miRNA expression was measured as described previously [10 (link)]. Briefly, 20 ng of total RNA was reverse transcribed using the Megaplex RT stem-loop primer pool (Applied Biosystems, Foster City, CA, USA), enabling miRNA specific cDNA synthesis for 430 different human miRNAs and 18 small RNA controls. Subsequently, Megaplex RT product was pre-amplified by means of a 14-cycle PCR reaction with a miRNA specific forward primer and universal reverse primer to increase detection sensitivity. Finally, a 1,600-fold dilution of pre-amplified miRNA cDNA was used as input for a 40-cycle qPCR reaction with miRNA specific hydrolysis probes and primers (Applied Biosystems). All reactions were performed on the 7900 HT (Applied Biosystems) using the gene maximization strategy [41 (link)]. Raw Cq values were calculated using the SDS software version 2.1 applying automatic baseline settings and a threshold of 0.05. For further data analysis, only those miRNAs with a Cq value equal to or below 35 (representing single molecule template detection [10 (link)]) were taken into account. For NB tumor samples all 448 miRNAs and small RNA controls were profiled. RT-qPCR assays were spread across two 384-well plates. Inter-run variation was accounted for by equalizing the mean Cq-value of the 18 small RNA controls that were profiled in both plates. For the remaining samples 366 miRNAs and 18 small RNA controls were profiled in a single 384-well plate.
>
Phenomena
>
Natural Phenomenon or Process
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Hydrolysis
Hydrolysis
Hydrolysis is a fundamental chemical process in which a molecule is cleaved into smaller components by the addition of water.
This reaction is crucial in a wide range of biological and industrial applications, including the digestion of nutrients, the production of biofuels, and the breakdown of complex polymers.
Hydrolysis can be catalyzed by enzymes, acids, or bases, and the specific conditions can greatly influence the rate and products of the reaction.
Understanding the mechanisms and optimization of hydrolysis is an active area of research, with implications for fields such as biochemistry, biotechnology, and environmental sciences.
Reserchers can leverage AI-driven tools like PubCompare.ai to efficiently locate and compare the most reliable hydrolysis protocols from the literature, helping to enhance the reproducibility and accuracy of their work.
This reaction is crucial in a wide range of biological and industrial applications, including the digestion of nutrients, the production of biofuels, and the breakdown of complex polymers.
Hydrolysis can be catalyzed by enzymes, acids, or bases, and the specific conditions can greatly influence the rate and products of the reaction.
Understanding the mechanisms and optimization of hydrolysis is an active area of research, with implications for fields such as biochemistry, biotechnology, and environmental sciences.
Reserchers can leverage AI-driven tools like PubCompare.ai to efficiently locate and compare the most reliable hydrolysis protocols from the literature, helping to enhance the reproducibility and accuracy of their work.
Most cited protocols related to «Hydrolysis»
Anabolism
Biological Assay
DNA, Complementary
Genes
Hydrolysis
Hypersensitivity
MicroRNAs
MIRN430 microRNA, human
Neoplasms
Oligonucleotide Primers
Stem, Plant
Technique, Dilution
Preparation of cDNA followed the procedure described in Mortazavi et al.2 (link), with minor modifications as described below. Prior to fragmentation, a 7 uL aliquot (∼ 500 pgs total mass) containing known concentrations of 7 “spiked in” control transcripts from A. thaliana and the lambda phage genome were added to a 100 ng aliquot of mRNA from each time point. This mixture was then fragmented to an average length of 200 nts by metal ion/heat catalyzed hydrolysis. The hydrolysis was performed in a 25 uL volume at 94°C for 90 seconds. The 5X hydrolyis buffer components are: 200 mM Tris acetate, pH 8.2, 500 mM potassium acetate and 150 mM magnesium acetate. After removal of hydrolysis ions by G50 Sephadex filtration (USA Scientific catalog # 1415-1602), the fragmented mRNA was random primed with hexamers and reverse-transcribed using the Super Script II cDNA synthesis kit (Invitrogen catalog # 11917010). After second strand synthesis, the cDNA went through end-repair and ligation reactions according to the Illumina ChIP-Seq genomic DNA preparation kit protocol (Illumina catalog # IP102-1001), using the paired end adapters and amplification primers (Illumina Catalog # PE102-1004). Ligation of the adapters adds 94 bases to the length of the cDNA molecules.
Acetate
Anabolism
Bacteriophage lambda
Buffers
Chromatin Immunoprecipitation Sequencing
DNA, Complementary
DNA Chips
Filtration
Genome
Hydrolysis
Ions
Ligation
magnesium acetate
Metals
Oligonucleotide Primers
Potassium Acetate
RNA, Messenger
sephadex
Tromethamine
Cells
Culture Media
Cycloheximide
Gene Expression
Genome
Granulocyte Colony-Stimulating Factor
HeLa Cells
Hematopoietic System
Hydrolysis
Males
Mice, Knockout
MicroRNAs
mRNA, Polyadenylated
RNA, Messenger
Stem Cell Factor
The use of a two-stage sulfuric acid hydrolysis for the analysis of lignin dates to the turn of the 20th century, although the use of concentrated acid to release sugars from wood dates to the early 19th century (7 ). Klason, in 1906, is often credited as the first to use sulfuric acid to isolate lignin from wood (7 −9 ). The method became named after Klason, and the insoluble residue from the test is known as “Klason lignin.” An English translation of a Klason paper, from this period (10 ), describes his attempt to determine the structure of spruce wood lignin. According to Brauns (7 ), Klason’s method originally used 72 wt % sulfuric acid; he later reduced this to 66 wt % to gelatinize the wood. He filtered the solids and subjected them to a second hydrolysis in 0.5 wt % hydrochloric acid.
