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Polysorbates 20 and 80 are the most used surfactants for the development of parenteral protein formulations, because of their beneficial safety and stabilisation profile. Although, showing excellent stabilisation properties, the stabilisation mechanism(s) of these surfactants for aqueous protein formulations are still unclear. Different stabilisation models have been discussed in the literature, among them the possible formation of protein-surfactant micelle complexes. This study focusses on the determination of the self-association properties of compendial grade polysorbate 20 HP (PS 20 HP) and polysorbate 80 HP (PS 80 HP) with regard to the formed micelle size (by dynamic light scattering, DLS), the concentration upon which the surfactant molecules self-associate (cmr, critical micelle concentration range) to form micelles, and the related surface tension. Surface tension and micelle size were determined as a function of temperature, as well as composition of the formulation (presence of a buffer salt and influence of ionic strength). The critical micelle concentration range values (cmr) at 25 °C are between 15–75 μM for PS 20 HP, but considerably lower for PS 80 HP with 7–16 μM, depending only slightly on the formulation composition. With increasing temperature, the cmr decreases slightly. PS 80 HP forms larger micelles (Rh = 4.5 nm) compared with PS 20 HP (Rh = 3.5 nm) (25 °C). The temperature dependency of the micelle size is more pronounced for PS 80 HP. Based on these results, the suggested stabilisation mechanism, especially for antibody formulations, by the formation of antibody-polysorbate micelle complexes, is critically discussed, and the current study shows that this stabilisation mechanism is not likely, for commonly used monoclonal antibody formulations.

Two of the most widely used surfactants to stabilize biologicals against e.g. interfacial stresses are polysorbate 20 (PS20) and polysorbate 80 (PS80). In recent years, numerous cases of hydrolytic polysorbate (PS) degradation in liquid formulations of biopharmaceuticals have been observed. Concomitant with the degradation of PSs, formulated proteins become inherently instable and more susceptible to aggregation. Furthermore, poorly soluble fatty acids (FA) are released from the PSs, which might lead to FA precipitation and the formation of visible and subvisible particles. Therefore, possible particle inducing factors have to be monitored closely. The major root cause of hydrolytic PS degradation in biologicals is the presence of enzymatic active host cell proteins (HCP), like lipases and esterases, which are co-purified with the active pharmaceutical ingredient. Such contaminants can be detected via their hydrolytic activity, either using ester-based substrates or PS itself. However, each approach has its up- and downsides, which makes the comparison of the results from other publications difficult. It was therefore the aim of the present study to investigate the impact of lipase specificities on the assay readouts. This study evaluates three different surrogate (model) lipases with distinctively different degradation kinetics and substrate specificities using specific analytical methods. The analytical panel contains on one hand two lipase activity assays with ester-based substrates, either detecting the release of para-nitrophenol or 4-methylumbelliferone, and on the other hand two PS-based monitoring analyses (fluorescence micelle assay and reverse phase high performance liquid chromatography - charged aerosol detection), which detect hydrolytic "activity" directly in the target substrate. Thereby, strengths and weaknesses of each assay are discussed, and recommendations are made for the respective use cases. Our results show that the determined lipase activities vary not only from assay to assay, but also significantly for the lipase tested, thus showing a different degradation fingerprint in the RP-HPLC-CAD chromatogram. This demonstrates that a comprehensive monitoring approach is essential to assess potential HCP contaminations.

The present study investigated the photodegradation of three different monoclonal antibodies (mAb) by visible light. Several chromatographic techniques, such as size-exclusion and hydrophobic interaction chromatography as well as mass spectrometry were used to measure relative changes of various oxidation related monoclonal antibody species. The results show that visible light is indeed capable of inducing the formation of protein photo-oxidation products, such as acidic, basic, hydrophilic, and several other protein species with altered physicochemical properties. Although, the formation rate of degradants of these three protein species was dependent on the light source’s intensity (I), their yield is clearly correlated to the applied light dosage (ld), which is defined as the product of light intensity I and irradiation time t (light dosage = I·t). Hence, our findings indicate that the degradation of monoclonal antibodies can be described according to the Bunsen-Roscoe reciprocity law. This correlation can be useful to assess the impact of photodegradation of biologics with regards to changes in light intensity and/or duration of light exposure of the protein, e.g. during the manufacturing of biologics.

Two of the most widely used surfactants to stabilize biologicals against e.g. interfacial stress are polysorbate 20 (PS20) and polysorbate 80 (PS80). In recent years, polysorbate degradation in biopharmaceutical formulations has been observed. Polysorbate (PS) is mainly composed of sorbitan and isosorbide fatty acid (FA) esters, varying in their FA composition. Especially hydrolysis, which can be induced chemically as well as enzymatically, leads to the release of FAs from PS. These FAs are poorly soluble in aqueous buffer systems due to their hydrophobic nature and therefore prone to precipitation and particle formation. Since the emergence of particles in liquid formulations has to be avoided, it is important to prevent their formation. This study evaluates the solubility limits of selected FAs, which are likely to be released during the degradation of PS20 and PS80 in the presence of defined PS concentrations. Our results show that the solubility is highly dependent on the pH, the temperature, the used PS concentration and the aliphatic chain of respective FAs. Solubility of FAs, such as palmitic and oleic acid under the conditions determined in this study, are in the range of 3–130 µg·ml−1 (12–460 µM). Furthermore, the results allow making an estimation to which extent PS may degrade before particle formation in the drug product may be expected.

