“…The higher-order structures of JS016 lots were characterized using an array of biophysical techniques. Results of the secondary structure analysis by microfluidic modulation spectroscopy (MMS), 17 tertiary structure analysis by ultraviolet circular dichroism (UV CD), and thermal stability analysis by differential scanning calorimetry (DSC) are shown in Figure 4(a) –(c), respectively. Figure 4.…”
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a serious public health crisis worldwide, and considering the novelty of the disease, preventative and therapeutic measures alike are urgently needed. To accelerate such efforts, the development of JS016, a neutralizing monoclonal antibody directed against the SARS-CoV-2 spike protein, was expedited from a typical 12- to 18-month period to a 4-month period. During this process, transient Chinese hamster ovary cell lines are used to support preclinical, investigational new drug-enabling toxicology research, and early Chemistry, Manufacturing and Controls development; mini-pool materials to supply Phase 1 clinical trials; and a single-clone working cell bank for late-stage and pivotal clinical trials were successively adopted. Moreover, key process performance and product quality investigations using a series of orthogonal and state-of-the-art techniques were conducted to demonstrate the comparability of products manufactured using these three processes, and the results indicated that, despite observed variations in process performance, the primary and high-order structures, purity and impurity profiles, biological and immunological functions, and degradation behaviors under stress conditions were largely comparable. The study suggests that, in particular situations, this strategy can be adopted to accelerate the development of therapeutic biopharmaceuticals and their access to patients.
“…The higher-order structures of JS016 lots were characterized using an array of biophysical techniques. Results of the secondary structure analysis by microfluidic modulation spectroscopy (MMS), 17 tertiary structure analysis by ultraviolet circular dichroism (UV CD), and thermal stability analysis by differential scanning calorimetry (DSC) are shown in Figure 4(a) –(c), respectively. Figure 4.…”
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a serious public health crisis worldwide, and considering the novelty of the disease, preventative and therapeutic measures alike are urgently needed. To accelerate such efforts, the development of JS016, a neutralizing monoclonal antibody directed against the SARS-CoV-2 spike protein, was expedited from a typical 12- to 18-month period to a 4-month period. During this process, transient Chinese hamster ovary cell lines are used to support preclinical, investigational new drug-enabling toxicology research, and early Chemistry, Manufacturing and Controls development; mini-pool materials to supply Phase 1 clinical trials; and a single-clone working cell bank for late-stage and pivotal clinical trials were successively adopted. Moreover, key process performance and product quality investigations using a series of orthogonal and state-of-the-art techniques were conducted to demonstrate the comparability of products manufactured using these three processes, and the results indicated that, despite observed variations in process performance, the primary and high-order structures, purity and impurity profiles, biological and immunological functions, and degradation behaviors under stress conditions were largely comparable. The study suggests that, in particular situations, this strategy can be adopted to accelerate the development of therapeutic biopharmaceuticals and their access to patients.
“…As we know, CD spectroscopy of protein is highly sensitive toward the secondary structure and the same applies for peptides. Therefore, the secondary structure of AR-9 was analyzed by CD spectroscopy using the reported method ( Liu et al, 2020 ). Moreover, the types of functional groups contained in AR-9 could be detected for FT-IR ( Hackshaw et al, 2020 ).…”
The dysbiosis of gut flora and its metabolites plays important roles in the progression of hyperlipidemia (HL), and some bioactive peptides are available for HL treatment. In this study, we aimed to isolate an active peptide (AR-9) from active peptides of E. sinensis (APE) and determine whether AR-9 could improve many symptoms of a HL rat induced by a high-fat diet (HFD) by modulating gut flora and its metabolites. Above all, AR-9 was derived from APE using ion-exchange chromatography, and its structure was deconstructed by Fourier transform infrared spectrometer (FT-IR), circular dichroism (CD) spectroscopy, and UHPLC-Q-Exactive-Orbitrap MS. Then, an HFD-induced HL model in SD rats was established and used to clarify the regulatory effects of AR-9 (dose of 3 mg/kg) on HL. Normal diet–fed rats were taken as the control. The plasma samples and liver were harvested for biochemical and histopathological examinations. 16S rRNA gene sequencing and untargeted metabolomics were sequenced to assess changes in gut flora and its metabolites from rat fecal samples. Finally, Spearman’s correlation analysis was used to assess the relationship between lipid-related factors, gut flora, and its metabolites so as to evaluate the mechanism of AR-9 against HL. The results of the separation experiments showed that the amino acid sequence of AR-9 was AVFPSIVGR, which was a fragment of the actin protein from Blattaria insects. Moreover, HFD rats developed exaltation of index factors, liver lipid accumulation, and simple fibrosis for 8 weeks, and the profiles of gut flora and its metabolites were significantly altered. After treatment, AR-9 decreased the levels of lipid factors in plasma and the extent of liver damage. 16S rRNA gene sequencing results indicated that AR-9 significantly increased the relative abundance of beneficial bacteria Bacteroidetes and reduced the relative abundance of the obesity-associated bacteria Firmicutes. Furthermore, AR-9 changed gut microbiota composition and increased the relative abundance of beneficial bacteria: Lactobacillus, Clostridium, Dehalobacterium, and Candidatus arthromitus. Fecal metabolomics showed that the pathway regulated by AR-9 was “arginine biosynthesis”, in which the contents were citrulline and ornithine. Spearman’s correlation analysis revealed that two metabolites (ornithine and citrulline) showed significantly negative correlations with obesity-related parameters and positive correlations with the gut genera (Clostridium) enriched by AR-9. Overall, our results suggested interactions between gut microbial shifts and fecal amino acid/lipid metabolism and revealed the mechanisms underlying the anti-HL effect of AR-9. The abovementioned results not only reveal the initial anti-HL mechanism of AR-9 but also provide a theoretical basis for the continued development of AR-9.
