Luke Deters is director Biologics at Catalent Pharma Solutions, Kansas City, Missouri, USA.

Luke Deters

The tendency of proteins to aggregate or to change conformation under certain conditions poses a problem for protein therapeutics such as monoclonal antibodies. If their physical form alters, then it is likely that their activity will also be impacted, so stability must be ensured throughout the product’s shelf life.

	A key technique in building stability into a protein therapeutic is to include a surfactant in the formulation. A suitable surfactant will help to minimize the aggregation, unfolding, and denaturation that the protein might otherwise undergo, which would affect its activity.

	Various surfactants are approved for use in therapeutic parenteral dosage forms, and by far the most frequently used is polysorbate 80 (PS-80), the common name for polyoxyethylene (20) sorbitan monooleate (

). This amphipathic, non-ionic surfactant is a hydrophilic polyoxyethylene (POE) sorbitan with about 20 ethylene oxide sub units, attached to a hydrophobic fatty acid ester tail that is, for the most part, oleic acid.

	This surfactant is also often found in both food products and cosmetics as an emulsifier. As it forms micelles, it helps to minimize the surface tension at the liquid interface. For the purposes of formulation, it helps to suppress aggregation and additionally aids solubility of the protein in solution.

	Although polysorbate 80 is the most common, the related polysorbate 20-the lauric acid analogue-is also used. Other surfactants that are sometimes found include the polyethylene glycol derivative, Triton X100, and Pluronic F-68, a polyoxyethylene/polyoxypropylene copolymer. 

	Polysorbate 80 predominates largely because it has been in use for so long, and scientists have decades of experience in its application in drug formulation. The use of PS-80 is not without drawbacks; perhaps the biggest challenge arises from the fact that it is very heterogeneous, with multiple different polymer chain lengths. Ensuring the purity of PS-80 poses a problem, because it contains variable quantities of oleic acid in unreacted form, in addition to structurally related materials and other impurities. This variance and the impurities will affect protein stability in formulation. For all of its benefits, characterizing PS-80’s composition precisely remains a challenge.

	PS-80 is also prone to a low level of hydrolysis, and there are many factors that can lead to this, including changes in pH and temperature, and the presence of peroxides. The protein in the formulation may even catalyse enzymatic hydrolytic degradation. 

	Fundamentally, the presence of hydrolysis can run the risk of damaging the protein so that it loses its efficacy, by affecting its conformational structure and the way that the molecule binds. But perhaps an even bigger issue is immunogenicity that results from protein aggregation or a change in conformation. Analytically, high aggregation levels can be detected via stability studies such as size exclusion chromatography, but unknown modifications of the protein may also be immunogenic.

	However, it is difficult to establish the exact purity of the polysorbate and analytical laboratories typically assay against a specification, looking at all the different components in the mix. For polysorbates, there are standards for the presence of free fatty acids but, generally speaking, it is difficult to determine what is considered ‘pure’.

	Another challenge facing analytical scientists studying polysorbate purity and composition is the reference standards themselves. As there can be significant variation for the material from lot-to-lot, batch-to-batch, and manufacturer-to-manufacturer, it is difficult to establish a solid reference for comparison to other products and lots.

	Historically, the regulators have asked for only limited information about polysorbates in a formulation, but in recent years they have started to increase the detail they require drug formulators to provide. Analytics have, therefore, been racing to catch up. Much of the additional work now required comprises of higher analytics that are not necessarily part of the release specifications, but it is crucial to determine what the differences from batch-to-batch are, and far more importantly, whether those differences are significant.

	When developing a protein therapeutic formulation, a large amount of early screening of potential formulation is required, with varying levels of the different additives. These are subjected to different stress conditions, such as temperature and time, as well as light stress, agitation stress, and pH changes. These can all give an insight into the behaviour of the PS-80, whether it is being degraded or not, and how it may impact the protein during that accelerated stability study. 

	This information can then be used in the selection of different grades of polysorbate, which are then subjected to longer-term stability studies to evaluate the product. Importantly, these data must be tied back to the toxicology studies and clinical studies.

	Various analytical methods are available for the characterization of PS-80. However, typically, a chromatography-based assay is required to give an accurate insight into all the various components. The tests cited in the United States Pharmacopeia (USP) and European Pharmacopoeia (Ph. Eur.) really only give high-level percentages of the components, and do not allow impurities to be resolved, or components that degrade or otherwise change over time. 

