Polysorbate stability

Surfactants are well-known protein denaturants. However, when sufficiently dilute, some surfactants (e.g. polysorbate) exert a stabilizing influence on some protein types. Proteins display a tendency to aggregate at interfaces (air—liquid or liquid—liquid), a process that often promotes their denaturation. Addition of surfactant reduces surface tension of aqueous solutions and often increases the solubility of proteins dissolved therein. This helps reduce the rate of protein... 

Keratinocyte growth factor is a 140 amino acid, 16.3 kDa member of the FGF family. It differs from native keratinocyte growth factor in that the first 23 N-terminal amino acids have been deleted, which improves its stability. After cell growth the product is recovered and purified by a multistep chromatographic protocol. It is presented in lyophilized format in single-use vials and containing mannitol, sucrose, polysorbate 20 and histidine as excipients. It is administered by daily i.v. injection, usually for several days. 

Hora et al. [3.19] described the complexity of protein stabilization by the example of recombinant, human Interleukin-2 (rhIL-2). Formulations with amino acids and mannitol/ sucrose are sensitive to mechanical stress e. g. by pumping. Hydroxypropyl-beta-cyclodextrin (HPcD) provides stability, but increases the sensitivity to oxygen. Polysor-bate 80 forms a mechanically stable product, but results in oxidation. In both cases contamination in the HPcD or traces of H202 in the Polysorbate may have been the starter for the oxidation. Brewster [3.20] reports, that HPcD stabilizes interleukin without forming aggregations and this results in 100 % biopotency. 

Mean particle size ranges 50 to 1000 mn. Depending on the type and concentration of the lipid, 0.5 to 5% emulsifier (surfactant) has to be added for physical stabilization. For dermal use, surfactants are very often poloxamer 188, polysorbate 80, lecithin, tyloxapol, polyglycerol methylglucose distearate (Tegocare 450), sodinm cocoamphoacetate (Miranol Ultra C32), or saccharose fatty acid ester. 

Purification entails use of an immunoaffinity column containing immobilized murine antifactor VII antibody. It is initially produced as an unactivated, single chain 406 amino acid polypeptide, which is subsequently proteolytically converted into the two-chain active factor Vila complex. After sterilization by filtration, the final product is aseptically filled into its final product containers and freeze-dried. The excipients present in the product include sodium chloride, calcium chloride, polysorbate 80, mannitol and glycylglycine. When freeze-dried in the presence of these stabilizing substances and stored under refrigerated conditions, the product displays a shelf-life of at least 2 years. It has proved effective in the treatment of serious bleeding events in patients displaying anti-factor VIII or IX antibodies. 

Allergic reactions to ESAs have been infrequent. There have been a small number of cases of pure red cell aplasia (PRCA) accompanied by neutralizing antibodies to erythropoietin. PRCA was most commonly seen in dialysis patients treated subcutaneously for a long period with a particular form of epoetin alfa (Eprex with a polysorbate 80 stabilizer rather than human serum albumin) that is not available in the USA. After regulatory agencies required that Eprex be administered intravenously rather than subcutaneously, the rate of ESA-associated PRCA diminished. However, rare cases have still been seen with all ESAs administered subcutaneously for long periods to patients with chronic kidney disease. 

The PEG could stabilize proteins by two different temperature-dependent mechanisms. At lower temperatures, it is preferentially excluded from the protein surface but has been shown to interact with the unfolded form of the protein at higher temperatures, given its amphipathic nature (57). Thus, at lower temperatures, it may protect proteins via the mechanism of preferential exclusion, but at higher temperatures possibly by reducing the number of productive collisions between unfolded molecules. PEG is also a cryoprotectant and has been employed in Recombinate, a lyophilized formulation of recombinant Antihemophilic Factor, which utilizes PEG 3350 at a concentration of 1.5mg/mL. The low-molecular weight liquid PEGs (PEG 300-600) can be contaminated with peroxides and cause protein oxidation. If used, the peroxide content in the raw material must be minimized and controlled throughout its shelf life. The same holds true for polysorbates (discussed below). 

Another aspect of polysorbates is that they are inherently susceptible to oxidative degradation. Often, as raw materials, they contain sufficient quantities of peroxides to cause oxidation of protein residue side chains, especially methionine (59). The potential for oxidative damage arising from the addition of stabilizer emphasizes the point that the lowest effective concentrations of excipients should be used in formulations. For surfactants, the effective concentration for a given protein will depend on the mechanism of stabilization. It has been postulated that if the mechanism of surfactant stabilization is related to preventing surface-denaturation, the effective concentration will be around the detergent s critical micellar concentration. Conversely, if the mechanism of stabilization is associated with specific protein-detergent interactions, the effective surfactant concentration will be related to the protein concentration and the stoichiometry of the interaction (39). 

However, for low-dose chug products, impurities from excipients can be more problematic for causing chemical stability issues with a drug product. Several commonly used excipients (including povidone, PEG400, polysorbate 80, and hydroxypropyl... 

Nassar MN, Nesarikar VN, Lozano R, Parker WL, Yande P, Palaniswamy V, Xu W, Khaselev N. 2004. Influence of formaldehyde impurity in polysorbate 80 and PEG-300 on the stability of a parenteral formulation of BMS-204352 identification and control of the degradation. Product Pharm. Dev. 9 189-195. 

Span 60 was found to cause the formation of a gel in the oil phase, which was attributed to the crystallization of span 60 within the oil phase. The net result is an extremely slow release rate from the span 60 v/w/o formulation [152]. These gelled span 60 systems may be stabilized by incorporation of polysorbate 20... 

Coffee Creamer. Emulsions of vegetable oil in an aqueous phase, including caseinates, are sold (as pasteurized liquid or as a spray-dried powder) as substitutes for cream for addition to coffee. Eor the liquid form, an emulsifier system of some combination of polysorbate 60, polysorbate 65, and sorbitan monostearate is used at a concentration of up to 0.4%. The spray-dried whitener uses the same emulsifier system, at a level of 1-3% of the dried powder. In both cases, sodium caseinate is probably the major stabilizer of the o/w emulsion. 

Buffers can also be provided in parenteral formulations to ensure the required pH needed for solubility and/or stability considerations. Other excipients included in parenteral products are preservatives (e.g., benzyl alcohol, p-hydroxybenzoate esters, and phenol), antioxidants (e.g., ascorbic acid, sodium bisulfite, sodium metabisulfite, cysteine, and butyl hydroxy anisole), surfactants (e.g., polyoxyethylene sorbitan monooleate), and emulsifying agents (e.g., polysorbates). An inert gas (such as nitrogen) can also be used to enhance drug stability. Stability and solubility can also be enhanced by the addition of complexation and chelating agents such as the ethylenediaminetetraacetic acid salts. For a more detailed list of approved excipients in parenteral products, the reader should consult the monographs within the USP. 

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