Although Klason is generally credited as being the first to use sulfuric acid for lignin analysis, Sherrard and Harris (11 ) credit the use of sulfuric acid to Fleschsig in 1883, Ost and Wilkening in 1912, and König and Rump in 1913. According to Harris (12 ), Fleschsig, in 1883, dissolved cotton cellulose and converted it nearly quantitatively into sugars using strong sulfuric acid followed by dilution and heating. According to Browning (13 ), Ost and Wilkening introduced the use of 72 wt % sulfuric acid for lignin determinations in 1910. A translated paper by Heuser (14 ) credited König and Ost and Wilkening for the sulfuric acid lignin method. Dore (15 ) described several improved analytical methods (cellulose, lignin, soluble pentosans, mannan, and galactan) for the summative analysis of coniferous woods. The discrepancies in attribution may be due to differing definitions for the method cited (e.g., first to use acid to determine lignin, first to use sulfuric acid, first to use 72 wt % sulfuric acid, etc.) and to missed citations across continental distances in the early 20th century.
Although Klason is generally credited as being the first to use sulfuric acid for lignin analysis, Sherrard and Harris (11 ) credit the use of sulfuric acid to Fleschsig in 1883, Ost and Wilkening in 1912, and König and Rump in 1913. According to Harris (12 ), Fleschsig, in 1883, dissolved cotton cellulose and converted it nearly quantitatively into sugars using strong sulfuric acid followed by dilution and heating. According to Browning (13 ), Ost and Wilkening introduced the use of 72 wt % sulfuric acid for lignin determinations in 1910. A translated paper by Heuser (14 ) credited König and Ost and Wilkening for the sulfuric acid lignin method. Dore (15 ) described several improved analytical methods (cellulose, lignin, soluble pentosans, mannan, and galactan) for the summative analysis of coniferous woods. The discrepancies in attribution may be due to differing definitions for the method cited (e.g., first to use acid to determine lignin, first to use sulfuric acid, first to use 72 wt % sulfuric acid, etc.) and to missed citations across continental distances in the early 20th century.
Acids
Cellulose
Galactans
Gossypium
Hydrochloric acid
Hydrolysis
Lignin
Mannans
Pentosan Sulfuric Polyester
Picea
Sugars
sulfuric acid
Technique, Dilution
Tracheophyta
Xylose
For our analysis, we employed publicly available read data (Supplementary Methods ) from: Saccharomyces cerevisiae (9 (link)), Arabidopsis thaliana (28 (link)), Mus musculus (11 (link)), the same Homo sapiens sample sequenced with two different RNA-Seq protocols, i.e. flowcell RT-Seq (FRT) and standard hydrolysis (STD) protocol (17), and RNA control sequences spiked-in in high concentrations (29 (link)). In a first step, we mapped and split-mapped non-redundantly all the reads to the respective reference genome sequence using the GEM library (http://sourceforge.net/projects/gemlibrary ); in the case of the cress data set, which is comparatively small, we also considered additional read mappings with long indels obtained with BLAT (30 (link)).
Subsequently, we focused on the distribution of reads that map to transcripts without alternatively processed forms. To define such transcripts, we considered a standard reference annotation of the transcriptome, i.e. the SGD annotation for yeast (31 (link)), the TAIR annotation for cress (32 (link)) and the murine as well as the human RefSeq annotation (33 (link)). This procedure provided us with mappings for 6 606 768 reads (47%) from yeast, 351 336 reads (65%) from cress and for 21 359 481 reads (68%) from mouse, and with 530 996 reads that map in proper pairs to the spike-in control sequences. Due to substantially different data set sizes (90 million versus 13 million reads), in the case of the human FRT- and the STD-Seq experiments, we extracted subsets of reads of suitable size before mapping to ensure comparability (Supplementary Table S1 ).
Subsequently, we focused on the distribution of reads that map to transcripts without alternatively processed forms. To define such transcripts, we considered a standard reference annotation of the transcriptome, i.e. the SGD annotation for yeast (31 (link)), the TAIR annotation for cress (32 (link)) and the murine as well as the human RefSeq annotation (33 (link)). This procedure provided us with mappings for 6 606 768 reads (47%) from yeast, 351 336 reads (65%) from cress and for 21 359 481 reads (68%) from mouse, and with 530 996 reads that map in proper pairs to the spike-in control sequences. Due to substantially different data set sizes (90 million versus 13 million reads), in the case of the human FRT- and the STD-Seq experiments, we extracted subsets of reads of suitable size before mapping to ensure comparability (
Arabidopsis thalianas
Brassicaceae
DNA Library
Genome
Homo sapiens
Hydrolysis
INDEL Mutation
Mice, House
Mus
RNA-Seq
Saccharomyces cerevisiae
Transcriptome
Most recents protocols related to «Hydrolysis»
Example 8
Characterization of Absorption, Distribution, Metabolism, and Excretion of Oral [14C]Vorasidenib with Concomitant Intravenous Microdose Administration of [13C315N3]Vorasidenib in Humans
Metabolite profiling and identification of vorasidenib (AG-881) was performed in plasma, urine, and fecal samples collected from five healthy subjects after a single 50-mg (100 μCi) oral dose of [14C]AG-881 and concomitant intravenous microdose of [13C3 15N3]AG-881.
