Protein PEGylation for the design of biobetters: from reaction to purification processes

The covalent attachment of polyethylene glycol (PEG) to therapeutical proteins is an important route to develop biobetters for biomedical, biotech and pharmaceutical industries. PEG conjugation can shield antigenic epitopes of the protein, reduce degradation by proteolytic enzymes, enhance long-term stability and maintain or even improve pharmacokinetic and pharmacodynamics characteristics of the protein drug. Nonetheless, correct information in terms of the PEGylation process from reaction to downstream processing is of paramount importance for the industrial application and processing scale-up. In this review we present and discuss the main steps in protein PEGylation, namely: PEGylation reaction, separation of the products and final characterization of structure and activity of the resulting species. These steps are not trivial tasks, reason why bioprocessing operations based on PEGylated proteins relies on the use of analytical tools according to the specific pharmaceutical conjugate that is being developed. Therefore, the appropriate selection of the technical and analytical methods may ensure success in implementing a feasible industrial process

Schedules of controlled substances: removal of naloxegol from control. Final rule.

With the issuance of this final rule, the Administrator of the Drug Enforcement Administration removes naloxegol ((5[alpha],6[alpha])-17-allyl-6-((20-hydroxy-3,6,9,12,15,18-hexaoxaicos-1-yl)oxy)-4,5-epoxymorphinon-3,14-diol) and its salts from the schedules of the Controlled Substances Act (CSA). This scheduling action is pursuant to the CSA which requires that such actions be made on the record after opportunity for a hearing through formal rulemaking. Prior to the effective date of this rule, naloxegol was a schedule II controlled substance because it can be derived from opium alkaloids. This action removes the regulatory controls and administrative, civil, and criminal sanctions applicable to controlled substances, including those specific to schedule II controlled substances, on persons who handle (manufacture, distribute, reverse distribute, dispense, conduct research, import, export, or conduct chemical analysis) or propose to handle naloxegol.

Peri- and postnatal rodent development of Hematide, an erythropoiesis-stimulating agent.

BACKGROUND: Aperi- and postnatal reproduction toxicity study was conducted in rats treated with Hematide, a synthetic PEGylated peptidic erythropoiesis stimulating agent (ESA). METHODS: Hematide, at IV doses of 0, 0.5, 3, and 15 mg/kg, was administered from implantation through lactation on gestation days (GDs) 5 and 18 and lactation day (LD) 13. RESULTS: Hematide induced pronounced polycythemia in all Hematide-treated dams. On LDs 2 and 21, hemoglobin (Hgb) increases above control levels were 3.1, 5.2, and 5.0 g/dL and 4.1, 5.1, and 5.5 g/dL at the 0.5, 3, and 15 mg/kg/dose, respectively. There were no effects on parturition, lactation, or maternal behavior in the F0 generation female rats. A slight decrease in pup viability on postpartum days 2-4 and lower body weights and/or body weight gain for the F1 generation were associated with pronounced polycythemia and decreases in maternal body weight gain and/or food consumption at > or =3 mg/kg/dose. Hematide fetal exposure was negligible. No Hematide effect, other than on growth and survival, was noted on developmental, functional, mating, and fertility end points in the F1 generation rats, and no effect on litter or fetal parameters was observed in the F2 generation. The maternal no-observed-adverse-effect level (NOAEL) for Hematide was 0.5 mg/kg, and the NOAEL for parturition and maternal behavior was 15 mg/kg. The NOAEL for F1 pup viability and growth was 0.5 mg/kg/dose. CONCLUSIONS: In conclusion, the Hematide-associated adverse findings were attributed to exaggerated erythropoiesis (pronounced and prolonged polycythemia) resulting from administration of an ESA to pregnant animals.

[Research on prerequisite for using bovine hemoglobin modified by polyethylene glycol as a red blood cell substitute].