Surfactants are used to stabilize biologics. Particularly, polysorbates (Tween® 20 and Tween® 80) dominate the group of surfactants in protein and especially antibody drug products. Since decades drug developers rely on the ethoxylated sorbitan fatty acid ester mixtures to stabilize sensitive molecules such as proteins. Reasons are (i) excellent stabilizing properties, and (ii) well recognized safety and tolerability profile of these polysorbates in humans, especially for parenteral applications. However, over the past decade concerns regarding the stability of these two polysorbates were raised. The search of alternatives with preferably less reservations concerning degradation and product quality reducing issues leads, among others, to poloxamer 188 (e.g. Kolliphor® P188), a nonionic triblock-copolymer surfactant. This review sums up our current knowledge related to the characterization and physico-chemical properties of poloxamer 188, its analytics and stability properties for biological formulations. Furthermore, the advantages and disadvantages as a suitable polysorbate-alternative for the stabilization of biologics are discussed.

The enzymatic hydrolysis of polysorbates, e.g. induced by specific host cell proteins in biologics, is a known risk factor regarding the potential particle formation in the product over time. One of the root causes for this observation is an increase in free fatty acids (FA) within the formulation, which indicates the need for convenient monitoring of FA release. This study presents a novel UPLC-QDa based method to evaluate the content of the FAs esterified to polysorbate 20 (PS20) after hydrolysis. The presented method is label-free, i.e. independent of elaborate fluorophore-labeling and able to directly measure the ionized FAs. Furthermore, the method allows the determination of released FAs as percentage of ester bond hydrolysis and as absolute concentration expressed in ng/mL. Additionally, we describe for the first time in FA analytics the application of an isolator column, to remove trace levels of FAs present in the eluents to improve the sensitivity of the method. Lastly, the capabilities of the newly developed method are proven in case studies with three different monoclonal antibodies, which display characteristic FA release patterns in PS20-containing formulations. In summary, we developed a reliable, sensitive method for FA quantification in biologics, which could also be used as a predictive tool, considering FA solubility, regarding the formation of particles.

Polysorbates are widely used as non-ionic surfactant in biopharmaceutical formulations. Recently, the degradation of polysorbate moved into the focus of attention, because in several published studies it was described, that stability issues in polysorbate containing formulations were observed leading to the formation and appearance of sub-visible and visible particles. For this reason, monitoring of polysorbate and its degradation products is of importance throughout the development of parenterals. The aim of the study was to develop a method for the selective marker-based quantification of adequate polysorbate 20 components of interest without the need to apply derivatization or complex detection techniques. A single quadrupole mass (QDa) detector was used coupled to an ultra-high performance liquid chromatography (UHPLC) system. Method development was based on a developed reversed phase-high performance liquid chromatography assay coupled to a charge aerosol detector (RP-HPLC CAD). Instead of a charge aerosol detector (CAD) a QDa detector was used in order to significantly improve the selectivity. The focus of this study is the development of the QDa based method for the analysis of polysorbate 20. Modifications of the mobile phase and the type of chromatography column allowed the separation of several components of polysorbate 20 from polar non-esterified to apolar higher order species. In addition, a multitude of components could be quantified by their individual m/z values. The peak assignment identified 676 compounds which originated from polysorbate 20. Some of these were selected and defined as marker components. It was shown that the developed method is capable to determine polysorbate 20 in different biopharmaceutical formulations. The proposed assay is based on an artful sample preparation as well as a unique calibration procedure that make the determination of several selected components achievable. Furthermore, it was successfully demonstrated that the analytical procedure is valid to reliably quantify several polysorbate 20 components at its 100% level (corresponds to 0.4 mg/mL intact polysorbate 20) and even at lower concentrations that occur e.g. in case of polysorbate 20 degradation. In conclusion, the method is beneficial to determine selected polysorbate 20 species during formulation development of biopharmaceuticals as well as during stability testing and trouble shooting.

Polysorbate is one of the most commonly employed non-ionic surfactant in protein containing biological formulations, whereby, it can stabilize these biomolecules under different stress conditions. Despite the fact that polysorbates are present in almost 70% of currently marketed parenteral biological drugs, polysorbate degradation in biopharmaceutical formulations has emerged as a specific quality concern. Different degradation pathways have been explored in the recent years with the aim of understanding the root cause for polysorbate degradation in biopharmaceutical formulations. In an attempt to explore hydrolytic degradation of polysorbates in accelerated degradation conditions, we studied extreme pH conditions. We investigated specific polysorbate degradation profiles depending on acidic or alkaline solution conditions. The acidic and alkaline hydrolysis of polysorbate is monitored for the total content using a fluorescence micelle assay (FMA). Additionally, the compositional changes in polysorbates were detected using reversed phase high performance liquid chromatography coupled to a charged aerosol detector (RP-HPLC-CAD). We show that the stability of polysorbate against chemical hydrolysis is dependent upon selected pH condition and differ for polysorbate 20 and polysorbate 80. Additionally, we were able to show that a degradation pathway dependent fingerprint may support the identification of the degradation root cause.