All-in-one stability indicating polysorbate 20 degradation root-cause analytics via UPLC-QDa.

Polysorbates (PS) are the most frequently used surfactants to stabilize biologicals. Ironically, these excellent stabilizing non-ionic surfactants have inherent structural properties, which lead to instabilities of their own. Such PS degradation can be triggered by multiple root-causes, like chemical and enzymatic hydrolysis or oxidative degradation. This can on the one hand reduce the concentration of surface-active PS and on the other hand lead to the formation of unfavorable degradants, like poorly soluble free fatty acids (FFA), which may phase separate and form visible FFA particles. Due to the potential criticality of PS degradation in biopharmaceutical formulations, various analytics have been established in recent years not only to monitor the PS content but also to evaluate specific PS markers and crucial degradants. However, in most cases sample preparations and several analytical assays have to be conducted to obtain a comprehensive picture of potential PS degradation root-causes. Here we show a novel approach for PS degradation UPLC-QDa based root-cause analytics, which utilizes previously established analytics for (i) most relevant polysorbate 20 (PS20) esters, (ii) PS20 free fatty acids and (iii) a newly developed method for the evaluation of PS20 specific oxidation markers. Thereby, this triad of analytical methods uses the same sample preparation and detector, which reduces the overall necessary effort, time investment and sample volume. Furthermore, the innovative PS20 oxidation marker method allows to quantify specific concentrations of the determined markers by external calibration and possible perception of oxidative degradation processes prior to relevant losses of PS20 esters, which could serve as an early indication during formulation development. The applicability of this method set was verified using several PS20 containing stress samples, which cover the most relevant root-causes, including acidic and alkaline hydrolysis, enzyme mediated hydrolysis, oxidative AAPH stress and Fe2+/H2O2 mediated degradation as well as autoxidation via long-term storage at elevated temperatures. Overall, this analytical setup has shown to deliver in-depth data about PS20 degradation, which can be used to narrow down the causative stress without the necessity of fundamentally different methods. Therefore, it can be seen as all-in-one solution during sometimes troublesome development of biopharmaceutical formulations, that supports the elucidation of the PS degradation mechanism(s) and thus establish mitigation strategies.

Visible light triggers the formation of reactive oxygen species in monoclonal antibody formulations.

Most monoclonal antibody formulations require the presence of a surfactant, such as polysorbate, to ensure protein stability. The presence of high concentrations of polysorbate have been shown to enhance photooxidation of certain protein drug products when exposed to visible light. The current literature, however, suggest that photooxidation of polysorbate only occurs when exposed to visible light in combination with UVA light. This is probable as peroxides present in polysorbate solutions can be cleaved homolytically in the UVA region. In the visible region, photooxidation is not expected to occur as cleavage of peroxides is not expected at these wavelengths. This report presents findings suggesting that the presence of one or more photosensitiser(s) in polysorbate must be a cause and is required to catalyse the aerobic oxidation of polysorbate solutions upon exposure to visible light. Our investigation aimed to clarify the mechanism(s) of polysorbate photooxidation and explore the kinetics and the identity of the generated radicals and their impact on monoclonal antibody (mAb) degradation. Our study reveals that when polysorbate solutions are exposed to visible light between 400 - 800 nm in the absence of proteins, discolouration, radical formation, and oxygen depletion occur. We discuss the initial formation of reactive species, most likely occurring directly after reaction of molecular oxygen, with the presence of a triplet state photosensitiser, which is generated by intersystem crossing of the excited singlet state. When comparing the photooxidation of PS20 and PS80 in varying quality grades, we propose that singlet oxygen possesses potential for reacting with unsaturated fatty acids in PS80HP, however, PS20HP itself exhibited no measurable oxidation under the tested conditions. The study's final part delves into the photooxidation behaviour of different PS grades, examining its influence on the integrity of a mAb in the formulation. Finally, we examined the effect of photooxidation on the integrity of monoclonal antibodies. Our findings show that the exposure to visible light in polysorbate-containing mAb solutions at high PS concentrations of 4 mg·ml-1 results in increased monoclonal antibody degradation, highlighting the need for cautious evaluation of the correct PS concentration to stabilise protein therapeutics.

Towards more tolerable subcutaneous administration: Review of contributing factors for improving combination product design.

Subcutaneous (SC) injections can be associated with local pain and discomfort that is subjective and may affect treatment adherence and overall patient experience. With innovations increasingly focused on finding ways to deliver higher doses and volumes (≥2 mL), there is a need to better understand the multiple intertwined factors that influence pain upon SC injection. As a priority for the SC Drug Development & Delivery Consortium, this manuscript provides a comprehensive review of known attributes from published literature that contribute to pain/discomfort upon SC injection from three perspectives: (1) device and delivery factors that cause physical pain, (2) formulation factors that trigger pain responses, and (3) human factors impacting pain perception. Leveraging the Consortium's collective expertise, we provide an assessment of the comparative and interdependent factors likely to impact SC injection pain. In addition, we offer expert insights and future perspectives to fill identified gaps in knowledge to help advance the development of patient-centric and well tolerated high-dose/high-volume SC drug delivery solutions.