal shift assay and DLS. In addition, this optimized formulation was also examined for viscosity, turbidity, and clarity; thermal stability using differential scanning calorimetry (DSC), freeze–thaw studies, and accelerated stability; and importantly, FSH binding. Structural integrity was also evaluated using FTIR and CD spectroscopy. These studies were carried out under Good Laboratory Practice (GLP) standards m), which is the temperature at which 50% of molecules are denatured42. Denaturation is observed by the increase in fluorescence of SYPRO Orange, which binds to hydrophobic residues that are exposed as globular proteins unfold37. Binding of ligand to the target protein results in increased stability and a higher Tm. Tm values that are higher (or more stable) indicate overall thermostability. To optimize the formulation, we increased MS-Hu6 concentration from 0.2 to 100 mg/mL , while Tm values dropped somewhat for the MS-Hu6 formulation compared with MS-Hu6 in PBS, the difference (or ΔTm) did not exceed 1°C—which provided assurance of thermostability even at 100 mg/mL.The ultra–high concentration of formulated MS-Hu6 was optimized using protein thermal shift for thermostability, size exclusion chromatography (SEC) for monomeric stability, and dynamic light scattering (DLS) for colloidal stability. Thermal shift determines a protein’s unfolding (melting) temperature  and dimer (peak 1) areas were monitored. Z–average values.We examined colloidal stability using DLS, a well–recognized, quick, and sensitive method to determine particle size and particle–size distribution. The method is based on light scattering due to Brownian motion. While maintaining the original structure of proteins, DLS measures the hydrodynamic diameter (rh) and polydispersity index (PDI). The samples were diluted with respective buffers (PBS or Formulation Buffer) prior to DLS. Data were collected in volume percent, with hydrodynamic radius (rh) presented for the prominent and smaller peaks . ParticlThe two MS-Hu6 (in PBS) samples displayed a prominent peak (volume % ~99%) with an average rh of 4.96 nm, which fell within the industry standard (rh <10 nm). However, there were two aggregated peaks with average volume percentages of <1%, but with an average rh of 91.4 and 2595 nm, respectively. The multiplicity of such peaks resulted in a high PDI, indicating varying particle size . In contH3) and Met 209(CH3), we opted also to use L–methionine to avoid methionine oxidation during manufacturing and storage. We examined the effect of disodium–EDTA and L-methionine on the stability of the MS-Hu6 formulation using protein thermal shift and DLS. Protein thermal shift showed that compared with MS-Hu6 (8.621 mg/mL) in PBS, formulated MS-Hu6 (100 mg/mL) plus 1 mM EDTA and 1 mM L-methionine showed slight increased ΔTm value for the Fc domain of 0.38 °C .We tested the effect of five concentrations of formulated MS-Hu6  on the dynamic viscosity, a surrogate measure for syringeability. Compared with water (3 cP) and formulation buffer (5.4 cP), formulated MS-Hu6 showed a concentration–dependent linear increase in viscosity  . The incWe also assessed turbidity  of various concentrations of MS-Hu6  using a microplate reader at 350 nm, as well as clarity at 350, 540 and 599 nm. Comparisons were made against MS-Hu6 in PBS (8.6251 mg/mL), PBS and formulation buffer—the latter showed very low absorbance at 0.068 (± 0.002), 0.072 (± 0.001) and 0.077 (± 0.001), respectively . There w48.Finally, there were also no differences in clarity at any wavelength except a minimal drop with formulated MS-Hu6 at 100 mg/mL . In all,51. Importantly, there was no alteration in any parameter in formulated MS-Hu6 versus MS-Hu6 in PBS (0.5 mg/mL).An alteration in the secondary structure or conformation of any protein indicates inactivation or denaturation. We used Circular Dichroism (CD) spectroscopy to evaluate the secondary structure of formulated MS-Hu6. As shown in −1, a surrogate for intra–molecular β-sheets, and the random coil structure peak at 1642–1657 cm−1 did not