Polysorbate 20/80 is a common nonionic surfactant in biologics and is commonly used in excipients for protein-based products. Compared to ionic surfactants (e.g., sodium dodecyl sulfate), nonionic surfactants avoid strong interactions with charged protein molecules and have low toxicity and good clinical tolerability.
Nearly 120 antibody-based products (monoclonal antibodies, bis-antibodies, and ADCs) that have been approved for marketing by the FDA have surfactants added to no less than 70% of the total number of products, and polysorbate 20 and polysorbate 80 additions again account for about 30% and 70% of these, respectively, with a small number of products with polysorbate added to the product.188 Polysorbate 80 was the first monoclonal antibody to be marketed in 1986. One of the excipients in the first monoclonal antibody product marketed in 1986 (Orthoclone OKT3, 1986), polysorbate surfactants are commonly used at concentrations ranging from 0.01 mg/mL to 1.0 mg/mL.
It is generally believed that the addition of surfactants has the effect of 1) surfactants competing with protein products for the gas-liquid interface, thus reducing the exposure of protein products; 2) reducing the non-specific adsorption of the product to its contact surfaces (e.g., tube walls, stoppers, etc.); and 3) increasing the solubility of the product.
Polysorbate is actually a mixture of amphiphilic nonionic surfactants consisting primarily of polyoxyethylene sorbitan fatty acid esters. The figure below shows an example of the synthesis process of polysorbate 80. Sorbitol is first dehydrated into dehydrated sorbitol (dehydrated sorbitol can be further dehydrated into isosorbitol), and the dehydrated product reacts with ethylene oxide and is further partially esterified with oleic acid (or lauric acid) to produce polysorbate 80 (or polysorbate 20), respectively, and there are usually many unesterified molecules in the product.
Polysorbate is a multi-phase mixture of different fatty acid esters, and the Chinese Pharmacopoeia requires that the oleic acid content of polysorbate 80 and polysorbate 20 should range from not less than 58.0%, and the lauric acid should range from 40.0% to 60.0%, respectively. A data sheet shows that there are variations in different batches (A1-E3) of polysorbate 80 between different suppliers.
The R&D and production process should take into account the effect of the product excipients themselves on the product to ensure the safety and efficacy of the final product, and the pharmacopoeias of various countries require that commercially available products must meet their minimum standards before they are allowed to be sold.
In addition to the polysorbate feedstock, polysorbates themselves present a risk of degradation which adds to the complexity of the substances they contain. In general, polysorbates are at risk of acid/base/enzyme-catalysed hydrolysis as well as oxidative degradation including metal ions. Exposure of polysorbates to air, light and transition metals are known to cause oxidation, and enzyme-mediated hydrolysis (generally mediated by host proteins) is thought to be the main cause of the degradation observed in biologics. The solvents used for antibody-based products are generally saline or sterile water for injection, and the poor solubility of free fatty acids in the degradation products of polysorbate is one of the main reasons for the formation of insoluble particles.
In response to hydrolysis, certain buffering excipients (e.g. histidine (salt), phosphate, etc.) are often added to the actual product, so the pH of the system in which the polysorbate is present should not change much. Host protein residue is a tricky problem, prompting the formulation to produce a variety of aggregates to the point of producing particles visible to the naked eye, such as protein disulfide enzyme (PD19), heat shock protein (DanK), etc. involved in the aggregation process, not only that, the host protein will also lead to degradation of the formulation occurs, so that the polysorbate loss of function and destabilisation of the formulation. To address host protein residues, upstream improvements can be made by knockdown of cell line related genes (without affecting the actual production conditions), optimisation of cell culture conditions, and downstream improvements can be made by optimisation of purification steps, it should be noted that highly concentrated antibody products may be accompanied by the presence of high concentrations of enzymatic impurities. Of course, the process may contain other substances with enzymatic activity, which, depending on their nature, should be targeted. For the oxidation problem caused by metal ions (usually copper and iron ions) within, it can generally be improved by adding disodium edetate, while citric acid, which is common in buffers, can also play a role in complexing metal ions.
Among several common buffer systems (histidine, citrate, succinate and phosphate), polysorbate showed the highest degradation rate in histidine buffer. The histidine buffer system and the concentration of polysorbate used in this experiment are close to the actual concentration used in the actual approved and marketed antibody products, which provides a certain significance. The choice of buffer in the formulation should be a factor to be considered in formulation screening of products that use polysorbate as a surfactant. It is worth noting that about 50% of the nearly 120 antibody products use histidine buffer systems, and more than 50% of the products with histidine buffer systems have polysorbate as the surfactant.
The actual composition of the formulation takes into account the addition of multiple ingredients as well as the proportions of the various ingredients and their actual dosages, and orthogonal experiments may be a good solution for such variable and complex experiments.
Common separation methods are chromatography (e.g., HPLC) and detection methods include UV/Vis and fluorescence spectroscopy, mass spectrometry, and so on. The heterogeneity of polysorbates themselves and the complexity of their degradation products pose challenges for their qualitative and quantitative characterisation. The development and validation of analytical methods for polysorbates is difficult due to their complex composition, heterogeneity and lack of chromophores.
Because of the potential problems with polysorbate described above, other surfactants may be good alternatives to its role as an excipient.There are also a number of products in the FDA-approved and marketed antibody class that use polysorbate as a surfactant instead of polysorbate, using polysorbate 188 (P188).The FDA has also approved and marketed a number of products in the FDA-approved antibody class.
In fact P188 is approved for use in a variety of formulations as an emulsifier, solubiliser, dispersant etc.
Grapentin C et al. studied the stability of four monoclonal antibodies in glass vials containing different liquid formulations of polysorbate or P188, and found that P188 provided protection comparable to that of polysorbate under conditions of stress (freezing and thawing, shaking)
The ester bond in polysorbate is subject to enzymatic or chemical hydrolysis, which is well circumvented by the structure of P188. However, it remains unclear whether degradation of the poly(ethylene oxide) and poly(propylene oxide) chains of P188 results in the formation of insoluble particles.
Wang T et al. showed that P188 did not perform as well as the citrate buffer system in terms of stability in the histidine buffer system, and the stabilised phenotype became poorer in the presence of metal ions.Substances identified by GC-MS included acetic acid, a typical autoxidation product of P188, ethylene glycol, its oligomers and alcohols, propylene glycol, its oligomers and alcohols, as well as a variety of ethylene glycol esters and propylene glycol esters. In addition, a number of PPO and PEO oligomers were observed, and the effect of these degradation products on different protein formulations needs to be further investigated.
Surfactants used as protein stabilisers in antibody-based injectable formulations, in addition to polysorbate 20/80 as well as P188, P407 from the Porosam family, PEG fatty esters (e.g., polyethyleneglycol (15)-hydroxystearate), and non-ester surfactants (e.g., dodecylglucoside and dodecylmaltoside) are all of potential use. Of course, these potential alternatives also have potential shortcomings, for example, ester surfactants may have problems similar to polysorbates including enzymatic degradation, but their biodegradability and biocompatibility should not be ignored, and the impact of these excipients on products including safety as well as efficacy is yet to be further explored by researchers.
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