This paper aims to raise concerns about the modification of bovine hemoglobin with the use of SC-mPEG as modifying reagent. The yield rates of SC-mPEG-BHB under dissimilar conditions are studied, and more important, a series of methods used to characterize the SC-mPEG-BHB mixtures are built. The best reactive system has been confirmed, i.e., at 4 degrees C, pH9; SC-mPEG and BHB are mixed according to the proportion of 60:1 and be submitted to reaction for 2 h.

Estimation of the theoretical pore sizes of the porcine oral mucosa for permeation of hydrophilic permeants.

The oral mucosa is an attractive alternative site for the delivery of macromolecules. However, hydrophilic macromolecules are generally not well absorbed through the oral mucosa because of their hydrophilicity and size. The transport of these molecules across the oral mucosa may occur via the aqueous route. The knowledge about the dimension of this route is essential to understand the permeation of these molecules across the oral mucosa. Polyethylene glycols (PEGs) were used as model hydrophilic permeants in this study to establish the relationship between permeability and molecular weight across the different regions of the oral mucosa. The radius of the pore (r(p)) for the aqueous pathway and the ratio of the area fraction of aqueous pathway to the barrier length of porcine mucosal membrane were calculated using Renkin's equation based on the experimentally determined permeability and theoretical diffusion parameters for PEG molecules. The pore sizes of aqueous pathway for buccal mucosa and sublingual mucosal were estimated as 18-22 and 30-53 A, respectively. Pore size estimation across different regions of oral mucosa indicated the potential of delivering therapeutically important macromolecules via oral mucosa.

Final report of the amended safety assessment of PEG-5, -10, -16, -25, -30, and -40 soy sterol.

