![]() Safety Ģ-Mercaptoethanol is considered toxic, causing irritation to the nasal passageways and respiratory tract upon inhalation, irritation to the skin, vomiting and stomach pain through ingestion, and potentially death if severe exposure occurs. Some carbamate protecting groups such as carboxybenzyl (Cbz) or allyloxycarbonyl (alloc) can be deprotected using 2-mercaptoethanol in the presence of potassium phosphate in dimethylacetamide. This prevents them from digesting the RNA during its extraction procedure. Numerous disulfide bonds make ribonucleases very stable enzymes, so 2-mercaptoethanol is used to reduce these disulfide bonds and irreversibly denature the proteins. Denaturing ribonucleases Ģ-Mercaptoethanol is used in some RNA isolation procedures to eliminate ribonuclease released during cell lysis. It is often used in enzyme assays as a standard buffer component. Preventing protein oxidation Ģ-Mercaptoethanol and related reducing agents (e.g., DTT) are often included in enzymatic reactions to inhibit the oxidation of free sulfhydryl residues, and hence maintain protein activity. Ģ-Mercaptoethanol is often used interchangeably with dithiothreitol (DTT) or the odorless tris(2-carboxyethyl)phosphine (TCEP) in biological applications.Īlthough 2-mercaptoethanol has a higher volatility than DTT, it is more stable: 2-mercaptoethanol's half-life is more than 100 hours at pH 6.5 and 4 hours at pH 8.5 DTT's half-life is 40 hours at pH 6.5 and 1.5 hours at pH 8.5. DTT is also a more powerful reducing agent with a redox potential (at pH 7) of −0.33 V, compared to −0.26 V for 2-mercaptoethanol. However, since 2-mercaptoethanol forms adducts with free cysteines and is somewhat more toxic, dithiothreitol (DTT) is generally more used especially in SDS-PAGE. Because of its ability to disrupt the structure of proteins, it was used in the analysis of proteins, for instance, to ensure that a protein solution contains monomeric protein molecules, instead of disulfide linked dimers or higher order oligomers. RS–SR + 2 HOCH 2CH 2SH ⇌ 2 RSH + HOCH 2CH 2S–SCH 2CH 2OHīy breaking the S-S bonds, both the tertiary structure and the quaternary structure of some proteins can be disrupted. In the case of excess 2-mercaptoethanol, the following equilibrium is shifted to the right: Some proteins can be denatured by 2-mercaptoethanol, which cleaves the disulfide bonds that may form between thiol groups of cysteine residues. This makes 2-mercaptoethanol useful as a protecting group, giving a derivative whose stability is between that of a dioxolane and a dithiolane. Reactions Ģ-Mercaptoethanol reacts with aldehydes and ketones to give the corresponding oxathiolanes. Thiodiglycol and various zeolites catalyze the reaction. ![]() Due to its diminished vapor pressure, its odor, while unpleasant, is less objectionable than related thiols.Ģ-Mercaptoethanol is manufactured industrially by the reaction of ethylene oxide with hydrogen sulfide. It is widely used because the hydroxyl group confers solubility in water and lowers the volatility. ME or βME, as it is commonly abbreviated, is used to reduce disulfide bonds and can act as a biological antioxidant by scavenging hydroxyl radicals (amongst others). This method enabled > 90 % overall recovery of unformulated DS at ≥ 150 g/L.2-Mercaptoethanol (also β-mercaptoethanol, BME, 2BME, 2-ME or β-met) is the chemical compound with the formula HOCH 2CH 2SH. Most of the rinse was combined with the retentate to hit the target protein concentration. After the retentate was collected, a minimal volume of buffer was added for the UF rinse. During the UF/DF process, the antibody was initially concentrated to 90 g/L, diafiltered, and concentrated to ≥ 180 g/L, then the retentate was collected. Analysis showed that the Capto S run removed the excipients with yields of ≥ 96%. In addition, Capto S has lower resin costs, takes less time to process, and uses milder elution conditions. A Capto S column was chosen over Protein A chromatography to remove excipients from formulated drug substance because of its higher binding capacity. Fortunately, a sufficient supply of formulated DS was available for reprocessing. Since the pilot plants were not available for large-scale campaigns, a creative alternative was needed to produce 2 kg of antibody from formulated DS for these studies. During process scale-up, the project team decided to make a high-concentration mAb drug substance for subcutaneous injection and change the formulation. The scope of this work is two-fold: 1) excipients removal from formulated mAb drug substance by Capto S chromatography and 2) UF/DF process development to make high-concentration drug substance (DS) for subcutaneous injection.
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