Plasma samples collected at selected time points from 0 through 336 hour postdose were pooled across subjects to generate 0—to 72 and 96-336-hour area under the concentration-time curve (AUC)-representative samples. Urine and feces samples were pooled by subject to generate individual urine and fecal pools. Plasma, urine, and feces samples were extracted, as appropriate, the extracts were profiled using high performance liquid chromatography (HPLC), and metabolites were identified by liquid chromatography-mass spectrometry (LC-MS and/or LC-MS/MS) analysis and by comparison of retention time with reference standards, when available.
Due to low radioactivity in samples, plasma metabolite profiling was performed by using accelerator mass spectrometry (AMS). In plasma, AG-881 was accounted for 66.24 and 29.47% of the total radioactivity in the pooled AUC0-72 h and AUC96-336 h plasma, respectively. The most abundant radioactive peak (P7; M458) represented 0.10 and 43.92% of total radioactivity for pooled AUC0-72 and AUC96-336 h plasma, respectively. All other radioactive peaks accounted for less than 6% of the total plasma radioactivity and were not identified.
The majority of the radioactivity recovered in feces was associated with unchanged AG-881 (55.5% of the dose), while no AG-881 was detected in urine. In comparison, metabolites in excreta accounted for approximately 18% of dose in feces and for approximately 4% of dose in urine. M515, M460-1, M499, M516/M460-2, and M472/M476 were the most abundant metabolites in feces, and each accounted for approximately 2 to 5% of the radioactive dose, while M266 was the most abundant metabolite identified in urine and accounted for a mean of 2.54% of the dose. The remaining radioactive components in urine and feces each accounted for <1% of the dose.
Overall, the data presented indicate [14C]AG-881 underwent moderate metabolism after a single oral dose of 50-mg (100 μCi) and was eliminated in humans via a combination of metabolism and excretion of unchanged parent. AG-881 metabolism involved the oxidation and conjugation with glutathione (GSH) by displacement of the chlorine at the chloropyridine moiety. Subsequent biotransformation of GSH intermediates resulted in elimination of both glutamic acid and glycine to form the cysteinyl conjugates (M515 and M499). The cysteinyl conjugates were further converted by a series of biotransformation reactions such as oxidation, S-dealkylation, S-methylation, S-oxidation, S-acetylation and N-dealkylation resulting in the formation multiple metabolites.
A summary of the metabolites observed is included in Table 2
Acetylation
AG 30
Biotransformation
Chlorine
Dealkylation
Deamination
Elements, Radioactive
Feces
Glutamic Acid
Glutathione
Glycine
Healthy Volunteers
High-Performance Liquid Chromatographies
Homo sapiens
Hydrolysis
Intravenous Infusion
Ions
Liquid Chromatography
Mass Spectrometry
Metabolism
Methylation
Parent
Plasma
Radioactivity
Retention (Psychology)
Tandem Mass Spectrometry
Urinalysis
Urine
vorasidenib
Example 1
<Step (A): Synthesis of porous particle having glycidyl group>
27.8 g of glycidyl methacrylate (trade name: Blemmer G (registered trademark) manufactured by NOF Corporation), 11.3 g of glycerin-1,3-dimethacrylate (trade name: NK Ester 701, SHIN-NAKAMURA CHEMICAL Co., Ltd.), and 1.9 g of 2,2′-azobis(2,4-dimethylvaleronitrile) were dissolved in 58.7 g of diethyl succinate as a diluent, and nitrogen gas was bubbled for 30 minutes to provide an oil phase.
Next, separately from the oil phase, 10.0 g of PVA-224 (manufactured by Kuraray Co., Ltd., polyvinyl alcohol having a degree of saponification of 87.0% to 89.0%) as a dispersion stabilizer and 10.0 g of sodium chloride as a salting-out agent were dissolved in 480 g of ion exchanged water to provide an aqueous phase.
The aqueous phase and the oil phase were placed in a separable flask and dispersed at a rotation speed of 430 rpm for 20 minutes using a stirring rod equipped with a half-moon stirring blade, then the inside of the reactor was purged with nitrogen, and the reaction was carried out at 60° C. for 16 hours.
After that, the resulting polymer was transferred onto a glass filter and thoroughly washed with hot water at about 50 to 80° C., denatured alcohol, and water in the order presented to obtain 100.4 g of a porous particle (carrier al).
The amount of glycidyl methacrylate used was 79.8 mol % based on the total amount of the monomers, and the amount of glycerin-1,3-dimethacrylate used was 20.2 mol % based on the total amount of the monomers.