“…The FT-IR spectra of the crude polypeptides with molecular weight between 3 and 5 kDa were dominated by the peaks at different wavelengths of 3908.35, 3406.39, 2918.37, 2855.62, 2364.81, 1657.87, 1542.62, 1459.88, 1051.33, 783.62, 699.28, 501.66 and 419.69 cm −1 (shown in Figure 2 ). According to the work of Long et al [ 33 ] and Liu et al [ 34 ], the absorption of β-sheet and α-helix were in the frequency area of 1610–1640 and 1650–1660, respectively; thus, bands around 1657.87 were expected for the crude polypeptides with α-helical and β–sheet structures, which is conducive to maintaining the secondary structure stability of polypeptides [ 35 ]. Absorbance at 3406.39 cm −1 wavenumbers was due to the stretching vibrations of the O–H group, indicating the presence of hydrogen bonds (intramolecular and intermolecular) in the polypeptide molecules [ 34 ].…”
Section: Resultsmentioning
confidence: 99%
“…According to the work of Long et al [ 33 ] and Liu et al [ 34 ], the absorption of β-sheet and α-helix were in the frequency area of 1610–1640 and 1650–1660, respectively; thus, bands around 1657.87 were expected for the crude polypeptides with α-helical and β–sheet structures, which is conducive to maintaining the secondary structure stability of polypeptides [ 35 ]. Absorbance at 3406.39 cm −1 wavenumbers was due to the stretching vibrations of the O–H group, indicating the presence of hydrogen bonds (intramolecular and intermolecular) in the polypeptide molecules [ 34 ]. Absorbance at 2918.37, 2855.62 and 2364.81 wavenumbers may be due to –CH 2 asymmetric and symmetric stretching vibrations of lipid in crude polypeptides [ 34 ].…”
Contamination by ochratoxigenic fungi and its prevention during the pile-fermentation of post-fermented tea have always been a concern. The present study aimed to elucidate the anti-fungal effect and mechanism of polypeptides produced by B. brevis DTM05 (isolated from post-fermented tea) on ochratoxigenic fungi, and to to evaluate their use in the pile-fermentation process of post-fermented tea. The results showed that polypeptides (produced by B. brevis DTM05) with a strong antifungal effect against A. carbonarius H9 mainly had a molecular weight between 3 and 5 kDa. The Fourier-transform infrared spectra of this polypeptide extract showed that it was a mixture consisting mainly of polypeptides and small amounts of lipids and other carbohydrates. The polypeptide extracts significantly inhibited the growth of A. carbonarius H9, and its minimum inhibitory concentration (MIC) was 1.6 mg/L, which significantly reduced the survival rate of spores. The polypeptides also effectively controlled the occurrence and ochratoxin A (OTA) production of A. carbonarius H9 on the tea matrix. The lowest concentration of polypeptides that significantly inhibited the growth of A. carbonarius H9 on the tea matrix was 3.2 mg/L. The enhancement of the fluorescence staining signal in the mycelium and conidiospore showed that the polypeptides with a concentration of more than 1.6 mg/L increased the permeability of the mycelium membrane and conidial membrane of A. carbonarius H9. The significant increase in the extracellular conductivity of mycelia suggested the outward leakage of intracellular active substances, and also further indicated an increase in cell membrane permeability. Polypeptides with a concentration of 6.4 mg/L significantly down-regulated the expression level of the polyketide synthase gene related to OTA production (acpks) in A. carbonarius H9, which may be the fundamental reason why polypeptides affect OTA production. In conclusion, reasonable use of the polypeptides produced by B. brevis can destroy the structural integrity of the cell membrane, make the intracellular active substances leak outward, accelerate the death of fungal cells and down-regulate the expression level of the polyketide synthase gene in A. carbonarius; thus, they can effectively control the contamination of ochratoxigenic fungi and OTA production during the pile-fermentation of the post-fermented tea.
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