首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
2.
3.

Objective

To explore the impact of taurine on monoclonal antibody (mAb) basic charge variants in Chinese hamster ovary (CHO) cell culture.

Results

In fed-batch culture, adding taurine in the feed medium slightly increased the maximum viable cell density and mAb titers in CHO cells. What’s more, taurine significantly decreased the lysine variant and oxidized variant levels, which further decreased basic variant contents from 32 to 27%. The lysine variant content in the taurine culture was approximately 4% lower than that in control condition, which was the main reason for the decrease in basic variants. Real-time PCR and cell-free assay revealed that taurine played a critical role in the upregulation of relative basic carboxypeptidase and stimulating extracellular basic carboxypeptidase activities.

Conclusion

Taurine exhibits noticeable impact on lower basic charge variants, which are mainly due to the decrease of lysine variant and oxidized protein variants.
  相似文献   

4.
Muscle mass is determined between protein synthesis and protein degradation. Reduction of muscle mass leads to bedridden condition and attenuation of resistance to diseases. Moreover, bedridden condition leads to additional muscle loss due to disuse muscle atrophy. In our previous study (Sato et al. 2013), we showed that administered lysine (Lys), one of essential amino acid, suppressed protein degradation in skeletal muscle. In this study, we investigated that the mechanism of the suppressive effects of Lys on skeletal muscle proteolysis in C2C12 cell line. C2C12 myotubes were incubated in the serum-free medium containing 10 mM Lys or 20 mM Lys, and myofibrillar protein degradation was determined by the rates of 3-methylhistidine (MeHis) release from the cells. The mammalian target of rapamycin (mTOR) activity from the phosphorylation levels of p70-ribosormal protein S6 kinase 1 and eIF4E-binding protein 1 and the autophagic–lysosomal system activity from the ratio of LC3-II/I in C2C12 myotubes stimulated by 10 mM Lys for 0–3 h were measured. The rates of MeHis release were markedly reduced by addition of Lys. The autophagic–lysosomal system activity was inhibited upon 30 min of Lys supplementation. The activity of mTOR was significantly increased upon 30 min of Lys supplementation. The suppressive effect of Lys on the proteolysis by the autophagic–lysosomal system was maintained partially when mTOR activity was inhibited by 100 nM rapamycin, suggesting that some regulator other than mTOR signaling, for example, Akt, might also suppress the autophagic–lysosomal system. From these results, we suggested that Lys suppressed the activity of the autophagic–lysosomal system in part through activation of mTOR and reduced myofibrillar protein degradation in C2C12 myotubes.  相似文献   

5.
Controlling acidic charge variants is critical for an industrial bioprocess due to the potential impact on therapeutic efficacy and safety. Achieving a consistent charge variant profile at manufacturing scale remains challenging and may require substantial resources to investigate effective control strategies. This is partially due to incomplete understanding of the underlying causes for charge variant formation during the cell culture process. To address this gap, we examined the effects of four process input factors (temperature, iron concentration, feed media age, and antioxidant (rosmarinic acid) concentration) on charge variant profile. These factors were found to affect the charge profile by modulating the cell culture oxidative state. Process conditions with higher acidic peaks corresponded to elevated supernatant peroxide concentration, intracellular reactive oxygen species (ROS) levels, or both. Changes in glycation level were the primary cause of the charge heterogeneity, and for the first time, supernatant peroxide was found to positively correlate with glycation levels. Based on these findings, a novel mathematical model was developed to demonstrate that the rate of acidic species formation was exponentially proportional to the concentrations of supernatant peroxide and protein product. This work provides critical insights into charge variant formation during the cell culture process and highlights the importance of modulating of cell culture oxidative stress for charge variant control.  相似文献   

6.
Microheterogeneity of monoclonal antibodies (mAbs) can impact their activity and stability. Formation of charge variants is considered as the most important source of the microheterogeneity. In particular, controlling the content of the acidic species is often of major importance for the production process and regulatory approval of therapeutic proteins. In this study, the preferential precipitation process was developed for reducing the content of acidic variants in mAb downstream pools. The process design was preceded by the determination of phase behavior of mAb variants in the presence of different precipitants. It was shown that the presence of polyethylene glycol (PEG) in protein solutions favored precipitation of acidic variants of mAbs. Precipitation yield was influenced by the variant composition in the mAb feed solutions, the concentration of the precipitant and the protein, and the ionic strength of the solutions. To improve yield, multistage precipitation was employed, where the precipitate was recycled to the precipitation process. The final product was a mixture of supernatants pooled together from the recycling steps. Such an approach can be potentially used either instead or in a combination with chromatography for adjusting the acidic variant content of mAbs, which can benefit in improvement in throughput and reduction in manufacturing costs.  相似文献   