<Step (B): Introduction reaction of alkylene group>
98 g of the carrier α1 was weighed onto a glass filter and thoroughly cleaned with diethylene glycol dimethyl ether. After cleaning, the carrier α1 was placed in a 1 L separable flask, 150 g of diethylene glycol dimethyl ether and 150 g (920 mol % based on glycidyl methacrylate) of 1,4-butanediol were placed in the separable flask, and stirring and dispersion were carried out.
After that, 1.5 ml of a boron trifluoride diethyl ether complex was added, the temperature was raised to 80° C. while stirring at 200 rpm, and the resulting mixture was subjected to the reaction for 4 hours.
The mixture was cooled, then the porous particle (carrier β1) bonded to a diol compound including an alkylene group in the structure thereof was collected by filtration and then washed with 1 L of ion exchanged water to obtain 152 g of a carrier β1.
The progress of the reaction was confirmed by the following procedure.
A part of the dry porous particle into which an alkylene group had been introduced was mixed with potassium bromide, and the resulting mixture was pelletized by applying a pressure and then measured using FT-IR (trade name: Nicolet (registered trademark) iS10, manufactured by Thermo Fisher Scientific Inc.) to check the height of an absorbance peak at 908 cm−1 due to the glycidyl group in the infrared absorption spectrum.
As a result, no absorbance peak at 908 cm−1 was observed by FT-IR.
<Step (C): Introduction Reaction of Glycidyl Group>
150 g of the carrier β1 was weighed onto a glass filter and thoroughly cleaned with dimethylsulfoxide.
After cleaning, the carrier β1 was placed in a separable flask, 262.5 g of dimethyl sulfoxide and 150 g of epichlorohydrin were added, the resulting mixture was stirred at room temperature, 37.5 ml of a 30% sodium hydroxide aqueous solution (manufactured by KANTO CHEMICAL CO., INC.) was further added, and the resulting mixture was heated to 30° C. and stirred for 6 hours.
After completion of the reaction, the obtained product was transferred onto a glass filter and thoroughly washed with water, acetone, and water in the order presented to obtain 172 g of a porous particle into which a glycidyl group had been introduced (carrier γ1).
The introduction density of the glycidyl group in the obtained carrier γ1 was measured by the following procedure.
5.0 g of the carrier γ1 was sampled, and the dry mass thereof was measured and as a result, found to be 1.47 g. Next, the same amount of the carrier γ1 was weighed into a separable flask and dispersed in 40 g of water, 16 mL of diethylamine was added while stirring at room temperature, and the resulting mixture was heated to 50° C. and stirred for 4 hours. After completion of the reaction, the reaction product was transferred onto a glass filter and thoroughly washed with water to obtain a porous particle A into which diethylamine had been introduced.
The obtained porous particle A was transferred into a beaker and dispersed in 150 mL of a 0.5 mol/L potassium chloride aqueous solution, and titration was carried out using 0.1 mol/L hydrochloric acid with the point at which the pH reached 4.0 as the neutralization point.
From this, the amount of diethylamine introduced into the porous particle A into which diethylamine had been introduced was calculated, and the density of the glycidyl group of the carrier γ1 was calculated from the following expression.
As a result, the density of the glycidyl group was 880 μmol/g.
Density(μmol/g) of glycidyl group={0.1×volume(μL) of hydrochloric acid at neutralization point/dry mass(g) of porous particle into which glycidyl group has been introduced}<Step (D): Introduction Reaction of Polyol>
150 g of the carrier γ1, 600 mL of water, and 1000 g (13000 mol % based on glycidyl group) of D-sorbitol (log P=−2.20, manufactured by KANTO CHEMICAL CO., INC.) were placed in a 3 L separable flask and stirred to form a dispersion.
After that, 10 g of potassium hydroxide was added, the temperature was raised to 60° C. while stirring at 200 rpm, and the resulting mixture was subjected to the reaction for 15 hours.
The mixture was cooled, and then the reaction product was collected by filtration and washed thoroughly with water to obtain 152 g of a porous particle into which polyol had been introduced (carrier 61).
The obtained carrier 61 was classified into 16 to 37 μm using a sieve to obtain 140.5 g of a packing material 1.
<Evaluation of Alkali Resistance>
The alkali resistance was evaluated by calculating the amount of a carboxy group produced by hydrolysis of sodium hydroxide according to the following procedure.
First, 4 g of the packing material was dispersed in 150 mL of a 0.5 mol/L potassium chloride aqueous solution, and titration was carried out using 0.1 mol/L sodium hydroxide aqueous solution with the point at which the pH reached 7.0 as the neutralization point. From this, the amount of a carboxy group before hydrolysis included in the packing material was calculated from the following expression.
Amount(μmol/mL) of carboxy group=0.1×volume(μL) of sodium hydroxide aqueous solution at the time of neutralization/apparent volume (mL) of packing material
Here, the apparent volume of the packing material is the volume of the packing material phase measured after preparing a slurry liquid by dispersing 4 g of the packing material in water, transferring the slurry liquid to a graduated cylinder, and then allowing the same to stand for a sufficient time.