	Both of the methods for PS-80 concentration determination and characterization that were developed at Catalent are based on high-performance liquid chromatography (HPLC), coupled with charged aerosol detection. This method of detection is suitable for both non-volatile and semi-volatile compounds that lack chromophores. The response is mass-dependent and independent of the chemical properties of the analytes. The quantitation method looks at the total amount of polysorbate present in the formulation; the characterization method gives more of an insight into the nature of the components in the mixture.

	To perform the experiments, PS-80 solution was subjected to various stress conditions to prepare degraded samples. A 0.01% w/v PS-80 solution in 25 mM histidine at pH 6.0 was variously exposed to thermal stress at 80 °C, base hydrolysis at pH 10 and 40 °C, oxidation with 2 mM 2,2’-azobis(2-amidinopropane) dihydrochloride (AAPH) at room temperature, and UV/fluorescent light exposure according to International Council for Harmonization (ICH) guidelines (1). The degraded samples were then spiked with bovine serum albumins or IgG monoclonal antibody, giving a final concentration of 5 mg/mL. The samples were then analysed using each of the methods.

	This fast, sensitive analytical method is based on the diluteâandâshoot approach, coupled with mixed-mode chromatography for the online separation of therapeutics and PS-80 components. PS-80 content is determined using a Waters Oasis Max column and a step gradient. 

	The result is a trace with one large peak, and the area of this can be used for quantitation against an external reference standard curve of polysorbate. Assay linearity was established in the range 12.5–100 µg/mL, or 0.00125–0.01%, with an R2 value of 0.99 by log–log plot [see 

	Degradation of PS-80 was manifested as a decrease in concentration, and this was observed in stressed samples. Calculated concentrations were normalized to their respective controls, either T = 0 or protected samples. The final relative PS-80 contents were found to be between 48% and 94%. Protected samples under light exposures had negligible changes in concentration. 

	Although it can give an insight into the degradation of polysorbate, in reality, substantial degradation will have to have occurred before it can be detected. It is, however, useful for routine product monitoring.

	The characterization method is broadly similar, as it also relies on HPLC with charged aerosol detection. However, in this case an off-line extraction is performed to remove the protein while preserving the hydrophilic POE sorbitan that elutes with protein.

	The polysorbate-containing sample is then injected straight onto an Agilent Zorbax SB-CN column, which has reverseâphase properties. This captures all the polysorbate in a long, shallow gradient, and allows all the different components to be separated. The result is a chromatographic profile or ‘fingerprint’ of the polysorbate and all its various components. This method is able to observe PS-80, POE sorbitan, various POE sorbitan esters, and oleic acid.

	The challenge is then to determine what all of those peaks are, and this can be achieved using mass spectrometry. One of the most important components to monitor is oleic acid, because of the stability problems it can cause. This method allows this individual component to be quantified. 

	Interestingly, there are noticeable differences in chromatographic profile between 7 and 16 minutes among PS-80 sourced from different vendors. This clearly shows that the chemical composition varies between the different products [

	Degradation affects all sorbitan POE esters. However, the POE sorbitan peak appeared to be unchanged under most conditions. The generation of the base hydrolysis products oleic acid and POE sorbitan was detected in the chromatographic profile (see 

	These two complementary techniques permit the quantitative and qualitative analysis of PS-80 in therapeutic products. Both methods have been shown to be sensitive and stability indicating. They are, therefore, suitable for the monitoring of the degradation of PS-80 in biopharmaceuticals as required by the regulators. Other applications for these methods include quality assessment of PS-80 raw material during manufacturing, and PS-80 content confirmation. They should also be applicable to the study of other polysorbates, notably PS-20. 

	There remain many unknowns about the behaviour of PS-80 in protein therapeutics, and future studies should include a detailed exploration of its degradation pathways, and the impact of PS-80 on the critical quality attributes of these important biopharmaceuticals.

Stability Testing: Photostability Testing of New Drug Substances and Products

	When referring to this article, please cite it as L. Deters, "Quantification and Characterization of Polysorbate-80 in Protein Formulations," 

Pharmaceutical Technology Europe’s Outsourcing Resources

Articles

HPLC coupled with charged aerosol detection is a suitable analytical technique to quantitate and characterize polysorbate-80 in therapeutic products; allowing quality assessment of raw material, content confirmation during manufacturing, and monitoring product stability.