7.
Glycation has been observed in antibody therapeutics manufactured by the fed-batch fermentation process. It not only increases the heterogeneity of antibodies, but also potentially affects product safety and efficacy. In this study, non-glycated and glycated fractions enriched from a monoclonal antibody (mAb1) as well as glucose-stressed mAb1 were characterized using a variety of biochemical, biophysical and biological assays to determine the effects of glycation on the structure and function of mAb1. Glycation was detected at multiple lysine residues and reduced the antigen binding activity of mAb1. Heavy chain Lys100, which is located in the complementary-determining region of mAb1, had the highest levels of glycation in both stressed and unstressed samples, and glycation of this residue was likely responsible for the loss of antigen binding based on hydrogen/deuterium exchange mass spectrometry analysis. Peptide mapping and intact liquid chromatography-mass spectrometry (LC-MS) can both be used to monitor the glycation levels. Peptide mapping provides site specific glycation results, while intact LC-MS is a quicker and simpler method to quantitate the total glycation levels and is more useful for routine testing. Capillary isoelectric focusing (cIEF) can also be used to monitor glycation because glycation induces an acidic shift in the cIEF profile. As expected, total glycation measured by intact LC-MS correlated very well with the percentage of total acidic peaks or main peak measured by cIEF. In summary, we demonstrated that glycation can affect the function of a representative IgG1 mAb. The analytical characterization, as described here, should be generally applicable for other therapeutic mAbs.  相似文献   

8.
This study describes the isolation of angiotensin I converting enzyme and antioxidative peptides from head protein hydrolysate of red scorpionfish (Scorpaena notata) prepared by treatment with a protease from the fungus Penicillium digitatum. After ultrafiltration, three peptides were isolated by a two-step procedure: size exclusion chromatography on a Toyopearl HW-40 followed by reversed-phase high performance liquid chromatography (RP-HPLC) with a high purification yield of 2.22 mg of peptide/g of initial protein. Active peptides were then identified by nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoLC/MS–MS), corresponding to the following sequences: Gln–Gln–Pro–His–Ser–Arg–Ser–Lys–Gly–Phe–Pro–Gly–Pro (1424.724 Da), Gly–Gln–Lys–Ser–Val–Pro–Glu–Val–Arg (1000.565 Da) and Val–Glu–Gly–Lys–Ser–Pro–Asn–Val (830.448 Da). Peptides D-I, E-I and F-I showed high angiotensin-I converting enzyme inhibitory activity with an IC50 values of 0.98, 1.69 and 1.44 µM, respectively as well as a synergistic antioxidant activity between the different fractions. Thus, we have demonstrated that underutilized wastes can be valorized by production of peptides that can be used as potential therapeutic compounds active against oxidative stress and hypertension.  相似文献   

9.
The ability to control charge heterogeneity in monoclonal antibodies is important to demonstrate product quality comparability and consistency. This article addresses the control of C‐terminal lysine processing through copper supplementation to yeast hydrolysate powder, a raw material used in the cell culture process. Large‐scale production of a murine cell line exhibited variation in the C‐terminal lysine levels of the monoclonal antibody. Analysis of process data showed that this variation correlated well with shifts in cell lactate metabolism and pH levels of the production culture. Small‐scale studies demonstrated sensitivity of the cells to copper, where a single low dose of copper to the culture impacted cell lactate metabolism and C‐terminal lysine processing. Subsequent analytical tests indicated that the yeast hydrolysate powder, added to the basal media and nutrient feed in the process, contained varying levels of trace copper across lots. The measured copper concentrations in yeast hydrolysate lots correlated well with the variation in lactate and pH trends and C‐terminal lysine levels of the batches in manufacturing. Small‐scale studies further demonstrated that copper supplementation to yeast hydrolysate lots with low concentrations of copper can shift the metabolic performance and C‐terminal lysine levels of these cultures to match the control, high copper cultures. Hence, a strategy of monitoring, and if necessary supplementing, copper in yeast‐hydrolysate powders resulted in the ability to control and ensure product quality consistency. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:463–468, 2017  相似文献   

10.