Subsequently, 4 g of the packing material was weighed into a separable flask, 20 mL of a 5 mol/L sodium hydroxide aqueous solution was added, and the resulting mixture was treated at 50° C. for 20 hours while stirring at 200 rpm. The mixture was cooled, then the packing material was collected by filtration, then washed with a 0.1 mol/L HCl aqueous solution and water in the order presented, and the amount of a carboxy group contained in the obtained packing material was calculated by the same method as above. From the difference between the amount of a carboxy group before and that after the reaction with the 5 mol/L sodium hydroxide aqueous solution, the amount of a carboxy group produced by the reaction with the 5 mol/L sodium hydroxide aqueous solution was calculated. As a result, the amount of a carboxy group produced was 21 μmol/mL.
If the amount of a carboxy group produced is 40 μmol/mL or less, the alkali resistance is considered to be high.
<Evaluation of Non-Specific Adsorption>
The obtained packing material was packed into a stainless steel column (manufactured by Sugiyama Shoji Co., Ltd.) having an inner diameter of 8 mm and a length of 300 mm by a balanced slurry method. Using the obtained column, a non-specific adsorption test was carried out by the method shown below.
The column packed with the packing material was connected to a Shimadzu Corporation HPLC system (liquid feed pump (trade name: LC-10AT, manufactured by Shimadzu Corporation), autosampler (trade name: SIL-10AF, manufactured by Shimadzu Corporation), and photodiode array detector (trade name: SPD-M10A, manufactured by Shimadzu Corporation)), and a 50 mmol/L sodium phosphate buffer aqueous solution as a mobile phase was passed at a flow rate of 0.6 mL/min.
Using the same sodium phosphate aqueous solution as the mobile phase as a solvent, their respective sample solutions of 0.7 mg/mL thyroglobulin (Mw of 6.7×105), 0.6 mg/mL γ-globulin (Mw of 1.6×105), 0.96 mg/mL BSA (Mw of 6.65×104), 0.7 mg/mL ribonuclease (Mw of 1.3×104), 0.4 mg/mL aprotinin (Mw of 6.5×103), and 0.02 mg/mL uridine (Mw of 244) (all manufactured by Merck Sigma-Aldrich) are prepared, and 10 μL of each is injected from the autosampler.
The elution time of each observed using the photodiode array detector at a wavelength of 280 nm was compared to confirm that there was no contradiction between the order of elution volume and the order of molecular weight size.
As a result, the elution volumes of the samples from the column packed with the packing material 1 were 8.713 mL, 9.691 mL, 9.743 mL, 10.396 mL, 11.053 mL, and 11.645 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced. When there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof, there was no non-specific adsorption, which is indicated as 0 in Table 1, and when there was a contradiction therebetween, non-specific adsorption was induced, which is thus indicated as X.
The porous particle (carrier al) obtained in the same manner as in Example 1 was subjected to the step D of Example 1.
<Step (D): Introduction Reaction of Polyol>
98 g of carrier al, 600 mL of water, and 1000 g (3050 mol % based on glycidyl group) of D-sorbitol (manufactured by KANTO CHEMICAL CO., INC.) were placed in a 3 L separable flask and stirred to form a dispersion.
After that, 10 g of potassium hydroxide was added, the temperature was raised to 60° C. while stirring at 200 rpm, and the resulting mixture was subjected to the reaction for 15 hours.
The mixture was cooled, and then the reaction product was collected by filtration and washed thoroughly with water to obtain 130 g of a porous particle into which a polyol had been introduced (carrier δ7).
The carrier δ7 was classified into 16 to 37 μm using a sieve to obtain 115 g of a packing material 7.
The alkali resistance of the obtained packing material 7 was evaluated in the same manner as in Example 1. As a result, the amount of a carboxy group produced in the packing material 7 was 120.3 μmol/mL, resulting in poor alkali resistance.
Further, the non-specific adsorption of the obtained packing material 7 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 8.606 mL, 9.769 mL, 9.9567 mL, 10.703 mL, 11.470 mL, and 12.112 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced.
Example 2
A porous particle (carrier al) was obtained in the same manner as in Example 1, and then a packing material 2 was obtained as follows.
98 g of the carrier α1 was weighed onto a glass filter and thoroughly cleaned with diethylene glycol dimethyl ether.
After cleaning, the porous particle was placed in a 1 L separable flask, 150 g of diethylene glycol dimethyl ether and 150 g (580 mol % based on the glycidyl group) of 1,4-cyclohexanedimethanol were placed in the separable flask, and stirring and dispersion were carried out.
After that, 1.5 ml of a boron trifluoride diethyl ether complex was added, the temperature was raised to 80° C. while stirring at 200 rpm, and the resulting mixture was subjected to the reaction for 4 hours.
The mixture was cooled, then the resulting porous particle (carrier $2) bonded to a diol compound including an alkylene group in the structure thereof was collected by filtration and then washed with 1 L of ion exchanged water to obtain 165 g of a carrier 32.
The progress of the reaction was confirmed by the following procedure.
A part of the dry porous particle into which an alkylene group had been introduced was mixed with potassium bromide, and the resulting mixture was pelletized by applying a pressure and then measured using FT-IR (trade name: Nicolet (registered trademark) iS10, manufactured by Thermo Fisher Scientific Inc.) to check the height of a absorbance peak at 908 cm−1 due to the glycidyl group in the infrared absorption spectrum.
