Leveraging mass detection to simultaneously quantify surfactant content and degradation mode for highly concentrated biopharmaceuticals.

Non-ionic surfactants are commonly used in parenteral protein formulations and include polysorbate 20, polysorbate 80 and poloxamer188. Recently, quantification and characterization of surfactants has generated considerable interest due to their connection to visible particle formation, a critical quality attribute for parenteral formulations. Typically, surfactant quantification is performed by mixed mode chromatography with evaporative light scattering detection (ELSD) or charged aerosol detection (CAD). However, these methods often suffer from loss of specificity in highly concentrated protein formulations. Here we present a mixed mode chromatography method using single quad mass detection, overcoming current limitations for highly concentrated proteins. In addition to content determination of intact surfactants, this method allows to quantify and characterize the predominant degradation patterns of polysorbates within a single measurement. Formulations with up to 200 mg/mL active pharmaceutical product (API) containing surfactant levels between 0.16 and 0.64 mg/mL were tested during method qualification. The obtained results for linearity (r > 0.99), precision (max. 3.8 % RSD) and accuracy (96-116 % recovery) meet current requirements for pharmaceutical products as defined in ICH Q2.

Screening techniques for monitoring the sub-visible particle formation of free fatty acids in biopharmaceuticals.

Free fatty acid (FFA) particles that originate from the enzymatic hydrolysis of polysorbate (PS) via co-purified host cell proteins generally appear abruptly in drug products during real-time (long-term) storage. Efforts were taken to understand the kinetics of FFA particle formation, aiming for a mitigation strategy. However, it is rather challenging particularly in the sub-visible particle (SVP) range, due to either the insufficient sensitivity of the analytical techniques used or the interference of the formulation matrices of proteinaceous drug products. In this study, we examined the feasibility of Raman microscopy, backgrounded membrane imaging (BMI) and total holographic characterization (THC) on the detection of FFA sub-visible particles (SVPs). The results indicate that THC is the most sensitive technique to track their occurrence during the course of PS hydrolysis. Moreover, with this technique we are able to distinguish different stages of FFA particle formation in the medium. In addition, a real time stability study of a biopharmaceutical was analyzed, demonstrating the viability of THC to monitor SVPs in a real sample and correlate it to the visible particles (VPs) occurrence.

Simultaneous quantification of polysorbate 20 and poloxamer 188 in biopharmaceutical formulations using evaporative light scattering detection.

Polysorbates and Poloxamer 188 constitute the most common surfactants used in biopharmaceutical formulations owing to their excellent protein-stabilizing properties and good safety profiles. In recent years, however, a vast number of reports concerning potential risk factors closely related with their applications, such as the accumulation of degradation products, their inherent heterogeneity and adsorption effects of proteins at silicon/oil interfaces have drawn the focus to potential alternatives. Apart from tedious efforts to evaluate new excipient candidates, the use of mixed formulations leveraging combinations of well-established surfactants appears to be a promising approach to eliminate or, at least, minimize and postpone adverse effects associated with the single compounds. Due to the similar molecular properties of non-ionic surfactants, however, baseline separation of these mixtures, which is mandatory for their reliable quantification, poses a great challenge to analytical scientists. For this purpose, the present work describes the development of a robust mixed-mode liquid chromatography method coupled to evaporative light scattering detection (mixed-mode LC-ELSD) for simultaneous determination of the (intact) Polysorbate 20 and Poloxamer 188 content in biopharmaceutical formulations containing monoclonal antibodies. Extensive qualification and validation studies, comprising the evaluation of method specificity, robustness, linearity, accuracy and precision according to ICH guidelines, demonstrated its suitability for quality control studies. A case study on the storage stability of a formulated antibody was conducted to underline the method's practical utility. Finally, the versatility of the developed approach was successfully tested by quantifying Polysorbate 20-related surfactants, such as Polysorbate 80 and super-refined Polysorbate.

Fast and Automated Characterization of Antibody Variants with 4D HPLC/MS.

Characterization of unknown monoclonal antibody (mAb) variants is important in order to identify their potential impact on safety, potency, and stability. Ion exchange chromatography (IEC) coupled with UV detection is frequently used to separate and quantify mAb variants in routine quality control (QC). However, characterization of the chromatographic peaks resulting from an IEC separation is an extremely time-consuming process, involving many cumbersome steps. Presented here is an online four-dimensional high performance liquid chromatography-mass spectrometry (4D HPLC/MS) approach, developed to circumvent these limitations. To achieve this, a 2D HPLC system was extended through the introduction of additional modules, hence enabling fully automated bioseparation of mAbs, fractionation of peaks, reduction, tryptic digestion, and reversed-phase (RP) separation of resulting peptides followed by MS detection. The entire separation and analytical process for an unknown peak is performed in less than 1.5 h, leading to a significant time savings, with comparable sequence coverage. To show the comparability with the traditional offline process, a proof of concept study with a previously characterized mAb1 is presented in this paper.

Tacky cyclic olefin copolymer: a biocompatible bonding technique for the fabrication of microfluidic channels in COC.

Cyclic olefin copolymer (COC) is widely used in microfluidics due to its UV-transparency, its biocompatibility and high chemical resistance. Here we present a fast and cost-effective solvent bonding technique, which allows for the efficient bonding of protein-patterned COC structures. The bonding process is carried out at room temperature and takes less than three minutes. Enzyme activity is retained upon bonding and microstructure deformation does not occur.

DC- and RF-GD-OES measurements of adsorbed organic monolayers on copper.

Our direct current (DC)- and radiofrequency glow discharge optical emission spectroscopy (RF-GD-OES) measurements of adsorbed organic monolayers were inspired by the work of Shimizu et al., who presented the first example of depth profile analysis of an adsorbed monolayer by RF-GD-OES in 2004. The great potential of RF-GD-OES for analyses of layers with thicknesses in the subnanometer range was surprising. Shimizu et al. discussed not only the qualitative detection of atoms of the organic monolayer (C, H, N, S), but also the determination of the different orientation of the molecules relative to the surface due to a significant peak sequence. This latter assumption was questioned in the analytical community. We intend to demonstrate the potential of the GD-OES technique for surface analysis in terms of reliability and reproducibility by using an advanced vacuum instrumentation and presputtering with silicon. It will be shown that comparable measurements can be reproduced not only with RF-GD-OES but, above all, also with DC-GD-OES. The experimental steps to adsorb thiourea molecules on a copper substrate are described in detail. Further experiments with other organic molecules, e.g. benzotriazole (BTA) or benzothiazole (BTH), disprove the predicted correlation between the orientation of the molecules relative to the surface and the occurrence of peak separation. Ultimately, a quantification of compounds of the organic monolayer in the case of adsorbed thiourea is achieved.