shift, confirming maintenance of the antibody’s native conformation in formulation  spectroscopy to further study the structure of formulated MS-Hu6 at 50 and 100 mg/mL compared with MS-Hu6 in PBS. The amide I band peak at 1637 cmmulation .ms). MS-Hu6 at 50 and 100 mg/ml were diluted to 5 mg/ml in formulation buffer. MS-Hu6 displayed multi–domain transitions, with the first transition representing unfolding of CH2 (Fc) domain (Tm >71 °C), the second representing Fab unfolding (Tm >80.30 °C), and a third CH3 (Fc) domain unfolding (Tm >85 °C)  to determine the protein unfolding temperatures (T >85 °C) . Both saProtein instabilities, such as aggregation, are of major concern during biopharmaceutical development. Aggregation and denaturation can both take place during several phases of the product’s lifecycle, including freeze–thaw, production, shipping and storage. We carried out a formal rapid freeze–thaw (F/T) study on our MS-Hu6 formulation (100 mg/mL) at −80°C/25°C and −80°C/37°C, over three cycles, to confirm stability at extreme conditions. Samples were analyzed using protein thermal shift (PTS) and dynamic light scattering (DLS).ms for formulated MS-Hu6 (100 mg/mL) without F/T were 69.55±0.12°C and 79.35±0.05°C for Fc and Fab, respectively , with PDIs being <1. The small increase in PDI in cycle III (−80/37°C) is likely due to the formation of reversible aggregates resulting from Fc–fc, Fc–Fab or air-liquid interface interactions and/or reduced pH during buffer crystallization57. Nonetheless, the data are in agreement with findings for commercialized monoclonal antibody products, and demonstrate no or minimal reversible aggregation . The formulation’s excellent stability meets industry standards for product development and may improve transportability and manufacturability of MS-Hu6.All rapid F/T samples were further evaluated by DLS to determine their colloidal stability. For three cycles, the major volume peak (>99%) rh was found to be between 3 to 7 nm . The Z–am shift for the Fab domain compared to day 0 , followed by relative good stability up to 90 days for Fab. However, during the last 90 days of incubation, there is a loss of thermostability for the Fc domain with a ΔTm 1.51°C. This data shows that formulated MS-Hu6 is thermostable at 4°C (refrigerated conditions) and has excellent Fab stability in accelerated conditions at 25°C for at least 90 days. These relatively stable Tms likely arise from the stabilizing effects of the salt, stabilizer and sugar utilized in the optimized formulation.Formulated MS-Hu6 (100 mg/mL) was evaluated for storage stability at 4°C and 25°C for 15, 30, 60 or 90 days. Samples were tested for thermostability using protein thermal shift and for monomer loss using size exclusion chromatography (SEC). At 4°C, there was no appreciable Tto day 0 . However59. These findings show that MS-Hu6 in formulation (100 mg/mL) maintained monomeric stability for more than 90 days. The formation of soluble reversible aggregates, normally as a result of antibody crowding in solution or weak Fc–Fc, Fc–Fab, Fab–Fab, hydrophobic or air–liquid interactions was almost non–existent, consistent with other robust IgG formulations. Moreover, no fragmentation was observed on SEC in any samples at 4°C for 90 days.We also assessed monomer loss by SEC. Samples stored at 4°C retained 99.36% of monomer area at baseline . After 9For samples stored at 25°C for 90 days, the average monomer area retained was 96.84±1.22% for three batches, and monomer loss was 3.16% on average . Howeverm.We evaluated formulated MS-Hu6 for its binding to human FSH. MS-Hu6 (1 μg/μL) in formulation buffer or PBS was incubated with and without 10 μg/μL FSH, and its thermostability was measured by protein thermal shift, as before. We expected that binding of FSH will stabilize the Fab, but not the Fc domain of MS-Hu6, and hence result in a right–shift of the Tm of 4.49±0.03 °C for MS-Hu6 in PBS and 4.80±0.07°C for formulated MS-Hu6  had comparable T59. We complemented SEC with DLS to ascertain aggregate formation and colloidal