Recombinant monoclonal antibodies are predominantly produced in mammalian cell culture bioprocesses. Post-translational modifications affect the micro-heterogeneity of the product and thereby influence important quality attributes, such as stability, solubility, pharmacodynamics and pharmacokinetics. The analysis of the surface charge distribution of monoclonal antibodies provides aggregated information about these modifications. In this work, we established a direct injection pH gradient cation exchange chromatography method, which determines charge heterogeneity from cell culture supernatant without any purification steps. This tool was further applied to monitor processes that were performed under certain process conditions. Concretely, we were able to provide insights into charge variant formation during a fed-batch process of a Chinese hamster ovary cell culture, in turn producing a monoclonal antibody under varying temperatures and glucose feed strategies. Glucose concentration impacted the total emergence of acidic variants, whereas the variation of basic species was mainly dependent on process temperature. The formation rates of acidic species were described with a second-order reaction, where a temperature increase favored the conversion. This platform method will aid as a sophisticated optimization tool for mammalian cell culture processes. It provides a quality fingerprint for the produced mAb, which can be tested, compared to the desired target and confirmed early in the process chain.

  相似文献   

11.
The serotonin 2A (5-HT2A) receptor has been implicated in obstructive sleep apnea (OSA). Single nucleotide polymorphisms (SNPs) in the 5-HT2A gene have been found in OSA, the most common being ?1438G/A and T102C; however, studies of the association between 5-HT2A SNPs and OSA risk have reported inconsistent findings. A meta-analysis was performed to quantitatively review the association between ?1438G/A and T102C SNPs and OSA. Five studies, including 791 subjects for ?1438G/A genotype and 1,068 subjects for T102C genotype, were selected. Pooled data analysis of the ?1438G/A genotype indicated a significantly increased OSA risk was associated with two variant genotypes (AA vs. AG+GG: OR 3.023, 95 % CI 2.169–4.213, P = 0.506 for heterogeneity; A allele carriers vs. GG: OR 1.938, 95 % CI 0.879–4.274, P = 0.012 for heterogeneity). Stratification analysis by gender supported the association in males, but not females. For the T102C genotype, no significantly increased OSA risk was associated with the two variant genotypes (CC vs. CT+TT: OR 1.065, 95 % CI 0.787–1.442, P = 0.361 for heterogeneity; C allele carriers vs. TT: OR 0.979, 95 % CI 0.737–1.3, P = 0.9 for heterogeneity).In conclusions, meta-analysis indicated that the ?1438G/A, and not T102C, polymorphism of 5-HT2A is a positive risk factor of OSA, especially in males.  相似文献   

12.
Charge variants, especially acidic charge variants, in recombinant monoclonal antibodies are critical quality attributes, which can affect antibodies’ properties in vitro and in vivo. Meanwhile, charge variants are cumulative effects of various post-translational modifications and chemical degradations on antibody. In this work, to investigate the effect of lowering culture pH in the stationary phase on acidic charge variant contents in fed-batch cultures and its mechanism, cell culture experiments in 2-L bioreactors were firstly performed to explore the changes in the charge distribution under the pH downshift condition using weak cation exchange chromatography. It is found that acidic charge variant contents were significantly decreased by pH downshift. Then, to reveal the mechanism by which the content of acidic charge variants is reduced under pH downshift condition, the variation of post-translational modifications and chemical degradations under the pH downshift condition was explored. Meanwhile, the structure of the acidic charge variants was characterized. Several analysis experiments including size exclusion chromatography, capillary electrophoresis-sodium dodecyl sulfate under non-reducing conditions, tryptic peptide map, and reduced antibody mass were applied in this study. The results show that the mechanism by which the content of acidic charge variants is reduced is that the contents of disulfide bond reduction, galactosylation, and asparagine deamination of the HC-N388 in the Fc domain were reduced by pH downshift.  相似文献   

13.
The aim of this study was to efficiently convert oil palm empty fruit bunch fiber (OPEFB), one of the most commonly generated lingo-wastes in Southeast Asia, into both cellulase and bioethanol. The unprocessed cellulase crude (37.29 %) produced under solid-state fermentation using OPEFB as substrate showed a better reducing sugar yield using filter paper than the commercial enzyme blend (34.61 %). Organosolv pretreatment method could efficiently reduce hemicellulose (24.3–18.6 %) and lignin (35.2–22.1 %) content and increase cellulose content (40.5–59.3 %) from OPEFB. Enzymatic hydrolysis of pretreated OPEFB using the crude cellulase with 20 % solid content, enzyme loading of 15 FPU/g OPEFB at 50 °C, and pH 5.5 resulted in a OPEFB hydrolysate containing 36.01 g/L glucose after 72 h. Fermentation of the hydrolysate medium produced 17.64 g/L ethanol with 0.49 g/g yield from glucose and 0.088 g/g yield from OPEFB at 8 h using Saccharomyces cerevisiae.  相似文献   