PEGs Soy Sterol are polyethylene glycol (PEG) derivatives of soybean oil sterols used in a variety of cosmetic formulations as surfactants and emulsifying agents, skin-conditioning agents, and cleansing and solubilizing agents. When the safety of these ingredients were first reviewed, the available data were insufficient to support safety. New data have since been received and the safety of these ingredients in cosmetics has been substantiated. Current concentration of use ranges from a low of 0.05% in makeup preparations to 2% in moisturizers and several other products. PEGs Soy Sterol are produced by the reaction of the soy sterol hydroxyl with ethylene oxide. In general, ethoxylated fatty acids can contain 1,4-dioxane as a byproduct of ethoxylation. The soy sterols include campesterol, stigmasterol, and beta-sitosterol. The distribution of sterols found in oils derived from common plants is similar, with beta-sitosterol comprising a major component. Impurities include sterol hydrocarbons and cholesterol (4% to 6%) and triterpine alcohols, keto-steroids, and other steroid-like substances (4% to 6%). No pesticide residues were detected. PEGS: Because PEGs are an underlying structure in PEGs Soy Sterols, the previous assessment of PEGs was considered. It is generally recognized that the PEG monomer, ethylene glycol, and certain of its monoalkyl ethers are reproductive and developmental toxins. Given the methods of manufacture of PEGs Soy Sterol, there is no likelihood of ethylene glycol or its alkyl ethers being present. Also, the soybean oil sterol ethers in this ingredient are chemically different from the ethylene glycol alkyl ethers of concern. PEGs are not carcinogenic, although sensitization and nephrotoxicity were observed in burn patients treated with a PEG-based cream. No evidence of systemic toxicity or sensitization was found in studies with intact skin. Plant Phytosterols: Intestinal absorption of ingested plant phytosterols is on the order of 5%, with 95% of the material entering the colon. Absorbed plant phytosterols are transported to the blood. Although there are some data suggesting that sulfates of beta-sitosterol can act as abortifacients in rats and rabbits, other studies of well-characterized plant phytosterols and phytosterol esters demonstrated no effect in an estrogen-binding study, a recombinant yeast assay for estrogen or estrogen-like activity, or a juvenile rat uterotrophic assay for estrogen or estrogen-like activity. In a two-generation reproduction study using rats, plant phytosterol esters in the diet had no effect on any parameter of reproduction or fertility. Subcutaneous injections of beta-sitosterol did reduce sperm concentrations and fertility in rats. Sitosterol inhibited tumor promoting activity of 12-O-tetradecanoylphorbol-13-acetate (TPA) in mice after initiation with 7,12-dimethylbenz[a]anthracene (DMBA), and reduced the tumors produced by N-methylnitrosourea in rats. Phytosterols were not genotoxic in several bacterial, mammalian, and in vitro assay systems. Phytosterols decreased epithelial cell proliferation in the colon of mice and rats, and were cytotoxic for human epidermoid carcinoma of the nasopharynx. PEGs Soy Sterols: The acute oral LD50 in rats of PEG-5-25 Soy Sterol was >10 g/kg. The acute dermal LD50 of a liquid eyeliner containing 2%PEG-5 Soy Sterol was >2 g/kg in rabbits. PEG-5-25 Soy Sterol was not a primary irritant in rabbits when applied undiluted. Undiluted PEG-5 Soy Sterol did not cause sensitization in guinea pigs. PEGs Soy Sterol did not produce ocular toxicity in rabbits. PEG-5 Soy Sterol was negative in the Ames mutagenicity test, with or without metabolic activation. PEG-5 Soy Sterol, at concentrations up to 2%in formulation, did not cause dermal or ocular irritation, dermal sensitization, or photosensitization in clinical studies. Because of the possible presence of 1,4-dioxane reaction product and unreacted ethylene oxide residues, it was considered necessary to use appropriate procedures to remove these from PEGs Soy Sterol before blending them into cosmetic formulations. Based on the systemic toxicity and sensitization seen with PEGs applied to damaged skin, it was recommended that PEGs Soy Sterol should not be used in cosmetic products applied to damaged skin. Although no dermal absorption data were available, oral studies demonstrate that phytosterols and phytosterol esters are not significantly absorbed and do not result in significant systemic exposure. Some small amounts did appear in the ovaries, however. This raises a concern about the potential presence of free phytosterols and beta-Sitosterol, which could have antiestrogenic, antiprogestational, gonadotrophic, antigonadotrophic, and antiandrogenic effects in PEG sterols. These concerns are alleviated by the extensive data showing that well-defined phytosterols and phytosterol esters are not estrogenic and do not pose a hazard to reproduction. Likewise, the absence of impurities in plant phytosterols and phytosterol esters and extensive data demonstrating the absence of any genotoxicity in bacterial and mammalian systems mitigate against the possibility of any carcinogenic effect with those same well-characterized materials. The Cosmetic Ingredient Review (CIR) Expert Panel concluded that the PEGs Soy Sterol are safe as used in cosmetic products.

Rationalization of membrane protein crystallization with polyethylene glycol using a simple depletion model.

Based on the importance of crystallizing membrane proteins in a rational way, cytochrome bc(1) complex (BC1) was crystallized using polyethylene glycol (PEG) as a sole crystallization agent. Interaction between protein-detergent complexes of BC1 was estimated by dynamic light scattering, and was compared with the numerical calculation using the Derjaguin-Landau-Verwey-Overbeek potential plus a depletion potential, without considering specific surface properties of the protein-detergent complexes. The experiments and calculation were found to be consistent and we obtained a relation between PEG molecular weight M and the range of depletion zone delta as delta approximately M(0.48+/-0.02). The stability of liquid phase of BC1 solutions was controlled by a ratio of (the range of depletion zone)/(the radius of a BC1 particle), which was consistent with recent theoretical predictions. The crystallization was most successful under a condition where the stability of the liquid phase changed from stable to unstable. The PEG molecular weight that fulfilled this condition coincided with the one used empirically to crystallize BC1 in the past by a number of groups. These results are compared to the fact that membrane proteins were often successfully crystallized close to the detergent cloud point.