stability. At each MS-Hu6 concentration in formulation, the major peak volume was >99% with a rh of 3–6 nm, and peak volumes for aggregated particles of <1%; this is consistent with that reported for several clinical–grade monoclonal antibodies61. Of note is that soluble reversible aggregates up to 10 μm are accepted in biotherapeutics development. However, due to their potential immunogenicity or adverse reactions, particulate matter or large aggregates may need to be monitored under strict FDA guidelines62.To assess for monomeric stability and potential aggregation, we used SEC to find that the monomeric peak was >99% at all MS-Hu6 concentrations—this suggested that formulated MS-Hu6 was monomerically stabile at higher concentrations with minimal aggregation and no fragmentation. Clinical–grade antibodies must display active protein loss of not more than 5% and secondary species <2% in a freshly reconstituted therapeutic formulationsH2 and CH3 domains of most IgG1 antibodies contains two conserved heavy chain (HC) methionine residues. The oxidation of these methionine residues reduces thermal and colloidal stability64, and in doing so, impairs long–term storage stability (shelf–life). The anti–oxidant L-methionine and chelating agent, disodium-EDTA are thus used to increase the shelf–life of commercial IgG1 biotherapeutics. Free L-methionine protects methionine residues from oxidation, while EDTA increases the effectiveness of anti–oxidants through its ability to complex heavy metal65. We found that the addition of 1 mM L-methionine and 1 mM disodium–EDTA added together resulted in thermostability in the protein thermal shift assay, and colloidal stability in DLS, with a major peak of 99.9% and rh 6.93 nm.Methionine oxidation of Fc domains is a common post–translational modification that can affect antibody bioactivity and potentially induce an immunogenic response. The interface of the C47, compounded at high concentrations, by protein–protein interactions. However, the viscosity at 100 mg/mL MS-Hu6 was considerably less than the industry maximum of 50 cPs67. The latter is known to affect syringeability. However, viscosity can vary between 30 and 50 cPs even at high antibody concentrations (>100 mg/mL). In recent years, viscosity control has been extensively studied as a function of concentration, temperature, pH, ionic strength, and ion type66.Viscosity is an essential parameter for any injectable dosage form, as highly viscous formulations increase back pressure and pain at injection sites. This issue is magnified for subcutaneously administered therapeutic antibodies at volumes <1.5 mL, making viscosity control a key determinant of highly concentrated formulations. We found an expected increase in viscosity with increasing concentrations of MS-Hu6, likely resulting from antibody crowdingPer European Pharmacopoeia, this would be categorized as Ref II  . In the case of clarity, percent transmittance of formulated MS-Hu6 was >99% at all wavelengths, indicative of clarity between 0.2 and 50 mg/mL, with a slight decrease at 100 mg/mL (350 nm). The clarity values fell into the categories of Ref I (3–6 NTU) and II. The majority of commercialized biopharmaceuticals have clarity values up to 12 NTU68.Turbidity and clarity are equally important parameters in biopharmaceutical development. Increases in turbidity and reduced clarity could result from antibody overcrowding or gelation and the formation of reversible aggregates due to weak protein–protein interactions, such as Fc–Fc, Fc–Fab, hydrogen bonding, and hydrophobic interactions. We found a small increase in turbidity (absorbance) of 100 mg/mL formulated MS-Hu6 at 350 nm. 51. These structures remained unaltered in the formulated version with no loss of active protein. Active protein loss in a newly reconstituted biotherapeutic formulations should be <5%, with secondary species at a maximum of 2%59. Intact secondary structure was confirmed by FTIR, in which the amide I band peak at 1637 cm−1 (representing intra–molecular β–sheets) and the random coil structure