14.
The different cultivation strategies of batch, fed-batch and continuous culture for the synthesis of biomass and carotenoids by Dietzia natronolimnaea HS-1 from waste molasses and its hydrolysate were compared. The efficiency of three various pretreatments (enzymatic, acidic and acidic at high temperature) for the determination of the best hydrolysate was also studied by evaluating the conversion rate of sucrose. The analytical procedures initially showed that canthaxanthin (CTX) and enzymatic hydrolysis were the most abundant pigment biosynthesized and the most suitable process for the substrate production, respectively. An increase in reducing sugar concentration of the enzymatic hydrolysate molasses (EHM) from 25 to 50 g/l led to a drastic decrease in biomass formation and substrate utilization. EHM (25 g/l) was a better substrate for the cell growth and product formation than the waste molasses (25 g/l). The application of EHM instead of molasses enhanced the biomass production in fed-batch culture more than batch and continuous cultures. However, the continuous cultivation had the highest biomass (12.98 g/l), carotenoid (27.33 mg/l) and CTX (25.04 mg/l) yields with 25 g/l of EHM. The CTX isolated from D. natronolimnaea HS-1 may be used as a natural antioxidant for possible production of healthy-functional foods in the future.  相似文献   

15.
Sugarcane bagasse is one of the low-cost substrates used for bioethanol production. In order to solubilize sugars in hemicelluloses like xylan, a new thermotolerant isolate of Candida tropicalis HNMA-1 with xylan-hydrolyzing ability was identified and characterized. The strain showed relative tolerance to high temperature. Our results demonstrated 0.211 IU ml?1 xylanase activity at 40 °C compared to 0.236 IU ml?1 at 30 °C. The effect of high temperature on the growth and fermentation of xylose and sugarcane bagasse hydrolysate were also investigated. In both xylose or hydrolysate medium, increased growth was recorded at 40 °C. Meanwhile, the efficiency of ethanol fermentation was adversely affected by temperature since yields of 0.088 g g?1 and 0.076 g g?1 in the xylose medium, in addition to 0.090 g g?1 and 0.078 g g?1 in the hydrolysate medium were noticed at 30 °C and 40 °C, respectively. Inhibitory compounds in the hydrolysate medium demonstrated negative effects on fermentation and productivity, with maximum ethanol concentration attained after 48 h in the hydrolysate, as opposed to 24 h in the xylose medium. Our data show that the newly thermotolerant isolate, C. tropicalis HNMA-1, is able to efficiently ferment xylose and hydrolysate, and also has the capacity for application in ethanol production from hemicellulosic sources.  相似文献   

16.
The association between xeroderma pigmentosum group D (XPD) Lys751Gln polymorphism and bladder cancer (BC) susceptibility was investigated by two meta-analyses, however, their results were contrary. We conjecture the reason might be the sample size, thus we performed this updated and cumulative meta-analysis using the Comprehensive Meta-Analysis software. We searched PubMed up to August 25th, 2013 and yielded 20 published articles with 21 case–control trails including 6,836 BC patients and 8,251 controls. The meta-analysis results showed that XPD Lys751Gln polymorphism was borderline significantly associated with BC susceptibility for overall population [Gln vs. Lys: OR 1.07, 95 % CI 1.01–1.12, P = 0.01; Gln/Gln vs. Lys/Lys: OR 1.15, 95 % CI 1.03–1.29, P = 0.01; Gln/Gln vs. (Lys/Gln + Lys/Lys): OR 1.13, 95 % CI 1.02–1.26, P = 0.02]. The cumulative meta-analysis according to the publication year showed the CI became increasingly narrower and tended to have statistical significance for the studies incessantly accumulated. In the subgroup analysis according to ethnicity, there was a significant association in Asian population and no association in Caucasian population. There was no publication bias detected. However, due to the limitations and cumulative analysis result of this meta-analysis, more well-designed and larger studies with risk factors adjusted are suggested to be performed to obtain a conclusive result on this topic.  相似文献   

17.
A recombinant IgG1 monoclonal antibody (mAb) showed multiple charge variants in a cation exchange chromatography profile. To better understand the correlation between charge heterogeneity and glycosylation, a rapid reversed phase ultra-performance liquid chromatography–mass spectrometry (UPLC–MS) method with integrated mass analysis has been developed for simultaneous determination of N-terminal pyroglutamate, C-terminal lysine truncation, and Fc glycosylation. The results show that various degrees and/or types of N-terminal pyroglutamate formation and C-terminal lysine (Lys) cleavage account for the majority of charge heterogeneity; and the charge variants showed Fc glycosylation patterns in relation to their terminal modifications. The amount of G1F decreased in the basic variants, whereas Man5 and G0F-GN increased. The complement-dependent cytotoxicity (CDC) activity of purified charge variants also suggested the potential impact of the charge differences on the glycosylation profile.  相似文献   