As a result, no absorbance peak at 908 cm−1 was observed by FT-IR.
<Step (C): Introduction Reaction of Glycidyl Group>
150 g of the carrier $2 was weighed onto a glass filter and thoroughly cleaned with dimethylsulfoxide. After cleaning, the carrier $2 was placed in a separable flask, 262.5 g of dimethyl sulfoxide and 150 g of epichlorohydrin were added, the resulting mixture was stirred at room temperature, 37.5 ml of a 30% sodium hydroxide aqueous solution (manufactured by KANTO CHEMICAL CO., INC.) was further added, and the resulting mixture was heated to 30° C. and stirred for 6 hours. After completion of the reaction, the porous particle was transferred onto a glass filter and thoroughly washed with water, acetone, and water in the order presented to obtain 180 g of a porous particle into which a glycidyl group had been introduced (carrier γ2).
The introduction density of the glycidyl group in the obtained carrier γ2 was measured in the same manner as in Example 1. As a result, the density of the glycidyl group was 900 μmol/g.
<Step (D): Introduction Reaction of Polyol>
150 g of the carrier γ2 was weighed onto a glass filter and thoroughly cleaned with diethylene glycol dimethyl ether. After cleaning, the carrier γ2 was placed in a 1 L separable flask, 150 g of diethylene glycol dimethyl ether and 150 g (5760 mol % based on the glycidyl group) of ethylene glycol (log P=−1.36) were placed in the separable flask, and stirring and dispersion were carried out. After that, 1.5 mL of a boron trifluoride diethyl ether complex was added, the temperature was raised to 80° C. while stirring at 200 rpm, and the resulting mixture was subjected to the reaction for 4 hours. The mixture was cooled, and then the reaction product was collected by filtration and washed thoroughly with water to obtain 152 g of a polyol-introduced porous particle (carrier δ2). The carrier δ2 was classified into 16 to 37 μm using a sieve to obtain 140.5 g of a packing material 2.
The alkali resistance of the obtained packing material 2 was evaluated in the same manner as in Example 1. As a result, the amount of a carboxy group produced was 15.2 μmol/mL, and it was confirmed that the packing material 2 had excellent alkali resistance.
Further, the non-specific adsorption of the obtained packing material 2 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 8.814 mL, 9.635 mL, 9.778 mL, 10.37 mL, 10.898 mL, and 12.347 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced.
A packing material 8 was obtained in the same manner as in Example 1 except that 150 g of ethylene glycol was used instead of 1,4-butanediol as an alkylene group-introducing agent.
The alkali resistance of the obtained packing material 8 was evaluated in the same manner as in Example 1. As a result, the amount of a carboxy group produced in the packing material 8 was 108.4 μmol/mL, resulting in poor alkali resistance.
Further, the non-specific adsorption of the obtained packing material 8 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 9.708 mL, 9.8946 mL, 10.6452 mL, 11.5374 mL, and 12.1656 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced.
Example 3
A carrier γ2 was obtained in the same manner as in Example 2.
150 g of the obtained carrier γ2 was weighed onto a glass filter and thoroughly cleaned with diethylene glycol dimethyl ether.
After cleaning, the porous particle was placed in a 1 L separable flask, 150 g of diethylene glycol dimethyl ether and 150 g of polyethylene glycol #200 (manufactured by KANTO CHEMICAL CO., INC., average molecular weight of 190 to 210, log P is unclear, but the close compound tetraethylene glycol (Mw of 194) has a log P of −2.02) (1790 mol % based on glycidyl group) were placed in the separable flask, and stirring and dispersion were carried out.
After that, 1.5 mL of a boron trifluoride diethyl ether complex was added, the temperature was raised to 80° C. while stirring at 200 rpm, and the resulting mixture was subjected to the reaction for 4 hours.
The mixture was cooled, and then the reaction product was collected by filtration and washed thoroughly with water to obtain 152 g of a porous particle into which a polyol had been introduced (carrier 63).
The carrier δ3 was classified into 16 to 37 μm using a sieve to obtain 140.5 g of a packing material 3.
The alkali resistance of the obtained packing material 3 was evaluated in the same manner as in Example 1. As a result, the amount of a carboxy group produced was 16.1 μmol/mL, and it was confirmed that the packing material 3 had excellent alkali resistance.
Further, the non-specific adsorption of the obtained packing material 3 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 8.517 mL, 9.241 mL, 9.47 mL, 10.034 mL, 10.484 mL, and 11.927 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced.
A packing material 9 was obtained in the same manner as in Example 2 except that no glycidyl group was introduced and no polyol was introduced. That is, the carrier $2 obtained in the step (B) of Example 2 was used as the packing material 9.
The non-specific adsorption of the obtained packing material 9 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 8.590 mL, 10.316 mL, 9.603 mL, 10.484 mL, 13.863 mL, and 12.861 mL, and it was confirmed that there was a contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that non-specific adsorption was induced. Because of this, the alkali resistance was not evaluated.