peak at 1642–1657 cm−1 were unaltered in formulated MS-Hu6 (100 mg/mL) compared with MS-Hu6 in PBS. The data rule out significant inter–molecular β–sheets resulting from protein aggregation or clustering. For further confirmation, we used the nano DSC to study multidomain transitions, as a surrogate for the conformation alterations of the Fab, Fc (CH2), and Fc (CH3) domains. Comparison between 50 and 100 mg/mL MS-Hu6 revealed almost overlapping peaks—further confirming retained conformational stability of the respective domains.Modification of secondary structure is a sign of protein inactivation or denaturation. In agreement with other IgG1 therapeutics, CD spectroscopy revealed that the predominant secondary structures in MS-Hu6 are β–sheets and unordered/random coils69. This could destabilize and degrade the product, and thereby reduce shelf–life. We thus carried out a rapid freeze–thaw (F/T) study at – 80°C/25°C and −80°C/37°C and for three cycles to confirm formulation stability at extreme conditions. ΔTms were found not to be significant (<1°C) for both Fc and Fab domains in all stressed conditions, indicating that formulated MS-Hu6 was thermostable under extreme stress conditions. The data also establish the absence of aggregation. For confirmation, the samples were further evaluated by DLS for colloidal stability. All samples showed excellent colloidal stability, with minimal reversible aggregation 55. Moreover, the PDI or rh under stressed conditions were broadly comparable for three F/T cycles, further confirming colloidal stability. However, a small increase in PDI was noted at the third F/T cycle at −80°C/37°C, likely attributable to reversible aggregates due to Fc–Fc, Fc–Fab, and air–liquid interface interactions57. For three cycles, these reversible aggregates remained in the 0.06 to 0.14% range, which is within acceptable limits (5–10%). This finding suggests that the ultra–high MS-Hu6 formulation retains its hydrodynamic radius at extreme storage conditions (−80 °C/37 °C for three cycles).Aggregation can occur at various stages of product development, including freeze–thaw, manufacturing, filling, shipping, and storagem <1°C on thermal shift). Moreover, at 25 °C, the Fc domain was stable for 60 days (ΔTm <1°C); however, after 90 days, the Tm fell slightly (>1°C), indicative of reduced stability. In contrast, the Tm for of the Fab domain did not change significantly (ΔTm–0.47°C). SEC was used to further confirm monomeric stability after 90 days. At 4 and 25 °C, all three batches retained >99% and 96.8% monomer, with loss of <1% and 3.16%, respectively (acceptable limit for active structure loss is 5%). No fragmentation was noted in any samples at 4°C (90 days). However, monomer loss was noted after 90 days at 25°C, due to the formation of soluble dimers, with molecular weights greater than 150 kDa; the latter are not considered critical due to their reversible and transient nature. Such dimerization is induced by antibody crowding in solution, weak interactions between Fc-Fc, Fc-Fab, and Fab-Fab, and hydrophobic and air–liquid interactions71. These soluble reversible aggregates are difficult to remove during antibody manufacturing and are therefore accepted in biotherapeutics (5–10%). However, and importantly, no fragmentation (low molecular weight species <150 kDa) was detected in any accelerated condition. Finally, MS-Hu6 in PBS and formulated MS-Hu6 were evaluated for their ability to bind FSH. Thermal shift assay revealed a right–shift for the Fab domain . The increased thermostability resulted from the effective binding of FSH to MS-Hu6. Expectedly, and as we have shown before, the Fc domain in either instance remained relatively unperturbed (ΔTm− 0.1°C)41.Accelerated stability refers to the extent to which a drug retains, within specified limits, the near same properties and characteristics that it possessed at the time of manufacturing. It is a surrogate for shelf life and determines optimum storage conditions. Both Fab and Fc domains of formulated MS-Hu6 remained stable at 4°C for 90 days  