Effect of Cyclodextrin on Preservative–Surfactant–Protein Interactions

Abstract

Particulate matter formation in parenteral protein formulations poses significant safety risks and quality challenges. In the multi-dose injectable products, therapeutic proteins, surfactants, and antimicrobial preservatives are present together, forming a complex system that is more prone to destabilizing interactions. Cyclodextrin (CD) has been proposed as a potential stabilizer, yet its role in such multiple-component systems remains insufficiently understood.
This project systematically investigated the effect of HP-β-CD on the interactions within a protein–surfactant–preservative model system, using bovine serum albumin (BSA) and human growth hormone (hGH) as model proteins, polysorbate 20 (PS20) as the surfactant, and phenol as the preservative. A stepwise experimental approach was adopted, progressing from binary to ternary systems. Instability was characterized by phenol- and salt-induced precipitation thresholds, detected simultaneously through absorbance, fluorescence, and light scattering signals using a Probe Drum titration instrument.
Results demonstrated that CD's effect on formulation stability is formulation-dependent, governed by the interplay of CD concentration, protein identity, and surfactant concentration. At high concentration, CD protected PS20 against phenol-induced clouding, whereas low CD concentration (10 mg/mL) showed destabilizing tendencies. Although CD slightly destabilized BSA, the overall performance of the CD-BSA ternary system was similar to that of PS20. CD provided no protective effect against phenol-induced hGH aggregation. In the hGH ternary systems, CD behaved as a destabilizer in the hGH system by disrupting PS20's protective role at intermediate surfactant concentrations. Consequently, in ternary systems, CD expressed its effect on formulation stability primarily through modulation of PS20 behavior.
These findings indicate that CD cannot be considered a universally beneficial stabilizer for complex protein formulations containing surfactants and preservatives, and its incorporation requires careful, case-by-case evaluation.

Popular Abstract

When the medicine that should protect you starts to fall apart, how a sugar ring might help
Imagine receiving an injection to treat a chronic illness. You expect the medicine to be pure, potent, and safe. But what if visible particles were forming inside the vial before it even reached you? This is a real and pressing challenge in the design of injectable protein medicines, and it's what this project set out to address.
Many biological drugs, such as insulin-like proteins, are packaged in multi-dose bottles: they are opened, used, refrigerated, and reopened multiple times over weeks. To keep the drugs inside safe between uses, manufacturers add preservatives, small antimicrobial molecules, often phenol-based, that kill bacteria and keep the environment sterile. They also add surfactants, molecules similar to soap, which could form a structure called a micelle to prevent the delicate protein drug from clumping together. These three components (protein, preservative, and surfactant) must coexist in the same tiny bottle, but they don't always get along.
The problem is that the preservative is hydrophobic (water-repelling). It tends to stick to other hydrophobic things in the bottle, including parts of the surfactant and the protein itself. When a preservative binds to the surfactant, it can cause the surfactant to cloud and clump at room temperature. When it binds to the protein, it can unfold and destabilize it. Both outcomes lead to visible or invisible particles in the injection, which is a quality defect not only regulated by pharmacopoeias but can also cause serious harm to patients if injected.
To address this, researchers have been looking at cyclodextrins (CDs), ring-shaped sugar molecules with a water-loving exterior and a water-repelling hollow interior, somewhat like a molecular bucket. The idea is that CD could "trap" preservatives inside its hollow core before it has a chance to cause damage. But does this actually work in a complex system with all three components present?
This project tested exactly that, using two model proteins (one stable, one prone to aggregate), a commonly used surfactant (polysorbate 20), phenol as the preservative, and HP-β-CD as the cyclodextrin candidate. The experiments used a titration instrument that could add tiny, precise amounts of phenol or salt solution to the system and simultaneously measure absorbance, protein fluorescence, and light scattering that could reveal exactly when and how the system became unstable.
The results were subtle and sometimes surprising. At high concentrations, CD did protect the surfactant from phenol-induced clouding not just by trapping phenol, but also by interacting directly with the surfactant molecules. However, at low CD concentrations, it could actually make things worse by loosening the surfactant's micellar structure.
For the protein that is prone to aggregate in the presence of preservatives (human growth hormone), CD offered no protection at all against phenol, because phenol binds so strongly to the protein that CD simply cannot compete. Worse, in its full three-component system, CD could undermine the surfactant's ability to protect the protein by pulling the surfactant away from the protein surface. And for the stable protein (bovine serum albumin), CD slightly provided destabilization at low concentrations, while in its three-component system, the effect of CD was similar to that on PS20 clouding induced by phenol.
The key takeaway: cyclodextrin is not a one-size-fits-all solution. Whether it helps, does nothing, or actually causes harm depends critically on the specific protein, the surfactant concentration, and the CD dose. For drug developers, this means that adding cyclodextrin to a formulation must be tested and justified carefully: what works for one product could fail or backfire for another.
This project adds important mechanistic insight into how these excipients interact with each other and with proteins in complex injectable systems, providing a scientific foundation for more rational formulation design of multi-dose biological drugs.

• author: Jia, Ruoyu
• supervisor: Marie Wahlgren
• organization: Pharmaceutical Technology (master); Food Technology and Nutrition (M.Sc.)
• course: KLGM06 20261
• year: 2026
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences; Technology and Engineering
• keywords: Cyclodextrin, Surfactant clouding, Protein aggregation, Preservatives, Formulation stability, Pharmaceutical formulation
• language: English
• id: 9234197
• date added to LUP: 2026-06-12 13:34:46
• date last changed: 2026-06-12 13:34:46