Example 4
A packing material 4 was obtained in the same manner as in Example 3 except that 33.2 g of glycidyl methacrylate (trade name: Blemmer G (registered trademark) manufactured by NOF Corporation), 5.9 g of glycerin-1,3-dimethacrylate (trade name: NK Ester 701, SHIN-NAKAMURA CHEMICAL Co., Ltd.), 58.7 g of diethyl succinate, and 1.9 g of 2,2′-azobis(2,4-dimethylvaleronitrile) were used to provide an oil phase. The amount of glycidyl methacrylate used was 90.0 mol % based on the total amount of the monomers, and the amount of glycerin-1,3-dimethacrylate used was 10.0 mol % based on the total amount of the monomers.
The alkali resistance of the obtained packing material 4 was evaluated in the same manner as in Example 1. As a result, the amount of a carboxy group produced was 11.5 μmol/mL, and it was confirmed that the packing material 4 had excellent alkali resistance.
Further, the non-specific adsorption of the obtained packing material 4 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 7.52 mL, 8.214 mL, 8.451 mL, 9.062 mL, 9.511 mL, and 11.915 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced.
A packing material 10 was obtained in the same manner as in Example 1 except that 150 g (480 mol % based on glycidyl methacrylate) of 1,10-decanediol was used instead of 1,4-butanediol as an alkylene group-introducing agent.
The non-specific adsorption of the obtained packing material 10 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 9.991 mL, 10.15 mL, 10.063 mL, 10.691 mL, 12.172 mL, and 11.531 mL, and it was confirmed that there was a contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that non-specific adsorption was induced. Because of this, the alkali resistance was not evaluated.
Example 5
A packing material 5 was obtained in the same manner as in Example 3 except that 21.5 g of glycidyl methacrylate (trade name: Blemmer G (registered trademark) manufactured by NOF Corporation), 17.6 g of glycerin-1,3-dimethacrylate (trade name: NK Ester 701, SHIN-NAKAMURA CHEMICAL Co., Ltd.), 58.7 g of diethyl succinate, and 1.9 g of 2,2′-azobis(2,4-dimethylvaleronitrile) were used to provide an oil phase.
The amount of glycidyl methacrylate used was 66.2 mol % based on the total amount of the monomers, and the amount of glycerin-1,3-dimethacrylate used was 33.8 mol % based on the total amount of the monomers.
The alkali resistance of the obtained packing material 5 was evaluated in the same manner as in Example 1. As a result, the amount of a carboxy group produced was 18.3 μmol/mL, and it was confirmed that the packing material 5 had excellent alkali resistance.
Further, the non-specific adsorption of the obtained packing material 5 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 8.692 mL, 9.434 mL, 9.625 mL, 10.236 mL, 10.759 mL, and 12.457 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced.
A packing material 11 was obtained in the same manner as in Example 3 except that 13.7 g of glycidyl methacrylate (trade name: Blemmer G (registered trademark) manufactured by NOF Corporation), 25.4 g of glycerin-1,3-dimethacrylate (trade name: NK Ester 701, SHIN-NAKAMURA CHEMICAL Co., Ltd.), 58.7 g of diethyl succinate, and 1.9 g of 2,2′-azobis(2,4-dimethylvaleronitrile) were used to provide an oil phase. The amount of glycidyl methacrylate used was 46.4 mol % based on the total amount of the monomers, and the amount of glycerin-1,3-dimethacrylate used was 53.6 mol % based on the total amount of the monomers.
The non-specific adsorption of the obtained packing material 11 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 8.872 mL, 10.131 mL, 9.82 mL, 10.422 mL, 12.782 mL, and 12.553 mL, and it was confirmed that there was a contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that non-specific adsorption was induced. Because of this, the alkali resistance was not evaluated.
It was confirmed that the exclusion limit molecular weights of the packing materials obtained in Examples 1 to 6 and Comparative Examples 1 to 5 were all 1,000,000 or more.
Example 6
A packing material 6 was obtained in the same manner as in Example 3 except that 33.2 g of glycidyl methacrylate (trade name: Blemmer G (registered trademark) manufactured by NOF Corporation), 5.9 g of ethylene glycol dimethacrylate (trade name: NK Ester 1G, SHIN-NAKAMURA CHEMICAL Co., Ltd.), 29.3 g of butyl acetate, 29.3 g of chlorobenzene, and 1.9 g of 2,2′-azobis(2,4-dimethylvaleronitrile) were used to provide an oil phase. The amount of glycidyl methacrylate used was 88.7 mol % based on the total amount of the monomers, and the amount of ethylene glycol dimethacrylate used was 11.3 mol % based on the total amount of the monomers.
The alkali resistance of the obtained packing material 6 was evaluated in the same manner as in Example 1. As a result, the amount of a carboxy group produced was 12.5 μmol/mL, and it was confirmed that the packing material 6 had excellent alkali resistance.
Further, the non-specific adsorption of the obtained packing material 6 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 9.613 mL, 10.427 mL, 10.444 mL, 11.066 mL, 11.582 mL, and 12.575 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced.