was provide by Genscript. Sodium chloride, potassium chloride and Tween 20 were purchased from Fisher Bioreagents, USA. Sodium dihydrogen phosphate, disodium hydrogen phosphate, and sucrose were from Alfa Aesar. L-Methionine and ethylenediamine-tetra-acetic acid (EDTA)  were procured from Sigma. Cell-culture grade water was obtained Corning. FSH (Cat # TLIH-A1110) was purchased from Golden West Biosolutions.All buffers were prepared in milli-Q water (Millipore) and filtered through 0.2 μm nitrocellulose filters (Millipore). The antibody was buffer-exchanged and concentrated by centrifugation at 3000 rpm and 4 °C in a Legend X1R Centrifuge  using Amicon Ultra-15 centrifugal filter units with a 30 kDa molecular weight cut–off . To obtain the required concentration, the antibody was diluted serially with the formulation buffer. Concentration of MS-Hu6 before and after buffer exchange was measured by the absorbance at 280 nm using a Nanodrop ND-1000 Spectrophotometer (ThermoScientific).per the Inactive Ingredient Limits Guidelines72. Our final optimized formulation contains 20 mM phosphate buffer (pH 6.2), 260 mM sucrose, 1 mM sodium chloride, 0.001 % w/v Tween 20, 1 mM L-methionine and 1 mM EDTA.The formulation for MS-Hu6 at ultra–high concentration was optimized using several FDA–approved excipients, used within the required range SEC was carried out to detect monomer loss and aggregate formation  using an AKTA Pure Fast Performance Liquid Chromatography System (Cytiva). Prepacked SEC columns  with TSKgel guard column SwXL (6 mm× 40 mm) were used. MS-Hu6 samples were diluted to 1 mg/mL using the formulation buffer, loaded onto the SEC column (500 μL), and eluted isocratically at a flow rate of 0.4 mL/min. The mobile phase is composed of sodium phosphate buffer at pH 6.2, 260 mM sucrose, 0.001% w/v Tween 20, and 1 mM NaCl at 25°C. Protein concentration was measured at 280 nm. The area under absorption curve of the chromatogram was used to determine monomeric loss. The experiment was repeated twice, and representative chromatographs are reported.Z–average of rh and PDI. All experiments were performed in duplicate, and representative particle size distribution (PSD) graphs are reported.To provide information on colloidal stability of formulated MS-Hu6, DLS was carried out to determine the size  and homogeneity . Briefly, 20 μL formulated MS-Hu6 was diluted into 1 mL using formulation buffer or PBS and transferred to disposable 1 mL microcuvettes . Using Zetasizer Nano-ZS 90 system , the diluted sample was analyzed for 60 seconds at 90° scattering angle at 25°C (5 cycles). The refractive index of the medium was set at 1.33 and dynamic viscosities were measured. Data were reported as m was calculated based on the inflection point of the protein thermal shift melting curve. The thermal shift (ΔTm) between sample A and sample B was calculated using The protein thermal shift assay was routinely used to assess the thermostability of formulated MS-Hu6. The assay utilizes Sypro Orange , which reports hydrophobic domains that are exposed during protein unfolding. Briefly, the 20 μL reaction mixture included 2.5 μL Sypro Orange dye (10X), 0.58 μL antibody samples and 16.92 μL protein thermal shift buffer (i.e. formulation buffer). The protein samples were heated from 25 °C to 95 °C at a rate of 0.3°C/sec in a StepOnePlus real-time PCR system (Applied Biosystems), and fluorescence was captured in the ROX channel. All experiments were performed in triplicate for each concentration. T100 to 500 μL of samples, including formulated MS-Hu6 at 1, 10, 20, 50, and 100 mg/mL, were analyzed using a Spindle S12 Viscometer  at 100 rpm and 25 °C to obtain dynamic viscosity values .Biotherapeutics are expected to experience extreme temperature conditions while being transported or stored. Freeze–thaw cycle testing is a type of stability testing that determines the robustness of an optimized formulation. Three batches of formulated MS-Hu6 (100 μL at 100 mg/mL) were subject to repeat freeze–thaws at −80°C/25°C or −80°C/37°C. Briefly, 100 