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Polysorbates HLB (PS) are a class of amphiphilic, non-ionic surfactants derived from the esterification of ethoxylated dehydrated sorbitol or isosorbide (derivatives of sorbitol) with fatty acids. Among them, Polysorbate HLB 20 (PS20) and Polysorbate HLB 80 (PS80) are the most widely used surfactants in biopharmaceutical formulations. They play a crucial role in preventing protein denaturation, aggregation, surface adsorption, and flocculation during the formulation process.
As protein stabilizers, polysorbates HLB are chemically diverse mixtures that can degrade via oxidation and hydrolysis pathways, with hydrolysis being either chemically induced or enzyme-catalyzed. The degradation of polysorbates HLB can unintentionally affect the quality, efficacy, safety, and stability of protein formulations. Therefore, regulatory agencies have become increasingly stringent in reviewing control strategies for polysorbates HLB to ensure their content remains stable throughout the shelf life of pharmaceutical products.
Polysorbates HLB 20 (PS20) and 80 (PS80) are used in most commercial therapeutic protein formulations as stabilizers due to the following factors:
Biocompatibility.
Low toxicity.
Effective protein stabilization.
Even at low concentrations, due to their high Hydrophilic-Lipophilic Balance (HLB) values and low Critical Micelle Concentration (CMC), both PS20 and PS80 provide sufficient protein stability.
Although the precise mechanism by which polysorbates HLB stabilize proteins is not fully understood, two major hypotheses have been proposed: interface competition and surfactant-protein complexation.
It is generally believed that Polysorbates HLB 20 and HLB 80 primarily stabilize proteins through interface competition. The surface activity of polysorbates HLB 20 and HLB 80 is much higher than that of typical therapeutic proteins (such as monoclonal antibodies, mAbs), allowing them to competitively block interfaces and inhibit protein adsorption onto gas-liquid interfaces. This characteristic effectively prevents protein unfolding at interfaces during manufacturing, sample handling, and storage (including mixing, filtration, pumping, shaking, stirring, and freeze-thaw processes). Similarly, it helps prevent protein adsorption and subsequent loss at product contact surfaces (e.g., filters, primary containers, sealed containers, and intravenous infusion tubing), which is crucial for ensuring accurate dosage delivery to patients.
Polysorbates HLB 20 and HLB 80 can also stabilize proteins through direct interactions, thereby increasing the colloidal stability of proteins. Therapeutic proteins can aggregate by combining hydrophobic patches, and polysorbates HLB can interact with these hydrophobic regions on the protein surface through hydrophobic interactions, thereby preventing protein aggregation and further unfolding. However, the direct interaction between surfactants and proteins, and the subsequent improvement in colloidal stability, may be protein-specific and not universally applicable. For example, a thermodynamic study demonstrated that Polysorbates HLB bind to human serum albumin, but very low binding was observed with three immunoglobulins studied, making their interaction negligible.
Polysorbates HLB are susceptible to degradation through oxidation and hydrolysis, which can occur via either chemical or enzyme-catalyzed hydrolysis. The causes and effects of this degradation will be discussed in detail below.
Hydrolysis of Polysorbates HLB involves the cleavage of fatty acid ester bonds, releasing free fatty acids that can form visible or non-visible particles. It has been suggested that enzyme-induced hydrolysis of Polysorbates HLB is a major cause of visible and sub-visible particle formation that can affect product quality. Under typical pH conditions for protein formulations, Polysorbates HLB hydrolysis is generally limited. It has been reported that 24 residual host cell proteins induce hydrolysis of Polysorbates HLB in protein formulations, including several isoforms of lysosomal phospholipase A2 (LPLA2), 25 identified phospholipase B-like-2 (PLBL2), and 26 liver carboxylesterases. Purified lipases, a subclass of esterases catalyzing lipid hydrolysis, often share physicochemical properties similar to the target proteins, making them difficult to remove. Attempts to knock out lipoprotein lipase (LPL) from Chinese hamster ovary (CHO) cell lines reduced LPL expression by over 95%, but Polysorbate HLB degradation was only reduced by 41-57%, suggesting that other lipases may be involved.
A range of carboxylesterases from different species were used to induce hydrolysis of Polysorbate HLB 20 and Polysorbate HLB 80. The degradation pattern was found to depend not only on the hydrolytic enzyme but also on the Polysorbate HLB subtype, such as the ester sequence, the identity of the hydrophilic head group, and the length of the fatty acid chains. No Polysorbate HLB subtype was found to be completely resistant to enzyme hydrolysis. Recently, presumed phospholipase B2 (PLBL2) was excluded as the fundamental cause of Polysorbate HLB hydrolysis in protein formulations through gene knockout and immune depletion studies. Discrepancies in reported results may be due to the fact that lipases promoting Polysorbate HLB hydrolysis are present in levels below the detection limit, making it difficult to trace, monitor, or associate them with Polysorbate HLB degradation.
Polysorbates HLB can also undergo oxidation due to temperature, light, or trace transition metals, resulting in the formation of peroxides that can lead to protein oxidation, while generated acids may lower the solution pH. Oxidation of Polysorbates HLB primarily occurs on the polyethylene oxide (POE or PEO) chains, leading to the formation of POE esters and other degradation products, such as short-chain alkanes, ketones, aldehydes, and acids. Oxidation may also occur on the fatty acid portion, particularly on "unsaturated fatty acids," such as oleic acid esters and linoleic acid esters, which are preferentially attacked. Oxidation in both parts is considered a free-radical chain process involving initiation, propagation, and termination steps. Interestingly, recent reports have indicated that oxidized Polysorbate HLB 80 improves its surface activity while maintaining its CMC characteristics and providing protection against mAb aggregation. In contrast, hydrolyzed Polysorbate HLB 80 results in slower surface adsorption. Free fatty acids released during hydrolysis can also form insoluble particles, negatively impacting protein quality and stability.
Histidine, a commonly used buffer for protein formulations, may have confounding effects on Polysorbate HLB degradation. L-histidine has been reported to have dual effects on the stability of Polysorbate HLB 20. In a 2,2'-Azobis(2-aminopropane) hydrochloride (AAPH) stress study, L-histidine acted as a scavenger of reactive oxygen species, protecting Polysorbate HLB 20. However, at 40°C, it accelerated the oxidation of Polysorbate HLB 20 during storage. Another study observed Polysorbate HLB oxidation in histidine placebo buffers, but it was found that trace metal contamination in histidine buffers was the root cause. When EDTA was added under similar conditions, Polysorbate HLB oxidation was inhibited. Additionally, histidine chloride buffers were found to promote histidine-imidazole-catalyzed hydrolysis of Polysorbates HLB in placebo formulations. However, when therapeutic proteins were present, histidine-catalyzed Polysorbate HLB hydrolysis was minimized.
In summary, Polysorbates HLB 20 and HLB 80, as effective protein stabilizers, are widely used excipients in parenteral protein formulations. However, commercial Polysorbates HLB 20 and HLB 80 are chemically diverse mixtures, and Polysorbates HLB lack distinct chromophores in their structures. This combination of unique physicochemical properties presents challenges in developing Polysorbate HLB control strategies and elucidating the structure-function relationships of Polysorbates HLB.