18.
The effects of 24-epibrassinolide under high temperature in eggplant (Solanum melongena L.) seedlings were studied by investigating the plant growth, chlorophyll content, photosynthesis and antioxidant systems. High temperature significantly inhibited the plant growth and markedly decreased the chlorophyll content, net photosynthetic rate, stomatal conductance and transpiration rate, while it increased intercellular CO2 concentration. In a similar manner, high temperature also decreased significantly maximum quantum efficiency of PSII, potential photochemical efficiency, the quantum efficiency of PSII, photochemical quenching, the excitation capture efficiency of open centers, and increased non-photochemical quenching. Application of 0.05–0.2 μM EBR remarkably promoted the plant growth and alleviated high-temperature-induced inhibition of photosynthesis. Under high temperature, reactive oxygen species levels and lipid peroxidation were markedly increased, which were remarkably inhibited by application of 0.05–0.2 μM EBR. The activities of antioxidative enzymes such as superoxide dismutase, peroxidase, catalase and ascorbate peroxidase, and contents of ascorbic acid and reduced glutathione were significantly increased during high-temperature treatments, and these increases were more pronounced than those of EBR at 0.05–0.2 μM treatment. The EBR treatment also greatly enhanced contents of proline, soluble sugar and protein under high-temperature stress. Taken together, it can be concluded that 0.05–0.2 μM EBR could alleviate the detrimental effects of high temperatures on plant growth by increasing photosynthetic efficiency and enhancing antioxidant enzyme systems. Addition of 0.1 μM EBR had the best ameliorative effect against high temperature, while the addition of 0.4 μM EBR had no significant effects.  相似文献   

19.
Terminal moieties of most proteins are long known to be disordered and flexible. To unravel the functional role of these regions on the structural stability and biochemical properties of AT2 lipase, four C-terminal end residues, (Ile–Thr–Arg–Lys) which formed a flexible, short tail-like random-coil segment were targeted for mutation. Swapping of the tail-like region had resulted in an improved crystallizability and anti-aggregation property along with a slight shift of the thermostability profile. The lipolytic activity of mutant (M386) retained by 43 % compared to its wild-type with 18 % of the remaining activity at 45 °C. In silico analysis conducted at 25 and 45 °C was found to be in accordance to the experimental findings in which the RMSD values of M386 were more stable throughout the total trajectory in comparison to its wild-type. Terminal moieties were also observed to exhibit large movement and flexibility as denoted by high RMSF values at both dynamics. Variation in organic solvent stability property was detected in M386 where the lipolytic activity was stimulated in the presence of 25 % (v/v) of DMSO, isopropanol, and diethyl ether. This may be worth due to changes in the surface charge residues at the mutation point which probably involve in protein–solvent interaction.  相似文献   

20.
Activated sludge of wastewater treatment plants carries a diverse microflora. However, up to 80–90 % of microorganisms in activated sludge cannot be cultured by current laboratory techniques, leaving an enzyme reservoir largely unexplored. In this study, we investigated carboxypeptidase diversity in activated sludge of a wastewater treatment plant in Shanghai, China, by a culture-independent metagenomic approach. Three sets of consensus degenerate hybrid oligonucleotide primers (CODEHOPs) targeting conserved domains of public carboxypeptidases have been designed to amplify carboxypeptidase gene sequences in the metagenomic DNA of activated sludge by PCR. The desired amplicons were evaluated by carboxypeptidase sequence clone libraries and phylogenetic analyses. We uncovered a significant diversity of carboxypeptidases present in the activated sludge. Deduced carboxypeptidase amino acid sequences (127–208 amino acids) were classified into three distinct clusters, α, β, and γ. Sequences belonging to clusters α and β shared 58–97 % identity to known carboxypeptidase sequences from diverse species, whereas sequences in the cluster γ were remarkably less related to public carboxypeptidase homologous in the GenBank database, strongly suggesting that novel carboxypeptidase families or microbial niches exist in the activated sludge. We also observed numerous carboxypeptidase sequences that were much closer to those from representative strains present in industrial and sewage treatment and bioremediation. Thermostable and halotolerant carboxypeptidase sequences were also detected in clusters α and β. Coexistence of various carboxypeptidases is evidence of a diverse microflora in the activated sludge, a feature suggesting a valuable gene resource to be further explored for biotechnology application.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号