μL formulated MS-Hu6 was incubated at −80 °C for 3 hours . Samples were thawed for 30 minutes at 25 °C in an Isotemp 250 thermostat (Fisher Scientific) or at 37 °C in a water bath (PolyScience), and analyzed by dynamic light scattering or protein thermal shift assay. The protocol was repeated to confirm antibody stability for up to three freeze-thaw cycles.73. At OD350, while protein molecules have no intrinsic absorbance, aggregated particles could scatter light and thus generate opalescence. Clarity was evaluated based on percent transmitted light. Briefly, 50 μL protein samples were transferred into an uncoated flat bottom 96–well plate  and absorbance at 350 nm (for turbidity) or transmittance at 350 nm, 540 nm and 595 nm (for clarity) was measured using a CLARIOstar microplate reader (BMG) at 25 °C after background subtraction (buffer without protein). All experiments were performed in duplicate.Turbidity was measured by the attenuation of incident light due to scattering−1 to 4500 cm−1. After subtracting the reference spectra from the sample spectra, smoothened second derivatives were generated using the Savitsky–Golay 7–point, 3rd order polynomial, 2nd derivative algorithm . For appropriate peak fitting, the 2nd derivative peaks were multiplied by −1. The region of structural interest, Amide I, was used to assess structural integrity.FTIR was carried out to confirm the structural integrity of formulated MS-Hu6 at high concentration. Our Nicolet iS10 FTIR Spectrometer (ThermoFisher) is equipped with an attenuated total reflectance (ATR) sampling accessory and a 45° ZnSe crystal. Briefly, 10–20 μL blank reference buffer or protein samples were loaded onto the ATR sample holder, and spectra were recorded from 500 cm74. Data analysis was conducted using Contin-LL (Provencher & Glockner Method) and reference set 774.Far-UV circular dichroism (CD) spectroscopy was used to assess secondary structure stability and integrity of formulated MS-Hu6. For this, 0.5 mg/mL (100 μL) of antibody samples were transferred to a cuvette. CD spectra were collected at 25 °C in a 10-mm path length with a 50-nm/min scan speed and a 0.45-sec response time using a Chirascan CD spectrometer (Applied Photophysics). A bandwidth of 2 nm was used to average five scans from 180 to 250 nm at 25 °C. Built–in functions were used to subtract the reference spectra from the respective sample spectra, followed by smoothening using the Savitzky-Golay function. In addition to protein thermal shift assays, DSC was also performed to evaluate the thermal stability of formulated MS-Hu6 at high concentrations. Briefly, samples were diluted with formulation buffer to 5 mg/mL, and 500 μL of buffer (reference) or diluted sample were loaded into 24 K gold cylindrical capillary cells in a Model-602000 Nano DSC (Waters), equilibrated for 10 minutes, and heated from 25 °C to 90 °C at a rate of 1° C/min at a constant pressure of 3 atm. To obtain the precise unfolding temperature, the specific heat capacity (Cp) of the references were subtracted from the respective protein samples followed by data fitting using the three-transition model . The respective thermograms for formulated MS-Hu6 is reported.Protein thermal shift assays and size exclusion chromatography were used to examine the stability of formulated MS-Hu6 at 4 °C (long term storage) or 25 °C (accelerated storage). Three batches for each condition were evaluated. Samples were stored in 0.5 mL Eppendorf tubes and collected at 0, 15, 30 and 90 days. Protein thermal shift assays were performed to assess thermal stability. To determine monomeric stability, size exclusion chromatography was carried out on samples that have been stored for 90 days at 4° C and 25° C. Experiments were run in triplicate and representative chromatographs are reported.To confirm binding, formulated MS-Hu6 or MS-Hu6 in PBS (1 μg/μL) was incubated with or without human FSH (10 μg/μL) at room temperature for 30 min. Protein thermal shift assays using Sypro Orange were performed as above. Experiments were carried out in triplicate.