A packing material 12 was obtained in the same manner as in Example 3 except that 37.1 g of glycidyl methacrylate (trade name: Blemmer G (registered trademark) manufactured by NOF Corporation), 2.0 g of glycerin-1,3-dimethacrylate (trade name: NK Ester 701, SHIN-NAKAMURA CHEMICAL Co., Ltd.), 58.7 g of diethyl succinate, and 1.9 g of 2,2′-azobis(2,4-dimethylvaleronitrile) were used to provide an oil phase. The amount of glycidyl methacrylate used was 96.7 mol % based on the total amount of the monomers, and the amount of glycerin-1,3-dimethacrylate used was 3.3 mol % based on the total amount of the monomers.
Packing into a stainless steel column using the obtained packing material 12 was attempted. However, the back pressure was high, making liquid feeding difficult, and this made it impossible to carry out the packing. Because of this, neither of the evaluations was able to be carried out.
Results of the above Examples and Comparative Examples are shown in Table 1.
From the above results, by adopting the configuration of the present invention, a packing material having suppressed non-specific adsorption and high alkali resistance can be obtained.
When no hydrophobic portion is provided or when the alkylene chain is short, the alkali resistance is low as shown in Comparative Examples 1 and 2. In addition, it was found that when the alkylene chain is too long or when no hydrophilic portion is provided, the hydrophobicity is strong, and non-specific adsorption is induced as shown in Comparative Examples 3 and 4. In addition, in Comparative Example 5 having many repeating units derived from a polyfunctional monomer, it was found that non-specific adsorption was induced, and in Comparative Example 6 having fewer repeating units derived from a polyfunctional monomer, it was found that the back pressure applied to the apparatus was high, making column packing difficult.
A 300
Acetone
Adsorption
Alkalies
Anabolism
Aprotinin
boron trifluoride
Buffers
butyl acetate
butylene
Butylene Glycols
chlorobenzene
COMP protocol
Cyclohexane
cyclohexanedimethanol
diethylamine
diethyl succinate
diglyme
Epichlorohydrin
Esters
Ethanol
ethylene dimethacrylate
Ethylenes
Ethyl Ether
Filtration
G 130
gamma-Globulin
Gel Chromatography
Glycerin
glycidyl methacrylate
Glycol, Ethylene
High-Performance Liquid Chromatographies
Hydrochloric acid
Hydrolysis
Nitrogen
Polyethylene Glycols
Polymers
polyol
Polyvinyl Alcohol
potassium bromide
Potassium Chloride
potassium hydroxide
Pressure
Ribonucleases
Sodium Hydroxide
sodium phosphate
Solvents
Sorbitol
Stainless Steel
Sulfoxide, Dimethyl
tetraethylene glycol
Thyroglobulin
Titrimetry
Uridine
Example 7
The synthesis of the methotrexate conjugates is described in (
Amination
Amines
Anabolism
Diamines
Esters
Ethylenediamines
Hydrolysis
Lipids
Methotrexate
Prodrugs
Pteridines
TERT protein, human
EXAMPLE 8
Rhizopus oryzae (RO) lipase was covalently bound to acrylic beads and contained in a device resembling a teabag. Enfalac infant formula (25 g) was combined with tap water (88 mL) at 37° C. Reactions were carried out in a glass bottle with 100 mL of infant formula and a tea bag containing either 100, 500, 1000, or 2000 mg of immobilized RO lipase. Each reaction was incubated at 37° C. for 30 minutes with inversion. Samples were taken at the following timepoints: 0, 1, 2, 3, 4, 5, 10, 20, and 30 minutes. Samples were analyzed for DHA and ARA by reverse phase high performance liquid chromatography (RP-HPLC).
At each concentration of immobilized RO lipase, the percent hydrolysis of DHA and ARA increased as the amount of immobilized RO lipase increased (FIGS. 27A-27D). These data demonstrate the feasibility of the tea bag device for pre-hydrolyzing formula with lipase.
Figs
High-Performance Liquid Chromatographies
Hydrolysis
Infant Formula
Inversion, Chromosome
Lipase
Medical Devices
Rhizopus oryzae
Example 8
Antibody-dependent cell-mediated cytotoxicity assays (ADCC assays) were performed for the characterization of anti-human CD25 antibodies using CD25-expressing SR786 cells, herein called target (T) cells, incubated for 20 minutes at 37 C with different concentrations of anti-human CD25 antibodies in a low-IgG FBS-supplemented medium (4% FBS in RPMI). ADCC effector (E) cells are then added to the cell-mAbs mixture at an E:T ratio of 1:1. The effector cells are Jurkat cells stably transfected with a luciferase reporter system and over-expressing CD16/FcgammaRIIIA (Promega). After overnight incubation at 37 C, the cells are lysed and luciferase activity is measured by mean of luminescence release from the hydrolysis of a specific luciferase substrate, following manufacturer instruction (Promega Bio-Glow protocol). Graphs of the raw data are produced using Graphpad Prism v7 to generate dose response curves. The Relative Luminescences Units (RLU) are plotted on a XY chart against the log of the antibody concentration, and the data fir to a non-linear regression curve from which the EC50 is calculated.
Anti-Antibodies
anti-c antibody
Biological Assay
Cells
Cytotoxicities, Antibody-Dependent Cell
FCGR3A protein, human
Homo sapiens
Hydrolysis
IL2RA protein, human
Immunoglobulins
Jurkat Cells
Luciferases
Luminescence
Monoclonal Antibodies
prisma
Promega
T-Lymphocyte
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